Annals of the Missouri Botanical Garden Volume I EL б With ‘Twenty-six Plates sed: One Figure Published quarterly by The Rumford Press, Concord, N. H. for the Board of Trustees of the Missouri Botanical Garden, St. Louis, Mo. 24085 v bur MEE 2 6 o ir^ Gi. rain: Annals of the Missouri. Botanical Garden A Quarterly Journal containing Scientific Contributions from the Missouri Botanical Garden and the Graduate Labora- tory and Faculty of the Henry Shaw School of Botany of Washington University in affiliation with the Missouri Botanical Garden. Editorial Committee George T. Moore Benjamin M. Duggar Jacob R. Schramm Information The Annals of the Missouri Botanical Garden appears four times during the calendar year, March, May, September, and November. Four numbers con- stitute a volume. 402.90, 44242 : Subscription Price Single Numbers . The following agent із" ўл William Wesle sex Street, Strand, London. E аара РА d o qu ам ағала s MISSOURI BOTANICAL GARDEN ЕРТЕ?! ао SiT i ATUM 8o £X $ масадан C xor. <>. ignc алы Ағалы тана TABLE OF CONTENTS ас ОИК S 1-1 Тһе effect of surface films and dusts on the rate of transpiration (with plate ЖЕН ODER 2 B. M. Duggar and J. S. Cooley Some pure culture methods in the alge......... The identifieation of the most characteristic sali- vary organism and its rela- tion to the pollution of air (with plate2) .......... Тһе Polyporacez of Ohio. . A contribution to our knowl- edge of the relation of cer- tain species of grass-green alge to elementary nitro- gen (with one figure and SEM usd The Thelephoraces of North America. I. (with plates 4 850 8)....... 2.22 Indieations regarding the source of combined nitro- gen for Ulva Lactuca.... 'The effect of certain condi- tions upon the acidity of Jacob R. Schramm August G. Nolte L. O. Overholts Jacob R. Schramm Edward A. Burt G. L. Foster tomato fruits....B. M. Duggar and M. C. Merrill А method for the differential staining of fungous and hos o8 .............. R. E. Vaughan 47-80 81-155 157-184 185-228 229—235 231—240 241-242 TABLE OF CONTENTS Two trunk diseases of the mesquite (with plates 6 BET ues sn А trunk disease of the Шас (with plates 8 and 9)... .. Descriptions of North Amer- ican Senecionee (with plates 10, 11, 12, 13, and Pte I A study of the physiological relations of Sclerotinia cin- erea (Bon.) Schréter..... The Thelephoraces of North America. II. Craterellus (with plates 15,16, and 17) The effects of surface films on the rate of transpira- tion: experiments with potted potatoes (with plate BR cin. B. M. Duggar and J. S. Cooley The Thelephoraces of North America. III. Craterellus borealis and Cyphella (with plate 19)......... Some oenotheras from Che- shire and Lancashire (with Hermann von Schrenk Hermann von Schrenk J. M. Greenman J. Б. Cooley Edward A. Burt Edward A. Burt plates 20, 21, and 22).... R. R. Gates A Texan species of Megap- terium (with plate 23).... R. R. Gates Diagnoses of flowering plants, chiefly from the southwest- ern United States and Mexico (with plates 24, 25 and 26) J. M. {Greenman and С. Н. Thompson Enzyme action in Fucus ve- siculosus L........ B. M. Duggar and A. R. Davis с index ido volume I..... ee PAGE 243-252 253-262 263-290 201-326 327-350 351-356 357-382 383-400 401-404 405-418 419-426 427—432 STAFF OF THE MISSOURI BOTANICAL GARDEN Director, GEORGE T. MOORE. ВемјЈАМІҸ M. DUGGAR, Epwarp A. Bort, Plant Physiologist. Mycologist and Librarian. HERMANN VON SCHRENK, JacoB В. SCHRAMM, Plant Pathologist. Assistant to the Director. JESSE M. GREENMAN, CHARLES Н. THOMPSON, Curator of the Herbarium. Assistant Botanist. Me tvin С. MERRILL, Research Assistant. BOARD OF TRUSTEES OF THE MISSOURI BOTANICAL GARDEN President, EDWARDS WHITAKER. Vice-President, DAVID 8. H. SMITH. Epwarp C. Error. SAUNDERS NORVELL. Свовав C. Нітснсоск. WiLLiAM H. H. PETTUS. P. СноотЕАО Marrirr. Рнішр C. SCANLAN. LEONARD MATTHEWS. Joun Е. SHEPLEY. EX-OFFICIO MEMBERS: EDMUND A. ENGLER, Henry W. Кв, President of the Academy of Science Mayor of the City of St. Louis. of St. Louis. Davip Е. Houston, Herman MavcH, Chancellor of Washington University. President of the Board of Public Schools- of St. Louis. DANIEL S. TUTTLE, Bishop of the Diocese of Missouri. А. D. Соммменам, Secretary. Annals of the Missouri Botanical Garden ---------- Vor. I MARCH, 1914 No. 1 INTRODUCTION In order to provide for the printing of scientific papers, which formerly constituted a large part of the volume known as the Annual Report, the Board of Trustees has authorized а new journal, to be known as the ANNALS OF THE Missouni BOTANI- CAL GARDEN. Тһе Annals will appear four times а year, in March, May, September, and November, and contain only scientific contributions from members of the staff of the Garden, from the faculty and graduate students of the Henry Shaw School of Botany of Washington University, and from vis- iting botanists doing all or a part of their work at the Garden. The increase in original contributions available for publication, due to the additions to the staff and the greater number of graduate students, makes it no longer possible to follow the practice of the past and print papers from sources other than the Garden. The publication of а monthly bulletin by the Missouri Botanieal Garden, in which appear promptly the annual re- ports of the officers of the Board, and of the Director, together with popular accounts of the various activities of the Garden; and the provision for the printing of scientific papers in the Annals, has made it advisable to discontinue the Annual Report, which was published each year from 1890 to 1912. Тһе Twenty- third Annual Report, therefore, marks the close of this series. The Annals will be maintained upon a strict subscription basis, using it in exchange only when its equivalent can be obtained. Some of the institutions and societies, the publi- cations of which have been received in exchange for the Annual (i) il INTRODUCTION Report, issue nothing of value to a botanical library and appar- ently are not interested in botany or related sciences. These have been stricken from the exchange list. On the other hand, the receipt of this number of the Annals is an indication that the Missouri Botanical Garden desires to continue the old exchange arrangement, the new journal being sent four times a year in place of the old Annual Report. Additional exchanges with publishers of journals dealing directly with botany are desired. Upon request, the monthly BULLETIN will be substi- tuted for the Annals as an exchange with those desiring a more popular and general account of the work and scope of the Missouri Botanical Garden. Сеовсе Т. Moore Direclor. THE EFFECT OF SURFACE FILMS AND DUSTS ОХ THE RATE OF TRANSPIRATION B. M. DUGGAR Physiologist to the Missouri Botanical Garden Professor of Plant Physiology in the Henry Shaw School of Botany of Washington University AND J. S. COOLEY Rufus J. Lackland Fellow in the Henry Shaw School of Botany of Washington University The fungicides commonly employed are either in the form of solutions (e. g., ammoniacal copper carbonate), suspensions (lime wash and Bordeaux mixture), and powders (sulphur). The use of spray mixtures or other fungicides has become world wide, and many problems of physiological interest have arisen respecting the effects of these substances on the plants which they are designed to protect. Bordeaux mixture has been under continuous observation for a period of about twenty years, and has proved interesting in both its toxic and other relations. The striking influence of this fungicide upon sound plants has awakened widespread interest, and numerous experiments have been made to determine the nature of the effects. Bordeaux mixture consists essentially of suspension films of copper hydroxid and certain other complex (mostly hydrated), largely insoluble, copper compounds; and when properly sprayed upon plant surfaces from the best nozzles, the particles are of extreme fineness, and there is realized an almost perfect surface film. In spite of the greatest care in preparation and application, it is injurious to certain plants, such as the peach and the plum, and may not be used satis- factorily in such cases for disease control. In recent years it has been shown that the extent of the injury to the apple and other plants may be considerable, and Bordeaux mixture is in such cases being supplanted. In this discussion, however, we may omit any detailed consideration of the toxic effects. of this mixture, a phase of the subject which has received much consideration in this country from Bain (2), Crandall (6), Clark. Ann. Mo. Bor. Garp., VoL. 1, 1914 (1) [Vor. 1 2 ANNALS OF THE MISSOURI BOTANICAL GARDEN (4), Swingle (21), and others. Moreover, with the exception of incidental references, we wish to deal at this time only with its physiological action in prolonging the vitality of leaves and plants. During the first years of the use of this spray mixture it was natural that any increased vitality of the sprayed plants would be attributed merely to the action of the fungicide in restrain- ing fungous or insect pests. Indeed, we find no authentic suggestion of any other effect than that mentioned for eight or ten years after the discovery of this fungicide. Since 1892 there have been frequent observations indicating beyond any reasonable doubt that in the absence of all disease-producing organisms there is often prolonged vitality of the sprayed plants as contrasted with the unsprayed. The increased longevity is partieularly noticeable in plants like the potato, in which, under normal conditions, the foliage frequently dies in advance of the first killing frost. Nevertheless, lengthened life in leaves of deciduous trees, notably of the apple, has like- wise been reported. It is not always possible to state definitely to just what extent any apparent increased vitality is to be attributed to the physiological action of the fungicide rather than to the control of pests, and it must be said that the fre- quency of the phenomenon and the reliability of the observers alone preclude the possibility of constant errors in this matter. In practical field experimentation the most significant differ- ences in yield and vitality as a result of spraying with Bordeaux mixture have been evident in the case of the potato, and with this crop it is a matter of common observation both in Europe and America. In recent years the consecutive reports on potato spraying by F. C. Stewart (19) and his associates at Geneva, New York, suggest in a decisive way the probable magnitude of the Bordeaux influence when disease is a minor factor. In general, observers are perhaps liberal in their estimates of the gain from fungous suppression. It will be pertinent to note a few observations and comments from the reports of the work done at Geneva. In 1904 the increase in yield from spraying potatoes five times was 233 bushels per acre. ‘Spraying prolonged the life of the plants 25 days. Late blight was the only trouble.” In his experiments 19141 DUGGAR AND COOLEY—FILMS AND TRANSPIRATION 3 of 1906 Stewart notes an increase in yield of 63 bushels per acre due to spraying five times. Не remarks: “Late blight, early blight, flea beetles and tip burn were all factors in this experiment, but none of them caused much damage." More striking were the results the following year when an increase in yield of 732 bushels per acre was obtained from spraying five times. In this case it is reported: Late blight and rot were wholly absent and early blight appeared only in traces. "There was some tip burn and a light attack of flea beetles. Considering the seemingly small amount of damage done by blight and insects, it is remarkable that spraying should have increased the yield so much." Іп 1909 the increase in yield in spraying six times was 492 bushels per acre, and the comment upon this result is as follows: * Early blight, late blight and rot were all absent. Some injury from flea beetles was noticeable through- out the season. After September 1 there was considerable tip burn. As late as September 24 the difference between sprayed and unsprayed rows appeared slight. Тһе sprayed rows held most of their foliage until killed by frost on October 14." Тһе senior author of this report visited the experimental plats which afforded these data in late September, 1911, prior to the killing frosts of October 27, and the contrast between the sprayed and unsprayed rows was pronounced; at the same time there was very little evidence of any disease on the un- sprayed plats. Regarding the condition of the plants Stewart says: "There was no late blight whatever, only a very little early blight, and very little flea beetle injury. Тһе unsprayed rows were affected by no disease of any consequence except tip burn, and even of that there was only a moderate amount. As the plants were still partially alive twenty weeks after plant- ing it is clear that they could not have been very much injured by anything. Yet spraying increased the yield at the rate of 93 bushels per acre. Plainly we have here a striking example of the beneficial influence of Bordeaux in the absence of diseases and insect enemies. ” Examining the comments of these and of other investigators regarding increased vitality as a result of spraying with Bor- deaux, we find that where the condition of the plant is well [Vor. 1 4 ANNALS OF THE MISSOURI BOTANICAL GARDEN defined at the close of the season, or at the time of the first killing frost, the sprayed plants are almost invariably more vigorous. Often, in the practical absence of any disease, sprayed plants may remain healthy until killed by frost, while unsprayed plants may have died from a few days to а few weeks in advance of frost. Following a recital of notable increases of yield in Connecticut as a result of spraying potatoes with Bordeaux, Clinton (5) ex- presses the conviction that an explanation must be found in the conservation of water. His statement follows: “The question naturally comes up, why did the sprayed potatoes give this increased yield over the unsprayed if there was no particular injury caused by the late blight fungus? Some little benefit was no doubt derived from the prevention of the early blight, but this must have been scarcely appreciable because this fungus was not at all conspicuous these years. Again, some very small benefit may have been due to lessening insect attack, since potatoes sprayed with both Bordeaux and Paris green keep off the insects somewhat better than where sprayed only with Paris green. This is especially true as regards the potato flea beetle. But here again the gain was of a very minor kind. .Ordinarily botanists have explained this increase as due to some stimulative effect the Bordeaux mixture has on the chlorophyll of the potato leaves in increasing starch pro- duction. Personally, the writer believes that the results are largely due to conservation of moisture in the leaves in dry seasons by clogging up the stomata and water pores with the sediment of the spray. The reasons for this belief are (1) that the potato leaves, through their numerous stomata and terminal water pores, lose water very easily, and are especially susceptible to what is known as tip burn in dry seasons; (2) that the un- sprayed vines uniformly suffered earlier and more severely from tip burn than the sprayed, which were green for about two weeks after the unsprayed were dead; (3) that in 1910, which was a season like the preceding years, except with а little injury from blight at the very end of the season, spraying with ‘Sulphocide’ and commercial lime-sulphur, sprays with comparatively little sediment, did not prolong the life of the 1914) DUGGAR AND COOLEY—FILMS AND TRANSPIRATION 5 vines or give increased yield, while spraying with Bordeaux mixture did." Although this theoretical explanation did not come to our attention until the experimental work reported in this paper was complete, it was, in modified form, the only possible opinion which we felt inclined to advocate, as а clue to the increased longevity caused by Bordeaux, until the contrary evidence yielded by our experiments. Review ОҒ LITERATURE Тһе experimental work undertaken in the past to determine the nature of the Bordeaux influence (apart from direct injury) has touched mainly upon (1) questions of increased photo- synthesis due either to ‘‘stimulation” of chloroplastid or chlorophyll development, or to a direct influence upon light quality; (2) changes in the respiratory rate, or surmised effects upon metabolism; and (3) a modification of the normal rate of transpiration. A few observations from the extensive liter- ature with particular reference to its bearing on transpiration may be cited. Rumm (16) finds that in sprayed grapes the chlorophyll content of the leaves increases and the fruit ripens earlier with a higher sugar percentage. Не attributed these phenomena to the higher *assimilatory activity," and in turn relates this to the following observation on transpiration:—that abscised, sprayed twigs remain fresh longer than those unsprayed, from which it is deduced that there is a falling off in transpiration as a result of spraying. Through independent observations made during the same year, Müller-Thurgau (15) and Bayer (3) subscribe to the view that lessened transpiration follows spraying. Moreover, this confirmation of Rumm is obtained by the former through an experiment which also proclaims that the reduction in transpiration as а result of spraying may be as much as forty per cent. Nevertheless, the report referred to is extremely brief and does not indicate clearly the condition of the plants during the period of observation, a matter most important in the final interpretation of the data afforded. Frank and Krüger (9, 10) reported some rather extensive [Vor. 1 6 ANNALS OF- THE MISSOURI BOTANICAL GARDEN quantitative experiments as a result of which they conclude, contrary to Rumm, that transpiration is accelerated by spray- ing. They state that sprayed leaves are in general more robust, thicker and stiffer. They also report an increased yield in pot experiments from spraying. АП these indications, as well as those of Leydheker (13) and others (1, 12) denote differences of yield which are so slight as to be of no fundamental impor- tance in the present consideration. Nevertheless, the trans- piration data of Frank and Krüger, as already observed, were obtained by satisfactory methods, and these are of greater interest when taken in conjunction with those of Zucker (22) who confirms their results entirely. Schander (18) in an extensive paper reports a comparatively small amount of experimental work on transpiration, but in the cases given his results indicate a retardation of water loss after spraying. His experiments with cobalt paper were incon- sistent, and twigs of Taxus baccata and potted bean plants were then employed, yielding the positive results noted. How- ever, his work embraced very few plants, and the transpiration differences observed are inconsiderable. He suggests that lessened transpiration of sprayed plants is to be expected, since the Bordeaux mixture must exert a shading influence as a result of the exclusion from the leaf of certain injurious rays. He attempts to verify this assumption of partial shad- ing by a study of leaf temperatures, but the experiments in this direction give no positive evidence for his theory. No adequate mention is made of the conditions surrounding these experiments, nor of the precautions observed. Ewert’s (8) experiments tend to substantiate the views of Rumm and Schander; but, unfortunately, the results are not satisfactory for accurate quantitative purposes, since evaporation from the pots was merely checked and not pre- vented,batting being employed to cover the soil surfaces. His experiments are of particular interest, however, with respect to his graph for comparative respiration in sprayed and un- sprayed plants. In the sprayed plants, respiration was found to be distinctly lower than in the unsprayed. It will be noted, however, that this diminished respiration is scarcely in keeping 19141 DUGGAR AND COOLEY—FILMS AND TRANSPIRATION 7 with the observation of Rumm and others regarding the higher assimilatory activity in sprayed plants. It is unnecessary in this report to review the considerable literature which has accumulated bearing on the question of increased starch formation as a result of the application of Bordeaux mixture, especially as it is proposed to discuss this phase of the subject in a later paper. METHODS As indicated in the title, the experimental work here reported is concerned merely with the transpiration of sprayed and un- sprayed leaves or plants. Other effects of sprays and dusts may be communieated in subsequent reports. In general, the methods involved are modifications of customary practices. The methods used were of two types, the experiments being carried out either by means of (1) leaves in burette potometers connected with side arm flasks, or (2) potted tomato plants. Potometer Experiments.—After much preliminary experimen- tation with a view to determining suitable leaves or twigs for potometer work, leaves of the castor bean were selected. Some of the preliminary experiments with other leaves are of interest, however, and will be referred to subsequently. Castor bean (Ricinus communis) leaves offer some special advantages, especially (1) large surfaces, (2) resistance towards Bordeaux mixture, and (3) prolonged vitality after abscision. The burettes were connected with the side arm flasks, as indicated in plate 1, and the flasks completely filled with water. The petioles of the leaves were cemented into the mouths of the flasks by means of “‘plastolina.” If a ring of this plastic substance is placed around the mouth of the flask when the glass is dry and a ball of the same material, larger than the mouth of the flask, is carefully attached around the petiole, then the petiole and plastolina may be plunged into the mouth of the flask and the two masses unite in a manner such as to give a perfectly air-proof, water-tight connection. It has been found desirable, for purposes of safety, to put on a second layer of the plastolina as soon as it is evident that the first permits no leakage. Even with these precautions, considerable diurnal changes in temperature may cause leakage, and it is particu- [Vor. 1 8 ANNALS OF THE MISSOURI BOTANICAL GARDEN larly important that each experiment should be carefully ex- amined prior to making all readings. The water columns in the burettes were so gauged as to eliminate the possibility of forcing water into the leaves. Тһе burettes were employed solely in order to get accurate readings of the water loss from hour to hour without shifting or disturbing the plants by weigh- ing; also rapidly to get data, should it seem necessary, under changing conditions. АП of these considerations proved very important, as it was found that a slight shifting of the position of the leaf affected materially the transpiration magnitudes. For each leaf used it was necessary to get its rate of transpira- tion in terms of some standard in order that the ratios might be established between certain leaves prior to the addition of the spray to some of them and the ratio between the same leaves after the applieation. At one time it seemed possible that the revolving table method of standardizing porous сарв might be applicable, but on further consideration it was believed that the use of this method in the laboratory, and the subse- quent disposition of the plants in the open, would lead to errors of considerable magnitude. For our purpose it was not consid- ered desirable to conduct the whole experiment on the revolving table, but this method will be employed in connection with our further studies. It was found very important to standardize the leaf in a given position and then permit it to remain in that position, as far as possible, throughout the experiment. This method was necessary largely because of the fact that it seemed wise to conduct the experiment in the open, during a considerable interval, at least. Further reference to the ar- rangement of the plants will be made in the discussion of the experimental work. Experiments with potted plants.—For the experiments with potted plants tomatoes were used. The pots were dipped in paraffin wax and the same sealing mixture was coated over the surface of the soil. In all the experiments reported there was no leakage in any case from improper sealing. Water was added daily, or twice a day, to supply the loss by trans- piration, the addition of water being made by means of a thistle tube fixed in each pot. The bell of the thistle tube was covered with paraffined paper during the entire interval. It was also 1914] DUGGAR AND COOLEY— FILMS AND TRANSPIRATION 9 found necessary to insert in each pot a small bent tube in order to provide for the changes in air pressure. The pots were weighed at the beginning and at the close of the experiment, but the condition of the plant and the amount of water entering readily from the thistle tube were found adequate to indicate the daily water requirements. То the total provisional transpiration quantities obtained from a summation of the quantities daily added, the differences in weight between the beginning and the close of the experiment were added or subtracted as required. From five to ten plants were employed with each kind of spray or dust used, and the plants of each lot were so distributed in the greenhouse that an equal number—so far as possible—from every group was subject to exactly the same influences. Moreover, positions in the greenhouse were shifted several times during the obser- vation intervals of from ten days to two weeks. As a result of a large amount of experimental work in the greenhouse it has become apparent that the points just referred to are important. Plants situated nearer the edges of the benches, or those which receive drafts from opening doors or from convection currents, show considerable differences in trans- piration rates, and this should be obviated. The leaves in the potometer work and the potted plants were sprayed or dusted liberally, and in the case of the sprays, in partieular, care was taken to cover completely with a fine spray of the material both surfaces of the leaves. Тһе dust applications were made in the late afternoon when the leaf surfaces were less dry, and after dusting the upper surfaces of the leaves the plants were inverted and the lower surfaces equally well treated. Тһе dusts were prepared by grinding to an impalpable powder in а mortar. Тһе Bordeaux mixture employed was made by the 4-6-50 formula, the weights of ingredients for making small quantities being approximately as follows: CuSO, 9.6 grams CaO 14.4 grams Water 1000 cc. The weak Bordeaux was one-half the strength of the above. The Са(ОН); was prepared by slaking gradually 60 grams of [Vor. 1 10 ANNALS OF THE MISSOURI BOTANICAL GARDEN CaO in 1 liter of water; and the mixture designated Al(OH) was prepared by mixing two solutions each of 900 cc., the one containing 26 grams of AlCl; and the other 30 grams of CaO (slaked as for the Bordeaux mixture). Тһе clay suspension consisted of 90 grams of fine air-dried clay in 1 liter of water. The lime-sulphur employed was the usual 1-25 strength. EXPERIMENTAL DATA AND DISCUSSION It will be observed from the brief review of earlier work that the evidence regarding the effect of Bordeaux mixture оп the transpiration rate is inconsistent. А majority of the observers adopt the view that the effect of this surface film is to reduce the transpiration. On a priori grounds this view would seem to be logical, since it would indicate a water conser- vation to which, in dry seasons at least, the plant might respond with increased vitality and yield. Nevertheless, it was believed that the experimental evidence at hand was of insufficient scope to establish this view of it. Contrary to expectations, all of our more important experimental evidence and observations are antagonistic to the a priori assumption as applied to the effects of Bordeaux mixture. Potometer experiments.—In attempting to secure leaves sat- isfactory for the work, some incidental observations were made which are of interest. Тһе work was begun during the winter, so that greenhouse-grown plants alone were available. Fur- thermore, in this work with potometers, Bordeaux mixture alone has been used by us. "Testing leaves of squash (Cucur- bita sp.), Pelargonium zonata, and Phytolacca, also shoots of potato and 1тезсепе, as to their behavior under the conditions required, it was found that of comparable leaves, sprayed and unsprayed, invariably the sprayed leaves were the first to wilt. This might be attributed either to an injurious action of the spray or to a greater water consumption. That the last men- tioned is the more probable explanation finds confirmation through a special observation on the potato. Owing appar- ently to some stoppage of the vascular system, abscised potato shoots are unsuitable for potometer work, wilting in a com- paratively short time even when cut under water; and sprayed potato shoots wilt more quickly than unsprayed, thus pointing 1914] DUGGAR AND COOLEY—FILMS AND TRANSPIRATION 11 to a more rapid water elimination after spraying. Potted potato plants from which the shoots were cut withstood the fungicide satisfactorily. Leaves of the large elephant’s ear (Caladium sp.) proved unsatisfactory on account of the excessive ‘‘bloom,” which interfered with the proper application of the spray. Canna leaves were similarly unfavorable, and leaves of the calla lily wilt soon after abscision. It has been stated above that the leaves of the castor bean proved most satisfactory in the potometer work. The experi- ments with these leaves were carried out in the open, except as otherwise noted, during the early fall. The plants were arranged for standardization and for subsequent observation at distances of about ten feet apart on an exposed lawn uni- formly sodded. No readings were made until the leaves had become adjusted to the conditions. Observations were made at frequent intervals when the water loss was rapid, in order to maintain the water column at a fairly uniform level, so that many of the data given in the tables which follow represent summations of several successive readings. Three series of potometer experiments were made, each series embracing six leaves, but in one series, accidents to some of the leaves, and the necessity of substituting new ones after the experiment began, resulted in such a shortening of the standardization intervals that it was thought necessary to discard the results, although they were in the same direction as the others obtained. The data are presented in full in the tables and all available data are used in computing the relations given. The relations may be more conveniently expressed if we first divide the leaves into classes, designated by letters, as follows: А-, three leaves (i. e., the transpiration quantities of these) in the standardization interval before spraying. A+, the same three leaves as in А--, but for апу interval after spraying. B, three control leaves (unsprayed throughout) during the standardization interval. В’, the control leaves as in B, after standardization. The ratio a =Q is to be compared with the ratio 2+0. [Vor. 1 12 ANNALS OF THE MISSOURI BOTANICAL GARDEN If Q' is greater than Q, then the spraying facilitates transpira- tion; if less, then the converse is true. If no accidents occurred : à; А- during the experiments, E would, of course, be a constant quantity, each term referring properly to the summed trans- piration quantities for three leaves during the standardization interval. Accidents are unavoidable, however, during the subsequent observations, and whenever these occur it is neces- sary to compute а new value of Q’ for any particular “run” in which the accident occurs. Тһе only consideration then is to have the same leaves (i. e., their summed transpiration quantities) in the ratios before and after standardization. If, for example, it is necessary to use a ratio, Q', of Nos. 1 and 3 to Nos. 2, 4, and 6 after spraying, then the new value of Q (in the standardization interval) for comparison must also be computed with Nos. 1 and 3 against Nos. 2, 4, and 6. TABLE I EFFECT OF FILMS OF BORDEAUX MIXTURE ON TRANSPIRATION OF STANDARDIZED CASTOR BEAN LEAVES; DATA FOR DAY PERIODS No. of leaf 1 2 3 4 5 6 Ratio Transp. 12:30-2:30P.M., | A— B А- В В A— |А- 72.9 1st day before spraying 10.8 | 17.3 | 28.5 | 45.9 | 33.8 | 33.6 В 97.0 Transp. 3:12-5:00 P. М., A+ В” | А+ В! В” | А+ А+ 70.3 1st day after spraying 7.6 | 20.4 | 23.2 | 26.2 | 26.1 | 39.5 dud - © 72.9 Relation, sprayed to un- Rate changed from 979 (= .75) to sprayed, Ist day 70.3 72.7 (7.9) Transp. 812-948АМ, | A+ | в | A+ | B' | в | А+ |A+ 104 2nd day after spraying 10.2 | 37.7 | 63.1 | 32.3 | 29.7 | 67.1 | В” 99.7 Rate changed from E (2.75) to Relation, sprayed to un- 97.0 sprayed, 2nd day, a. 140.4 prayed, y 10-4 (2 aj) Transp. 11:16-11:53 A.M., | A+ | В | A+ | В | В | A+ |А-- 61.7 2nd day after spraying 4.9 | 5.9 | 31.5 13.8, 7.5 | ре: H8 — 271.3 72.9 Ж Relation, sprayed to un- Rate changed from 97.0 (2.75) to sprayed, 2nd day, b. 61.7 375 (72.8) 19141 DUGGAR AND COOLEY—FILMS AND TRANSPIRATION 18 TABLE II EFFECT OF BORDEAUX MIXTURE ON TRANSPIRATION OF STANDARDIZED CASTOR BEAN LEAVES; DATA FOR DAY AND NIGHT PERIODS No. of leaf. 1 2 3 4 5 6 Ratio Transp. 4:04-5:25 P. M., Ist} А- B А- А- В day before spr. 7.5 7.6 10.9 11.7 7.6 9.4 Transp. 8:21-11:17 А. М.) А- В А- В А- В 2nd day before spr. 20.2 30.7 32.4 40.9 23.3 17.9 S veh B I= B P E B А-,101.9 Тоба! transp. before spr. 27.7 | 38.3 | 43.3 | 526 | 29.9 | 27.3 | B 118.2 Transp. 12:30-24:50 P. М.| А+ B’ A+ B' A+ в | A+_140.6 1st day after spr. 36.7 42.6 62.9 50,2 41.0 80.2 B’ 123 Relation, sprayed to un- 101.9... 140.6, sprayed, Ist day Rates changed from 125 5 (=.86) to 7193 (21.14) Transp. 8:56 A. M., to 4:44| А+ B' A+ В’ A+ В’ A+_ 66.6 P. M., 2nd day aft. spr. 20.7 —— 28.9 18.2 17.0 12.1 В’ 30.3 Relation, sprayed to un- 101.9 22 66.6 sprayed, 2nd day Rates changed from 79.9 (71.28) to 30.3 (22.2) Transp. 10:27 A. M., to 3:40] A+ В’ A+ В’ A+ В’ А+ 27.4 P. M., 3rd day aft. spr. 8.4 — T2 4.4 7.8 3.5 Ве Jo Relation, sprayed to un- 101.9 27:4. 3 sprayed, 3rd day Rates changed from 7,75 (21.28) to 7-5 (73.40) Ттапвр. 9:58 A. M., to 4:42 А-- B' А-- B’ A+ В’ А- 37.4 P.M., 4th day aft. spr. — — 22.1 14.1 15.3 7.9 оо 2.0 * Relation, sprayed to un- 74.2 37.4 sprayed, 4th дау | Rates changed from 79.9 (2.93) to 22.0 (21.7) : —— -- A+ B' A+ В’ A+ 206.2 Total transp. after spraying 125.1 86.9 811 58.7 1 ре Relation, sprayed to un- 74.2 ze 206.2 = нанел, totale Rates of totals changed from 79.9 (27.93) to 140.6 (21.47) Transp. 5:30 P. M., to 8:21} A— B A— B А- В А- 92.3 A. M., 1st night bef. spr. 6.9 6.3 9.0 | 11.2 64 | — | В "ИБ Transp. 4:50 P.M., to 8:40] A+ В’ А+ В’ А+ В’ A+ 15.8 А. M., 18% night aft. spr. 6.9 7.8 2T 7.5 6.2 8.5 F p3 Relation, sprayed to un- 22.8. 15.8 sprayed (night) Rate changed from 17.8 (21.27) to 15.8 (21.03) Transp. 3:45 P. M., to 9:30] A+ В’ А+ В’ A+ В’ A+ 40.9 А. M., 2nd night aft. spr. 20.0 ---- 5.1 4.9 5.8 4.2 F 49 Relation, sprayed to un- 22:9 ,— 40.9 ,_ sprayed (night) Rate changed from 112 (22.0) to Te (28.34) * For this “тап” the plants were transferred to а room in the building. [Vor. 1 14 ANNALS OF THE MISSOURI BOTANICAL GARDEN Summarizing the data for the rates in table rr, day intervals, we find that Q:Q', in the successive periods, as .86:1.14, as 1.28:2.2, as 1.28:3.46, and as .93:1.7. If we make the ratio before spraying equal in each case, to 1.0, then the value for the periods after spraying in the successive day intervals are respectively 1.33, 1.72, and 1.83. These differences in rate are so marked and consistent as to outweigh all considerations of individual differences, as disclosed by a detailed study of the figures in table п. It will also be noted that the less extensive data from table r are confirmatory; thus 0:0” in the successive intervals, as .75:.97, as.75:1.41, and as .75:2.3. On the basis of 1.0 for the ratio before spraying, we have for the periods after spraying, respectively, 1.29, 1.88, and 3.07. From the records of the potometer experiments it is obvious that only one conclusion may be drawn, namely, that the rate of transpiration is materially increased after spraying. Some points relative to environmental conditions, however, require special mention, and certain suggestive results must be left for further experimental study. Attention has been drawn to the fact that, in general, the potometer experiments were conducted in the open, during early October. During the last days of the work, cooler weather and danger from rain made it desirable to transfer the potometers to а room in the building, and the data for the third and fourth days after spraying, table п, were secured under these new conditions. In this room the shades were drawn and every precaution taken to secure uniformity. It will be noted that while the order of results is in the same direction as for the lawn exposure, the ratio is even higher than the average. No “shading action" of the Bordeaux, as postulated by беһалдег (18), could be considered a factor of importance in this case. The results in the laboratory suggest, further, that the ratio of sprayed to unsprayed will vary considerably with the con- ditions. Before removing the potometers to the laboratory, the night temperatures were so low that two night “runs” (ineluding the interval from about 6 Р.М. to 8 А.М.) were necessarily excluded on account of leakage. Other night “гип,” as shown in the tables, indicate the probability that under certain conditions unfavorable for evaporation, the surface 1914) DUGGAR AND COOLEY—FILMS AND TRANSPIRATION 15 fiim may actually effect à diminution in the rate of transpira- tion, although the transpiration data do not suffice to warrant more, at present, than an impression. In fact, the night “runs” should be considered apart from those of the day, for the latter are much more satisfactory. Experiments with potted plants.—The experiments with potted tomato plants were divided into two series which were consec- utive in time, and different only with respect to the substances applied to the leaves. Ав far as has been ascertained, this is the first time that tomato plants have been used in such work, but in our experience they are more satisfactory than potatoes. In the first series (table ш) 30 plants were used, іп lots of 10 each, for the applications of (1) strong Bordeaux mixture, (2) weak Bordeaux, and (3) controls. In the second series (table rv, v) 80 plants were used in 8 lots, and the substances employed as sprays or dusts are noted in the tables. In the second series it is to be noted that there are 3 substances of the nature of films (Ca(OH)2, АКОН)з, and lime-sulphur), 1 true suspension (clay), and 3 powders (charcoal, СаСОҙ, and powdered Al(OH)s). The methods of procedure involved in these experiments have already been outlined. It is necessary to add, however, that the plants used were about 12 inches high and as uniform in size as could be obtained. It was not possible satisfactorily to standardize plants for an experiment extending over several weeks: and it was necessary to rely in part upon numbers, and in part upon a rigidly accurate method of selecting the individual in each lot to eliminate any errors. Тһе method of selection consisted in getting together 8 plants so similar in size and vigor that no choice could be made between them, then distributing these at random to the 8 lots, this being continued until each lot embraced 10 plants. In each ease the experiments extend over 2 periods. At the close of the first period the plants were shifted in position and а second application of the spray mixture or dust was given. With the conclusion of the experiment the green weights of all plants were taken, thus enabling us to determine, in addition to the total transpiration quantities, the amount of transpiration per gram of green substance. 2 [Vor. 1 16 ANNALS OF THE MISSOURI BOTANICAL GARDEN TABLE III THE EFFECTS OF BORDEAUX MIXTURE ON THE RATE OF TRANSPIRATION; DATA IN GRAMS FOR 30 POTTED TOMATO PLANTS 1st period Oct. 18 to Nov. 4 Covering Strong Bord. Weak Bord. Check Plants nos. 1-10 11-20 21-30 Transpiration quantities 702 681 390 684 651 555 665 540 375 630 585 525 625 857 395 710 440 465 640 585 415 445 648 365 646 645 490 560 CMT 545 Total 6306 5622 4520 Ave. per plant 630.6 625 452.0 Second period Nov. 5-15 Covering Strong Bord. Weak Bord. Check Plants nos. 1-10 11-20 21-30 Transpiration quantities 571 628 356 559 549 554 575 442 368 574 603 518 515 720 385 740 453 420 514 499 437 570 534 417 495 702 439 546 564 Total 5659 5130 4458 Ave. per plant 565.9 570 445.8 Pt 1914) DUGGAR AND COOLEY—FILMS AND TRANSPIRATION 17 TABLE III (Continued) THE EFFECTS OF BORDEAUX MIXTURE ON THE RATE OF TRANSPIRATION; DATA IN GRAMS FOR 30 POTTED TOMATO PLANTS Green wts. of plants used. Plants nos. 1-10 11-20 21-30 Green weights in grams 55 80 40 58 58 63 51 41 46 39 51 56 50 60 39 67 45 53 46 40 48 46 53 40 39 74 48 49 ; 5 55 Total 500 502 497 Ave. per plant 50.0 55.8 49.7 TABLE IV THE EFFECT OF VARIOUS SPRAYS AND DUSTS ON THE RATE OF TRANSPIRATION; DATA IN GRAMS FOR 80 POTTED TOMATO PLANTS. 1ST PERIOD, OCT. 25 TO NOV. 8 Lime- Covering ^ (Са(ОН). АКОН),! Clay |AKOH):| Char- | CaCO, | sulfur |Check pwd. | coal 1-25 Plants nos. 30-39 | 40-49 | 50-59 | 60-69 | 70-79 80-89 100-109 90-99 Transpiration| 431 394 345 416 378 436 508 | 352 quantities 437 370 333 370 460 353 414 | 323 435 386 430 393 374 315 474 | 461 411 383 383 383 460 510 354 | 443. 358 329 347 645 273 375 526 | 490: 372 377 520 449 467 346 421 | 309: 314 398 437 365 320 471 352 | 330- 416 410 560 531 359 361 346 | 323. 375 517 362 408 386 456 285 | 343. 485 460 452 412 331 364 402 | 3177 Totals 4034 4024 4169 4372 3808 | 3987 | 4082 |3691 Ave.per plant) 403.4 | 402.4 | 416.9 | 437.2 | 380.8 | 398.7 | 408.2 369.1 . [Vor. 1 18 ANNALS OF THE MISSOURI BOTANICAL GARDEN TABLE V THE EFFECTS OF VARIOUS SPRAYS AND DUSTS ON THE RATE OF TRANSPIRATION; DATA IN GRAMS FOR 80 POTTED TOMATO PLANTS. 2ND PERIOD, OCT. 25 TO NOV. 8. Lime- Covering |Са(ОН), АКОН), Clay |АКОН):| Char- | ссу, | sulfur |Check pwd. coal 1-25 Plants nos. 30-39 | 40-49 | 50-59 | 60-69 | 70-79 | 80-89 |100-109 90-99 Transpiration| 494 812 624 665 573 560 846 | 590 quantities 463 587 582 601 610 543 641 | 569 592 653 693 609 530 553 667 | 564 539 587 615 654 744 700 622 | 701 604 654 594 754 556 512 680 | 764 457 614 579 606 617 552 616 | 546 544 665 694 476 569 706 604 | 496 647 605 753 704 664 585 478 | 558 597 806 579 641 596 620 601 | 514 582 810 590 711 562 617 428 | 601 Totals 5509 6793 6303 6421 6021 | 5948 | 6183 |5903 Ave. 550.9 | 679.3 | 630.3 | 642.1 | 602.1 | 594.8 | 618.3 | 590.3 *'Transp. per gm. 11.8 12.2 12.3 11.4 13.1 12.0] 12.8| 12.1 Green wts. of plants 30-109 at close of 2nd period. Green weights| 39 71 46 58 49 50 56 58 in grams 39 43 47 52 43 47 БІ 47 49 58 Бі 45 48 43 42 38 42 53 49 79 35 53 53 57 60 56 54 77 35 53 67 53 43 56 58 47 59 51 40 56 41 53 52 47 46 57 52 35 50 55 50 54 48 47 36 46 60 58 48 47 52 43 49 46 43 54 59 55 43 51 39 51 "Totals 466 557 514 561 458 495 485 487 Ave.perplant| 46.6 55.7 51.4 56.1 45.8 | 49.5 | 48.5 | 48.7 * Computed on the basis of green weights at the close of second period. 1914) DUGGAR AND COOLEY—FILMS AND TRANSPIRATION 19 Ап examination of the data in the several tables involved in the pot experiments serve to indicate that while there is а certain amount of individual variation in the transpiration quantities of the various plants in any group, general conclu- sions seem to be warranted. Тһе individual variations in transpiration in the Bordeaux series are in closer accord with the variations in green weight of the plants used than are those in the other series. Taking all factors into consideration, а film of Bordeaux mixture is found to facilitate transpiration. Other films and dusts employed do not seem to affect the rate of transpiration to the same extent. In a consideration of the results in detail it is to be noted that the Bordeaux series (table іп) is not strictly comparable with the other (tables rv, v), since they were not conducted simultaneously. If the transpiration in grams per gram of green weight for the control (Bordeaux series) is represented by 100, then the rate for weak Bordeaux on this basis is 113.2, and the rate for strong Bordeaux is 125.43. Тһе differences are in the same direction, but not so great as those obtained with the potometer experiments. Тһе use of both weak and strong Bordeaux mixture materially strengthens the conclu- sions to be deduced. Тһе series which gives the results with other sprays and dusts is not so easily interpreted. Тһе transpiration quantities vary slightly on either side of the control, and no covering gives a negative difference (contrasted with the control) greater than six per cent (this is the case of Al(OH)s), or a positive difference greater than about eight per cent (charcoal). These slight average differences may be no more than would be explained by the possible experimental error; but it is of interest to perceive that, with the exception of clay, those surface applications which give lower values than the control are those which might diminish the absorption of heat in direct sunshine. Тһе results might then be the resultant of two factors, (1) the direct effect of the surface film or dust on the rate of water loss, and (2) the indirect effect exerted through a modification of the temperature of the leaf. Accepting as а general conclusion an acceleration of trans- piration (specifically in the castor bean and in the tomato) [Vor. 1 20 ANNALS OF THE MISSOURI BOTANICAL GARDEN as a result of an application of a film of Bordeaux mixture, the following questions arise: (1) What is the physical or chemical basis of the increased evaporation from plant surfaces covered with Bordeaux mixture? (2) Is the increased evaporation in any way related to the increased vitality or longevity of sprayed leaves? Neither of these questions may be answered intelli- gently at present. With respect to the first, we have arranged experiments to determine the effects of Bordeaux on the passage of water vapor through non-living membranes; but the results are thus far conflicting, due possibly to the fact that we have not yet used membranes which are satisfactory analogues of leaves. Experiments in this direction will be reported later. No relation of transpiration to inereased longevity can be foretold, although it seems possible that the highest efficiency equilibrium relation of longevity may involve, in certain plants, a relatively high transpiration rate as either a direct or an indirect factor. Хо answer to the question will be satisfactory until a further study of other effects (*'stimulation," increased *assimilatory activity," etc.) of Bordeaux mixture shall have been made. BIBLIOGRAPHY 1. Aderhold, R. Der heutige Stand unserer Kenntnisse über die Wirkung und Verwertung der Bordeauxbrühe als Pflanzenschutz-mittel. Jahresb. d. Verein- igung der Vertreter der ang. Bot. 1: 12-36. 1904. 2. Bain, S. M. Тһе action of copper on leaves. Тепп. Agr. Exp. Sta. Bul. 15: 1-108. 1902. 3. Bayer, L. Beitrag zur pflanzenphysiologischen Bedeutung des Kupfers in der Bordeauxbrühe. Inaug-Dissert., Kónigsberg, 1902 [cf. Schander (18)]. 4. Clark, J. F. On the toxie properties of some copper compounds with reference to Bordeaux mixture. Bot. Gaz. 33:26-48. 1902. 5. Clinton, G. P. Spraying potatoes in dry seasons. Conn. Agr. Exp. Sta. Report 1909-10: 729-752. 6. Crandall, C. S. Bordeaux mixture. Ill. Agr. Exp. Sta. Bul. 135 : 201-296. 1909. 7. Duggar, B. M. Peach leaf curl and notes on the shot-hole effect of peaches and plums. Cornell Agr. Exp. Sta. Bul. 164: 371-388. 1899. 8. Ewert, R. Der wechselseitige Einfluss des Lichtes und der Kupferkalkbrühen auf den Stoffwechsel der Pflanze. Landw. Jahrb. 34: 233-311. pl. 2-4. 1905. 9. Frank, A. B., and Krüger, Fr. Ueber den direkten Einfluss der Kupfervitriol- kalkbrühe auf die Kartoffelpflanze. Arb. d. deut. landw. Ges. 1894: 1-46. 10. , Ueber den Reiz welchen die Behandlung mit Kupfer auf die Kartoffelpflanze hervorbringt. Ber. d. deut. bot. Ges. 12: 8-11. 1894. 11. Hawkins, L. A. Some factors influencing the efficiency of Bordeaux mixture. Bur. Pl. Ind. О. 5. Dept. Agr. Bul. 265: 1-29. 1912. 1914] 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. DUGGAR AND COOLEY—FILMS AND TRANSPIRATION 21 Kirchner, O. Ueber die Beeinflussung der Assimilationstátigkeit von Kartoffel- pflanzen durch Bespritzung mit Kupfervitriolkalkbrühe. Zeitschr. f. Pflanzenkr. 18:65-81. 1908. Leydheker, A. Die Bekümpfung der Kartoffelkrankheit durch die Verwendung von Kupfervitriol. Oesterr. landw. Wochenbl. 1893 : 163. [Reviewed in Zeitschr. f. Pflanzenkr. 4:33. 1894.) Lutman, В. Е. Тһе covering power of the precipitation membrane of Bordeaux mixture. Phytopathology 2: 32—41. 1912. Müller-Thurgau, H. Jahresb. der schweizerischen Versuchsstation und Schule f. Obst-, Wein- und Gartenbau in Wiidensweil 1892-93: 58-59. Rumm, C. Ueber die Wirkung der Kupferprüparate bei Bekämpfung der soge- nannten Blattfallkrankheit der Weinrebe. Ber. d. deut. bot. Ges. 11: 79-93. 1893. — — ——., Zur Kenntniss der Giftwirkung der Bordeauxbrühe. Inaug-Dissert. 1-76. 1 pl. 1895. Schander, R. Ueber die physiologische Wirkung der Kupfervitriolkalkbrühe. Landw. Jahrb. 33: 517-584. 1904. Stewart, F. C., French, G. T., and Sirrine, F. A. Potato spraying experiments in 1910. N. Y. (Geneva) Agr. Exp. Sta. Bul. 338: 115-151. 1910. i ч , Potato spraying experiments, 1902-1911. E. X. (Geneva) Agr. Exp. Sta. Bul. 349: 99-139. 1912. Swingle, W. T. Bordeaux mixture: its chemistry, physical properties, and toxic effects on fungi and algae. Div. Veg. Physiol. and Path. U. S. Dept. Agr. Bul. 9:1-37. 1896. . Zucker, A. Beitrag zur direkten Beeinflussung der Pflanzen durch die Kupfer- vitrio-Kalkbrühe. Inaug-Dissert. Stuttgart, 1896 (сі. Schander (18)!. Graduate Laboratory, Missouri Botanical Garden. ТТР ERE "EOM i MM EU ENS ққа» ў [Vor. 1, 1914] iments showing burette connected with scised leaf of Ricinus cemented into the mouth with PLATE I. tion ExPLANATION оғ PLATE ANNALS OF THE MISSOURI BOTANICAL GARDEN Potometer used in the t de arm flask, and ab 22 the si plastolina. ANN. Мо. Вот. GARD., Vor. 1, 1914 PLATE I р » s —á — - DUGGAR AND COOLEY —TRANSPIRATION SOME PURE CULTURE METHODS IN THE ALG JACOB R. SCHRAMM Assistant to the Director of the Missouri Botanical Garden Instructor in the Henry Shaw School of Botany of Washington University INTRODUCTION Too much confidence has frequently been placed by algolo- gists in their ability to recognize a given species of alga among varying numbers of other species, and in the various forms which it may assume—a fact which has led to much confusion and error, especially among members of the Protococcales. While it is now definitely known that in a number of alge a single species may present markedly dissimilar appearances, either as a result of varying environmental conditions, or because of the presence in the life history of several unlike stages, it is cer- tain that much of the so-called polymorphism, or pleomorphism, of alge finds its explanation in inadequate methods of study. It is becoming recognized that for life history studies in the algee it is necessary to employ cultures free from other species of alge. Even in cases where this is not, on first thought, necessary, as in the large, filamentous forms, it should be ob- served, for the possibility of introducing spores or sporelings of closely allied species is by no means excluded in all cases. Gratifying progress has already been made by some algologists, working especially with members of the Volvocales and Pro- tococcales, and it seems reasonably certain that the originally chaotic condition existing in the latter will be ultimately reduced to complete order by a careful observance of the necessity of working with pure cultures, or at least cultures containing but a single species of alga. In life-history studies where physio- logical differences between species are to be investigated, it is especially desirable and indeed necessary to employ pure cultures. Certain species of alge, especially representatives of the Chlorophycee, have been much used in physiological investi- gations—chiefly those concerning themselves with various ANN. Mo. Вот. Garp., Vor. 1, 1914 (23) [Vor. 1 24 ANNALS OF THE MISSOURI BOTANICAL GARDEN phases of nutrition. With the development of a clearer under- standing of the activities and life processes of the various micro-organisms, the necessity of working with rigorously pure cultures has become more and more evident. It is now generally appreciated that, in most cases, valid conclusions as to the physiology of a particular organism cannot be drawn with certainty where one or more foreign organisms have been present in the cultures. There can be no doubt that the frequent con- tamination of cultures of alge with bacteria, and even with fungi, has, in many cases, detracted markedly from the value of painstaking and otherwise careful physiological investigations. The readiness, however, with which many alge lend themselves to experimental purposes—on account of their small size and ease of handling and culture—will always make them favored objects of study; and it appears desirable at this time to bring together some of the experiences of the author in the prepara- tion of pure cultures of alge, with the hope that suggestions may be gained from them by those who desire to obtain such eultures for one purpose or another. An unfortunate use of the term “риге culture" has come into more or less general use and has frequently led to confusion and ambiguity. Ав used by many authors, it means simply a culture of a single species of alga not necessarily free from bacteria and fungi. Where the presence of other organisms is not specifically mentioned, it is clear that the above usage of the term may lead to serious misunderstandings. Indeed, it remains for the reader, in many instances, to decide for himself—from the technique employed—whether a culture of an alga free from all other organisms or only from other species of alge is meant. It is to be hoped, therefore, that the term pure culture shall come to have the same clearly defined mean- ing when used in connection with the alge that it has long had in the fungi and bacteria. In the following report the term is used to signify a culture of a single species of alga free from all other organisms. HISTORICAL Although incidental references to pure culture technique in the algæ are frequently found in the literature, relatively few con- tributions have appeared which deal extensively with the 1914] SCHRAMM—PURE CULTURE METHODS IN THE ALGJE 25 subject, or which outline in detail the methods employed. Beyerinek, in 1890 (4, 6), appears to have been the first to succeed in isolating species of alge in pure culture. Ditch water, boiled with ten per cent gelatin, and cooled, was mixed with a drop of water rich in protococcoid alge, poured into dishes, and allowed to cool. Numerous minute algal colonies appeared in course of time, and the number of bacterial colonies developing was so small that successful transfers of Scenedesmus acutus Meyen and Chlorella vulgaris Bey., were made, both organisms being subsequently cultured on a variety of media. In addition, the gonidia of Physcia parietina were obtained pure. Small pieces of the lichen thallus, carefully washed, were placed on solid gelatin plates. Those which showed themselves to be free from foreign organisms were transferred to gelatin plates containing malt-extract, the fragment being first torn to bits with needles and then dragged over the sterile surface. In a few days, small colonies of the algal symbiont appeared from which successful transfers were made. Іп a later paper (5), Beyerinck adds Stichococcus major and a second species of Chlorella to the list of alge previously cultured in a state of purity, the technique, in general, being the same. Miquel (16) was the first to isolate а diatom in pure culture. Subsequently, Richter (20, 21) isolated Nitzschia Palea (Kütz.) W. Sm., and Navicula minuscula Grun., by the use of synthetic agar plates. Attention is called by this author to the impor- tance of using agar which has previously been washed to free it from soluble impurities. A mixture of diatoms and other algee was placed on the surface of washed agar plates, and from the impure diatom colonies which developed transfers were made to other plates until at length pure cultures were obtained. In his isolations of certain protozoa in pure culture, Ogata (18) also obtained Polytoma uvella. While his method seems unnecessarily complex, it is of interest here. Sterile capillary tubes were filled in part with а column of sterile water, and subsequently а column containing the organisms was added below, care being taken not to separate the two by air. Both ends of the tube were then sealed. After sufficient time had elapsed for the movement of the motile Polytoma cells from the lower column into the upper sterile one, the tube was broken in [Vor. 1 26 ANNALS OF THE MISSOURI BOTANICAL GARDEN the region of the upper column. Тһе lower portion was dis- carded, and the upper one was sealed, subsequently transferred to а sterile medium, and broken to permit the organisms, free from contaminations, to enter the medium and begin their development. By the gelatin plate method, Krüger (13) prepared pure cultures of two new organisms—Chlorella protothecoides and Chlorothecium | saccharophilum—obtained from the exudation of Populus alba. Tischutkin (23) lists representatives from about eighteen genera of algee—including diatoms, green, and blue-green forms—as having been obtained in pure culture by the agar plate method. After three or four successive dilu- tions in liquid one per cent agar, the organisms were plated in Petri dishes. The filamentous forms he washed in sterile water, cut into short segments, and transferred to the liquid medium. The methods given by Ward (24) include plating in agar and silicic acid jelly, though as a whole the methods are applicable for the separation of algal species rather than for their isolation in pure culture. This is especially true of the plaster of Paris, and precipitated calcium carbonate methods. Gonidia from Xan- thoria parietina, and Gasparinia murorum (Hoffm.) Tornab., to- gether with Pleurococcus vulgaris and Scenedesmus caudatus were obtained in pure culture by Artari (1). Chodat and Goldflus (8), by the use of pieces of sterilized unglazed porcelain in contact with a mineral nutrient solution, claim to have isolated a species of Nostoc in pure culture. Тһе procedure was a simple one, consisting in repeated transfers to fresh sterile plates until a pure culture was at length obtained. Several years later Chodat and Grintzesco (9) reported that by essentially the same method, Oocystis elliptica, Dictyo- spherium pulchellum, Kirchneriella lunaris, Rhaphidium poly- morphum, Pediastrum tetras, Scenedesmus acutus, Pleurococcus vulgaris, Hematococcus lacustris, and Chlorella vulgaris had been obtained in pure culture. In cases where the number of algal individuals is small, but the bacteria and fungi relatively abundant, the authors point out the desirability of first increas- ing the number of the former by introducing the mixture into a mineral nutrient solution favorable for the growth of the alge but not so for the fungi. Where filamentous forms are 1914] SCHRAMM—PURE CULTURE METHODS IN THE ALG/E Ж concerned, the authors state that it is necessary to begin with the zoóspore, as a pure culture from filaments is extremely difficult to obtain. Му own experience does not bear out this statement in all eases as it was found that especially among the Ulotrichales pure colonies were regularly and easily obtained from filaments. Artari in 1902 (2) reports the isolation of Chlorococcum infusionum and Scenedesmus caudatus in pure culture. Chick (7) attempted to isolate Chlorella pyrenoidosa through the use of sterilizing agents such as hydrogen peroxide and sunlight. These trials, however, did not prove successful, as the alga failed to show a resistance sufficiently greater than that of the bacteria to make possible a successful separation. Тһе isolation was finally attained by placing a few drops of water containing the organism on a sterile synthetic agar plate, and spreading the same over the surface with а brush. Тһе same brush was used to distribute sterile water drops over the surface of other plates, no additional algal material being added. From the later dilutions pure colonies were obtained. Frank (10) was unable to obtain pure cultures of Chlamydomonas tingens by the agar plate method. Jacobsen (11) reports the isolation of Chlorogonium and Polytoma in pure culture. This author made use of an inter- esting method of separation of algal species based on their different degrees of resistance to drying. Discs of filter paper, on which drops of water containing Spondylomorum and Chlamydomonas variabilis had been placed, were dried in an incubator at 28°C. After twenty-four hours, the discs were placed in a suitable medium, but only the Chlamydomonas species developed, Spondylomorum having been killed. Chlo- rogonium euchlorum and Polytoma uvella also showed themselves very sensitive to drying, whereas Chlamydomonas usually sur- vived the desiccation. Old cultures of Chlorogonium euchlorum proved to be very resistant owing to the presence of zygospores which had been formed by the conjugation of gametes. While reference might be made to a number of other inves- tigations which deal in an incidental way with pure culture technique, it is believed that those given will serve to indicate, in a general way, the present status of the subject. (For further [Vor. 1 28 ANNALS OF THE MISSOURI BOTANICAL GARDEN information the reader is referred to Moore (17), Richter (21), Küster (14), and others.) It is apparent that the large majority of forms isolated in pure culture belong to the Protococcales. Only a few of the filamentous forms, several diatoms, and but one or two species of the blue-green alge have thus far yielded to pure culture technique. PURE CULTURE TECHNIQUE GENERAL Algæ, generally speaking, are provided with a more or less highly developed exterior mucilaginous investment which may be either a distinct, separable sheath, as in many of the Cyano- phycem, ог merely a gelatinization resulting either from a modification of the external portion of the membrane, or from an internal secretion, as in some of the desmids. In general, also, algæ are slow growing as compared with many fungi. In these two characteristics most of the difficulties encoun- tered in pure culture technique among the algæ find their explanation. Among the fungi, spores with non-gelatinous walls are readily obtainable in a majority of the forms, and usually in great abundance. When such spores are plated in the way ordin- arily employed in bacteriological technique, a large number of colonies free from bacteria are usually obtained. Among the algæ, however, such non-gelatinous, resistant spores are, if produced at all, generally present only in small quantities. When vegetative algal cells are plated on a suitable medium, algal colonies will often be obtained, but they usually form the nucleus of a larger bacterial colony which has developed from the bacteria adhering to the gelatinous surface of the algal cell. Among those fungi in which spores are not readily obtained, an isolation in pure culture may frequently be effected by allowing the fungus to grow on a suitable medium until the hyphæ have outstripped the bacteria in their growth, at which time pure mycelial transfers may be made from the terminal portions. If, however, a like procedure is attempted with the algæ it will usually be found that the bacteria adhere tena- ciously to the surface of the growing filaments and are carried 1914] SCHRAMM—PURE CULTURE METHODS IN THE ALG 29 along by the lengthening filaments. Except in rare cases, nothing is to be gained by this procedure in the alge. The task of isolating pure cultures of alge, therefore, becomes an individual problem for almost every species as it necessitates at once the determination of the period in the life history of any form at which the cells are free from bacteria or at which time the bacteria can be removed by one means or another. Having found a stage in which the alga is bacteria-free, it is of importance next to be able to bring about this stage more or less at will in order that the alga may be utilized when available. To obtain the above preliminary information, noth- ing is more serviceable than the usual plating method on a suitable medium. The Medium.—The requirements of a suitable solid medium for algal isolating purposes are, that it remain liquid down to a temperature at which delicate algal cells are not injured; that it be suitable for the growth of alge, and as unfavorable as possible for the growth of bacteria and fungi. For this purpose nothing was found so serviceable as the following, the mineral in- gredients being in the proportions recommended by Moore (17): Agar 10.0 grams NH; NO; 0.5 gram MgSO.. 7H;0 0.2 gram К.НРО, 0.2 gram CaCl: 0.1 gram FeSO. trace Dist. НО 1000 сс. The agar should be carefully washed, first in а stream of tap water and then in distilled water, as pointed out by Richter (20). An agar so prepared will remain liquid down to about 34.5- 35°C., and experience has shown that even the most delicate algal cells are uninjured by the short exposure to this temper- ature necessary in the plating process. From six to eight cc. of agar in a Petri dish eight em. in diameter is а suitable quan- tity with which to plate. Larger quantities so thicken the layer of agar in the dish that the higher powers of the microscope, with their objectives of short focal length, cannot be used in locating small developing colonies. [Vor. 1 30 ANNALS OF THE MISSOURI BOTANICAL GARDEN Material to be Plated.—The alga to be plated should be collected with as little adhering foreign matter as possible. If it is à filamentous form which can be manipulated with a platinum needle, it can be materially cleansed by washing in sterilized nutrient solution such as is used in the preparation of the agar. If the alga is a unicellular form, little can be done in the way of preliminary cleansing. Dilutions are made in the usual manner, the degree depending upon the number of algal organisms present. Тһе degree of dilution will depend in part, also, upon the number of bacteria and fungi present as determined by microscopic examination. It must be re- membered that the alge grow more slowly than most bacteria and fungi, and that unless the dilution, from the standpoint of the total number of organisms present, is great enough, the spread of bacterial and fungal colonies may be so great as to таКе the transfer of the later-appearing algal colonies impos- sible without contamination. The material should be introduced into the tube of liquid agar while the latter is still a few degrees above its congealing point, in order that the inoculated tube may be vigorously shaken for some time before its contents are poured into the Petri dish. Іп this way the algal cells are freed of large numbers of either accidentally or regularly adhering bacteria. Incubation and Transference.— The plates, after the agar has solidified, should be turned upside down in order to prevent the moisture which condenses on the cover from dropping, and spreading bacteria over the surface of the agar. Failure to do this often renders large numbers of platings worthless. The most favorable place to keep plates is in the light of a north window; and, as plates frequently remain under obser- vation for many weeks, it is further desirable to have them in a glass case to prevent outside contamination. In general it is not advisable to cover the plates with bell jars, as it increases the humidity in the Petri dishes and accelerates the growth of moulds present as contaminations. The plates should be examined frequently and when rapidly spreading colonies of fungi or bacteria appear, these should be dissected out in order to save the remainder of the plate. The length of time necessary for the appearance of the algal 1914] SCHRAMM—PURE CULTURE METHODS IN THE ALG/E 81 colonies varies greatly with the species, from one to three or four weeks usually being required, depending upon the particular form. In most cases it is not possible to wait until the algal colonies ean be seen macroscopically because spreading bacterial and fungal colonies usually encroach on the former to such an extent that a pure transfer is no longer possible. It becomes necessary, therefore, to look the plates over from time to time with the compound microscope in order to locate algal colonies in very earlystages of development. For this purpose а 12 mm. ob- jective is extremely serviceable, as its focal length is of sufficient magnitude to enable one to use it through the agar layer and glass bottom -of а Petri dish and at the same time obtain a magnification considerably greater than that afforded by the ordinary low-power objective. The colonies located are conven- iently marked by placing a small ink dot directly opposite them on the bottom of the Petri dish. Transfers should be made to agar slants by means of a minute platinum-foil spatula with which the agar directly over the ink dot can be neatly dissected out and transferred to the slant. It is not possible, in most cases, to make successful transfers with a platinum needle because the algal colony is usually composed of firmly cohering cells and, even in repeated attempts, not a single individual will adhere to the needle. Since many of the colonies are in the deeper strata, it is well to spread out the transferred agar fragment in a thin sheet in order to expose the contained algal cells directly to the air. Unless this is done, subsequent development in the slant may be extremely slow. Although bacteria grow slowly on this synthetic agar, their development is usually sufficient in a week to indicate whether the transfer has been successful or not. The purity of the culture may be further tested by making transfers to media more suitable for bacterial growth. With this brief preliminary consideration of some of the more general phases of pure culture technique in the alge, the isola- tion of single species will now be considered and attention called to the special problems and the technique involved in their isolation. [Vor. 1 32 ANNALS OF THE MISSOURI BOTANICAL GARDEN SPECIFIC CHLOROPHYCEJE Chlamydomonas pisiformis Dill forma minor Spargo.— Chlamydomonas species frequently occur in water rich in organic materials, and teeming with bacteria. When the alga was in the resting condition, the mucilaginous сей walls were found so impregnated with bacteria as to render isolation in pure culture impossible. Platings with motile cells, however, showed that the latter were absolutely free from regularly adhering bacteria, but the number of bacteria present rendered the plates worth- less. Then the gelatinous masses of resting cells were repeat- edly washed with sterile water and finally placed in distilled water where, after twelve to twenty-four hours, zoóspores appeared in great abundance and congregated on the side of the vessel nearest to the light. А minute portion of this liquid containing the zoóspores was removed with a fine capillary tube and introduced into a tube of liquid agar and plated. In platings thus made, numerous colonies of Chlamydo- monas appeared and the number of bacterial colonies was so small that a large number of successful pure transfers were made. Where the number of available motile cells is small and it is important that isolations be made from these, a modification of the method used by Barber (3) in the isolation of yeasts and bacteria was frequently used to advantage. А large num- ber of small, capillary pipettes were made and sterilized. After locating the cell or cells desired, they were removed with a pipette while being observed under the microscope, and trans- ferred to a drop of sterile nutrient solution or water. This process was repeated until it was certain that the number of bacteria had been reduced sufficiently to admit of successful plating. They were then taken up again by means of a sterile pipette, transferred to a tube of liquid agar, and plated. Numerous pure cultures were obtained in this way. Stichococcus bacillaris Nág., and S. subtilis (Kütz.) Klercker.— Preliminary platings with these forms showed that the cells, as obtained from the soil, yielded abundant bacteria-free colonies, and the problem of isolation became one of merely obtaining clean material and diluting sufficiently. Both of these species 1914) SCHRAMM—PURE CULTURE METHODS IN THE ALGJE 33 of Stichococcus are soil-inhabiting and сап be obtained—practi- cally free from other algee—on flower pots and greenhouse soils. The former species, because of its minute cells and the readiness with which the filaments resolve themselves completely into their constituent cells when placed in water, is а particularly easy one to obtain in pure culture. Rich material may be diluted until plates obtained from it show a sufficiently small number of bacterial colonies to admit of pure transfers and yet enough algal colonies for a number of transfers. S. subtilis is a larger species and the cells remain attached in rather long filaments. However, with vigorous shaking and previous teasing apart with needles, a sufficient number of single cells and small fragments of filaments are introduced to make possible numerous successful isolations. Тһе washing of the cells to remove adhering bacteria can, in these species and many others, be largely accomplished by introducing the raw material into test-tubes containing sterile mineral nutrient solution or water, stoppering, and shaking vigorously. Direct transfers from these to liquid agar, or to tubes of sterile water for further dilution, may then be made. This procedure fre- quently enables one to make successful platings where the direct transfer of raw material to liquid agar results in constant failure. Chlorella vulgaris Bey., and Chlorella sp.—Both of these species were isolated from soil in the open. An exterior gelat- inous investment is, as in the two above mentioned species of Stichococcus, conspicuously absent, and preliminary experi~ ments demonstrated that a large number of the vegetative cells were freed from all accidentally adhering bacteria by being shaken in the liquid agar before plating. The problem of isolating these species again becomes one of clean material and sufficient dilution. Species of Chlorella are perhaps the easiest among the alge to isolate in pure culture, the process requiring little more than a direct application of bacteriological methods. Attention should be called to another method—really a modification of the one just given—by means of which Chlorella species may be obtained in pure culture. Its application is not necessary in the species of Chlorella investigated, since [Vor. 1 34 ANNALS OF THE MISSOURI BOTANICAL GARDEN the vegetative cells can be so readily freed from adhering bacteria. But its general applicability to other forms justifies its mention at this place. Chlorella, like many other genera of the Protococcales, forms non-motile endogenous daughter cells which remain enclosed in the mother wall for varying lengths of time. The enclosed daughter cells are in all cases free from adhering bacteria. A group of daughter cells still enclosed within the mother membrane may be removed by means of a capillary pipette to a drop of sterile water, and from here to a succession of others until all readily removable bacteria have been left behind. The last transfer should be made to a drop of sterile water on a small sterile cover glass. By а slight pressure of a second cover glass, the mother membrane may be ruptured, liberating the enclosed, bacteria-free cells. The two cover glasses should then be introduced into a tube of liquid agar, the latter shaken vigorously, and finally poured into a Petri dish. Frequent isolations have been made in this way, and its importance in forms whose vegetative cells cannot be freed from adhering bacteria, and which do not form motile spores but only non-motile endogenous daughter cells, can hardly be overestimated. Pleurococcus vulgaris Menegh.—The majority of Plewrococcus cells, when thoroughly washed, will be found free from bacteria. A difficulty which frequently arises is that the alga grows so very slowly that fungi—which are persistently present in Pleu- rococcus cultures—take entire possession of the plates before a transfer can be effected. But with careful searching, minute colonies—often consisting of but a few cells—can usually be found and successfully transferred. The transferred colony, however, usually makes extremely slow progress in its growth on agar. Much better results are obtained when transfers are made to evaporimeters (as devised by Livingston (15)) supplied with the mineral nutrient solution. Scenedesmus sp., and Kirchneriella sp.—Both of these species were obtained in pure culture by washing and diluting clean, concentrated material іп sterile mineral nutrient solu- tion, and then plating. The great majority of the colonies of both species were contaminated with bacteria, pure colonies being very rarely found. This fact, together with the gelatin- 1914) SCHRAMM—PURE CULTURE METHODS IN THE ALGZE 35 ous exterior characteristic of the cells of both species, makes it probable that the pure colonies developed, not from mature individuals, but from autocolonies (produced within mature cells) which either had just escaped from the mother cell or had done so during the vigorous shaking,—in either of which cases they are free from adhering bacteria. Chlorococcum humicola (Nüg.) Rabenh.—This species was isolated in the zoósporie condition. Тһе alga, collected from soil, was placed in sterile mineral nutrient solution and after · twenty-four hours produced zoóspores in abundance. Platings with these yielded numerous pure colonies from which successful transfers were made. In this connection it should be men- tioned that all zoóspores thus far experimented with—including a considerable variety of forms—have been found free from bacteria. It is needless to say, therefore, that the presence of zoóspores in the life cycle of any alga provides a logical point of attack for its isolation in pure culture. While not all the attempts to isolate zoósporie forms in pure culture have proved successful, it is entirely probable that they will when the general technique is more closely adapted to individual forms. Protosiphon botryoides (Kütz.) Klebs.—The vegetative plant of Protosiphon, with its root-like process extending into the soil and the large aerial portion, is so persistently covered with bacteria that its isolation in pure culture in this condition is quite impossible. With slight desiccation, however, large numbers of chlamydospores with dry non-gelatinous mem- branes appear, which, at least so long as they remain enclosed within the mother membrane, are free from bacteria. From these, isolations in pure culture can be readily made according to the second method suggested for Chlorella—by carefully washing an individual plant filled with chlamydospores, liber- ating the latter by teasing with needles or by a slight pressure of the cover glass, and plating in the usual manner. Another method which has yielded pure cultures, but which is not to be recommended because it is far less reliable than the one just described, is based on the use of the motile gametes. When vigorous Protosiphon plants, growing on soil, are covered with distilled water, gametes, which congregate in the lighted [Vor. 1 36 ANNALS OF THE MISSOURI BOTANICAL GARDEN side of the vessel, are produced in large numbers. Plates made with this material yield an occasional pure culture, but most of the gametes fail to develop. It is impossible at present to say whether the colonies develop from newly formed zygotes or from gametes which fail to conjugate. Stigeoclonium tenue (Ag.) Kützing.— The ease and certainty with which zoóspores can be induced to develop in this form, and their extreme abundance, makes it, although a filamentous alga, an especially easy one to isolate. Freshly collected and thoroughly washed filaments of Stigeoclonium, placed in dis- tilled water or sterile nutrient solution, will, in from twelve to twenty-four hours, develop a great abundance of zoóspores. Cultures prepared in this way contain so small a number of bacteria that plates containing a hundred or more Stigeoclonium zoóspores are sufficiently free from bacterial colonies to render numerous successful pure transfers possible. Although a filamentous form, Stigeoclonium grows exceedingly well on the mineral nutrient agar. While other members of the Chetophoracee were not experimented with, it is reasonably certain that forms like Microthamnion, Chetophora, and Drapar- naldia, all of which readily yield large quantities of zoóspores, may be obtained in pure culture by a method identical with or similar to the one employed in the isolation of Stigeoclonium. Oedogonium sp., and Vaucheria sp.—While neither of these forms were obtained in pure culture, the observations made render it altogether likely that this will be possible when a little more attention is given to the cultural solutions. Repeated trials with the vegetative filaments demonstrated that from the latter no pure cultures could be obtained directly. The oóspore proved equally unsatisfactory because the oógonial wall is covered with adhering bacteria. Again, the odspore is, in most cases, so firmly and completely united with the oógo- nial wall that its separation from the latter is at present impos- sible. In both forms, however, zoóspores are readily obtained, and preliminary experiments demonstrated that these, like zoóspores in general, are bacteria-free. Where zoóspores could not be obtained in large quantities, individual ones were isolated with sterile pipettes, washed repeatedly in sterile water, and then either plated in the usual manner, or introduced into а 19141 SCHRAMM—PURE CULTURE METHODS IN THE ALG 97 tube of sterile mineral nutrient solution. Although the great majority of such isolations remained bacteria-free, the zoóspores failed to develop, and finally died. It is only necessary, there- fore, to find some medium in or on which the zoóspores will germ- inate and develop into plants, to effect а pure culture of Vaucheria or Oedogonium. Bulbochete was not used, but in all probability this form will lend itself to a similar technique. Conjugales.—Thus far it has not been possible to obtain 8 pure culture of any member of the Conjugales. Тһе repre- sentatives of this order, in their vegetative phases, are provided throughout with an exterior gelatinous investment which is very generally impregnated with bacteria. АП attempts to obtain pure cultures from vegetative individuals failed. Fur- ther, there is a complete absence in the order of motile spores and, in general also, of separable, asexual, endogenous spores. The zygospore, therefore, suggests itself as а possible means of solving the problem, especially in those forms where it is produced endogenously, and where it does not subsequently coalesce with the wall of the gametangium. While pure cultures were not obtained from these, the method used in Spirogyra setiformis is of interest and may prove serviceable in the ultimate isolation of these forms in pure culture. Filaments containing mature zygospores, but in which the zygospore-containing cell walls were still completely intact, were washed repeatedly in sterile water and then broken up as thoroughly as possible with needles; in this process, numer- ous zygospores were freed from the enclosing walls, later to be taken up with sterile pipettes, and transferred to sterile drops of water. Each zygospore was subsequently transferred from ten to twenty times to fresh, sterile water drops, and finally taken up with a sterile pipette. When a considerable number of zygospores had thus been isolated, they were introduced into a tube containing a few cc. of sterile water, vigorously shaken, and the entire contents poured out into a Petri dish containing a layer of sterile nutrient agar. After rocking the dish for a short time, it was allowed to remain quiet until the zygospores had settled down on the surface of the agar. The free water was then very slowly and carefully, but completely, drained from the surface of the agar, and the plate allowed to remain [Vor. 1 38 ANNALS OF THE MISSOURI BOTANICAL GARDEN in the light. While in a few cases bacterial colonies developed about the zygopsores, it was found that the great majority were free from all adhering bacteria. Such zygospores as were bacteria free were then transferred to test tubes containing sterilized mud and pond water. Although about sixty such transfers were made, not a single one yielded a growing culture, although zygospores kept in battery jars in the laboratory showed а high percentage of germination. It will require further experiments to find a suitable medium for the germina- tion and subsequent growth of isolated zygospores. However, the isolation of bacteria-free zygospores justifies the opinion that with them it will, sooner or later, be possible to culture Spirogyra in a state of purity. HETEROKONT/E Botrydium granulatum (1.) Greville.—This form is, in its general morphology, so similar to Protosiphon, that the tech- nique, as regards the use of chlamydospores, described for the latter, is entirely applicable here. Botrydium when submerged, however, forms an abundance of zoóspores instead of gametes, and from these pure cultures can be obtained with great ease when plated in the usual manner. Тһе method for using the chlamydospores can also be considerably abbreviated in Botryd- ium. When the plants form chlamydospores, the aerial globular portion of the plant collapses. Тһе cell, however, is so large that the aerial bag сап be torn open with fine sterile forceps, the spores removed under a hand lens with a needle and transferred directly to liquid agar. Platings made in this way show a very slight bacterial contamination, and pure transfers сап be made in abundance. While a direct, bacteria- free transfer has not been thus effected, it is altogether probable that it ean be done. Тһе pure transfers of Botrydium having been obtained, it was found that their development on agar was extremely slow, and ultimately all of the cultures died. Further experiments will be necessary in order to provide а favorable medium for growth. The clay-cup evaporimeter may perhaps prove of service in this connection as it did in the case of Pleurococcus. Botrydiopsis sp.—' This form was found abundantly during 1914] SCHRAMM—PURE CULTURE METHODS IN THE ALGJE 39 one season оп soil in the greenhouses. Тһе vegetative cells when placed in water readily produce zoóspores, and isolations were made from these with little difficulty. Unlike Botrydium, this form grows exceedingly well on the mineral nutrient agar. BACILLARIALES The diatoms were encountered only incidentally in connec- tion with other forms, and no particular effort was made to isolate forms in pure culture. Although diatoms, in general, have a gelatinous exterior, a small Navicula was on several occasions obtained in pure culture and grown successfully. It should be said, however, that the great majority of diatom colonies obtained were contaminated with bacteria. CYANOPHYCEJE In the class Cyanophycee, the most difficult problems of isolation are met. Тһе almost universal presence of an abun- dance of external mucilaginous material, the complete ab- вепсе of ciliated reproductive cells, and the virtually complete absence of free, endogenous spores, renders the technique particularly difficult. Тһе gelatinous investments аге, in all cases investigated, impregnated with bacteria which can- not be completely removed by the most vigorous washing. Among the forms studied were Aphanocapsa, several species each of Oscillatoria, Nostoc, and Anabena, Cylindrospermum, and Microcoleus. Of these, only two species, one of Oscilla- toria and one of Microcoleus, were obtained in pure culture. In the isolation of these two forms, silicic acid jelly was found to be indispensable. While directions for preparing this medium are to be found in many places in the literature, certain difficulties encountered in its preparation have made it desirable to give at this time, and in some detail, the method used. As regards the preparation and mixing of the sodium silicate and hydrochloric acid solutions, the directions given by Smith (22) may be followed. It is only necessary to point out in this connection that if Merck’s “sodium silicate pure crystals" is used, the solution should be made up with cold water. If hot water is used, an unidentified substance (insoluble in cold water) goes into solution, and frequently causes the coagulation [Vor. 1 40 ANNALS OF THE MISSOURI BOTANICAL GARDEN of the silicic acid-hydrochloric acid mixture before dialysis is complete. А point of very great importance is the preparation of the collodion dialyzing bags. Ав has been pointed out by Kellerman (12), and others, the degree of permeability of the bags depends, in а large degree, upon the way in which they are made. Ifthe guncotton solvent is made from equal parts of ether and absolute alcohol, the bags will, in most cases, have a very low permeability, and coagulation of the enclosed silicic acid solution will frequently result before dialysis is complete. Тһе degree of impermeability is further increased by drying the bags rapidly. If, however, 95 per cent (instead of absolute) alcohol is used, and the bags are allowed to dry spontaneously by invert- ing the test-tubes in which the bags are being prepared in suspended wire baskets, a much higher degree of permeability will be obtained. Bags prepared with 95 per cent alcohol were used, and the silicic acid-hydrochlorie acid mixture dialyzed in tap water until the chloride content was no greater than that of the water. The вШсіс acid solution was further purified by dialyzing in changes of ordinary distilled water and finally in triply distilled, nitrogen-free water. In this extended dialysis, а considerable portion of the silicic acid is lost, and it usually becomes necessary to concentrate the solution to obtain a jelly of sufficient firm- ness. This is best carried out in heavy, two-liter suction-flasks in which the pressure is reduced until the solution boils at from 35 to 409С. If the concentration is carried out at higher temperatures, coagulation sometimes results. In order to pre- vent the violent bumping which always takes place unless some Special precautions are taken, it is only necessary to bring through the rubber stopper at the top of the suction-flask a glass tube drawn out at the bottom to a very fine capillary, which dips into the solution. Тһе top of this tube, outside of the rubber stopper, should be provided with a piece of rubber tubing and pinch cock to regulate the intake of air. Тһе air thus admitted may first be washed to remove carbon dioxide, ammonia, or other impurities. The concentration should be continued until a sample, when congealed, has the proper consistency. Тһе directions given by Smith (22) for coagu- lating the medium apply here and it need only be mentioned 1914) SCHRAMM—PURE CULTURE METHODS IN THE ALGÆ 41 that the concentration of the mineral nutrients employed in the agar, 0.1 per cent, is quite sufficient to bring about coagu- lation. After it had become probable that no blue-green alga, in the ordinary vegetative condition, could be isolated by the usual plating method, tubes containing from two to three inches of solid, sterile, synthetic agar were inoculated at the surface with a species of Oscillatoria. The tubes were then completely wrapped in black paper, leaving only the very bottom exposed to the light, and inverted. It was hoped that in the rapid growth of the alga through the agar, the bacteria might be left behind. The growth toward the light in some cases amounted to eight mm., and more, per day. When the growth had approximately reached the bottom of the tube, the end of the latter was broken away, the surface of the agar seared, and transfers made from the interior of the agar plug. Although the experiment was repeated many times, and a total of at least fifty transfers made, a pure culture was never obtained, bacteria always being present. Large Petri dishes, containing a layer of sterile synthetic agar, were then inoculated at one edge with a species of Oscillatoria, and the dishes so placed that the point of inoculation was farthest away from the light. Тһе alga grew rapidly (on the surface of the agar) toward the light, and just before reaching the opposite edge of the dish, transfers were made from the farthest advanced filaments. Although transfers to fresh agar surfaces were continued to the number of six, a pure culture was never obtained. The experiment was then repeated, surfaces of silicic acid jelly replacing those of agar, with the result that numerous pure transfers were obtained from the second plate. A species of Microcoleus was obtained in pure culture in an identical manner. Most members of the Oscillatoriacee are provided with a sharply delimited, gelatinous sheath. Reproduction is effected by the formation of hormogonia which glide out of the sheath, move about slowly for a time, and then come to rest. In forms like Microcoleus, Lyngbya, and some species of Oscilla- toria in which the hormogonia escape from definite sheaths, [Vor. 1 42 ANNALS OF THE MISSOURI BOTANICAL GARDEN leaving the latter behind, it is fairly certain that the hormo- gonium is originally free from bacteria, but becomes contam- inated in passing through the older portion of the empty sheath and out of its terminal opening, both of which are more or less infected with bacteria. The persistence with which the bacteria cling to the hormogonium of Oscillatoria, once having infected it, is clearly shown by cultures on agar surfaces. Although a single hormogonium may have moved as much as two inches away from its parent filament, creeping all the while over a sterile agar surface, the hormogonium will be found covered with bacteria, and the path over which it moved will be clearly indicated by a continuous, linear colony of bacteria. With the use of silicic acid jelly, however, the multiplication of the bacteria is reduced to such an extent that, after a time, hormo- gonia escape uncontaminated, and begin the development. of pure colonies. Transfers from these, however, grow very slowly and in most cases eventually die. It seems probable, when Oscillatoria and Microcoleus have been completely sepa- rated from the invariably present bacteria, that the media which were favorable in the presence of the bacteria, become unfavorable in their absence. Further work will be necessary to grow these forms successfully after they have been isolated in pure form. The silicic acid jelly method was also attempted with the above mentioned heterocystic forms; however, up to the present time, no successful isolations have been made. Discussion It is apparent that the technique involved in the isolations just referred to depends entirely on mechanical separation of one kind or another. This method is reasonably efficient in those species in which zoóspores or other free endogenous spores are readily obtainable, or in which vegetative cells are either free from bacteria or can be rendered so by mechanical means. It is true that even in some species forming free endogenous spores, the above methods have not yielded pure cultures, as, for instance, in Vaucheria, Oedogonium, and Spiro- gyra. In these cases, however, it should be pointed out that it is not the isolation technique which is at fault but rather 1914) SCHRAMM—PURE CULTURE METHODS IN THE ALGZE 48 the cultural methods. Zoóspores and zygospores, respectively, free from other organisms, were obtained in these cases but failed to develop in the cultural media subsequently supplied. There can be little doubt, however, that the latter difficulty will be overcome in time. Except in the Oscillatoriacee, little progress was made in the Cyanophycee. The problem appears especially difficult in the Coccogoneales where all forms of motile reproductive bodies are absent, and in which the vegetative cells apparently cannot be rendered free from adhering organisms by mechan- lica means. Even in the heterocystic Hormogoneales, the situation is a difficult one, the more slowly moving hormogonia apparently being unable to escape the bacteria.! While no experiments were made along these lines, it appears highly desirable to attack the problem in the latter group through the spore. It is well known that the spores of blue-green alge are extremely resistant to heat, and it does not appear improbable that the bacteria—especially if they are all in the vegetative condition—could be killed by heat, leaving the algal spores unharmed. Chemical sterilizing agents may also prove of value here. The latter may also prove serviceable with members of the Coccogoneales and certain of the grass- green alge which have thus far failed to yield to the technique employed. CONCLUSIONS 1. By adapting methods of pure culture technique to indi- vidual species of alge, it has been possible to isolate in pure culture the following forms: Chlorophycee.—Chlamydomonas pisiformis Dill forma minor Spargo, Stichococcus bacillaris Näg., S. subtilis (Kütz.) Klercker, Ulothrix sp., Chlorella vulgaris Bey., Chlorella sp., Pleurococcus vulgaris, Scenedesmus sp., Kirchneriella sp., Chlorococcum humi- cola (Näg.) Rabenh., Protosiphon botryoides (Kütz.) Klebs, Stigeoclonium tenue (Ag.) Kützing, and a number of others of uncertain identity. 1In a contribution which has just appeared (Kulturversuche mit Chlorophyll- führenden Mikroorganismen, III. Zur Physiologie der Schizophyceen. Beitr. z. Biol. d. Pflanzen 12: 49-108. 1913), Ernest G. Pringsheim reports the isolation in pure culture of a species of Nostoc. The method used was that of repeated transfers to sterile silicic acid jelly plates. [Vor. 1 44 ANNALS OF THE MISSOURI BOTANICAL GARDEN Heterokonte.—Botrydium granulatum (L.) Greville, and Botry- diopsis sp. Bacillariales.—Navicula sp. Cyanophycee—Oscillatoria sp., and Microcoleus sp. 2. In addition, zoóspores from Vaucheria and Oedogonium, and zygospores from Spirogyra have been isolated free from other organisms. In conclusion, the author wishes to express his gratitude to Dr. Geo. T. Moore, at whose suggestion the work reported herein was undertaken, whose advice and interest have been a source of constant help; and to Mildred Spargo Schramm, for kindly assistance in many ways. LITERATURE CITED 1 Artari, Alexander, Ueber die Entwicklung der grünen Algen unter Ausschluss der Bedingungen der Kohlensüure-Assimilation. Bull. de la Soc. Imp. de Nat. de Moscou 1899: 39-47. 1899. 2. — — ——, Zur Frage der physiologischen Rassen einiger grünen Algen. Ber. d. deut. bot. Ges. 20: 172-175. 1902. 3. Barber, M. A. On heredity in certain micro-organisms. Kansas Univ. Sci, Bul. 4: 3-48. 1907. 4. Beyerinck, M. W. Kulturversuche mit Zoochlorellen, Lichengonidien und anderen niederen Algen. Bot. Zeit. 48: 725-785. 1890. 5.-----, Bericht über meine Kulturen niederer Algen auf Nahrgelatine. Centralbl. f. Bakt. 13: 368-373. 1893. 6. — — —-, Over gelatineculturen van eencellige groenwieren. [Reviewed іп Cen- tralbl. f. Bakt. 8: 460-462. 1890.) 7. Chick, Harriette. А study of a unicellular green alga, occurring in polluted water, with especial reference to its nitrogenous metabolism. Proc. Roy. Soc. 71: 458-476. 1903. 8. Chodat, R., et Goldflus, M. Note sur la culture des Cyanophycées et sur le devel-. oppment d’Oscillatoriées coccogénes. Bull. de |’ Herb. Boissier 5: 953-959. 1897. 9. Chodat, R., et Grintzesco, I. Sur les methodes de culture pure des algues vertes. Actes du Congrés Int. de Botanique, Paris, 1900, 157-162. 10. Frank, Theodor, Kultur und chemische Reizerscheinungen der Chlamydomonas. tingens. Bot. Zeit. 62: 153-188. 1904. ll. Jacobsen, H. C. Kulturversuche mit einigen niederen Volvocaceen. Zeitschr. f. Bot. 2: 145-188. 1910. 12. Kellerman, K. F. Тһе permeability of collodion tubes. Centralbl. f. Bakt. II. 34: 56-60. 1912. 13. Krüger, Wilhelm, Beitrüge zur Kenntniss der Organismen des Saftflusses (вор. Schleimflusses) der Laubbiume. Beitr. 2. Physiol. u. Morph. nied. Organismen 4: 69-116. 1894. 14. Küster, Ernst, Anleitung zur Kultur der Mikroorganismen. 105-107. 1907. 1914] 15. 16. 17. 18. 19. 20. 21. 22. 23. SCHRAMM—PURE CULTURE METHODS IN THE ALGJE 45 Livingston, B. E. А new method for cultures of alge and mosses. Plant World 11: 183-184. 1908. Miquel, P. De la culture artificielle des Diatomées. Le Diatomiste 8: 73-75. 1892: о: 93-99. 1892. [Reviewed in Compt. Rend. 114: 780-82. 1892.) Moore, С. T. Methods for growing pure cultures of alge. Journ. Appl. Microsc. 6: 2309-14. 1903. Ogata, M. Ueber die Reinkultur gewisser Protozoen (Infusorien). Centralbl. f. Bakt. 14: 165-169. 1893. Radias, M. Sur la culture des algues à l'etat de purité. Actes du Congrés Int. de Botanique, Paris, 1900. 163-167. Richter, Oswald. Reinkulturen von Diatomeen. Ber. d. deut. bot. Ges. 21: 493-506. 1903. ——————, Die Ernährung der Algen. Monograph. и. Abhandl. z. internat. Revue der ges. Hydrobiol. u. Hydrograph. 2: 31. 1911. Smith, E. Е. Bacteria in relation to plant diseases. Publ. Carnegie Inst. 271: 37-39. 1905. Tischutkin, N. Ueber Agar-Agarkulturen einiger Algen und Amóben. Cen- tralbl. f. Bakt. II. 3: 183-188. 1897. . Ward, H. Marshall, Some methods for use in the culture of alge. Ann. Bot. 13: 563-566. 1899. Graduate Laboratory, Missouri Botanical Garden. THE IDENTIFICATION ОЕ THE MOST CHARACTER- ISTIC SALIVARY ORGANISM, AND ITS RELATION TO THE POLLUTION OF AIR! AUGUST G. NOLTE INTRODUCTION Bacteriologists and sanitary engineers have, within the last score of years, given much attention to the detection of excre- mental pollution in water. They have shown that by making it possible to recognize certain characteristic accompanying organisms, bacteriological methods are capable of revealing this kind of pollution even when it exists to such a small degree as to be beyond the range of chemical detection. Small as these quantities of contaminating substances may seem, they may nevertheless endanger the health of a whole community by exposing it to possible pathogenic organisms derived from the excreta of a diseased host. It is not merely by the aggregate bacterial yield that the potability of a water in its relationship to disease is judged, but more specifically by the species of bacteria present, and their relative abundance. The micro-organisms which serve as an index of pollution, and for which special quantitative examination is made, are the members of the colon group. These, from their constant presence and relative abundance, are characteristic of material of excremental origin. Their presence in water in sufficient quantity indicates pollution, and their relative abundance serves as an index to the extent of the latter. Bacteriological technique has not as yet been applied to the same extent in the detection of pollution in air. Chemistry has, up to the present time, been of more practical value here. 1 An investigation carried out at the Missouri Botanical Garden in the Graduate Laboratory of the Henry Shaw School of Botany of Washington University, and submitted as a thesis in partial fulfillment of the requirements for the degree of master of arts in the Henry Shaw School of Botany of Washington University. ANN. Мо. Вот. GARD., Vor. 1, 1914 (47) 4 [Vor. 1 48 ANNALS OF THE MISSOURI BOTANICAL GARDEN The proportion of carbon dioxide is still the standard mainly relied on for estimating pollution of air by materials given off from the human body, although it is recognized that other factors may be of more importance. This method of exam- ining air, however, is of little or no value in furnishing an index to the probable or possible contamination with disease-pro- ducing germs, for there is at present no reason for believing that such organisms are given off in the breath during ordinary quiet breathing. Thus, M. Н. Gordon! calls attention to the following: Tyndall observed that expired air is optically purer than inspired air; Cornet found air expired by tubercular patients to be free from the tubercle bacillus; and Straus has shown that expired air is not only comparatively free from bacteria, but that it is considerably purer in this respect that inspired air. It nevertheless appears probable that bacteriology rather than chemistry will furnish à means of investigating the pollution of air by disease-producing germs. Тһе problem at hand is to devise, if possible, а method for estimating the degree of pollution of air by pathogenie organisms (given off from the human body) in a manner similar to that employed in estimat- ing the extent of pollution of water by similar organisms of excrementa] origin. HISTORICAL It appears that the present status of bacteriological analysis of air is comparable to that of bacteriological analysis of water some years ago, when the total number of bacteria in a given quantity was the chief factor determined. There are various ways in which pathogenic organisms may gain access to the air and ultimately to another individual. In addition to trans- fer by direct contact, disease-producing organisms may be given off in the urine, in feces, in sputum, or from the surface of the skin. Recently, also, attention has been called to the possi- bility of the pollution of air by the scattering of fine particles of mucus and saliva from the mouth in the acts of coughing, sneezing, and loud speaking. The latter methods of air pollu- 1 Report on a bacterial test for estimating pollution of air. Supplement to the Thirty-second Annual Report of the Local Government Board (London), containing the Report of the Medical Officer for 1902-3. 421-471. 1904. 19141 NOLTE— SALIVARY ORGANISMS AND AIR POLLUTION 40 tion are the ones to be considered in this investigation. They doubtlessly constitute an important means whereby patho- genie organisms enter the air from an infected person, subse- quently to be transmitted to other individuals. The discharge of sputum furnishes the most obvious way whereby pathogenic organisms may be expelled from the mouth. The expectorated mucus dries, and, in the form of dust, may later be inhaled to produce infection. Тһе work of Flügge and members of his school; however, has drawn attention to а more direct and по less important way by which germs may be aérially conveyed from the mouth. Тһе problem of trans- mission of micro-organisms by means of particles of mucus expelled from the mouth in various expiratory acts, was attacked in two ways by the investigators referred to above: 1. Тһе mouth was artificially infected with a culture of Bacillus pro- digiosus. This organism was chosen because the red pigmenta- tion of the colonies renders the identification easy. After agar plates had been placed at various distances from the person being experimented upon, the individual proceeded to speak, cough, sneeze, etc. At the end of the experiment the agar plates were covered and incubated at 25? C. for 3 days, during which time the characteristic red colonies of B. prodigiosus made their appearance. Тһе possibility of error due to the previous presence of this organism in the air of the room was excluded by exposing a series of agar plates immediately before the experiment began, with the result that in all cases the organism failed to appear. Тһе length of time that droplets of mueus remained suspended in the air after the several expir- atory acts was determined by exposing plates at various periods after the experiment had been completed. 2. Glass slides or empty Petri dishes were placed at various distances from a tubereular patient. Тһе droplets of mucus expelled during coughing, and deposited upon the glass slides, etc., were either examined microscopically or were washed off and injected intraperitoneally into guinea-pigs. In the former case a bacillus giving the characteristic staining reaction of the tubercle bacillus was found, and in the latter the development of tuber- 1 Gordon, loc. cit. [Vor. 1 50 ANNALS OF THE MISSOURI BOTANICAL GARDEN culosis in the inoculated animals resulted. In other experiments, guinea-pigs, instead of being inoculated, were directly exposed to the coughing of tubercular patients with the result that a number of the animals so exposed contracted tuberculosis. Varied and repeated experiments along these lines established the fact that in the acts of coughing, sneezing, and loud speak- ing, fine droplets of mucus are ejected into the air, that they float about and may be wafted by air currents, such as obtain in ordinary rooms, to a distance of from 24 to 40 feet. The most thorough investigation in recent years of the problem of air pollution with micro-organisms was made by Dr. М.Н. Gordon. This author believed that the positive recognition of disseminated saliva constituted an important step in the development of an applicable bacteriological method for the examination of air. By bacterial analyses of a number of samples of saliva obtained from normal individuals, Dr. Gordon determined that the streptococci are the organisms most abundantly present in saliva. Of these he was able to differentiate four morphologically different types—longus, me- dius, brevis, and conglomeratus. In endeavoring to differentiate these organisms on а physiological basis a study was made of their virulence, relation to oxygen, optimum growth temper- ature, pigment production, motility, gelatin liquefying power, indol production, action on litmus milk at 37°C., and action on various carbohydrates. It was found that the micro-organism which is most useful in the detection of droplets of saliva is Streptococcus brevis because it is the only one among the salivary cocci found which changes the color of neutral red broth to yellowish green, and produces acid and clot in milk. Having developed a means of differentiating the coccus most characteristic of saliva, Gordon next examined the open air for the presence of micro-organisms characteristic of saliva. In these experiments broth plates were exposed for a stated length of time and incubated anaérobically at 37°C. In but very few cases were the organisms isolated from the air. A further means of differentiating the characteristic salivary 1 Loc. cit. 1914) NOLTE—SALIVARY ORGANISMS AND AIR POLLUTION 51 coccus from the air cocci was sought in the action of the two on various organic substances. Іп this capacity the several broths containing lactose, syringin, and coniferin, proved especially serviceable. In lactose broth the typical salivary coccus was positive, i. e., it produced acid, whereas the air cocci were negative. In the syringin and coniferin broths, the air cocci were positive, the typical salivary coccus negative. To determine whether or not particles of saliva were dis- seminated through the air during the acts of coughing, sneezing, and loud speaking, Gordon performed experiments in a large and in a small room, using, at first, Flügge's method of arti- ficially infecting the mouth with a living culture of Bacillus prodigiosus and placing sterile agar plates at various distances in front of and behind the speaker. After 3-1 hour of loud speaking, it was found that B. prodigiosus had been disseminated to a distance of 40 feet in front of and of 12 feet behind the speaker. In other experiments in which no artificial infection of the mouth was resorted to, but in which the characteristic salivary coccus served as the index of dissemination, it was found that after 1-1 hour of loud speaking Streptococcus brevis appeared on broth plates placed as many as 12 feet in front of and behind the speaker. In similar experiments in which speaking was continued for one hour in an ordinary conver- sational tone, no dissemination of the salivary Streptococcus could be detected. From his experiments Dr. Gordon inferred that there were certain streptococci normally present in saliva which are appli- cable for the detection of droplets of saliva in air in much the same manner that Bacillus coli can be applied for the detection of fecal matter in water. THE IDENTIFICATION OF THE Most CHARACTERISTIC SALIVARY ORGANISM With a view of determining the organism most characteristic of saliva, I have undertaken, as a first step, a bacteriological analysis of the saliva of a normal individual. In this examina- tion special attention was paid to the type of organism most abundantly present. Having determined the type, i.e., whether bacillus, coccus, or spirillum, characteristic reactions for it were next sought in order to render its recognition easy. Since [Vor. 1 52 ANNALS OF THE MISSOURI BOTANICAL GARDEN а possible relation of the characteristic salivary organism to the pollution of air was to be investigated, it was necessary to examine the outdoor air free from human contamination for the presence of micro-organisms closely allied to those characteristic of saliva. Ав particles shed from the skin may be present in the air, it was further necessary to examine those micro-organisms found on the skin which were closely allied to the ones characteristie of saliva. In examining the saliva for the type of micro-organism · most constantly present, 1.е., whether bacillus, coccus, or spirillum, the dilution method was used. It is reasonably safe to assume, after repeated trials, that the type of micro- organism which persists longest in continued dilutions is the type most abundant in the material examined. This is true provided the medium on which the organism is grown is approx- imately equally favorable for the development of all the types present. Тһе dilutions were carried out as follows: A sample of saliva was collected in a sterile test-tube and 1 cc. introduced into а second tube containing 9 сс. of sterile distilled water. The contents of the latter were then thoroughly mixed and 1 ec. of the liquid introduced into a third tube likewise con- taining 9 cc. of sterile distilled water. This procedure was repeated until 6 dilutions had been effected. Obviously, 1 cc. quantities of each of the 6 successive dilutions contain re- 1 1 1 1 . spectively 5, i0» 990, 10,000) 100,000) 2Nd 10006 CC. Of saliva. One plate each from dilutions 4, 5, and 6 was made, 1 cc. of the respective dilutions being introduced into 10сс. of nutrient + 1 agar. After thorough mixing, the plates were incubated aérobically for 24 hours at 37°C. Тһе plate made from dilu- tion 5 produced 20 colonies, whereas the one from dilution 6 showed no growth. From each of the 20 colonies a cover-glass preparation stained with gentian violet was made. Microscopic examination revealed the fact that each of the 20 colonies was composed of micro-organisms of the coccus type. Transfers were then made to agar slopes which were incubated at 37°C., for 24 hours. The cultures obtained in this manner were num- bered from 1 to 20 and kept at 20°C., as stock cultures. In examining the open air, sterile agar plates were exposed as indicated in table 1. 1914] NOLTE—SALIVARY ORGANISMS AND AIR POLLUTION 53 TABLE I DATA ON THE COLLECTION OF AIR COCCI _ [388 [ази Time |95д |954 8 |Number of Puri © ons! given Place of exposure. сао арар! 6 Remarks expo- |-30,%./89,4. to sure | 32 920 оо 969| stock ECHO dx culture Window sill outside of lab-| 15 min- 21 to 27 oratory, 2nd floor. utes. 14 7 incl. On shelf, center of labora- One person in tory room. do. 2 0 room. Abun- dance of Mo- nilia present. On table, in reading room. do. 2 0 do. On table, in plating room of Monilia sup- laboratory. do. 0 0 pressed growth. On table, in basement do. 3 0 On lawn, in garden do. 9 1 28 to 31 incl. In living room. do. 7 6 32 to 37 incl. Window sill, 4th floor, Much soot оп downtown section do. 18 0 plate. On table, іп draughting One person room. do. 1 1 38 in room. After exposure the plates were covered and incubated aérobically for 24 hours at 37?C. Stained preparations of all colonies de- veloping were made and examined under the microscope. The coccus forms were transferred to agar slopes, and after incuba- tion for 24 hours at 37?C., were kept in stock at 20°C. Sev- eral of the plates exposed in various parts of the laboratory building were rendered worthless by an abundant growth of Monilia sitophila. The method of examining the skin for organisms closely allied to those characteristic of saliva, was as follows: Test- [Vor. 1 54 ANNALS OF THE MISSOURI BOTANICAL GARDEN tubes, each containing 10 cc. of distilled water and a piece of linen 2 inches square, were sterilized in the autoclav for 15 minutes at 15 pounds pressure. Samples were taken from three parts of the body of а normal individual, namely, the calf of the leg, the thigh, and the chest. "This was accomplished by briskly rubbing the portion of the body from which the sample was to be taken with the piece of linen held in sterilized forceps, and later replacing it in the tube of sterilized water. From these dilutions, after being thoroughly shaken, about 4 сс. quantities were plated in 10 cc. of nutrient agar. From each plate 2 coccus colonies were selected from which transfers were made to agar slopes. These, after 24 hours at 37°C., were kept as stock cultures at 20°C. There were now in stock a total of 44 pure cultures of Cocca- cee, 20 from saliva, 18 from the open air, and 6 from the skin. MORPHOLOGICAL CHARACTERS The form of the individual cell is of little value in differen- tiating the species of Coccacee, for under conditions favorable to their growth, all appear as regular spheres. Птершаг oval cells occur at times, but the form usually becomes normal after cultivation. Some writers lay considerable stress on the value of cell grouping in the Соссасее as a means of differentiation. With the utmost care in cultivation and staining, however, this could not be verified in the cultures under observation. All the cultures examined contained cells occurring singly, in pairs, in short chains, and in masses, but in no case did the cells of any specific culture exhibit a distinct tendency to occur іп any one form. А stained cover-glass preparation showed various cell groupings in different parts of the same microscopic field. Cell grouping was studied in the following manner: An oese of sterile +1 bouillon was placed on a sterile cover glass, inoc- ulated with а 24-hour culture of the organism to be examined, and inverted on a Van Tieghem cell containing a few drops of sterile distilled water. After sealing the cover glass on the cell with vaseline, the preparation was incubated for 24 hours at 37°C. At the end of this time the cover glass was removed and the drop of water containing the organism allowed to 1914] NOLTE—SALIVARY ORGANISMS AND AIR POLLUTION 55 evaporate. Then, 3 drops of mercuric chloride solution were applied and after 2 minutes washed off with distilled water. Following this, the preparation was treated with a few drops of 1 per cent acetic acid for 5 minutes, again washed in water, and finally stained for about 15 seconds with a few drops of gentian violet. After washing, and drying in the incubator at 37° C., the vaseline was removed from the cover glass with xylene, and the preparation mounted in balsam and examined under the microscope. Тһе relation to Gram stain was observed on 2 and 4-day agar cultures incubated at 20°C. The prepa- rations were treated with aniline oil-gentian violet for 1% minutes, with Gram’s iodine solution for 13 minutes, and finally with 95 per cent alcohol for 3 minutes. The reactions are recorded as “— —" (decolorized in both tests), “+ —’’ (stained in one test and decolorized in the other), and “+ +” (stained in both tests). CULTURAL CHARACTERISTICS All cultural characteristics were observed in streak cultures on agar slants after 14 days’ incubation at 20°C., and 37°C. Such differences as developed between the cultures were almost entirely variations in color and vigor of surface growth. Under the latter, 5 types were distinguished as follows: 1. Growth very faint and veil-like, or forming scattered translucent colonies. Growth better, but still meager. Growth good, but not abundant. Growth abundant. 5. Growth very heavy. i oo bo In the study of chromogenesis, apparent differences in pigment production, due to unequal vigor of growth or evaporation, were, so far as possible, eliminated. This was accomplished by exam- ining in each case the same amount of material—a loopfull— spread evenly on white drawing paper having a rough surface. After drying at room temperature, the color of the pigment produced was compared with the colors as given by Ridgway’. 1 Color standards and nomenclature. 1912. [Published by the author, Washing- ton, D. С.) [Yon 1 56 ANNALS OF THE MISSOURI BOTANICAL GARDEN This author uses as а basis the solar spectrum with its six fundamental colors and intermediate hues, augmented by a series between violet and red not in the spectrum. BIOCHEMICAL REACTIONS The production of indol was investigated in 5-day peptone broth cultures incubated at 37°C. One cc. of a 10 per cent sulphuric acid solution was thoroughly mixed with the broth culture, and then 1 сс. of a freshly prepared 0.01 per cent solution of sodium nitrite was carefully run in on top of the mixture. The appearance of a pink ring at the juncture of the nitrite solution with the acid-peptone solution, was regarded as an indication of the presence of indol. A blank determina- tion for purposes of comparison was made in each case. The action on neutral red broth as regards change in color was observed in cultures incubated for 5 days at 37°C., in the presence of hydrogen. The organisms were further grown in solutions of nitrate broth to determine whether or not reduction takes place, and if so, whether to nitrite or to ammonia. In carrying out the test a tube of nitrate broth was inoculated with the organism. to be tested, and incubated for 4 days at 37°C., an uninoculated tube of nitrate broth being similarly treated to serve as a check. At the end of 4 days, 3 cc. of the broth were removed to a clean test-tube, and 2 cc. each of a naphthylamine solution and of а sulphanilie acid solution added. Тһе development of a red color indicates the presence of nitrites, the intensity of the color being proportional to' the amount of nitrites present in solution. То test for ammonia in the remaining portion of the culture, а few drops of Nessler's solution were added. The appearance of a yellow color or precipitate indicates the presence of ammonia. Іп studying the liquefaction of gelatin by the cocci under observation, the extent of the action only was determined. This was accomplished by spreading а suspension of the organism over the surface of gelatin in 10 mm. tubes. It was found that the amount of material used in this inoculation did not affect the total amount of liquefaction, ie. whether the amount of transferred material was large or 1914] . NOLTE—SALIVARY ORGANISMS AND AIR POLLUTION 57 small the extent of the liquefaction after 30 days' growth at 20°C., was the same with any one organism. In the study of the action on sterile certified milk ее attention was paid to the coagulation of the milk and to the production of acid. Observations were further made on the effect of the organisms on lactose, saccharose, mannite, salicin, inulin, sorbite, raffinose, and rhamnose. Тһе medium in which these organic substances were used was prepared according to Dr. Houston’s formula, as follows: Liebig’s beef extract 1.0 per cent Peptone 1.0 per cent Organic compound to be tested 1.0 per cent Sodium bicarbonate 0.1 per cent 10 per cent litmus solution 1.0 per cent The medium, neutral in reaction to litmus, was sterilized for 15 minutes at 15 pounds pressure in 500 cc. containers, from which sterile fermentation tubes, provided with glass caps, were directly filled. In doing this it was necessary to take utmost precautions to obviate any possibility of contamina- tion. The various organie media thus prepared were inocu- lated, not from an agar slope, but from a 48-hour broth culture. Gas formation and the production of acid in the several media were observed after 3 days’ incubation at 37°C. DISCUSSION OF RESULTS A thorough study of the results will now be made with a view of finding, if possible, some characteristic or group of characteristics, morphological or biochemical, which may be used in differentiating the salivary cocci from the coccus forms of the air and the skin. The cell grouping varies throughout, there being no arrange- ment characteristic of any particular group. As observed, all the forms occur in groups, chains, and pairs. As regards the deportment of the various organisms toward the Gram stain, it was noted that all of the salivary cocci gave a positive reac- tion in both tests; of those from the air, 3 were positive in the two tests, 8 alternately negative and positive, and 6 negative throughout; of the skin cocci, 4 were positive and 2 negative [Vor. 1 58 ANNALS OF THE MISSOURI BOTANICAL GARDEN in both tests. This stain, as may readily be seen, is of no differential value here, for, although the salivary cocci react positively throughout, both positive and negative reactions occur among the air and skin forms. bs The production of indol among coccus forms is very uncom- mon. Of the salivary cocci under observation, none produced indol, and of the air and skin forms only one from each group produced it. The change of color in neutral red broth is, apparently, more frequently brought about by the salivary cocci than by the air and skin forms, but this difference is not sufficiently well marked to be of differential value. Of the 20 salivary cocci, 12 produced fluorescence, whereas only 1 of the air and none of the dermal forms produced this change. All of the forms under observation reduced nitrates to ammonia. Of the salivary forms, 14 out of 20; of the air cocci, 5 out of 18; and of the skin cocci, 5 out of 6, reduced nitrates to nitrites. It thus appears that the reduction of nitrates to ammonia is very common among members of the Coccacee, but that the reduction to nitrites only is variable and not characteristic of any one type. The average amounts of gelatin liquefied after 30 days' growth at 20°C., are as follows: by the salivary cocci, 2.8 се.; by the air forms, 1.9 се.; and by those of the skin, 1.4 cc. Fif- teen out of 20 of the salivary organisms, 15 out of 18 of the air forms, and 4 out of 6 of the skin cocci, liquefied gelatin. Sum- ming up the results obtained from the experiments on gelatin liquefaction, it is to be.noted that, in general, the salivary cocci liquefy gelatin more readily than do the air or skin forms, but aside from this it is apparent that there is nothing to warrant the use of gelatin as a differential medium. The results of the experiments on vigor of surface growth on agar slopes at 20°C., and 37°C., are given in table п. While it may be said, in general,—from the results given in this table—that the salivary cocci grow somewhat more vigor- ously at 37°C. than at 20°C., the air forms better at 20°C. than at 37°C., and the skin organisms about equally well at the two temperatures, the differences are not sufficiently pro- nounced to impart to the factor of vigor of surface growth any marked value as a differential characteristic. 1914] NOLTE—SALIVARY ORGANISMS AND AIR POLLUTION 59 TABLE II DATA ON THE VIGOR OF SURFACE GROWTH OF AIR, SALIVARY, AND DERMAL COCCI No. of Growth characteristics Source of | Temperature | cultures organism | of incubation used No | Very | Mea- | Good | Abun-| Very growth| faint | ger dant | heavy Saliva 20°C. 20 -- 3 7 10 — — Air 20?C. 18 1 — — 4 7 6 Skin 20°C. 6 — 1 2 2 1 -- Saliva 37°C. 20 — — 7 13 — — Air 37?C. 18 2 1 9 6 — — Skin 37°C. 6 -- 1 — 4 1 — In the following enumeration are listed the colors of the various pigments produced by the air, skin, and salivary cocci, the figure on the left having reference to the number in Ridgway corresponding to the particular pigment produced: Salivary cocci 15" d Light pinkish cinnamon 3 | 15” с Intermediate between light pinkish and pinkish cinnamon 15" b Pinkish cinnamon 15" a Intermediate between pinkish cinnamon and cinnamon 3 15" Cinnamon 1 21’ e Intermediate between massicot and straw yellow 23' f Naphthalene yellow 19 f Maize yellow 19’ b Mustard yellow 1 One gave too little growth for determination of the color. ны ны к сл н pi н [Vou. 1 60 ANNALS OF THE MISSOURI BOTANICAL GARDEN Air cocci н> 21’ f Massicot yellow 217 e Intermediate between massicot and straw yellow 217 d Straw yellow 21’ b Amber yellow 19' d Naples yellow 19’ b Mustard yellow 19' Primuline yellow 19 f Maize yellow 19 d Buff yellow 3’ b Light Jasper red One form did not grow. 16 кі m mL. ва іа ро Skin соссі 19 f Maize yellow 1 19 d Buff yellow 1 19 b Apricot yellow 2) 5 217 e Intermediate between massicot and straw yellow 1 White 1 At first glance the color of the pigments produced by the organisms would seem to furnish one mode of differentiation. In the majority of cases the salivary cocci produced cinnamon colored pigments, whereas pigments of a yellow color were usually produced by the air and skin forms. Closer inspection shows, however, that some of the salivary cocci, as well as the air forms, produce a maize yellow and a mustard yellow pigment; also that a maize yellow pigment and one inter- mediate between massicot and straw yellow are produced by representatives of both the salivary and skin cocci. It is apparent that these intergradations make the factor of pig- ment production largely inapplicable as a differential test. In milk the salivary соссі with one exception produced acid and coagulated the medium, whereas none of the air forms and but one of the skin соссі gave this combined reaction. This attaches to milk considerable value as a differential me- dium. In the media containing the various organic substances 1914) NOLTE—SALIVARY ORGANISMS AND AIR POLLUTION 61 —sugars, etc., none of the coccus forms produced gas. АП but one of the salivary cocci produced acid in the lactose medium, whereas none of the air cocci and but one of the skin forms deported themselves in this manner. This marks lactose broth as another medium of differential value. The salivary cocci with but one exception produced acid in saccharose, the single exception being the organism which produced no acid in the lactose medium. Two air cocci and one skin form also produced acid in saccharose, but notwith- standing these exceptions, it appears that saccharose is a third valuable differential medium. In the mannite, salicin, inulin, sorbite, raffinose, and rhamnose broths none of the organisms produced acid, thus marking these organic substances as of no value in differentiating the types of cocci under investigation. SUMMARY In reviewing the preceding discussion of results we find three media, namely, lactose and saccharose broths, and milk, which are of value in differentiating the cocci most character- istic of saliva from those of the air and the skin. One of the salivary coccus forms did not produce acid in lactose and saccharose broths and formed neither acid nor clot in milk. This may have been, and probably was, an air or skin form. Among the air cocci are two which vary somewhat from the remaining air and skin forms in that they produce acid in saccharose broth. Neither of them, however, produces acid in lactose broth, nor acid or clot in milk and in these respects they differ markedly from the characteristic salivary forms. Of the skin cocci one gave the characteristic reactions of the salivary organisms, and it is not at all unlikely that this was a salivary coccus. In general, then, it appears that the organism most characteristic of saliva is a coccus form which produces acid in lactose and in saccharose broths, and acid and clot in milk. FURTHER TESTS To further test the validity of the reactions above referred to as furnishing a reliable means of differentiating between salivary cocci and those of other origin, two additional samples of saliva, from two different individuals, were examined,— [Vor. 1 62 ANNALS OF THE MISSOURI BOTANICAL GARDEN one from а middle aged white person (A), the other from a colored person (B). The samples were collected and treated in à manner similar to that outlined in the early part of this paper. In the first case (A), transfers were made from all colonies on two plates, representing а dilution of one part saliva in ten billion. These subcultures, all of cocci, were numbered from 1 to 17 inclusive. In the second case (B), transfers were made from 36 colonies which developed on one-third of a plate representing a dilution of one part saliva in ten billion. The entire series of cultures, numbered from 1 to 36 inclusive, although made from 36 colonies from a plate containing a total of 100 colonies, were found to be made up of coccus organisms. After being incu- bated in + 1 nutrient broth for 2 days at 37°C., each of the cultures from samples (A) and (B) was transferred to the three differential media,—lactose and saccharose broths, and milk. The results recorded in tables пт and Iv were observed after 3 days’ incubation at 37°C. No gas was produced in any of the sugar media. A blank determination gave negative re- sults throughout on the three media. TABLE III REACTIONS OF SALIVARY COCCI (A) Ё £ Ig Eia S % Bl |81%1|4 5 С 3. "We в >. 2 . дыз . 5 12 3 мая S | |$ мясо) $ |$ | Ао ам |на на 2 14 11 ТІНҒІЗІЗТ Cl + pt tit EE SE ELEE, ЕЛ, ЛЕЛ, 4+) tit] + ft iti ti + УЕ 15 | +i ti ti + 4 0; 0; 0; O 10 |-Fi - | +] + 16 + +++ а ал Ж и ++ 11] ++ ЗЕ С; +t + 21+ + ++ -Findicates positive reaction. O indicates negative reaction. 1914) NOLTE—SALIVARY ORGANISMS AND AIR POLLUTION From the above table it is evident that all but one of the coccus forms in series (A) produced acid in lactose and saccha- rose broths, and acid and consequent clotting in milk. Тһе one exception was probably an air coccus. REACTIONS OF SALIVARY COCCI (B) = |e) stad) ШЫ Я НІ ва 51414 11+1+1+[ i13 f+] 4+] t+ ЖЕЖ Oe бал 210|-ғ-|0|0||-|-1- 26 |+ +1|+|+ +++ + 15 | +) +] + 27 | +/+ | | Sipe eis] te +i t+] + 8 | +i+i+t]+ 5} +t} +t] +} 4+) 17 +++ 29 | --| +/+ + 6ізі-і-|-1|181/0|-1|0 +++ + +++ |+ 4+] + 1 |+1+1|+|+ +++ +i ИЕ ЕЕ: 82 +1 +1 +1 + оо + [оо а о + о 33 | +[+1+ | + 10 -|-ic-|c-122 }+/]/+]4 34 | +} L| Fl п +++ 2+ ++ 35 | +l +i +t + 12 +++ 24 | +] ++ 36 ++ 4+] + +indicates positive reaction. In series (B), 32 out of the 36 cocci reacted positively through- out on the three differential media. Тһе remainder were positive with saccharose, but negative with lactose and milk, agreeing in this respect with the two air cocci to which reference has been made. The reactions of the organisms from saliva (A) and (B) 5 indicates negative reaction. [Vor. 1 04 ANNALS OF THE MISSOURI BOTANICAL GARDEN further indicate that the production of acid in lactose and вас- charose broths, and a similar production, together with clot, in milk, are characteristic reactions of the salivary cocci. CONCLUSIONS From the results of the preceding experiments it appears that а method applicable for the detection of the organisms characteristic of human saliva has been developed. It must be acknowledged that the number of organisms examined is comparatively small, especially where those of the air and the skin are concerned. Ап absolute test of the validity of the adopted mode of identification would necessitate the examination of many hundreds of strains of cocci from numerous sources. Nevertheless, the characteristie reactions of the salivary cocci examined seem to be sufficiently definite to warrant the assumption that the most characteristic salivary organism is a coccus form which produces acid in lactose and saccharose broths, and acid and clot in milk. Тнв RELATION OF THE Most CHARACTERISTIC SALIVARY ORGANISM TO THE POLLUTION OF AIR Having identified the most characteristic salivary organism, the next problem is to isolate it from the air. Its frequency of occurrence must also be determined, as this often serves as an index to the degree of pollution. The isolation of the organ- ism and the determination of its frequency of occurrence can be accomplished simultaneously. Then come the problems (1) of devising an air-collecting apparatus suitable for all occasions, and (2) of determining the quantity of air to be examined and the terms by which the sanitary quality of the air shall be expressed. In searching for a means of expressing the sanitary quality of air, let us consider the manner in which this is accomplished in drinking water. Authorities differ markedly on this subject. Shall a water be considered safe or unsafe for drinking purposes if B. coli is present in a 100 cc. sample, or shall its presence or absence in 10 cc. or 1 се. samples be taken as the basis for the eee ae 1914] NOLTE—SALIVARY ORGANISMS AND AIR POLLUTION 65 classification? Іп lieu of a definite standard let us assume the following table!: TABLE V PRESUMPTIVE TEST FOR B. COLI IN WATER BE quam oor | 01 | 10 | 100 | 100 Safe O О О О Reasonably safe О О о + -- Questionable O O -- = + Probably unsafe О + + + - Unsafe ud ue БЕ + + + indicates positive presumptive test for B. coli. We shall now endeavor to prepare a similar table for the purity of air, expressed in the number of salivary cocci present in given volumes. In the normal life processes, the volume of air inhaled is obviously much greater than the volume of water consumed, and this fact must be taken into consideration in establishing a criterion for the bacteriological examination of air. It has been estimated that the tidal air, i.e., the air taken in with each inspiration and given out with each expiration, amounts, in a normal adult when at rest, to one-half liter. Assuming the average frequency of respiration to be 15 per minute, the amount of air inhaled in one minute is 74 liters, in one hour, 450 liters, and in one day, at least 10,000 liters. Taking the average amount of unboiled water drunk in a day as 2 liters, it would appear that 5,000 times as much air as water is required daily. Hence, the following table, based on table v, may be used to. express the sanitary quality of air: ! Whipple, С. C. On the practical value of presumptive tests for B. coli in water. Techn. Quart. 16:18 e. m. 31. 1903. [Vor. 1 66 ANNALS OF THE MISSOURI BOTANICAL GARDEN TABLE VI TEST FOR CHARACTERISTIC SALIVARY COCCI IN AIR Safe О О O о + Reasonably safe О O O + + Questionable O O + + + Probably unsafe O + 4 + 4- Unsafe 4 + + + + + indicates positive reaction in the three differential media adopted. APPARATUS AND TECHNIQUE As it was the intention to collect samples of air in places other than the laboratory, a portable apparatus was necessary. As devised, it consists essentially of a sand filter, a support for same with an attachment for alternately opening and closing the exhaust and suction, and a bulb, having a capacity of 16 oz., with the required amount of rubber tubing. (See plate 2.) The sand filter is of the standard type. It consists of a glass tube 100 mm. long and 10 mm. in diameter, fitted with a one- hole rubber stopper, through which passes a piece of 6 mm. glass tubing. This stopper, with its tubing, forms the support for а cireular disc of bolting cloth with a 10 mm. layer of very fine clean quartz sand that passes through a 100, and is retained on à 140 mesh sieve. The support consists of a rectangular piece of wood 12 x 1 x 3 inches, fitted with a double pinch cock arrangement. Clamps for holding the filter in position are also provided. Тһе rubber bulb is connected to the apparatus in such а manner that when pressure is applied to the former and the pinch cock opened, the air contained in the bulb is expelled through the exhaust without disturbing the sand in the filter in any way. This operation occupies but a few seconds of time. Upon releasing the pinch cock, and immediately thereafter the bulb, the air is drawn through the sand. 1914] NOLTE—SALIVARY ORGANISMS AND AIR POLLUTION 67 The volume of air exhausted from the bulb at each pressure was determined as follows: Тһе bulb, filled with water, was weighed. Pressure was then applied, forcing out the water, after which the bulb was again weighed. The difference in weight in grams is approximately the volume of air in cc. ex- hausted by a similar pressure. In the calibrations the results varied but slightly. By placing the fingers on the bulb in a certain fixed position each time, it was found that the bulb could be made to deliver 300 cc. of air at each exhaustion and, conse- quently, to receive 300 сс. of air at each release of pressure. Tt was, of course, necessary to have all joints air-tight, this being accomplished by making all connections with rubber tubing and glass and using plenty of overlap. Тһе sand filter, after being plugged at both ends with cotton, was sterilized for 30 minutes at 15 pounds pressure. Тһе rubber stopper support was allowed to fit very loosely into the tube dur- ing sterilization in order to prevent setting of the rubber. After the apparatus was removed from the autoclav, the stopper was immediately fitted in tightly, thus rendering the connection air- tight. Тһе sand filter was always used within 24 hours after sterilization. It was connected to the support as shown in plate 2. When operated in public buildings or conveyances, the sup- port, with the filter, was wrapped in stiff paper in such a manner as to permit of the easy operation of the pinch cock and exhaust. Тһе apparatus thus wrapped was held in the left hand and from it heavy rubber tubing passed down the left coat sleeve and then diagonally across to the right coat pocket where it was connected to the bulb. "This rendered the whole apparatus inconspicuous. 'The bulb was operated with the right hand, the pinch cock with the left. А test tube with a sterile cotton plug was always carried, the latter being used to replace the plug which was removed from the intake of the filter at the beginning of the experiment. The plating was always carried out within 30 minutes after the sample was obtained. Тһе sand from the filter was carefully poured into a 100 се. flask containing 15 cc. of sterile distilled water. Тһе bolting cloth, which had a tendency to stick to the rubber stopper, was removed with sterile forceps and introduced [Vor. 1 68 ANNALS OF THE MISSOURI BOTANICAL GARDEN intotheflask. Тһе contents of the flask were thoroughly shaken and aliquot portions, as shown in table rx, were plated with 10 ec. of +1 nutrient agar. In plating, the introduction of much sand was avoided in the following manner: The end of the pipette was held immediately above the bottom of the flask while the liquid was being drawn up to a point slightly above the gradua- tion mark. After a few seconds, enough of the sandy liquid was allowed to run back into the flask to leave the water just at the mark. During this short interim a large proportion of the sand settled in the tip of the pipette and was returned to the flask as the liquid was lowered to the mark. Blanks were plated several times during the course of the experiments, but no growth developed in any case. The plates were іп all cases incubated for 4 days at 37°C., after which the number of bacterial colonies present in each was determined. Finally, all, or а representative number, of the colonies were examined for the presence of coccus forms. (See table rx.) The coccus colonies developing on agar are, as a rule, very small and often grow in the deeper strata of the medium. This renders the transfer difficult especially when two are to be made from the same colony—one for the stained preparation and one for the agar slope to be used as a stock culture. The difficulty was partially obviated by subculturing (from all the colonies in certain selected plates) to agar slopes, and incubating the latter at 37°C. After several days an examination served to eliminate the bacilli and moulds, leaving only the coccus cultures which were later examined for the presence of the sali- vary forms. In this examination the three differential media described above were used. SOURCES OF SAMPLES As the investigation in hand seeks to discover a relation be- tween the presence of a characteristic salivary organism and the pollution of air, it was thought best to collect the samples of air under normal conditions, i.e., conditions which are met with in every-day life. Public conveyances, on account of their usually crowded condi- tion and frequently inefficient ventilation, suggested themselves 1914] NOLTE—SALIVARY ORGANISMS AND AIR POLLUTION 69 as favorable places for tests. Hence, a local street car was chosen as a source for air samples. The often poorly ventilated but well filled motion picture theatres furnished another sup- posedly promising sampling place. The third locality chosen as a source for air samples was a local 5 and 10 cent store. It was thought that this would furnish an ideal source of contami- nated air because of the large crowds of people who are con- tinually voicing their sentiments and desires. In order to deter- mine whether or not the salivary organism is present in an at- mosphere which is not in immediate contact with human beings, and which is open to the ventilation of nature,the fourth sample was taken from the open air. DISCUSSION OF THE EXPERIMENTS The experiments in table rx are arranged according to the dates on which the tests were made. But for convenience in this discussion the experiments will be taken up according to the source of the samples. Experiment 1.—This experiment was carried out primarily to test the apparatus. The air sample was taken in a labora- tory on the second floor of an old building. There were usually at least two people present in the room, and practically no ventilation was provided, the doors and windows being con- stantly closed. The apparatus used differed from that used in the remaining experiments in that two sand filters were used in tandem instead of the usual one. During the 15 minutes of operation, 7,800 cc. of air were drawn through the sand of both filters at the rate of 520 cc. per minute. The sand of the first filter was introduced into 15 cc., that of the second into 6 ec. of sterile distilled water. Quantities of both solutions were plated with the following results: TABLE VII Filter Plate Quantity Total no. Coccus number number plated of colonies colonies 1 1 166, 2 0 1 2 106. 2 2 1 3 2-66. 4 2 2 4 5 cc. 0 0 [Vor. 1 70 ANNALS OF THE MISSOURI BOTANICAL GARDEN The reactions of the cocci isolated from the air in the first filter showed that there was one salivary coceus form present. The remaining three gave negative reactions on the three differ- ential media. It should be noted that out of the 15 cc. of solu- tion from the first filter, only 4 cc. were plated. Eight organ- isms were present in the quantity examined, making a total of 30 in the entire solution. One characteristic salivary coccus form developed in the portion examined, making, according to the law of averages, a total of 4 in the entire solution. The total volume of air examined being 7,800 cc., the frequency of occur- rence of the salivary coccus is 1 in 1,950. According to table vi, the sanitary quality of the air of the room was ‘probably unsafe" at the particular time at which the sample was taken. EXPERIMENTS 3, 5. 6, 8, 10 These experiments were carried out in a local street car. The same car line was chosen for all of the experiments in order to eliminate as many variables as possible, such as construction of ear, capacity, rate of locomotion, etc. Тһе car was of the ordinary ‘‘pay-as-you-enter” type now in use in St. Louis. It had a seating capacity of about 44 people, and could accom- modate approximately 40 more standing indoors. The air space in the car in question was about 2,500 cubic feet, or ap- proximately 30 cubic feet for each passenger when the car was filled to its capacity. As the samples were taken at a time when the outside tem- perature would not permit the windows to be open, the question - of ventilation was carefully studied. Ав is usually the case, the transoms were tightly closed, and only when the front and rear doors of the car were open at the same time was there an opportunity for a complete renewal of the air. This never happens when the car is in motion, and there is probably never a complete renewal of air unless a strong wind is blowing, thus causing a draught when the car is at a standstill, with both doors open. This particular car was provided with four vents in the roof which could be opened or closed at will. In several of the experiments some of the vents were open; in others, all were closed. The degree of pollution of the atmosphere in such a car de- 19141 NOLTE—SALIVARY ORGANISMS AND AIR POLLUTION 71 pends, of course, on the amount of coughing, sneezing, speaking, etc., of its occupants. А саг may be very crowded but if no coughing, etc., is going on, there will, theoretically, be no pollu- tion of the atmosphere from saliva. Again, if there is much talking, etc., among those present, the atmosphere may be greatly polluted by the dissemination of particles of saliva from the mouth. The samples were always taken in the early morning between the hours of six and seven, when the majority of the laboring class are on their way to work. "The tendency of the passengers at this time of the day is to be quiet, as the morning paper is of absorbing interest to a majority. Тһе samples of air were taken in the center of the car, the opening of the apparatus being about 4 feet from the floor level. In these experiments the apparatus described above was used. In all cases 10,800 cc. of air were drawn through the sand filter at the rate of 900 cc. per minute. The sand was introduced into 15 се. of sterile distilled water and plated as shown in table тх. The experiments carried out in street cars will now be taken up in order and the results discussed. If it can be shown that the characteristic salivary organism is present in the air of these cars in sufficient quantity, and if it can later be proved that this salivary organism is not present in the open air, it follows that the atmosphere in these cars is being polluted by the dissemina- tion of particles of saliva from the mouth. Experiment 3.—While the air sample was being taken for this experiment, 44 people were seated in the car, but none were standing. Out of the 20 colonies appearing on the plate (see table тх), 9 were of bacilli and 11 of coccus forms. Inoculated into the 3 differential media, 8 of the latter reacted negatively in all three media, 1 negatively on lactose and milk, but posi- tively on saccharose, and 2 gave positive reactions in all three media. It will be recalled that mention has been made of several organisms, both among the salivary and air cocci, which gave а positive reaction with saccharose, but reacted negatively with lactose and milk. 'The one above referred to as reacting in this manner is probably one of these unidentified coccus forms which seem to be present in both saliva and air. Out of (Уот 1 72 ANNALS OF THE MISSOURI BOTANICAL GARDEN the 11 cocci present, therefore, two were of the characteristic salivary type, and as only one-third of the sample was plated, a total of 6 may have been present in the entire volume of air examined, or a frequency of occurrence of 1 in 1,800. Accord- ing to table vr, the sanitary quality of the air was ‘probably unsafe." Experiment 5.—During the sampling process for this experi- ment, 44 persons were seated and approximately 30 standing. Of the 26 colonies which developed on plate 5 (see table rx), 14 were of bacilli, 2 of streptothrix, and 10 of cocci. When transferred to the three differential media, all of the latter gave negative reactions, indicating that the air in the car at the time of this experiment was ''safe." Experiment 6.—At the time of sampling, 44 persons were seated and 30 were standing. Оп account of the large number of colonies present, only a representative sector of plate 1—com- prising one-twelfth of the total area—was examined (see table тх). On this area 21 colonies were counted, 5 bacillus and 16 соссив. On the three differential media, 4 of the latter gave nega- tive reactions throughout, 6 were negative on lactose and milk but positive on saccharose, and 6 gave positive reactions on all three media. It follows that 6 salivary cocci were isolated from one-twelfth of the plate, making a total of 72 from the entire plate, or of 1,080 from the total volume of sand solution,—a frequency of occurrence of 1 in 10. According to table vr, the air in the car at the time of the experiment was ' unsafe." Experiment 8.—The number of persons seated and standing was the same as in experiment 6. Оп the plate examined (see table rx), 34 colonies developed—15 bacillus and 19 coccus. Оп the three differential media the coccus forms reacted as follows: Twelve gave negative reactions throughout, 6 were negative on lactose and milk but positive on saccharose, and 1 was nega- tive on lactose and saccharose but positive on milk. The last form was found, after again staining with gentian violet and examining under the microscope, to be a short bacillus. It is to be noted that 6 organisms of the unidentified coccus type were again present. No characteristic salivary cocci were present, thereby marking the air of this particular car as “safe” at the time of the experiment. 1914] NOLTE—SALIVARY ORGANISMS AND AIR POLLUTION 73 Experiment 10.—During this experiment, 44 persons were seated and 15 standing. It was noted that one transom was open. Тһе plate examined (see table rx) gave a total of 12 colonies, of which 5 were of bacilli and 7 of cocci. Of the latter, 6 reacted negatively on all three of the differential media, whereas 1 gave а positive reaction throughout. This makes the fre- quency of occurrence of the characteristic salivary coccus form 1 in 3,600, and, according to table vr, marks the air in this car as "questionable" at the time of the experiment. Summarizing the car experiments, it is to be noted that in three out of five cases the characteristic salivary coccus form was isolated, and in such quantity as to mark the air of one "unsafe," that of another “probably unsafe," and of а third “questionable.” EXPERIMENTS 9 AND 11 These experiments were carried out in a local vaudeville house. The construction of the building appeared modern in every respect. The lower floor had a seating capacity of about 2,000, while the balcony accommodated approximately 1,000 people. The house was filled with spectators on the occasions when the samples were taken. Upon inquiry, after the surprisingly good results given below were obtained, it was found that the build- ing was well ventilated by one of the modern appliances for this purpose, whereby the volume of air in the building (about 90,000 cubic feet) was being renewed to a greater or less extent every seven-tenths of a minute. For the collection of the air samples, the same apparatus was used as in the street car experiments, 10,800 cc. of air being drawn through the sand filter at the rate of 900 cc. per minute. The sand was introduced into 15 ce. of sterile distilled water and platings were made as indi- cated in table rx. Experiment 9.—The air sample was obtained near the center of the lower floor of the building about 60 feet from the stage. The entire lower floor was packed, and in addition about 100 or more persons were standing in the rear. On the plate ex- amined, a total of 14 colonies developed,—3 mold, 9 bacillus and 2 coccus. Molds were very abundant on the other plates. One of the coccus forms reacted negatively on lactose and milk but positively on saccharose, whereas the other gave negative [Vor. 1 74 ANNALS OF THE MISSOURI BOTANICAL GARDEN reactions on all three differential media. Тһе presence of the single unidentified coccus is again noted. Хо salivary coccus forms were isolated, from which fact it appears that the air in the particular location from which the sample was taken was “safe.” Experiment 11.—This sample was taken on the balcony of the building, about 10 feet from the rear wall. Every seat was occupied. As indicated in table тх, two plates were examined. On the first, 7 colonies developed—3 streptothrix, 3 bacillus, and 1 coccus. Оп the second plate 3 colonies appeared, all of which were of bacilli. The reaction of the coccus was negative on the three differential media, thereby indicating that the sample of air taken was free from salivary coccus forms and therefore “safe.” In summing up the results of the experiments carried out in the vaudeville house it is to be noted that in both cases no sali- vary coccus forms were found. Table rx further shows that the total number of organisms found per unit volume of air was smaller than in the street car experiments. EXPERIMENTS 4 AND 7 These samples were obtained in the basement of a local 5 and 10 cent store. The ceiling was rather low, being only about 9 feet from the floor level, the entire basement having a volume of about 72,000 cubic feet. Тһе samples in these experiments were taken in the midst of a crowd gathered to listen to a singer advertising songs. Little attention was given to the matter of ventilation until after the results of the experiments were obtained. Subsequently, however, investigation revealed the fact that ample provision had been made for ventilation. Transoms at the level of the sidewalk provide openings to the outside; along the inside wall and near the ceiling are revolving fans about 20 feet apart. These keep the air in circulation until it is drawn out by a suction fan situated in one corner, about 2 feet from the ceiling. Тһе same sampling apparatus was used as in the preceding experiments. As before, a total of 10,800 ec. of air was drawn through the sand filter in each sampling at the rate of 900 cc. per minute. Тһе samples were plated as shown in table rx. Experiment 4.—The air sample for this experiment was taken 1914] NOLTE—SALIVARY ORGANISMS AND AIR POLLUTION 15 in the midst of a crowd of about 100 people in front of a counter. On the plate examined, a total of 36 colonies developed—16 bacillus and 20 coccus. Of the latter, 18 gave negative reactions throughout on the three differential media, and 2 reacted nega- tively on saccharose and milk but positively on lactose, the latter sugar being fermented. No salivary coccus forms were isolated, indicating that the air in the basement at the time of the experiment was ''safe." Experiment 7.—This air sample was taken under practically the same conditions as in the previous experiment except that only about 50 people were in the crowd. The plate examined contained 2 streptothrix, 13 bacillus, and 8 coccus colonies. АП of the cocci gave negative reactions throughout on the three differential media. Хо salivary coccus forms were found, which fact leads again to the conclusion that the sanitary quality of the air during the experiment was ''safe." EXPERIMENTS 2, 12, 13, 14 These experiments were performed outdoors. Тһе air sample for experiment 2 was collected in a railroad switch yard at a time when there was no traffic. Тһе samples for experiments 12, 13, and 14 were collected in the immediate vicinity of large storage basins belonging to the local water works and located 300 or 400 feet from the bank of the Mississippi River. Тһе apparatus used was the same as that employed in the previous experi- ments. Experiment 2.—The outdoor temperature was 29?F., and while the air sample was being taken it was snowing. A total of 22,500 се. of air was drawn through the sand filter at the rate of 750 се. per minute—the operation extending over a period of 30 minutes. Samples were plated as shown in table Іх. Of the 3 plates examined, plate 1 yielded 2 bacillus colonies; plate 2, 1 streptothrix, 1 bacillus, and 6 coccus colonies; and plate 4, 2 mold, 1 streptothrix, 1 bacillus, and 2 coccus colonies. All of the coccus forms were grown on the three differential media, 7 giving negative reactions throughout, while 1 reacted positively on saccharose and negatively on lactose and milk. Тһе latter organism is one of the unidentified coccus forms previously referred to. Хо characteristic salivary cocci were found, in- [Vor. 1 76 ANNALS OF THE MISSOURI BOTANICAL GARDEN dicating that the sanitary quality of the air examined was “safe.” Experiment 12.—At the time the air sample was being taken, a slight drizzling rain was falling, accompanied by considerable wind and a temperature of 45°F. Prior to that time it had been raining continuously for about 24 hours. A total of 10,800 cc. of air was drawn through the sand filter at the rate of 900 cc. per minute, the apparatus meanwhile being held about 5 feet above the ground level. Тһе sand of the filter was introduced into 15 ce. of sterile distilled water, from which platings were made. ‘Table vim gives the details of the experiment, together with the results obtained. TABLE VIII No. of bac- Plate Quantity Total no. t а eed Coccus No. of sali- number plated of colonies melda colonies vary cocci 1 1 cc. 9 2 7 7 2 1 ес. 3 3 0 0 3 5 cc. 0 0 0 0 4 5 cc. 6 4 2 0 Attention should be called to the fact that on plate 1, in which only 1 сс. of the solution was used, 9 colonies developed—7 coccus and 2 bacillus—, while on plate 4,in which 5 cc. of the solu- tion were used, only 6 colonies appeared,—2 coccus and 4 bacillus. Furthermore, the 7 colonies in plate 1 proved to be of salivary cocci, whereas none of these organisms were present among the cocci of plate 5. These results unquestionably indicatelocal con- tamination. It is difficult to say just where the contamination took place. Obviously it did not occur during the collection of the sample or even during the mixing of the sand solution ; for had this been the case all of the plates should have shown salivary cocci, and the greater number should have occurred on those plates in which larger quantities of the solution were plated. In all probability plate 1 was locally contaminated. Experiment 13.—While the sample of air was being taken for this experiment, the temperature was 63°Е., a light breeze was blowing, and the sky was very cloudy although no rain had 1914] , NOLTE—SALIVARY ORGANISMS AND AIR POLLUTION 77 fallen during the preceding 18 hours. A total of 10,800 cc. of air was drawn through the apparatus at the rate of 830 cc. per minute. Samples of the sand solution were plated as shown in table rx. It is to be noted that in those plates containing 1 cc. of the solution no colonies developed, whereas in those contain- ing 5 cc., 1 bacterial colony appeared in each. Attention is called to the consistent results in this experiment to emphasize the fact that the inconsistencies in experiment 12 are due to local contamination. No salivary cocci were found. Experiment 14.—The air sample for this experiment was taken on a bright, clear day, with a rather strong wind blowing and a temperature of 55°F. А total of 10,800 cc. of air was drawn through the sand filter at the rate of 1,080 сс. per minute. Samples of the sand solution were plated as shown in table rx. Of the 3 coccus forms, 2 gave negative reactions on all three differential media, whereas 1 was positive on saccharose and negative on lactose and milk. The latter will be recognized as one of the unidentified coccus forms. No salivary cocci were isolated. Summarizing the open air experiments, it is to be noted that, barring the locally contaminated plate 1 in experiment 12, the characteristic salivary coccus form was not isolated; further- more, that the total number of organisms in the open air is comparatively low. SUMMARY AND CONCLUSIONS Examining the entire series of experiments it appears that in the majority of cases where ventilation was obviously inad- equate, the characterisitic salivary coccus form was isolated. On the other hand, the form could in no case be found where ample artifieial or natural ventilation existed. It has been shown that the most characteristic salivary organism can be differentiated and identified; also, that this characteristic organism can be isolated from the air. In the experiment carried on in one of the street cars in which there were many passengers, the characteristie salivary coccus form was found to be present in such quantities as to indicate that the air in this car was “unsafe.” It was later shown that [Vor. 1 78 ANNALS OF THE MISSOURI BOTANICAL GARDEN TABLE IX DATA ON THE COLLECTION AND EXAMINATION OF AIR SAMPLES No. of experiment 1 2 3 4 5 6 7 Date of collection 2/12/13 | 2/22/13 | 3/15/13 | 3/15/13 | 3/19/13 | 3/22/13 3/22/13 . 5 and 5 and Sampling place Lab. [Outdoors | Street car 10e. store Street car| Street car 10s wd» Outdoors 60 29 32 36 50 23 50 Temperature (?F.) Sampling pl. 80 29 45 70 48 45 70 БЕ i А Windy | Windy я a 9 Weather conditions Sunshine | Snowing snowing | snowing Sunshine | Sunshine | Sunshine Sitting 0 0 44 1 44 44 1 Approx. no. of persons Standing 2 0 0 100 30 30 50 Approx. volume of sampling pl. 3000 2500 72000 2500 2500 72000 (cu. ft.) Volume of air exam. (co.) 7800 22500 10800 10800 10800 10800 10800 Rate of filtration (cc. per min.) 520 750 900 900 900 900 900 Pl. I. (1 се.) 2 2 4 30 9 250 4 Pl. II. (1 се.) 2 8 3 36 6 250 3 No. of organisms|Pl. III. (2 cc.) 4 0 in following quantities of Too num- sand solution|Pl. IV (5 cc.) 6 12 230 25 | erous to |Spreader plated count. Too num- Pl. V. (5 со.) 20 200 26 | erous to 23 count, А I., П. and |I., II. and Plates examined II. IV. V. II. у. 1/12 of I. V. Total col. on plates exam. 8 16 20 36 26 21 ge 12 23 No. of bacilli, molds, eto, 4 8 9 16 "I > 1/ 12 i No. of cocci 4 8 11 20 10 16 on 1/12 8 of I. No. of salivary cocci 1 0 2 0 0 6 = y 12 0 Frequency of occurrence lin 1950 0 | 111800 0 0| 11іп10 0 . Е Probably Probably Sanitary quality vue Safe ndi Safe Safe Unsafe Safe No. of org. in total vol. of air 30 42 50 570 95 3750 58 1914] NOLTE——SALIVARY ORGANISMS AND AIR POLLUTION 79 TABLE IX (Continued) DATA ON THE COLLECTION AND EXAMINATION OF AIR SAMPLES No. of experiment 8 9 10 11 12 13 14 Date of collection 3/29/.3 | 3/29/13 4/1/13 4/1/13 4/8/18 4/9/13 | 4/10/13 А Pict ict Sampling place Street car jews Street car ue Outdoors | Outdoors | Outdoors Outdoors 41 59 50 77 45 63 55 Temperature(°F.) Sampling pl. 55 70 63 82 45 63 55 Rain, Sunshine, Weather conditions Sunshine | Cloudy | Sunshine | Sunshine very Cloudy very windy windy Sitting 44 3000 44 3000 0 0 0 Approx. no. of КЕЕ Standing 30 100 15 300 0 0 0 Approx. volume of sampling pl. 2520 90000 2500 90000 (cu. ft.) Rate of filtration (cc. per min.) 900 900 900 900 900 830 1080 Pl. I. (1 се.) 34 3 4 7 9 0 2 18 2 2 3 3 0 No. of organisms PL II. (1 ce.) 3 in following quantities сі РІ. III. (2 cc.) 24 sand solution F plated Рі. IV. (5 се.) ре = 14 | Spreader | Spreader 0 1 3 Pl. V. (5 се.) 8 12 35* 6 1 1 Plates examined Е ГУ. Ж; I. and II. I., IL. and IV. and V. TILIV., Ж; ала У, Total col. on plates exam. 34 14 19 10 18 2 8 No. of bacilli, molds, etc. 15 12 5 9 9 9 5 No. of cocci 19 2 "d 1 9 0 3 No. of salivary cocci 0 0 1 0 7 0 0 Frequency of occurrence 0 0 | 1 in 3600 0 | Lin 1235 0 0 Sanitary quality Safe Safe — Safe t Safe Safe No. of org. in total vol. of air 320 85 42 85 50 2 18 ж Abundance of molds. T Local contamination. [Vor. 1, 1914) 80 ANNALS OF THE MISSOURI BOTANICAL GARDEN the salivary coccus form could not be found in the open air devoid of the immediate presence of human beings. It thus appears that the presence of the salivary coccus form in air indicates the presence of man; furthermore, it indicates the pollution of air by particles of mucus from the mouth. Flügge ' and his school have shown that pathogenic organisms may be transmitted into the air, and other workers ? have shown that the tubercle organism is capable of being carried by even such feeble air currents as ordinarily exist in dwellings. The tubercle organism, as well as the characteristic salivary organism, is present in the saliva of tubercular patients. If, therefore, this salivary organism can be isolated from the air by means of the filter used in the above experiments, does it not follow that the tubercle organism could be isolated in a similar way? Since our manner of breathing is comparable to the operation of the apparatus used, it follows that the tuber- cle organism may be inhaled by man. It thus appears that the presence in the air of the most char- acteristic salivary organism is an index of the possible access of pathogenic organisms to the atmosphere. In conclusion, the writer wishes to express his thanks to Dr. Geo. T. Moore, for valuable suggestions and numerous courte- sies extended during the progress of the work; to Dr. J. R. Schramm, for suggestions, and aid in the preparation of the manuscript; and to Mr. Wilson F. Monfort, Chemist of the City of St. Louis Water Department, for advice given and op- portunities provided for the collection and examination of the samples. EXPLANATION OF PLATE PLATE 2 Air-sampling apparatus showing support, sand filter, pinch cock, exhaust and suction tubes, and pressure bulb. 1 Gordon, M. H. loc. cit. : Kolle and Wassermann, Handbuch der pathogenen Mikroorganismen 1: 169. ANN. Mo. Вот. GARD., Vor. 1. 1914 PLATE 2 ғ | м 2 A | я га ЧЫ pd NOLTE—SALIVARY ORGANISMS AND AIR POLLUTION THE РОГУРОВАСЕЖ OF OHIO L. O. OVERHOLTS Rufus J. Lackland Fellow in the Henry Shaw School of Botany of Washington University INTRODUCTION The Polyporacee, or “роге fungi," constitute a relatively small family of the Basidiomycetes, characterized by having the spores borne on the interior surfaces of tubes or pores which make up the hymenium of the fungus. In its most compre- hensive sense the family embraces the two subfamilies Boletee and Polyporee, including also such aberrant genera as Merulius, Porothelium, Solenia, etc. More often the Boleteg are made a separate family, the Boletaceew, usually distinguished from the true Polyporacee by the more fleshy nature of the plant and by the fact that the pores rather easily separate in a smooth layer from the flesh of the pileus. The true Polyporacee, on the other hand are more commonly leathery, corky, or woody in texture, and only in rare cases are the tubes separable from the context. More recently Dr. Murrill, who has monographed the North American species of the family for the North American Flora—now being issued by the New York Botanical Garden—, has still further limited the family so as to exclude not only the genera referred to above, but also certain of the true polypores which possess a more or less gelatinous or waxy hymenium. For the reception of certain of these forms he has erected the family X ylophagacee. С. а. Lloyd has published monographie papers on certain of the sections of the family, using for the most part as the generic names, the sectional names given by Fries. Within the past. year a third system of classification has been proposed by Miss. Ames, of Cornell University, who divides the family into groups on the character of the context, and these groups are separated into genera on the form of the fruit body, surface modifications, spore characters, etc. Various workers in Europe have at- ANN. Мо. Bor. GARD., VOL. 1, 1914 (81) [Vor. 1 82 ANNALS OF THE MISSOURI BOTANICAL GARDEN tempted to revise the genera of the Polyporacee but none of these classifications have been generally adopted by mycologists. Тһе family is here taken to include the following genera: Polyporus (including Polystictus), Fomes, Trametes, Dedalea, Lenzites, Cyclomyces, Favolus, Gleoporus, Merulius, and Гтрех. Distributed among these genera are practically one hundred species found within the state. Of these, 78 have been col- lected by the writer,4 others have been sent in by correspondents, and examination has been made of collections of 5 other species taken within the state and preserved either at the Lloyd Mu- seum at Cincinnati, or in the herbarium of the New York Botan- ical Garden. Of the remaining 12 species some are known only from the records left by Morgan, Lea, Montagne, Berkeley, and Kellerman, others are admitted because there is every reason to believe that they will be found within the state since they are known to have been collected in nearby counties of adjoining states. Тһе resupinate Polyporacee, usually included in the genus Poria, have been omitted from this paper. Very little is known in this country concerning these forms and very few authentic specimens were available for study and comparison. Most of the species that have been reported from this country have been based on scarcely more than a guess, and it is impossible for the amateur mycologist to determine his material from the confused and often fragmentary account that has been written. Until the genus has been thoroughly studied by a competent mycologist, only added confusion would result from anything more than a reference to it in this paper. In the preparation of the keys, relationships, both of genera and species, have been entirely ignored, the aim being to produce a usable key rather than to exhibit relationships. The writer believes that the color of the context is one of the most constant of the gross characters of these plants, and the genera are divided into sections on that basis. The presence or absence of a stipe, the duration of the plant, the hymenial configuration, the surface markings of the pileus, etc., are brought into the key in an order which the writer believes corresponds to their rela- tive importance as specific distinguishing characters. Spore characters, especially spore colors, are not used in the separation на ы. о ou Хазар». айық 1914) OVERHOLTS—THE POLYPORACEZ OF OHIO 83 of the genera, and in the separation of the species only where experience has shown that the spores are always easily obtained. In many cases it is impossible to obtain spores, especially if they be uncolored, from the hymenium of dried plants. However, when plants are taken in the fresh condition it is usually a simple matter to obtain them by leaving the fungus over night in a moist atmosphere and allowing the spores to fall upon a glass slide. Spores of the perennial woody forms may often be obtained by this method when an examination of the same material in the dried state does not reveal their presence. In this paper spore measurements have been freely taken from other publications, both European and American. This was done in order that the descriptions might be made more com- parable. Due credit is given to the author in every case where this was done. An effort has been made to make the descriptions exactly comparable one with another. For this purpose a definite sequence of presentation has been arranged for the different characters and this order preserved in all but a few instances in which entire descriptions were taken from the original sources. In the comments following each species the characteristic specific distinctions are pointed out and references are made to illustrations of one sort or another that give a good idea of the plant as the writer understands it. Practically all of these references are to papers published in this country. The writer has had access to all of the important publications on the family, both European and American. Most of the European writings are not available to a large part of those students for whom this paper is intended and it was believed that a careful selection of citations to the illustrations published in this country would be of more value than citations to the less known and often inac- cessible European publications. Those who are in a position to look up additional references will have access as well to volumes 19 and 20 of Saccardo’s ‘Sylloge Fungorum,’ where an exhaustive index to illustrations will be found. It is believed that there can be no question of the need of a paper worked out along the above indicated lines. No such publication exists for any state in the Union and the only aids that students have had in determining their collections have [Vor. 1 84 ANNALS OF THE MISSOURI BOTANICAL GARDEN been either the incomplete ‘“‘mushroom”’ books or such extensive works as 'Sylloge Fungorum' and in more recent years the mono- graph presented in the ‘North American Flora.’ In the matter of citation and nomenclature an attempt has been made to follow the rules and recommendations of the Inter- national Botanical Congress at Brussels. Since there has been little opportunity to compare specimens of our plants with those of Елторе or with type specimens, the procedure in the matter of synonymy has been very conservative. The only names cited as synonyms are those of which the writer has a personal knowledge gained from the examination of authentic material, usually species described from Ohio. Where there has been a doubt as to the identity of a plant in this country with that of one in the old world the procedure has been to use the name under which it has been described or known in this country. The first and therefore the most complete set of specimens is in the herbarium of the writer; a set of all of the more common forms is in the herbarium of Dr. Bruce Fink, of Miami Univer- sity, at Oxford, Ohio; a partial set is in the state herbarium, at Columbus; and a large number of species, sent to Dr. Murrill for determination and verification, are in the herbarium of the New York Botanical Garden. The writer is under deep obligations to the following persons in various ways: First of all to Dr. Bruce Fink, under whose direction the work was begun, whose aid, criticism, and advice has made this publication possible; to Dr. W. A. Murrill, of the New York Botanical Garden, for many kindnesses in verifying and determining specimens sent to him, and for the privilege of studying the specimens in the herbarium at that place; to Mr. C. G. Lloyd, of Cincinnati, for the privilege of working in the Lloyd Library and Museum and for determinations of speci- mens; to Rev. G. Bresadola, of Trient, Tyrol, for determination of specimens; to Dr. E. A. Burt, of the Missouri Botanical Garden, for access to his herbarium and for suggestions as to the final form of the paper; and to all who have aided in the work by sending specimens and in various other ways. It is hoped that the paper will be found useful not only to Ohio students but in the neighboring states of the Great Lakes 19141 OVERHOLTS—THE POLYPORACEJE OF OHIO 85 region and in the Ohio valley as well. It is with this idea in mind that the paper has been prepared. KEY TO THE GENERA. Sporophore entirely resupinate; pileus попе..................... Poria: Sporophore sessile or stipitate, sometimes effused-reflexed but not nor- mally entirely. meupinsie . —...... ere oorr trea 1 1. Hymenium composed of concentric lamelle; pileus stipitate..Cyclomyces р. 147 1. Hymenium not composed of concentric lamellz; pileus sessile or stipitate . . . 2 2. Hymenium not distinctly poroid, the pores reduced to shallow pits sep- arated by narrow ridges or reticulations................. Merulius p. 150 2. Hymenium distinctly poroid, irpiciform, dedaloid or lamellate, but not рімей.....................:............2..... hs sd Е 3 3. Hymenium more or less waxy or gelatinous, the layers of tubes separating smoothly from the context in fresh specimens or when moistened; pileus sessile, thin and НехіМе............................ Gleoporus p. 149 3. Hymenium not at all waxy or gelatinous and not separating smoothly from the context; pileus sessile or зіїріќафе............................ E 4. Hymenium either dedaloid, labyrinthiform or lamellate, at least in part 5 4. Hymenium poroid or sometimes broken up into teeth.............. 9 5 КЕК, 2.,22...................-ь.....2 5505 6 $. ОЕ... со ооо ПУРЕН 524. 8 6. Pileus minutely velvety to glabrous; context more than 1 mm. thick Е Dedalea p. 143 6. Pileus hirsute to villous; context 1 mm. ог less thick............... 7 7. Hymenium lamellate, at least in рагі......................... Lenzites p. 145 7. Hymenium dedaloid but never lamellate ..................... Dedalea p. 143 8. Plants woody and perennial, more than 1 cm. thick; hymenium not at all lameliate ......5 2] oo nb. Trametes p. 138 8. Plants coriaceous or corky, less than 1 cm. thick; hymenium often lamellate :.......:5 221—088 ЕКТІ ziles p. 145 9. Hymenium broken up into 4біі...,............4................... 10 9. Hymenium poroid, not broken up into teeth. ......................... 13 10. Tubes or teeth 5 mm. or more оп ........................ Irpex p. 151 10. Tubes or teeth less than 5mm. Іопр.............................. 11 11. Hymenium labyrinthiform at first and remaining so at the margin Dedalea p. 143 11. Hymenium never labyrinthiform............. еее cence eeeee 12 12. Pileus less than 1 cm. broad; fungus mostly resupinate...... ]трех p. 151 12. Pileus more than 1 cm. broad; fungus not mostly resupinate Polyporus p. 86 13. Pores large and hexagonal; stipe ргевепі............................ 14 13. Pores small and circular or angular; stipe present or absent.............. 15 14. Stipe lateral, often rudimentary; pores usually radiating and longer in the >мпариктеснда e. р erum ete Favolus p. 148 14. Stipe usually central or subcentral; pores not radiating....Polyporus p. 86 15. Tubes in a single layer; plants аппая!............................... 16 15. Tubes in two to several layers; plants perennial ...................... 17 1See Introduction p. 82. [Vor. 1 86 ANNALS OF THE MISSOURI BOTANICAL GARDEN 16. Tubes not in a distinct stratum but appearing to be sunken to differ- ent depths into the context.......................... Trametes p. 188 16. Tubes forming a well marked stratum entirely distinct from the MN есь с весь вова вов зоо во авс Polyporus р. 86 17. Hymenium bright yellowish brown; plants growing only on the wood of coniferous ігевв....................................... Trametes p. 138 17. Hymenium whitish, flesh-colored, dull brown, etc., but not bright yel- lowish brown; plants growing on the wood of either coniferous or decid- eene Келл ee ea eee ЕУ Fomes р. 126 DESCRIPTIONS AND KEYS ТО THE SPECIES POLYPORUS Mich. ex Fries, Syst. Myc. т: 341. 1821; Mich. Nov. Plant. Gen. 129. 1729. Plants annual or in rare cases persisting for two or three years, terrestrial or epixylous, sessile or stipitate; pileus fleshy, coriaceous or corky in texture, small or of immense size, often brightly colored; context white, yellow, red, or brown; tubes in a single layer, all sunken into the context to an equal depth so that their bases form a definite continuous straight line; mouths mostly cireular or angular, in rare cases showing a favoloid or daedaloid tendency and sometimes breaking up into teeth; stipe (when present) variable in position and texture; spores white (bluish in one species), or some shade of brown. KEY TO THE SPECIES Context white or уі... п.п... Section I Context reddish or уеПомінһ..........................222222277.. Section II Context brown or brownish...........................--.-0.. Section III, Section I, Sporophore stipitate ог substipitate. ........... Lilli ile e eee. 1 Sporophore sessile or sometimes effused-reflexed but never stipitate....... 21 1. Pileus and stipe covered with a reddish varnish......................... 2 1. Pileus and stipe not red-varnished..... 2.20.00... аа... 3 2. Varnish disappearing with age, the pileus then whitish or yellowish 61. P. Curtisii 2. Varnish persisting, the pileus not changing color.............. 60. P. lucidus 3. Plant small, not more than 1 em. high........................... 29. P. pocula 3. Plant always much larger. ..........LLuuulluulu e LLL 4 4. Stipe compound, branching near the base; pileoli usually severalormany. 5 4. Stipe simple or not branching more than once; pileus generally single.... 9 5. Pileoli small (usually less than 5 ст. broad) and numerous 5. Pileoli large (5 cm. or more broad) and few in number................... 7 19141 OVERHOLTS—THE POLYPORACEJE OF OHIO 87 6. Pileoli regular in outline and centrally attached; the branches of the stipe regular and cylindrical in іопп....................... 38. P. umbellatus 6. Pileoli always laterally attached; the stipe branches irregular. 39. P. frondosus 7. Spores roughly echinulate ................................. 41. P. Berkeleyi 7. Bposes поо... e e rrr ее, КИРЕК ЛЕУ 8 8. Pileus pallid or light brown; hymenium usually turning black where bruised and on drying. ............. eee E 40. P. giganteus 8. Pileus yellowish green; hymenium not turning black....... 37. P. flavovirens 9. Context soft and spongy above, firm next to the hymenium; plantsoften much distorted; usually growing about вішарв..................... 28. P. distortus 9. Context uniform; plants not Фініютіеі.................................. 10 10. Plants growing on the ртоппа.................................... 11 10. Plants growing on Уоойб......................................... 12 11. Stipe black and rooting at the base; pileus some shade of brown. .36. P. radicatus 11. Stipe not black and rooting at the base; pileus yellowish green. .37. P. flavovirens 12. Sporophore more or less globose; tubes concealed by a volva. .27. P. volvatus 12. Sporophore not globose; volva аБвепб............................. 13 13. Sporophore arising from а cup-shaped, sterile body that sometimes disap- pears; pileus white; found only on dead branches of Ulmus....... 6. P. conchifer 13. Sporophore not arising from a cup-shaped sterile һоду.................. 14 14. Margin of the pileus projecting 5 mm. or more beyond the hymenium; hymenium separating smoothly from the context in fresh specimens; growing only on Вейшіа................................ 26. P. betulinus 16. зканиеағазете......................%..........%%......- 15 15. Hymenium bright віірімш-уеПоУ......................... 42. P. sulphureus 15. Hymenium not bright вшШрһиг-уеПот................... ees 16 16. Mouths of the tubes minute, averaging 4-7 to а тап................. 17 16. Mouths of the tubes larger, averaging 1-3 to a mm..... ......... 18 17. Mouths of the tubes averaging 4 to a mm.; pileus rarely more than 5 cm. in diameter........... on ces eee 5-2... 35. P. elegans 17. Mouths of the tubes averaging about 6 to a mm.; pileus 4-20 cm. in diameter 84. P. picipes 18. Pileus large, more than 5 mm. thick; plant growing on living trees; stipe black at the bae. Е. Ao eere errore 33. P. squamosus 18. Pileus small or medium sized, not more than 5 mm. thick; stipe not black at the baee. ... сквер. › соло bean se VE v 19 19. Tubes long-decurrent on the stipe; context soft and friable when dry 32. P. pennsylvanicus 19. Tubes slightly or not at all decurrent; context not soft and friable when dry.. 20 20. Pileus yellowish brown; mouths of the tubes almost 1 mm. in diameter; (0 7* ое o 03 ee 31. P. arcularius 20. Pileus darker than above, sometimes sooty-black; mouths of the tubes averaging 2 to a mm.; walls at first thick............... 30. P. brumalis 21. Pileus red-varnished, at least when уошар............................. 22 31. Pilous Po Se eee See 23 22. Varnish disappearing with age, the pileus then whitish or yellowish. ..... 61. P. Curtisii 22. Varnish persistent, the pileus not changing color............ 60. P. lucidus 23. Sporophore more or less globose; tubes concealed by a volva....... 27. P. volvatus 23. Sporophore not globose; volva абвепі................................. 24 88 25. 25. 27. 27. 29. 29. 81. 81. 33. 33. 35. 35. 37. 37. 39. 39. 41. 41. [Vor. 1 ANNALS OF THE MISSOURI BOTANICAL GARDEN 24. Sporophore arising from the under side of a cup-shaped, sterile body; found only on dead branches of Ulmus.................. 6. P. conchifer 24. Sporophore not arising from a cup-shaped, sterile body............. 25 Margin of the pileus projecting 5 mm. or more beyond the hymenium; hymenium separating smoothly from the context in fresh specimens; found ev AL copes Я О. POPE TET 26. P. betulinus Plants not as аһоуе................................................. 26 26. Hymenium bright виіріш-уеПом...................... 42. P. sulphureus 26. Hymenium not bright sulphur-yellow.......... lle esses 27 Pileus distinetly brown in color; context usually light brown ; hymenium changing color when ітіней.............................. 46. P. resinosus Pileus not brown in color; hymenium never changing color when bruised .. 28 28. Hymenium more or less smoke-colored or Ыаек.................... 20 28. Hymenium not at all smoke-colored or black... o.oo aaa. 32 Pileus more than 4 mm. ӨМск........................................ 30 Pileus not more than 4 mm. ӨМек.................................... 31 30. Context fragrant, with the odor of anise.................. 23. P. fragrans 30. Context not fragrant, odor sometimes disagreeable........ 24. P. fumosus Mouths of the tubes angular, minute, averaging 5-7 to a mm. ; dissepiments QU I "LH 22. P. adustus Mouths of the tubes circular or subcircular, medium sized, averaging 3-5 to a mm.; dissepiments ӨМіек................................... 24. Р. fumosus 32. Context fibrous or coriaceous in fresh plants; pileus never more than 1.5 em. thick, and usually much thinner.................. 20.0 cee eee. 33 32. Context either soft, spongy and full of water or firm and corky, often fragile when dry; pileus often more than 1.5 em. thick.............. 43 Hymenium broken up into іееіһ.......................2............. 34 Hymenium entire or lacerate but not broken up into teeth. .............. 36 34. Context more than 1 mm. ӚНіск................22......... 9. P. biformis 34. Context not more than 1 mm. бһісек.................................. 35 Plants growing only on the wood of coniferous trees............. 2. P. abietinus Plants growing only on the wood of deciduous trees........... 8. P. pargamenus 36. Context 1 mm. or less бМісек.......................2.2..2..2........ 37 36. Context more than 1 mm. ӨНісе.................................. 40 Mouths of the tubes minute, averaging 4-6 to a mm.; hymenium never violet КЕШЕ Ы ee ны ИИ 20 38 Mouths of the tubes larger, averaging 2-3 to a mm.; hymenium often violet DEM. ERE ET rE or oe Se eee 39 38. Surface of the pileus villous or velvety; pileus multizonate, generally more than 2 om. Ьгоаф............................... 1. P. versicolor 38. Surface of the pileus densely hirsute; pileus azonate or with one or two zones, generally less then 2 ст. broad.................... 4. Р. hirsutulus Plants growing only on the wood of coniferous trees............. 2. P. abietinus Plants growing only on the wood of deciduous trees 222222... 3. P. pargamenus 40. Mouths of the tubes large, averaging 1-2 to a mm......... 9. P. biformis 40. Mouths of the tubes medium sized, averaging 3-4 to а mm.......... 41 Tubes more than 2 mm. long............... sess 7. P. pubescens Tubes not more than 2 mm. Іопр..................................... 42 42. Surface of the pileus velvety to ҺітеШе..................... 5. P. hirsutus 42. Surface of the pileus minutely pubescent or glabrous. ........ 8. P. Lloyd 1914) OVERHOLTS—THE POLYPORACEJE OF OHIO 89 43. Plants mostly төтіріпміе..........................................;. 44 48. Plants not mostly товарный в............... ось бо reap d 45 44. Pileus azonate, margin often іпгоЦеа.................. 10. P. semipileatus 44. Pileus zonate, margin always straight.................. ss. 21. P. zonalis 45. Pileus corky in texture when fresh, usually rather thick and firm... ........ 46 45. Pileus soft and spongy in texture when ітезһ.......................... 49 46. Pileus distinctly enerusted; hymenium and context pinkish or гову when fresh; plants usually growing on Ртатітив................ P. frazineus 46. Pileus not encrusted; hymenium and context whitish when fresh; plants au uiv on Frazénus. ....... Leere rr ouo a 47 47. Pileus more than 2 cm. thick; tubes more than 4 mm. long... .25. P. robiniophila 47. Pileus not more than 2 cm. thick; tubes not more than 4 mm.long.......... 48 48. Plants with a sweet anise ойбог........................... 23. P. fragrans 48. Plants with no odor, or odor disagreeable................ 24. P. fumosus 49. Mouths of the tubes minute, averaging 6-7 to a mm.; plants with a sweet acid «ао... t ue RE B 14. P. galactinus 49. Mouths of the tubes larger, averaging 1-4 to a mm. .................... 50 50. Pileus generally less than 4 сіп. Бговй............................. 51 50. Pileus generally more than 4 cm. Ьтояй........................... 53 51. Pileus pubescent; mouths of the tubes dentate, lacerate, or irregular....... 52 51. Pileus glabrous; mouths of the tubes entire; plants with a sweet acid odor 12. P. chioneus 52. Pileus and spores (in mass) often bluish or slate-colored; tubes equalling in length the thickness of the соһфбехб..................... 11. P. caesius 52. Pileus and spores pure white; tubes shorter in length than the thickness "om oc 0 M "————— 13. P. lacteus 53. Plants growing only on the wood of coniferous ітевв.................... 54 53. Plants growing only on the wood of deciduous ігеев..................... 55 54. Tubes usually more than 5 mm. long, the mouths averaging 2-3 to a mm. 19. P. borealis 54. Tubes usually less than 5 mm. long, the mouths averaging 4-5 to mm. 18. P. guttulatus 55. Margin of the pileus thick and тойпбей........................ 17. P. obtusus 55. Margin of the pileus thin and асшіе................................... 56 56. Mouths of the tubes large, averaging 1-2 to a mm.......... 16. P. delectans 56. Mouths of the tubes small, averaging 3-5 to а mm. ................ 57 57. Fresh plant with a disagreeable odor; context very hard when dry .20. P. Spraguei 57. Fresh plant with no disagreeable одог....................... 15. P. spumeus Section II Pileus and hymenium deep сіппаһат-гей.............................. 1 Pileus and hymenium not deep cinnabar-red (rosy or orange-colored in some ОЗОР ое... e 2 1. Pileus less than 5 mm. thick, often 2опайе.................... 44. P. sanguineus 1. Pileus more than 5 mm. thick, never zonate................ 46. P. cinnabarinus 2. Hymenium bright sulphur-yellow in fresh plants........... 42. P. sulphureus 2. Hymenium not bright вШрһш-уеПоу.............................. 3 3. Plant growing only on the wood of Quercus and Castanea; pileus yellowish or orange-ooloted Е о р. У аео ео с сеа 48. P. Pilote 1 For description see p. 130 under the genus Fomes. 90 ANNALS OF THE MISSOURI BOTANICAL GARDEN 3. Plant growing usually on Fraxinus; pileus usually stained more or less with ИР А РАНА ТОРЕ P. fraxineusi 3. Plant growing usually on coniferous wood; rose-colored without and within... P. carneust Section III Pileus stipitate or візвбірИдйе..............................2......... 1 Pileus sessile or effused-reflexed, not вбірйайе.......................... 9 1. Pileus and stipe covered with a reddish varnish at least when young. ...... 2 1. Pileus and stipe not гей-уатпінһей.................................... 3 2. Pileus and stipe at first red-varnished, the varnish disappearing and the pileus becoming whitish or yellowish when mature. ......... 61. P. Curtisii 2. Pileus and stipe strongly red-varnished, the varnish not disappearing with - £L еа RETI IM LL NE t 60. P. lucidus 3. Context not more than 1 mm. thick; plants growing on the ground........ 4 3. Context more than 1 mm. thick; plants growing on wood or attached to buried ры UE ALL EU. n 6 4. Surface of the pileus marked with silky вігіаііопв........ 59. P. cinnamomeus 4. Surface of the pileus not silky........ омоси 5 5. Mouths of the tubes small, averaging 2-4 to а mm.; tubes usually less than 3 BEN Е oe т. 68. P. perennis 5. Mouths of the tubes large, averaging 0.5-1 mm. or more in diameter; tubes usually more than 3 mm. Іопр............................... 67. P. focicola 6. Surface of the pileus distinctly encrusted..................... P. lobatus? 6. Surface of the pileus not at all епсгініед.................2......... 7 7. Context decidedly duplex, spongy above, firm next to the tubes. . .25. P. circinatus 7. Context not дарех.................................................. 8 8. Hymenium some shade of yellow (yellowish brown, yellowish green, etc.), quickly changing color when bruised; growing on or about trees and stumps of Pinus; spores "Һйе....................... 54. P. Schweinitzii 8. Hymenium cinereous to brownish, not changing color when bruised; growing on the ground or attached to buried wood; spores brown....... 66. P. obesus 9. Pilei forming a densely imbricate, globose or cylindrical mass. . ..... P. graveolens 9. Pilei not forming a densely imbricate, globose or cylindrical mass......... 10 10. Pileus гей-уагпізһей.................................... 60. P. lucidus 10. Pileus not геб-уагпівһей........................2222222222227277.. 11 11. Pileus distinctly encrusted... 2.0.0.0... на... Р. lobatuss 11. Pileus not distinctly епегініей................222222222222222222227.. 12 [Vor. 1 12. Plants growing on or about stumps or trunks of Pinus. . .64. P. Schweinitzit 12. Plants growing on wood of deciduous shrubs or trees, often on living ЕНЕ С, Eel crise. eu. EE 13 13. Context usually less than 7 mm. thick; plants small or medium sized ..... 14 13. Context more than 7 mm. thick; plants large... 17 Eos nus P ЕС 15 ПЕ. vrai ans О а E ое 16 1Бее p. 130 for a description of this plant. *For description of this plant see p. 131 under the genus Fomes. ‘This plant is described on p. 137 under the genus Fomes. *For description see p. 131 under the genus Fomes. *For description see p. 137 under the genus Fomes. 1914) OVERHOLTS—THE POLYPORACEJE OF OHIO 91 15. Pileus spongy and watery when fresh; context friable when dry; mouths of the tubes averaging 2-4 to à шіп............................ 27. P. nidulans 15. Pileus firm and rigid; context corky when dry; mouths of the tubes minute, averaging БҰЗСА И а фео КЕ 48. Р. gilvus 16. Plants growing on the wood of Alnus and Betula; spores light brown... 49. P. radiatus 16. Plants growing on the wood of Acer, Fagus, and other deciduous trees. . . 50. P. cuticularis 17. Context very light Бояп.................................. 46. P. resinosus 17. Context yellowish brown or багКег................................... 18 18. Surface of the pileus hirsute; plants growing on various diseased decid- TOE TOD. ow cece cece creer E 61. P. hispidus 18. Surface of the pileus fibrillose or glabrous; plants growing only on the wood of Өиетсив............................................... 19 19. Sporophore medium sized, less than 10 em. broad and 3 cm. ess ces ss 53. P. dryophilus 19. Sporophore large, more than 10 cm. broad and 3 cm. thick. . ..... 52. P. dryadeus 1. P. versicolor L. ex Fries, Syst. Myc. 1: 368. 1821. Boletus versicolor L. Sp. Plant. 1176. 1753. Pileus sessile or effused-reflexed, imbricate or single, dimid- iate or encircling twigs and then often orbicular by confluence, 2-5 x 2-7 x 0.1-0.3 cm., coriaceous, prevailing color grayish, but marked by many narrow, multicolored zones, ranging from white to yellow, brown, reddish, greenish, blackish, ete., villous or velvety, the margin thin and acute, usually sterile below; context white or whitish, fibrous, less than 1 mm. thick; tubes 1-2 mm. long, the mouths white or yellowish, sometimes somewhat glistening, circular to angular, averaging 3-5 to a mm., the walls thin, entire or slightly lacerate; spores white, smooth, oblong, sometimes curved, 1.2-2 х 5-6.3 и. On all kinds of dead wood. Common throughout the year. Easily distinguished by the multizonate, multicolored pileus. P. hirsutulus Schw. is often considered to be a form of this species. P. zonatus Fries, as reported by Morgan, is one of the many forms of it. The following references contain good illus- trations of our plant: Hard, Mushrooms f. 343., White, Hymen. Conn. pl. 36., and Moffatt, Higher fungi of the Chicago region рі: 17. 3.6. 2. P. abietinus Dicks. ex Fries, Syst. Мус. 1: 370. 1821. Boletus abietinus Dicks. Fasc. Pl. Crypt. Brit. 3:21. 1793. Pileus sessile or effused-reflexed, dimidiate and broadly attached, or flabelliform and attached by the attenuate base [Vor. 1 92 ANNALS OF THE MISSOURI BOTANICAL GARDEN of the pileus, 0.5-5 x 0.5-5 x 0.1-0.2 em., coriaceous, white to cinereous or almost black behind, villous, zonate, margin thin and acute; context white or pallid, fibrous, not more than 1 mm. thick; tubes less than 3 mm. long, the mouths white to bay and often violaceous toward the margin, averaging 2-3 to a mm., the dissepiments thin and soon lacerate and breaking up into teeth. Growing only on the wood of coniferous trees. In autumn. Rare. Closely related to P. pargamenus Fries, from which it is most easily separated by the habitat. The following spore dimensions are found in the literature: Karsten—“oblong 4-6 х 1-3 и”; Murrill—“ globose, smooth, hyaline, 4.5-5.5 шіп diam- eter"; Bresadola—“ hyaline, cylindrical, subcurved, 6-7 x 2.5 и.” 3. P. pargamenus Fries, Epicr. Syst. Myc. 480. 1838. Pileus sessile or effused-reflexed, imbricate, dimidiate or flabelliform, sometimes attached by an attenuate base, 1-7 x 1-7 x 0.1-0.4 cm., coriaceous, whitish to cinereous or yellowish brown, villous, zonate, the zones sometimes differently colored, margin very thin, acute, broadly sterile below, often violaceous in color; context white or whitish, fibrous, very thin, less than 1 mm. thick; tubes not more than 2.5 mm. long, the mouths whitish to bay and often violaceous toward the margin, angular, averaging 2-3 to a mm., the dissepiments thin and soon break- ing up into teeth; spores white, smooth, oblong, slightly curved, 2-2.5 x 5-6.3 и. Growing on the wood of deciduous trees, especially of Quercus and Prunus. September to December. Common. Close to P. abietinus Dicks. ex Fries, but usually found on dead wood of deciduous trees. Well represented by Hard (Mushrooms f. 345) as P. pergamenus. 4. P. hirsutulus Schw. Trans. Am. Phil. Soe. II. 4: 156. 1832. Pileus sessile or effused-reflexed, often imbricate, dimidiate, 0.5-2 x 0.5-2.7 x 0.1-0.2 cm., coriaceous, gray or cinereous to yellowish brown, hirsute or strigose, azonate or with 2-3 colored zones, margin thin and acute, usually sterile below; context white or whitish, membranous, less than 1 mm. thick; tubes less than 2 mm. long, mouths whitish to yellowish, rarely 1914) OVERHOLTS—THE POLYPORACEZ OF OHIO 93 glistening, circular or angular, averaging 3-5 to а mm., the dissepiments thin and entire. On dead branches of deciduous trees, more often on fruit trees. Found from August to December. Not common. Separated from P. versicolor L. ex Fries, by the more hirsute or strigose pubescence on the pileus, and by the smaller size. Specimens collected at Cincinnati by D. L. James and referred to P. velutinus Fries are now referred to this species. 5 P. hirsutus Wulfen, ex Fries, Syst. Myc. т: 367. 1821. Boletus hirsutus Wulfen, in Jacq. Coll. 2: 149. 1788. Pileus sessile, or effused-reflexed, dimidiate, 1.5-5 x 1,5-7 x 0.2-1 cm., flexible when moist, firm and sometimes rigid when dry, grayish to yellowish or smoky brown, hirsute or tomentose, sometimes zonate, sometimes concentrically sulcate, the margin thin or rather thick, acute, sometimes dark colored; context white or pallid, tough to soft-corky, 1-6 mm. thick; tubes 1-4 mm. long, the mouths white, grayish or fuliginous, circular to somewhat angular, averaging 3-4 to a mm., the walls rather thick and always entire; spores white, smooth, cylindrical, often curved, 2.5 x 5-8 и. On dead wood of deciduous trees. Found throughout the year. From closely related species with а conspicuous hairy covering this plant is perhaps most easily separated by the persistently thick walled tubes that never become torn or lacerate. Any plant with the characteristies of this group and possessing the dark-colored marginal band to which reference is made in the description may always with safety be referred to this species. From P. versicolor L. ex Fries, the plant is separated by the absence of the numerous multicolored zones. Нага’ѕ figure (Mushrooms f. 342) is not a good illustration of our plant. Murrill describes the plant under the name of Coriolus nigro- marginatus (Schw.) Murr. 6. P. conchifer Schw. ex Fries, Epicr. Syst. Myc. 463. 1838. Boletus conchifer Schw. Syn. Fung. Car. 98. 1822. Pileus sessile or attached by a lateral tubercle and then appearing substipitate, reniform to dimidiate in outline, 1-3 x 1-4 x 0.1-0.3 cm., coriaceous, white to yellowish, glabrous, [Vor. 1 94 ANNALS OF THE MISSOURI BOTANICAL GARDEN zonate or azonate, the margin very thin and acute; on the upper surface and at the base of the pileus a small cup-shaped or disk-like sterile structure is usually borne, white or brown and often zoned on the inside; context white, fibrous, less than 1 mm. thick; tubes not more than 2 mm. long, at first white, often yellowish on drying, the mouths angular and thin-walled averaging about З to а mm., the dissepiments often lacerate; stipe (?) rudimentary, tubercular; spores not obtained. Growing only on fallen branches of Ulmus. Common. This plant has somewhat the appearance of P. pubescens Schum. ex Fries, from which, however, it is easily separated by the much thinner pileus, the attenuate base, the presence of the sterile cup, and the habitat. 'The cup is sometimes absent. Тһе development of the cup has not been closely fol- lowed. Lloyd believes that the fertile pileus is first developed and from it the sterile cup arises, and that during the winter the fertile portion falls away, the cup persisting on the sub- stratum but not giving rise to new pilei the next season. Miss Ames comes to the conclusion that the sterile cups represent pilei whose marginal hyph: have been killed by unfavorable conditions and which as a result may develop a fruiting surface from the base of the dead cup-like pileus. "This would explain the occasional absence of the sterile cup, its presence depending upon the death of the marginal hyphe in the early stages of the production of a first pileus. Р. virgineus Schw. described from North Carolina is said to be this plant. The plant is exceptionally well illustrated by Lloyd (Мус. Notes, Polyporoid Issue 3 f. 365-66), and by Moffat (Higher fungi of the Chicago region pl. 16. f. 2). 7. P. pubescens Schum. ex Fries, Syst. Myc. т: 367. 1821. Boletus pubescens Schum. Enum. Pl. Saell. 2: 384. 1803. Polyporus Sullivantii Mont. Ann. Sci. Nat. II. 18: 243. 1842. Pileus sessile, dimidiate, 1.5-5 x 2.5-5 x 0.4-1 cm., fleshy- tough when fresh, firm when dry, white or yellowish in fresh specimens, sometimes umber or brown when dry, villous- tomentose, zonate or azonate, margin thin, acute; context white or pallid, fibrous-tough when fresh, more firm when dry, 1-5 mm. thick; tubes 1-4 mm. long, the mouths white, yellowish, or umber, angular, averaging 3-4 to a mm., the 1914] OVERHOLTS—THE POLYPORACEX OF OHIO 95 dissepiments thin, entire to dentate; spores white, smooth, cylindrical, curved, 2.7-3.6 x 5.4 и. On dead wood of deciduous trees. August to November. Common. Plants collected in the Miami valley by Morgan and referred by him to P. velutinus Fries belong here. Plants distributed by Kellerman in his 'Fascicles of Ohio Fungi’ as P. molliusculus Berk. are referred to this species. Р. fibula Fries as reported by Morgan is probably the same as P. pubescens var. Graii, here included under P. pubescens. Hard (Mushrooms f. 339) gives a good illustration of the plant. 8. P. Lloydii (Murr.) Overholts, n. comb. Coriolus Lloydii Murrill, М. Am. Flora 9: 23. 1907. Pileus rather thin, laterally connate, rigid, tough, cuneate to flabelliform, applanate, tubercular-sessile, 2-3 x 3-4 x 0.2-0.4 em.; surface white or isabelline, scabrous, somewhat rugose, marked with a few narrow, indistinct, pale latericeous zones; margin thin, fertile, irregular, lobed; context punky-fibrous, white, 1.5-2 mm. thick; tubes 1-1.5 mm. long, white within, mouths angular, subglistening, 4 to a mm., edges thin, firm, dentate, white or isabelline; spores globose, smooth, hyaline, 2 u; hyphe 5 y. On dead wood. Rare. The above description is taken from the ‘North American Flora.’ The type specimens were collected near Cincinnati, Ohio, by C. G. Lloyd, and to the writer’s knowledge the plant has not been found since. Тһе species appears to be distinct. 9. P. biformis Klotzsch, Linnaea 8: 486. 1833. P. molliusculus Berk. НооКегв Lond. Jour. Bot. 6: 320. 1847. Plants sessile, effused-reflexed or resupinate, often imbricate; pileus dimidiate or laterally confluent and elongate, 0-5.5 x 1.5-6 x 0.2-1.5 em., soft and pliable when fresh, slightly flexible to rigid when dry, white, pallid, bay, or ochraceous, appressed-fibrillose, usually rough, azonate or subzonate, the margin thin and acute; context white or whitish, fibrous-tough when fresh, soft-corky when dry, 1-5 mm. thick; tubes white, becoming bay on drying, 2-5 mm. long, the mouths circular to angular or sinuous, averaging 1-2 to a mm., the dissepiments 7 [Vor. 1 96 ANNALS OF THE MISSOURI BOTANICAL GARDEN rather thin and usually becoming lacerate and broken up into teeth at an early stage of growth, sometimes remaining poroid, especially toward the margin of the pileus; spores white, smooth, oblong, curved, 2-2.6 x 7-8 y. Growing on oldlogs. September to December. Common. The following group of characters will usually identify the species: the semi-resupinate habit of growth, whitish or tan-colored pileus, and the rather long tubes with large mouths, soon breaking up into teeth. P. molliusculus was named by Berkeley from specimens sent to him from Ohio by Lea. Mor- gan's determination of P. molliusculus was an error, his plants belonging to P. pubescens Schum. ex. Fries. Kellerman re- peated the error in distributing P. molliusculus in his “Оһо Fungi Fascicles.’ For illustration see Hard, Mushrooms f. 341. IO. P. semipileatus Peck, Ann. Rept. N. Y. State Mus. 34: 43. 1881. Plants resupinate or effused-reflexed, rarely strictly sessile; pileus dimidiate or elongate, 0-1.5 x 0.7-3.5 x 0.1-0.5 cm., soft and spongy when fresh, rigid when dry, white, yellowish, or reddish brown, slightly tomentose to glabrous, azonate, margin thin, acute; context whitish, soft when fresh, firm when dry, 1-4 mm. thick; tubes less than 2 mm. long, the mouths white, greenish or somewhat violaceous, angular, minute, averaging 4-6 to a mm., the walls entire; spores white, smooth, oblong, curved, 1 x 3-4 y. On old limbs on the ground. September to December. Rare. Easily recognized by the minute pores, the semi—resupinate habit of growth, and the often violet tinted hymenium. There is no previous record of the plant occurring in Ohio. Collec- tions were made at Oxford, in 1911, for the first time. тг. P. caesius Schrad. ex Fries, Syst. Myc. т: 360. 1821. Boletus caesius Schrad. Spic. Fl. Ger. 167. 1794. Pileus sessile or effused-reflexed, dimidiate, 1-3.5 x 2-6 x 0,3-2 em., soft and spongy when fresh, rigid when dry, whitish to cinereous, often with a bluish tinge, distinctly villous or tomentose especially behind; azonate, margin thin and acute; context white, soft, spongy and full of water when fresh, friable when dry, 0.3-1 em. thick; tubes 2-7 mm. long, mouths white, раШа, or bluish gray, angular, averaging 3-5 to а mm., the 1914] OVERHOLTS—THE POLYPORACEJE OF OHIO 97 walls thin and usually lacerate; spores minute, white, smooth, cylindrical, sometimes curved, 1.2-1.5 x 4.7-5.2 y. On dead wood of deciduous and coniferous trees. October to December. Rare. The bluish color of the pileus and hymenium is so often wanting that other characters must frequently be used in the identification of the plant. The slender tubes, usually longer than or as long as the thickness of the context, is apparently a rather constant character of the plant. The villous or tomen- tose pileus separates it from P. chioneus Fries and P. lacteus Fries and these are the only species with which it is likely to be confused. I2. P. chioneus Fries, Syst. Myc. І: 359. 1821. Pileus sessile, dimidiate, 1-3 x 2-5 x 0.5-3 cm., soft and spongy when fresh, rigid when dry, whitish to grayish or yel- lowish, azonate, glabrous or with a slight strigose tomentum towards the base, sometimes covered with a thin grayish or yellowish pellicle that becomes more evident on drying; margin acute, sometimes inflexed on drying; context white, soft and spongy when fresh, fragile when dry, 0.3-2 cm. thick, azonate, with a sweet acid odor; tubes 1-8 mm. long, mouths white or yellowish, usually glistening, angular, averaging 3-4 to a mm., the walls thin but entire; spores white, smooth, oblong, slightly curved, 1-1.7 x 4-5 y. On dead wood. September to November. From P. galactinus Berk. this plant is most easily separated by the oblong, curved spores. Тһе usually glabrous pileus and the absence of bluish tints separates it from P. caesius Schrad. ex Fries. Whether it is distinct from P. lacteus Fries may well be doubted. "The plant is much in dispute in Europe. Our plants have been described as P. albellus Peck. 13. P. lacteus Fries, Syst. Myc. т: 359. 1821. Pileus pure white, fleshy-fibrous, fragile, triangular, pubes- cent, azonate externally and internally, margin inflexed, acute; pores thin acute, dentate, becoming torn and labyrinthiform. Commonly small and thin but sometimes large and transversely elongate, often gibbous behind, becoming glabrate and uneven. (Adapted from Fries, Hymen. Eur. 546.) On dead wood of deciduous trees. Rare. [Vor. 1 08 ANNALS OF THE MISSOURI BOTANICAL GARDEN Until quite recently this and the preceding species have been held to be quite distinct. Of late years the European mycol- ogists are coming to believe that they cannot be regarded as distinct species. Murrill would separate them оп the ground that P. chioneus always has a distinct cuticle which is entirely lacking in P. lacteus. Тһе writer has endeavored to keep the plants distinct on the basis of the differences noted by Fries. If this proves unfeasible then the two must be united as one species under the name of P. chioneus, at least with reference to their occurrence in this country. 14. P. galactinus Berk., Hooker’s Lond. Jour. Bot. 6: 321. 1847. Pileus sessile, imbricate or single, dimidiate, 3-7 x 3-7 x 0.5-2 cm., soft and pliant when fresh, more or less watery, rigid and contorted on drying, white, grayish, or somewhat yellowish, tomentose to strigose-tomentose, especially at the base, be- coming glabrous with age, azonate, margin thin and acute; context white or pallid, watery and spongy when fresh, with a distinct sweet acid odor, firm when dry, sometimes more or less duplex, 8-8 mm. thick; tubes 2-7 mm. long, mouths white to bay, often glistening, circular to angular or sinuous, minute, averaging about 6 to a mm.; spores white, smooth, ellipsoid, 2-2.5 х 3.5-4 ир, uninucleate and with a very trans- parent wall. Growing on dead wood of deciduous trees. August to November. Common. The sweet acid odor mentioned in the description is a dis- tinguishing character of all collections of this species. No mention is made of the odor in any published work to the writer’s knowledge, except іп Peck's description of P. immitus in which the odor is described as subacid. Р. immitus is in all probability this plant. The odor is so constant that whenever it is noticed in connection with any minute-pored form of this section one can be sure that the plant belongs to this species. All of the collections that I have referred to this species are watery when fresh, have a sweet acid odor, and when dried shrink much in size and often become much contorted. The context becomes thin and hard and takes on a resinous, dark brown or black color. This appearance may be uniform through 1914) OVERHOLTS—THE POLYPORACEJE OF OHIO 99 the context or the dark resinous color may be limited to a narrow line next to the hymenium or confined to two or three narrow zones in the context. It is difficult to distinguish these species with a white watery context and the writer's presenta- tion of them may be open to criticism. 15. P. spumeus Sow. ex Fries, Syst. Myc. 1:358. 1821. Boletus spumeus Sow. Col. Fig. Eng. Fungi pl. 211. 1797. Pileus sessile, dimidiate, watery and fleshy-tough when fresh, firm when dry, 7-12 x 10-20 x 2-8 cm., much smaller on dry- ing, appearing appressed-tomentose, white or grayish, some- what yellowish or brownish on drying, azonate, margin rather thick but acute; context white, soft, spongy, and full of water, rather fragile on drying, more or less zonate, 1-3 em. thick; tubes 0.5-1.5 em. long, mouths white or yellowish on drying, angular, averaging 3 to à mm.; spores white, smooth, globose, ог subglobose, 4.5-5.2 v in diameter, distinctly uninucleate. Growing on injured or diseased deciduous trees, especially Ulmus and Acer. October and November. Rare. The plant is closely related to P. delectans Peck, with the same habitat and general appearance, but separated from that species by the smaller mouths of the tubes and by the distinctly uninucleate and more globose spores. Тһе plants so referred do not agree with the figure given by Sowerby, nor with Fries' description. My plants were determined by Bresadola. 16. P. delectans Peck, Bull. Torr. Bot. Club 11:26. 1884. Pileus sessile, sometimes imbricate, dimidiate in outline, 3-7 x 4-15 x 0.7-3 cm., rather spongy and watery when fresh, firm and rigid when’ агу, white or whitish, finely tomentose or glabrous, azonate, margin thin and acute; context white, in large specimens duplex, with a firm lower layer and a soft upper layer, in smaller specimens more uniform, 0.5-1.5 ст. thick; tubes 0.5-1.5 ста. long, mouths white, yellowish on dry- ing, circular to angular, large, averaging 1-2 to а mm.; spores white, smooth, ellipsoid to globose, 4.5-5.5 x 6.5-8.5 y. On diseased or injured trunks of deciduous trees, especially Acer; sometimes on logs of Fagus. September to December. Frequent. The species is separated from P. spumeus Sow. ex Fries by the larger tube mouths and the less globose spores that have [Vor. 1 100 ANNALS OF THE MISSOURI BOTANICAL GARDEN not been observed to be uninucleate as in that species. It is a large white fungus distinct from the other allied species in size, length of tubes, and habitat. 17. P. obtusus Berk. Ann. & Mag. Nat. Hist. I. 3:390. 1839. Plants annual, sessile, sometimes imbricate; pileus dimidiate, convex or ungulate, 3-9 x 4-15 x 3-6 cm., somewhat spongy when fresh, firm, rigid, and very light in weight when dry, cinereous to yellowish or darker in herbarium specimens, hirtose-tomentose, rarely becoming glabrous, azonate, margin thick, obtuse; context white or whitish, spongy to согЕу, some- times duplex, 1-3 cm. thick; tubes 1.5-3 em. long, the mouths white, bay or brown on drying, circular to angular and sinuous, 1 mm. or more in diameter; spores (teste Murrill) globose, smooth, hyaline, 6-8 м. On trunks of diseased deciduous trees, especially Quercus. Rare. Always easily recognized by the rounded and obtuse margin, and the long tubes with large mouths. Excellent illustrations are given by Spaulding (Ann. Rept. Mo. Bot. Gard. 16: pl. 13-19), 18. P. guttulatus Peck, in басс. Syll. Fung. 6: 106. 1888. P. maculatus Peck, Ann. Rept. N. Y. State Mus. 26:69. 1874. Pileus sessile, sometimes imbricate, dimidiate, 3-8 x 5-12 x 0.4-1.5 cm., soft and fleshy when fresh, firm and rigid when dry, white to yellowish or slightly brownish, glabrous, azonate or sometimes zonate on the margin, sometimes marked with rounded depressed spots, margin thin, acute; context white or pallid, soft and fleshy when fresh, soft-corky or friable when dry, 0.4-1 cm. thick; tubes 1-5 mm. long, the mouths white to yellowish or umbrinous, angular, averaging 4-5 to a mm.; spores white, smooth, oblong-ellipsoid, 2,5-3 x 3-5 u. (Cf. Mur- rill, globose, smooth, hyaline, 5 » in diameter.) Growing on wood of coniferous trees. Rare. The distinguishing character of the species is the presence of the round depressed spots on the pileus. 19. P. borealis Fries, Syst. Myc. 1: 366. 1821. Pileus sessile, dimidiate, sometimes with an attenuate base, 3-8 x 4-12 x 0.5-2.5 em., somewhat watery and spongy when fresh, rigid when dry, white or yellowish, sometimes brownish, hispid to tomentose, azonate, margin thin and acute; context 1914] OVERHOLTS—THE POLYPORACE OF OHIO 101 white or yellowish, distinetly duplex, firm and fibrous below, soft and floccose above, 0.5-2 em. thick; tubes 3-10 mm. long, the mouths white or yellowish, angular to irregular and uneven, rather large, averaging 2-3 to а mm.; spores (teste Murrill) ovoid, smooth, hyaline, 5-6 x 3-4 и. Growing only on trunks of coniferous trees. Rare. The species is most easily separated from its allies by the size and habitat. For illustrations see Atkinson, Mushrooms f. 9., Duggar, Fung. Dis. Plants f. 228., and Atkinson, Cornell Univ. Agr. Exp. Sta. Bul. 193: f. 63. 20. P. Spraguei Berk. & Curt. Grevillea 1: 50. 1872. Plants annual, sessile or decurrent, sometimes imbricate; pileus dimidiate, 4-12 x 4-10 x 0.6-2 cm., fleshy-tough when fresh, rigid when dry, white or cinereous, appressed-tomentose or glabrous, azonate or somewhat zonate, margin thin or rather thick, acute, often blackening on drying; context white, watery, tough-fibrous when fresh, sometimes very hard when dry, zonate, 0.3-1.5 em. thick, with a disagreeable odor in fresh specimens; tubes 0.3-1 em. long, mouths white or discolored, circular or angular, averaging 3—4 to a mm. ; spores (teste Murrill) ellipsoidal smooth, hyaline, бх 4 и. On dead wood of deciduous trees, especially on Fagus, Quer- cus, and Castanea. July to September. Common. Fresh specimens are always easily distinguished by the very disagreeable odor. Dried plants are characteristically very hard and rigid, the context almost bony in texture. 21. P. zonalis Berk. Ann. & Mag. Nat. Hist. I. то: 375. 1842. Plants annual, sessile, effused-reflexed, or entirely resupinate; pileus dimidiate or laterally confluent, 0-2.5 x 1-5 x 0.2-0.5 cm., fleshy and pliable when fresh, rigid and firm when dry, whitish to flesh-colored or isabelline, finely tomentose to gla- brous, at first azonate but becoming zoned when mature, the margin at first thick, thin with age; context white, fibrous when fresh, hard and rigid when dry, 1-2 mm. thick; tubes 1-3 mm. long, the mouths usually more or less flesh-tinted when fresh, angular, averaging 4-5 to a mm., the walls thick and entire, very firm and rigid on drying; spores white, smooth, globose, 2.5-5 u broad, with one large nucleus. [Vor. 1 102 ANNALS ОҒ THE MISSOURI BOTANICAL GARDEN On old rotting logs, especially of Liriodendron. August to December. Not common. The writer has collected this plant several times in the Miami valley, almost always on logs of Liriodendron tulipifera. The plant is usually entirely resupinate and has doubtless been de- scribed as a Poria, but good collections were made which showed beyond a doubt the pileate tendency of the plant. No dispo- sition could be made of the plant until Dr. Murrill suggested that it might belong to P. zonalis. Later, specimens were sent to Rev. Bresadola who pronounced it that species and an opinion recently received from Mr. Lloyd expresses the same view. It is, however, quite different from the usual forms of that plant and the name is used with some apprehension. The plant is also abundant in Missouri where the writer has found the pileate forms to be much more common than іп Ohio. P. zonalis has been supposed to be confined to the Gulf States in this country, although it is not surprising that semi-tropical forms found there should extend their range up the large river valleys to the north. 22. P. adustus Willd. ex Fries, Syst. Myc. т: 363. 1821. Boletus adustus Willd. Fl. Berol. 392. 1787. Plants annual, sessile, effused-reflexed, or resupinate; pileus dimidiate, often imbricate, 1-6 x 2-7 x 0.2-0.4 cm., fleshy- tough when fresh, coriaceous or rigid when dry, white to cin- ereous or pale tan, fibrillose-tomentose to almost glabrous, zonate or azonate, the surface usually rough, margin thick and broadly sterile below when young, becoming thin when mature; context white or pallid, rather soft when fresh, corky or fibrous-corky when dry, 1-3.5 mm. thick; tubes not more than 1 mm. long, the mouths grayish black to black, angular, even, minute, averaging 5-7 to a mm.; spores white, smooth, oblong to oblong- ellipsoid, 2-2,5 x 3.8-4.3 y. On stumps and trunks of dead deciduous trees. August to December. This species differs from P. fumosus Pers. ex Fries and P. fragrans Peck in the smaller size and the uniformly black hymenium. Lu NE UTEM VENE UE rd 1914] OVERHOLTS—THE POLYPORACEJE OF OHIO 103 23. P. fragrans Peck, Rept. N. Y. State Museum 30: 45. 1879. Plants annual, sessile or effused-reflexed; pileus dimidiate, imbricate, 2-8 x 4-10 x 0.5-2 cm., fleshy-tough when fresh, firm and rigid when dry, cinereous to reddish gray, finely tomen- tose to almost glabrous, subzonate or azonate, the margin thin and acute; context whitish or pallid, tough when fresh, soft- corky when dry, 4-8 mm. thick, with a sweet anise-like odor that persists in dried plants; hymenium sometimes separated from the context by a narrow, dark-colored line; tubes less than 4 mm. long, the mouths whitish or somewhat smoke-colored, blackish when bruised, angular, the dissepiments becoming dentate and the mouths unequal in size, averaging 3-4 to a mm.; spores (teste Murrill) white, globose to ovoid, smooth, 5-6 ит diameter. On stumps and trunks, especially of Ulmus. Frequent. The distinguishing characters of this species are the fragrant odor and the unequal and irregular pores—characters which separate it from P. adustus and P. fumosus. The name P. puberula Berk. & Curtis is sometimes applied to this plant. 24. P. fumosus Pers. ex Fries, Syst. Myc. 1:367. 1821. Boletus fumosus Pers. Syn. Fung. 530. 1801. Plants annual, sessile or effused-reflexed; pileus dimidiate, often imbricate, 2-7 x 3-8.5 x 0.3-2 cm., somewhat fleshy- tough when fresh, firm and rigid when dry, grayish to very pale tan-colored, finely tomentose, subzonate or azonate, margin thin and acute; context white to light umber, soft corky when fresh, eorky when dry, 0.3-2 cm. thick, with a rather disagreeable odor; hymenium separated from the context by a distinct, narrow, dark-colored line; tubes short,not more than 3 mm. long, the mouths whitish or smoky, blackish when bruised, circular to somewhat angular but thick-walled and entire, averaging 4—6 to а mm., spores white, smooth, elliptical to subcylindrical, 2.6-4 x 5.3-7.2 и. Growing on dead wood of deciduous trees. October to Decem- ber. Frequent. Distinguished from P. fragrans Peck by the more circular and entire tube mouths and, in our plants at least, by the absence of the fragrant, anise-like odor. The odor is disagreeable in [Vor. 1 104 ANNALS OF THE MISSOURI BOTANICAL GARDEN the fresh plants but disappears on drying. Bresadola ascribes а subanise odor to the plant at times. Тһе plants are, however, closely related and one may expect to find intermediate forms that are difficult to refer to either species. Thin, semi-resupi- nate forms are often scarcely distinguishable from P. adustus Willd. ex Fries. Тһе plant is illustrated by Bresadola (Fungi Trident. pl. 135). 25. P. robiniophila (Murr.) Overholts, n. comb. Trametes robiniophila Murr. N. Am. Flora 9:42. 1907. Plants annual, sessile, rarely imbricate; pileus dimidiate, fleshy-tough or somewhat coriaceous when fresh, firm and rigid when dry, 3.5-10 x 4-15 x 1-4 cm., white to cinereous or yel- lowish, finely tomentose to glabrous, azonate or rarely sub- zonate or concentrically sulcate in large specimens, margin at first thick and obtuse, becoming thin and acute when mature; context white, fleshy-tough when fresh, soft and punky when dry, 0.5-3 em. thick, usually with a sweet anise-like odor devel- oping in herbarium specimens; tubes 0.3-1 cm. long, mouths white, often bay or brownish in dried plants, circular to angular, averaging 4-6 to a mm., the walls thick and entire; spores white, smooth, ovoid to subglobose, 5.5-7 x 7-8.5 y. On deciduous trees, especially Robinia, Celtis, and Acer. August to December. Common. Dried plants are characterized by the tough, punky context and the sweet odor, as well as by the large size of the plant, the long tubes, the minute mouths, and the habitat. The plant was first described as a Trametes but it appears to belong rather to Polyporus. 26. P. betulinus Bull. ex Fries, Syst. Мус. 1:358. 1821. Boletus betulinus Bull. Herb. Fr. pl. 312. 1786. Pileus sessile or attached by a prominent lateral umbo, dimidi- ate to circular in outline, 3-9 x 3-15 x 1-5 cm., somewhat fleshy when young, firm and rigid when dry, glabrous, azonate, smooth, covered with a thin pellicle, margin more or lessincurved, with a wide sterile band on the lower surface; context white, somewhat fleshy when fresh, soft-corky when dry, 1-3.5 em. thick; tubes 3-8 mm. long, mouths white, circular to angular, averaging 3-4 to a mm.; hymenium at times covered by pro- jecting setae, sometimes as much as 2 mm. long; tubes separat- 19141 OVERHOLTS—THE POLYPORACEJE OF OHIO 105 ing in a smooth layer from the context; spores (teste Murrill) white, cylindrical, curved, 4-5 р long. Growing only on Betula. Not common. Always easily recognized by the habitat, the smooth, pellic- ulose surface and the inrolled, broadly sterile margin of the pil- eus. Good illustrations are given by Freeman (Minn. Plant Diseases f. 126), Hard (Mushrooms f. 337), White (Hymen. Conn. pl. 37), and Kellerman (Ohio Myc. Bul. 10: f. 43). 27. P. volvatus Peck, Rept. N. Y. State Mus. 27: 98. 1875. Plants annual, sessile or attached by a stem-like base; pileus globose or compressed-globose in form, 1-5.5 em. broad, 1- 3.5 em. thick, somewhat coriaceous-corky when fresh, hard and firm when dry, somewhat encrusted, whitish or yellowish, some- times tinged with red, glabrous, azonate, margin thick and rounded, extending downward and backward and forming a veil- like covering over the hymenium; context white or light colored, soft-corky, 0.2-1 em. thick; tubes 2-5 mm. long, the mouths whitish to brownish, circular, averaging 3-4 to a mm.; the cover- ing over the hymenium ruptures in from one to three places and allows the escape of the spores; spores (teste Peck) flesh- colored, elliptical, 5 х 7.5-9 и. On dead wood of coniferous trees. Rare. An aberrant form easily recognized by the veil-like covering of the hymenium. This is persistent, being coriaceous in tex- ture and as much as 1 mm. thick. Peck’s illustration (Rept. N. Y. State Mus. 27. pl. 2. f. 3-6) gives some idea as to the general form of the plant; Hard’s (Mushrooms f. 340) is not much better. von Schrenk gives a good illustration (U. 5. Dept. Agr., Div. Veg. Path. Bul. 25: pl. 1. f. 2). 28. P. distortus Schw. ex Fries, Elench. Fung. 1:79. 1828. Boletus distortus Schw. Syn. Fung. Car. 97. 1822. Poly- porus abortivus Peck, Bot. Gaz. 6: 274. 1881. Plants stipitate or substipitate, variable in form and size, sometimes with a distinct, well developed, centrally placed stipe, sometimes the whole plant distorted and the stipe rudi- mentary, often almost the entire surface of such forms covered with the tubes; pileus cireular to irregular in outline, fleshy- tough when fresh, firm and coriaceous when dry, variable in color, whitish, grayish, tan-colored, rufescent, or brownish, vil- [Vor. 1 106 ANNALS OF THE MISSOURI BOTANICAL GARDEN lous-tomentose, soft to the touch, azonate, margin thin and acute or thiek and obtuse; context white or whitish, with a firm corky layer next to the hymenium and a lighter colored, softer layer above, the whole 0.2-1 em. thick; tubes in well developed speci- mens 1-6 mm. long, whitish or rufescent when bruised, mouths angular to dedaloid and irregular, averaging 1-3 to a mm.; stipe central, lateral, or wanting, rarely well developed and up to 6 cm. long, more often rudimentary and tubercular, clothed like the pileus, soft on the outside and firm within; spores white, smooth, subglobose, 5.5-8.5 и in diameter; conidial (?) spores sometimes present, white, smooth, ovoid to elliptical, 3.34.2 x 5.2-7.8 и. Usually growing about stumps and probably always attached to buried wood. Common. Well developed specimens of this plant will be easily recog- nized by the duplex context and the soft, villous pileus; abnor- mal specimens by their distorted appearance. The duplex context is always more easily recognized in dried specimens. According to Lloyd our plant is identical with P. rufescens Fries of Europe. See Lloyd, Syn. Stip. Polyp. f. 458., for illustration of one form of the distorted plant. 29. P. pocula Schw. ex Berk. & Curt. Proc. Am. Acad. Arts Sci. 4:122. 1858. Sphaeria pocula Schw. Jour. Acad. Nat. Sci. Phil. 5:7. 1825. Enslinia pocula Schw. ex Fries, Summ. Veg. Scand. 2:399. 1849. | Pileus short-stipitate, pendant from dead branches, cireular in outline, 1-5 mm. in diameter, 1-3 mm. thick, coriaceous when fresh, rigid when dry, whitish to brown in color, pruinose or mealy, azonate; context coriaceous when fresh, hard when dry, less than 1 mm. thick; tubes not more than 0.5 mm. long, mouths at first appearing pruinose, whitish or brownish, cir- cular, very minute, averaging 5-6 to a mm.; stipe dorsally attached, concolorous with and expanding into the pileus, prui- nose, not more than 5 mm. long; spores (teste Murrill) globose, smooth, hyaline, 4 u in diameter. On dead branches, especially of Quercus and Castanea. Rare. This is the smallest known polypore and easily identified by its size and habit of growth. It was first decribed as an asco- 1914] OVERHOLTS—THE POLYPORACEJE OF OHIO 107 mycete (Sphaeria) by Schweinitz and later transferred to the genus Enslinia (Pyrenomycetes) by Fries. Excellent illustra- tions are given by Lloyd (Мус. Notes, Polyp. Issue 3: f. 369— 70; Syn. Stip. Polyp. f. 443). 30. P. brumalis Pers. ex Fries, Syst. Мус. 1: 348. 1821. Boletus brumalis Pers. Neues Mag. Bot. 1:107. 1794. Pileus stipitate, circular in outline, sometimes somewhat um- bilieate in the center, 1.5-5 cm. broad, 0.2-0.4 em. thick, fleshy-tough when fresh, rigid when dry, varying in color from yellowish brown to dark brown or almost black, minutely hispid to glabrous, rarely slightly squamulose, usually azonate but at times distinctly zoned, margin thin and entire, involute when young and incurved on drying; context white or pallid, soft-fibrous when fresh, firm when dry, 2 mm. or less thick; tubes 1-3 mm. long, usually slightly decurrent, the mouths white or whitish, at first circular and thick walled, later angu- lar and the dissepiments thinner, averaging 2-3 to a mm.; stipe central or subcentral, simple, cylindrical, grayish or brown- ish, minutely hispid or glabrous, 2-3 em. long, 0.2-0.3 em. thick; spores white, oblong, sometimes slightly curved at one end, smooth, 2.5 x 9 и. Growing on dead wood in the fall and early winter. Common. P. brumalis and P. arcularius are closely related species that are not always easy to separate. In general the forms occur- ring in the early spring and summer are likely to be P. arcularius, while those found in autumn and often late in winter are more likely to be P. brumalis. Hard (Mushrooms f. 335) gives a good illustration of the plant. 31. Р. агсшагіиѕ Batsch. ex Fries, Syst. Myc. 1: 342. 1821. Boletus arcularius Batsch. El. Fung. 97. 1783. P.arcular- iformis Murrill, Torreya 4:151. 1904. Pileus stipitate, circular in outline, convex to umbilicate, sometimes infundibuliform, 1-8 cm. broad, 1-4 mm. thick, fleshy-tough or coriaceous when fresh, rigid when dry, golden brown to dark brown, usually more or less squamulose, azonate, the margin usually distinctly ciliate, involute on drying; con- text white or pallid, fibrous-fleshy when fresh, compact-fibrous when dry, less than 2 mm. thick; tubes 1-2 mm. long, often decurrent, the mouths white, discolored on drying, angular and [Vor. 1 108 ANNALS OF THE MISSOURI BOTANICAL GARDEN often radially elongate, averaging 2 to a mm. in transverse direction and about 1 to a mm. in axial direction; stipe central or subcentral, concolorous with the pileus, fuscous-squamulose to glabrous above, often hispid at the base, 2-6 em. long, 2-4 mm. thick; spores white, smooth, elliptical-cylindrical, usually 2-3 guttulate, 2-3 x 6-8.5 y. On dead wood. Common. This species is much more common than the preceding and is distinguished from it by the lighter colored pileus, the ciliate margin, the hispid stipe base, and the larger and more alveolar tubes. Is is usually found in the spring and early summer. A small form of it with the pileus not more than 1 em. in diameter is especially common on twigs and bits of wood during the late spring and early summer. Murrill regards this form as a dis- tinct species and has named it P. arculariformis (Torreya 4: 151). It is here maintained as a form of P. arcularius. This spe- cies is well represented by Hard (Mushrooms f. 336). 32. P. pennsylvanicus Sumstine, Jour. Myc. 13: 137. 1907. Pileus stipitate, circular in outline, depressed, sometimes umbilicate or somewhat infundibuliform, 4-6.5 cm. in diameter, 0.2-0.5 em. thick, fleshy-tough, pale tan or ochraceous buff in color, with a thin cuticle, glabrous, azonate, margin thin and acute; context white, soft and watery when fresh, with a sweet acid odor, rather fragile when dry, 2-4 mm. thick; tubes white at first, discolored on drying, long decurrent on the stipe, 2-4 mm. long, mouths angular, thin walled, large, somewhat longer in the radial direction, 1-2 mm. long, 0.5 to 1 mm. wide; stipe central or excentric, whitish, glabrous, expanding above, 2-3 cm. long, 0.4-1 cm. thick; spores white, smooth, oblong-ellip- tical or fusoid, 4.2-5.7 x 10-14 и, often once to several times guttulate. Growing on old logs in July and August. Frequent. The above description is drawn from notes and specimens from two collections made at Oxford, Ohio, one in August, 1910, and the other in July, 1911. The odor of the fresh plant is described by the author as “nitrous”. Тһе large angular pores ally the species with P. arcularius Batsch. ex Fries and with Favolus canadensis Klotzsch. From the former it is eas- Пу separated by the much larger spores and from the latter by mE Ve акты. Re PL Men v 1914] OVERHOLTS—THE POLYPORACEJE OF OHIO 109 the well developed stipe with the decurrent tubes, the usually umbilicate pileus, and the friable context when dry. Possibly it should be referred to P. Rostkowii Fr. or to P. pallidus Schulz. & Kalchbr., both of which some regard as being small scaleless forms of P. squamosus Huds. ex Fries. Тһе spores agree well with those of P. squamosus, but although it can be shown to be related to that species, it is worthy of a distinct name. 33. P. squamosus Huds. ex Fries, Syst. Myc. 1:343. 1821. Boletus squamosus Huds. Fl. Angl. 626. 1798. [2nd ed.] Pileus short-stipitate or almost sessile, dimidiate to reniform in outline, 6-25 cm. in diameter, 0.5-4 cm. thick, fleshy when fresh, firm and rigid when dry, whitish to dingy yellowish or brownish, clothed, especially toward the center, with large, ap- pressed, brownish scales often concentrically arranged, azonate, margin thin and acute; context white, tough, soft-corky when dry, 0.5-3.5 em. thick; tubes 2-8 mm. long, decurrent, the mouths white or yellowish, large and angular, 1-2.5 mm. in diameter; stipe lateral, often rudimentary, black at the base, reticulate above by the decurrent pores, 1-5 cm. long, 1 cm. or more thick. Growing on injured or diseased deciduous trees. Rare. Lloyd gives the spores as ‘‘oblong, 5-6 x 12-15 u, hyaline, smooth.” Easily recognized by the large pores and the large, appressed, brownish scales. The plant is well illustrated by Bresadola (Fung. Trident. pl. 133), Freeman (Minn. РІ. Diseas. f. 125), Lloyd (Photograph. pl. 5), and Hard (Mushrooms f. 325). 34. P. picipes Fries, Syst. Myc. 1:353. 1821. P. fissus Berk. Hooker's Lond. Jour. Bot. 6:318. 1847. Pileus stipitate, circular to reniform in outline, convex or plane, when older usually becoming depressed or somewhat infundibuliform, 4-20 ста. broad, 0.1-0.8 em. thick, tough and leathery when fresh, very rigid and brittle when dry, sometimes yellowish brown but usually dark chestnut-brown to reddish brown, usually lighter in color towards the margin, azonate, margin very thin, usually wavy and often lobed; context white to somewhat ochraceous, leathery when fresh, firm when dry, 1-7 mm. thick; tubes not more than 2 mm. long, decurrent on [Vor. 1 110 ANNALS OF THE MISSOURI BOTANICAL GARDEN the stipe, the mouths white to brownish in color, circular to angular, very minute, invisible to the unaided eye, averaging 5-7 to а mm.; stipe central to lateral, dark brown or black on the lower half, glabrous, 1-6 em. long, 0.4-1.5 em. thick. On stumps and logs late in autumn. Common. The combination of black stipe base and minute pores char- acterizes this and the next species. The two are separated mainly on point of size. Murrill describes this plant under the name of P. fissus Berk., which was originally described from specimens collected in Ohio. Patouillard (Tab. Fung. No. 136) says the spores are ovoid. Lloyd now considers this plant to be a form of P. varius Fries, of Europe. A good illustration of our plant will be found in Hard, Mushrooms f. 319. 35. P. elegans Bull. ex Fries, Epicr. Syst. Myc. 440. 1838. Boletus elegans Bull. Herb. Fr. pl. 26. 1780. Pileus stipitate, circular to reniform in outline, convexo- plane or depressed, 1,5-7 cm. in diameter, 0.2-1 em. thick, leathery when fresh, rigid and firm when dry, pale ochraceous to dull orange-color, pruinose to glabrous, azonate, the margin rather thin, often radiate-striate, even or undulate; context white to light ochraceous, tough when fresh, soft corky when dry, 1-6 mm. thiek; tubes 1-3 mm. long, decurrent on the stipe, the mouth whitish to pallid, circular to angular, averaging 4-5 to a mm.; stipe central, excentrie or lateral, slender, black at the base, light colored above, pruinose or glabrous, 1-8 ст. long, 0.2-0.6 em. thick. On dead wood late in autumn. Not common. Spores were not obtained from the writer's specimens. Mur- rill gives them as “oblong, smooth, hyaline, 7-8 x 3-3.5y.” The species is closely related to P. picipes Fries but is separated from it by the smaller size and the uniform ochraceous color of the pileus that never takes on the darker chestnut shades assumed by P. picipes. Bulliard (Herb. Fr. pl. 124) gives an excellent illustration of the plant under the name of Boletus nummularius Bull. 36. P. radicatus Schw. Trans. Am. Phil. Soc. П. 4: 155. 1832. P. Morgani Peck, Ann. Rept. N. Y. State Mus. 32:34. 1879. Pileus stipitate, circular in outline, 3.5-20 em. broad, 0.3-0.8 ——— 2 aan 1914] OVERHOLTS—THE POLYPORACEJE OF OHIO 111 em. thick, fleshy or fleshy-tough when fresh, more or less friable when dry, yellowish brown or darker, finely tomentose or fibril- lose-scaly, often becoming glabrous, azonate; margin thin and acute, often involute on drying; context white or light yellow- ish, soft and spongy, 2-6 mm. thick; tubes 1-5 mm. long, decurrent on the stipe, the mouths white or brownish on drying, circular to angular and irregular, averaging 2-3 to а mm.; stipe central, simple or rarely branching once or twice, yellowish or brownish, prolonged below into a long, black, rooting base, velvety or rough-squamulose above, 6-15 cm. long, 0.5 to 2 cm. thick; spores white, smooth, ovoid-elliptical, 6-8 x 12-15 y. Growing on the ground, sometimes around stumps, and prob- ably attached to buried wood. Common. This plant is always easily recognized by the black and radi- cating base of the stem. The type specimens of P. Morgani Peck were collected іп Ohio by Morgan. For illustrations see Hard, Mushrooms f. 329., Lloyd, Syn. Sec. Ovinus f. 508; Syn. Stip. Polyp. f. 465., and Ohio Myc. Bull. 11: f. 46. 37. P. flavovirens Berk. & Curt. Grev. 1:38. 1872. Pileus stipitate, circular to irregular in outline, 4-10 cm. broad, 0.3-0.8 ст. thick, soft and fleshy when fresh, rigid but friable when dry, yellowish green or yellowish brown in color, the surface often cracked and areolate and the flesh showing yellowish in the cracks, slightly tomentose or glabrous, azonate, the margin thin and acute; context white or yellow, fleshy when fresh, soft and friable when dry, 1-4 mm. thick; tubes 1-5 mm. long, decurrent on the stipe, the mouths white or yellowish, sometimes reddish on drying, circular to angular, averaging 1-3 to а mm.; stipe simple or branched, usually excentrie but some- times central, often irregular in form, whitish or yellowish in color, 3-6 cm. long, 1-1.5 ст. thick; spores white, smooth, globose, ог subglobose, 3—4.7 шіп diameter. Growing on the ground in deciduous woods. Frequent in July and August. А species easily recognized by the color of the pileus. Тһе plant is fairly well represented by Hard (Mushrooms f. 327), and by Lloyd (Syn. Sect. Ovinus f. 501). According to Lloyd P. cristatus Pers. of Europe is not different from our plant, Murrill lists it under the name of Grifola poripes Fries ex Murr, 8 [Vor. 1 112 ANNALS OF THE MISSOURI BOTANICAL GARDEN 38. P. umbellatus Pers. ex Fries, Syst. Myc. 1:354. 1821. Boletus umbellatus Pers. Syn. Fung. 519. 1801. Plants stipitate, 7-20 cm. in diameter, the stipe branching repeatedly and giving rise to many centrally attached pileoli which are circular in outline, 1-4 ст. broad, less than 5 mm. thiek, fleshy in texture when fresh, rigid when dry, whitish to smoky brown in color, fibrillose or glabrous, azonate; mar- gin thin, acute, entire; context white, fleshy or fleshy-tough, rather brittle when dry, usually not more than 1 mm. thick; tubes less than 2 mm. long, decurrent on the stipe branches, the mouths white, angular, averaging 2-4 to a mm.; stipe compound, the branches cylindrical in form, central or subcentral, white, usually entirely covered with the decurrent tubes; spores white, oblong-elliptic, smooth, 2.3-3.5 x 7-9.4 y. Growing about the bases of stumps or trees, especially of Quercus. Rare. Easily distinguished from its allies by the more regular and cylindrical stipe branches, the small and centrally attached pilei which are more or less circular in outline, and by the ob- long-elliptic spores. Murrill describes it as Grifola ramosissima Scop. ex Murr. The plant is well illustrated by Lloyd (Syn. Stip. Polyp. f. 450), Hard (Mushrooms f. 320), and Atkinson (Mushrooms f. 183). 39. P. frondosus Dicks. ex Fries, Syst. Myc. 1:355. 1821. Boletus frondosus Dickson, Fasc. Pl. Crypt. Brit. 1: 18. 1785. Plant stipitate, the stipe many times branching and giving rise to. numerous overlapping pileoli, the whole plant forming a more or less globose mass often as much as 40 cm. in diam- eter; pileoli flabelliform or spathulate in outline, 2-7 cm. broad, 2-5 mm. thick, fleshy-tough when fresh, rigid when dry, grayish to mouse-colored, glabrous or minutely tomentose, azonate, the margin thin and acute; context white or whitish, fleshy-tough when fresh, fragile when dry, not more than 2 mm. thick; tubes 2-3 mm. long, decurrent оп the stipe, the mouths white, angular or irregular, averaging 1-3 to a mm.; stipe compound, short and thick; spores white, smooth, ovoid to elliptical, 4.5-6 x 6-9 u. Usually found at the bases of trees or stumps, preferably of Quercus and Ulmus. Common in late fall. 1914) OVERHOLTS—THE POLYPORACE OF OHIO 113 From P. Berkeleyi Fries and P. giganteus Fries this species is separated by the numerous small pileoli which in those species are large and few in number. Тһе irregular stipe-branches and the more spathulate pileoli separate it from Р. umbellatus Fries in which the stipe branches are cylindrical and the pileoli centrally attached and consequently more nearly circular in outline. The plant is illustrated in Atkinson, Mushrooms f. 181-82., Hard, Mushrooms f. 321., and МеПуаіпе, Am. Fungi pl. 128. 40. P. giganteus Pers.ex Fries, Syst. Myc. 1: 356. 1821. Boletus giganteus Pers. Syn. Fung. 521. 1801. Grifola Sums- tinei Murrill, Bull. Torr. Club 31: 335. 1904. Plants composed of а few broad pileoli, 6-15 em. in diameter and less than 0.5 em. thick, dimidiate to flabelliform or spathu- late in outline, fleshy-fibrous when fresh, more rigid when dry, grayish to brown, often black when dried—especially on the margin—,usually somewhat tomentose or fibrillose, azonate or subzonate, margin very thin and acute, often lobed, involute on drying; context white, fibrous, tough, 1-3 mm. thick; tubes 1-3 mm. long, at first white but blackish where bruised and on drying, the mouth angular to irregular, often torn, averaging 5-7 to а mm.; stipe short and thick; spores white, smooth, globose, 4-6 д broad. Growing on the ground around stumps. Frequent. Separated from P. Berkeleyi Fries by the smooth spores; from P. umbellatus Pers. ex Fries, and P. frondosus Fries, by the much larger and fewer pileoli, and distinct from all of these in the blackening of the margin or of the entire pileus and hymenium when bruised or in drying. In the ‘North American Flora’ it is described under the name of Grifola Sumstinei Murr. In this country it has always been held to be the same as the European plant P. giganteus Fries, and European specimens recently received from Bresadola confirm this view. А very good illustration will be found in Bresadola, Fungi Tridenti pl. 134., and in Boudier, Іс. Myc. 1: pl. 153. То the writer's knowledge it has not been illustrated in American mycology. 41. P. Berkeleyi Fries, Nov. Sym. 40. 1851. P. апат Berk. Grev. 12: 37. 1882. Pileus stipitate, the stipe sometimes branching and giving [Vor. 1 114 ANNALS OF THE MISSOURI BOTANICAL GARDEN rise to from 2 to 4 pileoli, sometimes simple with but one large pileus; pileoli fleshy-tough when fresh, becoming rigid on drying, more or less cireular in outline, 6-15 сіп. broad, 0.3-1.5 em. thick, light colored, whitish to yellowish, slightly tomentose or glabrous, azonate or obscurely zoned; margin rather thin, often lobed; context white, fleshy-tough, fragile when dry, 0.3-2 cm. thick; tubes 2-8 mm. long, decurrent on the stipe; mouths white or whitish, large and irregular, averaging 0.5-2 mm. in diameter; stipe short and thick, more or less tubercular, whitish in color, 4-7 em. long, 3-5 cm. thick; spores white, minutely echinulate, globose, 5.6-8.4 шіп diameter. Growing at the bases of trees and stumps, especially of Quercus. Frequent. This is one of the largest of our species and is easily distin- guished from all of its allies by the echinulate spores. Morgan’s description of P. апат Berk. applies to P. frondosus Fries and not to P. Berkeleyi for which Р. апат is a synonym. (See Lloyd, Mycological Notes 27: 341-342.) Тһе plant is well represented by the following illustrations: Lloyd, Photogr. pl. 9-10; Мус. Notes Polyp. Iss. 3: f. 362-63., and Hard, Mushrooms f. 323 and рі. 45. 42. P. sulphureus Bull. ex Fries, Syst. Myc. 1: 357. 1821. Boletus sulphureus Bul. Herb. Fr. pl. 429. 1788. P. cincinnatus Morgan, Jour. Cine. бос. Nat. Hist. 8: 97. 1885. Plants annual, often attenuate at the base and appearing substipitate, imbricate; pileus dimidiate to flabelliform in outline, 5-20 x 4-12 x 0.5-2.5 cm., fleshy and watery when young, becoming firm when old, yellowish to bright orange- colored, sometimes fading with age, finely tomentose to gla- brous, azonate or with broad colored zones, the margin thin and acute, sometimes lobed; context white or light yellow, fleshy when fresh, rather soft and friable when dry, 0.4-2 cm. thick; tubes 1-4 mm. long, the mouths bright sulphur-yellow, sometimes whitish or dull yellow with age or on drying, angular, averaging 2-4 to a mm.; spores white, smooth, ovoid to sub- globose, 4-5 x 5.5-7 и. Growing оп trunks and stumps of deciduous trees. Common. Specimens usually change color on drying and most of the red color of the pileus is lost. Тһе bright yellow of the hyme- eo 1914] OVERHOLTS—THE POLYPORACEZ OF OHIO 115 nium may or may not persist. The best colored representation of the fungus is that given by Rostkowius in Sturm, Deutschl. Flora 4: pl. 20. The plant is widely distributed and well known and has figured largely in American mycology. The following illustrations will aid in determinations: Atkinson, Mushrooms f. 184-85., Duggar, Fung. Dis. Pl. f. 226., Hard, Mushrooms f. 326., and von Schrenk, U. S. Dept. Agr., Div. Veg. Path. Bul. 25: pl. 11. f. 1-4. 43. P. Pilotae Schw. Trans. Am. Phil. бос. II. 4: 157. 1832. P. hypococcineus Berk. Lond. Jour. Bot. 6:319. 1847. Plants annual, sessile; pileus dimidiate, often subungulate, 5-12 x 6-15 x 1-5 cm., soft coriaceous or corky, buff or orange- colored, becoming whitish on drying, minutely tomentose or glabrous, azonate, margin usually obtuse; context pale buff, becoming carneous when dry, fibrous, sometimes very hard when dry, strongly zonate, 0.7-2 cm. thick; tubes 0.5-2 cm. long, the mouths orange-colored, becoming brownish on drying, angular, averaging 3-5 to а mm.; spores (teste Murrill) smooth, hyaline, 3-4 x 2-3 u. On dead wood of Quercus and Castanea. Rare. Easily distinguished from other species with a predominance of red or orange colors by the thick pileus and the long tubes. The plant is said to emit a strong odor when growing. The type specimens of P. hypococcineus Berk. were collected in Ohio by Lea. Р. castanophilus Atk., described from North Carolina, is said to be the same plant. 44. P. sanguineus L. ex Fries, Syst. Myc. і: 371. 1821. Boletus sanguineus L. Sp. Plant. 1646. 1762. [2nd ed.] Plants annual, sessile; pileus dimidiate to flabelliform, 2-5 x 2-8 x 0.2-0.5 cm., coriaceous, bright red, finely tomentose to glabrous, often zonate, the margin very thin and acute; context red or yellowish red, soft and floccose, scarcely more than 2 mm. thick; tubes 0.5-1.5 mm. long, the mouths red, more or less angular or cireular when young, averaging 2-4 to a mm.; pileus often attached by an attenuate base and then appearing substipitate. On dead wood of deciduous trees. September to December. Rare. [Vor. 1 116 ANNALS OF THE MISSOURI BOTANICAL GARDEN The species is distinguished from the following one by the much thinner pileus and the marked tendency to appear sub- stipitate. Otherwise it scarcely differs, and intermediate forms are found that are difficult to place satisfactorily It is usually considered to be a southern species, but Hard reports finding it in Ohio. His specimens were determined by Peck. 45. P. cinnabarinus Jacq. ex Fries, Syst. Myc. 1:371. 1821. Boletus cinnabarinus Jacq. Fl. Austr. 4:2. 1770. Plants annual, rarely reviving, sessile or effused-reflexed; pileus dimidiate or reniform, 2-6 x 2-10 x 0.5-2 cm., tough and leathery when fresh, more rigid when dry, orange-colored to cinnabar-red, often becoming paler or almost white with age, compactly tomentose or glabrous, usually azonate, margin thin or thick, acute; context red or yellowish red, floccose- fibrous to soft-corky, always zonate, 0.4-1.5 em. thick; tubes 1-4 mm. long, the mouths cinnabar-red, circular then angular and sometimes somewhat sinuous, averaging 2-4 to а mm.; spores white, smooth, oblong, 2-2.5 x 4.5-5.5 и. On dead wood of all kinds. September to December. Com- mon. The prevailing deep red color of both pileus and hymenium separates this species from all others of the genus except P. sanguineus Fries, from which this species differs only in being thicker and in having the context more strongly zoned. Р. cinnabarinus is à northern species and much more common in Ohio than is P. sanguineus. 46. P. resinosus Schrad. ex Fries, Syst. Myc. 1:361. 1821. Boletus resinosus Schrad. Spic. Fl. Ger. 171. 1794. Plants annual, sessile or decurrent, more or less imbricate; pileus dimidiate, 5-15 x 7-25 x 0.8-2.5 cm., somewhat fleshy and full of water when young, firmer when mature and soft- corky on drying, velvety-tomentose to glabrous, sulcate or with a few broad, colored zones, margin at first thick and somewhat obtuse, becoming thinner and acute; context pallid to light brown, fleshy and watery when young, soft-corky when dry, 0.5-2 ст. thick; tubes 1-6 mm. long, the mouths white to pallid, changing to a darker color on drying, circular to angular, averaging 4-6 to а mm.; spores white, smooth, cylindrical, curved, 1.2-2 x 5-6.3 y. 1914) OVERHOLTS—THE POLYPORACEJE OF OHIO 117 On old logs and stumps in October and November. Common. Distinguished by the brown pileus and the light brown, almost whitish context. For illustration see Hard, Mush- rooms f. 881. 47. P. nidulans Fries, Syst. Myc. 1:362. 1821. Plants sessile or effused-reflexed; pileus dimidiate, 1.5-6 x 2-8 x 0.5-2 cm., very soft, spongy, and full of water when fresh, firm and friable when dry, umber to cinnamon or tawny brown, finely villous-tomentose to glabrous, azonate, margin thin and acute, purplish or reddish where bruised; context con- colorous with the pileus, sometimes with a darker layer next to the hymenium, soft and watery when fresh, cheesy and friable when dry, 2-8 mm. thick; tubes 2-7 mm. long, mouths hoary when young, yellowish or reddish brown when mature, angular or sinuous, averaging 3-4 to a mm.; spores white, smooth, globose ог subglobose, 2-3.5 шіп diameter. On dead wood of deciduous trees, especially Quercus. June to September. Not common. Distinguished by the uniform umber brown color of the whole plant, the soft and watery context, etc. 48. P. gilvus Schw. ex Fries, Elench. Fung. 1: 104. 1828. Boletus gilvus Schw. Syn. Fung. Car. 96. 1822. Plants annual or reviving for two or three years, sessile or effused-reflexed, often imbricate; pileus dimidiate, 1-7 x 2-12 x 0.2-2 ста., leathery or corky when fresh, woody and rigid when dry, yellowish brown or reddish brown, in very young stages covered by a purplish, villous pubescence, otherwise glabrous, usually rough, more or less zonate, margin thin and acute; context yellowish brown, soft-corky to woody, 0.1-1.3 cm. thick; tubes 1-5 mm. long, the mouths reddish brown or darker, circular, then angular, averaging 6-8 to а mm., the walls rather thick and entire; spores white, smooth, oblong-ellipsoid, 3-4 X 5-6 м. On dead wood of all kinds. July to December. Common. Closely related to P. radiatus Sow. ex Fries and P. cuticularis Bull ex Fries, but distinet in the white spores, the lighter colored surface and the more woody context. Р. isidiodes Sehw. as reported by Lea belongs here. [Vor. 1 118 ANNALS OF THE MISSOURI BOTANICAL GARDEN 49. P. radiatus Sow. ex Fries, Syst. Myc. т: 369. 1821. Boletus radiatus Sow. Eng. Fungi pl. 196. 1799. Plants annual, sessile or decurrent; pileus dimidiate or flabel- liform and attached by an attenuate base, 2-5 x 2-7 x 0.3-2 cm., firm and rigid, yellowish brown or rust-colored, velvety to glabrous, sometimes conspicuously zonate, sometimes azonate, margin thin or thick, acute; context yellowish to rusty brown, corky and somewhat friable, 2-5 mm. thick; tubes 1-8 mm. long, the mouths grayish umber to rusty red, circular, then angular, averaging 4-5 to a mm.; spores (teste Bresadola) yellowish, elliptical, 3.5—4.5 х 5.5-6.5 и. Growing commonly on Betula and Alnus. Rare. A species intermediate between P. gilvus Schw. ex Fries, and P. cuticularis Bull. ex Fries, distinguished from the former by the habitat, the brighter color and the smoother surface of the pileus, and by the colored spores, and from the latter chiefly in the habitat. Тһе species was reported from Ohio by Lea but I have not examined the plants. 50. P. cuticularis Bull. ex Fries, Syst. Myc. 1:363. 1821. Boletus cuticularis Bull. Herb. Fr. pl. 462. 1809. Plants annual, sessile, often imbricate; pileus dimidiate or flabelliform and attached by an attenuate base, 3-7 x 3.5-10 X 0.3-1 em., spongy and fleshy-tough when fresh, leathery to rigid when dry, yellowish brown to rusty brown, compact wooly-tomentose, becoming fibrillose or almost glabrous, some- times subzonate on the margin, margin thin, acute, often inflexed; context yellowish brown or rust-colored, tough and watery when fresh, distinctly fibrous, 2-7 mm. thick; tubes 2-7 mm. long, the mouths hoary brown to rust-colored, angular, averaging 3-5 to а mm.; spores yellowish brown, smooth, subglobose to broadly elliptical, 4.2—5.7 x 5.5-7 и. On dead wood of deciduous trees. August to November. Common. This species is very closely related to P. radiatus Sow. ex Fries, but Ohio plants may be distinguished from that species _ by the habitat, the thicker and larger pileus, and by the more tomentose and spongy surface. Р. perplexus Peck, the types of which have been destroyed, is thought by some to be this species and our plants are frequently referred to it. 1914) OVERHOLTS—THE POLYPORACEZ OF OHIO 119 ңі. P. hispidus Bull. ex Fries, Syst. Myc. 1:362. 1821. Boletus hispidus Bull. Herb. Fr. pl. 210. 1791. Polyporus endocrocinus Berk. Hooker's Lond. Jour. Bot. 6:320. 1847. Plants annual, sessile, sometimes imbricate; pileus dimidiate, 6-20 x 9-25 x 2-6 cm., spongy and watery when fresh, firm and rigid when dry, yellowish brown to rusty red, soft from the covering of the dense hirsute or hispid tomentum or pubescence, azonate, margin thick or thin, obtuse or acute; context usually light yellowish brown above and dark reddish brown next to the hymenium, fibrous, firm when dry, 1-5 em. thick; tubes 0.6-1.5 em. long, mouths yellowish brown becoming darker where bruised, cireular, then angular, averaging 2-4 to а mm.; spores yellowish brown, smooth, broadly ovoid to ellipsoid, 6.5-7 x 7-9.5 и. On living trunks of deciduous trees. September to December. Rare. Much larger than P. cuticularis Bull. ex Fries, and P. radiatus Sow. ex Fries, and expecially distinet by the hirsute or hispid pubescence. Іп point of size it more nearly approaches р» dryadeus Pers. ex Fries, and P. dryophilus Berk., but easily dis- tinguished from them by the pubescence. 32. P. dryadeus Pers. ex Fries, Syst. Myc. 1: 374. 1821. Boletus dryadeus Pers. Obs. Myc. 3. 1799. Plants sessile; pileus dimidiate, applanate, 6-30 x 8-35 x 2-6 em., spongy and watery when fresh, more or less corky or woody when dry, grayish brown to dark brown or black in old specimens, glabrous, azonate, margin thick and obtuse, distilling drops of water when young and growing; context umber-brown to rust-colored, subshining when dry, soft and watery, corky or woody on drying, 1.5-4 cm. thick; tubes 0.3-2 cm. long, mouths grayish brown, darker on drying, circular, then angular, averaging 3-5 to a mm.; spores (teste Bresadola) globose or subangular, smooth, yellowish, 8-9 x 7-8 y. On living trunks of Quercus. September to November. Rare. Very closely related to P. dryophilus Berk., and probably the two have been confused in this country. Р. dryadeus is usually considered to be a more applanate form and much larger than P. dryophilus. There is also said to be a decided difference [Vor. 1 120 ANNALS OF THE MISSOURI BOTANICAL GARDEN in spore color in the two plants, P. dryadeus having much paler spores than P. dryophilus, but for this I cannot vouch. So far as known, P. dryadeus has not been collected in Ohio but the species has been reported from Michigan and Kentucky. Lloyd (Мус. Notes 36. f. 383) gives an illustration. 53. P. dryophilus Berk. Hooker's Lond. Jour. Bot. 6:321. 1847. Plants annual, sessile; pileus dimidiate, often ungulate, 3-12 x 7-20 x 1-10 ст., rather rigid, grayish brown, to reddish brown, scabrous with an innate, ferruginous pubescence, azo- nate or subzonate, margin thick and obtuse; context cinnamon or rusty brown, subshining, corky to hard and woody; tubes 0.3-2.5 em. long, ferruginous-yellow within, the mouths cin- namon-brown, angular, averaging 2-3 to а mm.; spores ferru- ginous, smooth, ellipsoid to subglobose, 5 x 6.5 y. On living Quercus and on logs. August to November. Rare. This species was originally described from specimens collected at Waynesville, Ohio, by Lea. To the description as given in Lea’s catalogue the following note was added: “Nearly allied to Polyporus dryadeus, but a smaller, more rigid species with larger, differently colored pores. It has also much resemblance - to P. gilvus." 54. P. Schweinitzii Fries, Syst. Мус. 1:351. 1821. Plants stipitate or sessile; pileus circular to dimidiate, 5-15 cm. broad, 0.5-1.5 cm. thick, spongy to soft-corky when fresh, firm, rigid, and sometimes friable when dry, ochraceous to orange-colored or brown in mature specimens, strigose-tomen- tose to almost glabrous, usually more or less zonate, margin thin or thick, acute; context yellowish to reddish brown, spongy when fresh, usually friable when dry, 0.2-1 em. thick; tubes 1-6 mm. long, the mouths yellowish, darker when bruised and sometimes dark brown on drying, circular to angular and soon irregular, averaging 1-3 to a mm.; stipe present and well developed or entirely absent, central or excentrie, agreeing in color, pubescence and consistency with the pileus, 0-6 cm. long, 1-2 сш. thick; spores (teste Lloyd) white, elliptical, smooth, 4 x 6 y. Growing on or about Pinus. Autumn. Rare. This species is а very variable one, yet quite distinct in habi- 1914] OVERHOLTS—THE POLYPORACEJE OF OHIO 121 tat, consistency, pubescence, color, etc. It is known in Ohio only from a collection made at Cincinnati (now in the Lloyd Museum) by Mr. Wm. Holden. For illustrations see Lloyd, Мус. Notes, Polyp. Issue 1: f. 208., aud von Schrenk, U. S. Dept. Agr., Div. Veg. Path. Bul. 25: pl. 1. f. 1., pl. 2. 55. P. circinatus Fries, Monogr. Hymen. Suec. 2:268. 1863. Pileus stipitate or substipitate, circular to spathulate or flabelliform, convex to depressed, 3-9 em. broad, 0.3-1 cm. thick, rather soft when fresh, firm when dry, yellowish to umber-brown, tomentose to velvety, azonate ог subzonate, margin rather thin, acute; context yellowish to cinnamon- brown, duplex, soft and spongy above, firm next to the tubes, 1-6 mm. thick; tubes 1.5-4 mm. long, the mouths whitish to cinnamon, subcircular to angular, averaging 2-4 to а mm.; stipe sometimes rudimentary, usually lateral or excentric, fulvous to dark brown, tomentose, soft, up to 5 cm. long, 0.5-1.5 em. thick; spores (teste Lloyd) pale color, 3 x 5 в. In coniferous and deciduous woods. The species has not been reported from Ohio. It is distin- guished by the duplex character of the context and by the poor development of a stipe. It is a question whether it is distinct from P. tomentosus Fr. Certainly P. dualis Peck is the same plant. Lloyd regards American plants in which the context is always duplex as belonging under P. circinatus Fries, and European plants with a uniform context as P. tomentosus Fries. The plant is illustrated by Lloyd (Myc. Notes Polyp. Issue f. 198-99). 56. P. obesus (Ellis & Ev.) Overholts, n. comb. Polystictus obesus Ellis & Ev. Bull. Torr. Bot. Club 24: 125. 1897. ' Stipitate. Stipe central, spongy, velutinous, dark cinnamon, 4-6 em. high, 0.5-1.5 em. thick above, enlarged below to 1-3 em.; pileus convex then depressed in the center, obconical at first with the margin obtuse, then spreading out with the margin acute, color lighter than that of the stipe, yellowish cinnamon, surface uneven, subcolliculose, not zonate, 4-6 cm. across; pores irregular, short (1 mm.), at first round with margins thick, finally irregular and subsinuous, 0.5-1 mm. across, margins acute; spores elliptical, ferruginous, 7-8 x 4-5 р. [Vor. 1 122 ANNALS OF THE MISSOURI BOTANICAL GARDEN On the ground, in contact with and partly attached to decay- ing pine limbs partly buried in the soil. (Тһе above description is according to Ellis and Everhardt, Bull. Torr. Bot. Club. 24: 125. 1897.) Distinguished from the next three species by the greater thickness of the pileus and stipe. From P. circinatus Fries, it is separated by the absence of a duplex context and by the slightly smaller pores. The plant is recorded by Morgan as P. Montagne Fries, but according to Lloyd the record is based on plants collected in Canada by Dearness. It is listed in Lea’s catalogue under the same name. 57. P. focicola Berk. & Curt. Jour. Linn. Soc. Bot. ro: 305. 1808. Pileus stipitate, circular in outline, convex-depressed to umbilieate, 2-4 cm. broad, 1-6 mm. thick, coriaceous when fresh, rigid when dry, grayish brown to cinnamon, finely tomen- tose, striate, zonate, margin thin and acute; context cinnamon- brown, fibrous, less than 0.5 mm. thick; tubes 1-6 mm. long, the mouths angular or irregular, cinnamon to rusty brown, averaging 1 mm. or more in diameter; stipe central, light to dark brown, minutely velvety, 1.5-3 em. long, 2-4 mm. thick 4 spores (teste Lloyd) pale colored, smooth, elliptical, 5 x 10 y. On burned earth in woods. July to November. Rare. The species differs from P. perennis L. ex Fries only in the much larger pores. Тһе plants were reported by Lea as P. connatus Schw. and by Morgan as P. parvulus Klotzsch. Тһе plant is well illustrated by Lloyd (Myc. Notes Polyp. Issue 1: f. 208-4). 58. P. perennis І. ex Fries, Syst. Myc. 1:350. 1821. Boletus perennis L. Sp. Plant. 1177. 1753. Pileus stipitate, cireular in outline, convex-depressed to umbilieate, 1.5-7 cm. broad, 1-3 mm. thick, coriaceous, rigid when dry, grayish brown to cinnamon or rust-colored but never silky, finely tomentose, zonate, margin thin and acute ; context cinnamon-brown, fibrous, less than 1 mm. thick; tubes 1-2.5 mm. long, the mouths grayish to cinnamon, angular, averaging 2-4 to a mm.; stipe central or subcentral, cylindrical, con- colorous with the pileus, velvety, 1.5-5 cm. long, 1-6 mm. thick; spores (teste Lloyd) pale colored, 4-5 x 8-10 ,. 1914] OVERHOLTS—THE POLYPORACE OF OHIO 123 Growing on burned earth. July to November. Not common. Тһе plant closely resembles the next species but is separated from it by the habitat and the dull cinnamon or cinnamon-gray color of the zonate pileus. Polystictus proliferus Lloyd is said by its author to be a form of this species. It was collected near Cleveland. This species is illustrated by Atkinson (Mush- rooms f. 187), Hard (Mushrooms f. 346), and Lloyd (Муе. Notes Polyp. Issue 1: f. 201). қо. P. cinnamomeus Jacq. ex. Fries, Epier. Syst. Мус. 429. 1838. Boletus cinnamomeus Jacq. Coll. Bot. ete. т: 116. 1786. P. subsericeus Peck, Ann. Rept. №. Y. State Mus. 33:37. 1880. Pileus stipitate, circular in outline, convex-depressed to umbilicate, 1-5 ст. broad, 1-3 mm. thick, pliant and tough, bright cinnamon-rufous to bright amber-brown, silky fibrillose, the fibrils sometimes suberect towards the center of the pileus, silky striate, sometimes zonate, margin thin and acute; context cinnamon or rusty brown, fibrous, less than 0.5 mm. thick; tubes not more than 2 mm. long, the mouths rufous-cinnamon, angular, averaging 2-4 to a mm.; stipe central, cylindrical, concolorous with the pileus, velvety to villous, 1-4 cm. long, 1-3 mm. thick; spores (teste Lloyd) pale colored, elliptical, smooth, 5-6 x 7-10 џ. Most frequently on clay banks, usually among moss. July to September. Not common. Distinguished from P. circinatus Fries, and P. obesus Ellis & Ev. by the very thin context; from P. perennis L. ex Fries by the silky pileus and the habitat; from P. focicola Berk. & Curt. by the much smaller pores. For illustrations see Lloyd, Myc. Notes Polyp. Issue 1: f. 200., and Bresadola, Fungi Trid. pl. 99. бо. P. lucidus Leyss. ex Fries, Syst. Myc. 1: 353. 1821. Boletus lucidus Leyss. Flora Halensis 300. 1783. [2nd еа] Ganoderma sessile Murr. Bull. Torr. Bot. Club 29: 604. 1902. Ganoderma subperforatum Atk. Bot. Gaz. 46: 337. 1908. Plants stipitate or sessile, annual; pileus dimidiate or reni- form in outline, 3-12 x 3.5-20 x 0.4-2.5 cm., coriaceous-corky when fresh, corky or woody when dry, the upper surface covered by an encrusting persistent layer of deep reddish chestnut varn- [Vor. 1 124 ANNALS OF THE MISSOURI BOTANICAL GARDEN ish, often wrinkled, glabrous or pruinose from a coating of brown conidial (?) spores, zonate or concentrically suleate, the margin thin and acute, sometimes lobed; context whitish to light brown, sometimes separated into an upper, light colored, soft layer, and a lower darker and firmer layer, but often uniform in color and texture, 0.2-1.5 em. thick; tubes 0.3-1.5 em. long, not decurrent, the mouths white or umber, darker when bruised, cireular to angular, averaging 3-5 to a mm.; the hymenium often with red-varnished patches on which no tubes are produced; stipe often entirely absent, lateral when present, covered like the pileus, 1-10 cm. long, 0.5-1 em. thick; spores yellowish brown, smooth or apparently slightly verrucose, ovoid with a truncate base, 5-6.3 х 9.4- 11g. On stumps and trunks of dead or injured deciduous trees. Common. The variation in the pileus from stipitate to sessile may be confusing at first, but the deep chestnut-red color, not changing to yellowish as in the next species, will usually be found to be the distinguishing character of the species. Тһе plant is de- scribed by Murrill under the name of Ganoderma sessile Murr. Atkinson has described a new species of Ganoderma from Ohio under the name of G. subperforatum. At the writer's request Professor Atkinson very kindly sent the type collection for examination. Under the ordinary high power of the microscope the spores of both P. lucidus and G. subperforatum appear to be praetieally smooth. Ву the use of the oil-immersion lens vary- ing degrees of apparent echinulation are to be made out in the ordinary forms of P. lucidus while in the type collection of G. subperforatum the spores do not have that appearance, although Professor Atkinson states that by first boiling the spores in potassium hydroxide solution the perforations in the spore walls are faintly visible. I am convinced, however, that the echinu- late appearance when present is not due to projections on the outer wall, but, as Atkinson has said, to perforations in the inner spore wall. An examination of the dozen or more col- lections of P. lucidus in my own herbarium have given evidence of a great variability in this character. Since G. subperforatum is not otherwise to be distinguished from P. lucidus, it has seemed best to consider the name as а synonym in this paper. Even 1914] OVERHOLTS—THE POLYPORACEJE OF OHIO 125 were the character constant one might well question the advisa- bility of separating the species on а character that requires the use of the oil-immersion lens for its detection. This and the following species are included in the genus Fomes by Saccardo, and many writers have followed his example. Why this should be done is not clear, for both species are always annual and the tubes are never stratified. Тһе following illustrations will aid in determination: Atkinson, Mushrooms f. 188; Bot. Gaz. 46: f. 2., and Hard, Mushrooms f. 332. 61. P. Curtisii Berk. Hooker's Jour. Bot. Kew Gard. Misc. 1: 101. 1849. Pileus stipitate, reniform or flabelliform in outline, 3-12 x 3-20 x0.7-2 cm., coriaceous-corky when fresh, corky when dry, covered with a thin chestnut or reddish varnish that soon begins to disappear, leaving the pileus yellowish or sometimes almost white, glabrous, zonate or concentrically sulcate, the margin rather thick, sometimes truncate; context in two layers, a yel- lowish or pallid upper layer, rather soft in texture, and a brown- ish lower layer next to the hymenium, firm or corky in texture, the whole 0.5-1 em. thick; tubes 0.3-1.2 сіп. long, not at all decurrent, the mouths white to brownish, mostly circular, averaging 3-5 to a mm.; stipe always lateral, cylindrical, per- sistently red-varnished and encrusted, sometimes bluish in color, the context in two layers as in the pileus, 2-10 cm. long, 0.5-3 em. thick; spores brown, ovoid to elliptic, smooth or appearing minutely echinulate, with a heavy outer wall, 4.6-7.2 х 8.5-11.8 y. On and about stumps and trunks of trees. Rare. This is typically a more southern plant and is rarely found north of the Ohio River. - It is distinguished from the preceding species by the yellowish color assumed by the mature pileus, the change in color being due to the disappearance of the reddish varnish. It is sometimes classed as a Fomes but is probably never truly perennial. For illustration see Atkinson, Bot. Gaz. 46: f. 1-8. [Vor. 1 126 ANNALS OF THE MISSOURI BOTANICAL GARDEN SPECIES DOUBTFUL OR EXCLUDED The following species reported by either Morgan or Lea are now believed to have been misdetermined, but the writer does not know to what species the plants should be referred: P. ovinus Schaeff. ex Fries; P. leucomelas Pers. ex Fries; P. lentus Berk.; P. fragilis Fries; and P. badius Schw. P. intybaceus Fries reported by Morgan is possibly а form of P. giganteus, P. frondosus, or a closely related species. P. pheoxanthus Berk. was originally described from material collected in Ohio by Sullivant. Тһе type specimen is said to be in fragments and the plant has never been collected since Sullivant's time. FOMES Fries, ex Gill. Champ. Fr. 682. 1878. Fries; Nov. Symb. 31. 1851. Plants typically perennial, epixylous, sessile (in our species) ; pileus corky or more often woody in texture, often becoming rimose, anoderm, or encrusted; context white, reddish, or brown- ish, soft and punky to hard and woody; tubes as in Polyporus except that they are arranged in definite or indefinite layers corresponding to periods of growth of the plant, the mouths circular or angular, never deedaloid or irpiciform; spores white or brown. The genus Fomes includes all of the perennial forms which have the tubes as in the genus Polyporus. Each season one layer of tubes is produced and plants of the first season’s growth are likely to be referred to the genus Polyporus. The key to that genus has been made to include a few such forms, the descrip- tions of which are always to be sought in the genus Fomes. A few species are so constantly annual iliduration that they might perhaps better be included in the genus Polyporus. KEY TO THE SPECIES Context white or only slightly colored (Species with wood-colored, flesh-colored, or rose-colored context included һете)............................... 1 Context yellowish brown or 4атКег................................... 7 1. Sporophore small, scarcely more than 2 cm. broad; context white.......... 2 1. Sporophore larger, more than 2 cm. broad; context whitish or somewhat О Ӛз СЕЕ А 8 1914) OVERHOLTS—THE POLYPORACEJE OF OHIO 127 2. Pileus entirely dark brown or black; plants growing only on the wood of Ainus amd Полное. афо 1. Е. scutellatus 2. Pileus not entirely black, the margin at least remaining white; plant growing on the wood of other deciduous trees, often on structural о JEREREESE О РЗ ss cn cs ve 2. F. ohiensis 3. Hymenium or context pinkish or гейдйівһ............................... 4 3. Hymenium or context whitish or уеШомівһ............................. 5 4. Tubes more than 3 mm. long; plants usually growing on stumps and онай of Frozima.................. edo n BI 6. F. fraxineus 4. Tubes not more than 3 mm. long; plants usually growing on the wood of ее Р е 7. Е. carneus 5. Hymenium distinctly stratified, the strata of tubes separated by distinct layers of context; mouths of the tubes angular, usually glistening. .4. F. connatus 5. Hymenium indistinctly stratified or if somewhat distinctly so the layers not separated by distinct layers of context; mouths of the tubes mostly circular, not giistening. ....................!......... 8.22. 7022... 6 6. Plant growing on dead wood, usually of coniferous trees; mouths of the tubes small, averaging 3-5 to а пап........................ 5. F. pinicola 6. Plant growing only on living Fraxinus; mouths of the tubes rather large, элект Ср. ee ere РОСТ 8. Р. fraxinophilus 7. Pilei forming a densely imbricate, globose or cylindrical mass. . ..8. F. graveolens 7. Pilei not forming a densely imbricate, globose or cylindrical mass. ........... 8 8. Surface of the pileus not distinctly епегивбей........................ 9 8. Surface of the pileus distinctly епегівбей........................... 13 9. Context less than 5 mm. thick; sporophore often effused-reflexed or entirely resupinate; growing usually on dead wood................... 9. F. conchatus 9. Context more than 5 mm. thick; sporophore generally sessile; often growing соаитлалныан;,,................................... 2 44 10 10. Sporophore found only on Robinia ....................... 11. F. rimosus 10. Sporophore found on some other һовб.............................. 11 11. Tubes in the older layers distinctly white encrusted orstuffed......... t2. + | 11. Tubes in the older layers not distinctly white encrusted or stuffed 12. F. Еъеграт 12. Surface of the pileus black, somewhat shining, and rimose; margin rather thin and аешіе................................ 18. F. igniarius 12. Surface of the pileus dull brown; margin thick and somewhat obtuse. . . . 14. Е. nigricans 13. Encrusting layer thin, easily indented; plants annual or sometimes reviving the second season but with the pileus distinct from and coming out below (0071 4 Ви 1 ......,................і.........222 17. F. lobatus 13. Encrusting layer thick and horny; plants strictly perennial.............. 14 14. Plant growing only on species of Ргитив.................... 10. F. fulvus 14. Plant growing on some other һові................................ 15 15. Content band and төсіу......................і............ 18. Е. igniarius ЕХ. 2.....................ӛ...).. C$ ma 5 I0 16. Mouths of the tubes medium-sized, averaging 3 to a mm.; spores white 15. F. fomentarius 16. Mouths of the tubes minute, averaging 5 to а mm; spores brown...... 16. F. applanatus [Vor. 1 128 ANNALS OF THE MISSOURI BOTANICAL GARDEN т, Е. scutellatus Schw. ex Cooke, Grevillea 14: 19. 1885. Polyporus scutellatus Schw. 'Trans. Am. Phil. Soc. II. 4: 157. 1832. Plants perennial, sessile, often attached by the apex of the pileus; pileus dimidiate or circular, convex, 0.5-1.5 x 0.5-2 x 0.1-0.5 em., corky when fresh, hard and woody when dry, dark brown or black at least when mature, velvety, azonate or some- what concentrically sulcate, margin rather thick, acute; context white to wood-colored, corky, not more than 2 mm. thick; tubes 1-2 mm. long, indistinctly stratified, the mouths white to umber, circular or subcircular, averaging 4—5 to а mm. Chiefly on dead limbs of Alnus and Hamamelis. Rare. This species is distinguished from F. ohiensis Berk. ex Murrill by the habitat and the black surface of the entire pileus including the margin. Specimens have been received from Mr. Claassen, Cleveland, Ohio. 2. F. ohiensis Berk. ex Murrill, Bull. Torr. Bot. Club 30: 230. 1908. Trametes ohiensis Berk. Grevillea 1: 66. 1872. Plants perennial, sessile, often attached by the vertex of the pileus; pileus dimidiate or shield-shaped, convex to ungulate, 0.5-3 x 0.5-4 x 0.2-1 cm., soft-corky when fresh, hard and woody when dry, at first pure white but becoming cinereous or yellowish and often black at the base but the margin remaining white, finely tomentose to glabrous, often zonate or concen- trically suleate, margin rather thick, acute or obtuse; context white to wood-colored, soft-corky to woody, 1-3 mm. thick; tubes 1-5 mm. long, often arranged in more or less definite rows, indistinctly stratified in two to six layers, the mouths white, circular, averaging 3-5 to a mm., the dissepiments almost as thick as the diameter of the pores. On dead wood of deciduous trees, especially on structural timber. Common. By its small size this species is separated from all perennial forms except F. scutellatus Schw. ex Cooke. It differs from that species in habitat and in the margin of the pileus always remain- ing white. 1914] OVERHOLTS—THE РОГУРОВАСЕЖ OF OHIO 129 3. F. fraxinophilus Peck ex Sacc. Syll. Fung. 6:172. 1888. Polyporus fraxinophilus Peck, Ann. Rept. N. Y. State Mus. 35:136. 1882. Plants perennial, sessile or effused-reflexed, often imbricate; pileus dimidiate, convex to compressed-ungulate, 2-25 x 3.5-40 x1.5-10 cm., woody, white at first, becoming blackish and often somewhat rimose with age, not encrusted, soon glabrous, concentrically sulcate, margin thick, obtuse or acute; context white to cinnamon wood-color, corky or woody, 0.5-1 сіп. or more thick; tubes 2-3 mm. long, indistinctly stratified in many layers, the mouths white to cinereous or yellowish, circular, averaging 2-3 to a mm., the walls thick and entire; spores white, smooth, ellipsoid to ovoid or pyriform, 5-6.3 x 7.3-8 y. Growing only on living trunks of Fraxinus. Common. In habitat the species corresponds closely to F. fraxineus Bull. ex Cooke, from which it differs in the entire absence of any rosy or reddish colors and in being always perennial. Ап excellent illustration is given by Hard (Mushrooms f. 350). 4. Е. connatus Weinm. ex Gill. Champ. Fr. т: 684. 1878. Polyporus connatus Weinm. Fl. Ross. 332. 1836. P. con- natus Fries, Epicr. Syst. Мус. 472. 1838. Plants perennial, sessile or effused-reflexed, sometimes imbri- cate; pileus dimidiate, convex, 2-10 x 3-15 х 0.5-4 ст., corky when fresh, somewhat woody when dry, whitish, cinereous, or slightly yellowish, sometimes blackish toward the base, not encrusted, velvety-tomentose to glabrous, usually azonate, margin thick, acute or obtuse; context white or pallid, punky to soft corky, 0.3-1 cm. thick; tubes 1.5-5 mm. long, distinctly stratified, the different strata separated from each other by a thin layer of context, the mouths whitish to yellowish, glis- tening, angular, averaging 4-5 to a mm., the walls entire to slightly dentate; spores (teste Bresadola) white, globose, 3-4 и in diameter. Growing on living deciduous trees, more often at the bases of species of Acer, and frequently covered with moss. Common. The distinguishing characters are the habitat, the layers of context interposed between successive layers of tubes, and the glistening mouths of the tubes. In but one other species of Fomes do we find the second character developed and that [Vor. 1 130 ANNALS OF THE MISSOURI BOTANICAL GARDEN is in F. applanatus Pers. ex Wallr. That species always grows on old logs and stumps and has a rusty brown context. Bresadola and Murrill regard F. populinus Schum. ex Cooke, to be the same plant as this species. This may be the case but the figure of F. connatus in Fries’ 'Icones' (f. 185) represents our plant much better than the figure of F. populinus in ‘Flora Danica’ (pl. 1791). Most of the specimens distributed in exsiccati in both this country and Europe are under the former name and that one is here given preference. А study of the types of both species should show whether they are the same or not, but from the evidence at hand our plants must be referred to F. connatus. қ. F. pinicola Sw. ex Cooke, Grevillea 14:17. 1885. Boletus pinicola Sw. Sv. Vet. Akad. Handl. 88. 1810. Pol- porus pinicola Fries, Syst. Мус. 1:372. 1821. Plants perennial, sessile; pileus plane to convex, rarely ungu- late, dimidiate, 4-15 x 6-20 x 3-10 cm., woody and rigid, gray- ish to black, partly or entirely covered with a reddish gluten that forms a crust over the surface, glabrous, sometimes con- centrically suleate, margin thin or thick, often obtuse; context pallid or wood-colored, corky to woody, 0.5-2 em. thick; tubes 3-5 mm. long, distinctly or indistinctly stratified, the mouths white to umber, cireular, averaging 3-5 to a mm., the walls thick and entire. On dead wood, usually of coniferous trees. Distinguished from closely related species by the resinous, somewhat sticky, reddish crust found on the pileus. The plant is common wherever coniferous woods are found. Hard (Mushrooms f. 348) gives а photograph of it but does not state that he ever collected it in Ohio. 6. F. fraxineus Bull. ex Cooke, Grevillea 14: 21. 1885. Boletus fraxineus Bull. Herb. Fr. pl. 433. f. 2. 1789. Poly- porus fraxineus Bull. ex Fries, Syst. Мус. 1:374. 1821. Plants annual or perennial, sessile, sometimes imbricate; pileus dimidiate, 4-10 x 6-15 x 0.6-2 em., согЕу when fresh, rigid and woody when dry, light colored, always with reddish or reddish brown stains, or altogether reddish, encrusted with a thin hard crust, minutely velvety to glabrous, more or less zonate, margin thin or thick, acute; context floccose-punky 19141 OVERHOLTS—THE POLYPORACE/E OF OHIO 131 to corky, whitish or pallid when dry, tinged pinkish or flesh- colored when fresh, 0.2-2 cm. thick; tubes 2-6 mm. long, usually in a single layer but sometimes stratified, mouths whitish, pallid, or flesh-colored, circular or subcircular, averaging 4-6 to a mm., the dissepiments rather thick and entire; spores (teste Murrill) subglobose, smooth, subhyaline, 5-6 x 6-7 и. Usually found on living Fraxinus but sometimes on other hosts. Rare. The habitat, the reddish blotches on the pileus, and the pink- ish hymenium and context in fresh specimens will identify the plant. But three collections are known from Ohio; one by Morgan, one by W. G. Stover near Columbus, in 1910, and one near Camden, Ohio, by the writer, in 1912. All of these collections are of the annual form. 7. Е. carneus Nees ex Cooke, Grevillea 14: 21. 1885, Polyporus carneus Nees, Nova Acta Acad. Leop. Carol. 13: pl. 3. 1827. Plants annual or perennial, sessile; pileus dimidiate, 1,5-5 x 1.5-10 x 0.3-1.5 em., soft-corky when fresh, firmer when dry, pinkish or rosy, sometimes blackish with age, velvety to gla- brous, usually azonate, margin thin and acute; context pinkish or rosy, floccose or punky to soft-corky, 0.2-1 em. thick; tubes 0.5-3 mm. long, usually in a single layer but sometimes strati- fied, mouths pinkish or rosy, circular or subcircular, averaging 3-5 to a mm., dissepiments thick and entire. Usually on wood of coniferous trees. Rare. The species will be recognized by the uniform pinkish color of the whole plant. The color is well retained on drying. Specimens are in the herbarium of the New York Botanical Garden, collected by James, in Ohio. Authorities disagree as to the identity of F. carneus and F. roseus Fries ex Cooke. 8. F. graveolens Schw. ex Cooke, Grevillea 13:118. 1884. Boletus graveolens Schw. Syn. Fung. Car. 97. 1822. Poly- porus conglobatus Berk. Hooker's Lond. Jour. Bot. 4:303. 1845. Plant composed of numerous overlapping pilei arising from a central solid core and forming a more or less globose or cylin- drical mass 5-12 cm. in diameter; pilei not more than 2 cm. broad, but connate laterally, corky when fresh, rigid and firm when dry, grayish brown to dull cinnamon-brown, becoming [Vor. i 132 ANNALS OF THE MISSOURI BOTANICAL GARDEN black in weathered specimens, slightly encrusted, pulverulent to glabrous, azonate or marked with fine grayish zones, margin thick, deflexed and almost concealing the pores; context fulvous to golden brown rust-colored, floccose-fibrous, 1-4 mm. thick; tubes 2-4 mm. long, the mouths grayish, hoary brown, or umber, cireular, averaging 3-4 to a mm., the dissepiments thick and entire. On logs or trunks of deciduous trees, especially Fagus, Quercus, and Acer. Rare. A characteristic plant that will be recognized at sight. It is commonly known as ‘‘sweet knot" from the sweet, powerful odor that it is said to give off. 'The writer has made four different collections of this rare plant in different stages of growth but has never been able to detect the slightest semblance of а sweet odor. Тһе plant is exceptionally well illustrated by Lloyd (Мус. Notes, Polyp. Issue 3: f. 367-68; Syn. Stip. Polyp. f. 455), and Hard (Mushrooms f. 334). Тһе first and the last of the figures cited are upside down. 9. F. conchatus Pers. ex Gill. Champ. Fr. 1:685. 1878. Boletus conchatus Pers. Obs. Myc. 1:24. 1796. Polyporus conchatus Fries, Syst. Myc. 1:376. 1821. Plants perennial, sessile, or more often effused-reflexed and frequently entirely resupinate; pileus dimidiate to conchate, 0-7 x 4-12 x 0.2-3.5 cm., woody, grayish brown, yellowish brown or black, rarely encrusted, tomentose at least on the margin, becoming glabrous behind, zonate or concentrically sulcate and sometimes somewhat rimose, margin thin, mostly acute; context yellowish brown to dark brown, woody, 1.5-3 mm. thick; tubes 1-2 mm. long, indistinctly stratified, mouths fulvous to dark brown, usually glistening, circular or subcircular, averaging 4-6 to a mm. On dead wood, rarely on living trees. Common. The plant is most frequently found entirely resupinate on old oak logs. Distinctly sessile forms are sometimes found, especially on living trees. The hymenium usually has a silky luster when viewed in changing positions, and on the whole the plant is so characteristic that when once recognized, the collector usually has no trouble with subsequent collections notwithstanding the fact that the species often presents great 1914] OVERHOLTS—THE POLYPORACEZ OF OHIO 133 differences in size and habit. In Europe the plant is usually known as F. salicinus Pers. ex Gill. and it was so reported from Ohio by Morgan. It is entirely different from all other species of Fomes except F. fulvus Scop. ex Gill. and F. ribis Schum. ex Fries in the thin pileus, often conchate in form and with a concave hymenium. Usually the pileus is not more than 1 em. thick. Р. fulvus Scop. ex Gill. is distinct in its habitat as is also F. ribis Schum. ex Fries. 10. F. fulvus Scop. ex Gill. Champ. Fr. 1: 687. 1878. Boletus fulvus Scop. Fl. Carn. 2:469. 1772. [2nd ed.] Poly- porus fulvus Fries, Epicr. Syst. Myc. 466. 1838. Plants perennial, sessile, effused-reflexed or entirely resupinate; pileus dimidiate, convex, 0-4 x 3-8 x 0.5-3 em., woody, fulvous to ferruginous when young, becoming grayish black or black in age, encrusted, minutely velvety to glabrous, sometimes suleate, margin rather thick, acute or obtuse; context dark brown, woody, 3-8 mm. thick; tubes 2-4 mm. long, rather distinctly stratified, the mouths circular to slightly angular, grayish brown to tawny, averaging 4-5 to a mm., dissepiments rather thick, entire. Growing only on wood of species of Prunus. Not common. One should have no trouble in identifying this species if the habitat is taken into consideration as it is the only perennial form that grows on Prunus. Morgan reported it under the name of F. supinus Schw. II. F. rimosus Berk. ex Cooke, Grevillea 14:18. 1885. Polyporus rimosus Berk. Hooker's Lond. Jour. Bot. 4:54. 1845. Pyropolyporus robiniae Murrill, Bull. Torr. Bot. Club 30:114. 1903. Plants perennial, sessile; pileus dimidiate, convex to ungulate, 3-20 x 6-30 x 1.5-10 em., woody, at first fulvous, becoming dark brown or black, not encrusted, velvety in young speci- mens, glabrous and very rimose in old plants, concentrically suleate, margin thick or thin, obtuse or acute; context fulvous to rusty brown, woody, 0.5-3 cm. thick, zonate; tubes 1-5 mm. long, indistinctly stratified in many layers, the mouths fulvous to rusty brown, circular, averaging 5-6 to а mm., walls rather thick and entire; spores brown, smooth, globose, 4-би in diameter. [Vor. 1 134 ANNALS OF THE MISSOURI BOTANICAL GARDEN Growing only on living trunks of Robinia. Common. The type locality for F. rimosus is given by Berkeley as the Swan River, Australia, and not Demerara and the Cape of Good Hope, as cited by Saccardo and by Murrill. If the speci- mens Murrill examined are from the two latter places, it is still possible that our plants belong under F. rimosus. Our species also oecurs in South Africa as specimens examined from that locality agree well with our plants. The plant is never found on any other host than the locust tree. This will distinguish it from all of its allies. Its closest relatives appear to be F. Everhartii Ellis & Gall. and F. igniarius L. ex Gill. The plant is well illustrated by Hard (Mushrooms f. 347), and by von Schrenk (Ann. Rept. Mo. Bot. Gard. 12: pl. 2). 12. Е. Everhartii Ellis & Gall. Mucronoporus Everhartii Ellis & Gall. Jour. Myc. 5:141. 1889. Plants perennial, sessile or decurrent; pileus dimidiate, convex, rarely ungulate, 2.5-10 x 4-20 x 2-6 cm., woody, entirely fulvous when young but becoming grayish brown or black and rough and rimose with age, velvety when young, glabrous when mature, scarcely encrusted, concentrically suleate with age, margin thin or thick, acute or obtuse, usually remaining fulvous in color; context fulvous to rusty brown, shining (at least in herbarium specimens), zonate, woody, 1-4 cm. thick; tubes 3-6 mm. long, indistinctly stratified, tubes of the older layers sometimes partly stuffed with mycelium, the mouths con- colorous with the context, circular, averaging 4-5 to а mm., the walls rather thin but entire, sometimes glistening; spores distinctly brown, smooth, globose, 4-5.3ш in diameter. On living trees, usually of Quercus. Not uncommon. Distinguished from F. igniarius L. ex Gill. and F. nigricans Fries ex Gill. by the absence of the distinct encrustation or stuffing of the tubes in the old layers, by the more shining context, the somewhat thinner dissepiments, the hyaline spores, and the absence of a distinct crust on the pileus. The two 1 Р. Everhartii was originally described under the genus Mucronoporus and as far as I have been able to find, no specific statement of transfer to the genus Fomes was ever made. At the present time I have not been able to satisfy myself as to who was the first to make (unknowingly, it seems) the new combination, and there- fore I do not know to whom credit for the transfer should be given. 1914] OVERHOLTS—THE РОГУРОВАСЕЖ OF OHIO 135 species are closely related, however, and without the spores it is sometimes difficult to decide between them. "Тһе species differs from F. fomentarius L. ex Gill. and F. applanatus Pers. ex Wallr. in the unencrusted pileus, the woody context, and the short tubes. 13. F. igniarius L. ex Gill. Champ. Fr. 1:687. 1878. Boletus igniarius L. Sp. Plant. 1176. 1753. Polyporus igni- arius Fries, Syst. Мус. 1:375. 1821. Plants perennial, sessile; pileus dimidiate, convex to some- what ungulate, 2.5-11 x 4-25 x 1.5-12 cm., woody, grayish black, or entirely black, encrusted, sometimes somewhat rimose in age, glabrous, concentrically suleate in older plants, margin rather thin, acute, usually grayish in growing specimens; context rusty red or rusty brown, scarcely shining, zonate, woody, 0.5-4 em. thick; tubes 2-5 mm. long, usually indis- tinetly stratified, the older layers becoming distinctly whitish encrusted, the mouths circular, grayish brown to dark brown, averaging 4-5 to a mm., the walls thick and entire; spores (teste Romell) hyaline, subglobose, 5-7.5x 4-7 и, often 1-guttate. On trunks of living deciduous trees. Not common. In no other species is the stuffing or encrusting of the tubes by à whitish substance so evident as in this and the next one. In F. Everhartii Elis & Gall. the tubes appear to be sometimes filled with a whitish mycelium but the character is scarcely evident except on close examination, while in F. igniarius and F. nigricans Fries ex Gill. in sections through the hymenium the whitish encrustation is plainly visible, and seems to be a distinguishing character. The plant is further to be distin- guished from F. Everhartii by the hyaline spores, and the thicker dissepiments. The pores are somewhat smaller, but in measuring them the thick dissepiments are included, so that the number per mm. is about the same in the two species. From F. fomentarius L. ex Gill. and F. applanatus Pers. ex Wallr. the species is separated by the more woody context, the thinner crust, and the much shorter tubes, as well as by the hyaline spores. In F. igniarius the pileus is darker in color and is usually much more rimose than in F. nigricans. For illustrations see Atkinson, Cornell Univ. Agr. Exp. Sta. Bul. 198: f. 73-4. [Vor. 1 136 ANNALS OF THE MISSOURI BOTANICAL GARDEN 14. F. nigricans Fries ex Gill. Champ. Fr., Hymen. 1: 685. 1878. Polyporus nigricans Fries, Syst. Мус. 1:375. 1821. Plants perennial, sessile; pileus dimidiate, convex to ungu- late, distinctly triangular in cross-section, 5-10 x 7-13 x 2-7 cm., woody, dull brown or becoming brownish black, not en- crusted, smooth or cracking somewhat in age but scarcely rimose, azonate or with one or two concentrie furrows, the margin thick, acute or obtuse, with a broad ferruginous band; context rusty brown, zonate, woody, 0.6-2 cm. thick; tubes 2-7 mm. long, distinctly or indistinctly stratified, becoming dis- tinetly white encrusted or stuffed in the older layers, the mouths dark brown, circular, minute, averaging about 5 to a mm., the walls thick and entire; spores white, subglobose or globose, 6.5u in diameter. On trunks of trees, especially on Betula. Not common. I have one collection of this fungus from W. A. Kellerman. The species has been confused with the preceding one from which it differs in the smoother and differently colored pileus and in being more decidedly triangular in cross-section. The best illustration is that given by Boudier (Ic. Мус. т: pl. 155). 15 F. fomentarius L. ex Gill. Champ. Fr. 1:686. 1878. Boletus fomentarius L. Sp. Plant. 1176. 1753. Polyporus fomentarius Fries, Syst. Myc. 1:374. 1821. Plants perennial, sessile; pileus dimidiate, convex to strongly ungulate, 3.5-15 x 6-20 x 2-9 em., hard and woody, grayish to cinereous, brownish, or black, covered with a thick horny crust that appears black and shining when cut, glabrous, smooth, never rimose, zonate or concentrically suleate, margin thick and obtuse; context fulvous to ferruginous, never shining, punky to soft-corky, zonate, 0.3-3 cm. thick; tubes 0.5-2.5 em. long, rather distinctly stratified, mouths grayish to cin- namon, averaging 3 to a mm., the walls thick and entire. On living deciduous trees. Not common. Distinguished from all of the preceding species by the punky or soft-corky context and the usually longer tubes. Most closely related to F. applanatus Pers. ex Wallr. but distinguished from it by the much longer pores and the hyaline spores. For 1914) OVERHOLTS—THE POLYPORACE/E OF OHIO 137 illustrations see Kellerman, Journ. Myc. 9: pl. 3., and White, Hymen. Conn. pl. 35. f. 2. 16. Е. applanatus Pers. ex Wallr. Crypt. Fl. Ger. 2: 591. 1833. Boletus applanatus Pers. Obs. Myc. 2: 2. 1799. Polyporus applanatus Fries, Epicr. Syst. Мус. 465. 1838. Р. leucopheus Mont. Syll. Crypt. 157. 1856. Plants perennial, sessile; pileus dimidiate, convex or plane, not ungulate, 3-30 x 5-50 x 1.5-7 ст., woody, usually grayish becoming brownish or blackish, glabrous, covered with a thick horny crust, zonate or concentrically sulcate, margin thin or thick, acute or obtuse; context dark ferruginous brown, floccose : to soft corky, 0.6-2 cm. or more thick; tubes 0.5-1.5 cm. long, distinctly stratified after the first season, the strata separated by thin layers of context, mouths whitish to umber, darker when bruised, circular, minute, averaging 5-6 to a mm. On dead wood of deciduous trees or on living trees. Common. This is our most common species of Fomes and may be found in every woodlot, usually on stumps or old logs. It is dis- tinguished from F. fomentarius L. ex Gill. by the more applanate pileus and the minute mouths of the tubes. For illustrations see Atkinson, Mushrooms f. 15., White, Hymen. Conn. pl. 35. f. 1., and Atkinson, Cornell Univ. Agr. Exp. Sta. Bul. 193: f. 82. 17. F. lobatus Schw. ex Cooke, Grevillea 14:18. 1885. Polyporus lobatus Schw. 'Trans. Am. Phil. Soc. II. 4:157. 1832. P. reniformis Morgan, Journ. Cin. Soc. Nat. Hist. 8:105. 1885. Plants annual, frequently reviving for two or three years but the second year's growth distinct from and coming out below that of the first year, sessile or more often appearing substipitate; pileus dimidiate or reniform, plane, depressed, or somewhat convex, never ungulate, 4-12 x 4-15 x 1-3 cm., corky or somewhat flexible when growing, usually umber to yellowish or dark rusty brown, glabrous, covered with a thin, easily indented crust, zonate or concentrically sulcate, margin thin and acute; context dark rusty brown, soft and floccose to punky, 0.3-1 cm. thick; tubes 0.4-1 cm. long, not stratified, mouths circular or subcircular, white, yellowish or umber- [Vor. 1 138 ANNALS OF THE MISSOURI BOTANICAL GARDEN brown, darker when bruised, averaging 4 to a mm., walls rather thin but entire. On dead wood of deciduous trees. Common. This species is easily separated from F. applanatus Pers. ex Wallr. in that it is not perennial, and in that, if the plant revives the second year, the pileus comes out below that of the first year, and the latter persists as a dead decaying pileus. The second difference is in the character of the encrusting layer of the pileus. In F. applanatus Pers. ex Wallr. the crust is hard and horny and one cannot indent it with the thumb nail, while in Р. lobatus the crust is thin, and often becomes cracked and brittle when old, but is always rather soft and easily indented. TRAMETES Fries, Gen. Hymen. 11. 1836. Plants annual or perennial, epixylous, sessile; pileus corky or woody in texture, small or medium sized ; context white or brown (never red), descending into and forming the walls of the tubes; tubes typically appearing sunken into the context to unequal depths so that their bases are not in a continuous straight line; mouths circular or angular, never breaking up into teeth and rarely showing a daedaloid tendency. One species here included in the genus is perennial, all the others are annual. The chief generic distinctions are the unequal depths to which the tubes are immersed in the context, and the homogeneous texture of the context and trama. The first distinction is often not apparent except on very close examina- tion, and at times appears to break down entirely. Conse- quently, students will meet with some difficulty at times in deciding between the two genera, Trametes and Polyporus. KEY TO THE SPECIES Context white or whitish |... LLL 1 Context brown or БгомлывЬ. 0000000 6 1. Pileus densely hirsute or РОО POL PC vin. vy cu van vd koe 7. T. Peckit 1. Pileus slightly pubescent to сз О E 2 2. Mouths of the tubes minute, averaging 4-6 to a mm....... Т. robiniophilas 2. Mouths of the tubes larger, averaging 1-3 to a mm..... TOME Io 3 3. Pileus rather large; context more than 5 mm. thick; plant growing only НЕЗ аа 3. Т. suaveolens 3. Pileus small, sometimes mostly resupinate; context less than 5 mm. thick; found on some other host. ..... 2,-0.0... а... 4 1 For description of this plant see p. 104 under the genus Polyporus. 19141 OVERHOLTS—THE POLYPORACEJE ОҒ OHIO 139 4. Hymenium light brown in сойют.......................... 4. T. malicola 4 Hymen white or WIE.. e] soares boa EO 5 5. Pileus white or light colored; mouths of the tubes averaging 1-2 to a mm. 1. T. sepium 5. Pileus cinnamon-brown; mouths of the tubes averaging about 3 to a mm. 2. T. serialis 6. Sporophore woody, perennial; hymenium bright yellowish brown in color; mouths of the tubes often somewhat daedaloid; growing only on PNE re NNA ERES (dis c 8. T. Pint 6. Sporophore coriaceous or corky; hymenium white or dull brown; growing in wood wd deciduous ігесв............... cdo les cov a 7 T PRENDE of hispid. ................... Ber E нь 8 7. Pileus finely tomentose or бі8іоОйв.................................... 9 8. Mouths of the tubes large, averaging 1 to a mm.; pileus more than 4 mm. с ee er ne Soe 7. T. Peckit 8. Mouths of the tubes medium sized, averaging 2-3 to а mm.; pileus less than 4 mm. ЯМек....,....................й../.// 2222 6. T. rigida 9. Context lesa than 1 mm. tbick................... 2... Sc n 6. T. mollis 9. Context more than 1 mm. thiek.................5. n 4. T. malicola I. Т. sepium Berk. Hooker’s Lond. Jour. Bot. 6:322. 1847. Plants annual, sessile or semirespuinate, imbricate or single; pileus dimidiate, 0.7-1 x 0.8-2.5 х 0.2-0.7 cm., flexible when fresh, eorky when dry, grayish to pallid or wood-colored, mi- nutely tomentose to glabrous, azonate, margin thin and acute; context white or pallid, tough when fresh, soft-corky when dry, less than 1 mm. thick; tubes 2-5 mm. long, mouths white or pallid, circular or rarely angular or sinuous, large, averaging almost 1 to a mm., the dissepiments thick and always entire; spores (teste Murrill) oblong, smooth, hyaline, 12 x 5 y. On fence posts, pickets, and other structural timber or dead wood. Distinguished from Т. serialis Fries by the short tubes, the whitish color of the pileus, and by the much larger mouths of the tubes; from T. rigida Berk. & Mont. by the lighter colored context, and the larger tube mouths. 2. Т. serialis Fries, Hymen. Eur. 585. 1874. [2nd ей.) Polyporus serialis Fries, Syst. Myc. 1:370. 1821. Plants annual, sessile, effused-reflexed, or resupinate; pileus dimidiate, 0-1 x 1-4 x 0.3-0.8 cm., corky when fresh, hard and firm when dry, cinnamon-brown to coffee brown, glabrous, zonate, margin rather thick but acute; context white, fibrous, not more than 1 mm. thick; tubes 2-6 mm. long, the mouths [Vor. t 140 ANNALS OF THE MISSOURI BOTANICAL GARDEN white or slightly discolored, sometimes slightly glistening, cireular to angular, averaging 3 to a mm., the walls firm and entire; spores (teste Bresadola) hyaline, elongate, 7-10 x 3-3.5 y. On dead wood. . Rare. The white pores and the internally white tubes contrast strongly with the rich brown color of the pileus. It is distinct from T. rigida Berk. & Mont. in the glabrous, thicker pileus. From T. верішп Berk. it differs in the much smaller pores and the brown pileus; T. malicola has no white color in the tubes. and the dissepiments are much thicker. 3. T. suaveolens L. ex Fries, Syst. Myc. 1:366. 1821. Boletus suaveolens L. Sp. Plant. 1177. 1753. Plants annual, sessile; pileus dimidiate, 3-9 x 6-14 x 1-3 ст., corky when fresh, firm and rigid when dry, white to grayish or slightly yellowish, finely villous-tomentose to glabrous, azonate, margin thin or thick, acute; context white or pallid, compact-corky to somewhat indurate, 0.5-2 cm. thick; tubes 0.2-1.5 em. long, the mouths white or cinereous, circular to slightly angular, averaging 1-3 to a mm. On dead or diseased Saliz. Rare. Distinguished from T. Peckii Kalchbr. by the prevailing whitish color and the more nearly glabrous pileus. 4. T. malicola Berk. & Curt. Journ. Acad. Nat. Sci. Phil. II. 3:209. 1856. Plants annual or reviving for two or three seasons, effused- reflexed or entirely resupinate; pileus very narrow, 0-1 x 1-5 X 0.3-0.8 cm., coriaceous and leathery when fresh, сотку when dry, avellaneous to cinnamon-brown or wood-colored, azonate, margin thick but acute; context wood-brown or lighter, soft- corky, 2-5 mm. thick; with a distinct pleasant odor when fresh; tubes 2-5 mm. long, sometimes indistinctly stratified in two or three layers, mouths wood-colored to cinnamon-brown, circular to angular or somewhat sinuous, averaging about 2 to a mm., the walls thick and entire; spores white, smooth, oblong, 2.8 -8.5 x 7.5-10 и. Growing on dead wood of deciduous trees, especially species of Acer. Common. Entirely distinct from T. sepium Berk. in the semiresupinate 1914) OVERHOLTS—THE POLYPORACEJE OF OHIO 141 habit of growth, the prevailing dull brown color of both hyme- nium and pileus, and the smaller-mouthed tubes. In this last respect the plant more nearly approaches Т. serialis Fries and T. rigida Berk. & Mont. From the former it is separated by the browner color of the hymenium, the lighter color of the pileus, the internally brown tubes, and the slightly larger and more irregular mouths. From the latter it differs chiefly in the more glabrous and less developed pileus and the longer tubes. The type specimens of Т. malicola were collected on the trunk of an apple tree by Schweinitz and referred by him to Р. popu- linus Fries. Murrill has placed the name as à doubtful syno- nym for P. galactinus Berk. Тһе writer has not examined the type of T. malicola, but our plants bear no resemblance to either P. populinus Fries or P. galactinus Berk. Our plants were determined by Lloyd and by Bresadola. 5. T. mollis Sommerf. ex Fries, Hymen. Eur. 585. 1874. Daedalea mollis Sommerf. Suppl. Fl. Lapp. 271. 1826. Plants annual or rarely reviving, rarely sessile, more often effused-reflexed or entirely resupinate; pileus dimidiate or elongate, 0-2.5 x 1-4 x 0.1-0.5 ст., coriaceous to rigid, umber- brown to almost black, finely tomentose to glabrous, zonate or multizonate, margin thin and acute; context light brown, fibrous, less than 1 mm. thick; tubes 2-3 mm. long, rarely in two or three layers, mouths light brown or grayish, subcircular to somewhat angular, often becoming sinuous or labyrinthi- form, averaging 1-3 to а mm.; spores (teste Bresadola) elongate- ellipsoid, smooth, hyaline, 9-11 x 4-4.5 и. On dead wood. Rare. Тһе species differs from T. rigida Berk. & Mont. in the distinetly brown and almost glabrous pileus. From T. serialis Fries it differs in the light brown context, the much thinner pileus and the usually larger and more irregular pores. Тһе context is much thinner than in T. malicola Berk. & Curt. and the general color is decidedly different. 6. T. rigida Berk. & Mont. Ann. Sci. Nat. III. 11:240. 1849. Plants annual or rarely reviving, sessile, effused-reflexed or entirely resupinate, sometimes imbricate; pileus dimidiate, [Vor. 1 142 ANNALS OF THE MISSOURI BOTANICAL GARDEN 0-3 x 2-6 x 0.1-0.3 cm., coriaceous when fresh, coriaceous or rigid when dry, cinereous to yellowish or slightly brownish, hirsute to hispid, usually zonate, sometimes with multicolored zones, margin very thin and acute; context light umber, fibrous, 0.5-3 mm. thick; tubes not more than 1 mm. long, the mouths white or brownish, circular to somewhat angular, averaging 2-3 to а mm., the walls rather thin but entire. On dead wood. Not common. Distinguished from all of its allies in the hirsute or hispid pubescence of the pileus. The pileus is thin and coriaceous and more nearly resembles the thin coriaceous species in Poly- porus. 7. T. Peckii Kalchbr. Bot. Gaz. 6:274. 1881. Plants annual, sessile or effused-reflexed; pileus dimidiate, 1.5-6 x 2.5-12 x 0.5-2 em., somewhat coriaceous when fresh, firm and rigid when dry, yellowish brown or reddish brown, densely hirsute or hispid, concentrically sulcate at times, margin thick or thin, acute; context light brown, fibrous, soft and spongy to firm and woody, 1-10 mm. thick; tubes 2-10 mm. long, the mouths dull brown or grayish brown, angular to irregular, averaging about 1 to a mm.; spores (teste Murrill) oblong or slightly curved, smooth, hyaline, 11-13 x 3.5-4 y. On dead wood of Populus, Liriodendron, and Salix. Sep- tember to December. Frequent. Easily recognized by the densely hirsute or hispid pubescence, the large pores, and the habitat. In Europe the species is known as T. hispida Fries. 8. T. Pini Thore ex Fries, Epicr. Syst. Myc. 489. 1838. Boletus Pini 'Thore, Essai Chlor. Dep. Land. 487. 1803. Plants perennial, sessile or effused-reflexed; pileus dimidiate, often ungulate, 3-15 x 5-20 x 1-6 cm., woody, yellowish brown to reddish brown or becoming black, the growing margin hir- sute to tomentose, glabrous behind, zonate or concentrically suleate, margin usually thick and somewhat obtuse; context yellowish brown to rusty brown, corky to woody, not more than 5 mm. thick; tubes 2-7 mm. long, indistinctly stratified, the mouths usually golden brown, subcircular to daedaloid and laby- rinthiform; spores (teste Bresadola) hyaline, subglobose, 5-6 x 4-5 y. 1914) OVERHOLTS—THE РОГУРОВАСЕЖ OF OHIO - 143 On coniferous wood. Rare. The bright color of the hymenium usually contrasts strongly with the darker colors of the upper surface. Р. piceinus Peck (=Trametes Abietis Karst.), which by some is regarded as a form of T. Pini, has never, to the writer's knowledge, been collected within the state. SPECIES DOUBTFUL OR EXCLUDED T. nivosus Berk. was erroneously reported from Ohio by Morgan. It is a tropical and subtropical species. DAEDALEA Pers. ex. Fries, Syst. Мус. т: 331. 1821; Pers. Syn. Fung. 499. 1801. Plants annual or rarely reviving for two or three years, ses- sile or effused-reflexed, growing on wood; pileus coriaceous to corky in texture, not encrusted; context white or whitish, fibrous or corky; hymenium typically daedaloid or labyrinthi- form, but sometimes poroid, irpiciform or lamellate; spores white. KEY TO THE SPECIES Pileus small, thin and coriaceous, hirsute or villous; hymenium at first sin- uous and dedaloid but soon breaking up into teeth....... 1. D. unicolor Pileus rather large and thick, corky, minutely velvety or glabrous; hyme- nium poroid, dedaloid, or somewhat lamellate but never breaking up iio ВИИ аа ое 224 000 2 2. Mouths of the tubes less than 1 mm. Ьгоай...................... 2. D. ambigua 2. Mouths of the tubes more than 1 mm. Әтодай........................... 3 3. Pileus less than 1.5 cm. thick; walls of the tubes thin; plant found abun- БЕР OR KUUS CQ OPER CORNER UESTRE. 3. D. confragosa 3. Pileus more than 1.5 em. thick; walls of the tubes thick; plant growing ба Quercus and Castanen. ......... Leurs ODER 4. D. quercina I. D. unicolor Bull. ex Fries, Syst. Myc. т: 336. 1821. Boletus unicolor Bull. Herb. Fr. pl. 408. 1788. Plants annual or sometimes the marginal hyphe reviving and continuing growth the second year, sessile, or effused-reflexed, imbricate; pileus dimidiate to flabelliform, 0.5-5 x 2-8 x 0.2- 0.5 em., eoriaceous, white to cinereous or light brown, some- times green from a covering of alge, villous or hirsute, zonate ог concentrically furrowed, margin thin, acute, sterile below; con- text white or pallid, fibrous, less than 1 mm. thick; tubes 1-4 mm. long, the mouths white to cinereous or umber, at first 10 [Vor. 1 144 ANNALS OF THE MISSOURI BOTANICAL GARDEN deedaloid and sinuous, but soon breaking up into teeth—though retaining the sinuous character at the margin of the pileus—, averaging about 2 to a mm. On dead wood. Common. This plant may at first prove puzzling to the collector, as it was to me when first collected, for the thin, flexible pileus and the usually toothed hymenium indicate a close relationship with the thin coriaceous species of Polyporus, or even with Гтрех. But the pores are decidedly sinuous, at least in young plants. Тһе thin pileus and the hirsute or villous pubescence separate the species from other members of the genus. 2. D. ambigua Berk. Lond. Jour. Bot. 4:305. 1845. Trametes lactea Berk. Hooker's Lond. Jour. Bot. 4: 305. 1845. Plants annual or rarely reviving for two or three years, sessile, sometimes appearing substipitate; pileus dimidiate to reniform, 3-14 x 5-20 x 0.3-1.5 cm., slightly flexible when fresh, сотку when dry, pure white to umbrinous, sometimes purplish black at the base, minutely velvety to glabrous, azonate or subzonate on the margin, margin rather thin, acute; context white or pal- lid, floccose-punky to corky, 0.2-1 em. thick; tubes 2-4 mm. long, sometimes stratified in two or three layers, mouths whit- ish or yellowish, circular to sinuous and dedaloid, never lamellate, averaging 2-3 to a mm. in transverse direction, walls rather thick and entire. On stumps and trunks of deciduous trees. Common. Distinguished from D. confragosa Bolt. ex Fries by the white color, the white context, the smaller pores and the habitat. Hard (Mushrooms /. 355-56) gives excellent illustrations of the plant. 3. D. confragosa Bolt. ex Fries, Syst. Мус. 1:336. 1821. Boletus confragosus Bolt. Hist. Fung. Suppl. 3:160. 1791. Lenzites Crategi Berk. Hooker's Lond. Jour. Bot. 6: 323. 1847. Plants annual, sessile; pileus dimidiate, 2-10 x 3-15 x 0.2-1.5 cm., slightly flexible to rigid, grayish or cinereous, rarely slightly brownish, minutely tomentose to glabrous, zonate, margin thin and acute; context whitish, floccose to corky, 0.2-1 cm. thick; tubes 0.1-1 em. long, mouths whitish to cinereous, sometimes slightly reddish, darker when bruised, subcircular at times but usually sinuous, dzedaloid, or labyrinthiform, sometimes becom- 1914] OVERHOLTS— THE РОГУРОВАСЕЖ OF OHIO 145 ing lamellate in old plants, 0.5-1.5 mm. broad; spores white, smooth, oblong, mostly curved, 1.5-2 x 6.2-7.5 u. On dead wood or on living trees, especially of байх. Com- mon. This is a very variable species. Sometimes very thin forms are found and such have been considered as species at different times. T'rametes rubescens Alb. & Schw. ex Fries is a thin form with a reddish hymenium. For illustrations, see Hard, Mushrooms f. 358., White, Hymen. Conn. pl. 34. f. 2., and Moffatt, Higher fungi of the Chicago region pl. 18. 4. D. quercina Г. ex Fries, Syst. Myc. т: 333. 1821. Agaricus quercinus L. Sp. Plant. 1176. 1753. Plants annual, or sometimes reviving, sessile; pileus dimid- iate, convex, 4-12 x 4-15 x 1.5-6 em., corky, whitish to umbri- nous or almost black, glabrous, margin usually thick and obtuse : context whitish, corky, 0.2-1 em. thick; tubes 1-2 em. long, the mouths whitish to umber, rarely circular, more often labyrin- thiform and elongate or lamellate, 1 mm. or more broad, edges thick and entire. On Castanea and Quercus, sometimes on the living trees. Rare. This species is distinct from all of the others in its habitat, the thickness of the pileus, and the larger sinuous pores. Hard (Mushrooms f. 357), and White (Hymen. Conn. pL. 84. f. 1) give illustrations of the plant. LENZITES Fries, Gen. Hymen. 10. 1836. Pileus coriaceous to corky, dry and floccose in texture. Lamelle coriaceous, firm, sometimes simple and unequal, sometimes anastomosing behind and forming pores; trama floc- cose and similar to the pileus, the edge subacute. Dimidiate, sessile, persistent fungi growing on wood and resembling Dedalea. (The above description is according to Fries, Ерісг. Syst. Myc. 403.) This genus is intermediate in position between the Agarica- cee and the Polyporacee and is sometimes included among the white spored genera of the former family. [Vor. 1 146 ANNALS OF THE MISSOURI BOTANICAL GARDEN KEY TO THE SPECIES Context «Мйе............... ЗР т т oe Pere у ОЕ 1. L. betulina Contest БОИ... аки токов еее sees 1 1. Tubes оғ interspaces 1 mm. or more broad; lamelle usually not much anas- UT erence Soe eka eae ek ee е” 3. L. sepiaria 1. Tubes or interspaces less than 1 mm. broad; lamell freely anastomosing. . . . . 2. L. vialis т. L. betulina L. ex Fries, Epicr. Syst. Myc. 405. 1838. Agaricus betulinus L. Sp. Plant. 1176. 1753. Plant annual, sessile, often imbricate; pileus dimidiate, 2-5 x 2-9 x 0.3-1 cm., coriaceous to somewhat corky, prevailing color grayish to brownish, marked with many narrow, multi- colored zones, tomentose; margin thin and acute; context white, usually not more than 1 mm. thick; hymenium usually lamellate but sometimes poroid, the lamelle coriaceous, about 1 mm. apart, 0.3-1 em. broad, white or whitish; spores globose, smooth, hyaline, 5-6 шіп diameter. On all kinds of dead wood of deciduous trees. Common. L. flaccida Fries as reported from Ohio is but a form of this species. The plant is well represented by Hard (Mushrooms f. 185-86), Lloyd (Photogr. pl. 14), and fairly well in Cooke, Illustrations pl. 1125. 2. L. vialis Peck, Ann. Rept. N. Y. State Mus. 26: 67. 1874. Dedalea pallido-fulva Berk. Hooker’s Lond. Jour. Bot. 6: 322. 1847. Pileus annual, sessile, imbricate or single, dimidiate or lat- erally connate, 1-4 x 2-7 x 0.2-0.8 cm., coriaceous or corky, grayish brown to cinnamon-brown, often darker at the base, subtomentose to glabrous, azonate or subzonate, margin acute; context brown, floccose-fibrous to soft-corky, 1-4 mm. thick; hymenium more or less poroid or labyrinthiform, rarely decid- edly lamellate, pores or lamelle averaging 2-3 to a mm., 1-3 mm. broad, concolorous with the surface of the pileus; spores cylindrical to elliptical, smooth, hyaline, 2.7—4 x 7-8.2 и. On dead wood of both deciduous and coniferous trees. Com- mon. The species was described from Ohio as Dedalea pallido- fulva Berk. and so reported by Morgan. 1914] OVERHOLTS—THE РОГУРОКАСЕЖ OF OHIO 147 3. L. saepiaria Fries, Epicr. Syst. Myc. 407. 1838. Dedalea sepiaria Fr. Obs. Мус. т: 105. 1815. Plants annual, sessile, often imbricate; pileus dimidiate or reniform, 1-5 x 2-7 x 0.3-1 cm., coriaceous to corky, bright yellowish red to dark ferruginous, often lighter or discolored with age, strigose-tomentose, zonate, margin thin; context fulvous to ferruginous, floccose to soft-corky, not more than 3 mm. thick; hymenium usually lamellate, the lamelle about 1 mm. apart, 2-5 mm. broad, rarely anastomosing, fulvous to rusty brown; spores cylindrical, smooth, white, 2.7-4 x 2-10.2 и. Always found on dead wood of coniferous trees. Frequent. Easily distinguished from the preceding species by the deeper color throughout and by the more distant lamelle that rarely anastomose. CYCLOMYCES Kunz. & Fries, Linnea 5: 512. 1830. Plants annual, terrestrial and stipitate in our species, coria- ceous, fuscous or cinnamon-colored; context brownish, some- times rusty brown, floccose to fibrous; hymenium poroid at first but soon breaking up into concentric lamelle. The genus is distinct from all others in the concentric arrange- ment of the lamelle. т. С. Greenei Berk. Hooker’s Lond. Jour. Bot. 4: 306. 1845. Pileus stipitate, circular in outline, usually depressed on бор, 2.5-9 em. broad, 0.5-2 em. thick, coriaceous when fresh, rigid when dry, yellowish brown to rusty or purplish brown, tomentose at first but becoming glabrous, more or less zonate, margin thin and acute; context fulvous to cinnamon-brown, soft floccose to fibrous or somewhat friable, thin at the margin, thicker next the stipe; tubes 5-8 mm. long, soon breaking up to form brownish concentric lamellz; stipe central or subcentral, expanding above into the pileus, velvety, somewhat spongy, 2-7 em. long, 0.7-2 cm. thick, fulvous to rusty brown in color. On the ground in woods. Rare. The species was reported from Ohio by Hard but I think has not otherwise been collected. For illustration see Hard, Mush- rooms f. 360-61. [Vor. 1 148 ANNALS OF THE MISSOURI BOTANICAL GARDEN FAVOLUS Fries, Elench. Fung. 1: 44. 1828. Plants annual, epixylous, more or less stipitate; pileus fleshy- tough when fresh, small or medium sized; context white, thin ; tubes in a single layer, the mouths angular, usually hexagonal, often radiating outward from the stipe and somewhat longer in the radial direction; spores white. In our species the stipe is much reduced and is usually lateral or at least eccentric. The genus is separated from Polyporus by the large favoloid pores, although some stipitate species of Polyporus closely approach in pore form the condition ascribed to this family. KEY TO THE SPECIES Plants about 2 cm. long and broad; hymenium more or less waxy or gelat- Е ee lI Loa 1. F. rhipidium Plants larger than above; hymenium not gelatinous or waxy.....2. F. canadensis 1. Е. rhipidium Berk. Hooker’s Lond. Jour. Bot. 6:319. 1847. Plants stipitate; pileus reniform, ceespitose-imbricate, 2 cm. long and broad, coriaceous, alutaceous to white, the cuticle breaking up into minute furfuraceous squamules, concentrically suleate; context whitish, thin; tubes short, less than 2 mm. long, more or less waxy and gelatinous, the mouths white, angular to elongate, denticulate, averaging 2-3 to а mm.; stipe lateral, pruinose, 6-7 mm. long. On dead wood. Rare. The above description is adapted from the original. Тһе species was originally described from Ohio from specimens collected by Lea. Morgan also probably collected it, but otherwise it is not known from the state. In habit and color it resembles Panus stypticus. 2. Е. canadensis Klotzsch, Linnea 7:197. 1832. F. ohiensis Berk. & Mont. Syll. Crypt. 171. 1856. F. striatulus Ellis & Ev. Am. Nat. 31: 339. 1850. Plants stipitate, the stipe often reduced to a lateral tubercle; pileus dimidiate to reniform, 1-4 x 1- 8x 0.1-0.7 cm., fleshy- tough when fresh, rigid when dry, at first reddish brown due to the presence of innate fibrils of that color, later becoming glabrous and fading to cream color or pure white, azonate, 19141 OVERHOLTS—THE POLYPORACEJE OF OHIO 149 margin thin and acute, often involute, especially on drying; context white or whitish, fleshy-tough, becoming firmer on drying, 0.5-2 mm. thick; tubes 1-5 mm. long, the mouths whit- ish to yellowish, distinctly angular, usually rhomboid or hex- agonal, often radiating outward from the stem and longer in the radial direction, very variable in size, 0.5-3 mm. long and averaging 1-3 to а mm. in transverse direction; stipe lateral or rarely subcentral, often rudimentary, not more than 1 cm. long, 1.5-7 mm. thick. On dead branches of deciduous trees, especially Hicoria. Common. F. striatulus Ellis & Ev. is supposed to differ from Р. cana- densis in having a pileus white in color from the first, and in the smaller pores. In Ohio both of these forms are found and the writer has come to the conclusion that F. striatulus is to be regarded as only a form of this rather polymorphic species, for the following reasons: First, specimens of F. canadensis fre- quently become whitish in color quite early in development; second, the small pores said to be characteristic of F. striatulus are also frequently found in specimens with the reddish brown pileus. In attempting to separate the plants into two species one finds reddish brown specimens with either large or small pores, and white specimens with either large or small pores. Тһе species is illustrated in Hard, Mushrooms f. 309. GLOEOPORUS Mont. Hist. Cuba 385. 1838. Plants annual, sessile or effused-reflexed; pileus small, thin and coriaceous; context fibrous, thin, usually white; tubes short, more or less gelatinous or waxy and in our species separating from the context in a thin, elastic layer when fresh or when moistened. Тһе genus is distinct from all others in the gelat- inous and at the same time separable hymenium. Опе species only is found in our flora. 1. G. conchoides Mont. Hist. Cuba pl. 15. f. 1. 1838. Sessile or effused-reflexed; pileus dimidiate or conchate, 0.5-3 x 1—4 x 0.1-0.5 cm., coriaceous when fresh, rigid when dry, white or cream-colored, velvety to glabrous, azonate, margin thin, acute, with a narrow sterile band below; context white, soft-fibrous, 1-4 mm. thick; tubes less than 1 mm. long, [Vor. 1 150 ANNALS OF THE MISSOURI BOTANICAL GARDEN gelatinous or waxy and separating from the context in a thin elastic layer when fresh or when moistened, the mouths flesh- colored to reddish purple or purplish black, circular, minute, averaging 5-6 to a mm. On dead wood of deciduous trees. Common. The waxy separating hymenium, reddish purple in color, will serve to distinguish this species. The plant has been known as Polyporus dichrous Fries. MERULIUS Haller ex Fries, Syst. Myc. т: 326. 1821; Haller, Hist. Stip. Helv. 3: 150. 1768. Hymenophore formed from a mycelial mucedinous context and giving rise to shallow irregular pores formed by the inter- section of obtuse folds of the hymenium; resupinate or pileate, more or less waxy in texture. Growing on rotting wood. This genus is a very natural one and forms a transition stage from the Polyporacee to the Thelephoracee through the genus Phlebia of the Hydnaceew. No special study of the genus has been made and only the two common species are included here, although several others have been reported from the state. KEY TO THE SPECIES Pileus always present, distinctly pinkish red when fresh. ........1. M. rubellus Pileus when present whitish or somewhat flesh-colored but not distinctly CoL o. Peg АМЕ D DEM 2. M. tremellosus I. M. rubellus Peck, Bot. Gaz. 7: 44. 1882. Pileus sessile or effused-reflexed, dimidiate, often imbricate, 8-5 x 5-7.5 x 0.2-0.5 cm., coriaceous-cartilaginous, scarcely waxy or gelatinous, deep pinkish red, often fading with age, finely tomentose, azonate, margin thin, acute; context white or light colored, tough when fresh, soft when dry, 1-4 mm. thick - tubes short, less than 1 mm. long, formed by anastomosing veins, averaging 1-2 to a mm., cream-colored or whitish; spores (teste Peck) minute, elliptical, hyaline 4-5 x 2.5-3 y. On dead wood of deciduous trees. Common. This plant is distinguished from the next one by the firmer consistency and the color, although the color of the pileus often fades in mature plants. Hard (Mushrooms f. 353) gives а good illustration of the plant. Шанағы”. PET NERONE, ue ete УВ " pe iiia ар = ыны қата АН ыса I Naa 1914] OVERHOLTS—THE РОГУРОВАСЕЖ OF OHIO 151 2. M. tremellosus Schrad. ex Fries, Syst. Myc. 1: 327. 1821. М. tremellosus Schrad. Spic. Fl. Ger. 139. 1794. Sessile, effused-reflexed, or entirely resupinate; pileus dimid- iate, 0-5 x 3-8 x 0.1-0.3 cm., fleshy or gelatinous-waxy, white or whitish, tomentose, azonate, margin thin and acute; context whitish, soft, 1-2 mm. thick; tubes very short, formed by anas- tomosing ridges or veins, averaging 1-2 to а mm., whitish or somewhat flesh-colored, in resupinate forms with a wide, thin, sterile border. On old logs in woods. Common. Quite often the plant is entirely resupinate and probably always so in young stages. Тһе form of the hymenium is exceptionally well shown in Atkinson, Mushrooms f. 191-92. Besides the above species, M. lacrymans Jacq. ex Fries has been included in practically every list of fungi reported from the states east of the Mississippi River, but its frequency of occurrence is probably in inverse ratio to the number of times reported. At any rate it is to be considered as a rare fungus in this country. I have never met with specimens in Ohio that I could so refer. IRPEX Fries, Elench. Fung. 1: 142. 1828. Hymenium inferior, dentate-lacerate from the first. Teeth concrete with the pileus, firm, subcoriaceous, acute, reticulately disposed or arranged in rows, in sessile forms connected at the base and gill-like, or favoloid in resupinate forms. Basidia 4-spored. Woody, subsessile or resupinate fungi allied to Lenzites апа Dedalea. (Adapted from Fries, Hymen. Eur. 619.) This genus is sometimes included in the Hydnacee but in at least one of the three species here described the hymenium is not toothed from the first, but is decidedly poroid and shows very close relationships to certain species of the thin pileate members of the genus Polyporus, e. g., P. biformis, P. prolificans etc., in which the hymenium soon becomes broken up into teeth. For this reason and because the plants are very common in our woods the three following species are described and most of the collections usually obtained will be found to answer to one of these descriptions. [Vor. 1 152 ANNALS OF THE MISSOURI BOTANICAL GARDEN KEY TO THE SPECIES Са, ХА; И re LIII 1 р с. 25552555. ымыз 2 1. Context less than 2 mm. thick; tubes or teeth less than 5 mm. long; pileus MEM SU cU ыс. ІЛЕ 1. I. tulipifera 1. Context more than 2 mm. thick; tubes or teeth more than 5 mm. long. .2. J. mollis 2. Hymenium сіппатоп-тоут.......................... 8. I. cinnamomeus 2. Hymenium grayish green to оПуасеойв.................... 4. I. farinaceus 1. I. tulipifera Schw. ex Fries, Epicr. Syst. Myc. 523. 1838. Boletus tulipifera Schw. Syn. Fung. Car. 99. 1822. Plants sessile, effused-reflexed, or entirely resupinate; pileus dimidiate to elongate in outline, 0-1 x 1-3 x 0.1-0.6 cm., cori- aceous, white or whitish, villous, zonate, margin thin and acute; context white, fibrous, 0.5-2 mm. thick; tubes 1-5 mm. long, the mouths light colored, averaging 2 to à mm., soon breaking up into compressed teeth that are connected at the base, and often with a concentric arrangement. On dead wood of deciduous trees. Common. From 1. cinnamomeus Fries, and 1. farinaceus Fries this plant is separated by the white or whitish color, and from 1. mollis Berk. & Curt. by the much thinner pileus and the shorter tubes or teeth. 2. I. mollis Berk. & Curt. Jour. Bot. & Kew Misc. 1: 236. 1849. Pileus sessile or effused-reflexed, dimidiate, 2-5 x 5-10 x 1-3 em., coriaceous, white or whitish, minutely tomentose to glab- rous, azonate, margin thin and acute; context white, 2-6 mm. thick, fibrous; hymenium usually irpiciform, the teeth white, coriaceous, 0.5-1.5 ст. long, compressed, united at the base. On dead wood of deciduous trees. This plant was reported from the Miami valley by Morgan. I have not collected it in Ohio. It is much thicker than J. tulip- ifera Schw. ex Fries, and the teeth are much longer. 3. I. cinnamomeus Fries, Epicr. Syst. Myc. 524. 1838. Pileus none, fungus usually entirely resupinate, coriaceous in texture, 2-5 mm. thick, entirely cinnamon-brown; context brown, not more than 1 mm. thick, fibrous; tubes or teeth 1-5 mm. long, becoming toothed at a very early stage, cinnamon- brown in color, more or less flattened, connected at the base. е7 РА АО 1914] OVERHOLTS—THE POLYPORACEJE OF OHIO 153 On dead wood, especially of species of Acer. Rather com- mon. Distinguished from the other species here listed by the uni- form brown color. 4. I. farinaceus Fries, Linnæa 5: 523. 1830. Pileus sessile, effused-reflexed, or resupinate, dimidiate, 0-0.5 x 1-3 x 0.1-0.3 em., coriaceous, deep brown, finely tomen- tose, zonate, margin thin and acute; context dark brown, fibrous, less than 1 mm. thick; tubes 0.5-1.5 mm. long, mouths usually grayish green or yellowish green, averaging 2-3 to а mm., soon breaking up into teeth. On dead wood of deciduous trees. Not common. Sometimes the fungus is entirely resupinate and then it usually has a narrow brown margin. It is distinct from all of the other species in having a greenish hymenium. INDEX TO THE SPECIES Names in italics are synonyms, rejected species, etc. Page Pag abietinus (Polyporus)............. 91 chioneus (Polyporus)............. 97 Abietis (Trametes) ............... 143 cincinnatus (Polyporus)........... 114 abortivus (Polyporus) ............. 105 cinnabarinus (Polyporus)......... 116 adustus (Polyporus).............. 102 cinnamomeus (Ігрех)............. 152 albellus (Робурогив).............. 97 cinnamomeus (Polyporus) ........ 123 ambigua (Dzdalea).............. 144 circinatus (Polyporus)............ 121 anar (Polgporu)........ eee X8 118 conchatus (Fomes)............... 132 applanatus (Fomes).............. 527 -conchatus (Póolyporus) 212222222. 182 applanatus (Роіуротиз)........... 187 conchifer (Polyporus)............ 93 arculariformis (Polyporus) ........ 107 conchoides (Glooporus).......... 149 arcularius (Polyporus) ............ 107 confragosa (Dedalea)............ 144 conglobatus (Роуротиз)........... 131 badius (Робуротизв)............... 126 connatus (Еотев)............... 129 Berkeleyi (Polyporus)............ 118 -connatus (Polyporus) . 03. 5.5 bcs 122 betulina (Lenzites)............... 146 Cralagi (Lensitet) ........... чаьа 144 betulinus (Polyporus)............ 104 cristatus (Polyporus) ............. 111 biformis (Polyporus)... -ssri i:s 95- Curtisii (Polyporus)... 2/22... 125 borealis (Polyporus).............. 100 cuticularis (Polyporus)........... 118 brumalis (Polyporus)............. 107 delectans (Polyporus)............ 99 cesius (Polyporus)............... 96 dichrous (Роїуротиз)............. 150 canadensis (Favolus)............. 148 distortus (Polyporus)............. 105 carneus (Ботев)... a 131 dryadeus (Polyporus)............ 119 carneus (Polyporue) ......-..- 29 131 dryophilus (Polyporus)........... 120 castanophilus (Polyporus)......... зе Oe CPolyporu8). 22. 121 154 ANNALS OF THE MISSOURI BOTANICAL GARDEN Page Page elegans (Ро]урогив)............. 110 malicola (Trametes)............. 140 endocrocinus (Polyporus) . ......... H5 moli (ires)... our 152 Everhartii (Еотев).............. 194 - жойы (Trametes)... erre 141 molliusculus (Робурогив).......... 95 farinaceus (Ігрех)............... 153 Morgani (РоЇуротиѕ)............. 110 fibula (Ройуроғив)................ 95 fissus (Робурогив)................ 109 nidulans (Polyporus)............. 117 flaccida (Гепгйеа)................ 146 nigricans (Ғотпев)................ 136 flavovirens (Polyporus). .......... 111 nigromarginatus (Coriolus)........ 93 focicola (Polyporus) .............. 142 поми (Trams) ... s 143 fomentarius (Fomes)............. 136 fragrans (Polyporus)............. 103 obesus (Polyporus)............... 121 fraxineus (Ғотев)............... 130 obtusus (Polyporus).............. 100 fraxineus (Робуротив)............. 130 ohieneis (Раюйи);............... 148 fraxinophilus (Fomes)............ 129 ohiensis (Ғотев)................. 128 fraxinophilus (Polyporus)......... 129 ohiensis (Ттатеғев)............... 128 frondosus (Polyporus)............ 112 ovinus (Polyporus) ............... 126 а OUO seen 133 fumosus (Polyporus)............. 103 pallido-fulva (Dedalea)............ 146 pallidus (Робурогив).............. 109 galactinus (Ро]урогив)........... 98 pargamenus (Polyporus).......... 92 giganteus (Polyporus)............ 113 parvulus (Робурогив)............. 122 gilvus (Роіурогив)............... 117 Peckii (Trametes)............... 142 graveolens (Еотев).............. 131 pennsylvanicus (Polyporus)....... 108 Greenei (Cyclomyces)............ 147 perennis (Polyporus)............. 122 guttulatus (Polyporus)........... 100 pergamenus (Polyporus)........... 92 perplexus (Робурогив)............ 118 hirsutulus (Polyporus)............ 92 piceinus (Робурогив)............. 143 hirsutus (РоГурогив)............. 93 рісірев (Polyporus).............. 109 Mepida (Гғотеіев)............... 149 -Pilote (Polyporup......... 115 hispidus (Polyporus)............. 19 Ra cM)... rg 142 hypococcineus (Роурогиз)......... H8 pua (Ром), 130 pocula (Епәйта)................ 106 igniarius (Еотев)................ 135 роса (Polyporus)............... 106 immitus (Робуротив).............. 98 pocula (брћтіа)................. 106 intybaceus (Роіуротиз)............ 126 populinus (Еотев)............... 130 isidioides (Роурогиз)............. iM робо ба... 111 proliferus (Polystictus)............ 123 lacrymans (Merulius)............. 151 prolificans (Polyporus)............ 151 duis ОУН)... 144 puberula (Роһуротив)............. 103 lacteus (Polyporus).............. 97 pubescens (Polyporus)........... 94 lentus (Робуротив)................ 126 leucomelas (Робуротив)............ 126 quercina (Dedalea).............. 145 leucopheus (Робурогив)........... 137 ибен Bec | 0.0.06: 137 radiatus (Polyporus)............. 118 lobatus (Роһюрона)............... 137 radicatus (Polyporus)............ 110 Lloydii (Polyporus).............. 95 ramosissima (Grifola) ............. 112 lucidus (Polyporus).............. 193 reniformis (Polyporus) . ........ 137 resinosus (Polyporus)............ 116 maculatus (Робуротив)............ 100 rhipidium (Favolus).............. 148 1914) OVERHOLTS—THE POLYPORACE OF OHIO 155 Page Page rigida (Trametes) ......... eee 141 striatulus (Favolus) .......... +, 148 rimosus (Ғотев)................. 133 suaveolens (Tramete8)...........- 140 robinie (Рутороіуротиз)........... 133 subperforatum (Ganoderma) . ....... 123 robiniophila (Роһурогав).......... 104 subsericeus (Робурогив)........... 123 robiniophila (Ттатейев)........... 104 Sullivantà (Polyporus)..........-- 94 roseus (Еотев).................. 131 sulphureus (Роһурогив)........... 114 Rostkowii (Роһурогив)............ 109 Sumstinei (Grifola) ........... 113 rubellus (МегаНив).............. 150 supinus (Еотев)................. 133 rubescens (Ттатейев).............. 145 rufescens (Робурогив)............. 106 tomentosus (Роїуротиѕ)............ 121 tremellosus (МегаШав)........... 151 &epiaria (Lenzites).........+-+++5 147 tulipifera (Ігрех)................ 152 salicinus (Еотев)................ 133 sanguineus (Polyporus)..........- 115 umbellatus (Polyporus)........... 112 Schweinitzii (Polyporus).......... 120 unicolor (Dzdalea)............-- 143 scutellatus (Fomes).......... 128 scutellatus (Робурогив)............ 198 varius (Ройурогив)............... 110 semipileatus (Polyporus)......... 96 velutinus (Робурогив)............. 93 sepium (Ттатпе(ев)............... 139 versicolor (Ро1урогав)............ 91 serialis (Робурогив).............. 139 vialis (Lenzites) .......... ees 146 gerialis (Trametes).......... 139 virgineus (Ройурогив)............. 94 sessile (бапойетта).............. 123 volvatus (Роурогив)............. 105 Spraguei (Роурогав)............. 101 gpumeus (Polyporus). ......... 99 zonalis (Роіурогив).............. 101 squamosus (Polyporus).........-- 109 zonatus (Роһуротив).............. 91 Graduate Laboratory, Missouri Botanical Garden. Annals of the Missouri Botanical Garden Vor. I MAY, 1914 No. 2 A CONTRIBUTION TO OUR KNOWLEDGE OF THE RELATION OF CERTAIN SPECIES OF GRASS- GREEN ALGÆ TO ELEMENTARY NITROGEN JACOB R. SCHRAMM Assistant to the Director of the Missouri Botanical Garden Instructor in the Henry Shaw School of Botany of ` Washington University А general survey of the literature pertaining to the relation of alge to free atmospheric nitrogen reveals the fact that com- paratively few forms have been experimented with under conditions which render the conclusions reached free from objection. Тһе principal fault which may be found with most of the work done is that the experiments were carried out with impure cultures. Representatives from not more than four or five genera of green alge have thus far been studied in pure eulture, and while the general conclusion reached is that these forms are unable to fix free atmospheric nitrogen either in the presence or in the absence of combined nitrogen and energy- furnishing materials, it is by no means certain that forms do not exist which, under one or all of these conditions, are able to utilize elementary nitrogen. This thought is especially justified when the small number of free-nitrogen-fixing species among the bacteria is considered. In the present investigation, there- fore, an attempt has been made to extend the observations over a greater variety of forms in pure culture, —understanding by the latter a single species of alga free from all other organisms. ANN. Мо. Вот. GARD., VOL. 1, 1914 (157) [Vor. 1 158 ANNALS OF THE MISSOURI BOTANICAL GARDEN HISTORICAL As early as 1854 Laurent (20, 21), and Morren (24) occupied themselves indirectly with the relation of alge to free atmos- рһегіс nitrogen. Morren was led to the conclusion that the sudden death of cultures of infusoria and alge was due to the insufficient quantity of combined nitrogen furnished when the number of organisms became considerable. The nitrogen requirement, he found, could be satisfied by ammonium car- bonate, organic nitrogenous compounds (decaying insects), and other nitrogenous substances in the water; but in no case did he find that free nitrogen from the atmosphere could serve as the source of nitrogen. While it is difficult to say with what organisms Morren worked, it is altogether probable that mem- bers of the Volvocacee were present among his "green," "brown," and “тей іпѓиѕогіа.” No additional contribution to the subject, so far as the author is aware, was made until the appearance of Frank’s paper (9) in 1888. In his investigation of the question of a possible fixation of free atmospheric nitrogen in natural soil without the instrumentality of cultivated plants, Frank exposed samples of unsterilized soil, poor in organic matter, in containers under a glass roof, watering them only with distilled water. During the 134 days that the experiment was continued, no phanero- gams appeared, but in all cases the surfaces of the soil samples became covered with a thin, crustlike, greenish layer composed of ‘‘zwei spangrüne Oscillariaformen, die eine dick-, die andere sehr dünnfádig; ferner grünes Chlorococcum humicola, viel- leicht auch Pleurococcus, sowie Vorkeimfüden von Моовеп, also kryptogame Gewáüchse . . . Diatomaceen waren nicht zu finden." Analysis showed an undoubted increase іп total nitrogen in the experiments. No increase in the nitrate content was observed,—the additional nitrogen being wholly in the form of organic nitrogenous compounds. These facts led the author to the conclusion that the abundance of algal cells, which are rich in protoplasm and therefore in organic nitrogen, accounts for the presence of the increased nitrogen in an organic form. That the appearance of the nitrogen in an organic form (algal substance) does not represent the primary fixation of free nitro- 1914] SCHRAMM—GRASS-GREEN ALGJE AND ELEMENTARY NITROGEN 159 gen and that the latter depends оп an inorganic process, the inor- ganic compounds thus produced being subsequently assimilated by the algz, is not rendered probable by later experiments. In these, Frank exposed samples of soil, kept free from vegetation, for long periods of time and at various temperatures. Plant growth was prevented by leaching the samples daily with hot water. In this manner any traces of nitrogen compounds formed were also obtained. Only at high temperatures—too high for plant growth—did he find a slight increase in total nitrogen and therefore believes that this process is of no impor- tance under conditions which admit of plant growth. From these observations Frank concludes that the alge themselves are the immediate agents in the fixation of free atmospheric nitrogen and inclines to extend this faculty to green plants in general. In the same year, Gautier and Drouin (11) ascribed an entirely different function to soil alge. Samples of artificial soils, free from organic material and containing only ammoniacal nitrogen, were exposed in a sheltered position for a considerable period of time. During the progress of the experiments the soil became more or less covered with a layer of green alge (Pleurococcus vulgaris, Protococcus viridis, etc.). Analysis showed, in every case, а loss in total nitrogen, an even greater loss іп ammoniacal nitrogen, and an intermediate gain in organic nitrogen. The authors assumed that the nitrogen lost was in the form of ammonia and that the amount of nitrogen appearing in the organic form was that part of the escaping ammoniacal nitrogen which, in bathing, so to speak, the algal cells on the surface, was absorbed, and subsequently built into organic nitrogen com- pounds. In support of this hypothesis the authors state that in proportion to the intensity of the algal growth loss in total nitrogen was diminished, and the amount of ammoniacal nitro- gen converted into organic nitrogen increased. Gautier and Drouin thus looked upon the alge as fixers of gaseous ammonia, which the soil tends to give off constantly, rather than as direct agents in the fixation of free atmospheric nitrogen. In 1889, Frank (10) made the fixation of elementary nitrogen by soil-inhabiting alge the subject of a special investigation. Four flasks containing sand moistened with distilled water and [Vor. 1 160 ANNALS OF THE MISSOURI BOTANICAL GARDEN plugged with cotton were treated as follows: Two were at once placed in the light; the third was covered with black paper and without further treatment placed with the first two; the fourth was exposed for six hours to a temperature of 100°C. and then placed with the rest. In the first two, rich algal growths de- veloped, composed of two species of Oscillatoria, a blue-green “ Nostoc-Form, " a yellowish green ‘‘Nostoc-Form,”’ a yellowish to pure green Microcystis, and a Gleocapsa. In the third and fourth flasks no growth of any kind developed. Analyses demonstrated that the total nitrogen content in the first two flasks had been doubled, whereas that in the latter two had suffered a distinet loss. Тһе experiments were repeated with unsterilized soil, all air gaining access to the flasks being first passed through sulphuric acid to remove any ammonia present. The same characteristic algal flora developed and analysis again showed a decided increase in total nitrogen. On the basis of these experiments, Frank makes the generalization that the soil, as such, is unable to fix free atmospheric nitrogen, and that when the process does take place, it is effected by means of the vege- tation of low alge which develop in the soil, and which pos- sess the ability of assimilating free gaseous nitrogen into vege- table, nitrogen-containing compounds. He goes still farther and states that the fact that low alge utilize free nitrogen makes it more and more probable that the assimilation of elementary nitrogen is a faculty appertaining to the entire plant world provided with chlorophyll, and that, since the simple algal cell is endowed with this faculty, the thought is justified that the assimilation of free atmospheric nitrogen is as absolute and fundamental a process of the entire plant kingdom as is the assimilation of carbon dioxide. Prantl (27), in cultivating fern prothallia in solutions with and without combined nitrogen, observed that whereas an abundant algal vegetation appeared in the former, only an Anabena, or a Nostoc, grew in the latter. When placed in nitrogen-free media, the blue-green alga always grew abundantly. From this observation, and without analytical data, Prantl assumes that free-nitrogen assimilation had taken place, either a direct one by the alga, or an indirect one in which the alga assimilated the ammonium nitrite which, according to the theory of Schoenbein, 1914) SCHRAMM—GRASS-GREEN ALG AND ELEMENTARY NITROGEN 161 is formed in the vaporization of water. Of interest are the observations by the same author on the unicellular grass-green alge, which he was unable to cultivate in solutions free from combined nitrogen. То these, therefore, he assigned the power of elementary-nitrogen fixation іп а much smaller degree than to Nostoc. Frank's conclusions were confirmed by the work of Schloes- ing and Laurent (31). These investigators supplemented the usual indirect method of analyzing the soil and harvest, with the direct method of determining at the beginning and at the end of the experiment the composition of the atmosphere in which the plants had been growing. To 2000 or 2500-gram quantities of a poor sandy soil 2.5 grams of limestone, 5 grams of a mixture of several rich soils, and a certain volume of a mineral nutrient solution containing, in some cases, a little potassium nitrate were added, and the whole placed in large flasks. In some, seeds of Jerusalem artichoke, oats, peas, and tobaeco were planted; others, to be used as checks, remained unplanted. То each flask were added 5 cc. of a liquid obtained by diluting 5 grams of rich soil with 20 cc. of water. After four- teen weeks, during which time the seeds germinated and pro- duced plants, the direct analytical method, confirmed by the results obtained by the indirect method, showed, except in two checks, an absorption of free atmospheric nitrogen. But the surfaces of the soils, during the progress of the experiments, be- came covered with green, cryptogamic plants, among which were mosses (Bryum, Leptobryum), and alge (Conferva, Oscillatoria, Nitzschia). This fact led the authors to repeat the first series of experiments, in every case suppressing the growth of chloro- phyllous cryptogams by covering the soils with a thin layer of dry, calcined, quartz sand. No trace of alge or mosses appeared, and, except in the case of the peas, no absorption of free atmos- pheric nitrogen was observed. This fact, together with the evident fixation of nitrogen in the checks of the first series (in which an abundant chlorophyllous cryptogamic vegetation but no phanerogamic vegetation developed), and the absence of fixation in those checks in which little or no algal growth devel- oped, led Schloesing and Laurent to conclude that there are some ‘‘inferior green plants” which are able to utilize free atmos- [VoL. 1 162 ANNALS OF THE MISSOURI BOTANICAL GARDEN pheric nitrogen. In the same year, Gautier and Drouin (12) reasserted their former conclusion as to the rôle of algæ in nitro- gen fixation, holding that the methods of those who adhere to the opinion that algæ fix free nitrogen are too faulty to make conclusions drawn from them convincing. In the work reported by Schloesing and Laurent in 1892 (32, 33) an attempt was made to reduce the complexity of the algal cultures by introducing into a single experiment only one or at most a few species of the algæ. All cultures were made on 600-gram quantities of either a subsoil or quartz sand to which was added (except in the two checks) a small quantity of an infusion prepared from soils. The cultures were allowed to de- velop for from three to six months, and, as in the previous experi- ments of these authors, analyses were made both of the contained atmosphere and of the soil and algal growth. The chlorophyl- lous plants which appeared in the various cultures are described as follows: т and 11—essentially a mixture of Nostoc puncti- forme Hariot and Nostoc minutum Desmazières, with a few colonies of Cylindrospermum majus Kuetz.; 111—almost а pure culture of Nostoc punctiforme; tv—Nostoc punctiforme (less pure than in ш), one colony of Phormidium papyraceum, and a small quantity of Nostoc minutum; v—two mosses—Brachy- thecium rutabulum and Barbula muralis; vi—an almost pure culture of an Oscillariee and Microcoleus vaginatus, with traces of T'etraspora, Protococcus, Stichococcus, Ulothrix, and Lyngbya; үп and vir—checks with no growths, or at most a few small patches of Phormidium autumnale Gomont and Nostoc punctiforme. Both analytical methods showed abun- dant nitrogen fixation in the first four cultures but not an ap- preciable one in the fifth,—a fact which the authors explain on the basis of specific differences in plants in their ability to fix free atmospheric nitrogen. The checks showed no appreciable fixation. Separate analyses were made of the top-soil layers, containing the algal growths, and the deeper layers, the in- creased nitrogen being found in the algal stratum,—a fact which the authors consider important in proving that the alge were responsible for the free-nitrogen fixation. In conclusion, Schloe- sing and Laurent admit the possibility that the bacteria present in the cultures had something to do with the fixation of free 1914) SCHRAMM—GRASS-GREEN ALGÆ AND ELEMENTARY NITROGEN 163 nitrogen, and state that it is not possible to affirm with certainty that the alge, free from other organisms, are able to effect fixation. Having observed, however, but few bacteria in the cultures they conclude that the alge after all are the active agents in the fixation of elementary nitrogen. Similar results were obtained by Koch and Kossowitsch (17). Sixty grams of washed, calcined sand were placed in large Erlen- meyer flasks and moistened with a mineral nutrient solution free from combined nitrogen. Since previous experiments had shown that algæ do not grow on sand free from combined nitro- gen, 0.04 gram of calcium nitrate dissolved in 50 cc. of water were added to each flask. After inoculation with a suspension of algal cells obtained from heaps of lime, а continuous slow stream of air, washed in sulphurie acid, was passed through all the flasks. Three cultures were placed in a north window, three in the dark (to determine whether the bacteria contained in the cultures fixed free nitrogen), and the remainder were used in determining the initial total nitrogen. After fifteen weeks, during which time a rich algal vegetation! developed on all cultures exposed to the light, the contents of the flasks were analyzed in toto. Those exposed to the light showed an un- doubted increase in total nitrogen, whereas those in the dark showed a slight loss in each case. Of particular interest was one culture which was brought into the light after it had remained in the dark for a considerable length of time. After the removal, a moderate growth of alge appeared, and analysis showed a slight gain in total nitrogen, which, however, was less than that found in the cultures which had been exposed to the light during the entire period. In agreement with the earlier workers, these authors ascribed to alge the faculty of free-nitrogen fixation, and emphasized the observation that the extent of this fixation was directly proportional to the intensity of the algal develop- ment. Petermann (26) reached a similar conclusion on the basis of experiments conducted on sterilized and unsterilized soils, which were respectively inoculated and uninoculated with alge. The former in each case showed a distinct gain in nitro- gen, whereas the latter showed either no increase or a slight loss. 1 The authors failed to state what асе developed, merely mentioning the presence of green and blue-green forms. [Vor. 1 164 ANNALS OF THE MISSOURI BOTANICAL GARDEN Incidental to his work on the respiratory quotient in alge, Sehloesing (30) reported that in а culture containing prin- cipally Protococcus vulgaris Ag., and smaller quantities of Chlorococcum infusionum Menegh., Ulothrix subtilis Kütz., and Scenedesmus quadricauda Bréb., there was at the end of two months no diminution of nitrogen in the supernatant atmos- phere. This fact led the author to place these alge among those forms which do not fix free atmospheric nitrogen. As will have been observed, the work reported upon in the contributions cited was done with impure cultures. While in some cases but a single species was used, bacteria were present in all cases. Although in many instances this is not expressly stated, the author’s experience convinces him that the technique employed by these earlier workers made the contamination of their cultures with bacteria very probable. It is evident, therefore, that in the work done thus far it is impossible to state with certainty whether the results obtained are due to the activity of the alge, or to the bacteria, or to both. The first work done on the fixation of free nitrogen by alge in which pure cultures were used was that of Kossowitsch (18), in 1894. The only form isolated in pure culture by this investi- gator was one which he states resembled both Cystococcus (Nägeli) and Chlorella vulgaris Bey. He leaves its identity uncertain but designates it, for convenience, Cystococcus. Pre- liminary experiments with impure cultures of this alga had demonstrated that asparagin and ammonium tartrate could not serve as the source of nitrogen and that growth took place only when nitrates were supplied. In the experiments with pure cultures, flasks containing 70 grams of clean sand moistened with a mineral nutrient solution containing a known amount of calcium nitrate were inoculated with a carefully tested pure culture of Cystococcus and allowed to remain four months. To a number of the cultures dextrose was added, and to others, in addition to this sugar, pea-tubercle bacteria. At the con- clusion of the experiments the cultures were carefully tested for purity. Analysis in every case showed an absence of free- nitrogen fixation, and demonstrated clearly for the first time that an alga, Cystococcus, under the conditions realized in the experiment, did not fix free atmospheric nitrogen. That the 1914) SCHRAMM—GRASS-GREEN ALGJE AND ELEMENTARY NITROGEN 165 same holds true for this alga in nature seemed probable to Kossowitsch, who found that it grew vigorously only so long as а nitrate was present. He further observed that after growth had ceased in any culture, it was promptly resumed upon the addition of a nitrate solution, but not when the nitrogen-free nutrient solution was added. Similar cultures were started in which the inoculation material was either a mixture of alge and bacteria derived from soil or lime, or à mixture of soil bac- teria with a pure culture of Cystococcus. In each case the cul- tures were set up with and without dextrose. Table gives the results of these experiments. TABLE I RESULTS OF KOSSOWITSCH'S EXPERIMENTS WITH PURE AND MIXED CULTURES Mg. of N in cultures + or — Sugar Content of cultures Initial Final — Cystococcus (pure culture) 2.6 rd РЕ 2.6 2.7 — Cystococcus, Phormidium, soil bacteria, 2.6 7.1 -- moulds 2.6 9.5 — Pure Cystococcus culture and bacteria 2.6 3.1 - 2.6 8.1 — Stichococcus and bacteria 2.6 2.3 4+ 2.6 2.7 -— Nostoc, large round alga, Scenedesmus, 2.6 ? -- soil bacteria 2.6 19.1 -- Nostoc, а Cylindrospermum (small 2.6 8.8 -- form), soil bacteria 2.6 25.4 Cystococcus, in pure culture, was again unable to fix free gaseous nitrogen, and the same conclusion is reached by Kosso- witsch for Stichococcus, which even in the presence of a mixture of bacteria failed to fix elementary nitrogen. Of especial in- terest are the cultures of pure Cystococcus with bacteria, as in these the fixation is ascribable only to the bacteria. Which of the organisms in the remaining cultures are responsible for [Vor. 1 166 ANNALS OF THE MISSOURI BOTANICAL GARDEN the marked fixation of free atmospheric nitrogen it is impossible to say, the author states. However, from his own results, and those of previous investigators, that the presence of algee exer- cises а favorable effect on the process of free-nitrogen fixation, and, further, that the alge thus far studied in pure culture do not possess this faculty of fixation, Kossowitsch concludes that the alge play an indirect róle. Не believes they do this by furnishing, through their photosynthetic activity, carbohydrates to the nitrogen-assimilating bacteria. He would look upon the alg: as occupying the same position with reference to free-living, nitrogen-fixing bacteria as the legumes do with reference to the nodule organisms. Stocklasa (35), while not making his conclusion very clear, leads one to believe that he considers certain algæ (which he fails to enumerate) capable of fixing free atmospheric nitrogen. Unfortunately, all of Stocklasa’s experiments were carried out with impure cultures. Molisch (23), in conducting experiments with alge relative to the necessary nutrient elements, attempted to cultivate Microthamnion Kiitzingianum Näg., Stichococcus bacillaris Nàg., S. major Rbh., Ulothrix subtilis (?) Kütz., and Protococcus sp.—all in impure culture—on a nitrogen-free mineral nutrient solution. In every case the alge failed to grow, and Molisch was led to the conclusion that alge require com- bined nitrogen for their development. Although no ехрегі- ments in which combined nitrogen was furnished to the algee were conducted, the author nevertheless makes the statement, based principally on the work of Kossowitsch just reviewed, that algee are not able to fix free atmospheric nitrogen. In the next year Bouilhac (4) reported that he had succeeded in isolating in pure culture Schizothrix lardacea, Ulothrix flaccida, and Nostoc punctiforme. "Unfortunately, this author does not give a detailed account. of his isolation methods. Six flasks containing а mineral nutrient solution free from combined nitrogen were inoculated with each alga, and to three of each a drop of soil suspension was added. Хо growth whatever devel- oped in any of the Schizothriz and Ulothrix cultures, nor in the Nostoc cultures to which the suspension had not been added. But in those cultures of the latter to which a drop of soil suspen- sion had been added, a splendid growth appeared and in each 1914] SCHRAMM—GRASS-GREEN ALGJE AND ELEMENTARY NITROGEN 167 culture analysis showed a nitrogen fixation of from 11 to 23 milligrams. From a second series (in which the cultural solu- tion contained per liter 0.1 gram arsenic acid in the form of potassium arsenate) a similar result was obtained, with fixation of nitrogen of from 5 to 60 milligrams. Тһе presence of Ulo- thrix or Pleurococcus in addition to the Nostoc and bacteria seemed to have no appreciable effect on the quantity of nitrogen fixed. Bouilhae thus concluded that Schizothrix lardacea and Ulothrix Лассіда (either alone or in the presence of soil bacteria) and Nostoc punctiforme (in the pure state) are unable to fix free atmospherie nitrogen in the absence of combined nitrogen. The abundant fixation in the cultures containing а mixture of Nostoc and soil bacteria is not ascribed by the author to the activity of either organism alone. Richter (28) observed pots of soil with and without plants, some placed in the dark, others in the light. While a rich algal vegetation developed in the latter, none appeared in the former. Only in a few cases was the growth accompanied by a marked free-nitrogen fixation, but in these instances the author believes it due to the alge. Pure cultures were not employed. Benecke (1) contributed some observations made on cultures of Hormi- dium, Vaucheria, Cladophora, and members of the Conjugales,— all containing bacteria. In nitrogen-free cultures there appeared what Benecke termed “nitrogen-hunger,” a condition which is characterized in Hormidium by the production of very long, pale filaments, the cells of which become extremely long and in which the development of the chloroplast is so meager that the cells are almost colorless. Stocklasa (36) found that the “Alinit” bacteria fix free gaseous nitrogen in much larger quantities when grown in the presence of species of Stichococcus and Nostoc. This influence he considers to be due to the pentosans which, according to his belief, are present in large quantities in various alge, and which, because of their ready solubility in water, serve as a favorable energy-furnishing medium for free-nitrogen- fixing bacteria. A noteworthy contribution to the subject is that of Krüger and Schneidewind (19). These authors for the first time con- ducted extensive experiments with a variety of alge in pure culture, including Stichococcus chloranthus, S. major, S. bacil- [Vor. 1 168 ANNALS OF THE MISSOURI BOTANICAL GARDEN laris, and S. sp., the latter isolated from five different sources; Chlorella sp., from the group of which Chlorella vulgaris Bey. is typical (also isolated from five different localities); Chlorella protothecoides and three other isolations of a form or forms belonging to the same group; Chlorothecium saccharophilum and five other isolations of forms belonging to the same group; and lastly, Cystococcus humicola. The media employed by the authors included the following: 1. One per cent dextrose, 0.2 per cent КзРО,, 0.04 per cent Mgs04, 0.02 рег cent CaCl», and 1 drop of a 2 per cent FeCl; solution to each 100 се. of solution. 2. Ignited sand moistened with solution 1. 3. Solution 1 plus 0.25 per cent (ХН.),5О,, and 0.25 per cent NaNO. 4. Ignited sand moistened with solution 3. 5. One-half per cent beef extract, } per cent peptone, and 1 per cent dextrose. Ignited sand moistened with solution 5. Diluted beerwort. Ignited sand moistened with solution 7. Humoüs clay soil plus 35 рет cent sand moistened with distilled water. есет The results obtained were uniform in that the media, free from combined nitrogen, failed to produce a healthy growth, whereas those containing nitrogen in a combined form showed an abundant growth,—some of the alge preferring the nitrogen in an organic and others in an inorganic form. Further, no fixa- tion of free atmospheric nitrogen was noted in any of the cultures. Krüger and Schneidewind conclude that there is a strong probability that all other chlorophyllous soil alge of this kind are unable to fix free atmospheric nitrogen, and, in general, agree with the opinion of Kossowitsch that the soil-inhabiting alge supply the free-living, nitrogen-fixing organisms with the necessary non-nitrogenous, energy-furnishing material. Conclusions similar to those of Kossowitsch were reached by Deherain and Demoussy (8), who succeeded in cultivating blue lupines free from root nodules in humus-free sand, the surface of which became covered with Phormidium autumnale and Ulothrix flaccida in the course of the experiments. The authors 1914) SCHRAMM—GRASS-GREEN ALG! AND ELEMENTARY NITROGEN 169 explained the growth of the lupines by supposing that the soil bacteria fixed free nitrogen at the expense of energy-furnishing organic materials supplied by the alge, and that the nitrogen so fixed in organic form became available to the legumes. A return to the conclusion that members of the Cyanophycee fix free atmospherie nitrogen is found in an investigation by Beyerinck (2). From 1} to 2-liter portions of tap or distilled water containing 0.02 per cent dipotassium acid phosphate were inoculated with 1-2 grams of garden soil, and placed in the light. After several weeks a characteristic growth of blue-green alge developed, containing, among other species, Anabena catenula, a form related to or identical with Nostoc paludosum, and Nostoc sphericum,—all non-motile species of Cyanophyceew. The devel- opment of the blue-green alge іп an almost nitrogen-free medium led Beyerinck, without analytical data, and in spite of the evident contamination of his cultures with soil bacteria, to the con- clusion that the Cyanophycee belong to the class of organisms possessing the faculty of free-nitrogen fixation. He regards the Cyanophycee as the only known organisms capable of synthe- sizing their organie materials from carbon dioxide and free nitrogen, and considers as significant in this connection the observations of Graebner (13) and Treub (37), who found that in the sequence of floras on fresh sand and lava soils, species of Cyanophycee are the first to appear. Cystococcus humicola was once more subjected to a careful investigation by Charpentier (7). His previous experiments had demonstrated that the dry weight of algal growth obtained in liquid glucose media was about one-half that of the weight of glucose consumed, and that 5.14 per cent of this dry weight was nitrogen. He then pointed out that the quantity of nitro- gen furnished by Kossowitsch to his pure cultures of Cystococcus humicola in the form of potassium nitrate was sufficient to produce at least 40 milligrams of growth (dry weight), and that while this growth was being produced it might not be neces- sary for the alga to seek nitrogen from the atmosphere. Once the dextrose was exhausted, the alga might, it is true, develop at the expense of atmospheric carbon dioxide, but the author holds the opinion that this would mean a double expenditure of energy for the assimilation of both carbon dioxide and free nitrogen and [Von 1 170 ANNALS OF THE MISSOURI BOTANICAL GARDEN that under these conditions growth would be difficult. Because of the vast amount of energy necessary for free-nitrogen fixation, as illustrated by Clostridium Pasteurianum, the author suggests that there is a strong probability that Cystococcus is capable of assimilating free nitrogen only when the expenditure of energy in carbon assimilation is reduced to a minimum,—that is to say, when abundant available organic materials are furnished. He further emphasizes the necessity of employing combined nitrogen in a less readily available form than nitrates, suggesting organic nitrogenous compounds. On media composed of a decoction of beans to which were added 1 per cent and 2 per cent of dextrose and gelatin, respectively, Cystococcus was grown and the entire culture analyzed for total nitrogen. Although care was taken to have an abundance of available organic material (dextrose) present, Charpentier found that in no case was there any indication of free-nitrogen fixation. He further found that ammonia, asparagin, and peptone were each able to serve as the sole source of nitrogen. The association of blue-green algæ and soil bacteria is again referred to as an effective agent in free-nitrogen fixation by Bouilhac and Giustiniani (5, 6). Buckwheat, white mustard, corn, and cress were planted in clean sand moistened with a mineral nutrient solution free from combined nitrogen, and the substrata inoculated with Nostoc punctiforme and Anabæna sp. covered with bacteria. The phanerogams grew to maturity and analysis showed a marked fixation of free atmospheric nitrogen. Of particular interest are the observations of Heinze (14), who, however, fails to state whether or not the Chlorella ex- perimented with was in pure culture. He found that no appre- ciable growth took place in cultural solutions free from combined nitrogen, but that in the presence of the latter a rich growth ap- peared, unaccompanied, however, by a definite fixation of nitro- gen. More important are his experiments with Nostoc in impure condition, a good growth of the form being obtained in a min- eral nutrient solution free from combined nitrogen and sugar. These cultures, as well as others on soil inoculated with a similar Nostoc culture contaminated with bacteria and fungi, showed a definite amount of free-nitrogen fixation. Heinze was unable to find Azotobacter present, and this, together with the observa- 1914) SCHRAMM—GRASS-GREEN ALGJE AND ELEMENTARY NITROGEN 171 tion that the contaminating fungus in pure culture was unable to fix free nitrogen, led the author to the conclusion that the Nostoc is, in all probability, directly responsible for the free- nitrogen fixation. Further, he would place Azotobacter in close relationship with the Chroococcacee, a family in which, he sug- gests, some forms capable of fixing free atmospheric nitrogen may be found. Richter (29), working with pure cultures of Nitzschia palea and Navicula minuscula, reached the conclusion that the former, and probably the latter also, is unable to assimilate elementary nitrogen in the absence of combined nitrogen. Heinze (15), in experimenting with a Nostoc culture which he had purified until it contained as a contamination only a Streptothrix, found that in solutions free from combined nitrogen and sugar but containing respectively mono, di, and tripotassium phosphate, a clearly demonstrable amount of free atmospheric nitrogen was fixed. Тһе Streptothrix was subsequently isolated and tested as to its ability to fix elementary nitrogen, both with and with- out sugar, but always with negative results. In conclusion, Heinze reasserts his former belief that Nostoc is capable of fixing elementary nitrogen. Mameli and Polacci (22) succeeded in growing Oedogonium, Spirogyra, Zygnema, and Protococcus in nutrient solutions free from combined nitrogen, and demonstrated by analysis an increase in total nitrogen. They ascribed to these forms, and to chlorophyllous cells in general, the faculty of synthesizing ammonia from free nitrogen and nascent hydrogen. Pure cul- tures were not used. Boresch (3) found that Phormidium corium Cohn became brown when grown in solutions containing very small amounts of combined nitrogen, but that the green color reappeared following the addition of potassium nitrate or organic nitrogen compounds. Several species of Oscilla- toria, Rivularia, and Chroococcus behaved similarly. But Anabena sp. did not change color even when the solution in which it was growing had become completely exhausted of its combined nitrogen. While the investigation concerns itself primarily with the relation of nitrogen to the color in alge, the observations point once more to species of Anabena as pos- sibly belonging to the class of free-nitrogen-fixing organisms, and [Vor. 1 172 ANNALS OF THE MISSOURI BOTANICAL GARDEN equally elearly to the conclusion that the remaining forms experimented with do not belong to this class. Oes (25) made the observation that Azolla with its endophytie Anabena Azolle grew exceedingly well in mineral nutrient solu- tion free from combined nitrogen. Analysis showed a distinct fixation of nitrogen. Attempts to cultivate the Anabena in pure culture failed. While calling attention to the possible direct róle of the associated bacteria in the observed fixation, the author inclines to the view that Anabena Azolle is itself capable of fixing free atmospherie nitrogen. The preceding survey of literature shows that in all of the earlier investigations, and in a considerable number of the later ones, impure cultures were used. In experiments conducted under these conditions, it is evident that negative results are, in general, more reliable than positive ones. Attention should therefore be called to the negative results which have been ob- tained from investigations with impure cultures. These, as will be seen from the literature cited, include a large number of genera and species from both grass-green and blue-green alge, and indicate in many cases with a reasonable degree of cer- tainty that the faculty of elementary-nitrogen fixation is absent in а very considerable number of species of both the Chloro- phycee and Cyanophycee. In the former class, all investiga- tions conducted with pure cultures have led without exception to the conclusion that these forms are unable to fix free atmos- pherie nitrogen. As regards the Cyanophycee, it should be stated at the outset that while many observations are on record both affirming and denying free-nitrogen fixation in the group, it is questionable whether experiments have been conducted with more than a single species in pure culture. Bouilhac, it is true, claims to have isolated Schizothrix lardacea and Nostoc punctiforme in pure culture. From the meager account given of the isolation technique, it appears very improbable that the latter form was actually obtained in culture free from bacteria, although the former may have been. However, the work of Heinze, while not conducted with pure cultures, renders free-nitrogen fixation in Nostoc probable, and it appears especially desirable, there- 1914) SCHRAMM—GRASS-GREEN ALG AND ELEMENTARY NITROGEN 173 fore, to study representatives from this genus as well as other members of the group Cyanophycee. In the progress of the work about to be reported, it soon be- came obvious that the development of pure culture methods would constitute a very considerable portion of the investigation, and it was deemed advisable to limit the nitrogen phase of the problem to the fixation of atmospheric nitrogen in the complete absence of combined nitrogen, leaving for a subsequent report the problem of elementary-nitrogen fixation in the presence of combined nitrogen. Тһе work concerning pure culture methods will be found reported elsewhere (34). EXPERIMENTAL MATERIALS AND APPARATUS Most of the alge isolated were soil-inhabiting species. Pre- liminary experiments showed that in nearly every case better growth was obtained on solid media than in liquid ones. For this reason it was decided to conduct all experiments concerning the fixation of free nitrogen in the complete absence of combined nitrogen on а solid medium. Agar could not be sufficiently freed from all traces of combined nitrogen, and the difficulties involved in preparing large quantities of silicic acid jelly suffi- ciently pure were so great that a No. 21 ground quartz was finally decided upon. Preparation of the Sand.—The sand, after being thoroughly washed, was boiled in concentrated hydrochlorie acid for two hours, subsequently washed free from chlorides with distilled water, and then heated almost to dull redness for from four to five hours. Тһе sand was then boiled a second time in chemi- cally pure concentrated hydrochlorie acid and again washed with distilled water until chlorides could no longer be detected. When this stage had been reached the washing with distilled water was continued a dozen times more, after which the sand was drained as thoroughly as possible and the washing completed with from five to ten changes of nitrogen-free water. After drying the sand in a clean evaporating dish, a sample was boiled in nitrogen-free water and the liquid tested for ammonia, nitrites and nitrates, but only uniformly negative results were obtained. i | [Vor. 1 174 ANNALS OF THE MISSOURI BOTANICAL GARDEN Nitrogen-free Water.—The distilling apparatus used was, in general, like that described by Jones and Mackay (16) for the preparation of water with a very low electrical conductivity, except that the water was triply distilled in place of doubly, and from glass throughout in place of being condensed in a block tin tube. Fig. 1 represents the distilling apparatus, and it need only be pointed out that flask іп was added to obviate any possibility of contaminating the distillate with spray from flask п. The water obtained from this still gave uniformly negative results when tested for ammonia, nitrites and nitrates. Fig. 1. Distilling apparatus for nitrogen-free water Cultural Apparatus.—One hundred се. flasks, carefully cleaned in acid-dichromate cleaning mixture, rinsed in nitrogen-free water and dried, were connected in series of ten each in the 1912 experiment (eight in the 1913 experiment) by means of glass tubing and rubber stoppers as shown in pl. 3 fig. 1. The glass tubing was cleaned in the same manner as the flasks, and the rubber stoppers were boiled in dilute alkali, then in dilute hydrochloric acid, and subsequently washed with distilled and nitrogen-free water. Into each flask of the 1912 experiment an accurately weighed 40-gram quantity (in the 1913 experiment 1914] SCHRAMM—GRASS-GREEN ALG] AND ELEMENTARY NITROGEN 175 30 grams) of sand was placed. For purposes of aération the separate series of flasks were joined together in groups of five, as shown in pl. 3 fig. 2, and the free end of the common connecting tube provided with three sets of triple wash-bulbs, --іһе two nearest the flasks containing nitrogen-free water, which served to moisten the air after passing through the third bulb containing 25 per cent sulphurie acid. In order to aérate any particular series of flasks it was only necessary to attach а filter pump to the rubber tube at the end of the series which it was desired to aérate and to open the pinchcock until the desired stream of air passed through the wash-bulbs. Chemicals.—The inorganic compounds used were all Baker and Adamson's analyzed chemicals; the organic compounds were Merck's highest purity chemicals. Cultural Solutions.—In the 1912 experiment, in which each series contained ten flasks, the following ten cultural solutions were used and in the following order, the flasks being numbered correspondingly: 1. ХН,ХО; 0.5 grams, MgSO,7H;O 0.2 grams, K;HPO, 0.2 grams, CaClh.?H;O 0.1 grams, FeSO, trace, Nitrogen-free water 1000 grams. 2. The same as No. 1, but containing 0.250 grams of NH,NO; in- stead of 0.5 grams. 3. The same as No. 1, but containing 0.100 grams of NH,NO; in- stead of 0.5 grams. 4. The same as No. 1, but containing 0.050 grams of NH4NO; in- stead of 0.5 grams. The same as No. 1, but free from combined nitrogen. The same as No. 5, but containing 2 per cent d-glucose. The same as No. 3, but containing 2 per cent d-glucose. The same as No. 5, but containing 2 per cent mannite. The same as No. 3, but containing 2 per cent mannite. The same as No. 3, but containing 2 per cent saccharose. ӘӘ o om 1 In the 1913 experiment, in which each series contained eight flasks, the following eight cultural solutions were used: [Vor. 1 176 ANNALS OF THE MISSOURI BOTANICAL GARDEN bt The same as No. 5 in the 1912 experiment. The same as No. 1 in the 1912 experiment. 3. Тһе same as No. 5 in the 1912 experiment, but with 5.26 grams of d-glucose (making a glucose solution isotonic жне а 1 per cent saccharose solution) added. 4. The same as No. 1 in the 1912 experiment, but with 5.26 grams of d-glucose (making a glucose solution isotonic with a 1 per cent saccharose solution) added. 5. Тһе same as No. 5 in the 1912 experiment, but with 5.32 grams of mannite (making a mannite solution isotonie with a 1 per cent saccharose solution) added. 6. The same as No. 1 in the 1912 experiment, but with 5.32 grams of mannite (making а mannite solution isotonie with a 1 per cent saccharose solution) added. 7. The same as No. 5 in the 1912 experiment, but with 10.00 grams of saccharose (making a 1 per cent saccharose solution) added. 8. The same as No. 1 in the 1912 experiment, but with 10.00 grams of saccharose (making a 1 per cent saccharose solution) added. nd Тһе solutions containing the organie compounds were all made isotonie in order to obviate possible differences in growth due to different osmotic pressures of the cultural solutions. The exact volume of solution necessary to just saturate the amount of sand used in each flask was determined and this amount of the various solutions added to the corresponding flasks. Тһе stoppers were then lightly inserted and one group sterilized at a time in a large Kny-Scheerer horizontal autoclav, at six pounds pressure for опе and one-quarter hours. INOCULATION Тһе groups of flasks were transferred directly from the auto- clav to the inoculating room which had previously been''steamed down." All inoculations were made with a DeVilbis atomizer, which, with the exception of the bulb, is made of metal and glass throughout. Тһе bulb was removed and the opening of the metal tip, to which the former is attached, plugged with cotton. After filling the glass container one-half full of solution No. 5 (1912 series), the whole (with the exception of the bulb) 19141 SCHRAMM—GRASS-GREEN ALG AND ELEMENTARY NITROGEN 177 was sterilized. After cooling, the liquid was inoculated with the desired organism (care being taken to avoid introducing any agar, which can readily be done if hard agar cultures are used from which to make the inoculation). Тһе DeVilbis atomizer is provided with an adjustable metal tip so that the spray may be directed downward. The metal tip further admits of steril- ization by flaming. By exercising care and keeping the hands moist with alcohol, comparatively few contaminations result, only four having appeared in a total of 320 inoculations. Attention should be called to the importance of inoculating in such а way that an approximately equal number of organisms are introduced and that they are uniformly distributed over the substratum. Unless this is done growth comparisons cannot be made with any considerable degree of accuracy, as differences may be due to localized and unequal inoculation. This is especially true in alge which do not form motile cells and which, therefore, are unable to spread rapidly over the substratum. GROWTH AND OBSERVATIONS All groups of flasks of the 1912 experiment were placed in the light of north windows at the ordinary room temperature and the cultures aérated at intervals of from three days to а week. The 1913 experiment was set up in duplicate, one-half being placed in a glass incubator kept constantly at from 29.5 to 30.5? C., and the other half in a similar incubator at the ordinary room temperature. Both series of cultures were placed directly in front of a north window and were aérated from time to time. Space will not permit the detailed tabulation of the observa- tions on growth. In the following tables, growth is indicated without reference to time. А few general statements may, however, serve to give some idea as to the relation of the com- position of the cultural medium to the time elapsing before a macroscopically visible growth appeared. In almost every case, growth was observed first on the glucose-containing medium and almost as soon or slightly later on the one containing sac- charose. It should be said, however, that а healthy growth was maintained on these two media, in most cases, for but а short time. Chlamydomonas pisiformis Dil forma minor Spargo is a marked exception in this respect, a splendid, healthy [Vor. 1 178 ANNALS OF THE MISSOURI BOTANICAL GARDEN TABLE II RESULTS OF THE 1912 EXPERIMENT (August 6, 1911—July 1, 1912.) Sol. 1* | Sol. 5 | Sol. 6| Sol. 7 |Sol. 8| Sol. 9 | Sol. 10 (with |(with-|(with-| (with | (with-| (with | (with Organism comb. | out | ош c.n.) out с.п.) с.п.) nitrogen)| c.n.) | c.n.) с.п.) Chlamydomonas pisifor- mis Dill forma minor| ++ – і = |++++| = [4444/4444 Spargo Chlorella sp., large form with clathrate chro-| +++ — - ++ - +++ ++ matophore Kirchneriella sp., a form without marked gela-| + - - ББ - 4- 4- tinous envelope Protosiphon botryoides x Е EN (Kütz.) Klebs ++ t t | +++ қ егі гі» eet ба ба ns Chlorococcum | humicola 22. _ (Nüg.) Rabenh. o4 TED. К-үеғжеғ Chlorella vulgaris Bey. | +++ | -t - ++ = 4- 4+4 Stichococcus bacillaris Nae +++] - | - | +4 ] - |] - - Slight, fair, good, and splendid growths are respectively indicated by +, ++, +++, and ++++. Хо growth is indicated by -. *The mention of growth in solutions 2, 3, and 4 is omitted because growth dif- ferences were not marked. These solutions were introduced in each series in order to note the effect of steadily decreasing quantities of ammonium nitrate on the in- tensity of growth. ТА scarcely detectable growth developed in these cases which, however, disap- peared in all cases within a short time. The growth was so slight as to be notice- able only when the flasks were compared with others absolutely free from growth. growth being maintained for a very long time. Growth on the mannite-containing medium was usually slower in making its appearance, but, in general, remained healthy for a longer period of time than those on glucose or saccharose. With very few exceptions, growth appeared last on the purely syn- thetic medium, but was maintained in a state of vigor longer A Қаш: $ ë : * T 2. LÀ ere И - > 1914] SCHRAMM—GRASS-GREEN ALG AND ELEMENTARY NITROGEN 179 TABLE III RESULTS OF THE 1913 EXPERIMENT (March 29-April 16, 1913.) Sol. 1 | Sol. 2 | Sol. 3| Sol. 4 | Sol. 5 | Sol. 6 | Sol. 7 | Sol. 8 Organism (with-| (with | (with-| (with |(with-| (with | (with-| (with out | c.n.) | out c.n.) out | c.n.) | out c.n.) c.n.) c.n.) c.n.) c.n.) Chlamydomonas pisi- formis Dill forma minor Spargo. Temp. 18.5-24?C. - | ++] =- | +++) | ++ - 4. EY. E 20.5. _ E +4 ds ix ый 4-4 Chlorella sp., large form with clath- rate chromato-| — + -— ++ - + - +++ phore. Temp. 18.5 -242С. Ditto. Temp. 29.5- 30.5°С. р = + т" ++ vd y b c ++ Stichococcus bacillaris AK Temp. 18.5- - + - + - + = ++ Ditto. Temp. 29.5- 30.5°С. Chlorococcum humi- cola (Nàg. Ra- 4 benh. Temp. 18.5 -24?C. Ditto. Temp. 29.5- 30.5°С. s =. йы +++ =" E xd +++ - |++4+4) - | + | - (+++ Protosiphon botryoi- des (Kütz.) Кез. — + - +++! - ++ - dM Temp. 18.5-24°С. Ditto. Temp. 29.5- 3 30.5*C. ES = =| tte] + = 144+ Chlorella vulgarisBey. Temp. т" ni a a eee v Ditto. Temp. 29.5- 30.5°C. Kirchneriella sp. Temp. 18.5-242С. Ditto. Temp. 29.5- 30.5°С. ЖА scarcely detectable and rapidly disappearing growth developed as іп the 1912 experiment. [Vor. 1 180 ANNALS OF THE MISSOURI BOTANICAL GARDEN than on any of the organic-compound-containing media. Glu- cose, вассһатове, and mannite were chosen as energy-furnishing compounds because of their general usefulness in this capacity among free-nitrogen-fixing bacteria, and also because they are representatives from three great classes of carbon compounds. Certain unpublished experiments, carried out by B. M. Dug- gar on nitrogen fixation in the fungi, indicate that, whereas no fixation takes place at ordinary temperatures, it does take place at elevated temperatures. It was thought desirable, therefore, to investigate the effect of elevated temperature on the process of elementary-nitrogen fixation by alge in the absence of combined nitrogen. However, the results tabulated in table пт show clearly that not only did no growth on any nitro- gen-free medium appear at the higher temperature, but also that that appearing on nitrogen-containing media was, in many cases, poorer than that obtained in cultures kept at ordinary temperatures. It should further be noted that growth was in some cases entirely suppressed. It would appear, therefore, that in the species investigated, growth at elevated temperatures is less vigorous than at ordinary temperatures and that, in all probability, no favorable effect on free-nitrogen fixation is to be expected by growing these species at the higher temperature maintained in the experiment. The incipient, ephemeral growth which was observed in a few cases where combined nitrogen was not furnished is be- lieved to be due to the minute quantity of combined nitrogen which was unavoidably introduced in the inoculation process. The inoculating material was, of necessity, derived from agar containing ammonium nitrate, and while no agar was trans- ferred it is altogether probable that enough combined nitrogen was carried over in the water adhering to the cells to account for the trace of growth. It should be emphasized again that in every case this growth was so slight as to have escaped detec- tion had not a comparison been made with a flask absolutely free from growth. In table ту the results of the two experiments are combined and show that in seven species complete results have been ob- tained. These results indicate with perfect uniformity that growth, under the conditions realized in the experiments, is impossible in the absence of combined nitrogen, even when readily ds ei enc oi асы м Ж” X 1914] SCHRAMM—GRASS-GREEN ALG AND ELEMENTARY NITROGEN 181 TABLE IV SUMMATION TABLE OF 1912 AND 1913 EXPERIMENTS (Solutions numbered as in 1913 experiment.) Sol. 1 80]. 2 801.3 | Sol. 4 |80]. 5 | Sol. 6 |Sol.7| Sol. 8 Organism (with-| (with | (with-| (with | (with-| (with |(with-| (with out | c.n.) | ош c.n.) out c.n.) out c.n.) c.n.) c.n.) c.n.) c.n.) Chlamydomonas pisiformis Dill forma im 2” ВЕ — |+ = 1++++| - +++ Spargo Chlorella sp. = MEE — T = +++ "- Bp ер Stichococcus bacil- laris Nag. е ТЕЧ! — TT - ^ = T Chlorococcum hu- micola (Näg.) = Hd = [+++ - t A 5 зи жө дік т Rabenh. Protosiphon botry- oides (Kütz.) = ger = qn = ЕЕ E +++ Klebs Chlorella vulgaris Bey. - ҒҒ = qe = T ме TE Kirchneriella sp. - + - + - + Е 4. assimilable energy-furnishing compounds like glucose, mannite, and saccharose are supplied; and that, therefore, these forms, under the conditions stated, are totally unable to fix free atmos- pheric nitrogen in the complete absence of combined nitrogen. CoNCLUSIONS 1. In agreement with all work that has previously been done on the assimilation of elementary nitrogen by grass-green alge in pure culture, it has been found that Chlamydomonas pisiformis Dill forma minor Spargo, Protosiphon botryoides (Kiitz.) Klebs, Chlorococcum humicola (Nàg.) Rabenh., Chlorella vulgaris Bey., Stichococcus bacillaris Nàg., Chlorella sp., and Kirchneriella sp., are unable to fix free atmospherie nitrogen in the complete [Vor. 1 182 ANNALS OF THE MISSOURI BOTANICAL GARDEN absence of combined nitrogen, under the conditions realized in the experiments. 2. Aslightly elevated temperature (from 5 to 10? C. above the ordinary range of room temperature—18-24?C.) does not, as is the case in certain fungi, enable the alge investigated to fix free gaseous nitrogen in the complete absence of combined nitrogen. In conclusion, the author wishes to express his sincere apprecia- tion and gratitude to Dr. George T. Moore, at whose suggestion the work reported upon in this paper was undertaken and under whose constant attention and generous aid it was carried to completion; to Dr. B. M. Duggar, for many valuable suggestions and innumerable courtesies; to Mildred Spargo Schramm, for kindly encouragement and help throughout the investigation; and to Dr. George R. Hill, Jr., for substantial aid during the last year of the work. BIBLIOGRAPHY 1. Benecke, W. Ueber Kulturbedingungen einiger Algen. Bot. Zeit. 56: 83-97. 1898. 2. Веуегіпек, M. W. Ueber oligonitrophile Mikroben. Centralbl. f. Bakt. II. 7: 561-82. 1901. 3. Boresch, Karl. Die Fürbung von Cyanophyceen und Chlorophyceen in ihrer Ab- hüngigkeit vom Stickstoffgehalt des Substrates. Jahrb. f. wiss. Bot. 52: 145-85. 1913. 4. Bouilhae, R. Sur la fixation de l'azote atmosphérique par l'association des algues et des bactéries. Compt. rend. acad. Paris 123: 828-30. 1896. 5. Bouilhac et Giustiniani, Sur une culture de sarrasin en présence d'un mélange d'algues et des bactéries. Compt. rend. acad. Paris 137: 1274-76. 1903. Я , Sur des cultures de diverses plantes supérieures en présence d'un mélange d'algues et des bactéries. Compt. rend. acad. Paris 138: 293- 96. 1904. 7. Charpentier, P. G. Alimentation azotée d'une algue, le Cystococcus humicola. Ann. Inst. Past. 17: 321-34, 369-420. 1903. 8. Deherain, P.-P., et Demoussy, E. Sur la culture des lupins bleus (Lupinus angustifolius). Compt. rend. acad. Paris 130: 465—69. 1900. | 9. Frank, B. Untersuchungen über die Ernährung der Pflanze mit Stickstoff und über den Kreislauf desselben in der Landwirthschaft. Landw. Jahrb. 17: 421-553. 1888. 10. —————, Ueber den experimentellen Nachweis der Assimilation freien Stick- stoffs durch erdbodenbewohnende Algen. Ber. d. deut. bot. Ges. 7:34-42. 1889. ll. Gautier, Arm., et Drouin, В. Recherches sur la fixation de l'azote par le sol et les végétaux. Compt. rend. acad. Paris 106: 754-57, 863-66, 944-47, 1098-1101, 1174-76, 1232-34. 1888. 12. Я , Sur la fixation de l'azote par le sol arable. Compt. rend. acad. Paris 113: 820-25. 1801. 1914] 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 28. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. SCHRAMM—GRASS-GREEN ALGJE AND ELEMENTARY NITROGEN 183 Graebner, P. Studien über die norddeutsche Heide. Bot. Jahrb. 20: 500- 654. 1895. Heinze, B. Einige Beiträge zur mikrobiologischen Bodenkunde. Centralbl. f. Bakt. II. 16: 640-53, 703-11. 1906. , Ueber die Stickstoffassimilation durch niedere Organismen. Landw. Jahrb. 35: 889-910. 1906. Jones and Mackay, A contribution to the study of water solutions of some of the elements. Am. Chem. Jour. 19: 83-117. 1897. Koch, A., und Kossowitsch, P. Ueber die Assimilation von freiem Stickstoff durch Algen. Bot. Zeit. 51: 321-25. 1893. Kossowitsch, P. Untersuchungen über die Frage, ob die Algen freien Stick- stoff fixiren. Bot. Zeit. 52: 97-116. 1894. Krüger, W., und Schneidewind, W. Sind niedere chlorophyllgrüne Algen im- stande den freien Stickstoff der Atmosphüre zu assimilieren und den Boden an Stickstoff zu bereichern? Landw. Jahrb. 29: 776-804. 1900. Laurent, M. P. Recherches physiologiques sur les animalcules des infusions végétales comparées aux organes élémentaires des végétaux. Paris. 1854. , Recherches sur les Infusoires. Réclamation de priorité addressée à loccasion d'une communication de M. Morren. Compt. rend. acad. Paris 39: 1034. 1854. Машей, E., and Polacci, б. Su l'asimilazione diretta dell’azoto atmosferico libero nei vegetali. Atti Istit. Bot. Pavia 15: 159-257. 1911. Molisch, H. Die Ernührung der Algen (Süsswasseralgen: I Abhandl.). Sitzungs- ber. d. k. Akad. d. Wiss., Wien, math.-naturw. Kl. 104: 783-800. 1895. Morren, M. Пе l'absorption de l'azote par les animalcules et les algues. Compt. rend. acad. Paris 38: 932-34. 1854. Oes, Adolph. Über die Assimilation des freien Stickstoffs durch Azolla. Zeit- schr. f. Bot. 5: 145-63. 1913. Petermann, A. Contribution à la question de l'azote. Bull. Acad. Belg. III. 25:267-76. 1893. Prantl, К. Die Assimilation freien Stickstoffs und der Parasitismus von Nostoc. Hedwigia 28: 135-36. 1889. Richter, O. Zur Frage der Stickstoffernáhrung der Kulturpflanzen. Landw. Vers.-Stat. 51: 221-41. 1898. — —— —., Zur Physiologie der Diatomeen. Sitzungsber. d. k. Akad. d. Wiss., Wien, math.-naturw. Kl. 115: 27-119. 1906. Schloesing, Th. Sur les échanges d'acide carbonique et d’oxygéne entre les plantes et l'atmosphére. Compt. rend. acad. Paris 117: 813-16. 1893. Schloesing, Th. fils, et Laurent, Em. Sur la fixation de l'azote libre par les plantes. Compt. rend. acad. Paris 113: 776-79. 1891. я , Sur la fixation de l'azote libre par les plantes. Compt. rend. acad. Paris 115: 659-61. 1892. j , Recherches sur la fixation de l'azote libre par les plantes. Ann. Inst. Pasteur 6: 65-115, 824-40. 1892. Schramm, J. R. Some pure culture methods in the algae. Ann. Mo. Bot. Gard. 1:23-45. 1914. Stocklasa, J. Studien über die Assimilation elementaren Stickstoffs durch die Pflanzen. Landw.Jahrb. 24:827-63. 1895. , Assimilieren die Alinitbakterien den Luftstickstoff? Centralbl. f. Bakt. II. 5: 350-54. 1899; 6: 22-24. 1900. Treub, M. Notice sur la nouvelle flore de Krakatau. Ann. Jard. Buitenzorg 7: 213-23. 1888. [Vor. 1, 1914| 184 ANNALS OF THE MISSOURI BOTANICAL GARDEN ExPLANATION OF PLATE PLATE 3 Fra. 1. Culture flasks containing quartz sand joined together in series of eight each, but before arrangement into groups. Ес 2. Five series of culture flasks arranged in a group with a common connect- ing tube (onthe left) and a series of three triple wash-bulbs. On the right, the rubber tubing, provided with pinchcocks, is shown attached to each series for use in aération. 3 5 PLATE 1914 $; Mo. Вот. GARD., Vor. ANN. BOSTON Б, COCKAYN THE THELEPHORACE OF NORTH AMERICA. I' EDWARD ANGUS BURT M ycologist and. Librarian to the Missouri Botanical Garden Associate Professor in the Henry Shaw School of Botany of Washington University INTRODUCTION This monographie study of the North American Thelephoracee was begun in 1894 as the author’s contribution towards a greatly needed manual of the Basidiomycetes of the United States,—a need that still confronts us. It has been necessary to carry on these investigations in connection with college and other work which required most of my time, but the long period covered has been an advantage; for during these two decades there has been such widespread interest in the Thelephoracee on the part of American students of fungi that it has been possible to study this family and its distribution from extensive series of freshly collected specimens from all the important regions of North America with the exception of Alaska, Mexico, and the Colorado- New Mexico region of the United States, from which but small collections have been received. "These specimens have been preserved unpoisoned in my herbarium in insect-proof tin boxes which receive herbarium sheets, and each will be cited by the number or other designation adopted by my cor- respondents in order that their specimens may be as useful for future reference as my own. Тһе quantity of material always awaiting examination has confined my work to a systematie treatment of this family. Except in the case of types of species, specimens of published exsiccati, and the specimens of Schweinitz’s herbarium, I cite but few specimens from the large herbaria. This is done on account of the difficulty and large amount of time involved in making a study of the material contained in them. Serious changes in the condition of the specimens in these herbaria have been occasioned partly by time but more largely by the poison- ous solutions with which the specimens were soaked for preser- vation under old-fashioned methods of herbarium procedure,— 1 Issued July 1, 1914. (185) ANN. Mo. Вот. Garp., Vor. 1, 1914 [Vor. 1 186 ANNALS OF THE MISSOURI BOTANICAL GARDEN methods well enough adapted for flowering plants but not for fungi. Early in the work it became apparent that the diagnoses of known species of resupinate Thelephoracee had failed utterly to enable the leading working mycologists of any country to recog- nize with certainty in the species about them those described in other eountries, or those described for their own country by earlier students. Тһе truth of this statement is shown by the errors and confusion in names of the common species which have been distributed in exsiccati, by the fact that in the large herbaria several different species are likely to bear the same specific name on the same or successive sheets, and by the vastly more important fact that the masters of mycology of each age, when relying wholly on the diagnoses published by their contemporaries or predecessors, have described as new species common апа conspicuous resupinate fungi which had been accurately described by immediate contemporaries or prede- cessors, and in very many cases just as accurately by still earlier students. All the mycologists concerned in these redescriptions have been earnest strivers after truth, I am convinced, and would have preferred to employ the earlier names for their plants, could they have known that those earlier names referred with certainty to their specimens. All these people were relying, as was the usage of their time, on a few words of published description in some other than their mother tongue. It is time to recognize generally that the resupinate Hymeno- mycetes, and especially the Thelephoracee, are extremely diffi- cult taxonomic problems. Descriptions must include more than a rather vague and generalized characterization of the mere superficial appearance and habit of the specimen with possibly a reference to spores which some one recorded for what was perhaps this species. The fungus itself is an individual of the species; the description in words and by illustration has merit in proportion to the success it has in producing in the mind of any educated stranger exactly the ideas which he could derive from the study in detail of the specimen itself. From the specimen, exact ideas may be had of coloration, of form, of dimensions, of texture, of consistency, of internal structure, of organs of minute size, of place of growth, and of host and һе АА шу Pe. e ы ы, Dept лға ag ae UNTERE 1914) BURT—THELEPHORACEJE OF NORTH AMERICA. I 187 substratum. If the description fails to give the color as exactly as if it had been noted by comparison with such a standard work as Ridgway’s ‘Color Standards’ or Saccardo’s ‘Chromo- taxia,’ then it is inferior to the specimen; if the description con- tains no information as to whether the basidia are simple or cruciate, making up the whole hymenium or arranged side by side with other organs of characteristic form, standing directly on the substratum or separated from it by densely or loosely interwoven hyphe or other form of subhymenial layer ;—if it does not contain all this information in exact terms and as much in addition as the specimen itself could afford, then it is an im- perfect description of the species. It may be so imperfect that a dozen different species of fungi could be assembled, to any one of which it would apply as well as to any other, as is the case with the supposedly common and cosmopolitan Corticium lacteum and C. calceum. Published exsiecati probably contain the full dozen under each of these names. In the case of resupinate Hymenomycetes, types and authentic specimens of the species are of the highest importance to supple- ment the prevailingly imperfect descriptions with full and exact data. Hence, the types of fungi on which the descriptions are based and the authentie specimens from the authors of the species are of importance in proportion to the degree in which these plants may yield data not afforded by the descriptions and existing illustrations of the species. In the case of the resupi- nate Hymenomycetes, the early descriptions are of slight prac- tical value except as they are backed up by types and specimens from their authors. For this reason, if there had been no other, the International Botanical Congress, at Brussels, acted for the best interests of mycology in fixing the beginning of the naming of Hymenomycetes with the publication of Fries’ ‘Sys- tema Mycologicum,'—the time when the preservation of types and authentie specimens of such fungi in herbaria became so prevalent that it was possible for later mycologists to distin- guish the resupinate species by taking the trouble to study the types, if authentie specimens could not be obtained. My method of becoming acquainted with our described species of T'helephoracee has been to study and arrange by species in my herbarium the specimens as they have accumu- [Vor. 1 188 ANNALS OF THE MISSOURI BOTANICAL GARDEN lated. In this arrangement due regard has been given to origi- nal descriptions of species and to all details of internal structure. Spore collections on glass slides have been made for each species whenever possible, and about five thousand mounts of sectional preparations in glycerin have been made from collections and preserved for reference in connection with internal structure of the specimens. From time to time I have taken my Thele- phoracee to herbaria where the types of our American species are stored and have there painstakingly matched them with the types. I have made sectional preparations from a frag- ment of each of these types in order to make sure that my specimens match the types not only in external characters but also in all details of internal structure. The sectional prepara- tions of type specimens have been preserved in glycerin. Speci- mens from my herbarium which have been so matched with type specimens have been used by me later for the determinations of subsequent collections. Such methods of investigation are probably too laborious and require too much time to become popular and they afford little opportunity for the inspirational flights attributed to genius, but they do afford a means of deter- mining within very narrow limits the species of North American T helephoracea. I am under especial obligation to Dr. W. G. Farlow for sug- gesting this work, for interest in its progress, and for frequent access to the Curtis Herbarium for comparisons with types. I am indebted also to Dr. C. H. Peck for opportunity to study his types in the New York State Herbarium, to the late Dr. L. M. Underwood for similar opportunity with the Ellis types in the Herbarium of the New York Botanieal Garden, to Dr. 8. W. Dixon and Professor 8. Brown, of the Philadelphia Acad- emy of Natural Sciences, for the privilege of studying in the Schweinitz Herbarium, to Sir W. T. Thistleton-Dyer and Mr. G. Massee for access to types and authentie specimens in Kew Herbarium, to the late Dr. T. M. Fries for the privilege of studying in the Herbarium of Elias Fries, at Upsala, and to Mr. Lars Romell, of Stockholm, Dr. P. A. Karsten, of Mustiala, and Abate G. Bresadola, of Trient, for many authentic speci- mens of their own species and for specimens which they had compared with types of early authors of Thelephoracem of 1914] BURT—THELEPHORACEZ OF NORTH AMERICA. I 189 Europe. In the later pages names of the many botanists who have participated in this work by the contribution of speci- mens from their respective regions are given in connection with the specimens. I feel my obligation to each of these cor- respondents. Having become thoroughly familiar with the species of a family of fungi, one then faces the task of deciding under what genera they shall be grouped in order that others may more easily recognize them. Our studies in systematie botany and the aecumulations of plants in herbaria are primarily for the purpose of enabling those who wish to obtain information about any particular plant, however obscure, to determine its name accurately and so be in a position to get at the world’s literature and knowledge concerning that species; and also to enable botanists so to entitle and index their researches that the results will be more available to the world at large. Stability in the nomenclature of plants is therefore important, and revo- lutionary changes in generic conceptions should not be lightly and frequently made. Whenever one proposes new genera to supersede a well-established genus which has satisfactorily embraced the related species of the world, the burden of proof should be on the one who makes the change to demonstrate that the advantages from the innovation will more than com- pensate for the confusion which would result as well as for the loss of knowledge indexed under the superseded name. Many new genera of fungi have been proposed during recent years. These have frequently come from students with a limited knowledge of the species of the world. It is not sur- prising that a botanist working on the few species of a limited region should be led to the establishment of new genera on the basis of what seem to be sharp differences in his species or groups of species. When, however, his knowledge encompasses just as definitely the structure of the many species of some large portion of the world, his perspective changes, and he may now find that the species which he formerly regarded as generically distinct are so closely connected by intermediate species that the contemplated generic separation would be unnatural and a hindrance to botanical progress. It is fundamental that genera be so sharply defined that any accurate observer who will make 3 [Vor. 1 190 ANNALS OF THE MISSOURI BOTANICAL GARDEN the study necessary for the application of the generic definition may be sure ninety-nine times out of a hundred that the fungus on which he is working is a Stereum, for example, and not а Thelephora, nor a Craterellus, nor a Cladoderris, nor a Corticium, nor a Pentophora, nor а Sebacina. It is an obligation on authors to group their species so accurately under genera that Stereum, for example, shall comprise all the species of this genus known to science, and no others. The synonomy of species in later pages will show how vaguely the genera of T'helephoracee have been comprehended. It is desirable that а genus should consist of but few species in those cases where the group is sharply and naturally set off from others, that is, where no intermediate species connect the genus with other groups. While such small genera are desir- able, if wholly natural, it is in the highest degree objectionable to create small artificial genera by arbitrarily segregating the species of a natural genus and so establishing indefinite lines of demarkation between genera. Under such a procedure the generic location of certain species becomes wholly arbitrary and always continues as a stumbling block for new students and this leads to the loading of our literature with so-called new species. A case in point is Saccardo’s scheme in the ‘Sylloge Fungorum’ in which he separates Hypochnus from Corticium and Peniophora without any natural generic planes of cleavage. In practical work one needs to know exactly what the generic limits of Corticium, Peniophora, and Hypochnus are. The question naturally arises as to just how loose and open the structure of the fructification must be to be included in the genus Hypochnus rather than in Corticium or Peniophora. Henning’s violation of the principle involved is still more fla- grant, for he separated the Hypochnacee as a new family from the Thelephoracee! and placed Hypochnus of Saccardo in the Hypochnacee, and Corticium and Peniophora in the Thelephora- сет. As all students of the T'helephoracee have found Hypoch- nus, as understood by Saccardo, wholly unworkable, it would increase the usefulness of the Sylloge Fungorum’ if Saccardo were to distribute among Corticium and Peniophora, the species which he now includes under Hypochnus. 1Engler und Prantl, Nat. Pflanzenfam. (I. 1**): 114. 1898. 1914) BURT—THELEPHORACE/E OF NORTH AMERICA. I 191 Probably all species of Corticium, as originally understood, have an hymenium composed of basidia arranged side by side between non-sporebearing organs termed paraphyses. In many species, it is difficult to distinguish between the basidia and the paraphyses except by prolonged study of special prepa- rations or by observations made at the time the basidia bear spores. In other species the sterile organs are conspicuous and distinct from the basidia either by their larger size, different form, or thicker or incrusted walls. Such conspicuous bodies are called cystidia, but if the paraphyses are merely finely but characteristieally branched near their tips, they are not called cystidia. Such branched paraphyses occur in the hymenium of occasional species of several genera of the Thelephoracee and are valuable characters for specific diagnosis. In 1880, Cooke proposed, from Kew Herbarium, to divide the old genus Corticium into two genera,—the name Corticium to be retained for those species having the non-sporebearing organs of the hymenium not distinguishable from the basidia, and the generic name Peniophora to be given to those species having cystidia. Ав the species of Corticium were very num- erous and extremely difficult taxonomically, this proposal was hopefully received, and for more than thirty years the transfer of species from Corticium to Peniophora has been going on and the end has not been reached yet. During this long period there has been confusion as to which species of the old genus Corticium belong in the emended Corticium and which in the genus Peniophora. Peniophora is an artificial rather than a natural genus, how- ever, and its adoption has given to many species а position intermediate between this genus and Corticium. These inter- mediate species have to be classed with the one genus or the other according to personal judgment, for no one can state just how conspicuous the sterile organs must be, nor of how constant occurrence, to merit the name cystidia. In Corticium Sambuci Fr., for example, cystidia are readily found in preparations from some collections, but several preparations may have to be made to demonstrate them in other collections. In the same species and in different parts of the same section, cystidia may some- times be sparingly and sometimes not at all incrusted. Some [Vor. 1 192 ANNALS OF THE MISSOURI BOTANICAL GARDEN species which I have placed in the genus Peniophora because of the presence of cystidia students may look for under Corticium when, by а more hasty study of their collections, they fail to detect these organs. Оп the other hand, students using more discriminating methods than mine may detect cystidia in species in which I have overlooked them, and such students will search in Peniophora for species which I have placed under Corticium. Species intermediate between genera always cause such trouble. There are many intermediates between Peniophora and Cortic- тит, yet in this particular case the advantage from the separa- tion undoubtedly more than compensates for the disadvantages occasioned by the intermediate species. The case of Peniophora has been considered at length, be- cause this genus is being regarded as a precedent for subdividing Stereum and grouping under Lloydella all those species which have conspicuous non-sporebearing organs between the basidia. Such a separation, however, would be artificial and give rise to a troublesome series of intermediate species, without the com- pensating advantage which accrued in the case of Peniophora and Corticium. Stereum is not a genus of difficult species nor does it comprise an immense number of species. It is just a fine, nat- ural group of species capable of being more sharply defined than it was by Fries, so as to receive some species from Тһе- lephora of Fries and to part with some toCorticium. So defined, even beginners will have no trouble in recognizing species of Stereum. Systematic work in mycology should strive to estab- lish and maintain just such natural, clean-cut genera as Sterewm. It seems to me best to work along constructive rather than destructive lines. Fries had a wonderful ability for the per- ception of the natural grouping of fungi on the basis of gross morphology and habit. Since his time, research has greatly enlarged the knowledge of the internal structure of fungi and of the organs of propagation. The value of such organs in the classification of seed plants is well known. It is feasible to modify somewhat the genera of T'helephoracem as defined by Fries, in accordance with the true relationships and differences shown by the present knowledge of internal structure, basidia, and spores, and a system results which is the natural evolution of taxonomic and morphologic study of T'helephoracem. This 1914] BURT—THELEPHORACEJE OF NORTH AMERICA. I 193 system has been communicated to my correspondents in con- nection with specimens. Its principal features are: 1. To restrict Thelephora to pileate species with simple basi- dia and colored spores. 2. To follow Karsten and Bresadola in placing under Hypoch- nus only resupinate species with colored echinulate spores. 9. To restriet Stereum to pileate species which have simple basidia and colorless spores and lack sets in the hymenium. 4. 'To include in Hymenochate all species having setze. 5. To include in Corticium species always resupinate, which have colorless spores and lack cystidia, excepting those species which for other reasons are placed іп Exobasidium. Include in Corticium hypochnoid as well as compact species. 6. To include in Peniophora all species which differ from Corticium merely by the presence of cystidia. I find this system workable and very satisfactory for the accurate location of species in genera, except in the case of the species intermediate between Peniophora and Corticium. The proposals to subdivide Peniophora into Gleocystidium, Penio- phorella, Gleopeniophora, etc., would create large numbers of species intermediate between the new genera, without compen- sating advantages. I have studied the species of my predecessors and co-workers sympathetically and have endeavored to find real differences between their species and those previously known so that the validity of theirs might be confirmed. The great area of land covered by the present work, the differences in climate and substratum, and the keen search by my correspondents have brought to hand a very large number of specimens. I have earnestly striven to place them under species already known, but it has been necessary to describe many as new. I regret that there are so many of these. Should any one have reason to believe that in any case I have described as new a species already known, I shall esteem it a favor to receive an authentic specimen of the older species or to be informed where such a specimen can be consulted. [Vor. 1 194 ANNALS OF THE MISSOURI BOTANICAL GARDEN Colors of specimens were noted and recorded during the first years of my work by comparison with Saccardo’s ‘Chromo- taxia' in accordance with his descriptive terms. Recently I have been using Ridgway’s ‘Color Standards and Nomen- clature,’ 1912, which has a greater variety of colors useful in the characterization of the species of Thelephoracee. In my own work with collections of living fungi I am endeav- oring to gather for each species a spore collection on a glass slip. Тһе spores adhere well so that they may be covered by paper and preserved in the envelope with the dried specimens from which the spores were obtained. Such collections give the exact color and dimensions of mature spores. These dimen- sions are generally rather larger than those obtained from spores of sectional preparations of dried herbarium specimens. The spores of dried specimens, i. e., those remaining attached to the specimens, are probably too immature to be of normal size, and sometimes there are so few of them that one must exercise caution to avoid errors due to the study of spores foreign to the fungus. Latex exists in many species of several of the genera and is more abundant and conspicuous in some species than in others, and its containing elements often extend to the hymenial surface. When specimens are in the vegetative condition, injury to the hymenium may liberate the fluid contents of the latex bodies so that this fluid exudes in colored drops at the edges of the wound, or discolors the bruised surface. For many of our species there is a lack of data concerning the color of this fluid or the discoloration. Тһе latex bodies are pale brown іп micro- scopic preparations made by my methods and must not be con- fused with setze or cystidia. Latex is well shown in Stereum spadiceum, S. sanguinolentum, and Corticium lactescens. There has been a disposition on the part of some authors to regard the Thelephoracee as not sharply separated from the Hyphomycetes. ‘The specimens which I have collected, in striv- ing to find all the Thelephoracee of my collecting region, and the specimens received from my correspondents afford no embar- rassment in recognizing the most hypochnoid species of T'hele- phoracee by the База which characterize the families of Hymenomycetes in general. 1914] BURT—THELEPHORACEJE OF NORTH AMERICA. I 195 The microscopical technique has been simplified as much as possible. Usually dried herbarium material had to be used for study and proved very satisfactory except in the case of speci- mens which had been subjected to poisoning processes for pres- ervation in herbaria. А small bit of the fructification having a promising hymenial surface 2 or 3mm. square—but smaller if the specimen is a valuable type—is first moistened with alcohol, then wet with water and cut out from the rest of the specimen and from the substratum. This bit is then placed in a holder of elder pith and oriented so that the sections may be cut per- pendicular to the surface of the hymenium and also contain as long hyphe as possible. The sections are cut as thin as possible, free hand, with a very keen section razor flooded with alcohol. The thinnest sections are placed on a slide in a drop of water and then a drop of seven per cent aqueous solution of potassium hydrate is added. Close observation of the sections should be made when the potassium hydrate solution comes in contact with them. For most species, the sections are merely cleared and the hyphe swelled to the normal size of vegetative hyphe. Ina few species, the alkaline solution may dissolve out the color of the section on coming in contact with it, or it may change this color to a violet, which finally disappears, or it may cause disorganization changes in certain structures leading to their disappearance or destruction. Such changes should be observed and noted, for they are of help in the determination of the species. In the cases in which potassium hydrate solution exerts a destructive action, lactic acid should be employed with other sections in the manner described for potassium hydrate. Lactic acid clears and swells sections well, but so much more slowly than potassium hydrate that I have used it only where the latter is not satisfactory. After the sections have been cleared, the potassium hydrate should be drained off, the sections lightly stained on the slide with alcoholic solution of eosin (but not overstained), mounted in water, and studied at once. For a thorough study of the species of the family at least one permanent preparation of each species should be retained for future comparisons. Permanent preparations may be made from the temporary water mounts by adding dilute glycerin— [Vor. 1 196 ANNALS OF THE MISSOURI BOTANICAL GARDEN two-thirds glycerin and one-third water—at the edge of the cover glass and allowing the glycerin to run under the latter as the water evaporates. When concentration of the glycerin is adequate, the excess should be wiped away with moist filter paper and the resulting smear removed to the very edge of the cover glass with a soft cloth moistened with 95 per cent alcohol. The preparations may then be sealed from the atmosphere by painting a ring of microscopical cement about the edge of the cover glass. At least two coats should be used for this ring, a light and very narrow one, and, after this dries, a very heavy, broad one. I have used Bell’s Microscopical Cement, made in London, and Brunswick Black Cement. A variable percentage of the rings crack in the course of a few years and allow the glycerin to escape from under the cover glass, but the sections in such preparations can be remounted. Dr. Thaxter has very recently informed me that he has been using King’s Transparent White Cement and King’s Amber Cement for fifteen years and that none of the rings made with these cements have cracked. By the use of circular cover glasses rather than square ones, а microscopist’s turn table may be used, thereby materially lessening the labor of preparing the rings. SYSTEMATIC ACCOUNT THELEPHORACEAE Thelephoree Persoon, Myc. Eur. т: 109. 1822; Fries, Hym. Eur. 629. 1872; Saccardo, Syll. Fung. 6:513. 1888. Hymenomycetes with the hymenium inferior or amphigenous (on the lower surface or surrounding the fructification), cori- aceous or waxy, even, rarely ribbed or papillate. Through several of the genera the Thelephoracee connect closely with all the other families of the Hymenomycetes. Hypochnus approaches Grandinia of the Hydnacee in the gran- ular hymenial surface of many of the species, but can be sepa- rated from this hydnaceous genus by the spore characters. Lachnocladium, with coriaceous structure, hairy stem, and colorless spores, is an intermediate genus between Clavaria, of the Clavariacee, and Thelephora but can be separated from the latter by the spore: characters. Craterellus connects with 19141 BURT—THELEPHORACE/E ОЕ NORTH AMERICA. I 197 Cantharellus, of the Адатсасее. Some species of Corticium must be cautiously separated from Меги ия, of the Polyporacee. The species of T'remellodendron, Hirneolina, and Setacina were formerly distributed among Thelephora, Stereum, and Corti- cium respectively, but are now separated from these genera by the cruciate character of the basidia,—such basidia as are pres- ent in many T'remellacem. АП these connecting genera will be included in the present monograph. Michenera and Heterobasidium are excluded genera. Lyman has shown! that Michenera artocreas B. & C. is only a stage in the life history of Corticium subgiganteum B. & C., and that the genus Michenera has ceased to be a genus of the Basidiomycetes. My own study of the type of Heterobasidium chlorascens Massee, which is the type species of the genus, failed to locate any basidia whatever. Very many Thelephoracee are of great economic importance on account of the dry rot induced by the growth of the mycelium in sills, floors, mine, bridge, and dock timbers, and other wooden structures located in moist, poorly ventilated places. Conio- phora puteana is а common species which rots coniferous wood. Only a very few Thelephoracee are classed as serious plant parasites. Of these the rhizoctonial stage of Corticium vagum is the most important. | KEY TO THE GENERA I. EU-THELEPHOREJE: Fructification not containing green lichen gonidia. Fructification fleshy or membranaceous, often infundibuliform, with the hymenium distinct, continuous, even, ribbed or at length rugose; ba- Coo f. s. ee a ae RET UU eS Craterellus Fructification submembranaceous, cup-shaped, often pendulous; hymenium typically concave, discoid; basidia віпіріе....................... Cyphella Fructification consisting of only a fleshy hymenium on the surface of living leaves and shoots; Бава вітріе......................... Exobasidium Fructification coriaceous or һага..................................... 1 1. Basidia globose or pyriform, longitudinally cruciately 4-septate or divided when mature; fructification erect, clavariform, more or less branched ....... Tremellodendron 1Cultural studies on the polymorphism of Hymenomycetes. Proc. Boston Soc. Nat. Hist. 33: 151-60. 1907. [Vor. 1 198 ANNALS OF THE MISSOURI BOTANICAL GARDEN 1. Ваза cruciate as in T'remellodendron; fructification effuso-reflexed or cup- shaped with the margin ігев................................... Hirneolina 1. Basidia cruciate as in T'remellodendron; fructification always resupinate . Sebacina 1. Basidia simple but with such large sterigmata as to resemble longitudinally divided Бавіізі............................................... Tulasnella 1. Basidia at first globose and simple, at length elongated and transversely sep- tate, straight or curved, bearing sterigmata on the convex side; fructification гевірішшаде................................................ Septobasidium 1. Ваза simple, usually 4-врогей....................................... 2 2. Spores colored; fructification рИеа4е........................... Thelephora 2. Брогев colored, rough-walled to echinulate; fruetification resupinate..... Hypochnus 2. Spores ochraceous, ferruginous or fuscous, even; fructification resupinate. . Coniophora 2. Spores white or rarely bright colored, even or rarely uneven.......... 3 3. Sete (brown, cylindric, rigid, even-walled bodies) present in the hymenium; fructifications range from pileate to resupinate................., Hymenochete 3. Cylindrie teeth composed of many consolidated hyphe protrude from the hymenium but are not covered by it. Our southern species was originally described as a Нуйтит........................2......222222.2... M ycobonia 3. Neither sets nor teeth present in the hymenium........................ 4 4. Fructification coriaceous, erect, clavariform; stem often hairy . . Lachnocladium 4. Fructification cup-shaped, resupinate with free margin or simply resupi- nate; hymenium pulverulent; with some two or three of the following characters: (1) large white spores ranging from 14-34 x 12-20 и; (2)much granular matter in the fructification; (3) prominent moniliform or branched paraphyses; (4) racemose organs in the hymenium which pro- duce a erop of conidia before basidiospores develop.......... Aleurodiscus 4. Fructification pileate ranging from infundibuliform and flabelliform to very narrowly reflexed forms; hymenium even. Some reflexed species may occur тезіріпаде....................................... Stereum 4. Fructification like that of an urn-shaped Stereum but hard and stuffed. One tropical species ...................................... Н ypolyssus 4. Fructification like that of Stereum but with ihe hymenium hardened and with radiating branched ribs. Species tropical.............. Cladoderris 4. Fructification always resupinate; structure not as in Aleurodiscus...... 5 5. Subhymenial tissue contains conspicuous brown stellate organs composed of several radiating агпв..........................222222222.... Asterostroma 5. Such brown stellate organs not ртевепі.............222222222222222222.. 6 6. Cystidia present in hymenium, or in subhymenial tissue, or in both; structure may be compact or hypochnoid..................... Peniophora 6. Cystidia not present; structure compact or hypochnoid........... Corticium ! With regard to the nature of these bodies see H. O. Juel, Bihang till K. Sv. Vet.-Akad. Handl. 23": Afd. III. 3-27. 1897. rc F 1914) BURT—THELEPHORACEJE OF NORTH AMERICA. I 199 П. HYMENO-LICHENS: Fructifieation regularly containing green lichen gonidia. Species tropical. Fructification pileate, coriaceous-membranaceous, with hymenium on the lower surface and somewhat waxy; gonidiallayer composed of some- what cubical masses of algal сейв................................. Cora Fructification like Cora in most respects but with the hymenium somewhat gelatinous and the gonidial layer composed of algal cells arranged in rows (cateniform) „(еее nn Rhipidonema THELEPHORA Thelephora Ehrhart [Crypt. Exsic. No. 178. 1785) Fries, Syst. Мус. 1: 428. 1821 (in part).-Persoon, Myc. Eur. 1: 110. 1822 (in part).-Saecardo, Syll. Fung. 6: 521. 1888 (in part).-Hennings, in Engl. &. Prantl, Nat. Pflanzenfam. [E 1: 125. 1898 (in part). The type species of the genus is Thelephora terrestris Ehrh. ex Fries. Fructifieations pileate or clavate, coriaceous; hymenium con- tinuous with the hymenophore and similar to it, inferior, or amphigenous in a few species, even or faintly ribbed or papillose; basidia simple, 4-spored; spores colored, typically muricate but even, or rough-walled in a few species. As more broadly defined by Fries and the other authors cited, Thelephora has been heterogeneous, consisting chiefly of the natural and homogeneous group of species defined above but also of some pileate species with simple basidia and hyaline spores, transferred to Stereum; also of some species with globose, longitudinally septate basidia, transferred to Tremellodendron, if with erect fructifications, or to Sebacina, if resupinate; and also of some resupinate species having simple basidia, of which those with muricate and colored spores may be found in Hypoch- nus, those with colored and even spores, in Coniophora, and those with hyaline spores, in Corticium and Peniophora. It is probable that the species of Patouillard’s section Dendrocladium of the genus Lachnocladium as understood by Patouillard ! might be transferred to Thelephora with advantage both to Thelephora and Lachnocladium, but these species are not within the geographical limits of my work. 1 Fragments Mycologiques (suite). Jour. de Bot. 3:33-37. 1889. О Д аса aia Our cal ан Тат” = t м Pe M em tenue [Vor. 1 200 ANNALS OF THE MISSOURI BOTANICAL GARDEN KEY TO THE SPECIES Erect species, usually with central stem and pileus divided into very narrow, branching, flattened or cylindric divisions; hymenium inferior or am- phigenous .................................................... 1 Егесі species, usually with central stem and more or less infundibuliform, cup-shaped or flabelliform pileus, which may be radially split into lobes and йіуікіопв.................................222222222222. 2 Species of incrusting, effuso-reflexed, dimidiate, or applanate habit ....... 5 . 2-6 em. high, much branched, glabrous, with fetid odor when growing, perhaps rarely ойог!еяв.............................222.22.... 1. T. palmata . 8-5 em. high, much branched, minutely pubescent; stem villose, without fetid odor. Compare Т. multipartita....... ls e e e sess. 2. T. anthocephala . Less than 2} em. high, branching at or below surface of ground, dusky drab except at Базе........................................... 8. T. cespitulans . Less than 2 em. high, very slender and fragile, cinereous. Known only from State of Х/аяһіпроп...........................22222222.2.... 4. T. scissilis . Large species, highly branched, with body of spore of regular obovoid form. Known only from Central Атпетіса........................... 5. T. angustata 2. Hymenium dark colored, i. e., brown to fuscous...................... 3 2. Hymenium light colored, i. e., pallid to штау........................ 4 . Small species, 13-3 сіп. high, upper surface usually drying pallid, usually deeply cleft or many-parted into narrow divisions; stem villose.6. T. multipartita . Small species, 6 mm.-23 em. high, infundibuliform or deeply divided into two or three triangular divisions, or flabelliform; stem villose. Closely related to T. тшйрағШа.............................2.....22....... 7. T. regularis . Fruetifieation 1 em. high, white; stem white, glabrous. Known only from Guadaloupe ...............................2.222222222222.... 8. T. pusiola . 13-5 em. high, larger species than the three preceding but with thinner pileus, fuscous purple (Rood’s brown) throughout, often with the thin lobes imbri- cate like the petals of a carnation; stem villose............ 9. T. caryophyllea . 2-4 em. high, somewhat tubular, hymenium vinaceous brown to drab ; stem suleate and pitted but not villose; spores 10-14 шіп diameter. Known only from Латаіса.......................................... 10. Т. magnispora . Large species, 23-7 cm. in diameter, with upper surface pallid except at the center and with the hymenium багЕ............................. 13. T. vialis 4. Small species, less than 2 cm. in height and in diameter, somewhat pallid іоБліек-гей........................................... 7. Т. regularis 4. Pileus with outer lobes forming a cup and with inner lobes distinet, crowded, erect, cinereo-fuscous. Known from Costa Rica and Brazil. 11. Т. corbiformis 4. Large species, 5-7 cm. broad, deeply infundibuliform, habit and color of Craterellus cornucopioides. Costa Rica and Jamaica. .12. Т. cornucopioides 5. Growing in applanate clusters, effuso-reflexed, or dimidiate................ 6 5. Always incrusting (Т. albido-brunnea is sometimes inerusting) ............. 8 6. Hymenium pale and colored like the pileus, einnamon-buff ; pileus spongy, more than 2 mm. thick; spores 8-10 x 6-8 u . . .14. Т. albido-brunnea 6. Hymenium and pileus yellowish, less than 2 mm. thick; spores 5-6 x 4 u 16. T. lutosa. 37 3 ен Po Wort MV ETT ess ims А009 1914] BURT—THELEPHORACEJE OF NORTH AMERICA. I 201 6. Hymenium drab, becoming sage-green when crushed in 7 per cent potas- sium hydrate solution; pileus pinkish buff to cinnamon-brown with a broad pale Dg. ..,.:............... n c 16. T. culicularis 6. Hymenium ferruginous brown (Rood’s brown) to fuscous ............ жер. d 7. Pileus, when squamulose, with the fibers matted and agglutinated into ap- pressed and wholly adnate squamules, margin dilated and whitish fimbriate becoming entire and сопсоіотойв........................... 17. T. intybacea 7 IPileus not zonate, fibrous-squamulose and usually strigose, margin fibrous- ЕКЕН І................................. 222 2 18. T.terrestris 7. Pileus zonate, in other respects resembling the preceding species.......... 19. T. griseozonata 8. Inerusting and ascending small plants, free branches somewhat terete but flattened towards the tips; spores umbrinous............... 20. T. fimbriata 8. Resupinate on leaves and twigson the ground and sending up free, simple or branching trunks; spores fuscous. Known from Cuba only 21. T. perplexa 8. Inerusting leaves, etc., on the ground and ascending as sessile flabelliform pilei which are dentate at the upper end or deeply divided, honey- yellow to tawny olivaceous throughout. Known from Cuba only.... 22. T. dentosa 8. Typically effused, rising obliquely upward from the support as a cluster of small trunks which branch and terminate in spiculous tips.23. T'. spiculosa I. Thelephora palmata Scop. ex Fries, Syst. Myc. 1: 432. 1821. Plate 4. fig. 4. Clavaria palmata Scop. Fl. Carn. 2: 483. 1760.- Ramaria palmata Holmsk. Fun. Dan. т: 106. pl.—.1799. —Merisma foetidum Pers. Syn. Fung. 584. 1801.-М. palmata Pers. Myc. Eur. 1:113. 1822.- Thelephora palmata americana Peck, Rep. N. Y. State Mus. 53: 857. 1900. Illustrations: Greville, Crypt. Fl. т: pl. 46.-Holmskiold, Fun. Dan. 1: pl. of Ramaria palmata.-Krombholz, Abbild. und Beschr. pl. 54. f. 24, 25.-Nees, System pl. 16. f. 151 B.-Baillon, Dietionn. de Botan. т: 737. f.?.-Loudon, Encyc. of Plants f. 16131.-Winter, Crypt. Flora т: 321. Fructification coriaceous-soft, fuscous purple, drying cinna- bar-brown or chestnut-brown, erect, very much branched, with very fetid odor; pileus with numerous somewhat fastigiate, palmate divisions which are even, flattened, dilated above, and with fimbriate and whitish tips; stem simple or soon branched; hymenium amphigenous; spores pale umbrinous under the microscope, sparingly echinulate,10 x 7-8 џи. Fructification of American specimens 2-6 cm. high, 1-3 cm. broad; stem 1-13 cm. long, 1-2 mm. thick. T Шалы анд 2-22 пены (өз MA [Vor. 1 202 ANNALS OF THE MISSOURI BOTANICAL GARDEN On moist ground in coniferous woods and also in grassy fields. Prince Edward Island to North Carolina and west to Illinois. June to October. In the American collections of this species the divisions of the pileus are narrow and a short stem is present. "The habit is so similar to that of Thelephora anthocephala that record of the fetid odor should always be made if observed. Тһе ultimate branches may be more or less terete, leading to the variety americana Pk. Specimens examined: Exsiceati: Ell. & Ev., N. Am. Fungi, 1937. Austria: G. Bresadola.! Sweden: L. Romell, 58. Canada: Rustico Bay, Prince Edward Island, J. Macoun, 324. New Hampshire: Chocorua, W. G. Farlow. ? Vermont: no locality data for specimen in Frost Herb., Univ. of Vermont. Connecticut: Manchester, C. C. Hanmer, 1398. New York: Fischer's Island, C. C. Hanmer, 196. New Jersey: C. G. Lloyd, 4612. Pennsylvania: Bethlehem, Schweinitz, Syn. N. Am. Fungi, 612 (in Herb. Schw.); Trexlertown, Dr. W. Herbst; Kitanning, D. R. Sumstine, 2; West Chester, B. M. Everhart, Ell. & Ev., N. Am. Fungi, 1937. Delaware: Newark, H. S. Jackson. Dist. of Columbia: Washington, O. F. Cook, comm. by P. L. Ricker, 1, 3. N. Carolina: Asheville, H. C. Beardslee, 924. Ohio: Connecticut, C. G. Lloyd, 4493. Illinois: Glencoe, E. T. and S. A. Harper, 664, 665. Missouri: St. Louis, N. M. Glatfelter (in Mo. Bot. Gard. Herb., 42560). ! With regard to the citation of specimens all except those of “Exsiccati” are in Burt Herb. which are cited without explicit reference to place in other herbaria. For example, the specimen cited, * Connecticut: Manchester, C. C. Hanmer, 196," isin Burt Herb. The data given is that received with the specimen and may identify a duplicate in another herbarium. Тһе location of all specimens іп herbaria other than my own is designated by the name of the herbarium in parenthesis with the prefix “in.” For example, the specimen cited, “Louisiana: St. Martinville, A. B. Langlois (in Lloyd Herb., 3000),” is in Lloyd Herb., but not in Burt Herb. 1914) BURT—THELEPHORACE/E OF NORTH AMERICA. I 203 2. T. anthocephala Bull. ex Fries, Syst. Мус. т: 433. 1821. Plate 4. fig. 1. Clavaria anthocephala Bull. Herb. dela France 2: 197. pl. 452. f.1. 17989. Illustrations: Bulliard, Ibid. pl. 452. f.1.-Sowerby, Col. Figs. Eng. Fun. pl. 156.-Berkeley, Outlines Brit. Fung. рі. 17. f. 4.-Dufour, Atlas des Champ. pl. 70. Fructification coriaceous-soft, somewhat ferruginous, drying fawn-color or cinnamon-brown, inodorous; pileus pubescent, divided to the stem into flaps which are dilated upwards and fimbriate and whitish at the apex or divided into irregular, branched, erect branches; stem simple, equal, villose; hymenium even; spores pale umbrinous under the microscope, ranging from angular-tuberculate to tuberculate-echinulate, 8-10 x 7-8y. Fructifications 3-5 cm. high, 1-3 em. broad; stem 1-13 cm. long, 1-2 mm. thick. On the ground in woods. Massachusetts and Ohio to Louisi- ana. June to August. Rare. Our specimens of Т. anthocephala and Т. palmata have the same habit but may be separated, even when dried, by the fine pubescence of the pileus visible with a lens, and by the villose- tomentose stem of the former. The spores of T. anthocephala are further slightly paler and have shorter spines with broader bases than those of Т. palmata. Specimens examined: Austria: G. Bresadola. Massachusetts: Newton, W. (7. Farlow (in Farlow Herb.). New York: Van Cortlandt Park, N. Y. City, L. O. Overholts (in Overholts Herb., 688). Pennsylvania: Kitanning, D. R. Sumstine, 10; Bethlehem, Schweinitz (in Herb. Schw.), the 614 of Syn. N. A. Fungi under the name T. flabellaris. North Carolina: Asheville, H. C. Beardslee, 0268. Louisiana: St. Martinville, A. B. Langlois, unnumbered specimen, and 1971, and by the same collector (in Lloyd Herb., 3000). Ohio: Norwood and Linwood, C. G. Lloyd, 1272 and 02164 re- spectively. Kentucky: C. 6. Lloyd, 1396. [Vor. 1 204 ANNALS OF THE MISSOURI BOTANICAL GARDEN Missouri: St. Louis, N. M. Glatfelter (in Mo. Bot. Gard. Herb., 42559). 3. T. caespitulans Schw. Trans. Am. Phil. Soc. N. S. 4: 166. 1831.1 Type: in Herb. Schweinitz. Fructification erect, coriaceous, dusky drab to olive-brown below, paler above, very much branched, forming clusters 21 em. high by 22 em. broad; pileus with numerous divisions joined together into а solid base but assurgent above and pressed to- gether closely, compressed, subcanaliculate, frequently obtuse and whitish at the apex; hymenium amphigenous; spores um- brinous under the microscope, sparingly tuberculate, 7-8 x 5—би. On the ground in mixed woods, Vermont to South Carolina, and in dense coniferous woods, Washington. September. Rare. This species is related to Т. palmata but is more olivaceous, and it is probably inodorous,—at least no odor has been noted. The dimensions for the clusters given above, as stated by Schweinitz, are probably maximum dimensions, for the speci- mens recently collected have been rather smaller. My Vermont specimens were growing with the thick, solid base buried in sandy ground in а wood road; they have dried pallid except at the base and are slightly pubescent. Тһе general habit of this species is somewhat suggested by a small cluster of Tremel- lodendron pallidum (Schw.) Atk. Specimens examined: Vermont: Lake Dunmore, E. A. Burt. Pennsylvania: Bethlehem, Schweinitz, type (in Herb. Schw., Acad. Nat. Sci., Phila.). South Carolina: Santee Canal, Ravenel, 1660 (in Curtis Herb. under name Т. vialis). Washington: Chebalis, C. J. Humphrey, 1287; Bingen, W. N. Suksdorf, 689. 4. T. scissilis Burt, n. sp. Plate 4. fig. 8. Type: in Burt Herb. Fructifications gregarious, coriaceous, erect, clavariform, branched, longitudinally ridged by the bases of numerous, 1 A figure will be given in Part II. 1914] BURT—THELEPHORACE/E OF NORTH AMERICA. I 205 small, appressed, acicular branches, the larger of which are at the apex of the fructification and spread slightly outward in fan-shaped manner; stem glabrous, castaneous; hymenium am- phigenous, on upper half of the fructification, avellaneo-ciner- eous; basidia simple, hyaline, 4-spored; spores pale umbrinous under the microscope, angular, 6-8 x 5-6y. Fructifications 13-2 cm. high; spread of branches at the top 2—6 mm.; stem 7-10 mm. long, 1 mm. thick. On the ground. Washington. January. This species is very distinct by its slender erect habit, cinereous color, and only slightly spreading branches. Specimens examined: Washington: Bingen, Klickitat Co., W. N. Suksdorf, 716, type. 5. T. angustata Fries, (Nov. Symb. Myc. 92.) Actis R. Soc. бе. Upsal. III. 1: 108. 1851. Type: in Herb. Fries. Fructifieation erect, cinereo-fuscous, pliant, becoming rigid and somewhat woody; stem elongated, radicated, rugose, gla- brous, compressed, irregularly divided at the upper end into unequal, fastigiate, compressed branches, which are clothed on the whole outer surface with the hymenium; hymenium amphi- genous, subrugose, gray ; basidia simple; spores umbrinous under the microscope, obovoid, apiculate at base, flattened on one side, echinulate, 14 x 7-94. On decaying wood. Central America. Substance, color, and hymenium exactly as in T. cornuco- pioides, but of the very different form of Clavaria rugosa and having highly branched forms; stem 5 em. long; color fuliginous. The fructification is fleshy-pliant when fresh, but on drying hardens much more than species of Stereum. Specimens examined: Costa Rica: Oersted (in Herb. Fries), type. 6. T. multipartita Schw. in Fries, Elenchus Fung. 1: 166. 1828. Plate 4. fig. 7a. Type: in Herb. Schweinitz. Fructifieations gregarious, erect, coriaceous, fusco-cinereous, usually drying pallid; pileus infundibuliform, sometimes cleft 4 [Vor. 1 206 ANNALS OF THE MISSOURI BOTANICAL GARDEN more or less deeply and unequally into a few lobes, sometimes divided to the stem and spreading so as to appear dimidiate, very often deeply divided and subdivided into many narrow and spreading divisions more or less dilated and whitish at the apex; stem erect or incurved, equal or tapering upward, some- times branched above, drying walnut-brown or pallid, villose; hymenium inferior, glabrous, even, fawn-color or vinaceous drab; spores unbrinous under the microscope, tuberculate, 7-9 x 5-бд. Fructification 11-33 em. high, 1-3 em. broad; stem 1-2 cm. long, 1-3 mm. thick. On ground in groves of broad-leaved trees, especially under oak. New York and Pennsylvania to Illinois. July to Sep- tember. The upper surface of the pileus was originally described as glabrous, but it is minutely pubescent under a lens, or sometimes fibrillose. "This species is very perplexing by its close relation- ship to T. regularis. 'The multipartite pileus is the only char- acter which seems available to separate collections of the former from the latter species. If a given collection consists wholly of specimens with pileus many-parted and subdivided into nar- row divisions, or if it contains some such specimens in addition to others with more regular infundibuliform pileus, I refer the collection to T. multipartita, as in the cases of the collections cited below from C. O. Smith and Dr. C. H. Peck respectively. As yet, I know of no characters by which to assort and separate into their respective species specimens mixed together of typical T. regularis and those specimens of T. multipartita which have the pileus infundibuliform or merely cleft more or less deeply and unequally into а few lobes. "Therefore it is my opinion that T. multipartita is a variety of T. regularis, but the collec- tions which have so far been submitted to me, have been com- posed of too few fructifications to assure me that this opinion is correct. Specimens examined: Exsiceati: Ell. & Ev., №. Am. Fungi, 2806, under the name T. caryophyllea. 1914) BURT—THELEPHORACE/E OF NORTH AMERICA. I 207 New York: Bolton, C. H. Peck, 3, 4, 5; Ithaca, C. O. Smith, Cornell Univ. Herb., 13359, and C. O. Smith and W. H. Long, Cornell Univ. Herb., 7743. New Jersey: Newfield, J. B. Ellis, Ell. & Ev., N. Am. Fungi, 2806. Pennsylvania: on island in Lehigh River, Schweinitz, type (in Herb. Schw.); Bethlehem, Schweinitz (in Herb. Schw.), the T. tuberosa of Syn. N. Am. Fungi, 613; Trexlertown, W. Herbst, 22, 36. Ohio: A. P. Morgan, Lloyd Herb., 2581, 2647; Oxford, L. O. Overholts (in Overholts Herb., 1685). Illinois: River Forest, E. T. and S. A. Harper, 666. 7. T. regularis Schw. Schrift. d. Naturforsch. Gesell., Leipzig, 1:105. 1822. Plate 4. figs. 6, 7b. Thelephora Ravenelii Berk. Grevillea т: 148. 1873.—T. hiscens Berk. & Rav. Grevillea 1: 148. 1873. Type: in Herb. Schweinitz, Acad. Nat. Sci., Phila. Pileus coriaceous, solitary, infundibuliform or divided to the stem into triangular divisions or flabelliform, fibrillose, drying pallid or tawny-olive, darker at center of the cup or at base of the divisions, margin lacerate; hymenium usually hair-brown, sometimes pallid; spores melleus to umbrinous under the micro- scope, angular-tuberculate, 6-7 x 4i-5y. Fructification 6 mm.-25 cm. high; pileus 5 mm.-23 em. broad; stem 3-15 mm. long, 1-13 mm. thick. In moss in wet places and on humus. Ontario to Alabama and westward to Kansas. Тһе differences in form of the pileus of T. regularis are well shown by the type in Herb. Schweinitz; this type consists of three fructifications, two of which are infundibuliform, the third and largest, flabelliform. Тһе hymenium is sometimes merely pallid, as in the case of the specimen which is the T. pannosa of Schweinitz, Syn. N. Am. Fungi, No. 606, but is not T. pannosa Fr. The cotypes of T. Ravenelit and T. hiscens agree in all respects with the authentic specimen of T. regularis in Curtis Herb. Specimens of Т. regularis which have the pileus infundibuliform and little cleft are suggestive of small specimens of T. caryophyllea but differ from the latter by the thicker pileus [Vor. 1 208 ANNALS OF THE MISSOURI BOTANICAL GARDEN and paler coloration of T'. multipartita which is wholly lacking in the rufescent coloration of T. caryophyllea. There is a diffe- rence of form between specimens of these two species which is brought out well by the figures in pl. 4. Specimens examined: Canada: Shannonville, Ontario, J. Macoun, 880. Maine: Portage, L. W. Riddle, 4. New Hampshire: Chocorua, W. G. Farlow (in Farlow Herb.). Massachusetts: near Boston, Sprague, 246 (in Curtis Herb. under the name T. anthocephala) ; Newton, W. G. Farlow (in Farlow Herb.). Pennsylvania: Bethlehem, Schweinitz, station cited by Schwei- nitz; also the specimen (in Herb. Schw.) under the name T. pannosa of Syn. N. Am. Fungi, No. 606; Trexlertown, С. 6. Lloyd; Kitanning, D. В. Sumstine. Delaware: Clayton, H. S. Jackson. North Carolina: Salem, Schweinitz, type (in Herb. Schw.); G. F. Atkinson, Cornell Univ. Herb., 23254. South Carolina: Greenville, Ravenel, 1498, type and cotype (in Kew Herb. and in Curtis Herb. respectively) of Т. Rav- enelii Berk.; Santee Canal, Ravenel, type and cotype (in Kew Herb. and in Curtis Herb. respectively) of Т. hiscens Berk. & Rav. Alabama: Peters, 576 bis (in Curtis Herb. under the name T. anthocephala). Wisconsin: Madison, W. Trelease (in Farlow Herb.); Lake Geneva, E. T. and S. A. Harper, 882, and (in Harper Herb., 883). Illinois: East St. Louis, N. M. Glatfelter (in Mo. Bot. Gard. Herb., 42563). Iowa: Johnson County, T. J. Fitzpatrick, 39. Missouri: St. Louis, N. M. Glatfelter (in Mo. Bot. Gard. Herb., 42564). Kansas: Bourbon County, A. O. Garrett, 80. 8. T. pusiola Pat. in Duss, Champ. Guad. & Martinique 12. 1903. Pileus with divisions triangular, white, hard, thin, entire or cut-lobed, glabrous, even or rugose, sometimes zonate, atten- 1014) BURT—THELEPHORACEJE OF NORTH AMERICA. I 209 uated into a slender stem; stem colored like the pileus, glabrous, cylindrie, woody ; hymenium inferior, even, brown; basidia clav- ate, 25 x 10и, four-spored; spores globose-angular, colorless or somewhat fuliginous, би in diameter; no cystidia. Fructification 1 em. high, divisions 5 mm. broad. Solitary or in clusters оп dead wood. Guadaloupe. Forest of Bains-Jaune, Duss, 289. Var. terrestris Pat. Ibid, has the divisions of the pileus nar- rower, laciniate, divergent, rigid. On the ground, Matouba, Guadaloupe, Duss. I have seen no specimens of either this species or its variety, neither of which have been reported since their original dis- covery. 9. T. caryophyllea Schaeffer ex Fries, Syst. Myc. т: 430. 1821. Plate 4. fig. 9. Elvella caryophyllea Schaeffer, Icon. Fung. 3: 115. pl. 826. 1762-1774.-Craterella ambigua Pers. Obs. Myc. т: 36. pl. 6. f. 8-10. 1796.-Thelephora caryophyllea Y ambigua Pers. Myc. Eur. 1:112. 1822. Illustrations: Schaeffer, Icon. Fung. pl. 325.—Persoon, Obs. Myc. т: pl. 6. f. 8-10.-Schnizlein, in Sturm, Deutsch. Flora 3: fasc. 81. pl. 6.-Lanzi, Fungi di Roma pl. 11. f. 4.-Saunders and Smith, Мус. Ill. pl. 41. f. 7-12.-Smith, W. G. Brit. Basid. 399. f. 96 a, b. Fructifications solitary or cespitose, coriaceous, fuscous purple but drying wood-brown; pileus infundibuliform, simple, or doubled by proliferous growth of smaller pilei from the disk of the principal pileus or of wedge-shaped lobes rising from its upper surface, upper surface radiately ridged or striate with masses of agglutinated fibers which are often dark colored, ob- scurely zonate when moist, margin incised; stem usually central, cylindric, villose, simple or branched; hymenium inferior, even, grayish olive to light yellowish olive; spores pale umbrinous, tuberculate, 7-8 x би. Fructification 13-5 em. high, 13-5 cm. broad; stem 1 cm. long, 2-3 mm. thick. On the ground under pines. Canada to South Carolina and west to Ohio, also in the Pacific states. August to November. Abundant locally. [Vor. 1 210 ANNALS OF THE MISSOURI BOTANICAL GARDEN T. caryophyllea may be distinguished from our other northern species which have a central stem and dark hymenium, by the thin lobes of the pileus which dry paler than the hymenium, and by the frequent occurrence of specimens with the pileus consisting of many lobes and pilei imbricately arranged in a manner suggestive of a double pink or carnation, as shown by Schaeffer's fig. 5, and Persoon's fig. 10 of the illustrations cited. Our specimens agree well with the figures of Schaeffer and Per- soon—those of Persoon are especially good but unfortunately occur in a work which is very rare. We find occasionally specimens which agree well with T. radiata (Holmsk.) Fr., but these specimens are connected so closely by intermediate forms—often in the same collection— with others which are undoubtedly T. caryophyllea that I refer them to the latter species. Specimens examined: Sweden: К. Starback, in Romell, Fun. Scand., 121. Canada: J. Macoun, 54 and 75 of 1903. Quebec: Hull, J. Macoun, 190. Ontario: London, J. Dearness (in Lloyd Herb.). New Brunswick: Restigouche River, T. F. Allen, comm. by Dr. Farlow. Maine: Orono, L. W. Riddle, 9. New Hampshire: Shelburne, W. G. Farlow. Vermont: Newfane, C. D. Howe; Middlebury, E. A. Burt, four collections. Massachusetts: Sprague, 47, Russell, 131, and D. Murray, 545 (all in Curtis Herb.); Worcester, G. E. Francis, 105. Connecticut: East Hartford, C. C. Hanmer, 1449; Central Vil- lage, J. L. Sheldon, 68, comm. by New York Bot. Gard. New York: Bolton, C. H. Peck; Ithaca, G. F. Atkinson, 9998, 9994; Saranac Lake, E. A. Burt; East Galway, E. A. Burt. Pennsylvania: Bethlehem, Schweinitz (in Herb. Schw.), the 608 of Syn. N. Am. Fungi. Dist. of Columbia: Zoélogical Park, Coville and Cook, No. A, comm. by P. L. Ricker. North Carolina: Schweinitz (in Herb. Schw.). 1914] BURT—THELEPHORACEJE OF NORTH AMERICA. I 211 Michigan: C. G. Lloyd, 4547; Sailor’s Encampment, E. T'. and S. A. Harper, 439, and Univ. of Wis. Herb., 2. Ohio: C. G. Lloyd, 1422, 2720; Cincinnati, A. P. Morgan, Lloyd Herb., 2641, and (in Lloyd Herb., 1152); Loveland, D. L. James (in Herb. U.S. Dept. Ag.). Kentucky: C. G. Lloyd, 1152. Washington: Bingen, W. N. Suksdorf, 717, 690. California: Jackson, J. H. Barber, comm. by W. A. Setchell; Stanford University, C. F. Baker, 255, distributed by Baker, Pacific Slope Fungi, 3743, under the name Т. radiata (Holmsk.) Fr. IO. T. magnispora Burt, n. sp. Plate 4. fig. 5. Type: in Burt Herb. Fructifications solitary or gregarious, coriaceous, stipitate; pileus irregularly infundibuliform, somewhat tubular, with ascending recurved lobes, drying avellaneous, becoming fuscous at the center with age, fibrous torn becoming radiately striate, margin incised ; stem equal, solid, drying hard, irregularly angled, suleate and pitted, vinaceous brown to drab; hymenium inferior, even, vinaceous brown; basidia simple; spores pale cinnamon, subglobose, echinulate, 10-14 шіп diameter. Fructification 2-4 cm. high; pileus 1-2 cm. in diameter; stem 7-12 mm. long, 2-5 mm. thick. On mossy ground. Chester Vale, Jamaica. December. In some of the specimens the pileus is decidedly eccentric through greater growth on one side than on the other, and it is not always lobed. The offensive odor of the dried specimens and the color of the hymenium are suggestive of Т. cuticularis. Specimens examined: Jamaica: Chester Vale, W. A. and Edna L. Murrill, type, New York Bot. Gard., Fungi of Jamaica, 295. 11. T. corbiformis Fries, (Nov. Symb. Mye. 92.) Actis. R. Soc. Sc. Upsal. III. т: 108. 1851.-Romell, Hymenomycetes Austro-Americani. Bihang till К. Sv. Vet.-Akad. Handl. 268: Аға. ПТ. 44. 1901. Type: in Herb. Fries. Fructification sessile, rigid, cinereo-fuscous, with cespitose lobes of which the outer ascend and coalesce into а rounded [Vor. 1 212 ANNALS OF THE MISSOURI BOTANICAL GARDEN eupulate pileus here and there lacunose-pervious, and the inner are distinct, erowded, erect, narrow; hymenium inferior, uneven, whitish ; basidia simple; spores slightly colored, becoming uneven, ovoid, 5-6 x 4-5 y. On the ground. Costa Rica and Brazil. January. “Тп substance, texture, color, etc., this species agrees exactly with Thel. cornucopioides and Thel. angustata but in form it ex- hibits a type unique in the Hymenomycetes. The clusters are regularly rounded, very dense, divided all the way to the base into innumerable lobes, of which the interior are free and erect, the exterior regularly ascendant, broader, compressed, clothed underneath by the hymenium and grown together into a cup here and there lacunose-pervious, undulate-crisped at the apex and fimbriate."—' Translation of the original comment on this species. In 1899, I found the type in Herb. Fries to be cinereo-pallid with a slight fuscous tinge and with basidia and spores as stated above but many of the spores even. Romell describes the spores of his specimens from Brazil as “hyaline, laeves, ellips., 5-7 x 3-4 mmm.,” and as agreeing with the type. I have reéxam- ined my sections from the type; the spores are certainly colored and many of them rough-walled. Specimens examined: Costa Rica: San José, Oersted (in Herb. Fries, Univ. Upsal.), type. 12. T. cornucopioides Fries, (Nov. Symb. Mye. 91.) Actis В. Soc. Se. Upsal. IIT. т: 107. 1851.1 Type: not known to be in existence; not in Herb. Fries, at Upsala, nor in Kew Herb. Pileus pliant becoming rigid, deeply infundibuliform, 5-71 cm. broad, radiately rugose, glabrous, fuscous; stem solid, rather glabrous, pallid; hymenium inferior, somewhat rugose, gray. On the ground. Near San José, Costa Rica. This species bears so singular a resemblance to Craterellus cornucopioides that from pictures they are scarcely to be dis- tinguished. The present species has the stem truly solid and the substance fleshy pliant when living, nearly stony-woody when dry; stem 5-73 cm. long, 4-6 mm. thick, equal or attenu- 1A figure will be given in Part II. 1914) BURT—THELEPHORACEJE OF NORTH AMERICA. I 213 ated at the base, compressed, rather glabrous, very tough, pallid; pileus membranaceous-cartilaginous, when dry quite rigid, radiately rugose, with the ridges elevated towards the undulate and at first fimbriate margin, not zonate after the manner of species of Stereum; hymenium inferior, hardened. Related to Cladoderris. I refer to Т. cornucopioides a collection made in Jamaica by Prof. F. S. Earle, in 1902, the specimens of which agree well with the original description, as translated above, except in size. They are 3-34 cm. high and 2 cm. broad with stem about 1 cm. long by 2-4 mm. thick. The dried fructification is very hard and stony and softens so little with water that the edge of the razor is turned in sectioning. The spores are colorless and even at first and become slightly colored and angular, 9-10 x би. Specimens examined: Jamaica: Castleton Gardens, F. S. Earle, New York Bot. Gard., Plants of Jamaica, 238. 13. T. vialis Schw. (Syn. N. Am. Fungi) Trans. Am. Phil. бос. N.S. 4:165. 1834. Plate 5. fig. 15. T. tephroleuca В. & C. Grevillea 1:149. 1873. Type: in Herb. Schweinitz. Fructification coriaceous, dirty whitish or pallid, sometimes wood-brown at the center, upper surface usually radiately pli- cate or rough with masses of agglutinated fibers; pileus polymor- phic, sometimes composed of ascending lobes or small pilei which arise from а common base and grow together above to forma broad cup, or sometimes with the whole interior of the cup filled with small pilei and lobes many of which arise proliferously from the upper surface of the outer lobes; stem central when present; hymenium inferior, rugose, somewhat рарШове, yel- lowish pallid becoming avellaneous or somewhat fuscous; spores olive-buff under the microscope, bluntly angular (i. e., tips of the angles obtuse), 43-7 x 43-би. Fructification 23—5 or 6 em. high, 21-7 cm. broad. On ground in frondose woods. Vermont to South Carolina and west to Illinois. September. This is a fine, large species well marked by the dirty whitish or yellowish, fibrillose upper surface of the pileus, thick substance of the same color unless the specimen is old, and the brown, [Vor. 1 214 ANNALS OF THE MISSOURI BOTANICAL GARDEN slightly wrinkled hymenium. As in the otherwise very different Т. caryophyllea, large specimens sometimes resemble a double flower from the great number of small pileoli and lobes present in the center. Schweinitz described the species as sometimes having dimidiate pilei, but I have seen no such specimens. My collection assumed a disagreeable odor in drying but no such odor has been noted by others. Specimens examined: Exsiccati: Ell. & Ev., N. Am. Fungi, 1110, and Fun. Col., 1598, in both under the name 7’. caespitulans. Vermont: Lake Dunmore, Е. A. Burt. New Jersey: Newfield, J. B. Ellis (in Mo. Bot. Gard. Herb., 5155), also in the exsiccati cited. Pennsylvania: Bethlehem, Schweinitz, type (іп Herb. Sehw.); Michener, 1504 (in Curtis Herb. and in Kew Herb.), the собуре and type respectively of Т. tephroleuca; Trexlertown, W. Herbst, 43, C. G. Lloyd and W. Herbst, 2866, 3088 (both in Lloyd Herb.) ; N. M. Glatfelter (in Mo. Bot. Gard. Herb., 42561). Dist. of Columbia: Washington, F. J. Braendle, comm. by C. H. Peck. North Carolina: G. Ғ. Atkinson (in Cornell Univ. Herb., 23253); Asheville, H. C. Beardslee; Schweinitz cited North Caro- lina as a station. South Carolina: Caesar's Head, Ravenel, one of the types (in Curtis Herb. and Kew Herb.) of T. tephroleuca. Ohio: C. G. Lloyd, 4000. Illinois: Glen Ellen, E. T. and S. A. Harper, 669. 14. T. albido-brunnea Schw. Trans. Am. Phil. Soc. N. В. 4:166. 1834. Plate 5. fig. 13. Stereum Micheneri B. & C. Grevillea т: 162. 1873 (in part).- Stereum spongiosum Massee, Jour. Linn. Soc. Bot. 27: 172. 1889.—Thelephora odorifera Peck, Rep. №. Y. State Mus. 44: 132 (22). 1891. Type: in Herb. Schweinitz. Pileus sessile or with very short stem, coriaceous, spongy when dry, uniformly cinnamon-buff or with the older portions chest- nut-brown, sometimes assuming mesopod form when encircling small twigs or shrubs, sometimes effuso-reflexed, usually dimidi- 1914) BURT—THELEPHORACEJE OF NORTH AMERICA. I 215 ate and somewhat imbricated, fibrous-tomentose, margin thick and entire; substance concolorous with the upper surface, spongy, more than 2 mm. thick, with hyphae 4%-би in diam- eter; hymenium inferior, even, not polished, cinnamon-buff ; basidia simple; spores deep olive-buff under the microscope, echinulate, 8-10 x 6-8y. Pileus 2-4 em. in diameter when circular, or 1-23 cm. long, 2-4 em. broad, often 1 cm. thick at base when dimidiate. Running up and encircling twigs on the ground and against the base of shrubs. Canada to Louisiana and west to Wiscon- sin. August. Peck describes the odor as quite fragrant at first but states that it is lost after а few weeks; I did not notice any especial odor for my collection. Т. albido-brunnea may be distinguished from our other dimidiate and reflexed species of Thelephora by its even and pale hymenium and thick spongy pileus. Schwei- nitz confused one collection of this species with T. biennis Fr., from the specimen of which in the Fries Herbarium, at Upsala, itis clearly distinct. Тһе types of Stereum spongiosum Massee, viz., Curtis, 3582, and Ravenel, 1732, in Kew Herbarium, have colored echinulate spores 8-10 x 6-74, although described by Massee as “еШірвоідеге 6-7 x 4u” without mention of color and projections of the wall. The type of T'helephora odorifera Peck, in Coll. N. Y. State, is somewhat bleached or faded but quite typical. Specimens examined: Exsiccati: Ravenel, Fun. Car. IV, 12, the type distribution of 7. Micheneri B. & C.; Ell. & Ev., N. Am. Fungi, 1599, and Fun. Со!., 1209, under the name Т. Micheneri in both. Canada: Toronto, J. Dearness (in Lloyd Herb.). Vermont: Lake Dunmore, Е. A. Burt. New York: Selkirk, C. H. Peck (in Coll. N. Y. State), the type of Т. odorifera Pk.; Alcove, С. L. Shear, 1010, 1163, 1184; Jamesville, L. М. Underwood. Pennsylvania: Bethlehem, Schweinitz (in Herb. Schw.), the type, and also the Nos. 627 and 625 of Syn. N. Am. Fungi under the names respectively of T. biennis and T. laciniata; Michener (in Curtis Herb., 3582, and also in Kew Herb., same number), type of Stereum spongiosum Massee; Trex- lertown, W. Herbst, 18, and (in Lloyd Herb., 3052). [Vor. 1 216 ANNALS OF THE MISSOURI BOTANICAL GARDEN North Carolina: Blowing Rock, G. F. Atkinson, 4322. South Carolina: Ravenel, 790 (in Curtis Herb. and in Kew Herb.), under the name Thelephora biennis; Santee Canal, Ravenel, 1732 (in Curtis Herb. and in Kew Herb.), type of Stereum spongiosum Massee. Louisiana: Bogalusa, C. J. Humphrey, 466. Ohio: Cincinnati, A. P. M organ, Lloyd Herb., 2627. Michigan: Saugatuck, E. A. and S. A. Harper, 654. Wisconsin: Milwaukee Co., comm. by Mrs. F. W. Patterson. 15. T.lutosa Schw. Trans. Am. Phil. Soc. №.8.4:166. 1834.1 Туре: in Herb. Schweinitz. Pilei cespitose, densely imbricated, at first somewhat fleshy but at length hard, undulate-plicate, yellowish, almost sub- tomentose with pulverulence, somewhat horizontally attenu- ated behind, margin sublobate, at length inflexed; pileus less than 2 mm. thick, with hyphae Зи in diameter; hymenium be- coming yellowish, even; spores olive-buff under the microscope, angular, 5-6 x 32-4. Cluster about 13 cm. high and broad. On the ground in roads and in woods. North Carolina. The type is distinct from T. albido-brunnea, having thinner pileus, finer hyphae, and smaller and paler spores. "The pilei were crowded together into a small buff-colored cluster about 15 em. high and broad, somewhat as in Tremellodendron palli- dum (Schw.); I failed to find stems at their bases. Specimens examined: North Carolina: Salem, Schweinitz (in Herb. Schw.), type. 16. T. cuticularis Berk. Hooker’s Lond. Jour. Bot. 6: 324. 1847. Republished in Lea, Catalogue of Plants in Vicinity of Cincinnati 66. d. 1849. Plate 5. fig. 14. Type: in Kew Herb., and a portion of it from Berkeley in Curtis Herb. Pilei coriaceous-soft, effuso-reflexed or dimidiate, imbricate, sometimes laterally confluent, drying pinkish buff to cinnamon- brown, with a broad, pale margin, surface radiately rugose, soft, silky fibrillose; substance of the same color as pileus; hy- menium inferior, concave, even, drab to brownish drab; spores umbrinous under the microscope, flattened on one side or some- what kidney-shaped, not angular, echinulate, 8-9 x 6-7и. ! А figure will be given in Part II. 1914) BURT—THELEPHORACEZ OF NORTH AMERICA, I 217 Pileus 1-15 cm. long, 2-4 ст. broad, 1 mm. thick. On mossy bark at the base of trees and on fallen twigs in groves. Vermont to Texas and west to Missouri. June to August. In his description Berkeley noted that the odor of this species is strong and unpleasant; my specimens retained such an odor for several years but I did not notice it before they were dried. T. cuticularis may be distinguished from our other species by its drab hymenium, portions of which become sage-green when crushed under a cover glass in a 7 per cent solution of potassium hydrate, and by its spores, which are not at all angular or irreg- ular as regards the main body of the spore, but ovoid and flat- tened on one side or slightly kidney-shaped and sparingly studded with slender spines. Specimens examined: Vermont: Middlebury, E. A. Burt. Rhode Island: Olney, 1851 (in Kew Herb. and in Curtis Herb.). Pennsylvania: Bethlehem, Schweinitz (in Herb. Schw.), the Nos. 628 and 629 of Syn. N. Am. Fungi, under the names respec- tively of T. fuscocinerea, апа Т. gausapata; Kitanning, D. R. Sumstine, 1. Delaware: Newark, H. S. Jackson. North Carolina: Asheville, H. C. Beardslee, 03195. Florida: Mrs. Sams, comm. by C. G. Lloyd. Texas: W. H. Long, Jr., 351, 387 (in Cornell Univ. Herb.). Ohio: Waynesville, T. G. Lea (in Kew Herb.), type; Preston, А.Р. and L. V. Morgan, comm. by C. G. Lloyd, also C. G. Lloyd, specimen dated July 26, 1896; Cincinnati, C. G. Lloyd, 4492. Wisconsin: Blue Mounds, E. T. and S. A. Harper, 861. Missouri: Columbia, B. M. Duggar, 289. 17. T. intybacea Pers. ex Fries, Syst. Myc. 1:431. 1821. Plate 5. fig. 11. T. intybacea Pers. Syn. Fung. 567. 1801-1807; Myc. Eur. т: 110. 1822. Illustrations: Bulliard, Champ. de la France pl. 278.-Bigeard et Guillemin, Champ. Super. France 436. pl. 44. f. 1. Fructifications cespitose, soft, whitish, then rufous-ferru- ginous, drying chestnut-brown to Rood's brown, with stems [Vor. 1 218 ANNALS OF THE MISSOURI BOTANICAL GARDEN somewhat lateral and growing into one another; pilei imbricated, fibrous, usually with the fibers matted and agglutinated into appressed and wholly adnate squamules, margin dilated and whitish-fimbriate at first, at length becoming entire and colored like the rest of the pileus; hymenium inferior, concolorous with the upper surface, papillose; spores concolorous with hyme- nium, snuff-brown under the microscope, angular-tuberculate, 7-9 x 6-84. Clusters often 5-8 cm. іп diameter; individual pileus 2-3 em. long, 2-4 cm. broad, 1 mm. thick. On the ground in pine woods, growing up from the layer of fallen leaves. Ontario to North Carolina and westward to Ohio and Michigan. August to October. The clusters are sometimes central but more often with the pilei lateral and triangular; sometimes the mass ascends small sticks and then extends out from this support in reflexed forms; the upper surface is usually uneven and dries somewhat depressed between the adnate squamules. This species is distinguished from ferruginous specimens of T. terrestris by the thicker and entire margin of the pileus and by the absence of free squamules. Specimens examined: Exsiccati: Ell. & Ev., Fun. Col., 1410. Austria: G. Bresadola. Ontario: Toronto, J. Dearness, comm. by C. G. Lloyd; Harraby, Lake Rosseau, E. T. and S. A. Harper, 682. Maine: Portage, L. W. Riddle, 3. New Hampshire: Shelburne, W. G. Farlow. Vermont: Middlebury, Sudbury, Grand View Mt., E. A. Burt. Massachusetts: A. P. D. Piguet, comm. by Dr. Farlow; Natick, G. E. Morris, No. E. Connecticut: East Hartford, C. C. Hanmer, 1434. New York: Alcove, C. L. Shear, 1009; East Galway, E. A. Burt; Ithaea, G. F. Atkinson, Cornell Univ. Herb., 3050, 19652. Dist. of Columbia: Takoma Park, C. L. Shear, 799, 796; Wash- ington, O. F. Cook, 4, comm. by P. L. Ricker. North Carolina: Asheville, H. C. Beardslee, 0341. Ohio: A. P. Morgan (in Lloyd Herb.). Michigan: C. G. Lloyd, 4546; Lawton, L. A. Hawkins; Sailor's Encampment, Allen and Stuntz, 1, Univ. of Wis. Herb. 1914] BURT—THELEPHORACE® OF NORTH AMERICA. I 219 18. T. terrestris Ehrh. ex Fries, Syst. Мус. 1: 431. 1822. Plate 5. fig. 10. T. terrestris Ehrh. Crypt. Exsiec. No. 178. 1785.-Persoon, Syn. Fung. 566. 1801; Myc. Eur. т: 113. 1822.—Stereum lacin- таит Pers. Obs. Myc. т: 36. 1796.- T helephora laciniata Pers. Syn. Fung. 567. 1801.-T. caryophyllea 8 laciniata Pers. Myc. Eur. 1: 112. 1822.-T. laciniata Fries, Syst. Мус. т: 431. 1821. Illustrations: Batsch, Elenchus Fung. pl. 24. f. 121.-Nees, System der Pilze pl. 34. f. 251.-Bolton, Hist. Fung. pl. 173.— Sowerby, Col. Fig. of Eng. Fungi pl. 213.-Cooke, Handbook I: 310.-Stevenson, Brit. Hym. 2: 2061.-Smith, Brit. Basid. 399. f. 96 С-Е. Fructifications dark fuscous to fawn-color, coriaceous-soft, cespitose, obconic, with a short stem-like base, or dimidiate and sessile, or incrusting and effuso-reflexed; pileoli more or less imbricated, sometimes laterally confluent, fibrous-squamulose and usually strigose, thin, margin fibrous-fimbriate and lacin- iate; hymenium inferior, papillose, fuscous to fawn-color; spores pale fuscous, irregular, angular, sometimes slightly tuberculate, 6-9 x би. Clusters 5-8 cm. in diameter, with single pileolus about 3 cm. long and broad; obconic pileus 2-3 ст. in diameter; dimidiate pileolus 13-2 em. long, 2-3 cm. broad, about 1 mm. thick. On sandy ground in bare fields and at base of trunks and from fallen twigs and leaves in pine woods. Canada to South Carolina, and in Michigan, Jamaica, and Alaska. July to De- cember. My observations of this species acquired from specimens received and from seeing it growing abundantly near Middle Grove, N. Y., seem to show that the medium from which this fungus derives its food produces an interesting effect on the fructification. Growing from bare, sandy ground the fructifica- tions are dark fuscous in color, and may be flattened clusters of imbricated pileoli, or of the obconic-pileus type composed of ascending pileoli confluent laterally, or dimidiate, sessile pileoli. When growing on abundant woody matter, as is the case in the specimen in Sowerby’s illustration already cited, the fructifica- tion assumes à redder color and replaces its dimidiate, sessile pileus on earth by a reflexed one on the wood. With regard to [Vor. 1 220 ANNALS OF THE MISSOURI BOTANICAL GARDEN other forms of the clusters and pileoli, the covering of the upper surface, and the spore characters there is no difference between those fructifications produced without woody food and those having it. There is no sharp color separation between these color extremes. Specimens growing on the ground usually have a short stem- like base, while those growing on wood are reflexed; the same col- lection may show both these conditions, as, for example, that from Skagway, Alaska, if some of the fructifications start from sticks and others directly from the ground. Persoon regarded the stem in T. terrestris as the chief character separating that species from his T. laciniata, as may be seen from his own de- scriptions contrasting the two in his ‘Synopsis Fungorum, pp. 566 and 5067, as follows: “3. THEL. TERRESTRIS: subimbricata obscure fusca, pileo applanato fibroso-strigoso.”’ "Hab. in arenosis ad terram. Stipes breuis, lateralis omnino adest. Substantia submollis, non ita coriacea sicca, vti in ceteris speciebus." “4. THEL, LACINIATA: imbricata obscure fusca, pileo tenui laciniato crispo subtus papillis congestis scabro.” “Hab. ad radices truncorum. Cespitem difformem efformat, 2 упс. lata, tenuis. Stip. vix adest distinctus." These descriptions supplement each other as a description for one species; each has special application to fructifications growing side by side under such conditions as to show that they аге from а common mycelium. Persoon never claimed that his species differed from 7. terrestris in color. Fries gave a different description of T. laciniata in his works cited—to the injury of T. intybacea—, but the characters he gives are not satisfactory. European mycologists with a wide knowledge of the Thele- phoracee as they grow are unable to distinguish these two species. In letters to me, Bresadola regards Т. laciniata as a synonym of T. terrestris; and Romell does not know T. terrestris if it is distinct from T. laciniata. Specimens examined: Exsiccati: Ellis, №. Am. Fungi, 511; ЕП. & Ev., №. Am. Fungi, 2732, under the name 7. intybacea. Austria: G. Bresadola. Sweden: G. Romell, 52, 55, 56, 57. Newfoundland: A. C. Waghorne, 276 (in Mo. Bot. Gard. Herb.). 1914) BURT—THELEPHORACEJE OF NORTH AMERICA. I 221 Quebec: Gaspe, J. Macoun, 229. Ontario: Ottawa and Belleville, J. Macoun. Maine: Wells, J. Blake, comm. by P. L. Ricker. New Hampshire: Chocorua, W. G. Farlow. Massachusetts: Magnolia and Woods Hole, W. G. Farlow; Ips- wich, G. E. Morris, No. F. Connecticut: South Windsor, East Hartford, and Rockville, C. C. Hanmer, 1227-29, 944, 1067. New York: East Galway and Middle Grove, E. A. Burt, three collections from the latter station; Ithaca, G. F. Atkinson, Cornell Univ. Herb., 22976. New Jersey: Belleplain, C. L. Shear, 1246; Newfield, J. B. Ellis, Ellis, N. Am. Fungi, 511. Pennsylvania: Schweinitz (in Herb. Schw.), the 624 of Syn. N. Am. Fungi. North Carolina: Asheville, H. C. Beardslee, 02280; Salem, Schwei- nitz (in Herb. Schw.), the 624 of Syn. N. Am. Fungi. Alabama: Tuskegee, Beaumont, 199 (in Curtis Herb.). South Carolina: Society Hill, M. A. Curtis, 2693 (in Curtis Herb.). Michigan: Agricultural College, G. H. Hicks, Ell. & Ev., Fun. Col., 2732. Alaska: Skagway, J. Macoun, 47; Evans, 410 (in Mo. Bot. Gard. Herb.). Jamaica: Cinchona, W. A. and E. L. Murrill, New York Bot. Gard., Fun. of Jamaica, 451. 19. T. griseozonata Cooke, Grevillea 19: 104. 1891. Plate 5. fig. 12. Type: in Ravenel, Fun. Amer., 444. | Fructifications cespitose, coriaceous-soft ; pileoli extended into a short sublateral stem, imbricate; applanate, silky-strigose, zonate with alternating cervine (Rood's brown) and light buff zones, margin subfimbriate; hymenium inferior, castaneous when fresh, drying Rood's brown, rugose, somewhat papillose; spores pale fuscous, angular, 6-9 x 6-7џ. Cluster 3—6 cm. in diameter; obconic pileus and single pileo- lus each 2-3 cm. in diameter. 5 [Vor. 1 222 ANNALS OF THE MISSOURI BOTANICAL GARDEN On sandy ground in pine woods. New Jersey to Louisiana. August to November. This species is closely related to 7. terrestris and has the same habitat, habit of growth, and spore characters, but is distin- guished from that species by its zonate pileus. Тһе fructifica- tions usually occur in flattened clusters with spreading pileoli; sometimes the individual pileoli acquire an infundibuliform appearance by the growing together for part of their length of opposite edges of individual pileoli; sometimes a small оһсопіс pileus occurs composed of two or more pileoli with adjacent edges confluent. In the collection cited below from Mississippi, small lobes are present in the cavity of the cup, as in T. vialis and T. caryophyllea. Specimens examined: Exsiccati: Ravenel, Fungi Am., 444, type distribution; Ravenel, Fun. Car. II, 28, under the name 7’. caryophyllea; Ellis, N. Am. Fungi, 714; Ell. & Ev., Fun. Col., 1305. New Jersey: Newfield, J. B. Ellis, in his exsiccati cited. South Carolina: Aiken, H. W. Ravenel, Fungi Am., 444, type collection. Alabama: Auburn, C. F. Baker, Lloyd Herb., 3462. Mississippi: Biloxi, Mrs. E. S. Earle, 32. Louisiana: St. Martinville, A. B. Langlois, by. 20. T. fimbriata Schw. ex Schweinitz, Trans. Am. Phil. Soc. М. 8.4: 166. 1834. Plate 4. fig. 3. Merisma fimbriatum Schw. (Syn. Fung. Car., No. 1067) Schrift. d. Naturforsch. Gesell., Leipzig, т: 110. 1822.-Tele- phora scoparia Peck, Rep. N. Y. State Mus. 42: 123 (27). pl. 2. f. 20, 21. 1889. Illustrations: Peck, Rep. N. Y. State Mus. 42: pl. 2. f. 20, 21. Type: in Herb. Schweinitz. Fructification coriaceous-soft, incrusting and ascending small plants (mosses, etc.), here and there emitting fascicles of branches united below, subterete, acuminate or fimbriately incised, at first pale or whitish, soon ferruginous brown, dry- ing Rood's brown; hymenium even, pruinose-pubescent; spores umbrinous, tuberculate, 7-11 x 6-9 и. КЫ p wenige cd: 9 T: UE баны еда ы Шыт рысты ха, E n E А eal gS ак А, сансан MR UP oe petere euet 4 1914] BURT—THELEPHORACEJE OF NORTH AMERICA. I 223 Inerusting and ascending upward 1-3 сіп.; free branches 5-10 mm. long, 1 mm. thick, sweep of fascicle about 5-10 mm. In moist places. New York to South Carolina, and west to Illinois. July and August. Тһе type is an incrusting specimen, covering as its main axis a small twig in one specimen and a moss in the other, and send- ing out а few lateral branches which are flattened towards the free ends and subfimbriate; main trunk is cylindric, latericius (of ‘Chromotaxia’), ends of branches paler; spores umbrinous under the microscope, tuberculate, 7-8 x би. Schweinitz de- scribed the species as becoming hard and cartilaginous, but this is an error probably due to the foreign matter surrounded by the main trunk. Several other specimens are present in his herbarium under various names. Specimens examined: Exsiccati: Ellis, N. Am. Fungi, 512, under the name Т. cristata. Massachusetts: Weston, A. B. Seymour, T 1 (in Mo. Bot. Gard. Herb., 45573). New York: Bethlehem and Selkirk, C. H. Peck (in Coll. N. Y. State), type of Т. scoparia; Syracuse, from Herb. Cornell Univ., 19474. New Jersey: Newfield, J. B. Ellis, N. Am. Fungi, 512. Pennsylvania: Bethlehem, Schweinitz (in Herb. Schw.), the 615 of Syn. N. Am. Fungi, under the name Т. stabularis. North Carolina: Salem, Schweinitz (in Herb. Schw.), type, and also the 1063 of Syn. Fung. Car., under the name Merisma fuscescens. Indiana: Millers, E. Т. and S. A. Harper, 670. Illinois: Havana, H. С. Beardslee; Riverside, E. T. and 8. А. Harper, 668. 21. T. perplexa Burt, n. sp.! Type: in Curtis Herb. Fructification incrusting, coriaceous, consisting of a resupinate membrane from the central portion of which arise cylindric trunks either simple or digitately branched; resupinate portion spongy, firm, separable, fuscous at the center, margin thin, determinate, pinkish buff; ascending portions spongy, firm, 1 A figure will be given in Part II. [Vor. 1 224 ANNALS OF THE MISSOURI BOTANICAL GARDEN fuscous, simple and tapering upward or soon branching and terminating in paler either subulate tips or somewhat flattened ends; spores fuscous, subglobose, echinulate, 8-10 x 8-9. The resupinate membrane may be 3 cm. in diameter; ascending portion of fructification 2-3 em. high, 15-2 mm. thick. On decaying leaves and sticks оп the ground. Cuba. Berkeley & Curtis based their description of Thelephora den- tosa on two collections made in Cuba by C. Wright in different years; these collections are different specifically. Тһе original description applies chiefly to the earlier collection, made in 1857, which is unnumbered. I take my type of T. perplexa from the later collection, C. Wright, 238. Specimens examined: Exsiecati: Fungi Cubenses Wrightiani, C. Wright, 238, under the name T'helephora dentosa B. & C. Cuba: C. Wright, 288, type (in Curtis Herb.). 22. T. dentosa Berk. & Curtis emend Burt.! T. dentosa B. & C. (Fungi Cubenses) Jour. Linn. Soc. Bot. 10: 329. 1807. Type: type and cotype in Kew Herb. and Curtis Herb. re- spectively. Fructification coriaceous-soft, incrusting leaves and small twigs on the ground and ascending as free, sessile, dilated, tri- angular, flabelliform pilei which are dentate at the upper end or deeply divided into а few finger-shaped divisions, honey- yellow to tawny olivaceous throughout, minutely hairy under a lens; spores honey-yellow, globose to ovoid, weakly echinulate, 6-10 x 6-84. Pileus 1 em. high, 5 mm.-1 em. broad. On rotten vegetation. Cuba. June. As already stated in connection with T. perplexa, Berkeley & Curtis cited for types of their T'. dentosa specimens from two collections made in Cuba by C. Wright. "These collections were made with an interval of several years between the collections, which differ specifically. Ав noted by Berkeley & Curtis, their description applies better to the earlier collection, to which I now 1 A figure will be given in Part II. 1914] BURT—THELEPHORACEJE OF NORTH AMERICA. I 225 restrict their species. This earlier collection was distributed by C. Wright, unnumbered, under the name Thelephora dentosa B. & C. before the publication of the description of this species, and the cotype in Curtis Herb. is unnumbered also. By what was apparently a slip of the pen, Berkeley cited this type as C. Wright, 507. By the kindness of Dr. Farlow I have been permitted to examine the manuscript records which show that Wright collected only one No. 507, which was determined by Berkeley as Xylaria obovata Berk. and is cited under this species by Berk. & Curtis, Jour. Linn. бос. Bot. 10: 380. 1867. I find in Curtis Herb. such a specimen labelled Xylaria obovata Berk., Cuba, C. Wright, 507. I conclude that the type and собуре of T. dentosa B. & C., first cited in their description, are from the collection distributed by C. Wright, unnumbered, under the name Thelephora dentosa В. & С. Specimens examined: Exsiccati: Plantae Cubenses Wrightianae, unnumbered, under the name Thelephora dentosa B. & C. Cuba: C. Wright, cotype (in Curtis Herb.). 23. T. spiculosa Fries, Syst. Myc. 1: 434. 1821; Epicr. Syst. Мус. 539. 1836-38. Plate 4. fig. 2. Illustrations: Persoon, Syn. Fung. pl. 3. f. 16. Type: an authentic specimen from Fries, in Kew Herb. Fructifications cespitose, from byssoid becoming fleshy, vari- able by incrusting habit, pale buff at first, main portions becoming purplish-fuscous (Rood's brown) with age, ramose- spiculous, tips penicillate and whitish; spores umbrinous under the microscope, irregular, echinulate, 8-9 x 6-7 y. Clusters 1-2 cm. high, 2-4 cm. in diameter, single fructifica- tion 1-2 cm. high, about 1 mm. in diameter, with branches spreading 4-6 mm. On leaves on ground in moist groves. Ohio to Wisconsin. July. Rare. The best specimens which I have seen have main trunks of the fructifications running side by side over partially decayed beech leaves and confluent into an effused mass. "These trunks ascend obliquely from the leaves to a height of 1-2 ст., branch sparingly, and terminate in spiculous tips. Тһе fructification [Vor. 1, 1914] 226 ANNALS OF THE MISSOURI BOTANICAL GARDEN must be inconspicuous in the woods since the general color of the mass is the same as that of the leaves on which it is effused, although the main trunks may be darker. Specimens examined: Exsiecati: Kunze, Fun. Sel. Exsic., 560. Sweden: specimen from Fries (in Kew Herb.). Austria: G. Bresadola. Ohio: Preston, C. G. Lloyd. Michigan: Glen Lake, C. G. Lloyd, 02471. Wisconsin: Lake Geneva, E. T. and S. A. Harper, 883. (To be continued.) ExPLANATION оғ PLATE PLATE 4 All figures of plates 4 and 5 have been reproduced natural size from photographs of dried herbarium specimens of species of Thelephora. Fig. 1. Thelephora anthocephala. From specimen collected at Linwood, Ohio, by C. G. Lloyd, No. 02164. Fig. 2. Т. spiculosa. а, from specimen on leaves of Fagus collected in Europe by Bresadola, which I compared with the specimen from Fries in Kew Herbarium; b, from specimen collected at Glen Lake, Mich., by С. б. Lloyd, No. 02471. Fig. 3. Т. fimbriata. From specimen incrusting living strawberry (Fragaria) plant, collected at Riverside, Ш., by Е. Т. and 8. A. Harper, No. 668. Fig. 4. T. palmata. From specimen from New Jersey, from С. С. Lloyd, No. 4612. Fig. 5. Т. magnispora. From type specimens collected at Chester Vale, Jamaica, by W. A. and Edna L. Murrill, No. 295. a shows upper surface and side of pileus, and 5, the hymenium. Fig. 6. T. regularis. From a sketch of the type in Herb. Schweinitz. Fig. 7 a. T. multipartita. From specimens collected at Trexlertown, Pa., by Dr. W. Herbst. Fig.7 b. T. regularis. From specimens collected at Clayton, Del., by H. S. Jack- son. Fig. 8. T.scissilis. From type specimens collected at Bingen, Wash., by W. N. Suksdorf, No. 716. Fig. 9. T. caryophyllea. From specimens collected in Michigan, by C. С. Lloyd, No. 4547. ANN. Мо. Вот. GARD., VoL. 1, 1914 ! И E i E > : “! æ ) È | і с 4 i L^ Ес ttn Or ir aes BURT— THELEPHORACEAE OF NORTH AMERICA 1. THELEPHORA ANTHOCEPHALA.—2. Т. SPICULOSA.-—3. T. FIMBRIATA.— 4.Т. PALMATA.—5. T. MAGNISPORA.-—6 AND 7 b. T. REGULARIS.— 7 a. T. MULTIPARTITA. — 8. T. SCISSILIS.— 9. T. CARYOPHYLLEA. COCKAYNE. BOSTON, таулы [Vor. 1, 1914 228 ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 5. Fig. 10. 7. terrestris. From specimens collected on ground in open fields at Middle Grove, М. Y. а shows the fibrose-strigose upper surface and fimbriate margin of the pileus, and b, the hymenium of lower surface. Fig. 11. Т. intybacea. From specimens collected in pine woods incrusting fallen pine leaves and twigs at Middlebury, Vt. a shows upper surface with matted, ` adnate squamules and whitish, thick, entire margin; b, the hymenium of lower surface. Fig. 12. Т. griseozonata. From specimen of type collection, distributed in Ravenel, Fun. Amer., Хо. 444. Fig. 13. Т. albido-brunnea. a, upper side of specimen collected at Saugatuck, Mich., by Е. Т. and 8. A. Harper, No. 654. The specimen is about 2 cm. thick; b, hymenium of specimen collected at Lake Dunmore, Vt. Fig. 14. T. cuticularis. From specimens collected at Blue Mounds, Wis., by E. T. and 8. A. Harper, No. 861. а, viewed obliquely from above; b, viewed from under side to show hymenium. Fig. 15. Т. vialis. From specimen collected at Lake Dunmore, Vt. Ths foe. ROVE ee hs IE мут gg жүл АГ E dex | | BH Bi bi | d МҮЛТ £ M ых = TER ТҰТ етее ol Wem E ANN. Mo. Bor. GARD., Vor. 1, 1914 PLATE 5 ВОЕТ — THELEPHORACEAE OF NORTH AMERICA 10. THELEPHORA TERRESTRIS.—-11. T. INTYBACEA.— 12. T. GRISEO-ZONATA. — 13. Т. ALBIDO-BRUNNEA. — 14. T. CUTICULARIS.— 15. T. VIALIS. COCKAYNE, BOSTON, INDICATIONS REGARDING THE SOURCE ОҒ COM- BINED NITROGEN FOR ULVA LACTUCA G. L. FOSTER Teaching Fellow in the Henry Shaw School of Botany of Washington University INTRODUCTION Very little attention has been given the question of the sources of nitrogen for marine alge. Nevertheless, the ques- tion is an interesting one both physiologically and ecologically, because of the extremely small amount of nitrogen supposed to be present in sea-water, and because of the very noticeable change in the type of algal flora when the nitrogen content of the environment is increased, as by the presence of sewage. The literature bearing on the subject is practically limited to a debate between a few authors as to the amount and form of nitrogen in sea-water, and the way in which the supply is maintained. This dispute involves some questions of funda- mental importance for marine biology; consequently, а brief statement of the different views is pertinent. Natterer (13) reports that careful analyses of water from the high seas show scarcely a trace of nitrates. Nitrites are somewhat more abundant, but not sufficiently so to admit of quantitative determination. Ammonium compounds, on the other hand, according to Thoulet, are present in sufficient amount to be quantitatively determined, and vary from .13 to .34 mg. per liter (.013-.034 per cent) according to the locality. Reinke (15) considers these amounts of nitrogen reported to be insufficient for the production of the enormous amount of living material in the sea, especially when the activity of nitrifying and denitrifying bacteria is taken into account. He considers as of prime importance in this question the nitro- gen-fixing bacteria which have been demonstrated in sea-water by Benecke and Keutner (4), and others. Reinke found Azo- tobacter embedded in the gelatinous material on the surface of Laminaria fronds and argues for a symbiotic relation between the alge and bacteria. ANN. Mo. Bor. GARD., Vou. 1, 1914 (229) [Vor. 1 230 ANNALS OF THE MISSOURI BOTANICAL GARDEN Brandt (7), however, attaches little or no importance to Reinke's view, and maintains that the nitrogen content of sea-water is determined by a balance between the activity of denitrifying bacteria, on the one hand, and the great amount of nitrogenous material carried to the sea by the rivers, on the other. Brandt (5, 6) considers that the nitrogen content of sea-water is аба minimum" and is the limiting factor in the production of marine organisms. Considering especially the plankton life, he finds that the amount of plankton is propor- tional to the nitrogen content of the water, and correlates the comparative poverty of tropical seas in plankton life with the relatively greater activity of denitrifying bacteria in the warmer waters. More recently Pütter (14) bas reported that analyses of the water from the Gulf of Naples give per liter .18 mg. of nitrogen in nitrates and nitrites, and .56 mg. in ammoniacal nitrogen. Furthermore, he claims that these figures represent less than half the total combined nitrogen actually present in sea-water. In his opinion there is no need for considering the nitrogen content to be at a “minimum” since it is present in greater concentration than the carbon dioxide. It is impossible to say which of these views is the correct one, and further work in this field is much needed. The above named authors incidentally assume that nitrogen is available for the alge only in the form of nitrates or ammo- nium salts. This is entirely an a priori assumption, as no data are offered in support of such a view. On the contrary, it seems more likely that the alge can use many organic ni- trogen compounds. This would seem probable in the light of recent work which has been done on the fresh-water alge. In regard to the nutrition of the fresh-water forms we have departed far from the old idea that green plants are strictly autotrophic. Thus, by the work of Beyerinck (3), Charpentier (8), Chick (9), Artari (1, 2), and others, it has been established that many of the fresh-water alge have, with respect to nitrogen, distinct saprophytic tendencies,—preferring organic to inor- ganic nitrogen. Artari, especially, has shown that several alge (Chlamydomonas, Stichococcus, Chlorella, Scenedesmus, and others) can grow and retain the chlorophyll under completely 1914) FOSTER—SOURCE ОҒ COMBINED NITROGEN FOR ULVA 231 saprophytie conditions, as in solutions containing amino acids and glucose in the absence of light and carbon dioxide. In all these cases, however, growth is more rapid under so-called mixotrophie conditions, i. e., with both organie nitrogen and carbon present in addition to sunlight and carbon dioxide. Artari takes up the question of the relative value of different nitrogenous compounds, and shows that they vary greatly with different algee,—some preferring peptones, others, amino acids and ammonium salts, and a few, nitrates. On the whole, the majority of forms investigated grow best in the presence of amino nitrogen. Many alge, especially of this last class, are often found in water polluted with sewage or decaying organic matter. Among the marine alge, there is a more or less definite flora characteristic of sewage-polluted waters. Most conspicuous among the plants of this group are the species of Ulva. Letts and Richards (11), in their reports on sewage in British har- bors, state that Ulva latissima grows in excessive quantities in polluted waters, and they find that the nitrogen content of this seaweed varies with the degree of pollution of the water. Cultural experiments conducted by Letts and Richards showed that Ulva latissima grows more rapidly in a mixture of sewage and sea-water than in pure sea-water alone. EXPERIMENTAL Preliminary experiments were made at the Woods Hole Laboratory to determine the sources of available nitrogen for Ulva lactuca. The algal material used in the experiments was collected at the mouth of an inlet where the water was at all times highly polluted with sewage. The cultures were main- tained in the laboratory in glass tumblers containing 150 сс. of solution. When brought in, the fronds of Ulva were well rinsed in clean sea-water and cut into strips exactly 3 cm. in length and about 2 cm. wide. Three such strips were placed in each vessel, and the cultures kept at a temperature of 21°C. by placing the vessels in a tray of running water. In each case the solution was renewed at the end of 5 days. After 10 days the strips were again measured and the increase in length recorded. [Vor. 1 232 ANNALS OF THE MISSOURI BOTANICAL GARDEN Two main types of nutrient solution were used,—one (solu- tion A) being natural sea-water, the other (solution B) being an artificial sea-water minus nitrogen. These stock sea-waters were made double strength and subsequently diluted by the addition of distilled water and the stock solution of the nitro- genous compounds to be tested. The following nitrogen com- pounds were used in the experiments: ammonium nitrate, urea, acetamid, sodium asparaginate, acetanilid, and dimethyl- anilin. Parallel experiments were run, adding these compounds to solution A and solution B. Preliminary tests roughly determined the maximum поп- toxic concentrations of these compounds when added to sea- water to be: Ammonium nitrate............ 0.011 gram molecular с, реа 0.010 gram molecular Acetamid..................... 0.250 gram molecular Asparagin..................., 0.080 gram molecular The table presents the results of the experiments. All figures for concentrations represent fractions of gram molecules per liter, except in the case of dimethylanilin. Here the solubility was not known and the figures represent fractions of a saturated solution in distilled water at 20°C. In the column headed "growth" is recorded the increase in length in millimeters of the strips of Ulva after 10 days in the solution. In each case the figures for growth represent the average of three or more cultures. Checks show the growth in solutions A and B with no additional nitrogen. It is apparent from the following table that, under the condi- tions of the experiment, ammonium nitrate and urea are consider- ably better nutrients for Ulva than the other compounds used. These two cause a marked increase in growth over that of the controls, in both the artificial and natural sea-waters. The nutritive value of these compounds was also indicated by the healthy appearance of the cultures. Тһе alge were of a deep green color, very turgid, and considerably curled by rapid growth. Judging from the growth and general appearance of the cultures, there is little choice between the nutrient values of ammonium nitrate and urea. 1914] FOSTER—SOURCE OF COMBINED NITROGEN FOR ULVA 233 COMPARATIVE TABLE SHOWING GROWTH OF STRIPS OF ULVA IN VARIOUS NITROGEN-CONTAINING SOLUTIONS Ammonium nitrate Urea Acetamid Growth Growth Growth Cone. | Sote] Sole | 09. Sol*| So.*| C9? [sa | Sol* A B А В А В Check 0.8, 0.3 Check 0.8| 0.3) Check 0.8 | 0.3 0.00005 1.0| 0.5 0.0005 1.4| 0.5 0.001 0.8 | 0.4 0.0001 1.4 1.5 0.001 1.6 0.6 0.005 Lo 0.4 0.0005 9-20 0.005 1.6 1.4 0.01 0.7 0.5 0.001 1.4 1.2 0.01 0.9 1.3 0.10 1.0 10 Sodium asparaginate Acetanilid Dimethylanilin Growth Growth Growth Con. [Sol*|Bol* | Ce [ба | soe) 60064 (вое | вое А В А В А В Check 0.8 | 0.3 Check 0.8 | 0.3 Check 0.8 | 0.3 0.002 0.7 | 0.6 0.0005 0.6 | 0.5 0.002 0.3 | 0.4 0.01 0.9 | 0.2 0.0025 0.0 | 0.0 0.02 0.0 | 0.5 0.05 0.7 | 0.0 0.0125 0.0 | 0.0 0.10 0.0 | 0.0 * Sol. А = natural sea-water; sol. В = artificial sea-water. 1 Conc. under dimethylanilin represents fractions of а saturated solution in dis- tilled water at 2090. Acetamid has а somewhat lower nutrient value than ammo- nium nitrate or urea, but still it causes a greater growth than do the control solutions to which no foreign nitrogen was added. The alga in acetamid solutions appeared normal in every way. The results with the sodium asparaginate were rather unex- pected. This compound is well known to be a good nutrient for many fungi and fresh-water alge. For Ulva, on the other hand, sodium asparaginate appears to have no appreciable nutrient value. In no case did it cause any notable increase in growth, although the algal material appeared perfectly normal. [Vor. 1 234 ANNALS OF THE MISSOURI BOTANICAL GARDEN Acetanilid and dimethylanilin are in a separate class,—being decidedly toxic at all the concentrations used. At the lowest concentrations there was slight growth at first, but in ten days all cultures were dead and discolored. The results with these last two compounds are comparable to those obtained with similar substances by Czapek (10) and by Lutz (12) working on fungi and fresh-water alge. They found that compounds having the nitrogen attached directly to a benzene nucleus are toxic. Pure culture methods were not attempted on account of the brief time available for this work, and the question of the pos- sible interaction of ammonifying bacteria is therefore pertinent. However, the rapid augmentation of growth upon the addition of the amido compounds, and the comparative absence of bacteria both suggest a direct absorption of these substances. Moreover, since rapid growth of the alga occurs in concentra- tions of the amido compounds considerably greater than the toxic limit for ammonium salts, and since, further, no evi- dence of toxicity of fairly strong solutions of urea and aceta- mid developed during the interval of these experiments, no support is given to the thought that ammonification may be an important factor. However, in further continuation of this work it is proposed to control this possibility by quantitative tests. It seems probable from the facts brought out here, as well as from the work of Letts and Richards, that Ulva is not lim- ited to an inorganic nitrogen supply, since growth occurs with urea or acetamid as the sole source of nitrogen, and, as Letts and Richards have shown, that it grows more rapidly in sewage- polluted water than in pure sea-water. Undoubtedly, further experiments would show that other organic compounds can supply available nitrogen for Ulva. The results also indicate that for Ulva, at least, the amount of available nitrogen in the water is the limiting factor in growth. This is shown by the fact that growth is more rapid in sea- water containing additional nitrogen (ammonium nitrate, or urea) than in pure sea-water. The above mentioned results of Letts and Richards also point to the same conclusion, as does 1914] FOSTER— SOURCE OF COMBINED NITROGEN FOR ULVA 235 the abundant growth of Ulva in nature in waters polluted with sewage. In conclusion, the writer is pleased to express his thanks for the generous assistance given during this study by Prof. B. M. Duggar, under whose direction the work was carried out while occupying a research table maintained by Dartmouth College at the Marine Biological Laboratory, Woods Hole, Massachusetts. Graduate Laboratory, Missouri Botanical Garden. LITERATURE CITED 1. Artari, A. Zur Frage der physiologischen Rassen einiger griinen Algen. Ber. d. deut. bot. Ges. 20: 172-75. 1902. 2. —— ——, Zur Physiologie der Chlamydomonaden. Jahrb. f. wiss. Bot. 52: 410-66. 1913. 3. Beyerinck, M. W. Culturversuche mit Zoochlorellen, Lichenengonidien und anderen niederen Algen. Bot. Zeit. 48: 725-54, 757-67, 781-85. 1890. 4. Benecke, W., und Keutner, J. Über stickstoffbindende Bakterien aus der Ostsee. Ber. d. deut. bot. Ges. 21: 333-46. 1903. 5. Brandt, K. Ueber den Stoffwechsel im Meere. Wiss. Meeresunters. N.F. Abt. Kiel 4: 215-30. 1899. 6. ————, Ueber den Stoffwechsel im Meere. 2. Abhandl. Ibid. 6: 25-79. 1902. 7. ————, Ueber die Bedeutung der Stickstoffverbindung für die Produktion im Meere. Beih. bot. Centralbl. 16: 383-402. 1904. 8. Charpentier, P. С. Alimentation azotée d'une algue, le Cystococcus humicola. Ann. Inst. Pasteur 17: 321-34, 369-420. 1903. 9. Chick, H. А study of a unicellular green alga occurring in polluted water, with special reference to its nitrogenous metabolism. Proc. Roy. Soc. 71: 458-77. 1903. 10. Czapek, Fr. Zur Kenntniss der Stickstoffversorgung und Eiweissstoffwechsel bei Aspergillus niger. Ber. d. deut. bot. Ges. 19: 130-39. 1902. 11. Letts, E. A., and Richards, E. H. On green sea weeds (especially Ulva latissima) in relation to the pollution of the waters in which they occur. Seventh Report of the Roy. Comm. on Sewage Disposal. Appendix 3: 72-100. 1911. 12. Lutz, L. Sur l'action exercée sur les végétaux par les composés azotées organ- iques а noyau benzenique. Compt. rend. Cong. Soc. Sav. Paris 1903: 65-69. 13. Natterer, К. Chemische Untersuchungen von der Expedition Б. M. Schiff “Pola.” Denkschr. К. Akad. Wiss., Wien, math.-naturw. КІ. 65: 445. 1898. 14. Pütter, A. Der Stoffhaushalt des Meeres. Zeitschr. f. allgem. Physiol 7: 321-68. 1907. 15. Reinke, J. Die zur Ernührung der Meeresorganismen disponiblen Quellen an Stickstoff. Ber. d. deut. bot. Ges. 21: 371-80. 1903. THE EFFECT OF CERTAIN CONDITIONS UPON THE ACIDITY OF TOMATO FRUITS B. M. DUGGAR Physiologist to the Missouri Botanical Garden Professor of Plant Physiology in the Henry Shaw School of Botany of Washington University AND M. C. MERRILL Research Assistant to the Missouri Botanical Garden In а recent communication the senior author (4) has referred to the possibility that the total acid content of tomato fruits ripened at a temperature of 30°C., or above, may be related in some way to the failure of lycopersicin development at that temperature. It was determined that the ''total acidity for green, ripening, and ripe fruits, grown under the same condi- tions, is unexpectedly uniform, amounting to .57 to .58 per cent citric acid." The fruits just referred to were of the same variety picked at the same time. Тһе tests of acid con- tent of incubated fruits were made later in the season, and these indicated a lower acidity than that of normally green or ripe fruits. At that time the requisite material was obtained from the Department of Horticulture, Cornell University. During the past summer several varieties of tomatoes were grown in the Missouri Botanical Garden in order to furnish material for further pigment studies, and incidentally this material has enabled us to determine with greater care the acid content of tomato fruits, especially of different varieties, and likewise the comparative acidity of fruits direct from the field and of those of the same picking incubated for various intervals. The tests included below were made by pulping thoroughly а weighed quantity of the tissue (15 gm.), diluting with 150 сс. distilled water, employing for each titration 25 сс. of this solution diluted with distilled water to 50 cc., and titrating with n/10 NaOH, using phenolphthalein as indicator. Not less than two titrations were made in any case, and these were from one or more samples of tissue. Тһе accompanying table ANN. Мо. Вот. GARD., Vor. 1, 1914 (237) 6 [Vor. 1 298 ANNALS OF THE MISSOURI BOTANICAL GARDEN indicates the variety and condition of the fruit; quantities of n/10 NaOH required to neutralize; and the per cent of acidity in terms of citric acid. TABLE SHOWING ACID CONTENT OF TOMATO FRUITS Condition Average no. : Total per Variet ot ot of cent of acid y When Interval When n/10 NaOH, s cit - picked or incubation titrated* to neutralize "— Dwarf Stone Ripe 0 Red 1.695 .52 Dwarf Stone Half grown 0 Green 1.82 .56 Dwarf Stone Half grown | Incub. 32? C. 10 days | Artif. yellow 2.135 .66 Dwarf Stone Half grown Lab. 24 days Red 1.375 .42 Dwarf Stone Half grown | Incub. 32? C. 10 days Green 1.485 .46 | Sparks' Earliana Ripe 0 Red 1.695 .52 Sparks' Earliana | Half grown 0 Green .87 .58 Truckers' Favorite | Half grown | Incub. 32? C. 22 days | Artif. yellow 2.56 .79 Truckers' Favorite | Half grown Lab. 24 days Red 1.66 .51 Red Peach Half grown | Incub. 32? C. 22 days | Artif. yellow 2.115 .65 Red Peach Half grown Lab. 24 days Red 1.675 .52 Yellow Peach Half grown | Incub. 32° C. 22 days | Artif. yellow 2.47 .76 Yellow Peach Half grown Lab. 24 days Yellow 2.065 .64 Yellow Plum Ripe 0 Yellow 2.12 .65 Yellow Plum Half grown 0 Green 1.92 .59 Yellow Pear Half grown | Incub. 32? C. 20 days | Artif. yellow 1.60 .49 Yellow Pear Half grown Lab. 24 days Yellow 1.395 .43 * All fruits designated “тей,” “yellow,” and “artificial yellow" were, at the same time, ripe. The results above reported may not yet be as extensive as might be desired in order to follow closely the changes in acid- ity under different conditions; but they consistently point out certain relations of interest which may be briefly enumerated as follows: (1) A comparison of the acid content of green and normally ripened fruits was made, using Dwarf Stone, Sparks’ Earliana, and Yellow Plum, all direct from the field. There were no marked differences between the green and ripe stages within the variety; yet the acidity of the green fruits of the red varieties in these tests is somewhat higher, while the acid eontent of the green fruits of the one yellow variety tested is somewhat lower. (2) Fruits of Dwarf Stone, Truckers' Favorite, Red Peach, Yellow Peach, and Yellow Pear which 1914] DUGGAR AND MERRILL—ACIDITY OF TOMATO FRUITS 239 were picked green and ripened in the incubator at 32-33°С. (10-22 days) exhibit a higher acid content than either those ripened on the vines or those ripened at the temperature of the laboratory. (3) There are considerable differences in the acid- ity of varieties, but judging from the results of these tests the normally ripened fruits of yellow varieties commonly con- tain as much acid as those of red varieties. The several facts brought out by these tests render it obvious that there is now no sufficient evidence to justify relating pigmentation to total acidity. Тһе acidity changes are, how- ever, interesting in themselves, in these as well as in other fruits. No attempt was made to follow progressively any changes in acidity induced by conditions; but in titrating on one occasion, after an interval of two days, new samples of both red and yellow fruits which had been ripened in the labo- ratory, it was found that the acidity had noticeably declined since the previous titrations from the same lots of fruits. We have reckoned the acidity of the tomato in terms of сігіс acid, as is customary. It should be noted, however, that while Bowman (3) and others report citric as the chief acid of the tomato, Albahary (1), on the contrary, gives .48 per cent as the malic acid content and .09 per cent as that of citric acid in the fresh fruits. The author last mentioned gives no indications respecting the variety or condition of the fruit employed. In a later contribution (2) he reports the results of analyzing tomato fruits in different stages of matura- tion, as follows: “1916 fruit vert avant l'apparition de la graine dans la pulpe; 2? le fruit vert au moment oü la graine est complètement formée; 3? le fruit rouge arrivé à sa pleine matu- ration." Іп the second stage, corresponding to practically full grown, green, he finds .58, and in the ripe fruits .42 per cent of organic acids. This is in complete agreement with our findings. In the earliest stage of fruit development Alba- hary finds an acid content of only .116 per cent. Wehmer (5), after quoting Albahary (1) as to the percentage of the various acids in the fruit, remarks, “Піс Aciditüt wechselt stark je nach dem Reifestadium (von 0,06-0,697% des Saftes auf Citronensáure berechnet)." He does not indieate the (Vor. 1, 1914] 240 ANNALS OF THE MISSOURI BOTANICAL GARDEN source of these data, and certainly the smaller percentage given can refer only to the youngest stages of fruit development. Graduate Laboratory, Missouri Botanical Garden. LITERATURE CONSULTED .l. Albahary, J. M. Analyse compléte du fruit du Lycopersicum esculentum ou Tomate. Compt. rend. acad. Paris 145: 131-33. 1907. 2. ————, Étude chimique de la maturation du Lycopersicum esculentum (To- mate). Compt. rend. acad. Paris 147: 146-47. 1908. 3. Bowman, W. Tomatoes: chemical examination of fruits. Va. Agr. Exp. Sta. Bul. 9: 16-18. 1891. 4. Duggar, B. M. Lycopersicin, the red pigment of the tomato, and the effects of conditions upon its development. Washington Univ. Studies 1: 22-45. 1913. 5. Wehmer, C. Die Pflanzenstoffe 685-86. 1911. А METHOD FOR THE DIFFERENTIAL STAINING OF FUNGOUS AND HOST CELLS R. E. VAUGHAN Assistant in Plant Pathology, University of Wisconsin Exchange Fellow in the Henry Shaw School of Botany of Washington University In making histological studies of fungi on living or dead plant tissues the use of the stain known as “Pianeze IIIb” has been found very satisfactory in differentiating the fungus from the plant substratum, this differentiation occurring both in lignified and unlignified cell walls. Тһе host tissue stains green and the mycelium a deep pink. This stain, devised by Dr. Pianeze for the study of cancer tissue,! is made up as follows: Malachite адтгееп.......................... 0.50 gm. nr С 0.10 gm. CORPOS | gn ar 0.01 gm. Water, distilled........... BS es .. .. 150.00 ce. Alcohol, 95 pet се. Е. ........... .. 50.00 се. Dr. Pianeze reports that it gives the following staining reactions: green in chromatin of resting or dividing nucleus, rose in cell protoplasm and membrane, and red in cancer bodies. For use with plant tissues the procedure is as follows: Wash in water or alcohol, stain in the undiluted mixture 15-45 minutes, remove excess stain in water, and decolorize in 95 per cent alcohol to which a few drops of hydrochloric acid have been added. For permanent mounts, clear with a carbol- turpentine mixture, remove clearer in xylol, and mount in balsam. Preparations of Stereum, Corticium, and Polystictus have been made with great success. This stain is also valuable for staining germinated spores on the surface of a leaf. The procedure in this case is as follows: Infect marked portions of a leaf with a suspension of spores applied with a pipette, and place the plant under suitable conditions for fungous growth for 24-48 hours. Then permit 1Pianeze, G. Beitrag zur Histologie und Aetiologie des Carcinoms. Beiträge 2. path. Anat. и. z. allg. Path. Supplement т: 1-193. 1896. [ef. p. 58.) ANN. Mo. Вот. GARD., Vor. 1, 1914 (241) [Vor. 1, 1914] 242 ANNALS OF THE MISSOURI BOTANICAL GARDEN the leaf to dry in the air, remove the area desired from the balance of the leaf, and place in a killing fluid. Тһе best com- bined killing and tissue-clearing mixture for this purpose is one recommended by Dr. Duggar, composed of glacial acetic acid and 95 per cent aleohol. I have used equal parts of these agents most advantageously. This dissolves the chlorophyll, renders the leaf transparent or nearly so, and at the same time fixes the fungus with little plasmolysis. Allow the killing mix- ture to act for 24-36 hours; wash in 50 or 70 per cent alco- hol, to remove the acid; and pass successively through the stain (15-30 minutes), water (2 minutes), acid alcohol (as short a time as possible), carbol-turpentine (until clear), xylol (until clearing agent is removed), and then mount in balsam. This process of differential staining has been successfully used with Ascochyta Pisi on pea, Helminthosporium sativum on barley, and Phoma Brassice on cabbage. Pianeze’s stain has not given as good results with the rusts as Durand’s combination of Delafield’s haematoxylin and eosin. Durand's stain! was not uniformly successful, however, and it was found that one of the chief diffieulties often experienced finds its explanation in the killing solution which the stain follows. Flemming's solution, which was first used, gave very poor results. А modification of Gilson's mercuric chloride solution was found most satisfactory. This solution, as recom- mended by Dr. Durand, is made up as follows: bic AD 007 КАССИНИ ee 60 ce. Alcohol, 95 рег сепі........................... 42 се. Acetic acid, шізсізі............................ 18 ce. Nitric acid, concentrated...................... 2 ce. Mercurie chloride, sat. ад. өо!................... 11 ce. Diseased tissue may be fixed from 6 to 24 hours, then washed in 65 per cent alcohol, run through the alcohols, infiltrated with cedar oil, and imbedded in paraffin. This method is undesirable for nuclear structures, but gives excellent prepara- tions for gross histological work. Graduate Laboratory, Missouri Botanical Garden. ! Durand, E. J. The differential staining of intercellular mycelium. Phyto- pathology 1: 129-30. 1911. SiC Eee, -i "жа a E Е so mR Mica EE тав E AA T EES. TWO TRUNK DISEASES OF THE MESQUITE HERMANN VON SCHRENK Pathologist to the Missouri Botanical Garden The diseases of the mesquite (Prosopis glandulosa Torr.) hitherto recorded are comparatively few in number; Heald and Wolf (5) enumerate seven from southern Texas as due to fungi. The pods are frequently affected by an anthracnose, Gleosporium leguminum (Cke.) Sacc.; the leaves are attacked by Cercospora prosopidis Heald and Wolf, a species of powdery mildew (Ету- siphe ?), and by a rust, Ravenelia arizonica Ell. & Ev.; and a leaf blight due to some unknown cause is also mentioned. The large limbs and smaller branches show galls, evidently not due to insect attack, and the mistletoe (Phoradendron flavescens (Pursh) Nutt. is sometimes destructive. In addition to the above, the writer has frequently noted the weakening effect, particularly near the ends of branches, brought about by vigor- ous growths of the ball moss (Tillandsia recurvata L.). Birge (1) has given a good description of the effects of this plant on trees in Texas. Of the insect injuries of the mesquite, that of the mesquite borer (Cyllene antennatus White) is of interest. The insect is described by Horn (6) as attacking mesquite wood in Arizona, but no description of its work is given. While I have not seen the insect at work in Texas, the holes found in the mesquite trees are so like those described for other species of Cyllene, notably Cyllene robinie Forster (10)—which attacks the locust —that the assumption seems warranted that the Texas insect is the one referred to by Horn. The tunnels extend straight through the bark into the heart-wood, and up and down in the latter, thus forming ideal channels for the entrance of fungous spores. The only reference to trunk diseases which has been found is a brief statement by Havard (4), in an account of the mesquite, in which he mentions that ‘‘unfortunately it too often happens that the zones of the heart-wood are fissured, decayed or de- ANN. Mo. Вот. Garp., Vou. 1, 1914 (243) [Vor. 1 244 ANNALS OF THE MISSOURI BOTANICAL GARDEN tached from each other, so that it is diffieult to get flawless boards." In 1912 the writer found the older mesquite trees in the vicinity of San Antonio, Texas, seriously affected by a trunk disease, caused by one of the polyporous fungi. In one small field some twenty or more trees were found bearing the fruiting bodies of this fungus. Its distribution in the vicinity of San Antonio was general, and it is probable that it extends over а wider range, as evidenced by the finding of a sporophore by Underwood in the vicinity of Austin, in 1891. Where the mesquite develops into a bush with several trunks, sometimes only one of the several trunks is affected, but in other cases several or all of them contract the disease. Тһе age of the affected trees was difficult to estimate. "The mesquite grows rather rapidly at first, but very slowly after eight or ten years. According to Sargent, trunks thirty years old may be seven to eight inches in diameter, while trees one foot in diam- eter are probably over one hundred years old. Тһе trees found affected were from two to ten inches in diameter and all over twenty years of age, some of them probably very much older. The decay is confined entirely to the heart-wood of the main trunks, extending from the ground up into the trunk for varying distanees. Тһе distribution is such that it is obvious that the fungus gains entrance through wounds in the trunk above the ground, chiefly through old branch stubs and borer holes, as is во frequently the case with trunk diseases of this kind. One instance was found which made it appear obvious that the holes made by the borer had served to give the fungus a start. Sections of diseased trunks showed that the heart-wood was decayed to a greater or less degree (pl. 6 fig. 2). Mesquite wood has very sharply defined heart and sap-wood. The latter is light yellow or almost white and very narrow, being composed of but a few rings of wood, whereas the heart-wood is rich brown orreddish. Тһе decay of the heart-wood begins near the center, and gradually spreads outward towards the bark; there is very little, if any, change in color (except that the decayed wood is a lighter shade of brown), and here and there irregular, thin lines of undecayed wood can be seen extending through the diseased 1914) VON SCHRENK— TWO TRUNK DISEASES OF MESQUITE 245 part. The decayed wood isvery brittle, but still remains fibrous, that is, it does not crumble into powder like charcoal. It splits like sound wood, but is spongy and soft. The wood of the mesquite is very hard and heavy, a cubic foot weighing 47.69 pounds when absolutely dry. It consists of numerous, distinct medullary rays, and distinct but irregularly distributed bands of very thick-walled wood fibers, between which occurs a thinner-celled wood parenchyma. In the heart-wood the lumina of the cells of the latter tissue are usually completely filled with a yellow-brown substance, largely composed of tan- nin. McMurtree (8) found tannic acid in large quantities in mesquite wood, 6.21 per cent in the heart-wood, 0.5 per cent in the sap-wood, and 0.5 per cent in the bark. Besides tannin he found of materials other than tannin, insoluble in water but extracted by ether, 0.6 per cent in the heart-wood, 6.7 per cent in the sap-wood, and 1.84 per cent in the bark. A considerable number of large, open ducts are found in the early part of each wood ring. These also are filled with a yellow-brown substance similar to that found in the wood parenchyma. The fine, colorless mycelium of the fungus spreads throughout the wood substance. Unlike Polyporus rimosus in locust wood (10), the fungus does not destroy the wood as a whole, but attacks only the heavily lignified groups of wood fibers. "These are wholly destroyed, leaving holes or gaps between the vessels and wood parenchyma. The dissolution of the wood fibers evi- dently proceeds with great rapidity, starting with the secondary thickening of each cell. The cells disappear entirely, and in advanced stages of decay small masses of mycelium are the only evidence of their former presence. Although the wood paren- chyma and the vessels are filled with hyphe, they resist destruc- tion almost completely,—a fact which may be connected with the very high tannin content of both of these tissues. The recent results of Wehmer (11), who found that for certain species of fungi tannin exerts a retarding influence on develop- ment, and the similar findings of Knudson (7), and of Cook and Taubenhaus (3), who state that “tannin has a tendency to retard or inhibit the growth of fungi," and that “the parasitic forms are more sensitive to the action of tannin than the sapro- phytic forms,” lend support to this idea. Cook and Taubenhaus [Vor. 1 246 ANNALS OF THE MISSOURI BOTANICAL GARDEN also found that for the parasitie fungi tested, concentrations of from 0.1 per cent to 0.6 per cent were sufficient to retard growth. While the mere presence of considerable tannin may not entirely prevent the development of a fungus, it may retard its growth, and in the mesquite may explain the comparative immunity of the wood parenchyma to its attacks. The selective destruction of the wood fibers will serve to distinguish this form of decay from the other types of hardwood decay. From the material found it was not possible to judge of the ultimate stages of the disease. In view of the fact, however, that sporophores four years old were observed, it seems that the resistance of a part of the wood structure is more or less per- manent. Хо mesquite trees were found broken off as a result of the action of the fungus. It is conceivable, however, that very severe storms might break off trees weakened by the disease. ` The fungus which causes the decay is Polyporus texanus (Mur- rill) Saec. & Trott. The sporophores, which are annual and very distinet and easily recognized, develop around old knots. At the end of one year the sporophore dries and cracks (pl. 6 fig. 1, and pl. 7 figs. 1, 2), and many of them become badly eaten by inseets. Тһе latter may completely destroy the fruiting structure, thereby preventing the formation of new pilei from the original one. Тһе sporophores occur either singly or in groups. In the latter case the oldest sporophore of the group is situated near the trunk, and gives rise during the second year to another pileus; from the latter a third one may grow out during the following year. This habit is well shown in pl. 6 fig. 1, and in pl. 7 fig. 1. The photograph reproduced in pl. 6 fig. 1 shows а group of three sporophores from below ; the oldest one (in the back), dried and cracked; the second one formed immedi- ately below the oldest one; and the youngest one developed at the side. "This condition is also evident in pl. 7 figs. 1,2. On the trees observed there was usually only one sporophore or a single group of sporophores, and while the internal decay ex- tended in some cases for ten to twelve feet up and down in the trunk, in no case did the sporophores develop at more than one point. Polyporus texanus (Murrill) Sacc. & Trott., was first described by Murrill (9) in 1904 from a specimen collected by Under- 1914] VON SCHRENK—TWO TRUNK DISEASES OF MESQUITE 247 wood on a mesquite (?) tree near Austin, Texas, in 1891. Mur- rill’s description of this fungus is as follows: “Pileus ungulate, attached by the vertex, 8 x 5 x 4 ст., surface fulvous to fuliginous, concentrically and radially rimose, especially in age, the separated areas imbricated; margin very obtuse, concolor- ous, context corky, concentrically banded, fulvous to umbrinous, very thin, only one-tenth the length of the tubes in thickness; tubes 3 em. long, 2-3 to a mm., tawny chestnut, polygonal, edges thin, entire; spores ovoid, smooth, very dark brown, 1-2 guttulate, 8 x 10u.” While this description was made from one specimen, the characterization is a good one and well defines the sporophores recently collected, and now in the herbarium of the Missouri Botanical Garden. One of the marked characters of the fruit- ing structure is the concentrically and radially rimose surface (pl. 7 figs. 1, 2) with imbricated areas, particularly in the older specimens. The tubes are very long, 2-35 cm. (as stated by Murrill), and make up the larger part of the mass of the sporo- phore. The largest specimen found measured 9.5 ст. in width, 7 em. in length, and 5 em. in thickness. Using Ridgeway's color scale, the top is avellaneous gray, the tubes tawny, the substance antique brown (umbrinus of Saccardo’s scale); near the margin the color is verona brown to warm sepia. Murrill's statement that the sporophore is attached by the vertex should be amplified, as many of the sporophores are practically dimidi- ate. With the additional material now available for study, the modified description of the fungus in question is as follows: Polyporus texanus (Murrill) Saec. & Trott. Syll. Fung. 21: 272. 1912. Inonotus texanus Murrill, Bull. Torr. Bot. Club 31:597. 1905. Pileus ungulate, attached by the vertex or dimidiate, 4-9.5 ста. wide, 3-7 em. long, and 4-5 em. thick; surface avellaneous gray to fulvous, concentrically and radially rimose, especially in age, the separated areas imbricated; margin very obtuse, verona brown to warm sepia; context corky, concentrically banded, antique brown, very thin, only one-tenth the length of the tubes in thickness; tubes 2-31 cm. long, 2-3 to a mm., tawny, polygonal, edges thin, entire; spores ovoid, smooth, very dark brown, 1-2guttulate, 8x104. Parasitic on living mesquite trees. [Vor. 1 248 ANNALS OF THE MISSOURI BOTANICAL GARDEN In the same locality in which Polyporus texanus occurred, one mesquite tree was found bearing a sporophore of Fomes rimosus Berk. This fungus causes the heart rot of Robinia Pseudo—Acacia (10), and it is of interest to note its occurrence on a new host. The specimen found is a typical sporophore of Fomes rimosus, measuring about two inches in length ; unfortunately it was not recognized at the time of collection, and sections of the affected tree were therefore not made. In view of the destructive char- acter of this fungus when found on Robinia, however, it is prob- able that it causes a similar heart rot of the mesquite. Further search will be made in the San Antonio region for additional evidences of its occurrence. The wood of the mesquite is usually described as being very resistant to decay after it has been cut from the tree. For many years mesquite posts have been used in the southwest in prefer- ence to other kinds. Mesquite ties, foundation posts, etc., have also proved that the wood is very resistant to decay. This applies only to the heart-wood, however. The sap-wood is very short-lived, and where small trunks are cut, as is now frequently the case, and used for fence posts, the length of life is very short, —sometimes not over two to three years. The destruction of the sap-wood is due to a number of insects and saprophytic fungi, all of which are common on dead branches, posts, etc., in the vicinity of San Antonio. Of the more common fungi, the following were recently collected: Polystictus Lindheimeri B. & C., Stereum Leveillianum Fr., Schizophyllum commune Fr., Len- zites protractus Fr., and Stereum albobadium Schw. The author acknowledges assistance from the following: Mr. Kearney Mason, of San Antonio, for permission to fell trees on his land and assistance in doing so ; Dr. E. A. Burt, Mycologist and Librarian to the Missouri Botanical Garden, and Mr. C. G. Lloyd for aid in the identification of species of fungi. 1914] VON SCHRENK—TWO TRUNK DISEASES OF THE MESQUITE 249 LITERATURE CITED 1. Birge, W. L. The anatomy and some biological aspects of the “Ball Moss," Tillandsia recurvata L. University of Texas Bul. 194: 1-24. 1911. 2. Bray, Wm. L. Тһе mistletoe pest in the Southwest. U. S. Dept. Agr., Bur. Pl. Ind. Bul. 166: 1-39. 1910. (еі. рр. 18, 25.) 3. Cook, M. C., and Taubenhaus, J. J. The relation of parasitic fungi to the con- tents of the cells of the host. Part I. The toxicity of tannin. Del. Ag. Exp. Sta. Bul. 91: 1-67. 1911. 4. Havard, V. The Mezquit. Am. Nat. 18: 451-59. 1884. (сі. p. 457.) 5. Heald, F. D., and Wolf, F. A. A plant disease survey in the vicinity of San An- tonio, Texas. U.S. Dept. Agr., Bur. Pl. Ind. Bul. 226: 1-112. 1912. (сї. p. 72.) . Horn, С. H. American Coleoptera. Trans. Am. Entom. Soc. 8:135. 1880. . Knudson, L. Tannic acid fermentation. Jour. Biol. Chem. 14:159-202. 1913. MeMurtree, Wm. Rept. U. S. Commissioner of Ag. for 1875 182. 1876. . Murrill, Wm. Тһе Polyporaceae of North America—IX. Bull. Torr. Bot. Club 31: 593-610. 1904. (еі. p. 597.) 10. von Schrenk, Hermann. А disease of the black locust. Ann. Rept. Mo. Bot. Gard. 12:21-31. 1901. 11. Wehmer, C. Der wachstumshemmende Einfluss von Gerbsüuren auf Merulius lachrymans in seiner Beziehung zur Resistenz des Eichenholzes gegen Haus- schwamm. Mycologisches Centralbl. т: 138-48. 1912. а: "TES T y Y dida ites ie е RA [Vor. 1, 1914] 250 ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 6 Disease of the mesquite due to Polyporus texanus Еа. 1. View showing the manner in which a group of sporophores of Polyporus іетатиз grows on the trunk; also the lower surfaces of the sporophores. Fic. 2. Two sections of diseased mesquite trunk showing the manner in which the wood is destroyed. et РО Т | А ANN. Мо. Вот. GARD., VOL. 1, 1914 ' Prats 6 T vy n ym 3 { FIG. 2. VON SCHRENK — TRUNK DISEASES OF MESQUITE COCKAYNE. BOSTON, [Vor. 1, 1914 252 ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 7 Disease of the mesquite due to Polyporus texanus Еа. 1. Side view of a group of sporophores of Polyporus tezanus growing on a living mesquite tree. Ета. 2. Front view of a group of sporophores of Polyporus tezanus growing on a living mesquite tree. WWE VIG S x dup ee eee эта қылы асылы TN PLATE 1914 Mo. Bor. Garp., Vor. 1, ANN. FIG VON SCHRENK BOSTON, COCKAYNE, EM ысу; А TRUNK DISEASE OF THE LILAC HERMANN VON SCHRENK Pathologist to the Missouri Botanical Garden A general discussion of diseases of the common lilac (Syringa vulgaris L.) was recently published by Klebahn (3). This author enumerates a number of diseases, such as the one of bacterial origin ascribed to Pseudomonas Syringe, various leaf diseases due to species of Microsphera, Gleosporium, and other leaf parasites, and a disease due to Botrytis cinerea. The major part of the work, however, deals with a disease due to Heterosporium Syringe Oud., affecting the leaves, and a serious twig blight due to Phytophthora Syringe Klebahn. Subsequent papers by various writers deal with one or the other of the diseases mentioned by Klebahn. During recent years а destructive trunk disease of the com- mon lilac (Syringa vulgaris L.) has been noted a number of times in the Missouri Botanical Garden, and in grounds in the vicinity of St. Louis. The affected plants were usually old bushes which had been more or less neglected, and the tops of the leading trunks were frequently dead. Long shoots from the root and others from the part of the trunks near the ground made a dense tangle around the main stem; on the latter sporophores of Polyporus versicolor were found in various stages of develop- ment, sometimes isolated, but more frequently in groups. Sec- tions were made of the trunks on which this fungus was growing and it was found that such trunks were invariably diseased, while those close by, either from the same root system or from adjacent bushes—which were free from the fungus—were al- ways sound. In pl. 8 fig. 1 two affected trunks are shown cut at points about three feet from the ground. In both cases the larger part of the stem was alive, as evidenced by the presence of vigorous shoots along the entire length. РІ. 8 fig. 2, and pl. 9 figs. 1, 2 represent sections of lilac trunks taken from different bushes to show different stages of the disease. Ann. Мо. Вот. GARD., Vou. 1, 1914 (253) 7 [Vor. 1 254 ANNALS OF THE MISSOURI BOTANICAL GARDEN The wood of the lilac is white in color, hard, and close-grained. In younger trunks there is no appreciable difference between heart-wood and sap-wood; as the trunks grow older, however, the heart-wood turns darker, and in those twelve years old, or thereabouts, it is distinetly darker than the rather thin,white sap- wood. The disease first manifests itself in the inner heart-wood, frequently in close proximity to the holes made by the lilac borer. This lepidopterous insect (Podosesia syringe Harris) (for whose identification I am indebted to Dr. E. P. Felt) has been found very destructive to lilac bushes, and, according to Beutenmüller (1), occurs from New England and the middle states westward to Colorado and southwest to Texas. Quoting from Beutenmüller's account: “Тһе female deposits her eggs in patches on roughened or knotty places on the bark of ash and lilac. The eggs, according to Hulst, hatch in about six days, and the newly born larve at once eat their way through the bark into the solid wood. They run their channels longitudi- nally for about 8-10 inches through the wood. The larve pupate in slight cocoons after cutting their way to the bark, of which they leave only a thin outer skin. The pupation usually takes place early in May, and the moths emerge in about three weeks." Felt (2) briefly described the habits of the larva, stating that ‘‘a sign of its presence in midsummer being largely the sudden wilting of a shoot." He quotes from an observation made by Dr. Kellicott in which the latter states that he “watched 20 or more issue from a single tree in one day, and found that often there were more than one hundred in one tree." Felt recommends cutting and burning all infested wood in the early spring. In the vicinity of St. Louis the lilac borer has been very active in recent years, judging from the fact that very few lilac bushes over five years old were found free from its attacks. Without much doubt the fungous spores get into the interior of the lilac trunks through the borer holes, and start to develop within the heart-wood on the edges of the borer holes. In pl. 9 fig. 1 two borer holes, still filled with pieces of the borings, can be seen in the lower right-hand trunk, and one small hole in this same sec- tion occurs in the sap-wood. The fungus, after it has begun to 1914) VON SCHRENK—A TRUNK DISEASE OF LILAC 255 grow in the hole, rapidly spreads up and down in the heart- wood, and soon grows out from the center toward the bark. Ав the disease progresses, the wood is converted into a soft, pithy, white mass, having the consistency of corn-stalk pith. Тһе line of demarcation between the sound and completely destroyed wood is very sharp (see pl. 9), resembling in this respect the type of decay caused by this same fungus in living catalpa trees ((5), pl. 26). Тһе line between sound and decayed wood is so sharp that entirely decayed fibers adjoin perfectly sound ones. Between the wholly unaffected wood and the completely de- stroyed fibers, is a narrow ring of darker wood, which is, to all intents and purposes, sound; the wood cells are partially invaded by the mycelium of the fungus, and the lumina are filled with a yellow-brown liquid, which when seen in mass gives the section the dark color referred to. This liquid dries out in some places and leaves а brown amorphous substance, such as has frequently been found in the early stages of decomposition of hardwood wood fibers (6). It probably consists of decomposition products which are infiltrated into the sound wood immediately in ad- vance of the fungus. In cases where the fungus starts in several centers, rings of the darker colored wood surround each decayed portion, à condition which is well shown in pl. 9 figs. 1, 2, where the fungus is growing in the center of the trunk and in addition in three more peripheral localities. In the lilae the brown sub- stance referred to is ultimately destroyed (see the middle trunk of the lower tier, pl. 9 fig. 2, where the wood is destroyed up to the bark). | The completely decayed wood, which readily absorbs water, resembles pith, and in general is very similar to catalpa wood destroyed by Polyporus versicolor (5). It has some of the attributes of wood, i.e., it can be split, is fairly compact, and cannot be crumbled into powder. Sound lilac wood is very heavy and hard, and is composed almost wholly of very thick- walled wood cells, with small vessels scattered with considerable regularity throughout the annual ring; wood parenchyma is almost wholly absent. Тһе hyphs of Polyporus versicolor attack and very rapidly destroy the layers of secondary thick- ening of the wood cells. The middle laraell: retain the nature of lignified fibers and resist destruction almost entirely, although et [Vor. 1 256 ANNALS OF THE MISSOURI BOTANICAL GARDEN here and there some of them are dissolved, giving rise to small separated cell groups. Entire dissolution rarely takes place (this was also found to be true for diseased catalpa wood ((5) pl.52)). With the removal of the secondary thickening, the resulting decayed wood has a skeletonized appearance. It has all of the elements, but these are very thin-walled. Тһе fine medullary-ray cells are destroyed here and there, producing radial, isolated masses, but more frequently the decayed mass hangs together firmly. Тһе dissolution of the layers of secon- dary thickening goes forward very evenly, bringing about the sharp dividing line between sound and decayed wood already referred to. Тһе only difference between the catalpa and Шас diseases is that in the catalpa the entire wood mass is skeletonized, whereas in the Шас hard areas of undestroyed wood fibers are left here and there, surrounded by decayed wood (pl. 9 figs. 1, 2). "These masses are either entire rings (pl. 9 fig. 1) or irregular areas lying detached within the decayed parts, and represent portions of the heart-wood which for some reason have temporarily es- caped total destruction; the wood fibers are filled with the yellow- brown substance, but do not otherwise differ from normal wood fibers. As the disease progresses, however, they are finally destroyed. This was made evident by the fact that in the upper parts of diseased trunks these immune areas were always found coexistent with the early stages of the disease, while lower down in the trunks, where the advanced stages of decay had been reached, they were practically absent. The temporary immunity may be due to the presence of more resistant groups of wood fibers, possibly also to a high concentration of decom- position products. 'The development of the fungous mycelium from the center of the trunk out toward the bark differs radically from that of any other disease known to the writer. In most trees the destruc- tion of wood by a fungus growing in the dead heart-wood is confined to the latter,—further growth ceasing as soon as the mycelium reaches the sap ring. Ав has been suggested by Münch (4), this is probably due to the fact that most mycelia of wood-destroying fungi require a balance between the amounts of oxygen and water contained in the wood fiber. Any undue 1914) VON SCHRENK—A TRUNK DISEASE OF LILAC 257 percentages of either may make the conditions unfavorable for further development. In the Шас disease the fungus may grow outward concentrically in а regular manner (pl. 9 fig. 1). Very frequently, however, the fungus grows out into the sap ring at one side, at first slowly, then more rapidly. This is well shown in pl. 9 fig. 2, where four successive stages are represented by photographs. In the upper left-hand trunk the fungus has almost reached the bark, and in the three lower ones it has reached the bark and is gradually killing it. "The probable explanation for this behavior is to be sought in the water content of the wood fibers. It was found that in many cases where the fungus actually grew up to the bark and through it, that on that side the lilae borer had been active; the wood fibers in the vicinity of the holes dried sufficiently to make growth possible for the mycelium, and as the destruction took place more drying occurred in adjacent areas until ultimately the whole sap region on that side was invaded and destroyed. A number of water determinations were made of the wood fiber in the immediate vicinity of the growing mycelium, and the results compared with those obtained from normal sap-wood. In all eases the sap-wood about to be invaded was found to have a very much lower water content than the normal sap- wood. Unfortunately, it was impossible to get exact data which would indicate accurately the highest moisture content at which growth was possible; infected wood had obviously already reached and gone beyond that point, and as to sound new wood, even that which was near the borer holes, nothing could be postulated with certainty concerning its susceptibility or non- susceptibility to fungous attack. It would be an interesting problem to test the water susceptibility of Polyporus versicolor in its relation to lilae wood. It seems probable, however, that the drying out of one side of the trunks was at least one of the determining factors in the rather striking and exceptional method of growth of the fungus. Whether the fungus would eventually have destroyed the entire trunk it is impossible to state, because no such wholly destroyed trunks were found, There seems to be no reason, however, why this should not [Vor. 1 258 ANNALS OF THE MISSOURI BOTANICAL GARDEN occur; in faet, the right-hand trunk of the lower tier in pl. 9 fig. 2 has very little live wood left. After the mycelium has reached the bark it grows through it, and fruiting bodies develop on the outside. Тһе latter some- times occur singly, but more commonly in linear groups parallel to the long axis of the trunk. Frequently one or more fruiting bodies grow out from the holes made by the borer. In the right-hand trunk in pl. 8 fig. 1 sporophores are shown growing out at the base of vigorous, live shoots; in the left-hand trunk the shoot is dead, having been killed during the year as the fungus invaded the wood from which the shoot was growing. The sporophores found were typical of Polyporus versicolor L. This fungus is so common on the dead wood of various hard- woods that a detailed description is hardly necessary. It is interesting to note here that this is the second instance where this fungus attacks living plants. In the case of the catalpa the fungus grows vigorously only in the live tree; infected wood rarely, if ever, is decayed after itis cut from the tree. Many thousand posts of catalpa, the heart of which had been partially destroyed by Polyporus versicolor, have served as fence posts during the last ten years without showing a sign of decay of that part of the wood which was sound at the time of cutting. With the lilac it is different; the dead wood is just as subject to attack as is dead oak, beech, or gum wood. The age at which lilac bushes are attacked has not been defi- nitely determined. Those examined were about 15-20 years old (23 inches in diameter). The trunk shown in pl. 8 fig. 2 was over thirty years old. It is probable that the disease is not serious until the bushes are ten or more years old, although this will depend somewhat on the rate of growth. Trunks 11-2 inches in diameter were frequently found diseased. The effect of the disease is to gradually kill the top of the trunk; side shoots then develop farther down, which in turn are killed by the fungus, and eventually the trunk is broken off by the wind or snow. The prevention of the disease is possible by continued atten- tion to the borers. A careful examination for the latter should be made in June or July, and if any are present these should be killed by means of a wire, and the holes—after antiseptic treatment with some coal-tar compound—plugged. Painting 19141 VON SCHRENK—A TRUNK DISEASE OF LILAC 259 or brushing the lower parts of trunks with whale oil soap, or its equivalent, should also prove of value. Wherever a diseased trunk is found it should at once be cut out and burned. All dead wood in the neighborhood of lilac bushes should be cleaned up, so that the chances for infection may be reduced. LITERATURE CITED 1. Beutenmüller, Wm. Monograph of the Sesiide. Mem. Am. Mus. Nat. Hist. 1:244. 1903. 2. Felt, E. P. Insects affecting park and woodland trees. Rep. N. Y. State Mus. 59:: 8-332. 1905. [cf. p. 104.) [For other insects attacking lilac see Rep. М. Y. State Mus. 59%: 839. 1905.) 3. Klebahn, Н. Krankheiten des Flieders 1-60. Berlin. 1909. [Шивё.] 4. Minch, Ernst. Untersuchungen über Immunität und Krankheitsempfánglichkeit der Holzpflanzen. Naturwiss. Zeitschr. f. Forst u. Landw. 7:54-75, 87-114, 129-60. 1909. 5. von Schrenk, Hermann. The diseases of the hardy catalpa. U.S. Dept. Agr., Bur. Forestry Bul. 37:51-58. 1902. 6. ——————, A disease of white ash. U.S. Dept. Agr., Bur. Pl. Ind. Bul. 32: 1-20. 1908. [ef. p. 14.) [Vor. 1, 1914] 260 ANNALS ОҒ THE MISSOURI BOTANICAL GARDEN ExPLANATION OF PLATE PLATE 8 Trunk disease of lilac due to Polyporus versicolor Fra. 1. View of two diseased Шас trunks showing the sporophores of Polyporus versicolor, and the manner in which living branches grow from diseased trunks. Fra. 2. Sections of an old diseased lilac trunk showing the decayed heart-wood. po unm Чет ИВ 1914 Т; И: ту р Вот. Сак ANN. Мо. рет, рта E! 4 TRUNK 3ENK- CHI VON S 3OSTON. COCKAYNE, (Vor. 1, 1914] 262 ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 9 Trunk disease of the lilac due to Polyporus versicolor Fra. 1. Sections of three diseased trunks showing early stages of the disease. Fra. 2. Sections showing progressive stages of the lilac trunk disease. ANN. Мо. Вот. Garb., VOL. Ы 1914 COCKAYNE BOSTON, PLATE о на ий Annals of the Missouri Botanical Garden ——=——д—— ЕВСЕН ———_ я Vor. I SEPTEMBER, 1914 No. 8 DESCRIPTIONS OF NORTH AMERICAN SENECIONE/E: J. M. GREENMAN Curator of the Herbarium of the Missouri Botanical Garden Associate Professor in the Henry Shaw School of Botany of Washington University The following descriptions and notes are the results obtained from a critical study of material in several herbaria during the preparation of a monograph of the North American species of the genus Senecio. Some of the species here described have been in manuscript a number of years and a few of them have been withheld from publication, because of incomplete speci- mens, hoping that additional material might be brought together before publication. In many cases supplementary and sub- stantiating material has been obtained from which it is now possible to make fairly complete diagnoses. In one or two instances a reconsideration of certain natural groups within the genus, in the light of recent collections, has made it possible to combine forms which formerly were taken to represent distinct species. Very few new species have resulted from recent col- lections, but there are still many regions, particularly in Central America, which are inadequately explored. Тһе writer would welcome material in this genus from any part of North America in order that the geographical range of species may be recorded as accurately as possible in his forthcoming monograph. Тһе sections indieated in parentheses immediately following the generic name are in accordance with my preliminary paper to which reference is made under the species. ! Issued September 30, 1914. Амм. Мо. Вот. GARD., Vor. 1, 1914 (263) [Vor. 1 264 ANNALS OF THE MISSOURI BOTANICAL GARDEN Senecio (5 Aurei) hyperborealis Greenm. Monogr. Senecio, pt. 1, 24. 1901; in Engl. Bot. Jahrb. 32: 20. 1902, nomen. S.resedifolius Hook. Fl. Bor. Am. 1 : 333. pl. 117. 1833, not Less. Herbaceus perennis; caule simplice vel ramoso suberecto 1-2 dm. alto plus minusve foliaceo juventate glabro vel parce floc- culoso-tomentuloso ѕере ad basin et in axillis foliorum per- sistenter lanato-tomentoso ; foliis inferioribus petiolatis indivisis vel plerumque irregulariter lyrato-pinnatifidis 4-10 em. longis 1-2.5 cm. latis, lobis remotis; foliis superioribus multum reductis sessilibus et bracteiformibus; capitulis paucis terminalibus radi- atis 10-12 mm. altis 2-3.5 em. (radii inclusis) diametro; floribus femineis 10-12, ligulis flavis 10-12 mm. longis ca. 2 mm. latis; disci flosculis numerosis; acheniis sepe paulo hispidulis. Specimen examined: Canada: Arctic America, Hooker (Gray Herb.), ТҮРЕ. Var. columbiensis (Gray) Greenm. Monogr. Senecio, pt. 1, 24. 1901; in Engl. Bot. Jahrb. 32: 20. 1902, nomen. S. resedifolius var. columbiensis Gray, Syn. Fl. 1?: 390. 1884. Habitu formae typies; capitulis heterogamis, ligulis floris femineis quam squamis involucri paulo brevioribus; achsniis glabris. Specimen examined: British Columbia: Mucklung River, 25 July, 1882, Mr. Mackay (Gray Herb.). Senecio (5 Lobati) prolixus, comb. nov. S. diffusus Greenm. Monogr. Senecio, pt. 1, 24. 1901; in Engl. Bot. Jahrb. 32: 20. 1902, nomen, not Linn. f. Herbaceus perennis glabrus vel in axillis foliorum albo- tomentosus; caule tereti striato simplici vel ramoso erecto 2-5 dm. alto; foliis petiolatis vel sessilibus inferioribus lyrato-pin- natifidis petiolo incluso usque ad 15 cm. longis 1.5-5 cm. latis utrinque glabris, segmentis lateralibus oblongo-cuneatis cum sinis altis rotundatis disjunctis granditer dentatis, superioribus remotis sessilibus pinnatifidis sursum multum reductis; inflores- centiis laxe corymboso-cymosis 1-2.5 dm. diametro; capitulis circiter 1 em. altis radiatis; involucris campanulatis parce cal- yeulatis glabris; involucri squamis plerumque 21 lanceolatis vel lineari-lanceolatis 5-6 mm. longis acuminatis acutis; flos- culis liguliferis са. 13, ligulis oblongis 5-6 mm. longis flavis; жена. > 1914] GREENMAN—NORTH AMERICAN SENECIONEJE 265 floribus disci numerosis 50-60; acheniis maturitate 2-3 mm. longis striatis glabris. Specimens examined: California (?): “Mohave Region," April-May, coll. of 1884, J. 6. Lemmon, 3130 (Gray Herb.), түре. Arizona: Wickenburg, valley of the Hassayampa River, April, 1876, Dr. Edward Palmer, 614 (Gray Herb. and Mo. Bot. Gard. Herb.). The specimens cited may be looked for in herbaria under S. multilobatus Torr. & Gray, to which the species here proposed is related, but from which it differs in well developed specimens in the outline and size of the leaves, loose inflorescence, and larger heads with 21 instead of 13 involucral bracts. 8. pro- lixus has rather more the aspect of S. Breweri Davy. Senecio ($ Tomentosi) appendiculatus Greenm. Monogr. Senecio, pt. 1, 24. 1901; in Engl. Bot. Jahrb. 32:20. 1902, nomen. S. neo-mexicanus Gray, Proc. Am. Acad. то: 55. 1883, in part; Syn. КІ. 1°; 392. 1884, in part, as to plant of Thurber. Herbaceus perennis ubique plus minusve albo-tomentosus; caulibus subcespitosis erectis 1.5-3 dm. altis striatis ssepe foli- aceis; foliis radicalibus oblanceolatis vel oblongo-obovatis petiolo incluso 3.5-10 cm. longis 0.5-2 cm. latis dentatis ad basin in petiolum paulatim angustatis integris, eis caulinis petio- latis vel sessilibus 2-7 сіп. longis ad basin plerumque ampliatis irregulariter dentatis subamplexicaulibusque; inflorescentiis ter- minalibus corymboso-cymosis 6-12-cephalis; capitulis 10-12 mm. altis radiatis; involucris campanulatis minute calyculatis; involueri squamis plerumque 21 lanceolatis 5-7 mm. longis acutis sparsissime tomentulosis; flosculis liguliferis ca. 13, ligulis flavis; floribus disci numerosis са. 70; acheniis glabris. Specimens examined: New Mexico: Mule Spring, May, 1851, Geo. Thurber, 280 (Gray Herb.), TYPE; Organ Mountains, Dona Ana Co., 25 April, 1907, E. O. Wooton, 3370 (Mo. Bot. Gard. Herb.). This species is related to S. neo-mexicanus Gray, to which it has been usually referred, but from which it differs in having a more leafy stem, undivided leaves, and with the stem-leaves commonly ampliated into a more or less dentate half-clasping base, and finally in having glabrous instead of hirtellous achenes. [Vor. 1 266 ANNALS OF THE MISSOURI BOTANICAL GARDEN Senecio ($ Tomentosi) convallium Greenm. Monogr. Senecio, pt. 1, 24. 1901; in Engl. Bot. Jahrb. 32: 20. 1902, nomen. Herbaceus perennis ubique sericeo-pubescentes; caulibus cæspitosis erectis З dm. altis; foliis inferioribus rosulatis petiolatis elliptico-lanceolatis vel oblongo-oblanceolatis 2.5-6 cm. longis 5-19 mm. latis acutis integris vel supra mediam partem pauci- dentatis basi longe cuneatis integriusculis juventate utrinque sericeo-pubescentibus ætate supra plus minusve glabratis, foliis superioribus spathulato-oblanceolatis angusti-petiolatis; inflores- centiis corymboso-cymosis paucicapitatis; capitulis circiter 1 em. altis subradiatis; involucri bracteis 13-15 lineari-attenuatis 7-9 mm. longis acutis sparse sericeo-tomentulosis; floribus femineis subligulatis; floribus disci 30-35; achaeniis 3.5 mm. longis striatis glabris. Specimen examined: Utah: Rabbit Valley, altitude 2130 m., August, 1875, L. F. Ward, 704 of the “U. S. Geological and Geographical Sur- vey of the Territories" (Gray Herb.), ТҮРЕ. Тһе species here characterized has been hitherto confused with S. canus Hook., from which it is readily distinguished by the subsericeous pubescence and technical characters of the head. Senecio ($ Tomentosi) kernensis Greenm. Monogr. Senecio, pt. 1, 24. 1901; in Engl. Bot. Jahrb. 32: 20. 1902, nomen. Herbaceus perennis ubique dense lanato-tomentosus; caule tereti erecto ca. 1 dm. alto; foliis inferioribus rosulatis petiolatis elliptico-oblongis vel oblongo-rotundatis 1-3 em. longis 3-10 mm. latis apice obtusis vel rotundatis basi abrupte angustatis vel subtruneatis utrinque dense lanato-tomentosis, marginibus integris vel suberenato-dentatis revolutisque, foliis superioribus bracteiformibus multum reductis; inflorescentiis terminalibus со- rymboso-cymosis paucicapitatis; capitulis 8-10 mm. altis radiatis 5-8 mm. (radii exclusis) diametro parce calyculatis; involucri squamis ca. 13 lineari-lanceolatis 5-6 mm. longis acutis floccoso- tomentulosis subglabratis; achaeniis glabris. Specimen examined: California: South Fork of Kern River, altitude 3760 m., Septem- ber, 1875, Dr. J. T. Rothrock, 334 of the ‘Explorations and Surveys west of the 100th Meridian" (Gray Herb.), ТҮРЕ. 1914) GREENMAN—NORTH AMERICAN SENECIONE/E 207 Senecio ($ Tomentosi) macropus Greenm. Monogr. Senecio, pt. 1, 24. 1901; in Engl. Bot. Jahrb. 32: 20. 1902, nomen. S. arizonicus Gray, Syn. Fl. 17: 392. 1884, in part, as to plant of Rusby. Radix robusta in sieco 2.5 cm. diametro; caulibus erectis usque ad 7.5 dm. altis glabris vel in axillis foliorum albo-tomentulosis striatis plus minusve purpurascentibus; foliis radicalibus petio- latis lyrato-pinnatifidis petiolo incluso 10-14 em. longis 4-5 em. latis, segmentis paucijugis ineequalibus terminali majore ovato- oblongis 5-6 em. longis grosse dentatis, ceteris cunPatis et den- tatis vel linearibus et integris; foliis caulinis remotis sessilibus pinnato-lobatis semiamplexicaulibusque sursum sensim reductis; infloreseentiis terminalibus corymboso-cymosis; (apitulis ca. 1 em. altis radiatis, ligulis flavis; involucris campaniilatis minute calyeulatis; involucri squamis circiter 21 lineari-lanceolatis 6.5- 8 mm. longis acutis glabris maturitate retrorsis; oribus disci numerosis; acheniis glabris. | Specimen examined: | Arizona: without definite locality, coll. of 1883, H. H. Rusby, 175 (Gray Herb.), TYPE. Professor Rusby's plant was referred by Dr. Gray to S. arizonicus Greene, but from the very large root, the sublyrate, smooth and even somewhat glaucous radical leaves, and nearly naked stem it seems amply distinct. Senecio ($ Tomentosi) oreophilus Greenm. Monogr. Senecio, pt. 1, 24. 1901; in Engl. Bot. Jahrb. 32: 20. 1902, nomen. S. neo-mexicanus Gray, Proc. Am. Acad. 19: 55. 1883, in part; Syn. Fl. 1?: 392. 1884, in part, as to plant of Greene. Herbaceous perennis juventate ubique tomentulosus denique plus minusve glabratus; caule tereti erecto striato 2-3 dm. alto subnudo 2-3-bracteato; foliis rosulatis petiolatis oblongo- oblanceolatis vel oblongo-cuneatis petiolo incluso 3-10 em. longis 0.7-2.5 em. latis supra mediam partem crenato-dentatis basi in petiolum sensim angustatis integriusculis juventate utrinque albo-tomentulosis mox glabratis; braeteis caulinis linearibus apice basique parum ampliatis dentatisque; inflorescentiis laxe corymboso-cymosis usque ad 1 dm. diametro; capitulis 10-12 mm. altis calyculatis radiatis; involueris campanulatis basi tomentulosis ceteris glabris; involueri squamis plerumque 21 [Vor. 1 268 ANNALS OF THE MISSOURI BOTANICAL GARDEN lanceolatis 6.5-8 mm. longis acutis; flosculis liguliferis ca. 12, ligulis oblongis 8 mm. longis 3 mm. latis 4—5-nerviis; floribus disci numerosis са. 50; acheniis in angulis sursum hispidulis. Specimen examined: New Mexico: Pinos Altos Mountains, 6 May, 1880, Edward Lee Greene (Gray Herb.), TYPE. A plant similar in habit to S. neo-mexicanus Gray, to which Dr. Greene's specimen was referred by Professor Gray in estab- lishing that species. А careful study of all the original material, which has been made possible through the courtesy of Dr. B. L. Robinson, has shown that the S. neo-mexicanus of Dr. Gray consisted of at least three recognizably distinet forms of which Wright's No. 1415, as the first specimen cited, must be taken as the type. With the Wright plant several specimens at hand аге almost the exact counterpart. The Greene plant in question, namely S. oreophilus, differs in several important particulars, notably in its essentially naked stem, oblong-cuneate leaves with subentire or sinuate-dentate margin, and a marked ten- dency for the foliage to become glabrous with age. Senecio (§ Tomentosi) oreopolus Greenm. Monogr. Senecio, pt. 1, 24. 1901; in Engl. Bot. Jahrb. 32: 20. 1902, nomen. Plate 11. Herbaceus perennis ubique albo-tomentosus; caulibus cæs- pitosis simpliee vel ramosis 0.8-3 dm. altis; foliis inferioribus petiolatis ovato-ellipticis vel elliptico-lanceolatis vel rarius sub- obovatis 0.8-3.5 ст. longis 5-18 mm. latis obtusis vel supra mediam partem paucidentatis basi abrupte vel longe cuneatis integriusculis juventate utrinque albo-tomentosis state supra paululo subinde glabratis, petiolatis 1-6.5 em. longis, foliis supremis grosse reductis petiolatis vel sessilibus integris vel rarius irregulariter dentatis basi sepe expansis et subauricu- laribus; inflorescentiis corymboso-cymosis; capitulis plerumque са. 1 em. (8-14 mm.) altis radiatis parce calyculatis; involucri squamis plerumque 13 (9-13) lanceolatis vel lineari-lanceolatis 5-7 mm. longis acutis glabris vel leviter tomentulosis ; flosculis liguliferis 5-13; floribus disci 20-30; pappi setis albis bracteis involucri longioribus; achzeniis 3-3.5 mm. longis glabris. 1914] GREENMAN—NORTH AMERICAN SENECIONE 269 Specimens examined: California: Rock Creek Сайоп, Basin of the Upper Kern River, Tulare Co., altitude 3050 m., July, 1904, H. M. Hall & H. D. Babcock, 5526 (Gray Herb.), түре; Natural Bridge, Voleano Creek, Basin of the Upper Kern River, altitude 2985 m., July, 1904, H. M. Hall & H. D. Babcock, 5438 (Gray Herb.); gravelly slopes, Little Kern River, altitude 3045-3350 m., April-September, 1897, C. A. Purpus, 5240 (Gray Herb. and Mo. Bot. Gard. Herb.); Castle Peak, near the highest point, altitude 2740 m., 5 August, 1903, A. A. Heller, 7102 (Gray Herb. and Mo. Bot. Gard. Herb.); Sierra Nevada, coll. of 1875, John Muir, 4452 (Mo. Bot. Gard. Herb.); near the summit of Silver Mountain, altitude 3350 m., coll. of 1863, W. H. Brewer, 2050 (Gray Herb.); Ebbett’s Pass, W. H. Brewer, 2005 (Gray Herb.); Sonora Pass, W. H. Brewer, 2686 (Gray Herb.); Mono Pass, coll. of 1866, H. N. Bolander, 6140 (Gray Herb.). Nevada: Mt. Rose, Washoe Co., altitude 3200 m., 26 August, 1911, A. A. Heller, 9882 (Mo. Bot. Gard. Herb.). Forma aphanactis, forma nova. Caulis сігейег 1 dm. altus; foliis petiolo incluso 1.5-2.5 cm. longis 5-7 mm. latis; capitulis discoideis. Specimen examined: California: mountain peak near Sonora Pass, altitude 3200 m., coll. of 1863, W. H. Brewer, 1905 (Gray Herb.), TYPE. Senecio ($ Tomentosi) Wrightii Greenm. Monogr. Senecio, pt. 1, 24. 1901; in Engl. Bot. Jahrb. 32: 20. 1902, nomen. S. fastigiatus Gray, Pl. Wright. ii. 99. 1853, not Nutt. Herbaceus perennis ubique subtomentosus; caule erecto 1-4 dm. alto foliato; foliis oblongo-oblanceolatis vel lanceolatis indivisis et integris vel supra mediam partem paucidentatis juventate albo-tomentosis plus minusve glabratis, inferioribus basi integriusculis in petiolum sensim angustatis, eis caulinis sessilibus basi sspius ampliatim et irregulariter dentatis amplexicaulibusque; inflorescentiis terminalibus subeorymboso- cymosis multicapitatis; capitulis 8-10 mm. altis minute calycu- latis radiatis; involucris campanulatis basi subincrassatis tomentosis, bracteis involucri plerumque 13 lanceolatis 5-7 mm. longis acutis tomentulosis; flosculis liguliferis 6-8, ligulis anguste 774 [Vor. 1 270 ANNALS OF THE MISSOURI BOTANICAL GARDEN oblongis ca. 8 mm. longis 4-5-nerviis; floribus disci са. 30; achaeniis glabris. Specimens examined: New Mexico: ravines between the copper mines and the Mim- bres, October, 1851, Charles Wright, 1289 (Gray Herb. and Mo. Bot. Gard. Herb.), түре; Santa Rita del Cobre, 22 September, 1880, E. L. Greene (Mo. Bot. Gard. Herb.); among spruce, Lookout Mine, Sierra Co., altitude 2680 m., O. B. Metcalfe, 1179 (Mo. Bot. Gard. Herb.). Senecio ($ Amplectentes) subauriculatus Greenm. Monogr. Senecio, pt. 1, 25. 1901; in Engl. Bot. Jahrb. 32: 21. 1902, nomen. Plate 14. Herbaceus perennis; caule erecto ramoso striato glabro; foliis in partibus superioribus caulinis anguste lanceolatis 5-15 cm. longis 0.5-1.5 em. latis acuminatis acutis integris vel remote apiculato-denticulatis sessilibus et auriculo-semiamplexicauli- bus vel basi іп petiolum sensim angustatis et subdecurren- tibus membranceis supra glabris juventate subtus floccoso- tomentosis denique plus minusve glabratis ; inflorescentiis ter- minalibus laxe subeorymboso-eymosis; pedunculis bracteatis, bracteis lineari-attenuatis; capitulis radiatis 12-14 mm. altis heterogamis; involucris campanulatis calyculatis albo-floccoso- tomentulosis, bracteolis calyculatis linearis acutis suberoso- marginatis; involucri squamis plerumque 21 lineari-lanceolatis са. 1 сіп. longis acutis et atro-penicillatis; flosculis liguliferis ca. 13, ligulis oblongis flavibus; floribus disci numerosis (50-60); pappi setis albis; achzeniis pubescentibus. Specimen examined: Mexico: State of Oaxaca, mountains southeast of Miahuatlan, altitude 2750-3170 m., coll. of 1895, E. W. Nelson, 2526 (Gray Herb.), TYPE. A well marked species related to S. Warszewiczit A. Br. & Bouché and to S. prionopterus Rob. & Greenm. Senecio ($ Mulgedifolii) alatipes Greenm. Monogr. Senecio, pt. 1, 25. 1901; in Engl. Bot. Jahrb. 32: 21. 1902, nomen. Herbaceus perennis ubique glabrus; caule tereti striato erecto l m. vel ultra alto; foliis parte inferiori ignotis, eis caulinis petiolatis vel sessilibus amplexieaulibusque oblongo-ovatis vel oblongo-lanceolatis 0.5-1.5 dm. longis 2-5 ст. latis acutis vel 1914] ; GREENMAN— NORTH AMERICAN SENECIONEÆ 271 acuminatis indivisis vel subpanduriformibus utrinque glabris subtus pallidoribus, margine irregulariter calloso-dentatis; peti- olis usque ad 12 em. longis anguste alatis ; inflorescentiis terminalibus paniculatis; capitulis 8-10 mm. altis discoideis 20—25-floris; involucris anguste campanulatis calyeulatis glabris; involucri squamis plerumque 13 lineari-lanceolatis acutis penicil- latis ca. 6 mm. longis; achzniis striatis glabris. Specimen examined: Mexico: State of Chiapas, between Teneapa and Yajalon, alti- tude 900-1520 m., 13 October, 1895, E. W. Nelson, 8277 (U. S. Nat. Herb., fragments and tracing in Gray Herb.), ТҮРЕ. Senecio ($ Mulgedifolii) callosus Schz. Bip. in Flora 28: 408. 1845. S. eximius Hemsl. Biol. Cent.-Am. Bot. 2: 239. 1881, as to synonomy.—S. doratophyllus Hemsl. І. c., in part, as to Bour- geau's No. 1086, not Benth.—S. viejensis and S. latipes Greenm. Monogr. Senecio, pt. 1, 25. 1901; in Engl. Bot. Jahrb. 32: 21. 1902, nomen.—Cacalia Toluccana DC. Prodr. 6: 328. 1837.— C. prenanthoides Gray, Proc. Am. Acad. 10: 58. 1883, in part, as to Bourgeau’s No. 1086, not HBK.— ErecAthites runcinata Нет]. Biol. Cent.-Am. Bot. 2: 234. 1881, in part, as to Bour- geau's No. 1086, not DC. Herbaceus perennis ubique glabrus vel sparsissime tomen- tellus; caule tereti erecto circiter 1 m. alto striato plus minusve purpurascenti; foliis radicalibus et eis caulinis infimis petiolatis vel sessilibus amplexieaulibusque runcinato-pinnatifidis, lobis remotis, usque ad 4 dm. longis 3-18 em. latis utrinque glabris subtus pallidioribus calloso-dentatis, summis sessilibus et auri- eulato-amplexicaulibus indivisis lanceolato-attenuatis ; inflores- centiis terminalibus laxe paniculatis polycephalis; capitulis discoideis 10-12 mm. altis calyculatis 15-34-floris; involucri squamis plerumque 13 (8-13) lineari-lanceolatis 8-10 mm. longis acutis glabris et corollis plus minusve purpurascentibus; pappi setis albis; achzniis striatis glabris. Specimens examined: Mexico: State of Mexico, Désierto Viejo pres Mexico, Bourgeau, 1086 (Gray Herb. and Berlin Herb.); near Guapimalpam, coll. of 1855, Schaffner (Gray Herb.); fir woods, Sierra de las Cruces, 11 December, 1892, C. G. Pringle, 5818 (Gray [Vor. 1 272 ANNALS OF THE MISSOURI BOTANICAL GARDEN Herb.) ; Sierra de las Cruces, altitude 3350 m., 11 February, 1899, C. G. Pringle, 7709 (Mo. Bot. Gard. Herb.); Mt. Ixtaccihuatl, altitude 2430-3350 m., С. A. Purpus, 100 (Gray Herb.); fir forests, Mt. Ixtaccihuatl, altitude 3350- 3650 m., February, 1903, C. A. Purpus, 45 (Mo. Bot. Gard. Herb.). State of Vera Cruz, Las Vigas, near Jalapa, 2 December, 1903, С. 6. Pringle, 11869 (Gray Herb.), forma. State of Oaxaca, without definite locality, Cuming (Gray Herb.) State of Colima, coll. of Jan. 9-Feb. 6, 1891, Dr. Edward Palmer, 1145 (Gray Herb.), distributed as ‘‘Erechthites runcinata DC." The examination of a large suite of herbarium specimens, partieularly in the light of recently acquired material, has led the writer to place a somewhat different interpretation on this species than formerly; hence, a brief description is here given and a few specimens from widely distributed exsiccati, well illustrating the species, are cited. Senecio (5 Mulgedifolii) Coulteri Greenm. Monogr. Senecio, pt. 1, 25. 1901; in Engl. Bot. Jahrb. 32: 21. 1902, nomen. Cacalia runcinata Less. Linnea 5: 162. 1830, not HBK. Herbaceus perennis; caulibus erectis 3-6 dm. altis striatis paulo tomentulosis plus minusve purpurascentibus; foliis inferioribus petiolatis runcinato-pinnatifidis usque ad 3 dm. longis 1,5-6 em. latis supra glabris subtus arachnoideo-tomentu- losis inequaliter et obtuse calloso-dentatis, foliis superioribus gradatim reductis sessilibus amplexicaulibusque; inflorescentiis terminalibus subcorymboso-cymosis; capitulis numerosis dis- coideis ca. 1 em. altis brevi-calyculatis; bracteis involucri ple- rumque 13 lanceolatis acutis 8 mm. longis glabris et purpura- scentibus; floribus disci 30-40; acheeniis glabris. Specimens examined: Mexico: State of Vera Cruz, Real del Monte, Dr. Thomas Couller, 429 (Gray Herb.), түре, C. Ehrenberg, 381 (Berlin Herb. and Gray Herb.); Mt. Orizaba, Schiede, 363 (Berlin Herb.). State of Mexico, on Nevada de Toluca, 15 October, 1903, J. №. Rose & J. N. Painter, 7940 (U. S. Nat. Herb. and Gray Herb.). Senecio ($ Mulgedifolii) iodanthus Greenm. Monogr. Senecio, pt. 1,25. 1901; in Engl. Bot. Jahrb. 32:21.1902, nomen. Plate 12. ааа ыы ee ТОМАР 1914] GREENMAN—NORTH AMERICAN SENECIONEJE 278 Herbaceus perennis; caulibus 5-9 dm. altis foliaceis striatis glabris plus minusve purpurascentibus; foliis inferioribus pler- umque lyrato-pinnatifidis oblongo-lanceolatis 1.5-3 dm. longis 3.5-9 em. latis acutis vel acuminatis sinuato-calloso-dentatis supra glabris subtus juventate arachnoideo-tomentulosis et заре crispo-puberulentis fere glabratis, foliis superioribus sur- sum gradatim reductis sessilibus amplexicaulibusque; inflores- centiis racemoso-panieulatis 2-5 dm. longis 0.3-1.2 dm. latis; capitulis 10-12 mm. altis discoideis calyculatis; bracteis involucri circiter 13 lanceolatis 8 mm. longis acutis vel obtusis penicillatis glabris vel sparse puberulentis purpurascentibus; floribus disci cà. 24; pappi setis albis quam corolla brevioribus; corollis albis vel purpurascentibus; acheniis glabris. Specimens examined: Mexico: State of Mexico, in pine woods, Nevada de Toluca, altitude 3000-3600 m., 26 September, 1892, C. G. Pringle, 4302 (Gray Herb. and Mo. Bot. Gard. Herb.), түре. State of Morelos, Tres Marias Mts., altitude 2895 m., 5 November, 1903, C. G. Pringle, 11498 (Gray Herb.). This species is closely related to 8. Coulieri Greenm. but differs in having а smooth and more leafy stem, nearly glabrous leaves, and distinctly racemose-paniculate inflorescence. Senecio purpurascens Klatt, Leopoldina, Heft 24, p. 126. 1888. Var. fossanervius Greenm. Monogr. Senecio, pt. 1, 25. 1901; in Engl. Bot. Jahrb. 32: 21. 1902, nomen. Forme typice habitu simili; foliis inferioribus petiolatis, peti- olo incluso, usque ad 11 em. longis 1.5 cm. latis sinuato-dentatis vel ad basin sublyratis supra glabris fossanerviis subtus tomen- tellis et in nerviis pilosis; involucri squamis fere glabris. Specimen examined: Mexico: without definite locality, E. W. Nelson, 1308 in part (U. S. Nat. Herb., fragments in Gray Herb.), TYPE. Senecio ($ Suffruticosi) carnerensis Greenm. Monogr. Sene- cio, pt. 1, 25. 1901; in Engl. Bot. Jahrb. 32: 21. 1902, nomen. Perennis basi suffrutescens ubique plus minusve lignescens; caule tereti erecto simplici vel ramoso; foliis indivisis petiolatis vel sessilibus lanceolatis vel oblanceolatis 1.5-5 em. longis usque ad 1 em. latis acutis denticulatis juventate utrinque tomentosis [Vor. 1 274 ANNALS OF THE MISSOURI BOTANICAL GARDEN supra plus minusve glabratis subtus persistenter albo-tomen- tosis, superioribus subauriculatis; inflorescentiis terminalibus paucicapitatis; capitulis ea. 1 em. altis brevi-calyculatis radiatis; bracteis involueri plerumque 13 anguste lanceolatis apice atratis acutis glabris vel parce tomentulosis; flosculis liguliferis pler- umque 8, ligulis flavis 4-nerviis; floribus disci 30-40 acheniis sursum brevi-sericeo pubescentibus. Specimen examined: Mexico: State of Coahuila, mountains, Carneros Pass, altitude 3050 m., 8 September, 1889, C. G. Pringle, 2857 (Gray Herb., photograph in Mo. Bot. Gard. Herb.), ТҮРЕ. This species was originally referred to 8. longilobus Benth., but it is more closely allied to S. stoechadiformis DC. and 8. Picridis Schauer; it is readily separated from both these species by having fewer involucral bracts, short, appressed and black-tipped bracteoles suggesting those of S. vulgaris L. Senecio (§ Suffruticosi) filicifolius Greenm. Monogr. Sene- cio, pt. 1, 25. 1901; in Engl. Bot. Jahrb. 32: 21. 1902; Contr. U. S. Nat. Herb. 16: 19. 1912, nomen. Herbaceus perennis (?) erectus ramosus 1.5-4 dm. altus ubique glabrus; caule tereti ad basin plus minusve lignescenti; ramis ramulisque striatis stramineis; foliis sessilibus vel subalato-peti- olatis pectinato-pinnatifidis 1.5-8 ст. longis 1-6 em. latis; seg- mentis linearis attenuatis acutis; inflorescentiis subcorymboso- cymosis oligocephalis; capitulis са. 12 mm. altis ligulatis; involu- cris campanulatis ealyeulatis; involucri squamis plerumque 21 bracteolis calyculatis duplo longioribus lineari-lanceolatis acutis glabris vel juventate parce tomentulosis mox glabratis; flosculis liguliferis ca. 12, ligulis flavis; floribus disci 50-60; pappi setis albis; acheeniis sursum sericeo-hispidulis. Specimens examined: Arizona: Valley of the Santa Cruz River, 11 May, 1881, C. G. Pringle, 316 (Gray Herb.), түре; Tucson, 12 March, 1892, J. W. Toumey, 708 (Gray Herb.); Tempe, coll. of 1892, Ganong & Blaschka (Gray Herb.); Hart’s Ranch, 17 miles south of Tucson, 11 April, 1903, J. J. Thornber, 436 (Mo. Bot. Gard. Herb.); Ft. Huachuea, coll. of 1894, Maj. T. Е. Wilcox (Mo. Bot. Gard. Herb.) ; open сайопв, San Francisco Mts., April, 1887, H. H. Rusby, 214 in part (Mo. Bot. Gard. Herb.). 1914] GREENMAN—NORTH AMERICAN SENECIONEJE 275 Mexico: Sandy plains near Altar, State of Sonora, 4 April, 1884, C. G. Pringle (Gray Herb.). This species has been hitherto included with S. Douglasii DC. from which it differs in being essentially glabrous through- out, in having usually more numerous and shorter lateral leaf-segments, fewer, shorter, and less conspicuous calyculate bracteoles. Senecio (§ Suffruticosi) teliformis Greenm. Monogr. Senecio, pt. 1, 26. 1901; in Engl. Bot. Jahrb. 32: 22. 1902, nomen. Herbaceus perennis; caule erecto tereti superne striato stramineo floccoso-tomentoso plus minusve glabrato; foliis su- premis sessilibus lanceolato-attenuatis 3-6 ст. longis ad basin ampliatis usque ad 1.5 em. latitudine semiamplexicaulibusque supra juventate floccoso-tomentulosis plus minusve glabratis subtus persistenter albo-tomentosis, margine dentatis vel denti- culatis revolutisque; foliis inferioribus ignotis; inflorescentiis terminalibus corymboso-cymosis multicapitatis bracteatis floc- coso-tomentulosis; capitulis 8-10 mm. altis radiatis ealyculatis, bracteolis calyculatis lineari-attenuatis conspicuis subflaccidis floccoso-pubescentibus; involucri bracteis plerumque 21 lineari- lanceolatis 5-6 mm. longis acutis glabris penicillatis; flosculis liguliferis sepius 8, ligulis oblongis 5-6 mm. longis flavis; floribus disci са. 40 quam bracteis involucri longioribus, pappi setis albis; acheniis sursum adpresso-sericeo-pubescentibus maturitate 3 mm. longis. Specimen examined: Mexico: State of Oaxaca, mountains of Telixtlahuaca, altitude 2500 m., 10 December, 1894, Rev. Lucius C. Smith, 367 (Gray Herb., photograph and fragments in Mo. Bot. Gard. Herb.), TYPE. Although only the upper part of the plant is at present known to the writer, nevertheless it evidently belongs to the section Suffruticosi and appears to be most closely related to S. Picridis Schauer and S. alvarezensis Greenm. From the former it differs by the usually broader base of the upper stem leaves, more numerous heads and conspicuous bracteoles, while from the latter it is readily separated on foliar characters alone. Senecio ($ Palmatinervii) albonervius Greenm. Monogr. Senecio, pt. 1, 26. 1901; in Engl. Bot. Jahrb. 32: 22. 1902, nomen. [Vor. 1 276 ANNALS OF THE MISSOURI BOTANICAL GARDEN Arborescens 2-4 m. altus; caule tereti primo albo-tomentuloso maturitate glabrato et cortice brunneo tecto; foliis petiolatis basi palmatinerviis late ovatis 3-5 cm. longis latisque sinuato- 9-ll-lobatis remote calloso-mucro-denticulatis basi cordatis juventate utrinque tomentulosis plus minusve glabratis supra in nerviis persistenter albo-tomentulosis, petiolis plerumque 3-10 (usque ad 14 em.) longis; inflorescentiis terminalibus pani- eulatis multicapitatis; capitulis 10-12 mm. altis radiatis; in- volueris anguste campanulatis vel subcylindricis brevicalyculatis; involucri squamis circiter 8 lineari-lanceolatis vel oblongis ob- tusis 5-6 mm. longis glabris vel parce tomentulosis ; flosculis liguliferis plerumque 5, ligulis 5-7 mm. longis flavis 4-nervatis, pappi setis tubo corolle longioribus; floribus disci 8-10 ; ach- æniis glabris. Specimens examined: Mexico: State of Mexico, Valley of Temascaltepec, April, 1831, Schiede (Berlin Herb. and Gray Herb.), TYPE; open woods, Ixtaccihuatl, altitude 2430-3350 m., March-July, 1903, C. A. Purpus, 201 (Gray Herb. and Mo. Bot. Gard. Herb.). State of Vera Cruz, Mineral del Monte, Ehrenberg, 324 (Berlin Herb. and Gray Herb.). State of Morelos, Sierra de Tres Marias, altitude 3050 m., 15 April, 1904, C. G. Pringle, 8903 (Gray Herb. and Mo. Bot. Gard. Herb.). State of Michoacan, north slope of Mt. Tancitaro, altitude 2280-3200 m., 24 February, 1903, E. W. Nelson, 6904 (U. S. Nat. Herb. and Gray Herb.). The broadly ovate, shallowly sinuate-lobed leaves with per- sistent white tomentum on the veins of the upper leaf-surface, together with a terminal many-headed panicle and yellow ray- flowers, render this species distinet and easily recognized among all those of the palmately veined section to which it belongs. Senecio angulifolius DC., var. ingens, var. nov. Habitu et foliis forme typice; inflorescentiis compactis pauci- vel multi-capitatis, bracteis bracteolisque perconspicuis; capitulis 1.5-2 em. altis 40-45-floris radiatis vel discoideis. Specimens examined: Mexico: Mt. Ixtaccihuatl, above timber line, March-July, 1903, C. A. Purpus, 193 (Mo. Bot. Gard. Herb.), TYPE; rocky slopes, Mt. Ixtaccihuatl, altitude 5790-6090 m., 1914] GREENMAN—NORTH AMERICAN ЗЕМЕСТОМЕЮ 977 November, 1905, C. А. Purpus, 1517 (Мо. Bot. Gard. Herb.). State of Puebla, Mt. Orizaba, near Chalchicomula, 25 February, 1892, Jared G. Smith, 473 (Мо. Bot. Gard. Herb.). On account of the conspicuous more or less foliaceous bracts of the inflorescence S. angulifolius DC. is a very characteristic species and is almost always recognized without difficulty. There is, however, a considerable variation in the size of the heads and in the number of flowers of the disk, as well as in the degree of development of the ray-flowers. In fact the latter may be well developed, more or less reduced, or entirely absent. Тһе extremely large headed form, which is well exemplified by the specimens cited above, seems well worthy of varietal rec- ognition. Doctor Purpus's No. 1517 is somewhat intermediate between the species and the variety. Senecio (5 Palmatinervii) brachyanthus Greenm. Monogr. Senecio, pt. 1, 26. 1901; in Engl. Bot. Jahrb. 32: 22. 1902, nomen. Verisimiliter frutex; caule tereti cortice brunneo tecto juven- tate hirtello-puberulento glabrato; foliis longipetiolatis sub- peltatis palmatinerviis suborbieularis circiter 7-lobatis mem- branaceis utrinque parce hirtellis subtus pallidioribus mucro- denticulatis, petiolis usque ad 13 em. longis minute puberulentis; inflorescentiis terminalibus subglanduloso-hirtellis; capitulis sub- cylindricis 10-12 mm. altis heterogamis; involucri bracteis 8 lanceolatis 8-10 mm. longis acutis vel obtusis plus minusve purpurascentibus extus subglanduloso-hirtellis; flosculis fem- ineis 5 multum reductis, ligula nulla, tubo gracili squamis invol- ист! breviore; floribus disci 8-10; pappi setis albis; acheniis glabris. Specimen examined: Mexico: State of Guerrero, between Ayusinapa and Petatlan, altitude 1540-2155 m., E. W. Nelson, 2137 (Gray Herb. апа U. S. Nat. Herb.), ТҮРЕ. Тһе leaves and reduced ray-flowers of this species are similar to those of S. cordovensis Hemsl., but the character of the involucre indicates a closer relationship with S. chapalensis Watson. Senecio ($ Palmatinervii) chapalensis Watson, Proc. Am. Acad. 25: 155. 1890. [Vor. 1 278 ANNALS OF THE MISSOURI BOTANICAL GARDEN Var. areolatus Greenm. Monogr. Senecio, pt. 1, 26. 1901; in Engl. Bot. Jahrb. 32: 22. 1902, nomen. А forma typica recedit foliis utrinque glabratis subtus areo- latis, petiolis usque ad 15 em. longis plus minusve purpur- ascentibus ; flosculis liguliferis granditer reductis. Specimen examined: Mexico: State of Morelos, on shaded bluffs of a wet canyon above Cuernavaca, altitude 1980 m., 15 February, 1899, C. G. Pringle, 8010 (Gray Herb. and Mo. Bot. Gard. Herb.), түре. Senecio ($ Palmatinervii) Chrismarii Greenm. Monogr. Senecio, pt. 1, 26. 1901; in Engl. Bot. Jahrb. 32: 22. 1902, nomen. Frutex; caule primo parce pubescenti maturitate glabro; foliis petiolatis palmatinerviis circumscriptione triangulari- ovatis 7-10 em. longis 5-8 cm. latis hastatis 3-5-lobatis ciliatis mucro-denticulatisque granditer cordatis supra sparse hirtello- puberulentis subtus glabris vel in nervis puberulentis, lobiis mucronato-acutis; petiolis gracilibus 4-9 em. longis parce hir- tellis vel glabris; inflorescentiis terminalibus laxe paniculatis paucicapitatis dense glanduloso-puberulentis, pedunculis gra- cilibus remote bracteatis; capitulis 1.2-1.5 em. altis discoideis paucicalyculatis; involueri squamis sepius 8 lanceolato-oblongis са. 1 em. longis acutis penicillatis extrinsecus hirtello-puberu- lentis plus minusve purpuraseentibus interioribus Scarioso- marginatis; floribus disci plerumque 20 involucri bracteis longioribus; pappi setis albis; achzniis glabris. Specimen examined: Mexico: without definite locality, Chrismar (Berlin Herb., tracing and fragments in Gray Herb.), туре. The affinity of this species is with SS. hederefolius Hemsl., S. anisophyllus Klatt, and S. alienus Robinson & Seaton. From the first two it differs in having deeply cordate leaves with more or less reflexed lateral lobes, and from the last it is readily sep- arated by the deeply cordate leaves and absence of peltation. Senecio (§ Palmatinervii) hypomalacus Greenm. Monogr. Senecio, pt. 1, 26. 1901; in Engl. Bot. Jahrb. 32: 22. 1902, nomen. Plate 10. 1914] GREENMAN—NORTH AMERICAN SENECIONE® 279 Frutex erectus; caule tereti primo dense sordido-puberu- lento, sepissime lenticellis intermixtis, maturitate cortice brunneo tecto; foliis petiolatis vel supremis sessilibus circum- scriptione ovato-rotundatis vel ovato-oblongis palmato-3-5- nerviis distincte 5-11-lobatis supra crebe crispo-hirtellis subtus lanato-tomentosis basi cordatis vel subtruncatis, margine sinua- tis calloso-denticulatis ciliatis; petiolis usque ad 6 em. longis; inflorescentiis terminalibus paniculatis polycephalis subglandu- loso-hirtellis; capitulis 10-12 mm. altis parce calyculatis radiatis; bracteis involucri plerumque 8 (non-nunquam 7) oblongis vel subobovatis 5-6 mm. longis obtusis vel acutis extus crebe sub- glanduloso-hirtellis, interioribus late scarioso-marginatis; flos- culis femineis liguliferis, ligulis anguste oblongis 5-6 mm. longis flavis; floribus disci circiter 10 (7-13) quam involucrum bis tanto fere longioribus; pappi setis albis; achzeniis glabris. Specimens examined: Mexico: State of Oaxaca, mountains of Telixtlahuaca, altitude 2375 m., 10 December, 1894, Rev. Lucius С. Smith, 368 (Gray Herb., photograph and fragments in Mo. Bot. Gard. Herb.), түре; Sierra de San Felipe, altitude 2130-2440 m., 17 November, 1894, Charles L. Smith, 210 (Mo. Bot. Gard. Herb.) ; Cerro de San Felipe, altitude 1900 m., 25 September, 1895, C. Conzalli, 119 (Gray Herb.). This species is related to S. oaxacanus Hemsl., but differs from it in having distinctly lobed leaves which are thicker in texture, densely subglandular-hirtellous above and soft tomen- tose beneath; moreover, the leaf-margin of S. hypomalacus is markedly sinuate and the lobes show a tendency to become again lobate. С. and E. Seler’s No. 1581 from Tillantongo, which has been referred to S. oaxacanus Hemsl., is somewhat intermedi- ate between the two species, but it has the leaf-outline and thinner texture of Mr. Hemsley’s species. Senecio (§ Palmatinervii) Kerberi Greenm. Monogr. Senecio, pt. 1, 26. 1901; in Engl. Bot. Jahrb. 32: 22. 1902, nomen. Herbaceus robustus perennis usque ad 3m. altus; caule tereti erecto glabro vel parce tomentuloso; foliis petiolatis pal- mato-5-7-nerviis ovato-oblongis 5-10 em. longis 5-8 cm. latis 5-7-lobatis carnoso-denticulatis reticulato-venosis supra sparse hirtellis subtus subarachnoideo-tomentulosis, lobis obtusis vel 2 [Vor. 1 280 ANNALS OF THE MISSOURI BOTANICAL GARDEN subrotundatis et mucronato-acutis; petiolis 2-2.5 em. longis; inflorescentiis terminalibus paniculatis multi-capitatis pubes- centibus, pedunculis minute bracteatis; capitulis 7-8 mm. altis radiatis; involucris campanulatis minute calyculatis fere glabris : involucri squamis 13 lineari-laneeolatis vel lanceolato-oblongis 4.5-5 mm. longis acutis glabris; flosculis femineis 5 liguliferis, ligulis oblongis 4-5 mm. longis flavis; floribus disci ca. 14, pappi setis albis; achzeniis glabris. Specimen examined: Mexico: "Tromptero, Mesa del Arrero," 21 November, 1880, Kerber, 94 (Berlin Herb., fragments and tracing in Gray Herb.), TYPE. This species is known at present from a single specimen in the Royal Botanical Museum of Berlin. From this specimen the writer was permitted, as in a number of other cases, while making a study of the genus several years ago, to make a tracing and take fragments for the Gray Herbarium of Harvard Uni- versity. Тһе species is related to S. Hartwegi Benth. and S. reglensis Greenm., but from these and from other species of the section Palmatinervii to which it belongs, it is readily distin- guished by the somewhat elongated more or less fan-shaped and bluntly lobed leaves. Senecio ($ Palmatinervii) velatus, sp. nov. Plate 13. Frutex; caule tereti carnoso ramoso ad apicem sordido-tomen- toso cetero glabro in sieco cortice brunneo tecto; foliis petiolatis palmato-7-nerviis circumscriptione ovato-rotundatis ca. 10 em. longis latisque angulato-7-9-lobatis membranaceis integris ju- ventate utrinque plus minusve albo-tomentosis subtus persis- tenter arachnoideo-tomentulosis, lobis triangulari-ovatis mu- cronato-acutis; petiolis ca. 8 em. longis floccoso- pubescentibus; inflorescentiis terminalibus dense cymoso- corymbosis minute bracteatis multicapitatis glabris vel in axillis ramulorum floccoso- tomentulosis; capitulis ca. 1.5 cm. altis radiatis; involucri sub- cylindrici squamis ssepius 8 lanceolato-linearis vel lanceolato- oblongis 7-10 mm. longis acutis vel obtusis; flosculis liguliferis 3-5, ligulis anguste oblongis са. 1 em. Тоқты floribus disci 6-7, pappi setis albis; achæniis glabris striatis. 19141 GREENMAN—NORTH AMERICAN ВЕМЕСІОХЕЖ 281 Specimen examined: Mexico: State of Jalisco, on bluffs of barranca, near Guadalajara, 20 May, 1891, С. 6. Pringle, 5160 (Gray Herb., photograph and fragments in Mo. Bot. Gard. Herb.), ТҮРЕ. The writer has withheld publication of this species for several years with the hope that additional material might be secured. Mr. Pringle's specimen, from which the above description is drawn, is in the Gray Herbarium and consists of a terminal portion of a flowering stem and two detached leaves. In stem and inflorescence characters it corresponds very well with typical specimens of 8. precox DC. except that the terminal portion of the stem and branches are covered with a tawny pubescence, not glabrous as is usually the case with the DeCandollean spe- cies. On account of the similarity of stem and inflorescence and because of the detached leaves the plant has been referred doubtfully to the peculiarly characteristic and well known S. precoz DC. The extreme care with which Mr. Pringle prepared his plant material and the fact that the leaves on the specimen under consideration, although detached from the stem, accord with the type of foliage of the section Palmatinervii lead me to believe that we have to deal in the present case with an unrecorded species related to but distinct from S. precoz DC., and in all probability one of limited geographical distribution. Senecio Klattii, nom. nov. S. roseus Klatt, Ann. k. k. Naturhist. Hofmus. Wien 9: 366. 1894, not. S. roseus Schz. Bip. in Flora 28: 498. 1845. 282 (Vor. 1, 1914) ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 10 Senecio hypomalacus Greenm., Mexico From the type specimen, Rey. Lucius C. Smith No. 368, in the Gray Herbarium of Harvard University. MI. ee ee PLATE 10 CIONEAE А МЕ 2 BOSTON COCKAYNE, + мы z a x = E br y. 7 i = б; -— ал m = > < e cá c г | | Fa < Z A A c e Mo. Bor. GARD., VOL. ANN, MUI a a oe ia TATTOO DE ME uo Ша ыы сом 4 A a ағын s v йе — -— МРГ. ес А ve UBER UR, оралар i o ОДИН барысы тант $5 ees, ee тұсу! TU TS Lo Gs (Vox. 1, 1914] 284 ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION or PLATE PLATE 11 Senecio oreopolus Greenm. California From the type specimen, Hall and Babcock No. 5526, in the Gray Herbarium of Harvard University. амы ER E e TNR o co a Қалы а аза 2) ANN. Мо. Вот. GARD., VOL. 1, 1914 GREENMAN —NORTH AMERICAN SENECIONEAE COCKAYNE, BOSTON PLATE 11 CVM 20 а ee ape [Мог 1, 1914] 286 ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 12 Senecio iodanthus Greenm. Mexico From the type specimen, Pringle No. 4302, in the Gray Herbarium of Harvard University. NT. ei? Ls De N 24 ш E < ы > = = = — я ы o - & я < © - > ш 5 ж 2 " gh ON a ARS GENE Gg Хе 4 b SENECIONEAI AMERICAN REENMAN — NORTH r^ ( BOSTON COCKAYNE, (Уот. 1, 1914) 288 ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 13 Senecio velatus Greenm. Mexico From the type specimen, Pringle No. 5160, in the Gray Herbarium of Harvard University. ANN. Mo. Вот. GARD.. Vor. 1, 1914 GREENMAN — NORTH AMERICAN SENECIONEAE COCKAYNE, BOSTON PLATE 18 [Vor. 1, 1914] 290 ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 14 Senecio subauriculatus Greenm. Mexico From the type specimen, E. W. Nelson No. 2526, in the Gray Her- barium of Harvard University. NEPOTES s Seo ee MT NOTER us a ee a ы NE % u H жы N aS e ) ANN. Мо. Вот. GARD., VOL. 1, 1914 PLATE 14 4 ME Come tiu TERE AL 2 $ e Mi GREENMAN —NORTH AMERICAN SENECIONEAE COCKAYNE, BOSTON А І А STUDY ОҒ THE PHYSIOLOGICAL RELATIONS OF SCLEROTINIA CINEREA (BON. SCHROTER J. S. COOLEY Formerly Rufus J. Lackland Fellow in the Henry Shaw School of Botany of Washington University INTRODUCTION | This paper reports the results of an experimental study regard- , ing certain physiological activities of the brown-rot fungus of 4 stone fruits. The investigation concerns itself primarily with the conditions influencing the penetration and infection of green and ripe fruits by the fungus in question, the action of the parasite on the host cell, and the secretion of the enzymes which act upon the cellulose and pectic substances of the host. The work was undertaken with the hope of throwing some further light upon the factors concerned in fungous parasitism. Our present conception of this subject is based upon fragmentary and, in some respects, contradictory evidence. However, each year there are acquired new facts, or new applications of known facts, bearing upon this exceedingly involved and complex question. An examination into the history of investigations concerning the interaction of host and parasite shows that the study of this subject dates back to the work of the pioneers in. . VN T he plant pathology ; modern methods and recent discoveries + ve, D however, given an added impetus to research along this fine. ~ Progress in combating fungous diseases depends | ot | upon a familiarity with the life history of the parasite, especially upon an intimate knowledge of the metabolism of the parasite and the nature of the changes which it induces in the host. Indeed, many of our recommendations for controlling parasitie diseases of plants will perhaps be modified when a more exact knowledge of the interrelations of host and parasite is gained. Furthermore, a more intimate knowledge of the physiological aspects of plant pathology will undoubtedly throw much light on the question of immunity and susceptibility. We should, of course, like to know more about the factors favoring or inhibiting parasitic action, as well as the conditions ANN. Mo. Вот. GARD., Vor. 1, 1914 . (291) [Vor. 1 292 ANNALS OF THE MISSOURI BOTANICAL GARDEN which influence the infection and the penetration of parasitic fungi. It would also be interesting to know why some fungi are so virulent and rapid in their destructive action on the host; for instance, it would be instructive to know whether it is due to the secretion of an enzyme, or a toxie substance (e. g., some acid), or to the disturbance of the osmotic relations of the host cells, or to some other perhaps unknown factor. For a study of some of these problems the writer has chosen as the organism Sclerotinia cinerea (Bon.) Schróter, the fungus causing the brown rot of stone fruits. This form is particularly suitable for the pur- pose since it is а virulent parasite, yet grows well as a saprophyte —readily lending itself to cultivation in the laboratory. HistoricaAL REVIEW Space will permit only a brief review of some of the more important papers dealing with certain aspects of this subject. Much of the literature that is indirectly concerned with the problem, or that is fully reviewed or superseded by subsequent publications, will not be discussed here. In the period from 1858 to 1878 little experimental evidence appeared concerning the nature of the action of fungous para- sites, although several writers make mention of the penetration of host cells by fungous hyphae. Penetration was then fre- quently spoken of as merely a process of boring through (‘‘durch- bohrung") the host tissue, Kühn (34), as early as 1858, men- "1 о: .2 this fact in a discussion of the potato-blight fungus. _A few years later, in 1863, de Bary (1) speaks of the penetration of the host by Peronospora, and further makes mention of this faet in connection with his work on the rusts (2); again in his work 'Morphologie und Physiologie der Pilze, Flechten, und Myxomyceten’ (3) he discusses the penetration of the host, but says he has no knowledge of the force that causes this boring into the host tissue. Hartig (26), in his early work on wood-destroying fungi, as well as in his later investigations, emphasizes the fact that fungi are able to destroy cellulose. Ву а microscopical study of diseased wood he found that the properties of the latter are very materially c»anged by the fungus; he did not, however, attempt to isolate an enzyme. 1914] COOLEY—SCLEROTINIA CINEREA 293 De Bary (4), in 1886, gives us the first important contribu- tion to our knowledge concerning the action of parasites on host cells. This author, in his epoch-making research on the fungus now known as Sclerotinia libertiana, reports that the organism secretes a substance that discolors, plasmolyzes, and finally kills the host cells. This toxic secretion penetrates the host cells in advance of the fungus, killing them before they are actually pierced by the fungous filaments. De Bary was able to isolate this toxic substance, which he considered as probably an enzyme, and found that it would cause an injury to the host tissue similar to that produced by an attack of the fungus itself. He holds that the fungus will not grow on living tissues, for it attacks only through a wound and kills the cells in ad- vance of itself, thus not actually growing upon the living tissue. The product resulting from the disintegration of the cell wall of the host was thought to be a sugar that served as food for the fungus. In this connection de Bary also mentions finding oxalic acid encrusting the older fungous filaments. / Тһе next important paper on the interaction of host and Е parasite was that of Marshall Ward (51) published just two years after de Bary's work and concerning itself with a species of pd Botrytis causing a lily disease. In this excellent piece of work | the author showed that the fungous hyphae on coming in contact with such solid substances as sections of a lily bulb, or even a cover glass, secrete from the tips drops of a substance that а: has a very peculiar effect on the host cell. He ound аба water extract of this secretion when applied to sections of а lily bulb will cause the cell walls to swell and to assume an ab- аз normal appearance; the middle lamella is first dissolved and finally the entire cell wall is disorganized. Ward does not con- sider that this toxic secretion is stimulated by starvation. Several investigators have held that the penetration of many fungi is due to chemotropism, i. e., that penetration of the fungous hyphae is due to some stimulus which the constit- uents diffusing slowly from within the host cells exert. Biisgen (16), Miyoshi (89), Behrens (6), Schmidt (44), and others have adhered to the view that chemotropism is important, but more recent work, such as that of Fulton (25), does not uphold the theory. [Vor. 1 294 ANNALS OF THE MISSOURI BOTANICAL GARDEN Behrens (6) investigated some of the physiological relations of saprophytes in comparison with parasites, using Mucor sto- lonifer, Penicillium sp., Botrytis cinerea, and Oidium ( — Sclero- tinia!) fructigenum. This author holds that Sclerotinia does not produce a cellulose-dissolving enzyme, and that the fungus merely forces its way through the host tissue by a purely me- chanieal force, or that, in some cases, it splits the middle lamella but does not dissolve it. In the case of the other fungi mentioned above he believes that an enzyme is secreted which dissolves the middle lamella. The cause of the injury due to Sclerotinia, he holds, is not that the cellulose walls or the pectin of the middle lamella is dissolved, but that the turgor and the osmotic relations of the penetrated cells are materially modified. Ac- cording to this author some substance diffuses through the walls and stimulates the fungus to bore through or between the сей walls. He demonstrated in Botrytis and Penicillium, more- over, a thermo-stable toxic body which disintegrated the host cells, and believes that these fungi secrete a pectin-dissolving enzyme which is different from that which acts upon cellulose. Nordhausen (40), at about the same time, made similar studies on Botrytis cinerea and comes to similar conclusions. He finds that the enzyme does not cause a strong swelling of either the Idle lamella or the cellulose cell walls, the action in this respect g more like that of de Bary's Леска. Smith (46) stud- Ве parasitism of Botrytis cinerea, but in certain particulars dot get the same results as de Вагу and Ward. Like them e finds that the parasite secretes some soluble substance that penetrates and kills the living cells in advance of the fungous filaments, but unlike Ward he could detect no swelling of the cell wall. Smith believes that this toxic substance is not an мы enzyme, for boiling does not inactivate it, but thinks that it is perhaps oxalie acid, since this substance is always present in the cultures and amounts in some cases to as much as two per cent. The analytieal methods whereby the oxalie acid was determined, unfortunately, are not given. Schellenberg (43) investigated the action of several sapro- phytic and parasitic fungi on hemicelluloses from a number of ! Wehmer, C. Ber. d. deut. bot. Ges. 16: 298-307. 1898; Saccardo, Syll. Fung. 4: 34. 1886. 1914) COOLEY—SCLEROTINIA CINEREA 295 different sources. He claims that these fungi act differently toward different celluloses, dissolving some and having no effect on others. The nature of the penetration and the action of certain parasites on the host tissue were also studied. There was no case in which Botrytis dissolved true cellulose, but it readily dissolved the hemicellulose part of the cell, leaving the cellulose intact. According to this author, therefore, the pene- tration and dissolving action of such parasites as Botrytis vul- garis is due to their ability to dissolve hemicelluloses. Не considers that the middle lamella is largely composed of hemi- celluloses or closely allied substances. According to this view, therefore, organisms that dissolve the middle lamella are essen- tially hemicellulose-dissolving forms. As a result of his studies on Sclerotinia fructigena and S. cinerea, Schellenberg finds a different action on different fruits, but in no case does he report a splitting of the cells along the line of the middle lamella, as some previous investigators have reported. He believes that there is a slight dissolving action on that part of the cell wall which is in immediate contact with the fungous filament, but that the rest of the cell wall remains intact. In the twigs also he finds that the fungus dissolves the hemicellulose and leaves the true cellulose unacted upon. An extensive literature has developed concerning the enzymes of importance in the nutrition of fungi, but since these investi- gations either deal with saprophytes, or are only indirectly concerned with the work to be reported in this paper, it will be unnecessary to do more than mention some of the papers here. Among the more important contributors may be mentioned Ward (50, 52), who was the first to use pure cultures of a wood- destroying fungus (Stereum), Biffen (9), who studied the biology of Bulgaria polymorpha, Bourquelot and Hérissey (13), who in- vestigated the enzymes in sporophores of Polyporus sulphureus, Czapek (18), who made his investigations with natural infections of Merulius lacrymans and with other fungi, Kohnstamm (33), who worked on some species of Merulius, Buller (14, 15), who in- vestigated sporophores of Polyporus squamosus, Van Iterson (28), who developed methods for isolating cellulose-dissolving bac- teria and fungi, and Dox (19), who investigated the enzyme action of species of Penicillium and Aspergillus. It is interest- 3 [Vor. 1 296 ANNALS OF THE MISSOURI BOTANICAL GARDEN ing to note that although we have every reason to believe that cytase is present in timber-decay organisms yet its presence has been demonstrated only indirectly by cytological methods. It is true, however, that many of the investigators mentioned above who found no cytase used the sporophores in their experiments and not the mycelium. Тһе status of the subject of the enzymes concerned in the metabolism of parasitie fungi is given in Reed's recent publica- tion (42), which concerns itself with the enzymes produced by the parasitic fungus Glomerella rufomaculans. This author has proved that the parasite produces many of the enzymes that had previously been reported for saprophytes, and by quan- titative methods has demonstrated different enzymes acting on the several classes of nutritive substances, such as carbo- hydrates, glucosides, fats, and proteins. He did not, however, investigate the cytolytic activity of the fungus but states that the nature of the diseased host would indicate that cytase very probably is not produced by this fungus. Peltier (41), as a re- sult of his investigations with Botrytis Fuckeliana, finds that the host cells are killed in advance of the fungous penetra- tion, and that the parasite secretes a thermo-stable toxic sub- stance, but, unlike Smith, finds no oxalic acid. Тһе method of testing for oxalic acid unfortunately is not given. The action of bacteria on cellulose and other plant products has been extensively studied by a number of investigators, but for the purpose at hand it will suffice to cite some of the more recent publications in which the earlier literature is reviewed. The work of Jones (29, 30), which gives a good resumé of the early work on this subject, is reviewed below under the dis- cussion of pectin. McBeth and Scales (38) report that а number of bacteria and fungi hydrolyze cellulose and claim that filamentous fungi play a very important róle in the destruction of cellulose in soils. Тһе cellulose-destroying fungi, according to these authors, act differently toward different kinds of cellulose, but their experi- ments do not seem to support this conclusion. Kellerman and McBeth (32) have also contributed to our knowledge of the cyto- lytie activity of fungi. Kellerman (31) has employed a method 1914) COOLEY—SCLEROTINIA CINEREA 297 by which it is demonstrated that cytase diffuses in agar consid- erably beyond the region of hyphal penetration, and that a portion of the agar containing the enzyme dissolves cellulose in a manner similar to that of the fungus itself. The organism employed in my work was isolated from an infected plum twig, at Madison, Wisconsin. The original cul- tures were taken from a single colony in a Petri dish, this pro- cedure giving reasonable assurance that I was working with a single strain of the organism. Regarding the systematic relations of this organism a word may not be out of place here, since considerable confusion has arisen in the literature regarding the specific name of the organism causing the brown rot of stone fruits (27, 53, 37). Woronin (56) has made an important contribution designed to establish the systematic position of the two species Sclerotinia cinerea and S. fructigena. It has gener- ally been held that S. fructigena causes the brown rot of stone fruits in this country, while in Europe this fungus is found only on pome fruits; but Matheny (37) has recently given good evi- dence tending to show that it is S. cinerea which causes the brown rot of stone fruits both in this country and in Europe. EXPERIMENTAL STUDIES INFECTION Some investigators, as, for instance, Zschokke (57), have held that Sclerotinia cinerea is unable to penetrate sound fruit, while Smith (45), among others, has held that the fungus rapidly pene- trates and infects sound and unwounded fruit (peaches). Casual observation in the field would seem to justify the former view, for those fruits in contact with other fruits or twigs, and there- fore liable to puncture or abrasion, are the ones that are usually found infected; indeed, field observations and laboratory exper- iments point to the conclusion that infection takes place much more readily, especially with immature fruits, when the cuticle is broken. One would, therefore, naturally raise the question as to whether or not infection can take place when the cuticle is unbroken, and if so under what conditions and in what stages of the development of the fruit. During the summer of 1913 [Vor. 1 298 ANNALS OF THE MISSOURI BOTANICAL GARDEN the writer performed a number of experiments which throw more light on the question of the infection of the host. Methods and Resulis.—The methods employed were as fol- lows: Plum twigs bearing leaves and fruit were broken off and brought into the laboratory, washed with a mercuric chloride solution (1-1000) and in sterile water. They were then sus- pended in sterile moist chambers prepared by placing moistened absorbent cotton in the bottom of wide-mouthed one-liter Erlen- meyer flasks that had previously been plugged and sterilized. Twigs having one or more green leaves were used in every case, for in this way green plums hang on the twigs and remain alive for some time. This method was especially applicable here, for it enabled one to maintain absolutely sterile conditions in а moist atmosphere and at the same time keep the host living and in a normal condition. Тһе results of these infection ex- periments are given in table т. Discussion of Results.—From these results it is evident that plums were infected as early as June 27, at which time they were immature, in fact not more than half-grown. Infection did not take place when a spore suspension was placed on very green and immature plums unless the epidermis was broken or punc- tured. There were, however, some instances where plums re- mained healthy in the flask for two or three weeks and became infected only after the lapse of time had brought about an artificial maturity. On the other hand, plums that were ap- proaching maturity, though not mature, as well as mature fruits, may be infected by applying a spore suspension to the natural surface, i. e., a surface which has not been punctured or injured in any way. In this connection it should be mentioned that infection was much more readily accomplished when two plums were hanging so as to be in contact with each other than when they were not touching. This, no doubt, was due to the fact that a drop of water containing spores may be held between the plums long enough for spore germination and infection to take place. These results also indicate that infection takes place readily without puncturing when a portion of the mycelial felt is laid on the surface of either green or ripe fruit. It should be noted here that one can sometimes find plums in the field only half-grown which are affected with the brown-rot быт. сы MOD QU НТ 1914] n COOLEY—SCLEROTINIA CINEREA 299 TABLE I RESULTS OF INFECTION EXPERIMENTS WITH SCLEROTINIA CINEREA Date Fruit Inoculating Treatment Method of НА material of surface inoculation Green Spore Cuticle killed | Surface қ June 27 plums suspension by steam application Eom Green Skin punetured | Needle June 27 plums Spores with needle puncture m ә (Green Skin punetured | Needle p - plums Е with needle puncture ++ Green Skin punetured | Needle do plums ылы with needle puneture et Green Spore Surface E 2 plums suspension Untreated application Sour Skin punctured | Needle 8 Ў қ July cherries Spores with needle puncture + Green Skin punctured | Needle July 23 plums Spores with needle puncture tt July 23 Green Spore d Surface E y | plums suspension Untreate application | Green Spore ue Surface | July 23 plums suspension Skin cut application | SUE July 23 ре Mycelium Untreated mane | ++ dii plums ied — application | Green | Surface | July 23 Di Mycelium Untreated applitation | te Green . Е Surface | July 30 pitaa Mycelium Untreated application | ++ Aug. 5 еа — . Untreated Surface E + plums suspension application | | Nearly | Aug. 13 | ripe Spore — Untreated Surface 0| ++ suspension application | plums à | | Ripe Spore Surface | Aug. 13 plums suspension Untreated applicatic n | Tn *4 + indicates that practically every inoculated fruit became infected. +indicates that only a portion of the inoculated fruits became infected. — indicates that none of the inoculated fruits became infected. [Vor. 1 300 ANNALS OF THE MISSOURI BOTANICAL GARDEN fungus, but so far as the writer's observation indicates, infection in these cases takes place through the twig, or, in some cases, through another plum with which it is in contact and which in turn is infected through the twig. Nevertheless, field observa- tions also verify the laboratory work in that plums (especially certain varieties, such as Wood) when approaching maturity may be infected in the field without being in contact with other fruits and without having any visible punctures or wounds іп the skin. All these experiments and observations point to the conclusion that penetration of the cuticle is a very important factor in the infection of fruits, especially immature fruits; that infection of very green fruits without punctures is rare; and, on the other hand, that maturing fruits without punetures may be readily infected both by spores and by a mycelial felt in the field and in the laboratory. PENETRATION The nature of penetration and the course of the hyphe of parasitie fungi in piercing host tissue is an interesting and im- portant question in connection with a study of the nature of parasitic action. In the case of the brown-rot fungus growing on the plum it is of importance to know whether or not the hyphe merely follow the middle lamellæ or whether they enter the cells wherever they come in contact with them. Previous investigators differ very widely in their opinions as to the nature and course of the penetration of the fungus in question, a condi- tion which is perhaps partly explained by the fact that different hosts were employed in the various investigations. Further- more, it appears that the methods employed in some of the researches were not of such a character as to readily yield com- plete information concerning all the facts in the case. In my own work a study of the penetration of the host tissue by the fungus was made by examining a number of sections of infected tissue in which the disease had reached various stages of development, and comparing them with sections of healthy tissue from the same fruit. For this purpose a special method was employed. Methods and Results.—Small pieces of fruit composed of diseased and sound tissue were cut from plums inoculated with 1914) COOLEY—SCLEROTINIA CINEREA 301 a pure culture of the fungus. These segments were immersed in 70 per cent alcohol just long enough to partially kill the fungous filaments and the host cells, yet not long enough to discolor the sound tissue or to modify or change the color of the diseased tissue in any way. From this material razor sections, containing both diseased and healthy tissue, were made, stained for a short time in eosin, and then partially destained with alcohol. If the pieces of plum had not remained in the alcohol for a sufficient length of time, the razor sections were immersed in 70 or 95 per cent alcohol before staining. By employing this method it is possible to stain the fungous filaments deeply, while the host tissue remains unaffected. Indeed, this method permits of a rather sharp color differentiation between the healthy and the diseased tissue, the latter being blackened by the disease. This method, though quite applicable for the pur- pose at hand, was primarily developed for another purpose, which will be discussed below. Since every fungous filament is very sharply differentiated, one may readily study the course of the hyphæ with reference to the host cells. By staining, sectioning, and examining dis- eased material taken from the margin of the infected area, one finds the fungous һурһе penetrating the cells at any point of contact; indeed, after examining а number of specimens by the method reported above, the writer finds no indications that the fungous hyphe follow the middle lamellze, as has been reported by other investigators (57, 6) for pears and other fruits. The above method also enables one to contrast the cell walls of infected and penetrated cells with those of normal tissue. It is entirely possible that the fungous filaments, on coming in contact with a cell wall, secrete just enough enzyme to dissolve their way through the cell walls, leaving the walls of the host cells surrounding the hyphæ entirely normal, i. e., without swelling or disorganization. Another and somewhat different experiment was performed to get additional evidence on this point. From sound plums which had previously been rendered sterile by washing in bi- ehloride of mereury solution (1-1000) and sterile distilled water, free-hand sections were cut with a razor sterilized in 50 per cent aleohol. Тһе sections were arranged in hanging drop cultures [Vor. 1 302 ANNALS OF THE MISSOURI BOTANICAL GARDEN and each inoculated with a drop of a very dilute spore suspen- sion containing two or three spores per drop. Тһе progress of the fungus and the condition of the host cells were noted from day to day but no visible disintegration of the cell walls could be observed, nor did the fungus show any particular affinity for the middle lamelle. Conclusions.—We would conclude, therefore, as a result of direct observation on the host tissue, that the fungus penetrates the host very readily and rapidly, that it does not necessarily follow the middle lamelle in the plum and the peach, and that there is no visible general disintegrating action on the middle lamell: or on the cell walls of the living host. ACTION OF THE FUNGUS ON THE LIVING HOST CELLS А significant fact in the metabolism of the brown-rot fungus is that it induces such an exceedingly rapid decay in the infected fruits. This rapid decay might be connected both with a rapid growth of the fungus and with a pronounced power which the organism possesses of breaking down and changing the constituents of the host. Moreover, several representatives of the genus Sclerotinia have been reported to have the power of secreting an enzyme or some other substance which kills the host cells in advance of penetration. Were this the case, it would be expected that rapid decay would accompany the action of the parasite. Is this view applicable to the action of Sclero- tinia cinerea? Тһе investigators who have made a study of this organism differ very widely in their views regarding the effect which it has on the host tissues, and it seemed desirable, therefore, to determine the relation of hyphal penetration to the death of the cells. Methods and Resulls.—In order to fix the material for this study, it was found satisfactory to proceed as follows: Small pieces of the host tissue were taken from the margin of the dis- eased area and placed in 95 per cent alcohol for a short time. Free-hand sections were made of this material so as to include both diseased and healthy cells, and the sections stained for a short time in eosin and subsequently decolorized in part with alcohol, if necessary to give the desired contrast. Ву this Мей -----------.. 1914] COOLEY—SCLEROTINIA CINEREA 303 method the fungus may be distinctly differentiated from the host tissue, the killing and staining agents having little or no effect on the host cells. There is a more or less sharply differ- entiated line of demarcation between the injured and the sound cells, as indicated by the darker color of the former. The effect of the fungus is readily discerned by the blackening of the host tissue, this being especially noticeable in green plums. Тһе discolored and poisoned cells are not at first plasmolyzed, and it is to be noted here that discoloration rather than plasmolysis should be taken as the index of the toxic action of this fungus оп its host. It should perhaps be mentioned here, too, that the blackened cells shade off somewhat gradually into the hyaline healthy ones, and that, therefore, there is not always a sharp line of demareation between the diseased and the healthy cells. However, in spite of these difficulties, I was convinced, after having examined a large number of sections of diseased and healthy tissue, that there is no positive evidence that the host cells are discolored, and therefore injured and poisoned, in advance of actual penetration by the fungus. The indirect method employed to determine the same point consisted in applying to sound fruits an extract from decayed plums. Fruits were disinfected with mercuric chloride solution, washed in sterile distilled water, and inoculated with Sclerotinia cinerea. When the plums had become thoroughly decayed the juice was extracted and filtered under sterile conditions through a Chamberlain filter. The juice thus obtained was incubated for one week at a temperature of 22-259 C., and also tested on nutrient agar plates, and found to be sterile by both methods. From sound plums, which had been disinfected in the usual manner, a cone-shaped plug was cut out and the resulting cavity filled with this sterile extract,—the controls being prepared in a similar manner, using sterile water instead of the plum extract. The results were negative, that is, the controls were not unlike those treated with the extract from decayed plums. The same experiment was repeated in a modified form by using thin razor sections of both green and ripe plums, the sections being made under sterile conditions as before, and ob- served in a hanging drop of sterile juice from decayed plums. [Vor. 1 304 ANNALS OF THE MISSOURI BOTANICAL GARDEN By means of this method one could readily observe any changes that might take place in the cells and make accurate compari- sons with controls. Frequent observations were made, and throughout this experiment, which continued for several days, one could not distinguish between the appearance of those sections in a drop of sterile water and those in the sterile extract from decayed plums. 16 is possible and perhaps probable that this fluid, being merely the juice of the fruit, was too dilute to be effective, but the experiment was made because of the possibility of positive evidence. Discussion of Results.—The initial stage in the injury caused by this fungus is shown by discoloration only and not by plas- molysis, and therefore one cannot draw conclusions with ab- solute certainty as to the poisoning effect of the extract on the cells of a cut surface, for the latter turn brown as soon as exposed to the air, just as when infected with the organism. It was comparatively easy, however, to observe that the extract had no effect on the cell walls, for no difference could be observed between the cell walls of the tissue thus treated and those of the control specimens. Even where the sections were left in the extract for several days neither swelling nor disorganization of the cell walls or middle lamelle was noted. When sections of plum tissue were inoculated with one or more spores of the brown-rot fungus no cell-wall disintegration resulting from the growth of the fungus could be observed. A comparative study of sections of tissue, respectively exposed and not exposed to the action of the extract from decayed fruit, showed that no dif- ference could be detected between the two, and that, therefore, no enzyme with a perceptible cytolytic action exists under these conditions. It has been held by some, notably by Behrens (6), that the injury to the host cell is largely physical in that the fungus penetrates at such a prodigious rate that the fluids of the host cell are allowed to escape with loss of turgor to the latter; furthermore, that the osmotic equilibrium is soon destroyed, with plasmolysis and death ensuing. It is very probable that part of the rapid injury to the host can be explained on purely physical grounds, but this may not be the only factor involved, although we do not now know what chemical activity of the fungous cells may be concerned in the rapid killing of the host tissue. | очи И 1914] COOLEY—SCLEROTINIA CINEREA 305 ACTION OF THE FUNGUS ON CELLULOSE A number of investigators have regarded cellulose dissolution as a very important factor in the parasitism of many fungi; indeed, some of the earlier workers seemed to consider this the prime factor involved. While it is a well known fact that there are many fungi, especially saprophytes, which hydrolyze, or dis- solve, certain celluloses, research extending over a wide field has revealed the nature of parasitism to be a very complex one in which other factors are as important as the dissolution of cel- lulose and the cell wall. It has been the writer’s purpose to study from two different points of view the action of the brown-rot organism on celluloses, (1) by observing the action of the fungus on pure cellulose iso- lated from the host tissue, and (2) by studying microscopically its action on the host cell walls themselves. In the former study cellulose agar was used, the cellulose being isolated from plums by the methods discussed below. Methods апа Results.—In the above mentioned study of the action of the fungus on pure cellulose, a variety of reagents, media, and methods for the preparation of cellulose were em- ployed, a brief account of which follows. Schweizer’s reagent was prepared by adding a slight excess (40 grams to the liter) of copper carbonate to dilute ammonium hydroxide solution composed of three parts of water to ten parts of ammonium hydroxide (sp. gr. 0.90). The copper solution was then shaken vigorously, allowed to stand over night, and the supernatant solution siphoned ой. This is the procedure employed by Mc- Beth and Scales (38). Paper cellulose from filter paper was prepared according to the method given by McBeth and Scales (88) by dissolving 15 grams of sheet filter paper in Schweizer’s reagent, diluting about ten times with water, and precipitating the cellulose with a solution of one part of hydrochloric acid to five parts of water. This mixture was then further diluted to 15 or 20 liters, the supernatant liquid siphoned off, and the residue washed re- peatedly with water until the precipitated cellulose was free from both copper and chlorine. After standing quietly for several days the clear liquid was siphoned off and the precipitate used for the preparation of cellulose agar. [Vor. 1 306 ANNALS OF THE MISSOURI BOTANICAL GARDEN Cellulose agar was made by adding about one per cent (esti- mated by the weight of the paper before treating with Schwei- zer's reagent) of precipitated paper cellulose, prepared as stated above, to а mineral nutrient solution, the complete medium having the following composition: Monopotassium phosphate, 1 gram \ Magnesium sulphate, 1 gram Sodium chloride, 1 gram Ammonium sulphate, 1 gram Calcium carbonate, 2 grams Tap water, 1000 ec. The insoluble precipitate appearing in the mineral nutrient solution was filtered off before the cellulose suspension and agar were added. Good results were also obtained by using 0.5 gram of calcium nitrate instead of 2 grams of calcium carbonate, in which case filtering is unnecessary. The mineral nutrient solution having the composition tabulated above will be referred to as nutrient “А.” Another nutrient solution very low in organie matter was also employed in the cellulose agar, but with rather unsatis- factory results. This solution, which will be referred to as nutrient “В,” is that employed by Reed (42), and is made up as follows, the only organic material present being the small amount of sodium citrate: ее 10 grams Dipotassium phosphate....................... 5 grams Magnesium вйірһа4е......2................... 1 gram SO Белі Т ТЕСТ КГ ЯМЕН ЕЕ 1 gram (bn co а ыса ye eighteen с с, 1000 cc. In making the cellulose agar this nutrient solution was used in exactly the same way as nutrient “А.” Since previous investigators have held that the celluloses from various sources differ in their resistance to hydrolyzing enzymes, an attempt was made in this investigation to prepare a cellulose from a natural host—plums—of the parasite. In order to secure a cellulose that is modified as little as possible in the process 1914) COOLEY—SCLEROTINIA CINEREA 307 of isolation three different methods were employed in preparing cellulose from plums, the resulting products being designated, for convenience in reference, respectively as soda cellulose, washed cellulose, and potassium chlorate cellulose. In the preparation of soda cellulose ripe plums were squeezed through cheese cloth and the pulp was washed thoroughly with water. The pulp was then treated with an 8 per cent solution of sodium hydroxide and heated in the autoclave аё ten pounds pressure. After thoroughly washing the pulp with water the heating with alkali was repeated and the product given final washings until free from alkali. The second method of isolating cellulose—washed cellulose— consisted in washing the fruit pulp with water until free from substances soluble in cold water. Water was then added and the mixture heated in the autoclave at 15 pounds pressure, and washed. Тһе operation was repeated as long as any water- soluble substances could be detected. This method, of course, gives an impure cellulose, yet the product is one that is free from water-soluble substances. The third method consisted in oxidizing, dissolving, and wash- ing out the plum pulp until a pure cellulose—potassium chlorate cellulose—was obtained. Pulp, secured from ripe plums in the manner stated above, was washed with cold water until the wash water was free from solutes, and then treated with a cold solution composed of 30 grams of potassium chlorate dis- solved in 520 ce. of cold nitric acid (sp. gr. 1.1). This mixture was kept in the ice box for about three weeks, at the end of which time the pulp was entirely white. This method! is said to yield a product that differs only very slightly from the original cellulose. The product obtained by these various methods was not al- lowed to dry, for it is possible that drying changes the nature of cellulose so that it is more resistant to the action of cytolytic enzymes. А part of the cellulose obtained by each of the pre- ceding methods was treated with Schweizer’s reagent and pre- cipitated with hydrochloric acid and washed as stated above under the preparation of filter-paper cellulose. These three cellulose preparations thus treated with Schweizer’s reagent, as 1 Fowler, G. J. Bacterial and enzymatic chemistry. 159. 1911. [Vor. 1 308 ANNALS OF THE MISSOURI BOTANICAL GARDEN well as the three corresponding untreated portions, were used in the preparation of cellulose agars, according to the method given above. Тһе media were placed іп test tubes of very small (8 mm.) diameter, and sterilized. Тһе tubes of melted agar were then cooled rapidly in cold water in order to bring about the hardening of the agar before the cellulose had had time to settle to the bottom of the tubes. Tubes of the various cellulose agars were inoculated with Sclerotinia cinerea and others with a species of Penicillium, which will be designated as P. expansum', isolated from decay- ing peaches and apples. Since these two fungi, viz., Sclerotinia cinerea and Penicillium expansum, act very differently toward the host, a word contrasting their action may not be out of place here. Аз a result of inoculating apples, peaches, or pears with a pure culture of Sclerotinia the host tissues are promptly killed, while the fruits remain practically as firm after complete decay as before inoculation. On the other hand, the fruits inocu- lated with the Penicillium become very soft and watery, develop- ing a pustule or sunken area where the infection took place. One may assume, therefore, that the Sclerotinia does not materi- ally affect the celluloses and pectic substances that make for the firmness of the fruit, while, on the other hand, Penicillium does affect these substances, causing the fruit to lose its firm consis- tency. Since these two fungi show such entirely different and opposing characteristics as regards their effect on the same host, it is interesting to compare their action in pure cultures on cellulose and pectin-like substances. Such a comparative study was made, the results of which are given in table 1. Discussion of Results.—The results given in table m indicate that both Sclerotinia cinerea and Penicillium expansum exhib- ited in general a very slight hydrolytic action when grown on cellulose isolated from the plum, there being very slight action with both fungi on the soda cellulose and also on the potassium chlorate cellulose and no action on the washed plum cellulose. On the other hand, both fungi very readily dissolve filter-paper ` А culture of this organism was sent to Dr. Chas. Thom, who very kindly examined it and gave as his opinion that it was P. erpansum, or perhaps a strain of that species. The organism in question, when grown on the media employed by Thom, showed characters very similar to those of P. expansum, as given by Thom (48). 1914] COOLEY—SCLEROTINIA CINEREA 309 TABLE II ACTION OF SCLEROTINIA CINEREA AND PENICILLIUM EXPANSUM ON CELLULOSE TES P B iiis MESS Penicillium expansum Eu c nd Ed Growth » de Growth = жеті Soda cellulose А +-+T =F +4 + Soda cellulose В 2 ЕРГЕ E Potassium chlorate cellulose A ++ ++ + Potassium chlorate cellulose B ++ Washed ligno-cellulose* | A + | = Washed ligno-cellulose* | B — - Washed cellulose E A - - Soda cellulose (Schweizer's) | B | а= а Soda cellulose (Schweizer's) | A ++ | + Washed cellulose (Schweizer’s) A - Soda cellulose Pen eh +++ — TT 2H juice Filter paper strips P +++ - ons Е Filter paper strips A ++ - ++ -- Filter paper strips B - ++ - 0.5% Filter paper strips glucose | +++ — +++ - solution Filter-paper cellulose A ++ +++ ++ -++ *Ligno-cellulose is the name here given to cellulose from the vascular tissues of the plum, i. e., that part of the pulp which did not go through the cheese cloth. +Growth and cellulose hydrolysis are indicated by +, the relative intensities of growth and degrees of hydrolysis being indicated by one or more + marks. Absence of growth and absence of hydrolysis are indicated by —. [Vor. 1 310 ANNALS OF THE MISSOURI BOTANICAL GARDEN cellulose, and, strange to say, Sclerotinia is just as active in this respect as Penicillium. Іп many cases the growth was as good on the plum cellulose as on the filter-paper cellulose, yet the hydrolytie action of the fungi was very much weaker on the former medium. No cellulose hydrolysis oceurred where peach juice or some soluble carbohydrate, such as glucose, was added. It seemed probable at first that a very small amount of glucose, or peach juice, or sodium citrate would give the fungus a vigor- ous start and thus accelerate its eyto-hydrolytie activity, but the quantities of these substances employed was sufficient to exert a protective influence, there being a vigorous growth but no apparent cellulose hydrolysis. The fact that these fungi do not dissolve cellulose, derived either from the host or from paper, when other organie nutri- ents are supplied, verifies the writer's observation that Sclerotinia cinerea does not disintegrate the cell walls of the host tissues. Furthermore, the fact that the fungus dissolves paper cellulose very readily when it is the only carbohydrate supplied, leads one to conclude that the action of the fungus on paper cellu- lose in a nutrient solution low in carbohydrates is not neces- sarily a good criterion for judging the behavior of the fungus in the host tissue. In the host tissue there may be a form of cellulose different from that of paper, and it is furthermore very evident that there is present in the fruit an abundance of organie material evidently operating in a protective manner. The fungus fails to produce cytolytic enzymes when grown on plum or paper cellulose to which peach juice or even a very little sugar has been added, but acts vigorously on paper cellulose to which no organic nutrient has been added. It is rather | peculiar that both fungi aet much more readily on paper cellu- lose than on cellulose isolated from the fruits which are natural hosts for these organisms. Sclerotinia cinerea grows very slowly when first transferred to a nutrient medium poor in soluble carbohydrates, very few spores and no aérial mycelium being produced. At the expira- tion of a week or more one may observe that the fungous myce- lium has penetrated the surface layer of the agar, and at the expiration of two to three weeks, in ease the fungus is growing on paper-cellulose agar, a clear translucent ring may be observed 1914] COOLEY—SCLEROTINIA CINEREA 811 іп the agar just below the fungous filaments, thus indicating that the cellulose is being hydrolyzed. With increasing age of the fungus, this clear and almost transparent area gradually enlarges downward, although the fungus shows little or no cor- responding penetration. At the expiration of three weeks or a month, there is а very distinct, clear, and nearly transparent zone in the medium below the region occupied by the fungous mycelium. Since one could see very distinctly how far the fungous filaments had penetrated into the substrate, it was very evident that the cyto-hydrolytic enzyme had diffused beyond the limits of the mycelium. The method employed in this investigation for the demonstra- tion of cellulase was the same as that used by Kellerman in his recent work (31) and was utilized to demonstrate the fact that the cyto-hydrolytic enzyme secreted by this fungus penetrates the substrate considerably beyond the limits of the filaments themselves. Tubes containing cellulose agar, in which the fungus had been growing for four weeks, were disinfected exter- nally by washing with a bichloride of mercury solution, and cut off at à point about 12 mm. below the clear portion of the me- dium. Тһе cotton plug was then flamed and pushed into the tube with a glass rod until the agar was partially shoved out of the eut end of the tube. Тһе clear portion of the agar was then cut into disks about 12 mm. in thickness, which were laid on plates poured with nutrient cellulose agar, great care, of course, being exercised throughout the operation to maintain aseptic conditions. The plates so prepared were then placed in an incubator at 25°C. where they remained for two weeks, at the expiration of which time the cellulose was very distinctly hydrolyzed in a ring about the sterile slices of agar. Micro- scopic examination confirmed the macroscopic observation that these agar disks were free from any infection. As might be expected, the activity of the secretion of the enzyme cellulase is influenced by temperature, a fact which is well illustrated by the following experiment: Tubes containing cellulose agar inoculated with the brown-rot fungus were kept at temperatures of 10-12, 16-20, and 24-26°С. respectively, and at the end of twelve days the following results were noted: In the cultures maintained at 10-12°C. no apparent growth or 4 (Vor. 1 312 ANNALS OF THE MISSOURI BOTANICAL GARDEN hydrolysis had taken place; those kept at 16-20°С. showed a good growth but no visible cellulose hydrolysis; and in those maintained at 24-26?C. there was about the same extent of growth as in the preceding series but accompanied by a very evident cellulose hydrolysis, a distinctly clear zone of dissolved cellulose surrounding the region occupied by the fungous myce- lium. It is therefore evident that even with approximately the same amount of growth cellulose hydrolysis is much more rapid at the higher temperature. An effort was made to determine whether or not it is possible to “train up" more active cyto-hydrolytic strains of the Sclero- tinia and Penicillium in question. On the one hand, these fungi were grown for several successive generations on peach-juice agar—a medium in which the organisms show no cytolytic ac- tivity. On the other hand, these fungi were cultivated for sev- eral successive generations on paper-cellulose agar—a medium which is low in soluble carbohydrates, and one in which the fungi exhibit considerable cytolytic activity. Tubes of paper- cellulose agar were then inoculated with the fungi grown in these two ways and careful observations were made to detect any differences in cyto-hydrolytie activity. No differences developed, however, from which it would appear that the source of cultures of Sclerotinia or of Penicillium does not materially affect the cellulose-dissolving capacity of these organisms, 1. e., each fungus shows the same cellulose-hydrolyzing power wheth- er the organism was cytolytically active during the immedi- ately preceding generations or not. EFFECT OF THE FUNGUS ON PECTIC SUBSTANCES The power of organisms to change pectic substances has been considered an important factor in the disintegration and softening of host tissue by certain plant parasites. Before enter- ing into a discussion of the experimental phases of this subject, it will perhaps be well to give some idea of the present status of this question, as well as a very brief resumé of the extensive literature which has accumulated about it. Fremey (23, 24), in 1840, was the first to report an enzyme act- ing on pectic substances. This enzyme, which he isolated and called pectase, induced the coagulation of pectin, Fremey attrib- ut рр Bui? 1914] COOLEY—SCLEROTINIA CINEREA 313 uting this action of the enzyme to the presence of calcium salts. It is of interest to note that pectase was one of the first plant enzymes to be described. Bertrand and Mallévre (7, 8) con- cluded that pectose and pectase are almost universally present in green plants, being especially abundant in the leaves. These authors showed that acidity is an important factor in the inhi- bition of coagulation of pectie bodies by pectase, and also that either barium, calcium, or strontium is necessary for the action of pectase. Mangin (35, 36), by mieroscopie tests, has thrown much light on the nature of the middle lamella and holds that pectose is very pronounced in the cell walls of young tissue. In the older cell walls, on the other hand, this author believes that calcium pectate predominates in the middle lamella, considering that the latter is largely if not entirely composed of this sub- stance and that it frequently collects on the surface of the cell walls adjoining intercellular spaces. Bourquelot (11), and Bourquelot and Hérissey (12) secured a thermo-labile enzyme from barley malt extract which acted upon a solution of pectin (taken from the gentian root), changing the latter in such a way that it was no longer coagulated by pectase. The action of this enzyme, which they called pectinase, was thought by them to be that of converting the pectin into reducing sugar. They also designated as pectinase an enzyme which dissolves the pectic coagulum (the latter has been supposed to be calcium pectate). A good resumé of the status of the chemistry of pectic substances is given by Bigelow and others (10). A number of investigators have reported upon the action of bacteria on plant cells, including the effects of the organisms on the middle lamella. Winogradsky (55), Behrens (5), and others attributed the changes taking place in the flax plant dur- ing retting to the dissolving action which the bacteria exert on the middle lamella. It will be unnecessary to review here any more of the earlier work which has been done along this line, since it has been so thoroughly discussed in the comprehensive publications by Jones (29), and Jones, Harding, and Morse (30) on the soft rot of vegetables. These authors studied the effect of the soft-rot bacillus (Bacillus carotovorus) on the host and find that the organism is identical with what has been [Vor. 1 314 ANNALS OF THE MISSOURI BOTANICAL GARDEN designated as B. oleracee Harrison, and B. omnivorous Van Hall, and that it may possibly be identical also with Potter's B. destructans. By many tests Jones has shown that this organism secretes an enzyme which causes the disintegration of the host cells by dissolving the middle lamella, which, according to the majority of investigators, is composed of salts of pectic acid. This author has further isolated from pure cultures of the or- ganism an extra-cellular enzyme, which he designated pectinase, that destroys the middle lamella of the cells just as does the erowing organism. Jones, therefore, considers this enzyme responsible for the disintegrating action of the bacillus. In my own work I shall adopt the nomenclature used by Jones (29, 30) and Ешег (21), namely, employing pectinase as the term to designate the enzyme inducing coagulation of a pectin solu- tion and also the hydrolysis of calcium pectate, or pectinate. Methods.—In order to determine the effect of the fungus on the middle lamella I have used two methods, (1) a microscopic study of the effect of the fungus on the host cells, and (2) a study of the effect of the organism on the substances (isolated from the host) which are commonly reported to be constituents of the middle lamella. The first method has been discussed above and may be dismissed here by stating that it yielded no positive evidence that the fungus dissolves the middle lamella. By the second method the problem was studied by isolating pec- tin from the host and studying the effect of the fungus on it and also on its salts, as, for instance, calcium pectinate. Pectin was isolated from plums by the following method: Thoroughly ripe fruits were steamed—no water being added, the juice filtered off and treated with Almen’s reagent! (to precipi- tate the protein) and with a very dilute solution of oxalic acid (to precipitate the calcium). It was found that under these conditions neither а calcium nor a protein precipitate was thrown down either by Almen’s reagent or the oxalic acid, and this pro- cedure, therefore, was deemed unnecessary and was abandoned. The plum juice was carefully filtered through a Buchner filter 1Abderhalden, E. Handbuch d. biochem. Arbeitsmethoden 2: 391-92. 1910. Almen’s tannic acid solution is made by treating 4 grams of tannic acid with 8 cc. of a 25 per cent solution of acetic acid, and making up to 190 сс. with 40 or 50 per cent alcohol. 1914] COOLEY—SCLEROTINIA CINEREA 315 and the filtrate treated with 95 per cent alcohol until a floccu- lent coagulum of pectin was produced. This pectin was sepa- rated by means of a Buchner filter, redissolved in water, reprecip- itated with alcohol, again separated by means of a Buchner funnel, and finally dried at a temperature slightly higher than room temperature,—the reprecipitation being for the purpose of purification. It should be noted here that the plums were sufficiently acid to make the addition of hydrochloric acid to the aleohol unnecessary. | Experiments with pectin and pectinase.—From the pectin isolated by the above method a saturated aqueous solution was prepared—some of the mineral nutrient solution! minus calcium being added, and the resulting solution rendered sterile by frac- tional sterilization. Test-tubes of this pectin solution were inoculated with Sclerotinia cinerea and Penicillium expansum with the result that both organisms produced a rather vigorous growth of mycelium and a few spores. At the expiration of one week the inoculated tubes showed a slight clear area just below the fungous felt due to the coagulation and settling out of the pectin in that part of the solution. The coagulation was at this time somewhat more pronounced in the Penicillium cul- tures than in those of Sclerotinia, yet very noticeable in both cases, beginning directly below the fungous felt and progressing toward the bottom of the tube. After two weeks the greater part of the pectin solution was coagulated, the flocculent coagu- lum, or precipitate, being very different from the precipitate produced in a pectin solution by a calcium salt. It should be emphasized here that every precaution was taken to maintain a calcium-free solution, and when it is considered that the addi- tion of calcium develops a reaction very different from that produced by the enzyme, and, furthermore, that the check gave no coagulation whatever, not even when allowed to stand a month or more, the conclusion would seem to be warranted that calcium is not necessary for the production of a gel by pectinase. Both Sclerotinia and Penicillium, therefore, produced a coag- ulum in an aqueous solution of pectin, while no such results were obtained in the controls, thus justifying the conclusion iNutrient solution employed was the same as mineral nutrient solution A used in preparing cellulose agar, but without the calcium. [Vor. 1 316 ANNALS OF THE MISSOURI BOTANICAL GARDEN that these two fungi are capable of producing pectinase. The cultures were kept at a temperature of 18-20?C. Experiments with calcium pectinate.—Caleium pectinate was prepared by treating a water solution of pectin with freshly- made limewater (care being exercised to avoid an excess of lime), the product thus obtained being filtered off and thoroughly washed until it was no longer alkaline. The calcium pectinate thus prepared was used in making a pectinate agar in a manner similar to that employed in the preparation of cellulose agar, the same mineral nutrient solution (nutrient A) being used and the whole rendered sterile by fractional sterilization. After the last heating, care was taken to distribute the pectinate, which quickly settles to the bottom of the tubes, uniformly throughout the agar by stirring the medium with a sterile glass rod. These tubes were then inoculated with Sclerotinia and with Penicillium, the object being to compare the action toward pectic substances of two fungi that have entirely dif- ferent effects on the host cells, the former producing no soften- ing effects, while the latter causes a very rapid softening and disorganization of the host tissue. The inoculated tubes of pectinate agar prepared by the above method were kept at a temperature of 22-24°C. Contrary to expectations, there was very little growth when no soluble carbohydrate was supplied, and, furthermore, no dissolving action on the calcium pectinate. On the other hand, when 0.5 per cent glucose was added, both fungi produced a vigorous growth, but neither one gave any indication of pectinate hydroly- sis, or dissolution. Here again, as in the cellulose hydrolysis, the two fungi, Sclerotinia and Penicillium, behave alike. This is not in accordance with the observed behavior of these two organisms toward the host tissue. ACID RELATIONS OF THE FUNGUS Some investigators have held that the content of tannin (47) and of malic and other acids of the host determines whether or not the fungus can grow in the tissues and rot the fruit. In accordance with this view a fungus may not so readily attack green as ripe fruit, the former being supposed to exhibit a higher 1914] COOLEY—SCLEROTINIA CINEREA 317 content of these restraining agents. The question of the acid relation of the host tissue is one of fundamental significance and one that is worthy of considerable investigation; it is im- portant to know to what extent acidity may be a limiting factor in parasitism. A case in which a certain acid content is favorable for the fungus is developed by Falck (22). He finds the acidity of the substrate to be a conditioning factor for the growth of several species of Merulius. In this connection the author observes that Coniophora, in particular, acts to pave the way for Merulius in that the former organism renders the nutrient substrate deci- dedly acid, and thereby provides favorable conditions for the germination of the spores and the subsequent growth of myce- lium and fruit bodies of Merulius. In connection with the in- vestigation of the plum disease here discussed it would be well to know if the acidity of the fruit changes during the progress of its growth, and if so in what direction. It is also essential to know whether or not a change in the acidity of the host can account for the fact that ripe fruit is more susceptible to the disease than green fruit. Some experiments were planned, therefore, to determine to what extent the acidity of the host influences the attack of the parasite, and also to investigate what effects, if any, the fungus has with respect to the acid content of the host. In order to determine the changes in acidity which take place during the growth of the fruit (plums), several analyses for acid- ity were made at intervals during the summer. The plums for all of the analyses were taken from the same tree, a known weight of pulp being ground up in a mortar and squeezed through muslin. The acidity was reckoned in the number of ec. of N/10 NaOH required to neutralize one gram of plum pulp. Тһе results were as follows: June 28, 1 gram plum pulp required 0.66 cc. N/10 NaOH for neutralization, Aug. 2, 1 gram plum pulp required 2.12 cc. N/10 NaOH for neutralization, Aug. 19, 1 gram plum pulp required 2.46 cc. N/10 NaOH for neutralization, [Vor. 1 318 ANNALS OF THE MISSOURI BOTANICAL GARDEN the fruit being market ripe on August 19. In these tests my results agree with those obtained by Bigelow and Gore (10) for peaches, and with those of Thompson and Whittier (49) for some other fruits. Тһе last mentioned investigators, how- ever, found that the acidity of peaches decreases toward matu- rity. I have been unable to secure data covering the acidity of plums throughout the season. The above results show that the acid content of plums in- creases rather than diminishes toward the maturity of the fruit. The results of the experiments and field observations show that mature and ripe fruit is much more susceptible than the green and immature fruit. Тһе above facts, showing that as the fruit approaches maturity the acidity increases while the suscepti- bility to the disease also increases, indicate that there is no close relationship between the low acid content of the host and susceptibility to the brown-rot fungus, and that we must look to other factors to explain infection as observed in the field. Ав pointed out, my experiments indicate that penetration is a TABLE III RELATION OF THE GROWTH OF SCLEROTINIA CINEREA TO THE REACTION OF THE MEDIUM Meum | Acidity | Orb after | Growth atter | Spe Cherry juice | +2.3* -1 +t + Cherry juice | +1.5 ++ ++ ++ Cherry juice | +1.0 +++ ++ ++ Cherry juice | +0.15 - + + Cherry juice | —0.15 | 0 ++ 0 Cherry juice | | —0.30 0 ++ 0 *Acidity is given in ec. of N/10 NaOH necessary to neutralize 1 cc. of the juice. {The + sign indicates a fairly good mycelial growth, or spore formation, and the — sign indicates that the growth was just perceptible; 0 indicates no growth, or no spore formation. — 1914] COOLEY—SCLEROTINIA CINEREA 319 very important factor. It is possible that a study of the tannin content' might yield some relation of interest. A preliminary experiment was planned to determine the acidity at which the optimum growth and spore production of the fungus occurs. For this purpose the juice from ripe sour cherries was used. The juice was squeezed out of the cherries (no water being added) and a portion titrated to determine the acidity. Then 50 cc. of this liquid were put into each of a number of Erlenmeyer flasks of 125 cc. capacity; some of the flasks were left untreated, while others received various quanti- ties of N/10 NaOH to bring each to the desired acidity or alka- linity. Тһе flasks were then sterilized and inoculated. Тһе results are given in table rrr. It is clear, therefore, that although the fungus eventually grows on а medium as acid as the natural juice of sour cherries, it grows more luxuriantly on a somewhat less acid medium. 1% is à rather significant fact that on the media near the neutral line the fungus at first shows no perceptible growth, but at the expiration of two weeks has produced nearly as much mycelial growth as on the acid medium. It is also of interest to note that we find spore formation abundant on the very acid media but entirely lacking on the alkaline media. This experiment indicates that the fungus can adjust itself to a slight degree of alkalinity. OXALIC ACID PRODUCTION BY THE FUNGUS The first important reference to охаПе acid production by fungi is in the publication by de Bary reviewed in a preceding section. He reports that the older hyphe of the fungus were encrusted with crystals of oxalic acid, and he attributed some of the poisonous action of the parasite to the production of this substance; in fact, he mentions oxalie acid fermentation. Since the appearance of de Bary's paper a limited number of investi- ! Cook and Bassett and their associates (17) believe that there are enzymes in the host plant which may act upon cell constituents and play the réle of alexins. They are of the opinion that tannin, as such, is not abundant in fruits, but that it may be formed by the action of oxidizing enzymes upon certain phenols. Injuries produced by parasitic fungi may accelerate the activity of the host in the production of tannin, the latter perhaps being toxic to the growth of parasitic fungi. [Vor. 1 320 ANNALS OF THE MISSOURI BOTANICAL GARDEN gators have reported the presence of oxalic acid resulting from the growth of both fungi and bacteria, but unfortunately much of this work is of little value, because methods of analysis are not given. The detection of this acid by some methods is very unsatisfactory. A few years after de Bary's work, Wehmer (54) published an extensive series of articles on this subject. He studied a number of fungi (mostly saprophytic) with reference to oxalic acid excre- tion, and of these he found Aspergillus to be the most active and Penicillium next, and, therefore, he confined his studies to these two fungi. Some of the factors concerned in the production of oxalie acid or its salts, aecording to Wehmer, may be summed up here: (1) A large yield of oxalic acid is not produced in the presence of free organic or inorganic acids, not being found in the medium when free acids exceeded 0.2-0.3 per cent, while, on the other hand, it can be formed in the presence of as much as 2-3 per cent of the salts of these acids. (2) The sources of nitrogen are very important, for the amount of the oxalie acid produced varies aecording to the kind and quantity of nitrog- enous compounds supplied. (3) Abundant oxalie acid forma- tion is favored by the addition of some basie phosphate, or at least some compound with which the acid can combine to form a soluble salt. (4) The effect of light or darkness on oxalie acid formation is inappreciable. (5) Temperature is an influ- encing factor in oxalate production, for the latter is inhibited by a high temperature, the temperature for a maximum oxalate production being, in fact, very near the minimum for the growth of the organism. Wehmer's analytical method consisted in precipitating out the oxalic acid, or its soluble oxalate, as the calcium salt, which was filtered off, dried to a constant weight, and weighed. Al- though this method is perhaps as well suited for this purpose as any other reported, it is open to criticism. А detailed dis- cussion, however, will not be given here. Wehmer holds that oxalic acid is a type of excretion, and that it is in some way connected with respiration, that is, with CO; elimination. Не considers that the variability in the amount of oxalie acid produced is due to its use in the metabolism of the fungus. Emmerling (20), in his contribution to this subject, 1914] COOLEY—SCLEROTINIA CINEREA 321 emphasizes the influence of such nitrogenous substances as proteins, amino acids, and amides in the nutrient. Не finds that Aspergillus niger when grown in non-amino acids, for example, tartarie, lactie, etc., produces no oxalie acid, whereas an abundant oxalic acid production results on such substances as peptone or aspartic acid. Smith (46) and Peltier (41) both conducted experiments to determine whether or not oxalic acid is present in media in which Botrytis has been growing. Peltier reported negative results, but Smith found oxalic acid and thinks that the pois- oning effect of the fungus is perhaps due to the presence of this acid. Unfortunately, neither of these authors gives his methods of analysis, and, with the exception of one incident in Smith’s publication, the quantity of oxalic acid found is not reported. Peltier and others have been able to produce an injury with oxalic acid similar to that produced by certain parasitic fungi, such as Botrytis, yet this is not conclusive evidence that oxalic acid is the toxic substance secreted by the organism. The articles mentioned above constitute the chief publica- tions that have to deal with the production of oxalic acid by fungi. The publications on the production of oxalic acid by bac- teria and other plants will not be reviewed here. Whether ox- alic acid production is a phenomenon peculiar to certain genera or to certain species of the fungi, whether it is purely the result of external conditions, or whether it results primarily from cer- tain constituents of the medium, has not been clearly demon- strated. A series of experiments was planned in the hope of throwing some light on its production in the fungus here studied. The method of analysis employed was a modification of Weh- mer’s method of precipitating the oxalate with calcium chloride and determining the amount of oxalate thus precipitated. This method, however, is not well adapted to the purpose at hand, especially when quantitative methods are used, and fruit juice is employed for the medium on which to grow the fungus. An attempt is being made to develop a method that will be better suited to our purpose. Culture media were prepared from peaches and plums by filtering the juices of these fruits through a Hill pressure filter under sterile conditions. The product thus obtained was . [Vor. 1 322 ANNALS OF THE MISSOURI BOTANICAL GARDEN placed іп flasks and incubated for a week and found to be sterile, after which the flasks were inoculated with Sclerotinia cinerea. At the expiration of thirty-seven days these cultures were ana- lyzed and were found to contain the following amounts of oxalic acid per 50 сс. of the respective juices: Plum Шісе.................. 0.0019 grams of oxalie acid, Peach јиісе. ................ 0.0077 grams of oxalic acid, Peach juice. ................. 0.0094 grams of oxalic acid, emo No trace of oxalic acid. Plum and peach juices that had been sterilized by heat, thereby precipitating some of the contained proteinaceous material, were also used as culture media, and here, too, every culture containing the fungus gave a positive test for oxalic acid. For investigating the production of охаПс acid by the fungus in the unaltered fruit, lots of 500 grams each of peaches were disinfected with bichloride of mercury solution, inoculated respectively with Sclerotinia, Penicillium, and Aspergillus niger, and kept under sterile conditions until the fruits were decayed, or, in the case of the Penicillium and Aspergillus, until partially decayed. Тһе decayed fruits were then digested with hy- drochlorie acid and analyzed for their oxalic acid content with the following results: Peach inoculated with Penicillium ..No trace of oxalic acid, Peach inoculated with Aspergillus. ..No trace of oxalic acid, Peach inoculated with Sclerotinia сіпегеа.................. 0.0087 grams of oxalic acid, Peach сопіто!.................... No trace of oxalic acid. The results of these experiments with oxalic acid show that Sclerotinia cinerea when grown either on fruit juices or on peaches produces more or less oxalic acid as a result of its meta- bolism. It is also significant that the other two fungi employed, namely, Aspergillus and Penicilliwm, which are not natural parasites on the plum or the peach, produced no oxalic acid under the conditions in which the experiments were carried out. SUMMARY 1. The brown-rot organism will infect fruits which are im- mature, even penetrating those which are not more than half- grown or those in which the pits are still soft, provided the 1914] COOLEY—SCLEROTINIA CINEREA 323 skin is punctured. Infection of green fruits is also effected when a portion of the mycelial felt of the fungus is laid on the surface of the plum. On the other hand, ripe or nearly mature fruits may be readily inoculated by sowing a spore suspension on the unpunctured surface. 2. The fungus does not show any particular affinity for the middle lamella, but penetrates and permeates with equal avidity any part of the host tissue. 3. A study of the effect of the organism on the host gives no positive evidence that a toxic substance is abundantly secreted in advance of penetration. 4. The fungus shows very slight cytolytic action with respect to cellulose isolated from the plum, while, on the other hand, the organism readily hydrolyzes cellulose from filter paper when this is the only carbohydrate supplied. No general cytolytic action of the organism on the cell wall of the host is perceptible. 5. An aqueous solution of pectin isolated from plums was со- agulated by Sclerotinia, thus indicating the secretion of the enzyme pectinase. In respect to its action on pectic substances, Sclerotinia cinerea behaves in a manner similar to that of Pen- icillium expansum, yet these two organisms produce very dif- ferent effects on the host, the former producing a firm rot and the latter a soft one. Neither organism will dissolve calcium pectinate. 6. The experiments on the acid relations of the fungus indi- cate that the changing acidity of the host as the fruit reaches maturity does not explain the fact that ripe fruit is more sus- ceptible to the disease than green fruit. 7. The brown-rot fungus produces oxalic acid when grown either on a fruit juice medium or on peaches. The writer takes pleasure in acknowledging his indebtedness to Professor B. M. Duggar for his advice and helpful criticism in this investigation. Part of this work was done during the summer of 1913 in the Laboratory of Plant Pathology of the University of Wisconsin, and the writer wishes to express his gratitude to Professor L. R. Jones for the courtesy extended to him while at Madison. Graduate Laboratory, Missouri Botanical Garden. [Vor. 1 324 ANNALS OF THE MISSOURI BOTANICAL GARDEN BIBLIOGRAPHY . de Bary, А. Recherches sur le développement de quelques champignons para- sites. Ann. d. Sci. Nat. Bot. IV. 20: 5-148. 1863. --------, Neue Untersuchung ueber Uredineen II. Monatsber. d. Akad. d. Wiss. z. Berlin. 1886. --------, Morphologie und Physiologie der Pilze, Flechten, und Myxomyceten. Handbuch der physiologischen Botanik 2: 1-316. 1866. [cf. pp. 212-19.] . ————, Ueber einige Sclerotinien und Sclerotienkrankheiten. Bot. Zeit. 44: 377-87, 393-404, 409-26, 433-41, 449-61, 465-74. 1886. . Behrens, J. Untersuchungen über die Gewinnung der Hanffaser durch natürliche Róstmethoden. Centralbl. f. Bakt. II. 8: 114-20, 131-37, 161-66, 202-10, 231-36, 264-68, 295-99. 1902. --------, Beiträge zur Kenntnis der Obstfáulnis. Centralbl. f. Bakt. II. 4: 514-22, 547-53, 577-85, 635-44, 700-706, 739-46, 770—77. 1898. . Bertrand, G., et Mallévre, A. Recherches sur la pectase et sur la fermentation pectique. I. Jour. de Bot. 8: 390-96. 1894. ; , Sur la diffusion de la pectase dans le régne végétal et sur la préparation de cette diastase. Jour. de Bot. 10: 37-41. 1896. . Biffen, R. H. On the biology of Bulgaria polymorpha, Wett. Ann. Bot. 15: 119-34. pl. 7. 1901. . Bigelow, W. D., Gore, H. C., and Howard, B. J. Studies on apples. U. 8. Dept. Agr., Bur. Chem. Bul. 94: 1-99. pl. 1—5. 1905. . Bourquelot, Em. Sur la physiologie du gentianose; son dédoublement par les ferments solubles. Compt. rend. acad. Paris 126: 1045-47. 1898. .—— ——, et Hérissey, Н. Sur l'existence dans l'orge germée d'un ferment soluble agissant sur la pectine. Compt. rend. acad. Paris 127: 191-94. 1898. , › Les ferments solubles du Polyporus sulfureus (Bull). Bull. Soc. Мус. Fr. 11: 235-39. 1895. . Buller, A. H. R. The enzymes of Polyporus squamosus, Huds. Ann. Bot. 20: 49-59. 1906. .————, The destruction of wood paving blocks by Lentinus lepideus. Fr. Jour. Econ. Biol. 1: 1-10. pl. 1—11. 1905. . Büsgen, M. Ueber einige Eigenschaften der Keimlinge parasitischer Pilze. Bot. Zeit. 511: 53-72. pl. 2-3. 1893. . Cook, M. T., Bassett, H. T., Thompson, F., and Taubenhaus, J. J. Protective enzymes. Science N. S. 33: 624-29. 1911. . Czapek, F. Zur Biologie der holzbewohnenden Pilze. Ber. d. deut. bot. Ges. 17: 166-70. 1899. . Dox, A. W. Тһе intracellular enzyms of Penicillium and Aspergillus. Т. 8. Dept. Agr., Bur. РІ. Ind. Bul. 120: 1-70. 1910. . Emmerling, O. Oxalsiurebildung durch Schimmelpilze. Centralbl. f. Bakt II. 1o: 273-75. 1903. . Euler, H. General chemistry of enzymes. 1-319. 1912. . Falck, В. Hausschwamforschungen. Zeitschr. f. Forst. u. Jagdwesen. Heft 6. p. 1-405. pl. 1-17. 1912. (а. pp. 273-80.] . Fremey, E. Premiers essais sur la maturation des fruits. Recherches sur la pectine et l'acide pectique. Jour. de Pharmacie 26: 368-93. 1840. [Original not consulted.] 1914] 24. 25. 26. 27. 28. 29. 30. 81. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. COOLEY—SCLEROTINIA CINEREA 325 Fremey, E. Memoire sur la maturation des fruits. Ann. Chim. et Phys. ПІ. 24: 1-58. 1848. [Original not consulted.] Fulton, H. R. Chemotropism of fungi. Bot. Gaz. 41: 81-108. 1906. Hartig, R. Die Zersetzungserscheinungen des Holzes der Nadelholzbiume und der Eiche. Berlin. 1-151. pl. 1-21. 1878. Humphrey, J. Е. On Monilia fructigena. Bot. Gaz. 18: 85-93. pl. 7. 1893. Van Iterson, C., Jr. Die Zersetzung von Cellulose durch aérobe Mikroorgan- ismen. Centralbl. f. Bakt. II. 11: 689-98. pl. 1. 1904. Jones, І, В. Тһе cytolytic enzyme produced by Bacillus carotovorus and cer- tain other soft rot bacteria. Centralbl. f. Bakt. II. 14: 257-72. 1905. — —— —, Harding, Н. A., and Morse, W. J. The bacterial soft rots of certain vegetables. I. N. Y. (Geneva) Agr. Exp. Sta. Tech. Bul. 11: 251-968. 1909. (сі. pp. 291-368.] Ibid, Vermont Agr. Exp. Sta. Bul. 147: 243-360. 1910. [ef. pp. 283-360.) Kellerman, K. F. Тһе excretion of cytase by Penicillium Pinophilum. U. 8. Dept. Agr., Bur. Pl. Ind. Cire. 118: 29-31. 1913. ——— —, and McBeth, I. б. The fermentation of cellulose. Centralbl. f. Вас. II. 34: 485-94. pl. 1-2. 1912. Kohnstamm, P. Amylolytische, glycosidspaltende, proteolytische und cellulose losende Fermente in holzbewohnenden Pilzen. Beih. 2. bot. Centralbl. 1o: 90-121. 1901. Kühn, J. Die Krankheiten der Kulturgewiichse, ihre Ursachen und ihre Ver- hütung. Berlin. 1-312. pl. 1-7. 1858. Mangin, L. Propriétés et réactions des composés pectiques. Jour. de Bot. 6: 206-12, 235-44, 363-68. 1892. --------, Recherches sur les composés pectiques. Jour. de Bot. 7: 37-47. pl. 1., 121-31. pl. 2., 325-43. 1893. Matheny, W. A. А comparison of the American brown-rot fungus with Sclero- tinia fructigena and S. cinerea of Europe. Bot. Gaz. 56: 418-32. f. 1-6. 1913. McBeth, I. G., and Scales, F. M. Тһе destruction of cellulose by bacteria and filamentous fungi. U. S. Dept. Agr., Bur. Pl. Ind. Bul. 266: 1-52. pl. 1-4. 1913. Miyoshi, M. Die Durchbohrung von Membranen durch Pilzfáden. Jahrb. f. wiss. Bot. 28: 269-89. 1895. Nordhausen, M. Beitrüge zur Biologie parasitiirer Pilze. Jahrb. f. wiss. Bot. 33: 1-46. 1899. Peltier, G. L. А consideration of the physiology and life history of a parasitic Botrytis on pepper and lettuce. Rept. Mo. Bot. Gard. 23: 41-74. pl. 1-6. 1912. Reed, H. S. Тһе enzyme activities involved in certain fruit diseases. Ann. Rept. Va. Agr. Exp. Sta. 1011-1012: 51-77. 1912. Schellenberg, H. C. Untersuchungen iiber das Verhalten einiger Pilze gegen Hemizellulosen. Flora 98: 257-308. 1908. Schmidt, E. W. Uber den Parasitismus der Pilze. Zeitschr. f. Pflanzenkrankh. 19: 129-43. 1909. Smith, E. F. Peach rot and peach blight. (Monilia fructigena Pers.) Jour. Myc. 5: 123-34. 1899. Smith, R. E. The parasitism of Botrytis cinerea. Bot. Gaz. 33: 421-36. 1902. von Schrenk, H. A disease of the black locust (Robinia pseudacacia L.). Rept. Mo. Bot. Gard. 12: 21-31. pl. 1-3. 1901. [Vor. 1, 1914) 326 ANNALS OF THE MISSOURI BOTANICAL GARDEN 48. Thom, C. Cultural studies of species of Penicillium. U. S. Dept. Agr., Bur. Animal Ind. Bul. 118: 1-109. /. 1-36. 1910. 49. Thompson, F., and Whittier, A.C. Fruit juices. Del. Agr. Exp. Sta. Bul. 102: 1-28. 1913. 50. Ward, H. M. On the biology of Stereum hirsutum. Phil. Trans. Roy. Soe. Lond. 189: 123-34. 1897. 51. —————, A lily-disease. Ann. Bot. 2: 319-82. pl. 20-24. 1888. 52. —— — —, Penicillium аз a wood-destroying fungus. Ann. Bot. 12: 565-86. 1898. 53. Wehmer, C. Monilia fructigena Pers. ( —Selerotinia fructigena m) und die Monilia-Krankheit der Obstbüume. Ber. d. deut. bot. Ges. 16: 208-307. pl. 18. 1898. 94. ————, Entstehung und physiologische Bedeutung der Oxalsüure im Stoff- wechsel einiger Pilze. Bot. Zeit. 49: 233-46, 249-57, 271-80, 289-98, 305-13, 321-32, 337-46, 353-63, 369-74, 385-90, 401-7, 417-28, 433-39, 449-56, 465- 78, 511-18, 531-39, 547-54, 563-69, 579-84, 596-602, 611-20, 630-28. 1891. 55. Winogradsky, S. Sur le rouissage du lin et son agent microbien. Comot. rend. acad. Paris 121: 742-45. 1895. 56. Woronin, M. Uber Sclerotinia cinerea und Sclerotinia fructigena. Mem. de l'Acad. Imp. d. Sei. de St. Petersbourg, Classe Phys. Math. VIII. то: 1-38. pl. 1-6. 1899. 57. Zschokke, A. Ueber den Bau der Haut und die Ursachen der verscl windenen Haltbarkeit unserer Kernobstfrüchte. Landw. Jahrb. d. Schweiz 11: 153-97. pl. 1-2. 1897. THE ТНЕ,ЕРНОВАСЕЖ OF NORTH AMERICA. II! CRATERELLUS EDWARD ANGUS BURT M ycologist and Librarian to the Missouri Botanical Garden Associate Professor in the Henry Shaw School of Botany of Washington University CRATERELLUS Craterellus Pers. Мус. Eur. 2:4. 1825.—Fries, Epicr. 531. 1838; Hym. Eur. 630. 1874.—Saccardo, Syll. Fung. 6: 514. 1888.—Hennings, in Engl. & Prantl, Nat. Pflanzenfam. (1. 1**): 127. 1898. The type species of the genus is Craterellus cornucopioides L. ex Pers. Fructifications fleshy or membranaceous, pileate, often tubi- form, infundibuliform, or flabelliform, sometimes clavate; hymenium waxy-membranous, distinct, continuous, adnate to the hymenophore, even or rugose; basidia simple; spores usually white. Craterellus is closely related by its fleshy C. Cantharellus, C. odoratus, C. lutescens, etc., with the genus Cantharellus. These species resemble so closely in coloration and habit species of the latter genus that careful examination of the hymenium should be made for generic determination. Craterellus has its hymenium even or slightly rugose. In exceptional con- necting species, such as C. clavatus, it is somewhat lamelliform for a part of the distance from margin of the pileus to the stem. The clavate C. pistillaris and C. unicolor connect Craterellus closely with Clavaria. Craterellus cornucopioides, C. ochrosporus, C. clavatus, С. Cantharellus, and С. odoratus are edible species, which are often abundant locally. 1 Issued September 30, 1914. Norr.—Explanation in regard to the citation of specimens studied is given in Part I, Ann. Mo. Bot. Gard. 1: 202, footnote. Тһе technical color terms used in this work are those of Ridgway, Color Standards and Nomenclature. Washington, D. С., 1912. ANN. Мо. Bor. GARD., Vou. 1, 1914 (327) 5 [Vor. 1 328 ANNALS OF THE MISSOURI BOTANICAL GARDEN KEY TO THE SPECIES Hymenium somewhat radiately lamelliform—at least near the margin; stem solid... III 1 Hymenium plane, rugose-wrinkled, or ribbed and rugose-wrinkled......... 2 1. Fructification large, 4-10 em. high; stem about 1 ст. thick; spores 10-13 x 4-44и..................................................... 1. C. clavatus 1. Fructification small, 1-1} cm. high; stem 1 mm. thick; pileus umbilicate; spores 9х7рң........................................... 11. C. delitescens 2. Fructification with pileus infundibuliform and pallid rose; hymenium and stem white. In М. Carolina in moss near Kalmia bushes. . . .4. C. roseus 2. Fructification entirely egg-yellow, about 3-9 cm. high, 22-9 em. broad... 3 2. Fructification neither entirely egg-yellow nor with pileus pallid rose and hymenium and stem white......... lesse 4 3. Pileus convex, then depressed ог infundibuliform; stem solid. . .2. C. Cantharellus 3. Pileus convex, then depressed or cyathiform; stem hollow or cavernous; fructification sometimes Вгалеһей............................ 8. C. odoratus 4. Pileus tubiform with cavity extending nearly or quite to the base of BENED MM MET CAMP UC qp I 5 4. Pileus not tubiform, but instead infundibuliform, depressed, truncate, convex, or НаһеШіога.......................................... 6 5. Pileus and stem smoky brown to blackish; hymenium cinereous drab; spores 1210 acs | er ЕСЕТ Е 5. С. cornucopioides 5. Pileus drying avellaneous to snuff-brown; stem black with chamois-colored pubescence at its base; hymenium chamois-colored or colored like the pileus; spores 12-15 x 7-8 м.................................... 6. C. ochrosporus 5. Pileus somewhat tubiform; hymenium dark cinereous; spores 6-73 x 42-5 и 7. C. dubius 5. Pileus somewhat tubiform or umbilicate, yellowish brown to fuscous; hyme- nium and stem yellow; spores 10-12 x 6-8 и.................... 8. С. lutescens 6. Pileus infundibuliform, 2-3 cm. broad; hymenium pallid cinereous; spores 10-12 х6-7и.................................... 9. C. sinuosus 6. Pileus deeply cup-shaped, 4-8 mm. broad; hymenium cream-buff; spere Sx БИТ 10. C. calyculus 6. Pileus convex, then umbilicate, 5 mm. broad; hymenium sometimes obscurely lamelliform, chamois-colored; stem chamois-colored; spores БЕТЕГЕ eer 11. C. delitescens 6. Pileus merely depressed, truncate, convex, or сЇіауаќе................. 7 Е с ES occ ТТГ ТТТ Soh ig fee ae 8 7. Fructification small, 1-3 cm. high, 4-9 mm. broad, narrowly obconic, white; spores 3-4 и in йізтебег............................... 12. С. taxophilus 7. Fructification 2-5 cm. high, from obconic often becoming abruptly enlarged and somewhat cerebriform at the upper end but with the stem remaining comparatively нелдег..................................... 13. C. unicolor 7. Fructification large, 6-15 cm. high, clavate or оһсопіс and truncate, tapering downward; stem often bulbous at the base. Fructification dries sorghum- brown to Таясойв....................................... 14. C. pistillaris 8. Pileus ligulate at first, then spreading laterally and becoming somewhat palmately cleft into & few branches, fawn-color shading into bone- brown. Known from Ohio.............. esses 15. C. palmatus: 1914] BURT—THELEPHORACEJE OF NORTH AMERICA. II 329 8. Pileus somewhat triangular, drying a dirty pinkish buff ; hymenium drying Isabella-color to clay-color. Known only from Florida. .16. C. dilatus 8. Fructification entirely white; pileus reniform, dimidiate, attached laterally to a slender erect stem. Known only from Washington 17. C. Humphreyi I. Craterellus clavatus Pers. ex Fries, Еріст. 533. 1836- 1838. | Plate 15. fig. 6. Merulius clavatus Pers. Obs. Мус. 1:21. 1896.—Cantharellus clavatus Fries, Syst. Myc. 1: 322. 1821.—Nevrophyllum clava- tum Fries ex Patouillard, Tab. Anal. Fung. 1: 193. f. 434. 1883- 1886.—Cantharellus brevipes Peck, Rep. N. Y. State Mus. 33: 21. pl. 1. f. 18-20. 1879. Illustrations: Scheffer, Icon. Fung. pl. 164, 276.—Kromb- holz, Abbild. und Beschr. pl. 45. f. 18-17.-Егіев, Sverig. Atl. Svamp. pl. 91.—Richon et Roze, Atlas Champ. pl. 50. f. 10-14.— Bresadola, Funghi Manger. pl. 82.—Peck, Вер. N. Y. State Mus. 33: pl. 1. f. 18-20.—Harper, Mycologia 5: pl. 93, 94. Fructifieations solitary or cespitose, fleshy, flesh whitish; pileus narrowly obconic, turbinate, truncate or depressed, gla- brous, ochraceous buff, attenuated into the stem, the margin thin and erect; stem short, solid, tomentose at the base; hyme- nium lamelliform near the margin, rugose-wrinkled elsewhere, becoming pruinose with the spores, light vinaceous drab, drying drab; spores pale ochraceous in the mass, 10-13 x 4-41 ,.. Fructifieations 4-10 em. high; pileus 3-8 em. broad; stem 1-2 сіп. long, 8-15 mm. thick. On the ground in coniferous woods. Maine to Connecticut and west to Minnesota, and in Montana. July to September. This species is intermediate between Craterellus and Cantharel- lus. Тһе marginal portion of the hymenium is like that of a Cantharellus, and the remainder of the hymenium, like that of a Craterellus. There is good authority for including this species in Cantharellus and there is the authority of Fries and herbarium usage for classing it in Craterellus. С. clavatus is edible but too rare, at least in the east, to be common in herbaria. Specimens examined: Exsiecati: De Thuemen, Myc. Univ., 1807. Austria: G. Bresadola. Maine: Sprague (in Curtis Herb., 5786). [Vor. 1 330 ANNALS OF THE MISSOURI BOTANICAL GARDEN New Hampshire: Shelburne, W. G. Farlow (in Mo. Bot. Gard. Herb., 4868). Vermont: Lake Dunmore, Е. А. Burt. Connecticut: Rainbow, C. C. Hanmer, 1454 (in Hanmer Herb.). New York: Ballston, C. H. Peck, the type of Cantharellus brevipes (in Coll. N. Y. State). 2. C. Cantharellus Schw. ex Fries, Еріст. 534. 1836-1838. Plate 15. fig. 7. Thelephora Cantharella Schw. Schrift. d. Naturforsch. Gesell., Leipzig, 1: 105. 1822.—Craterellus lateritius Berk. Grevillea 1: 147. 1873. Illustrations: Peck, Rep. N. Y. State Mus. 49: pl. 44. f. 1-5; Mem. N. Y. State Mus. 3*: pl. 26. f. 17-21.--Нага, Mushrooms f. 378.—Marshall, Mushroom Book 73. f. Type: in Herb. Schweinitz. Fruetifieations single or cespitose, fleshy, firm, egg-yellow; pileus convex, becoming depressed or infundibuliform, glabrous, yellow, the margin often lobed or irregular; stem solid, cylindrie or tapering downward, glabrous, yellow; hymenium nearly even or rugose wrinkled, yellow, or with a reddish salmon tinge and drying ochre-red; spores 7-10 x 33-5} и. Fructifications 4-9 em. high; pileus 23-8 cm. broad; stem 21-5 em. long, 5-10 mm. thick. On the ground in open woods. Massachusetts to Alabama and westward to Ohio; also in Mexico. June to September. Abundant locally. This species is so similar to Cantharellus cibarius in habit, coloration, size and form—differing from the latter only in the more even hymenium, that figures of C. cibarius will serve very well for Craterellus Cantharellus, if allowance is made for the different hymenium. Тһе firm and solid stem of С. Cantharellus distinguishes this species from C. odoratus easily. The latter species sometimes has its pileus greatly branched. My illus- tration of this species is photographed from the dried herbarium specimen of the cotype of C. lateritius Berk. In this specimen the lobes of the pileus were pressed together above before drying. The hymenium of this specimen is now ochre-red and agrees in color with that of the authentic specimen of C. Cantharellus in Curtis Herb.; both these specimens have been poisoned. I 1914] BURT—THELEPHORACEJE OF NORTH AMERICA. II 331 found the spores of the type in Herb. Schw. 8-9 х 35-4 и, or a little slenderer than in northern specimens. Hard states that the spores are yellowish or salmon colored in the mass when collected. This species is edible. Specimens examined: Exsiceati: Ell. & Ev., N. Am. Fungi, 1921. Massachusetts: Sprague (in Curtis Herb.) ; Milton, H. Webster. Connecticut: East Hartford, C. C. Hanmer, 2391, 2468 (both in Hanmer Herb.). Pennsylvania: West Chester, B. M. Everhart, Ell. & Ev., N. Am. Fungi, 1921. West Virginia: Eglon, C. G. Lloyd, 02292. North Carolina: „Schweinitz, type (in Herb. Schweinitz); Blowing Rock, G. F. Atkinson, 4318. South Carolina: Clemson College, P. H. Rolfs, 1830. Alabama: Peters (in Curtis Herb., 4539, and in Kew Herb.), the cotype and type respectively of C. lateritius; Auburn, Ғ. S. Earle (in Mo. Bot. Gard. Herb., 4928). Ohio: A. P. Morgan (in Lloyd Herb.). Kentucky: С. G. Lloyd (in Lloyd Herb.). Mexico (?): Botteri, 27 (in Curtis Herb.). If the stem is hollow this specimen is C. odoratus. 3. C. odoratus Schw. ex Fries, Epicr. 532. 1836-1838. Plates 15, 16. figs. 8-10. Merulius odoratus Schw. Schrift. d. Naturforsch. Gesell., Leipzig, 1: 91. 1822.—Cantharellus odoratus Fries, Elenchus Fung. т: 51. 1828.—C. confluens Berk. & Curtis, Jour. Linn. бос. Bot. 9: 423. 1867. Type: in Herb. Schweinitz. Fructifications gregarious, sometimes cespitose, simple or branched, egg-yellow; pileus thin, convex, then depressed and somewhat cyathiform, sometimes pervious, yellow, the margin deflexed, often lobed or irregular; stem cylindric or somewhat tapering towards the base, concolorous with the pileus, hollow or cavernous; hymenium even or somewhat rugose-wrinkled, ochraceous orange or with a reddish tinge approaching San- ford’s brown; spores even, 7-9 x 4-5 и. Fructifications 3-7 ст. high; pileus 2-9 em. broad; stem 2-4 em. long, 3-8 mm. thick. [Vor. 1 332 ANNALS OF THE MISSOURI BOTANICAL GARDEN In moist places in woods. North Carolina and Georgia to Ohio and Missouri. June to October. Specimens of this species have sometimes been confused in recent years with the better known C. Cantharellus, which ranges farther north. The color and general habit of these species is the same; both have the egg-yellow color and the characteristic fragrance of Cantharellus cibarius when moistened after drying, and all three are edible. Craterellus odoratus is more membra- naceous than C. Cantharellus and it differs from both this species and Cantharellus cibarius in having a hollow or cavernous stem whose pliant walls may be pinched together, like those of a rubber tube, before the specimens are dried. Highly branched forms may occur as shown in pl. 16 fig. 10a; this character was unduly emphasized in the original description. The ample collections in the Glatfelter Herbarium seem to show that Cra- terellus odoratus is the most frequent Craterellus in the vicinity of St. Louis. Dr. Glatfelter notes on his collection that he has eaten this species and found it quite good. In pl. 15 fig. 8, I give a figure, natural size, from a photograph of the dried her- barium cotype of C. confluens B. & C., to show how close the resemblance is to the specimens of C. odoratus, collected at St. Louis and figured in the following plate. Тһе type of C. con- Лиепв has the hymenium rugose-wrinkled, as is often the case in specimens of C. odoratus; its habit, dimensions, structure, coloration, and spores are quite those of C. odoratus. Specimens examined: North Carolina: Salem, Schweinitz, type (in Herb. Schweinitz). South Carolina: Society Hill, Ravenel, 192 (in Curtis Herb.). Georgia: Station cited by Schweinitz. Alabama: Auburn, L. M. Underwood. Ohio: Oxford, L. O. Overholts, 1721 (in Overholts Herb.). Missouri: near St. Louis, N. M. Glatfelter, 348 (in Mo. Bot. Gard. Herb., 42590), and J. B. S. Norton (in Mo. Bot. Gard. Herb., 4926). Mexico: near Orizaba, Botteri, 6 (type and cotype in Kew Herb. and Curtis Herb., respectively, of C. confluens). 4. C. roseus Schw. ex Fries, Epier. 533. 1836-1838. Merulius roseus Schw. Schrift. d. Naturforsch. Gesell., Leip- 1914] BURT—THELEPHORACEJE OF NORTH AMERICA. II 333 zig, 1: 91. 1822.—Cantharellus roseus Fries, Elenchus Fung. 53. 1828. Fructifications solitary, somewhat fleshy; pileus infundibuli- form, somewhat strigose, pallid rose, the margin lobed and inflexed; stem apparently stuffed, attenuated downward, white; hymenium somewhat rugose, white. In mosses, especially in proximity to Kalmia. North Carolina. Specimens of this species have the habit of Cantharellus cibarius but are thinner. Fries received a specimen of Craterel- lus roseus from Schweinitz and expressed the opinion in 'Elen- chus’ that the species is good. І have seen no specimens of C. roseus and base the above on the original description and the comments by Schweinitz and Fries. 5. C. cornucopioides L. ex Pers. Myc. Eur. 2: 5. 1825. Plate 17. fig. 17. Peziza cornucopioides L. Sp. Pl. 1181. 1753. [156 ed.]— Elvella cornucopioides Scop. Fl. Carn. 2: 476. 1760.—M erulius cormucopioides Pers. Syn. Fung. 491. 1801.— Cantharellus cornucopioides Fries, Syst. Мус. 1: 321. 1821. Illustrations: Vaillant, Botan. Paris. pl. 13. f. 2, 3.—Bolton, Hist. Fung. pl. 103.—Flor. Dan. pl. 384, 1260.—Holmskiold, Fung. Dan. 2. pl. 5.—Sowerby, Brit. Fung. pl. 74.—Scheeffer, Icon. Fung. pl. 165.—Bulliard, Herb. de la France pl. 150.-- Schnizlein, in Sturm, Deutsch. Flora 3: fasc. 31. pl. §.—Bresa- dola, Funghi Manger. 75. pl. 83.—Cooke, Brit. Edible Fung. pl. 11. f. 89.—Dufour, Atlas Champ. pl. 70. f. 157.—Hard, Mushrooms 451. f. 379.—Peck, Rep. N. Y. State Mus. 48: pl. 24. f. 7-10.—cf. Saccardo, Syll. Fung. 19: 478, for other refer- ences to illustrations. Fruetifications gregarious or somewhat cespitose; pileus thin, somewhat membranaceous, tubsform, pervious, sometimes granular or minutely squamulose, smoky brown to blackish, usually drying Prout’s brown, with the erect, spreading, or de- curved margin generally lobed, wavy, or irregular; stem short, hollow, even, blackish brown; hymenium even or rugose- wrinkled, cinereous drab; spores hyaline, even, 12-16 x 6-10 u. Fructification 5-8 em. high; pileus 23-5 cm. broad; stem 1-3 em. long, 3-5 mm. thick. [Vor. 1 334 ANNALS OF THE MISSOURI BOTANICAL GARDEN On earth in mixed woods. Canada to South Carolina and westward to Missouri. June to September. The cornucopia craterellus is well characterized by its cornu- copia-shaped or narrowly trumpet-shaped pileus ashy to sooty brown in color, by thin flesh which is somewhat tough and flexile, cinereous drab hymenium which sometimes has a brown- ish tinge, and black stem. This species is too infrequent to afford more than a few herbarium specimens in the regions where I have collected fungi, but it is reported so plentiful in some states as to be highly regarded as an edible species. Specimens examined: Exsiccati: Ravenel, Fung. Car. II. 27; Ellis, N. Am. Fungi, 321; Ell. & Ev., Fung. Col., 1723; Shear, N. Y. Fungi, 49; Rabenhorst-Winter, Fung. Eur., 3640. Sweden: L. Romell, 48. Canada: J. Macoun, 72, 73. Ontario: Casselman, J. Macoun, 347. Vermont: Grand View Mt., E. A. Burt. Massachusetts: Sprague, 211 (in Curtis Herb.). Connecticut: W. A. Setchell. New York: Sand Lake, C. H. Peck (in Coll. N. Y. State); Aleove, C. L. Shear, Shear's N. Y. Fungi, 49; Ithaca, H. von Schrenk (in Mo. Bot. Gard. Herb., 4763, 42584), W. H. Long, Jr., Ell. & Ev., Fung. Col., 1723. New Jersey: Newfield, H. Leahy, Ellis, N. Am. Fungi, 321. Pennsylvania: locality cited by Schweinitz, Syn. N. Am. Fungi; W. Herbst (in Lloyd Herb.). North Carolina: (in Curtis Herb., 502); locality cited by Schweinitz, Syn. Fung. Car. Sup. South Carolina: M. A. Curtis (in Curtis Herb.). Ohio: Loveland, D. L. James, comm. by U. 8. Dept. Agr. Kentucky: Mammoth Cave, C. G. Lloyd. Missouri: Perryville, C. H. Demetrio, Rabenhorst-Winter, Fung. Eur., 3640; Meramec Highlands, P. Spaulding (in Мо. Bot. Gard. Herb., 4869). 6. C. ochrosporus Burt, n. sp. Plate 17. fig. 15. Ап С. ocreatus Pers. Муе. Eur. 2:5. pl. 18. f.2. 1825? Туре: in Mo. Bot. Gard. Herb., 42585. Fructifications gregarious or cespitose; pileus thin, somewhat 1914] BURT—THELEPHORACEX OF NORTH AMERICA. II 335 membranaceous, tubzform, pervious, minutely floccose-squam- ulose, drying avellaneous to snuff-brown, the margin erect or decurved; stem short, hollow, black, with chamois-colored pubescence at the base; hymenium even or somewhat rugose, sometimes colored like the pileus but in the type chamois- colored; spores straw-yellow in the mass, even, obtuse, 12-15 x 7-8 | Fructifications 4-7 cm. high; pileus 1-33 em. broad, 1-21 ста. long, 2-4 mm. thick. On the ground among mosses in woods. New York and Missouri. Juneto September. Probably abundant in Missouri. Dr. Glatfelter noted a pleasant minty odor for the specimens. This species closely resembles C. cornucopioides in form, but differs from that species in having hymenium, spores, and base of stem yellow. А collection from the same spot from which the type collection came, but made in June two years later, has the hymenium snuff-brown and approaches C. cornucopioides in this respect. Iam not aware of any data on C. ocreatus Pers. except that based on the original description which is cited above. That species has presumably not been collected by European mycologists since the original collection from the environs of Paris a century ago. Our specimens differ from that description in having the stem yellow pubescent at the base and the hymenium somewhat rugose, and they may differ in other characters, e. g., spore colors, etc., not given in the brief description of C. ocreatus. Hence I give to our American specimens a distinct name. Specimens examined: New York: East Galway, E. A. Burt. Missouri: Meramec Highlands, N. M. Glatfelter (in Mo. Bot. Gard. Herb., 42585, type, and 42586-87); Columbia, В. M. Duggar, 134. 7. C. dubius Peck, Rep. N. Y. State Mus. 31: 38. 1879. Illustrations: Hard, Mushrooms f. 380. Type: in Coll. New York State. Fructifications solitary or cespitose; pileus thin, infundi- buliform or subtubiform, subfibrillose, dark brown or lurid brown, pervious, the margin generally wavy and lobed; stem short, hollow, colored like the hymenium; hymenium dark [Vor. 1 336 ANNALS OF THE MISSOURI BOTANICAL GARDEN cinereous and rugose when moist, the obscure crowded irregular wrinkles abundantly anastomosing, nearly even and paler when dry; spores broadly elliptical or subglobose, 6-75 x 42-5 и. Fructification 5-71 em. high; pileus 23-5 ст. broad, 4 mm. thick. On ground in woods. Ontario and New York to Illinois. August to October. Rare. Тһе specimens of this species have the same coloration as those of C. cornucopioides but differ from the latter in having a shorter and more funnel-shaped pileus, and smaller spores. Moffatt reported C. dubius as abundant at Glencoe, Illinois. Specimens examined: Ontario: Belleville, J. Macoun, 228 (in Coll. N. Y. State). New York: Adirondack Mts., C. H. Peck, type (in Coll. N. Y. State). Michigan: Sailor's Encampment, Univ. of Wis. Herb., 46. 8. C. lutescens Pers. ex Fries, Epicr. 532. 1838. Plate 17. fig. 20. Merulius lutescens Pers. Syn. Fung. 489. 1801; Albertini & Schweinitz, Consp. Fung. 234. 1805.—Cantharellus lutescens Fries, Syst. Myc. 1: 320. 1821.—Merulius xanthopus Pers. Mye. Eur. 2: 19. pl. 13. f. 1. 1825. Illustrations: Vaillant, Botan. Paris. pl. 11. f. 9, 10.— Scheffer, Icon. Fung. pl. 157.—Bolton, Hist. Fung. pl. 105. f. 2. Persoon, Myc. Eur. 2: pl. 13. f. 1.—Hennings, in Engl. & Prantl, Nat. Pflanzenfam. (1.1**): 129. f. 70 H.—Stevenson, Brit. Hym. 2: 259. Fructifications solitary to cespitose; pileus thin, somewhat membranaceous, varying from convex and umbilicate to tubi- form or funnel-shaped, often pervious, yellowish brown to fuseous, with margin often lobed or irregular; stem flexuous, cylindric, hollow, yellow, drying ochraceous buff, often hairy at the base; hymenium remotely ribbed, even or rugose-wrinkled, yellow, drying cadmium-yellow to ochraceous buff; spores even, 10-12 x 6-8 y. Fruetifieations 21-5 em. high; pileus 15-3 em. broad, stem 11-4 em. long, 2-4 mm. thick. On moist ground in woods and swamps. Newfoundland to North Carolina and westward to Michigan. August to October. L айлалы ыы 1914) BURT—THELEPHORACE OF NORTH AMERICA. II 337 This species probably ranks next to C. cornucopioides in frequency in the United States. The long and yellow stem readily distinguishes this species from C. ochrosporus. Speci- mens of Cantharellus infundibuliformis resemble those of Craterel- lus lutescens in form, size, and color, but those of the former species have true lamelle. Specimens examined: Exsiecati: Ellis, N. Am. Fungi, 1302; De Thuemen, Myc. Univ., 404. Sweden: Stockholm, L. Romell, 49; Femsjé, L. Romell. Newfoundland: Bay of Islands, A. C. Waghorne, 34 (in Mo. Bot. Gard. Herb.). New Hampshire: Shelburne, W. С. Farlow, Ellis, М. Am. Fungi, 1302, and (in Mo. Bot. Gard. Herb., 4932). Vermont: Lake Dunmore, Е. A. Burt. Massachusetts: Worcester, G. Е. Francis, 100. New England: Sprague, 1689 (in Curtis Herb.). New York: Sand Lake and Helderberg Mts., C. H. Peck (in Coll. N. Y. State); East Galway, Е. A. Burt. Pennsylvania: locality cited by Schweinitz, Syn. N. Am. Fungi. North Carolina: locality cited by Schweinitz, Syn. Fung. Car. Sup. Michigan: Glen Lake, C. G. Lloyd, 02462. 9. С. sinuosus Fries ex Fries, Epicr. 533. 1836-1838. Cantharellus sinuosus Fries, Syst. Myc. 1: 319. 1821. Illustrations: Vaillant, Botan. Paris. pl. 11. f. 11-23.—Fries, Icon. Hym. 2: pl. 196. f. 2.—Dangeard, Le Botaniste 4: 147. f.— Gillet, Champ. France Hym. рї. Fructifications cespitose, slightly fleshy; pileus infundibuli- form, undulate and floccose, light drab; stem cylindric, stuffed, pallid cinereous; hymenium at length with interwoven wrinkles, pallid cinereous; spores 10-12 x 6-7 м. Fructifications 2-3 cm. high; pileus 2-3 сіп. broad; stem 13-2 em. long, 2-4 mm. thick. On ground in mixed woods. South Carolina. Rare. I have seen only dried herbarium specimens of Craterellus sinuosus. The spore measurements are those of a specimen from Sweden received from Romell. In this specimen the hymenium has dried somewhat chamois-colored. [Vor. 1 338 ANNALS OF THE MISSOURI BOTANICAL GARDEN Specimens examined: Exsiecati: Rabenhorst, Fung. Eur., 208 (in Kew Herb.). Sweden: L. Romell, 50. South Carolina: Ravenel (in Curtis Herb., 2982). C. crispus Fr., sometimes regarded as a variety of C. sinu- osus, was reported from New England, Sprague, by Berkeley & Curtis, Grevillea 1: 147, but the specimen is not satisfactory for study. I do not, therefore, like to include C. crispus as one of our species. 10. C. calyculus (B. & C.) Burt, n. comb. Stereum calyculus Berk. & Curtis, Hooker's Jour. Bot. and Kew Gard. Misc. 1: 238. 1849; Grevillea 1: 161. 1873. Type: type and cotype in Kew Herb. and Curtis Herb. respectively. Fructifications somewhat fleshy-membranaceous; pileus thin, deeply cup-shaped, minutely tomentose, drying Saccardo’s umber, opaque; stem apparently hollow, cream buff, attenuated below, tomentose at the base; hymenium even or slightly venose, cream buff; spores slightly yellowish under the microscope, even, 8 x 6 y. Fructifications 2-3 em. high; pileus 4-8 mm. broad; stem 1 em. long, 1-2 mm. thick. On ground in damp shady woods. North and South Caro- lina. August and September. Upon moistening, the type in Kew Herbarium proved too soft and fleshy and the hymenium too waxy for a Stereum. The sections have the structure of Craterellus. The species is near C. sinuosus and may prove to be a small form of this when ample material gives more complete knowledge of the species, but, for the present, I regard C. calyculus as a distinct species. I refer to C. calyculus a collection made by Professor Atkinson at Blowing Rock, North Carolina, the rough-dried and cespitose specimens of which show a somewhat tubiform pileus and spores 7-8 x 43 и. Specimens examined: North Carolina: Blowing Rock, G. F. Atkinson, 4200. South Carolina: Santee River, Ravenel, Curtis Herb., 1716 (the type and собуре in Kew Herb. and Curtis Herb. respectively). 1914] BURT—THELEPHORACEJE OF NORTH AMERICA. II 339 11. C. delitescens Burt, n. sp. Plate 17. fig. 18. Type: in Burt Herb. Fructifications gregarious, cespitose, somewhat fleshy; pileus thin, convex, then umbilicate, dry, fibrillose, sepia-colored, the margin inrolled; stem equal, solid, glabrous, chamois-colored ; hymenium even or sometimes obscurely lamelliform, chamois- colored; spores white, even, broadly ovoid, 9 x 7 p, borne four to а basidium. Fructification 10-15 mm. high; pileus 5 mm. broad; stem 10-15 mm. long, 1 mm. thick. Growing among mosses on very thin soil on rocks by water- fall. Vermont. August. This species is intermediate between Cantharellus and Craterel- lus in its hymenial structure, but, as some of the specimens have the hymenium even and bearing mature spores, I include the species in Craterellus. The specimens are much smaller than those of C. calyculus and have the pileus becoming merely umbilicate. The little fructifications were well concealed among the mosses; I have found them but once. Specimens examined: Vermont: Falls of Lana, Lake Dunmore, Е. A. Burt, type. 12. C. taxophilus Thom, Bot. Gaz. 37: 215-19. f. 1-8. 1904. Plate 17. fig. 21. Illustrations: Thom, ibid. f. 1-8. Type: in Cornell Univ. Herb., 15445. Fructifications single, rarely gregarious, fleshy - membra- naceous, entirely white when young, becoming pallid to ochra- ceous buff with age, drying cinnamon buff; pileus narrowly obconic, slightly viscid, the apex truncate, plane, or depressed and with a thin margin which is erect or expanded; stem solid, equal or tapering downward, flexuous, pruinose, with scattered white hairs at the base; hymenium even, becoming longitudi- nally rugose-wrinkled with age or upon drying; spores white, even, subglobose, 3-4 шіп diameter, borne four to a basidium. Fructifications 1-3 em. high; pileus 4-9 mm. broad; stem 1-2 ст. long, 1-1 mm. thick. On rotten twigs and leaves under prostrate branches of Taxus canadensis. New York. October and November. This delicate fungus was under observation by Dr. Thom [Vor. 1 340 ANNALS OF THE MISSOURI BOTANICAL GARDEN for a month and is described in detail and beautifully illustrated in connection with his original description in the work cited above. I reproduce merely some simple outline sketches of C. taxophilus; this is a very distinct species. The specimens were found in Fall Creek Gorge and nowhere except under prostrate branches of Taxus, yet they grew on rotting twigs and leaves of other species as well as on pieces of T'azus. Specimens examined: New York: Ithaca, C. Thom, Cornell Univ. Herb., 15445. I3. C. unicolor Rav. Grevillea 1: 148. 1873. Plate 16. fig. 11, 12. C. corrugis Peck, Bull. Torr. Bot. Club 26: 69. 1899. Type: in Ravenel, Fung. Car. II. 26. Fructifications solitary or cespitose, fleshy, with the flesh white, soft, soon shrinking and leaving the pileus hollow; pileus at first, elavate, obtuse, flesh-colored tinted with violet, soon obconic or turbinate, broadly convex or truncate, and often abruptly cerebriform at the upper end, glabrous, ochra- ceous buff, drying Rood's brown to Natal-brown, the margin obtuse, corrugated by the hymenial wrinkles; stem short, equal or tapering downwards, colored like or a little paler than the pileus; hymenium wrinkled or corrugated, colored like the pileus; spores white, 8-12 x 4-6 y. Fructifications 2-5 em. high; pileus 11-5 em. broad; stem 1-21 em. long, 5-8 mm. thick. On ground in thin woods. Massachusetts, Pennsylvania, and South Carolina. October to January. This fungus presents strikingly the vagaries in the distri- bution of fungi. It was originally collected at Black Oak, South Carolina, in 1850, by Ravenel, in sufficient quantity so that he distributed the type collection in his exsiccati. Appar- ently, this fungus, whenever collected, was referred to other species until 1898, when members of the Boston Mycological Club found it in several localities in Massachusetts and it was adequately described by Peck, as C. corrugis, from specimens received from Dr. Francis. I have received no speciniens of this species since that season; I searched for it in vain for several years in the adjoining state, Vermont. I have compared the specimens of C. corrugis, received from Dr. Francis, with Peck's 19141 BURT—THELEPHORACEJE OF NORTH AMERICA. II 341 type and with the specimens of C. unicolor in five different copies of Ravenel’s ‘Fungi Caroliniani.’ C. corrugis is certainly the same species as C. unicolor. It is very strange that in the interval of nearly half a century from the time of the original collection, C. unicolor did not attract attention from an inter- mediate station. Specimens examined: Exsiccati: Ravenel, Fung. Car. II. 26; Ell. & Ev., N. Am. Fungi, 1922a under the name C. pistillaris. Massachusetts: Worcester, G. E. Francis, 61, 84, and col- lection dated Nov. 2, also the type (in Coll. N. Y. State) of C. corrugis; Lynn, H. Webster; Medford, Mrs. Page and Mrs. De Long, ex Herb. Boston Mycological Club, 420; Arlington Heights, E. A. Burt. Pennsylvania: Trexlertown, W. Herbst, the C. clavatus of his *Fungal Flora'; West Chester, B. M. Everhart, Ell. & Ev., N. Am. Fungi, 1922a. South Carolina: Black Oak, Ravenel, 1406 (in Curtis Herb. and in Kew Herb.), and type, Ravenel, Fung, Car. II. 26. 14. C. pistillaris Fries, Epicr. 534. 1836-1838. Plates 16, 17. figs. 13, 14. Illustrations: Scheffer, Icon. Fung. pl. 169.—Harper, Мусо- logia 5: 263. pl. 95. Fructifications gregarious, fleshy-spongy, drying sorghum- brown to fuscous; pileus somewhat clavate to turbinate or narrowly obconic, truncate, or somewhat convex, at first yel- lowish cinnamon, then becoming tinged with fuscous, the edge obtuse; stem solid, paler than the pileus, often bulbous at the base; hymenium corrugated and rugose-wrinkled, colored like the pileus, drying sorghum-brown to fuscous; spores even, 10-12 x 6-8 и. Fructifications 6-12 em. high; pileus 2-33 сіп. broad; stem 3-6 em. long, 4-12 mm. thick. On ground in woods under coniferous trees. New Hampshire, Vermont, and Michigan. August to October. Specimens of this species have so nearly the coloration of C. unicolor that those, small and undeveloped, in a collection of C. pistillaris cannot readily be distinguished from partially developed specimens of C. unicolor; but with age, those of C. [Vor. 1 342 ANNALS OF THE MISSOURI BOTANICAL GARDEN unicolor—or at least some of them—have the pileus enlarge abruptly in diameter near the upper end and become abruptly globose-cerebriform on a slender stem, as shown in figs. 11 and 12, while C. pistillaris increases in length as well as in diameter, tapers downward more uniformly from the truncate upper end, and may have the stem bulbous at the base. It is à vexed question with mycologists whether Craterellus pistillaris Fr. is Clavaria pistillaris L. The specimens which I refer to Craterellus pistillaris agree well with specimens of this species in Curtis Herbarium, collected at Upsala, Sweden, in 1853, and communicated by E. P. Fries. Pl. 16 fig. 13 is from a photograph, natural size, of these specimens. Their spores are 9x64. The Friesian specimens have the same dark color as our American specimens. Only one of the former shows a bulbous tendency at the base of the stem ; in this respect our specimens are more like the illustration of Scheeffer, cited above. I believe, therefore, that we have Craterellus pistillaris Fr. in our flora. I have collected in mixed frondose woods in Mis- souri what I refer to Clavaria pistillaris as understood by Euro- pean mycologists. As compared with the former species it is of softer structure, much paler in color, more regularly clavate in form, sometimes splitting at the apex. The illustrations of most European authors agree well in regard to Clavaria pis- tillaris. Тһе colored figures of this species in Batsch, Bulliard, Sturm, Dufour, Flora Danica, Hussey, Krombholz, Quelet, and Sowerby present fructifications of the same habit and bright coloration which we have by Peck, Bull. N. Y. State Mus. 94: pl. 93. f. 1-4. and Mem. N. Y. State Mus. 4: pl. 66. f. 15-17. Specimens examined: Sweden: Upsala, E. P. Fries (in Curtis Herb.). Austria: G. Bresadola. New Hampshire: Shelburne, W. G. Farlow (in Mo. Bot. Gard. Herb., 4933). Vermont: Middlebury, E. A. Burt. I5. C. palmatus Burt & Overholts, n. sp. Plate 17. fig. 19. Type: in Mo. Bot. Gard. Herb. and in Overholts Herb. Fructifications gregarious or perhaps cespitose, fleshy-soft ; pileus fawn-eolor shading into bone-brown towards the stem, 1914) BURT—THELEPHORACEZ OF NORTH AMERICA. II. 343 glabrous, flattened and ligulate at first, then spreading out laterally at the apex, and at length somewhat palmately cleft into 2-12 unequal, obtuse, finger-shaped branches; stem curved, solid, equal or somewhat tapering towards the base, bone- brown, sometimes swollen where attached to the substratum; hymenium even or but slightly venose, inferior, colored like the pileus; spores white, even, pyriform, tapering to the base, 6-8 x 3-4 и. Fructifications 1-23 cm. high; pileus 3-15 mm. broad, 1 mm. thick; stem 8-15 mm. long, 1-14 mm. thick. On rotten chunks of wood in frondose woods. Ohio. June. All specimens of the collection except one have the pileus flabelliform; in this exceptional specimen, the pileus is narrowly turbinate, depressed, and with the finger-shaped branches arranged in a circle on the margin, pl. 17 fig. 19b. This species makes for Craterellus the same connection between the central- stemmed, cup-shaped type of pileus and the flabelliform type that T'helephora multipartita shows in Thelephora, and that is common in Stereum. The hymenium of the flabelliform speci- mens of Craterellus palmatus is so similar to the upper surface of the pileus in color and consistency that one cannot readily distinguish between these surfaces in the dried specimens. For these reasons, the present species cannot be referred to either Skepperia or Friesula, and it is of especial interest in showing that Craterellus has a natural section of species with flabelliform pileus. Тһе spores of C. palmatus are noteworthy. Specimens examined: Ohio: Oxford, L. O. Overholts, 1649, type (in Mo. Bot. Gard. Herb. and in Overholts Herb.). 16. C. dilatus Burt, n. sp. Plate 17. fig. 16. Type: in Farlow Herb. Fruetifieations single, fleshy; pileus flabelliform, somewhat triangular, glabrous, drying a dirty pinkish buff, the margin somewhat irregularly lobed, crisped, and curving upward; stem solid, equal, flexuous, drying Natal-brown, with white myce- lium at the base; hymenium even, drying Isabella-color to clay- color; spores white, even, broadly ovoid, obtuse, 8-10 x 6-7 и. Dried fructification 4 em. long; pileus 15 mm. long, 15 mm. broad, $ mm. thick; stem 23 cm. long, hardly 1 mm. thick. 6 [Vor. 1 344 ANNALS OF THE MISSOURI BOTANICAL GARDEN On sandy ground in swamp. Florida. September. Only a single fructification was collected; the description is based upon this dried specimen. The species is distinguished by its fan-shaped, triangular pileus and the comparatively long and slender stem. Its characters are those of a true Craterellus and yet such that we cannot regard it as a flabellate form of any other species. Specimens examined: Florida: Sorrento Swamp, R. Thaxter, type (in Farlow Herb.). 17. C. Humphreyi Burt, n. sp. Plate 17. fig. 22. Type: in Burt Herb. and in Humphrey Herb. Fructifications gregarious, fleshy, moderately tough and flexible, entirely white, usually with the pileus standing out horizontally at the apex of the erect stem; pileus reniform, dimidiate, sometimes clasping behind, convex, becoming plane or somewhat depressed, usually even, dry, minutely pubescent, the margin entire, even or slightly crisped; stem lateral, erect, often bent at right angles just before joining the pileus, cylindric below, equal, solid, pubescent; hymenium nearly even, some- times radiately venose near the stem, brittle when fresh; spores white, even, subglobose, 33-4} x 35 и. Fructifications 3-7 cm. high; pileus 6 mm. - 2 em. long, 1-32 em. broad, 2 mm. thick; stem 22-6 em. long, 2 mm. thick. On humus and among mosses in low swampy thicket. Wash- ington. October. Тһе habit of this curious species is very suggestive of Hydnum auriscalpium; many of the specimens have the erect stem bent at right angles near the apex so that the pileus extends out in a horizontal plane. Sometimes the stem branches at its upper end and bears two pilei. "The pubescence on the stem is rather coarse and is most abundant towards the base. АП parts of the fructification were rather brittle in vegetative condition, and broke when bent too far. It is а connecting species be- tween Craterellus and Arrhenia, but with the hymenium rather too even for Arrhenia, in my opinion. Specimens examined: Washington: Hoquiam, C. J. Humphrey, 1886, type. 1914) BURT—THELEPHORACEJE OF NORTH AMERICA. II 345 Berkeley & Curtis, Jour. Linn. Soc. Bot. 10: 328, described three species of Craterellus from Cuba, which have been trans- ferred to other genera by Patouillard, Bull. Soc. Myc. France 15: 193-94. pl. 9, as follows: С. spathularius to Skepperia and C. marasmioides and C. pulverulentus to Cymatella. Ihave received no collections referable to these genera and defer their consideration to the final part of my monograph in the hope that some specimens may be received in the meantime. Craterellus canadensis Kl. ех Saccardo, Syll. Fung. 6: 519. 1888, was published by Berkeley, Ann. Nat. Hist. 3:380. 1839, under the name Cantharellus canadensis КІ. from a specimen in Hooker Herb. bearing manuscript notes by Klotzsch. The specimen was collected in Canada by Richardson. In connec- tion with the original description, Berkeley noted that the nearest affinities of C. canadensis are with C. clavatus. In 1856, after studying the specimens in Herb. Schweinitz, Berkeley & Curtis, Jour. Acad. Nat. Sci., Phila. N. S. 3: 206. 1856, note that Cantharellus canadensis Kl. is apparently the same species as Cantharellus floccosus Schw. I have seen no specimens of C. canadensis and follow Berkeley's final disposition of the species. (To be continued.) [Vor. 1, 1914] 346 ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 15 All figures of this plate have been reproduced natural size from photo- graphs of dried herbarium specimens. Fig. 1. T'helephora cespitulans. From authentic specimen in Curtis Herb., col- lected by Schweinitz in North Carolina. Fig. 2. T.lutosa. From authentic specimen in Curtis Herb., collected by Schwei- nitz in North Carolina. Fig. 3. T. dentosa. From cotype in Curtis Herb., colleeted in Cuba by C. Wright. Fig. 4. T. perpleza. From type in Curtis Herb., collected in Cuba by C. Wright, 238. a shows a resupinate portion, and b, an ascending portion of the specimen. Fig. 5. T. cornucopioides. From specimen collected in Castleton Gardens, Jamaica, by F. S. Earle, 238. Fig. 6. Craterellus clavatus. From specimen collected at Lake Dunmore, Vt. Fig. 7. C. Cantharellus. From the cotype in Curtis Herb., 4539, of C. lateritius, collected in Alabama, by Peters. Fig. 8. C. odoratus. From the собуре in Curtis Herb. of C. confluens, collected near Orizaba, Mexico, by Botteri, 6. Fig. 9. C. odoratus. From the specimens in Curtis Herb., collected at Society Hill, S. Carolina, by Ravenel, 192. ANN. Мо. Вот. GARD.. VOL. 1, 1914 PLATE 16 BURT— THELEPHORACEAE OF NORTH AMERICA 1. THELEPHORA CAESPITULA NS. — 2. T. LUTOSA.—3. T. DENTOSA.—4. T. PERPLEXA — 5. T. CORNUCOPIOIDES. — 6. CRATERELLUS CLAVATUS. — 7. C. CANTHARELLUS. — 8 AND 9. C. ODORATUS. COCKAYNE, BOSTON [Vor. 1, 1914] 348 ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION оғ PLATE PLATE 16 АП figures of this plate have been reproduced natural size from photo- graphs of dried herbarium specimens, but in the case of fig. 10 the specimens were moistened. Fig. 10. C. odoratus. From specimens collected near St. Louis, Mo., by N. M. Glatfelter, 348. Тһе rough dried specimens were moistened before being photo- graphed. а shows a branched specimen; b, a fructification split longitudinally to show extent of depression of the pileus and the hollow stem; c, view of hymenium. Fig. 11. C. unicolor. From authentic specimen in Curtis Herb., collected at Black Oak, S. Carolina, by Ravenel, 1406. Fig. 12. C. unicolor. From specimen of C. corrugis collected at Medford, Mass., by Mrs. Page and Mrs. DeLong. Fig. 13. С. pistillaris. From specimen in Curtis Herb., collected at Upsala, Sweden, by E. P. Fries. ANN. Мо. Вот. GARD., VOL. 1, 1914 PLATE 16 4 BURT — THELEPHORACEAE OF NORTH AMERICA 10. CRATERELLUS ODORATUS.-—11 AND 12. С. UNICOLOR. — 13. С. PISTILLARIS. COCKAYNE, BOSTON [Vor. 1, 1914] 350 ANNALS OF THE MISSOURI BOTANICAL GARDEN ExPLANATION OF PLATE PLATE 17 All figures are natural size. Figures 14-20 are from photographs of dried herbarium specimens, but which were moistened before being photographed in case of specimens used for figs. 15 and 17. Fig. 14. C. pistillaris. From specimen collected under hemlock (Tsuga) tree, at Middlebury, Vt. Fig. 15. C. ochrosporus. From type specimens in Mo. Bot. Gard. Herb., col- lected near St. Louis, Mo., by N. М. Glatfelter, 1253. а is split longitudinally to show the depth of depression of the pileus; b, side view. Fig. 16. C. dilatus. From type in Farlow Herb., collected at Sorrento Swamp, Florida, by R. Thaxter. a shows upper surface of pileus, and 6, the hymenium. Fig. 17. C. cornucopioides. From specimen collected in Canada, by J. Macoun, 72. Fig. 18. C. delitescens. From type specimens collected at Lake Dunmore, Vt. Fig. 19. C. palmatus. From type specimens in Mo. Bot. Gard. Herb. and Over- holts Herb., collected at Oxford, Ohio, by L. O. Overholts, 1649. а shows specimens having flabelliform pileus, and b, a specimen with turbinate pileus. Fig. 20. C. lutescens. a shows hymenium of specimen collected at Shelburne, New Hampshire, by W. G. Farlow, and 5, upper surface of specimen collected at Lake Dunmore, Vt. Fig. 21. C. tazophilus. From sketches of photographs of type specimens when in vegetative condition, collected at Ithaca, New York, by C. Thom. Fig. 22. C. Humphreyi. From sketches of the type specimens when in vegetative condition, collected at Hoquiam, Wash., by C. J. Humphrey, 1386. йылы” қа. ANN. Мо. Вот. GARD., VOL. 1, 1914 PLATE 17 g 4 th te ^s P 129. % zx са MESA СУРА BURT —THELEPHORACEAE ОЕ NORTH AMERICA 14. CRATERELLUS PISTILLARIS. — 15. С. OCHROSPORUS. — 16. С. DILATUS. — 17. C. CORNUCOPIOIDES. — 18. C. DELITESCENS. — 19. С. PALMATUS. — 20. C. LUTESCENS. --21. C. TAXOPHILUS. —22. C. HUMPHREYI. COCKAYNE, BOSTON $ THE EFFECTS ОЕ SURFACE FILMS ОХ THE RATE OF TRANSPIRATION: EXPERIMENTS WITH POTTED POTATOES B. M. DUGGAR Physiologist to the Missouri Botanical Garden, in Charge of Graduate Laboratory Professor of Plant Physiology in the Henry Shaw School of Botany of Washington | University AND J. S. COOLEY Formerly Rufus J. Lackland Fellow in the Henry Shaw School of Botany of Washington University In a previous report! we have presented data which is be- lieved to justify the conclusion that an application of a surface film of Bordeaux mixture to the leaves of the castor bean or the tomato increases materially the rate of transpiration. The importance of a careful determination of various physiological effects of this spray mixture was suggested primarily by the increased vitality and yield exhibited by potatoes (Solanum tuberosum) treated with this fungicide during seasons when fungi and insects were unimportant factors. In our previous experiments the potato was not included, and it seemed most important, as a next step, to ascertain the effects of certain sprays upon the transpiration of this plant. Experience has demonstrated that the potato may not be used satisfactorily in potometer experiments. Moreover, it was desired to arrange the experiment so that the transpiration quantities obtained might represent an interval of a week or more. On the other hand, it had been found as a result of our previous work with potted tomatoes that a very considerable amount of labor is required when it becomes necessary to add measured quantities of water every day to a series of fifty or more potted plants. Accordingly, for this and for other work proposed, a method was devised whereby we were able to employ a self-watering device based on a principle often used in the laboratory. 1 Duggar, B. M., and Cooley, J. S. The effect of surface films and dusts on the rate of transpiration. Ann. Mo. Bot. Gard. 1:1-22. pl. 1. 1914. ANN. Mo. Вот. Garp., Vor. 1, 1914 (351) [Vor. 1 352 ANNALS OF THE MISSOURI BOTANICAL GARDEN The apparatus is shown in pl. 18. The rack, or support, is made of a single sheet of galvanized iron 18 ст. wide and 55 em. long, these dimensions being adequate for a stand 33 cm. high. Besides cutting a hole in the upper part for the insertion of the neck of the bottle, the operation of making a stand will be clear from the plate and involves merely a few slits with the shears, the balance being accomplished by bending. Two or four rivets may be used if additional strength is required. With regard to other features of the apparatus it is well to note that (1) the shoulder of the flower pot rests on the rim of a tin сар somewhat deeper than the pot, the latter containing the im- mediate supply of water; (2) there is an inverted bottle with a capacity of about 1500 сс. serving as a reservoir of water and aspirator; and (3) the bottle is connected with the cup by glass and rubber tubing. In setting up an experiment the exposed area of the pot (above the shoulder) and the soil are covered with paraffin or parawax; the cup is filled with water to such height that when the pot is inserted the water will rise to the height of about 2 cm. on the side of the pot, thus insuring adequate absorption; while a notch in the side of the cup makes it possible to introduce the rubber tube connecting with the bottle, this tube being adjusted to reach just below the new level of water in the cup. With a tube of proper diameter, the water level in the cup is kept practically constant so long as the bottle contains water. This apparatus, complete, may be quickly and sufficiently accurately weighed on the Troemner scales. То prevent up- setting, after arranging in the experimental area, it is well to make the stand secure by providing a small hole in the base, through which a bamboo stick may be thrust into the soil. То this stake, also, for further support, the bottle may be fastened by соға or rubber band. Тһе deviee above described has saved much time and has enabled us to obtain a soil moisture content practically uniform in all the pots used in the experiment. It possesses the disad- vantage of tending to maintain а moisture content which “ог long-term cultures is too high for the best growth of the potato. A slight modification of the method would seem to be practi- cable in several aspects of transpiration work. 1914] DUGGAR AND COOLEY—FILMS AND TRANSPIRATION 353 Three weeks before the experiment began the plants were repotted, new 5-inch pots of good quality being used, and at the time of the installation of the experiment the drainage holes in the pots were carefully corked, so that all transfer of water would be through the porous walls. Тһе potato plants em- ployed were grown in the greenhouse during the early spring, but on April 20, about two weeks before the test was made, they were placed outside, to insure hardiness. When used, the plants were from 25 to 45 ст. high, each plant with from about 15 to 30 leaves. Some plants were blossoming, and tubers were forming. The experiment embraced 7 series, or lots, of 10 plants each, sprayed with mixtures as follows: (1) strong Bordeaux, (2) control, no spray, (3) weak Bordeaux, (4) lime wash, (5) lime sulfur, (6) strong Bordeaux and lampblack, and (7) lime wash and lampblack. The strong Bordeaux (designated hereafter Bordeaux) contained 12 grams CuSO, and 14.4 grams СаО per liter of water, being approximately the 5-6-50 formula of agricultural practise. It was made up in the usual way. The weak Bordeaux was one-half the strength of the stronger mixture. Тһе lime wash was a Са(ОН): suspension consisting of 60 grams of CaO per liter of water. A commercial prepara- tion of lime sulfur was used, and this was diluted, as usual, to about 1-25. The Bordeaux-lampblack and the lime-wash- lampblack preparations were made by rubbing into small quantities of the Bordeaux and lime wash 5 and 10 grams re- spectively of lampblack, then diluting to one liter. The method of selecting the plants for the different lots was precisely that described in the previous report, that is, selecting at one time 7 plants (as many as there were lots) between which there could be little or no choice, and distributing these at random, 1 to each lot until each included 10 plants. АП plants (except controls) were sprayed on May 5, but a rain that night, before protection was provided, necessitated respraying the following day. After spraying, the plants were placed on the stands and each connected with its water supply. They were arranged on an exposed lawn, each lot occupying a row, with the plants 4 feet apart. Moreover, several rows of potted pota- toes were arranged around the entire area in order that all [Vor. 1 354 АХМ:! OF THE MISSOURI BOTANICAL GARDEN plants in the experimental area might have equal exposure. Over the expernental plot а frame was provided, so that the whole area т... $ be protected by tarpaulins in case of rain. Fortunately, however, no rain occurred during the period of the experiment. After a preliminary exposure of 24 hours, which enabled us to determine that the 70 plants of the experimental area were in good condition, the initial weighings were made. А definite order was established, this being crosswise of the different lots. The same order was observed at the close of the period, and similarly in the second period a consistent scheme was followed, in order that the time interval might be as uniform as possible. After the weighings at the close of the first period, all plants were discarded which showed any signs of weakness or injury arising from the conditions of the experiment. It should be stated, too, that these conditions were taxing. Тһе weather was bright and warm, the pots were severely exposed, and, as already noted, the water content of the pots was necessarily fairly high. With the plants remaining in a condition appar- ently normal and vigorous from the first period, a second “тип” was made, the latter including from 4 to 7 plants in TABLE SHOWING WATER LOSS AND GREEN WEIGHT OF THE PLANTS Ist period, May 6-11, | 2nd period, May 11-15, 10 plants | 7 plants Lot Film covering Ave. Ave. | Water | Ave. Ave. Water water | green loss per| water | green | loss рег loss рег| weight |g. green| loss рег | weight |g.,green plant |per plant| weight | plant |per plant| weight 1 | Bordeaux, strong 526.6 | 50.3 | 10.46 | 463.3 | 55.0 8.42 2 | Control 413.8 61.0 6.78 | 433.3 63.1 6.86 3 | Bordeaux, weak 642.4 60.9 10.54 | 574.0 61.7 9.30 4 | Lime wash 584.5 70.7 8.27 | 6013.6 76.3 8.04 5 | Lime sulfur 443.0 | 62.8 7.06 | 450.7| 70.0 6.44 6 | Bordeaux and lamp- | black 792.1 66.1 11.97 | 653.0 75.2 8.69 7 | Lime and lampblack | 596.6 | 58.3 | 10.20 | 585.6 | 66.8 | 8.78 each lot. In selecting plants for this second period, the size factor was again taken into consideration, as far as possible. 1914] DUGGAR AND COOLEY—FILMS AND TRANSPIRATION 355 More stress should, however, be laid upon the data from the first period. Тһе green weights of the plants discarded at the close of the first period were taken immediately, while those plants used in the second period could not be weighed until the close of that interval. This small interval of time, however, could eause no material change in the weights. In the accom- panying table there are given in grams the average water loss per plant, the average green weight per plant, and the water loss per gram of green matter. From the data exhibited it is obvious that with potted potatoes, as with castor bean leaves and potted tomatoes in our earlier experiments, there is a marked acceleration of transpira- tion induced by spraying with Bordeaux mixture, as also with some other films. Of the several films employed, lime sulfur alone yields an average water loss comparable with that of unsprayed plants. Of all lots showing increased transpiration those treated with weak Bordeaux and lime wash were in some respects most satisfactory, inasmuch as the plants used, like those in the control, were, in general, in very good condition throughout the period of the experiment. On the other hand, those treated with the stronger Bordeaux, the Bordeaux and iampblack, and the lime and lampblack gave, towards the close of the periods, evidences of the injurious effects of the increased transpiration (apparently) upon the vitality of the plants. Тһеве statements may not seem to be in entire aecord with the figures presented, for during the second period of the ex- periment, for example, the transpiration quantity is relatively greatest in the case of those plants sprayed with weak Bordeaux mixture. Nevertheless, our observations enabled us to predict that certain lots, especially numbers 1 and 6, would give in the second period, particularly, transpiration values less than might be anticipated. The smaller quantities in the lots re- ferred to, as contrasted with the weak Bordeaux, are to be explained, in fact, as a direct result of incipient wilting and slight injury, brought about by the higher transpiration capacity induced under conditions already accentuating transpiration. It is believed, in the first place, that the experiments here reported confirm our earlier conclusion, namely, that a film of Bordeaux mixture facilitates water loss; but, in the second place, Қ-сы шыға m P uir тар [Vor. 1, 1914] 356 ANNALS OF THE MISSOURI BOTANICAL GARDEN treatment; with a fairly thick lime wash or lime wash and lamp- black also increases the transpiration rates, the latter more than the former. Lampblack added to Bordeaux seems also to give a higher rate than the Bordeaux alone. It is to be emphasized, however, that the strength of the lime wash employed is four times as great as the lime in the stronger Bordeaux mixture ; likewise, more lampblack is used with the lime wash than with the Bordeaux. It seems to be definitely established that cer- tain specific characters of the film are important, but these results suggest, further, that the additional quality of color is а factor requiring consideration. Тһе fact that injury may result from the accelerated transpiration induced by a heavy film of Bordeaux under the conditions of our experiment does not mean that under normal conditions of growth in the field a benefit may not accrue to certain plants—from factors asso- ciated with a high transpiration rate. Graduate Laboratory, Missouri Botanical Garden. EXPLANATION OF PLATE PLATE 18 View of the apparatus (with tomato plant) by means of which watering was automatically controlled. It has been found convenient to have both stand and cup painted green. For description see text, p. 322. ANN. Mo. Bor. GARD., VOL. 1, 1914 PLATE 18 DUGGAR AND COOLEY — TRANSPIRATION СОСКАҮХЕ BOSTON Annals of the Missouri Botanical Garden Vor. I NOVEMBER, 1914 No. 4 THE THELEPHORACEZE OF NORTH AMERICA III! CRATERELLUS BOREALIS AND CYPHELLA EDWARD ANGUS BURT Mycologist and Librarian to the Missouri Botanical Garden Associate Professor in the Henry Shaw School of Botany of Washington University Since the publication of Part II, on Craterellus, Dr. Farlow has very kindly called my attention to, and permitted me to study, a specimen of a rare species from Labrador which was not included in my account of our North American species. This species is now described here so as to bring its description and illustration continuous with those of our other species of Craterellus. The following is suggested for insertion in “Кеу to the Species," on page 328 (Ann. Mo. Bot. Gard. 1: 328. 1914). 6. Pileus membranaceous, infundibuliform, pale buff; hymenium pale buff; spores 5-7 x 4-54 u; from Labrador. See page 357 (Ann. Mo. Bot. Gard. 1:357. ТОЛА) ЕЕ rH T sm C. borealis. Craterellus borealis Burt, n. sp. Plate 19. fig. 1. Type: in Farlow Herb. Fructifications solitary, small; pileus infundibuliform, taper- ing uniformly to the stem, glabrous, drying between cartridge buff and cream-buff, the margin entire; stem nearly equal, Notre.—Explanation in regard to the citation of specimens studied is given in Part I, Ann. Mo. Bot. Gard. 1:202. 1914, footnote. Тһе technical color terms used in this work are those of Ridgway, Color Standards and Nomenclature. Washington, D. C., 1912. 1 [Issued January 30, 1915. ANN. Мо. Вот. GARD., Vor. 1, 1914 (357) [Vor. 1 358 ANNALS OF THE MISSOURI BOTANICAL GARDEN slender, minutely downy, pale mouse-gray; hymenium colored like the pileus, remotely ribbed, with the ribs radiating from the stem, thin, branching; spores colorless, even, 5-7 x 4-53 и. Fructification 2 cm. high; pileus 1 em. broad, 13 mm. long; stem 7 mm. long, 3 mm. thick, enlarging to 1 mm. where joining the pileus. In moss. Labrador. August 8, 1908. The above description is based on the single dried specimen collected by the Bryant Labrador Expedition. Тһе small size, regular obconie form, and very pale color of the membranaceous pileus and the slender stem are characters making C. borealis clearly distinct from other species of Craterellus. Specimens examined: Labrador: Gready Harbor, Gready Island, Owen Bryant, type (in Farlow Herb.). CYPHELLA Cyphella Fries, Syst. Myc. 2: 201. 1823. Fructifications somewhat membranaceous, cup-shaped, rarely plane, adnate behind, commonly extended in stem-like form, pendulous; hymenium typically concave or disk-shaped, defi- nitely inferior in the pendulous species, even or at length rugu- lose; basidia typically four-spored; spores subovate or globose, hyaline, rarely colored. C. digitalis Fries is the type species of this genus. The fructifications of all our North American species are com- paratively small, ranging in diameter from a fraction of a milli- meter for some species to five to fifteen millimeters for those of the largest species. Тһе fructifications are produced оп the bark of small rotting twigs on the ground and on dead herb- age, and can only be distinguished from small Pezize by dem- onstrating basidia rather than asci in the hymenium. This demonstration is simply made by crushing under a cover glass & portion of a fructifieation in water containing a little seven per cent solution of potassium hydrate, and then examining the preparation with the compound microscope. Тһе basidia are usually four-spored; in a few species I have as yet been able to detect only two-spored basidia. Cyphella is closely related to Solenia by such species as C. fasciculata and C. mellea, but is separated from it in such cases 1914) BURT—THELEPHORACEJE OF NORTH AMERICA. III 359 by the absence of a hyphal subiculum over the area on which the fructifications are distributed, and by the less cylindric form of fructifications of Cyphella. Cyphella is allied to Meru- lius by C. muscigena and also to Craterellus by this species, speci- mens of which were described as a Craterellus. А few species of Cyphella are common and widely distributed, but most of our North American species are apparently ex- tremely local and are known only from their respective type collections. The lack of specimens available for carrying about to compare with types has been a serious disadvantage in my study of this genus. Ваза and basidiospores have not as yet been found for some species which, although originally referred to Cyphella, have to be regarded as even doubtful Basi- diomycetes. I have supplemented the original descriptions with measurements of dried fructifications and with such data in regard to basidia and spores as the specimens afford. In the case of very scanty types, the few fructifications are too precious for gross comparison to be used for microscopic study. For such species, it seems to me that the descriptions should stand on the original data, without prejudice, until new collections become available. Such imperfectly known and partially de- scribed species are grouped together under the heading “Species Imperfectly Known." Cyphella convoluta Cke., C. Cupressi (Schw.) Fries and C. subcyanea Ell. & Ev. are excluded species. KEY TO THE SPECIES Fructifications sulphur-colored ; hymenium even; spores 43x21-3 и 1. C. sulphurea Fructifications sulphur-colored; hymenium minutely pitted; spores 6-8 x КЕНІ... bees ОЛЧЕ uses E a 2. C. leta Fructifications white or whitish; on поввев............................ 1 Fructifications white; not on шоввев................................. 2 Fructifications neither white nor вшрһиг-сојогей....................... 3 1. Fructifications helmet-shaped; hymenium slightly wrinkled; spores 10 x vou ТЕККЕ РТ M UT 8. C. galeata 1. Fructifications flattened, irregular in form, sometimes stipitate; spores 3-5 РР A sae ОР N mee ERE RR 4. C. muscigena 1. Fructifications seated upon or developing from webby strings of mycelium 5. C. arachnoidea 2. Fructifications villose, not easily crushed, with a firm base or a short gem; done 12-15 х6-бім................:.....,/2...... 6. C. Tilie 2. Fructifications villose, easily crushed, sessile; spores 10-12 x 5-7 и 7. C. villosa 2. Fructifications whitish, minutely webby-hairy, easily crushed, sessile; ккге9-І8кім....................................... 8. С. caricina [Vor. 1 360 ANNALS OF THE MISSOURI BOTANICAL GARDEN 2. Fructifications glabrous, with an oblique stem; spores 44-6 x 3-33 и 9. C. capula 2. Fructifications villose, snow-white, sessile, very minute and delicate; spores 5-6 x 4-43 ш; from New England............ 10. C. minutissima 2. Compare with C. cinereo-fusca, C. Palmarum, C. Peckii, C. perexigua, C. pezi- zoides and C. trachycheta of "Species Imperfectly Known." 3. Fructifications wholly pale ivory-yellow, downy-pubescent, cup-shaped, ses- sile; spores 4-7 х3-4н..........................:....11. C. Langlois 3. Fructifications wholly cream-color, not hairy, helmet-shaped, sessile, re- supinate-reflexed; hymenium wrinkled; spores 7} x 4} 4; on prickle-bear- ing stems, Jamaicà..... iiie nmn 12. C. porrigens 3. Fructifications mineral-gray, tomentose, cup-shaped, sessile; hymenium fus- cous; spores angular, 41-6 x 4j и; on Лиирегиз.......... 13. C. cupuleformis 3. Fructifications wholly gray-pallid, flocculose, sessile; spores 4 x 3 и........ 14. C. griseo-pallida 3. Fructifications externally cinereous, farinaceous, flattened, sessile; hymen- ium convex, brown; spores 8 х 3$ и; on Alnus............ 15. C. subgelatinosa 3. Fructifications darker colored than the аһохе.......................... 4 4. Fructifications vinaceous-buff, hairy, sessile, $ mm. broad; spores 10-12 x 6-8 ш; on bark of Сағтуа............................. 16. C. Ravenelii 4, Fructifications drying Isabella-color, hairy, sessile, 1-1} mm. broad; spores 13 x 8 м; on Оиегсив............................. 17. C. texensis 4. Fructifications Isabella-color, hairy, sessile, 3-4 mm. broad; some spores colored, 5-6 x 4-43 и; оп баЙт........................... 18. C. mellea 4. Fructifications tawny-olive, tomentose, stipitate; often cespitose; spores 7-9 x 2-2} и; usually on Айпи8....................... 19. C. fasciculata 4. Fructifications fuscous when moist, drying mouse-gray, cespitose and ses- sile оп a common short trunk, glabrous, structure gelatinous 20. C. conglobata 4. Fructifications sepia or olive-brown, cup-shaped, probably glabrous, ses- sile or with a very short stem; spores 6-8 x 33-4 u; on rotting leaves of СПафйойшв.......................................ЛҺл0.0л...... 21. C. fumosa 4, Compare C. Banana, C. filicicola and C. musecola in "Species Imperfectly Known." 1. C. sulphurea Batsch ex Fries, Hym. Eur. 665. 1874. Peziza sulphurea Batsch, Elenchus Fung. Contin. і: 209. pl. 27. f. 146. 1786.—P. campanula Nees, System d. Pilze 268. f. 295. 1816.—Cyphella sulphurea Batsch, in Patouillard, Tab. Anal. Fung. 114. f. 256. 1883; Peck, Rep. N. Y. State Mus. 31: 38. 1879. Illustrations: Batsch, Elenchus Fung. Contin. pl. 27. f. 146. —Nees, System d. Pilze f. 295.—Patouillard, Tab. Anal. Fung. f. 256.—Oudemans, Ned. Kruidk. Archief III. 2: pl. 8. f. 1-6. Fructifications scattered or gregarious, membranaceous, broadly campanulate, somewhat irregular, extended into a short stem, even, glabrous, sulphur-yellow, the margin somewhat re- pand; hymenium even; База cylindric, 16 x 4$ u, 4-spored; 1914] BURT—THELEPHORACEJE OF NORTH AMERICA, Ш 361 spores colorless, even, broadly ovoid, somewhat flattened on one side, 43 x 23-3 y. Fructifications about 2-3 mm. high; pileus 1-2 mm. broad; stem 1 mm. long, mm. thick. On living stems of herbs іп damp places. New York. Sep- tember. Rare. Тһе minimum dimensions given above for the fructifications are about those of European specimens of this species as figured; the American specimens run rather larger in Peck's collection. Peck noted that some of his specimens were white when col- lected, but that they dried yellow like the others of the collec- tion. In other respects our American specimens agree closely with the figures and description of European specimens. Oude- mans gives the spore dimensions as 10-12 x 4-5 y, but Patouil- lard gives them as they are in American specimens. Specimens examined: New York: Griffins, Delaware Co., C. H. Peck (in Coll. N. Y. State). 2. C. læta Fries, Epicr. 568. 1836-1838. Illustrations: Patouillard, Tab. Anal. Fung. f. 362. Fructifications membranaceous, obliquely cup-shaped, ex- tended at the vertex into a stem, pendulous, entire, everywhere glabrous and sulphur-colored; stem straight or somewhat flexu- ous, hymenium minutely pitted; spores colorless, even, 6-8 x 3-4 џ, borne four to a basidium. Fructifications 3-5 mm. high, 2-4 mm. broad; stem 1-2 mm. long, about 3 mm. thick. On dead stems of large herbs lying on the ground. New York. August. Fries described the fructifications as 6-8 mm. broad; the dimensions given above are those of Patouillard’s figures and of the specimens collected by Peck. Patouillard notes that the specimens blacken when old; Peck states, “The beautiful sulphur-color is lost in drying.” The pitted surface of the hymenium is a noteworthy character of C. leta and this and the larger spores of C. leta distinguish it from C. sulphurea. Specimens examined: New York: East Berne, C. H. Peck (in Coll. N. Y. State). [Vor. 1 362 ANNALS OF THE MISSOURI BOTANICAL GARDEN 3. C. galeata Schum. ex Fries, Ерісг. 567. 1836-1838. Plate 19. fig. 2. Merulius galeatus Schum. Plant. Sællandiæ 2: 371. 1803.— Cantharellus galeatus Fries, Syst. Myc. 1: 524. 1821; Flor. Dan. 12: fasc. 34. 11. pl. 2027. f. 1. 1830. Illustrations: Flor. Dan. pl. 2027. f. 1. Fructifications membranaceous-soft, somewhat sessile, ob- versely cup-shaped and then dimidiate, helmet-shaped, even, whitish, the margin entire; hymenium at length rufescent, slightly wrinkled; spores ovate or obovate, 10 x 8 y. Fructifications 4-15 mm. in diameter. On mosses. Ohio. When young entire, cup-shaped; gray when moist, snow- white when dry, then rufescent. Тһе above description is that given in European works. Тһе species has been reported from Ohio by Morgan but I have not studied his specimens nor any European specimens of this species. Тһе form and coloration of the pileus and the large spores should distinguish C. galeata from the other species which occur on mosses in North America. 4. C. muscigena Pers. ex Fries, Epicr. 567. 1836-1838. Plate 19. fig. 3. Thelephora muscigena Pers. Syn. Fung. 572. 1801; Fries, Syst. Myc. 1:524. 1821.—T. vulgaris a candida Pers. Myc. Eur. 1:115. pl.?. f. 6.1822.—Cantharellus levis Fries, Syst. Myc. 1 :524.1821; Elenchus Fung. 55. 1828.—Craterellus Pogonati Peck, Bull. Torr. Bot. Club 33: 218. 1906. Illustrations: Persoon, Myc. Eur. 1: pl. ?. f. 6.—Patouillard, Tab. Anal. Fung. f. 465.—Oudemans, Ned. Kruidk. Archief ПІ. 2: pl. 11. f. 2. Pileus membranaceous-soft, sessile, stipitate or attached by upper surface, irregular, flattened, white, externally minutely tomentulose or silky under a lens; stem when present lateral or eccentric, slender, white; hymenium even or sometimes rugulose, drying pinkish buff; spores white in collection on slide, even, apiculate at base, flattened on one side, 41-5 x 21-3 и but only 3-43 x 2-3 шіп preparations of the hymenium, borne four to а basidium. Pileus 2-6 mm. in diameter; stem when present 3-5 mm. long, 3 mm. thick. 1914) BURT—THELEPHORACE OF NORTH AMERICA. ІП 363 On Polytrichum and other mosses. New England and New York. August and September. The fructifications are very variable in form and they are attached in various ways to the moss plants; they may be some- what incrusting but at some distance above the ground. Тһе substance of the pileus is very soft and its upper surface is some- what bibulous and shows its interwoven fibers under a lens. The spores of this species are given in Saccardo’s ‘Sylloge’ as 8-10 x 5 y, but the European specimens of exsiccati cited below have small spores of the dimensions which I give for American specimens, and Bresadola, Ann. Муе. 1: 111. 1903, gives the spore dimensions as 3-4 x 3 и. Тһе specimens of C. Родопай were described as sterile by Peck; I find them to be rather im- mature but bearing spores 3 x 2 y. Specimens examined: Exsiccati: Karsten, Fung. Fenn., 441; Krieger, Fung. Sax., 1564. Finland: Karsten (in Herb. Fries), and Fung. Fenn., 441. Germany: Saxony, W. Krieger, Krieger, Fung. Sax., 1564. Vermont: near Falls of Lana, Salisbury, E. A. Burt. Connecticut: South Windsor, C. C. Hanmer, 1956, the type collection of Craterellus Pogonati Pk. New York: Floodwood, E. A. Burt. 5. C. arachnoidea Peck, Rep. N. Y. State Mus. 44: 184 (22). 1891. Type: in Collection New York State. Fructifications membranaceous, very thin, tender, white, externally downy, irregularly cup-shaped; hymenium some- what uneven in large specimens; spores colorless, even, some- what flattened on one side, 4-5 x 31-4 y, borne at least two toa basidium. Fructifications 2-4 mm. in diameter. On bark and mosses. Vermont and New York. September. Тһе cups are seated upon or developing from fine, white, loosely branching, webby strings of mycelium. This is a marked character in the type and is the chief character for separating this species from C. muscigena. The spores are slightly more globose than in the latter and it may be that the hymenium of C. arachnoidea is superior; in C. muscigena it is inferior. The hyphe are about 2 шіп diameter in each species. [Vor. 1 364 ANNALS OF THE MISSOURI BOTANICAL GARDEN Specimens examined: Vermont: South Lincoln Notch, near Middlebury, E. A. Burt. New York: Carrollton, C. H. Peck, type (in Coll. N. Y. State). 6. C. Tiliz Peck ex Cooke, Grevillea 20: 9. 1891. Plate 19. fig. 16. Peziza Tilie Peck, Rep. N. Y. State Mus. 24: 96. 1872.— Trichopeziza Те (Peck) Sacc. Syll. Fung. 8: 428. 1889; Seaver, Proc. Iowa Acad. Sci. 12: 116. 1905; Mycologia 1: 110. 1909. Type: in Collection New York State and a portion from it in Kew Herbarium. Fructifications gregarious, rather fleshy, minute, sessile or nearly so but with firm base, white, globose, then expanded and concave, drying cup-shaped, densely white villose; hairs straight, cylindric, granular incrusted, 200 x 6 4; hymenium concave, even, ivory-yellow to vinaceous buff; spores white in a collec- tion on a slide, simple, even, ovate, somewhat curved, 12-18 x 6-64 м, borne four to a basidium. Fructifications 3-1 mm. high, 1-1 mm. broad; stem, when present, about one-half the height of the whole fructification. On bark of dead branches of Tilia Americana and Ulmus on the ground. Canada and Vermont westward to Missouri. March to October. Probably common. C. Tilie has somewhat the habit of C. albo-violascens but differs from the latter in having no violaceous tints, in being more hairy, in having slenderer spores, and in having at the base а very firm tubercle which offers considerable resistance when the fructification is crushed under a cover glass or sec- tioned. While not cespitose the fructifications of C. Ті о are often so near together that seven or eight have been counted on an area а centimeter square. Ireferto C. Tiliæ many American specimens which have been distributed under the name C. pezi- zoides Zopf. The European specimens which Sydow has dis- tributed under the latter name seem to me from the studies and comparisons which I made in Kew Herbarium to be С. Currey В. & Br. rather than C. Tilia. Specimens examined: Exsiecati: Shear, N. Y. Fungi, 55; Ell. & Ev., N. Am. Fungi, 2316a, under the name C. pezizoides; Ell. & Ev., Fung. 1914) BURT—THELEPHORACEJE OF NORTH AMERICA. III 365 Col., 5, under the name C. pezizoides; Rabenhorst, Fung. Eur., 3942, under the name C. pezizoides. Quebec: Hull, J. Macoun, 672. Ontario: Ottawa, J. Macoun, 318, 430; London, J. Dearness, Ell. & Ev., N. Am. Fungi, 2316a, and Fung. Col., 5. Vermont: Middlebury, C. O. Smith, and also E. A. Burt. New York: Knowersville (Altamont), C. H. Peck, type (portion in Kew Herb.); Alcove, C. L. Shear, Shear, N. Y. Fungi, 55. Ohio: Oberlin, F. D. Kelsey (in Mo. Bot. Gard. Herb., 4942). Michigan: Agricultural College, С. Н. Hicks, comm. by W.G. Farlow, 6 (in Mo. Bot. Gard. Herb., 43807). Wisconsin: Blue Mounds, 1. E. Melhus, comm. by C. J. Hum- phrey, 2410 (in Mo. Bot. Gard. Herb.). Missouri: C. H. Demetrio, Rabenhorst, Fung. Eur., 3942. 7. C. villosa Pers. ex Karsten, (Mycol. Fenn. 3) Bidrag Finska Vet.-Soc. 25: 325. 1870. Plate 19. fig. 13. Peziza villosa Pers. Syn. Fung. 655. 1801; Fries, Syst. Myc. 2: 104, pr. p. 1823.—An Cyphella pezizoides Zopf, in Morgan, (Myc. Fl. Miami Val.) Jour. Cincinnati Soc. Nat. Hist. 10: 202. 1888? Illustrations: Patouillard, Tab. Anal. Fung. f. 257. Fructifieations gregarious, membranaceous, sessile, drying globose or obconic and with the pore nearly closed by the hairs, white, externally white-villose; the hairs granular incrusted, cylindric, 200 x 5-6 ш; hymenium even, concave; spores hya- line, even, ovoid, flattened on one side, broadest near the base, 10-12 x 5-7 и. Fructifications about $ mm. high, $—+ mm. broad. On dead stems of Artemisia, Helianthus, and Solidago. South Carolina, Missouri and California. June and July. The fructifications of C. villosa resemble those of C. Tilie in form, color, and hairiness but are much smaller than those of C. Tile, more membranaceous and easily crushed under a cover glass, and have smaller spores. Тһе hymenium is very pale with not more than a very slight yellowish tint. Specimens examined: Exsiccati: Krieger, Fung. Sax., 1457; Ravenel, Fung. Am., 459; Ell. & Ev., N. Am. Fungi, 2316b, under the name Cyphella pezizoides Zopf. [Vor. 1 866 ANNALS ОҒ THE MISSOURI BOTANICAL GARDEN South Carolina: Aiken, Ravenel, Ravenel, Fung. Am., 459. Missouri: Emma, C. H. Demetrio, Ell. & Ev., N. Am. Fungi, 2316b. California: Half-moon Bay, San Mateo Co., E. B. Copeland, Baker, Pacific Coast Fungi, 3611 (in Mo. Bot. Gard. Herb., 4944). 8. C. caricina Peck, Rep. N. Y. State Mus. 33: 22. 1880. Plate 19. fig. 8. Type: in Collection New York State. Fructifications scattered, membranaceous, sessile, wholly white, externally minutely webby-hairy; hymenium glabrous, uneven in large specimens; basidia cylindric, 20 x 5 u, 4-spored; spores colorless, even, lanceolate or subclavate, pointed at base, 8-13 x 4 м. Fructifications 1-2 mm. broad. On culms and leaves of carices, New York. August. The spores of the type are noteworthy by their tapering base. Specimens examined: New York: Verona, C. H. Peck, type (in Coll. N. Y. State). 9. С. сарша Holmsk. ex Fries, Epicr. 568. 1836-1838. Plate 19. fig. 4. Peziza Сарша Holmsk. Nov. Act. Havn. 1: 286. f. ?; Fung. Dan. 2: 41. pl. 22. 1899. Illustrations: Holmskiold, Nov. Act. Havn. 1: 286. f. 7; Fung. Dan. 2: pl. 22.—Flor. Dan. 33: pl. 1970. f. 3.—Patouillard, Tab. Anal. Fung. т: f. 35. Fructifications membranaceous, obliquely campanulate, ex- tended into an oblique stem, glabrous, whitish, the margin sin- uate, irregularly shaped; hymenium even. . . . On dead stems of herbaceous plants. —Translation of description in Fries’ ‘ Epicrisis.’ Fructifications in the figures of Holmskiold 4-9 mm. high; pileus 2-7 mm. long, 2-4 mm. broad; stem 1-2 mm. long. On dead stems of Feniculum and other herbs. New York and South Carolina. I have not been able to study any European specimens of this species. In the copy of Cooke’s ‘Fungi Britannici’ in the herbarium of the Missouri Botanical Garden the packet labeled C. capula, 112, contains only some pieces of stubble. The Amer- 1914] BURT—THELEPHORACEJE OF NORTH AMERICA. ІП 367 ісап specimens distributed іп Ravenel's (Fungi Americani, 458, were determined by Cooke. In their present dried condi- tion these specimens agree well with Holmskiold's illustrations in form; the stem of these specimens is now hair-brown and the pileus pale olive-buff; their dimensions are: fructifications 1-3 mm. long, pileus 1-2 mm. long and broad; stem $-1 mm. long х 100 » thick. Тһе basidia are 16-20 x 31-41 и; spores color- less, even, flattened on one side, 41-6 x 3-31 и. Specimens examined: Exsiecati: Ravenel, Fung. Am., 458. South Carolina: Aiken, Ravenel, Ravenel, Fung. Am., 458. IO. C. minutissima Burt, n. sp. Plate 19. fig. 5. Type: in Mo. Bot. Gard. Herb. and in Farlow Herb. Fructifications gregarious, very minute, membranaceous and very delicate, sessile, globose, snow-white, externally villose, often with mouth oblique, margin inrolled; hairs white, in- crusted, 75-90 x 4 и; hymenium concave, white; База cla- vate, 16 x 4 u; spores colorless, even, 5-6 х 4-44. Fructifications 200-500 џи broad, about 200-500 м high. On inner bark of Populus. New Hampshire. August. The characters of this species agree in some details with those in the incomplete description of C. globosa Pat., the specimens of which were collected on the under side of leaves of ferns in Ecuador by von Lagerheim, but as no mention is made of spore characters for C. globosa and as other species of Cyphella have not been found to vary widely with regard to kind of substra- tum, it seems best to regard our New England species as proba- bly distinet. С. punctiformis (Fries) Karst. is а small white Cyphella, described by Karsten as having spores 5-8 x 2-4 y; I have not been able to study authentic specimens of C. punc- tiformis, but comparison of C. minutissima with this species of northern Europe should be made. I refer to C. minutissima a collection made by myself in Ver- mont on bark of rotting locust limbs. Тһе fructifications of this collection lack spores but agree in all other respects with the type. Specimens examined: New Hampshire: Chocorua, W. G. Farlow, 8, type (in Mo. Bot. Gard. Herb., 43803, and in Farlow Herb.). Vermont: Middlebury, E. A. Burt. [Vor. 1 868 ANNALS ОҒ THE MISSOURI BOTANICAL GARDEN 11. C. Langloisii Burt, n. sp. Plate 19. fig. 6. Type: in Farlow Herb. and Burt Herb. Fructifications gregarious, membranaceous, cup-shaped, ses- sile, drying pale ivory-yellow, externally downy pubescent, the margin inrolled; hairs colorless, somewhat crinkled together, granular incrusted, 100-150 x 33-44 и; hymenium concave, even, pale ivory-yellow to cream color; spores colorless, even, pointed at the base, 4-7 x 3-4 u; basidia clavate, 20 x 5u, 2-spored. Fructifications about 1 mm. high; }-} mm. broad. On dead stems of Arundinaria and on decaying pieces of wood lying on the ground. Louisiana. September and April. The fructifications of C. Langloisii are about as small as those of C. minutissima but differ from them in being somewhat ex- tended laterally and occasionally somewhat laterally confluent rather than always globose, in having an ivory-yellow rather than snow-white color, and in having the hymenium colored and the hairs longer than in C. minutissima. Comparison should be made with C. fraxinicola В. & Br., of which I have studied no specimens but which seems distinct by some characters of the incomplete published description. Specimens examined: Louisiana: St. Martinville, A. B. Langlois, 1802, type (in Farlow Herb.), and cz, type, in Burt Herb., and cy, and from the same collector but comm. by W. G. Farlow, 5 (in Mo. Bot. Gard. Herb., 43791). 12. C. porrigens Burt, n. sp. Plate 19. fig. 7. Type: in Burt Herb. and New York Bot. Gard. Herb. Fructifications scattered, membranaceous, thin, wholly cream- color, sessile, obversely cup-shaped or helmet-shaped, resupinate by the upper surface of one side but with the greater portion of the pileus extended and reflexed; hymenium inferior, somewhat wrinkled when moistened, concave, basidia clavate, 20-25 x 4-44 y, with four sterigmata; spores colorless, even, flattened on one side, obovate, 73 x 44 y. Fructifications 1-1 mm. broad. On dead prickle-bearing stems, possibly Rubus sp. Wet mountainous region at altitude 4500-5200 feet. Cinchona, Jamaica. About January 1. This species does not appear closely related to any other 1914] BURT—THELEPHORACEJE OF NORTH AMERICA. ІП 369 species; it is marked by the resupinate-reflexed habit of most fructifications; only rarely is a fructification attached by its vertex. The dried specimens are externally minutely fibrillose under a lens but do not show hairs in microscopic preparations. When the fructifications are moistened the hymenium shows two or three minute wrinkles radiating from an eccentric point. Specimens examined: Jamaica: Cinchona, W. A. and Edna L. Murrill, N. Y. Bot. Gard., Fungi of Jamaica, 607, type. 13. C. cupuleformis Berk. & Rav. Grevillea 2: 5. 1873. Plate 19. fig. 9. Type: type and собуре in Kew Herb. and in Curtis Herb. respectively. Fructifications scattered, rarely in clusters of two or three, sessile, cup-shaped, somewhat globose, externally mineral gray and obscurely tomentose, the margin incurved; hymenium concave, even, fuscous; basidia clavate, 20-25 x 4-6 y, having 2-4 sterigmata which become finely attenuated; spores colorless, angular, 43-6 x 4% м. Fructifications 4 mm. high, 3-1 mm. broad. On bark of Juniperus virginiana. South Carolina and Georgia. Тһе hairiness of the exterior of the pileus is due to the irregu- larly curved and interwoven һурһе which form the surface layer of the pileus; these hyphe are colorless and about Зи in diameter, and they bear scattered but large incrusting granules. The angular spores of this species are often octahedral in form and are noteworthy for Cyphella; at maturity, they are attached to the basidium by sterigmata becoming 6 и long and so finely attenuated that the attachment of the spores to the basidia is made out with difficulty. This species may be readily known by its occurrence on bark of Juniperus virginiana and by its angular spores. Specimens examined: Exsiccati: Ravenel, Fung. Am., 224. South Carolina: Ravenel, 1403, type (in Kew. Herb.). Georgia: Darien, Ravenel, Ravenel, Fung. Am., 224. 14. C. griseo-pallida Weinm. Hymeno- et Gastero-mycetes in Rossico. 522. 1836. Illustrations: Patouillard, Tab. Anal. Fung. f. 255. TUM 3 [Vor. 1 370 ANNALS OF THE MISSOURI BOTANICAL GARDEN Fructifications gregarious, adnate-sessile, membranaceous, wholly gray-pallid, externally flocculose; hymenium glabrous, even. At first having the form of globose, closed granules, soon open, campanulate or crateriform, often dimidiate in old stages. Fructifications 3 mm. high, 2-2 mm. broad. On moist ground and on pine wood thinly covered with earth and on old cracked trunks of Lonicera tartarica (in Europe). — Translation of original description. On bark, twigs and leaves lying on the ground. New York and Ohio. November. I have not seen the type of C. griseo-pallida nor any European specimens which have been compared with it, but Peck, Rep. N. Y. State Mus. 30: 48. 1879, has referred to this вресіев а collection which he made at Sand Lake, New York. Peck notes that his specimens sometimes have a very short stem. I found the spores of these specimens hyaline, even, somewhat flattened on one side, 4 x 3 и; basidia 12 x 4 y. Specimens examined: New York: Sand Lake, C. H. Peck (in Coll. N. Y. State). I5. C. subgelatinosa Berk. & Rav. Grevillea 2: 5. 1873. Type: in Kew Herb. Fructifications scattered, somewhat gelatinous, sessile, flat- tened, externally cinereous and farinaceous, the thin margin inflexed; hymenium slightly convex, even, brown ; basidia cla- vate, about 25 x 5-6 u, probably 2-spored; spores colorless, even, ellipsoidal, 8 x 31 y. Fructifications about 14 mm. broad. On Alnus serrulata. South Carolina. The fructifications of the type have dried with the slightly convex hymenium so prominently visible that they resemble brown apothecia of lichens with a pale margin (exciple). The most of the basidia are immature; I found one showing two sterig- mata distinctly. No spores were found attached to basidia ; the spore characters, which are given above, are those of loose spores in the preparation. С. subgelatinosa is so very distinct from our other species of Cyphella that it will probably be over- looked by botanists collecting Basidiomycetes only, unless es- pecially kept in mind. 1914) BURT—THELEPHORACE/E OF NORTH AMERICA. Ш 371 Specimens examined: South Carolina: Aiken, Ravenel, 1714, type (in Kew Herb.). 16. C. Ravenelii Berk. Grevillea 2: 5. 1873. Plate 19. fig. 14. Type: type and cotype in Kew Herb. and in Curtis Herb. respectively. Fructifications single or gregarious, sessile, subglobose, some- what flattened, depressed at the pore, minutely hairy under a lens, vinaceous buff; hairs minutely rough, about 300 в long, 4 y thick, tapering towards the free end, olive-yellow under the microscope; spores hyaline, or perhaps very slightly colored, even, broadly ellipsoidal, 10-12 x 6-8 и. Fructifications 0.6 mm. high, 0.8 mm. broad; pore 0.15 mm. in diameter. On bark of Carya. South Carolina. Тһе specimens of this species which I have seen have been on thick and cracked portions of bark apparently from large branches or the main trunk of the tree. Sometimes only one fructification occurs on а piece of bark a centimeter square; sometimes such a piece bears from 3 to 6 fructifications with some of them barely in contact with one another. The type specimen contains so few fructifications that I made a micro- scopie preparation at Kew Herbarium from the specimen dis- tributed by Ravenel in Ellis, N. Am. Fungi, 721, which seems to me to be certainly the same species as the type. Berkeley described the spores in his original description as “elliptic, .00025 (in.) long"; I found them about twice this length in my preparation referred to and also in a preparation recently made from the specimen in Ravenel, Fung. Am., 130, in the Mo. Bot. Gard. Herb. Specimens examined: Exsiccati: Ravenel, Fung. Am., 130; Ellis, N. Am. Fungi, 721. South Carolina: Aiken, Ravenel, 1755, the type and cotype (in Kew Herb. and in Curtis Herb. respectively); and also Aiken, Ravenel, Ravenel, Fung. Am., 130, and Ellis, N. Am. Fungi, 721. 17. C. texensis Berk. & Curtis, Grevillea 20: 9. 1891. Plate 19. fig. 10. Type: in Kew Herb. Fruetifieations scattered, sessile, pallid but at present time [Vor. 1 372 ANNALS OF THE MISSOURI BOTANICAL GARDEN Isabella-color (melleus of “Chromotaxia’), cup-shaped, at length flattened and disk-shaped, externally hairy; hairs olive-ocher under the microscope, granular incrusted, cylindric, 300-400 x 43-6 р; basidia clavate, 25-30 x 6-8 и, 4-spored; spores hyaline, even, broadly ellipsoidal, 13 x 8 y. Fructifications 1-14 mm. broad. On Quercus. Texas. The type is scanty, consisting of three fructifications, but these fructifications are in fine condition and present well the characters of the species. С. texensis now impresses me as more closely related to C. Raveneli? than I observed when studying the specimens of both in Kew Herbarium. The fructifications of С. texensis are the melleus of Saccardo’s ‘Chromotaxia’ and the hairs are of a little greater diameter and have larger incrust- ing granules than those of C. Ravenelii, but the spores and basidia are very similar in form and dimensions in both species. Specimens examined: Texas: Wright, 3779, type (іп Kew Herb.). 18. C. mellea Burt, n. sp. Plate 19. fig. 12. Type: in Burt Herb. and in U. S. Dept. Ag. Herb. Fructifications closely gregarious, sessile, Isabella-color, spher- ical and with margin inrolled in the dried state, sometimes obconic, externally hairy; hairs granular incrusted, baryta- yellow under the microscope, cylindric, 80-100 x 31-4 и; hy- menium even, whitish or pale olive-buff; basidia clavate, 12-16 x 6 п; spores mostly colorless but some pale baryta-yellow, even, broadly ellipsoidal, 5-6 x 4-41 u. Fructifications about 3-3 mm. high and broad. On rotten wood of Salix nigra. Louisiana. December. In the specimen upon which the description is based, the most of the fructifications are about à mm. high and broad and are distributed on the rotten wood at the rate of about 200 per square centimeter. Rarely a short stem-like base is visible when the fructifications emerge from the bottom of small crevices between the fibers of the wood, but the fructifications are gen- erally sessile. The species is intermediate between Cyphella and Solenia but is included in the former genus because the fruc- tifications do not arise from a common subiculum and are more globose than in Solenia. The description of C. mellea suggests 1914) BURT—THELEPHORACEJE ОҒ NORTH AMERICA. ІП 373 those of C. Ravenelii and C. texensis in many respects, but the fructifications are much smaller and more numerous than in either of these species, and their various parts are also much smaller and some of the spores are colored. Specimens examined: Louisiana: Bohemia, Plaquemines Co., A. B. Langlois, 864a, type, in Burt Herb. and also (in U. S. Dept. Ag. Herb.); А. B. Langlois, 864 (in U. S. Dept. Ag. Herb.). I9. C. fasciculata Schw. ex Berk. & Curtis, Jour. Acad. Nat. Sci. Phila. 3: 207. 1856. Plate 19. fig. 17. Cantharellus fasciculatus Schw. Trans. Am. Phil. Soc. N.S. 4: 153. 1831.—C. fasciculatus Schw. in Saccardo, Syll. Fung. 5: 495. 1887.—Cyphella fasciculata Berk. & Curtis, Grevillea 2: 6. 1873.—Solenia anomala Pers. var. orbicularis Peck, Rep. N.Y. State Mus. 47: 168 (42). 1894.—Cyphella fulva Berk. & Rav. Grevillea 2: 5. 1873.—C. Ravenelii Saccardo, Syll. Fung. 6: 672. 1888.--С. Saccardoi Sydow, in Saccardo, Syll. Fung. 14: 233. 1900.—C. furcata Berk. & Curtis, Grevillea 2: 5. 1873. Type: in Herb. Schweinitz. Fructifications gregarious, sometimes fascicled, pezizoid, tawny olive; pileus stipitate, cup-shaped, extended vertically or pendulous, tomentose with tawny-olive, even-walled hairs which are flexuous or somewhat spirally curved towards the tips, the margin strongly inrolled; stem short, variable in length, cylindric, tomentose, colored like the pileus ; hymenium concave, even, drying olive-buff; spores hyaline, even, cylindric, slightly curved, 7-9 x 2-21 u, borne four to a basidium. Fasciculate clusters about 2 mm. in diameter, 1 mm. high; fructifications 3-1 mm. in diameter, 1-2 mm. high; stem 1-1 mm. long, 3-1 mm. thick. On bark of twigs of Alnus in swamps and rarely on Prunus virginiana and Pyrus Malus. Canada and Newfoundland to South Carolina and westward to Wisconsin. Throughout the year, more highly fasciculate from autumn to spring. Common. This fungus is very common on dead twigs of Alnus in swamps. The color is similar to that of Solenia anomala but the fructifi- cations are rather larger and more cup-shaped than those of the latter and have the hymenium merely concave rather than lining a tube. "The fructifications burst out through the outer bark 2 [Vor. 1 374 ANNALS OF THE MISSOURI BOTANICAL GARDEN either singly or in clusters of from two to twenty individuals more or less connected together at the base. The differences in habit between the extremes of highly fascicled forms and those with fructifications gregarious and largely single, impress one as of specific weight at first and I should like to recognize these extremes as two species but they intergrade too completely. The dated collections which I have seen, indicate that the speci- mens become highly fasciculate in autumn and winter. I do not understand why Berkeley attempted authorship for this species. Тһе C. fasciculata B. & C. is certainly that of Schweinitz both in description and in fascicled form of types; and as for C. fulva B. & Rav., it is noted in the original descrip- tion that it is the same as Cantharellus fasciculatus Schw. Specimens examined: Exsiceati: Ellis, №. Am. Fungi, 936, fascicled form; Ell. & Ev., Fung. Col., 1818, fascicled form under the name C. Ravenelii Berk.; Shear, N. Y. Fungi, 308, fascicled form under the name Solenia anomala (Pers.) Fr. var. orbicularis. Pk. Peck det.; Ravenel, Fung. Car. IV., 16, the type distribu- tion of C. fulva B. & Rav.; Ravenel, Fung. Am., 129 (bear- ing spores in abundance); Shear, N. Y. Fungi, 56. Newfoundland: Headquarters, B. L. Robinson & H. von Schrenk (in Mo. Bot. Gard. Herb., 4764 and 43789, the latter com- municated by W. G. Farlow); Bay of Islands, A. C. Wag- һотпе, 127 (in Mo. Bot. Gard. Herb., 42593). Quebec: Hull, J. Macoun, 356. Ontario: Ottawa, J. Macoun, 23. Maine: J. Blake (in Curtis Herb., 6926, and in Kew Herb.). New Hampshire: Conway, W. G. Farlow; North Conway, W. G. Farlow (in Mo. Bot. Gard. Herb., 43786); Shelburne, H. von Schrenk (in Mo. Bot. Gard. Herb., 4765), W. 6. Farlow (in Mo. Bot. Gard. Herb., 43787); Franklin Falls, Mrs. J. B. Harrison, Ellis, N. Am. Fungi, 936. Vermont: Middlebury, on Alnus and on Prunus virginiana, Е. A. Burt. Massachusetts: Newton, W. G. Farlow (in Mo. Bot. Gard. Herb., 42591, 42592 and 43788). New York: Torrey, type (in Herb. Schw.); Sartwell, cotype and type of C. fasciculata B. & C. (in Curtis Herb., 2659, and in 1914] BURT—THELEPHORACE® OF NORTH AMERICA. ІП 975 Kew Herb. respectively) and specimen (іп Mo. Bot. Gard. Herb., 4937); Ithaca, G. F. Atkinson; East Galway, E. A. Burt; Keeseville, C. O. Smith, Ell. & Ev., Fung. Col., 1818; Alcove, C. L. Shear, Shear, N. Y. Fungi, 56 and 308; Albany, C. H. Peck, comm. by H. D. House (in Mo. Bot. Gard. Herb., 43821); Karner, C. H. Peck, comm. by H. D. House (in Mo. Bot. Gard. Herb., 43820). South Carolina: Ravenel, 1683 (in Curtis Herb. and in Kew Herb.), and in Ravenel, Fung. Car. IV., 16; Aiken, Ravenel, Ravenel, Fung. Am., 129. Alabama: Beaumont, the cotype and type of C. furcata (in Curtis Herb., 4022, and in Kew Herb. respectively). Wisconsin: Madison, W. Trelease (in Mo. Bot. Gard. Herb., 42594). 20. C. conglobata Burt, n. sp. Plate 19. fig. 15. Type: in Mo. Bot. Gard. Herb. and in Farlow Herb. Fructifications cespitose, 10-30 together, sessile on a common short trunk which is erumpent through the bark; individual fructifications subglobose, fuscous and glabrous when moist, drying mouse-gray and with the margin inrolled; hymenium concave, black or nearly black; basidia simple, with four sterig- mata; spores colorless, even, cylindric, slightly curved, 8-10 x 21-3y. Cluster 1-2 mm. in diameter, emerging about 2 mm. from the bark; cups 400—500 џ broad, nearly as high. Clusters scattered on small limbs of Alnus. New Hampshire and New York. July and September. The clusters of this curious fungus are distributed at the rate of about 5 or 6 clusters to the square centimeter on what I con- clude to have been the under side of a horizontal limb—perhaps a limb prostrate on the ground; for cups in clusters exactly on this presumably under side have the pore central while in the clusters which emerged more obliquely from the limb the cups are somewhat auriform with oblique pore and are arranged in imbricated manner. The outer surface of the cups is composed of irregularly branched and interwoven pale brownish hyphe about 2 шіп diameter. The substance of the fructifications and common trunk-like base is composed of colorless hyphe with walls gelatinously modified. [Vor. 1 376 ANNALS OF THE MISSOURI BOTANICAL GARDEN One might regard this fungus as the type species of а new genus distinet from Cyphella or Solenia by the common central mass on which the individual eups are borne, but in Cyphella fasciculata the cups sometimes occur singly and sometimes branching from a common central or basal mass. For this reason it seems best to include the present species in Cyphella through its relationship in plan of structure to C. fasciculata, from which it is specifically distinct in other respects, however. Both these species are excluded from Solenia by their short and globose fructifications and by the absence of a subiculum on the general area over which the clustered fructifications are distributed. Specimens examined: New Hampshire: Lower Bartlett, В. Thaxter, comm. by W. С. Farlow, 4, type (in Mo. Bot. Gard. Herb., 43806, and in Farlow Herb.). New York: Adirondack Mts., C. H. Peck, comm. by H. D. House (in Coll. N. Y. State and in Mo. Bot. Gard. Herb., 43818); North Elba, C. H. Peck, comm. by H. D. House (in Mo. Bot. Gard. Herb., 43819). 21. C. fumosa Cooke, Grevillea 20:9. 1891. Plate 19. fig. 11. Type: in Kew Herb. Fructifications gregarious, membranaceous, cup-shaped, flexuous, sepia or olive-brown and blackening, even, attenuated below into a very short stipe, or sessile; hymenium even; basidia cylindrie-clavate, 20 x 4-5 и; spores colorless, even, somewhat flattened on one side, 6-8 x 33-4 и. Fructifications 1-2 mm. broad. On rotting leaves of Gladiolus. South Carolina. Cooke described the spores of this species as globose, 4 и in diameter, but I found no such spores in my preparation from the type. Spores 6-8 x 31-4 и are abundant and are probably the spores of this species, although I could not find any spores still attached to the basidia. I conclude from my microscopical preparations that the fructifications are glabrous. Specimens examined: South Carolina: Aiken, Ravenel, 3071, type (in Kew Herb.). 1914) BURT—THELEPHORACEZ OF NORTH AMERICA. Ш 977 SPECIES IMPERFECTLY KNOWN C. cinereo-fusca Schw. ex Saccardo, Michelia 2: 303. 1881. Peziza cinereo-fusca Schw. Schrift. d. Naturforsch. Gesell., Leipzig, 1: 119. 1822; Fries, Syst. Myc. 2: 97. 1823.—Cy- phella cinereo-fusca (Schw.) басс. Syll. Fung. 5: 674. 1888. —Lachnella cinereo-fusca (Schw.) Васе. Syll. Fung. 8: 399. 1889. Fructifications minute, gregarious, sessile, externally fari- naceous-hirsute and ash-green, the margin incurved; hymenium fuscous-bay. On decorticated branches of Cercis. [North Carolina.] 3 mm. broad. Cups often closed. —"Translation of original description. I have not seen an authentie specimen of this species nor any- thing on Cercis which seems referable to it. The species is given here on the authority of Saccardo, l. c., who refers to this species a Cyphella collected on Vitis vinifera near Toulouse, France, by Roumeguere. Saccardo does not state that he made comparison with an authentic specimen from Schweinitz, and he has entered the species in the ‘Sylloge Fungorum’ in both the Basidiomycetes and the Discomycetes. С. Palmarum Berk. & Curtis, (Fung. Cub.) Jour. Linn. Soc. Bot. 10: 337. 1867. Type: type and cotype probably in Kew Herb. and Curtis Herb. respectively. White, pileus cyathiform, externally obscurely pruinose; stem short, tomentose, rather thick. Scarcely 2 mm. high; stem rather thick for the size of the pileus, often oblique. On petioles of palms. Cuba. June. C. Wright, 753. —Arranged from original description. C. Peckii Sacc. Syll. Fung. 6: 684. 1888. С. candida Peck, Вер. N. Y. State Mus. 27: 99. 1875. Type: in Coll. N. Y. State. Fructifications scattered or gregarious, membranaceous, soft, obconic, nearly or quite sessile, sometimes deflexed, wholly white, externally tomentose; hairs tapering to a sharp point, rough-walled, 60-70 x 33 и. [Vor. 1 878 ANNALS OF THE MISSOURI BOTANICAL GARDEN Fructifications about 1 mm. broad. On dead stems of ferns, Osmunda cinnamomea. New York. September. The type specimens of this species are immature. I could make out neither distinet asci nor basidia in the hymenium. In a crushed preparation I found one spore, colorless, even, pointed at one end, 6 x 23 и. It may have been а basidiospore of this species or it may have been a foreign spore. Specimens examined: New York: Forestburgh, C. H. Peck, type (in Coll. N. Y. State). C. perexigua Sacc. Michelia 2: 136. 1880. Cups bell-shaped, very short and obliquely stipitate, small, -4 mm. long, thin-membranaceous, internally and externally whitish cinereous, externally minutely puberulent; spores not seen. Appears related to С. eruceformis and cupuliformis but is one-third as large. . . . On decorticated branches. South Carolina. Ravenel.—Translation of original description. I have not seen the type of C. perexigua, which is probably in Saccardo Herb. As basidia and basidiospores have not been found for American specimens, it is uncertain whether this species is a Cyphella. Patouillard, Tab. Anal. Fung. 19. f. 34. 1883, referred to C. perexigua a species of Cyphella which he collected at Poligny, France, but that reference is doubtful in the absence of knowledge in regard to basidia and basidiospores for American specimens. C. pezizoides Zopf, in Morgan, (Мус. Fl. Miami Val.) Jour. Cincinnati Soc. Nat. Hist. 10: 202. 1888. Type: probably in the State Univ. of Iowa Herb. “Fructifications membranaceous, nearly sessile, globose then cup-shaped, clothed externally with long erect white hairs. Hymenium even, brownish; spores obovate, .012-.013 mm. in length. “On old herbaceous stems; not common, cupule pezizoid, scarcely pedicillate, about half a line in diameter. The long hairs are erect and connivent over the hymenium; they are hya- line and incrusted with crystals of calcium oxalate.” —Original description. The type is not accessible at present. 1914) BURT—THELEPHORACEJE OF NORTH AMERICA. ІП 379 С. trachycheta Ell. & Ev. Jour. Мус. 4:73. 1888. Type: in New York Bot. Gard. Herb. Fructifications gregarious, sessile by а narrow base, white, cup-shaped, clothed outside with appressed hairs; hairs subhy- aline, very rough, with a smooth tapering tip 12-15 u long; hairs paler around the base of the fructification and coarsely roughened by irregularly shaped tubercles, some of which are prolonged into short spines; hymenium nearly white with a slight tinge of slate color; basidia and spores could not be well made out, but the latter are apparently very minute. Fructifications 300-4004 high and broad, occasionally 1 mm. broad and with the margin distinctly lobed. On fallen leaves of Quercus. Louisiana. July. The above description is arranged from that originally pub- lished. Iam under obligation to Dr. Murrill for recently send- ing to me a portion of the type for study, but the specimen proves too immature to show whether this species is а basidio- mycete. The hymenium of this specimen is now pale olive- buff; the hairs are 50-75 x 6 u, heavily encrusted except near the tips, but I failed to find any hairs roughened by tubercles or bearing spines. Specimens examined: Louisiana: A. B. Langlois, 1424, type (in N. Y. Bot. Gard. Herb.). C. Banana Cooke, Grevillea 6: 132. 1878. Type: probably in Kew Herb. Fructifications fuliginous or wood-brown, finger-shaped, pendulous-extended behind, glabrous, the margin entire; hy- menium white, rugose; spores linear, obtuse, curved, 10-12 x 3 y. —Translation of original description. On dead leaves of Musa. Gainesville, Florida. Ravenel. C. filicicola Berk. & Curtis, Grevillea 2: 5. 1873. Type: type and cotype probably in Kew Herb. and Curtis Herb. respectively. Stem very short; cups irregular, sometimes oblique, externally very obscurely tomentose, umber. On dead fern. North Carolina. Curtis Herb., 4934, type. Тһе above contains all the items of the original description; I overlooked this species when studying in Curtis Herb. and in Kew Herb. [Vor. 1 380 ANNALS OF THE MISSOURI BOTANICAL GARDEN C. musecola Berk. & Curtis, Jour. Linn. Soc. Bot. то: 337. 1867. Type: type and cotype in Kew Herb. and Curtis Herb. respectively. Pileus erucible-form, pallid purple, with very short stem or sessile, externally tomentose; hymenium luteus (cadmium- yellow). — Translation of original description. About 2 mm. across. On sheaths of plantain leaves. Cuba. C. Wright, 761. By the kindness of Dr. Farlow I have been permitted to examine a specimen from the type collection. I fail to find any fructifieations of a Cyphella present. A leaf-spot fungus has caused some dark purple discolorations 1-2 mm. in diameter at various points in the surface of the leaf. Specimens examined: Cuba: C. Wright, ?51, comm. by W. G. Farlow (in Mo. Bot. Gard. Herb., 43790). EXCLUDED SPECIES C. convoluta Cooke, (Fungi of Texas) Ann. М. Y. Acad. Sci. I: 179. 1878. Type: In Kew Herb. “Scattered, cup-shaped, then flattened, 1 to 2 mm. wide, margin membranaceous, involute, externally white, internally fleshy-red ; spores oblong (.007 mm. long). “On trunks. Ravenel (295)."— The original description. I examined the type of this fungus, which was collected at Houston, Texas, and do not regard it as a Cyphella. The “basidia”’ are filiform and only l-spored; spores are abundant, hyaline, even, 4-5 x 2-21 u. C. Cupressi Schw. ex Fries, Еріст. 567. 1836-1838. Merulius Cupressi Schweinitz, Schrift d. Naturforsch. Gesell., Leipzig, 1: 92. 1822. This species is an insect gall, not a Basidiomycete. Its true nature seems to have been first pointed out by Berkeley & Curtis, Jour. Acad. Nat. Sci. Phila. 3: 207. 1856. C. subcyanea Ell. & Ev. Jour. Mye. 2: 37. 1885. As this species is not mentioned in Saccardo’s 'Sylloge Fun- gorum’ and as the early numbers of the Journal of Mycology аге rare, I quote the original description as follows: еее жібі t eo TT EU 1914] BURT—THELEPHORACE OF NORTH AMERICA. ІП 381 “Оп living leaves of Sabal Palmetto, Louisiana, Nov. 1885. Rev. A. B. Langlois, No. 57. Shallow cup-shaped, thin, substi- pitate, oblique, less than 1 mm. across, whitish and nearly smooth outside, hymenium bluish or lead colored. Spores filiform multinucleate, upper end thickened, curved into a semicircle, 40-60 u long by 13 и thick, on short (11-12 x 11-2 и) subcylindrical sporophores, which are a little thickened below.” This species was distributed in 1891 in Ell. & Ev., N. Am. Fungi, 2602, the specimens having been collected on living stems of Smilax in Louisiana by Mr. Langlois. Mr. Langlois communicated to me still better specimens on dead canes of Arundinaria. Тһе fructifications occur scattered here and there in grayish areas 2-4 mm. long by 1-1 mm. broad on the surface of the stems. Dr. Farlow informs me in a letter as the proofs are at hand that the above species is the lichen Heterothecium Augustini Tuckm. (To be continued.) [Vor. 1, 1914 382 ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 19 Тһе figures of this plate have been reproduced natural size from photo- graphsof dried herbarium specimens except in the cases noted otherwise. Fig. 1. Craterellus borealis. From the type specimen collected at Gready Island, Labrador, by Owen Bryant. Fig. 2. Cyphella galeata. From photograph, natural size, of the figure in Flor. Dan. pl. 2027. f. 1. Fig. 3. С. muscigena. Тһе two figures on the left are from specimens collected at Floodwood, New York, by E. A. Burt; the two on the right are from the type collection of Craterellus Pogonati collected at South Windsor, Connecticut, by C. C. Hanmer, 1956. Fig. 4. C.capula. From photograph, natural size, of the figure in Fung. Dan. 2: pl. 22. Fig. 5. C. minutissima. From the type specimens collected at Chocorua, New Hampshire, by W. G. Farlow, 3. Drawings of, a, two fructifications, X14; b, spores, x510; c, a hair from outer wall of fructification, x510. Fig. 6. C. Langloisii. From the type specimens collected at St. Martinville, Louisiana, by A. B. Langlois, ez. Drawings of, a, two fructifications, x17; b, spores, x510; c, a hair from outer wall of fructification, x510. Fig. 7. C. porrigens. From the type specimens collected at Cinchona, Jamaica, by W. A. and Edna L. Murrill, 607. Drawings greatly enlarged of, a, a fructification showing attachment to a piece of woody stem; b, diagrammatic section of the same fructification; c, two spores, x510. Fig. 8. С. сагісіпа. Three spores, X510, from the type specimen collected at Verona, New York, by C. H. Peck. Fig. 9. C. cupuleformis. From the specimens in Ravenel, Fung. Am., 224, collected at Darien, Georgia, by Ravenel. Drawings of, a, two fructifications, x6; b, а basidium, x510; c, four spores, x510. Fig. 10. С. texensis. Three spores, x510, from the type specimens collected in Texas, by C. Wright, 3779. Fig. 11. C. fumosa. Three spores, x510, from the type specimens collected at Aiken, South Carolina, by Ravenel, 3071. Fig. 12. C. mellea. From the type specimens collected at Bohemia, Louisiana, by A. B. Langlois, 864a. Photograph, a, of a piece of wood bearing many fructi- fications, and drawings of, b, median longitudinal section of a fructification, x60; с, three spores, x510; d, a hair from outer wall of fructification, x510. Fig. 13. C. villosa. Three spores, x510, from the specimens in Krieger, Fung. Sax., 1457, collected at Kónigstein, Germany, by W. Krieger. Fig. 14. C. Ravenelii. From the specimens in Ravenel, Fung. Am., 130, collected at Aiken, South Carolina, by Ravenel. Drawings of, a, a fructification on a piece of bark, x6; b, two spores, x510. Fig. 15. С. conglobata. From the type specimens collected аб Lower Bartlett, New Hampshire, by R. Thaxter. Photograph, a, of a portion of a branch bearing many clusters of fructifications, and drawings of, b, a median vertical section through one cluster of fructifications, x6; c, two spores, x510. Fig. 16. C. Tilie. From specimens collected at Middlebury, Vermont, by E. A. Burt. Photograph of, a, a piece of limb bearing many fructifications, and drawing of, b, three spores, x510. Fig. 17. C. fasciculata. From specimens collected at Ottawa, Canada, by J. Macoun, 23. Photograph of, a, a piece of bark bearing many fructifications, and drawings of, b, a cluster of fructifications, x6; c, three fructifications, x10; d, two spores, x510. ANN. Mo. Вот. Garp., Vor. 1, 1914 z PLATE 19 BURT— THELEPHORACEAE OF NORTH AMERICA 1. CRATERELLUS BOREALIS.—2. CYPHELLA GALEATA.—3. C. MUSCIGENA.— 4. C. CAPULA.— 5. C. MINUTISSIMA.—6. C. LANGLOISII.—7. C. PORRIGENS —8. C. CARICINA.—9. С. CUPULAEFORMIS.— 10 C. TEXENSIS.—11. C. FUMOSA.—12. C. MELLEA.—13. C. VILLOSA.—14. C. RAVENELII.— 15. C. CONGLOBATA.—16. C. TILIAE.—17. С. FASCICULATA. COCKAYNE, BOSTON. SOME CENOTHERAS FROM CHESHIRE AND LANCASHIRE:! R. R. GATES University of London Formerly Research Assistant to the Missouri Botanical Garden OBSERVATIONS (Enotheras are known to have been naturalized on the Lan- eashire coast since 1805, and probably existed there much earlier. They are now found on the sand dunes in many places, from Liverpool and the vicinity of Birkenhead northwards along the coast to Southport and Blackpool. They are not- ably abundant at St. Anne’s-on-Sea, where they have been described by Bailey (707), and in certain localities near Birken- head (MaeDougal '07). I have grown, chiefly at the Mis- souri Botanieal Garden, extensive cultures of plants from the latter region, from seeds obtained through Dr. D. T. MacDougal in 1907, and have visited the Lancashire coast in 1910 and again in July, 1914, when I travelled along the coast from Liver- pool to Southport and from Blackpool to St. Anne’s. The cenotheras everywhere appear to be spreading, although chil- dren gather the flowering shoots in armfuls. The profusion of individuals is greatest at St. Anne’s, where acres of waste land in the town are dotted over with them. Smaller colonies occur in various other places, notably at Bidston Junction, near Hightown and at Formby. Small groups of half a dozen plants are sometimes found in isolated places on the dunes. I will first refer to some of these colonies as I saw them during my last visit, and will then describe a few of the many forms observed in cultures. The Bidston Junction colony, referred to in MacDougal (97), is a compact and almost uniform one occurring on a triangular piece of ground between railway tracks, about five minutes’ walk down the foot path from Bidston Junction towards Wal- lersy, on the right-hand side. Some years ago, quantities of sand were dumped here from the coast between Wallersy and New Brighton. Soil from neighboring gardens has also been ! [ssued January 30, 1915. ANN. Мо. Bor. GARD., Vou. 1, 1914 (383) [Vor. 1 384 ANNALS OF THE MISSOURI BOTANICAL GARDEN deposited here, and the advent of the cenotheras is doubtless from one or other of these two sources. The plants closely resemble the “Isle of Wight" race of Œ. Lamarckiana (to be described in a book now in process of pub- lication) and the species as it generally appears in English gardens. Тһе rosettes in this colony differ in having green midribs (both dorsally and ventrally), or pink midribs (both dorsally and ventrally), but the depth of red varies. Тһе same applies to the stem-leaves. This is curiously different from other races, such as Œ. mut. rubrinervis, in which the midribs are red dorsally and green ventrally. Тһе rosette leaves are usually nearly or quite smooth, but some may be crinkled. The plants were short, their average height being about twenty- two inches, though some reached a height of over three feet. The stems bear many red papilla. The smaller plants were unbranched, the lower stem-leaves being closely crinkled and curled while the upper leaves and bracts are often quite smooth. A peculiarity of the race was the irregular disposition on the stem of much-crinkled and nearly smooth leaves, without gradual transitions between them such as usually occur in de Vries's гасе of (Е. Lamarckiana. Not infrequently crinkled and smooth leaves alternate. Тһе buds have fewer long hairs than in the above mentioned race, and the sepals have uniformly the red color pattern 5-7 of (Е. mut. rubrinervis, though they vary somewhat in depth of shade. Тһе dimensions of the flowers were as follows: bud cone 50 mm., hypanthium 43 mm., ovary 11 mm., diameter of cone at base 11 mm., length of petals 50 mm., width 60 mm. One plant was identical with the race of de Vries, except in its larger flowers, reddish sepals and fewer long hairs. In most plants there is also a strong distinction between the smooth and crinkled leaves. This colony differs, therefore, in minor peculiarities from any race of Œ. Lamarckiana previously observed, and it ex- hibits а relatively narrow range of variation. Along the electric railway tracks north of Liverpool, between Crosby and Hightown, an equally extensive and uniform colony of Œ. biennis was found. Thousands of plants, in flower and rosettes, Were growing on uncultivated land with a nearly pure sandy soil, behind the coast range of sand hills in a long narrow 19141 GATES—SOME (ENOTHERAS FROM CHESHIRE AND LANCASHIRE 385 area near а clump of small poplar trees. Near the upper end of this area the plants differed in having smaller flowers (petals 21 mm.) and narrow leaves (20 mm. broad). Тһе remainder of the plants had somewhat larger flowers (petals usually 25-27 mm. long), and broader leaves (extreme width 50 mm.).! This was almost the only variation observed, and the race comes very close to the type of Œ. biennis L. The dimensions of the buds were as follows: bud cone 20 mm., hypanthium 25 mm., ovary 11 mm., anthers surrounding the stigma. The rosette-leaves and stem-leaves all have red midribs both dorsally and ventrally. On the same stem some leaves are smooth and some more or less crinkled. The buds are green, devoid of red, with some long hairs, and there are no red рарШ on any part of the plant. Some of the larger plants are well-branched and with very stout stems, a huge pith and a very narrow ring of wood. This colony is even more uniform than the previous one, and must have originated from one or a very few plants. Small colonies of (E. biennis were seen at Formby, near the station and in other places. А race of Œ. Lamarckiana also grows here on the dunes, although I did not succeed in finding the spot, but local gardens cultivate it. The species is depicted, however, in a rose window erected in St. Luke’s Church, Formby, in 1898, containing representative plants of the local flora. The central portion of the window is divided hexagonally and in the six sections the evening primrose alternates with the sea holly. The foliage and large flowers of the former are distinctly shown. Around the margin of the window are Pyrola rotundi- folia and irises. At Blundell Sands, near Crosby, a small colony of Œ. Lamarck- jana was seen on waste ground, and again on the extensive sand dunes between Birkdale and Ainsdale, near Southport. In the latter case there were only three plants, and these pos- sessed red sepals, color pattern 7, green midribs, crinkled leaves, and about 2 long hairs. By far the greatest abundance of plants was found at St. 1These apparently correspond to Lysimachia virginiana altera, foliis latioribus, floribus luteis majoribus, Cat. Altdorff. See Gates, R. R. The mutation factor in evolution [pp. 61, 65, 70]. Macmillan. London. [Vor. 1 386 ANNALS OF THE MISSOURI BOTANICAL GARDEN Anne's. In addition to those in the town, which are in great profusion, numerous smaller colonies are scattered along the adjacent sand dunes. Тһе great majority of the plants is the same as at Bidston Junction except in the crinkling of the leaves, having foliage closely resembling that of de Vries's (Т. Lamarck- tana, midribs red both above and below, the red absent in some individuals. 'The flower measurements were, length of petals 50 mm., hypanthium 45 mm., ovary 10 mm. Several aberrant individuals were also observed. One dwarf mutant was found growing in the shade of a large plant. It resembled (Е. mut. nanella but had red midribs. One large rosette, having leaves very obtuse and pale pink midribs, probably belonged to (Е. mut. brevistylis. А number of plants represented a shorter spindling type with very narrow rosette-leaves (18 mm. wide X 14 em. long). Another plant belonged to a new type, large and branching with thicker, narrower leaves (33 mm. x 13 em.), stiffer and narrowly pointed, midribs white, and later in beginning to flower (buds only half developed, July 16). In addition to these probable mutants, there were found in one field à few plants of a small-flowered (E. biennis race grow- ing with the @. Lamarckiana. They differed from the latter only in the small flowers (petals 22 mm., style short), and hence were unlike the @. biennis race previously described. Near by were also found plants, evidently hybrids of these two races, with petals about 30 mm. in length. CULTURES Some of my cultures of cenotheras from near Birkenhead have already been described in a general way (Gates, 713). Here I wish to describe a few of these forms in detail, and also to refer to my experiments with plants from St. Anne’s. I have not seen the colony from which the Birkenhead seeds were obtained, but it evidently contains a great profusion of forms belonging to both Œ. Lamarckiana and Œ. grandiflora, while all the colonies I have observed have a much more uniform population. (GE. MULTIFLORA One of the distinct races in these cultures I have already (Gates, '10) referred to as Œ. multiflora. It is descended 1914] GATES—SOME CENOTHERAS FROM CHESHIRE AND LANCASHIRE 387 entirely from one individual from a sowing of Birkenhead seeds at Woods Hole in 1908. From this individual an F; of 376 plants was grown in the two following years. About 4 per cent of these plants showed virescence, as described in the above paper. In 1910 a total of 297 plants were grown, most of which belonged to the Ез. An Е; numbering 193 plants in nine fam- ilies was grown in 1911, and an F, of 356 plants in eight fam- ilies in 1912. Тһе plants were by no means uniform, and they varied considerably from year to year. Тһе description given is therefore a generalized one, and the condition of variability is no doubt similar to that of many wild "species." Ву isol- ating the offspring of а larger number of individuals, no doubt this variation could have been further analyzed, but more pressing problems have prevented this being done. Plate 20 fig. 1 shows a typical rosette of my 1909 culture, pl. 20 fig. 3 the full-grown plant, and pl. 20 fig. 6 а flowering shoot on a larger scale. Specimens of this species are preserved in the herbarium of the Missouri Botanical Garden from my cultures of 1909, and in the British Museum (Natural History) from the 1912 families. Description: Rosette of few leaves, broad and obtuse-pointed, somewhat crinkled. Full-grown plant pyramidal in outline, with lateral branches and persisting rosette leaves. Average height about 88cm. Stems slender, stem-leaves smooth, lance- olate, bracts broadly cuneate at base with a very short petiole, tip long-pointed, more or less curled, margin irregularly repand- denticulate. Inflorescence compact, flowers numerous; buds squarish, slender with very long and slender sepal tips, sepals thin, bud cone 35 mm. long, hypanthium 37 mm., sepal tips 7 mm., ovary 10 mm., petals 43 mm., very broad and overlapping when flower is open, long hairs fairly numerous. Few red papilla on main stem, many on side branches. In 1909 culture the buds were all green, but in 1911 they had the red color pattern of (E. mut. rubrinervis and the stems were also reddish. As regards variations, virescence appeared in the first two generations but not in the last two. On the other hand, a var. elliptica was first observed in F; and further studied in F; and F,. This variety differs essentially in being smaller and having narrower leaves and narrow, more or less elliptical petals. Plate [Vor. 1 388 ANNALS OF THE MISSOURI BOTANICAL GARDEN 20 fig. 2 shows a rosette of this variety in Е, ( 1911). One family of 50 plants in 1910 contained 5 of this variety. Usually these plants show partial variability, some flowers having broad petals and others narrow and elliptical ones. Even the differ- ent petals of the same flower may show these differences. Flowers with elliptical petals are invariably smaller and are fre- quently found on the side branches when those of the central stem have normal petals. Hence this variation may be a matter of strength in the plant. Тһе variation, from petals which are broad and truneate or emarginate to those which are narrow and elliptical, or even almost cruciate, is continuous. Thus on one plant in 1911, the dimensions of the petals in two flowers were as follows: Flower 1. Petal (1) 31 mm. x 21 mm. Petal (2) 25 mm. x 17 mm. Petal (3) 20 mm. x 12 mm. Petal (4) 22 mm. x 13 mm. In this flower the petals are very small and very unequal in size but all elliptical. Flower 2. Petal (1) 38 mm. x 39 mm. Petal (2) 37 mm. x 37 mm. Petal (3) 34 mm. x 36 mm. Petal (4) 35 mm. x 36 mm. In this flower the petals were nearly full size, nearly equal, and scarcely elliptical. The inheritance of this condition is on a sliding scale, plants with only broad petals giving some offspring with elliptical petals, and plants with elliptical petals giving some offspring having only broad petals, though in the latter case the plants bearing elliptical petals are more numerous than in the former case. Thus the F; family from a normal plant contained 14 specimens having broad petals only and 15 having some ellipt- ical petals; while another F; family of 44 plants derived from a plant having elliptical petals contained only 5 plants having exclusively broad petals. These peculiarities of the petals are probably to a large extent under the control of environmental features such as temperature and water supply. The difference between broad and narrow leaves is much sharper. Thus in my Е, cultures in 1912 certain families contain 1914) GATES—SOME (ENOTHERAS FROM CHESHIRE AND LANCASHIRE 389 both the broad or normal type (pl. 20 fig. 5) and the elliptica variety (pl. 20 fig. 4). The latter had a number of flowers with elliptical petals and it also had a different method of branching. Plate 21 fig. 12 is representative of a uniform F, culture of 49 plants of the variety elliptica. This photograph is taken on a larger scale, and the nodding of the stem is merely due to wilting. This differs from typica (pl. 20 fig. 5) constantly in having nar- rower leaves and short branches, as well as in the occasional elliptical flowers which appear to be largely under environ- mental control. The variability of this race is therefore as interesting as are the features, such as the general bud and leaf characters, in which it is constant. The fact should also be mentioned that a lata-like mutant, doubtless having 15 chromosomes, appeared in the Е, generation, and also a mutant resembling (Е. mut. albida. СЕ, RUBRINERVOIDES This race resembles Œ. mut. rubrinervis in many features, and yet differs from it constantly throughout. I have pre- viously referred to this Birkenhead race as No. 25 (Gates, '11, p. 350) and studied the variation of the red stripes on the buds. In all, 1968 plants of this race have been grown in the years 1909-1912, so that four generations of offspring from a single individual have been cultivated. An illustration of that indi- vidual has already been published (Gates, 712, pl. 3). One fam- ily of offspring was grown in 1909, two in 1910, eight in 1911 and nine іп 1912. Usually the variability of families progressively decreased, since each family was derived from the selfing of one individual of the previous generation. Тһе discussion of the precise ancestry of this race is of course out of the question, but its characters bear nearly though not quite the same rela- tion to the Œ. Lamarckiana from this region that the Lamarck- iana and rubrinervis of de Vries's cultures bear to each other. The 1909 family, or F;, numbered 111 plants. Plate 21 fig. 8 shows one of these as а rosette. Тһе leaves are narrower and more pointed than in mut. rubrinervis, and nearly smooth. About 20 of the plants in this culture omitted the rosette stage altogether and shot up a stem directly from the seedling stage (pl. 20 fig. 7). A normal mature plant of this family is shown 3 [Vor. 1 390 ANNALS OF THE MISSOURI BOTANICAL GARDEN in pl. 21 fig. 11. It will be seen that there is no indication of a rosette, and the branching is quite different from that of Œ. mut. rubrinervis. In many cases, however, a rosette is formed. When the rosette is omitted the branching is changed. Plate 21 fig. 10 shows on a larger scale another individual in flower. The stem-leaves differ from those of GZ. mut. rubrinervis in being narrower, more pointed and smoother. In this race the red papille on the stem were very numerous, and the buds likewise were slightly more red than in (7. mut. . rubrinervis. The modal color pattern of the whole population was 6 as in Œ. mut. rubrinervis, but plants with their mode at 7 were much more numerous than in the latter (see Gates, '11, p. 351). The race as à whole inherited the capacity for pro- ducing a slightly greater amount of pigment. The ovary usu- ally bore many long hairs arising from red papille; on the hy- panthium were few long hairs from slight green mounds; and on the bud cone scattered long hairs from conspicuous red papille. In occasional buds, when the color pattern was only 8, the green papille were more numerous. Іп addition to the color pattern of the sepals there was usually weak red on the hypanthium.! The same conditions as regards pigmentation have been main- tained in later generations. Тһе plants were, however, by no means uniform in all respects, and this was not to be expected since they were derived from one individual of a freely inter- crossing population. Plate 21 fig. 9 represents a rosette of one of the Е, plants. The latter differs obviously from the one represented in pl. 21 fig. 8, but the race retained in this and subsequent generations the long, narrow, smoothish leaves as well as the pigmentation. The various Ез and Е, families, each derived from a selfed individual, produced sub-races differing more of less from each other and varying within narrower limits. It does not appear that the Mendelian theory of the sorting out of factors, or "genes," affords an adequate explanation of all these phenomena. 1 Since this condition of bud-pigmentation resembles that obtained in certain F4 and Ез hybrids of Œ. mut. rubricalyz and Œ. grandiflora (see Gates '14), it is possi- ble that it may have arisen in a similar way, i. e., by the appearance of a red-budded mutation which subsequently crossed with other species, in which crosses some blend- ing of pigmentation occurred giving rise to the present condition. 19141 GATES— SOME (ENOTHERAS FROM CHESHIRE AND LANCASHIRE 391 (E. TARDIFLORA This name I have used for another race having many peculiar- ities and showing more resemblance to Œ. grandiflora in its flowers and foliage. It is race No. 52 from the same source as the above. А single individual produced in 1909 nineteen plants which were fairly uniform. Тһе rosettes contained only a few leaves, but large plants were formed, one of which is shown in pl.22 fig. 17. Although this photograph was taken on August 21, the plants with one exception had not begun to flower. The leaves resembled those of @. grandiflora. They were large with long and acute tips, tapering to the bases, often: bearing reddish blotches, sometimes much curled, somewhat crinkled along the midrib. The margin was conspicuously serrately toothed (see pl. 22 fig. 17). At the end of the season (Septem- ber) these plants came into bloom, and pl. 22 fig. 20 shows a plant photographed on October 2. The buds resembled those of (Е. grandiflora but were small. The bud cones were pointed, smooth and rounded, the petals slightly larger than in @. bien- nis, or in a few cases much larger. The petals were also deeply emarginate, strongly cuneate and narrow; and the bracts were very small, narrowly lanceolate and yellowish, giving a peculiar appearance to the flowering shoot. The margins of the bracts were nearly entire or in some cases distantly denticulate. The offspring of the plant in pl. 22 fig. 20 were grown and showed the same peculiarities. The race has not been culti- vated further. It was doubtless of hybrid origin and was more nearly allied to G7. grandiflora than to the Lamarckiana complex. CE. RUBRITINCTA Reference may be made to one further race which was known as type M.’’ It originated from one plant in a sowing of the Birkenhead seeds in 1909. It will be understood that scarcely two plants from this sowing were alike, but some were much more distinct than others. Тһе plant in question was a hand- some one with very narrow leaves and bright red midribs. Its offspring, grown in 1911, were lost with the exception of one plant which was the same as the parent. It is shown in pl. 22 fig. 16. Тһе basal leaves were very long with long petioles, the stem leaves very narrow, smooth, with margin closely repand- [Vor. 1 392 ANNALS OF THE MISSOURI BOTANICAL GARDEN denticulate, blade narrowing gradually to a very short petiole, midribs and petioles bright red dorsally and ventrally; lower- most bracts 17 mm. in width by 9 em. in length, upper bracts 11 mm. wide by 58 mm. in length. .The buds most resemble those of (E. grandiflora, being nearly devoid of long hairs, slender and somewhat rounded, with setaceous sepal tips and some red on the sepals; length of petals 32 mm., hypanthium 43 mm., sepal tips 9 mm., ovary 10 mm. In 1912 three families of Е, offspring, numbering in all 236 plants, were grown from the plant just described. АП three families agreed in containing several types exhibiting a remark- able degree of variability. An attempt was made to place the plants in five classes, but the categories overlapped and made classification for the most part impossible. Тһе majority of the plants resembled the parent individual in their main features but they varied enor- mously in width of leaf from broad (21 mm.) to very narrow (8-6.5 mm.). These conditions were connected by interme- diates, and, moreover, there were considerable variations within the individual, one braneh with very narrow leaves being found on a plant with broad leaves. In addition to these variants, the three families contained 35 dwarfs, or 14.8 per cent, and the latter varied in leaf-width in the same remarkable manner. The dwarfs agreed only in having short internodes. Two of them are shown in pl. 21 figs. 13, 14, the former having narrow leaves and extremely short internodes, the leaves of the latter being quite linear. Тһе plant would never be taken for an cenothera. Тһе advent of a large percentage of dwarfs in this family is similar to their occurrence in other G7. grandiflora races from that loeality (see Gates, '14, p. 246). Тһе precise manner in which this capacity for producing dwarfs is inherited, is a diffi- cult question which need not be considered here, particularly as it has been discussed elsewhere (Gates, '14). Plate 22 fig. 15 represents one of the Lamarckiana-like rosettes from this source, grown in 1909. Others approached de Vries’s race more closely, to the point of identity. Plate 22 figs. 18, 19 represent selected rosette-leaves taken from this cul- ture to show the range of types exhibited. Such leaves as the 19141 GATES—SOME (ENOTHERAS FROM CHESHIRE AND LANCASHIRE 393 two on the right in pl. 22 fig. 18 were greatly overgrown and were far larger than ever appear even in Œ. mut. gigas. These forms have not been sufficiently studied since to give an ade- quate account of them. It will be obvious that the forms described here under the names multiflora, multiflora elliptica, rubrinervoides, tardiflora and rubritincta are not pure species or even true-breeding races. They are undoubtedly as diverse from each other as average species, however, and many systematie species if bred experi- mentally would probably not breed true within narrower limits than these races have done. One feature of interest attaching to these races is the fact that the main type persists essentially unchanged, though various mutants and heterozygous forms are thrown off. The behavior is not, in the main, like the Men- delian process of recombination. Repeated selfing of each race usually decreases its variability by eliminating various hybrid elements. But this process does not extend to the basal differ- ences between the races, which, as we have seen, remain as unlike as they were before. In this aspect the hereditary behavior of these races resembles that of Œ. Lamarckiana. But there are a number of differences which I need not fully consider. Thus (E. multiflora gives rise to its variety elliptica much as though it were split off from a heterozygous condi- tion, and the variability of rubritincta in leaf-width, as well as its production of numerous dwarfs, is unlike anything in the behavior of (Е. Lamarckiana. Many other equally distinct types were derived from this locality (see, e. g., pl. 22 figs. 18, 19), but they have not been cultivated in subsequent generations. Œ. LAMARCKIANA FROM ST. ANNE'S Іп 1910 I obtained seeds from a colony of Œ. Lamarckiana growing by the Manchester Children's Hospital Convalescent Home, at St. Anne’s-on-Sea. Many of these were found in later cultures to agree exactly with the Lamarckiana of de Vries except in the red color pattern of the sepals. I was formerly in- clined to lay little stress on this difference but there is no doubt that it is inherited. Тһе fact therefore remains that a precise duplicate for de Vries's race of Œ. Lamarckiana is relatively [Vor. 1 394 ANNALS OF THE MISSOURI BOTANICAL GARDEN infrequent on the Lancashire coast, although many forms ap- proach it very closely and differ only in this one feature. As will be seen below, certain other plants agreed with de Vries's Lamarckiana except in the shape of the buds. In 1911 a sowing of the seeds yielded 22 plants. "The rosettes were for the most part uniform and very similar to (E. La- marckiana, two, however, having red midribs and lighter green leaves (rubrinervis type). One plant was aberrant, resembling Œ. mut. semilata in its buds, which were, however, small as in (Е. biennis. Тһе bud cone was also somewhat rounded and barrel-shaped, length of ovary 11 mm., hypanthium 37 mm., cone 19 mm., petals 22 mm., style short so that anthers sur- round base of stigma. The features of this plant make it scarcely likely that it arose as a hybrid. It produced plenty of pollen and seeds. | Another sowing of these seeds in 1912 yielded 140 plants, which ineluded one mut. lata with bad pollen (doubtless having 15 chromosomes) and one variegated Lamarckiana plant. The variegation was noticed when the plant was a young seedling. It reached maturity and proved to be a periclinal chimera. Nearly all the leaves were variegated green and yellow. Many leaves were green bordered with yellow, showing the absence of chloroplasts from the epidermal and probably also the hypo- dermal layer. Occasional leaves were almost entirely yellow, and some were yellow on one side of the midrib and green on the other. There were also broad white bands on the margin of the sepals. The pollen was abundant and plenty of seeds were set. Two sowings of seeds from this plant were made in 1912. The seeds numbered respectively 121 and 145. Only two seeds in one pan were observed to germinate, and the seedlings quickly died, probably from lack of chlorophyll. Regarding the origin of this periclinal mutation, it would appear to have originated in the embryo after fertilization through the loss of chloroplasts from the outer layers of the growing point. The foliage in the rest of the culture agreed with the type of (Е. Lamarckiana. One plant differed in having stem-leaves more or less pointed at the base, not crinkled, midribs pink, and smaller flowers (petals 29 mm. long x 38 mm. broad, style short, buds 19141 GATES—SOME (ENOTHERAS FROM CHESHIRE AND LANCASHIRE 395 squarish). Two other plants agreed exactly with Œ. Lamarck- iana except in the buds. The petals were 35 mm. long x 48 mm. broad, emarginate, anthers reaching nearly to top of stigma lobes, sepals green and with the same pubescence as in Œ. Lamarckiana, from which these two plants therefore differed only in the somewhat smaller flowers and shorter style. One mut. nanella also occurred in this culture, and several other slightly aberrant individuals, including a plant with broadly elliptical foliage. The ‘‘Lamarckiana foliage” was also more variable than in cultures from de Vries, this no doubt being due to the continued inbreeding in the latter case. It will be understood that the new forms described here are scarcely to be looked upon as ‘‘new species” according to the usual interpretation at the present time. They merely represent a partial analysis of a complex interbreeding colony of forms, and their variability is one of their most interesting features. Nearly all if not all the differences observed are inherited, how- ever, and the mutations can in many instances be separated from the characters arising through hybridization. The forms are, moreover, as distinct from each other as many species of (Enothera. In conclusion, I am indebted to the Missouri Botanical Garden and the John Innes Horticultural Institution for the facilities provided for growing the plants, and to Mr. Е. J. Allard for several of the photographs. А portion of the expenses of my second visit to Lancashire was defrayed by a grant from the Royal Society. LITERATURE CITED Bailey, С. (07). De Lamarck’s evening primrose (GZnothera Lamarckiana) on the sandhills of St. Anne's-on-the-Sea, North Lancashire. (Address, Annual Meeting Manchester Field Club.) 1-28. pl. 1—6. 1907. Gates, В. В. (710). Abnormalities in (Enothera. Rept. Mo. Bot. Gard. 21: 175-84. pl.29-31. 1910. , (11). Studies on the variability and heritability of pigmentation in Gino- thera. Zeitschr. f. induct. Abst.- u. Vererbungsl 4: 337-72. рі. 6. f. 1-6. 1911. ‚ (12). An onagraceous stem without internodes. New Phytologist 11: 50-53. pl. 2-8. 1912. , (13). A contribution to a knowledge of the mutating cenotheras. Trans. Linn. Soc. Bot. 8: 1-67. pl. 1-6. 1913. [Vor. 1, 1914] 396 ANNALS OF THE MISSOURI BOTANICAL GARDEN Gates, К. В. (14). Breeding experiments which show that hybridization and mutation are independent phenomena. Zeitschr. f. induct. Abst.— u. Vererbungsl. 11: 209-79. f. 1-25. 1914. MacDougal, D. T., Vail, A. M., and Shull, G. H. (07). Mutations, variations, and relationships of the cenotheras. Carnegie Inst. of Washington Publ. 81: 1-92. pl. 1-22. f. 1-78. 1907. EXPLANATION OF PLATE PLATE 20 Fig. 1. (E. multiflora, rosette, 1909. Fig. 2. (%. multiflora elliptica, rosette, 1911. Fig. 3. Œ. multiflora, full-grown plant, 1909. Fig. 4. (E. multiflora elliptica, 1912. Fig. 5. Œ. multiflora, 1912. Fig. 6. (E. multiflora, flowering shoot, 1909. Fig. 7. (Е. rubrinervoides, young plantlet showing absence of rosette, 1909. Ann. Mo. Вот. GARD., VoL. 1, 1914 PLATE 20 GATES—OENOTHERAS COCKAYNE. BOSTON. 898 Кір. Гір. Кір. Fig. Fig. Fig. Fig. [Vor. 1, 1914 ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 21 (Е. rubrinervoides, rosette, 1909. (E. rubrinervoides, rosette, 1910. (Е. rubrinervoides, showing nearly smooth, pointed leaves, 1909. (Е. rubrinervoides, no rosette, 1909. (E. multiflora elliptica, 1912. (Tip of plant drooped from wilting.) Linear-leaved dwarf in offspring of (E. rubritincta, 1912. Dwarf offspring of Œ. rubritincta, 1912. PLATE 21 ANN. Mo. Bor. GARD., Vor. 1, 1914 ERAS S—OENOTH ATE * y C 400 Кір. 15. Fig. 16. Fig. 17. 21, 1909. Fig. 18. Fig. 19. Fig. 20. [Vor. 1, 1914] ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 22 (Е. Lamarckiana-like rosette, 1909. СЕ. rubritincta, 1911. (Е. tardiflora, showing serrated leaves and absence of flowers, August Selected leaves from various rosettes, 1909. Selected leaves from various rosettes, 1909. (Е. tardiflora, showing late appearance of buds, October 2, 1909. ANN. Мо. Вот. GARD., Vor. 1, 1914 PLATE 22 GATES—OENOTHERAS COCKAYNE, BOSTON. А TEXAN SPECIES OF MEGAPTERIUM' R. R. GATES University of London Formerly Research Assistant to the Missouri Botanical Garden While looking over some material in the herbarium of the Missouri Botanical Garden, a sheet was found containing three specimens which were so distinctive that it seemed desirable to describe them. The interest in them was enhanced by the fact that one of the specimens differs strikingly from the other two in such a way as to suggest that it may be a mutation. The plants in question were collected at Amarillo Creek, in Northern Texas, by J. Reverchon, who had recognized them as representing a new species of Megapterium. I am indebted to Dr. Greenman for suggesting а very appro- priate name for this species. Тһе diagnosis is as follows: Megapterium argyrophyllum, sp. nov. Plate 23. figs. 1 and 2. Herba cespitosa; foliis lanceolatis, petiolatis obseuré gland- uloso-denticulatis, utrinque dense canescento-pubescentibus; caulibus et alabastris (hypanthio et ovario incluso) canescente pubescentibus; ovarium quadrialatum, pedicellatum; hypan- thium 9-10 cm. longum, paulatim ad basin coni dilatum; petala 3-4 cm. longa. Var. retusifolium, var. nov. Plate 23. fig. 3. A forma typica differt foliis subrotundis bis oblongo-obo- vatis, retusis, mucronatis; flore grandiore (petala 45 mm. longa). Specimens examined: Texas: on rocky bluffs at Amarillo Creek, in northern Texas, 29 May, 1902, J. Reverchon, 2749 (Mo. Bot. Gard. Herb.), TYPE; stony bluffs along Red River, Randall Co., northern Texas, 12 August, 1900, H. Eggert (Mo. Bot. Gard. Herb., 4 sheets). Two of the specimens, one slightly older than the other (see pl. 23 fig. 1, 2), represent the type of the species. Тһе plants are esspitose or with very short internodes, leaves coriaceous, lanceolate, broad-pointed, tapering below to a petiole, about 1 Issued January 30, 1915. Ann. Mo. Вот. GARD., Vor. 1, 1914 (401) [Vor. 1 402 ANNALS OF THE MISSOURI BOTANICAL GARDEN 8 сіп. long by 2 em. in greatest width, margin distantly and obscurely glandular-denticulate, very densely and uniformly covered on both surfaces with an appressed canescent pubescence of long, pointed, tuberculate hairs. Stems and buds less densely covered with the same type of pubescence, ovary four-winged, 10-15 mm. in length, densely canescently pubescent, pedicel- late; hypanthium 9-10 cm. in length, 2-2.5 mm. thick, grad- ually widening to base of cone; bud cone 30-35 mm. in length, diameter at base 8 mm., sepal tips appressed, 3-4 mm. in length, petals 3-5 em. long, stigma surrounded by or slightly exceeding the stamens; capsules immature. The remarkable canescent pubescence covering the whole plant, as well as the cæspitose habit, distinguish this species from Megapterium missouriensis (Sims) Spach, and M. macro- carpum.' The flowers are also smaller, there are no purple spots on the sepals, and the hypanthium is shorter than in these species, which differ in foliage as well. Тһе present species is apparently perennial. Its nearest relative is M. Fremontii (Watson) Britton, from which it differs in the more esspitose habit, larger flowers, and much broader leaves. The variety retusifolium is founded on the third specimen on the sheet (see pl. 23 fig. 3). It differs sharply from the species in the shape of the leaves, which are very broad and blunt at the point, subrotund to oblong-obovate, retuse, and distinetly mucronate. The margin of the leaves is also nearly or quite entire. Тһе flowers are larger (petals 45 mm., bud cone 9 mm. in diameter at base). Microscopic examination of the hairs disclosed considerable variation in size, but apparently no con- stant difference from those of the species. The Eggert specimens, while obviously belonging to the same species, show much more variability in foliage. Тһе leaves vary on different specimens from narrowly lanceolate (9 mm. in width) to broad oblong-lanceolate (30-36 mm. wide) and acuminate. Тһе latter resemble var. retusifolium except the leaf tips, which are only slightly retuse in one specimen. One of the broad-leaved specimens also has a smaller flower (petals 20 mm.). Cultures from seeds from this locality would doubt- ! Megapterium macrocarpum (Pursh), comb. nov. Gnothera macrocarpa Pursh, Fl. Am., Sept. 2: 734. 1814. 1914] GATES—A TEXAN SPECIES OF MEGAPTERIUM 403 less disclose a considerable number of forms. The ripe fruits from these specimens are broadly winged, nearly orbicular, about 35 mm. long and 25 mm. wide, retuse or acuminate at the apex. Examination of herbarium specimens of M. missouriensis (Sims) Spach makes it evident that the polymorphism in this species as now understood is quite as great as in many species of GEnothera. There are included races varying in amount and character of pubescence, in width of leaf from broadly lanceolate to almost linear, in presence or absence of purple spots on the sepals, in size of flower, and other features. [Vor. 1, 1914] 404 ANNALS OF THE MISSOURI BOTANICAL GARDEN ExPLANATION OF PLATE PLATE 23 Figs. 1 and 2. Megapterium argyrophyllum. From the type specimens, J. Rever- chon, No. 2749 in part, in the Herbarium of the Missouri Botanical Garden. Fig. 3. М. argyrophyllum var. retusifolium. From the type specimen, J. Rever- chon No. 2749 in part, in the Herbarium of the Missouri Botanical Garden. Ann. Mo. Вот. GARD., Vor. 1, 1914 PLATE 23 GATES—MEGAPTERIUM COCKAYNE, BOSTON. DIAGNOSES ОҒ FLOWERING PLANTS, CHIEFLY FROM THE SOUTHWESTERN UNITED STATES AND MEXICO! J. M. GREENMAN Curator of the Herbarium of the Missouri Botanical Garden Associate Professor in the Henry Shaw School of Botany of Washington University AND C. H. THOMPSON Assistant Botanist to the Missouri Botanical Garden The present paper is the result of a study of several collec- tions of plants from the southwestern United States and Mexico, especially the relatively large series of specimens secured by Mr. Harley P. Chandler at Rio Hondo, Texas, and by Mr. Charles Russell Orcutt along the Texas-Mexican boundary and in vari- ous parts of Mexico. These collections have been received at the Missouri Botanical Garden for identification and incidental to the work thereto the following plants seem to the writers to be worthy of record and characterization. Anthericum (Hesperanthes) Chandleri Greenman & Thomp- son, sp. nov. Fibre radicales carnoss apice clavate, collo radicis parce fibroso; foliis plurimis 12-15 graminoideis planis lanceolato- linearibus sensim attenuatis acutis 3.5-4.5 dm. longis 7-10 mm. latis circiter 24-nerviis cum venis transversis conjunctis utrinque glabris integerrimis; scapo 1 m. alto tereti glabro bracteato, bracteis plus minusve foliiformibus sursum gradatim reductis; inflorescentiis panieulatis usque ad 3.5 dm. longis glabris, racemo terminali 2-2.5 dm. longo, racemis lateralibus 1-1.5 dm. longis, bracteis triangulari-aeuminatis vel lanceolato-attenuatis sub- всагіовів 3-20 mm. longis; floribus 2-4 in axillis bractearum; pedicellis 10-12 mm. longis infra medium articulatis; perianthio pallido-flavo vel stramineo, laciniis oblongo-lanceolatis triner- viis circiter 1 cm. longis; staminibus perianthio duplo breviori- bus, filamentis muricatis; stylo 8 mm. longo glabro; capsula matura ignota. 1 [ssued January 30, 1915. ANN. Мо. Вот. GARD., Vou. 1, 1914 (405) 4 [Vor. 1 406 ANNALS OF THE MISSOURI BOTANICAL GARDEN Specimen examined: Texas: vicinity of Rio Hondo, Cameron County, September, 1918, Harley P. Chandler, 7059 (Mo. Bot. Gard. Herb.), TYPE. This species belongs to the subgenus Hesperanthes according to Baker's treatment of this group (Jour. Linn. Soc. Bot. 15: 253-363. 1876); it is apparently most nearly related to А. stenocarpum Baker, а co-type of which is in the herbarium of the Missouri Botanical Garden, from which it is readily distin- guished by the broader leaves, entire leaf-margins, the presence of anastomosing cross-veins, and by the leafy scape and yellow flowers. Zephyranthes chrysantha Greenman & Thompson, sp. nov. Bulbus subglobosus 2-2.5 em. diametro tunicis brunneo- nigrescentibus vestitus, collo 3-5 em. longo 6-8 mm. diametro; foliis 2-4 sub anthesi evolutis linearibus 2.5-4.5 dm. longis 2-3 mm. latis glabris; scapis 2-3 dm. altis glabris; spatha mem- branacea 2.5-3.5 ст. longa inferne tubulosa, tubo 1-1.5 em. longo, lobo unilaterali lanceolato 1.5-2 cm. longo; pedicellis 2.5-3.5 ст. longis gracilibus; perianthio infundibuliformi 3-3.5 em. longo flavo 6-lobato, tubo cylindraceo circiter 5 mm. longo, lobis oblanceolatis 3-3.2 ст. longis 5-12 mm. latis acutis staminibus ad apices tubi perianthii insertis segmentis perian- thii duplo brevioribus; stylo brevitrilobato staminibus subæ- quantibus; capsula depresso-globosa 10-12 mm. longitudine et diametro, seminibus numerosis irregulariter compressis 5-6 mm. longis 2-5 mm. latis atratis et sæpe nitidis. Specimen examined: Texas: Rio Hondo, Cameron County, September, 1913, Harley P. Chandler, 7056 (Mo. Bot. Gard. Herb.), түре. The species here characterized is allied to Z. Eggersiana Urb., particularly in the size and color of the flowers, but differs in having more numerous and broader leaves, shorter perianth- tube and longer spathes. Sisyrinchium angustissimum (Rob. & Greenm.) Greenman & Thompson, comb. nov. Plate 24. S. alatum Hook. var.? angustissimum Rob. & Greenm. Am. Jour. Sci. 50: 166. 1895. Radices carnoso-fibrosi fasciculati; caulibus erectis strictis vel 1914) GREENMAN AND THOMPSON— FLOWERING PLANTS 407 subflexuosis 2.5-9 dm. altis multo-ramosis angustissime ancipiti- alatis foliosis glabris vel obscure hirtello-puberulentis basi reliquiis brunneis fibrosis squamarum et foliorum primorum obtecto; foliis radicalibus linearibus gramineis usque ad 4.5 dm. longis 1-4 (rarius 6) mm. latis crebrenerviis glabris vel margin- ibus hirtellis, eis caulinis conformibus sed sursum gradatim reductis; spatha diphylla, bracteis foliiformibus 1.5-2 cm. longis glabris marginibus plus minusve purpurascentibus, pedicellis 2-4 ex eadem spatha 1.5-2.7 cm. longis gracilibus glabris; perian- thio profunde 6-partito verisimiliter flavo, lobis ovato-ellipticis acutis vel emarginatis et submucronatis 5-7-nerviis; ovario oblongo-obovato juventate sepe pubescenti glabrato; capsula matura oblonga 5-10 mm. longa 4-6 mm. diametro glabra, seminibus subglobosis circiter 1.5 mm. diametro in sicco nigres- centi. Specimens examined: Mexico: State of Oaxaca, Sierra de San Felipe, altitude 2895 m., 22 June and 29 August, 1894, C. G. Pringle, 4703 (Mo. Bot. Gard. Herb.), со-түреЕ; Sierra de San Felipe, altitude 8048 m., August-September, 1894, Charles L. Smith, 758 (Mo. Bot. Gard. Herb.). State of Morelos, lava beds above Cuernavaca, altitude 2590 m., 19 November, 1902, C. G. Pringle, 11191 (Mo. Bot. Gard. Herb.). State of Puebla, vicinity of San Luis Tultitlanapa, near Oaxaca, June, 1908, C. A. Purpus, 3356, 3357 (Мо. Bot. Gard. Herb.). After a careful reéxamination of the original material on which this variety was based, particularly in the light of addi- tional specimens from subsequent collections, it seems unde- sirable to retain the plant as a variety of 8. alatum Hook. Mr. Hooker's species was founded on specimens collected in Deme- rara, British Guiana, by Dr. Hancock; and specimens secured by Mr. Gardner in the Organ Mountains of Brazil and by Tweedie on the marshes of the La Plata River were considered conspecific. While the writers have not seen any of these specimens, yet from the original description and the illustration accompanying it that species is interpreted as having a broadly winged stem, short and relatively broad ensiform leaves, and broad spathes. These characters can not be applied properly [Vor. 1 408 ANNALS OF THE MISSOURI BOTANICAL GARDEN to the Mexican plant in question. It seems advisable, there- fore, to regard the south Mexican plant as a distinet species which may be further characterized as above. OECOPETALUM Greenman & Thompson, gen. nov. Jcacinacee Calyx 5-lobus. Petala 5 hypogyna valvata intus costata, margine et apice inflexa. Stamina 5 hypogyna petalis alterna et iis basi coherentia, filamentis dilatis glabris apice contractis; anther erect: lanceolate basi sagittatæ connectivo latiusculo; thece laterale remote et in cavitatibus petalorum recepte. Discus obsoletus. Ovarium uniloculare, stylus erectus conicus, stigma terminale. Ovulum 1 pendulum. Fructus ignotus.— Frutices vel arbores. Folia alterna coriacea integerrima. Flores cymis brevibus axillaribus dispositi. О. mexicanum Greenman & Thompson, sp. nov. Plate 25. Frutex (?) vel arbor (?); ramis cortice griseo tectis; ramulis juventate sericeo-pubescentibus mox glabratis; foliis alternis petiolatis elliptico-lanceolatis 1-2,5 dm. longis 3.5-10 ет. latis brevi-acuminatis obtusis integerrimis utrinque glabris vel prae- sertim in nerviis sparsissime adpresso-puberulentis subtus palli- dioribus basi sensim angustatis acutis, petiolis 7-15 mm. longis supra canalyculatis; inflorescentiis in axillariis superioribus cymosis plus minusve adpresso-sordido-pubescentibus, pedun- culo usque ad 2 em. longo; floribus cum pedicello articulatis et caducis; calyce griseo-tomentoso parvo circiter 2 mm. alto 5-lobato, lobis ovatis obtusis 1 mm. longis; petalo 5 oblongo- lanceolato 8 mm. longo 2 mm. lato verisimiliter albo utrinque glabro intus longitudinaliter insigniter unicostato; ovario et stylo glabro; fructu et seminibus ignotis. Specimen examined: Mexico: State of Vera Cruz, Sierra Madre near Miscantla, August, 1912, С. А. Purpus, 6159 (Mo. Bot. Gard. Herb.) TYPE. Specimens of the plant here described were submitted to the Missouri Botanical Garden for determination by Mr. Т. 8. Brandegee who suggested its probable relationship with Mappia. After a careful study of the material at hand it seems unmis- takably to belong to the Icacinaceae, but until the fruit is known its exact position in the family must remain doubtful. 19141 GREENMAN AND THOMPSON—FLOWERING PLANTS 409 In habit and in the structure of the flower it possesses certain characters in common with Mappia, Kummeria and Poraqueiba, but in a combination of the floral characters, particularly in the free or merely coherent glabrous and strongly ribbed petals, the broad smooth filaments, elongated anthers, which in cross section are distinctly x-shaped, and in the single suspended ovule the plant in question differs from the genera above men- tioned. Generic rank is therefore given to it and we propose the name Oecopetalum, from oikos house and zéradoy petal, in reference to the little recesses or pockets formed by the adjacent petals in which the anthers rest. Stemodia linearifolia (Morong) Greenman & Thompson, comb. nov. Stemodiacra linearifolia Morong, Ann. N. Y. Acad. Sci. 7:183. 1893. Stemodia tomentosa (Mill. Greenman & Thompson, comb. nov. Erinus tomentosus Mill. Dict. 1768. [8th ed.]— Herpestes tomentosa Schlecht. & Cham. Linnea 5: 106. 1830.—Stemodia lanata Ruiz & Pav. in DC. Prodr. то: 383. 1846; Hemsl. Biol. - Cent.-Am. Bot. 2:450. 1882.—Stemodiacra tomentosa О. Kuntze, Rev. Gen. 2: 466. 1891. Siphonoglossa Greggii Greenman & Thompson, sp. nov. Suffruticosa; caulibus erectis vel adscendentibus 0.5-2 dm. longis subcylindratis et sepe quadrisulcatis pubescentibus in lineis decussatis cum pilis reflexis; foliis oppositis brevipetio- latis lanceolatis vel obovatis 0.5-2.5 em. longis 3-7 mm. latis acutis vel obtusis vel rotundatis integris basi in petiolum grada- tim angustatis supra glabris subtus paulo pallidioribus juventate secundum nervos venasque adpresso-puberulentis; floribus in axillis supernis solitariis sessilibusque, bracteis subspathulatis; calyce profunde 5-partito 4 mm. longo, laciniis lineari-lanceolatis glabris; corolla 1.5-2 cm. longa bilabiata, labio anteriore horizon- taliter patenti trilobulato, labio posteriore suberecto emarginato, tubo gracili 9-14 mm. longo extus pubescenti; ovario et stylo glabro; capsula circiter 7 mm. longa glabra, seminibus suborbic- ularibus compressis verrucosis circiter 2 mm. diametro. Specimens examined: Mexico: State of Tamaulipas, Matamoras, 7 June, 1847, Dr. J. [Vor. 1 410 ANNALS ОҒ THE MISSOURI BOTANICAL GARDEN Gregg, 916 (Mo. Bot. Gard. Herb.), Tv PE; Cervallo, 29 Мау, 1847, Dr. J. Gregg, $45 (Mo. Bot. Gard. Herb.). Texas: Rio Hondo, Cameron County, September, 1913, Harley P. Chandler, 7081 (Mo. Bot. Gard. Herb.). The species here proposed is nearly related to S. Pilosella Torr. from which it is distinguished by the pubescence of the stem, namely reflexed hairs disposed in decussating lines, somewhat narrower leaves, and uniformly shorter fruit. Siphonoglossa Pilosella Torr. Bot. Mex. Bound. 124. 1859. This species is well represented in the herbarium of the Mis- souri Botanical Garden by a suite of more than thirty specimens. To it should be referred one of Lindheimer’s Texas plants, namely number 1065, collected in 1851, which by clerical error was distributed as “Ruellia Parryi Gray." Randia Gaumeri Greenman & Thompson, sp. nov. Frutex ramosus; caule ramisque cortice griseo glabro tectis; spinis axillaribus usque ad 1.5 em. longis divaricatis; foliis obovatis 0.5-1.5 em. longis apice plerumque rotundatis integris basi in petiolum marginatum contractis utrinque glabris vel supra in nervis ad basin puberulentis; floribus axillaribus sessi- libus; calyce toto 1-1.5 mm. longo 4-lobato glabro; lobis triangu- laribus acutis ciliatis; corolla hypocraterimorpha parva 4-lobata, tubo cylindraceo circiter 2.5 mm. longo extrinsecus glabro, lobis contortis ovatis tubo subaequantibus; antheris ad faucem сого sessilibus exsertis; ovario biloculari; Басса ignota. Specimen examined: Mexico: State of Yucatan, at Izamal, coll. of 1895, Dr. Geo. F. Gaumer, 589 (Mo. Bot. Gard. Herb.), түре. The divarieately spreading axillary spines, relatively small obovate leaves, and the minute flowers amply distinguish this species from all others of the genus. It is with pleasure that the authors dedicate this new species to Dr. Gaumer, who has done so much to further our knowledge of the flora of Yucatan. Randia Purpusii Greenman & Thompson, sp. nov. Verisimiliter frutex; ramis ramulisque cortice brunneo vel griseo tectis; spinis ad apices ramorum plerumque quaternis vel binis, vel rarius nullis, 3-6 mm. longis; foliis lanceolatis vel obovato-lanceolatis 1.5-5.5 em. longis 0.8-2 cm. latis obtusis 1914) GREENMAN AND THOMPSON—FLOWERING PLANTS 411 vel acutis integris basi in petiolum marginatum gradatim angus- tatis supra hirsutis subtus paulo pallidioribus et subtomentosis; stipulis triangulari-ovatis utrinque pubescentibus; floribus sessil- ibus axillaribus terminalibus; calyce toto 6-7 mm. longo 4- lobato, tubo 1.5 mm. longo sericeo, lobis linearibus vel anguste spathulatis 3-3.5 mm. longis patentibus parce pubescentibus; corolla hypocraterimorpha profunde 4-lobata, tubo cylindraceo fere 1.5 em. longo extus parce piloso, lobis oblongo-lanceolatis tubo subzequantibus; antheris ad faucem corolle paulum exser- tis; ovario biloculari, ovulis plurimis; fructu ignoto. Specimen examined: Mexico: State of San Luis Potosi, Minas de San Rafael, May, 1911, C. A. Purpus, 5208 (Mo. Bot. Gard. Herb.), ТҮРЕ. Randia truncata Greenman & Thompson, sp. nov. Plate 26. Frutex erectus 3-4 m. altus ramosus; eaule ramisque tereti cortice griseo tectis juventate parce strigulosis mox glabratis; spinis 0.5-1 em. longis binis ad apices ramorum; foliis obovatis vel spathulatis 0.5-3 cm. longis 0.3-1.7 cm. latis ad apicem rotundatis obtusis vel submucronato-acutis integris utrinque glabris basi in petiolum marginatum plus minusve abrupte contractis; floribus sessilibus axillaribus terminalibus; calyce toto 1.5-2 mm. longo, limbo cupuliformi truncato; corolla hypocraterimorpha in sieco atrato, tubo cylindraceo 1-1.5 ет. longo extus glabro intus sparse piloso, lobis subovatis 4-5 mm. longis 3-4 mm. latis apice rotundato vel brevissime acuminato; antheris ad faucem corollae sessilibus semiinclusis; baeca im- matura globulosa circiter 0.5 cm. diametro. Specimens examined: Mexico: State of Yucatan, vicinity of Izamal, coll. of 1895, Dr. Geo. F. Gaumer, 713, TYPE, and 506 (both in Mo. Bot. Gard. Herb.); road to Progresso north of Merida, 7 April, 1865, Schott, 262 (Mo. Bot. Gard. Herb.), distributed as “E. aculeata." Co-types of the above species may be looked for in herbaria under R. xalapensis under which name Dr. Gaumer's material cited above was distributed. From this species, however, В. truncata differs in the more obovate outline and the less conspic- uous veins of the leaf, the somewhat longer and more slender corolla-tube, and in the smaller truncate calyx. [Vor. 1 412 | ANNALS OF THE MISSOURI BOTANICAL GARDEN Sclerocarpus elongatus (Greenm.) Greenman & Thompson, comb. nov. S. Schiedeanus var. elongatus Greenm. Proc. Am. Acad. 32:309. 1897. Herbaceus; caule tereti ramoso erecto vel adscendenti 1-1.5 m. alto striato sparse strigoso plus minusve purpurascenti basi lignescenti; foliis brevipetiolatis trinerviis inferioribus oppositis superioribus alternis anguste lanceolatis 2.5-13 em. longis 0.3- 1.5 em. latis acuminatis acutis integris vel remote denticulatis basi in petiolum gradatim angustatis supra tuberculato-hispidis subtus paulo pallidioribus seeundum nervos venasque hirsutis; inflorescentiis laxe panieulatis, pedunculis gracilibus 0.5-8 cm. longis strigosis; capitulis 6-8 mm. altis; involueris subeampanu- latis circiter 5 mm. altis, squamis biseriatis oblongo-lanceolatis ovatis vel subobovatis extus strigoso-pubescentibus ciliatis leviter atratolineatis; flosculis liguliferis 5-8, ligulis oblongis 6-10 mm. longis flavis; floribus disci circiter 30; achzeniis matur- itate obliquis striatis glabris. Specimens examined: Mexico: State of Morelos, fields around Cuernavaca, altitude 1585 m., 31 October, 1896, C. G. Pringle, 6606 (Mo. Bot. Gard. Herb.), со-түрЕ; valley, near Cuantla, altitude 1370 m., 28 October, 1900, C. G. Pringle, 9061 (Mo. Bot. Gard. Herb.). State of Vera Cruz, Ojapa, 30 June, 1910, C. R. Orcutt, 5156 (Mo. Bot. Gard. Herb.). Venezuela: without definite locality, A. Fendler, 685 (Mo. Bot. Gard. Herb.). А further study of co-type material of this species, supple- mented by subsequent collections, and a careful comparison of it with S. Schiedeanus (DC.) Benth. & Hook. f., as represented by Schiede’s number 225 preserved in the herbarium of the Missouri Botanical Garden and Pringle's number 8338 from the type locality, shows several important differences between the species and the plant referred to it as variety elongatus. Тһе latter has narrowly lanceolate leaves, a much-branched stem, open inflorescence, and more numerous and smaller heads which altogether indicate that the plant in question should be regarded as of equal specifie rank rather than a variety of S. Schiedeanus, hence it is here raised to specific rank and a somewhat amplified description is appended. 1914] GREENMAN AND THOMPSON—FLOWERING PLANTS 413 Flaveria longifolia Gray, Pl. Fendl. 88. 1849. Var. subtomentosa Greenman & Thompson, var. nov. Forme typice habitu simili; caule plus minusve tomentoso; folis lanceolato-attenuatis basi plerumque ampliatis amplexi- caulibusque utrinque subtomentosis. Specimens examined: Mexico: State of San Luis Potosi, Minas de San Rafael, Novem- ber, 1910, C. A. Purpus, 4776 (Mo. Bot. Gard. Herb.), TYPE; Rio Verde, 17 November, 1910, C. R. Orcutt, 5421 (Mo. Bot. Gard. Herb.); Rio Verde, 2-8 June, 1904, Dr. Edward Palmer, 26 (Mo. Bot. Gard. Herb.). (Уот. 1, 1914] 414 ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 24 Sisyrinchium angustissimum (Rob. & Greenm.) Greenm. & Thomp. Mexico From the type number, Pringle No. 4703, in the Herbarium of the Missouri Botanical Garden. CONES ol odds ааа 2 ік tege ida. Ағ ANN. Mo. Вот. GARD., Vor. 1, 1914 PLATE 24 GREENMAN AND THOMPSON—DIAGNOSES OF FLOWERING PLANTS COCKAYNE, BOSTON. [Vor. 1, 1914 416 ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 25 Oecopetalum mexicanum Greenm. & Thomp. Figs. 1 and 2, flowering branches; 3, flower; 4, inner face of petal; 5 and 6, front and side view of stamen; 7, longitudinal section of pistil; 8, diagrammatic cross- section of flower bud; 9, diagrammatic cross-section of anther before and after dehiscence. Mexico From the type specimen, Purpus No. 6159, in the Herbarium of the Missouri Botanical Garden. > - зы сыйына Е ГОЧ РТИ eS IE i ыны, алыса да IE o n. —— m. dim у а. ANN. Mo. Bor. Garp., Vor. 1, 1914 PLATE 25 GREENMAN AND THOMPSON-—DIAGNOSES OF FLOWERING PLANTS COCKAYNE, BOSTON. [Vor. 1, 1914 418 ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION оғ PLATE PLATE 26 Randia truncata Greenm. & Thomp. Mexico From the type specimen, Gaumer No. 713, in the Herbarium of the Missouri Botanical Garden. í і Амм. Мо. Вот. GARD., VoL. 1, 1914 ” PLATE 26 GREENMAN амр THOMPSON--DIAGNOSES OF FLOWERING PLANTS 2317 vv COCKAYNE, BOSTON. ENZYME ACTION IN FUCUS VESICULOSUS L. B. M. DUGGAR Physiologist to the Missouri Botanical Garden, in Charge of Graduate Laboratory Professor of Plant Physiology in the Henry Shaw School of Botany of Washington University AND А. R. DAVIS Rufus J. Lackland Fellow in the Henry Shaw School of Botany of Washington University Little is known regarding the metabolism of the Fucacee. The chemical nature of the chief accumulation products has not yet been sufficiently investigated. Іп fact, prior to 1905 very little work of importance had been contributed on the products of any group of the marine alge. Even the chemical deter- mination of the carbohydrates, for example, in some of the larger groups of alge, afforded no suggestion as to the nature of these produets. More activity in this general field of work has been manifest, however, since the date referred to. Diverse views prevailed regarding the nature of the various granules which had been long detected microscopically. In the earlier literature Hansteen's (792, '00) opinion has generally dominated, by which it was claimed that the granular bodies of the cell—and par- ticularly the larger vesicular forms—contain fucosan, а carbo- hydrate, which was considered the first visible product of photosynthesis. On the other hand, Crato (792, '93) main- tained, from microchemical reactions, that the larger vesicles, physodes as he called them, contained phloroglucin, or some derivative of this body. Miither and ТоПепв (704) found а methylpentosan in Fucus and Laminaria, while Koenig and Bettels (705) among others found glucose and fructose, as well as pentoses and methyl pentoses, in Laminaria after hydrolysis. Swartz (711) gives an extensive summary of the previous work on carbohydrate occurrence in the alge, and contributes much data on the digestion of the hemicelluloses, but she studied no brown alge. The existence of reducing sugars in Ғисасев was clearly shown by Tihomirow (710). Recently the carbohydrates have been more completely investigated by Kylin (712, 718). Nevertheless, much remains to be done on these products, while ANN. Mo. Вот. GARD., Vou. 1, 1914 (419) [Vor. 1 420 ANNALS OF THE MISSOURI BOTANICAL GARDEN the proteins (aside from agar agar and related compounds) and other organie substances are scarcely known. In view of the very considerable data on the carbohydrate metabolism in higher plants, it seems particularly desirable to investigate further this relation in the brown alge. Moreover, no general study having been made, as far as we could learn, of the enzymes of the Ғисасев, it seemed possible that a determi- nation of the more characteristic enzymes, and of their distri- bution in Fucus, might lead to a better comprehension of the nature of the metabolism of these plants. Accordingly, during the summers of 1913-14 we have made an examination of F'ucus vesiculosus with respect to its enzyme content. In preparing the Fucus material for study we have followed several of the customary methods which have been found satisfactory in yielding enzymes of a high degree of efficiency. білсе our results with Ғисив have been so generally negative with respect to the presence of the commoner enzymes of plant metabolism, it may be well to indicate briefly how the material was handled. Тһе Fucus plants were obtained in quantity, apparently in a condition of active growth, and the material was carefully pieked over to avoid the contamination of attached animals and smaller alge, then washed, and finally treated by one of several methods. Some of it was hung in a shaded, warm room until quite dry and brittle, then ground in a mill to an extremely fine powder, the latter being preserved in dry bottles for extraction, as subsequently indicated. For other phases of the work the plants fresh from the water were ground almost to a pulp in a meat grinder, sometimes passing the material twice or oftener through the machine. In some cases this fresh pulp, further comminuted in a mortar, or an extract from it, was used directly, while in other cases an alcohol-acetone dry preparation was made from it—the latter by treating alternately with 95 per cent alcohol (15 minutes) and acetone (5-10 minutes) until practically dehydrated, with a final brief treatment with absolute alcohol or ether, when the material was spread out on filter paper to dry. The alcohol-acetone material was thoroughly pulverized in a mortar for further use. In the preparation of extracts the dry material was treated with distilled water (usually 10 parts of water to 1 part of A EI rdiet eae 1914] DUGGAR AND DAVIS—FUCUS VESICULOSUS 421 material), or in some cases with sea-water, using commonly 20 per cent alcohol or 2-3 per cent toluene as a preservative. In general, toluene has proved the most satisfactory antiseptic. Тһе filtered extract was then precipitated with 95 per cent alcohol, the precipitate caught on a filter, washed with alcohol and dried. In a few cases the extract was used direct, and in certain respects the common practices were variously modified in the hope of detecting some simple explanation of the large number of negative results. The hydrolytic experiments were carried out in small Erlen- meyer flasks or test-tubes, and always in duplicate or tripli- cate. In addition, nearly every series was repeated once or oftener. А special effort was made to determine the presence of carbohydrases, and for this purpose weak solutions, usually 0.5 per cent, of starch, glycogen, dextrin, saecharose, maltose, and lactose were employed in numerous tests. Хо reduction, or no change in the reducing value of the substrate, by the Fehling method, was found in any case in our final experiments, although in some cases a relatively large quantity of the sup- posedly enzyme-containing material was used. We found it necessary to purify the best dextrin obtainable by precipitation with 95 per cent alcohol from a strong aqueous solution. In the preliminary experiments, and chiefly with опе preparation, traces of reduction were found with glycogen, but in many later experiments this finding was not confirmed. Owing to the consistently negative results with these carbo- hydrates it seemed possible that there might be an adjustment of enzyme action in Fucus such that a relation of the mineral salts, as in sea water, might be requisite for highest action. Consequently the enzyme solution in one large series of experi- ments was diluted with double strength sea-water; in another case the material was extracted with sea-water; and finally, fresh material was used, making with it a diffusion in sea water. In every instance the result was negative. Another possibility then suggested itself, namely, that the presence of certain inhibiting substances might account for the absence of hydrolytic change. Accordingly, the effect of the Fueus material on the activity of taka diastase was determined in this way: To 10 grams of ground fresh material 100 cc. of 5. — мен [Vor. 1 4221 ANNALS ОҒ THE MISSOURI BOTANICAL GARDEN water and 1 gram of taka diastase were added, this being per- mitted to stand for 5 hours, as in extraction, and the filtrate from this extraction was tested upon starch solution. Тһе results were positive, indicating that no free substances were present which could inhibit diastase action. In another test 1000 grams of Fucus material were divided into two lots of 500 grams each. To one of these, 5 grams of commercial malt diastase were added, and both were then treated by the alcohol- acetone method, and subsequently extracted and precipitated in the usual way. The material to which diastase had been added gave positive tests for the hydrolysis of carbohydrates in an extensive series with dextrin, glycogen, saccharose, and laminarin; but a solution of the precipitate from the lot receiving no diastase produced no changes in these substrates. These experiments included controls of several kinds. With every substrate, boiled material was also used, and it is interesting to note that the “enzyme” material increased in reducing power with boiling. The tests referred to in the previous paragraph seemed all the more important inasmuch as the Fucus material had been found to be strongly acid, and it seemed possible that this acidity alone might prove an injurious factor. From the experiments just mentioned it is seen, however, that acidity could scarcely have been an important consideration. A quantitative deter- mination of the acidity was nevertheless made, by titration with NaOH, and it was found to be about .0565 N HCl. There із а slight increase in the acidity, if the pulp is permitted to remain in water 12 hours. Owing to the determination by many, as, for example, Miither and Tollens (704), Kylin (13), Swartz (711), and others of the presence of hemicelluloses, especially pentosans, in the marine alge, and, further, since the commoner carbohydrate enzymes had not been identified by us, it seemed desirable to examine the material for pentosanase. The most available pentosan was that of cherry gum, accordingly this material in fresh condition was obtained and utilized in many tests with the Fucus prepara- tion, the flasks being maintained at temperatures ranging from 27—40° C. Although the experiments were permitted to run for a period of several days, no reduction above the amount found 1914] DUGGAR AND DAVIS—FUCUS VESICULOSUS 423 in the controls was obtained, and certainly no pentosanase active on this material could be assumed to occur abundantly in Fucus tissues. Only one series of tests has been made to identify cellulase in the material here reported upon, and the results are presented with much reserve. Precipitated cellulose, prepared from filter paper, was employed, and the experiments were conducted at 40° C. The indications were that slight cellulase activity may occur. By means of the action of the alcohol-acetone preparation upon a 4 per cent olive oil-casein emulsion, the lipolytic activity was investigated in the usual way. With the emulsion used alcohol is most serviceable as a preservative. In the tests referred to there was no indication of hydrolysis after one week; so the preparations were permitted to stand for two months, but still without change. That the conditions in the above case were otherwise favorable for lipolytic action is shown by the fact that the same substrate yielded with an alga of another family a decidedly positive test in two days. Several series of experiments were likewise carried out for the determination of esterases. With methyl acetate, ethyl acetate, and ethyl buty- rate the Fucus material produced no change, irrespective of the concentrations employed. In some of our preliminary experiments it had appeared that urease was present, but a careful investigation of this point demonstrated an error in the earlier results, and no amidases were discovered through the action upon 0.5 per cent solutions of urea, acetamid, methylamine, asparagin, diphenylamine, and acetanilid. In these experiments NH; determinations were made according to the method of Folin. No liquefaction of gelatin or of agar occurred during a ten-day interval in a large series of test-tubes arranged with these two substrates. In the different tests these media were made neutral, alkaline, and slightly acid. In the neutral and slightly acid tubes no observable change occurred; but in those tubes containing a higher percentage of acid — both in those contain- ing the Fucus preparation and in the controls — general lique- faction occurred. It is obvious, therefore, that these gel- forming proteins are not noticeably affected by any enzymes [Vor. 1 424 ANNALS OF THE MISSOURI BOTANICAL GARDEN occurring in the Fucus material. More extensive series of tests were arranged to determine the presence of proteinases which might act upon some more widely distributed native proteins, such as albumin, casein, and legumin. No tests were made to determine the transformation of these bodies into proteoses or peptones, but the formaldehyde method of determining amino acids was employed, and in no case had any transformation of these substances proceeded to the amino acid stage. Glucose, levulose, and galactose were used in two series of experiments designed to determine the presence of zymase in the aleohol-acetone Fucus powder. No sufficient evidence, however, of the occurrence of this enzyme was obtained even when the most delicate tests were employed to determine the liberation of СО. The action of Fucus extract from the alcohol-acetone preparation upon tannin was tested by means of quadruplicate experiments. Two concentrations of tannin were used, 1 per cent and 2.5 per cent. The determinations were made by means of Jean’s iodine method, but in no case did the flasks receiving the Fucus extract exhibit hydrolysis greater than that shown by the controls. Neither prepared nor fresh Fucus material gave sufficient evidence of oxidase or peroxidase action to be considered positive. Negative results were obtained both by the direct method with gum guaiacum, and by the in- direct method, in which the reagent mentioned is used with hydrogen peroxide, and apparently acidity is not a determin- ing factor. The use of benzidine seemed to indicate oxidase activity, but it has been clearly shown that the ease with which this reagent undergoes ‘‘spontaneous”’ oxidation in boiled solutions necessitates caution in using it as a test of oxidase activity. Tests for catalase by the usual method, evolution of oxygen on the addition of hydrogen peroxide, have clearly in- dicated that this enzyme is widespread in Fucus. It should be noted that the findings with respect to oxidase and catalase activity are in agreement with those of Atkins (714). Catalase was very generally identified by him in the alge, but evidence of oxidase in the Fucacee was obtained only with benzidine as a reagent. The unexpectedly negative character of the experimental work here briefly outlined prompted us to make many repeti- E NE^ 1914) DUGGAR AND DAVIS—FUCUS VESICULOSUS 425 tions of experiments and minor modifications in technique not referred to in this preliminary account. Тһе nature of the results, furthermore, made it seem desirable that a much more general study be made of the abundance and distribution of the enzymes in the various families of the marine alge, and such an investigation is now in progress by one of us. It would seem idle to attempt here an explanation of the nega- tive results obtained, yet two or three possibilities have occurred to us which may be mentioned. Тһе conditions of life of the Fucacee, especially the temperature relation, make it possible to suspect that metabolic changes occur at a very slow rate. If this is the case, it might be assumed that the commoner metabo- lic enzymes might be present in such small quantity that an indication of their presence would not be apparent by utilizing the methods ordinarily employed. The very fact that the capacity for food accumulation, that is to say, the "storage" of food materials, has not become highly developed in these forms suggests that the usual enzymes might not be found in abun- dance. Nevertheless, if such is the case, it may be pointed out that the present methods of enzyme work are very inadequate when applied to metabolic processes in general dealing with the transformation of products which do not accumulate in some quantity in the cell. In this connection attention may be drawn to Arber’s (’01) observation on the slow rate of trans- formation of starch in the thallus of Ulva latissima, where a darkening period of from three to five weeks was required for the disappearance of this product. The other possibility which has suggested itself is that in the cells of the Fucacee there may occur inhibiting substances which upon the death of the cell may form with the enzymes com- pounds from which the ferments cannot be again recovered. We have no evidence of the existence of any such bodies. Fur- ther investigation of Fucus and related alge should perhaps throw some light upon the negative evidence produced by our extensive data. Graduate Laboratory, Missouri Botanical Garden. [Vor. 1, 1914] 426 ANNALS OF THE MISSOURI BOTANICAL GARDEN LITERATURE CITED Arber, E. А. М. (01). On the effects of salts on the assimilation of carbon dioxide in Ulva latissima. Ann. Bot. 15:39-69. 1901. Atkins, W. В. С. (14). Oxydases and their inhibitors in plant tissues. Part III: The localization of oxydases and catalase in some marine alge. Scientif. Proc. Roy. Dublin Soe. N. S. 14: 199-206. 1914. Crato, E. (792). Die Physode, ein Organ des Zellenleibes. Ber. d. deut. bot. Ges. то: 295-302. pl. 18. 1892. —— ——, (93). Ueber Ше Hansteen’schen Fucosankórner. 164. 11:235-241. 1893. Hansteen, В. (92). Studien zur Anatomie und Physiologie der Fucoideen. Jahrb. f. wiss. Bot. 24:317-862. pl. 7-10. 1892. ‚ (00). Ueber das Fucosan als erstes scheinbares Product der Kohlensäure- assimilation bei den Fucoideen. bid. 35:611-625. pl. 14. 1900. Koenig and Bettels (05). Die Kohlenhydrate der Meeresalgen und daraus herge- stellter Erzeugnisse. Zeitschr. f. Unters. d. Nahrungs- u. Genussmittel 10:457- 473. 1905. Kylin, Н. (12). Ueber die Inhaltskórper der Fucoideen. Arkiv f. Bot. 115:1-26., pl.1. 1912. ‚ (13). Zur Biochemie der Meeresalgen. Hoppe-Seyler's Zeitschr. f. physiol. Chem. 83:171-197. 1913. Müther and Tollens ('04). Über die Producte der Hydrolyze von Seetang (Fucus), Laminaria, und Carragheenmoos. Zeitschr. d. Ver. d. deut. Zucker Ind. 54:59. 1904. [Cited by Swartz.] Swartz, M. D. (11). Nutrition investigations on the carbohydrates of lichens, alge, and related substances. Trans. Conn. Acad. Arts and Sci. 16:247-382. 1911. Tihomirow, W. A. (710). Sur la valeur de la réaction microchimique de la phényl- hydrazine: pour la constatation du sucre dans les tissus des plantes. Ann. Jardin Bot. de Buitenzorg, Suppl. 3?: 537-582. pl. 13-15. 1910. аһ” GENERAL INDEX New scientific names of plants and the final members of new combinations are printed in bold face type; synonyms and page numbers having reference to figures or plates, in italic; and previously published scientific names and all other matter, in ordinary type. A Acid, oxalic, produced by Sclerotinia cinerea, 319 Acidity: The effect of certain conditions upon the, of tomato fruits, 229; of plum fruits, 317; relation of growth of Sclerotinia cinerea to, 318 Agaricacez, 197 Agaricus betulinus, 146; quercinus, 145 Air, The identification of the most char- acteristic salivary organism, and its relation to the pollution of, 47 Air-sampling apparatus, bacteriological, 80; construction and use of, 66 albido-brunnea (Thelephora), 214, 216, 228 albo-violascens (Cyphella), 364 Aleurodiscus, 198 Alge: A contribution to our knowledge of the relation of certain species of grass-green, to elementary nitrogen, 157; Some pur? culture methods in the, 23; Indications regarding the source E EN nitrogen for Ulva Lactuca, angustata (Thelephora), 205 ur mE (Solenia), 373, var. arbicularis, 3 Antherieum Chandleri, 405; stenocar- pum, 406 anthocephala (Thelephora), 202, 203, 226 arachnoidea (Cyphella), 363 Arrhenia, 344 artocreas (Michenera), 197 Asterostroma, 198 Augustinii (Heterothecium), 381 B Bananæ (Cyphella), 379 biennis (Thelephora), 215 Boletus abietinus, 91; adustus, 102; applanatus, 137; arcularius, 107; betu- linus, 104; brumalis, 107; caesius, 96; cinnabarinus, 116; cinnamomeus, 123; ANN. Мо. Bor. Garp., Vor. I, 1914 conchatus, 132; conchifer, 93; con- fragosus, 144; culicularis, 118; dis- tortus, 105; dryadeus, 119; elegans, 110; fomentarius, 136; fraxineus, 130; frondosus, 112; fulvus, 133; fumosus, 103; gilvus, 117; giganteus, 113; grave- olens, 131; hirsutus, 93; hispidus, 119; igniarius, 135; lucidus, 123; nummu- larius, 110; perennis, 122; Pini, 142; pinicola, 130; pubescens, 94; radiatus, 118; resinosus, 116; sanguineus, 115; spumeus, 99; squamosus, 109; suave- olens, 140; sulphureus, 114; tulipifera, 152; umbellatus, 112; unicolor, 143; versicolor, 91 Bordeaux mixture, effect of, on trans- piration, 12, 351 borealis (Craterellus), 357, 382 Botrydiopsis sp., isolation of, in pure culture, 38 Botrydium granulatum, in pure culture, 38 brevipes (Cantharellus), 329 Burt, E. A. Тһе 'Thelephoracez of North America, I, 185; II, 327; III, 357 isolation of, C Сасайа prenanthoid^s, 271; runcinata, 272; Toluccana, 271 caespitulans (Thelephora), 204, 346 ealeeum (Corticium), 187 calyculus (Craterellus), 338 campanula (Peziza), 360 canadensis (Cantharellus), 345 canadensis (Craterellus), 345 candida (Cyphella), 377 Cantharellus, 197; brevipes, 329; cana- ео ада 345; floccosus, 345; levis, 362 Cartharellus (Craterellus), 330, 332, 346 capula (Cyphella), 366, 382 caricina (Cyphella), 366, 382 earyophyllea (Thelephora), 207, 209, 226 Castor bean leaves, use of, in potometer experiments, 7, 11 (427) 428 Cellulose, methods of preparation of, 305 Chlamydomonas pisiformis forma minor, isolation of, in pure culture, 32; rela- tion of, to elementary nitrogen, 178, 179, 181 Chlorascens (Heterobasidium), 197 Chlorella sp., and C. vulgaris, isolation of, in pure culture, 33; relation of, to elementary nitrogen, 178, 179, 181 Chlorococcum humicola, isolation of, in pure culture, 35; relation of, to elementary nitrogen, 178, 179, 181 cinereo-fusca (Cyphella), 377 cinereo-fusca (Peziza), 377 Cladoderris, 198 Clavaria pistillaris, 342 clavatus (Craterellus), 329, 345, 346 confluens (Craterellus), 332 conglobata (Cyphella), 375, 382 Coniophora, 198, 199 convoluta (Cyphella), 380 Cooley, J. S.: A study of the physiolog- ical relations of Sclerotinia cinerea (Bon.) Schröter, 291; Duggar, В. M., and, The effect of surface films and dusts on the rate of transpiration, 1, The effects of surface films on the rate of transpiration: experiments with potted potatoes, 351 Cora, 199 corbiformis (Thelephora), 211 кене ШМоуай, 95; nigromarginatus, 3 e Me (Craterellus), 327, 333, 850 cornucopioides (Thelephora), 212, 346 corrugis (Craterellus), 340 Corticium, 190, 191, 192, 193, 197, 198, 199; calceum, 187; lactescens, latex in, 194; lacteum, 187; Sambuci, 191; sub- giganteum, 197; vagum, 10” Craterellus, 190, 196, 197, ^ 27; borealis, 357, 382; calyculus, 258; canadensis, 845; Cantharellus, 330, 332, 346; clavatus, 329, 345 346; confluens, 332; согписоріоіг ев, 327, 333, 350; corrugis, 340; erispus, 338; delitescens, 339, 350; dilatus, 343, 350; dubius, 335; Humphreyi, 344, 350; lateritius, 330; lutescens, 336, 350; Pogonati, 362, 882; marasmioides, 345; ochrosporus, 327, 334, 350; ocreatus, 334; odoratus, 327, 331, 346, 348; palmatus, 342, $50; pistillaris, 327, 341, 348, 350; Pogonati, 362, 382; pulverulentus, 345; roseus, 332; sinuosus, 337; spathu- larius, 345; taxophilus, 339, 820; uni- color, 327, 340, 341, 342, 348 crispus (Craterellus), 338 cupuleformis (Cyphella), 369, 382 Cupressi (Cyphella), 380 cuticularis (Thelephora), 216, 228 Cyclomyces, 82, 147; Greenei, 147 [Vor. 1 ANNALS OF THE MISSOURI BOTANICAL GARDEN Cymatella, 345 Cyphella, 197, 358; albo-violascens, 364; arachnoidea, 363; Banans, 379; can- dida, 377; capula, 366, 382; caricina, 366, 882; cinereo-fusca, 377; conglo- bata, 375, 382; convoluta, 380; Cup- ressi, 380; cupuleformis, 369, 382; digitalis, 358; fasciculata, 373, 376, 382; filicicola, 379; fraxinicola, 368; fulva, 373; fumosa, 376, 382; furcata, 373; galeata, 362, 382; globosa, 367; griseo- pallida, 369; læta, 361; Langloisii, 368, 382; mellea, 372, 382; minutis- sima, 367, 368, 382; musæcola, 380; muscigena, 362, 363, 382; Palmarum, 377; Peckii, 377; perexigua, 878; pezi- zoides, 364, 365, 378; porrigens, 368, 882; punctiformis, 367; Ravenelii, 371, 372, 373, 882; Ravenelii Sacc., 373; Saccardoi, 873; subcyanea, 380; sub- gelatinosa, 370; sulphurea, 360; tex- ensis, 371, 373, 382; Tiliæ, 364, 365, 882; trachychæta, 379; villosa, 365, 882 D Daedalea, 82, 143; ambigua, 144; con- fragosa, 144; mollis, 141; pallido-fulva, 146; quercina, 145; sæpiaria, 147; unicolor, 143 Davis, A. R., Duggar, B. M. ала. ке action in Fucus vesiculosus, 19 delitescens (Craterellus), 339, 350 Dendrocladium, 199 dentosa (Thelephora), 224, 346 digitalis (Cyphella), 358 dilatus (Craterellus), 343, 350 dubius (Craterellus), 335 Duggar, B. M.: and Cooley, J. S., The effect of surface films and dusts on the rate of transpiration, 1, The effects of surface films on the rate of transpira- tion: experiments with potted pota- toes, 351; and Davis, A. R., Enzyme action in Fucus vesioulosus, 419; and Merrill, M. C., The effect of certain conditions upon the acidity of tomato fruits, 229 Dusts, the effect of surface films and, on the rate of transpiration, 1 E Enslinia pocula, 106 Enzymes: cellulase in Selerotinia cinerea and Penicillium expansum, 305; рес- tinase in Sclerotinia cinerea and Pen- icillium expansum, 312 Erechthites runcinata, 271, 272 Erinus tomentosus, 409 Eu-Thelephores, 197 Exobasidium, 193, 197 “К.А ais > cn —— кта 1914] ANNALS OF THE MISSOURI BOTANICAL GARDEN 429 F fasciculata (Cyphella), 373, 376, 382 Favolus, 82, 148; canadensis, 148; ohiensis, 148; rhipidium, 148; striatu- lus, 148 filieicola (Cyphella), 379 Films and dusts, the effect of surface, on the rate of transpiration, 1 Films, the effect of surface, on the rate of transpiration: experiments with potted potatoes, 351 fimbriata (Thelephora), 222, 226 Flaveria longifolia, 413, var. subtomen- tosa, 413 Flowering plants, Diagnoses of, chiefly from the southwestern United States and Mexico, 405 Fomes, 82, 126; applanatus, 130, 135, 136, 137, 138; carneus, 131; conchatus, 132; connatus, 129; Everhartii, 134, 135; fomentarius, 135, 136, 137; frax- ineus, 129, 130; fraxinophilus, 129; fulvus, 133; graveolens, 131; igniarius, 134, 135; lobatus, 137; nigricans, 134, 135, 136; ohiensis, 128; pinicola, 130; populimus, 130; ribis, 133; rimosus, 133, cause of trunk disease of mesquite, 248; roseus, 131; salicinus, 133; scutellatus, 128; supinus, 133 Foster, G. L. Indications regarding the source of combined nitrogen for Ulva Lactuca, 229 fraxinicola (Cyphella), 368 Fucus vesiculosus, Enzyme action in, 419 fulva (Cyphella), 373 fumosa (Cyphella), 376, 382 Fungous and host cells, A method for the к differential staining of, 241. furcata (Cyphella), 373 G galeata (Cyphella), 362, 382 Ganoderma sessile, 123; subperforatum, 123 Gates, R. R. A Texan species of Mega- ‚ pterium, 401; Some cenotheras from Cheshire and Lancashire, 383 globosa (Cyphella), 367 Gloeocystidium, 193 Gloeopeniophora, 193 Gloeoporus, 82, 149; conchoides, 149 Grandinia, 196 Greenman, J. M.: Descriptions of North American бепесіопеге, 263; and Thomp- son, C. H., Diagnoses of flowering plants, chiefly from the southwestern United States and Mexico, 405 Grifola poripes, 111; ramosissima, 112; Sumstinei, 113 griseo-pallida (Cyphella), 369 griseozonata (Thelephora), 221, 228 H Herpestes tomentosa, 409 Heterobasidium, 197; chlorascens, 197 Heterothecium Augustinii, 381 Hirneolina, 197, 198 hiscens (Thelephora), 207 Humphreyi (Craterellus), 344, 350 Hydnaces, 196 Hymenochaete, 193, 198 Hymeno-lichens, 199 Hyphomycetes, 194 Нуросһпасеге, 190 Hypochnus, 190, 193, 196, 198, 199 Hypolyssus, 198 I Infection and penetration of plums by Sclerotinia cinerea, 297 Inonotus texanus, 247 Introduction, i intybacea (Thelephora), 217, 220, 228 Irpex, 82, 151; cinnamomeus, 152; fari- naceus, 152, 153; mollis, 152; tulipi- fera, 152 K Kirchneriella sp., isolation of, in pure culture, 34; relation of, to elementary nitrogen, 178, 179, 181 Kummeria, 409 L Lachnocladium, 196, 198, 199 laciniata (Thelephora), 219 laciniatum (Stereum), 219 lactescens (Corticium), 194 lacteum (Corticium), 187 læta (Cyphella), 361 lævis (Cantharellus), 362 Langloisii (Cyphella), 368, 382 lateritius (Craterellus), 330 Lenzites, 82, 145; betulina, 146; Cra- taegi, 144; flaccida, 146; protractus, destruction of sap-wood of mesquite by, 248; saepiaria, 147; vialis, 146 Lilac, A trunk disease of the, 253 Lloydella, 192 lutescens (Craterellus), 336, 350 lutosa (Thelephora), 216, 346 M magnispora (Thelephora), 211, 226 Mappia, 408 marasmioides (Craterellus), 345 430 Megapterium, А Texan species of, 401; argyrophyllum, 401, 404, var. retusi- folium, 401, 404; Fremontii, 402; macrocarpum, 402; missouriensis, 402, 403 mellea (Cyphella), 372, 382 Merrill, M. C., Duggar, B. M., and. The effect of certain conditions upon the acidity of tomato fruits, 229 Merulius, 82, 150, 197; lacrymans, 151; rubellus, 150; tremellosus, 151; tremel- losus, 151 Mesquite, Two trunk diseases of the, 243 Michenera, 197; artocreas, 197 Micheneri (Stereum), 214 Microcoleus sp., isolation of, in pure cul- ture, 39 minutissima (Cyphella), 367, 368, 382 Moore, G. T. Foreword, 1 Mucronoporus Everhartii, 134 multipartita (Thelephora), 205, 208, 226 mus:ecola (Cyphella), 380 muscigena (Cyphella), 362, 382 Mycobonia, 198 N Navicula sp., isolation of, in pure culture, Nitrogen: A contribution to our knowl- edge of the relation of certain species of grass-green alge to elementary, 157; Indications regarding the source of combined, for Ulva Lactuca, 229 Nolte, A. G. The identification of the most characteristic salivary organism, and its relation to the pollution of air, 47 O obovata (Xylaria), 225 ochrosporus (Craterellus), 334, 350 ocreatus (Craterellus) 334, 335 odoratus (Craterellus), 331, 346, 848 odorifera (Thelephora), 215 Oecopetalum, 408; mexicanum, 408, 416 Oedogonium, isolation of zoospores free from bacteria in, 36 Oenothera Lamarckiana, 393, 400; macro- carpa, 402; multiflora, 386, 396; var. elliptica, 387, 396, 398; rubrinervoides, 389, 396, 898; rubritincta, 391, 398, 400; tardiflora, 391, 400 Oenotheras, Some, from Cheshire and Lancashire, 383 Ohio, The Polyporaceæ of, 81 Oscillatoria sp., isolation of, in pure cul- ture, 39 Overholts, L. O. The Polyporaceæ of Ohio, 81 [Vor. 1 ANNALS OF THE MISSOURI BOTANICAL GARDEN P palmata (Thelephora), 201, 226 palmata americana (Thelephora), 201 Palmarum (Cyphella), 377 palmatus (Craterellus), 342, 350 pannosa (Thelephora), 207 Peckii (Cyphella), 377 Penicillium expansum, studies in, 308, 315, 322 Peniophora, 190, 191, 192, 193, 198, 199 Peniophorella, 193 perexigua (Cyphella), 378 Permeability of collodion membranes, perplexa (Thelephora), 223, 346 Peziza campanula, 360; Ті, 364 pezizoides (Cyphella), 364, 365, 378 pistillaris (Clavaria), 342 pistillaris (Craterellus), 341, 348, 350 Pleurococcus vulgaris, isolation of, in pure culture, 34 Pogonati (Craterellus), 362, 382 Polyporaceæ, 197 Polyporaceæ of Ohio, The, 81; index to species of, 153; key to genera of, 85 Polyporus, 82, 86; abietinus, 91, 92; abortivus, 105; adustus, 102, 103, 104; albellus, 97 ; апат, 113, 114; applanatus, 137; arculariformis, 107; arcularius, 107; badius, 126; Berkeleyi, 113; betulinus, 104; biformis, 95; borealis, 100; brumalis, 107; caesius, 96, 97; carneus, 131; castanophilus, 115; chion- eus, 97; cincinnatus, 114; cinnabarinus, 116; cinnamomeus, 123; circinatus, 121, 122, 123; conchatus, 132; conchifer, 93; conglobatus, 131; comnatus Vries, 129; connatus Schw., 122; connatus Weinm., 129; cristatus, 111; Curtisii, 125; eutieularis, 117, 11S, 119; delec- tans, 99; dichrous, 150; distortus, 105; dryadeus, 119, 120; dryophilus, 119, 120; dualis, 121; elegans, 110; endocro- cinus, 119; fibula, 95; fissus, 109; flavovirens, 111; focicola, 122, 123; fomentarius, 136; fragilis, 126; fragrans, 102, 103; fraxineus, 130; fraxinophilus, 129; frondosus, 112, 113, 114, 126; fulvus, 133; fumosus, 102, 103; galac- tinus, 97, 98, 141; giganteus, 113, 126; gilvus, 117, 118, 120; guttulatus, 100; hirsutulus, 91,92; hirsutus, 93; hispidus, 119; hypococcineus, 115; igniarius, 135; immitus, 98; intybaceus, 126; isidioides, 117; lacteus, 97; lentus, 126; leuco- melas, 126; leucophaus, 137; lobatus, 137; Lloydii, 95; lucidus, 123; macula- tus, 100; molliusculus, 95, 96; Morgani, 110; nidulans, 117; nigricans, 136; obesus, 121, 123; obtusus, 100; ovinus, 126; pargamenus, 92; parvulus, 122; pennsylvanicus, 108; perennis, 122, physiological 1914) ANNALS OF THE MISSOURI BOTANICAL GARDEN 123; perplexus, 118; phaeoxanthus, 126; рісірез, 109, 110; Pilots, 115; pini- cola, 130; pocula, 106; populinus, 141; puberula, 103; pubescens, 94, 96, var. Стауй, 95; radiatus, 117, 118, 119; radicatus, 110; reniformis, 137; resin- osus, 116; rimosus, 133; robiniophila, 104; Rostkowii, 109; rufescens, 100; sanguineus, 115, 116; Schweinitzii, 120; scutellatus, 128; semipileatus, 96; serialis, 139; Spraguei, 101; spumeus, 99; squamosus, 109; subsericeus, 123; Sullivantii, 94; sulphureus, 114; texa- nus, 247, cause of trunk disease of mesquite, 246, 250, 252; tomentosus, 121; umbellatus, 112, 113; varius, 110; velutinus, 93, 95; versicolor, 91, 93, eause of trunk disease of lilae, 253, 260, 262; virgineus, 94; volvatus, 105; zonalis, 101; zonatus, 91 Polystietus, 82; Lindheimeri, destruc- tion of sap-wood of mesquite by, 248; obesus, 121; proliferus, 123 Poraqueiba, 409 Poria, 82 porrigens (Cyphella), 368, 382 Potato plants, transpiration of, 351 Potometer, 22; experiments with, 7 Prosopis glandulosa, trunk diseases of, Protosiphon botryoides, isolation of, in pure culture, 35; relation of, to elemen- tary nitrogen, 178, 179, 181 pulverulentus (Craterellus), 345 punctiformis (Cyphella), 367 Pure culture methods in the Alg:e, Some, 23 pusiola (Thelephora), 208, var. terrestris, 209 puteana (Coniophora), 197 Pyropolyporus robinie, 133 R radiata (Thelephora), 210 Randia aculeata, 411; Gaumeri, 410; Purpusii, 410; truncata, 411, 418; xalapensis, 411 Ravenelii (Cyphella), 371, 372, 382 Ravenelii (Cyphella), 373 Ravenelii (Thelephora), 207 regularis (Thelephora), 206, 207, 226 Rhipidonema, 199 roseus (Craterellus), 332 Ruellia Parryi, 410 5 Saccardoi (Cyphella), 373 Salivary organism, The identification of the most characteristic, and its relation to the pollution of air, 47 Sambuci (Corticium), 191 431 sanguinolentum (Stereum), 194 Scenedesmus sp., isolation of, in pure culture, 34 Schizophyllum commune, destruction of sap-wood of mesquite by, 248 Schramm, J. R. A contribution to our knowledge of the relation of certain species of grass-green alge to ele- mentary nitrogen, 157; Some pure culture methods in the alge, 23 von Schrenk, Hermann. A trunk dis- ease of the lilac, 253; Two trunk diseases of the mesquite, 243 scissilis (Thelephora), 204, 226 Sclerocarpus, elongatus, 412; Schied- nr a 412; Schiedeanus var. elongatus, 12 Sclerotinia cinerea (Bon.) Schréter, A study of the physiological relations of, 291 scoparia (Thelephora), 222 Sebacina, 190, 197, 198, 199 Senecio, 263; alatipes, 270; albonervius, 275; alienus, 278; alvarezensis, 275; angulifolius, 277, var. ingens, 276; anisophyllus, 278; appendiculatus, 265; arizonicus, 267; brachyanthus, 277; Breweri, 265; callosus, 271; canus, 266; carnerensis, 273; chapalensis, 277, var. areolatus, 278; Chrismarii, 278; convallium, 266; cordovensis, 277; Coulteri, 272, 273; diffusus, 264; doratophyllus, 271; Douglasii, 275; ex- imius, 271; fastigiatus, 269; filicifolius, 274; Hartwegi, 280; hederzfolius, 278; hyperborealis, 264, var. columbiensis, 264; hypomalacus, 278, 282; iodanthus, 272, 286; Kerberi, 279; kernensis, 266; Klattii, 281; latipes, 271; longilobus, 274; macropus, 267; multilobatus, 265; neo-mexicanus, 265, 267, 268; oaxa- canus, 279; oreophilus, 267; oreopolus, 268, 284, forma aphanactis, 269; Picridis, 274, 275; præcox, 281; prion- opterus, 270; prolixus, 264; purpuras- cens, 273, var. fossanervius, 273; reglensis, 280; resedifolius, 264, var. columbiensis, 264; roseus, 281; roseus, 281; stoechadiformis, 274; subauricu- latus, 270, 290; teliformis, 275; velatus, 280, 288; viejensis, 271; vulgaris, 274; Warszewiezi, 270; Wrightii, 269 Senecionez, Descriptions of North Ameri- can, 263 Septobasidium, 198 Silicic-acid jelly, preparation of, 39 sinuosus (Craterellus), 337 Siphonoglossa, Greggii, 409; Pilosella, 410 Sisyrinchium alatum, 407; alatum var. angustissimum, 406; angustissimum, 406, 414 Skepperia, 343, 345 [Vor. 1, 1914] 432 ANNALS OF THE MISSOURI BOTANICAL GARDEN Solenia, 358, 372; anomala, 373, var. arbicularis, 378 —— (Craterellus), 345 phaeria pocula, 106 iculosa (Thelephora), 225, 226 pirogyra setiformis, isolation of zygo- spores free from bacteria in, 37 spongiosum (Stereum), 215, 216 Staining, A method for the differential, of fungous and host cells, 241 Stemodia, lanata, 409; linearifolia, 409; tomentosa, 409 Lkw linearifolia, 409; tomentosa, 409 Stereum, 192, 193, 198, 199; albobadium, destruction of sap-wood of mesquite by, 248; calyculus, 338; Leveillianum, destruction of sap-wood of mesquite by, 248; sanguinolentum, latex in, 194; spadiceum, latex in, 194; spongio- sum, 215, 216 Stichococcus bacillaris, isolation of, in pure culture, 32; relation of, to elemen- tary nitrogen, 178, 179, 181; Б. subtilis, isolation of, in pure culture, 32 Stigeoclonium tenue, isolation of, in pure culture, 36 subcyanea (Cyphella), 380 subgelatinosa (Cyphella), 370 subgiganteum (Corticium), 197 sulphurea (Cyphella), 360 Syringa vulgaris, trunk disease of, 253 x taxophilus (Craterellus), 339, 350 Temperature, effect of: on growth of alge in pure culture, 180; on acidity of ripening tomato fruits, 239; on cellulose hydrolysis by Sclerotinia cin- erea, 311 tephroleuca (Thelephora), 213 terrestris (Thelephora), 199, 218, 219, 222, 228 texensis (Cyphella), 371, 382 Thelephora, 193, 196, 197, 198, 199; albido-brunnea, 214, 216, 228; angus- tata, 205; anthocephala, 202, 203, 226; biennis, 215, 216; caespitulans, 204, 826; caryophyllea, 207, 209, 226; corbiformis, 211; cornucopioides, 205, 212, 346; cuticularis, 216, 228 ; dentosa, 224, 346; fimbriata, 222, 226; griseo- zonata, 221, 228; hiscens, 207; inty- bacea, 217, 220, 228; laciniata, 219; lutosa, 216, 346; magnispora, 211, 226; multipartita, 205, 208, 226; odorifera, 214; palmata, 201, 203, 226; palmata americana, 201; pannosa, 207; perplexa, 223, 346; pusiola, 208, var. terrestris, 209; radiata, 210; Ravenelii, 207; regularis, 206, 207, 226; scissilis, 204, 226; scoparia, 222; spiculosa, 225, 226; tephroleuca, 213; terrestris, 199, 218, 219, 222, 228 ; vialis, 218, 228 Thelephoracee of North America, The, I, 185; II, 327; III, 357. Thompson, C. H., Greenman, J. M. and. Diagnoses of flowering plants, chiefly from the southwestern Vnited States and Mexico, 405 Tilie (Cyphella), 364, 382 Таш (Peziza), 364 Tomato fruits, The effect of certain con- ditions upon the acidity of, 229 Tomato plants, transpiration of, 15 trachycheta (Cyphella), 379 Trametes, 82, 188; Abietis, 143; hispida 142; lactea, 144; malicola, 140, 141;, mollis, 141; nivosus, 143; ohiensis, 128; Peckii 140, 142; Pini, 142; rigida, 139, 140, 141; robiniophila, 104; rubes- cens, 145; sepium, 139, 140; serialis, 139, 141; suaveolens, 140 Transpiration, Тһе effect of surface films and dusts on the rate of, 1, 351 Tremellodendron, 197, 199 Tulasnella, 198 U betas sp., isolation of, in pure culture, Ulva Lactuca, Indications regarding the source of combined nitrogen for, 229 unicolor (Craterellus), 340, 341, 342, 348 V vagum (Corticium), 197 Vaucheria sp., isolation of zoospores free from bacteria in, 36 Vaughan, R. E. A method for the differential staining of fungous and host cells, 241 vialis (Thelephora), 213, 228 villosa (Cyphella), 365, 382 W Watering device, automatie, 356; con- struction and use of, in transpiration experiments, 351 X xanthopus (Merulius), 336 Xylaria obovata, 225 Z Zephyranthes chrysantha, 406; Egger- siana, 406 pmo" = УЕЗ ааа. DA -B. M. Duggar, and J. S. Cooley - ` Some Pure Culture , Methods ir in the Algae. : Jacob R. ‘Schramm DT The Identification of the Most Characteristic Salivary Organ- 3 e Pies ism and its Releton.t to the. ies а А August G. Nolte | 2. E 9. ore Ma E: ташы отта BY THE RUMFORD: PRESS, IA or TRUSTEES оғ г ТЕЕ. MISSOURI BOTANICAL GARTEN St. Pets. Mo. 5 The Effect of Sürfate Films а ud Baste on me Site: of а. pe e N. н. FOR THE sob. i оғ THE MISSOURI BOTANICAL GARDEN Cle ; М? 4 A Tea ; GEORGE 7. MOORE, eed СЫҒА АЗА den ON Ты ok Sear Minoa. DvacaR,- = `` EDWARD А. BURT, 3 fa © Plant Physiologist. Be " Mycologist and Librarian, T HERMANN уох SecHRENE, г © JACOB В. SCHRAMM, ` а ~~ Plant Pathologist. 3 Assistant to'the Director. 222 У aeo Jess "M. GREENMAN, i. CmanpEs Hy THOMPSON, ae йел Jean wes Ы "Curator of the Herbarium... А Assistant Botanist. mut. f EE e “Меһуін- C. MERRILL, Research Agsistant; is e pr BOARD. OF TRUSTEES а | OF THE MISSOURI =. GARDEN | AWO EAR 222 bp oc A gi із, S EDWARDS WHITABER. — ы. у Vice-President, ` ? СЯ A -DAVID 8. В. МР Уа а Ержан) с; Enron. m ceo : ЫЫ! Мовы, Xm овоне. C.. Hirccock. ENSIS UN палам H. Н, Pirros. s S x LEONARD Marraews. | А ~ PHILIP e. Baitan, Dos | a Jonk F. SumpLev. EX-OFFICIO ME MBERS: .EbpMUND А. ENGLER, . х ‘Henry. W. Kien, | EPs oe ет of the Academy. of Belénee- Mayor ot the es of St. Lonis: І deni. қс 4 ^ot St. Louis. ; А S «Dam Е. HousTON, - EDWARD A; заны ah “Chancellor, af АМ ЖА University. Baier of 2s Board: of Publie Schools ae mi} te >». : о; yf 4 DixxL S. отк, Bishop of the Diocese of Missouri, ^ pak. 5 de А. D. CUNNINGHAM, Secretary.