ANN. Mo. Bor. GARD., VOL. 2, 1915 PLATE 2 Fig. 2 BRITTON—VEGETATION OF MONA ISLAND COCKAYNE, BOSTON ANNALS. yet Tepe: ate ae ite: ca i ABE abe: ur, ar KEN I Z = = O D = = rs 5 £ oO © = z oO n = y Ir i A mE 5 t Taa ee oe oP t, y naar. E AT eel ) ae g ER a ae i NEE E A MV ee T Annals of the Missouri Botanical Garden Volume II 1915 With Twenty-seven ;Piates and Seyenty-nine Figures Published quarterly by the Board of Trustees of the Missouri Botanical Garden, St. Louis, Mo. Entered as second-class matter at the Post Office at St. Louis, Missouri, under the Act of March 3, 1879. 24086 Annals of the Missouri Botanical Garden A Quarterly Journal containing Scientific Contributions from the Missouri Botanical Garden and the Graduate Labora- tory 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 Information The Annals of the Missouri Botanical Garden appears four times dur- ing the calendar year, February, April, September, and November, Four numbers constitute a volume. Subscription Price - - - $3.00 per volume. Single Numbers - - - - 1.00 each. The following agent is authorized to accept foreign subscriptions: William Wesley & Son, 28 Essex Street, Strand, London. Doty N Laora ates ° . o 66 nr etot “ee . > STAFF OF THE MISSOURI BOTANICAL GARDEN GEORGE T "MOORE. BENJAMIN M. ER Epwarp A. Bur Physiologist charge of Mycologist and ae Graduate Se Risen ALVA R. Dav HERMANN VON SCHRENK, Research Luis Pathologist. X C. E. HUTCHINGS, JESSE M. GREENMAN, Photogtaphes, Curator of the Herbarium. KATHERINE H. LEIGH, Secretary to the Director. BOARD OF TRUSTEES OF THE MISSOURI BOTANICAL GARDEN ` President, EDWARDS WHITAKER Vice-President DAVID S. H. SMITH. Epwarp C. ELIOT. LEONARD MATTHEWS. GEORGE C. HITCHCOCK. WILLIAM H. H. PETTUS. P. CHOUTEAU MAFFITT. PHILIP C. SCANLAN, EDWARD MALLINCKRODT. JOHN F. SHEPLEY. EX-OFFICIO MEMBERS: EDMUND A. ENGLE Davip F. HOUSTON a of = BE of Science Chancellor of Washington University. f St. Lou ` Henry W. KIEL, JAMES P. HARPER Mayor of the City of St. Louis. President of the Board of Public Schools of St. Louis. ANIEL S. TUTTLE, Bishop of the Diocese of Missouri. A. D. CUNNINGHAM, Secretary. TABLE OF CONTENTS The Twenty-fifth Anniversary Celebra- CD a ore renee mere rrr. oe eo SS The Vegetation of Mona Island...... N. L. Britton The Flora of Norway and its Immigra- REN N. Wille The Phylogenetie Taxonomy of Flower- A C. E. Bessey The Botanical Garden of Oaxaca...... C. Conzatti The Origin of Monocotyledony...... J. M. Coulter The History and Functions of Botanic Gardens ne Rew ee ne A. W. Hill Recent Investigations on the Proto- plasm of Plant Cells and its Colloidal Properties, isc. 5m 2000 F. Czapek The Experimental Modification of Germ- gia Se Saree Sey eee oe D. T. MacDougal The Relations between Scientific Botany SRG PRYEOBALHOIOBT «cas sca neehees O. Appel The Law of Temperature Connected with the Distribution of the Marine PAGO IEE en Sta ee W. A. Setchell Phylogeny and Relationships in the BOB i er ee . F. Atkinson A Conspectus of Bacterial Diseases of Plants ee 5 ava ke etn eee E. F. Smith 109-164 165-174 175-183 185-240 241-252 253-274 275-285 287-305 307-313 315-376 377-401 TABLE OF CONTENTS Rhizoctonia Crocorum (Pers.) DC. and R. Solani Kühn (Corticium vagum B. & C.) with Notes on Other Species OU ER NT eee eT Cee B. M. Duggar Some Relations of Plants to Distilled Water and Certain Dilute Toxie So- in 3 2 Rp ee EHE ant oe M. C. Merrill Electrolytic Determination of Exosmo- sis from the Roots of Plants sub- jected to the Action of Various Agents Pere eee Tee Teer eer reer Cee SF M. C. Merrill Monograph of the North and Central American Species of the Genus Se- necio—Part II................. J. M. Greenman The Thelephoraceae of North America. RE ERDE NER: ONE E. A. Burt Toxieity of Galactose for Certain of the Higher Plants..................Lewis Knudson Comparative Studies in the Polypora- a Se ae EOT red ET L. O. Overholts The Thelephoraceae of North America. h ee er rere Ts REP eee K. A. Burt Enzyme Action in the Marine Algae....A. R. Davis General Index to Volume II...................40. PAGE 403-458 459-506 907-572 979-626 627-658 659-666 667-730 731-770 771-836 837-841 Annals of the Missouri Botanical Garden Anniversary Proceedings Vor. 2 FEBRUARY-APRIL, 1915 Nos. 1 anD 2 THE TWENTY-FIFTH ANNIVERSARY CELEBRATION The twenty-fifth anniversary of the organization of the Board of Trustees of the Missouri Botanical Garden was cele- brated at the Garden on October 15 and 16, 1914. A list of the American and foreign scientists in attendance, the com- plete program of the anniversary exercises, the banquet pro- ceedings, and the papers presented at the scientific meetings will be found respectively on pages 1-3, 4-5, 6-27, and 29-401. DELEGATES AND VISITING SCIENTISTS S. ALEXANDER DR. I. W. BAILEY MR. Detroit, Michigan DR. FRANK M. ANDREWS Indiana University, Bloomington, Indian DR. O. APPEL kaiserlichen Biologischen Anstalt, Berlin, Germany DR. CHARLES O. APPLEMAN Maryland Agricultural em Station, College Park, Maryland DR. J. C. ARTHUR aay University, Lafayette, Indi- DR. GEORGE F. ATKINSON Cornell University, Ithaca, York DR. C. B. ATWEL een University, Evanston, Illin ANN, Mo. BoT. GARD., New Vor. 2, 1915 Bussey Institution, Jamaica Plain, Massachusetts DR. H. M. BENEDICT University of Cineinnati, Cincin- o DR. CHARLES E. BESSEY University of Nebraska, Lincoln, Nebraska PROF. MABEL BI SHOP Rockford College, Rockford, Illinois DR. CAROLINE A. BLACK New zum College, Durham, New Hampsh DR. FREDERICK H. BLODGETT Texas Agricultural ee Sta- tion, College Station, Texas DR. N. L. BRITT New York Botanical Garden, New York City (1) NEE EON TET ALTOS EEE eS LR ore hPa pe ae RUS UT BAY aT ia An d aaa oe a 2 ANNALS OF THE MISSOURI BOTANICAL GARDEN MRS. E. G. BRITTON New York Botanical Garden, New York City DR. SEVERANCE BURRAGE Indianapolis, Indiana R. T. J. BURRILL University of Illinois, Urbana, Ill- inois DR. OTIS W. CALDWE University of Chisago. "Chicago, Ill- R. H. S. CONARD Grinnell College, Grinnell, Iowa DR. JOHN G. COULTER loomington, Illinois DR. JOHN M. COULTER University of Chicago, Chicago, Ill- inois DR. STANLEY COULTER zu University, Lafayette, Ind- DR. HENRY C. COWLES hor pa of Chicago, Chicago, Ill- ois REV. JOHN DAVIS Hannibal, Missouri H. DENNISTON University of Wisconsin, Madison, PROF. H. B. DORNER oe of Illinois, Urbana, Ill- DR. FREDERICK DUNLAP University of Missouri, Columbia, Missouri DR. E. J. DURAND University of Missouri, Columbia, DR. R. A. EMERSON Cornell University, Ithaca, New York ROF. A. T. ERWIN State College, Ames, Iowa DR. Wine ae G. FARLOW vard University, Cambridge, + poh etts DR. MARGARET C. FERGUSON Wellesley College, Wellesley, Massa- chusetts ae F. D. FROMME Indiana Agrieultural Experiment Station, Lafayette, Indiana (Vou, 2 DR. GEORGE D. FULLER er of Chicago, Chicago, Ill- sage P. AIN Kansas “Agricultural College, Man- hattan, Kansa DR. REGINALD R. GATES University of London, London, Eng- land PROF. A. H. GILBERT =. University, Lexington, Ken- ucky DR. RICHARD ne ead, Department of Animal Gen- etics, Kaiser Wilhelm institut, Ber- lin, Germany DR. ROBERT F. GRIGGS Ohio State University, Columbus, DR. H. A. HARDING University of Illinois, Urbana, Ill- inois DR. J. ARTHUR HARRIS Station for Experimental Evolution, d Spring Harbor, New York DR. L, H. HARVEY State Normal School, Kalamazoo, Michigan DR. ANSEL F. HEMENWAY Transylvania University, Lexington, DR. HENRI HUS University of Michigan, Ann Arbor, Michigan . D. KERN Pennsylvania re EHEN State College, Pennsylva DR. J. S. KINGSLEY University of Illinois, Urbana, Ill- DR. J. E. KIRKWOOD University of Montana, Missoula, Montana DR. LEWIS KNUDSON Cornell University, Ithaca, New York f DR. EDWARD KREMERS University of Wisconsin, Madison, Wisconsin DR. W. J. AND University of Chicago, Chicago, Ill- ois DR. monas LEFEVRE University of Missouri, Columbia, 1915] ANNIVERSARY CELEBRATION—VISITING SCIENTISTS 3 DR. MICHAEL LEVINE nae High "School, New York DR. I. F. LEWIS University of Missouri, Columbia, issouri DR. D. T. MACDOUGAL Carnegie Institution, Tucson, Ari- zona DR. J. N. MARTIN Iowa State College, Ames, Iowa MR. FRED A. MILLER Indianapolis, Indiana DR. Er F. Sea eld Museum of Natural History, Tllinois DR. D. M. MOTTIER In FER University, Bloomington, Indian DR. poen NELSON University of Wyoming, Laramie, Wyoming DR. W. A. NOYES University of Illinois, Urbana, Ill- inois DR. LULA PACE aylor University, Waco, Texas R. L. H. PAMME Iowa State College, Ames, Iowa MR. GEORGE L. PELTIER University of Illinois, Urbana, Ill- inois DR. WANDA MAY PFEIFFER University of Chicago, Chicago, Ill- 1no1s DR. A. J. PIETERS University of Michigan, Ann Arbor, ichigan DR. C. V. PIPER partment of Agriculture, Wash- ington, D DR. RAYMOND J. POOL University of Nebraska, Lincoln, Ne- braska PROF. J. L. PRICER State Normal School, Normal, Ill- inois R. M. J. PRUCHA University of Illinois, Urbana, Ill- inois DR. FRANCIS RAMALEY University of Colorado, Boulder, Colorado DR. GEORGE M. REED University of Missouri, Columbia, Missouri W. A. SETCHELL University of California, Berkeley, Califor = ter rg SHIMEK a State University, Iowa City, DR. ALEXANDER SMITH Columbia University, New York City DR. ERWIN F. SMITH Department of Agriculture, Wash- ington, D. C. DR. LAETITIA M. SNOW Wellesley College, Wellesley, Massa- chusetts DR. HERMAN A. SPOEHR Desert Laboratory, Tucson, Arizona PROF. W. C. STEVENS University of Kansas, Lawrence, Kansas DR. S. M. TRACY Department of Agriculture, Biloxi, Mississippi DR. E. N. TRANSEAU State Normal School, Charleston, Ill- inois MR. A. G. VESTAL University of Colorado, Boulder, Colorado DR. ELDA R. WALKER University of Nebraska, Lincoln, Ne- braska DR. HENRY B. WARD University of Illinois, Urbana, Ill- inois DR. JOHANNA WESTERDIJK ee e Laboratory, Am- sterdam, Hollan DR. KARL M. WIEGAND Cornell University, Ithaca, New York DR. E. MEAD WILCOX University of Nebraska, Lincoln, Ne- braska DR. N. WILLE University = Christiania, Chris- iania, way DR. WILLIAM L. WOODBURN Lh meg gi e University, Evanston, Illin DR. R. = WYL State University, Iowa City, Iowa [VoL. 2 ANNALS OF THE MISSOURI BOTANICAL GARDEN PROGRAM Thursday, October 15 10:30 A.M. AUTOMOBILE RIDE THROUGH THE CITY FOR DELEGATES AND VISITING SCIENTISTS 1:00 P.M. LUNCH A HE GARDEN 2:00 P.M. FIRST SCIENTIFIC PROGRAM (Graduate Lecture Room) ÄDDRESS oF WELCOME - - Director George T. Moore THE VEGETATION OF Mona [SLAND DIRECTOR-IN-CHIEF N. L. BRITTON New York Botanical Garden, Bron® Park, New York Tue Fora or Norway AND Its IMMIGRATION PROFES N. WILLE University of Christiania, Christiania, Norway Tur PHyLocEeNetic TAXONOMY OF THE FLOWERING PLANTS ESSOR CHARLES E. BESS University of Nebraska, Lincoln, Nebraska Tue BOTANICAL GARDEN OF OAXACA DIRECTOR CASSIANO CONZATTI Botanical Garden of the State or Oaxaca, Mexico (Read by Title THE SCIENTIFIC SIGNIFICANCE OF THE [MPERIAL BOTANIC GARDEN OF PETER THE GREAT, WITH SPECIAL REFERENCE TO THE FLORA or ASIA DR. WLADIMIR I. LIPSKY Jardin Impérial Botanique de Pierre le Grand, St. Petersburg, Russia (Read by Title) CoMPARATIVE CARPOLOGY OF CRUCIFERAE WITH VESICULAR FRUITS—SoME GENERAL BIOLOGICAL AND SYSTEMATIC CONCLUSIONS DIRECTOR J. BRIQUET Jardin Botanique de la Ville Genève, Geneva, Switzerland (Read by Title) THE ORIGIN OF eg PROFESSOR JOHN M. COULTER University of Chicago, Chicago, Tilinois Tue History AND FUNCTIONS OF vage GARDENS ASSISTANT DIRECTOR ARTHU oyal Botanic Gardens, Kew, England (Read by Title) 8: 30—11: 30 P.M. RECEPTION. DIRECTOR’S RESIDENCE 1915] ANNIVERSARY CELEBRATION—PROGRAM Procram (Continued) Thursday, October 16 10:30 A.M. SPECIAL PERSONALLY CONDUCTED TRIP THROUGH THE CONSERVATORIES AND UNDS OF THE GARDEN; IN- SPECTION OF Be LIBRARY, AND HERBARIUM. 12: 30 P.M. LUNCH AT THE GARDE 1:30 P.M. SECOND SCIENTIFIC RE (Graduate Lecture Room) RECENT INVESTIGATIONS ON THE PROTOPLASM OF PLANT CELLS AND ITS COLLOIDAL + One PROFESSOR SS ee APEK Physiologisches Institut der K. schen Univertiähh: Prag, Austria (Rend Be Title) EXPERIMENTAL MODIFICATION OF THE GERM-PLASM DIRECTOR D. T. MACDOUGAL Department of Botanical Pa re Institution of Washington, Tucson, Ariz HORMONE IM pain DIRECTOR HANS FITTING Botanische Anstalten der Universität Bonn, Bonn, Germany (Read by Title) THe RELATIONS oF SCIENTIFIC Botany to PHYTOPATHOLOGY HEIMER REGIERUNGSRAT DR. O. APPEL Kaiserlichen N Anstalt für Land- mee Forstwirtschaft, Berlin-Dahlem, German Tue Law or TEMPERATURE CONNECTED Wire THE DISTRIBUTION oF MARINE ALGAE PROFESSOR WILLIAM A. SETCHEL Unwersity of California, Berkeley, California UEBER FORMBILDUNG UND RHYTHMIK DER PFLANZEN OR GEORG KLEBS Botanisches Institut Universität Heidelberg, Heidelberg, Germany by Title) PHYTOPATHOLOGY IN THE u IRECTOR JOHANNA WEST Phytopathological Laboratory, ee Holland PHYLOGENY AND RELATIONSHIPS IN THE me PROFESSOR GEORGE F. ATKINSO Cornell University, Ithaca, New Fork. THE ORGANIZATION OF A MUSHROOM INALD BULLER R A. H. REG University of Manitoba, Winnipeg, Canada Read by Title) PROFESS ( A Consprectus or BACTERIAL DISEASES In PLANTS WIN F. SMITH Bureau of Plant Industry, U. 8. Department of Agriculture, ington, D. ©. DR 7:30 P.M. TRUSTEES’ ene LIEDERKRANZ CLUB [voL. 2 6 ANNALS OF THE MISSOURI BOTANICAL GARDEN BANQUET MR. EDWARDS WHITAKER Toastmaster Ladies and Gentlemen: This being an epoch in the history of the Missouri Botanical Garden, it was thought that a short biography of its founder and benefactor would be interesting. Henry Shaw was born in Sheffield, England, July 24, 1800. He received his primary education at Thorne, a village a few miles distant from his birthplace, and at this early age developed a fondness for flowers and plants. Completing his course at Thorne, he continued his education at Mill Hall, twenty miles distant from London, where he was a student for six years. In 1817 he entered the service of his father, who was a manufacturer and dealer in metal wares, such as andirons, grates, ete. In 1818 his father sailed from England with his family for America, landing in Canada. We are without reliable inform- ation as to the exact place in which he located. The same year, probably in the late fall, he sent his son to the city of New Orleans to familiarize himself with the planting and growing of cotton. The climate of New Orleans did not suit him and the business was not to his liking, and his stay in Louisiana was short. He decided to seek his fortunes else- where, and so took passage on the ‘‘Maid of Orleans,’’ and landed at St. Louis, May 3, 1819. With the assistance of his uncle, James Hoole of Sheffield, he started a cutlery and hardware business in a room on the second floor of a building in the business district, which served as warehouse, show-room, office, and dwelling, doing his own cooking and housework, as he was without, and never was blessed with, a better half. His business was successful and uniformly profitable, and, at the age of 39, he had amassed a fortune, as he thought, large enough for any one and sufficient to gratify his taste for botany and the sciences. 1915] ANNIVERSARY CELEBRATION—BANQUET 7 He retired from business in 1840, and took a trip abroad, the first since leaving his native shore. This trip was evi- dently of short duration, as in 1842 he arranged his affairs and sailed a second time for the Old World, remaining three years, traveling extensively and making the acquaintance of botanists and scientists. Holding the English idea that a gentleman of fortune and leisure should maintain a town house and country home, he commenced the erection of his country home on the Garden grounds in 1848, completing it in 1849, and in 1851 built his town house at the corner of Seventh and Locust Streets, the site now occupied by the Mercantile Club. His last trip abroad was in 1851, and in 1858 he commis- sioned Dr. George Engleman of this city, a noted botanist then traveling in Europe, to procure material and information that he thought would be of service to a botanical garden; and at the suggestion of Sir William J. Hooker, then Director of Kew Gardens, began to prepare a laboratory and erected a museum building, and this was the commencement of Shaw’s, now the Missouri Botanical, Garden. While constructing the garden along the lines suggested by Sir William J. Hooker, he commenced the improvement of a tract of land immediately south of the Garden, now known as Tower Grove Park. In 1857 he had an act of the legisla- ture passed authorizing the city to receive, under certain con- ditions, as a donation this tract for a park. Among them was that the park was to be managed and controlled by a board of park commissioners of his appointment; secondly, that appro- priations were to be made sufficient to complete it in accord- ance with the plans already adopted; and the third condition, that an annual appropriation sufficient for its maintenance should be made; and in 1868 he deeded the property to the City. 7 Having in mind the conveying to Trustees of his estate to be administered by them for the benefit of the Garden, and a question having arisen whether such a trust was legal and could be administered in this state, he had an act of the legis- lature passed declaring his intentions, and authorizing him to [VoL, 2 8 ANNALS OF THE MISSOURI BOTANICAL GARDEN transfer his property to trustees and further declaring it lawful. Shortly afterwards, the Supreme Court of the state decided in the case of Chambers vs. The Mullanphy Relief Fund Bequest, that such trusts were legal and could be admin- istered in this state, thereby removing the doubt entertained by some of the legal profession. In 1866 Mr. Shaw secured the services of Mr. James Gurney from the Royal Botanical Garden in Regents Park, London, who was Head Gardener during Mr. Shaw’s lifetime and for several years afterward, and is now Head Gardener Emeritus, and also Superintendent of Tower Grove Park. There is no record of Mr. Shaw ever having had a public opening of the Garden, and a committee of trustees appointed to investigate and report on the date the Garden was estab- lished, decided that the Missouri Botanical Garden began its existence in 1889, upon the organization of the trust declared by Mr. Shaw’s will. Mr. Shaw executed his will January 26, 1885, devising his estate, with the exception of a few minor bequests, to a board of trustees of seventeen, the original members of which were designated in the will, and the board thus constituted, ex- clusive of certain ex-officio members, was to be self-perpetu- ating. The five trustees by virtue of the offices they hold were the Mayor of the City of St. Louis, the Chancellor of Washington University, the Episcopal Bishop of the Dio- cese, the President of the Board of Public Schools, and the President of the St. Louis Academy of Science. There were two honorary trustees appointed, Professor Asa Gray of Har- vard University, and Professor Spencer F. Baird of the Smithsonian Institution. Before the death of Mr. Shaw, on August 25, 1889, and the probating of his will, on September 17, both of the honorary trustees had passed away as well as two of the active members of the Board. The remaining trustees met October 14, 1889, at Mr. Shaw’s late residence, Seventh and Locust Streets, and effected an organization of the Board, electing Mr. Rufus J. Lackland President, Mr. Henry Hitchcock Vice-President, Mr. A. D. 1915] ANNIVERSARY CELEBRATION—-BANQUET 9 Cunningham, Secretary and Treasurer, and appointing Pro- fessor William Trelease Director. Immediately thereafter, by-laws were adopted and com- mittees appointed so that the estate could be efficiently managed. There were four committees—the Garden Com- mittee, the Auditing Committee, the Lands Committee, and the Ways and Means Committee—the President of the Board being ex-officio member of all committees. All actions of the committees require the approval of the Board before becom- ing operative. There have been three Presidents, three Vice-Presidents, one Secretary and Treasurer, and two Directors of the Garden since the organization of the Board. Of the original trustees named in the will but one survives, Mr. William H. H. Pettus, whose feeble health prevents his being with us this evening. There are but two salaried officers connected with the estate, the Secretary and Treasurer, and the Director, the trustees serving without compensation. And I wish here to correct an impression prevailing among many that this estate is exempt from taxation. That is erroneous. With the exception of the Garden grounds proper, the estate pays taxes the same as any citizen, and I may add that this item consumes about one-fourth of the gross income, the remainder being used for the maintenance of the Garden and other objects of the trust. Mr. Shaw was a man of independent thought and action, and while devising his estate to trustees, he at the same time appointed the public administrator of the City of St. Louis the executor of his will. Among provisions of the will was an annual appropriation for a flower sermon to be preached at such church and by such minister as the Bishop of the Diocese may select; an annual banquet for florists and gardeners in and about St. Louis, at which the Director of the Garden was to preside; a banquet for the trustees and the guests they may invite—literary and scientific men and friends and patrons of the natural sciences. Another provision of the will was that his residence at Seventh and Locust Streets was to be taken down and rebuilt [voL. 2 10 ANNALS OF THE MISSOURI BOTANICAL GARDEN upon the Garden grounds. It also provided that the Garden should be open to the public every day in the week, excluding holidays and Sundays with the exception of the first Sunday of June and September in each year, when the Garden should be open from 2:00 p. m. to sundown. This latter provision was literally carried out until the spring of 1912, when the Board thought the best interests of the Garden would be promoted by adding additional Sundays, and, having legal advice that there was no objection to their so doing, it was opened from April 1 to December 1, from 2: 00 o’elock until sundown. This action proved to have been wise, as the attendance at the Garden increased threefold. Such, briefly, ladies and gentlemen, were the objects and the accomplishments in the life of Henry Shaw, a man of whom any City, State or Nation might well be proud, and I request that this assemblage rise and drink with me, in silence, to the memory of Henry Shaw. [This toast was then drunk by those assembled.] The Toastmaster then presented as follows the be ro cng of the Hon. Henry W. po Mayor of the City of St. Louis, who had been expected to respond to a - Mr. Shaw in his wisdom appointed as one of the Trustees the highest official of the City of St. Louis. He was with us a short time this evening and was compelled to leave, owing to a previous engagement that he thought it would be impos- sible to break, and I have promised him to make his apologies for not remaining. n introdueing the next speaker of the evening, Dr. Johanna Westerdijk, Director of the Ph hei u aia Laboratory, Amsterdam, Holland, the Toast- aster spoke as follow We are complimented by the presence this evening of a lady from a foreign shore, whose achievements have given her a high position in the botanical world. It is my privilege to introduce Dr. Johanna Westerdijk, of Amsterdam. DR. JOHANNA WESTERDIJK Mr. Chairman, Ladies and Gentlemen: This is a delightful day, but I am sorry that not our great Holland botanist is in 1915] ANNIVERSARY CELEBRATION—BANQUET 11 our midst. He would be so much more able to express his feelings for America and for the Missouri Botanical Garden, which I know he loves so well. But since he is not here, I think it is a great honor for me to express my feelings, and I know that these feelings are the feelings of all the Dutch botanists, who all love botanical gardens, from the day of Boerhaave up to recent times. Mr. Chairman and Trustees, and Mr. Director Moore of the Botanical Garden, I thank you in the name of Holland for the delightful day, for the splendid reception I have had here; and if I may express myself in a bit of your American slang at a most solemn banquet, I thank you for the most jolly time I have had in this most delightful bunch of interesting American botanists. Geheimer Regierungsrat Dr. O. Appel, of the Kaiserlichen Biologischen Anstalt, Berlin-Dahlem, Germany, was next called upon by the Toastmaster in the following We have been favored by the presence of a number of for- eigners, among them a neighbor of Dr. Westerdijk, and I trust that, being in the Liederkranz Club, he will feel sufficiently at home to give us his impressions of our country, through which he has travelled extensively. I take pleasure in intro- ducing Dr. O. Appel, of Berlin. DR. O. APPEL Ladies and Gentlemen: If I should speak to you of my botanical or phytopathological work, I could use your lan- guage; but to express my feelings I must use my mother tongue, the German language! Sie haben zu dem Tage, den Sie heute festlich begehen, auch eine Anzahl europäischer Fachgenossen eingeladen und die Beteiligung von einer groszen Anzahl erwartet. Die Gründe, die die meisten Ihrer europäischen Gäste am Erscheinen ver- hindert haben, sind Ihnen bekannt und werden wohl von Ihnen allen bedauert. Dasz eine grosze Anzahl hervorragender Vertreter des Auslandes hier erwartet wurde, hat seine Berechtigung, denn in den Jahren seines Bestehens hat der Botanische Garten von [Vou, 2 12 ANNALS OF THE MISSOURI BOTANICAL GARDEN St. Louis sich würdig in die Reihe der gröszeren derartigen Institute eingegliedert, und trotz der groszen Entfernung hat schon mancher europäische Botaniker diese Stätte der Wiss- enschaft ausgesucht und die Kunde von seiner raschen Ent- wickelung in ferne Länder getragen. Aber nicht nur durch den Beweisz sind die Bande zwischen unseren deutschen Botanikern und den am Shaw’s Garden arbeitenden Fachgenossen geknüpft worden, auch dureh man- nigfachen Austausch von Material und Gedanken haben sich viele Beziehungen ergeben, die heute eigentlich ihren Ausdruck durch das Erscheinen einiger unserer bedeutendsten Fach- genossen, Klebs und Fitting, ihren Ausdruck haben finden sollen. Da dies nun nicht sein konnte und äuszere Umstände mir als dem einzigen deutschen Botaniker die Teilnahme an Ihrer Feier vergönnt haben, so möchte ich nicht versäumen, Ihnen im Namen der deutschen Botaniker die besten Wünsche aus- zusprechen. Fünfundzwanzig Jahre erscheinen als eine kurze Spanne Zeit und doch haben Sie ein Recht den Abschlusz dieser fünf- undzwanzig Jahre zu feiern. Dieser erste Zeitabschnitt ist einer der wichtigsten, vielleicht überhaupt der wichtigste, denn in ihm sind die Grundlagen für die ganze Zukumft des Gartens geschaffen worden. Was in diesen Jahren geschaffen worden ist, das haben Sie alle gesehen. Noch erkennt man da und dort die kleinen und einfachen Verhältnisse, unter denen die Arbeit begonnen worden ist, aber daneben und sie überragend hat schon die neue Zeit dem Garten und seinen Gebäuden ihr Gepräge aufgedrückt. Uberall sieht man, mit welcher Plan- mäszigkeit und Groszzügigkeit die Entwickelung gefördert worden ist und wie sowohl der wissenschaftlichen Arbeit, wie der Nutzbarmachung für die grosze Allgemeinheit in jeder Weise Rechnung getragen wird. Aber auch denen, die nicht in der Lage sind, die Schätze des Gartens, der Laboratorien direkt zu benutzen, haben Sie eine Quelle der Belehrung und Anregung gegeben durch die Herausgabe der beiden periodischen Schriften ‘Annals of the Missouri Botanical Garden’ und ‘Missouri Botanical Garden 1915] ANNIVERSARY CELEBRATION—BANQUET 13 Bulletin,’ von denen die erste fiir die Gesamtheit der bo- tanischen Welt bestimmt ist, wahrend die letztere sich an alle die in Ihrer eigenen Heimat wendet, die fiir die Botanik als scientia amabilis Sinn und Verständnis haben. So gehört denn keine grosze Prophetengabe dazu, dem Shaw’s Garden eine weitere gedeihliche Entwickelung vorher- zusagen. Dasz aber auch die deutschen Botaniker immer da, wo sie können, und wo ihre Mitwirkung erwünscht ist, gerne mit Ihnen Hand in Hand arbeiten werden, dafür bringt Ihnen die Art der deutschen Wissenschaft, die stets die Förderung jeglicher Forschung zum allgemeinen Besten im Auge gehabt hat und auch in der Zukunft als höchstes Ziel im Auge behalten wird, den Beweis. Meine Wünsche aber erlauben Sie mir zusammenzufassen in den Ruf: Hortus botanicus Shawensis vivat, crescat, floreat! (A translation of Dr. Appel’s address follows.) For this day which you are celebrating, you had invited also a number of European colleagues and expected that of these a large proportion would participate in the exercises. The causes which have prevented most of your European guests from being present are known to all of you and doubtless are regretted by you all. The expectation of a larger number of foreign representa- tives is justified, for during the years of its existence, the Botanical Garden of St. Louis has deservedly taken its place in the ranks of the larger institutions of its kind, and, despite the great distance, many a European botanist has already sought out this scientific center and carried the message of its rapid development to distant lands. But the ties that exist between our German botanists and their colleagues working at Shaw’s Garden have been estab- lished not alone by such visits, but also by the abundant ex- change of material and ideas, in which relationships have developed which to-day were to have found expression through [VoL 2 14 ANNALS OF THE MISSOURI BOTANICAL GARDEN the appearance and participation of two of our most note- worthy colleagues, Klebs and Fitting. But since this could not be, and circumstances have gra- ciously willed it that I should be the only German botanist to participate in your celebration, I wish to express to you on behalf of the German botanists our best wishes. Twenty-five years appear as a short interval of time and yet you have a right to celebrate the completion of these twenty-five years. This first period is one of the most impor- tant, if not the most important, for in it have been established the foundations for the entire future of the Garden. You have all seen what has been created in these years. One still recog- nizes here and there the simple conditions under which the work was started, but these are eclipsed by the imprint which later years have left on the Garden and its buildings. One sees everywhere with what ability and foresight the develop- ment of the Garden has been promoted and every provision made for the scientific work and the increased usefulness of the Garden to the public. But you have also provided a source of information and stimulation to those who are not in a position to directly make use of the resources of the Garden by the publication of the two periodicals, ‘Annals of the Missouri Botanical Garden’ and ‘Missouri Botanical Garden Bulletin,’ of which the former is intended for the entire botanical world, whereas the latter goes to those in your home who have an interest in, and an understanding for, botany as a scientia amabilis. It does not, therefore, require a great gift of prophecy to predict for Shaw’s Garden a further deserving development. Wherever German botanists can help and wherever their cooperation is desired, they will always gladly work hand in hand with you, proof of which is furnished by the very char- acter of German science, which has always sought to further each and every investigation for the greatest general good, an ideal which will not be lost sight of in the future. My wishes you will permit me to express thus: Hortus botanicus Shawensis vivat, crescat, floreat!” 1915] ANNIVERSARY CELEBRATION—BANQUET 15 The Toastmaster next called upon are N. m of the University of Christiania, Christiania, Norway, as follow We have also a friend and ah tots Norway, who, I understand, had a rather peculiar experience in this country. He told me that he had for forty-eight hours or more lost his better half by having the tickets and she starting without any Pullman accommodations. I know he can talk to us interest- ingly, and we will be glad to hear from Professor N. Wille, of Christiania. PROFESSOR N. WILLE The Members of the Board of Trustees, Fellow Scientists, Ladies and Gentlemen: I am deeply grateful to the members of the Board of Trustees of the Missouri Botanical Garden for the kind invitation to participate in this celebration. Had it not been for this I should perhaps never have known America. In the short time that I have been here I have learned much, and I only regret that it is not possible for me to remain in your country longer. When I see the splendid botanical equipment of the Missouri Botanical Garden, I can only lament that it has not been possible for me to prosecute my work under such unusually favorable circumstances. My best wishes for the continued scientific development of the Missouri Botanical Garden. In introducing Captain Henry King, the Toastmaster spoke as follows: We have now reached one of the very many interesting sub- jects of the evening, namely the press. Who is there among us who has not, at some time and some place, received flattering notices at its hand, while again, hard knocks, administered without warning and at the most unexpected moment. If I may be permitted to make a suggestion to! the speaker who is to follow me, it is that he go easy with us scientists and delvers in the soil, and in the language of a son of Erin’s Isle, “If you can’t go easy, go as easy as you can.” It is my privilege to introduce Captain Henry King, Editor of the ‘St. Louis Globe-Democrat.’ CAPTAIN HENRY KING It is the paramount duty of the newspaper editor to tell the truth. I do not mean literally and completely, but approxi- [VoL. 2 16 ANNALS OF THE MISSOURI BOTANICAL GARDEN mately and within the rule of reason and the zone of safety. Less is expected of other people, apparently, or the editor would not so often find it so hard to get the truth when he wants to print it. Take, for example, the tremendous and de- plorable situation now presented in Europe. With all our anxiety and all our facilities, we can not be certain how much or how little of the wild and whirling daily reports from there —news from hell, so to speak—is dependable. We have not yet even found out definitely what it is all about, and why hun- dreds of thousands of industrious and inoffensive citizens have been taken from their homes and affairs, and sent forth with all kinds of murderous weapons to slay one another as fast as possible. The most that we can be sure of is that a war of unparalleled dimensions and appalling severity is raging, and that about the only really good thing in it is that white mes- senger of pity and succor, the Red Cross nurse. And yet I am assured by a leading St. Louisan just returned from the seat of war that the reports in the St. Louis papers are more rational, consistent and enlightening, after all, than those in the papers of any of the cities on the other side of the Atlantic. This man’s word is good and his judgment accurate. You all know him. I refer to the Hon. Charles Nagel. The lesson of Mr. Nagel’s gratifying statement is a timely and an important one. It goes to show that in a case of world- wide interest and illimitable consequences, where the truth veritably lies at the bottom of a well, the St. Louis press gets nearer to it by care and candor, by unprejudiced analysis and fair-minded discrimination, than the press of Europe. This example is an extreme one, perhaps, but I feel safe in saying that it is characteristic and relatively prevalent in all cases. I am here, as you have been advised, to talk about the press, or at least to use it as atext. You do not expect me, I am sure, to stand here on this festive and botanical occasion and confess the sins of my esteemed contemporaries, or to acknowledge my own, for that matter. So, if you please, I am going to side- step the sins, for the present, and declare from personal knowl- edge and daily comparison that St. Louis has ample reason to be proud of her newspapers. They are not perfect, to be sure, 1915] ANNIVERSARY CELEBRATION—BANQUET 17 which is only saying that human life is not perfect, for they are made out of life as life is lived in this goodly city and else- where from day to day. They tell you the current history of the community, of the country, of the universe, and they tell it as correctly as the limitations of human nature permit. They have defects of temperament, faults of accident and mis- information, I frankly admit. If they had not these delin- quencies, mingled with their excellences, as has the life out of which they are made, they would soon become too good for this world and their home would be in heaven, and you would not have any use for them here on this rolling and imperfect planet. They make mistakes, yes—just as you do, and all men (and some women) do, just as the busy life out of which they are made is in great measure a matter of mistakes, which con- stitute what we call experience, and experience is only another name for news. Bear with me, I beg you, if I seem to be too ardent in this topic of the St. Louis press. But I am putting aside, for this occasion, the proverbial modesty of my profession, with a view to telling you the naked truth as if I were under oath. And let me remind you, while I think of it, that the only monument in the world to ‘‘The Naked Truth’’ stands only a short distance from where we are assembled, and its purpose is to typify and commemorate the lives and services of three great St. Louis newspaper editors, Schurz, Preetorius and Daenzer. I am talking to you of the successors of those men, whom I know like a book— my neighbors, my friends, my fellow-workers—,the men who direct and adorn and give tone and influence to the St. Louis press. I know them to be tireless in their pursuit of facts, in their zeal for the public welfare, in their ambition to promote the growth and progress of this admirable city. It is sometimes said in criticism of them that they are governed mainly by commercial considerations, and one of the pestif- erous sort of professional reformers has lately sent forth a book in which he goes so far as to charge that their policies are absolutely dictated by their big advertisers. Well, if it wasn’t for the big advertisers, you would hardly be able to get the modern wonder and recognized necessity of a daily newspaper [VoL. 2 18 ANNALS OF THE MISSOURI BOTANICAL GARDEN for the absurd price of a penny a copy, the cheapest of all known commodities of general use; and I often think that the advertisements constitute the most interesting and serviceable part of the paper. That is not the only reason why we print so many of them, I am bound to admit, but it certainly tends to ameliorate the condition and to make the habit almost in- nocent. As for the advertiser as a dictator of editorial policy, we do not find him very insistent or obstreperous. In a life- time of experience, I have never yet known an advertiser to solicit any selfish advantage or assert any right of arbitrary interference on account of his patronage; but it is a common thing to have them come forward in earnest and practical support of projects for the common good. We owe it largely to the advertisers, the business men, that we have the Veiled Prophet with us every year; that we had an incomparable World’s Fair; that we produced the unequaled Pageant and Masque; and I don’t believe they will permit the reproach of failure to overtake the Symphony Orchestra. And I’m going to include the Free Bridge in this assurance, though just now I do not see any practicable way to connect with it. This brings me to the point of chief interest, to the Missouri Botanical Garden, with its immense display of floral splendor, its infinite sources of delight and instruction, of admonition and of consolation. I wish I could botanize about it in the thorough and skillful manner of our distinguished scientific visitors. But alas, [ have to make the bashful admission that I probably know less about botany as a science than any other person on your program to-night—unless it may be your Toastmaster. The fact is, I have had less to do with flowers than with quadrupeds, such as the Donkey, the Bull Moose, and the Elephant—God bless him—begging the pardon of those of you who don’t happen to like him as well as others of us do. But I am tolerably familiar with the part which flowers have played in the affairs of the world. I know how all literature is pervaded by their fragrance and their symbolism. I am not unmindful of their cherished associations in the lives of all classes, from the cradle to the grave. I know how, in many instances, when wisdom reaches its limit and language fails, 1915] ANNIVERSARY CELEBRATION—BANQUET 19 they have the gift of talking to us and for us, in a form of expression which we can grasp only with our feelings and emotions, and which our hearts rather than our heads must interpret and utilize. But I must not deviate too far from the relation of the editor to floriculture, which is similar to that of the boy in Mr. Lincoln’s story who, being asked if he liked gingerbread, re- plied, you remember, ‘‘I reckon I like gingerbread better than any boy in this town, and get less of it.’’ So it is mainly with the editor and the bouquets. He is more apt, as a rule, to have stale vegetables thrown at him, figuratively speaking, and to be condemned to wait for his flowers until he reaches that point in his career where he no longer has use for anything else. But, happily, the editor is nothing if not a philosopher. The discipline of his profession teaches him patience and tolerance and sweet reasonableness. In the nature of things, he gives more attention to other people’s affairs than to his own—so much so, indeed, that now and then he is accused of being over- zealous, not to say over-inquisitive, in that respect. If a bouquet comes his way it surprises and confuses him, since it contradicts his personal experience that if virtue be not its own reward, then it usually remains unrewarded. Nevertheless, he goes on boosting instead of knocking, because it is his mission to spread the gospel of good cheer and make more room in the sun for those who inhabit the earth. He welcomes particularly an occasion like this, where he can help to cele- brate the choice taste, the fine civic spirit, the munificent public benefaction of a man like Henry Shaw. And his pleasure is doubled when to such an opportunity is added the chance to compliment the Missouri Botanical Garden upon having for President of its Board of Trustees a man with the many ex- cellent qualities of Edwards Whitaker. Science is the basis of the great enterprise which Mr. Shaw founded, of course, but science needs trained business sense to invest its service with the highest practical usefulness. Mr. Whitaker has shown in a marked degree his realization of the possibilities of his posi- tion, and the steps by which the benefits of Shaw’s Garden, as we familiarly call it, can be materially multiplied. I feel [VoL. 2 20 ANNALS OF THE MISSOURI BOTANICAL GARDEN authorized to say that in this important work he will have the hearty coöperation of the St. Louis press; and I am sure that he will in turn see to it that the editors get all the floral tributes that are due to them, at least when the time arrives for them to confront the ultimate River of Separation, and each of them shall need something of that sort to waft aloft in his behalf the beautiful message of Tennyson— “For though from out our bourne of time and place, The flood may bear me far, I hope to see my Pilot face to face When I have crossed the bar.” In the following words the Toastmaster called upon the next speaker of the evening, Dr. William G. Farlow We have with us this evening a guest who, I can truth- fully say, is loved by every botanist in America, and I can also assert without fear of contradiction that he is recognized as their dean. I am proud to introduce Dr. William G. Farlow, of Harvard University. DR. WILLIAM G. FARLOW Mr. President and Ladies and Gentlemen: As I look upon this company and see how many there are here, all of whom are interested in the St. Louis Botanical Garden, I can’t help asking myself the question: ‘‘Why are they interested in the Garden?’ Some have one reason; some have another. Some like it for the flowers that are shown there; some like it for the scientific work done there. But whatever their reasons may be, I would like to take advantage of this occasion to say a few words about what seems to me to be the true object and aim of botanical gardens. Let us go back to history. The first garden on record, I believe, was the Garden of Eden. That garden unfortunately was obliged to be closed to the general public only a short time after it was opened. But we learn some lessons even from the Garden of Eden. In the first place, do not mix zoölogy and botany. The Garden of Eden was not purely a botanical gar- den. You know what the snake did and will always do in botanical gardens. There is another curious thing about the 1915] ANNIVERSARY CELEBRATION—BANQUET 21 Garden of Eden. It is the only garden I ever heard of from which people were excluded because they had just begun to learn something, and it seems to be exceedingly cruel that they should have been turned out into a cold world merely because they knew something. But it is a long step from the Garden of Eden, and history is a little more accurate in recent times than it was then. The traditional botanical garden, the one which has existed for centuries in Europe and to a less extent in this country, was a place where the seeds of a great many plants were sown; some came up and some did not, but they were all labelled. Now many plants are annual but labels are perennial and the un- fortunate result in many of the older gardens was that there was a luxuriance of labels and a comparative poverty of plants corresponding to the labels. The ideal garden is nature. We can never equal nature in anything like proximate perfection. Go up in the mountains or go out into the woods. You see nature where it has existed for ages, the result of centuries of work. What we see is not what has been planted a few years before. It is the result of the conflict of ages going on between natural forces, and what we see is the final result, such as can not be obtained by man. We find plants which grow where they naturally grow; we see moss where moss should grow; we see trees where trees should be. Ina botanical garden of the present day, such as the Mis- souri Botanical Garden, we should imitate nature as far as is possible in a limited space and offer to the general public and the special students of botany an epitome of the vegetation of the world. Those of our botanists who visited the Garden yesterday and to-day saw a superb display of cosmos. I don’t know that St. Louis people fully appreciate what a fine exhibition of flowering plants we have seen here, but the cosmos are per- fectly magnificent and you have reason to be proud of them. I hope your spring flowers are equally splendid, and there is no reason why in the summer you can’t have groups of equally fine character. The old-fashioned botanical gardens had no beauty whatever. They were simply artificial and repulsive, [VoL. 2 22 ANNALS OF THE MISSOURI BOTANICAL GARDEN but at present a botanieal garden must in the first place be beautiful. Although beauty is not the end of everything, we begin with beauty and end with science both practical and theoretical. Besides the flower beds and hothouses the casual visitor notices certain buildings of considerable size scattered here and there. What they are for is not perhaps known to many of those attracted by the floral display. Without these buildings and their contents and the experts in charge of them there could be no floral display of any real importance. Al- though they add little to the beauty of the Garden, in these buildings is done the work which gives to the Garden its scien- tific value and entitles it to recognition throughout the botan- ical world. The very valuable library and herbarium are, in a sense, the soul of the Garden, since from them is obtained a knowledge of the plants cultivated, and they are a necessity to those carrying on research in the laboratories. At this late hour I cannot enter into details. It should be said, however, that for the library and herbarium, fire-proof buildings, always expensive, are necessary since if destroyed they could not be replaced by insurance. The laboratories for research are in a somewhat different position. The value of research in vegetable physiology and pathology and other sub- jects other than systematic botany, which is, of course, carried on in the herbarium, cannot be overestimated. Convenient and well-equipped laboratories are a necessity in a modern garden. They do not, however, require expensive fire-proof buildings. The outfit of the laboratories should be up to date, but new and improved instruments are invented from year to year and an occasional conflagration is not to be dreaded since the insurance on the older instruments can be used for pur- chasing better new ones. Furthermore, the trend of original research is constantly changing and, in trying to adapt them- selves to the current demands of the scientific public, the na- ture of the work done in research laboratories and in conse- quence their equipment vary from time to time. As I look at my audience, I am reminded of something I saw in the train coming to St. Louis the other day. I picked up what I believe was the last number of ‘Life,’ and glanced at 1915] ANNIVERSARY CELEBRATION—BANQUET 23 a cartoon, a crowd of persons seemingly very much pleased, and wishing to know why they were hilarious, I saw that the title was the ‘‘ Millenium Celebration in Honor of the Abolish- ment of After Dinner Speeches.’’ Your faces remind me somewhat of those of the crowd I saw in ‘Life,’ and I now close, fearing that you may be hoping that the millenium will arrive before we have another twenty-five year dinner, when I shall not be with you. The Toastmaster next called upon the Hon. Charles Nagel, of St. Louis, in the following words: Our city has had a number of her sons, and adopted sons, called to occupy positions of responsibility at the National Capital, one of whom, after four years’ service in the Cabinet, has returned to the city of his adoption; and I am proud to introduce the Hon. Charles Nagel, ex-Secretary of Commerce and Labor. HON. CHARLES NAGEL Mr. Toastmaster, Ladies and Gentlemen: It appears to me that the last speaker was both wise and unkind in referring to the illustration from ‘Life’ at the close of his speech. I can assure him that my embarrassment was sufficient without that reference. In endeavoring to account for my presence in a family of botanists, I have been compelled to go pretty far back in my life and to recall an incident when an aged grandmother, whom I never knew, sent me what Dr. Appel will pardon me for call- ing a ‘‘Botanisirbuechse,’’ to encourage me in the collection of plants. It was only a trifle at the time, and yet I imagine I share the experience of most people in tracing my interest in nature to that early incident in my life. I would say that my love of nature is such that I would rather have my child love the virility and strength of an oak leaf than all the bouquets and flowers that can be gathered. I believe that real love for the strength of nature is what we need, and not the pampering influences of the selected flower. I believe in the forests of New Hampshire that my friend, Dr. Farlow, loves; and I can see him now searching [VoL, 2 24 ANNALS OF THE MISSOURI BOTANICAL GARDEN for his specimens there, but never unmindful of the grandeur of all nature to which his specimen furnishes only a clue. But studying of the plant means more than that. It means the reason of nature. I have sometimes thought that if we knew more of the reason for the decline of one plant and the triumph of another, we would have a better understanding of the meaning of the inevitable and unavoidable conflicts that are now tearing the world apart; and we Americans, if we knew more of the generating influence of the one and the survival of the other, would appreciate that it takes conflict and danger to make strong men and women. I do not want to go too far, but while a park need not be a botanical garden, no park can succeed unless it has applied to it the science and the work of the school of botany. We can not have the city beautiful, with all our preaching, until we understand the true meaning of a school of botany and of our, botanical gardens. If any one doubts it, let him look abroad. He who has seen the beautiful forests about Paris, the splendid forests about Berlin, the wonderful forests about the Hague, will say to himself, ‘‘ Yes, this is nature, profound and beauti- ful’’; but it is not an accident. It is the result of nature’s force guided by experience and science. That is what we need—politics, government, must take into counsel the man of wisdom and experience to produce those wonderful results which so far we cannot imitate. There is more than that. Abroad, not only the government utilizes the information which these men and women of science have to give, but every man and woman throughout the land con- sciously or unconsciously is influenced by the same teaching. Wherever you look and whatever you see demonstrates to you the result of that kind of work. It means not only the flowers and the plants, but it evidences the happiness of family life. Every field shows it; every home and every garden patch shows it. That is the lesson we have to take unto ourselves in this, our new country. That is what we have to do if, as a people, we are to succeed; and it is for this reason that we welcome the greetings of the distinguished guests who have joined us to-night. 1915] ANNIVERSARY CELEBRATION—BANQUET 25 True, all the countries are not represented; but we have the right to say to ourselves that science and civilization stand above all the conflicts of the day. Ultimately, the very nations who are now engaged in this conflict will again have to unite their hands to bear the standard of civilization jointly upon the Continent; and we have a right to say to-night that while only a few countries are represented, from the standpoint of science and civilization broadly speaking, the few representa- tives are here to speak to us for all the civilized nations of the world. The last speaker of the evening, Dr. George T. Moore, was called upon by the Toastmaster as follows: Mr. Shaw’s will requires the Board to appoint a Director of the Garden, who is to reside upon the Garden grounds. He is virtually the executive of the board and the Garden Committee so far as Garden matters pertain, and he might be compared to the man behind the gun, as much of the success of the Gar- den depends upon him. The Director is known to so many of you, an introduction seems hardly necessary; but for form’s sake I take pleasure in introducing Dr. on T. Moore, Di- rector of the Missouri Botanical Garden. DR. GEORGE T. MOORE It was my pleasant task on yesterday to weleome those who honored us with their presence at the first formal exereises celebrating the passing of a quarter century in the life of the Missouri Botanical Garden. To-night has been delegated to me the duty of closing what at least for the Garden has been a most memorable festival, one which long will remain that delight which, joined with memory and hope, constitutes a perfect occasion. An after-dinner speech is sometimes regarded as a sort of verbal culture medium for the propagation of words, and it is remarkable with what rapidity those who confine their efforts to media containing no solidifying substance can cloud an otherwise clear situation. With the example set me to-night, it behooves me to speak directly to the point and not spoil an evening which thus far has been faultless. (Vor. 2 26 ANNALS OF THE MISSOURI BOTANICAL GARDEN That the Missouri Botanical Garden was fortunate in its founder, I have tried to indicate early on this anniversary occasion, and it is not necessary, even if it were possible, for me to add anything to the appreciative words which have been spoken at this table. I do feel, however, that perhaps not enough emphasis has been placed upon the fact that it is the organization of the Board of Trustees which furnishes the real reason for this anniversary, and that in honoring Mr. Shaw and in praising the courage and skill which he displayed, we are apt to forget the prolonged efforts of those men who have unselfishly given of their time and thought to make the dream of Henry Shaw come true. You botanists present know that he who would keep up his scientific fire must also have the means of keeping up his ma- terial woodpile. Certainly no place in this country has a trust been so closely and so successfully administered as by that body of men who, from the very first, have labored without remuneration or recognition from those they served, the Board of Trustees of the Missouri Botanical Garden! Every citizen of St. Louis, every visitor to the Garden, every botanist or individual who may have been assisted by the fa- cilities of the Garden, library or collections, has reason to echo the words of George Washington, which, slightly altered, are just as applicable to the Board of Trustees of the Missouri Botanical Garden throughout its existence, as they were to Benjamin Franklin: “Tf to be venerated for wisdom, if to be admired for talents, if to be esteemed for service, if to be loved for devotion, can gratify the human mind, they must have had and have the pleasing consolation that they had not and will not have lived in vain.” In the long run, which is a sort of mathematical name for Providence, such services have their reward, but every twenty- five years, I think the Board of Trustees as a body—for the individuals wouldn’t permit such a thing—should at least be entitled to a public statement of the facts. We are grateful to all who, through their active participa- tion or by their presence at the sacrifice of valuable time and by long journeyings, have contributed to the success of this 1915] ANNIVERSARY CELEBRATION—BANQUET 27 occasion. Especially do we owe thanks to those who by presenting such splendid papers have made the programs such as will be difficult to surpass in the future. The celebration of an anniversary is a ground of congratu- lation or regret according as it marks the progress or decline of the event it commemorates. My only hope at this time is that on the next anniversary occasion of the Missouri Botan- ical Garden the advances made along the lines of the various activities in which the Garden is interested, may be far beyond those of the present, and that the celebration will exceed the twenty-fifth as many times as the Garden is years older. The Toastmaster concluded the program of the evening with the following remarks: The hour is growing late. A few words before parting. On behalf of the Board of Trustees of the Missouri Botanical Gar- den, I wish to thank one and all for their presence here this evening, especially those who have journeyed far to be with us, and to express the hope that we may enjoy this pleasure many years to come. Good night! ADDRESS OF WELCOME GEORGE T. MOORE Director of the Missouri Botanical Garden It becomes my pleasant duty at the beginning of the pro- gram celebrating the twenty-fifth anniversary of the organiza- tion of the Missouri Botanical Garden to formally do what I am sure has already been done over and over again by each member of the staff—welcome most heartily those guests who have done us the honor of coming to share with us the simple, yet I hope adequate, ceremonies which have been arranged for this occasion. At one time it was expected that this welcome would be extended by Mr. Houston, who, because of his triple offices, as a member of President Wilson’s cabinet, a Trustee of the Missouri Botanical Garden, and Chancellor of Wash- ington University, as well as the grace with which he would have addressed you, would most suitably have performed this duty. Pressure of public work has prevented the Secretary of Agriculture from being with us, however, and I can only hope that you will feel that the welcome extended to you now carries with it as much cordiality and good will as if it came from an officer of the Government, the Garden, and the University. Nothing could be more fitting at this time than some account of the life and work of the founder of this Garden, who de- serves, both because of his far-sighted planning and his mag- nificent gift, to rank as America’s foremost patron of botany. Most of you are no doubt familiar with the simple but im- pressive biographical facts concerning Mr. Shaw. How he came to this country from England with his father in 1818, being eighteen years of age, and after brief stays in Canada and New Orleans, settled in St. Louis. With a small stock of hardware he began business in one room, which also served as his bedroom and kitchen. From such a small beginning— and this on borrowed capital—scarcely more than twenty years were required by this pioneer merchant to amass a ANN. Mo. Bor. GARD., Vor. 2, 1915 (29) [VoL. 2 30 ANNALS OF THE MISSOURI BOTANICAL GARDEN fortune, for at forty years of age Mr. Shaw retired from active business to devote the remaining forty-nine years of his life to travel, and later to the active and remarkably intimate crea- tion and management of a garden—that garden of which, be- cause of his intelligent planning and unprecedented fore- thought and liberality, we are to-day celebrating the silver anniversary. The advice and counsel of such men as Dr. George Engel- mann, Sir William Hooker and Professor Asa Gray was freely sought and as freely given. In this connection I should like to read a letter from Sir Joseph Hooker, written June 17, 1888: “The Camp, Sunnydale, England. “My Dear Mr. Shaw :— “T have just received your most handsome present of Engelmann’s Botanical Works, edited by our dear late friend, Dr. Gray, and I do thank you most heartily, no less for your kind gift than for the effective service to botany that this most valuable contribution to the science renders. It is indeed a noble tribute to a man whose labors as a most conscientious and painstaking botanist have never been surpassed, and I prize it for the sake of the man whom I knew so well and esteemed so highly. I shall never forget my visit to him and to you and the afternoon I spent in ur aes and museum at St. Louis, in company with Dr. and Mrs. G ave been most interested in all that Dr ae told me last year Ren the noble botanical institution that you have founded and in his hopes that it would be a center of diffusion of knowledge, the influence of which would be felt far and wide. “I think that he was more proud of your consulting him in the matter of its organization than of any of the many services which he had rendered to American botany, and he certainly regarded his labor with you as the most pleasant episode of his later years and by far the most importan “Believe me, my dear sir, most faithfully and gratefully yours, JosEPH D. HOOKER.” The country home of Mr. Shaw was built on these grounds in 1849, and the breaking of the prairie for his garden is said to have begun in 1857. There is no record of any formal opening of the Garden to the public, however, the date 1858 on the entrance of the main gate probably being the year it was erected rather than the time it was first opened to visitors. The small ‘‘ Museum and Library,” as it is designated in the 1915] MOORE—ADDRESS OF WELCOME 31 stone over its entrance, was built in 1859, and this same year the installation of the Bernhardi Herbarium, previously pur- chased in Europe, marked Mr. Shaw’s intention to make the Garden a center for scientific investigation and research. How successfully the founder of the Missouri Botanical Gar- den incorporated this idea in the document intended for the guidance of those who should administer this bequest, is evi- denced by the remark of Judge Medill, one of the first members of the Board of Trustees, who, after the reading of the will, exclaimed: ‘‘That is a scientific institution and much should come of its services to botany!’’ Mr. Shaw died August 25, 1889, and on September 10 the formal organization of the Board of Trustees, created by his will, took place. This is the anniversary we celebrate, for, as I have indicated, it is the only definite anniversary we have. Certainly as a ‘‘ botanical institution, public in character,’’ the Missouri Botanical Garden began its existence upon the organ- ization of the trust declared by Mr. Shaw’s will. Two other notable bequests of Mr. Shaw require brief men- tion at this time, one indicating his desire for further scientific investigation in botany, the other the love for the beautiful in nature and his wish that all might have unlimited oppor- tunity for acquiring and indulging this same passion. I refer, of course, to the endowment of the Henry Shaw School of Botany of Washington University, and the gift of Tower Grove Park to the city of St. Louis. The first is, owing to the broad-minded liberality of the Board of Trustees of the Gar- den and the untiring and unselfish efforts of place among similar schools of the kind of would not himself be ashamed. The latter, 1 care of Mr. Gurney, its first and only Supe we are proud to call the Head Gardener Em souri Botanical Garden, is nobly fulfilling which it was created. It is proper, then, that this company of s semble here to do honor to the memory of rejoice with us for the successful complet: years of usefulness of the Missouri Botanic: its staff, taking a which Mr. Shaw ınder the fatherly rintendent, whom eritus of the Mis- the purpose for cholars should as- ‘ Henry Shaw, to ion of twenty-five ıl Garden. [Vor. 2, 1915] 32 ANNALS OF THE MISSOURI BOTANICAL GARDEN Both personally and in my official capacity I welcome you not only to these ceremonies, but as coöperators in an era of even greater effort and achievement for the cause of the science which Mr. Shaw loved and honored and encouraged. THE VEGETATION OF MONA ISLAND! N. L. BRITTON New York Botanical Garden During the progress of the scientific survey of Porto Rico, organized by the New York Academy of Sciences with the aid of the American Museum of Natural History, the New York Botanical Garden and Columbia University, in coopera- tion with the Porto Rican Insular Government, exploration has been carried out not alone on the mainland of Porto Rico but on several small islands adjacent and politically a part of that colony. Two of these islands lie in the Mona Passage between Porto Rico and Santo Domingo, and being scientific- ally almost unknown, were made points of examination in February, 1914, when I visited them in company with Mr. John F. Cowell, Director of the Buffalo Botanic Garden, Dr. Frank E. Lutz, Assistant Curator of Invertebrate Zodlogy in the American Museum of Natural History, and Mr. W. E. Hess, Plant Propagator of the Porto Rico Agricultural Ex- periment Station at Mayaguez. The trip was made in a sloop chartered at Mayaguez. Desecheo Island, lying about eighteen miles northwest of Mayaguez, was first visited, and explored during two days; this island is somewhat more than one square mile in area, bordered by rocky coasts, rising abruptly into several hills, and covered with low trees and shrubs. Its flora is essen- tially identical with that of the drier parts of Porto Rico and of Santo Domingo; the small tree Morisonia americana and the snowy cactus (Mamillaria nivosa) have, however, not yet been found on the Porto Rican mainland, although both occur on the Island of Culebra east of Porto Rico, and neither of them is known on Santo Domingo. The cactus Opuntia haitiensis, plentiful there, is otherwise known only in His- paniola, and the shrub Torrubia discolor of Hispaniola and Cuba has not been found on Porto Rico. The collection made 1 Issued May 17, 1915. ANN. Mo. Bor. GARD., VOL. 2, 1915 (33) [Vou, 2 34 ANNALS OF THE MISSOURI BOTANICAL GARDEN by us on Desecheo, together with one made by Professor F. L. Stevens and Mr. W. E. Hess in May, 1913, shows that the spermatophytes of Desecheo number about 90 species; further intensive exploration might reveal a few more. A single species of fern was seen, four species of mosses, and two species of hepatics. As there is no probability of this little island ever having been a part of the Porto Rico main- land, its plants must have reached it by natural agencies; there are probably as many fungi and lichens as of other land plants collectively, so the total land flora of Desecheo probably includes at least 200 species. Mona Island, lying about thirty miles to the southwest of Desecheo, in the middle of the Mona Passage between Santo Domingo and Porto Rico, has an area of approximately twenty square miles. Prior to our visit, only one botanical collection had been made there, when it was visited by Professor F. L. Stevens in 1913, at which time he obtained specimens of about 150 species of flowering plants, and gave especial atten- tion to the parasitic fungi. The considerable land area of this island made a complete knowledge of its flora desirable, from the standpoint of geographical distribution of West Indian plants, and we were able to devote five days to collect- ing. The greater portion of Mona is a limestone plateau elevated from 125 to 175 feet, the surface of this plateau being nearly level and devoid of hills; its soil is very sparse, con- sisting altogether of reddish loam in depressions of the lime- stone surface, and not of considerable extent at any point visited by us. The limestone is evidently very porous, and there are no streams or ponds, and only a single spring was seen; the limestone is honeycombed with caves and caverns, some of them of considerable size. The rainfall is evidently considerable, but there are no records of its amount. Despite the paucity of soil, the whole plateau is rather densely covered with shrubs and low trees of a considerable number of species, their roots, for the most part, penetrating into crevices of the limestone. Herbaceous vegetation is restricted to comparatively few species. Eight species of cacti inhabit this plateau, and in places are very abundant, the snowy cactus 1915] BRITTON—VEGETATION OF MONA I (Mamillaria nivosa) being more plentiful other island visited by us; Opuntia Taylo from Hispaniola, Culebra and the Virgin Isl a single colony; this has not yet been dete Rican mainland. The limestone plateau of Mona is border out by steep escarpments and is accessible except along the southwestern side, where t several miles long and averaging about half which the plateau is reached at a number At the foot talus of large limestone blocks. and of the talus on this southwestern side, tions of Mona occur, and several species c large size, notably the manchioneel (Hippi and two species of Ficus. Here also gr ferns, several bryophytes, and a number infesting dead wood. The soil of the nar abundant than that of the plateau, perm operations on a small scale and supporting up of a considerable number of kinds of tr baceous vegetation than exists on the plat elements of this vegetation are two orchid: nodes, hitherto known from Hispaniola and lucayanum, of Porto Rico, Anagada and t coastal sands, which extend almost uninter shore of the plain, are inhabited by charact: sand-dune species. Lichens are quite abundant on tree trun the talus, including a considerable numbe fessor Lincoln W. Riddle has examined the submitted the following report upon them: “The exploration of Mona Island has yie lichens, i collected by Dr. N. L. Britton, i WwW s, and 2 collected incidentally by D 42 has represent 26 species in condition "fo “The species growing on the limestone rock striking and interesting part of the collection. species of Omphalaria, a species of Collema, and matocarpaceae, which is, unfortunately, sterile further determinable. The omphalarias are all SLAND 35 here than on any ri, hitherto known ands, was found as ected on the Porto ed nearly through- at but few points, here is a low plain a mile wide, from of points over a of the escarpment the moistest condi- f trees here reach mane Mancinella) ow two species of of Polyporaceae row plain is more itting agricultural a low forest made ees, with more her- teau. Among rare 3, Domingoa hyme- Cuba, and [bidium he Bahamas. The ruptedly along the eristic West Indian ks and on rocks of r of species. Pro- » collection and has lded 42 numbers of These r determination. s constitute the most These include four 1 a species of the Der- 2 and, therefore, not | little known species. [Vou. 2 36 ANNALS OF THE MISSOURI BOTANICAL GARDEN O. polyglossa Nyl., collected from limestone rocks in Cuba by Charles Wright, and not otherwise known, is apparently common on Mona Island, as it is represented by two numbers, each with several well- developed specimens. There occur also O. lingulata Tuck., previ- ously known from Cuba and Bermuda; a sterile omphalaria related to O. Wrightu Tuck., but apparently not identical; and one other species of the genus, probably new. It has not yet been possible to identify the species of Collema, and that may also prove to be new. Curiously enough, none of these calciphile species has yet been de- tected among the material collected in Porto Rico. “In marked contrast to the rock-lichens, the bark-inhabiting lich- ens are all common species, widely distributed in Tropical America. The genus Trypethelium is best represented, with the species T. Eluteriae (four numbers), T. ochroleucum, and its variety pallescens, and T. mastoideum (two numbers). There are also such character- istic species as Graphis Afzelii, Melanotheca cruenta, Pyxine picta, Physcia alba and P. speciosa, Parmelia sulphurata and P. tinctorum, and Ramalina complanata and R. Montagnei. Probably owing to the comparatively unfavorable conditions on Mona Island, the foliose and fruticose lichens are mostly small specimens, not well-developed.” The total flora of flowering plants, as indicated by the col- lection made by Professor Stevens and our own, includes about 230 species; some of them are found only in cultivated grounds on the coastal plain and have probably been introduced by man. The total flora of land eryptogams is probably as great or greater than that of flowering plants, so we may conclude that the land flora of Mona consists of as high as 500 species. So far as the investigation of the collections has proceeded, the only apparent endemic species are a Chamaesyce, which Dr. ©. F. Millspaugh has described as new, a Tabebuia, the description of which is herewith included, and two very inter- esting riccias, here described by Dr. Marshall A. Howe. One or more of the lichens may be undescribed. Further explora- tion in Porto Rico and in Hispaniola may very well reveal their presence on these larger islands. It is interesting to have ascertained that the flora of this isolated limestone island is not more highly specialized. It is not necessary, in my opinion, to assume a former land connection between Mona and either Porto Rico or Santo Domingo, because all its native species may readily have reached it through natural agencies. 1915] BRITTON— VEGETATION OF MONA IS I append a list of the species collected a mined, and have indicated in this list their kı except that of the lichens and Uredinales, Rico, Curacao, Hispaniola and the Bahamas, to Mona. LAND 317 is thus far deter- 10wn distribution, as regards Porto the nearest lands The names of new species, and new binomials, are printed in heavy face type. List or SPECIES INHABITING Mona MONOCOTYLEDONS VALOTA INSULARIS (L.) Chas Common on u coastal slain and on the plateau: P Bahamas; Curaca SYNTHERISMA DIGITATUM (Sw.) Hitche. Frequent in cultivated ground, coastal plain: Bahamas. PASPALUM CAESPITOSUM Fluegge oo nt on the coastal plain and on the plateau: P Baham oe Seabee SIMPSONI Nash Collected by Professor era not found by us: Port PANICUM UTOWANAEUM Scribn. requent on the coastal plain and on the plateau: [Cuba; Guadeloupe]. PANICUM BARBINODE Trin. m f soil, Playa de Fajaro: native of South Ameri West Indie PANICUM ADSPERSUM Tri Moist soil, coastal plain: Berto Rico; Bahamas. PANICUM MAXIMUM Jac a on the coastal plain: West Indie LASIACIS DIVARICATA (L.) Hitche. a nt in thickets, coastal plain and plateau: P Po Native of tropical Af Baham CHAETOCHLOA SETOSA (Sw.) Serib Frequent on the coastal plain: Porto Rico; Hispaniola; CHAETOCHLOA CAUDATA Occasional on the coastal plain: rg IMBERBIS (Poir.) Seribn. Frequent on the coastal plain: Porto Rico; Hispanic CENCHROPSIS MYOSUROIDES (HBK) Nash Frequent in cultivated ground on the coastal plain: Lam.) Scribn. Desecheo; [Jamaic ca. IsLAND orto Rico; Hispaniola; ` rto Rico; Hispaniola; orto Rico; Hispaniola; o Rico; Bahamas. Porto Rico; Desecheo; Naturalized in the rica; naturalized in the orto Rico; Hispaniola; a; Bahamas; Curacao. a; Cuba; St. Thomas]. la; Bahamas. Bahamas; Cuba. [VoL, 2 38 ANNALS OF THE MISSOURI BOTANICAL GARDEN bsg sore ECHINATUS Com on ee coastal plain and on sand dunes: Porto Rico; Hispaniola; Bea Gare CENCHRUS CAROLINIANUS Walt. Collected = a Stevens, not found by us: Porto Rico; Hispaniola; Bahamas; Cura ARISTIDA BROMOIDES HBK. Cc n on the coastal plain: Porto Rico; Bahamas; Curacao. SPOROBOLUS VIRGINICUS (L.) Bea ze on coastal sands ie on ‘he vn! plain: Porto Rico; Hispaniola; Baham j SPOROBOLUS ARGUTUS (Nees) Kunth Frequent in moist soil on the coastal plain: Porto Rico; Hispaniola; Curacao. CHLORIS PARAGUAIENSIS Steud Coastal plain, Sardinera: Porto Rico; Hispaniola; Bahamas; Curacao. mgr eaaa PETRAEA (Sw.) D Common on coastal ca and pol ‘the coastal plain: Porto Rico; Hispaniola; Hikes ELEUSINE INDICA (L.) Gaertn. Cultivated ground, Brome plain: Porto Rico; Hispaniola; Bahamas; Curacao. DACTYLOCTENIUM AEGYPTIUM (L.) Willd Cultivated ground, coastal plain: Porto Rico: Hispaniola; Bahamas; u PAPPOPHORUM LAGUROIDEUM Schrad. Wet soil, coastal plain, between Sardinera and Ubero: Desecheo [Cuba; St. Eustatius]. .—. CILIARIS (L.) Link Com on the coastal plain: Porto Rico; Hispaniola; Bahamas; Curacao. CYPERUS ELEGANS L. Border of a marsh on the coastal plain: Porto Rico; Hispaniola; Bahamas; Curacao. CYPERUS TENUIS Sw. Occasional on the coastal plain: Porto Rico; Hispaniola. er LIGULARIS L. , Sardinera: Porto Rico; Hispaniola; Bahamas; Curacao. at at BRUNNEUS Sw on on coastal sands: Porto Rico; Bahamas; Hispaniola; Curacao, regen SPATHACEA Rot Com n the coastal plain: Porto Rico; Bahamas; Hispaniola. SCLERIA ee (L.) Sw Frequent on the coastal plain and on the plateau: Porto Rico; Hispaniola; Bahamas. ? THRINAX PONCEANA 0. F. Cook Apparently this species, but determined from leaves only. Rare in thickets on the coastal plain, and not found either in flower or in fruit: Porto Rico. ar plain between Sardinera and Ubero: Porto Rico; Hispaniola; Baham ANAMOMIS FRAGRANS (Sw.) Griseb. nn on the coastal plain: Porto Rico; Hispaniola. Recorded from the Baham JACQUINIA BARBASCO (Loefl.) Mez. ommon in coastal thickets and occasional on the coastal plain: Porto Rico; Hispaniola; Curacao ? DIPHOLIS s Coastal plain, Sardinera. A tree about 12 m. hìgh, in foliage only. BUMELIA OBOVATA (Lam.) DC. Frequent on the coastal plain. ie in flower or fruit at the time of our visit: Porto Rico; Hispaniola; Curaca SS. OBTUSA L. Common on the coastal plain and on the plateau: Hispaniola; Bahamas. agak iiair TETRAPHYLLA L. (R. nitida Jacq.) vie a t on the coastal plain and on the plateau: Porto Rico; Hispaniola; Baham ECHITES AGGLUTINATA Jacq. Occasional on the coastal plain and on the plateau: Porto Rico; Hispaniola. URECHITES LUTEA (L.) Britton Occasional on the coastal plain: Porto Rico; Hispaniola; Bahamas. METASTELMA (undetermined) oastal rocks, 0. METASTELMA (undetermined) Occasional on the coastal plain and on the plateau. EVOLVULUS GLABER Spreng. Moist soil, coastal plain: Porto Rico; Hispaniola; Bahamas; Curacao. ee ee >= 1915] BRITTON—VEGETATION OF MONA ISLAND 47 N. JAMAICENSIS (Jacq.) Hall. f. casional on coastal sands: Porto Rico; ana Bahamas. JACQUEMONTIA PENTANTHA (Jacq.) D. Frequent on aa coastal plain and on the any Porto Rico; Hispaniola; Bahamas; Cura OPERCULINA AEGYPTIA (L.) House Cultivated ground, coastal plain: Porto Rico; Hispaniola; Curacao. ? EXOGONIUM MICRODACTYLUM (Griseb.) Hous Oceasional on the plateau. Specimen Fa Wr for certain determination. IPOMOEA PES-CAPRAE (L.) Roth. Common on coastal sands: Porto Rico; Hispaniola; Bahamas; Curacao. en TRILOBA L. requent in cultivated ground on the coastal plain: Porto Rico; Bahamas. 0. GRANDIFLORUM (Jacq.) Choisy. (Ipomoea tuba G. Don.) Frequent in coastal thickets: Porto Rico; Hispaniola; Bahamas; Curacao. VARRONIA GLOBOSA Jacq. Occasional on the coastal plain: Porto Rico; Hispaniola; Bahamas; Curacao. sake Per SUCCULENTA Jacq. on the coastal plain and on the plateau: Porto Rico; Hispaniola; je ee GNAPHALODES (L.) Britton. (Tournefortia gnapha- 6 R as on “coastal sands: Porto Rico; Hispaniola; Bahamas; Curacao. en HIRSUTISSIMA L. se of limestone cliffs, Se Porto Rico; Hispaniola. rn MICROPHYLLA Ber mon on the coastal plain and on | the plateau: Porto Rico; Hispaniola. HELIOTROPIUM CRISPIFLORUM Urban Moist soil, coastal plain: Porto Rico. Closely resembles the Porto Rico plant but is lower and with shorter internodes; no flowering specimens were obtained. a PARVIFLORUM L. Freq on the coastal plain: Porto Rico; Hispaniola; Bahamas; Curacao. LANTANA E Ait. Collected by Professor Stevens, not found by us: Porto Rico; Hispaniola. Apparently e aly distinct from L. Camara L. LANTANA an LUCRATA L. om ur a plain and on the plateau: Porto Rico; Hispaniola; Bahamas; Gara en nn: (L.) Med on astal plain and on the le Porto Rico; Hispaniola; Feen Pre VALERIANODES STRIGOSA (Vahl) Kuntz Frequent on the coastal plain and on the plateau: Porto Rico; Hispaniola. 1Mallotonia a Britton, gen. nov. Tournefortia Section Mallotonia Grise b. Fl. Brit. W. I. 483. 1861. Type species: F i gnaphalodes (L.) R. Br. [VoL 2 48 ANNALS OF THE MISSOURI BOTANICAL GARDEN SALVIA SEROTINA L. (8. micrantha Vahl) u on the coastal plain and on the plateau: Porto Rico; Hispaniola; Baham HYPTIS PECTINATA (L.) P Cultivated ground on the id plain: Porto Rico; Hispaniola; Bahamas; Curacao SOLANUM NIGRUM L. (8. americanum Mill. ) Cultivated ground, Sardinera: Porto Rico; Hispaniola; Bahamas; Curacao. SOLANUM EDEN L. Occasional a A e bases of cliffs and on the coastal plain: Porto Rico; Hispaniola; Bah s. BRAMIA MONNIERIA (L.) Drake. (Herpestis Monniera HBK.) Border of a pool, ae Porto Rico; Hispaniola; Bahamas. CAPRARIA BIFLORA I Common on lei Sönstai plain and on the plateau: Porto Rico; Hispaniola; Bahamas; Curac a mer DULCIS L. moist soil on the coastal plain: Porto Rico; Hispaniola; Bahamas, Ls ear HETEROPHYLLA (DC.) Britton. (Raputia (?) heterophylla -3 da bebuia triphylla DC., not Bignonia triphylla L.) Fre requent on the EUR plain and on the plateau, Leaves 1-foliolate to 5-foliolate: =, Rie TABEBUIA m Britton, sp. tree up to 5 m. high. Leaves 3- 5. foliolate; petioles slender, lepidote, 6 em. long or less; veliotilen of the larger, ont leaflets slender, lepidote, 8-20 mm. long; lower leaflets sessile or nearly so; leaflets thin-coriaceous, narrowly oblong he base x abo 2-lipped; corolla pink, glabrous, about 5 m. fong, its cylindric tube 5-6 m lon long, the lobes nearly e Limestone cliffs, ht heal Mona Island, Porto Rico (Britton, Cowell and Hess, 1686). SESAMUM ORIENTALE L Cultivated ground, aad plain. Native of the East Indies. BLECHUM BROWNEI . Shaded rocks, an Porto Rico; Hispaniola; Bahamas. een PERIPLOCIFOLIA Jacq. onal on the coastal plain, a narrow-leaved race: Porto Rico; His- paniola JUSTICIA PECTORALIS Jac order of pool, ia ‘Porto Rico; Hispaniola. PLANTAGO MAJOR L. Cultivated ground, coastal plain. Not collected. Native of the Old World. u Sii MA CARIBAEUM (Jacq.) R. & S. Frequent on the coastal plain and on the plateau: Porto Rico; Hispaniola; Bahamas. er Arche ATA on ig ni; plain and on the plateau: Porto Rico; Hispaniola; ikea u. 1915] BRITTON—VEGETATION OF MONA ISLAND 49 GUETTARDA ELLIPTICA Sw Occasional on the coastal plain: Porto Rico; Hispaniola; Bahamas. STENOSTOMUM ACUTATUM DC. Frequent on the coastal plain and on the plateau: Porto Rico; Curacao. u e mn L. Com and dunes, on om a pas and occasional on the plateau: Porto ie: | fdepabtols: Bahamas; Curac CHIOCOCCA ALBA (L.) Hit Oceasional on the coastal oi and on the plateau: Porto Rico; Hispaniola; Bahamas STRUMPFIA MARITIMA Jacq. Limestone plateau near Ubero, frequent: Porto Rico; Hispaniola; Bahamas; Curaca PSYCHOTRIA UNDATA Jacq. Occasional on the coastal plain: Porto Rico; Hispaniola; Bahamas. ERNODEA LITTORALIS Sw Common on saat sands: Porto Rico; Hispaniola; Bahamas; Bonaire. SPERMACOCE TENUIOR L Frequent on the coastal plain: Porto Rico; Hispaniola; Bahamas; Curacao. CUCUMIS ANGURIA L Cultivated ground, Sardinera: Porto Rico; Hispaniola; Curacao. EUPATORIUM ODORATUM L. Common on the coastal plain: Porto Rico; Hispaniola; Bahamas. EUPATORIUM ATRIPLICIFOLIUM Lam Coastal rocks, Sardinera: Porto Rico; recorded from Hispaniola and from the Bahamas. se rere EN zu (Nutt.) Britton in wa Be and cultivated grounds, coastal plain: Porto Rico; His- paniola (2); Baham LEPTILON BONARIENSE (L.) Small Cultivated ground, Sardinera: Pons Rico; Hispaniola. PLUCHEA PURPURASCENS (Sw.) D Borders of marshes, coastal plain: Porto Rico; Hispaniola; Bahamas. BORRICHIA ARBORESCENS (L.) D Occasional on coastal rocks: Borto “Rico; Hispaniola; Bahamas. WEDELIA PARVIFLORA L. C. Rich Common on the coastal plain: Porto Rico. e a RUDERALIS (Sw.) Sch. Bip. ultivated ground, SPUTA plain: Porto Rico; Hispaniola. Erroneously recorded pies the Baham BIDENS en HBK. Collected by ent Stevens, not found by us: Porto Rico; Hispaniola; Bahamas; PTERIDOPHYTA (Determined by Miss Margaret Slosson ) ADIANTUM FRAGILE Sw. Limestone cliff, Sardinera: Porto Rico; Hispaniola. [vor. 2 50 ANNALS OF THE MISSOURI BOTANICAL GARDEN en AUREUM L. of pool near ee Determined from barren leaf specimen: Porto Rioo; Hispaniola; Bahamas; Curacao CYCLOPELTIS SEMICORDATA (Sw.) J. Smith Shaded limestone rocks, Sardinera: Porto Rico; Hispaniola. MUSCI (Determined by Elizabeth G. Britton and R. S. Williams) ner INVOLVENS (Hedw.) Mitt n dead wood and shaded rocks: Porto Rico; Hispaniola. TORTULA AGRARIA Sw. e ground near Sardinera: Porto Rico; Hispaniola; Bahamas. i ge GUADELUPENSIS Broth. Wet soil on the coastal plain between Sardinera and Ubero: Guadeloupe; a er. MICRODECURRENS E. G. soil on the coastal plain En pre R and Ubero: St. Thomas. CALYMPERES RICHARDI C. Muell. On tree trunks, base of cliff, Sardinera: Porto Rico; Hispaniola; Bahamas. CALYMPERES (an apparently undescribed species) n Bourreria, Ubero; Hispaniola. Be esyy JU. RMANNIA h ARER i ir Professor Fa AW. Evans) Se BAHAMENSIS Evans O estone, Ubero; on trunk of Gymnanthes, Sardinera: Bahamas. MASTIGOLEJEUNEA AURICULATA (Wils. & Hook.) Schiffn. n shaded limestone and on dead wood, Sardinera: Porto Rico; Bahamas. LEJEUNEA (barren and undeterminable) On shaded limestone, bark and dead wood. ae giao SQUARROSA (R. B. & U.) Dum On trunks and logs on the aa plain: Porto Rico; Bahamas. FRULLANIA (barren and Fu On dead wood, Sardin RICCIACEAE (Contributed by Dr. Marshall Avery Howe) RICCIA BRITTONII, sp. n Thallus simple or once ae: forming irregularly gregarious patches, oblong-ovate, linguiform, or obovate, 2- 5 mm. x 1-2 mm., subacute or obtuse, con- border 80-175 u wide, >. orous or very commonly br pg laterally and ven- trally; median sulcus dee cute except in older part ntral scales small, inconspicuous, hyaline, a exceeding the thin membranous peter ae thallus m s; transverse sections mostly 1.5-2.0 tim s wid , the ventral outlines semi-orbicular in younger parts, becoming flattened in the older; cells of the primary dorsal epidermis cylindric dome-shaped or subhemispheric, soon col- lapsing, leaving shallow slightly indurated more or less persistent cup-like ves iges; monoecious; antheridial ostioles scarcely elevated; spores brown, be- coming subopaque, soon exposed, 100-145 u in maximum diameter, rather ob- ee ee ee ee 1915] BRITTON—VEGETATION OF MONA ISLAND 51 scurely or sometimes distinctly angled, pa flattened, destitute of wing- margins, almost uniformly areolate over the whole surface, with age showing in profile obtuse or truncate papillae 3-5 u long, areolae mostly 10-18 u wide. wet, sunny soil, accompanied by R. violacea, ee Sardinera and Ubero, gy „Island, Februar ry, 1914, Britton, Cowell, & Hess, 1749a a Brittonii TR certain gr of contact with Riccia ad a Bisch. an 3 "R. eo ora M. A. Howe.! It is close to = sorocarpa in vegetative char- acters, though differing in the wider, es pronounced, scarious-albescent Ba margins and re in er sense of the ee but it departs widely fr sibs this p spona in the s hic een la pose Are -145 u vs. 70-90 u diam.), are destitute ty win vamos eects nd c only have the me "of. the inner faces almost as well and regularly developed as those of t ts un ee: dictyospora, the species differs in the less elongate tha I us cz 4-10 mm.), the albescent instead of dark purple thallus-margins and seal es, u more = ruhen and less parabolie outlines of transverse sections of the thallu us, and in the Er De (100-145 u vs. 95-116 u, max. diam.), with larger areolae (10-18 u 12 u. RICCIA VIOLACEA, + nov Thallus simple or 1-3 times di chot otomous, irregularly gregarious, 1. 5-4.0 mm. ong, the main segments en or linguiform, 0.65-1.15 mm. broad, rather fi © 5 d at the ses; except at apex Bier 2. very short or a dark violet, rarely ov or oc nal vi e sube i papillae 30-110 u long and en Br broad at base; cells of the primary dorsal iene n wet, sunny soil, accompanied te ‘Riccia he ge between gpl and Ubero, Mona Island, February, 1914, Br pee Cowell, & Hess, 17496 In size, habit, and color, R. violacea is s wg Rel of R. nigre rella DC., = Her ane oo erjen or very short cilia = the margins, which are wanting n R. nigrella, the scales are much smaller, more rudimentary and more div then in R. nigrella, and the cells of the Be ‘epidermis are much less persistent. Its nearest affinity is doubtless wit . atro paee seria which is known from Sicily, Sardinia, and Greece; from this pees o differ T one ed judge from the descriptions alone) in the ee Fe pami margins, the very s iae A divided, rarely o overlapping scales, ye the commonly Filet Me which are confined t o the margins and sides w hile in R. ara the hyaline LICHENES (Determined by Professor Lincoln W. Riddle) ARTHOPYRENIA On Coccolobis obtusifolia, Ubero. PYRENULA On bark, Sardinera. MELANOTHECA CRUENTA (Mont.) Muell. Arg. n Gymnanthes, Sardinera. TRYPETHELIUM ELUTERIAE Spreng. On Pithecolobium, Sardinera, and on Coccolobis obtusifolia, Ubero. 1Bull. Torr. Bot. Club 28: 163. 1901. [VoL. 2 52 ANNALS OF THE MISSOURI BOTANICAL GARDEN TRYPETHELIUM MASTOIDEUM Ach. On Pithecolobium, Sardinera. TRYPETHELIUM OCHROLEUCUM Nyl. On Zanthoxylum, between Sardinera and Ubero. OPEGRAPHA On Ficus, Sardinera; on Calyptranthes, Ubero. GRAPHIS AFZELII Ach. On Zanthoxylum, between Sardinera and Ubero; on Pithecolobium, Sardinera. GRAPHIS Collected by Professor Stevens. CHIODECTON On Plumiera, Sardinera. LEPTOTREMA On dead wood, Sardinera. CLADONIA diag seer var. CONIOCRAEA (Floerke) Wainio On dead log, Sardiner OMPHALARIA LINGULATA Tuck. n limestone, Sardinera ee POLYGLOSSA ii exposed limestone, Ubero OMPHALARIA On limestone, Übero. COLLEMA On limestone rocks, Sardinera. LEPTOGIUM (sterile > ne On Torrubia, Sardin PARMELIA TINCTORUM Despv. On a tree trunk. PARMELIA SULPHURATA Nees and Flot. O dead log, Sardinera RAMALINA MONTAGNEI De Not. On a twig, Sardinera. Collected also by Professor Stevens. RAMALINA COMPLANATA (Sw.) Ach. On a twig, Sardinera. PYXINE PICTA (Sw.) Tuck On Pithecolobium, le on Zanthoxylum, between Sardinera and Übero. PHYSCIA en re Nyl. (A small form) O cus, Sardine PHYSCIA ALBA Fee On Calyptranthes, Ubero; also, not typical, on Torrubia, Sardinera. The collection also contains a sterile plant near Omphalaria Wrightu Tuck., from wet, sunny soil between Sardinera and 1915] BRITTON—VEGETATION OF MONA ISLAND 53 Ubero, a sterile species of the Dermatocarpaceae growing on limestone at Ubero, and three other sterile and undetermin- able specimens. BASIDIOMYCETES (Determined by Dr. W. A. Murrill) LENTINUS CRINITUS (L.) Fries On dead wood, Ubero: Porto Rico; Bahamas. SCHIZOPHYLLUM ALNEUM (L.) Schroet. Frequent on dead wood: Porto Rico; Bahamas. DAEDALEA AMANITOIDES Beauv. On dead wood, Ubero: Porto Rico; Bahamas. INONOTUS CORROSUS Murr n dead wood, Sardinera: Porto Rico; Bahamas. i tetera DEPENDENS Murr. On dead wood: Porto Rico; Bahamas. POGONOMYCES HYDNOIDES (Sw.) Murr. On dead wood: Porto Rico; Bahamas Ba SANGUINEUS (L.) Mur requent on dead wood at base of C Porto Rico; Bahamas. weg at EN RIGIDA (Berk. & Mont.) Mur n dead wood, Sardinera: Porto Rico; fait SOOLON PINSITUS (Fries) Pat. ead wood: Porto Rico; Bahamas. XYLARIA On dead log, Ubero. UREDINALES (Determined by Professor J. C. Arthur) COLEOSPORIUM Fe Pat. On Plumiera obtu KUEHNEOLA GOSSYPII nn ) Arth. On Gossypium barbade PUCCINIA CENCHRI Dietr. & Holw. On Cenchrus. PUCCINIA CRASSIPES Pa & C. On Ipomoea triloba PUCCINIA EUPHORBIAE P. Henn On Aklenea petiolaris wo Millsp. PUCCINIA INFLATA Art On Stigmaphylion RE (Poir.) Small PUCCINIA LATERITIA B. & C. n Ernodea littoralis Sw. [vor, 2 54 ANNALS OF THE MISSOURI BOTANICAL GARDEN PUCCINIA URBANIANA P. Henn On Valerianodes a (Vahl) Kuntze UREDO sr agence Art ea ee (Sw.) Kuntze zu. CAMELIAE Mayor. On Chaetochloa setosa. Many parasitic fungi collected by Professor Stevens have not yet been determined. ALGA (Determined by Professor N. Wille) SCYTONEMA OCELLATUM Lyngb. Flat limestone plateau, Ubero. RECAPITULATION Species indicated in the foregoing list..................00005 292 Deduct thallophytes (distribution little known)............. 47 245 Deduct undetermined and doubtfully determined species....... 12 233 Deduct certainly introduced species.................0.00005 8 225 Deduct-endemic BOON, ie 4 221 In common with Pos Weisse in 211 mo il) We VE ee 185 "= BEER oy toi) er 155 $ Š “o GULIGA RE En ET keni 87 SPECIES OTHER THAN ENDEMIO ONES AND THALLOPHYTES NOT KNOWN ON Porto Rico (INCLUDING DESECHEO, CULEBRA AND VIEQUES) Cenchropsis myosuriodes: Bahamas; Cuba. Domingoa hymenodes: Hispaniola; ‘Cub a. Caesalpinia domingensis: Hispaniola. Guilandina melanosperma: St. Croix. Dodonaea Ehrenbergii: Bahamas; Hispaniola; Cuba. 1915] BRITTON—-VEGETATION OF MONA ISLAND Sarcomphalus Taylori: Bahamas. Plumiera obtusa: Hispaniola; Bahamas; Cuba. Brachiolejeunea bahamensis: Florida; Bahamas. Hyophila guadelupensis: Guadeloupe; Montserrat. Bryum subdecurrens: St. Thomas. 55 56 plateau. 2. ANNALS OF THE MI (Vou, 2, 1915] SSOURI BOTANICAL GARDEN EXPLANATION OF PLATE art of Mona Island PLATE 1 Fig. 1. Escarpment, Mona Island, showing openings of caves. Fig. f rom the ocean, showing escarpments and Ann. Mo. Bor. GARD., VOL. 2, 1915 PLATE 1 BRITTON—VEGETATION OF MONA ISLAND COCKAYNE, BOSTON 58 ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 2 Fig. 1. Escarpment and tables, Mona Island. Fig. 2, Coastal thicket, Mona Island. [vor. 2, 1915] THE FLORA OF NORWAY AND ITS IMMIGRATION N. WILLE Professor at the Christiania University The phytogeographical investigations in a country may be carried on in the following three main directions: Floristic phytogeography, or an investigation into the geo- graphical distribution of the plant species. The result of this work should be charts of the distribution in the country of the various species. In a country with such varied condi- tions of life as Norway, this is a very comprehensive and very arduous task, requiring an infinitude of detailed investi- gations in all parts of the country. Ecological phytogeography, which endeavors to find out how and why the different species of plants in various places and under various conditions of life come together in plant- communities. This branch of science, which was founded by Professor E. Warming, must be based upon phytoanatomy and phytophysiology, as the connection between the organi- zation of the vegetable species and their external conditions of life must be investigated. Investigations such as these may yield interesting results in all countries, and are most easily carried on where the conditions of life are uniform over wide areas; but in a country like Norway, with its varied conditions, they present very great difficulties. Historical phytogeography has for its aim the investiga- tion of the changes that in the course of time have taken place in the vegetation of a country—to find out, for instance, when and whence important species have immigrated, how quickly they have spread, why others, that had formerly been more widely distributed, had a more restricted distribution in a later period, ete., ete. With regard to this last branch of science, the Scandin- avian countries, Denmark, Finland, Norway, and Sweden, present peculiarly favorable conditions; for there is no doubt that these countries were formerly buried under a continu- ANN. Mo. Bor. GARD., Vor. 2, 1915 (59) [VoL. 2 60 ANNALS OF THE MISSOURI BOTANICAL GARDEN ous covering of ice, which destroyed all vegetation except perhaps the most hardy. All other species of plants have immigrated subsequently from the neighboring countries, which were not covered with ice during the Glacial Epoch, and could therefore afford a dwelling-place for a more or = less abundant flora. In the following pages I shall endeavor to give an account of the results at which historical phytogeography may be said to have arrived as far as Norway is concerned. SURVEY OF THE DISTRIBUTION OF THE NORWEGIAN FLORA It will first be necessary, however, to give a general ac- count of the most important points regarding the composi- tion and distribution of the Norwegian flora throughout the country. I shall here consider only the vascular plants (about 1,500 species), however, as the distribution of the lower plants is not sufficiently known to enable us to draw definite conclusions. The area of Norway is about 125,000 square miles, stretch- ing from latitude 57° 58’43” north to latitude 71° 10’ 20” north. The conditions for plant life will thus be very differ- ent in the southern and northern parts of the country. But in addition to this, there is a great difference between the climate in the east and that in the west of southern Norway. In the valleys of the East Country, there is a very pronounced inland climate, with hot summers and a winter temperature that falls below —40°C., while on the west coast region there is a low summer temperature, but a mean January tempera- ture of sometimes more than 2°C. The most important condition affecting the distribution of plants in Norway is the temperature. In this connection we shall in the first place speak of the lowest winter tempera- ture that the plants can survive. J. Holmboe (’13) has shown that the distribution of Ilex aquifolium in Norway coincides closely with the January isotherm for 0°C. Herb- aceous plants which die down in the winter may of course be independent of the lowest winter temperature, as they are covered with snow; but they are not entirely independent of 1915] WILLE—FLORA OF NORWAY 61 the spring and autumn temperature. Plants are also in a great measure dependent on the height of the temperature in the period of vegetation, which, in Norway, comprises in the main the four months, June, July, August, and September. Temperature de lur moyenne de Janner Contuqrade Niveau delamer ah 10° j 1.32 / IPOT TN / N A “> En Fig. 1. Isotherms for January. In this way, the conditions prevailing in Norway are very varied, the July isotherm for Christiania being 17°C., while for the west coast it is only from 12 to 14°C. A. Helland (’12) has calculated that where the mean sum- mer temperature in Norway is less than 13°C., the fruit [VoL. 2 62 ANNALS OF THE MISSOURI BOTANICAL GARDEN trees yield nothing worth mentioning; and where it is less than 11°C., the eultivation of grain is uncertain. The mini- mum limits of the necessary mean summer heat for the fol- lowing wild Norwegian trees and shrubs appears to be as Tenperature de lair | Ka 213 Meyenne de Juillet as ty te a, A Pas Codligrade Mean de la mer SH I X RX LA 5 a SR IRA) rtp sy x Kl A A I ERS N Br Nr er | eS AAS SOK ON XZ < Pe, EDL Le EI h >< T ma : A z N i y N : FR gar y Y) eS Bhp: À \ 9 4 i A R ; DR. EN ye he Ä N CKS | | & YG § LE V4 Blas Gi lh 24 W NW Fig. 2. Isotherms for July. follows: for Fagus sylvatica, 13.4°C.; Quercus pedunculata, 12.6°C.; Corylus Avellana, Acer platanoides, and Tilia cor- data, 12.5°C.; Alnus glutinosa and Fraxinus excelsior, 12.4° C.; Sorbus Aria and Ulmus montana, 11.2°C.; Picea excelsa and Pinus sylvestris, 8.4°C.; Alnus incana, Prunus Padus, 1915] WILLE—FLORA OF NORWAY 63 and Sorbus Aucuparia, 7.7°C.; Populus tremula, 7.6°C.; Betula odorata, 7.5°C.; Juniperus communis var. nana, 5.3° C.; and Betula nana, 4.3°C. As the mean temperature of summer decreases with in- creasing height above sea-level very nearly 0.6°C. per 330 feet, the distribution of plants is greatly influenced by the circumstance that Norway is a mcuntainous country, its highest mountain, Galdhöpiggen, being 8,095 feet in height, and thus within the region of perpetual snow. But a pecu- liarity of the Norwegian mountains is that they form broad (as much as sixty-two miles broad}, undulating mountain plateaus, which are intersected by deep or shallow valleys, where there are narrow lakes or small rivers. The edge of these mountain plateaus, in the south of Norway, lies at a height of from 2,950 to 3,280 feet above the sea, so that Picea excelsa and Pinus sylvestris disapp2ar slightly below this height, the edge of the plateaus and the lowest valleys that intersect them being covered with Betula odorata. The great mass of the mountain plateaus, which rise above the birch- limit, is thus treeless. It has been calculated that there are 26,333 square miles of forest land in Norway, of which 73 per cent consists of Picea excelsa and Pinus sylvestris, ‘while the remaining 27 per cent is mainly Betula odorata with a little Betula verru- cosa, Quercus pedunculata, and Q. sessiliflora, and a very little Fagus sylvatica in the south. The vegetation limits are lower not only toward the north, as one would expect, but also towarc. the west, as they are lower near the sea than inland. This will be seen from the following height-limits in feet: Snow-line a u Gausta in Telemarken (south of N nee Pane 3450 3024-3113 Vos (west of Norway).......... 3936 3359 1994 Snehaetta, in the Dovre Mountains (central Norway) ........... 5375 3464 2880 Rödö in Helgeland (just within the Arctic Circle)........... 3280 so 777 Alten in Finmark (70° N. Lat.). 3516 1476 777-1023 [VoL. 2 64 ANNALS OF THE MISSOURI BOTANICAL GARDEN The distribution northward and height above sea-level of the various vegetable species, will be dependent mainly upon the temperature during the summer months. The rainfall, which in various other countries plays so important a part as a factor in vegetation, is of less import- Z [>] 2 4 b 8 w _ 7 7 1b {a 20 22 z2 26 16 30.5% J | \ 4 3 €A tA c Po x ho Y Tu 1 O Lok [4 is sg) t Ad Ni Mi ‘ A -AN Pd “yO k RA A i x ABER Wi PS ra nae di yor yw A A Ad IN An 9 t 4 i © ar io EA i ic is 30 7% Fig. 3. The annual rainfall in Norway (in eentimeters) —After M. Mohn. ance in Norway, as even on the Dovre Mountains, where the rainfall is smallest (about 300 mm. per annum), there is sufficient rain to occasion, on account of the inconsiderable evaporation, swamps and peat-bogs, where even entirely hydrophilous communities thrive. 1915] WILLE—FLORA OF NORWAY 65 It was formerly supposed that the largest rainfall was on the outermost islands off the west coast of Norway, and that this was the cause of the Atlantic vegetation that is found there, with such characteristic plants as Hymenophyllum peltatum, Erica cinerea, Scilla verna, Vicia Orobus, ete. But more recent investigations have shown that the rainfall is greater a little way in from the coast, where the mountains begin. In Hovlandsdal, near the Sogne Fjord, a mean rain- fall has been observed of 3,178 mm., and at Skaanevik, near the Folgefon, 2,945 mm., whereas the outermost islands off Bergen show a rainfall of only 1,300 mm., and off Florö of 1,900 mm. It is, therefore, clear that the occurrence of the above-mentioned Atlantic plants on the outermost islands is due not to a larger rainfall, but to a milder winter tempera- ture. There are, of course, species of plants that cannot thrive in the great humidity of the West Coast; but as there are also localities with comparatively dry soil, it may be rather the low summer temperature than the large rainfall that pre- vents them from thriving. The importance of the soil for vegetable growth appears to depend, in Norway, mainly upon whether the soil is rich, or deficient, in lime. In addition to its chemical influence, a calcareous subsoil, especially when consisting of calcareous slate or limestone, is of consequence from the fact that it forms a warm soil. In Norway, therefore, most of the southern species are found only in the limestone country surrounding the Skien Fjord and the upper part of the Kris- tiania Fjord. The terrestrial plants of Norway may be divided into five zones, according to the ability of the plants to ascend the mountains and extend northward in their growth, that is to say, according to their dependence on the mean temperature of the summer. These zones are here indicated by the upper limit of a characteristic species of plant. I. THE QUERCUS PEDUNCULATA ZONE In the east of Norway this tree is found as far as Lake Mjösen (60° 45’ N.), and in the west up to Nordmöre (62° (VoL, 2 66 ANNALS OF THE MISSOURI BOTANICAL GARDEN 99°); but it is nowhere known to have reached a greater height above sea-level than 1,722 feet. The oak can stand a winter temperature as low as —33.8° C., but requires a mean summer temperature of 12.6°C. It is now comparatively rare, and seems to be decreasing. It occurs in large quantities only on the Silurian and along the lower parts of the coast. A number of deciduous trees that are susceptible to cold have about the same distribution as the oak, both in height and in northward extension. These are: Acer platanoides, Alnus glutinosa, Betula verrucosa, Crataegus Oxyacantha, Fraxinus excelsior, Prunus avium, P. insititia, Pyrus Malus, Sorbus Aria, S. fennica, Taxus baccata, and Tilia cordata. There are also a number of species of eryptogams. It may on the whole be said that the zone here designated the Oak Zone is that of Norway’s most abundant flora. Within the Oak Zone, large districts may in their turn be marked off that possess a characteristic flora, the occurrence of which is especially conditioned by circumstances of tem- perature and soil. 1. The Region of the Silurian Flora.—This is developed in an especially characteristic manner on the calcareous slate along the Langesund Fjord, the west side of the Kristiania Fjord, in Ringerike and Hadeland, and around Lake Mjösen. In some of these districts it is fairly cold in the winter, but very hot in the summer;! and as the soil is calcareous and warm, a xerophilous steppe-flora, with its characteristic Labiatae, Boragineae, and Centaurea species, and thistles, such as Carlina vulgaris, Carduus acanthoides, ete.—species which also occur in the steppe-regions of South Russia, can thrive well on southern slopes. For the rest, the flora is rich in characteristic species, e.g., Artemisia campestris, Brachypodium pinnatum, Carex prae- cox, Cephalanthera rubra, Cirsium acaule, Fragaria collina, Libanotis montana, Ononis campestris, Phleum phalaroides, 1 In Kristiania, the 30-years’ average minimum atmospheric temperature for the month of January is —16.5°C., and the average maximum for the month of June +28,9° 1915] WILLE—FLORA OF NORWAY 67 Spiraea Filipendula, Thymus Chamaedrys, Trifolium mon- tanum, Veronica spicata, ete. Where the soil is deep and not too dry, the above-mentioned deciduous trees that are susceptible to cold form forests or copses, intermingled with Corylus Avellana, Prunus spinosa, species of Rosa and Rubus, and a luxuriant ground vegeta- tion, among which are several orchids. A few of these trees and the more hardy species of the Silurian flora, such as Origanum vulgare and others, may, like an advance-guard, overstep the boundaries of the Silurian regions, but then they generally occur in warm localities, in talus at the foot of cliffs, or in steep slopes that face south- ward, even high up the sides of the valleys, or in the upper parts of the West Country fjords. But the number of species diminishes with increasing dis- tance from the lowland Silurian regions, and there are only a few species that have advanced as far as north of the Dovre Mountains. 2. The Region of Fagus sylvatica.—This region is situated along the southeast coast of Norway, from the Swedish border to Grimstad, where it extends as far north as Holmestrand. There is a small beech-wood a little to the north of Bergen, but this is a solitary instance, and has nothing to do with the real distribution area of the beech. The beech is purely a lowland plant, as there is only one place in which it goes to a height of 886 feet above the sea, its usual height being not more than 525 feet. When culti- vated, it can grow almost as far north as Quercus pedunculata, but prefers a rather higher summer temperature (13.4°C.) and thrives best on comparatively warm gravel banks. The beech is one of those plants which has recently ap- peared to spread to new regions; and there is no doubt that it has not yet nearly reached the limits of distribution to which it will little by little attain, especially along the low land of the south coast. This is due to the fact that it must have immigrated in fairly recent times. The following plants may also be mentioned as occurring chiefly in the region of the beech: Cladium Mariscus, Coron- [VoL. 2 68 ANNALS OF THE MISSOURI BOTANICAL GARDEN illa Emerus, Epilobium obscurum, Laserpitium latifolium, Ligustrum vulgare, Luzula nemorosa, Melampyrum cristatum, Rubus corylifolius, R. Lindebergii, Selinum carvifolium, Sium latifolium, Viscum album, Vicia cassubica, V. lathyroides, ete. A few of these have a rather larger distribution than the beech has at present; others, which must have immigrated recently, are found only within quite a small area. The region for the cultivation of wheat in Norway coin- cides in the main with that of the beech, but extends a little farther, namely westward as far as Mandal, and to a height of 1,246 feet above sea-level. 3. The Region of Ilex Aquifolium.—This region is situated a little to the west of that of the beech, and does not have a lower mean temperature for January than 1°C. It extends from Arendal to Christianssund (63° 7’ N. Lat.), but does not include the outermost islands on the west coast. A large number of vegetable species occur in this region. As especially characteristic may be mentioned Aeropsis prae- cox, Asplenium Adiantum nigrum, Cardamine hirsuta, Cen- taurea decipiens, C. nigra, C. pseudophrygia, Cerastium tetrandrum, Chrysosplenium oppositifolium, Circaea lutetiana, Conopodium denudatum, Corydalis claviculata, Cynosurus cristatus, Digitalis purpurea, Drosera intermedia, Gentiana Pneumonanthe, Geranium columbinum, Hedera Helix, Her- acleum australe, Hydrocotyle vulgaris, Hypericum pulchrum, Hypochaeris radicata, Juncus squarrosus, Leontodon hispidus, Luzula sylvatica, Lysimachia nemorum, Meum athamanticum, Quercus sessiliflora, Pilularia globulifera, Polygala depres- sum, Primula acaulis, Rosa pimpinellifolia, Rumex obtusi- folius, Sagina subulata, Scirpus setaceus, Sedum anglicum, Senecio Jacobaea, Stellaria Holostea, Teesdalia nudicaulis, Triticum acutum, T. junceum, and Weingartneria canescens. A few of these species, however, can bear a January isotherm that lies a little lower than 1°C. These species, among which are Hedera Helix and Quercus sessiliflora, occur, therefore, also in the beech region in the southeast of Norway, but have their chief distribution in the Ilex Region, and must therefore be assigned to that region. 1915] WILLE—FLORA OF NORWAY 69 4. The Region of the West-European Coast Flora.—This includes the outermost islands in the province of Bergen. The characteristic feature of the climatic conditions here, as we have already stated, is not the large rainfall, for this is in reality smaller than in certain parts of the Ilex Region; but it is the extremely mild winter temperature, and a com- paratively low summer temperature. For purposes of comparison we will here give the mean minima for February and the mean maxima for July, for Kristiania, which forms a center for the Silurian flora, Larvik, the center of the beech region, Mandal of the holly, and Utsire of the West-European coast flora. aximum temperature for February temperature for July Kristiania ........ 15.5° C. 28.8° C. TL os Rin SS 14.5° C. 25.8° C. Mandal leks anes 11.970, 24.8° C. MMNIO Fick E 5.7° C. 19.9° C. On these outermost islands in the province of Bergen, the mean temperature for January is 2°C. Among the plant species that are especially characteristic of this region may be mentioned Asplenium marinum, Erica cinerea, Hymenophyllum peltatum, Scilla verna, and Vicia Orobus. These species are found in England, and some of them southward along the shore of the Atlantic. II. THE PINUS SYLVESTRIS ZONE In the east of Norway Pinus sylvestris goes right down to the sea, and occurs in many places in the Oak Zone; but in speaking here of a special zone for Pinus sylvestris, we refer to the great continuous forests of Pinus sylvestris and Picea excelsa, which cover wide tracts of country from the upper limit of the Oak Zone to a height of 3,116 feet in the south of Norway, 1,640 in the central part, and 623 in the north. Pinus sylvestris avoids the sea, and is therefore absent from the outermost belt of islands; but inland it forms, either alone or together with Picea excelsa, a more or less continuous region of distribution below the above-stated height-limits up to latitude 70°N. [VoL. 2 70 ANNALS OF THE MISSOURI BOTANICAL GARDEN Picea excelsa, which immigrated much later than Pinus sylvestris, supplants the latter in favorable localities in the east of Norway; but in the west its field of distribution is very small, and extends only to latitude 69°N. Farther north, in the interior of Finmark, small spruce forests do indeed occur, but they are formed of Picea obovata. The forests that are formed of Pinus sylvestris are light, but as they often grow upon dry, poor soil, they are poorly furnished with vegetable species. There may occur scattered specimens of Betula odorata, Alnus incana, Juniperus com- munis, Sorbus Aucuparia, and Populus tremula, and then a poor ground vegetation of mosses (e.g., Polytrichum juni- perinum), and lichens (e.g., Cladonia rangiferina, Cetraria islandica, and Peltigera), among which grow some easily contented higher plants, especially Aira flexuosa, Arctosta- phylos officinalis, Calluna vulgaris, Empetrum nigrum, Fes- tuca ovina, Luzula pilosa, Melampyrum sylvaticum, Pteris aquilina, Trientalis europaea, Vaccinium Myrtillus, V. ulig- mosum, and V. Vitis-Idaea. Where this forest, from some cause or other, has been de- stroyed, extensive heath-lands are often formed, consisting chiefly of Calluna vulgaris, among which occur Empetrum nigrum and species of Vaccinium, as also Antennaria dioica, Aira flexuosa, Campanula rotundifolia, Festuca ovina, Nardus stricta, and others. Picea excelsa forms forests on more fertile soil; but as they are very dense and dark, other trees have difficulty in foreing an entrance, and even the ground vegetation is as a rule very poor, owing to the want of light. A thick carpet of mosses (especially Hylocomium splendens) covers the ground, and the only plants that thrive are fungi, Polystichum spinulosum and some other ferns, Linnaea borealis, Milium effusum, Oxalis Acetosella, Pyrola uniflora, and others. Where the forests of Picea are less dense, or where Pinus sylvestris grows upon a more fertile soil, these conifers may be mingled with various deciduous trees, and in the lower districts even with less hardy deciduous trees, which other- wise belong to the Oak Zone. The ground vegetation in such 1915] WILLE—FLORA OF NORWAY 71 places is also much more abundant, and the ordinary lowland flora may be found fairly well represented. Almost all cultivated land in Norway lies in the Oak and Pine Zones. Rye and oats ripen up to latitude 69°N., barley even up to 70°N.—in the south it can be grown up to a height of 2,066 feet above the sea. The potato is cultivated rather farther north and a little higher above sea-level than barley. Side by side with the growing of grain is that of forage plants, of which the most important species are Trifolium pratense and Phleum pratense. III. THE BETULA ODORATA ZONE Betula odorata also occurs in the lowlands, and extends farther toward the sea than Pinus sylvestris, but by its zone, as here defined, is meant the region above the height limit of Pinus sylvestris upon the mountains and north of its distri- bution. In the very south of Norway, Betula odorata goes up to about 3,600 feet above the sea, and northward as far as latitude 71° 10’ N. Thus beyond the Birch Zone there is only the northeastern part of Finmark and the highest mountain regions. In the south of Norway the great proportion of the so-called ‘‘saeters’’ lies in the Birch Zone, as this tree generally oc- cupies the margin of the mountain wastes, and fills the little valleys that intersect them with a short-stemmed forest of Betula odorata subsp. alpigena. Side by side with this mountain form of birch, there may also grow Alnus incana, Populus tremula, Prunus Padus and Sorbus Aucuparia. The ground vegetation will be somewhat variable according to the degree of moisture in the soil. On dry gravelly slopes, especially if they face the south, the following species of higher plants are generally found in addition to a few species of lichens, such as Cetraria island- ica, Stereocaulon, ete.: Arctostaphylos officinalis, Agrostis vulgaris, Aira flecuosa, Alchemilla alpina, A. vulgaris var. pubescens, Antennaria dioica, Anthoxanthum odoratum, Astragalus alpinus, Botrychium Lunaria, Betula nana, Call- una vulgaris, some species of Carex, Empetrum nigrum [Vor. 2 72 ANNALS OF THE MISSOURI BOTANICAL GARDEN Euphrasia officinalis, Festuca ovina, Gnaphalium norvegicum, Juniperus communis, Lotus corniculatus, Luzula campestris, L. pilosa, Maianthemum bifolium, Melampyrum sylvaticum, Nardus stricta, Pedicularis Oederi, Peristylis viridis, Phleum alpinum, Poa alpina, Pyrola minor, Rhinanthus minor, Soli- dago Virgaurea, Trientalis europaea, Vaccinium Myrtillus, V. uliginosum, V. Vitis-Idaea, and Vicia Cracca. Where the soil is deeper and damper, and along streams and in shady places, Salix glauca, S. hastata, S. lanata, S. lap- ponum, S. Myrsinites and their hybrids make their appear- ance. The vegetation here is more luxuriant, as in addition to most of the above-named, the following species are found: Aconitum septentrionale, Agrostis rubra, Alchemilla vul- garis var. alpestris, Aira alpina, A. caespitosa, Bartschia alpina, species of Carex, Equisetum hiemale, Geranium sylva- ticum, Gymnadenia conopea, Montia fontana, Mulgedium alpinum, Myosotis sylvatica, Orchis maculata, Polygonum viviparum, Pinguicula vulgaris, Polemonium caeruleum, Ran- unculus platanifolius, Rumex Acetosa, Saussurea alpina, Selaginella spinulosa, Soyera paludosa, Spiraea Ulmaria, Viola biflora, and others. Many of these species occur right down to sea-level, some also higher up in the next zone; but as they are always found in the Birch Zone and have their most abundant development there, it is best to refer them to that zone. IV. THE ZONE OF DWARF WILLOWS This zone occupies the northeast part of the Varanger peninsula in Finmark and the mountains above the birch limit, up to a height which, in the southernmost point, may be put at 4,133 feet above the sea. It is thus only the tops of the highest mountains which rise like islands above this zone. The mean summer temperature here will be from 8.5 to 4.3°C., according to the height and situation in higher latitudes. The composition of the vegetation varies greatly according to the moisture conditions of the soil, which in their turn to some extent depend on exposure to the sun, south slopes being dry, north slopes damp. 1915] WILLE—FLORA OF NORWAY 73 On the drier tracts there are low copses of Betula nana and Juniperus nana, with a ground vegetation of mosses and lichens and a poor selection of mountain plants, such as An- tennaria alpina, Arctostaphylos alpina, Azalea procumbens, Carex rigida, Hieracium alpinum, Juncus trifidus, Erigeron alpinus, E. uniflorus, Festuca ovina, Gnaphalium supinum, Luzula arcuata, Luzula nivalis, L. spicata, Lycopodium alpi- num, L. Selago, Nardus stricta, Pedicularis lapponica, Poly- gonum viviparum, Rhodiola rosea, Salix herbacea, S. reticu- lata, Trientalis europaea, Vaccinium Myrtillus, V. uliginosum, V. Vitis-Idaea, Viscaria alpina, and others. Where the soil is very poor and the climate during the vegetation period very dry, as on the mountain moorlands in the east of Norway—’round the lake Faemundsoe, and between the valleys Oesterdal and Gudbrandsdal—there occur great lichen-covered heaths consisting of Cladonia rangi- ferina, Cetraria nivalis, C. cucullata, Alectoria divergens, and A. nigricans, which give a grayish white appearance to the mountains. Among the masses of lichens there are found only a few very easily satisfied mountain plants such as Festuca ovina, Nardus stricta, Solidago Virgaurea, ete. Where, on the other hand, the soil abounds in lime, and the conditions otherwise are favorable, as in certain places on the Hardanger Plateau in the south, Lom and Dovre in the center, and several places in the north of Norway, rare mountain plants occur, such as Alsine biflora, A. hirta, Dryas octopetala, Primula scotica, P. stricta, Oxytropis lapponica, Papaver radicatum, Rhododendron lapponicum, Salix polaris, Veronica saxatilis, ete. If the soil, on the contrary, is deep and damp, as in mor- asses and along streams, or where water trickles down the sides of mountains, there is quite a different and more abund- ant vegetation, consisting of mosses with thickets of Salix glauca, S. lanata, S. lapponum, and S. Myrsinites, often with an undergrowth of Aira alpina, Andromeda hypnoides, Car- damine bellidifolia, Cerastium trigynum, Eriophorum capi- tatum, E. vaginatum, Juncus biglumis, J. castaneus, J. tri- glumis, Koenigia islandica, Oxyria digyna, Petasites frigida, [VoL. 2 74 ANNALS OF THE MISSOURI BOTANICAL GARDEN Ranunculus glacialis, R. nwalis, R. pygmaeus, Saxifraga aizoides, S. caespitosa, S. rivularis, S. stellaris, Silene acaulis, Tofieldia borealis, Vahlodea purpurea, Veronica alpina, ete. V. THE LICHEN ZONE This embraces the often stony tracts above the preceding zone, i.e., the highest mountain tops and the ground from which they rise. Rocks and stones are here covered with the blackish yellow Lecidea geographica and other lichens. Where there is a little soil, some hardy mosses grow, and under favorable con- ditions a very few species of higher plants. I may mention, as an illustration, that in 1877, when visit- ing the mountain Haarteigen (5,546 feet) in Hardanger, 1.e., in the south of Norway, I noted the following higher plants upon the comparatively flat top of the mountain: Carex rigida, Luzula arcuata, L. spicata, Lycopodium Selago, Poa alpina, Polygonum viviparum, Ranunculus glacialis, and Rhodiola rosea. As already repeatedly stated, all plant species are not strictly confined to the zone under which they are mentioned as especially characteristic factors. It is very general for species somewhat to overstep the boundaries of their true zone, either upward or downward. Certain species are even found in all zones from the sea to the snow, since they have a remarkable ability of adapting themselves to all kinds of soil and to all kinds of climatic conditions. As instances of such species we may mention Calluna vulgaris, Empetrum mgrum, Eriophorum vaginatum, Festuca ovina, Nardus stricta, Polygonum viviparum, and the species of Vaccinium. Another circumstance is that typical mountain plants are sometimes found in the lowlands right down to the sea, e.g., in Jaederen, Alchemilla alpina, Arctostaphylos alpina, Part- schia alpina, Saxifraga aizoides, and Selaginella spinulosa. Betula nana occurs in the southeast of Norway down to fifty feet above the sea, and Dryas octopetala occurs at Lange- sund and at Varaldsö in Hardanger at sea-level. These occur- rences were formerly often explained as relics of a previ- 1915] WILLE—FLORA OF NORWAY 75 ous age with a colder climate, but I do not think we need have recourse to such an explanation. In all the steep-sided valleys, typical mountain plants spread downward along streams and rivers, and often appear far below their real habitat. Whether they will remain there depends only upon their ability to compete with lowland plants and to withstand the night frosts in the spring after the snow has melted. I assume, therefore, that the occurrence of the above- mentioned mountain plants in the lowlands is due to a chance carrying of seed to places that were favorable to the welfare of the species, e.g., limestone at Langesund and Varaldso for Dryas octopetala, a peat-bog for Betula nana, and so forth. THE IMMIGRATION OF THE NORWEGIAN FLORA Geologists have long been agreed that Scandinavia and great parts of adjacent lands have once been covered with one entire ice-cap, as the interior of Greenland is at the present time. By degrees the view obtained that there have really been two such glacial epochs, separated by an inter- mediate warm period, in which the conditions probably more or less resembled those of the present day. During the first, called the Great Glacial Epoch, the ice- cap extended as far as central Germany, over almost the whole of England, over the whole of Finland, and over a great part of northern Russia. It follows that under such conditions, all, or almost all, vegetation must have disap- peared from the Scandinavian peninsula, from Norway and Sweden. I am inclined to believe that in places in Norway, the tops of high mountains rose above the ice-covering, and that a very few species of plants may have survived there; but this is a matter of no interest in the question upon which I shall now endeavor to throw light, namely, the immigration of the flora of Norway after the Last Glacial Period. This was of considerably smaller extent. On the south the ice reached only as far as Mecklenburg, and the ice- boundary then ran obliquely northward up through Jutland in Denmark, of which, therefore, only a part was entirely covered with ice. There can be no doubt that the whole of F [VoL, 2 76 ANNALS OF THE MISSOURI BOTANICAL GARDEN Sweden was covered by this ice-cap, but as regards Norway, the conditions are still a matter of dispute. Some geologists maintain that the ice went right out into the sea on all sides; others assume that in some parts there was an iceless coast- region, where only here and there great glaciers ran out into the sea. The great majority of the species in the Norwegian flora must, however, have immigrated after the last Glacial Period; but with regard to their immigration and the conditions under which it took place, various theories have been advanced. The first to take up this question, especially with regard to Sweden, was F. W. Areschoug (’66), who, in 1866, main- tained that the present vegetation of Scandinavia was made up of at least three elements of different period and origin, namely : (1) An arctic vegetation, which immigrated from the east during the latter part of the Glacial Period, and, from its origin, may be called the North Siberian Flora; (2) A northeastern and eastern vegetation, which came into Europe from Siberia after the Glacial Period, but before the immigration of the beech. From its origin, it may be called the Alta Flora; (3) A southeastern and southern vegetation, which came simultaneously with the beech, partly from the Caucasus and the countries ’round the Caspian and Black Seas, partly from the countries of the Mediterranean. This may be called the Caucasian and Mediterranean Flora. Areschoug also pointed out that a number of arctic species in the north German and south Swedish lowlands must be regarded as relics of the vegetation of the high north, which, after the melting of the ice-cap, withdrew toward the north or up into the mountains. This view received strong support in the discovery by A. G. Nathorst (’71) in 1870, in the fresh-water clays of the south of Sweden, of remains of typical arctic plants which do not grow there now, but only very much farther north, namely, Betula nana, Dryas octopetala, Salix herbacea, S. polaris, and S. reticulata. ~ 1915] WILLE—FLORA OF NORWAY 77 In 1875, Axel Blytt (’76) first brought forward his well- known theory on the immigration of the flora of Norway during alternate wet and dry periods. According to Blytt’s theory, the wild plants of Norway should be arranged in the following six groups: (1) the arctic (the mountain flora) ; (2) the subarctic (the vegetation of mountain and wooded slopes), which is more frequent in the arctic than in the more southern, lower regions; (3) the boreal (the vegetation of the rocky slopes covered with foliage trees), which has its widest distribution in the low land, but not the coast districts; (4) the Atlantic (Bergen coast vegetation), with distribution in the coast district, especially between Stavanger and Kristianssund; (5) the sub-boreal, which occurs in the southeast of the country, especially ’round the Kristiania Fjord; and (6) the sub-Atlantic (Kristianssand coast vegeta- tion), which has its widest distribution in the coast district between Kragero and Stavanger. The arctic, boreal, and sub-boreal species of plants are warmth-loving, continental plants, while the subarctic, At- lantic, and sub-Atlantic keep chiefly to the coast districts and are insular in character. The former have immigrated dur- ing dry periods, the latter during damp periods, in the order in which they have been placed. Blytt assumed that within the period of history it is scarcely probable that any very great changes have taken place in climate or vegetation, and that the present is a dry period. Blytt (’83) subsequently maintained that these changes of climate were due to cosmic causes, namely alterations in the eccentricity of the earth’s orbit and alternate changes in the earth’s position with regard to the sun, occupying periods of about 21,000 years. By the aid of this hypothesis he calculated the period from the conclusion of the Glacial Epoch down to the present time to be between 80,000 and 90,000 years. The damp and dry periods were thus of equal duration, namely 10,500 years. As Blytt moreover started with the assumption that the plants could advance only step by step in their migrations, and could not be transferred direct from Denmark or England (VoL, 2 78 ANNALS OF THE MISSOURI BOTANICAL GARDEN to Norway, he supposed that the six different flora-elements had immigrated from the south through Sweden to the places in which they are now found, but during the subsequent change of climate had died out in the intermediate regions, in which they do not grow now. Since then, Gunnar Andersson (’96, ’06) has discussed this question with special reference to Sweden. He builds more particularly upon paleontological studies of the plants pre- served in peat-bogs. He assumes that the climate, after the melting of the ice, continued to grow warmer until—since Corylus Avellana, according to fossil occurrences, had a far more northerly distribution area than at the present time— it showed a mean temperature in August that was about 2.5° C. higher than at the present time. The temperature has, therefore, fallen to that of the present day. Gunnar Andersson designates the various periods after the Glacial Epoch according to the most characteristic plant, and assumes that the immigration has taken place in the follow- ing order: (1) The Dryas Flora includes certain arctic species, e.g., Dryas octopetala, Salix herbacea, S. polaris, S. reticulata, Oxyria digyna, Arctostaphylos alpina, and others, which are supposed to have migrated into Sweden when the melting of the ice had begun, and followed this northward. The most northerly place, however, where these arctic plants are found in Sweden is in West Gothland, in about the latitude of Gothenburg. They have not been found, from this period, farther north. (2) The Betula odorata Flora is more subalpine. With it came also Salix aurita, S. caprea, and S. cinerea, ete. (3) The Pinus sylvestris Flora immigrated during a some- what warmer period, which continued to grow warmer. In the lower, and thus older, part of the Pine Zone are found Prunus Padus, Rubus idaeus, Rhamnus Frangula, Sorbus Aucuparia, and Viburnum Opulus ; in the upper, and therefore more recent, part, which has had a warmer climate, we find Alnus glutinosa, Cornus sanguinea, Crataegus monogyna, 1915] WILLE—FLORA OF NORWAY 79 Corylus Avellana, Tilia europaea, Ulmus montana, ete. Here we come to the transition to the next flora. (4) Quercus Flora, which immigrated during the warmest period after the Glacial Epoch, when the mean summer temperature was about 2.5°C. higher than at the present day. In addition to Quercus pedunculata and Q. sessiliflora, there immigrated during this period Acer platanoides, Fraz- inus excelsior, Hedera Helix, Viscum album, and a great num- ber of warmth-loving plants, which have since kept to the warm slates and limestones. As the last immigrants during the steady decrease of the summer temperature, Gunnar Andersson gives (5) The Fagus Flora and (6) the Picea excelsa Flora. What is new in this theory is that there is assumed to have been only one period with higher temperature since the Glacial Epoch. This, too, is supported by the results at which W. C. Brogger (’00) has arrived in his investigations of the Quaternary fossil molluse fauna in the south of Norway. Since then, the question of the immigration of the flora into Sweden has been treated in a series of papers by R. Ser- nander (710), who rather inclines to A. Blytt’s theory, and in Norway by J. Holmboe (’03), who subscribes to that of Gunnar Andersson. _ The geological basis, however, upon which all investigations of the immigration of the flora into the Scandinavian penin- sula must be built, has of late years undergone considerable alteration. A number of recent discoveries of fossil plants also give new points of support. There is still, however, uncertainty concerning many points, so that the opinions of geologists and phytogeographers by no means coincide. In the first place, by counting the layers in stratified clay deposits in Sweden, Gerhard de Geer (’08) has succeeded in proving that not more than about 12,000 years have elapsed since the ice-cap of the last Glacial Period extended as far as Skaane in the south of Sweden. The ice had taken about 4,000 years to withdraw thus far from its southernmost boundary in Germany, and it afterwards took as much as about 3,000 years to withdraw to a range of terminal moraines [VoL. 2 80 ANNALS OF THE MISSOURI BOTANICAL GARDEN in central Sweden, and in the south of Norway to the morainie ridges that extend from Fredrikshald to Moss, Horten, Arendal, etc., and are designated by the Norwegian word “Ra.’ According to G. de Geer, these great terminal moraines must have been formed about 9,000 years ago when the inland ice stood still along that line for a period of about 350 years. It is a matter of indifference to us that other geologists be- lieve that this ‘‘Ra’’ period occurred somewhat earlier. What is of great importance in the immigration of the flora, however, is that the extreme southeast of Norway and the center of Sweden, at the time of the ‘‘Ra’’ formation, lay much lower than at the present time, and sank still lower some time after the ice withdrew. It is supposed that the sea near Kristiania, during the ‘‘Ra’’ period, was about 660 feet higher than it now is, and a little later rose to 720 feet above its present height, which is the highest limit of the late glacial sea. But this limit differs in different parts of the country; it falls toward the coast, especially toward the west coast of Norway. At Larvik, for instance, it is about 426 feet; at Arendal, 246 feet; at Kristianssand, about 130 feet; at Mandal, 82 feet; and at Farsund, only 28 feet. Farther north it increases again, so that at Kristianssund it is about 246 feet, and at Trondhjem, 650 feet, or almost as great as at Kristiania. THE DRYAS PERIOD I have previously (’05) endeavored to show by Dryas and Salix polaris, which A. G. Nathorst has found in a fossil state in the south of Sweden, that the arctic flora cannot have made its way thence into Norway; for during the ‘‘Ra’’ formation the masses of ice went right out into the sea, and when the ice had withdrawn far enough to leave open land within the “Ra” line, the climate had already altered to such an extent that the arctic flora was extinct in the south of Sweden. The earliest plants of which J. Holmboe (’03) has found remains in the southeast of Norway, prove also to be sub- 1915] WILLE—FLORA OF NORWAY 81 alpine; but farther west fossil arctic plants have been found in a number of places. D. Danielsen (’09, ’12) has found, between Kristianssand and Mandal, fossil leaves of Salix polaris from 46 to 59 feet, Dryas octopetala from 46 to 52 feet, and Betula nana from 46 to 52 feet above the sea. The uppermost marine bound- ary here is from 137 to 141 feet above sea-level, but the leaves are supposed to have been carried out by currents and de- posited at a depth of perhaps 65 feet. Something similar may have taken place with most of those subsequently mentioned, as they are sometimes found covered with more or less loose material. C. F. Kolderup (’08) has found, near Bergen, Dryas octo- petala, Salix polaris, and S. reticulata, from 115 to 130 feet above the sea, while the marine boundary lies at a height of about 190 feet above sea-level. J. Rekstad (’05, ’06, ’07, ’08) has found Salix polaris 130 feet above the sea in Söndfjord, 187 feet above the sea in Nordfjord (marine boundary 250 feet above sea-level), and in Nordmöre sometimes 82 feet, sometimes from 344 to 377 feet, above sea-level; and Salix herbacea in Nordfjord 220 feet above the sea (marine boundary 360 feet above the sea), in Söndmöre 85 feet. K. O. Björlykke (’00) has found Salix reticulata near Krist- iania 540 feet above the sea, and near Trondhjem 340 feet above sea-level. P. A. Oeyen (’04, ’07) has found Dryas octopetala and Salix reticulata near Trondhjem at a height of 557 feet above sea- level, and Salix polaris in Asker, near Kristiania, 600 feet above the sea (the marine boundary at the latter locality is 692 feet above sea-level). Remains have also been found of species that may have a subalpine occurrence, such as Betula nana, Juniperus nana, and Salix phylicifolia; but as they are less conclusive, they are not included here. The point of especial interest is that these fossil plants on the west coast are found with remains of the high arctic molluse Yoldia arctica, which is not now found on the shores [VoL, 2 82 ANNALS OF THE MISSOURI BOTANICAL GARDEN of Norway, but on the coast of Spitzbergen, and indicates a mean temperature of from —3 to —7°C. and thus quite an arctic climate. At Kristianssand these arctic plant- remains are found together with remains both of Yoldia arctica and Mytilus edulis, while Salix polaris, near Kristi- ania, is found with Mytilus and far below the highest marine boundary. Two questions now present themselves, (1) did Salix polaris and other arctic vegetation continue to live during the Last Glacial Period upon a stretch of coast in the west and north of Norway that was not covered with ice, or (2) did Salix polaris and the other arctic plants immigrate from Jutland —where they lived during the Last Glacial Period—to the first land from which the ice disappeared at Kristianssand, and thence spread along the edge of the ice on both sides as the latter disappeared? I have previously endeavored to uphold the first of these views as the more probable, having found (’05, p. 337) that the discoveries hitherto made of the remains of arctic plants favored the belief that ‘during the Last Glacial Period there lived in Norway a high-arctic vegetation upon a strip of coast that was free from ice and must have extended about as far down as the Sogne Fjord. Subsequently, as time went on, several species of high-arctie plants that had immigrated from Russia and Siberia made their way for a greater or smaller distance southward in the north of Scandinavia.’ Various later discoveries of arctic plants all the way down to the south point of Norway go to prove that the iceless margin of coast may have extended thus far, at any rate par- tially. The isolated occurrence of Saxifraga Aizoon, growing upon the mountains in inner Ryfylke, east of Stavanger, is also difficult to understand unless it is assumed that it mi- grated thither from an iceless margin of coast, as this species, beyond being found in the Alps, is only known in Nordland in Norway, and in Iceland and Greenland. But it seems probable that here a number of vegetable species from the interglacial period may have survived the Last Glacial Period. This must have been the case with 1915] WILLE—FLORA OF NORWAY 83 Artemisia norvegica, whose province of distribution in Nor- way is on the Dovre and adjoining mountains in the north- west (Troldheimen), some of which could scarcely have been covered with ice during the Last Glacial Period. It is even possible to name, with considerable accuracy, some of these plants, as they form the ‘‘Greenland element’’ in the arctic flora of Norway. I designate as such those plants which Norway has in common with Iceland, Greenland, or the north of North America, but that are not found in western Siberia. These are as follows: Arnica alpina is found in the north of Norway from Salten to Alten, and also in the north of Sweden, on the Kola Penin- sula and Novaja Semlja, but not again until the east of Siberia is reached. It is also found in Greenland and on the Alps. Campanula uniflora is found in Norway from Lom to Reisen, in Swedish Lapland and Novaja Semlja, but elsewhere only in Greenland and arctic North America. Carex nardina is found in Norway from Salten to Kvaen- angen, and in Swedish Lapland, but elsewhere only in Ice- land, Greenland, and arctic North America. Carex scirpoidea is known in Norway in Salten, and else- where only in eastern Siberia and western Greenland. Draba crassifolia is found in Norway, ’round Tromsö, but otherwise only in Greenland. Pedicularis flammea is found in Norway from Salten to Lyngen, and in Swedish Lapland, but elsewhere only in Ice- land and Greenland. Platanthera obtusata is found in Norway in Reisen and Alten, but otherwise is known only from eastern Siberia and arctic North America. A fact that possesses peculiar interest in the study of the occurrence of these and other similar species of plants in the Norwegian mountains, is the discovery in central Norway of interglacial remains of Elephas primigenius and Ovibos moschata. These great mammals became extinct at the begin- ning of the Last Glacial Period, but some of the plants that lived at the same time found a dwelling place upon the iceless [voL. 2 84 ANNALS OF THE MISSOURI BOTANICAL GARDEN coast margin and there managed to survive that period, and then to some extent followed the retreating ice up to the mountains where they are now found. Andr. M. Hansen (’04, ’04*) even assumes that at least 300, perhaps as many as 500, kinds of vascular plants may have lived upon this supposed iceless strip of coast, which he as- sumes to have been fairly broad. These figures are perhaps rather high, but it is not possible to make more exact state- ments until paleobotanical investigations have been carried out in the peat-bogs in these regions. Against the second possibility, namely, that the arctic plants may not have immigrated from Denmark to Kristians- sand until after the ice had withdrawn, several facts may be eited. These arctic plants, farther up the west coast of Norway (e.g., in Nordfjord), are found together with Yoldia arctica, and thus in a decidedly arctic climate, while those near Kristi- anssand, though, indeed, found with Yoldia, also occur with Mytilus, which indicates that the climate was somewhat milder and that the plants originated at a more recent period than those in Nordfjord. Thus the arctic plants, e.g., those in Nordfjord, cannot have immigrated thither from Kristians- sand, but may be assumed to have been there during the Last Glacial Period. On the other hand, Salix polaris near Kristiania, which ap- pears to have originated at a somewhat later period, may have been able to immigrate thither along the margin of the ice from Kristianssand; but this cannot at present be stated with certainty, as no fossils have been found between the two points. THE BETULA ODORATA PERIOD As the ice-cap withdrew and the climate became milder, the land began to rise. In the center and south of Sweden, this took place so rapidly that a land connection was formed between Sweden and Denmark, and also between south and north Sweden, very much as it is at present. The Baltic thereby became a lake, its waters becoming gradually fresher and containing fresh-water animals, especially Ancylus fluvia- Bi ee gez We e 1915] WILLE— FLORA OF NORWAY 85 tilis, which has given to this geological period the name of the Ancylus Period By this upheaval of the land, a broad migration road for Mis of ig reaps: oe white area a represents indicated by par s lake > ag —n by oblique cross-lines neh land.—Chiefly afte ing the Ancylus Period: the remainder of the a ice sheet; region rallel ee Ya represen (water) ; plants was and from the southeast and east to Norway Seeds were probably carried over now and again before this upheaval of the land—as soon as land was vacated by the re u [VoL. 2 86 ANNALS OF THE MISSOURI BOTANICAL GARDEN ice in the southeast of Norway; but the direct land connection facilitated the spread of all species of plants. Betula odorata was an early immigrant, and with it were a number of other plants of which fossil remains have been found, especially in peat-bogs in the southeast of Norway,! namely, Betula nana, Carex ampullacea, C. filiformis, Cicuta virosa, Comarum palustre, Empetrum nigrum, Equisetum fluviatile, Hippuris vulgaris, Juniperus communis, Meny- anthes trifoliata, Myriophyllum spicatum, Nymphaea alba, Potamogeton natans, Scirpus lacustris, Vaccinium Vitis- Idaea, Zannichellia polycarpa. But in addition to these, it may probably be assumed that the following species, which are found as subfossil remains from the subarctic or partially arctic period in Swedish peat- bogs,? may have migrated into Norway by this southeastern road as soon as some of the nearest land areas were free from ice. These are Andromeda polifolia, Arctostaphylos alpina, A. Uva Ursi, Batrachium confervoides, Diapensia lapponica, Montia fontana, Myrtillus uliginosus, Oxyria digyna, Phrag- mites communis, Polygonum viviparum, Populus tremula, Potamogeton filiformis, P. praelongus, Salix aurita, S. caprea, S. cinerea, S. phyllicifolia, S. repens, Scheuchzeria palustris, and Stachys sylvatica. During this period Hippophaë rham- noides also immigrated to Sweden, but as it spread along the east coast of that country and thence through Jemtland to the north of Norway, this could not have taken place until much later, after the last of the central inland ice had melted. THE PINUS SYLVESTRIS PERIOD After Betula odorata, but during the so-called Ancylus Period in Sweden, Pinus sylvestris migrated to the south- east of Norway, while the climate was still comparatively cold; but, as we may gather from some of the plants that occur, especially in the latter part of the pine zone, the tem- perature became rather rapidly warmer. J. Holmboe has found in the peat-bogs of Norway the fol- lowing fossil plants in the pine zone: Alisma Plantago, Alnus 1 By J. Holmboe (°03). ? By Gunnar Andersson (’96). 1915] WILLE—FLORA OF NORWAY 87 glutinosa, A. incana, Andromeda polifolia, Betula verrucosa, Carex Pseudocyperus, Cladium Mariscus, Corylus Avellana, Eriophorum vaginatum, Isoetes lacustris, Linnaea borealis, Lycopus europaeus, Naias marina, Nuphar luteum, Oxycoccus microcarpus, Rhamnus Frangula, Rubus Idaeus, Salix aurita, Scheuchzeria palustris, Solanum Dulcamara, Spiraea Ulmaria, and Ulmus montana. In addition to these, Gunnar Andersson has found in Swedish peat-bogs from the pine period the following species: Calla palustris, Caltha palustris, Carex riparia (?), C. vesi- caria, Ceratophyllum demersum, Cornus sanguinea, Crataegus monogyna, Eriophorum angustifolium, Galium palustre, Iris Pseudacorus, Myriophyllum alterniflorum, Naias flexilis, Myrtillus nigra, Naumburgia thyrsiflora (?), Oxalis Aceto- sella, Pedicularis palustris, Potamogeton pectinatus, Prunus Padus, Ranunculus repens, Rubus saxatilis, Rumex Hydro- lapathum, R. maritimus, Sorbus Aucuparia, Sparganium ramosum, Thalictrum flavum, Tilia cordata, Viburnum Opulus, and Viola palustris. But several of these latter species did not get as far as Norway until the succeeding warmer period, and we shall therefore find them again in the list of fossils that have been found in peat-bogs from the Oak Period. A few of them may also have immigrated by other routes, as a land connec- tion with Sweden was established not only in the south but also in the east, the ice having withdrawn to the interior of the country, and at the close of the Ancylus Period prob- ably melted away entirely. Various discoveries go to prove, for instance, that Alnus glutinosa migrated into Norway from the south, while Alnus incana came from the east. There are in Norway two quite distinct forms of Pinus sylvestris L., which by some botanists are given as species, namely, var. septentrionalis Schotte, and var. lapponica (Fr.) Hn. The second of these, which is found in abundance in Finland and the far north of Sweden, also grows in Norway, especially in the north, and on the mountains farther south, where here and there it pushes down into the valleys. It may be assumed that this P. sylvestris var. lapponica did not im- [VoL, 2 88 ANNALS OF THE MISSOURI BOTANICAL GARDEN migrate from the northeast until much later—after the ice- cap had melted in the north of Norway and Sweden, and then made its way southward. The common Pinus sylvestris, on the contrary, as we have said, undoubtedly migrated into Norway from the southeast through Sweden, which is prob- ably the way by which most of those species immigrated which are now found growing with it in the southeast of Norway. THE QUERCUS PEDUNCULATA PERIOD The climate gradually becomes warmer, the inland ice has quite disappeared, and simultaneously with its disappearance the land in a belt across central Sweden begins once more to sink (the Littorina Subsidence). When this subsidence cul- minated, the south of Sweden was a great island which, on the south, was separated—as it now is—from Denmark by Oeresund and by a broad arm of the sea, which ran from Skagerak through the district in which the lakes Venern and Vettern now lie right to the Baltic. This sea thus acquired an opening into the North Sea, and its waters gradually be- came salt. This subsidence of the land, which took place when the land around Kristiania was about 230 feet lower than it now is, did not greatly affect Norway, for it amounted in the latter to only a few yards. But it may probably be assumed that so great an arm of the sea, with a current of Gulf Stream water that even brought Gulf Stream nuts (Entada giga- lobium) with it to the shores of Bohuslaen—whence they are not carried at the present day—must have made the climate warmer and more insular than it now is. Before the sub- sidence, then, the climate must have been warm and dry, after the subsidence, warm and damp. How much warmer the climate must have been is apparent from Gunnar Andersson’s investigations—following the dis- covery of fossils—on the distribution of Corylus Avellana at that time, compared with its present distribution. It appears that the mean temperature of the summer months must have been about 2.5°C. higher than it now is. In the sea off the coast of Norway there lived at that time species of the more 1915] WILLE—FLORA OF NORWAY 89 @ Locality of fossil hazel i 4 it gr RT Northrrn limit of the ear Vi ee HR eS lier common occurrence es A, Í 5 ics eo PFT wa, ae VEO orthern limit of the pre- SDR“ SS sent common occurrence ose A X of the hazel \ ARN - RLM Belial i Di; VAC | I VD 1\6 8 ö.6r, Oo K Fig. 5. Map of the present and the former distribution of Corylus Avellana in Sweden. The entire area from which Corylus has disappeared is about 32,42 square miles. (For key, see upper left-hand corner of figure.)—After Gunnar Andersson — [VoL, 2 90 ANNALS OF THE MISSOURI BOTANICAL GARDEN southern mollusc genus Tapes, which shows that the average annual temperature must have been between 8 and 9°C. (Brögger, ’00). Various opinions have been expressed as to whether the warmest period was before, at, or a little after, the maximum of the Littorina Subsidence in Sweden. This is of little im- portance here, but what is more important is that the earliest remains of stone implements in Norway date from this warmest period (the Tapes Period), which, therefore, in the opinion of archaeologists, must be assumed to have been about 7,000 years ago. This accords well with G. de Geer’s calcu- lations from the number of clay strata. J. Holmboe has found the following species of plants, to- gether with Quercus pedunculata, in Norwegian peat-bogs: Acer platanoides, Aspidium Thelypteris, Bidens cernua, B. tripartita, Calla palustris, Carex stellulata, C. vesicaria, Cer- atophyllum demersum, Crambe maritima, Fraxinus excelsior, Galeopsis Tetrahit, Iris Pseudacorus, Myrica Gale, Naias flexilis, Naumburgia thyrsiflora, Oxalis Acetosella, Peuce- danum palustre, Potamogeton praelongus, Ranunculus repens, Rubus fruticosus, Ruppia rostellata, R. spiralis, Scirpus ma- ritimus, Sorbus Aucuparia, Sparganium ramosum, Stachys sylvatica, Thalictrum flavum, Tilia cordata, Viola sp., Zostera marina. It will at once be seen that a good many of these species were enumerated as having been found in the south of Sweden during an earlier period, i.e., with Pinus sylvestris. This agrees very well with the assumed immigration route through Sweden, as it must have taken a considerable length of time for these plants to spread through Sweden into southern Norway. It must not, however, be forgotten that the occur- rences of plants in the peat-bogs indicate only the minimum length of time of their existence in the place in question, as they may very well have lived there for a long time before being deposited in a peat-bog, to be found there through the investigations of a botanist. In addition to the above, Gunnar Andersson has found the following fossil species in the Oak Period in Sweden, these 1915] WILLE—FLORA OF NORWAY 91 species being either unknown in Norway or found only in later deposits, some of them probably not having immigrated until later, together with Picea excelsa. They are Angelica sylvestris, Cakile maritima, Cornus suecica (?), Helianthus peploides, Hedera Helix, Ledum palustre (?), Potamogeton crispus, Ranunculus Flammula, R. sceleratus, Sagittaria sag- ittifolia, and Viscum album. A. Blytt (’82) assumed that a great many warmth-loving species, constituting what he called the ‘‘boreal flora,’’ must have immigrated at this time, especially several xerophilous plants, such as a number of Labiatae, Boragineae, etc. (some of which are now commonly found on the steppes of southern Russia), which still keep especially to warm slates and lime- stones in the Norwegian lowland in the east, the west, and the province of Trondhjem. Andr. M. Hansen (’04) draws especial attention to the fol- lowing among these species, constituting what he calls the ‘‘Origanum community,’’ and which grow on open slopes with a very sunny exposure: Agrimonia Eupatoria, Androsace septentrionalis, Arenaria serpyllifolia, Calamintha Acinos, Campanula Cervicaria, Carex muricata, Centaurea Scabiosa, Dianthus deltoides, Echinospermum lappula, Origanum vul- gare, Plantago media, Polygala amara, Ranunculus Polyan- themos, Torilis Anthriscus, Trifolium medium, Turritis glabra, Verbascum nigrum, and V. Thapsus. As they grow upon dry slopes, it is not very probable that remains of them will be preserved in peat-bogs or elsewhere. Paleontologic- ally, therefore, their immigration cannot be determined, but something may be concluded as to their occurrence in the present day; for it appears that this warmth-loving plant community has its most connected province of distribution from the lowlands of the southeast of Norway, on the warm slates through Valdres and Gudbrandsdal, and are then met with once more on the low land of the western fjord valleys, and in the province of Trondhjem. To this last locality there is evidently also an immigration road through Jemteland from the east coast of Sweden. On the other hand, this plant community is wanting throughout so great a part of the [VoL. 2 5° Oreaur 10° i 50° M\ D I ° i \ DR N SAREE, N Ca AS HD Qu ‘h A ie ~ D A # Au 9g opt: j fo up upi W \ fy N, ut i è i W y fy Hae iil fi N Fig. 6. -B aora commun montane re ketch-map showing the distribution = a re of t ity (vertical en lines) in Seandi gion during the es) i via g o black- dotted areas.—After Bar M. Han xtent er rmest post a ia is indiana 1915] è WILLE—FLORA OF NORWAY 93 southwestern lowlands, that it can hardly be imagined that it migrated along the coast to the west and Trondhjem. It must therefore be taken for granted that these plants migrated by way of the mountain passes, some of which now lie at such an altitude that even Pinus sylvestris cannot live in the highest localities. But I have already mentioned that the summer temperature during this period was about 2.5°C. higher than it now is. We see, moreover, that remains of pine forests are found on the mountains in Norway, e.g., on the Dovre Mountains in central Norway, and on the Hard- anger plateau in the south of Norway, respectively 990 and 1,470 feet above the present highest limit of Pinus sylvestris. Under the then existing climatic conditions, the now treeless passes were clothed with forest, and warmth-loving plants were able to spread through them. A. Blytt, and after him R. Sernander, distinguishes between a boreal and a sub-boreal flora, the members of which are supposed to have been lovers of warmth and dryness, but separated in their immigration by an Atlantic flora that loved humidity and warmth. With this I cannot agree. Several of the species that Blytt (’82) classes under ‘‘sub-boreal’’ are found in a fossilized state together with those he calls ‘‘bor- eal’’; and around Kristiania many species of these so-called different floras grow together under exactly the same condi- tions in the same localities. It seems likely, however, that the elimate was more humid during the Littorina Subsidence, when the water of the Gulf Stream could make its way directly into the Baltie across central Sweden. A. Blytt (’82, p. 23) says: ‘‘Man darf des- halb mit einem hohen Grad von Wahrscheinlichkeit be- haupten, dass die atlantische Flora in dieser Regenzeit einge- wandert ist, und ihren Weg rund um den Christianiafjord gefunden hat (in derselben Weise, wie unter der folgenden Regenzeit die subatlantische).’’ I cannot agree in all respects with this. Those forms which Blytt calls ‘‘atlantische Arten’’ include a great number of species, of which some occur in what I have here called the ‘‘region of Ilex Aquifolium,’’ others constitute what I have called the ‘‘west European [vor. 2 94 ANNALS OF THE MISSOURI BOTANICAL GARDEN coast flora,’’ while among other species belonging to Blytt’s group Rhynchospora alba, Alnus glutinosa, Myrica Gale, Arnica montana, Erica Tetralix, Ranunculus Flammula, Ly- chnis Flos-cuculi, etc., may be mentioned, which grow on the low-lying land in many parts of southern Norway. Asa rule, they prefer, it is true, damp places, but some species go right up to the Birch Zone on the mountains, so they may be pre- sumed to have immigrated from the southeast through Sweden; but there is nothing to prove that this took place just at the maximum of the Littorina Subsidence. As in- stances, indeed, of the contrary, Alnus glutinosa from the Birch Period and Myrica Gale from the Oak Period are found in Norwegian peat-bogs and were, therefore, much earlier. I believe that the west European coast flora on the west coast of Norway immigrated directly, by fits and starts, from England; but we will return to this later on. THE PICEA EXCELSA PERIOD According to archaeological ealeulations, the Scandinavian Stone Age lasted about 3,000 years, so that the Bronze Age in Scandinavia began about 4,000 years ago. During this period the elimate was undoubtedly warmer than it now is, and it was not until the Bronze Age that any noticeable fall seems to have taken place. At the beginning of the Stone Age the land around Kristi- ania lay 230 feet lower than at present, but during the Stone Age it was elevated about 184 feet, and during the Bronze Age it rose to about its present height above sea-level. In the Bronze Age, or perhaps in the latter part of the Stone Age, Picea excelsa migrated into Norway from the east, from Finland through Sweden. In Finland it is still found as a fossil in the Oak Period, and in Sweden, especially in the north and east, it is so found, while spruce is not found fossilized in the south of Sweden or Denmark after the Glacial Epoch. In the north of Norway (Finmark) there are occurrences of spruce that are entirely independent of the spruce’s great province of distribution in the south of Norway. It appears 1915] WILLE—FLORA OF NORWAY 95 that these northern occurrences are of a distinct form (Picea excelsa [Lam.] Link f. obovata Ledeb.), which is classed by some botanists as a separate species, and has its distribution through the north of Finland and Russia. There can, of course, be no doubt that the spruces in these northernmost occurrences immigrated independently from Finland, and probably at a later period, as there is a tradition that they were imported thither by human beings (Lapps). According to J. Holmboe, Calluna vulgaris came into Nor- way during the same recent period in which Picea excelsa made its appearance, but there is no doubt that the former immigrated from the west and then spread eastward, i.e., in the direction opposite to that in which Picea excelsa spread. Both these species have now a very wide distribution in Norway. Strange to say, there has not been found in the deposits from the Pine Period in Norwegian peat-bogs a single plant that is not to be found in the earlier periods. In Sweden the only new species found is Rubus Chamaemorus, which, how- ever, undoubtedly grew there long before, as it must on the whole be considered to be a subalpine species. This is suffi- cient to show that special conditions are necessary in order that parts of plants may be preserved in bogs, and that it will, therefore, always be only a small proportion of the plants growing around the bogs which will be so preserved. It is strange, for instance, that Taxus baccata is not found in Norwegian peat-bogs. It is found as a fossil from the Oak Period in Sweden, and must have been far more common in Norway in the early Iron Age than it is at the present time, as H. Conwents found, on examining twenty-three vessels in the Archaeological Museum in Kristiania, that eighteen of them were of Taxus and only one of Picea excelsa. According to R. Sernander (’10) the period of greatest warmth must have occurred in the Bronze Age, and he believes that it was then that Corylus Avellana was most widely dis- tributed northward. The Bronze Age, however, judging from the molluscs that were then found off the south coast of Norway, seems to have had a cooler climate than that of the (Vou, 2 96 ANNALS OF THE MISSOURI BOTANICAL GARDEN Tapes Period, i.e., the Scandinavian Paleolithic Age. On the other hand, R. Sernander believes that at the beginning of the Iron Age—about 2,400 years before our own day—so great a decline in temperature ensued that the montane plants made their way into the lowlands in many places. He inter- prets the present occurrences of alpine plants in the lowlands as relics from that period. This can, however, be the case only to a certain extent, for there is no doubt that at the present day alpine plants spread down to the lowlands and continue to grow there, provided the conditions are favorable. R. Sernander gives to his assumed cold, damp period at the beginning of the Iron Age the name employed by A. Blytt, the ‘‘sub-Atlantic period’’; but the two have, in reality, very little to do with one another. A. Blytt states that his sub- Atlantic period occurred when the south coast of Norway lay from 30 to 50 feet lower than its present level, which would answer to the beginning of the Bronze Age. He men- tions, among other species that immigrated during the sub- Atlantic period, Carex Pseudocyperus and Cladium Mariscus, which had already immigrated in the Pine Period, and Cera- tophyllum demersum, which had immigrated in the Oak Period, besides two or three species that were certainly im- ported later in foreign grain and grass seed. I do not yet consider R. Sernander’s cold, damp ‘‘sub- Atlantic period’’ at the beginning of the Iron Age to have been clearly proved, although there are a few facts that speak in its favor. But even if such a cold, damp period did super- vene, its principal effect would have been to decimate the oak flora—in localities that were not especially warm—more rapidly than if the climate had gradually become colder from the Stone Age to the present time, as most people believe. Similarly, it may have promoted the occasional descent of montane plants to the lowlands, but it appears that this can also take place under the present climatic conditions, without the necessity of having recourse to relic occurrences from the ‘¢sub-Atlantic’’ or even from the ‘‘Dryas Period.’’ An instance of such an occurrence is that of Dryas octo- petala at Langesund in southeastern Norway. This species 1915] WILLE—FLORA OF NORWAY + 97 is found there right down to the level of the sea, and is very common on the limestone of the locality. Together with J. Holmboe (’03), I have endeavored to prove that over the whole of the area in which Dryas appears, the latter can scarcely have existed for more than 100 years. I cannot ascribe any convincing power to the objections that have been raised against this line of argument. THE FAGUS SYLVATICA PERIOD In Norway, as already mentioned, Fagus sylvatica grows upon the southeast coast, with Larvik as a center. There is, in addition, an isolated beech-wood in Seim, to the north of Bergen, 280 miles from the nearest occurrence of beech. It was formerly believed by A. Blytt that this beech-wood in Seim was a relic of a connected distribution of beech along the coast; but no discovery of fossils favors this idea. On the contrary, these two occurrences of beech appear to be per- fectly independent of one another. J. Holmboe (’05, ’09) has endeavored to find out when the beech appeared at Larvik and in Seim. He has come to the conclusion, judging from what has been found in the peat- bogs, that at Larvik the beech immigrated considerably later than Picea excelsa. It can thus actually be assumed to have immigrated in the Iron Age, or perhaps as late as the time of the Vikings. This late immigration is in harmony with the fact that in the southeast of Norway the beech is making very rapid advance at the present time. Holmboe says that the beech-wood in Seim, from a geological point of view, is very recent, but that in any case its age should scarcely be put lower than about 1,000 years. It seems to me most probable that the beech was introduced into Norway by man in the time of the Vikings, when there was ample communication with those countries in which this so generally useful tree formed extensive forests. In Seim, near Bergen, where the beech grows, the Norwegian King Haakon the Good, who was educated in England at the court of King Athelstan, and reigned from 935 to 961, had one of his estates; and it is not unnatural to suppose that he may [VoL. 2 98 ANNALS OF THE MISSOURI BOTANICAL GARDEN have tried to introduce a tree that he knew so well from his childhood and youth in England. It is certain that in the course of time man has assisted in introducing many species of plants, some consciously, as, for instance, plants for cultivation, others by chance and unconsciously. In the famous Viking ship from Oseberg, which is believed with certainty to have originated in the first half of the ninth century, fruit, seeds, and other remains of plants have been found, and have been determined by J. Holmboe (’06). The following cultivated plants were among them: Avena sativa, Corylus Avellana, Isatis tinctoria, Juglans regia, Lepidium sativum, Linum usitatissimum, Pirus Malus, and Triticum vul- gare. As Isatis tinctoria is found growing apparently wild, in certain places in Norway, there can scarcely be any doubt that it has found its way thither from localities where it had previously been cultivated as a dye-plant. This is probably also the case with Serratula tinctoria in Jaederen, near Stav- anger. The weeds found in the Oseberg ship were as follows: Capsella Bursa-pastoris, Chenopodium album, Galeopsis Tet- rahit, Lamium (purpureum?), Polygonum Convolvulus, Stell- aria media, and Urtica urens. Several of these, it is true, had immigrated earlier, as has been said of Galeopsis Tetrahit; but it shows that as early as the time of the Vikings, Gua were opportunities of importing foreign weeds. In monastery gardens various medicinal, OE TRT and ornamental plants were cultivated, and one i inclined to be- lieve that several of these which now have quite a wide dis- tribution in Norway, e.g., Aquilegia vulgaris, Berberis vul- garis, Sambucus nigra, etc., originally spread with the mon- asteries as centers. It is still easier to demonstrate a number of species of weeds that have been imported recently, and of which some appear to have a really astonishing power of spreading. J. Holmboe (’00) has traced the spread of the following weeds from the year when they were first observed in Norway: Alyssum calycinum (1857), Anthemis tinctoria (1772?, 1807), Barbarea vulgaris (1790), Berteroa incana (1826), Bunias 1915] WILLE—FLORA OF NORWAY 99 orientalis (1812), Campanula patula (1870), Cerastium arv- ense (1817), Chrysanthemum segetum (1704), Cotula coron- opifolia (1875), Conringia orientalis (1859), Erigeron can- adensis (1874), Galinsoga parviflora (1880), Lepidium per- foliatum (1875), L. virginicum (1889), Matricaria discoidea (1850), Rudbeckia hirta (1880), Senecio viscosus (1804-1808), Thlaspi alpestre (1874), and Xanthium spinosum (1872). Some of these plants are now among the most troublesome weeds in large and small areas in Norway. There can, I suppose, be no doubt that man, directly and indirectly, in the 7,000 years in which he has lived in Norway and maintained a lively intercourse—especially during the last 2,000 years—with the rest of Europe, must have assisted in introducing a great number of plants in addition to the above named. Among the former may be mentioned Agros- temma Githago, Anchusa arvensis, Anthemis arvensis, Avena fatua, Brassica campestris, B. migra, Bromus secalinus, Car- duus crispus, Centaurea cyanus, Chenopodium capitatum, C. hybridum, C. glaucum, C. polyspermum, C. rubrum, Circium arvense, Convolvulus arvensis, Euphorbia Helio- scopia, E. Peplus, Fagopyrum tataricum, Fumaria officinalis, Galeopsis angustifolia, G. Ladanum, G. speciosa, Galium Aparine, G. Mollugo, Lolium temulentum, Matricaria Cham- omilla, Raphanus Raphanistrum, Sinapis alba, S. arvensis, Sonchus asper, S. oleraceus, Spergula arvensis, Spergula vernalis, Thlaspi arvensis, ete. In addition to these there are a great many species that are generally classed in the floras under the heading ‘‘run wild’’ or ‘‘perhaps originally run wild,’’ and concerning which it may certainly be assumed that they have been introduced by man’s mediation in some way or other. It is no longer possible to maintain the old dogma which held that the entire plant community migrated step by step, like a regiment of soldiers, and took possession of the country under climatic conditions that were favorable to the various species, while the previous vegetation was decimated and only survived in especially favorable localities; for vegetable [Vou, 2 100 ANNALS OF THE MISSOURI BOTANICAL GARDEN species generally immigrate singly and independently of one another. It is not only man that assists in carrying plants across large sea surfaces; the wind, ocean currents, and especially birds from time to time transport seeds and other parts of plants, which, under favorable conditions, continue to grow. I will not here go further into this complex question in its entirety, but will refer to R. Sernander’s (’01) detailed work on the conditions for spreading in a great number of Scandin- avian plants. I must, however, mention a few examples of probable, or certain, chance distribution. At Vaage Lake, far up the valley Gudbrandsdal, 990 feet above sea-level and separated from the innermost fjords of the west coast by 56 miles of very high mountains, grows the typical sea-shore plant, Elymus arenarius. That this occurrence represents a relic is absolutely out of the question, for the sea cannot have reached the height of Vaage Lake since the Silurian Period. But I have seen gulls flying over the lake, and they may pos- sibly have carried seeds with them, which have found a favor- able soil in the long sandy shores. In 1837, Coleanthus subtilis was found upon a flooded river bank a little north of Kristiania, and in 1842 a great number of specimens were collected in the same locality, probably all that existed there, for in spite of the most careful search for a number of years, the plant has never subsequently been found in that or in any other place in Norway. As its nearest habitat is in Bohemia, it can only be supposed that some wad- ing bird, in rapid flight from Bohemia to Norway, brought the seed with it; and, furthermore, that as the seed fell upon favorable soil, the plant grew up and had already begun to spread when the collection was made in 1842. I have already (’05) endeavored to show that Campanula barbata, which occurs in a limited area on the mountains of central Norway, and is not again found until we come to the mountains of Central Europe, cannot be a glacial relic, but must have been accidentally introduced into Norway (by birds?) in recent times. 1915] WILLE—FLORA OF NORWAY 101 Judging from the distribution in the present day of a num- ber of plants on the south and west coasts of Norway, it seems natural to assume that they have been brought directly over the sea from the nearest country, Denmark or England. It was thus not necessary for them to move step by step by the long route through Sweden, or even round the Kristiania Fjord, to reach their present habitats. The latter is all the less probable from the fact that certain of them seem to have been imported quite recently, when the climatic conditions cannot have been very different from those which exist at the present time. The following are instances of these: Aera setacea grows in Norway from Kristianssand to Stavanger. The species is common in Jutland in Denmark, but in Sweden is found only in the extreme south. Airopsis praecox is found from Kragerö to Nordmöre. It oceurs, it is true, in Sweden, from the south up to Vester- gothland and Bohuslän; but from that region to Kragerö is considerably farther than from Jutland, where the plant is found in abundance. Corydalis claviculata is found from Kristianssand to Haugesund. It grows wild in Denmark and England, but not in Sweden; I assume, therefore, that it immigrated from one of the former countries. Galium saxatile is found from Kristianssand to Nordmöre. It grows in Sweden from Skaane to Bohuslän, but it is far more probable that it came from Jutland, where it is common. Genista tinctoria is found only at Brevik, and must have been recently imported, as there are only a few specimens of it. It is found wild only in southern Sweden, but is common in Jutland. Geranium columbinum is found in the district extending from Kragero through the west of Norway to the Trondhjem Fjord. In Sweden it has an eastern distribution from Skaane to Upland. It is common in Denmark. Heracleum australe is found from Kragerö to Söndfjord. It occurs in Sweden from the south right up to Vermeland, but the distance from this district to Kragerö is considerably greater than that from Jutland, where it also occurs. [VoL. 2 102 ANNALS OF THE MISSOURI BOTANICAL GARDEN Hydrocotyle vulgaris grows here and there from Larvik to Bergen. In Sweden it does not extend farther than to Dals- land, but it is exceedingly common in Jutland. Hypericum pulchrum grows in the region extending from Larvik through the west of Norway to the Trondhjem Fjord. In Sweden it is found from Halland to Bohuslan, but it is more natural to suppose that it immigrated from Denmark or England, where it is common. Luzula sylvatica grows along the coast from Arendal to Lofoten. It is found wild only in the south of Sweden, but is common in Jutland. Rubus Radula is found from Kragerö to Mandal. In Sweden it is found from Skaane to Bohuslän, but is very com- mon in Jutland. Sarothamnus scoparius grows between Grimstad and Mandal. In Sweden it is wild only in the east. It is very common in Denmark. Scirpus multicaulis grows at Arendal and in Jaederen. It is found in Sweden from Skaane to Vestergothland. It is com- mon in Denmark. Scirpus setaceus is found to the west of the Kristiania Fjord and more recently has been found also along the coast almost as far as Bergen. It is found in Sweden from Skaane to Bohuslan, but it can scarcely be supposed to have migrated thence to its most easterly occurrence in Norway, as the center of its distribution in Sweden lies farther south, and in Norway farther west. It seems, therefore, more probable that it has been brought to Norway directly from Denmark. * Since writing the above, I have discovered Hydrocotyle vulgaris in a locality n Kirkeöen (Hvaler) in southeastern Norway. The locality lies about midway between the easternmost of the previously known Norwegian stations and the Swedish localities and might be looked upon as proof that the species in question had immigrated step by step through Sweden and not directly from Denmark. This, however, is not the case. On an excursion in 1907, I visited the exact spot where I later found Hydrocotyle vulgaris and I can maintain with certainty that Hydrocotyle was not growing there at that time. The plant has, therefore, been introduced into the locality in question since that date. My opinion, therefore, that an has immigrated by leaps and bounds direetly from Denmar into Norway, is only strengthened by this discovery. 1915] WILLE—FLORA OF NORWAY 103 Stellaria Holostea grows along the coast from Grimstad to Bergen. It is found in Sweden from Skaane to Bohuslan, but must have migrated into southern Norway from Denmark, where it is common. Teucrium scorodonia is found from Lyngör to Flekkefjord. In Sweden it has probably only become wild, but in Denmark it is common. Vicia cassubica is found from Kragerö to Kristianssand. In Sweden it is found from Skaane to Dalsland, but it is common in Denmark. Vicia lathyroides grows along the coast from the Hvaler Islands farthest east off Norway, to Kristianssand. In Sweden, however, its distribution is easterly from Skaane to Upland, so it must be assumed that it migrated into Norway directly from Jutland in Denmark, where it is not uncommon. It will be noticed that most of these plants which I assume to have immigrated directly from Denmark (Jutland) to the south of Norway, are either bog or leguminous plants, or are such as have small seeds or stone-fruits. The carriage across water surfaces of such plants as these one would imagine could most easily take place through chance transport by birds. The distance across the Skagerak from Denmark to Norway is about 93 miles, and according to J. A. Palmén (’76) there are regular lines followed by birds of passage from Jutland to Jaederen, as also one almost to Kristianssand and another to Risör, the very places which appear to be the center of the distribution of the majority of the above-named species which I assume to have come directly from Denmark. It is still less probable that a number of plants that belong to the coast flora of Western Europe, and in Norway are found only in the extreme west, where the winter temperature is unusually mild (from +1 to +2°C.), should have immi- grated from England via Denmark and Sweden, where they do not now grow, or at any rate grow only in the extreme south. If they did make such a journey, the climate must have been so much milder in the southeast of Norway that the warm period that is proved in the Stone Age would not have gone nearly far enough. A climatic change as violent [VoL. 2 104 ANNALS OF THE MISSOURI BOTANICAL GARDEN as this would have been, and that in a comparatively very recent geological period, is not probable, nor is it necessary to assume it in order to explain the occurrence of these plants in the west of Norway, if only one does not blindly adhere to the dogma that plants can migrate only step by step. As instances of plants which I assume have migrated from England direct to the west of Norway, the following may be mentioned : Asplenium Adiantum nigrum is rare from Jaederen to Kristianssund. It is found in England, but only in the very east of Denmark and the extreme south of Sweden; immi- gration from the two last-mentioned countries seems, there- fore, to be out of the question. Asplenium marinum grows in the west of Norway from Mosterö to Stadt. It is found in England, but neither in Sweden nor Denmark. Erica cinerea grows on the outermost islands from Farsund to Söndmöre. It is found in England, but in neither Sweden nor Denmark. Hymenophyllum peltatum grows in the outermost coast dis- tricts from Farsund to Nordfjord. It is found in England, but neither in Sweden nor Denmark. Scilla verna grows in the extreme coast regions from Sönd- fjord to Söndmöre. It is found in England, but neither in Sweden nor Denmark. Scolopendrium vulgare is found in two or three places between Hardanger and Söndfjord. It is common in England, but it is doubtful whether it has grown in Denmark, and in Sweden it is found only in the extreme east, in Gothland. Vicia Orobus grows farthest west, from Lister and Jaederen to Söndmöre. It is common in England, but is not found in Sweden, and only here and there in Jutland. It might thus be supposed to have come from Denmark direct to Norway, but in that case it would probably grow a little farther south than it does. I consider it, therefore, most probable that it came over from England to the coast of Norway, and then spread 1915] WILLE— FLORA OF NORWAY 105 along the coast southward and northward to its present limits. It also appears, according to I. Hagen (’12), that the case is similar with regard to a number of mosses, a direct migra- tion from England to Norway being assumed. Hagen has so little faith, however, in the ability of these plants to migrate by leaps and bounds, that he supposes a post-glacial land con- nection with England, over which migration might gradually take place. This land bridge between Norway and England was origin- ally hypothetically constructed for the pre-glacial times by L. Stejneger (’07), who considers it necessary on zodgeo- graphical grounds. At the conclusion of his paper he says: “T think I may safely claim to have made it appear probable: “1. That if the characteristic and important portion of the animals and plants of west Norway, called the ‘Atlantic’ biota, in- vaded that country from Scotland, it came by way of a land bridge connecting northern Scotland with western Norway north of 59° north latitude. “2. That this land bridge existed after the first (Scandinavian) great glaciation. “3. That part of this biota surely survived the second (Scandin- avian) glaciation along the west coast of Norway, and that possibly the climate was not too severe for all to survive. “4. That there is a possibility of a reéstablishment of the land bridge during the ‘Upper Forestian’ stage with its congenial, more continental climate, during which the tenderer species may have immigrated, in case it should be proven that they could not have come with the hardier ones.’ As will appear from the foregoing pages, I have also main- tained (’05) that during the Last Glacial Period there was a stretch of coast in Norway that was free from ice, where some arctic plants, and, of course, also animals, were able to survive that period. Since then Gunnar Andersson and Selim Birger (’12) have endeavored to give to the facts that favor this view the inter- pretation that the entire arctie flora element must have im- migrated through Sweden, and followed the receding margin of ice. I consider their arguments on this point so unconvine- [VoL, 2 106 ANNALS OF THE MISSOURI BOTANICAL GARDEN ing, especially in view of the most recent discoveries of fossil arctic plants, and my own observations of the rock formations in the west and north of Norway, that I have come to the con- clusion that this iceless strip of coast was broader than I formerly supposed, and extended to the extreme southern point of Norway. In this respect my view is thus in perfect accordance with that of Stejneger. As to whether there was an interglacial direct land connec- tion between England and Norway, as Stejneger assumes, I cannot express an opinion, but I do not, in any case, consider it necessary for botanical reasons, although I am inclined to believe that the assumption of Stejneger will prove to be cor- rect. On the other hand, I consider a post-glacial land con- nection between England and Norway, concerning which Stejneger himself is much in doubt, to be quite out of the question. There is nothing that can be brought forward to prove that previous to the post-glacial subsidence the land lay high enough for any real land bridge between Norway and England to exist. On the other hand, there are several facts that go to show that the southern part of the North Sea has lain higher than it now does, so that even considerable por- tions that are now under the sea were clothed with forest. This may possibly to some extent have diminished the distance between England and Norway; but the deep Norwegian Chan- nel outside the coast of Norway has certainly been in existence ever since the Last Glacial Period. But a land connection is not necessary to explain why the few species of plants that Norway and England have in com- mon, and that must be assumed to have migrated over the North Sea, were able to come over in the course of the last 7,000 years. It must not be forgotten that according to J. A. Palmén (’76) there are two lines followed by birds of passage between England and the west of Norway; and that there may also have been other chance means of transport. All things considered, I am inclined to believe that in trying to explain the distribution of vegetable species and the paths they have followed, we shall arrive at better results by study- ing the ways in which they spread at the present time than 1915] FLORA OF NORWAY 107 WILLE by setting up hypotheses of tremendous convulsions of nature such as elevated and depressed land connections, climatic changes from cosmic causes, the oscillatory movement of the poles, etc., which can neither be proved nor disproved, as they lie beyond the spheres in which our present knowledge has a firm foundation on which to stand. LITERATURE CITED Andersson, G. (’96). Svenska vaextvaerldens historia. Stockholm, 1896. —_——, (’06). Die Entwicklungsgeschichte der Skandinavischen Flora. Con- gres ‘Internat. Bot., Wien, 1905, Resultats scientifiques 45-97. f. 1-30. 1906. —, (09). Swedish climate in the late eg ee Sveriges Geo- logiska Undersökning. Aarbok 1909:1-88. 2. f. 1-11. 1909. , och Birger, S. (’12 Den norrlandska florans geografiska foerdelning och invandringshistorie med saerskild haens syn till dess sydskandinaviska arter. Norrlaendskt Handbibliothek v. Upsala & Stockholm, 1912. Areschoug, F. W. (’66). Bidrag till den skandinaviska vegetationens historia. Lunds Univ. Aarsskrift 1866:—. j6. Björlykke, K. O. (’00). Glaciale plantefossiler. Naturen 1900:—. 1900. Blytt, A. (°76). Essay on the ae Se of p ea flora during alter- nating rainy and dry periods. Kris tiania, 187 —————., (’82). Die Theorie = ee kontinentalen und insularen Kli- mate. Bot. Jahrb. 2:1-50. ——, (’82). Nachtrag zu der Abhandlung: Die Theorie der wechselnden kontinentalen und insularen Klimate. Ibid. 2: 177-184. pl. 1. 1882. (’83 . Om vexellagring og dens mulige betydning for tidsregningen i geolog ien og laeren om arternes forandringer. Videnskabsselskabets For- ee 1883°:1-31. f. 1-2. 1883. , (06). Haandbog i Norges Flora. Udgivet ved Ove Dahl. Kristiania, 1906. Brögger, W. ©. (700). Om de senglaciale og postglaciale nivaaforandringer i “Kr ristianiafeltet (molluskfaunan). Norges geologiske undersögelse 3] :—. 1900-1901. —, (’05). Strandliniens beliggenhed Under: stenalderen. I. Det sydoestlige Norge. Norges geologiske undersögelse 41: Kristiania, 1905. en D- (709). ee undersökelser omkring Kristianssand. t Mag. 47: 23-95. pl. 1- —, (12). Kvartaergsologiake streiftog paa Soerlandet. Nyt Mag. 50: 263-382. pl. 7-9. 1912 de Geer, G. (°08). On late Quaternary time and climate. Geologiska föreningen i Stockholm foerhandlingar. 30:—. 1908. ————,, (’10). A thermographical record of the late Quaternary climate. Die Verändrung des Klimas. Stockholm, 1910. [VoL. 2, 1915] 108 ANNALS OF THE MISSOURI BOTANICAL GARDEN Hagen, I. (712). Geografiske grupper blandt Norges loevmosser. Naturen Aarg. 36:—. 19 Hansen, A. M. (’04). Hvorledes har Norge faaet sit Plantedaekke. Naturen 1904:—. 1904. ———,, (04a). Landnaam i Norge. En Utsigt over Bosaetningens Historie. Kristiania, 1904, ————-, (’13). Fra istiderne. Soerlandet. Videnskabsselskabets Skrifter. I. Math.- nat. Kl. 1913°:—. 191 Helland, A. (712). Traegraenser og Sommervarmen. Tidsskrift for Skogbruk. Aarg. 20:—. 1912. ig 2 (700). Nogle ugraesplanters indvandring i Norge. Nyt Mag. 38: 2. f. 1-3. 1900. ———, (03). Planterester i Norske torvmyrer. Et bidrag til den norske vegetations historie efter g“ . istid. Videnskabsselskabets Skrifter. I. Math.- nat. Kl. 1903°:— ‚ (705). Studier over norske planters historie. II. Nyt Mag. 43:33-60. 1905. ———., (’06. Ibid. III. Ibid. 44:61-74. 1906. —, (0). a ved Lygrefjord i Nordhordland. Bergens Mus. Aarbog 1908** :— ——, (’138). BE i Norge. En plantegeografisk undersoekelse. Ibid. 1913's. 19 Kolderup, C. 8). Bergensfeltet og tilstoedende trakter i Er og (’0 cot i | tid. Bergens Mus. Aarbog 1907"; 1-266. pl. 1. f. 1-38. Nathorst, A. G. x ’7l). Om naagra arktiska yaextlemningar i en soettvattenslera vid Alnar p i Skaane. Lunds Univ. Ars-skrift 1870: —. 1871. Oeyen, P. A. (’04). Dryas octopetala L. og Salix reticulata i vort "e foer indsjoeperioden. Videnskabsselskabets Forhandlinger 1904': 1-6. 190 —, pan Skjaelbanke-studier i Kristiania omegn. Nyt Mag. 45:27-67. f. 1-3. Palmén, J. A. am Ueber die Zugstrassen der Vögel. Leipzig, 1876. Rekstad, E Shige a fra terrasser og str Ne - ag vestlige Norge. G ns Mus. Aarbog 1905°:1-46. pl. I. f. 1-12. u, go Ibid. II. Ibid. 1906':1-48. pl. 1. f. 1-19. 1906. —, (07). Ibid. III. Ibid. 1907°: 1-32. pl. 1. f. 1-15. 1907. , (08). Bidrag til kvartaertidens historie for Nordmör, 1908. Sernander, R. (’01). es skandinaviska vegetationens spridningsbiologi 1-459. f. 1-32. Upsala, 1 ——, (710). Die en Torfmoore als Zeugen postglazialer Klima- schwankungen. Die Verändr. des Klimas. Stockholm, 1910. Stejneger, L. (’07). The origin of the so-called Atlantic animals = ae of western Norway. Smithsonian Mise. Coll. 48:458-514. pl. 67-70. f. 1-2. 1907 Wille, N. (’05). Om a pas a af det arktiske Floraelement til Norge, Nyt Mag. 43:315-338. 1905 ———, und Holmboe, J. (’03). Dryas a bei Langesund. Fine gla- ciale Snake Hie. Nyt Mag. 41:27-43. 19 i THE PHYLOGENETIC TAXONOMY OF FLOWERING PLANTS CHARLES E. BESSEY University of Nebraska I. GENERAL Discussion Seventeen years ago in presenting a somewhat similar paper! to a smaller body of botanists, I began by saying that ‘it is as yet impossible to present a complete phylogeny of the angiosperms,’’ and then a little later, ‘‘it will be many a year before the direct evidence we so much desire will leave no considerable gaps,’’ and I am impelled to use the same words now as I begin this discussion to-day. For, while in this interval paleontology has uncovered many important facts whose significance is unmistakable, it is still true that there are ‘‘considerable gaps’’ in the record of the evolution of plants, both before and after the attainment of flower produc- tion. In other words, we are still in quest of direct testimony as to how flowers came into existence in particular, and as to the details of how and when they were modified afterwards. Yet we are not wholly without the direct testimony of the rocks in our inquiry as to the phylesis of the higher plants. And I may be permitted here to enter a defense of such a discussion as I propose to make in this paper, in reply to those who think that since much of what I shall have to say is reached by a process of deduction, or, as it is more commonly called, speculation, it can have little scientific value. And I grant that in those fields where direct observation, experiment, and induction are possible there can be no defense of the ex- clusive deductive or speculative method. There are, however, many fields of botanical inquiry in which experiment is im- possible, and observation is reduced to a minimum, and this essey, C. E. The phylogeny and taxonomy of angiosperms. (Address of the retiring president of the Botanical Society of America, at its third annual meeting, at Toronto, Canada, August 17, 1897.) Bot. Gaz. 24: 145-178. f. 1-3. 1897. ANN. Mo. Bot. GARD., VOL. 2, 1915 (109) (von. 2 110 ANNALS OF THE MISSOURI BOTANICAL GARDEN is necessarily the case when we are dealing with questions which relate to periods of time long past, as must be those involving phylogeny. Moreover, it must not be forgotten that what I propose to do is after all much like what is done in even those sciences which we sometimes call the exact sciences. The ether of space, the undulatory theory of light, the tentative hypotheses as to the nature of electricity and gravitation, the form and extent of the universe, and the constitution of matter itself, are a few of the familiar speculations which physicists, astron- omers and chemists have made parts of the conceptions of their respective sciences. To be sure, one can go but a short distance indeed in any science without finding it necessary to erect a speculative framework upon which to arrange his ob- served facts. As Jevons has so aptly expressed it in his ‘Principles of Science’ (2: p. 131): “When facts are already in our possession, we frame an hypoth- esis to explain their mutual relations, and by the success or non-success of this explanation is the value of the hypothesis to I a judged. In the framing and deductive treatment of hypotheses, we must avail ourselves of the whole body of scientific truth already accumulated, and when once we have obtained a probable hypothesis, we must not rest until we have verified it by comparison with new facts. * Out of the infinite number of observations and u eidi which are pos- sible at every moment, theory must lead us to select those few critical ones which are suitable for confirming or negativing our anticipations.” A little later (p. 137) he remarks: “The true course of inductive procedure is that which has yielded all the more lofty and successful results of science. It consists in anticipating Nature, in the sense of forming hypoth- eses as to the laws which are probably in operation; and then observing whether the combinations of phenomena are such as would follow from the laws supposed. The investigator begins with facts and ends with them. He uses such facts as are in the first place known to him in suggesting probable hypotheses; de- ducing other facts which would happen if a particular hypothesis is true, he proceeds to test the truth of his notion by fresh obser- vations or experiments. If any result prove different from what he expects, it leads him either to abandon, or to modify his hypothesis; but every new fact may give some new suggestion as to the laws in action.” 1915] BESSEY—PHYLOGENETIC TAXONOMY 111 I may quote one more sentence from the Manchester logician (p. 138): ‘‘Agreement with fact is the one sole and sufficient test of a true hypothesis.”’ So I come with a general hypothesis of the evolution of living things, and of plants in particular. This hypothesis is based upon observed facts, which are here given such a uni- form interpretation as will make my general hypothesis, and it is this latter that I wish to discuss to-day, making such ap- plication as will enable us to arrange the flowering plants in accordance with it. I am going to confine my discussion pretty largely to the plants of the highest phylum, here restrieted to those that bear flowers. Since the discovery of the pteridosperms, it is manifestly untenable to regard all seed-bearing plants as members of one phylum. In other words, the Spermatophyta of the books constitute not one phylum, but several phyla. Briefly, I shall exclude first of all the cycad phylum which began in the Paleozoic period with the pteridosperms, and has extended with many losses to the present. I shall also exclude the conifer phylum, related to but not included in the cycad phylum. These two phyla are commonly associated in a group under the name of gymnosperms, but I have no hesi- tation in keeping them as distinct phyla, the eycads lower, and the conifers higher. The remaining seed-bearing plants, whose seeds are en- closed in carpels, constituting the old group of angiosperms, I regard as a distinct phylum, and because the flower is the dominant and characteristic structure, I designate them as the Phylum Anthophyta, and they are the flowering plants about which I speak to-day. So in clearing the way for this discussion, let me show the relationship of these three phyla of higher plants by means of an analytic key, as follows: A, Gametophyte generation larger, and longer-lived than the a sporo- phyte generation. Here are set off the liverworts and mosses. Gametophyte rag smaller and shorter-lived than the independent phyte generatio (a) Hire we set off ee up: in fier both Ss are mostly holo d independent of o nother, the megagametop ve still con- taining chlorophyll, Sind eben calamites, and ee w [vVor. 2 112 ANNALS OF THE MISSOURI BOTANICAL GARDEN ~ b) Gametophytes hys kR he dependent upon, and nourished by, the sporophytes, the megagametophyte not containing chlorophyll. (1) Megagametophyte a fully developed cellular mass before the forma- tion of the eggs; microgametophytes few-celled; antherids basicidal; sperms =». and motile; megasporophylls open, in simple spirals to simple robili; seeds fleshy; microsporophylls . multisporangiate; bundles ities heidal, in a small, ATEEN cylinder; pith and cortex large; stems simple; leaves ample, mostly bi doers persistent, vein | ey EL re err eee eee eer CYCAD PHYLUM (2) Megagametophyte a fully developed cellular mass before the forma- tion of the eggs; microgametophytes few (to one) -celled; antherid apicidal ; sperms non-ciliated and not visibly motile; megasporophylls ls open, in well- developed strobili; seeds not fleshy; microsporophylls with few (2-8) sporangia ; bundles tracheidal, in an enlarging cylinder; pith and cortex small; stems branched; leaves small, simple, persistent, veins parallel FRE CT eC CO EEC TEE ERECT CCT TE Cae reer ree CONIFER PHYLUM (3) Megagametophyte fully developed as a cellule ar mass (endosperm) only after the fertilization of the egg; microgametophytes one-celled; antherids apicidal; e non-ciliated d t visibly ile; mega- 3 arpels), in floral strobili (flowers), often much reduced; seeds not fleshy; microsporophylls (stamens) with four sporangia; bundles fibrovascular, in an enge va. eylinder; pith and cortex eg ht m © © 5 S gz) p" = © = © zn © fu T bundles scattered and stem non-enlarging) ; stems branched; leaves tly large, simple to compound, casita, to re veins net si d to parallel E S CCITT TEE ee Cee FLOWERING PLANT PH In the foregoing analysis, I have emphasized the similarities rather than the dissimilarities between the plants of these phyla, and such a statement will serve to show that they are related, and yet no one can compare them and not be forced to the conclusion that they must have diverged from one an- other at an early period in their evolution. And this diver- gence is to be interpreted as involving the eycad phylum as the primitive group from which have sprung the conifers on the one hand and the flowering plants on the other. Following the plan which I adopted in my earlier paper, I may here designate a number of generally accepted principles of classification as they apply to the flowering plants. While generally accepted, these principles have rarely if ever been formulated by taxonomists or others, so that as here formu- lated they may create some surprise and perhaps some oppo- sition. For the sake of brevity I give them in the form of dicta, as follows: A. GENERAL DICTA 1. Evolution is not always upward, but often it involves degradation and degeneration. 1 Loe. cit. 1915] 2. ~~ P qn > ~ QO Ne) Bá = eur rm en bo n Go pi TN BESSEY—PHYLOGENETIC TAXONOMY 113 In general, homogeneous structures (with many and similar parts) are lower, and heterogeneous structures (with fewer and dissimilar parts) are higher. Evolution does not necessarily involve all organs of the plant equally in any particular period, and one organ may be advancing while another is retrograding. Upward development is sometimes through an increase in complexity, and sometimes by a simplification of an organ or a set of organs. Evolution has generally been consistent, and when a par- ticular progression or retrogression has set in, it is per- sisted in to the end of the phylum. In any phylum the holophytic (chlorophyll-green) plants precede the colorless (hysterophytic) plants, and the latter are derived from the former. . Plant relationships are up and down the genetic lines, and these must constitute the framework of phylogenetic taxonomy. B. DICTA HAVING SPECIAL REFERENCE TO THE GENERAL STRUCTURE OF THE FLOWERING PLANTS . The stem structure with collateral vascular bundles ar- ranged in a cylinder is more primitive than that with scattered bundles, and the latter are to be regarded as derived from the former. . Woody stems (as of trees) are more primitive than her- baceous stems, and herbs are held to have been derived from trees. The simple, unbranched stem is an earlier type, from which branching stems have been derived. . Historically the arrangement of leaves in pairs on the stem is held to have preceded the spiral arrangement in which the leaves are solitary at the nodes. . Historically simple leaves preceded branched (‘‘com- pound’’) leaves. . Historically leaves were first persistent (‘‘evergreen’’) and later deciduous. . The reticulated venation of leaves is the normal structure, 114 fond er wur ~] = de) bo > bo rat i) bo bo Oo bo m 2 cn 2 D> 2 ~] [VoL, 2 ANNALS OF THE MISSOURI BOTANICAL GARDEN and the parallel venation of some leaves is a special modification derived from it. DICTA HAVING REFERENCE TO THE FLOWERS OF FLOWERING PLANTS The polymerous flower structure precedes, and the oligo- merous structure follows from it, and this is accom- panied by a progressive sterilization of sporophylis. . Petaly is the normal perianth structure, and apetaly is the result of perianth reduction (aphanisis). . The apochlamydeous perianth is earlier and the gamo- chlamydeous perianth is derived from it by a symphysis of the members of perianth whorls. . Acti phy is an earlier structure than zygomorphy, and the latter results from a change from a similar to a dissimilar growth of the members of the perianth whorls. . Hypogyny is the more primitive structure, and from it epigyny was derived later. Apocarpy is the primitive structure, and from it synearpy was derived later. . Polyearpy is the earlier condition, and oligocarpy was de- rived from it later. . The endospermous seed is primitive and lower, while the seed without endosperm is derived and higher. . Consequently, the seed with a small embryo (in endo- sperm) is more primitive than the seed with a large embryo (in scanty or no endosperm). In earlier (primitive) flowers there are many stamens (polystemonous) while in later flowers there are fewer stamens (oligostemonous). The stamens of primitive flowers are separate (apostem- onous), while those of derived flowers are often united (synstemonous). The condition of powdery pollen is more primitive than that with coherent or massed pollen. . Flowers with both stamens and carpels (monoclinous) pre- cede those in which these occur on separate flowers (diclinous). 1915] BESSEY—PHYLOGENETIC TAXONOMY 115 28. In diclinous plants the monoecious condition is the earlier, and the dioecious later. Let us now endeavor to apply these principles candidly in an attempt to secure a phyletic taxonomy of the flowering plants. As a consequence, we begin with the plants that are primi- tively opposite-leaved, as shown by their first leaves (‘‘cotyle- dons’’) that are always opposite. These are what we have known as dicotyledons. But this name, which was once sig- nificant, is no longer useful, and in fact has become somewhat misleading, so that I propose to substitute for it the name Oppositifoliae for the first class of the Anthophyta. Like- wise for the other class, hitherto known as the monocotyle- dons, in which the leaves are alternate from the first, and con- tinue so throughout the whole plant body, I propose the more appropriate name of Alternifoliae. In considering these two classes, it is quite evident that the first is not only the larger in the number of its species, but also that it includes many more important modifications of struc- ture than does the other. Yet there is much similarity in the kinds of modification of structure in the two classes, the larger class, from its very largeness, including many more details of modification and variation. In both classes we begin with apocarpous plants, and pro- ceed toward those that are syncarpous. So the Ranales on the one hand, and the Alismatales on the other, are near the point of beginning. In one class syncarpy is attained after the passing of a few hundred species (Alismatales, 409 species), while in the other it is not reached until much beyond the limits of the order Ranales, for it is well known that the synearpy of many Malvales and Geraniales is distinctly in- complete, the coherence between the carpels being so feeble that they readily separate at maturity. All told, fully 10,000 species of this class are passed before complete syncarpy is attained. The strobiloid flower structure, in which the axis is elong- ated, cylindrical, spheroidal, or flattened, bearing on its sur- [vor. 2 116 ANNALS OF THE MISSOURI BOTANICAL GARDEN face the fertile and sterile sporophylls, prevails in the earlier orders of both classes, in the smaller, continuing through the Alismatales, Liliales, Arales, Palmales, and Graminales, and aggregating more than 11,700 species. In the larger class the strobiloid structure prevails throughout fourteen orders, from the Ranales to the Lamiales, and aggregating more than 53,000 species. In these strobiloid flowers, as a result of the domi- nance of the strobilar structure, we have what has been known as the hypogynous form of flower. In both classes the strobi- loid flowers show progressive modifications involving the perianth (actinomorphy to zygomorphy, diplochlamydy to achlamydy), the stamens (polystemony to oligostemony), the carpels (polycarpy to oligocarpy), the ovules (multiovulate to rariovulate). In the larger class the perianth modifications proceed with such regularity that we may recognize lower (apopetalous), and higher (sympetalous) groups of orders, but this is not observed in the smaller class, where indeed sympetaly is never more than sporadic, and does not become a fixed structure. In summary fashion I may now outline the taxonomy of the flowering plants: The opposite-leaved class (Oppositifoliae, or dicotyledons) is the first to emerge from the eycadean phylum, appearing as the ranalean complex. From this Ranalean type arises the alternate-leaved class of flowering plants (Alternifoliae, or monocotyledons) as apo- carpous Alismatales, and these soon merge into the syncarpous Liliales, which are successively more and more modified in the Arales, Palmales and Graminales. From Liliales by a cotyloid modification the mostly actinomorphie epigynous Iridales are derived, and from these again the zygomorphic epigynous Orchidales. Returning to the Ranales, we find that they give rise first to five apopetalous, polycarpellate orders with gradually in- creasing syncarpy, namely Malvales, Geraniales, Guttiferales, Rhoeadales, and Caryophyllales. From the last arise three orders of sympetalous, polycarpellate plants, the Ebenales, Ericales and Primulales, and the latter have developed the 1915] BESSEY— PHYLOGENETIC TAXONOMY 117 dicarpellate orders Gentianales, Polemoniales, Scrophulariales and Lamiales, constituting a series which shows diminishing numbers of stamens, carpels and seeds, and increasing zygomorphy. This phyletic sequence from Ranales to Lami- ales constitutes the sub-class Strobiloideae, or cone-flowers. Returning again to the Ranales, we find that they give rise to the simpler, cotyloid, apopetalous, polystemonous, poly- carpous, hypogynous Rosales (sub-class Cotyloideae), from which by the early deepening of the cotyloid structure we have the mostly polystemonous, polycarpous, epigynous Myrtales, Loasales and Cactales as a strongly developed side line. The oligostemonous Celastrales continue the main phyletic line with reducing numbers of stamens, carpels and seeds, and a gradual deepening of the cup, to the side-line of the Sapin- dales, which are eventually epigynous, and the mostly dicar- pellate Umbellales. The sympetalous, epigynous Rubiales with reduced calyx, few carpels and few seeds, pass easily into the Campanulales, and the Asterales, the latter with but one seed in the dicarpellary, one-celled, one-seeded, inferior ovary, and with its calyx, when not obsolete, transformed into bracts, spines or bristles to form a ‘‘pappus’’ for the efficient distribu- tion of the seeds. II. Taxonomy or FLOwERING PLANTS Phylum XIV. ANTHOPHYTA. The Flowering Plants. Typically chlorophyll-green plants (a few colorless hystero- phytes), ranging from small or even minute plants to great trees a hundred or more meters in height; alternation of gen- erations obscured by the extreme reduction of the gameto- phyte to a condition of dependence upon the long-lived, leafy- stemmed sporophyte. Spores of two kinds (heterosporous), produced on sporophylls which are borne in modified, often much reduced strobili (flowers); microsporophylls (stamens) normally with four sporangia (pollen sacs); the microspores being set free (as ‘‘pollen’’) when mature; megasporophylls (carpels) folded lengthwise (constituting the ‘‘pistil’’) en- closing the sporangia (ovules) in which the megaspores [VoL, 2 118 ANNALS OF THE MISSOURI BOTANICAL GARDEN remain and develop the minute gametophyte; archegones very much reduced, including little more than the egg, which is SCROPHULARIALES xb / 4 £K ig. 1. Chart to show relationship of the orders. Relationship is indicated by position; the areas are approximately proportional to the number of species in the orders. fecundated by the non-ciliated sperms (male nuclei) from the tubular antherids, resulting in the formation of an embryo 1915] BESSEY—PHYLOGENETIC TAXONOMY 119 sporophyte; megasporangia surrounded by one or two en- veloping indusial coats (seed coats); mature seed with or without endosperm (gametophyte tissue). The flowering plants are here held to have sprung from cycadean strobiliferous ancestors, probably of the general type of the Bennettitineae, and as a consequence those antho- phyta are considered to be primitive in which the sporophylls are many and distinct. Symphylly and syncarpy are later structural conditions than apophylly and apocarpy. So also, fewer sporophylls in the anthostrobilus is a later condition derived from the earlier polyphyllous structure. The sym- physis of sporophylls is a mode of evolution, and so is their aphanisis. Ä The plants constituting this phylum are those commonly termed angiosperms, in contrast with the gymnosperms, in- cluding the cycads (Cycadophyta) and conifers (Strobilo- phyta). It appears to the writer, however, that these are more properly three pretty distinct phyla, and that the rela- tionship of the gymnosperms to the angiosperms is so remote that the treatment here given them is more nearly in accord- ance with what is known as to their phylogeny. There are two classes, Alternifoliae (monocotyledons) and Oppositifoliae (dicotyledons), of which the second was quite certainly the earlier, as it is now much the larger numerically. Indeed, it is becoming more probable that the monocotyledons are to be regarded as a peculiar side branch which sprang from the primitive dicotyledons after the latter had become well established. Yet the monocotyledons have not developed to as high a rank in any of their orders as have some of the dicotyledons. Although I have here changed the technical names of these two classes, there is no objection to the retention of the old terms for the English names in popular usage: accordingly on the following pages I shall frequently make such use of the old names. Class 32. ALTERNIFOLIAE (MONOCOTYLEDON- EAE). The Monocotyledons. Leaves of young sporophyte [vor. 2 120 ANNALS OF THE MISSOURI BOTANICAL GARDEN alternate; leaves of mature sporophyte alternate, and usually parallel-veined; fibro-vascular bundles of the stem scattered, usually not arranged in rings. (Species about 23,700.) Sub-Class ALTERNIFOLIAE-STROBILOIDEAE. Axis of the flower from spheroidal to flattened, bearing on its sur- face the hypogynous perianth and stamens (or the stamens may be attached to the perianth), and the many or few, su- perior, separate or united carpels. Order ALISMATALES. Carpels separate, superior to all other parts of the flower; endosperm scanty or none (species about 409). Related to and probably derived from the Ranales of the dicotyledons. Family 1. Alismataceae. Water Plantains. Aquatic or paludose herbs with mostly radical, often large leaves; flowers small to large; perianth in two whorls of three leaves each (calyx and corolla); placenta sutural; ovules mostly solitary. Alisma, Sagittaria. (Pf. 21: 227.)! Family 2. Butomaceae. Aquatic or paludose herbs, bear- ing narrow or broad leaves, with convergent veins; flowers large; perianth in two whorls, of three leaves each (calyx and corolla); placenta parietal; ovules many. Butomus, Lim- nocharis. (Pf. 21: 232.) Family 3. Triuridaceae. Very small, pale, leafless ea growing in wet places in tropical countries. Triuris. (Pf. 235.) Family 4. Scheuchzeriaceae. Aquatic or paludose herbs with rush-like leaves, and small flowers, with a two-whorled perianth, each 4-6-parted. Triglochin, Scheuchzeria. (Pf. 21 ; 222.) Family 5. Typhaceae. Cat-tails. Aquatic or paludose herbs, with linear, sheathing leaves and eylindrical-erowded flowers; pistil 1-celled; ovule 1. Typha. (Pf. 2': 183.) Family 6. Sparganiaceae. Aquatic or paludose plants with creeping rootstocks and erect stems, bearing linear 1The abbreviation “Pf.” has reference to Engler and Prantl’s ‘Natiirlichen Pflanzenfamilien,’ and the bold face, exponent, and Roman figures following refer respectively to “Abteilung,” “Teil,” and page of this publication. 1915] BESSEY—PHYLOGENETIC TAXONOMY 121 leaves; flowers monoecious in dense globose heads. Spar- ganium. (Pf. 21:192.) Family 7. Pandanaceae. Screw-pines. Shrubs or trees with spirally crowded, narrow, stiff leaves on the ends of the branches; pistil 1-celled; ovules one or many. Pandanus. (Pf. 21: 186.) Family 8. Aponogetonaceae. Aquatic plants with petioled, oblong, translucent leaves, with convergent veins; flowers small, spicate. Aponogeton. (Pf. 21:218.) Family 9. Potamogetonaceae. River-weeds. Aquatic or paludose herbs with mostly alternate stem-leaves; flowers mostly small and inconspicuous; perianth none, or of 1-6 leaves in 1 or 2 whorls. Potamogeton, Zostera, Zannichellia. (Pf. 21.194.) Order Littates. Carpels united (usually 3), forming a com- pound pistil, superior; perianth (usually of 6 parts) in two similar whorls, delicate and corolla-like; endosperm copious. (Species about 3370.) Family 10. Liliaceae. The Lilies. Pistil mostly 3-celled; stamens 6; perianth of two similar whorls, each of three sim- ilar leaves. Lilium, Erythronium, Tulipa, Yucca, Asparagus, Allium. (Pf. 25:10.) Family 11. Stemonaceae. Pistil 1-celled; stamens 4; peri- anth of two similar whorls, each of two similar leaves. Stem- ona, Croomia. (Pf. 2°:8.) Family 12. Pontederiaceae. Aquatic herbs with 3 or 1- celled pistil; stamens 6 or 3; perianth of two similar whorls, each of three similar or dissimilar leaves. Pontederia, Heter- anthera. (Pf. 24:70.) Family 13. Cyanastraceae. Tropical African rhizomatous plants. Cyanastrum. (Syllabus, 141.)! Family 14. Philydraceae. Pistil 3-celled; stamen 1; peri- anth of two similar whorls, each of two dissimilar leaves. Philydrium. (Pf. 24:75.) + “Syllabus” has reference to Engler and Gilg’s ‘Syllabus der Pflanzenfamilien,’ and the numbers following refer to pages of this publication, [vor. 2 122 ANNALS OF THE MISSOURI BOTANICAL GARDEN Family 15. Commelinaceae. Spiderworts. Succulent herbs with 3 or 2-celled pistil; stamens 6; perianth of two dissimilar whorls of three similar leaves. Commelina, Tradescantia. (Pf. 24: 60.) Family 16. Xyridaceae. Rush-like plants with a 1-celled or incompletely 3-celled pistil; stamens 3; perianth of two dissimilar whorls, each of three similar leaves. Xyris. (Pf. 21:18.) Family 17. Mayacaceae. Slender, creeping, moss-like plants with 1-celled pistil; stamens 3; perianth of two dissimilar whorls, each of three similar leaves. Mayaca. (Pf. 2*: 16.) Family 18. Juncaceae. Rushes. Herbs with narrow leaves; pistil 1-3-celled; ovules solitary or many; fruit a dry 3-valved pod. Juncus, Luzula. (Pf. 2°:1.) Family 19. Eriocaulonaceae. Rush-like herbs with flowers in close heads; perianth segments 6 or less, small; pistil 3 or 2-celled; ovules orthotropous, pendulous. Eriocaulon. (Pf. 24: 21.) Family 20. Thurniaceae. South American herbs, with small, 1-nerved leaves, and small axillary flowers. Thurnia. (Syllabus, 139.) Family 21. Rapateaceae. Tall, sedge-like marsh herbs with 3-celled pistil; stamens 6, in pairs; perianth of two dis- similar whorls, each of three similar leaves. Rapatea. (Pf. 21.28.) Family 22. Naiadaceae. Slender, branching, wholly sub- merged aquatics, with sheathing, mostly opposite leaves, and monoecious or dioecious flowers. Naias. (Pf. 21: 214.) Order Arates. Compound pistil, mostly tricarpellary, su- perior; ovules one or more; perianth reduced to scales or entirely wanting; endosperm usually present. (Species about 1052.) Family 23. Cyclanthaceae. Mostly herbaceous plants with broad, petioled leaves having parallel venation; pistil 1-celled ; ovules many, on four parietal placentae. Cyclanthus. (Pf. 23:93.) 1915] BESSEY—PHYLOGENETIC TAXONOMY 123 Family 24. Araceae. Arums. Mostly herbaceous plants with broad, petioled leaves, having reticulate venation; pistil 1-4-celled; ovules 1 or more. Anthurium, Acorus, Monstera, Symplocarpus, Calla, Philodendron, Calocasia, Caladium, Arum, Arisaema. (Pf. 2°: 102.) Family 25. Lemnaceae. Duckweeds. Very small, floating, aquatic herbs; pistil 1-celled; ovules 1 or more. Lemna, Spiro- dela. (Pf. 23: 154.) Order Patmates. Compound pistil mostly tricarpellary, superior; ovule solitary; perianth reduced to rigid or herbace- ous scales; endosperm copious. (Species about 1085.) Family 26. Palmaceae. Palms. Trees or shrubs with pin- nate or palmate leaves; pistil 1-3-celled; fruit a 1-seeded berry or drupe (rarely 2-3-seeded). Phoenix, Chamaerops, Calamus, Oreodoxa, Cocos. (Pf. 23:1.) Order GRAMINALES. Compound pistil reduced to 2 or 3 car- pels; ovule solitary; perianth reduced to small scales or en- tirely wanting; endosperm copious. (Species about 5795.) Family 27. Restionaceae. Rush-like herbs or undershrubs, with spiked, racemed, or panicled mostly diclinous flowers; perianth segments 6 or less, chaffy; pistil 1-3-celled; ovules orthotropous, pendulous. Restio. (Pf. 24: 3.) Family 28. Centrolepidiaceae. Small rush-like herbs with mostly monoclinous flowers in spikes or heads; perianth none; pistil 1-several-celled; ovules orthotropous, pendulous. Cen- trolepis. (Pf. 24:11.) Family 29. Flagellariaceae. Erect or climbing herbs with long narrow leaves, and panicled flowers; pistil 3-celled; ovules solitary, anatropous, ascending; fruit a 1-2-seeded berry. Flagellaria. (Pf. 24:1.) Family 30. Cyperaceae. Sedges. Grass-like herbs with 3-ranked leaves; perianth segments bristly or none; pistil 1-celled; ovules anatropous, erect. Cyperus, Scirpus, Fim- bristylis, Rhynchospora, Carex. (Species 1959.) (Pf. 22:98.) Family 31. Poaceae. Grasses. Mostly erect herbs with hollow, jointed stems, and 2-ranked leaves; perianth segments [voL. 2 124 ANNALS OF THE MISSOURI BOTANICAL GARDEN of 2-6 scales or vestiges; pistil 1-celled; ovules anatropous, ascending. Bambusa, Bromus, Triticum, Bouteloua, Avena, Agrostis, Phalaris, Oryza, Panicum, Andropogon, Zea. (Species 3545.) (Pf. 22:1.) In the Poaceae the hypogynous, tricarpellary monocotyle- dons reach their culmination, as a highly specialized side line. In grasses the specialization involves plant-body, inflorescence, and flowers. Their nodose, mostly hollow, elongated stems, and long, narrow, tough leaves; the spreading paniculate ar- rangement of their spikelets; and their 1-celled, tricarpellary 1-ovuled pistils, producing caryopsis-fruits, are some of the more obvious indications of high specialization, suggesting the possibility that these plants, rather than the orchids, are the highest of the monocotyledons. With the Poaceae the hypo- gynous monocotyledonous phylum ends. Grasses have not given rise to other groups of plants. Sub-Class ALTERNIFOLIAE -COTYLOIDEAE. Axis of the flower normally expanded into a cup, bearing on its margin the perianth and stamens (or the latter may be at- tached to the perianth). The carpels are thus inferior. Flow- ers from actinomorphic to zygomorphic. Order Hyprauses. Flowers diclinous; compound tricarpel- lary pistil inferior to all other parts of the flower; perianth segments in each whorl alike in shape (flower regular) ; seeds without endosperm. (Species about 53.) Family 32. Vallisneriaceae. Tape-grasses. Small aquatic herbs mostly inhabiting the fresh waters of temperate cli- mates. Vallisneria, Hydrocharis, Philotria. (Pf. 2! : 238.) Order Inmates. Compound tricarpellary pistil inferior; flower-leaves in each whorl mostly alike in shape (flower reg- ular, actinomorphic) ; seeds with endosperm. (Species about 4419.) Family 33. Amaryllidaceae. Amaryllises. Leaves nar- row, or the blade broad, with longitudinal veins; pistil 3-celled; ovules many; stamens 6 or 3. Amaryllis, Crinum, Narcissus, Agave, Hypoxis. (Pf. 25:97.) 1915] BESSEY— PHYLOGENETIC TAXONOMY 125 Family 34. Haemodoraceae. Leaves sword-shaped; pistil 3-celled; ovules 1 to many; stamens 6. Haemodorum. (Pf. 2° :92,) Family 35. Iridaceae. Leaves sword-shaped; pistil 3- celled; ovules many; stamens 3. Crocus, Iris, Tigridia, Sisy- rinchium, Ixia, Tritonia, Gladiolus, Freesia. (Pf. 2°: 137.) Family 36. Velloziaceae. Woody-stemmed, leafy plants, with a 3-celled pistil containing many ovules, stamens 6 or more. Vellozia. (Pf. 25:125.) Family 37. Taccaceae. Stemless herbs, with broad pin- nately parallel-veined leaves; pistil 1-celled; ovules many; stamens 6. Tacca. (Pf. 2°: 127.) Family 38. Dioscoreaceae. Yams. Mostly twining herbs, with broad, petioled, longitudinally-veined leaves; pistil 3- celled; ovules 2 in each cell; stamens 6. Dioscorea, Testudin- arta. (Pf. 2°:.130.) Family 39. Bromeliaceae. Pineapples. Leaves mostly rosulate; external perianth whorl calycine; pistil 3-celled; ovules many; stamens 6. Tillandsia, Dendropogon, Ananas. (Pf. 2*. 32.) Family 40. Musaceae. Bananas. Large herbs, the stem often composed of the sheathing leaf-bases; perianth petaloid of 6, often dissimilar segments; stamens 6; pistil 3-celled; ovules 1 to very many. Strelitzia, Musa. (Pf. 2°: 1.) Family 41. Zingiberaceae. Gingers. Perennial, medium- sized herbs, with creeping or tuberous rootstocks; perianth irregular; stamen 1, anther 2-celled, with several ‘‘stamin- odes’’; pistil 3-celled; ovules 1 or more in each cell. Curcuma, Zingiber, Amomum. (Pf. 2°:10.) Family 42. Cannaceae. Cannas. Perennial herbs of medium size, with simple pinnately-veined leaves; perianth irregular; stamen 1, anther 1-celled, with several ‘‘stamin- odes’’; pistil 3-celled; ovules 1 to many. Canna. (Pf. 2°: 30.) Family 43. Marantaceae. Perennial herbs of variable habit; leaves parallel or pinnately veined; perianth irregular ; functional stamen 1, with several ‘‘staminodes’’; pistil 3- [Vou. 2 126 ANNALS OF THE MISSOURI BOTANICAL GARDEN celled; ovules 1 in each cell. Calathea, Maranta. (Pf. 2°: 33.) Order OrcuimwaLes. Compound tricarpellary pistil inferior; flower-leaves in each whorl mostly unlike in shape (flower irregular, zygomorphic); seeds numerous, minute, without endosperm. (Species about 7578.) Family 44. Burmanniaceae. Flowers irregular; stamens 3 or 6. Burmannia. (Pf. 2°: 44.) Family 45. Orchidaceae. Orchids. Flowers irregular; stamens 1 or 2. Cypripedium, Orchis, Platanthera, Vanilla, Spiranthes, Epidendrum, Dendrobium, Oncidium. (Species 7521.) (Pf. 2%: 52.) In the Orchidales, and especially in the Orchidaceae, we have what is generally regarded as the highest development of monocotyledonous plants, and yet it must be acknowledged that many of their most striking flower structures are rather easily made entomophilous modifications of the perianth, the most mobile portion of the plant. In many ways the ‘‘grassy’’ plants (especially the Poaceae) show greater and more pro- found structural modifications than do the much more con- spicuous orchids. With the orchids the epigynous monocoty- ledonous phylum ends. Class 33. OPPOSITIFOLIAE (DICOTYLEDONEAE). The Dicotyledons. Leaves of young sporophyte opposite; leaves of mature sporophyte opposite or alternate, usually reticulate-veined; fibrovascular bundles of the stem in one or more cylindrical layers. (Species about 108,800.) As indicated above the dicotyledons are here considered to have had their beginning earlier than the monocotyledons, which must be regarded as having diverged very early from the primitive dicotyledons, and developed into a relatively small lateral branch. The point of divergence of the mono- cotyledons from the dicotyledons must have been in the order Ranales, probably in the neighborhood of the Ranunculaceae. It is not probable that the early (woody) magnoliads or ano- nads gave rise to the monocotyledonous divergence; it is much more probable that this modification arose after the reduction had taken place from the ligneous to the herbaceous Ranales. 1915] BESSEY— PHYLOGENETIC TAXONOMY 127 Here we have a possible explanation of the marked herbaceous- ness of monocotyledons as contrasted with the general ten- dency toward a more ligneous structure in dicotyledons. Sub - Class OPPOSITIFOLIAE - STROBILOIDEAE. ‘‘Cone flowers.’? Axis of the flower normally cylindrical, spherical, hemispherical or flattened, bearing on its surface the hypogynous perianth, stamens and pistils (or the stamens may be attached to the corolla). Super - Order STROBILOIDEAE-APOPETALAE-POLYOARPELLATAE. Carpels typically many, separate or united; petals separate. Flowers mostly actinomorphic. This super-order has much in common with the Alismatales, and also with the Cotyloideae- Apopetalae. In fact, these three groups appear to have diverged from a common point of origin. Order Ranates. All parts of the flower mostly spirally arranged (acyclic), free (not united) ; carpels typically many, separate (or rarely united), rarely reduced to 1; stamens gen- erally indefinite; embryo mostly small, in copious endosperm. (Species about 5551.) The twenty-four families here included in the order Ranales naturally group themselves about three centers, the magnolias (Magnoliaceae), the anonas (Anonaceae), and the buttercups (Ranunculaceae). The plants in these centers are typically diplochlamydeous, polycarpellate, hermaphrodite, and actino- morphic, and the modifications in the surrounding families have been such as to result in an achlamydeous structure, which may be monocarpellate, diclinous, and even zygomorphie. Ranalean evolution has thus been one of more and more marked simplification of flower structure. It is interesting to observe that while the families of Ranales have thus been evolved, the order has given rise to no less than five phyletic groups of full ordinal rank. One of these (Malvales) has produced a further modification (Sarracent- ales), for three of them the evolutionary development came to a stand-still with the ordinal limits (Geraniales, Guttiferales and Rhoedales), while the virile Caryophyllales continued a development beyond its ordinal limits into the Ebenales, Eri- [vor. 2 128 ANNALS OF THE MISSOURI BOTANICAL GARDEN cales and Primulales, and through the latter into Gentianales, Polemoniales and Scrophulariales to the end of this phyletic line in the Lamiales. Family 46. Magnoliaceae. Magnolias. Petals present, usually many; receptacle usually elongated; shrubs and trees with alternate leaves and usually large flowers. Magnolia, Liriodendron. (Pf. 37: 12.) Family 47. Calycanthaceae. Petals present, usually many; seeds without endosperm; shrubs with opposite leaves. Caly- canthus. (Pf. 37:92.) Family 48. Monimiaceae. Petals absent; carpels many, 1-ovuled, embedded in the receptacle; trees and shrubs with opposite or whorled leaves, and diclinous flowers. Kibara, Monimia, Siparuna. (Pf. 37: 94.) Family 49. Cercidiphyllaceae. Trees with naked dioe- cious flowers, many stamens, and a single whorl of 2-5 free carpels. Cercidiphyllum. (Pf. 3?: 21.) Family 50. Trochodendraceae. Trees and shrubs with naked flowers, many stamens, and a single whorl of 5 to many partly connate carpels. Trochodendron. (Pf. 32:21.) Family 51. Leitneriaceae. Shrubs with alternate leaves and dioecious flowers in catkins; perianth minute or 0; pistil 1-celled, 1-ovuled; endosperm minute. Leitneria. (Pf. 3': 28.) Family 52. Anonaceae. Papaws. Petals present, in two whorls of 3 each; stamens and carpels many; endosperm ruminated; trees or shrubs with alternate leaves. Asimina, Anona. (Pf. 3°: 23.) Family 53. Lactoridaceae. Much-branched shrubs of the South Pacific Islands, with alternate leaves, and apetalous flowers. Lactoris. (Pf. 37:19.) Family 54. Gomortegaceae. Large trees of South America, with opposite evergreen leaves, and acyclic flowers; carpels 2-3, each with 1 ovule. Gomortega. (Pf. Nachträge zu Teil u-v, 172.) Family 55. Myristicaceae. Nutmegs. Sepals 3; petals absent; pistil 1 (or a second rudiment), 1-seeded; endosperm 1915] ‘ BESSEY— PHYLOGENETIC TAXONOMY 129 ruminated; trees or shrubs with alternate leaves and small, inconspicuous, dioecious flowers. Myristica. (Pf. 37:40.) Family 56. Saururaceae. Rhizomatous marsh herbs, with alternate leaves; flowers perfect, small, spicate; perianth 0; carpels 3-4, more or less united. Saururus. (Pf. 31:1.) Family 57. Piperaceae. Peppers. Herbs, shrubs, and trees with alternate (or opposite) leaves; flowers perfect or diclinous, mostly spicate; perianth 0; pistil 1-celled, 1-ovuled; endosperm present. Piper, Macropiper. (Pf. 31:3.) Family 58. Lacistemaceae. Tropical American shrubs and trees with alternate leaves, and perfect flowers; perianth mostly 0; stamen 1; pistil 3 or 2-carpellary. Lacistema. (Pf. 31:14.) Family 59. Chloranthaceae. No perianth whatever; pistil 1, with 1 ovule; mostly tropical trees and shrubs, with opposite leaves, and small flowers. Chloranthus. (Pf. 31:12.) Family 60. Ranunculaceae. Buttercups. Petals present in one whorl, or absent; sepals mostly deciduous; stamens and carpels indefinite, the latter usually separate; mostly herbs with alternate leaves. Myosurus, Ranunculus, Anemone, Cle- matis. (Pf. 37:43.) Family 61. Lardizabalaceae. Petals and sepals 6 each; stamens 6; twining or erect shrubs, with alternate leaves. Akebia, Lardizabala. (Pf. 3°: 67.) Family 62. Berberidaceae. Barberries. Petals usually present, in 1-3 whorls; stamens few; carpel 1 (rarely more), with many ovules; mostly shrubs with alternate leaves and perfect flowers. Podophyllum, Berberis. (Pf. 3°: 70.) Family 63. Menispermaceae. Moonseeds. Petals present, in 2 whorls; carpels 3 or more; twining shrubs with alternate leaves and small dioecious flowers. Menispermum, Cocculus. (Pf. 32: 78.) Family 64. Lauraceae. Laurels. Aromatic trees and shrubs with alternate simple leaves and small flowers; petals 0; carpel 1; ovule 1, pendulous; endosperm 0. Cimnamomum, Persea, Ocotea, Umbellularia, Sassafras, Litsea, Laurus. (Pf. 32: 106.) [vor. 2 130 ANNALS OF THE MISSOURI BOTANICAL GARDEN Family 65. Nelumbaceae. Lotuses. Large aquatic herbs with peltate leaves, large acyclic flowers, with many stamens, and many separate carpels, the latter immersed in the flattish axis (‘‘receptacle’’); seeds 1 or 2, endosperm 0. Nelumbo. (PE 3? +1.) Family 66. Cabombaceae. Water-shields. Small aquatic herbs with floating, sometimes peltate leaves, and few to many stamens, and separate carpels (not immersed) ; seeds 2 or 3; endosperm present. Cabomba, Brasenia. (Pf. 3°: 2.) Family 67. Ceratophyllaceae. Aquatic herbs with verti- cillate, divided leaves; flowers diclinous; perianth 0; stamens 12-16; carpel 1, 1-ovuled; endosperm scanty. Ceratophyllum. (Pf. 37:10.) Family 68. Dilleniaceae. Petals present, in one whorl; sepals persistent; stamens numerous, indefinite; carpels from many to 1, with 1 or more seeds; endosperm copious; mostly shrubs and trees with alternate leaves, and perfect flowers. Dillenia, Actinidia. (Pf. 3°: 100.) Family 69. Winteranaceae. Aromatic trees with alternate leaves; flowers perfect; sepals 4-5; petals 4-5 (or 0) ; stamens 20-30; pistil 2-5-carpellary, with as many parietal placentae; endosperm copious. Winterana, Cinnamodendron. (Pf. 3°: 314.) Order Matvatss. Pistil usually of 3 to many weakly united carpels, with as many cells (sometimes greatly reduced) ; ovules mostly few; stamens indefinite, monadelphous, branched, or by reduction separate and few; endosperm present or ab- sent. (Species about 3829.) Family 70. Sterculiaceae. Trees and shrubs with alternate leaves; flowers perfect or diclinous, with or without petals; stamens monadelphous or polyadelphous, 2-celled; pistil 4-many-celled; endosperm present or 0. Theobroma, Ster- culia. (Pf. 3°: 69.) Family 71. Malvaceae. Mallows. Herbs, shrubs, and trees with alternate leaves; flowers perfect, with petals; stamens monadelphous, 1-celled; pistil 5-many-celled ; endosperm little 1915] BESSEY— PHYLOGENETIC TAXONOMY 131 or 0. Abutilon, Althaea, Malva, Hibiscus, Gossypium. (Pf. 3°: 30.) Family 72. Bombacaceae. Tropical trees with alternate, palmate leaves; sepals and petals present; staminal column 5-8-cleft. Adansonia, Bombax. (Pf. 3°: 53.) Family 73. Scytopetalaceae. Trees of the southern hemi- sphere, with alternate leathery leaves; sepals small; petals much larger, valvate; stamens many. Scytopetalum. (Pf. Nachträge zu Teil m-1v, 242.) Family 74. Chlaenaceae. Madagascar trees and shrubs with alternate leaves; inflorescence dichotomous; petals con- torted. Rhodochlaena, Leptochlaena. (Pf. 3°: 168.) Family 75. Gonystylaceae. East Indian trees with leath- ery, evergreen leaves, pentamerous flowers, and a berry-like fruit. Gonystylus. (Pf. Nachträge zu Teil ı-ıv, 231.) Family 76. Tiliaceae. Lindens. Trees, shrubs (and herbs) with mostly alternate leaves; flowers mostly perfect, with petals; stamens free, 2-celled; pistil 2-10-celled; endosperm present or 0. Corchorus, Tilia, Grewia. . (Pf. 3°: 8.) Family 77. Elaeocarpaceae. Tropical trees and shrubs, with alternate or opposite simple leaves; sepals and petals present; stamens distinct, many; pistil of 2-several carpels. Elaeocarpus, Aristotelia. (Pf. 3°: 1.) Family 78. Balanopsidaceae. Australian trees and shrubs with alternate leaves; flowers dioecious, apetalous, the stam- inate in catkins, the pistillate solitary, producing acorn-like, 2-celled, 2-seeded fruits; seeds endospermous. This family is doubtfully given place here, and it may be that it should be placed near the Fagaceae, as is done by Baillon. Balanops. (Pf. Nachträge zu Teil ı1-ıv, 114.) Family 79. Ulmaceae. Elms. Trees and shrubs with al- ternate, simple leaves, small apetalous flowers, a 1-celled (rarely 2-celled) ovary, which develops into a samara, drupe or nut. Ulmus, Celtis, Zelkova, Planera. (Pf. 31:59.) Family 80. Moraceae. Figs. Trees, shrubs, and herbs, mostly with a milky juice, and alternate or opposite leaves; [voL, 2 132 ANNALS OF THE MISSOURI BOTANICAL GARDEN flowers apetalous, diclinous (monoecious or dioecious) ; ovary 1-celled, 1-ovuled. Morus, Toxylon (Maclura), Broussonetia, Dorstenia, Artocarpus, Castilloa, Antiaris, Ficus, Humulus, Cannabis. (Pf. 3': 66.) Family 81. Urticaceae. Nettles. Herbs, shrubs, and trees with alternate or opposite leaves; flowers mostly diclinous, apetalous; stamens few, 2-celled; pistil monocarpellary, 1- celled, mostly 1-seeded; endosperm none. Urtica, Boehmeria. (Pf. 31: 98.) Order SArRRACENIALES. Pistil of 3-5 carpels united; pla- centae parietal or central; seeds small, numerous, endosperm- ous; herbs with ‘‘insectivorous’’ leaves; related to the mal- lows, with which they should possibly be included. (Species about 66.) Family 82. Sarraceniaceae. Pitcher-plants. Herbs with pitcher-shaped leaves, and perfect flowers; sepals 4-5; petals 5, rarely 0; stamens indefinite; pistil 3-5-carpellary. Sarra- cenia, Darlingtonia. (Pf. 3*: 244.) Family 83. Nepenthaceae, Pitcher-plants. Tropical un- dershrubs with pitcher-shaped leaves and dioecious flowers; sepals 4 or 3; petals 0; stamens 4-16; pistil 4~3-carpellary. Nepenthes. (Pf. 3°: 253.) Order GERANIALES. Pistil of several (5-2) mostly weakly united carpels; ovules 1-2 (or many), mostly pendulous, at- tached at the inner angle of the carpel. (Species about 9268.) Family 84. Geraniaceae. Geraniums. Herbs, shrubs, and trees, with opposite or alternate (compound or simple) leaves; torus elongated; stamens 10; pistil mostly 5-celled; ovules few; endosperm sparse or 0. Geranium, Pelargonium, Erodium. (PE 3*1) Family 85. Oxalidaceae. Sorrels. Herbs, rarely shrubs or trees, the juice sour; leaves mostly 3 or more foliate; flowers pentamerous, regular; stamens 10; ovules many; endosperm fleshy. Oxalis. (Pf. 34:15.) Family 86. Tropaeolaceae. Nasturtiums. Succulent, prostrate or climbing herbs, with alternate, peltate leaves, and 1915] BESSEY—PHYLOGENETIC TAXONOMY 133 irregular, long-peduncled, spurred flowers; stamens 8; ovary tricarpellary; ovules solitary; endosperm 0. Tropaeolum. (Pf. #223.) Family 87. Balsaminaceae. Touch-me-nots. Succulent herbs, mostly erect, with opposite or alternate leaves, and ir- regular, spurred axillary flowers; stamens 5; ovary penta- carpellary, ovules many; endosperm 0. Impatiens. (Pf. 35.383.) Family 88. Limnanthaceae. Succulent marsh herbs, with alternate, pinnate leaves; flowers pentamerous; stamens 10; carpels 5; endosperm 0. Limnanthes. (Pf. 3°: 136.) Family 89. Linaceae. Flaxes. Herbs and shrubs, with al- ternate simple leaves; pistil 3-5-celled; endosperm fleshy (or rarely 0). Linum. (Pf. 34: 27.) Family 90. Humiriaceae. Trees with alternate simple leaves; pistil 5-7-celled; endosperm copious. Humiria, Sac- coglottis. (Pf. 34:35.) Family 91. Erythroxylaceae. Shrubs and trees, with mostly alternate, simple leaves; flowers pentamerous; stamens 10; ovary 3-4-carpellary; fruit a drupe; endosperm fleshy. Erythroxylon. (Pf. 3*: 37.) Family 92. Zygophyllaceae. Herbs and shrubs with usually opposite, compound leaves; pistil lobed, 4~5-celled ; endosperm copious (or rarely 0). Zygophyllum, Guaiacum, Larrea. (Pf. 34:74.) Family 93. Cneoraceae. Shrubs with alternate entire leaves, trimerous or tetramerous flowers; pistil 3 or 4-celled, each cell with one ovule; endosperm fleshy. Cneorum. (Pf. 34 : 93.) Family 94. Rutaceae. Oranges. Herbs, shrubs, and trees with glandular-dotted, opposite, simple, or compound leaves; pistil lobed, 4-5-celled; endosperm fleshy or 0. Xanthoxylum, Ruta, Dictamnus, Ptelea, Limonia, Citrus. (Pf. 34:95.) Family 95. Simarubaceae. Trees and shrubs with gener- ally alternate, non-glandular, simple, or compound leaves; pistil lobed, 1-5-celled; endosperm fleshy or 0. Simaruba, Quassia, Holacantha, Ailanthus. (Pf. 3*: 202.) [voL. 2 134 ANNALS OF THE MISSOURI BOTANICAL GARDEN Family 96. Burseraceae. Balsamic trees and shrubs with alternate compound leaves; pistil 2-5-celled; endosperm 0. Protium, Canarium, Bursera. (Pf. 3*: 2381.) Family 97. Meliaceae. Trees and shrubs with alternate compound leaves; pistil 3-5-celled; endosperm present or 0. Swietenia, Melia. (Pf. 3*: 258.) Family 98. Malpighiaceae. Trees and shrubs with usually opposite, simple or lobed leaves; pistil tricarpellary; endo- sperm 0. Stigmatophyllon, Malpighia, Byrsonima. (Pf. 3%: 41.) Family 99. Trigoniaceae. Climbing shrubs with opposite simple leaves and irregular flowers; pistil tricarpellary; seeds many, endospermous. Trigonia. (Pf. 34: 309.) Family 100. Vochysiaceae. Shrubs and trees with oppo- site or whorled leaves; sepals 5; petals 1, 3, or 5; stamens several, usually but one fertile; pistil tricarpellary; seeds few; endosperm 0. Vochysia, Qualea. (Pf. 34: 312.) Family 101. Polygalaceae. Herbs, shrubs, and trees with alternate leaves; flowers irregular; sepals 5; petals 3-5; stamens usually 8; ovary 2-celled; ovules solitary; endosperm present or 0. Polygala, Xanthophyllum. (Pf. 34: 323.) Family 102. Tremandraceae. Small shrubs with alternate, opposite, or whorled leaves; flowers regular; sepals and petals 3, 4, or 5 each; stamens twice as many; ovary 2-celled; ovules mostly solitary; endosperm fleshy. Tremandra, Tetratheca. (Pf. 34: 320.) Family 103. Dichapetalaceae. Trees and shrubs with alter- nate simple leaves; pistil 2-3-celled; endosperm 0. Dicha- petalum, Tapura. (Pf. 3+: 345.) Family 104. Euphorbiaceae. Spurges. Herbs, shrubs, and trees, mostly with a milky juice and alternate or opposite leaves; flowers diclinous, with a perianth of 1 or 2 whorls, or wanting; stamens 2-celled, free or united; pistil usually 3- celled; ovules mostly solitary ; endosperm copious. Euphorbia, Pedilanthus, Phyllanthus, Croton, Mallotus, Acalypha, Ricinus, Jatropha, Manihot, Stillingia. (Species 4319.) (Pf. 3°: 1.) 1915] BESSEY—PHYLOGENETIC TAXONOMY 135 Family 105. Callitrichaceae. Floating herbs with opposite sessile leaves; flowers diclinous, sessile in the leaf-axils; peri- anth none; stamens 1 or 2; ovary 2-celled; endosperm fleshy. Callitriche. (Pf. 3°: 120.) Order Gurtirerates. Pistil mostly of 2 or more carpels, 2-several-celled, with axile placentae; stamens usually in- definite ; despir usually wanting. (Species.about 3138.) Family 106. Theaceae. Teas. Trees and shrubs usually with alternate leaves; inflorescence various; petals imbricated; seeds few; endosperm scanty or 0. Thea, Stuartia. (Pf. 3°: 175.) Family 107. Cistaceae. Herbs and shrubs with opposite (or alternate) leaves; sepals 3-5; petals 5; stamens many; pistil 3-5-carpellary, with as many parietal placentae; seeds usually many, endospermous. Cistus, Helianthemum, Hud- sonia. (Pf. 3°: 299.) Family 108. Guttiferaceae. Trees, shrubs, and rarely herbs, with opposite or whorled, glandular-dotted leaves; in- florescence often trichotomous, with flowers mostly diclinous; petals 2-6, or more, imbricated or contorted; stamens many; carpels mostly 3-5; endosperm 0. Hypericum, Mammea, Clusia, Garcinia. (Pf. 3°: 194.) Family 109. Eucryphiaceae. Evergreen trees of the south- ern hemisphere, with opposite leaves; flowers large, tetramer- ous; stamens many; pistil many-celled; seeds endospermous. Eucryphia. (Pf. 3%: 129.) Family 110. Ochnaceae. Tropical shrubs and trees with alternate, coriaceous, simple leaves; pistil lobed, 1-10-celled; endosperm fleshy or 0. Ochna. (Pf. 3°:131 Family 111. Dipterocarpaceae. Tropical, resiniferous trees and shrubs with alternate leaves; inflorescence panicled; flow- ers regular, perfect; petals contorted; fruiting calyx enlarged, and wing-like; carpels few (3-1); seeds 2 in each cell; endo- sperm 0. Dipterocarpus. (Pf. 3°: 243.) Family 112. Caryocaraceae. Tropical trees and shrubs, with alternate trifoliate leaves, large showy flowers, and many (VoL, 2 136 ANNALS OF THE MISSOURI BOTANICAL GARDEN long stamens; seeds solitary; endosperm scanty or 0. Cary- ocar. (Pf. 3°: 153.) Family 113. Quiinaceae. South American trees and shrubs, with opposite or whorled simple leaves; sepals 4-5; petals 4-5; stamens 15-30. Quiina. (Pf. 3°: 165.) Family 114. Marcgraviaceae. Tropical trees and shrubs, with alternate, simple leaves; sepals 2-6; petals as many; stamens as many or more; ovary 3-5-celled; seeds many; en- dosperm 0. Marcgravia. (Pf. 3%: 157.) Family 115. Flacourtiaceae. Mostly tropical trees and shrubs with alternate leaves; sepals 2-15; petals 10-0; stamens indefinite; carpels 2-10; seeds endospermous. Pangium, Fla- courtia, Samyda. (Pf. 3%: 1.) Family 116. Bixaceae. Tropical shrubs with alternate leaves; sepals 3-7; petals large; stamens indefinite; pistil bi- carpellary; seeds endospermous. Bixa. (Pf. 3°: 307.) Family 117. Cochlospermaceae. Tropical trees and shrubs with alternate lobed or compound leaves; petals large; sta- mens indefinite; pistil 3-5-carpellary; endosperm copious. Cochlospermum. (Pf. 3°: 312, and Nachträge zu Teil n-ıv, 251.) Family 118. Violaceae. Violets. Herbs and shrubs with alternate (or opposite) leaves; sepals and petals 5, irregular; stamens 5; pistil 3-carpellary with 3 parietal placentae; en- dosperm copious. Rinorea, Hybanthus, Viola. (Pf. 3°: 322.) Family 119. Malesherbiaceae. South American branching herbs or undershrubs, with perfect, regular, pentamerous flowers; endosperm fleshy. Malesherbia. (Pf. 3% : 65.) Family 120. Turneraceae. Tropical herbs and shrubs with alternate leaves; flowers perfect; sepals and petals dis- similar; stamens definite; ovary tricarpellary; endosperm copious. Turnera. (Pf. 3°: 57.) Family 121. Passifloraceae. Passion flowers. Climbing herbs and shrubs (a few trees) with alternate leaves; flowers perfect, regular; sepals and petals similar, distinct; stamens definite; ovary free; endosperm fleshy. Adenia, Passiflora. (Pf. 3%, 69.) 1915] BESSEY— PHYLOGENETIC TAXONOMY 137 Family 122. Achariaceae. South African herbs and under- shrubs, related to the Passifloraceae; but with the petals united. Acharia. (Pf. 3%; 92.) Family 123. Caricaceae. Papaws. Succulent-stemmed tropical trees, mostly with palmate leaves and milky juice; flowers pentamerous; fruit a many seeded berry; endosperm fleshy. Carica. (Pf. 3%: 94.) Family 124. Stachyuraceae. Asiatic shrubs and trees with alternate leaves; sepals 4; petals 4; stamens 8; endosperm fleshy. Stachyurus. (Pf. 3°: 192.) Family 125. Koeberliniaceae. Leafless, thorny Texan and Mexican shrubs, with tetramerous flowers; pistil bicarpellary ; seeds many; endosperm scanty. Koeberlinia. (Pf. 3°: 319.) Order Ruogapates. Pistil of 2 or more united carpels, mostly 1-celled, with parietal placentae; stamens indefinite or definite; endosperm none or copious. (Species about 2856.) Family 126. Papaveraceae. Poppies. Mostly milky-juiced plants, with alternate leaves, and regular or irregular flowers; sepals 2-3; petals 4 or more (or 0); stamens indefinite; pistil many-carpellary; seeds usually many; endosperm fleshy. Eschscholtzia, Sanguinaria, Argemone, Papaver, Bicuculla, Fumaria. (Pf. 37: 130.) Family 127. Tovariaceae. Annual herbs of the tropics, with alternate leaves; 8-merous flowers, and many seeds, with scanty endosperm. Tovaria. (Pf. 37: 207.) Family 128. Nymphaeaceae. Water-lilies. Aquatic herbs with floating leaves, and regular flowers; petals present, in 1-many whorls (really acyclic); pistils closely united; seeds many, endospermous. Victoria, Castalia, Nymphaea. (Pf. $*:1.) Family 129. Moringaceae. Trees of the tropics, with de- compound leaves and pentamerous, zygomorphic flowers, and producing bean-like tricarpellary pods; endosperm 0. Mor- inga. (Pf. 37: 242.) Family 130. Resedaceae. Mignonettes. Herbs and shrubs with scattered leaves and zygomorphie flowers; sepals 4-8 [VoL 2 138 ANNALS OF THE MISSOURI BOTANICAL GARDEN (or 2 or 0); stamens 3-40; pistil 2-6-carpellary; seeds many; endosperm 0. Reseda. (Pf. 3°: 237.) Family 131. Capparidaceae. Capers. Herbs, shrubs, and trees with alternate or opposite leaves, and regular or irreg- ular flowers; sepals 4; petals 4 (or 0); stamens 4 (or many) ; pistil 2-6-carpellary, endosperm 0. Cleome, Capparis. (Pf. 3? : 209.) Family 132. Brassicaceae. Mustards. Herbs, rarely shrubs, with alternate (or opposite) leaves, and regular flowers; sepals 4; petals 4; stamens 6 or 4; pistil 2-carpellary; endosperm 0. Sinapis, Brassica, Raphanus, Bursa, Alyssum. (Pf. 3? : 145.) Order CARYOPHYLLALES. Pistil usually of 3 or more united carpels, mostly 1-celled, with a free-central placenta, and many ovules (sometimes reduced to a one-celled, one-ovuled ovary) ; stamens as many or twice as many as the petals; flowers reg- ular; seeds mostly endospermous, usually with a curved em- bryo. (Species about 4330.) The general arrangement of the families of the order Caryophyllales may be understood by placing the Caryophyl- laceae centrally at the base; from this, one line runs off to the diplochlamydeous, hermaphrodite Frankeniaceae and Tamari- caceae to the achlamydeous, diclinous Salicaceae, while on the other hand another line passes from the diplochlamydeous, many-ovuled Caryophyllaceae to the apetalous, 1-ovuled Amaranthaceae, Chenopodiaceae and Polygonaceae. Family 133. Caryophyllaceae. Pinks. Herbs (and shrubs) with opposite leaves; petals 3-5, stalked or not; ovules many on a central placenta; seeds endospermous. Silene, Lychnis, Dianthus, Alsine, Paronychia, Illecebrum. (Pf. 3™: 61.) Family 134. Elatinaceae. Small marsh herbs or under- shrubs, with small, opposite or whorled leaves; inflorescence axillary ; petals imbricated; stamens 4-10; endosperm 0. Ela- tine. (Pf. 3°: 277.) Family 135. Portulacaceae. Purslanes. Herbs, or some- what woody plants, usually somewhat succulent, with alternate or opposite leaves; sepals usually 2; petals 4-5; seeds many, endospermous. Claytonia, Portulaca. (Pf. 3': 51.) 1915] BESSEY—PHYLOGENETIC TAXONOMY 139 Family 136. Aizoaceae. Herbaceous or shrubby plants with mostly opposite or verticillate, often fleshy leaves; calyx tetramerous or pentamerous; corolla often wanting; ovary mostly 2-5-celled with few to many ovules in each cell; seeds endospermous. Mollugo, Sesuvium, Mesembrianthemum. (Pf. 31°; 33.) Family 137. Frankeniaceae. Herbs and undershrubs with opposite leaves, and perfect flowers; petals 4-5, long-stalked ; ovules many, on 2-4 parietal placentae; seeds endospermous. Frankenia. (Pf. 3°: 283.) Family 138. Tamaricaceae. Tamarixes. Shrubs and herbs with minute, alternate, deciduous leaves and mostly racemose, perfect flowers; petals 5; ovules many, on 2-5 parietal pla- centae; seeds hairy-tufted; endosperm 0. Tamarix. (Pf. 3°: 289.) Family 139. Salicaceae. Willows. Shrubs and trees with large alternate leaves and racemose flowers; perianth 0; ovules many, on 2-4 parietal placentae; seeds hairy-tufted; endo- sperm 0. Here regarded as reduced, dioecious, apetalous, Tamaricaceae. Salix, Populus. (Pf. 31: 29.) Family 140. Podostemonaceae. Riverweeds. Small aquatic, sometimes thallose, plants; flowers perfect or diclinous; peri- anth 0; pistil 1-3-celled; ovules many, centrally attached; en- dosperm 0. Podostemon. (Pf. 3*:1.) Family 141. Hydrostachydaceae. Large tuber-forming Madagascar plants, with naked, dioecious flowers, single stamens, and numerous ovules on 2 parietal placentae; en- dosperm 0. Hydrostachys. (Pf. 3?*: 22.) Family 142. Phytolaccaceae. Pokeweeds. Herbs, shrubs, and trees with usually alternate leaves; petals 0 (or 4-5); carpels several, distinct or nearly so, 1-ovuled; seeds endo- spermous. Phytolacca. (Pf. 3™:1.) Family 143. Basellaceae. Herbaceous climbing plants, with mostly alternate leaves; calyx dimerous; corolla pentamerous ; stamens 5; ovary tricarpellary, 1-celled, with one ovule; en- dosperm scanty. Basella, Boussingaultia. (Pf. 3™ : 124.) [VoL. 2 140 ANNALS OF THE MISSOURI BOTANICAL GARDEN Family 144. Amaranthaceae. Amaranths. Herbs, shrubs (and trees) with opposite or alternate leaves, and regular, mostly perfect flowers; perianth of scarious sepals; petals 0; ovules 1 or more, basal, campylotropous; endosperm copious. Celosia, Amaranthus, Froelichia. (Pf. 31°: 91.) Family 145. Chenopodiaceae. The Goosefoots. Herbs, shrubs (and trees) with mostly alternate leaves, and regular, perfect or imperfect flowers; perianth of herbaceous sepals; petals 0; ovule 1, basal, campylotropous; endosperm fleshy. Beta, Chenopodium, Spinacia, Atriplex, Sarcobatus, Salsola. (Pf. 31*: 36.) Family 146. Polygonaceae. Buckwheats. Herbs, shrubs, and trees with mostly alternate leaves and regular, perfect flowers; perianth often petaloid; petals 0; pistil tricarpellary, 1-celled; ovule 1, erect, orthotropous; endosperm copious. Eriogonum, Rumex, Rheum, Polygonum, Fagopyrum, Coc- coloba. (Pf. 31*:1.) Family 147. Nyctaginaceae. Four o’clocks. Herbs and rarely shrubs and trees, with opposite or alternate leaves; flowers mostly perfect; petals 0; sepals often petaloid; pistil seemingly monocarpellary; ovule 1, erect; endosperm copious to scanty. Mirabilis, Bougainvillea, Allionia. (Pf. 31:14.) Family 148. Cynocrambaceae. Annual, succulent herbs, with petioled leaves, opposite below, alternate above; flowers monoecious, apetalous, small, axillary; pistil monocarpellary ; endosperm fleshy. Cynocrambe. (Pf. 3!1*: 121.) Family 149. Batidaceae. Maritime shrubs with opposite fleshy leaves and small, dioecious flowers; petals 0; ovary 4- celled; ovule solitary, erect; endosperm 0. Very doubtfully placed here. Batis. (Pf. 31°: 118.) Super-Order SrroprLomEAE-SyMPETALAE-POLYCARPELLATAE, Carpels typically many, united; petals united. Flowers actinomorphic. Order EBenaues. Flowers regular, perfect, or dielinous; stamens mostly isomerous with, and opposite to, the corolla- lobes, or in several series; ovary 2—many-celled; seeds mostly 1915] BESSEY— PHYLOGENETIC TAXONOMY 141 solitary or few, usually large, centrally attached. (Species about 1136.) Family 150. Sapotaceae. Sapodillas. Tropical trees and shrubs with a milky juice, and mostly alternate leaves; flowers mostly perfect; sepals and petals 4-8 each; stamens in 2-3 whorls, attached to the corolla; ovary superior, several-celled; endosperm from fleshy to 0. Achras, Sideroxylon, Chrysophyl- lum, Mimusops. (Pf. 4':126.) Family 151. Ebenaceae. Ebonies. Tropical and subtropical trees and shrubs, with very hard wood, and mostly alternate leaves; flowers mostly dioecious; sepals and petals 3-7 each; stamens usually many and free from the corolla; ovary 3- many-celled, superior; endosperm copious. Diospyros, Maba. (Pf. 41: 153.) Family 152. Symplocaceae. Tropical and subtropical trees and shrubs, with mostly perfect flowers; sepals usually 5; petals usually 5; stamens many, attached to the base of the corolla; ovary 2-5-celled, inferior; seeds few, endospermous. Symplocos. (Pf. 4':165.) Family 153. Styracaceae. Styraxes. Trees and shrubs of warm climates with alternate leaves; flowers mostly perfect, sepals and petals 5 each; stamens usually many, attached to the base of the corolla; ovary 3-5-celled, usually inferior; seeds few, endospermous. Halesia, Styrax. (Pf. 41: 172.) Family 154. Fouquieriaceae. Mexican shrubs with small leaves (becoming thorn-like), and panicled tubular flowers; sepals 5; petals 5, united into a tube; stamens 10-15, free; ovary tricarpellary; placenta central; seeds few; endosperm scanty. This small family is given place here with some con- fidence that it is much more closely related to these families than to those of the Caryophyllales and Polemomales, with which it has been associated. Fouquieria. (Pf. 3°: 298.) Order Ertcatzs. Flowers regular, perfect, pentamerous or tetramerous; stamens alternate with the corolla-lobes, and as many or twice as many; cells of the mostly superior ovary (or placentae) 2 to many; seeds minute. (Species about 1730.) [voL.'2 142 ANNALS OF THE MISSOURI BOTANICAL GARDEN Family 155. Clethraceae. White alders. Shrubs and trees of warm climates, with alternate deciduous leaves and pen- tamerous flowers; stamens 10; pistil tricarpellary ; endosperm fleshy. Clethra. (Pf. 4':1.) Family 156. Ericaceae. Heaths. Shrubs and small trees with mostly evergreen alternate or opposite leaves; ovary typically superior (sometimes inferior), 2-10-celled; anthers usually dehiscing by an apical pore; endosperm fleshy. Rho- dodendron, Kalmia, Gaultheria, Arctostaphylos, Gaylussacia, Vaccinium, Calluna, Erica. (Pf. 41:15.) Family 157. Epacridaceae. Shrubs and small trees (mostly Australian) with mostly alternate evergreen leaves; ovary superior, mostly 2-10-celled; fruit capsular or drupaceous; anthers dehiscing by a slit; endosperm fleshy. Epacris. (Pf. 41: 66.) Family 158. Diapensiaceae. Low undershrubs, with alter- nate evergreen leaves; ovary superior, 3-celled; fruit a cap- sule; anthers dehiscing by a slit; endosperm fleshy. Diapen- sia, Shortia. (Pf. 41:80.) Family 159. Pirolaceae. Wintergreens. Low evergreen, or chlorophylless herbs, with pentamerous or tetramerous (rarely hexamerous) flowers; stamens twice as many as the petals; ovary 4-6-celled; endosperm fleshy. Pirola, Chima- phila, Monotropa. (Pf. 4':3.) Family 160. Lennoaceae. Parasitic, leafless herbs; ovary superior, 10-14-carpellary, 20-28-celled ; ovules solitary ; anth- ers dehiseing by a slit; endosperm copious. Lennoa. (Pf. 41.12.) Order PrımuLaues. Flowers regular, mostly perfect and pentamerous; stamens epipetalous, mostly opposite to the corolla-lobes; ovary pluricarpellary, mostly 1-celled, with a free-central placenta. (Species about 1581.) Family 161. Primulaceae. Primroses. Herbs with alter- nate or opposite leaves; stamens attached to the upper portion of the corolla tube; pistil 2-6-carpellary, one-celled; ovules many; fruit a capsule dehiseing longitudinally from the apex, 1915] BESSEY—PHYLOGENETIC TAXONOMY 143 or circumscissilely; endosperm fleshy. Primula, Androsace, Lysimachia, Cyclamen, Dodecatheon. (Pf. 41. 98.) Family 162. Plantaginaceae. Plantains. Herbs with clustered radical leaves, or alternate or opposite stem leaves; stamens alternate with the petals; ovary mostly 2-celled; ovules many; placenta axile; fruit a capsule dehiscing circum- scissilely ; endosperm fleshy. Plantago. (Pf. 4°»; 363.) Family 163. Plumbaginaceae. Leadworts. Herbs with alternate or clustered leaves; stamens opposite the petals; pistil 5-carpellary, one-celled, with one basal, anatropous ovule; fruit capsular; dehiscence valvate or irregular; endo- sperm copious. Plumbago, Armeria. (Pf. 41:116.) Family 164. Myrsinaceae. Trees and shrubs with mostly alternate leaves; stamens attached to the ae part of the corolla tube; ovules usually few; fruit a drupe or berry; endo- sperm fleshy. Myrsine, Ardisia. (Pf. 4': 84.) Family 165. Theophrastaceae. Tropical trees and shrubs closely related to the preceding family, and sometimes in- cluded in it, but with many ovules. Theophrasta, Jacquinia. (Pf. 41:88.) Super-Order STROBILOIDEAE-SYMPETALAE-DICARPELLATAE. Carpels typically two, united; petals united. Flowers mostly perfect, from actinomorphie to ee Order GENTIANALESs. Corolla actinomorphic (regular), mostly pentamerous; stamens alternate with the corolla-lobes, and usually of the same number and attached to the tube; leaves opposite (rarely alternate). (Species about 4664.) Family 166. Oleaceae. Olives. Shrubs and trees (rarely herbs) with mostly opposite leaves, and tetramerous flowers; corolla-lobes mostly valvate or 0; stamens 2 (or 4); ovary 2-celled; ovules 1-3; endosperm present or 0. Syringa, Olea, Jasminum, Fraxinus. (Pf. 47:1.) Family 167. Salvadoraceae. Mostly tropical shrubs and trees, with opposite undivided leaves, and tetramerous or pen- tamerous flowers; corolla-lobes imbricated; stamens 4; ovary 2-celled; ovules 2; endosperm 0. Salvadora. (Pf. 4*:17.) [VoL, 2 144 ANNALS OF THE MISSOURI BOTANICAL GARDEN Family 168. Loganiaceae. Herbs, shrubs, and trees with mostly opposite simple leaves and pentamerous or tetramerous flowers; corolla-lobes imbricated or contorted; stamens mostly 4-5; ovary 2-celled (rarely 4-celled); ovules I-many; endo- sperm fleshy. Gelsemium, Logama, Spigelia, Strychnos. (Pf. 4? : 19.) Family 169. Gentianaceae. Gentians. Mostly herbs, with usually opposite undivided leaves and pentamerous or tetram- erous flowers; corolla-lobes contorted, valvate, or induplicate ; stamens 4-5; ovary bicarpellary, usually 1-celled; ovules many; endosperm copious. Erythraea, Gentiana, Eustoma, Menyanthes. (Pf. 4°: 50.) Family 170. Apocynaceae. Dogbanes. Milky-juiced trees, shrubs, and herbs, with opposite or whorled, simple leaves and mostly pentamerous (rarely tetramerous) flowers; corolla-lobes contorted or valvate; stamens 5 (or 4), with granular pollen; ovary 2-celled or the carpels separating; ovules many; endosperm fleshy. Vinca, Apocynum, Nerium. (PE # +109.) Family 171. Asclepiadaceae. Milkweeds. Milky-juiced herbs and shrubs, with opposite, whorled (or alternate) leaves and pentamerous flowers; corolla-lobes contorted; stamens 5, with agglutinated pollen; ovary of two separated carpels with one discoid stigma; ovules many; seeds usually comose; endosperm fleshy. Asclepias, Enslenia, Ceropegia, Stapelia, Hoya. (Pf. 4°: 189.) Order Potemoniates. Corolla actinomorphic, becoming somewhat zygomorphic in the later families; stamens alter- nate with the corolla-lobes, of the same number and attached to the corolla tube; leaves alternate (rarely opposite). (Species about 4112.) The relationship of this order to the Primulales, and through it to the Caryophyllales, is so obvious as to make it scarcely necessary to point it out here. Family 172. Polemoniaceae. Phloxes. Herbs (and shrubs) with alternate leaves (rarely opposite below) ; flowers pentam- erous; corolla-lobes 5, contorted; ovary tricarpellary, 3-celled ; 1915] BESSEY— PHYLOGENETIC TAXONOMY 145 ovules 1 or more in each cell; endosperm fleshy. Cobaea, Phlox, Gilia, Polemonium. (Pf. 4°*: 40.) Family 173. Convolvulaceae. Morning-glories. Herbs (often climbing), shrubs (and trees) with alternate leaves and pentamerous flowers; corolla-limb more or less plicate (rarely imbricated); ovary 2 (3-5)-celled; ovules few; endosperm fleshy. Evolvulus, Quamoclit, Ipomoea, Convolvulus, Cus- cuta (parasitic). (Pf. 4°*:1.) Family 174. Hydrophyllaceae. Herbs with radical or alter- nate (rarely opposite) leaves and pentamerous flowers; corolla-lobes imbricated (or contorted); ovary 1 or incom- pletely 2-celled; ovules 2 or more; endosperm fleshy. Hydro- phyllum, Phacelia, Nama. (Pf. 4°*: 54.) Family 175. Borraginaceae. Forget-me-nots. Herbs, shrubs, and trees with alternate leaves and pentamerous flowers; corolla-lobes imbricated (or contorted); ovary bicarpellary, 4-celled, 4-lobed; ovules solitary in each lobe; endosperm fleshy or 0. Heliotropium, Cynoglossum, Oreocarya, Borrago, Myosotis, Mertensia, Lithospermum. (Pf. 4°: 71.) Family 176. Nolanaceae. Herbaceous or suffrutescent pros- trate South American plants, with alternate, entire leaves; calyx 5-parted; corolla long funnel-shaped ; stamens 5, inserted on the corolla; carpels 5, distinct or united; endosperm fleshy. Nolana. (Pf. 4:1.) Family 177. Solanaceae. Nightshades. Herbs, shrubs (and trees) with alternate leaves and pentamerous, mostly regular, but sometimes irregular flowers; corolla-limb more or less plicate (rarely imbricated); ovary mostly 2-celled; ovules many; endosperm fleshy. Lycium, Atropa, Hyoscyamus, Physalis, Capsicum, Solanum, Datura, Nicotiana, Petunia. (Pf. 4°»: 4.) Order SCROPHULARIALES. Corolla mostly zygomorphie (ir- regular or oblique); stamens fewer than the corolla-lobes, usually 4 or 2; ovules numerous; fruit mostly capsular (i. e., dehiscent). (Species about 7081.) Family 178. Scrophulariaceae. Snapdragons. Herbs (or shrubs and small trees) with alternate, opposite, or whorled i [VOoL. 2 146 ANNALS OF THE MISSOURI BOTANICAL GARDEN leaves; ovary 2-celled with an axile placenta; seeds numerous, with endosperm. Verbascum, Linaria, Antirrhinum, Maur- andia, Collinsia, Scrophularia, Mimulus, Veronica, Digitalis, Gerardia, Castilleia, Pedicularis. (Pf. 4: 39.) Family 179. Bignoniaceae. Catalpas. Trees, shrubs (and herbs) with opposite or whorled leaves; ovary 1 or 2-celled with parietal or axile placentae; seeds numerous, without endosperm. Bignonia, Catalpa, Tecoma. (Pf. 4°”: 189.) Family 180. Pedaliaceae. Mostly tropical herbs with gen- erally opposite leaves; ovary 1, 2, or 4-celled with axile pla- centae; seeds l-many, with but little endosperm. Pedalium, Sesamum. (Pf. 48°: 253.) Family 181. Martyniaceae. Mostly tropical herbs with generally opposite leaves; stamens 2 or 4; ovary 1-celled with projecting parietal placentae; endosperm 0. Martynia. (Pf. 48> ; 265.) Family 182. Orobanchaceae. Broom-rapes. Leafless para- sitic herbs; ovary 1-celled; placentae 4, parietal; ovules minute, numerous; endosperm fleshy. Orobanche, Thalesia, Conopholis. (Pf. 4°: 123.) Family 183. Gesneraceae. Tropical and subtropical herbs, shrubs (and trees) with usually opposite leaves; ovary in- ferior or superior, 1-celled, with 2 parietal placentae; seeds numerous; endosperm scanty or 0. Streptocarpus, Gesnera, Gloxinia. (Pf. 4°: 133.) Family 184. Columelliaceae. South American trees and shrubs with opposite, evergreen leaves and nearly regular flowers; stamens 2; ovary inferior, 2-celled, with an axile pla- centa; endosperm fleshy. Columellia. (Pf. 4°”: 186.) Family 185. Lentibulariaceae. Bladderworts. Aquatic or marsh herbs with basal, entire or dissected leaves and irreg- ular flowers; ovary 1-celled, with a globose basilar placenta; seeds numerous; endosperm 0. Pinguicula, Utricularia. (Pf. 48>: 108.) Family 186. Globulariaceae. Shrubs and undershrubs or evergreen herbs, with alternate leaves, and a terminal capitate 1915] BESSEY— PHYLOGENETIC TAXONOMY 147 cluster of small irregular flowers; ovary 1-celled, with a single ovule; endosperm fleshy. Globularia. (Pf. 4°: 270.) Family 187. Acanthaceae. Herbs (shrubs and trees) with opposite leaves; ovary 2-celled; placentae axile; fruit a dry pod which splits open vertically; seeds 2-many, without endo- sperm. Thunbergia, Ruellia, Acanthus, Justicia. (Pf. 48> :274.) Order Lamiatzs. Corolla mostly zygomorphic (irregular or oblique) ; stamens fewer than the corolla-lobes, usually 4 or 2; ovules mostly 2 in each carpel; fruit indehiscent. (Species about 4119.) Family 188. Myoporaceae. Mostly Australasian shrubs and trees, with usually alternate leaves; flowers axillary; fruit a 1-4-seeded drupe; endosperm scanty. Myoporum. (Pf. 48> ; 354.) Family 189. Phrymaceae. Erect, perennial herbs, with opposite leaves, and small spicate flowers; calyx and corolla cylindrical, 2-lipped; stamens 4; ovary 1-celled, 1-ovuled; stigma bifid; endosperm 0. Phryma. (Pf. 4°”: 361.) Family 190. Verbenaceae. Verbenas. Herbs, shrubs, and trees, with usually opposite leaves; ovary of 2 carpels, but 2-8-celled, with 1 ovule in each cell; stigma usually undivided; endosperm scanty or 0. Verbena, Lantana, Lippia, Tectona, Vitex. (Pf. 4°: 132.) Family 191. Lamiaceae. Mints. Mostly aromatic herbs, shrubs (and trees) with opposite or whorled leaves; ovary 4-celled, 4-lobed with 1 ovule in each cell; stigma usually bifid; endosperm scanty or 0. Lavendula, Nepeta, Stachys, Salvia, Thymus, Mentha, Coleus. (Pf. 4°: 183.) With this order (Lamiales), and especially with this family (Lamiaceae), we attain the summit of the cone-flowers (Stro- biloideae). We next return almost to the point of beginning, and there start on a new phyletic line. Sub-Class OPPOSITIFOLIAE - COTYLOIDEAE. “(Cup Flowers.’’ Axis of the flower normally expanded into a disk or cup, bearing on its margin the perianth and stamens (or the latter may be attached to the corolla). [VoL. 2 148 ANNALS OF THE MISSOURI BOTANICAL GARDEN Super-Order COTYLOIDEAE - APOPETALAE. Petals separate. Carpels many to few, separate to united, superior to inferior. This super-order appears to have originated near the begin- ning of the Strobiloideae, and therefore the orders Ranales and Rosales are to be regarded as closely related. Their relationship to Alismatales, also, has already been pointed out. Order Rosaues, Flowers cyclic, usually perfect, dichlamy- deous (rarely apetalous), actinomorphie to zygomorphie (regular to irregular) and mostly pentamerous; carpels usually several to many, separate or more or less united, some- times united with the axis-cup (rarely reduced to 1); styles usually distinct. (Species about 14261.) Family 192. Rosaceae. Roses. Herbs, shrubs, and trees with mostly alternate leaves; stamens usually indefinite, on the cup-margin; carpels several to many (rarely 1), free (but they may be enclosed in the deep cup) ; ovules usually 2, ana- tropous; endosperm 0. Potentilla, Fragaria, Spiraea, Rosa. (Species about 2700.) (Pf. 3°: 1.) Family 193. Malaceae. Apples. Shrubs and trees with alternate leaves; stamens usually many on the cup-margin; carpels few, more or less united, and adnate to the axis-cup, so as to be ‘‘inferior’’; endosperm 0. Sorbus, Pirus, Malus, Crataegus. (Pf. 3°:1, 18.) Family 194. Prunaceae. Plums. Shrubs and trees with alternate leaves; stamens many, on the cup-margin; carpel one, in the bottom of the deep cup, becoming a drupe; endo- sperm 0. Prunus, Amygdalus. (Species 150.) (Pf. 3°: 1, 50.) Family 195. Crossosomataceae. Southwest North American shrubs, with small leaves and a bitter bark; sepals and petals 5 each; stamens 20 or more; carpels 3-5; seeds many, reni- form; endosperm scanty. Crossosoma. (Pf. Nachträge zu Teil ı1-ıv, 185.) Family 196. Connaraceae. Tropical trees and shrubs with alternate compound leaves; stamens definite (5-10); pistils mostly 5, free; ovules 2, ascending, orthotropous; endosperm fleshy or 0. Connarus, Cnestis. (Pf. 3°: 61.) 1915] BESSEY— PHYLOGENETIC TAXONOMY 149 Family 197. Mimosaceae. The mimosas. Mostly tropical trees, shrubs, and herbs, with alternate mostly compound leaves; flowers actinomorphic; stamens 10 or more, usually separate; carpel 1; fruit a legume; seeds mostly without endo- sperm. Acacia, Mimosa. (Species 1483.) (Pf. 33:70, 99.) Family 198. Cassiaceae. The sennas. Mostly tropical trees, shrubs, and herbs, with alternate mostly compound leaves; flowers zygomorphic; stamens 10 or less, usually sepa- rate; carpel 1; fruit a legume; seeds with or without endo- sperm. Cassia, Caesalpinia, Gleditsia, Gymnocladus. (Species 1172.73 (Pf. + : 70, 125.) Family 199. Fabaceae. The beans. Mostly herbs of tem- perate climates, but with many shrubs and trees; leaves alter- nate, mostly compound; flowers zygomorphic; stamens 10 or less, usually more or less united; carpel 1; fruit a legume; seeds usually without endosperm. Lupinus, Medicago, Tri- folium, Robinia, Astragalus, Arachis, Vicia, Pisum, Phaseolus. (Species 6948.) (Pf. 3°: 70, 184.) This family constitutes a well-marked side-line in the order Rosales, with zygomorphic, entomophilous flowers. It is not obvious what relation, if any, exists between this form of the flower, and the legume structure of the fruiting carpel. Family 200. Saxifragaceae. Saxifrages. Herbs with alter- nate leaves, regular 4 or 5-merous mostly perfect flowers, with 8 or 10 stamens, and usually 2 more or less united carpels which are superior; seeds many; endosperm copious. Saxi- fraga, Heuchera, Mitella. (Pf. 3°: 41.) Family 201. Hydrangeaceae. Hydrangeas. Shrubs and trees with mostly opposite leaves, and regular 4 or 5-merous mostly perfect flowers, with few (8) to many (40) stamens, and 2-5 united carpels, which are more or less overgrown by the axis-cup; seeds many; endosperm copious. Philadelphus, Hydrangea. (Pf. 37*: 41.) Family 202. Grossulariaceae. Gooseberries. Shrubs with alternate leaves, regular 4 or 5-merous perfect flowers, usually 5 stamens, and 2 to several united carpels which are wholly [Vou, 2 150 ANNALS OF THE MISSOURI BOTANICAL GARDEN overgrown by the fleshy cup (ovary inferior) ; seeds few, endo- sperm copious. Ribes. (Pf. 3°": 41.) Family 203. Crassulaceae. Stonecrops. Mostly fleshy herbs, with opposite or alternate leaves and perfect flowers; stamens definite (4-10 or many); pistils several, free or little united; ovules many; placentae central or axile; endosperm fleshy. Sedum, Cotyledon, Crassula, Penthorum. (Pf. 3%: 23.) Family 204. Droseraceae. Sundews. Gland-bearing marsh herbs with perfect flowers; stamens mostly definite (4-20); pistil synearpous, 1-3-celled, superior; ovules many, on basal, axile, or parietal placentae; endosperm fleshy. Drosera, Dionaea. (Pf. 37: 261.) Family 205. Cephalotaceae. Pitcher-plants. Perennial Australian herbs with a rosette of elliptic, and pipe-shaped radical leaves, and a central, erect, spicate flowering stem; flowers regular, perfect, apetalous; sepals 6; ovules solitary; endosperm copious. Cephalotus. (Pf. 37°: 39.) Family 206. Pittosporaceae. Trees and shrubs of the southern hemisphere, with alternate leaves; sepals, petals, and stamens 5 each; ovary 2-carpellate; endosperm copious. Pitto- sporum, Marianthus. (Pf. 3°*: 106.) Family 207. Brunelliaceae. South American trees, with opposite or whorled leaves and diclinous flowers; sepals and petals 4-5 or 7 each; stamens twice as many; carpels usually 4-5, free; endosperm fleshy. Brunellia. (Pf. Nachträge zu Teil ı1-ıv, 182.) Family 208. Cunoniaceae. Shrubs and trees, mostly of the southern hemisphere, with opposite or whorled leaves and small, perfect flowers; sepals and petals 4-6 each; stamens twice as many; carpels 2-5, united; endosperm fleshy. Belangera, Cunonia. (Pf. 3°*: 94.) Family 209. Myrothamnaceae. Small, rigid, balsamic South African and Madagascar shrubs, with opposite leaves, and dioecious, achlamydeous flowers; ovary tricarpellary; seeds many, with fleshy endosperm. Myrothamnus. (Pf. 37*; 103.) 1915] BESSEY—PHYLOGENETIC TAXONOMY 151 Family 210. Bruniaceae. Heath-like shrubs of the southern hemisphere, with small leaves and small, perfect, regular, pentamerous flowers; stamens definite; pistil 2-3-celled, in- ferior or superior; ovules 1 to many, pendulous; endosperm copious. Bruma. (Pf. 3°: 131.) Family 211. Hamamelidaceae. Witch-hazels. Shrubs and trees with mostly alternate leaves and perfect or imperfect, mostly pentamerous flowers; stamens few or many; pistil bicarpellary, its ovary inferior; ovules solitary or many ; endo- sperm thin. Liquidambar, Altingia, Hamamelis. (Pf. 321; 115.) Family 212. Casuarinaceae. Beefwood trees. Shrubs and trees with striate stems bearing whorls of reduced scale-like leaves; flowers diclinous; petals 0; pistil bicarpellary, 1-celled; ovules 2, lateral, half anatropous; endosperm 0. Casuarina. (Pf. 31:16.) This family, which has puzzled botanists from the first, is doubtfully placed here, on the theory that these plants are leafless relatives of the Hamamelidaceae. Family 213. Eucommiaceae. Chinese trees, with alternate leaves, and achlamydeous diclinous flowers; stamens 6-10; pistil bicarpellary, 1-celled, 2-seeded; endosperm present. Eucommia. (Pf. Nachträge zu Teil m-1v, 159.) Family 214. Platanaceae. Plane-trees. Trees with alter- nate leaves, and monoecious flowers in globular heads; peri- anth 3-8-merous; stamens 3-8; pistils 3-8, each 1-celled, 1-ovuled; endosperm scanty. Platanus. (Pf. 3°: 137.) Order Myrraues. Flowers usually actinomorphic (regular) or nearly so, usually perfect; pistil of united carpels, usually inferior; placentae axile or apical (rarely basal); style 1 (rarely several) ; leaves simple, usually entire. (Species about 7323.) Here again we shall soon reach the end of a phyletic side- line, consisting principally of the order Myrtales, with the Loasales and Cactales as the ultimate branches. Family 215. Lythraceae. Herbs, shrubs, and trees usually with opposite leaves and 4-angled branches; flowers mostly 4-6-merous; stamens definite (8-12), or indefinite; pistil 2-6- [VoL, 2 152 ANNALS OF THE MISSOURI BOTANICAL GARDEN celled, free; ovules numerous, on axile placentae; endosperm 0. Lythrum, Cuphea, Lagerstroemia. (Pf. 37:1.) Family 216. Sonneratiaceae. Tropical trees with opposite leaves; ovary sunken in the axis-cup, many celled (4-15); stamens many; endosperm 0. Sonneratia. (Pf. 37:16.) Family 217. Punicaceae. Pomegranates. Small tropical and sub-tropical trees with opposite leaves and 5-7-merous flowers; stamens many; ovary inferior, 4-15-celled, producing a pulpy, many-seeded fruit; endosperm 0. Punica. (Pf. 37 ; 22.) Family 218. Lecythidaceae. Tropical trees, with alternate leaves and usually 4-6-merous flowers; stamens many; ovary inferior, 2-6-celled; endosperm 0. Barringtonia, Napoleona, Lecythis, Bertholletia. (Pf. 3°: 26.) Family 219. Melastomataceae. Mostly tropical herbs, shrubs, and trees with generally opposite or whorled leaves; stamens usually double the number of petals; pistil 2-many- celled, inferior; ovules minute, numerous, on axile or parietal placentae; endosperm 0. Melastoma, Osbeckia, Rhexia, Tamonea. (Pf. 37:130.) Family 220. Myrtaceae. Myrtles. Trees and shrubs with opposite or alternate leaves, and perfect, regular flowers; stamens many; pistil 2-many-celled, inferior; ovules 2 to many; plancentae basal or axile; endosperm 0. Myrtus, Piménta, Eugenia, Jambosa, Eucalyptus, Malaleuca. (Species 2556.) (Pf. 37: 57.) Family 221. Combretaceae. Trees and shrubs often climb- ing, with opposite or alternate leaves; stamens usually definite (4-10); pistil 1-celled, inferior; ovules 2-6 or solitary, pen- dulous; endosperm 0. Terminalia, Combretum, Laguncularia. (Pf. 37: 106.) Family 222. Rhizophoraceae. Mangroves. Mostly tropical trees and shrubs with opposite leaves and regular, 4-8-merous flowers; stamens 2-4 times the number of petals; pistil 2-6- celled, usually inferior; ovules 2, pendulous; endosperm fleshy. Rhizophora, Carallia. (Pf. 37:42.) 1915] BESSEY—PHYLOGENETIC TAXONOMY 153 Family 223. Oenotheraceae. Evening primroses. Herbs (shrubs and trees) with opposite or alternate leaves, and perfect, 2-3-4-merous, regular flowers; stamens 1-8, rarely more; pistil usually 4-celled, inferior; ovules 1 to many on axile placentae; endosperm scanty or 0. Epilobium, Anogra, Oenothera, Meriolix, Gaura, Fuchsia, Circaea. (Pf. 37 : 199.) Family 224. Halorrhagidaceae. Aquatic or terrestrial herbs with opposite or alternate leaves and perfect or im- perfect, sometimes apetalous flowers; pistil 1—4-celled, in- ferior; ovules solitary, pendulous; endosperm present. Hal- orrhagis, Myriophyllum. (Pf. 37:226.) Family 225. Hippuridaceae. Aquatic perennial erect herbs, with whorled leaves, and small, reduced, axillary apetalous flowers; ovary 1-celled, 1-ovuled; endosperm scanty. Hip- puris. (Pf. 37:237.) Family 226. Cynomoriaceae. Parasitic rhizomatous fleshy plants with spicate, small, apetalous, diclinous flowers, each with a single ovule; endosperm fleshy. Cynomorium. (Pf. 31; 250.) Family 227. Aristolochiaceae. Dutchman’s-pipes. Her- baceous or shrubby plants, with alternate leaves and large, apetalous, perfect, irregular flowers; stamens 6, rarely more; pistil 4 or 6-celled, inferior; ovules numerous, on axile (or protruding parietal) placentae; endosperm copious. Asarum, Aristolochia. (Pf. 31: 264.) Family 228. Rafflesiaceae. Fleshy, parasitic herbs, of warm climates, leafless, or nearly so, with mostly imperfect flowers; petals 0, or rarely 4; stamens 8 to many; pistil 1-celled or imperfectly many-celled, inferior; ovules minute, very num- erous, on parietal or pendulous, folded placentae; endosperm present. Raflesia, Cytinus. (Pf. 31: 274.) Family 229. Hydnoraceae. Parasitic, succulent, tropical herbs with perfect, 3-4-merous flowers; perianth single, val- vate; stamens 3-4, but anthers many; seeds very numerous; endosperm copious. Hydnora. (Pf. 31: 282.) [VoL, 2 154 ANNALS OF THE MISSOURI BOTANICAL GARDEN Order Loasates. Flowers usually actinomorphie, perfect or diclinous; pistil mostly tricarpellary, 1-celled, its ovary usually inferior; placentae parietal and with many ovules; styles free or connate; leaves ample, entire, lobed or dissected. (Species about 1392.) Family 230. Loasaceae. Star-flowers. Herbs (rarely climbing) with opposite or alternate leaves; flowers perfect; sepals and petals dissimilar, mostly 5 each; stamens indefinite, 5-10 or more; ovary 3-7-carpellary, 1-celled; endosperm mostly 0. Mentzelia, Loasa. (Pf. 3°:100.) Family 231. Cucurbitaceae. Melons. Mostly climbing or prostrate herbs and undershrubs, with alternate leaves; flowers mostly diclinous and pentamerous; stamens definite (usually 3); ovary mostly tricarpellary; endosperm 0. Melo- thria, Momordica, Luffa, Citrullus, Cucumis, Lagenaria, Cucurbita. (Pf. 4:1.) Family 232. Begoniaceae. Begonias. Mostly erect herbs with alternate leaves; flowers diclinous, more or less zygo- morphic; stamens indefinite and numerous, ovary tricar- pellary, 3-celled, usually 3-angular; endosperm little or 0. Begonia. (Pf. 3%: 121.) Family 233. Datiscaceae. Herbs or large trees, with alter- nate leaves; flowers small, and diclinous; stamens 4 to many; ovary 3-8-carpellary;; placentae on the walls; seeds small, and many; endosperm scanty. Datisca. (Pf. 3%: 150.) Family 234. Ancistrocladaceae. Climbing plants of tropical Asia, with alternate leaves, and small, regular, perfect flowers; petals 5; stamens 5-10; ovary 1-celled, many-seeded; endosperm present. Ancistrocladus. (Pf. 3°: 274.) Order Cacrates. Flowers actinomorphic or very slightly zygomorphic, perfect; stamens many; pistil 4-8-carpous, in- ferior, 1-celled, with 4-8 parietal placentae; style single, with 2 to many stigmas; endosperm scanty or 0; embryo curved. Fleshy-stemmed plants with leaves mostly small or wanting. (Species about 1168.) Family 235. Cactaceae. Cactuses. Mostly natives of the warmer portions of America; from small herbs to tree-like 1915] BESSEY—PHYLOGENETIC TAXONOMY 155 dimensions. Peireskia, Opuntia, Cereus, Carnegiea, Echino- cactus, Melocactus, Cactus, Rhipsalis. (Pf. 3°: 156.) Order Crenastrates. Receptacle often developing a gland- ular, annular or turgid disk, which is sometimes adnate to the pistil, in which case the pistil is more or less inferior; pistil 1 to many-celled (rarely apocarpous) ; ovules 1-3, pendulous or erect; endosperm present or 0. Flowers actinomorphic and mostly perfect. (Species about 2741.) Family 236. Rhamnaceae. Buckthorns. Trees and shrubs often climbing, with alternate or opposite, simple leaves; petals present; disk more or less adnate to the 2-4-celled pistil; ovules 1 or 2, erect; endosperm fleshy. Zizyphus, Rhamnus, Ceanothus, Phylica, Colletia. (Pf. 3°: 393.) Family 237. Vitaceae. Grapes. Climbing shrubs (and trees) with alternate, simple or compound leaves; petals coher- ent, valvate; pistil superior, 2-celled, 2-ovuled (or 3-6-celled, 1-ovuled) ; endosperm often ruminate. Vitis, Parthenocissus, Cissus. (Pf. 35: 427.) Family 238. Celastraceae. Bittersweets. Shrubs (often climbing) and trees, with usually alternate, simple leaves; petals present, imbricated; disk more or less adnate to the 2-5-celled pistil; ovules usually 2, erect or pendulous; endo- sperm fleshy. Euonymus, Celastrus, Cassine. (Pf. 3°: 189.) Family 239. Buxaceae. Boxes. Evergreen shrubs and trees, with alternate or opposite leaves, and usually monoe- cious, small, apetalous flowers; stamens 4; pistil tricarpellary, superior; endosperm fleshy. Pachysandra, Buxus. (Pf. 3°: 130.) Family 240. Aquifoliaceae. Hollies. Trees and shrubs, with alternate or opposite, simple leaves and small, perfect flowers; pistil superior, 3 to many-celled; ovule 1, pendulous; endosperm fleshy. Ilex, Nemopanthes. (Pf. 3°: 183.) Family 241. Cyrillaceae. South American evergreen shrubs or small trees, with alternate leaves; sepals 5; petals 5; stamens 5-10; carpels 2-5, united, superior; endosperm fleshy. Cyrilla. (Pf. 35:179.) [VoL. 2 156 ANNALS OF THE MISSOURI BOTANICAL GARDEN Family 242. Pentaphylacaceae. Chinese trees, with alter- nate, leathery leaves and small, perfect flowers; sepals 5; petals 5; stamens 5; pistil superior, of 5 carpels, each 2-ovuled; endosperm scanty. Pentaphylax. (Pf. Nachträge zu Teil u-ıv, 214.) Family 243. Corynocarpaceae. New Zealand trees, with alternate, fleshy, leathery leaves; sepals 5; petals 5; stamens 5; pistil superior, of 2 carpels; endosperm 0. Corynocarpus. (Pf. Nachträge zu Teil m-1v, 215.) Family 244. Hippocrateaceae. Tropical trailing and climbing woody plants with opposite leaves; sepals 5; petals 5; stamens 3 or 2 or 5; pistil of 3 carpels more or less adnate to the disk; endosperm 0. Hippocratea, Salacia. (Pf. 3°: 222.) Family 245. Stackhousiaceae. Australian herbs and shrubs with simple alternate leaves and perfect flowers; petals 5; stamens 5; ovary 2-5-celled; ovule 1 in each cell, erect; endo- sperm fleshy. Stackhousia. (Pf. 3°: 231.) Family 246. Staphyleaceae. Bladder-nuts. Erect shrubs and trees, with opposite, compound leaves and pentamerous perfect flowers; sepals 5; petals 5; stamens 5; pistil of 2-3 superior carpels; seeds few to many; endosperm fleshy or 0. Staphylea, Turpima. (Pf. 35 : 258.) Family 247. Geissolomataceae. South African evergreen shrubs, with opposite sessile leaves; sepals 4; petals none; stamens 8; pistil superior, of 4 carpels, each 2-ovuled; endo- sperm fleshy. Geissoloma. (Pf. 3%: 205.) Family 248. Penaeaceae. South African evergreen heath- like shrubs, with small, opposite leaves and regular, perfect flowers; petals 0; pistil superior, 4-celled; ovules 2-4, erect; endosperm 0. Penaea. (Pf. 3%: 208.) Family 249. Oliniaceae. African shrubs and trees, with thick, leathery, opposite leaves, and small, regular, perfect flowers; sepals 4-5, large; petals 4-5, very small; stamens 4-5; pistil inferior, of 3-5 carpels; endosperm 0. Olinia. (Pf. 38 ; 213.) Family 250. Thymelaeaceae. Shrubs, small trees (and herbs), with alternate or opposite, usually coriaceous, simple 1915] BESSEY— PHYLOGENETIC TAXONOMY 157 leaves and small petalous or apetalous, mostly perfect flowers; pistil superior, 1-5-carpellary, 1-celled; ovule 1, pendulous; endosperm fleshy, sparse, or 0. Gnidia, Thymelaea, Daphne, Dirca. (Pf. 3%; 215.) Family 251. Hernandiaceae. Tropical trees and shrubs, with alternate leaves; flowers perfect or monoecious, regular; sepals 4-10; petals none; stamens 3; pistil 1-celled, inferior; ovule 1, pendulous; endosperm 0. Hernandia. (Pf. 32: 126.) Family 252. Elaeagnaceae. Oleasters. White or brown- scurfy trees and shrubs, with alternate or opposite, simple leaves and perfect or diclinous flowers; petals 0; pistil 1-celled; ovule 1, ascending; endosperm 0 or scanty. Elaeagnus Lepargyraea. (Pf. 3°: 246.) Family 253. Myzodendraceae. South American parasitic shrubs, with alternate, rather small leaves; flowers dioecious, apetalous; stamens 2-3; pistil 1-celled, inferior; endosperm fleshy. Myzodendron. (Pf. 31:198.) Family 254. Santalaceae. Sandalwoods. Parasitic herbs, shrubs, and trees, with alternate or opposite, simple leaves and small, perfect, or diclinous flowers; epigynous; petals 0; pistil inferior, 1-5-carpellary, 1-celled; ovules 2-5, pendulous; endosperm present. Santalum, Comandra, Thesium. (Pf. 31: 202.) Family 255. Opiliaceae. Shrubs of tropical climates, with alternate leaves, and perfect flowers; sepals, petals and sta- mens 4-5 each; pistil superior, 1-celled, 1-ovuled; endosperm fleshy. Opilia. (Pf. Nachträge zu Teil ı-ıv, 142.) Family 256. Grubbiaceae. South African shrubs with op- posite leaves, and epigynous, apetalous flowers; ovary 2-celled; ovules 2; endosperm fleshy. Grubbia. (Pf. 31; 282.) Family 257. Olacaceae. Trees and shrubs, often twining, mostly tropical, with usually alternate, simple leaves and mostly perfect, apetalous flowers; pistil superior or inferior, 1-3-celled; ovules 2-3, pendulous; endosperm fleshy. Olaz. (Pf. 31: 231.) Family 258. Loranthaceae. Mistletoes. Parasitic ever- green shrubs with opposite (or alternate) leaves, often re- 2 [VoL. 2 158 ANNALS OF THE MISSOURI BOTANICAL GARDEN duced to bracts; flowers perfect or dielinous; petals 0; pistil 1-celled, inferior; ovule 1, erect; endosperm fleshy. Loranthus, Viscum, Phoradendron, Razoumowskia. (Pf. 3: 156.) Family 259. Balanophoraceae. Parasitic, leafless herbs, all tropical, with much reduced, apetalous, monoecious or dioecious flowers; pistil 1-celled, inferior; ovule 1, pendulous; endosperm fleshy. Balanophora. (Pf. 3': 243.) Order Sarınpaues. Flowers mostly actinomorphic, perfect, or diclinous; pistil 1 to several-celled, superior to inferior; ovules 1-2, erect, ascending, or pendulous; endosperm mostly 0. (Species about 2903.) The Sapindales lie wholly in a phyletie side-line, and the order has been developed from some part of the intermediate order Celastrales, which constitutes a transition from the lower hypogynous cup flowers to those in which epigyny is fixed. In the lower Sapindales hypogyny still persists, but in the higher families this gives way to complete epigyny. Family 260. Sapindaceae. Soapberries. Trees and shrubs, mostly tropical, with alternate (or opposite), mostly com- pound leaves and mostly perfect, irregular flowers; disk pres- ent or 0; petals 3-5 or 0; pistil 1-3-celled; ovules 1 or 2, ascending; endosperm usually 0. Paullinia, Sapindus, Talisia, Litchi, Koelreuteria, Dodonaea. (Pf. 3°: 277.) Family 261. Hippocastanaceae. Horsechestnuts. ‘Trees and shrubs, with opposite, palmately compound leaves; flowers mostly regular; sepals 5; petals 4-5; stamens 8-5; pistil su- perior, tricarpellary; endosperm 0. Aesculus. (Pf. 3°: 273.) Family 262. Aceraceae. Maples. Trees and shrubs, with opposite, simple or compound leaves and small, regular flow- ers; sepals 4-10; petals as many or none; pistil superior, bi- carpellary, winged in fruit; endosperm 0. Acer. (Pf. 3°: 258.) Family 263. Sabiaceae. Trees and shrubs of the tropics, with alternate, simple or compound leaves, and perfect or diclinous flowers; petals 4-5; pistil 2-3-celled; ovules 1 or 2, horizontal or pendulous; endosperm 0. Sabia, Meliosma. (Pf. 35: 367.) 1915] BESSEY— PHYLOGENETIC TAXONOMY 159 Family 264. Icacinaceae. Tropical trees and shrubs, with alternate or opposite leaves and regular, perfect or diclinous flowers; sepals 5; petals 5; stamens 5; pistil superior, 1-celled, and tricarpellary; endosperm fleshy. Icacina. (Pf. 35:233.) Family 265. Melianthaceae. Tropical trees and shrubs, with alternate leaves, and pentamerous, mostly perfect, zygo- morphic flowers; endosperm fleshy. Melianthus. (Pf. 3°: 374.) Family 266. Empetraceae. Heath-like shrubs, with small alternate leaves; flowers small, regular, mostly dioecious, solitary or in heads; petals present; stamens 2-3, 2-3-celled ; pistil 2 to many-celled; seeds solitary, endospermous. Corema, Empetrum. (Pf. 3°: 123.) Family 267. Coriariaceae. Shrubs with opposite, sessile leaves and perfect or diclinous flowers; 5 sepals; 5 petals; 10 stamens; 5-10 carpels, slightly united; seeds few; endosperm scanty. Coriaria. (Pf. 35:128.) Family 268. Anacardiaceae. Sumachs. Trees and shrubs, mostly tropical, with alternate, usually compound leaves and small, perfect flowers; petals 3-7 or 0; pistil 1-5-celled, superior, but surrounded by the fleshy cup; ovules solitary, pendulous (or erect); endosperm 0. Mangifera, Anacardium, Schinus, Cotinus, Metopium, Rhus. (Pf. 3°: 138.) Family 269. Juglandaceae. Walnuts. Trees and shrubs, with alternate, compound leaves and small, diclinous, apet- alous flowers; pistil bicarpellary, 1-celled, adnate to the fleshy cup, and so inferior; ovule 1, erect, orthotropous; endosperm 0. Engelhardtia, Juglans, Hicoria. (Pf. 31:19.) Family 270. Betulaceae. Birches. Trees and shrubs, with alternate, simple leaves, and monoecious or dioecious flowers, which are in aments; petals none; calyx small or none; sta- mens 2-10; pistil inferior, bicarpellary, 1-2-celled; endosperm 0. Carpinus, Ostrya, Corylus, Betula, Alnus. (Pf. 31:38.) Family 271. Fagaceae. Beeches. Trees and shrubs, with alternate, simple leaves and small, diclinous flowers; petals 0; pistil mostly tricarpellary, 2-6-celled, inferior; ovules 2 in each cell, erect or pendulous; fruit usually 1-seeded; endo- sperm 0. Fagus, Castanea, Pasania, Quercus. (Pf. 31:47.) [VoL. 2 160 ANNALS OF THE MISSOURI BOTANICAL GARDEN Family 272. Myricaceae. Bayberries. Shrubs and trees, with alternate, simple leaves and small, achlamydeous, diclinous flowers; petals 0; pistil free, bicarpellary, 1-celled; ovule 1, erect, orthotropous; endosperm 0. Myrica. (Pf. 31; 26.) Family 273. Julianaceae. Dioecious, tropical trees, with alternate leaves; flowers small, apetalous, dioecious; stamens 4-8; pistil of 3-5 carpels; endosperm 0. Juliana. (Pf. Nach- träge zu Teil n-ıv, 335, and Syllabus, 161.) This family is given place here very doubtfully. Family 274. Proteaceae. Shrubs, trees (and herbs) of the southern hemisphere, with mostly alternate, simple, usually coriaceous, evergreen leaves; flowers perfect or diclinous; sepals petaloid; petals 0; stamens 4; pistil monocarpellary, 1-celled; ovule 1, erect or pendulous; endosperm little or none. Protea, Leucadendron, Grevillea, Hakea, Banksia. (Pf. 31: 118.) This puzzling family is given place here very doubtfully. Order UMBELLALES. Flowers actinomorphic (regular), usually perfect, 4-5-merous; calyx small to minute; stamens usually definite (4-5) ; pistil syncarpous, 1 to many-celled, its ovary inferior; ovules solitary, pendulous; styles free or united at the base; endosperm copious; embryo usually minute. (Species about 2809.) Family 275. Araliaceae. Aralias. Trees, shrubs (and herbs), mostly tropical, with alternate leaves; flowers in umbels, heads, or panicles; ovary 2-15-celled; fruit a berry with a fleshy or dry exocarp. Hedera, Aralia, Panax. (Pf. 38:1 Family 276. Apiaceae. Parsleys. Herbs (shrubs and trees), with alternate leaves; flowers small, pentamerous, mostly umbellate; ovary 2-celled; fruit splitting into two dry indehiscent mericarps. Hydrocotyle, Sanicula, Eryngium, Coriandrum, Conium, Apium, Cicuta, Carum, Foeniculum, Angelica, Ferula, Heracleum, Daucus. (Species 2177.) (Pf. 38 : 63.) Family 277. Cornaceae. Cornels. Shrubs and trees (rarely herbs), with usually opposite leaves; flowers larger, 4-5- 1915] BESSEY—PHYLOGENETIC TAXONOMY 161 merous, umbellate, capitate, or corymbose; ovary 2-4-celled, fruit drupaceous. Garrya, Nyssa, Cornus, Aucuba. (Pf. 38 ; 250.) Super-Order CoTYLoIDEAE - SYMPETALAE. Petals united. Carpels few, united, inferior; stamens usually as many as the corolla-lobes, mostly attached to the corolla. Order Rusıues. Flowers 4-5-merous, actinomorphie (rarely zygomorphic) ; stamens 4-5, attached to the corolla; calyx small; ovary 2-8-celled; ovules 2 to many in each cell. (Species about 5063.) Family 278. Rubiaceae. Madders. Trees, shrubs and herbs, mostly tropical, with opposite or whorled leaves; flowers usually perfect, and regular, with valvate, contorted, or im- bricate corolla-lobes; carpels mostly 2; style simple, bifid, or multifid; fruit a capsule, berry, or drupe; endosperm from fleshy to 0. Houstonia, Cinchona, Bouvardia, Cephalanthus, Randia, Coffea, Mitchella, Galium, Rubia. (Pf. 4*:1.) Family 279. Caprifoliaceae. Honeysuckles. Mostly woody -plants with opposite leaves; flowers usually zygomorphic, with imbricate corolla-lobes; carpels 2-5, with 1 or more pendulous ovules; style usually with a capitate undivided stigma; fruit a berry; endosperm fleshy. Sambucus, Viburnum, Linnaea, Lonicera. (Pf. 4*: 156.) Family 280. Adoxaceae. Moschatels. Slender herbs with scaly rootstocks, bearing ternately compound leaves; flowers small, regular, greenish, in heads; stamens about 10; ovary 3-5-celled; fruit drupaceous; endosperm cartilaginous. Adoxa. (Pf. 4*:170.) Family 281. Valerianaceae. Valerians. Herbs (and shrubs) with opposite leaves; flowers somewhat irregular, cymose, corymbose, or solitary; stamens 1-4, the anthers free; ovary 1-3-celled, the ovules pendulous; fruit with 1 fertile cell, 1-seeded; endosperm scanty, or 0. Valerianella, Fedia, Valeriana. (Pf. 44: 172.) Family 282. Dipsacaceae. Teasels. Herbs (and shrubs) with opposite or whorled leaves; flowers zygomorphic, in [VoL. 2 162 ANNALS OF THE MISSOURI BOTANICAL GARDEN involucrate heads; stamens 2-4, the anthers free; carpels 2, but pistil 1-celled; ovule 1, pendulous; endosperm scanty. Cephalaria, Dipsacus, Scabiosa. (Pf. 4*:182.) Order CAMPANULALES. Flowers actinomorphic to zygo- morphic; stamens mostly free, their anthers free or connate; ovary 1 to several-celled; ovules 1-8. (Species about 1539.) Family 283. Campanulaceae. Bellflowers. Mostly milky- juiced herbs (shrubs and small trees), with alternate (or oppo- site) leaves; flowers regular or irregular; stamens usually 5, free, or more or less united; carpels 2-5; ovules many; endo- sperm fleshy. Campanula, Lobelia. (Pf. 4:40.) Family 284. Goodeniaceae. Mostly Australian herbs and shrubs, with alternate (or opposite) leaves; flowers usually irregular; stamens 5, free, or cohering above; ovary 2—4- celled; ovules many; endosperm fleshy. Goodenia, Scaevola, Brunonia. (Pf. 45:70.) Family 285. Stylidiaceae. Mostly Australian herbs, with tufted, radical, or scattered and sometimes crowded stem- leaves; flowers usually irregular; stamens 3-2, mostly con- nate with the style; ovary 2-celled, many-ovuled; endosperm fleshy. Stylidium, Levenhookia. (Pf. 45:79.) Family 286. Calyceraceae. South American herbs, with alternate leaves; flowers regular or irregular in involucrate heads; stamens attached to the corolla-tube, anthers free; ovary 1-celled; stigma capitate; ovule 1, pendulous; endo- sperm fleshy. Tone Calycera. (Pf. 4°: 84.) Order AstEraLus. Composites. Flowers actinomorphie or zygomorphic, collected into involucrate heads; calyx small, and often forming a ‘‘pappus’’; stamens 5, epipetalous, mostly with their anthers connate, dehiscing introrsely; carpels 2, united, inferior, with one style which is 2-branched above; ovule one, erect, anatropous; endosperm 0. An immense order (commonly regarded as a family) of about 14,324 species, which are usually distributed among fourteen tribes, all of which are here raised to families. In the following arrangement the Helianthaceae are regarded as the lowest, from which the two principal phyletie lines have arisen, cul- Wie Sse Bs cae te 1915] BESSEY— PHYLOGENETIC TAXONOMY 163 minating on the one hand in the Eupatoriaceae, and on the other in the Lactucaceae. (Pf. 4°: 87.) Family 287. Helianthaceae. Sunflowers. Calyx not capil- lary; receptacle chaffy ; usually with ray flowers; mostly large and coarse plants, with leaves usually opposite. Helianthus, Zinnia, Rudbeckia, Silphium. (Species 1364.) (Pf. 4°: 210.) Family 288. Ambrosiaceae. Ragweeds. Calyx not capil- lary; receptacle chaffy; without ray flowers; mostly large and coarse plants, with leaves usually alternate, flowers diclinous. Ambrosia, Xanthium. (Species 74.) (Pf. 4°: 220.) Family 289. Heleniaceae. False sunflowers. Calyx not capillary; receptacle usually naked; with or without rays; anthers tailless; medium-sized plants with opposite and alter- nate leaves. Helenium, Gaillardia. (Species 449.) (Pf. 45.251.) Family 290. Arctotidaceae. Gazanias. Calyx not capil- lary; receptacle naked; anthers tailless. South African plants with mostly alternate leaves. Gazania, Arctotis. (Species 278.) (Pf. 4°: 307.) Family 291. Calendulaceae. Marigolds. Calyx not capil- lary; receptacle naked; anthers tailed. Old World plants, mostly tropical, with alternate leaves. Calendula. (Species 125.) (Pf. 4°: 303.) Family 292. Inulaceae. Everlastings. Calyx from bracteose to capillary; receptacle usually naked; anthers tailed; usually rayless; mostly low plants, with alternate leaves. Inula, Antennaria, Gnaphalium, Helichrysum. (Species 1580.) (Pf. 4°:172.) Family 293. Asteraceae. Asters. Calyx from bracteose to capillary; receptacle naked; usually with rays. Medium- sized plants, with alternate leaves. Aster, Solidago, Erigeron, Bellis. (Species 1815.) (Pf. 4°: 142.) Family 294. Vernoniaceae. Ironweeds. Calyx from brac- teose to capillary; receptacle naked; without rays; style branches hispidulous. Medium-sized plants, with mostly alter- nate leaves. Vernonia. (Species 788.) (Pf. 45:120.) [VoL, 2, 1915] 164 ANNALS OF THE MISSOURI BOTANICAL GARDEN Family 295. Eupatoriaceae. Blazing-stars. Calyx from bracteose to capillary; receptacle naked; without rays; style branches papillose. Medium-sized plants, with opposite and alternate leaves. Lacinaria, Eupatorium. (Species 944.) (Pf. 4°: 131.) Family 296. Anthemidaceae. Camomiles. Calyx a short crown or wanting; involucral bracts with scarious margins; receptacle chaffy or naked; usually with white ray flowers. Medium-sized plants, with alternate leaves. Anthemis, Chrysanthemum, Artemisia. (Species 915.) (Pf. 4°: 267.) Family 297. Senecionidaceae. Groundsels. Calyx capil- lary; involucral bracts mostly 1-seriate; receptacle naked; flowers mostly yellow, with or without rays. Medium-sized to large plants, with alternate leaves. Senecio, Arnica. (Species 1982.) (Pf. 4°: 283.) Family 298. Carduaceae. Thistles. Calyx mostly capillary; involucral bracts multiseriate; anthers tailed; receptacle usually bristly (not chaffy); without rays. Mostly stout plants, with alternate leaves. Carduus, Arctium, Cnicus. (Species 1563.) (Pf. 4°: 312.) Family 299. Mutisiaceae. Mutisias. Calyx mostly capil- lary ; receptacle usually naked; flowers all two-lipped. Medium to large (even woody) plants, of tropical or warm regions, with mostly alternate leaves. Mutisia, Chaptalia. (Species 550.) (Pf. 4°: 333.) Family 300. Lactucaceae. Lettuces. Calyx mostly capil- lary; receptacle usually naked; flowers all strap-shaped. Small to medium-sized plants, mostly with a milky juice, and with alternate leaves. Lactuca, Hieracium, Cichorium, Leontodon, (Taraxacum). (Species 1701.) (Pf. 4°: 350.) THE BOTANICAL GARDEN OF OAXACA : ZATTI Director of the Botanical Garden of Oaxaca, Mexico I. GENERAL At the end of the year 1909, when I was at the head of the Teachers’ Normal School of the State of Oaxaca, a post which I had held since the middle of 1891, I was asked by the Min- istry of Improvements, Colonization and Industry, at that time under Sr. Lic Don Olegario Molina, to assume the man- agement of the Botanical Garden which was to be established on the grounds of the Agricultural Experiment Station of the same state. This station is situated about four kilometers from the city and had been in operation for only a few months. Professor Don Félix Foéx, the first director of the station, was entrusted with the establishment of the Garden. He had several interviews with me; however attractive the proposition appeared to me, I could not decide to accept it. Finally, after much hesitation, I accepted the new position, and since then I have devoted myself to it entirely, even though success is doubtful; without fear of being contradicted, I can say boldly that I have been everything in the Botanical Garden, laborer, manager, topographer, landscape gardener, clerk, gardener, excursionist, and a hundred other things besides. At the beginning of 1910 there was a general suspension for several months of the activities of the Station. As soon as the work could be resumed I devoted, with the half dozen men that I had at my command, the rest of that year and the whole of 1911 to the preliminary task of levelling, cleaning and adapt- ing, in general, the ground for the new branch of the Station. This was a mistake; I recognize it now when it is too late. I should have insisted that the Botanical Garden, which was to be established on the grounds of the Station, be absolutely independent of the latter, or else I should have refused its management. Unfortunately, I did neither, and to this date ANN. Mo. BoT. GARD., VoL, 2, 1915 (165) [VoL. 2 166 ANNALS OF THE MISSOURI BOTANICAL GARDEN I deplore the consequences of such a serious lack of fore- thought, since, depending on the wills of others with ideas differing from mine, the Garden will never be able to prosper, or will prosper with great difficulties on account of lack of freedom. Having finished the preliminary tasks which I had under- taken, I proceeded to make a sketch of the Garden as shown in fig. 1, which is here reproduced as approved by the authori- ties. As may be seen in the sketch, the Botanical Garden of Oaxaca is still in the process of formation. The tract of land assigned to it consists approximately of nine hectares, an area extensive enough to contain all the most prominent specimens of the mundane flora and all the characteristic specimens of the national flora. Of the three valleys of the Station to the east of the Oaxaca and Ejutla Railroad, the Garden occupies the middle one, which is the one best suited for that purpose and at the same time most accessible. At the beginning it was subdivided into five departments, somewhat unequal in size, together compris- ing a rectangle 400 meters in length (from north to south) by 200 meters in width (from east to west); but later this area was increased by an addition of 3,000 square meters, which was annexed to the southwest corner, and again by a sixth department, semilunar in outline, comprising 5,000 square meters, annexed at the middle part of the west side. Deduct- ing from this total area about two hectares which will be taken up by the prospective lake, walks, and lanes, there remain not more than seven hectares of land which can be utilized for the cultivation of plants. As I have shown in a recent work, the Botanical Garden of Oaxaca is the first and only one worthy of the name in the whole of the Republic. This fact alone, signifying a positive progress, should have been sufficient to enlist the support of the authorities, as well as the public in general; but contrary to what might be expected, its existence has been, especially recently, extremely neglected. I have made this clear in the opinion expressed in my reports to the higher authorities, as may be seen from the following: CONZATTI—BOTANICAL GARDEN OF OAXACA 167 = WILA DLL RAET AN I ‘Sta DE ODW TODLIVAHDOFDAL-LE Il LT 9 $ » IK 3 T EJI = IN 14 3 33 o NSS [è | Ses 2a RIE 3 ae FMA Ble ls |S lz az] SC GERERR i X i x . y j I F BIA::: 1); 2 See EEEE) i ADEM FNE % Salg z "UOIJEULIOF JO ssao0id UT BORXBQ JO UAPICH [VoIULJOg oY} JO YoYoYG WLS Y WPWAVO ec SL oe@eeee# eee tRt o # w e . e ee e ' “LIMITE PROVISIONAL ‘T am not at all satisfied with the progress of the Botanical Garden, especially during the second half of the fiscal year, 1913-1914. f [VoL, 2 168 ANNALS OF THE MISSOURI BOTANICAL GARDEN Receiving no encouragement, lacking entirely means and workmen, its existence has been extremely diflicult, so much so that it would be practically impossible for it to continue under the same conditions for any length of time without failing for want of support. I must not cherish any illusions in this respect, and I consider it my duty to make this clear with all frankness.’ In the same report I point out: ‘Such a difficult situation is due especially to the deplorable condi- tions which have depleted the Public Treasury, and that as soon as the present sad state of affairs disappears (which, fortunately, seems to be already taking place), all the branches of the administration will again receive that encouragement of which they are in such great need.’ And this I believe sincerely, since I have faith in the move- ment which is being started for the salvation of the country and for the restoration of peace. After all, this is the history of the development of every new idea; it is obliged to struggle on its own merits—with danger of being suppressed—against all kinds of difficulties. One of these, and certainly not the least which I have en- countered, has been the predominating instability everywhere, due to the political disturbances which have been ravaging the country for a long time. This circumstance and the abso- lute lack of means have prevented me from making the trips which I had planned in order to bring to the Garden some living plants, which to-day constitute the most pressing need of our institution. I am convinced that the life of the Botanical Garden depends essentially on providing it with plants. Since the departments are really well prepared, the essential thing now is to fill them with plants, preferably with the greatest possible number of specimens of the Mexican flora which are found in the mountains; and the only effec- tive way of obtaining them is to go and get them. As long as this cannot be done, the work of the Garden must be limited to the routine work of preserving what is already there. II. DETAILED DESCRIPTION At the end of 1913, according to the compilation made at that time, the Botanical Garden contained the following 1915] CONZATTI—BOTANICAL GARDEN OF OAXACA 169 plants: 1,099 in the systematic department, 101 in the arbo- retum, 1,158 in the propagation department, and 1,035 in the geographical department and the fruticetum, or a total of 3,393 specimens. For reasons already mentioned, the Botan- ical Garden from then until now has not only remained stationary, since it has received no appreciable additions, but it has also deteriorated a great deal, partly because a great number of plants have dried up from lack of water, and partly because its personnel—reduced to only four workmen— is insufficient to attend to the varied duties which are required. In fig. 1 some of the plants are indicated by black dots as occurring in the outer departments, arboretum and fruticetum, neither of which have any particular shape. GEOGRAPHICAL DEPARTMENT This department, on the contrary, is meant to represent in its main outlines the political map of the State of Oaxaca, the divisions of which are marked with the initial letters of the districts which constitute it. These districts at present are grouped, primarily on the basis of their climatic conditions, into six natural regions, as follows: Central, Cuicateca, Ser- rana, Istmica, Costena, and Mixteca, separated from one another by lanes two meters in width. The edges of these regions have already begun to receive—as a kind of an en- closure—the typical plants of each region, while the interior of each will receive the most characteristic vegetable produc- tions of the exuberant soil (see fig. 2). In accordance with this plan, the central region (fig. 2), which consists of the districts (see fig. 1) E—to the right of O— (Etla), Zi (Zimatlan), M (Miahuatlan), E—to the left of C—(Hjutla), Tla (Tlacolula), and O (Ocotlan), all border- ing on, or similar by their products to, district C (Center), shows now on its perimeter 121 specimens of Ceanothus azureus, a vigorous and elegant shrub of the hills which sur- round the Capitol. The point corresponding to Santa Maria del Tule, a small village in the same region and situated about two leagues east of Oaxaca, is planted with a shrub ‘‘Sabino del Tule’’ 4 [VoL. 2 170 ANNALS OF THE MISSOURI BOTANICAL GARDEN (Taxodium distichum) two meters in height and a direct off- spring—by seed—from its historic parent. Among other things, it has the merit of being the oldest member of the department. The Cuicateca region, consisting of the districts Cui (Cui- catlan), Teo (Teotitlan), and Tu (Tuxtepec), is limited now to 65 specimens of Vallesia glabra, or ‘‘Tree of the Pearls,’’ native of the Canyon of Tomellin. This small collection is characterized by its exuberant growth and uniform size. Of the districts which constitute this region, only Cuicatlan has received a supply of plants—twenty-two different specimens from Quiotepec. Among these are six plants of Bursera sucedanea from Linaloé, called ‘‘ Palo Hediondo’’ (fetid stick) by the natives of that place. Three districts form the Serrana region, Ix (Ixtlan), V. A. (Villa Alta), and Ch (Choapam); only very recently I have planted around these, 81 specimens of Cerocarpus fother- gylloides, a beautiful rustic little tree which is native of this region. The perimeter of the Istmica region, composed of the dis- tricts J (Juchitan) and Te (Tehuantepec), was also planted in a similar manner with some 34 specimens of an arboreal Pereskia, new to science, from the coast of Salina Cruz. In the district of Tehuantepec I have planted 30 plants coming from the same region and belonging to about a dozen species in several genera—Stemmadenia, Pedilanthus, Mimosa, ete., and in the district of Juchitan species of several genera of the Cactaceae—Opuntia, Cereus, Mamillaria, Selenicereus, Echinocactus, ete.—have been planted. On the southern side of this department there are planted 40 palm-trees, species of Phoenix, about two meters high, bordering a walk which bears the name of the famous Bra- zilian botanist, Barbosa Rodrigues; while on the north side runs another walk, five feet wide, called ‘‘ Andres Cesalpino,’’ along the edges of which we have planted 148 specimens of Poinciana Conzattui Rose, brought from Tehuantepec. Finally I shall mention the collection of Mexican agaves 1915] 171 BOTANICAL GARDEN OF OAXACA CONZATTI -—Kaisehg — i CALZADAS Y ei, > ae CENTROS DE IRRIGACION S \ wi [ J Pro agac i Wiad; amada Bon Piang | Fraticetum 4 àa t= D L Tolipetalas m Tol! pe talag 4 Tol! petalas 4 ev £y = vod 2 MM 5 Region Cantray a Ù Y x S r Diesti-/ Yedorne, g Lago bg + +9 Region Mixteca y Dapartmerde 2 Geografico LIEFERT Frutceran |lDETARTMENTO LAGO |NEPARTMENTO ARRBorTtTaum GEOGRAPHICO SISTEMALTICO. Fig. 2. Walks and irrigation centers in the Botanical Garden of Oaxaca. which are in the district H (Huajuapam) of the Mixteca region, as well as the fact that it is planned to introduce into [VoL, 2 172 ANNALS OF THE MISSOURI BOTANICAL GARDEN this department various groups of practically useful plants —industrial, tinctorial, poisonous, medicinal, ete. DEPARTMENT OF PROPAGATION This department is situated in the middle eastern part of the Botanical Garden and comprises an area of not more than half a hectare. Its shape is that of a semicircle bounded on its convex side by the Adolf Engler walk; this is the name of the famous author of the classification adopted by the Garden, with few very slight exceptions suggested by the ‘Lexicon Generum Phanerogamarum’ of von Post and O. Kuntze. The sides of this walk are planted for the time being with various specimens of Melia Azedarach, but in the near future these will be replaced by specimens of ‘‘Rosa-Cacao,’’ an imposing pyramid-like tree with horizontal and vertical branches. As indicated by the name, this department is devoted to the propagation of plants for this Garden and similar establishments in this and other countries. WALKS AND IRRIGATION CENTERS Of the walks of the Garden, the one called ‘‘Carlos Linneo’’ forms the western boundary line of the Garden and serves it, so to speak, as a base. It is a straight line 420 meters long, running from north to south, parallel to the Oaxaca and Ejutla Railroad, and throughout its length there are, five feet apart, 84 specimens of Casuarina stricta about three meters in height. Two other walks worth mentioning on account of their width (10 meters) are the Asa Gray and the John Lindley walks; these run along the outer side of the systematic department and have as a border 105 laurels from India, as yet rather small. One of the far-reaching improvements for the progress of the Botanical Garden has been the establishment of a prac- tical irrigation system, which was first introduced at the end of 1913 and developed later as shown in fig. 2 For this purpose we first laid under the ground 400 meters of 24-inch pipe through the center of the Garden from the large circular tank, situated on the southern slope, to the wide 1915] CONZATTI—BOTANICAL GARDEN OF OAXACA 173 avenue leading from the Station building on the north. This was the main artery and at fixed points, which were carefully selected beforehand, crosses were placed to mark the respec- tive connections. These consisted of lateral ramifications of smaller pipe which were to carry the water to the 35 irriga- tion centers, 50 meters apart, into which the Garden is sub- divided. All these centers must have nozzles, and at present there are 18 of them in working order; these are marked with crosses in fig. 2. To install them we have used 500 meters of smaller piping, so that a similar amount, if not a little more, would be required to complete the network. Of these irrigation centers eight belong to the arboretum, twelve to the systematic department, seven to the geographical depart- ment, five to the fruticetum, and three to the propagation department. As soon as the Botanical Garden has completed its irrigation system and has a sufficient supply of water for all seasons, we shall be able to consider its existence as assured. SYSTEMATIC DEPARTMENT Together with the two preceding departments, the geo- graphical and propagation departments, the systematic de- partment constitutes the central part of the Garden, and from the botanical point of view is the most interesting of them all. Many plants have already been planted in it, as may be seen in pl. 3, which represents the central part of the department ; but the empty places are still numerous, and the need of having them planted is great. The shape of this department is that of an immense cup, 200 meters long and measuring 145 meters at its widest part. As I have shown in a previous paper, which was published some time ago in the ‘Memorias y Revista de la Sociedad Cientifica ‘‘ Antonio Alzate,” ’ of Mexico, and to which I now refer for a better presentation of this subject, “its interior is subdivided into 45 large squares approximately equal, among which are distributed the 277 phanerogamie families of the ‘‘Syllabus’’ of Dr. Engler.” The plants in this department, therefore, are arranged strictly in the order of affinity, [VoL. 2, 1915] 174 ANNALS OF THE MISSOURI BOTANICAL GARDEN namely, vascular cryptogams and monocotyledons at the base, followed in order by the dicotyledonous groups, Apetalae, Polypetalae, and finally the Gamopetalae. With the latter the lineal series is closed, since according to the consensus of modern opinion they constitute the most highly differentiated group of flowering plants. In the preceding lines I have endeavored to condense the most prominent features relative to the life of the Botanical Garden of Oaxaca. They are totally without pretense on my part, although they would wish to carry to the minds of all those who may read them the same high concept which I myself have formed of such a progressive institution. In spite of the discouragement that I often feel about the Garden, I have confidence in its final success. Everything indicates that to-day the Republic is approaching rapidly a better era, which will be effected through organic peace and progress in its truest sense, since the horizon appears already free from the dark clouds. In concluding, I wish to say that the Botanical Garden of Oaxaca, after showing itself in the preceding lines in all its smallness, has the honor of sending its older brother, the Missouri Botanical Garden of St. Louis, its most cordial con- gratulations for the Twenty-fifth Anniversary, wishing it long life and abundant prosperity. EXPLANATION OF PLATE PLATE 3 General view of the Botanical Garden of Oaxaca, Mexico, particularly of its Systematic Department. PLATE 3 5 2, 191 Ann. Mo. Bor. Garp., VoL. 4t 2 CONZATTI—BOTANIC GARDEN OF BOSTON COCKAYNE, OAXACA THE ORIGIN OF MONOCOTYLEDONY II. Monocotyledony in Grasses J. M. COULTER The University of Chicago Recently Dr. Land and I published! the results of an in- vestigation suggested by a specimen of Agapanthus umbel- latus, one of the South African Liliaceae, possessing two good cotyledons. It seemed to us that if the seedlings of the same species are indifferently monocotyledonous or dicotyledonous, there must be some evident relationship between the two con- ditions. These two conditions of the seedling of Agapanthus were compared critically, and Sagittaria was included in the investigation because it has stood, along with Alisma, for the typical monocotyledonous embryogeny, in which the terminal cell of a filamentous proembryo is said to give rise to the single cotyledon, in contrast with the dicotyledonous embry- ogeny, in which the corresponding terminal cell produces the stem tip, and the cotyledons are distinctly lateral. No con- trast would seem sharper and less capable of being confused with intergrades. The result of the investigation, as recorded in the paper referred to, was to show us that there are no such rigid cate- gories for cotyledony; that the cotyledonary apparatus is al- ways the same structure, arising in the same way, and vary- ing only in the details of its final expression. Briefly stated, the situation is as follows: In the embryogeny of both mono- cotyledons and dicotyledons, a peripheral cotyledonary zone gives rise to two or more growing points, or primordia; this is followed by zonal development, resulting in a cotyledonary ring or sheath of varying length. If both growing points con- 1 Coulter, John M., and Land, W. J. G. The origin of monocotyledony. Bot. Gaz. 57; 509-519. pl. 28-29. 1914. ANN. Mo. Bot. GARD., VOL. 2, 1915 (175) [VoL. 2 176 ANNALS OF THE MISSOURI BOTANICAL GARDEN tinue to develop equally, the dicotyledonous condition is at- tained; if one of the growing points ceases to develop, the con- tinued growth of the whole cotyledonary zone is associated with that of the other growing point, and the monocotyledon- ous condition is attained. In like manner, polycotyledony is simply the appearance and continued development of more than two growing points on the cotyledonary ring. It fol- lows that cotyledons are always lateral structures, arising from the peripheral zone developed at the top of a more or less massive proembryo. This reduces cotyledony in general to a common basis in origin, the number of cotyledons being a secondary feature. The constancy in the number of coty- ledons in a great group is no more to be wondered at than the same constancy in the number of petals developed by the petaliferous zone. This is a brief statement of the thesis of our previous paper, detached from the evidence upon which it was based. It was our purpose to extend the investigation far enough to include all of the representative regions of monocotyledons, so that the conclusion could be tested sufficiently to lead either to its abandonment or to its establishment. This second paper deals with a study of the embryos of grasses, which have been examined more extensively, perhaps, than the embryos of any other monocotyledonous group. As a result of this extensive study there are available many accurate records in the form of good figures, giving the details of embryogeny in such a way that interpretation is almost as satisfactory as it would be from the actual material. Of course this use of illustra- tions has been checked by the direct inspection of more or less material. The embryo of grasses early attracted special attention be- cause it does not seem to conform to the plan of the ordinary monocotyledonous embryo. Certain structures appear that could not be accounted for, but they enriched terminology. As a consequence, the nature of scutellum, epiblast, and coleoptile became subjects of discussion. It was to be expected that 1915] COULTER—ORIGIN OF MONOCOTYLEDONY 177 the embryo of grasses, with all of its unusual structures, would be interpreted in terms of a rigid conception of the monocotyledonous embryo; in other words, that the conventional monocotyledonous em- bryo would be read into the grass embryo. There is no better illustration of the com- pelling power of a preconception than this treatment of the grass embryos, for it so happens that they show all the intermedi- ate stages between dicotyledony and mono- Fig. 2. Em a: of Zizania aquat- ica: s, scutellum; e, epiblast ; c, cole- optile; s iL — After Bruns. cotyledony. Very early in the history of this subject, the scutellum came to be recognized as a cotyledon. The corollary to this proposition, however, was that it must be recog- nized also as a terminal structure. Any one who has seen the vascular system of the embryo of corn (fig. 1), the most highly specialized of all grass embryos, with its distinct axial cylinder, made up of stem cylinder and hypocotyl cylinder, and the cotyledonary strands lead- ing off from the intermediate cotyledonary plate, just as do the strands of any lateral cotyledons, will understand the great difficulties in the way of interpreting this cotyledon as a terminal structure. The structure which pre- Fig. 1. Embryo of Zea Mays: s, scutellum ; c, cole- (scutellum) from the cotyledonary vascular plate; op- posite the vascular connection of the cotyledon ae ap- oup of nie gpa cotyle Baal); x 18. sented the greatest difficulty, however, was the epiblast, usually defined as a small scale ‘‘opposite’’ or ‘‘over against’’ the [VoLr. 2 178 ANNALS OF THE MISSOURI BOTANICAL GARDEN cotyledon. The definition is accurate, for the epiblast occupies exactly the place of a second cotyledon opposite the large and functional one (fig. 2). If some one had found an epiblast vigorous enough to establish vascular connections, this debated structure would long since have been accepted as a second cotyledon, for the definition of it al- ways emphasized the fact that it is a scale in the right position for a cotyledon, but with ‘‘no vascular strands.’’ So obvious is the interpretation of the grass embryo when an epiblast is developed that Porteau in 1808, Mirbel in 1809, Turpin in 1819, and Bischoff in 1834, all called the epiblast a rudimentary cotyledon. The sub- mergence of this idea seems to have been due to Schleiden, who in 1837 dissented from this view, and it disappeared from literature. It reappeared in 1897, when Van Tieghem, in his paper on the embryo of grasses and sedges,! reiterated it, based chiefly upon the study of vascular connections. Any series of sections, cross or longitudinal, through the em- bryos of grasses, shows the fol- lowing facts: the so-called scutel- lum or functional cotyledon arising from the peripheral coty- ledonary ring or sheath which surrounds the apex of the em- bryo, and establishing vascular connections laterally with the Fig. 4. Embryo of Oryea setiva: cotyledonary plate; the epiblast s, scutellum; e, epiblast; o, cole- A u : optile; X22.—After Bruns. in a similar relation to the coty- ledonary ring on the opposite side, and varying in develop- ment from a structure somewhat smaller than the large cotyledon, to complete suppression; and the apex of the After Bruns. ıVa eghem, Ph. Morphologie de l’embryon et de la plantule chez les Graminées et les Cypéracées. Ann. d. Sci. Nat., Bot. VIII. 3: 259-309. pl. 14-16. 1915] COULTER—ORIGIN OF MONOCOTYLEDONY 179 embryo, continuing beyond the cotyledonary ring or sheath, and producing a variable number of leaves. The early appearance and rapid develop- ment of these leaves seems to account for the abortion of one of the growing points. I am convinced that if grass embryos had been the only monocotyledonous embryos studied; we should never have heard of terminal cotyledons. ome common grasses, whose embryos have been figured by Bruns,! may be used to illustrate stages in the abortion of the second cotyledon. The abortion always is accompanied by the diver- sion of the growth of the Fig. 5 Embryo of Spartina cyno- After Bruns. Fig. 6. Embryo of Leptochloa arab- ica: s, scutellum; e, epiblast; o, > optile; X 44. After Brun ipheral cotyledonary ring as does the so- called scutellum, and attaining at least one- quarter to one-third of its length. This unusual development of the second cotyledon is associated with the fact that the stem axis above the cotyledons develops a long internode, so that the first leaves begin to appear at an unusual distance from the origin of the cotyledons. In fact, in this whole cotyledonary zone in connection with the growing point that remains active; so that growing tissue is not suppressed, but develops as one structure rather than as two. In Zizania aquatica (fig. g-.-.-.. 2), the so-called epiblast is very conspicuous, arising as distinctly from the per- case the length of the second cotyledon is Fig. 7. Embryo of approximately the length of the first inter- Triticum gorge s, seutellu en um; pi- node, and where the leaves begin this coty- blast; c, coleoptile; x22.— After Bruns. ledon ends. In Leersia clandestina (fig. 3), the second cotyledon (epi- blast) approaches the large cotyledon in length even more 1 Bruns, Erich, Der Grasembryo. Flora 76: 1-33. pl. 1-2. 1892. 180 (VOL, 2 ANNALS OF THE MISSOURI BOTANICAL GARDEN than does that of Zizania, and all the connections of the various organs show a lateral origin for the cotyledons, and a terminal origin for the ‘‘coleoptile,’’ a structure made up chiefly of leaves arising from an indistinctly differen- tiated stem-tip region. Oryza sativa (fig. 4) is interesting in the relation of the parts of the embryo, the ‘‘scutellum’’ and ‘‘epiblast’’ being opposite and well-balanced structures, between which the prominent plumule Transverse section n (s), show- Fig. 8. sep cotyledo ing it embracing the plumule d that a bundle opposite that of the te is miss- ing, but = rudiment is evi- dent in lower section; x 20. (a name ex- pressing the real char- acter of the tina cyno- suroides blast) is less promi- nent, but its relation to the functioning cotyle- don, and the relation of both to the plumule are evident. In Leptochloa arabica (fig. 6) and in Triticum vulgare (fig. 7), the epi- blast remains very small, but the significant connections are evident. It is in the embryo of Zea Mays that this reduction series reaches its extreme expression in the complete disappearance of the epiblast or second cotyledon (fig. 1), whose position is indicated merely by more or less protuberant Fig. 9. Transverse section through the pp Song ee plate Zea Mays: the functioning cotyledon (s) Ps not overlap small are pe so rhe pert the site of the miss- otyledon (epiblast), as in- dicated also by the Sea of a procambium ma (a), which is the enp Ternary of former vascular connection; x 20. 1915] COULTER—ORIGIN OF MONOCOTYLEDONY 181 tissue and by the very obvious vascular relations. A cross- section of this very specialized embryo is instructive (figs. 8 and 9). The large functional cotyledon is seen originating on one side, embracing the vascular axis of the embryo and more or less overlapping the other side, where in most grasses the second cotyledon (epiblast) appears. Moreover, in the section of the centrally placed plumule, with its succession of leaves, a section of the stem tip may be seen, clearly representing the axis of the embryo, with no suggestion of a lateral origin. A transverse section through the cotyledonary plate (fig. 9) shows some tissue developed at the site of the missing cotyle- don (not overlapped by the functioning cotyledon). This is emphasized by the appearance of a mass of procambium at the base of the protuberance, which in other grasses develops into the epiblast. This procambium is distinctly a rudiment of a former vascular connection. Some idea of the frequency with which the second cotyledon appears among the grasses may be obtained from the excel- lent work of Bruns on the grass embryo, published in 1882, and from the work of Van Tieghem, already cited, published in 1897. Bruns examined 82 genera, representing 12 tribes. In 29 of these genera epiblasts were present, and the genera represented 9 of the 12 tribes. The tribes in which no epiblasts were found were Oryzeae, Agrostideae, and Aveneae. The situation in the Agrostideae is noteworthy, for 13 genera were examined, and no trace of an epiblast found. Festuceae may be mentioned, for 20 of its genera were examined, and only 4 of them were found to possess epiblasts. Taking Bruns’ re- sults as a whole, they indicate that approximately 40 per cent of the grasses still develop a second cotyledon to a stage that enables it to be recognized under ordinary inspection as a definite structure. The work of Van Tieghem included a somewhat wider range of forms, 91 genera being examined, and 61 of these showed epiblasts. This suggests that perhaps in as many as two- thirds of the grasses a second cotyledon is more or less ob- vious. In any event, it is certain that the grasses as a whole exhibit a remarkable number of transition stages from dicoty- (VoL, 2 182 ANNALS OF THE MISSOURI BOTANICAL GARDEN ledony to monocotyledony; and this fact strongly supports the view that grasses are a comparatively primitive assem- blage of monocotyledons. It is not difficult to explain the prolonged misconception concerning monocotyledony. When the first detailed studies of monocotyledonous embryogeny were made by Hanstein, and supplemented by Famintzin, a form (Alisma) with a fila- mentous proembryo was selected. If a form with a massive proembryo had been selected for these early investigations, there would probably have been no misconception, for in such proembryos the peripheral (that is, lateral) cotyledonary zone is so evident that it could hardly have escaped recognition. Since that time, embryogeny that starts with a filamentous proembryo has been regarded as the typical embryogeny, and all other kinds of proembryos have been dismissed as excep- tions. In the case of this filamentous proembryo, it was ob- served that the terminal cell passed into the quadrant and octant stages, and later a terminal cotyledon appeared. It seemed safe to conclude that the terminal cell had developed the terminal cotyledon. The inference was true so far as it went, but it failed to recognize the fact that the terminal cell develops other structures as well. With the origin of the terminal cotyledon disposed of, the conclusion was confirmed by the appearance at its base of a notch, from which arose the stem tip. What could be more obvious than that the stem tip is lateral in origin, and therefore must arise from the cell of the proembryo behind the terminal one? In this way the conventional embryogeny of monocotyledons was established, and the relation of monocotyledony to dicotyledony became completely obscured. The facts not observed in these earlier investigations are as follows: The terminal cell of the proembryo forms a group of cells; the peripheral cells of this group develop the cotyle- donary ring or sheath, on which two growing points appear. One of these growing points soon ceases to be active, and the whole zone develops in connection with the other growing point; but at the base of the growing cotyledon a notch is left by the checking of the other growing point. This notch 1915] COULTER—ORIGIN OF MONOCOTYLEDONY 183 is really the space between the two very unequal cotyledons, which surround the real apex of the embryo. The apex of the embryo is at the bottom of the notch, and not at the tip of the large embryo. This apex soon begins to form leaves, and the so-called stem tip appears issuing from the bottom of the notch, in a relation apparently lateral only because the two cotyledons are so unequal. Furthermore, when the stem tip is examined, it is found not to be a stem tip, but a cluster of leaves whose rapid development has aborted one of the grow- ing points on the cotyledonary zone. All this is very obvious in grasses, and is equally obvious in any massive proembryo, but it escaped the earlier observers of filamentous proembryos. The general conclusion is that monocotyledony is simply one expression of a process common to all cotyledony, gradually derived from dicotyledony, and involving no abrupt transfer of a lateral structure to a terminal origin. This paper was prepared in collaboration with Dr. W. J. G. Land, who also supplied the material and made the illus- trations. THE HISTORY AND FUNCTIONS OF BOTANIC GARDENS ARTHUR W. HILL, M.A., F.LS. Assistant Director, Royal Botanic Gardens, Kew There are three things which have stimulated men through- out the ages to travel far and wide over the surface of the globe, and these are gold, spices and drugs. It is to the two latter of these universal needs of man that we may trace the origin and foundation of botanic gardens. The value of spices has led to the foundation of more than one botanic garden in the tropics, while to the necessity for drugs must be attributed the formation of the earliest botanic gardens in Europe. Before entering more fully into the history of the found- ing of the various botanic gardens it may be pointed out that progress in the science of botany and the establishment of gardens were by no means contemporaneous. To the Greeks, for instance, we owe the foundation of our knowledge of the classification of plants, and these early botanists were assidu- ous in collecting plants from all available sources and in drawing up accurate descriptions. Little interest, however, would appear to have been aroused in them to cultivate the plants they so carefully de- scribed, and the only record we have of the existence of any- thing of the nature of a botanic garden is the mention of Aristotle’s Garden at Athens which he bequeathed to Theo- phrastus, by whom it was newly equipped and improved. Prior to the interest displayed by the Greeks in the vegeta- tion of the earth and quite independent of their influence we find evidence of the formation of gardens in Egypt, Assyria, China, and subsequently in Mexico—gardens not strictly botanic in our more modern sense but enclosures’ set apart 1 See Greene, E. L. Landmarks of botanical history. Smithsonian Mise. Coll. 54': pp. 56-57. 1909. No doubt Theophrastus (370-286 or 262 B. C.) gained his intimate knowledge of plants very largely from the specimens cultivated in this early Athenian garden. (185) ANN. Mo. Bot. GARD., VOL. 2, 1915 [VoL, 2 186 ANNALS OF THE MISSOURI BOTANICAL GARDEN for the cultivation of plants of some definite economic or aesthetic value. In considering the history of this subjeet we look back to the earliest history of mankind, with which gardening in some form is inseparably connected, for, as Francis Bacon reminds us: “God Almightie first pes a Garden and indeed it is the Purest of Humane Pleasures. It is the greatest refreshment to the spirits of man; Aai which Buildings and Palaces are but grosse Handyw orkes: and a man shall ever see that when ages grow to civility and elegancie men come to Build stately sooner than to garden finely as if gardening were the greater Perfection.’ We are still exercised to seek out and grow ‘‘every tree that is pleasant to the sight and good for food,’’ and the ‘‘tree of life’’ also in the midst of the garden is ever the object of our inquiries. It would be well indeed if at this present time we could discover that tree whose leaves were to be ‘‘for the healing of the Nations.’’ The earliest garden of which we have any representation is the Royal Garden of Thotmes mt of about the year 1000 B. C., which was planned by Nekht, head gardener of the gardens attached to the Temple of Karnak.! This Royal Garden, rectangular in outline, with its rows of date and branched doum palms and with its vine pergola and lotus tanks, was probably in the nature of a pleasure garden, while those at- tached to the temples may well have been of more economic importance. The Chinese,? however, should, as might be supposed, be credited with being the real founders of the idea of botanic gardens, since it is clear that collectors were de- spatched to distant parts and the plants brought back were cultivated for their economic or medicinal value. The semi- mythical Emperor Shen Nung, of the twenty-eighth century B. C., is considered to be the Father of Medicine and Hus- bandry and is said to have tested the medical qualities of herbs and discovered medicines to cure diseases. If this be 1 See Holmes, E. M. Horticulture in relation to medicine. Roy. Hort. Soc., Jour. 31: pp. 44-45. f. 11. 1906. * Bretschneider, E. Botanicon sinicum. China Branch Roy. Asiatic Soc., Jour. N. S. 25: p. 24. 1893. 1915) HILL—BOTANIC GARDENS 187 correct, it was but a repetition of history which led to the foundation of the monastic herb gardens in the ninth century A. D., and the subsequent institution of botanic or herb gar- dens in connection with the medical faculties of the earliest European universities. We learn from Bretschneider also that the Han Emperor Wu Ti (140-86 B. C.) planted a number of rare herbaceous plants and trees brought from the southern regions in the garden of his palace and the following plants have been identified from the list enumerated: Nephelium Litchi, N. logan, Areca Catechu, the banana, Quisqualis indica, Cana- rium album, C. Pimela, Cinnamomum Cassia, Canna indica, and sweet oranges. He also despatched officers to the north- western frontiers of China, who brought back reports on the productions of this region. Ancient Chinese authors ascribe to Wu Ti the introduction of the vine, pomegranate, saff- flower, common bean, cucumber, lucerne, coriander, walnut, ete. It is a fact of no small interest in this connection to remem- ber that the modern world has turned to China and that her vast botanical treasures have only recently been seriously explored through the enterprise of British, French, and American botanists for the enrichment of our botanic gar- dens and pleasure grounds. The establishment of gardens in Mexico is a noteworthy fact—though we have but little information about them— since their origin must have been autochthonous and inde- pendent of such institutions in the Old World. Prescott? tells us, and we have reason to believe his account to be true, that Montezuma had extensive gardens filled with fragrant shrubs and flowers and especially with medicinal plants. New Spain, indeed, furnished more important species of medicinal plants perhaps than any other part of the world, and their virtues were understood by the Aztecs, who are credited with having studied medical botany as a science. The gardens at Iztapalan? and Chalco? are said to have been stocked with * Prescott, W. H. Conquest of Mexico 2: pp. 110,111. 1847. [3rd ed. London.] * Ibid. pp. 60 and 61. Ibid. 3: p. 37. 1847; Clavigero, D. F. S. Stor. del Messico 2:p. 153. [VoL. 2 188 ANNALS OF THE MISSOURI BOTANICAL GARDEN trees and plants scientifically arranged, and the gardens at Chaleo, which were preserved after the Conquest, furnished Hernandez with many of the specimens described in his book." The cases cited, however, have little more than an academic interest for us and have in no way influenced the founda- tion of modern botanic gardens. These we can trace back to monastic institutions and probably to the famous injunc- tions of Charlemagne,” the direct outcome of which was the establishment, among others, in the ninth century, of the “hortus” at St. Gall with the attendant ‘‘herbularis,’’ or Physic Garden, this latter being the precursor of the physic gardens established in connection with the medical faculties of the Italian and other universities in the sixteenth century. It is fortunate that we have preserved to us exact details of the ““hortus’’ and ‘‘herbularis’’ at St. Gall, with lists of the plants cultivated therein.? The hortus was an oblong enclosure containing eighteen rectangular beds, while the Physic Garden, or herbularis (see fig. 1), formed a square set with similar beds and having the doctor’s house close at hand. The monks being bound to live on pulse, vegetables and fruits and to gather the same for themselves, the garden and its cultivation were of especial importance in the monastery. To the fostering care of the monks and to their knowledge of drugs, horticulture and botany, in common with other arts and sciences, we owe a debt the magnitude of which it is difficult to estimate. We do well to recall at this point the services rendered in recent years to the biological sciences by the labors of Gregor Mendel in the monastic garden at Brunn, if only to emphasize how widespread and far-reaching are the functions involved in the true idea of the botanic garden. The fourteenth and fifteenth centuries, as is well known, were times of a great revival and interest in learning, and 1 Hernandez, F. Nova plantarum animalium et mineralium Mexicanorum historia. Rome, 1651. Holmes, E. M. Horticulture in relation to medicine. Roy. Hort. Soc., Jour. 31:p. 50. 1906 ® Archaeological Inst., Jour. 5: p. 113; see also Amherst, A. History of gardening in England p. 5. 1896. [2nd ed.] 1915] HILL—BOTANIC GARDENS 189 the science of botany received its due share of attention. Unfortunately, energy was chiefly employed in attempting to identify the plants named by the Greek writers with those of Western Europe and progress in the science was only fitful. The compilation of herbals was the main occupation a 2 : ees Bi =, gis 3 b T D SA eee i See | 45 16 Fig. 1. Monastery of St. Gall. Physic Garden: 1, Fasiolo; 2, Sat aregia; 3, Rosas; 4, Sisimbria; 5, eig 3 Lu bestico; ts Penieulum; 8, eae 9, Lilium; 10, Salvia; 11, Ruta , Gladiola; 13, Pulegium 14, Fenugraeca; 15, Men Ar 16, ann no. The Ce emetery contai ned apples, pears, peaches, mulberries, plums, laurels, figs, hazelnuts, service, chestnuts, medlars, quinces, almonds, and walnuts of industrious botanists and many of these works, though of little botanical value to-day, can be treasured by us as store- houses of artistic beauty. With the real growth in the knowledge of plants and their uses there grew up also a mass of superstitious information, [VoL. 2 190 ANNALS OF THE MISSOURI BOTANICAL GARDEN partly founded on old tradition, increased with the importa- tion of strange drugs,! and partly no doubt invented by the herbalists and drug-sellers to prevent any infringement of their monopoly in plants of real or supposed medicinal virtue, and to frighten the ignorant from attempting to collect the plants for themselves. The faint resemblance of the mandrake root to the human form, for instance, probably suggested its use as a remedy for sterility; it is still sold to-day in Egypt as a charm. Its use may have led to the discovery of its anaesthetic qualities since it was used in ancient times for this purpose, and the legends which abounded as to the danger of death to those who gathered the root may have been circulated in order to try to prevent its use for criminal purposes. It was largely owing to the need of protecting the doctor and apothecary against the drug-sellers that the growing of ‘‘simples’’? in recognized gardens had its origin. As the seats of the medical profession were established in the uni- versities and monasteries, these institutions set apart definite enclosures for the cultivation of medicinal herbs, the ‘‘sim- plicia”’ or ‘‘simples’’ from which the ‘‘remedia composita’’ were prepared by the apothecaries. Since the universities and monasteries were generally situated in towns, their physic gardens were usually small, and on the continent of Europe we still see these ancient gardens, which have been gradually transformed into the botanie gardens of the universities. In connection with the growth of learning and increase of observation which is noticeable in the arts and sciences at this time of renaissance, it is strange that biology was still so largely under the thrall of superstition and curious invention. Reference to the early herbals, such as the ‘Buch der Natur’ (1475), the ‘Herbal of Apuleius’ (1484), and the ‘Grant Her- bier’ (1526), shows both as regards text and illustration a per- sistent state of ignorance of facts, which could easily have been remedied by observation, and possibly does not represent * Medicinal plants were imported from the Continent in a dry state, hence the English word “drug,” which is part of the Anglo-Saxon verb “drigan,” to dry. 1915] HILL—BOTANIC GARDENS 191 the true state of the knowledge of the more competent medical botanists of the period. The herbal of Brunfels (1530), with its beautiful and accurate illustrations but indifferent text, and those of Bock, Fuchs, Cordus, and many others, may be taken as evidence of the rapid advance that was taking place in the knowledge of plants, though the fabulous and mythical still found adherents even amongst the most learned. Private physic gardens, as distinct from the monastic herb- aries, existed towards the end of the fifteenth century, and some of these developed into municipal gardens for the grow- ing of ‘‘simples.’’? The botanic garden at Padua, which ap- pears to have been one of the earliest of these gardens, was founded in 1545 on the exact spot which it now occupies near the church of S. Antonio and S. Giustino. The garden owes its origin to the sound suggestion put forward at the end of the year 1542 by Francesco Bonafede, who in 1533 had founded the chair of ‘‘simples’’ (Lectura Simplicium)—the first in Europe—at the University of Padua. This garden is of especial interest, as not only have we an excellent account of it written by de Visiani,' but also because it is preserved very largely in its original condition. The cir- cular wall by which it is enclosed, though not the original one built in 1551, oceupies the same site, and was rebuilt between 1700 and 1707; within the wall the garden is laid out in numerous little beds with stone edgings. The garden under- went many vieissitudes and fell into considerable decay, but in the year 1837 it was thoroughly restored, and the arrangement of the beds may well be a restoration of the original condition of the garden. In any case it affords an excellent example of the type of geometrical garden illus- trated in horticultural books published at the end of the sixteenth and beginning of the seventeenth centuries,” which for so long a time dominated garden design on the Continent. 1 de Visiani, R. Dell’ origine ed anzianita dell’ orto botanico di Padova. Padua, 1839. Saccardo, P. A. L’orto botanico di Padova nel 1895. pl. 1-8. f. 1. Padua, 1895. 2 See illustrations of the gardens of De Vries, 1580-1583, reproduced by Sir F. Crisp in ‘Illustrations of some mediaeval gardens,’ 1914; and cf. Mariani, Flori- legium renovatum et auctum. Frankfurt, 1641. 192 [VoL. 2 ANNALS OF THE MISSOURI BOTANICAL GARDEN It is to be regretted that the principal features have been somewhat obscured by the growth of trees, but the ground plan fortunately remains unaltered. Pisa in 1544, Bologna in 1547, and others,! quickly followed the lead given by Padua. We are fortunate in possessing an elaborate plan of the Pisan garden published in Tilli’s catalogue? of 1723 with a list of the plants cultivated in the various beds and enclosures, the latter being here reproduced. Pe ae O RER ER N EXPLICATIO Topiarium magnum instar Tentorii, rh ie Arboribus flexilibus, & ferreis .. circumdatum. Umbraculum primum venustum, opere topiario, Citris Ar- boribus, ac Citroidibus poma suaveolentia ferentibus instruc- tum, & fontibus ornatur mbraculum alterum ET hae repletum. Baron pro Plantis Americar um. Locus pro Plantis Aegyptiis aquam respuentibus. aporarium cum laminis vitreis xum ad semina bk sepa . Platea cum variis Aloés Plantis. Nemus exoticarum, & en ala Arborum. Hydrophylacia, seu Caste Locus pro Plantis ehe & Sylvestribus. . Laboratorii Chimici, in quo Anthlia Pneumatica reperitur, pars externa Hortum respiciens. Supra verö extat infundi- bulum ad pluviam Be de qua fuse D. G. Derham in suis Pea a irp ibis , ac etiam in Demonstr. cap. iii. pag. 23. mentionem facit; plien pars externa variis fontibus, ac lapidibus figuratis est ornata; ibi scilicet reperiuntur As- troites, qui in Metallotheca Mercati pag. 235. & Corallites, Sardinia, aut ex Jamaica, ut Rayus Hist. Tom. iii. pag. ex Sloanis verbis, adhuc nescimus. 1 Botanic gardens were founded in Zurich, 1560; ip 1568; Leyden, 1577; Leipzig, 1579; Montpellier, 1598; Paris, 1597, known as Jardin des Plantes after 1635; Heidelberg, before 1600; Giessen, 1605; Strasburg, a Oxford, 1621; Jena, i Upsala, 1657; Chelsea, 1673; Berlin, 1679; Edinburgh, 1680; Amster- dam, 168 See also foot-note, p a er M. A. Catalogus Saiar Horti Pisani. Florence, 1723. 1915] HILL—BOTANIC GARDENS 193 16. Pergulae Laurorum. ' 17. Prunorum diversae Sp murum tegentes. 18. Aurantiorum Arbo ivisiones Plantarum secundum earum propriam naturam, areolis 19. Locus Herbis tantum Hortensibus repletus. 20. Locus Acanaceis Plantis. 21. Locus Plantis Umbelliferis. 22. Locus Plantis Palustribus. 23. Locus Plantis Venenatis. 24. Locus Plantis Odoratis. 25. Locus Plantis Bulbosis. 26. Florilegii locus. 27. Vaporarium fixum, ac fimo An repletum, ubi Ananas, & similes Plantae and ae aluntur 28. Ostium 29. Ostium Eaborakoris Chimici, ubi Anthlia reperitur, aditum respiciens.! 30. Ostium alterum Horti publiei: intus insignium Botani- corum Virorum effigies visuntur. 31. In Tecto Infundibulum pluviam recipiens. 32. Paries Aurantiis Hermaphroditis ornata. 33. Platea. 34. Ubi Muscae odoratae D. Chimentelli oriuntur. 35. Aditus qui ad ostium Viae publicae dueit: ibi Balenae, & Physeteris ossa suspensa, ut pagina 4. hujus Catalogi, ubi de Agarico agitur 36. Fenestrae Domus Custodis. 37. Fenestrae Musei In earum medio Inscriptio haec legitur. The beds at Pisa are arranged on the geometrical plan and the pieture of the garden shows a perfect specimen of the typical formal garden of the end of the sixteenth century. The plants were grouped chiefly according to their properties and morphological characteristics: Thus one finds beds for poison- ous plants, prickly plants, smelling plants, bulbs and marsh plants. ‘‘Aloes’’ (Aloe, Gasteria, etc.) were also grown and are figured in the catalogue and there was a ‘‘vaporarium pro plantis Americanis.’’ The lectures on ‘‘simples’’ delivered at the early Italian universities were not at first accompanied by demonstrations upon living specimens, but the growing of the plants in 1 This and the remaining buildings, etc., are shown on a separate plan which is not reproduced here. [VoL, 2 194 ANNALS OF THE MISSOURI BOTANICAL GARDEN definite gardens led to the establishment of demonstrations upon living specimens of the medicinal plants, and at Padua sixteen years after the foundation of the garden, a separation was made of the ‘‘Lectura’’ from the ‘‘Ostensio simplicium,’’ or demonstration of living plants. Botany, however, in all these early universities to which gardens were attached was merely ancillary to medicine. At Montpellier, for instance, the same professor taught anatomy in winter and botany in summer, and as late as 1773 anatomy, surgery and botany formed the subjects for one and the same professor at Jena. Very soon after the founding of the gardens at Padua and Pisa, plants other than those of strictly medicinal value were introduced into the physic gardens. This was due to the revival of interest in the plant world which took place about the middle of the sixteenth century and to the desire for travelling and interest in collecting which then sprang up. Conrad Gesner, writing in 1561 in the ‘Horti Germaniae,’? mentions that in botanic gardens not only medicinal herbs were cultivated but also other plants, especially rare ones, for the purpose of observing and admiring nature: “Hortorum alii vulgares sunt, utilitatis tantum gratia confiti: in quibus olera, legumina, vites, fructus qui edendo sint, & gramen, usum homini aut pecori praebent. Alii Te B ut Medicorum & Pharmacopolarum: in quibus non hortenses tantum stirpes, sed etiam sylvestres omnis generis, & peregrinae quoque coluntur, propter remedia quae ex ipsis earúmve parti- bus homini fiunt. Alii similes istis, sed magis varii, in quibus non solium plantae remediis nobiles, sed aliae etiam quae uis rariores ee coluntur, propter admirationem & contem- plationem naturae John Ray visited both Padua and Pisa early in 1664; refer- ring to the garden at Padua, he says: ‘‘Here is a public Physick garden, well stored with simples but more noted for its prefects, men eminent for their skill in Botanics.’’ The Pisan garden at this time would not appear to have been in a very flourishing condition since Ray merely remarks, “The 1Gesner, Conrad. Horti Germaniae p. 237 verso. Strasburg, 1561. 1915] HILL—BOTANIC GARDENS 195 Physick Garden at our being there but meanly stored with simples. ’’4 In particular, Gesner? alludes to several gardens at Padua and mentions the one under the charge of Anguillara, which was no doubt the Botanic Garden, as having a fine collection of plants with representatives from Syria, Crete and other dis- tant places. He refers in the first place to the Garden of Caspar à Gabrielis ‘‘vir inter nobiles Patavinos longe nobi- lissimus,’’ and then to ‘‘Priulanus hortus magnificus,’’ which was under Aloisius Anguillara (Romanus). Gesner’s account is as follows: er Priulanus hortus magnificus, plantis variis & raris e etiam et admirationi est. Omnes vero omnium, ni fallor, hortorum magnificentia simul, & stirpium in eo vari- arum omnis Ba, e Creta etiam & aliunde peregrinarum, numero laudes facile vineit publieus ille Patavii ın medico gratiam inclyti Senatus Veneti u institutus Be cul hoc tempore Aloisius Anguillara Romanus, vir in stirpium historia nostro seculo exercitatissimus atque peritissimus omnium, magna cum laude praeest.” According to Saccardo,’? Luigi Squalermo (detto Anguil- lara) was the first prefect ‘‘dell ’orto padovano ed ostensori dei semplici’’ from 1546 to 1561. . From this time onwards, no doubt, the tendency was to grow as many plants as possible, and a healthy rivalry commenced between the various botanical establishments as to who could show the greatest number of different species in cultivation. Travels through the Low Countries 1: p. 182. 1738. [2nd ed. Ray mentions the following eminent men at Padua: Aloysius Mundella, Aloysius Anguillara, Melchior Guilandinus, Jacobus Antonius Cortusus, Prosper Alpinus, da Vesling ardo, m cit. p- 7, gives the following list of Prefects of the Paduan are on 1546-1561 Luigi Squalermo (detto Anguillara). 1561-1589 Melchiore Guilandino. 1590-1603 Giacom’ Antonio Cortuso. 1603-1616 Prospero Alpini (o Alpino). 1616-1631 Giovanni Prevozio (Prevot). 1631 Giovanni ee iosto rinunciatario. 1631-1637 Alpino Alpini 1638-1649 Giovanni lan 2 Gesner, ©. De Hortis Italiae. Loc. cit. p. 239 verso. ® Loc. cit. p [VoL. 2 196 ANNALS OF THE MISSOURI BOTANICAL GARDEN In the botanic garden at Paris, for example, in the year 1636, there were about 1,800 species under cultivation and the num- ber had risen in 1640 to 2,360, and in 1665 to as many as 4,000 species. With the interest aroused in the collection and cultivation of plants came also the interest in their description and illus- tration, and many bulky and costly works were produced to illustrate the plants grown in botanic gardens. In Great Britain the foundation of the botanic gardens at Oxford, Chelsea, and Edinburgh, was preceded by the estab- lishment of several interesting private gardens devoted to the cultivation of medicinal herbs and plants of botanical interest, catalogues of which were published. The Rev. Wil- liam Turner (1510-1568), who has been called the ‘‘ Father of English Botany,’’ had a garden somewhere at Kew and after- wards a renowned garden at Wells, when he was Dean of the Cathedral. Then there was the noted physic garden of John Gerard (1545-1612) in Holborn, at that time the most fashion- able district in London, the catalogue of which—published in 1596—enumerates 1,030 plants and is of interest as being the first complete catalogue ever published of the contents of a single garden. His ‘Herball,’ published in 1597, was not his own work, but was simply a translation by a certain Dr. Priest of the ‘Stirpium Historiae Pemptades’ of Dodoens, which Gerard adopted and published as his own. On the title page of the edition of 1597, a garden is figured which has been generally considered to represent Gerard’s own garden in Holborn, but as Sir Frank Crisp! points out, he obviously bor- rowed his illustration from an engraving by A. Collaert, representing a garden of A. D. 1590, in April, much in the same unscrupulous manner as he borrowed his text. Among other early private physic gardens of interest in connection with the history of such institutions in England may be mentioned the garden of Thomas Johnson, M.D., the apothecary who had a garden on Snow Hill, in 1633—he it ! Guide for the use of visitors to Friar Park, Henley-on-Thames, Pt. Il. Illustrations of some mediaeval gardens p. 87. 1914 The illustration reproduced by Gerard is to be found on the title page of Tabernaemontanus, J. T. Kreuterbuch. [eds. of 1664 and 1687.] 1915] HILL—BOTANIC GARDENS 197 was who brought out the improved and enlarged edition of Gerard’s ‘Herball’ in 1638. The garden of John Parkinson (1567-1650), apothecary to James I, and King’s Herbalist in Long Acre, and that of John Tradescant (died 1638) the elder, at Lambeth, are also worthy of particular mention. John Tradescant, his father and his son were all of them botanists, collectors, and travellers. Tradescant the elder, who was gardener to various noblemen and also to Queen Elizabeth, was appointed Gardener to Charles I and founded a garden at Lambeth. This garden, after that of Gerard, was probably the most important early botanic or physic garden in England, and a catalogue of the plants therein was pub- lished in the ‘Museum Tradescantianum’ by his son in 1656. In addition to the garden, the Museum is worthy of notice in passing, since the curiosities it contained were bequeathed by the younger Tradescant to Mr. Ashmole, and formed the nucleus of the collection in the Ashmolean Museum at Oxford.! Parkinson was created King’s Herbarist, ‘‘ Botanicus regius primarius,’’ by Charles I. He was a horticulturist rather than a pure botanist, and his well-known book on garden plants, ‘Paradisi in sole Paradisus Terrestris,’ published in 1629, probably did much to stimulate interest in the cultivation of new and rare ornamental plants. Parkinson it was who had the boldness to depict the Garden of Eden on the title page of his ‘Paradisus,’ and includes among other remark- able products, the ‘‘Vegetable Lamb,’’ a pineapple, and an opuntia, the two latter plants being, as far as we are aware, unknown in the Eastern Hemisphere before the discovery of America. Reference need only be made in passing to garden illus- trations from 1580 and onwards, and to such works as the ‘Hortus Floridus’ of Crispian de Passe, published in Hol- land in 1614, and to the numerous herbals that were being produced to show the great strides that had been made in horticulture and botany in Elizabethan and early Stuart times. The establishment of a botanic garden in Oxford in the year 1621, the nineteenth year of the reign of James I, is an 1See Johnson, G. W. History of English gardening p. 98. London, 1829. [VoL. 2 198 ANNALS OF THE MISSOURI BOTANICAL GARDEN important landmark in the history of botanical progress in Kngland and follows the lead already given by the founding of university botanic gardens on the Continent. Like them, it was ‘‘primarily founded for a Nursery of Simples, and that a professor of Botanicey should read there and shew the use and virtue of them to his auditors.’’ The founding of the Oxford Garden! was due to the munifi- cence of Henry, Lord Danvers, Earl of Danby, who acquired the lease of five acres of meadow land by the River Cherwell, near Magdalen College, and arranged that the University should lease the ground from the College, to whom it belonged. The land was considerably raised to prevent flooding, at great expense, and was surrounded by a wall which was completed about 1632. l Access to the Garden was by means of the Danby gateway, the foundation stone of which was laid with all fitting cere- mony on St. James’ Day, 1621, by the Vice-Chancellor of the University.” The following is taken from Vines and Druce :3 “Botanic Lectures. “The next Lecture that must be mentioned is that of Botan- icey: but before I speak anything of its institution and settle- ment, I think it convenient that somewhat should be said of the Physic Garden, because ’twas primarily founded for a Nursery of Simples, and that a Professor of Botanicey should read there, S *Daubeny, C. The Oxford Botanic Garden, popular guide. Oxford, 1850; Günther, R. T. Oxford Gardens. Oxford, 1912; Vines, S. H., and Druce, G. C. An account of the Morisonian Herbarium, ete. [Introduction.] Oxford, 1914. *The date of the founding of the Garden has usually been incorrectly given as 1632, the year of the completion of the gateway, and in the account given by Wood of the foundation of the Garden there is a mistake of 1622 for 1621, but in their interesting epitome of the history of the garden, Vines and Druce show clearly that 1621 is the correct date when the ground was handed over and dele- gates were appointed. ® Loc. cit. pp. IX-X. 1915] HILL—BOTANIC GARDENS 199 ee in ancient times been a Cemitery for the Jews of Oxon, gave the University £250 to make a purchase of it. Upon the Ren: of it they bought out the present possessor thereof, Mar. 27, 19 Jac. Dom. 1622; and not long after the University took a lease of the said ground from Magdalen College (for to them it did belong) in their own name July 28 following, by pay- ing yearly for it 40s. Afterward much soil being conveyed thither for the raising of the ground to prevent the overflowing of the waters, the first stone of the fabric was laid on the day of St. James the Apostle (July 25) an. 1622, after this manner: About two of the clock in the afternoon, the Vicechancellor with certain Heads, Doctors, and both the Proctors, went solemnly from St. Mary’ s Church to that place; where being settled, Mr. Edward Dawson, a Physician of Broadgates, spoke an elegant Oration ; which being done, Dr. Clayton, the King’s Professor of Medicine, spake another. Afterward the Vicechancellor laid the first stone with the offering of money thereon, er to the ancient custom; then several Doctors and both the Proctors; at ee done, the Vicechancellor ee with a brief Orat a the said Earl proceeding in building and encom- passing it with a stately free-stone wall; which being almost finished, set up in front thereof, next to the East ‚Bridge, a comely Gatehouse of polisht stone; on which for the perpetua- tion of his name, he caused this Inscription to be engraven on the out and inside thereof: GLORIAE DEI OPT. MAX. HONORI CAROLI REGIS IN USUM ACAD. et REIPUB. HENRICUS COMES DANBY D. D. MDCXXXI. In the year 1633 all the wall being finisht, and soon after the floor raised, which cost the Earl £5,000 and more, he caused to be planted therein divers simples for he ent of the Faculty of Medicine. All which and several hundred more may now compare with any in the kingdom or elsewhere.” An interesting plan of the Garden by Loggan, made in 1675, shows four main enclosures within the boundary wall, each containing four series of geometrically arranged beds according to the formal arrangements then in vogue. Thomas Baskerville! gives the following description of the early condition of the Garden (about 1670-1700): “Amongst ye severall famous structures & curiosities where- with ye flourishing University of Oxford is enriched, that of ye Publick Physick Garden deserves not ye last place, being a 1 Account of Oxford Collectanea (c. 1670-1700). [VoL. 2 200 ANNALS OF THE MISSOURI BOTANICAL GARDEN matter of great use & ornament, prouving PENE PEE Pe not only to all Physitians, Apothecaryes, and those who are more immedi- ately concerned in the practice of Physick, but to persons of all qualities seruing to help ye diseased and for ye delight & pleasure of those of perfect health, containing therein 3,000 seuerall sorts of plants for ye honor of our nation and Univer- sitie and service of ye Commonwealth A further interesting piece of information given by Basker- ville is as follows: “Anno 1670. Here was built by the Income of the money given by the ffounder a fair greenhouse or Conservatory to pre- serve tender plants and trees from the Injury of hard winter.” This conservatory covered with a roof of stone slates i shown in Loggan’s plan and was of sufficient solidity to = transformed early in the eighteenth century into the her- barium, library, and professorial residence, but it was subse- quently demolished. The conservatory was heated in severe weather by means of a four-wheeled fire-basket, or wagon filled with burning charcoal, which was drawn eee irds and forwards along the path by a gardener.! Similar conservatories, or orangeries, were common in English gardens, and the building now used as a Museum (No. III) at Kew, was erected as an orangery in 1760. The first wooden greenhouses ever made were those erected at Oxford, in 1734, on either side of the Danby Gate.? Although the Garden was founded in 1621, it appears that some twenty years elapsed before Jacob Bobart was appointed the first gardener, owing probably to delays caused in prepar- ing the site. Under his supervision the Garden attained a con- siderable reputation and was visited by many distinguished people, including Evelyn and Pepys. Bobart’s catalogue of the plants cultivated, published in 1648, enumerates 1,600 plants, 600 of which were British, and many Canadian; it may e taken as evidence of his successful management of the Garden. 1 See Gardeners’ Chronicle N. S. 23: 732. f. 163. 1885. The figure is repro- duced in Giinther, R. T. Oxford oe dens p. 92. Oxford and London, 1912. 2 See engraving in Oxford Almanac, 1766; reproduced in Günther, loe. cit., plate facing p. 153. 1915] HILL—BOTANIC GARDENS 201 Owing to the outbreak of the Civil Wars and the death of the Earl of Danby, in 1644, his intention to provide the Uni- versity with a Professor of Botany as well as with a physic garden and a gardener, was long delayed, and the first profes- sor, in the person of Dr. Robert Morison, was not elected to fill the office until December 16, 1669. Morison’s first lecture was given in the Medicine School on September 2, 1670, and on September 5, he ‘‘translated himself to the Physic Garden where he read in the middle of it (with a Table before him) on herbs and plants for five weeks space, not without a con- siderable Auditory.’’! Space does not permit us to follow the fortunes of the Ox- ford Garden or to make mention of the many distinguished professors associated with it since its foundation, but it is of interest to remember that Sir Joseph Banks was a student at Christchurch, from 1760 to 1763, in the days of Sibthorp’s professorship, a time when no lectures on botany were given and the subject was much neglected in the University. Banks was so keenly interested in botany that he applied to Sibthorp for permission to procure a qualified lecturer to be paid entirely by the students. This request being acceded to and a sufficient number of students having been obtained, Banks went to Cambridge and secured the services of a Mr. Lyons, a botanist and astronomer, for the purpose.” The assistance rendered by the sister university in the botanical education of one who was to achieve such great things for the science and to have so large a share in directing the fortunes of the Royal Gardens at Kew, is worthy of more particular notice since botany was not officially recognized in Cambridge until 1724, when a professor was appointed, and there was no botanic garden there until the year 1762. The Botanic Garden at Edinburgh, which now claims atten- tion, has had a somewhat involved history, as the present Royal Botanic Garden is the sixth and only remaining botanic garden in the Scottish capital, though in the early years of ı Vines, S. H., and Druce, G. C. loc. cit., p. XXIV. ® Anonymous, Sir Joseph Banks and the Royal Society p. 62. London, 1844. [voL. 2 202 ANNALS OF THE MISSOURI BOTANICAL GARDEN the eighteenth century there were three distinct gardens in Edinburgh. The original Edinburgh Garden was founded by Sir Robert Sibbald and Sir Andrew Balfour, physicians, for the cultiva- tion of medicinal plants in order ‘‘to safeguard the Practi- tioner against the Herbalist and to enable him to have a cor- rect knowledge of the plants which were the source of the drugs he himself would have to compound.’’! For this purpose they acquired the lease of a small area of ground near Holyrood, and James Sutherland was secured to look after it and instruct the apprentices and lieges in botany. Such success attended the venture that a piece of the Royal Flower Garden at Holyrood was assigned to the cultivation of medicinal plants and this with the title of Physic Garden be- came the Royal Botanic Garden in Scotland. In 1767 the same physicians acquired from the Town Council of Edinburgh a lease of the Garden of Trinity Hos- pital and adjacent ground—a site now partly occupied by the Waverley Station—and Sutherland was appointed to lec- ture on botany as Professor in the Town’s College, now the University, and to be in charge of this new Physic or Town’s Botanic Garden. Then in 1702 another botanic garden was established by the University—the College Garden—of which Sutherland was also placed in charge. The distance of the two existing gardens being too great from the University, Sutherland resigned the care of the Town’s Garden and Col- lege Garden in 1706, but remained King’s Botanist, retaining the Keepership of the Royal Botanic Garden, and the Town Council appointed a professor to take charge of the Town and College Gardens. There were thus two rival botanical schools with their gardens in Edinburgh, and it was not until the year 1739 that the rivalry was terminated by the appointment of Dr. Charles Alston, the then Keeper of the Royal Botanic Garden, to the University Chair—a combination which holds to the present day by consent of the Crown and the University. * Balfour, I. Bailey, History of the Royal Botanic Garden, on Notes of the Roy. Bot. Gard., Edinburgh 4; 1904. Historie Notice. pp. 1915] HILL—BOTANIC GARDENS 203 Between the years 1760 and 1786 a new site was found for a botanic garden and the other gardens were abandoned. This new garden, formed during John Hope’s keepership, eventu- ally became unsuitable owing to the growth of the town, and the present site (twenty-seven acres) was selected about 1820, during the keepership of Professor Graham. The Edinburgh Garden, through the University, still retains its connection with the Medical School, and the instruction of the medical student is one of the functions of the Professor and his staff. With its fine collections of living plants, its herbarium, library, laboratories, and remarkable series of specimens in the museums, the Edinburgh institution may well serve as an example of the ideal botanic garden. The Chelsea Physic Garden,' which next claims attention, was founded as the Garden of the Society of Apothecaries? in London in the year 1673. The earlier garden of the Society had been at Westminster, but this had no river frontage, and the ground at Chelsea was leased from Charles Cheyne, in 1673, as a convenient spot for building a barge house for their processional barge in which they attended city functions, as was customary for city companies. In 1676 the plants at Westminster were moved to the Chelsea Garden, which had already been suitably enclosed with a wall. The freehold of the Manor of Chelsea, including the Physic Garden, was purchased in 1712 by Dr. (after- wards Sir Hans) Sloane, who in the year 1722 conveyed the Garden by deed to the Society of Apothecaries. The convey- ance was made ‘‘to the end that the said garden might at all times thereafter be continued as a Physick Garden, and 1 Field, H., and Semple, R. H. Memoirs of the Botanic Garden at Chelsea. London, 1878. 2 The Society of Apothecaries itself was formed in 1617 “that the ignorance and rashness of presumptuous Empirics and unexpert men might be restrained, whereby many discommodities, inconveniences and perils do daily arise to rude and incredulous people.” See Blunt, R. Cheyne Walk and thereabout p. 99. London, 1914. Certain continental botanic gardens, such as the ancient garden at Salzburg were founded in connection with local pharmaceutical schools and have had no connection with any university. [VoL, 2 204 ANNALS OF THE MISSOURI BOTANICAL GARDEN for the better encouraging and enabling the said Society to support the charge thereof, for the manifestation of the power, wisdom, and glory of God in the works of the creation, and that their Apprentices and others might better distinguish good and useful plants from those that bore resemblance to them, and yet were hurtful and other the like good purposes.’’? The utilization of the Garden for the sole purpose of grow- ing medicinal plants to be converted into drugs for the Society’s use was prohibited by Sir Hans Sloane’s deed of gift, and he definitely encouraged the science of botany by making it a condition that fifty specimens of distinct plants, well dried and preserved, which grew in their garden that same year, with their names and reputed names, were to be delivered yearly to the President and Fellows of the Royal Society of London, ‘‘until the number of two thousand had been attained.” He also enjoined that the plants so presented in each year were to be specifically different from those pre- sented in every former year; and this injunction was more than faithfully carried out by the Society.? The Garden achieved some notoriety in having been the first garden in England where the Cedar of Lebanon was planted; the final survivor of the four placed there in 1683 was only removed in the year 1904. John Evelyn, who visited the Garden in 1685, was impressed by the heating arrangement of the greenhouses, then quite an innovation. ‘‘What was very ingenious,’’ he remarks in his diary, ‘‘was the subterranean heate conveyed by a stove under the conservatory, which was all vaulted with bricks, so as he* has the doores and windows open in the hardest frosts, seclud- ing only the snow.’’ An arrangement far more efficient and useful than the remarkable open fire-baskets formerly in use at Oxford. 1 Perrédés, P. É. F. London Botanic Gardens. Wellcome Chemical Research Laboratories, London, Publ. 62: p. 57. London, 1906 (transferred from the present to the past tense). ? Johnson, G. W. History of English gardening p. 150. London, 1829. 3 John Watts, appointed gardener in 1680. 1915] HILL—BOTANIC GARDENS 205 The appointment of Philip Miller,! in 1723, as Head Gard- ener, is an important event in the history of the Garden, both for the value of his services to the Garden itself and for his widespread influence on botany and horticulture. At the time of Miller’s appointment, exotic plants were pouring in from every clime under the patronage of a general taste for their acquisition. Hothouses were multiplying and their inhabitants accumulating to a hitherto unheard-of ex- tent, and a man of Miller’s practical skill and botanical knowl- edge was needed not only to demonstrate his skill, but also to impart his knowledge for the use of others. From his ‘Dic- tionary’ it can be seen that many plants were grown and flowered at Chelsea for the first time under cultivation. William Aiton (1731-1793), the first Curator of the Royal Gardens at Kew, was a pupil under Miller at Chelsea, nor must Nathaniel Bagshaw Ward, Examiner to the Society of Apothecaries from 1836 to 1854, the inventor of Wardian cases, be forgotten. His invention made possible the intro- duction of the tea plant to India by Robert Fortune (Curator of the Chelsea Garden, 1846-1848), of Cinchona from South America to Kew by Markham, and thence to India, and of many other valuable products to botanic gardens which have subsequently been disseminated for the use of mankind. Not the least useful of the activities of the Chelsea Physic Garden were the herborizing excursions around London, under the charge of the Demonstrator of Plants, which were maintained for some two hundred years. The Physic Garden has suffered many vicissitudes in the course of its existence, and towards the end of the last century almost ceased to exist, hut for- tunately a new arrangement for its maintenance was made in 1899.2 Reorganized under the new scheme and with its modern greenhouses and laboratory, the Chelsea Garden has entered on a sphere of usefulness in connection with the teach- ing of botany and the provision of material and opportunity E Charles Miller, son of Philip (who had aided in the selection of the site), was made first Curator of the original Cambridge University Botanical Garden founded in 1762. 2 The Chelsea Physic Garden. First Report of Committee of Management, 1905, with plan of the Garden in 1753. [VoL. 2 206 ANNALS OF THE MISSOURI BOTANICAL GARDEN for botanical investigation as great if not greater than at any time in the past. The origin of the Royal Botanic Gardens, Kew, was due to the interest in botany displayed by Princess Augusta, Princess Dowager of Wales, under the guidance of Lord Bute, an enthu- siastic botanist; and a piece of the Royal Garden attached to Kew House was set apart in 1760 for the purpose of forming a physic garden. =. space allotted consisted originally of nine acres, enclosed by walls (the ornamental building now standing, called the Temple of the Sun, being then nearly the centre of the Garden), which was laid out and scientifically planted in two divisions, one containing a collection = herbaceous plants, arranged ac- cording to the Linnean system, then in its infancy, but with which Aiton had become ny acquainted ee under Miller. This division was called the Physic Garden “The second division was called the ee containing all the then known introduced hardy trees and shrubs scien- tifically arranged. Within the area were several Glass houses, and in 1761 a large hothouse, nei feet long, was wo by Sir m. Chambers in afte years known the Great ete In the same year an ae 130 feet ieee was also erecte No doubt several of the old and interesting trees now stand- ing near the Temple of the Sun were planted in Princess Augusta’s arboretum soon after the foundation of the Garden. William Aiton was placed in charge of the Garden under the direction of Lord Bute, and was Chief Gardener from 1759 to 1793. Sir W. Chambers, the designer of the Pagoda and most of the Temples still to be seen in Kew, gives the follow- ing account of Princess Augusta’s Physic Garden: “The Physic or exotic garden was not begun before the year 1760; so that it cannot possibly be yet in perfection; but from the great botanical learning of him who is the principal man- t Smith, John. Records of the Royal Botanic Gardens, Kew. p. V. 1880. See also Kew Bull. Misc. Inf. 1891; 289-294. 1891. The Great Stove stood near the Temple of the Sun and was removed in 1861. Its site is marked by an old wistaria, trained on an iron cage which grew upon its walls. The method of ventilating the house was designed by William Hales, the physiologist, who described his method in a letter to Linnaeus written in d is in use at Kew to-day and was devised independently by Sir W. T. Thiselton-Dyer. The Orangery is now Museum No, III. 1915] HILL—BOTANIC GARDENS 207 ager and the assiduity with which all curious productions are collected from every part of the globe without any regard to expense, it may be concluded that in a few years this will be the amplest and best collection of curious plants in Europe.’ With the death of Princess Augusta in 1772, George III in- herited the Kew property and united the gardens of Kew House with those lying contiguously, which formed the gar- dens of the Palace of Richmond, and so produced the extensive domain now occupied by the Royal Botanic Gardens. To the great benefit of Kew, George III chose Sir Joseph Banks as his botanical adviser, and for forty-eight years Sir Joseph directed the affairs of the Gardens. During his term of office the practice of sending out collectors was established, a prac- tice fraught with discoveries of wide-spread interest and value for horticulture and botany. Of the many Kew collectors? it is well to mention in particular the following: Francis Masson, the famous collector of Cape plants; David Nelson, assistant botanist on Cook’s third voyage, who subsequently died from exposure after the mutiny of the Bounty; Archibald Menzies, who travelled in Australia and Chili and introduced Araucaria imbricata; William Ker, the collector in China, who in 1812 became Superintendent of the Royal Botanic Garden, Ceylon; and Allan Cunningham, whose travels took him to Brazil, the Cape, Australia, Tasmania, New Zealand and Norfolk Island. Cunningham returned to Australia, in 1836, to fill the post of Superintendent of the Botanic Garden at Sydney. The days of Sir Joseph Banks were indeed the Golden Age of Kew, and under his direction the Royal Gardens became a center of botanical exploration and horticultural experiment unparalleled before or since. The well-known lines of Eras- mus Darwin? refer to the Kew of Sir Joseph Banks’ day, en- riched by the labors of her collectors: 1 Chambers, Sir W. Plans, elevations, sections and perspective views of the gardens and buildings at Kew in Surrey, the seat of Her Royal Highness, the Princess Dowager of Wales p. 3. Brentford, 1765? 2For the complete list of Kew collectors, see Kew Bull. Mise. Inf. 1891: 295-311. 1891. 3 The Botanic Garden. 1791. [VoL, 2 208 ANNALS OF THE MISSOURI BOTANICAL GARDEN “So sits enthroned, in vegetable pride, Imperial Kew by Thames’ glittering side; George III and Sir Joseph Ka both Auð in 1820, and for some twenty years the Royal Gardens gradually fell into a condition of sad neglect. In the early years of the reign of Queen Victoria, however, the Royal Gardens were restored to their proper position as the National Botanical Garden, thanks to the devoted labors of the committee of which John Lindley and Sir Joseph Paxton were the distinguished mem- bers, and Sir William Hooker was appointed Director of the Royal Botanic Gardens in 1841. Thence onwards, under Sir Joseph Hooker, and Sir William Thiselton-Dyer, the history of Kew has been one of steady progress and usefulness and the Royal Botanic Gardens have played a prominent part in connection with all matters of botanical enterprise in the British Colonies. 1 The erg nt at Kew comprises: I. The Botanie Gardens and Arbo- retum (288 es); II. The Herbarium and Library; III. The Museums devoted to (i and ii). prha e r and 1 and their economie products, (iii) exotic timbers and conifers, (iv) British forestr ry, and (v) The North Gallery of paintings by Miss Marianne North; IV. The Jodrell Laboratory for scientific research; V. The Pathological Laboratory; . Director’s Offi The more important books dealing with the history of Kew and its collec- tions are: l. Aiton, W. Hortus Kewensis, 3 vols. London, 1789 2. een W. T. Hortus Kewensis, 5 vols. London, 1810- 13. [2nd ed.] 3. Scheer, F. Kew and its Gardens. Richmond 4. Historical account of Kew to 1841. Kew Bull. P Mise, Inf. 1891: 279-327. 189 5. Royal ER Gardens, Kew, Reports on progress and condition. 1855-1882. 6. Perredes, P. E. F. London Botanic Gardens. Wellcome Research Labora- — London, Publ. 62: 17-40. (e W. J. The Ro ag! Botanic Gar London, 1908. 8. Popular Official Guide t o the oe Botenie RR He Kew, 1912. 9. Kew Bull. Mise. Inf. 10. Kew Plant Lists and Museum Guides 11. Smith, J. Records of the Royal Botanic Gardens, Kew. London, 1880. 1915] HILL—BOTANIC GARDENS 209 Kew, having no connection with any university or educa- tional establishment,' differs markedly in this respect from the botanic gardens to which allusion has been made. Her sphere of usefulness is largely concerned with the economic aspect of botany, and it is her aim and object to encourage and assist, as far as possible, scientific botanists, travellers, merchants and manufacturers, in their varied botanical in- vestigations. Space does not permit of more than a brief mention being made of the new Berlin Garden at Dahlem and of many other important gardens on the Continent and in Great Britain and Ireland. The Berlin Botanic Garden? was founded in 1679 in the heart of the city, and in 1801 it was reorganized and im- proved. The removal of the Garden to its present site at Dahlem was completed in 1909. The new Garden with its geo- graphical and ecological arrangements of the plants and the splendid Botanical Institute and Museums, now forms one of the finest schools of botany in the world. In her aims and objects she compares more closely to Kew than to any other botanic garden. The pre J notes refer to other important gardens not specifically men- tioned in the tex e Upsala Gaetan (founded 1655-57) was injured by the great fire in 1702, and remained neglected until 1741. The restoration was begun by Rosen and energetically taken up by Linné. (See Swerderus, M.B. Botaniska Tragarden, Upsala, 1655-1807. Falun, 1877.) The Imperial Botanic Garden of Peter the Great, Petrograd (St. Petersburg), was founded in 1713 (see Kew Bull. Misc. Inf. 1913: 243-252. 1913), and that of Vienna in 1754. The Cambridge Botanic Garden was founded in 1762 by Richard Walker, D.D., formerly Vice-Master of Trinity College. The Garden was transferred to its present site in 1846 and occupies about twenty acres. It is in close connection with the Botany School at Cambridge and provides abundance of material for research work and for the teaching purposes of the Botany School. The Garden is also fitted with a small laboratory. Some eighteen acres are available for extension au 1 Lectures and demonstrations in chemistry and physics, => botany, systematie and geographical botany, economie botany, plant pathology and on soils and manures are given in the Gardens to the young gardeners at Kew. 2See Urban, I. Geschichte des Königl. botanischen Gartens und des Kön nigl. rbariums zu Berlin, nebst einer Darstellung des augenblicklichen Zustandes dieser Institute. Festschr. naturwiss. u. med. Staatsanst. Berlin, 1881. Engler, A., and others. Der Kgl. bot. Garten und das Kgl. bot. Museum zu Dahlem. Berlin, 1909. (vor. 2 210 ANNALS OF THE MISSOURI BOTANICAL GARDEN The Royal Botanic Gardens, egie Dublin, were founded in 1790, through the influence of Dr. Walter Wade and the Hon. Dublin Society, and in 1877 were transferred to the Science and Art ok The Botanic Garden of Trinity College, Dublin, was established in 1998-98, (See Notes from the Botanical School of Trin. Coll., Dublin 1: p. 896 The garden at Breslau was den in ‘1811, The Geneva Garden, founded in 1817, has recently been transferred to a new site. The Munich Garden was founded in 1822 (see Martius, Hort. Bot. R. Acad. Monacensis p. 5. 1825.) It is now one of the most interesting anda on the Continent and forms an integral part of the new and magnificently equipped Botanical Institute. The Glasgow Botanic Garden was established in 1817, having been preceded by an < 4 Q } Q 3 -A - a -_ a a © > < oO H N 3 + m 2 | 3 Š = Zz Z Er de SHigkle volpar ci. HILL—BOTANIC GARDENS COCKAYNE, BOSTON [Vor. 2, 1915] ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 8 The Oxford Botanic Garden, founded 1621. Reproduced from Log- gan’s plan of the Garden in 1675. (See p. 197.) Ann. Mo. Bor. GARD., Vor. 2, 1915 PLATE 8 South Elevation of the Conservatory — X PeR A > Er we P | 4 SE & j | 3 d D w Tr i] 1 I Eg ' | - \ i ~ r East Bridge HILL—BOTANIC GARDENS COCKAYNE, BOSTON [VoL. 2, 1915] 234 ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 9 Royal Er Gardens, Kew, showing dates and extent of successive additions to the area open to the publie and site of the original Botanic nn of 1760. Photograph of plan in W. J. Bean’s “The Royal Botanie Gardens. Kew,’ London, 1908. (See p. 206.)—Pub- lished by permission of Cassell & Co., Ltd. London, England. ad op bi at ©. el . ee a ne 4 = O NDP. OE en Zu Ts FF ae 2 | Ann. Mo. Bor. GARD., VoL. 2, 1915 PLATE 9 i E CE Gr 2 En or r H MERLIN’S CAVE S ' Ss} A H A 1897 QUEEN’s COTTAGE OLD Gases Lei DEER PARK HILL—BOTANIC GARDENS COCKAYNE, BOSTON 236 [Vor. 2, 1915] ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 10 The Herbaceous Ground, Royal Botanic Gardens, Kew, showing beds arranged according to the natural orders. PLATE 10 Mo. Bor. GARD., VOL. 2, 1915 ANN. HILL—BOTANIC GARDENS COCKAYNE, BOSTON ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 11 The Rhododendron Dell, Royal Botanic Gardens, Kew. [Vor. 2, 1915] PLATE 11 N ELSE IP ALP Ann. Mo. Bot. GARD., VOL. 2, 1915 HILL—BOTANIC GARDENS COCKAYNE, BOSTON KENNY w at a, Er F ite rn t te i ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 12 The Lake, Royal Botanic Gardens, Kew. PLATE 12 NS em 4 4 ARDI BOTANIC ( COCKAYNE, BOSTON 5 Ti HILL Ann. Mo. Bor. GARD., VOL. 2, 1915 RECENT INVESTIGATIONS ON THE PROTOPLASM OF PLANT CELLS AND ITS COLLOIDAL PROPERTIES FREDERICK CZAPEK Pflanzenphysiologisches Institut der K. K. Deutschen Universitat, Prague, Austria I have the honor of publicly congratulating the Representa- tives of the Missouri Botanical Garden upon the Twenty-fifth Anniversary of Henry Shaw’s magnificent foundation,—the unique memorial of a magnanimous citizen of this great metropolis. I shall endeavor to show to the members of this splendid assembly how plant physiologists at present attempt to reach a satisfactory understanding of the wonderful mechanism which in never-ceasing variation is unfolded to us in myriads of phenomena characteristic of nutrition, reproduction, adap- tation, growth, and stimulation, in the lower as well as in the higher plant organisms. Wherever science is following these various processes to their mysteriously hidden roots, the physiologist has to face the complex problems associated with the living content, the so-called protoplasm of the plant cell. Without this singular matter plant cells are mere dead bodies able neither to grow, to take up food, nor to assimilate their nutriment. It was not until 1841 that Hugo von Mohl, the well-known botanist of Tiibingen, discovered the important fact that all phenomena in cell life are strictly confined to the thin layer of slimy material which clothes the inside of each growing and living plant cell. He stated that this protoplasmic slime was stained deeply yellow by means of iodine, and he expressed the opinion that protein substances in particular were the con- stituents of this living material, from which all other parts and organs of the cell were believed to take their origin. We shall not be surprised to learn that biologists felt in- clined to suppose that the protoplasm might contain some ANN. Mo. Bot. GARD., Vou. 2, 1915 (241) [VoL. 2 242 ANNALS OF THE MISSOURI BOTANICAL GARDEN peculiar and highly complex proteins constituting the living matter in the proper meaning of the word, whose chemical qualities we should have to make responsible for the whole complex of life phenomena. Therefore, it appeared a most attractive problem to subject protoplasm to a thorough chemical investigation. The names of Reinke and Rodewald are connected with this work. These two botanists, in 1880, then in Gottingen, analyzed the protoplasmic mass, the so- called plasmodium, of Fuligo septica, a common species of the Myxomycetes. The result was that a part, about three- quarters, of the material was recognized to belong to the pro- tein group in the widest sense; while 25 per cent was a mix- ture of diverse carbohydrates, fatty bodies, organic acids, and inorganic materials. No evidence of the presence of any peculiar protoplasmic substances was found. Reinke, there- fore, laid emphasis on the point that protoplasm could not be regarded as a single chemical body of peculiar qualities, but that it should be considered as a mixture of various sub- stances, of which not even one was unknown to the chemists. The consequence of this view was that Reinke inclined to the hypothesis that the peculiarities of protoplasm were not due to its chemical nature but rather to its peculiar structure. The stuff-hypothesis had to be replaced by a structure-theory of protoplasm. At present, however, we can scarcely accept all conclusions drawn by Reinke from his famous analysis of protoplasm. Reinke thought that all the vital properties of living proto- plasm were destroyed when cells were killed, in the same way as the mechanism of a watch is destroyed by grinding it down in a mortar. The chemical substances, however, may remain unchanged while the mechanism is forever destroyed. The first experiments which proved that Reinke’s simile is not quite an exact one were obtained from studies on the various enzyme effects which continue in a mass of finely comminuted tissue. Among those effects we know a series of processes which undoubtedly belong to the complex of vital metabolism, —as, for example, to those of respiration and digestion. And these effects may be followed for weeks and for months after 1915] CZAPEK—PROTOPLASM AND ITS COLLOIDAL PROPERTIES 243 trituration of the cells, if precaution is taken to prevent change in the material by bacterial action. But the essential difference between such autodigestion and the life-process consists in the fact that the first is not ruled by the laws of correlation and regulation, which are so peculiar to life pro- cesses. Nevertheless, we cannot say that the whole of the life-mechanism is destroyed by grinding down living organs. At least a part of it cannot immediately be transformed by this type of disintegration. From this we may draw the con- clusion that there are certain chemical substances present in protoplasm which are responsible for certain activities of the living tissue. Such substances are the enzymes, which are entirely unknown in inanimate nature, and absolutely dis- tinctive of cell protoplasm. Further, we cannot suppress some scruple that in Reinke’s analysis there were examined not the original protein-bodies of protoplasm, but only substances artificially produced during the treatment of the original material. Our chief objection against the ‘‘Engine-Theory”’ of proto- plasm is that no mechanism has hitherto been known which may be destroyed by heat as easily as is protoplasm, whilst on the other hand one cannot immediately and entirely destroy it merely by pounding to an impalpable pulp. Besides this, recent investigations on the proteids of animal organs—in which great care was taken to dry the pulp quickly at a tem- perature as low as possible—have shown that there really exist highly compounded protein bodies of hitherto unknown constitution which have to be considered as real constituents of protoplasm. Can such discoveries in some way explain the vital prop- erties of the cell? It seems as if we may not understand the wonderfully accurate working-together of all organs in cells without supposing trans-microscopical structural qualities; but we need not assume any mysterious new forces or struc- tures. Most of the well-known characteristics of protoplasm can be understood by considering further the colloidal state of the constituents of the cells. [VOL. 2 244 ANNALS OF THE MISSOURI BOTANICAL GARDEN The first naturalist who turned his attention to the great importance of colloidal substances in cells was Biitschli, the zoologist of Heidelberg. A great number of his admirable papers deals with the microscopical features of cell plasma, which he described as a framework of jelly-like substances containing interstices, or meshes filled with fluid substances. Biitschli emphasized the view that the foam structure de- scribed by him is not peculiar to living matter, because a mix- ture of oil and gelatin solution shows the same microscopical structure which he attributed to protoplasm and to all colloids. But later on it became more and more probable that such a foam structure in protoplasm indicates nothing more than certain gross features which are by no means identical with the real colloidal structure of plasmatic constituents. Not even in gels, or solid colloids, apparently, is the foam structure a dominant characteristic. Zsigmondy’s recent work on gela- tinous structure clearly showed that while forming the gel the colloidal particles, which are distinctly visible in the ultra- microscope, do not arrange themselves in a network, but settle quite irregularly; so that we cannot assume that meshes are formed in the precipitation of colloids. On the other hand, biologists of rank, as Lepeschkin, after a careful study of the microscopical structure and the physical properties of protoplasm, have arrived at the conclusion that we should not regard it as a foamy mass, or jelly-like substance, but rather as a liquid colloid with the characteristics of protein sols of certain higher concentrations. We can easily confirm the observation that protoplasm, examined by means of the highest power of the microscope, often appears merely as a homogeneous liquid, or transparent mass, sometimes mod- erately turbid from the presence of small distinct drops or corpuscules which are collectively known under the name of ‘“microsomata.’’ Even though we do not accept Bütschli’s idea with respect to specific structure, we fully share his more general point of view that living protoplasm owes its peculiar activities to colloidal qualities. And this represents our attitude to-day towards protoplasmic investigation. 1915] CZAPEK—-PROTOPLASM AND ITS COLLOIDAL PROPERTIES 245 The chemistry of colloids is not a descriptive science. To the utmost extent it has to use experimental physical methods. So we cannot advance in knowledge of protoplasm by mere microscopical observation, but mainly by experimental investi- gation. A long time even before colloidal chemistry became domin- ant as the basis for the physiology of protoplasm, a memorable epoch in plant physiology had opened, developing from the ingenious work of Pfeffer and De Vries on the osmotic prop- erties of living cells. These investigations unveiled the fundamental fact that living protoplasm alone is in posses- sion of those peculiar properties of permeability which are responsible for the whole complex of nutrition. Dead proto- plasm behaves quite differently. Since, however, differences in respect of the penetration of different solutions can be detected to a certain extent in colloidal membranes, it be- came probable that the so-called semipermeability of living protoplasm is a colloidal phenomenon, due to the constituent colloids in living protoplasm; whilst after the death of the cells the coagulation of these colloids completely changes the peculiar permeability of the protoplasmic layer. It was, however, Ernest Overton, in 1899, then at Zurich, who acquired the merit of placing colloidal chemistry in fundamental relation to the phenomena of diosmosis in living cells. The well-known theory of Overton consists in the hypothesis that fatty substances play an important röle as constituent elements in the protoplasmic matrix. It is due to such substances, generally comprised under ‘‘lipoid bodies,’’ that living cells show quite distinctive diosmotie qualities. Overton’s hypothesis is founded upon the fact that only those substances which readily dissolve in fatty oils are easily diffus- ible in living cells; whilst all substances which are insoluble in oily media, as sugar or mineral salts, easily produce plas- molysis, because they penetrate into cells only very slowly. The leading physical idea in this theory was the so-called ‘‘Partition-Rule’’ of Berthelot and Jungfleisch. This law states the fact that there exists a constant relation between the quantities of a certain solute dissolved in two immiscible [VoL. 2 246 ANNALS OF THE MISSOURI BOTANICAL GARDEN solvents. Overton considered the endosmosis of dissolved substances into living cells as merely a question of solubility. It is known how fertile this idea has proved in physiology, particularly in the phenomenon of narcosis, where it is still the leading hypothesis in animal physiology. But recently experimental work, including my own, has shown that it is scarcely quite correct to consider the endos- mosis of solutions into living cells as a typical solution phenomenon. According to Loewe even the partition of methylene-blue or of chloroform between oil and water cannot readily be explained by means of the principle of Henry and Berthelot. Rather, the oily solution of such substances is not a true solution, but only a colloidal solution; so it is not ruled by the laws of osmotic pressure, but by the laws of adsorption. A striking fact was discovered by Traube and by myself in studying the effects of alcohols and other capillary-active substances on living cells. Their injurious action clearly and exclusively depends upon the relative capillary activity. Every one of these substances kills the cells at a concentration corresponding exactly to a certain value of surface tension. The main importance of this observation consists in the evi- dence that in narcotic effects capillary phenomena must be a dominant factor. This cannot be interpreted by the supposi- tion that the entrance of narcotics into cells is due to true solution phenomena. The observed capillary effects distinctly show that the factor of real moment is to be found in altera- tions of contact-surface; but such surface-phenomena are met with only in colloids and in their adsorption. A prominent feature of our experiments involves the fact that cells of higher plants are constantly killed by concentra- tions of narcotics such that the capillary activity reaches about two-thirds of the surface tension of pure water in contact with air. It is remarkable that saturated and neutral emulsions of triolein or other typical fats always show approximately the same surface tension value. This result I tried to explain by means of the hypothesis enunciated in the following sentences: Alcohol and other narcotics are taken up by ad- 1915] CZAPEK—PROTOPLASM AND ITS COLLOIDAL PROPERTIES 247 sorption into living protoplasm. According to the theorem of Willard Gibbs the surface in liquid systems which consist of different fluids and contain some capillary-active sub- stances is always occupied by those substances which show the greatest reduction in the surface tension of the medium. If subsequently another substance with greater capillary activity is added to the system it displaces all other substances from the surface. Narcotics may displace certain plasmic substances in an analogous way, provided that the surface tension of the concentration applied is just a little lower than the surface tension of the plasmatic substances referred to. The fact that the fatal narcotic pressure value coincides with the maximum surface tension in fat emulsions may be ex- plained by the hypothesis that fatal effects of alcohols on living cells consist in destroying the emulsion structure of protoplasm, by displacing some fatty substances. So our experiments to a certain extent uphold the view that the surface layer of protoplasm really contains fat, and thus far is in accordance with Overton’s hypothesis. In the course of time the lipoid-theory of Overton has met with sharp criticism. Among other renowned physiologists, Ruhland strongly denied the presence of fatty bodies in the plasmatic membrane of plant cells. On the other hand, we are aware that animal physiologists, such as Fühner, Höber, and Vernon still firmly adhere to the old lipoid-theory. However, since according to Overton sugars and mineral nutrient salts are believed to penetrate only poorly into the living cell, it is obvious that Overton’s hypothesis stands in direct contrast to the common experiences in respect to plant nutrition. The substances referred to are materials which the cells have to take up as among their most important nutrients. Nevertheless, there have been developed some sup- plementary theories which permit us to lessen the difficulties of the lipoid-theory, for example, that of Nathansohn, accord- ing to which the lipoid membrane of protoplasm is not a con- tinuous film of fat, but a kind of mosaic of fat and protein which is able to permit the penetration of both fat-soluble substances and mineral salts. [voL. 2 248 ANNALS OF THE MISSOURI BOTANICAL GARDEN Ruhland’s experiments especially were not at all favorable to the lipoid-hypothesis. They show decidedly the error of the opinion that only those aniline dyes penetrate into living cells which are soluble in oil. Many aniline dyes have been found which are easily taken up by cell protoplasm in spite of their insolubility in fat, while other coloring matters which easily dissolve in fat do not penetrate at all through the living plasmatic layer. Ruhland, as well as Küster, drew from such experiments the convincing conclusion that substances readily soluble in lipoids may not always be readily taken up by the living cells. But in other respects it seems as if Ruhland had gone too far when he denied that protoplasm possesses any fat content. He emphasized that he never could detect any microscopical trace of plasmatic substances which may be stained by means of such aniline dyes as are readily stored by fat. Since our own experiments seem to be in some accord with the view that fatty matter really is present in protoplasm, I wanted to compare some chemical systems which are entirely free from fat with protoplasm in respect to its behavior toward alcohols. It could be taken as a proof of the view that protoplasm does not contain fatty bodies, if there were noticed no difference between the effects of alcohols on the physical properties of such systems and on protoplasm. The investi- gations of Mr. Geo. H. Chapman in our laboratory were begun in order to examine the influence of different narcotics on enzymes. Surprisingly, the results were opposed to the above- mentioned view of similar action with respect to these systems. This work clearly showed that the capillarity-rule which is so distinctive of the effects of narcotics on living protoplasm does not apply to the effects of narcotics on enzymes. While the deleterious influence of methyl, ethyl, and propyl alcohol gradually increases with the molecular weight of these homol- ogous substances, the higher members such as butyl and amyl alcohol act considerably less on enzymes, and both heptyl and octyl aleohol have practically no weakening influence on these ferments. In respect to their coagulation by diluted alcohol protein solutions show relations corresponding to 1915] CZAPEK—PROTOPLASM AND ITS COLLOIDAL PROPERTIES 249 those just discussed. In consequence of this result we can hardly explain the effects of narcotics on protoplasm by the view that only plasmatic protein bodies are influenced by such toxic agents. Besides this, for the coagulation of protein bodies there is required not less than five mols of ethyl alcohol while a little more than two mols is sufficient to kill living protoplasm. Therefore, some other substances in protoplasm besides the protein bodies must be affected by the alcohols, and these substances must differ from the latter in their physical properties. So it seems that the view ac- cording to which the plasmatic membrane is constructed ex- elusively of hydrocolloids, viz., proteins, as Ruhland believes, cannot be considered to be quite satisfactory. Our attention must be directed anew to the possibility that some lipoids play the part of important constituents of the protoplasmic mem- brane. On the other hand, I have to state that several lines of ex- perimental work have led us to the conclusion that the endos- mose of solutions into living cells never does take place by way of plasma lipoids, but only through hydrocolloidal con- stituents of the cell plasma. The work of Mr. Krehan, which dealt with the influence of highly diluted hydrocyanic acid on plant cells, distinctly showed that in the presence of this agent the permeability of cells to certain salts, such as sulphates, and to sugar, is raised, so that the threshold of plasmolysis for these substances is raised. When the effects of different salts on plasmolysis were compared it became manifest that just those salts causing the greatest rise of the plasmolytie limit, are those which were strongly adsorbed, and which display a most marked effect on the precipitation or coagulation of albumen. Such salts are sulphates, citrates, tartrates—by their anionic effects, and the salts of am- monium, calcium, and magnesium—by their cationic effects. These phenomena are only to be understood upon the supposi- tion that hydrocolloids are the media through which different substances must pass when taken up by the living cell plasma. There has been discovered not the faintest indication that [VoL. 2 250 ANNALS OF THE MISSOURI BOTANICAL GARDEN lipocolloids can play an important part in endosmose, as Overton originally suggested. If there really are plasmatie lipoids present, they probably have no significance as the path of nutrient substances into cells. But, on the other hand, lipoids certainly participate in narcotic effects, because the more soluble is this narcotic in fat the more of the narcotic substance is stored by the plas- matic substances. Consequently, the higher members of the series of alcohols are more injurious for cells than the lower, because the lipoid constituents of pretoplasm become satur- ated with the narcotic and can discharge these narcotics only slowly. So the protoplasm succumbs to the influence of the narcotic agent. On this point I share the opinion of Boeseken and Waterman. The capillarity-rule can scarcely be explained otherwise than by the hypothesis that lipoids are present in the surface layer of protoplasm. So we are forced to continue our work as an exploration designed to determine if lipocolloids are present in protoplasm. A plan was devised and a decision was sought in the following manner: Emulsions of pure trio- lein or of olive-oil were prepared which had about the same surface tension value as have solutions injurious to proto- plasm. To a series of samples arranged from such a fat emulsion alcohol in gradually increasing amount was added. The question now was whether there were effects produced on the emulsion in some way comparable to the action of alcohol on cells. Cell plasma contains also protein bodies and mineral salts. So our model of emulsion had to be compounded by adding a solution of mineral salts, as a physiologically balanced mixture, and by adding also albumen solution. The mineral salts were added as in the Van’t Hoff mixture in 0.1 molar concentration. An alkali is indispensable, so that 0.1 mol of sodium carbonate was used in order to produce a fine and stable emulsion upon shaking the mixture with oil. The results were in brief the following: When a fat emulsion from olive oil was prepared by mixing only oil, water, and sodium carbonate, the decomposing effect of alcohol on the emulsion was noticed at a concentration of 3 mols, i. e., about 15 per 1915] CZAPEK—PROTOPLASM AND ITS COLLOIDAL PROPERTIES 251 cent. When concentrations higher than this were used then the emulsion, examined capillarimetrically, did not differ from a mixture of pure alcohol and water of the same concen- tration (but without oil). Then we added to the emulsion Van’t Hoff’s solution 0.1 mol instead of water. The decompo- sition of the emulsion by ethyl alcohol was now observed at 2 mols, i. e., about 10-11 per cent. This is just the concentra- tion of alcohol which kills cells of the higher plants. The addition of sodium chloride 0.1 mol instead of Van’t Hoff’s liquid showed the critical concentration of alcohol to be 3 mols, about the same concentration as in the absence of mineral salts. On the other hand, the addition of magnesium chloride induced the fatal effect of alcohol at 1 mol, much lower than in living cells. Magnesium sulphate showed the same effect as magnesium chloride, and the sulphate of sodium the same as the chloride. Therefore, it does not seem probable that the differing solubility in alcohol is responsible for the various effects of the salts. One may endeavor to explain these phenomena in the following way: Emulsions are only stable when the droplets of the emulsified fat remain suspended in a soap solution of approximate concentration. Substances which alter the limiting surface between the soap solution and the suspended oil must prove fatal as soon as their capillary activity surpasses the capillary effect of the soap solution. Bivalent cations, such as Mg and Ca, which form insoluble salts with fatty acids, lower the concentration of soap, so that alcohol must exhibit a decomposing action on the emulsion, even in lower concentrations. From such experiments it seems as if the critical concen- tration of alcohol for living cells would not be so sharply determined by proteins contained in protoplasm as by the mineral salt and the lipoid constituents of the protoplasm. Since we suppose that the various mineral salts in protoplasm are present in about the same concentration as they are found in sea water, or as they are mixed together in Van’t Hoff’s solution, we have to face the question whether the destructive effect of alcohol on living cell plasma consists in some decom- position of colloidal fat emulsoids in protoplasm. [VoL, 2, 1915] 252 ANNALS OF THE MISSOURI BOTANICAL GARDEN That protein bodies are not primarily affected by alcohol and other narcotics seems to be sufficiently proved by the fact that ethyl alcohol coagulates protein solution at a concentra- tion not lower than 5 mols, and that while the higher alcohols show fatal effects on living cells, they do not produce any protein coagulation. So we are brought, I think, by several facts to the conclu- sion that living protoplasm must be considered as a colloidal emulsion of lipoids in hydrocolloidal media, the latter con- taining proteins and mineral salts. For the endosmotic pas- sage of dissolved substances the fatty constituents of proto- plasm have no significance. The narcosis, however, and the deleterious effects of aleohols clearly show how lipoids, more than the protein constituents of the surface layer of proto- plasm, participate in such phenomena. The more we advance in the disclosure of the details regarding colloidal mixtures and structures in living protoplasm, the more indispensable it is to be reserved when applying the new results to the var- ious problems to which an approach is so tempting to the physiologist. Many may feel inclined to be disappointed when they ob- serve how much time and mental energy are needed to study only so small a question as that about the presence of fat in protoplasm. But now after some years’ work on this sub- ject it may be seen how important a part is to be attributed even to the combination of mineral salts contained in the plasma colloids. And so we may hope that in the progress of research new and unexpected paths may become visible and open to the indefatigable investigator. Further, we shall not be discouraged if when after long and patient work some results and ideas are won which subsequently are proved untenable. We are all common soldiers in the great battle for truth in science, and we know that few will attain the happi- ness of planting the flag of victory upon the battlements of the conquered fortress. Bas THE EXPERIMENTAL MODIFICATION OF GERM-PLASM D. T. MACDOUGAL Department of Botanical Research, Carnegie Institution of Washington The doctrine of an inviolable germ-plasm has formed the foundation of many imposing edifices in biological thought, and facilitated many advances in genetics and heredity during the last two decades. The authors who have rigidly adhered to the principles of the hypothesis and reasoned from its tenets have exposed many fallacies which have been offered in ex- planation of problems in evolution. This prevalence of theoretical considerations over mistaken experiences has laid the foundation for an unreasoning devo- tion to the idea of an independent germ-plasm, carrying agents which may not be seen, measured, or tested in any practicable manner, and which might consequently be termed ‘‘idealo- plasm’’ with attributes approaching the supra-physical. The desperate straits of those who voluntarily consign them- selves to the bondage of such a conception is well exemplified by the group of writers who subscribe to the conclusion that all evolutionary movement is due simply to recombination and re- arrangement of qualities or factors already present in the protoplasm. An additional illustration of the futile extremes to which this view may be pushed is to be found in the recent utterances of Bateson, who has arrived at the conclusion that evolution is mainly and essentially loss of inhibitors, and re- lease of activities previously latent or suppressed, an hypoth- esis which predicates premutation. If it be allowed that the non-appearance of a character is a direct loss of its determiner and that the appearance of a new feature is the loss of a retarder or inhibitor which held it in abeyance, then the answer to the question as to the method by which organisms have arrived at their present condition is obvious, but of a simplicity that is metaphysical instead of actual and hence of little value, even tentatively, as a frame- ANN. Mo. Bor. GARD., Vor. 2, 1915 (253) [VoL. 2 254 ANNALS OF THE MISSOURI BOTANICAL GARDEN work on which new concepts in biological science may be for- mulated. The group of problems with which we are endeavor- ing to make headway are in the domain of physiology and their solution may be reached only by experimentation, the re- sults of which are to be interpreted in terms of physico-chem- ical activities and their correlated functional manifestations in the living organism. That phylogenetic advance in the main lines of descent in the plant kingdom at least reflects, or harmonizes with, the ex- pectancies of somatic experience is tacitly admitted on all hands, but that the direct response of a shoot to the environ- ment, or conversely stated, that the impression on the soma made by environic agencies is communicated to successive gen- erations in a constant manner has not been demonstrated, although it seems fairly established that certain experiences of individual plants are reflected directly or indirectly to the next generation, and in lesser degree to the next or second generation. How are lasting or permanent changes brough about? Functional adequacy and architectural suitability present themselves on every hand, yet about all of our reliable evi- dence is against anything like a direct or functional adaptation becoming hereditary or continuously transmissible. Two methods of experimental attack on the problem are available. Species showing measurable features and of sim- ple genetic constitution may be taken from their habitual or known environment to other localities in which the climatic and soil characters may be calibrated and the response of the organism, somatically and hereditarily, determined. Hun- dreds of thousands of introductions and acclimatization opera- tions have been carried out in agriculture, horticulture, and especially in botanic gardens during the last century, yet neither the genetic constitution nor the response of the or- ganism has been followed by trained observers who compared the plants in their different habitats. The exposure of the organism to any climatic complex, of course, might affect the germ-plasm directly, and any departure detected in such ex- perimentation must be evaluated by controlled cultures under 1915] MACDOUGAL— MODIFICATION OF GERM-PLASM 255 laboratory conditions in which both the nature of the reaction and the identity of the inciting agent may be found. The most notable series of experiments of this character which have as yet been carried out are those of Tower with the potato beetles. Over two hundred species of seed-plants selected for their suitability and promise of response have been taken into the series of cultures of the Department of Botanical Research on mountain top, desert, and at the sea-shore, less than eighty of which have survived and about a score continue in all three locations. The most notable feature in the behavior of these plants put under stress in unaccustomed habitats consists in divergences in sexual reproduction and seed-formation. Con- jointly with this decrease of the sexual reproduction, vegeta- tive propagation assumes a greater importance. Shoots are variously affected. The measurement of these departures and their fate when the nth generation is returned to the original habitat, or to a place in which the habital tension is changed, will be necessary to determine whether or not perma- nent impress on the species has been made. The second method would include all forms of experimenta- tion in which inciting agents would be applied directly to the reproductive bodies, in which case any deviation from the usual or typical would be more clearly attributable to changes in the germ-plasm. It is pertinent to call attention to the necessity for new viewpoints and new standards in the evaluation of any results which may be obtained in such manner. We are not likely to go far or progress easily into the region of the unknown if we attempt to interpret these effects too directly, with the idea that determiners, inhibitors, genes, ete., are ultimate or even penultimate units. In brief, the time has come for testing the performances of lineal series of organisms by methods in which attention will be centered upon the physico-chemical complex and an open eye will be kept for cleavage lines which may cut across directly or obliquely the limits of all of the arbitrary concepts of alternate inheritance. The house of the living thing is inclusive of walls, doors, roofs, windows, floors, (Vou, 2 256 ANNALS OF THE MISSOURI BOTANICAL GARDEN ceilings, rafters, and plumbing, but the materials used may be bricks, stones, metals, sand, lime, boards, glass, and paint. Our present mee lasd us to experiments with these com- ponents rather than to trials of the possible combinations and inhibitions, possibilities and impossibilities of sets of builders’ blocks, no matter how complete or full these may be. Living material is a colloidal complex with its enmeshed re- actions highly fluctuant, its combinations unstable and its types of energy transformation multifold. It is concrete, however, and amenable to experimentation of many kinds. Its physical qualities and form undergo changes of phase which have some correspondence with the mechanism of mor- phogeny, reproduction, and heredity. Thus, for instance, in the higher plants the germinal protoplasm in the earlier stages of the individual is in the form of meristematic tracts made up of highly distended plasts in which absorption of water, hydra- tation, auxetic enlargement, and division of the separate ele- ments is very marked and rapid. Elements at the peripheries of these masses are separated which undergo differentiation and pass into the permanent tissues of the individual. These separating cells may be modified to an enormous extent by external agencies; thus conditions of aridity acting upon an individual may cause the tissues formed from its embryonic tracts to make such structures as to give the organs which they make up a xerophytic aspect. This final xerophytie or other character of the soma, how- ever, is in the permanent tissue, and the modifications which have resulted in its specialization ensued after the cells were pushed away from the meristem, and there seems to be no reflection of the final fixed qualities back to the embryonic tract, although there are many promising possibilities to be considered. Of these none are more interesting than the regenerative processes by which highly specialized cells reassume embryonic activity and reproduce members or indi- viduals vegetatively. Actual tests of the transmission and permanence of the specializations under these conditions have not yet been made with that exactitude which would allow any serious conclusion to be formulated. At certain stages of the 1915] MACDOUGAL—-MODIFICATION OF GERM-PLASM 257 ontogeny, generally much later in the plant than in the animal, and this is a matter which may be determined by the environie agencies, the germ-plasm or meristem tract undergoes such change of phase that instead of all of its separating elements passing into somatic cells a few become reproductive masses from which sexually specialized elements may be differenti- ated, and in which the number of chromosomes, the metabolic balance, degree of hydratation, auxetic energy and mechanism of division suggest physico-chemical conditions widely dif- ferent from those of somatic elements; furthermore, the repro- ductive elements are highly individualized. The meristem in its myriad cells may at any moment present all of the phases of growth and differentiation. The egg nucleus or the fer- tilized egg, a single element of the plasma, may include the fate of the individual and its unending line of progress, and it may be affected in its entirety by agencies impinging upon it. The reaction of such specialized cells to external agencies would of course be different from those of the meristem tracts, which are made up of plasmatic units of the most generalized form. The experiments of Tower with the Leptinotarsae, which have been carried on under widely diverse conditions in southern tropical Mexico, in the arid semi-tropical climate of the Desert Laboratory, and under controlled conditions at the University of Chicago, furnish a great series of cultures of these beetles in which it is possible to demonstrate logically by exclusion and analysis that certain climatic features, notably moisture, may affect the germ-plasm, or the entire organism when the germ-plasm is in a certain stage, in such manner as to induce disturbances in hereditary lines. These experi- ments show the vulnerability of the germ-plasm. That the germ-plasm is directly responsive to the action of foreign substances which are introduced into the embryo-sae was demonstrated when (early in 1905) I was so fortunate as to hit upon an experimental method of treatment of the ovaries of seed-plants which resulted in the formation of em- bryos developing into individuals not entirely identical with the parental types. The essential feature of the discovery [VoL, 2 258 ANNALS OF THE MISSOURI BOTANICAL GARDEN consisted in the successful introduction of various substances into the neighborhood of the embryo-sacs at the time that fertilization was imminent, and when the first trials were made I had two main purposes in mind: first, to ascertain whether or not foreign substances could be introduced into ovaries in such manner as to affect the ovules with a minimum of trau- matic effects, so that the ovaries might reach maturity; and secondly, to ascertain whether or not such changes could be produced in an early stage of sexual specialization, before the development of the embryo-sac or after the union of the sexual elements in fertilization. The first results were obtained with pure strains of Oeno- thera biennis and Raimannia odorata at the time mentioned, but the transfer of my activities from the New York Botan- ical Garden to the Desert Laboratory made it impossible to carry out cultures of the progeny or to repeat similar experi- ments upon this material. Meanwhile, Col. R. H. Firth, of the Royal Medical Corps of Great Britain, duplicated’ my general results with Raimannia and other plants in 1908, al- though the fact that I had previously done this work was unknown to him. New material was selected from the vicinity of the Desert Laboratory and the tests were begun anew in 1906. The diff- culties to be overcome in such experiments are fully commen- surate with the importance of the problem upon which they bear. It is a necessary preliminary that the plants chosen for the operations should be an elementary strain, a matter which may need two or three years for determination, if not already known. Next, not all ovaries will withstand the shock and injury inflicted in the operations. The chances of ulti- mate success will be greatest in many-seeded ovaries in which the number, however, does not extend much beyond that of ovules which may be affected by a single operation, giving some opportunity for differentiation of effects and not entail- ing large cultures. Lastly it is advantageous to deal with perennial species which come quickly to maturity. This gives 1 Firth, R. H. Roy. Med. Corps, Jour. 16:497-514. 1911. y p 1915] MACDOUGAL—MODIFICATION OF GERM-PLASM 259 the operator opportunity to preserve the original material alive and to have it for comparison with succeeding genera- tions. The numerous cacti in the vicinity of the Desert Laboratory lead them to be selected for some tests, and the mechanical conditions for operation which they offer are unexcelled. As much as 1 ce. of solution may be introduced into the ovary of an opuntia without traumatic effects, but as all are under suspicion as to their genetic complexity, and as they germi- nate and develop slowly, the investigator must wait the greater part of a decade to obtain decisive results. Striking depart- ures were obtained with Echinocereus Fendleri, a small cylin- drical form native to southern Arizona, and the changed characters grouped in one derivative have not been obtained in nature or in cultures of the original. This derivative has been obtained a second time. The species, however, presents such a complexity of characters that definite conclusions are difficult. Similar conditions were encountered in Penstemon Wrightii, about which an announcement was made in 1909. Some of these, however, furnished material from which the greatest sources of error might be eliminated. The search for suitable subjects for experimentation was continued and the results with Penstemon led to a closer ex- amination of other members of the Scrophulariaceae. Finally, an undescribed species of Scrophularia from the pine-forest area on the Santa Catalina Mountains in Arizona was brought into the environic series of the Laboratory of this Department in 1909. Rootstocks were taken to the Coastal Laboratory, and seeds were germinated at various localities. After hav- ing seen many hundreds of plants taken from various parts of its range and having followed them thoroughly two and three generations, it was found that the species is a simple one and not readily separable into elementary forms or strains. The only noticeable feature suggestive of complications was the fact that the broad-bladed nepionic leaf-forms are sometimes carried nearly to the summits of stems grown under certain conditions, giving the appearance of a robust race. [VoL. 2 260 ANNALS OF THE MISSOURI BOTANICAL GARDEN Another feature that received attention was the fact that branches formed in the closing part of the cycle of develop- ment of shoots bear leaves very much smaller than those aris- ing from the median part of the main stem during the first part of the season. The flowers borne on these branches are also much paler than those on the more robust branches. Pelorie flowers sometimes appear near the apices of the in- florescences in this as well as in other species of the genus. It is to be noted also that the divisions of the corolla are variously and irregularly ineised on individuals at times dur- ing the season, but these are not heritable and do not appear in any regular manner. This scrophularia appearing to offer some promise, several ovaries of a plant at Carmel were treated with solution of potassium iodide, one part in forty thousand, in July, 1911, and the ripened capsules were collected in September of that ear. No record was made as to the time of day (see page 268) and nothing may therefore be said as to the possibilities of the action of the reagent on egg or pollen nuclei, singly, together, or after fertilization. No other species of Scro- phularia grew near the cultures at that time. The seeds were sown in suitable pans of screened soil, and in February three plantlets had survived. In May these were set in the open and their development followed. One formed a shoot fairly equivalent to the normal, finally producing flowers in which the anthocyans of the flowers were of a notice- ably deep hue. The two remaining plantlets were character- ized by a succulent aspect of the leaves, and by a lighter or yellow color of the leaves and stems. Inflorescences were matured late in 1912, and the flowers on one of the derivatives, as they may be called, were so completely lacking in color as to be a cream-white, this derivative being designated as albida, while the other showed some marginal color and a rusty tinge, and was designated as rufida. Some disturbance of the relative velocities of development of the fibrovascular elements and mesophyll had taken place in both forms, so that the leaves were variously bowed and con- vexed and the two halves of the laminae were unequal and the 1915] MACDOTIGAT MODIFICATION OF GERM-PLASM 261 whole blade was more oblique in outline. The elongation of the lamina had been checked and the ratio of width to length of the leaves was greater than in the parental stock. If cor- respondent leaves of rufida and the originals were laid side by side it could be seen that the basal veins on the side away from which the tips were curved were different in the two cases, the derivative showing two strong veins in the place in which one lateral with a thin branch occurred in the original (fig. 1). The water relations of derivatives and normal were not identical, and when young shoots or branches developed D O Fig. 1. O, branching lateral vein in parental Scrophularia; D, branching vein replaced by two laterals in leaf of modified Scrophularia. under similar conditions were detached, those of the deriva- tives flagged and wilted much more quickly than those of the normal. The auxetic departures noted above also extended to the inflorescences, which in the original show a fairly regular basipetal development into thyrses. The derivatives, how- ever, exhibited a rather irregular maturation of clumps of buds and the thyrses were very irregular, not reaching the spread of the parental forms. The fragility of the leaves does not seem to extend to the flowers, which opened very slowly, and in some cases the distended corolla persisted for a few days. The amount of color in the corolla was largely a matter of illumination, but under equivalent circumstances the derivatives always showed less than the parental form. As noted above the color persists to some degree in the deriva- [VoL, 2 262 ANNALS OF THE MISSOURI BOTANICAL GARDEN tives along the margins of the uppermost lobes of the corolla, while that on the broad upper surface disappears. It is to be recalled that it is the color of this region which is variously disposed in other species of the genus. The corolla lobes were irregularly incised in the flowers of the first and second seasons of the F;, as they have been seen to be in the original, but in the second generation of both derivatives cultivated at the Desert Laboratory this effect per- sists as a regular wedge-shaped incision of the lower lip only, and is not seen in every individual of both derivatives, although the seeds were from plants which may have been pollinated by the parental form. Seedlings from the original stock grown from seeds gath- ered on the Santa Catalina Mountains in Arizona were sowed early in 1910 at the Desert Laboratory and the plantlets pre- served on April 15 furnished the data: First pair of leaves smaller than in the derivative, being only 13-15.5mm. wide and 16-18mm. long, obscurely dentate with not more than two or three blunt teeth showing on each side. The petioles were 12-16mm. long. The third pair of leaves above the cotyledons, which probably were not quite mature, had petioles 20mm. long, and laminae 22-25x50-52mm. Mar- ginal stalked glands were so numerous that 15-20 appeared in the field of the microscope at one time, and these structures were very numerous on the petioles. It is to be noted that differences in the last-named feature between this original and the derivatives disappear in the adult, or on the leaves appear- ing in the later stages. Seeds from the original two derivatives matured at Carmel late in the summer of 1913 were sowed in the greenhouse at Tucson in November, 1913. But one plant of albida, the ex- tremest departure, survived, while four of rufida were secured. These, of course, represented the F of the departures. The measurements of rufida correspondent with those of the original are as follows: First three leaves deeply incised, five or six teeth on a side, abruptly pointed. Petioles 18-22mm. long, laminae 21-26mm. wide and 41-45mm. long. Mature leaves on sixth, seventh and eighth internodes, with petioles 1915] MACDOUGAL——-MODIFICATION OF GERM-PLASM 263 36-45mm., and laminae 36-56x85-100mm. Marginal glands showing 6-10 in field, few on the petioles. The single plantlet of albida bore leaves, the first pair of which were not deeply cut, the three or four teeth on each side being abruptly but sharply pointed, the petioles 15mm. long, and the laminae 24-26x35-38mm. The leaves from the sixth, seventh and eighth internodes had petioles 30-40mm., and laminae 45-51x90-100mm. Not more than four stalked glands might be seen in the field at any one time. These trichomes were very sparsely distributed over the under surface of the petioles only. The greater relative width of these leaves was correlated with a greater angle of divergence of the lateral veins from the midrib, a feature which, as will be shown later, was to be observed in adult plants. The three plants representing the progeny of the treated individual were established in a row within a half meter of each other at Carmel in 1912. Irregular clusters of long thickened roots were formed, and these, as is customary with the species, bear buds and are a means of propagation of the plant. The three plants were taken up in November, 1913. While the main clumps could be identified, yet broken frag- ments of roots were preserved which could not be assigned to any one of the three, and although these were and are still pre- served they are not taken into account here. Albida was divided in May and June, 1914, and portions were sent to codperators in New York, St. Louis and Chicago, but all failed to survive this unseasonable transplantation, so that at the present time this strain is represented by only two clumps, one of which is at the Desert Laboratory and the other at the Coastal Laboratory. The single plant of albida bloomed at Tucson early in the year, while the one at Carmel reached that stage too late to mature seeds. Rufida was divided into three clumps and reset in the garden at the Coastal Laboratory in November, 1913. The shoots from these began to open flowers in July, 1914, which corre- sponded in all essential particulars with those of the previous seasons except that they were more highly regular. Two were enclosed in small glass cages for protection and to insure [voL. 2 264 ANNALS OF THE MISSOURI BOTANICAL GARDEN close pollination, a strong individual of the original being similarly enclosed for purposes of control. Conditions being favorable for a minute comparison of these plants with the parental type, colored illustrations of flowers and buds and diagram of structure were prepared. The inequality of the leaves was recorded by direct prints. The dimensional relations noted above were again seen. The readiness with which the leaves flag was noted and in these organs, as well as in the stems, it was seen that rigidity is maintained by tur- gidity rather than by stiffness of the mechanical tissues. The development of the bast-fibers is less marked in the derivative, Fig. 2. Lower line shows outline of angle of stem of parent Scrophularia; upper line outline of same feature in derivative. and a similar deficiency of wood-formation is noted. A cor- respondent difference is apparent in the wings of the angles of the stems, which are thick with their sides parallel in the original, while in the derivative these decrease in thickness gradually toward the margin, with the effect in cross-section seen in fig. 2. The actual value or importance of these dif- ferences is not a matter of moment in the present connection. The chief interest lies in the fact that recognizable effects have been produced by the introduction of foreign substances into ovaries and that the differences shown by the first generation, F, are borne by the second generation, F2. The original ob- servations with the plant in which this was demonstrated 1915] MACDOUGAL— MODIFICATION OF GERM-PLASM 265 began in 1909, the treatments were made in 1911, and now first and second generations of the derivatives are alive, as well as the original stock. Much irrelevant comment and inconclusive experimentation has followed the original announcement of the discovery of the methods used in this work. The necessity for a careful genetic analysis of the material for treatment has already been noted, and it may be well to call attention to some of the features of operation which might appear simple, yet are not easily carried out. No better way has yet been found for in- troducing solutions into the region of the embryo-sac than by injection into ovaries with an all-glass syringe fitted with gold needles (14 karat). The wounding of the ovary produces abortion in some species, and in almost all treatments some of the ovules are crushed. This, however, is a matter of no mo- ment if some reached by the reagent survive and come to maturity. The extent and mode of diffusion of the reagent is in fact one of the most important features of the treatment, and the experimenter will do well to make control tests for the purpose of finding out whether or not there is some possibility of success. A test of the ovaries of Carnegiea previously described showed that the liquid was taken up by the placental vessels and conducted to a point near the egg cell in a very short time if the reagent were introduced into the ovaries of flowers fully open and mature. Operations made at an earlier stage re- sulted in the accumulation of the reagent in the inner walls of the locule, in the integument of the ovule and especially at the micropylar orifice. The pollen tube would be subject to the action of the accumulated substance in the micropyle and integument in this case.! It being my present intention to extend experimentation in the Scrophulariaceae, tests have been made with methylene blue in the ovaries of Penstemon Torreyi, the solution being one part of the dye to ten thousand of distilled water. 1 MacDougal, D. T. Alterations in heredity induced by ovarial treatments. Bot. Gaz. 51; 241-256. 1911. [VoL. 2 266 ANNALS OF THE MISSOURI BOTANICAL GARDEN Three hours later but little of the color could be found in sections of the ovary. Next, five ovaries of Oenothera 3.21 A” reer Fig. 3. Diagram of flower of Scrophularia, showing mechanical features of plige treatment: s, sepals; c, corolla; g, nectar gland; p, placenta; st, style; , ovule; rec, receptacle; p, tip of hollow needle thrust through the ovarial wall mee penetrating the placenta. The stippling shows the diffusion of a solution of methylene blue introduced by the needle.—Drawn by F. E. Lloyd. (a stable cruciate hybrid) were injected with a solution of one 1915] MACDOTIGQAT MODIFICATION OF GERM-PLASM 267 part inathousand. Fifteen to forty ovules had been touched by the color in young flowers not yet open. A much larger number had been colored in the ovaries of mature flowers. This solution was introduced into ovaries of the scrophularia under examination (fig. 3). Young ovaries in this plant showed very few ovules affected, none in a few cases. Older ovaries in which fertilization had probably taken place showed as many as 15-20 colored ovules. Probably only a small pro- portion of the ovules affected would have survived and de- veloped into viable seeds, so that many of the treated ovaries would have yielded nothing but normal seeds. This condition is to be taken into account by those who do not recognize the technical difficulties in the way of duplication of any particular treatment. The recent results of Churchman and Russell! in securing stimulation of the growth of animal tissues with methylene blue suggest that this substance might produce some effects on the embryo-sacs of plants, and also the advantage of using a reagent the diffusion and penetration of which are visible and obvious. It was desirable to use this dye in obtaining some knowledge of the probable action of other solutions in Scrophularia, so tests were made with this plant. A number of ovaries on a detached shoot in the laboratory were placed in a solution of one to a thousand at 9:30 a.m. Material was taken for ex- amination at suitable intervals. The placental walls and funicles were stained in part within a half hour. Two hours later the color had advanced well along the conducting tract in the funicular stalk. Five hours after treatment a notable amount of the dye had been carried clear to the embryo-sac, where it stained the nucellus and the antipodal region deeply. It is to be noted that the material was still alive and that this material if left attached to the plant would have developed some mature seeds in all proba- bility (fig. 3). 1 The effect of gentian violet on protozoa and on growing adult tissue, Soc. Exp. Biol. and Med., Proc. 2:124. 1914. [vor. 2 268 ANNALS OF THE MISSOURI BOTANICAL GARDEN Professor F. E. Lloyd, of MeGill University, who kindly came to my aid in this matter, now made a brief study of the intra-vitam staining in the ovules of Scrophularia and found that the reagent accumulated throughout the embryo-sac in- clusive of the egg cell, demonstrating the possibility of the direct action of introduced solutions on the entire egg appa- ratus as well as upon the endosperm. The micropylar orifice was closed and was not stained in the ordinary treatments and took up only a small amount of the dye when laid separately in a solution of it. Professor Lloyd also showed me prepara- tions in which pollen tubes deeply stained had entered the micropyle and had elongated, reaching the egg.! These experi- ments made clear the immediate possibility of reagents reach- ing the egg apparatus through the funicle and of the staining of the pollen tube and nucleus in the cavity of the ovary before fertilization. It is also possible that the pollen tube might be affected by reagents which had accumulated in cells through which it penetrates to the egg nucleus (fig. 4). These facts would make it probable that treatments before pollination has taken place would affect the embryo-sac and its inclusions only, while introductions of solutions at a later stage would be likely to affect the pollen tubes and nuclei. These generalizations are to be taken to be applicable to Scro- phularia, and to species which present similar arrangements for reproduction. The egg in ovules in which the micropyle is open might be even more readily exposed to the action of a reagent, and if the ovule is porogamous the pollen tube would also inevitably be affected, and still many other combinations may be encountered which need not be enumerated at this time. It is of course to be understood also that not all of the ovules in any pistil are in equivalent stages of development at any given moment, and this applies also to the penetration by the pollen tubes. Pollination of Scrophularia takes place in the morning, and substances introduced before mid-forenoon * See Lloyd, F. E. The intra-vitam absorption of methylene blue in ovules of Scrophularia. Report of the department of botanical research for 1914. Carnegie Inst. Washington, Yearbook 13:77-81. 1914. 1915] MACDOUGAL—MODIFICATION OF GERM-PLASM 269 would be taken up and diffused through the tissues, especially through the funicle before the pollen tubes had reached the cavity of the ovary. Introductions timed to meet the elongat- _ = raphe--77 (SAER oe C6738 EN 4 Apt Opens NAE canes) InN PATE Fig. 4. Diagram of longitudinal section of ovule of Scrophularia: fun, funicle; chal, chalaza; ant, antipodal cells; tap, tapetum; end, endosperm; mic, micro- pyle. The shading shows the course of a solution of methylene blue diffusing through the funicle from the placenta (see fig. 3) and its selective fixation in the tapetum and nucellus. The solution finally reaches the ovum. ing pollen tubes would of course be more liable to affect the pollen nuclei, and a number of lots of seeds matured in ovaries [VoL, 2 270 ANNALS OF THE MISSOURI BOTANICAL GARDEN treated at various stages of development now await germina- tion and test. The differences between the two surviving derivatives of Scrophularia described in this paper may well be due to such differential action. It is to be seen that if egg or sperm were affected singly the resultant seed into which these elements might enter would be hybrid. Even if both were acted upon, it is by no means to be taken for granted that the effects in the two would be equivalent. The Fə of rufida was identical in the cultures described, while the Fa of albida presented some modifications, the status of which is not yet established, as both were open pollinated in the Fı. Very little informa- tion as to hybrids in Scrophularia is available. Goddijn and Goethart! report that S. Neesit Wirtg. X S. vernalis L. is a unified, stable, intermediate type and that the reciprocal is of a similar character. The behavior of the original stock, and the facts of fertiliza- tion, yield nothing suggestive of parthenogenesis, and the de- rivatives may be taken to be produced by a typical fertiliza- tion. No cytological examination has yet been made for the purpose of ascertaining possible differences induced in the chromosomes. This discussion may be fittingly brought to an end by a brief reconsideration of the salient ideas which have been touched upon. The point of view taken throughout all of the work which has been described is one in which the conception of a theoretical or idealized germ-plasm has been relegated to secondary position, and attention has been concentrated upon the concrete germ-plasm of the higher plants. This physical basis of heredity is seen to present two distinct phases. In one it takes the form of a meristem or embryonic tract of highly distended cells in which auxesis and division are both rapid and the elements which are separated from it pass by differentiations into the permanent tissues of the soma. En- vironie agencies affect only the development of the somatic cells which are being formed from the meristem, and the ex- 1 Ein künstlich erzeugter Bastard Scrophularia Neesii Wirtg. X S. vernalis L. Van’s Rijks Herb., Mededeel. 1913+": 1-9. 1913 1915] MACDOUGAL— MODIFICATION OF GERM-PLASM 271 perience of these cells are not reflected back to the embryonic tract, so far as available facts may be considered. Sexually specialized reproductive elements with a reduced number of chromosomes are developed from the embryonic tracts in a late stage of the ontogeny, and these elements present a meta- bolic balance different from that of the meristem stage, the colloids having a greater density, and some of the energy transformations having altered velocities. The embryonic tract or meristem of a higher plant at any given moment includes an enormous number of primitive or initial cells and of separating elements in all stages of division, growth, and differentiation toward the specialized tissues which are derived from it. The tract as a whole could there- fore not react in a unified manner to any climatic or environic agency which would impinge upon the plant. Such forces, as a matter of fact, visibly affect only the manner in which the dif- ferentiation of the resting tissues takes place. The rejuven- escence of such differentiated cells might carry the effects into the organ or individual produced by the regeneration, but no test has yet been made of this matter, or of the transmission of such supposititious characters to a second sexually produced generation; neither has the proposal, that repeated or long continued exposure of the germ-plasm to any environic stimu- lus may result in the fixation of effects, been tested out. The continuation of introduced species in the mountain, desert, and coastal plantations of the Department of Botanical Research for the term of years during which any one person might con- duct such experiments, may not be taken as an adequate test of this phase of the matter, although these cultures are carried on for the express purpose of determining what permanent changes may be induced by the tension of unusual environic complexes. So far these have been confined to alterations in sexual and asexual reproductive procedure, and to alterations in structure and aspect of the shoot, while no tests have been made upon the fixity of the changes. Aberrant behavior of the chromosomes in certain determi- native or initial cells may possibly be responsible for bud- mutations or bud-variations, and theoretically it is conceivable [VoL. 2 272 ANNALS OF THE MISSOURI BOTANICAL GARDEN that special stimuli might be applied to such cells in a manner that might bring about similar results. Practically, however, it would be enormously difficult to localize initial cells with sufficient certainty so as to give any slight chance of success. The second stage of germ-plasm in which it is in the form of sexually specialized elements offers far more promising conditions for experimental modification of the genetic con- tent of the species which it represents. Solutions may be in- troduced into the ovaries in such manner as to affect the egg bearing the entire group of qualities of the species, and fur- thermore the direct action of such reagents may be ascertained to some extent. The present-day aspect of the mechanism of heredity is one which increases momentarily in complexity. The greater part of the researches in genetics during the last fifteen years has been devoted to the interaction of factors, determiners, in- hibitors, or qualities in the organism. If these conceptions may be taken to be the expression of the reactions of either chemical groupings or to rest upon a physico-chemical founda- tion of any kind, the reagents which have been used have not been of a selective character, but would affect practically the entire colloidal mass of the protoplast in some manner and to varying extent, neutralizing or coagulating proteins, and their general tendency would be to inhibit or check energy trans- formations. In the case of the iodine treatments the free ions from potassium iodide or the iodie acid formed would cause a neutralizing effect, as it does not seem from the results of Czapski and Adler! that this element would form any com- pound with the proteins. The experimenter is dealing with an actual physico-chemical complex of highly unstable compounds in which many types of energy transformation are occurring. Introduced sub- stances may slow down or inhibit some of these, and accelerate others or start new reactions. The morphological possibili- ties in any given strain of plants are somewhat limited, how- ever, and in this sense the direction of the departures is al- 1 Beiträge zum Chemismus der Jodwirkung. Biochem. Zeitschr, 65: 117. 14. 1915] MACDOUGAL—MODIFICATION OF GERM-PLASM 273 ready determined. This limitation of the possibilities of morphogenesis is the chief one in any expectancy of duplica- tion of results in successive treatments, outwardly mechanic- ally identical. The variables in any experimental setting are many, and the briefest consideration of the physical effects consequent upon the introduction of a foreign solution to the vicinity of the embryo-sac, reveals at once the lack of probability of exact repetitions in a mechanism so complex. The conditions are much different from those which would be presented if free floating eggs or sperms were immersed in a solution. If we are able to induce other changes in Scrophularia besides those shown, they will be quite as important in demonstration of the fact that germ-plasm had been modified as if they were exact repetitions of previous inductions. If previous results were exactly recalled there might be some suggestion of premuta- tion. It is evident that the experimenter who wishes to proceed with the greatest precision and least loss of effort will first test the genetic strictness of his living material, ascertain the rate and manner and diffusion of solutions in the ovary and ovules, the time of pollination and the rate of development of the tube in reaching the egg. Next, the structure and number of ovaries and the traumatic reactions of the entire pistil are to be taken into account. Having also traced out the simpler features of pollination and fertilization, the operator should test the effects of various reagents which may neutralize pro- teins, including enzymes, or act as excitators or catalyzers. Without enlarging too much upon the difficulties to be encount- ered in the experiments described in this paper, they may be illustrated by the fact that over fifty operations upon Scrophu- laria in July, August and September, 1914, at Carmel, Cali- fornia, were total failures, as the ovaries perished before reaching maturity. Finally, many present interests in phylogeny and genetics will be concerned with the nature of the evolutionary move- ment which is simulated by the alterations which have been induced experimentally by the method described. Some of [VoL. 2, 1915] 274 ANNALS OF THE MISSOURI BOTANICAL GARDEN these would unquestionably be designated as of a retrogres- sive character, such, for example, as the defection of a part of the color pattern of the corolla; others, such as the accentuated ineision of the leaves and corollas and the development of the venation, as progressive alterations; while still others may not with any substantial reason be assigned to either class. With reference to taxonomic criteria, it may be said that the di- vergent individuals are distinguishable at sight from the parental stock, but the real test of the characters presented is not their degree or kind of departure, but their stability and permanence indicative of actual modifications of the germ- plasm. THE RELATIONS BETWEEN SCIENTIFIC BOTANY AND PHYTOPATHOLOGY DR. O. APPEL Mitglied der Kaiserlichen Biologischen Anstalt fiir Land- und Forstwirtschaft, Berlin-Dahlem The ever-increasing importance of phytopathology is the result of the steady development of agriculture, forestry, and horticulture. In this way phytopathology has become a part of each of these sciences. In former times well-known botanists, such as Gleditsch, Martius, Caspary, de Bary, and Sachs did not estimate themselves too highly to concern themselves at times with phytopathological problems. In modern times, however, it is not often that a university professor of botany occupies himself with such problems. This is due partially to the specialization which has become a necessity in modern science. Above all, however, this is due to a peculiar concep- tion which looks upon the applied branches of applied natural science as something inferior to the pure natural sciences. It must, however, be said that we find exceptions even here, if we think of such scientists as Brefeld and De Vries. Agriculture has within a short time presented many problems to phytopathology, and of these the principal ones have been those of disease control. These problems were often solved in a hasty way, which, I must admit, lacked scientific thoroughness. But even in the solution of these problems many interesting facts were brought to light. But with the progress in working out these questions it became more and more evident that many of these problems could not be ultimately solved unless investigated in a thoroughly scientific manner. In criticising the plant pathologists it should not be for- gotten that most of them are for the greater part autodidacts. Until recent times there were no places where scientific phytopathology was taught. In Germany it was only the ANN. Mo. Bot. GARD., Vou, 2, 1915 (275) [VoL. 2 276 ANNALS OF THE MISSOURI BOTANICAL GARDEN University of Munich, in which von Tubeuf has been and is still teaching the subject. In Austria, Hecke has been giving lectures for some years. In the United States there has been much progress in this line, due, no doubt, to the fact that plant diseases are of greater importance here than in any other country. As the number of chairs in phytopathology in our institutions of learning increases, however, the rela- tion between scientific botany and phytopathology will be- come more and more intimate. j Among the factors which favor unusual ravages by vegeta- ble and animal parasites, I wish to mention the rapid develop- ment of agriculture by way of growing the same varieties or races over vast areas, the great fertility of the previously uncultivated soils, which often induced people to crop the soil and neglect rotation, and lastly the favorable climatic condi- tions, which not only favor the cultural plants but also their parasites. One of the oldest problems of phytopathology is the smut- problem. Since ancient times smuts have been among the most important plagues of our cereals, and long before we knew the cause of these diseases people tried to control them. But rational measures of control could not be developed before the cause of the disease was known. Julius Kühn succeeded in clearing up the life-history of the stinking smut. This was the first distinct step in advance, but here, unfortunately, progress ceased for some time, principally because of the lack of knowledge concerning the taxonomy of the smut-fungi. All loose smuts of oats, wheat, barley, and the close smuts of oats and barley were united under the single species Ustilago carbo. This prevented the investiga- tions of the biology of the smuts, and it was not until the fact was demonstrated that various species of smuts were concerned that the way was opened for the proper investiga- tion of the biology and subsequently also of the control of the parasite. The development of our knowledge of the smuts was due to the biological facts demonstrated by Brefeld and Hecke. They discovered that infection takes place through the 1915] APPEL—PHYTOPATHOLOGY AND SCIENTIFIC BOTANY 277 flowers. This fact pointed out the way of control. The problem was to kill one organism, the smut-fungus, within another organism, the grain seed, without doing damage to the latter. Jensen by his empirical work had demonstrated that such a procedure was possible. The correct method of control, however, could not be worked out because of the lack of knowledge concerning the fundamental scientific facts involved. In order to establish such a firm basis, I, together with my assistant, Riehm, studied the resistance of the smut- fungi to external conditions, primarily to the effects of tem- perature. When the mycelium was grown in water and other substrata we demonstrated the fact that the thicker-celled mycelium as well as the spores are more resistant to external influences than is the vigorously growing mycelium. How- ever, not only the smut but also the grain is more resistant in the resting period than when germinating. Therefore, we tried to bring the infected grain seed under conditions which cause the fungus to grow and which at the same time do not allow the seed to germinate. We succeeded in doing this by allowing the seeds to remain for about four hours in water at 25-30°C. If one then subjects the seeds to a temperature at which the mycelium is killed but which does not yet induce germination in the grain, it is possible to kill the mycelium in the seed without injuring the latter. In these investigations the key to the so-called hot water and hot air treatment was found, and it was then only a technical problem to build apparatus with which the desired results could with certainty be realized. For our conditions in Germany this latter problem has also been solved. We have constructed several pieces of apparatus of this sort, and the treatment of grain against loose smut has been introduced on many farms. But the smut-problem has not been solved for all cases. This is especially true in the case of the stinking smut in the United States. This disease is of the greatest importance in the wheat districts of Idaho. In Germany Tilletia Tritici is spread by the seeds and is controlled by seed disinfection. In Idaho it occurs so generally in the soil that disinfection is [VoL. 2 278 ANNALS OF THE MISSOURI BOTANICAL GARDEN of no avail. Losses of 25 per cent of the crop are not uncom- mon. The solution of this problem seems possible only by the breeding of disease-resistant varieties. It is certain that smut-resistant races of wheat exist. The problem is to find these varieties and, in case they are not sufficiently produc- tive, to cross them with other varieties until races which com- bine the desired characteristics are obtained. In the districts where smut occurs every year it is possible to find these races in an empirical way. But in general it is my opinion that all work of selecting and breeding should be prosecuted along fundamental scientific lines. It is therefore first of all necessary to determine to what characters the plant owes its disease-resistant qualities. When this has been accomplished it is next necessary to deter- mine to what extent the characters are heritable, that is to say, whether they appear in crosses as dominant or recessive. The great advantage of this method lies in the fact that it makes it possible to recognize resistant races (by the presence of the specific characters to which resistance is due) without infection experiments, which are uncertain owing to the influ- ence of external and unknown conditions. I have shown to you by this example that in the solution of a single phytopathological problem such diverse branches of botany as taxonomy, biology of the flower, fungus-biology, and inheritance are involved. The following examples will show that in addition other branches of botany are of import- ance in phytopathology. In exact phytopathological investigations it is a primary factor that one know the host plants and the parasites in detail. This information must be based upon thorough systematic knowledge. This seems to be very easy in culti- vated plants, the species of which are generally well distin- guished. Some cases, however, are more complicated. When we want to make studies of cereal rusts, it is not sufficient to know the races of cereals by their agricultural names. We must know to what botanical species they belong; our eulti- vated wheats, for instance, comprise species of different sus- ceptibilities. 1915] APPEL—PHYTOPATHOLOGY AND SCIENTIFIC BOTANY 279 Much more difficult are the systematic relations of the fungi. Many experiments and publications are valueless because the identity of the fungus was not made sure of in every single case. These difficulties are greater in case the fungi in question belong to the Fungi Imperfecti, where very often only the name of the genus has been determined, while the species name was simply made from the name of the host. Moreover, the descriptions of these imperfect fungi are often so insufficient that it is impossible to identify the fungi after- wards, especially when they occur on other plants or on a different or unrecognizable substratum. Within a genus that is rich in species there have sometimes been erected so many species that there is no possibility of identification. We find an instance of this in the genus Fusarium. Several hundreds of species have been described; which of these are identical has not yet been made clear, and in many cases this may never be possible. We cannot always solve the problem by making use of the exsiccata of the author of the species. Moreover, on one species of host several species of Fusariwm may be harbored, and the author has often considered them identical. It is further often impossible to find what fungus was the type of the author’s description. In such a case the only alternative is a thorough reworking of the taxonomy. How extensive a work this may often involve is instanced by the genus Fusarium. To establish the fundamental facts regard- ing the taxonomy within this genus required four years of work on my part as well as on the part of my assistant, Dr. Wollenweber, who devoted all of his time to the subject. Even after the establishment of these fundamental facts, only a very small part of the species had been determined, and for another two years Wollenweber has been working up the remaining species. I wish only to point out in addition that there exist more genera of this type: Botrytis, Gloeosporium, and Alternaria and its relatives. Modern taxonomy of fungi cannot limit itself to the morphology of the species casually collected. It must have the help of pure cultures on various media, for in artificial culture additional differences show themselves. These differ- [voL. 2 280 ANNALS OF THE MISSOURI BOTANICAL GARDEN ences are not only biological, such as color formation and changes in the culture media, but also morphological, such as the form of the ‘‘Fuszellen’’ or basal cells of species of Fus- arium, and even gross, as, for instance, differences in form of colonies, ete. In the first place, artificial culture is of enormous value as it furnishes the proof of the presence or absence of a relation between different forms of fungi. This knowledge not only gives us a better insight into the development of the organism, but also gives us most important information as to the methods of control. In the identification of bacteria cultural methods are abso- lutely necessary as these organisms cannot be determined otherwise. The determination of the host and its enemies is not only desirable on the ground given above, but also because it gives us opportunity for ecological observations. A disease occurs only when conditions are favorable to its development, and these conditions are often pointed out by the composition of the flora of the locality, of which my studies upon the dying-out of alder trees in Germany give you a clear proof. In different localities these trees are killed by a fungus, Valsa oxystoma. The fungus grows into the wood through wounds, especially where branches or twigs are broken off, and kills out parts of the cambium and the bark. The parts into which it does not penetrate remain alive. There was no doubt about the fungus being the cause of the disease, but there were groups of trees which, though the fungus was present, were not quite killed out, the damage done in these localities being much smaller. I hit upon the correct explanation of this con- dition through a study of the special character of the flora under the trees. It was a typical flora of pastures, in which occurred specimens of Iris pseudacorus and retreating areas of Carex paniculata. These two plants are typical inhabitants of the peats, or water borders. It was clear that the locality had been formerly of a peaty character. I could determine that recently the water level had been lowered for the forma- tion of artificial meadows. Without a knowledge of the flora this relation would never have been found, as these meadows 1915] APPEL—-PHYTOPATHOLOGY AND SCIENTIFIC BOTANY. 281 were situated behind a chain of hills. The depth of the ditches had changed the water level and prepared the right conditions for an attack of Valsa oxystoma. In another paper I have shown the importance of the work of E. Münch.! This work is a model as to the manner in which investigations of plant diseases upon a scientific basis should be prosecuted. And, therefore, I wish to come back in a more detailed way to the work of Miinch. The fungous diseases of our trees belong, in general, to the most im- portant diseases, and we yearly lose millions on their account. But we did not know the factors upon which the appearance of such diseases rested until these were demonstrated by the work of Miinch. It was known that many fungi attack woody plants under definite conditions. Sometimes closely related species of one genus of hosts behave differently and some- times only definite tissues are attacked. Lastly these rela- tions vary in different years or seasons in different localities. The difficulty has been that the cause of this variability was sought in the different soil conditions which might have an influence on the constitution of the tissues of the host, in external injuries—such as sunburn or frost, and in the period of development of the fungus. These factors, however, are not of fundamental importance in the question of the produc- tion or suppression of a fungous attack. Miinch has proved through numerous experiments that the content of air in the tissues is the determining factor. The greater part of the wood-decaying fungi have a large air re- quirement and are able to grow only when a maximum of air is furnished. In the first place the content of air is dependent on the quantity of water, and the occurrence of this large class of plant diseases depends upon the water supply. Simi- larly, the quantity of solid substance may be of influence. Specimens with narrow annual rings are more resistant than those with broad ones, because there is less room for air in the former. The different annual rings of the same wood 1Untersuchungen über Immunität und Krankheitsempfänglichkeit der Holz- pflanzen. Naturwiss. Zeitschr. f. Forst- u. Landw. 7:54-75, 87-114, 129-160. [VoL, 2 282 ANNALS OF THE MISSOURI BOTANICAL GARDEN may be attacked differently, which is supported by the evi- dence of many observers. Not infrequently do we find tree trunks in which only some annual rings have been infected, or in which the same ring is diseased on one side and healthy on the other. The decayed rings are always the broad ones. The same varieties have a different air content in different localities. In the neighborhood of water sprouts or vigorous branches, the tissues are rich in water and poor in air, and infections very often do not penetrate into such regions. We know now that poorly fed and crippled specimens are likely to be attacked; on the other hand, it seems clear that fruit trees which are richly fed with nitrogen are very sus- ceptible to canker. An abundance of nitrogen induces the development of a very loose tissue, which during drought is more subject to diseases than a firm tissue. We recog- nize the periodicity in the occurrence of many plant diseases, for we know the fluctuations in the water content of a tree. The air content of the healthy bark of beeches in winter-rest is 19-20 per cent, and diminishes at the time of budding to 11 per cent, rising afterwards. This is correlated with the fact that the canker, which in Europe is caused by Nectria ditissima, does its damage from autumn until spring, while this damage ceases during the vegetative period. This was pointed out by Aderhold, who, however, failed to recognize the cause. If once we know the absolute percentage of air necessary for fungous growth in the different kinds of wood, we may decide through direct investigation whether in certain locali- ties the danger of infection is large or small. We may test the different varieties and try to avoid the danger. By this method the control is not directed against the fungus, but against the conditions which make its growth possible. In other words, we use instead of direct control, measures which prevent the outbreak of epidemic diseases. You see by this example what an exactly planned scientific investigation may do, and you can recognize the application of these facts to the American conditions. In the irrigated districts the fruit trees have but few die-back diseases due 1915] APPEL—PHYTOPATHOLOGY AND SCIENTIFIC BOTANY 283 to species of Valsa and other fungi. When, however, such diseases occur, you will find the cause in defective irrigation methods, which may be remedied by changing the irrigation system. It is of the greatest importance that the land be irrigated at the time the trees contain less water and plenty of air, and that the next irrigations be made in time to prevent an excessive decrease of the water in the tissues. Not all fungi, however, are dependent upon the air contained in the wood. This is, for instance, the case with Armillaria mellea, where the rhizomorphs bring a sufficient quantity of air into the inner tissues. Whoever has cultivated the fungus artificially knows that after a short time rhizomorphs are formed which grow deep into the medium. But the rhizo- morphs are not formed on all kinds of trees and it may be possible that the fungus in these cases depends on the air already in the wood. Another question of great importance for American con- ditions is the question whether the growth of bacteria, prin- cipally of Bacillus amylovorus, is dependent upon the air con- tent of the host or not. These experiments must be sup- ported by thorough physiological investigations. That manner of control which seeks to remove the bacteria by cutting out the branches does not guarantee success for the future. I have been convinced of this in my trip through the United States, where I visited districts in which this control measure was thoroughly carried out. It may be possible that not only trees, but also herbaceous plants, show relations between fungous growth and air con- tent. I think it must be so for the organisms which cause the wilt diseases and the rhizoctonia disease of the potato, both of which have a high air requirement. On media poor in air these fungi grow only on the surface and absorb very eagerly the oxygen of hydrogen peroxide. The growth of Rhizoctonia in the well-aérated peat soil of the Stockton Delta and the forest soil of Germany is more marked in the dry years than in the years when the plants get a sufficient supply of water. In the United States these diseases are wide-spread, princi- pally through the irrigated lands. In my trip I came to the [vor. 2 284 ANNALS OF THE MISSOURI BOTANICAL GARDEN conclusion that these diseases are not to be controlled by fighting the fungi, but by influencing the potato plant. Though caused by a fungus, the production of the conditions favora- ble to the progress of the disease is attributable to irrigation. In many cases the root system was poorly developed, the dif- ferent kinds of irrigation showing an influence upon the growth of the underground parts of the plants. We know very little of the conditions of growth of the potato in spite of a few publications on this subject by Müller-Thurgau, De Vries, and Vöchting. Moreover, we know nothing about transpiration and water requirements in these plants or about their ability to form roots, or the factors that influence these processes. It is, therefore, very important that Shantz, of the Depart- ment of Agriculture, has actually undertaken the investigation of these problems. Others must follow him as soon as possible to solve these questions for the irrigated lands. The chemical-physiological side of the phytopathological questions also needs more attention, as has been pointed out recently by me and others in work upon the freezing problem. For a true judgment of the resistance to frost, in the case of cereal diseases, Gossner has apparently found the right way. The earlier stated fact that the cells of small pieces of tissue floating on a sugar solution are less quickly killed by frost than when floating in water, made it probable that the young plant is protected by sugar against frost in- jury. The investigation of the winter and summer rye shows that the sugar content of the former is several per cent greater than of the latter. The same is the case for frost-susceptible races of wheat. We may thus find out the relative frost re- sistance of closely related races of plants by determining the sugar content. But other phases of chemistry are of importance in phy- topathological investigations, as, for instance, the chemistry of colloids, which, as Ruhland showed in his work, is of great value. The microchemical reactions are also of great importance. We know today that cork formation in the potato is a protection against bacterial invasion. I could show by using the reaction of Tisson that the deposition of 1915] APPEL—PHYTOPATHOLOGY AND SCIENTIFIC BOTANY 285 cork in the cell walls near the places of infection occurs earlier than the formation of cork plates. Of special interest is the physiology of inheritance. In this lecture I wish merely to emphasize that the inheritance of the unit characters and their behavior in the next generation is one of the fundamentals of breeding resistant races. Finally, I must speak of anatomy. The necessity of the examination of series of sections oblige the pathologist to make use of the latest discoveries in histology. It is by way of anatomy that we shall approach the problem of leaf-roll of the potato. Onemjer has shown that the sieve tubes, which have the function of providing the plant with albumen, are destroyed in the leaf-rolling plants, similar symptoms oceur- ring in plants which suffer from other diseases only when the plants are nearly dead. In leaf-rolling plants, however, we find these changes from the very beginning, and we may use them in diagnosis. Anatomy, likewise, points out relations be- tween external disease symptoms and inner changes of struc- ture. For instance, the three inner diseases of the potato, leaf- roll, wilt, and bacterial ring disease, have distinguishable anatomical characters; the leaf-roll is a disease of the phloem; wilt, of the secondary wood vessels; and bacterial ring, of the spiral vessels. A thorough anatomical knowledge is of pri- mary importance in all investigations concerning the inner structure of healthy and diseased plants, the formation of excretions and tyloses, and the different ways of recovery. I hope that it has been possible for me to show you that phytopathology has many fundamental relations to scientific botany, and that it further presents many important problems for scientific investigation which deserve attention from the botanical departments of universities. Should I have succeeded hereby in winning new friends to phytopathology in this sense it would be a source of genuine satisfaction and pleasure to me. THE LAW OF TEMPERATURE CONNECTED WITH THE DISTRIBUTION OF THE MARINE ALGAE WILLIAM ALBERT SETCHELL University of California What I have to bring before you is simply a preliminary consideration of the general subject of the geographical dis- tribution of the marine algae together with some inquiry into the conditions immediately affecting such distribution and as possibly effecting a segregation into the larger units. In accordance with such an intention, I have started a tabula- tion of all the marine species and varieties, which is far from being completed as yet, but which has, however, reached a stage at which certain general statements may be made as to probable results. The geographical distribution of the marine algae has been treated of in various ways and in many papers. It is more or less customary to make a comparison between a particular flora and other more or less corresponding floras in com- parative tables, percentages of common and endemic species, ete. Certain speculations, based on such data, as to the origin of certain algal floras have also been indulged in. The result is that we have certain geographical areas fairly well marked out and certain others more or less indistinctly out- lined or surmised. Certain ecologic classifications have been proposed, particularly as to zonal occurrence in varying depth, influence of varying degrees of salinity, character of the sub- stratum, influence of surge, quiet waters, ete. Very little attention, however, has been paid to general factors control- ling distribution over larger areas. We speak broadly of tropical species, or of arctic or antarctic species, of temperate species, etc., but no attempt has been made to survey the distribution of marine algae in general throughout the oceans and seas of the world and to attempt to determine the limit- ing factors segregating one large area from another. An attempt to determine how far our present knowledge of ANN. Mo. BoT. GARD., VOL. 2, 1915 (287) [VoL, 2 288 ANNALS OF THE MISSOURI BOTANICAL GARDEN species and their distribution may further such an inquiry is the object of the present paper. Among the more general discussions, there are to be men- tioned first those connected with the geographical distribu- tion in the Arctic Ocean. Kjellman’s extensive and funda- mental paper ‘Algae of the Arctic Sea’ (’83) led the way and placed at the disposal of future students a very con- siderable amount of data and brought forward certain funda- mental points of view as to a division of the arctic marine flora into provinces, as well as a consideration of the condi- tions underlying this division. This work was the result of the working over of very considerable collections of the vari- ous Swedish expeditions into the Arctic Ocean and a careful examination of all other existing data. Later, Rosenvinge (’93, ’98, ’98*, ’98") published a series of papers dealing with the marine flora of Greenland, and Jöns- son (’03, ’03°, ’04, ’12) has also published on the same subject as well as on the algae of Iceland and Jan Mayen. Finally, somewhat over twenty years after Kjellman’s paper, Simmons (’05) surveyed the whole matter, revised all tabulations of the Arctic flora and brought forward further views together with a full discussion of all literature bearing upon the subject. In these various papers and others not referred to specific- ally, the North Polar Sea is defined and delimited from the Northern Atlantic and Northern Pacific Oceans. The condi- tions under which marine algae occur in the Polar regions as well as the differences between the conditions of the various portions of its waters are also determined and discussed. The North Atlantic has also been treated of, but more flor- istically than as to uniformity, or differences, of physical con- ditions affecting the flora. A considerable part of the discus- sion regarding the North Atlantic Ocean has centered about the Faeroes. Simmons (’97), Börgesen (’02, ’05), Por- sild and Simmons (’04), and Börgesen and Jónsson (’05), have discussed the marine flora of these islands together with its relation to other North Atlantic floras and ocean cur- rents. Reinke (’89), Svedelius (’01), and Kylin (’06, ’07), 1915] SETCHELL—DISTRIBUTION OF MARINE ALGAE 289 have considered the algal flora of the Baltic Sea and its rela- tion to that of the North Atlantic from points of view both floristic and as to physical conditions. Harvey (’58), Far- low (’81), and Collins (’00), have dealt similarly with the algal flora of the northeastern coast of North America, and Börgesen and Jönsson (’05) have made an extended floristic comparison between the floras of the North Atlantic and those of the polar or arctic seas. For the antarctic and subantarctic regions, the work even of floristic comparison is still hampered by incomplete knowl- edge. The foundations were laid by Hooker (’45) in the ‘Cryptogamia Antarctica’ in which there are scattered notes on distribution. Skottsberg (’06) published his ‘Observa- tions on the Vegetation of the Antarctic Sea’ and later (’07) the first part of his antarctic and subantarctic work. The latter has only floristic details with notes on distribution. Gain (712) has given a detailed discussion of the distribution of the marine algae thus far credited to either the antarctic or the subantarctie regions of the western hemisphere. Mur- ray and Barton (’95) have given a comparison between the arctic and antarctic marine floras, and Mme. Lemoine (’12) has made a similar comparison limiting it, however, to the species of erustaceous Corallinaceae. The distribution of marine algae in the warmer portions of the oceans, Atlantic, Pacific, and Indian, has not been so much considered as that of the colder portions, although very considerable floristic work has been done. Murray (’93) pub- lished a comparison of the marine floras of the warm Atlantic, Indian Ocean, and the Cape of Good Hope. Yendo (’02) has made definite statements about the distribution on the coasts of Japan. Saunders (’01) and Setchell and Gardner (’03) have dealt with the northwest coast of North America, and Schmitz (’96) and Schroeder (’12) have called attention to the relations between the marine flora of East Africa and those of the East Indies and of the central Pacific Ocean. Various papers and floras have considered distribution, such as bathymetric zonal distribution or according to vary- ing substratum, salinity, etc., within limited regions, prov- [vor. 2 290 ANNALS OF THE MISSOURI BOTANICAL GARDEN inces, or districts, but no general paper has as yet appeared dealing with the distribution over the oceans in general or any definite suggestions as to the factors concerned. The nearest approach to an attempt to account for the gen- eral facts of distribution is my own attempt (cf. Setchell, 93) to explain the main facts of the geographical distri- bution of the Laminariaceae. The plants of this family are rather inhabitants of the colder than of the warmer waters, proceeding, as it were, from the poles towards the equator, but lacking in strictly tropical waters. It was found that the Laminariaceae flora changed its facies with every increase or decrease of 5°C. of summer temperature, thus forming latitudinal zones controlled by temperature relations. This idea was extended to explain the demarcations of the floras of the west coast of North America by Gardner and myself (ef. Setchell and Gardner, ’03) with apparent adequate reason. In attempting to discuss the more general facts of distri- bution we first necessarily consider the various marine floras and their subdivisions. While the term flora has been used in all sorts of senses, both wider and narrower, to include any aggregation of plants of any region under discussion, whether larger or smaller, it generally carries a certain idea of uni- formity of composition with it when used in connection with the floristics of distribution. This uniformity may, however, be only as regards region. It is desirable, here, to use the word for the aggregation of species of marine algae found in a certain region, province, or district, having a certain fairly considerable percentage of species in common through- out its extent, even of the more extended region. The world’s surface, whether land or water, is usually divided into zones of temperature, these in turn into regions, the regions into provinces, and the provinces into districts. For marine floras, the districts must be still further divided into formations, and these in turn into bathymetric or littoral belts. The bathymetric belts, in their turn, show different algal associations. 1915] SETCHELL—DISTRIBUTION OF MARINE ALGAE ~ 291 Considerable work has been done in the description of various floristic associations occurring in various depth belts and of various formations, and the special ecological relation- ships have been discussed and made reasonably plain. My intention, however, is to discuss the broader distribution and segregation of floras, particularly as to regions and perhaps provinces and to attempt to determine the factor, or factors, governing these. In attempting to mark out the various floristic regions and their provinces, we are met with certain difficulties. The flora of the Arctic or Boreal region is fairly definite and has been the most carefully studied and tabulated. The prov- inces of the Arctic region are the Asiatic, the American, that of West Greenland, and the extended province of Spitzbergen (cf. Simmons, ’05). The North Atlantic Ocean as distin- guished from the Arctic has five regions, viz., those of North- western Europe, Southwestern Europe, and the Mediterraneo- Northwest African region on the east and Northeastern North America and Middle eastern North America on the west. The Antarctic or Austral region possesses a fairly consistent flora and is not so readily divided into provinces, but the Antarctic- Magellanic province may be contrasted with the Indo-Pacific province. The South Atlantic Ocean has a flora as yet little understood, but, for the present at least, may be considered to have the regions of Southwest Africa and Southeast South America. The Northern Pacific has Bering Sea probably representing a province of the Arctic or Boreal region. Otherwise it is divided into five regions, viz., those of North- west North America, Middle West North America, and South- west North America on the east and the Ochotsk-Yeso region and that of East and West Honshu (or Nippon) on the west shores. The South Pacific Ocean has five regions, viz., those of Southwest South America, Middle West South America on the east and those of New Zealand and South and South- east Australia on the west coasts. The southern portion of the Indian Ocean has two regions, viz., that of Southwest Australia and the South Africa or Cape region. The tropical waters may probably be divided into two regions, viz., the [voL. 2 292 ANNALS OF THE MISSOURI BOTANICAL GARDEN Tropical Atlantic and the Indo-Pacific regions with their proper subdivision into provinces. Concerning these various regions, it may be said that some seem to possess very dis- tinct and characteristic species content while others are more or less related to one another. However, it is expected that there will be a possibility of discussing this segregation at another time in more extended fashion. Of particular interest and importance in connection with the marking off of floristic regions, are the points or areas of demarcation. Some of these are well established while others may be only more or less accurately surmised. One of these much referred to in the literature (cf. Harvey, ’58; Farlow, ’81; etc.) is Cape Cod on the eastern coast of Massa- chusetts which divides so clearly and so accurately the flora of northern New England from that of southern New England. Cadiz in Spain appears to be another point of de- marcation, or possibly indication of an area, where the flora of the Southwestern European region stops, or mingles with that of the Mediterranean-Northwest African region. At Clare Island on the west coast of Ireland (Cotton, ’12, p. 160) the flora ‘‘resembles that of the southwest of England,” but it has elements also of a distinctly northern character. It is probably in or near a demarcation area. Similarly southern Norway and the west coast of Sweden (Kjellman, ’02, ’06; Svedelius, ’01; Kylin, ’06, ’07) have a mixed flora and are in a transition region. In Japan Cape Inuboi on the east coast of Honshu (cf. Yendo, ’02, p. 181) is a demarcation point and the Strait of Sangar (ef. Yendo, ’02, p. 182) is also a region of demarcation or transition. On the opposite side of the Pacific Ocean, along the western coast of North America, Cape Flattery or just south of it, Point Conception, and the region about the mouth of the Gulf of California are demarcation points or indicate transition areas (cf. Setchell, ’93, p. 370; Saunders, ’01, p. 393; Setchell & Gardner, ’03, p. 170). In the southern hemi- sphere the marine flora of the Cape Region is definitely de- limited both to the southwest and to the northeast and in 1915] SETCHELL—DISTRIBUTION OF MARINE ALGAE 293 Australia the marine flora of the southeastern region is defi- nitely set off from that of the southwestern region. These various points and regions will doubtless become more definite and more of them will become established as careful investigations of the floras are made. They un- doubtedly indicate that thereabouts are changes in the con- ditions regulating the separation of the general flora into its larger divisions and are of great importance in any inquiry as to the general factors affecting the distribution of marine algae. Along with the mapping out of floras into regions, provinces, etc., it seems best to consider, next, the factors which seem to regulate the distribution. These have been considered by Kjellman (’83) and by others, and are summed up by Oltmanns (’05). Particularly is it desirable to consider which may be chiefly responsible for the limiting of the species within the regions or provinces. The substratum exercises an important influence on the attached flora or benthos and that is particularly the part of the marine flora I intend to limit this paper to, since the plankton brings in certain particular factors having to do with its floating habits. Of course, benthos can only exist on its proper firmer substratum and different species differ in the nature of this. However, it is sufficiently evident that the character of the substratum limits species only locally and can by no means be considered as a factor in controlling floral regions or even floral provinces. The motion of the water is a limiting factor in distribu- tion, some algae preferring quiet water, some flowing, some surge, etc., but this factor, too, is clearly a local and not a general one in the distribution of the marine algal benthos. The specific gravity of sea-water varies and with it, of course, its salt content. This variation, so far as marine algae are concerned, varies from water only slightly brackish to that (in case of exposed and shallow tide pools) of an almost concentrated solution. There is a latitudinal zonal difference here also, but it is not so great as may be found in localities at no considerable distance from one another. It [VoL, 2 294 ANNALS OF THE MISSOURI BOTANICAL GARDEN certainly seems impossible that this can be a general factor. Its local effect, however, may be very considerable. Light varies from the equator, where it is most intense, to the poles where it is least. It very decidedly limits the dis- tribution as to depth. Marine algae of the benthos need light and are, therefore, limited to the neritic portion of the photic zone as to their general distribution. Outside of this general limitation, however, it does not appear that the varying in- tensity of light can be considered as a prime factor in limiting floral regions and floral provinces, i.e., not alone. Varying temperature, however, does act directly upon algae to limit their distribution, both locally and generally. It can easily be recognized to be the one most important factor in controlling the distribution of benthos over wide areas as well as, at times, in smaller districts or spots. We recognize that, in general, the species of the frigid zones, of the tem- perate zones, and of the tropical zones are sufficiently different to give an entirely different facies to each. Yet, in consider- ing general regions, we find that they are not marked out by the same parallels as are used to mark these zones geographic- ally. These geographical zones, however, are established more particularly as regards direction of the sun’s rays and the temperature of the air rather than that of the water. The waters concerned with the life and persistence of the algae, even of the benthos, are, relatively speaking, the sur- face waters, since algae seldom grow lower than at a depth of 100 meters and for the most part cease at 20-30 (or at times 40) meters. The normal decrease in temperature at such depths is slight even in temperate waters, although, at times, sufficient to account for special sporadic anomalous distribution. The range in temperature under which algae, in general, may carry on their full course of vegetative and growth activities is from —2°C. up to the neighborhood of 90°C., but that for marine algae is only from —2°C. up to 30°C. (or possibly 32°C.), this being the extent of ranges for all surface waters of the ocean. A comparison between charts in which the isotherms for surface temperature of the water of the oceans are laid off 1915] SETCHELL—DISTRIBUTION OF MARINE ALGAE 295 shows a definite correspondence between certain of these lines and the boundaries of different marine floral regions as previ- ously laid out and indicated in this paper. From the point of view of the distribution of the marine benthos, so far as algae are concerned, it is found by practice to be satisfactory to divide the surface waters of the ocean into nine zones, as follows: Upper Boreal, Lower Boreal, North Temperate, North Subtropical, Tropical, South Sub- tropical, South Temperate, Lower Austral, and Upper Aus- tral. The limiting isotherms of surface temperature chosen are those of the summer month or maxima, viz., the isotheres, which are those of February (or possibly March) for the southern hemisphere and those of August (or possibly Sep- tember) for the northern hemisphere. These lines are laid down with approximate accuracy in the charts of the atlases of the different oceans published by the ‘‘ Deutsche Seewarte’’ of Hamburg (’92, ’96, 02). These isotherms are more accurate and explicit for the open ocean than for the neritic zone where the algal benthos occurs, but, with certain allowances, the zones as indicated are sufficiently accurate. Each of the zones I have proposed covers 5°C. range of surface temperature with the exception of the Upper Boreal and the Upper Austral, each of which includes a range of 10°C. or slightly over. The zones, then, more or less arbi- trarily adopted, are the Upper Boreal and Upper Austral, between the isotheres of 0°C. (or even —2°C.) to 10°C., Lower Boreal and Lower Austral between the isotheres of 10°C. and 15°C., North Temperate and South Temperate between the isotheres of 15°C. and 20°C., North Subtropical and South Subtropical between the isotheres of 20°C. and 25°C., and the Tropical between 25°C. and 30°C. (or above). These 5°C. zones are thus laid out according to the 5° isotheres, because on inspection these isotheres approach most closely or touch the shores at the division points of floras and principal floral provinces. They have been de- termined empirically, and indicate, as it seems from experi- ence in working with them, that they coincide with floral boundaries the oceans over more exactly than do any of the [Vou 2 296 ANNALS OF THE MISSOURI BOTANICAL GARDEN winter isotherms or isocrymes, or any of those in the inter- mediate seasons. For example the isothere of 20°C. passes somewhat south of Cape Cod to the eastern end of Long Island, but the shal- low and more or less protected waters of Long Island Sound, Narragansett Bay, Buzzard’s Bay and Vineyard Sound carry a higher temperature eastward even to the Cape Cod region. At exposed points, however, the somewhat colder waters of the ocean outside exist and exercise their influence at exposed points or in deeper waters. Again at Cadiz, the isothere of 20°C. abruptly curves up to the coast. At Cape Inuboi, Japan, the isothere of 25°C. touches land and at the Strait of Sangar, that of 20°C. The Cape Region of South Africa is included between the isotheres of 20°C. and 25°C. Similar relations hold good on the coast of Ireland, for the 15°C. isothere comes in just north of Clare Island at about Annagh Head. On the south coast of Australia, the isothere of 20°C. touches the east coast just above Cape Howe and the south coast about Cape Arid, thus leaving the southeastern coast below 20°C. of average summer temperature and the southwestern coast above it. Although the western coast of North America has its tem- perature relations very much disturbed, as I shall indicate later, yet there is a fairly definite relationship to the isotheres of 10°C., 15°C., 20°C., and 25°C. The arctic or boreal floristic region has a definite southern boundary in the 10°C. isothere and the subarctic in that of 15°C., while those of the North Atlantic are bounded to the south by that of 25°C. The strictly tropical species are found almost entirely between the isotheres of 25°C. and 30°C. (or 32°C.). It is expected that a later paper will deal more definitely and in more detail with the reasons for selecting the isotheres as bounding lines for the temperature zones. Two seeming disturbances of those zonal areas may be noted in passing; one is that the polar zones (Upper Boreal and Upper Austral) are for 10°C. interval rather than 5°C. This is in accordance with what is known of the distribution of the marine flora in the higher Arctic and the higher Ant- 1915] SETCHELL—DISTRIBUTION OF MARINE ALGAE 297 arctic regions, where there seems to be no useful purpose served in segregation by assuming two zones rather than one. The second disturbance of zonal areas is through the occurrence of local areas, of greater or less extent, of water of a higher or lower temperature than is normal for the gen- eral zone. Colder waters occurring among warmer waters are found along the west coasts of North and of South America, of northwestern and southwestern Africa, and of northeastern Africa. These are due to currents or to up- wellings of cold water. Their existence is well substantiated but their cause is still a matter of discussion among ocean- ographers. When warm waters exist among colder waters, they occur as ‘‘spots’’ or small areas where the higher tem- perature is due to comparatively local factors apart from general oceanographic conditions. Such disturbances as up- wellings and spots may bring about a puzzling discontinuity in the distribution, very puzzling, indeed, until the immediate cause is discovered. Another matter causing seeming disturbance of the limits of temperature zones proposed is the seasonal variation of the temperature of the surface waters. This is variable, but in general may be considered to hold true as follows: The seasonal surface temperature variation as platted for 2° squares is least in the Upper Boreal, Upper Austral and Tropical zones, where it is not over 5°C. in range; is greatest in the Temperate zones where it averages nearly 15°C. and may be as great as 27 or 28°C., and is medium in the Subtropical zones and in the Lower Boreal and Lower Austral zones where it approximates 10°C. These, then, are the principal features of temperature dis- tribution with which we may be concerned. In connection with the empirical establishing of the temper- ature zones previously outlined, I have attempted to arrange each and every species of marine algal benthos thus far described in the zone or zones to which it has been accredited. The work is not as yet by any means completed, but a general view has been obtained for the Rhodophyceae, Phaeophyceae, Chlorophyceae, and Myxophyceae, and the greater part of the [VoL. 2 298 ANNALS OF THE MISSOURI BOTANICAL GARDEN Rhodophyceae have been worked out in fair detail, although no percentages of absolute accuracy can be given at present. The general results are as follows: (1) The greater part of the species are known from one zone of temperature. (2) A considerable number of species are known from two zones of temperature. (3) A comparatively small number are credited to three zones of temperature. (4) Species credited as occurring in four or five zones of unlike temperature are extremely few and almost always doubtfully so accredited. (5) There is a change of facies of the flora in each suc- cessive zone, i.e., with every increase or decrease of 5°C., excepting in the cases of the Upper Boreal and the Upper Austral. This means that most species are, so far as known, confined to zones of amplitude of 5°C. of summer temperature, that certain species extend over zones representing 10°C. ampli- tude, while a few may extend over zones representing 15°C. amplitude of summer temperature, and extremely few defin- itely known in zones covering over 20°C. amplitude of sum- mer temperature. To mention the results of the preliminary survey of the marine Rhodophyceae so far listed and checked, may give approximate conditions which also seem to exist in other groups. The species and varieties thus far accredited to this group number about 3,350. Of these the northern hemi- sphere has about 34 per cent in its extratropical waters, the southern hemisphere approximately 44 per cent, while the tropical waters have approximately 22 per cent. Of the entire number, approximately 71 per cent are confined to one zone of temperature; about 21 per cent extend over two succes- sive zones of different temperature; about 6 per cent are accredited to three successive zones of different temperature; while between 1 and 2 per cent are accredited, but with more or less, generally very considerable, doubt, to four, or even to five, successive zones of different temperature. 1915] SETCHELL—DISTRIBUTION OF MARINE ALGAE 299 Commenting on the above, it may be surmised that the percentage in one zone is high on account of many new or little known species which have been collected only once, while the percentage of species occurring in two successive zones of different temperature is low because of our incomplete knowledge. Concerning the species credited to three zones, the percentage is small but perhaps not much lower than will be found on final careful revision. Here seasonal occurrence and ‘‘spot’’ distribution will undoubtedly be found to be con- cerned in the overlapping, as it will be also in the case of overlapping in two zones. Concerning the occurrence in four or five successive zones of different temperatures the percent- age although small will, with very little doubt, be decidedly decreased or even entirely erased when the doubtful cases are investigated and cleared up. There may be a fraction of one per cent still left, however, and if there is, I doubt not that some fairly simple physiological explanation of their toleration of such an extreme range of temperature will be found. The disturbances in the uniformity of regular in- crease or decrease in the temperature of surface waters, as referred to latitude, have already been mentioned as due to cold upwellings and spot variation according to local physical peculiarities. These disturb, of course, the zonal distribution. Where such intrusive areas of colder or warmer water are extensive, the distribution in those areas must be considered in connection with the nearest zone of similar temperature. Spot distribution also, may be so referred but only in general considerations of distribution. Otherwise it must be con- sidered specially. The disturbance of regular zonal distribution which must have special consideration from the zonal point of view is that which arises from seasonal variation in the surface tem- perature accompanied by seasonal occurrence of a certain ele- ment of the flora in some district or province of a region of the particular zone. Seasonable amplitude varying on an average from about 5°C. to 15°C. in extent, as I have mentioned before, is found in the various temperature zones. Seasonal duration, or, at [voL. 2 300 ANNALS OF THE MISSOURI BOTANICAL GARDEN least increased seasonal vigor in certain elements of the flora is found in all zones, a phenomenon of mixed dependence upon light and temperature. It is most marked in the Temperate zones but is to be found in the Subtropical, Lower Boreal and Lower Austral zones as well. In the Upper Boreal and Upper Austral zones its appearance is perhaps more associated with varying intensity of light than with temperature, and it is least pronounced in the Tropical zone, where it seems to be wholly dependent upon light variation. It is certain that many boreal summer species appear as winter or early spring species in the Temperate zone and like- wise certain temperate species appear during the colder sea- son in the Subtropical zone. There is some, but apparently not very much, overlapping between the upper portions of the Subtropical zones and the Tropical zone. From the very incomplete studies thus far made, it seems that most species range through from 5 to 10°C. of temperature, that each zone has its own characteristic species and that extensions up to 15°C. for active growth and reproduction are few, if at all existent. More careful examination, however, is neces- sary to satisfactorily demonstrate this last point. While the limits of the temperature zones have been founded on the isotheres or lines of average daily summer temperature, seasonal phenomena cause us to consider also the isocrymes or lines of average daily winter temperature, especially as to overlapping or transitions between the zones. The isocrymes are of especial importance in those portions of certain zones where, especially on account of strong currents, the seasonal variation is extreme, e.g., on the eastern coast of North America and on the eastern coast of Asia. In such regions there may be expected extreme expression of seasonal change of flora. The disturbances of distribution due to upwellings cause confusion in the tabulated results unless they are to be defi- nitely accounted for. This confusion is greatest at present in connection with the species of the central coast of California. Spot distributions also cause the species concerned to be tabu- lated in more than one, or, if combined with seasonal disturb- 1915] SETCHELL—DISTRIBUTION OF MARINE ALGAE 301 ance, over three zones. Spot distributions are less easy to detect than other anomalous distributions but enough are sufficiently known to make apparent their influence and im- portance in any scheme of representation of geographical distribution. While the distribution of any particular species of plant depends upon a complex of conditions controlling continued existence, both vegetative and reproductive, certain more general factors may be distinguished as prevailing over larger areas, while others, less general, may account for local and usually discontinuous distribution within particular provinces and districts, and as components of various formations, bathy- metric belts, and associations. Temperature has come to be considered as one of the most important of the conditions controlling, or governing, the dis- tribution of plants and animals (cf., e.g., Merriam, ’94, ’98, ete.; Livingston and Johnson, 713; and others). Any bio- logic factor has, of necessity, two variables (cf. Livingston and Johnson, ’13, p. 351), intensity and duration, and these two variables present considerable range, especially in the case of land plants. For marine plants, particularly for those species constantly submerged, the amplitude of these variables is less than for the land plants. The surface waters of the ocean, while influenced by the temperature of the air, change slowly and only within certain limits. More con- siderable is the variation through the influence of varying, especially seasonal, currents or upwellings. Yet on the whole the temperature variables are seemingly, at least, much less in amplitude than are those of the land. For those plants exposed during tidal changes the temperature variables may be considerable in amplitude. Yet such exposures are only occasional and of short duration, except, perhaps, for the plants of the uppermost tide limits. One matter of import- ance as to all factors in plants submerged entirely or for the greater portion of the time, is the uniformity of exposure to the same conditions. While the land plant may have its roots buried in the soil of one temperature and its aerial organs exposed to a considerably different temperature, the entire [voL. 2 302 ANNALS OF THE MISSOURI BOTANICAL GARDEN surface of the submerged plant is exposed to one and the same temperature. The problem, therefore, of temperature as a physiological factor in controlling the distribution of algae, in general, and of marine algae in particular, is, as com- pared with that of land plants or of land animals, compara- tively simple. Any attempt to unravel the physiological basis for the con- trol of distribution must be, at this point of the progress of the work, lacking sufficient data for conviction. The state- ments presented merely represent approximate optimal con- ditions for the duration, succession, and, therefore, continued persistence of the species of the various life zones. It seems certain that the coefficients for continued existence vary among the different species, but are restricted in the case of each species to about 10°C. in amplitude. There must be for each species a certain minimum and a maximum of optimal temperature for continued life and reproduction. It is pos- sible that certain species may continue to exist outside these, especially if they possess powers of vegetative reproduction. Thus far, it has been in mind to attempt to determine co- efficients of efficiency as Livingston and Johnson have sug- gested in the case of climatic factors controlling the distribu- tion of land plants, but no real beginning has, as yet, been made. The interval of 10°C. certainly suggests the working of the van’t Hoff-Arrhenius principle as applied to vital phenomena. Taking the variation of 10°C. as the control- ling interval of temperature and regarding it as an index to the summation of temperature, it may be possible in a later paper to definitely estimate the coefficients of temperature- efficiency in a fashion similar to that already suggested by Livingston and Johnson (’13) for land plants. If the rate of the vital activities are, in general, doubled or nearly so with each increase of 10°C., then, judging from the results of the Rhodophyceae, thus far tabulated, it would seem that marine algae cannot endure an acceleration greater than 2, that each species has its own definite initial tempera- ture for efficient vegetative and reproductive activity and that such initial efficient activity may be accelerated up to the 1915] SETCHELL—DISTRIBUTION OF MARINE ALGAE 303 doubling point, but not beyond it. In this way may be ex- plained the fact that from 0°C. (or —2°C.) to 10°C. of mean summer temperature marks the limits of the Upper Boreal and Upper Austral zones. The marine algae inhabit- ing these zones are subjected to a range of not over 10°C. at any, or all, times. The species of the Temperate zones, enduring a mean summer temperature of 10°C. to 15°C. have a range of 10 to 12°C., probably not over, at any or all times. Similarly those of the Subtropical and Tropical zones endure a range of not over 10°C. If, therefore, tentatively, a temperature efficiency coefficient be estimated according to the formula of Livingston and Johnson (713, p. 365) but modi- fied by leaving out the assumption of an initial temperature higher than 0°C., viz., u = 2 T the efficiency coefficient in the case of the Upper Boreal and the Upper Austral zones (0 to 10°C.) will be unity to 2, in case of the Lower Boreal and also the Lower Austral (10 to 15°C.), will be 2 to 3, for the Temper- ate zones (15 to 20°C.), the coefficients will be 3 to 4; for the Subtropical zones (20 to 25°C.), the coefficients will be 4 to 5, and for the Tropical zones (25 to 30°C.), the coefficients will be 5 to 6. Incidentally to carry out this idea of temperature efficiency coefficients, it may be said that the application to the case of thermal algae, where I find the 10°C. amplitude rule also to apply, would carry the coefficient index up as high as 16, i.e., in the case of those species enduring highest temper- atures (80°C.), and even to 18 in the case of thermal bacteria (90°C.) In ei I may say that while much detail remains to be considered and brought into order before the final data and conclusions may be published, I have reason to believe that the statements and conclusions I have either made or brought forward in this preliminary account, will probably not need be changed, at least to any great extent. List or Works RererreD To Börgesen, F. (’02). Marine algae. Botany of the Faeröes 339-532. f. 51-110. 1902. ———, (’05). The algae-vegetation of the Faeröese coasts with remarks on the phyto-geography. Ibid. 683-834. pl. 13-24. f. 151-164. 1905. [voL. 2 304 ANNALS OF THE MISSOURI BOTANICAL GARDEN , and Jénsson, H. (’05). The distribution of the marine algae of the Arctic Sea and of the northernmost part of the Atlantic, Ibid. Appendix: I-XXVIII. 1905. Collins, . (700). rad lists of New England plants,—V. Marine algae. Rhodora 2;41-52. Cotton, A. D. (’12). Marine algae. Clare Island Survey, Part 15. Roy. Irish cad., Proc. 31:1-178. pl. 1-11. 1912. Deutsche Seewarte-Hamburg (’92). Indische Ozean, ein Atlas. —, (96). Stiller Ozean, ein Atlas. ————, (’02). Atlantischer Ozean, ein Atlas. [2nd ed.] Farlow, W. G. (’81). Marine algae of New England and adjacent coast. U. S. Fish Comm. Rept. 1879: 1-210. pl. 1-15. 1881. Gain, L. (712). La flore algologique des régions antarctiques et ee Deuxiéme ce ewe er eee Francaise 1908-1910. Sei. Nat. Doe. Sei 1-218. pl. 1-7. f. 1-98. Harvey, W. H. (’58). Nereis Boreali- a Part I. Melanospermeae. Smithsonian Contr. 1-149. pl. 1-12. 185 Hooker, J. D. (’45). The cryptogamic botany of the Antarctic voyage of H. M. Discovery Ships Erebus and Terror, ete. 1-258. pl. 57-198. 1845 Jónsson, H. (’01). The marine algae of Iceland (I. Rhodophyceae). Bot. Tids- skrift 24: 127-155. f. 1-4. 1901. ———., (03). Ibid. (II. Phaeophyceae). Ibid. 25:141-195. f. 1-25. 1903. ————, (’03a). Ibid, (III. Chlorophyceae. IV. Cyanophyceae). Ibid. 337-385. f. 1-19. 1903. — . — (04 The marine ape of East Greenland. Meddelelser om Grön- land 30: 1- is. f. 1-13. 1904 ‚ (12). The marine algal vegetation. In Warming, E., and Rosevinge, L. K. The Botany of Iceland 1: 1-186. f. 1-7. 1912. aag eer F. R. (’83). The algae of the Arctic Sea. Kongl. Sv. Vetensk.-Akad. andl. 20": 1-351. pl. 1-31. 1883 (02). Om Algenvegetationen i Skelderviken och angränsande Katte- gatts omräde. Meddelanden fran Kongl. Landbruksstyrelsen 2:71-81. 1902. —, (06). Om främmande alger ilandrifna vid Sveriges västkust. Arkiv f. Bot. 5**:1-10. 1906. Kylin, H. (’06). Biologiska jakttagelser rörande algfloran vid svenska väst- kusten. Bot. Notiser 1906:125-138. 1906. ———., (’07). Studien über die ie Tg der schwedischen Westkiiste. Inaug. Diss. 1-287. pl. 1-7. f. 1-48. Lemoine, Mme. Paul (712). Sur les caracteras des genres Melobesiees arctiques et antarctiques. Compt. rend. acad. Paris 154:781-784. 1912 sari ren B. E., and Johnson, Grace (’13). ie pe ~~ in plant eae and et Bot. Gaz. 56:349-375. f. 1-3 Merriam, C. (794). Laws of temperature control of the geographic distri- ution of et eo and plants. Nat. Geog. Mag. 6:229-338. 3 col. maps. 1894. 1915] SETCHELL—DISTRIBUTION OF MARINE ALGAE 305 ——, (98). Life zones and crop zones of the United States. U. S. Dept. Agr., Biol. Survey, Bull. 10:1-33. 1898. Be G. (93). A comparison of the marine floras of the warm Atlantic, Indian Ocean, and the Cape of Good Hope. Phycological Memoirs 2:65-69. and Barton, E. S. (°95). A comparison of the Arctic and Antarctic marine floras. Ibid. 3:88-98. 1895. Oltmanns, F. (°05). Morphologie und Biologie der Algen 2; 1-443. f. 468-617. 1905. Porsild, M. P., och Simmons, H. G. (’04). Om Faeröernes Havalgevegetationen og dens Oprindelse. En Kritik. Bot Notiser 1904: 149-180. 1 map. 1904. Reinke, J. (°89). Algenflora der westlichen Ostsee, deutschen Antheils. Ber. d. omm. z. wiss. Unters. d. deut. Meere in Kiel 6:III-XI and 1-101. f. 1-8. 1 col. map. 1889. Rosevinge, L. K. (’93). Grönlands Havalger. Meddelelser om Grönland 3; 765- 981. pl. 1-2. f. 1-57. 1893. —, (’98). Om Algevegetationen ved Grönlands Kyster. Ibid. 20: 131-242. f: i 1898. ————, (’982). Deuxième mémoire sur les Algues du Groenland. Ibid. 1-125. pl. i. \ 1-25. 1898. —, (’98>). Sur la végétation d’algues marines sur les cotes du Grönland. Ibid. 339-346. Saunders, De A. (’01). Papers from the Harriman Alaska Expedition. XXV. The Algae. Wash. Acad. Sci., Proc. 3: 391-486. pl. 43-62. 1901. Schmitz, Fr. (96). Marine Florideen von Deutsch-Ostafrika. Bot. Jahrb. 21: 137-177. 1896 Schroeder, B. (’12). Zellpflanzen Ostafrikas gesammelt auf der akademischen Studienfahrt, 1910. Hedwigia 52:288-315. 1912. Setchell, W. A. (’93). On the classification and geographical rt of the Laminariaceae. Conn. Acad. Arts and Sci., Trans. 9:333-375. 1893. ————-, and Gardner, N. L. (’03). at of northwestern America. Univ. Cal. ‘Publ, Bot. 1:165-418. pl. 17-27. 1903 Simmons, H. G. (97). Zur Kenntniss der Meeresalgen der Färöer. Hedwigia 36:247-276. 1897. ——, (’05). Remarks about the relations of the floras of the northern At- lantie, the Polar Sea, and the northern Pacific. Beih. bot. Centralb. 19°: 149-194. 1906 Skottsberg, K. (706). Observations on a BER of the Antarctic Sea. Bot. Studier 245-264. pl. 7-9. 1 map. 190 — ( Zur Kenntniss der subantarktischen und antarktischen Meeres- algen. I. haeophye ceae. Wiss. = d. Schwedischen Südpolar-Exp. 1901- 1903. 4: nn 172. pl. 1-10. f. 1-187. 1907. Svedelius, N. (’01). Studier öfver Osterjöns Hafsalgflora. Inaug. Diss. 1-132. f. 1-26. Upsala, 1901. Yendo, K. (’02). The distribution of marine algae in Japan. Postelsia 1:177- 192. pl. 19-21. 1902 PHYTOPATHOLOGY IN THE TROPICS JOHANNA WESTERDIJK Director of the Phytopathological Laboratory, Amsterdam, Holland Tropical life is a luxurious life. Nowhere does plant and animal life show itself in such variety and abundance as on the equator. As the conditions in those regions are uncommonly favor- able to plant growth, it would appear that the plant parasites also have a good chance of living. In several tropical coun- tries plant diseases have been studied in a more or less ex- tensive way, but the general features of plant diseases in the tropics, unlike those of the temperate regions, have hardly been touched. I have been for some time studying plant diseases in our colonies of the East Indies, the so-called Malayan Archipelago, and I wish to give you some general impressions on fungous diseases in those countries. My remarks can be only suggestions, as thorough investigations on these tropical problems have never, so far as I know, been made. The Malayan Isles have an average temperature of 30°C. in the lower parts, accompanied by a humidity of 80-100 per cent. The climate is a monsoon climate. In the time of the wet season it pours every afternoon, but in the dry time the rains are very scarce in the lowlands but not infrequent in the forest-covered mountains. One would be inclined to think that this combination of high temperature and moisture would be extremely favorable for fungous growth, and that therefore fungous diseases would play a large part in the culture of economic plants. This, however, is not the case. We find that insect troubles prevail, and that, compared with our temperate regions, few diseases exist. We would not conclude these facts from the literature, as a large number of diseases caused by fungi have been described. But in visiting the countries it struck me that only a few diseases are of real importance; a great ANN. Mo. Bor. GARD., Vor. 2, 1915 (307) [VoL, 2 308 ANNALS OF THE MISSOURI BOTANICAL GARDEN many of those described must have been found occasionally, and have had no serious influence upon the cultivation of plants. Not only among the cultivated plants do we find little fun- gous growth, but also in the natural vegetation. In the virgin woods the trees have few enemies among the fungi, and even the flora of mushrooms on the ground, so characteristic of our woods, is absent. Everything seems to point to the con- clusion that conditions are unfavorable to fungous growth. Why is this so? As has already been said, there are two conditions which characterize a tropical climate: (1) a high temperature which is about equal through all seasons, and (2) a high humidity, the latter varying somewhat in the dif- ferent monsoons, but being altogether much higher than in our climates. It seems to me that the tropical temperature is too high for many fungi. I cultivate in my laboratory over 600 fungi, and this collection shows clearly that the temperature of opti- mum growth of the greater part of the fungi lies beneath 30° C., often under 25°C. An exposure to high temperature prevents many parasites from forming their spores or fruit- ing bodies, whereas others require a change of temperature for normal growth. The Polyporaceae, for instance, bear exposure to frost very well, but many of them scarcely develop at 30°C. High temperature very often gives rise to an abnor- mally abundant mycelial growth, combined with an absence of spores. On the other hand, the high moisture content of the air must be favorable to fungous development. But every fungous disease of plants involves two organisms, the parasite and the host, and the same conditions may in- fluence these two in a very different way. The heavy rain- falls, combined with the abundant transpiration—owing to the intense heat, must cause a high water-content and a small air-content, of the wood-vessels of the trees, thereby making a substratum poor in air. We know that this is an important factor in fungous growth. This fact, combined with the high temperature, would explain the rare occurrences of Hymeno- mycetae and other wood-destroying fungi in the tropies. 1915] WESTERDIJ K—PHYTOPATHOLOGY IN THE TROPICS 309 I shall begin the consideration of the different groups of fungi which cause plant diseases in the tropics by mention- ing one biological group of hymenomycetous fungi the mem- bers of which attack tropical cultivated plants. These are the so-called root fungi. It is certain that the root parasites belong to different species of Hymenomycetae, and that one species of host-plant may be attacked by a number of species of these fungi. Several of the latter, if not all, are charac- terized by the peculiar mycelium characteristic of the Hymenomycetae; in many cases, however, fruiting bodies have never been found. Practically all cultural woody plants —tea, coffee, rubber, quinine, cacao, coca—may suffer from the attacks of root-fungi, these attacks occurring mostly on virgin soil. The fungi develop on the decaying stumps of the forests, grow through the soil, and reach the roots and stem bases of the young tea, coffee, or quinine plant. The bark is pene- trated and the mycelium destroys both bark and wood (the mycelium strands can be very clearly seen between bark and wood). Whereas young plants up to three or four years old nearly always are killed, older ones may resist; different species of plants, however, behave differently in this respect. In some districts the fruiting bodies of Fomes semitostus appear on the dying plant or on the dead roots, but in others fruiting bodies have never been found. A second biological group of fungi, so common in our lati- tudes, has only a few repr tatives in the tropics under dis- cussion. I am speaking of those ascogenous or imperfect fungi which cause the die-back diseases of our orchard, forest, and park trees, e.g., Valsa, Diplodia, and others. These fungi kill the branches by penetrating into the bark and sometimes into the wood. They appear on our trees when these are in a dry condition, and in dry climates or in dry years such dis- eases are of importance. Not so, however, in the tropics. The only die-back disease which is common is caused by Corticium javanicum, which, however, belongs to the Hymen- omycetae and forms red layers on twigs, branches, and even trunks of all cultural woody plants, e. g., rubber, coffee, quinine, tea, cacao, coca, and fruit trees. We find the disease [voL. 2 310 ANNALS OF THE MISSOURI BOTANICAL GARDEN mostly in very moist valleys, where the wind has no free play. The fruiting bodies of many Ascomycetae develop in dry air, and it is not remarkable that that type of disease is found in some parts of the West Indies, which have a drier climate. A group which has no representative in the tropics is that of the powdery mildews (Erysiphaceae). These fungi occur only in colder climates. The so-called false mildews or Perono- sporaceae, on the other hand, are of considerable impor- tance, these fungi seeming to thrive well under the moist and hot weather conditions. We find the canker of rubber and cacao (caused by Phytophthora Faberi) of far-reaching im- portance. In both the rubber and the cacao the disease at- tacks the bark and, in the case of the cacao, also the fruit. The growth of this fungus depends upon a very moist air. This is proved by the fact that when the trees are cut back severely so that the trunk is exposed to sun and wind, the wounds often heal and the disease is stopped. A plantation in which the trees are planted far apart also suffers less. Another fungus—Phytophthora Nicotianae—belonging to the Peronosporaceae is the cause of a dangerous tobacco disease. The parasite kills the seedlings in the beds, the plants ‘‘melt,’’ and even the mature tobacco plants are at- tacked. The fungus penetrates into the pith of the lower part of the stem and the ‘‘tobacco-tree’”’ falls. A third member of this family destroys a large part of the Indian corn, so widely grown by the natives. It is Peronospora Mayidis, unknown, so far as I am aware, in the large corn areas of the United States. The exceedingly moist climate, combined with the excessive heat, evidently favors the attack by the fungus. In the potato fields of the mountain districts of Java we find a friend of our countries, Phytophthora infestans. Potatoes are grown in the tropics between 1500 and 6000 feet altitude. In the lower areas we find phytophthora-infected regions only rarely, but the higher we ascend, the lower the temperature (frosts may even occur in the nights) and the more destruc- tive the phytophthora becomes. The spores of the fungus (it has been proved) cannot germinate at a high temperature, which explains the occurrence of the disease only in the higher 1915] WESTERDIJ K—PHYTOPATHOLOGY IN THE TROPICS 311 altitudes. It is very remarkable that in the tropics tubers are never, so far as I observed, affected. This fact might help us to discover the cause of the difference in susceptibility of the tubers of different potato varieties in our climate. Speaking of potatoes, I wish to point out another disease of ous regions which I found in the tropics and which has the greatest influence upon tropical potato culture. I am speak- ing of the internal brown spot, the nature of which has not been recognized. Nearly every potato tuber shows this disease and in a much more striking way than in the tem- perate regions. The brown spot is accompanied by a soft consistency of the tuber and a small amount of solid sub- stance. As far as we know to-day, this trouble is a physiologi- cal one, caused by particular conditions of ‘‘climate and soil,’’ the nature of which is unknown to us. The cause of the disease may be different in the tropics and in our regions, but a care- ful study of it in warm climates might give us an indication as to what conditions favor it. Among the large group of rust-fungi, there is only one representative which is of importance to tropical agriculture. This is the coffee-leaf disease, due to Hemileia vastatria, a rust which to a considerable degree ruined a large part of the coffee culture of Eastern Asia, and obliged the growers to introduce other species, which, unhappily, are of poorer quality. On other cultural plants, however, no rust of any importance occurs. The important cereal crop of the tropics, the rice, has no rust enemy. The rust of the sugar-cane is of no consequence in cane growing. The same is true of the smut diseases. Rice smut is found exceptionally, and smut of sugar-cane is a rarity; smut of corn is even rarer than in our regions. Leaf spot diseases, belonging to ascogenous or imperfect frzgi, are much less frequent than in Europe or the United States. The leaf spots of sugar-cane (Leptosphaeria Sacchari, Cercospora Sacchari, and Cercospora Kophei) are widely spread but have little influence on cane production. They are of more importance in the moist western part of Java than in the drier east. The tea blights (Pestalozzia palmarum and [Von, 2 312 ANNALS OF THE MISSOURI BOTANICAL GARDEN Laestadia Theae) cause but small losses of tea leaves in our colonies. The sugar-cane evidently is the crop which is most subject to the attack of fungi. This becomes clear when we look upon the method of propagating the saccharum. Small pieces of the cane stem are used as cuttings, which are put into the soil. The soft pith, rich in sugar, is an ideal substratum for fungous growth, and we must not be astonished that even saprophytes enter it. Thielaviopsis ethaceticus and Colle- totrichum falcatum are two typical destroyers of sugar-cane cuttings. Bacterial diseases are scarcely to be found. I will admit that more bacterial diseases may be discovered, but up to the present time the only bacterial disease of importance is the tobacco wilt due to Bacillus Solanacearum, the same trouble which occurs in the United States. The same bacillus also causes a disease of peanuts. Probably the gum-disease of sugar-cane is also caused by bacterig. It is curious that algae in some cases (Cephaleuros virescens) cause diseases of tea and coffee plants, as they kill not only leaves but, as is true in the case of tea, also the twigs. Here I have come to the end of the list of fungous troubles. Compared to the fungous diseases of the United States and even to those of Europe, those of the tropics are smaller in number. Tropical agriculture might be compared to the agriculture of the United States more than to that of the Old World. Vast areas are covered with one crop and often with one variety of a crop, so far as we know anything definite about varieties and races of tropical plants. In the subtrop- ical regions of the United States, where at certain times the temperature equals that of the tropics, the air is much drier and there is a certain change of temperature, even in the region of eternal spring in California, which is foreign to the tropical climate. In the tropics of Asia and the subtropies of the United States insect troubles have assumed immense proportions, but as to fungous diseases, these are of more importance in the subtropics of the New World. 1915] WESTERDIJ K—PHYTOPATHOLOGY IN THE TROPICS 313 Different groups of fungi are much less restricted in their geographical distributions than are phanerogams. Up to the present time, no special tropical families among the fungi are known, and, as far as I know, the only fungus group that has no representatives in the tropics is that of the Ery- siphaceae. The secondary part which fungi play in the plant diseases of the tropics is not caused by the absence of fungi, but by the particular conditions which influence both the host and the parasite, and their relations to each other. To estab- lish the exact nature of these influences is a problem for the future. PHYLOGENY AND RELATIONSHIPS IN THE S CETES! GEO. F. ATKINSON Cornell University Part I. ARGUMENT Perhaps there is no other large group of plants whose origin and phylogeny have given rise to such diametrically opposed hypotheses as the fungi. The presence of chlorophyll and the synthesis of carbohydrates from inorganic materials are such general and dominant characteristics of plants, that many students regard them as the fundamental traits which pri- marily marked the divergence of plant from animal life. Ac- cording to this hypothesis all plants possess chlorophyll or were derived from chlorophyll-bearing ancestors. No one questions the origin of the chlorophylless seed plants from chlorophyll bearing ones by the loss of chlorophyll and reduction of photosynthetic organs.” What is more natural then, than the hypothesis that the fungi have been derived from chlorophyll-bearing ancestors? It is not my purpose to discuss the question as to whether or not the Phycomycetes, or lower fungi, had an independent origin, or were derived from one or several different groups of the green algae. I wish to consider some of the evidence which points to the origin of the Ascomycetes from fungus ancestry, rather than from the red algae. 1 The first part of this paper is the abstract or argument as read at the anni- versary proceedings. Because of the brief character of the abstract which renders many of the statements more or less categorical, while some therefore will appear dogmatic, the subject is further elabóratoð, and illumined by examples in a series of Notes which follow as an appendix in Part II. ? The ehlorophylless seed plants constitute ees ee be small, isolated groups of separate origin from different families or orders of the spermatophytes. They do not constitute a phylum. The situation is quite different with the Ascomycetes, which make up a great phylum with ascending and diverging lines, as well as descending branches. They do not give evidence of many isolated groups derived by degeneration from many separate families of the red algae. ANN. Mo. Bor. GARD., Vor. 2, 1915 (315) [VoL. 2 316 ANNALS OF THE MISSOURI BOTANICAL GARDEN In this abstract the statements must be more or less cate- gorical, and some will therefore appear rather dogmatic. 1. The phylogenetic relation of the oöblastema filaments of the red algae, and the ascogenous threads of the sac fungi.— The nuclear history in the two structures is very different. In the red algae there is a single fusion of one pair of sex nuclei in the egg, forming a true diploid nucleus which multiplies by division in the oöblastema filament providing the primary nucleus for each cystocarp. The oöblastema filament fuses with vegetative auxiliary cells to furnish attachment and base for food supply of the eystocarp, but the diploid and haploid nuclei of the fusion cell repel each other. The attempt to show a phyletic relation between the copulation of short oöblastema filaments with cells of the procarp, or the fusion of the procarp cells, after the union of haploid gametic nuclei, in some groups of red algae, and the communication of func- tional archicarp cells of certain sac fungi, as well as entertain- ing the notion that fusions of approximate cells of the asco- genous hyphae are phyletically related to the fusion of odblas- tema filaments and auxiliary vegetative cells, introduces additional confusion into a doctrine already overburdened with questionable hypotheses. The odblastema filaments and ascogenous threads are parallel developments. They present an example of morphological homology or analogy, not of phylogenetic affinity. 2. The phylogenetic relation of the ascus and carpospore, or tetrasporangium (see Part 1, Notes m and m1).—There are two horns to the dilemma here, and either one requires several additional supporting hypotheses. The origin of the ascus from a coenocytic zygote, in some cases by reduction, in others terminating a progressive splitting of the same, is far more comprehensible. The nuclear fusion in the ascus is not vege- tative (see Note m1). It takes place in all forms thus far in- vestigated and is to be considered the final stage of the sexual act, however modified this may be. Were it merely vegetative fusion there would be no need of conjugate division in the ascus hook to avoid the union of sister nuclei. The nucleo- cytoplasmic relation, or balance, would be just as easily at- 1915] ATKINSON—PHYLOGENY IN THE ASCOMYCETES 317 tained by fusion of sister nuclei, or even by contemporaneous growth of nucleus and cytoplasm, such as is well known to occur in many other cases, for example in sexual cells, gonoto- konts, ete. 3. The phylogenetic relation of the ascocarp and cystocarp. —If this principle of the resemblance between different types of cystocarp and ascocarp has any force, it would mean that the sac fungi had as many points of origin from the red algae as there are points of resemblance between their fruit struc- tures. I presume no one at the present time holds any such view of the polyphyletic origin of the Ascomycetes. 4, The phylogenetic relation of the trichogyne and sexual apparatus of the Ascomycetes and those of the red algae.— The sexual apparatus of some of the Ascomycetes, particu- larly the trichogyne, and the so-called spermatia, is generally conceded to be the strongest evidence in support of their phyletic relation to the red algae. This theory, however, re- quires a jump from the simple trichogyne, a continuous pro- longation of the egg of the red algae, to the complex, multi- septate one of the Ascomycetes. It requires further the re- duction of this trichogyne to a unicellular one, and then to the simple gamete. It also requires the transition from free an- theridia, or spermatia, to fixed ones, and from this specialized condition to the simple gamete, thus finally attaining the gen- eralized condition of the copulation of simple gametangia. This appears to me to be a rather strained backward reading of the evidence. ORIGIN OF THE ASCOMYCETES FROM FUNGUS ANCESTRY Although Sachs’ suggestion of the relation of the Ascomy- cetes to the red algae was received with favor by many stu- dents at that time, and the doctrine has received a fresh im- petus in recent years, it was not accepted by some of the foremost students of the fungi at that time (Winter, ’79; deBary, ’84). DeBary plead for the application of the theory of descent which had come to be used as the basis of classification for the higher plants. As a result of his ex- tensive studies of development in the Phycomycetes and As- [VoL, 2 318 ANNALS OF THE MISSOURI BOTANICAL GARDEN comycetes he was led to the conclusion that the Ascomycetes were derived from the Phycomycetes. This doctrine is based chiefly on the evidence of a phyletic relation between the sex- ual organs of the two groups. In spite of the persistence of the belief in the origin of the sac fungi from the red algae, deBary’s doctrine of their descent from the Phycomycetes has had many adherents. Nowhere in deBary’s writings have I been able to find any statement which can be construed as favoring the origin of the sac fungi from the red algae. The esteem in which his judgment is held, even at the present day, has led to the republication of a rumor of an ante mortem statement by deBary to the effect that he was inclined to the view that the procarps of the two groups pointed to the origin of the Ascomycetes from the Rhodophyceae! Our present knowledge of the cytology of the ascus would not perhaps favor such close contact between the Ascomycetes and Phycomycetes as would appear from the knowledge pos- sessed in deBary’s time. Unfortunately we are not yet in possession of any cytological knowledge of spore production in the zygote of the Phycomycetes which we can use for com- parison. But at any rate, the difficulties in this relation are no greater than are met with in attempting to derive the ascus from the carpospore or tetrasporangium of the red algae. Origin of the ascogenous threads.—The ascogenous threads are outgrowths of the zygote or oögonium and represent one method of splitting up and proliferation of the same in accord- ance with recognized principles of progression in the same direction of increase in the output of spores following the sexual process, or its equivalent, and terminating the diploid phase. One of the most instructive forms suggesting a mode of transition from the Phycomycetes to the Ascomycetes, is Dipo- dascus. Its sexual organs are strikingly like those of certain Mucorales or Peronosporales in their young stages. The sexual organs, which can be recognized as antheridium and oögonium, arise either from adjacent cells of the same thread, or from different threads. After resorption of the wall at the point of contact, the fertilized oögonium (or zygote) grows 1915] ATKINSON— PHYLOGENY IN THE ASCOMYCETES 319 out into an elongate stout ‘‘ascus’’ or zygogametangium with the production of numerous spores. While all phases of the nuclear phenomena have not yet been made clear, the gametes are multinucleate, and multiplication either of the sex nuclei, or of the fusion nucleus, takes place in the gen- eralized ‘‘ascus.’’ This so-called ascus is an outgrowth of the undif- ferentiated oögonium or ascogonium. The split- ting up of such a generalized ascus by fil- amentous outgrowths, the ascogenous threads, which branch and pro- Fig. 1. Dipodascus albidus: A, copulation f $ of gametangia; B, communication established duce terminal asci Con- between antheridium and oö jgonium; ©, the two taining fewer spores, °x nuclei approaching each other; D, fusion would be a very natural growth of generalized ascus from oögonium side course in progressive of copulating gametes, early stages of, in C and evolution, specialization, G, G, spore mass crowded out of end of ascus. a, and increase in spore antheridium; o, oögonium.—A-E, after Juel; F and @, after Lagerheim. output. Origin of the ascus in the Endomycetaceae.—The tendency of generalized forms to split up in different directions, often giving rise to divergent lines or series, is a well founded prin- ciple in the doctrine of descent. These series are often of different character in respect to numbers and diversity of forms, as well as to progression or reduction in one or more structures. One of the directions in which descent from such a generalized, coenocytic, germinating zygote (or ascus) as represented by Dipodascus has taken place is that of reduction in size of the generalized ascus and in the number of spores. Evidence of this reduction is furnished by Dipodascus itself ; for, as the culture ages the asci become smaller and smaller and the spores fewer in number. In this way by reduction in number of spores to 8 and 4, just permitting the meiotic nuclear divisions, forms like Eremascus and Endomyces have [VoL, 2 320 ANNALS OF THE MISSOURI BOTANICAL GARDEN arisen. Further reduction of one of the gametes, or of the vegetative stages, would result in apogamous forms of En- domyces, the Exoasceae,! the Saccharomycetes, or yeasts, ete. By reduction and loss of one of the gametes without reduction in size of the generalized ‘‘ascus,’’ such forms as Ascoidea, Protomyces, Taphridium, ete., may have arisen. Origin, progression and sterilization of the so-called tricho- gyne.—There is no well developed trichogyne-like structure in any of the known Phycomycetes. But there is evidence in a few of the forms, like certain species of Cystopus, of a tendency of the odgonium, probably under chemotactic stimulation and a softening of the wall, to develop a short process directed toward the antheridium. This has been suggested by a number of stu- dents (Lotsy, ’07, p. 468) to be an indication of the origin of the trichogyne in the As- comycetes. It does not mean that Cystopus? is to be regarded as an ancestral form of the Be though certain species do pos- ess a number of peculiarities which may be ae u. attributed to sucha hypothetical form. This of the archicarp of peculiar feature of the odgonium of some cha species of Cystopus is, however, of impor- The fertile part is tance as it indicates one probable method as lc te of origin of the trichogyne in the Ascomy- the so-called “tricho- cetes. The trichogyne is not a character haste aig triche nossessed by all Ascomycetes, even of those which still retain two functional gametangia. This, I believe, is strong evidence of the independent origin of the trichogyne in the Ascomycetes. It arose as a copulating process or beak from the oögonium Archicar TY Ascogorivm 1 Such an origin for the Exoasceae is more comprehensible than the theory that their mycelium may represent ascogenous hyphae which have migrated from the condition of parasitism in the vegetative portion of a former ascocarp, to parasitism on their present hosts, as suggested by Harper (’00, p. 392). ? One of these features is the generalized character of the sexual organs, which are polyenergid, but particularly the great variation in number of func- tional egg nuclei in different species as described by Stevens (’99, ’01). 1915] ATKINSON—PHYLOGENY IN THE ASCOMYCETES 321 under chemotactic stimulation, combined with a transverse splitting of the oögonium or archicarp. The failure of the antheridium to perform its function in the sexual process, its reduction or loss, are well known fea- tures in the life history of a number of Ascomycetes. In many cases where the antheridium or its supposed equivalent, the spermatium, is to all appearance potentially functional, its failure to function appears to be due to the sterilization of the terminal portion of the archicarp.* Analogous situations are known in the seed plants. I need only cite the case of Elatostoma acuminatum (see Strasburger, 09). The nucleus of the embryo sac mother cell enters the preliminary phases of the heterotypic division. After synap- sis the further stages of the heterotypic division are inhibited, and by typic or ‘‘vegetative’’ division the eight-nucleated embryo sac is formed. The egg, therefore, ripens with a dip- loid nucleus, and, without fertilization, develops the embryo. The walls of the inner integument grow together at the micro- pylar end of the ovule and harden, thus forming an effectual barrier to the entrance of the pollen tube (Treub, ’05; Stras- burger, 09). While great disturbances occur in pollen de- velopment and most of the pollen grains are empty or un- developed, some pollen is formed which appears normal. In some cases the mother cell, which usually forms the diploid embryo sac, undergoes a true reduction division forming a row of four cells, the lower one of which forms a normal em- bryo sac with a haploid egg. The few male plants of this species, Strasburger thinks, result from fertilization of such 1 While the “trichogyne” or terminal portion of the archicarp assumed vegeta- tive characters in an increasing degree, it seems that it did not in every case lose all of the features appropriate to a receptive organ. It appears in a few cases at least to still respond to chemotactic or analogous stimuli, seeking the fixed spermatia as in Collema pulposum (according to Bachmann, ’13) an Zodiomyces vorticellarius (Thaxter, 96). In a number of cases there seem to be receptive areas on the trichogyne where the free sperms become fixed, where fusion of sperm and trichogyne takes place. The perforation of the transverse walls of the trichogyne, which is said to occur after fusion with the sperm, also appears to be another example of the retention of an ancestral character of the archicarp which primarily permitted the passage of sperm nuclei through the terminal segment, or the association of nuclei of different segments as partheno- genesis or apogamy was introduced. [VoL, 2 322 ANNALS OF THE MISSOURI BOTANICAL GARDEN haploid eggs by sperms from the normal pollen. This sterility of the archicarp, I believe, has been brought about by its assumption more and more of a vegetative char- acter. The formation of septa at the base of the ‘‘trichogyne’’ in such forms as Pyronema and Monascus, which primarily may have been the beginning of a transverse splitting of the oögonium, would make more difficult the fertilization of the basal portion of the archicarp. In Aspergillus repens the so- called ‘‘trichogyne,’’ or terminal cell of the archicarp, some- times gives rise to ascogenous hyphae! (according to Miss Dale, ’09). The basal portion of the two-celled archicarp, or the basal or central portions of the several-celled archicarp, seem to be the portions which have retained the function of ascogenic cells where that function still resides in the archi- carp. As the archicarp becomes longer, the sterile portion, which is non-ascogenic, becomes longer and more septate. This only increases the difficulties of the passage of the sperm nuclei. The increasing vegetative character of the terminal portion of the archicarp has given rise to the long, simple, multisep- tate ‘‘trichogyne’’ of the lichens and many Pyremomycetes and Discomycetes, as well as to the profusely branched multi- septate trichogyne of certain Laboulbeniales.* It is an inter- esting fact that in many of the cases of the extraordinary vegetative development of the terminal portion of the archi- carp (the ‘‘trichogyne’’), antheridia and spermatia are en- tirely wanting.? The degeneration changes of the sterile portion of the archi- carp (multiseptate and often also much branched ‘‘tricho- gyne’’) which are described as taking place after connection of the spermatium with the receptive terminal cell (for lichens see 1 It is worthy of note in this connection that Olive’s studies (’05) of Mon- ascus led him to regard the “trichogyne,” or terminal cell of the archicarp, as the ascogonium, and the second cell, or ascogonium according to others, as a nurse = Thaxter (’96) says that when the spermatia do not become attached to the receptive cell of the trichogyne the vegetative growth of the trichogyne is greatly increased. * (Lachnea cretea, according to Fraser, ’13; in Teratomyces actobii, Thaxter, ’96, was not able to find antheridia.) 1915] ATKINSON— PHYLOGENY IN THE ASCOMYCETES 323 Stahl, ’77, Baur, ’98, Bachmann, ’13; for the Laboulbeniales, Thaxter, ’96, p. 225), may be classed as secondary or accom- panying sexual phenomena. It does not necessarily follow that the sperm nucleus reaches the egg or fertile portion of the archicarp. The trichogyne changes taking place after the entrance of the sperm into, or its connection with the re- ceptive terminal cell, are not dependent on the final fate of the sperm, i. e., whether it reaches the egg or not. They are ante- cedent phenomena and in no sense a proof that fertilization has taken place. These disintegration changes, initiated, it would seem, by the influence of the sperm on the receptive cell of the archicarp, terminate the vegetative growth of the archi- carp and thus the reflex upon the fertile portion at the middle or base releases the ascogenic cells from the inhibiting influ- ence of the vegetative phenomena, and they then proceed with the modified sexual process among the ascogonial nuclei which may be now associated in sexual pairs, or this pairing be post- poned to some period in the development of the ascogenous hyphae. Origin of spermatia in the Ascomycetes.—The presence of the so-called spermatia in many lichens and other Ascomy- cetes, associated at the same time in numerous instances with the trichogyne-like termination of the archicarp, is one of the major pieces of evidence brought forward in supporting the doctrine of the red algal origin of the sac fungi. If we accept this doctrine, then in the Ascomycetes we must read the his- tory of the antheridia in the following order: They appeared first as free structures, spermatia, abjointed from spermatio- phores, large numbers of which were crowded in highly spe- cialized receptacles. At the next step there were few, imbedded, isolated anther- idiophores to which a few spermatia remained attached, until finally the stage was reached where spermatium and anther- idiophore were merged into the simple antheridium. This doctrine also requires that along with the change from free spermatia to the simple antheridium, there was a transition from the condition in which the spermatia do not function to [Vou 2 324 ANNALS OF THE MISSOURI BOTANICAL GARDEN that where the sperm nuclei of the simple antheridium are functional. Notwithstanding this interesting course of evolution of the antheridium and of sexuality which we trace if the red algae are accepted as the source of the Ascomycetes, I believe, just as in the case of the archicarp and trichogyne, the evidence warrants us rather in reading it in just the opposite direction; and that in the last stages of progressive development of the sexual apparatus in the Ascomycetes, the resemblances to the sexual apparatus of the red algae are merely those of mor- phological homology and analogy, not phylogenetic homology and affinity. According to this view, then, the ancestral forms of the Ascomycetes were fungi with well developed, simple but gen- eralized gametangia. This condition is retained in a number of existing Ascomycetes, in many of which true sexuality exists.! In connection with the specialization of the antheridium and the origin of the spermatia of the Ascomycetes, Monascus is an extremely interesting form. The antheridium is an elongate terminal cell of a hypha. The archicarp arises as a branch below the septum. It curves closely against the an- theridium, bending it over more or less at right angles, and copulates at any point along the side of the antheridium, there being no portion of the latter especially selected as a copula- tion place. The conidia in Monascus are formed in chains by constriction and septation of terminal portions of hyphae similar in diameter to the antheridium. The archicarp some- times copulates with a conidium of the chain before their final separation (Barker, ’03). A chain of conidia is thus homol- ogous with the antheridium, and a conidium with any section of the antheridium. It would be but a step from this condi- 1 Examples of generalized, simple (non-septate) gametangia are rage in Dipodascus and Gymnoascus. Examples of simple specialized gametangia, i. e., — en are found in the powdery mildews (Erysiphaceae) ge Eremascus. A second stage is presented in forms where the antheridium remains where it is split transversely into two cells, the terminal one (trichogyne) func- PERE as a copulating organ and zes tube for the sperm nuclei. Examples e found in Pyronema and Mon 1915] 3 ATKINSON—PHYLOGENY IN THE ASCOMYCETES 325 tion to the copulation of the archicarp with free conidia. The situation in Collema pulposum (Bachmann, ’13), Ascobolus carbonarius (Dodge, ’14), and Zodiomyces vorticellarius (Thaxter, ’96), is sim- ilar where the tricho- gyne copulates with spermatia (conidia) still attached to the sperma- tiophore. These cases are very strong evidence suggesting the homology of conidia (or pyeno- spores as the case may be) and spermatia! in the Ascomycetes. Progression in the di- rection of multiplication of antheridia, or sper- matiophores, and their association in groups followed from the sim- ple and more or less isolated situation, pro- gressing along the same course which is recog- nized in the association and massing of conidiophores into bundles, cushions, or pyenidia. It is the same course which is universally recog- nized as a striking indication of progression in other groups of plants, a cephalization of fruiting or reproductive struc- tures, as in the bryophytes, lycopods, conifers, and angio- sperms. In the latter it has given us the flower, and further cephalization of the flower has resulted in the head of the com- m ascospores.—Upper row of wer after Barker; lower De after Schikorr 1 Their function in the ancestral or early forms may have been generalized enough to permit of their performing as conidia or sperms, as in the case of Ectocarpus, — Ulothrix, ete. Strasburger (’05, p. 25) has expressed the idea that the pyenospores of the Ascomycetes might have been spermatia, and that the process of fructification now presented by these fungi is a secondary adaptation in place of the a, “fertilization by spermatia. [Vou 2 326 ANNALS OF THE MISSOURI BOTANICAL GARDEN posites, the highest stage of phyletic evolution in the plant world. In conclusion, the Ascomycetes present a very rich variety of form, structure, and adaptation with very marked diverging series. Some of these series present evidences of progres- sion from simple, generalized forms to highly specialized forms, while others indicate descent by reduction. The evi- dences of progression are of the same kind and value as are generally recognized in other groups of plants. A Sachs, in his later writings, agreed with deBary in recog- nizing the Ascomycetes as a distinct phylum, with an as- cending series from simple and generalized forms to com- plex and specialized ones. He Fig. 4. Gymnoascus Reessii: A-D, never mentioned the tricho- formation of sexual organs, fusing at C; ‘ f E, sexual organs in uninucleate condi- SYNE as evidence of their phy- tion; 7, op s reap ie in multi- letic relation to the red algae. ER But his theory was based on the presence of a procarp whether with or without a tricho- gyne. He selected Gymnoascus, where the sexual apparatus consists of simple copulating gametangia, as the simplest ascomycete known at that time. It is only in recent years that the trichogyne has been seized upon as evidence of the phyletic relation of the two groups and has forced this anomalous backward reading of the history. Part II. ELUCIDATION NOTE I The red algae are remarkable for the great constancy in the form of the procarp (procarpie branch, carpogonial branch, ete.) and the very great divergence in the processes subse- quent to the fertilization of the egg (terminal cell of the pro- carp, carpogonium) and ending in the production of the carpo- spores. The general character of this divergence may be shown by a brief presentation of several types, as follows: 1915] ATKINSON—PHYLOGENY IN THE ASCOMYCETES 327 1. The simplest type of eystocarp development occurs in the Nemalionales where the carpogonium, or egg cell, after fertilization, gives rise to several branched sporogenous threads in a compact cluster, bearing terminally the carpo- spores (Nemalion, Lem- anea, ete.), or in some species the sporogenous threads are more widely extended in the thallus, the branches producing separated clusters of carpospores (Dermo- Fig. 5. A and B, Lemanea; O, Batrachosper- i mum: cpbr, procarp or carpogonial branch; nema dichotomum, see cpg, carpogonium or egg; tr, trichogyne; sp, Schmitz and Haupt- spermatium; g, gonimoblast; csp, nig i —A and B, after Atkinson; C, after Davis. fleisch, ’97). Fertiliza- tion by the fusion of a sperm nucleus with the egg nucleus after entrance into the trichogyne and migration down into the carpogonium has been described in Nemalion (Wolfe, ’04) and in Batrachospermum (Schmidle, ’99; Osterhout, ’00). 2. In Polysiphonia (Rhodomeniales) the procarp branch of four cells is curved around so that the carpogonium is in contact with an auxiliary cell lying between the carpogonium and the pericentral cell which gave rise to the procarp. After fusion of the sperm and egg nucleus in the carpogonium, the fusion nucleus divides once. The carpogonium now connects with the auxiliary cell mentioned, which fuses with the peri- central cell. The two diploid nuclei migrate into the peri- central cell, the carpogonium separates from the auxiliary cell, while it and the remaining cells of the procarp degenerate. The pericentral cell now fuses with several other auxiliary cells, which arose from it as a branch, forming the central cell. The diploid nuclei remain in the upper part of the central cell, while the haploid nuclei from the auxiliary cells, some having divided, now degenerate (Yamanouchi, ’06). 3. A somewhat different situation exists in Erythro- phyllum delesseroides (Gigartinales). The odblastema fila- ment from the fertilized egg connects with the auxiliary cell which is the basal cell of the seven or eight-celled pro- [voL. 2 328 ANNALS OF THE MISSOURI BOTANICAL GARDEN carp. This in turn fuses with the two other large cells of the basal portion of the procarp, thus forming the large fusion cell from which the gonimoblasts, or sporog- enous threads arise (Twiss, 711). 4. In Harveyella mi- rabilis! a large cell which gives rise to the four-celled procarp is LOSS the auxiliary cell. A | short oöblastema_ fila- Fig. 6. A, Harveyella mirabilis; B and 0, ment from the egg con- Erythrophyllum delesseroides; D, E, F, and €, nects with the latter, ae o oöblastema filament; ac, auxiliary cell; g, tral cell. lic ns basal cells of the Hed in Ery- 5. In Callithamnion throphyllum which fuse with the a (Ceramiales) the fusion à m e or . . . tions are diploid; note that in the fusion cell (diploid) nucleus in the ee one (MR: egg divides into two. nucleus—A, after Sturch; B and C, after Two short odblastema Twiss; D, E, F, and G, after Oltmanns. filaments proceed from the carpogonium, each containing a diploid nucleus, and fusing with an auxiliary cell at the side of the base of the procarp. Each of the two auxiliary cells now contains two nuclei. A wall divides each cell into two. The upper daughter cell con- tains the diploid nucleus and becomes the central cell, giving rise to the sporogenous threads, while the haploid nucleus in the lower cell degenerates (Oltmanns, ’04). 6. The most complicated type may be represented by Dudresnaya purpurifera (Cryptonemiales) where several oöblastema filaments arise from the sterilized egg cell. These fuse with auxiliary cells which are either certain cells of the procarp branch, or terminal cells of its branched system, or of more distant ‘‘secondary procarp branches.’’ An oöbla- — © = Ru 1 H. mirabilis is parasitic on certain species of Polysiphonia, and is devoid of chlorophyll. For this reason it is regarded by some as indicating a step in the direction of an ascomycete. 1915] ATKINSON—PHYLOGENY IN THE ASCOMYCETES 329 stema filament after fusing with one auxiliary cell may grow forward and fuse with another and so on. The diploid nucleus formed in the egg multiplies by division in the odblastema filaments. In the fusion cell, resulting from the union of the filament and auxiliary cell, the diploid and haploid nuclei repel each other so that the former lies on the filament side while the latter lies in the base of the auxiliary cell. An outgrowth Dudresnaya purpurifera: A, odblastema filaments rig with auxiliary cells; B, C and D, outgrowth from the fusion cell to m the central cell; O, diploid nucleus dividing; D, Ren cell of eystocarp oh a wall. Note that the nucleus of the auxiliary cell remains distant from the diploid nucleus of the oöblastema filament. Shaded portions are diploid. one een -e P9, carpogonium; tr, trichogyne; of, odblastema for f cell; ac, auxiliary cell; an, auxiliary cell nucleus; cy, ern se of as ie Oltmanns. arises from the oöblastema filament at the point where the diploid nucleus lies. The latter divides, one nucleus migrat- ing into the outgrowth, while a wall separates it from the fusion cell. This new cell with its diploid nucleus becomes the central cell (Oltmanns, ’04). . In Cruoriopsis cruciata the situation is similar. The oöblastema filament by coursing widely through the thallus, fuses with the terminal cell (auxiliary cell) of ‘‘secondary procarp branches.’’ Each of these fusion cells, or auxiliary cells, then gives rise to one or two simple rows of 2-4 spores Schmitz, ’79, ’83), or a single 2-4-celled spore chain (Olt- manns, ’04). [VoL 2 330 ANNALS OF THE MISSOURI BOTANICAL GARDEN Relation between the fusions of procarp and auxiliary cells, and those of archicarp cells—Several persons have made the interesting suggestion that certain similarities between the events which take place in the fusion of one or more of the middle or basal cells of the procarp with an outgrowth from the carpogonium, either direct, or through the medium of an auxiliary cell, as represented in Erythrophyllum, Harveyella, Callithamnion, ete. (third, fourth and fifth types mentioned above), and those occurring in the fusion among themselves of the middle or basal cells of the archicarp prior to the forma- tion of the ascogenous threads, may be evidence of a phylo- genetic relationship between the red algae and Ascomycetes. Thus Baur (’98) suggests that the first fertile cell of the sev- eral-celled ascogone of Collema crispum may be the egg cell, that this may be fertilized by the entrance of the sperm nu- cleus and its fusion with the egg nucleus. This fusion nucleus may now divide. The other cells of the ascogone below the egg are conceived of as auxiliary cells into each one of which a nucleus resulting from the division of the fertilized egg nucleus migrates after pore formation in the intervening walls. In an interesting paper on the morphological relationships of the Florideae and Ascomycetes, Dodge (’14) emphasizes this theory by pointing to a number of cases in the lichens and other Ascomycetes where fusion, or pore connections, are known to occur between the ascogenous cells of the archicarp where more than one cell gives rise to ascogenous hyphae. Examples among the lichens are Collema crispum (Baur, ’98), Physcia pulverulenta (Darbishire, 00), Anaptychia ciliaris (Baur, ’04), and Collema pulposum (Bachmann, 713), while among the other Ascomycetes may be mentioned the follow- ing: Ascobolus (Harper, 96. Here there is but one asco- genous cell which gives rise to the ascogenous hyphae, but pore formation in intervening walls permits intereommunication between several adjacent cells in the middle of the archicarp. The species is not given), Ascophanus carneus (Cutting, ’09), Lachnea cretea (Fraser, 713), Polystigma rubrum (Nienburg, 14). 1915] ATKINSON— PHYLOGENY IN THE ASCOMYCETES 331 Now as to the suggested relationship between the phenom- enon of broad or narrow pore formation in the walls of certain cells near the middle or base of the archicarp in certain lichens and other Ascomycetes, and that shown in the communications taking place between the carpogonium and auxiliary cells (often including one or more of the other procarp cells), it may be said (1) that in the red algae this communication of the carpogonium (terminal procarp cell) with other procarp cells when it does take place is not direct, but by a roundabout method, either through a distinct outgrowth from the carpogonium, or through the medium of one or more auxiliary cells, or by a combination of both, to form the central cell; (2) no evidence of any similar round- about method has been observed in the archicarp of the sac fungi. The intercommunication between the middle or basal cells of the archicarp is always direct, and no communication in the multicellular archicarp occurs by means of which either a fertilized nucleus, or a sperm nucleus has been observed to migrate from the terminal cell to the middle or basal cells; (3) that in a number of the fungi where pore formation occurs between cells of the fertile portion of the archicarp, the ‘‘trichogyne’’ is either absent, or admittedly degenerate, or the antheridium is absent. Examples are: Ascobolus, studied by Harper (’96), antheridium and trichogyne absent; Asco- phanus carneus, antheridium absent, trichogyne doubtful or degenerate; Lachnea cretea, no antheridium observed, tri- chogyne not functional; Polystigma rubrum,! trichogyne not functional, from a multicellular cell at base of archicarp one nucleus migrates into the adjacent uninucleate archicarp cell, which is regarded as the ascogonium (Nienburg, ’14). In none of the lichens has a sperm or other nucleus been observed to move down into the fertile part of the archicarp. Pore formation in the archicarp of the Ascomycetes has no phyletie relation to the fusions of auxiliary cells among themselves or with a short oöblastema thread or the egg cell. It occurs in- 1 Blackman and Welsford (’12), who earlier investigated the cytology of Polystigma rubrum, are of the opinion that the “spermatia” as well as the archi- carps degenerate, and that certain vegetative cells become transformed into as- cogones [voL. 2 332 ANNALS OF THE MISSOURI BOTANICAL GARDEN dependently in different groups of the fungi as a means of permitting the association of nuclei, often in conjunction with the association of sex nuclei or their equivalent modified sex nuclei (see the situation in Basidiobolus, Eidam, ’86; Raci- borski, ’96; Fairchild, ’97; Olive, ’07; Woycicki, ’04). Relation of odblastema filaments and ascogenous hyphae.— In the Ascomycetes the processes in the growth of the zygote or ascogenic cell present to a certain extent a somewhat analo- gous course of progression to that of the carpogenie cell of the red algae. In the less complicated process, as shown in the Laboulbeniales, the carpogenic cell may undergo a few divi- sions, the subterminal cell of the series forming the as- cogonium. The ascogonium then usually divides to form two or four ascogenic cells, or without division forms the single ascogenic cell (Thaxter, ’96; Faull, ’12). The ascogenie cells give rise directly, by budding, to the asci. They are, there- fore, somewhat comparable or analogous to the gonimoblasts of the red algae. In Sphaerotheca (Harper, ’95*, p. 475) there is a single short ascogenous thread of a few cells (arising from the one-celled oögonium or ascogonium) forming a single ascus from the subterminal cell. Where the process is more complex, as in Pyronema (Harper, ’00; Claussen, ’12), several long ascogenous hyphae arise from the large single-celled zygote or ascogonium, giving rise ultimately to numerous terminal asci. In other forms the ascogonium is several- celled, a number of the cells developing ascogenous hyphae (Collema, Stahl, ’77; Baur, ’98; Bachmann, ’12, ’13; Anap- tychia ciliaris, Baur, ’04; Physcia pulverulenta, Darbishire, ’00; Ascophanus carneus, Cutting, ’09; Lachnea cretea, Fraser, ’13; ete.). Some of the chief objections in the way of accepting the theory of a phylogenetic relation between the odblastema fila- ments of the red algae and the ascogenous threads of the sac fungi are as follows: 1. The fusion of a free sperm and the egg nucleus in the single uninucleate oögonium or carpogenie cell. So far as we know this is universal in the red algae. In the Ascomycetes the odgonium is usually multinucleate or multiseptate. In no 1915] ATKINSON— PHYLOGENY IN THE ASCOMYCETES 333 case has fertilization by a free sperm been determined, and in forms with a multiseptate ‘‘trichogyne,’’ or oögonium, the so- called spermatia, or antheridia, do not, so far as we know, play the usual röle in fertilization, not even a modified röle by asso- ciation with the oögonial nuclei. 2. The individual nuclei of the oöblastema filaments are of the usual diploid character, and there is no fusion of these nuclei prior to the formation of the carpospores. The indi- vidual nuclei of the ascogenous threads, or ascogenic cells, are probably haploid in character, and sooner or later form the so-called synkarion, an association of two nuclei, together equivalent to a diploid nucleus. Fusion of the paired nuclei takes place before the formation of the ascospores. 3. It has been suggested that the complex processes in the extensive migration, branching and fusions of the odblastema filaments with auxiliary cells as is known to occur in the Cryptonemiales (as in Dudresnaya, Cruoriopsis, Gloeosi- phoma, ete.), may furnish still more important evidence of the ancestry of the Ascomycetes than that suggested in the fusions of procarp and auxiliary cells on the one hand, and archicarp cells on the other (Dodge, 714). The fusions of the oöblastema filaments with auxiliary cells and the production of sporogenous threads from the central cells thus formed, are supposed to be represented by the fusions which are known to occur between the ultimate and antepenult cells of the ascus hook prior to the formation of additional asci. The processes in both groups result in the multiplication of spore origins and consequently in an increase in spore output. Perhaps the nearest analogue to the process in the Ascomycetes which re- sults in the formation of the ascus with its four to eight spores, is found in Cruoriopsis, where one or two spore chains of two to four spores each are produced as a result (Schmitz, ’79, ’83; Oltmanns, ’04). The theory of ‘‘second sexual fusions’’ in the red algae was founded on the discovery of these fusions of the odblastema filaments with auxiliary cells, since it was sup- posed that a fusion occurred between the nucleus of the oöblastema filament (derived from the diploid nucleus of the fertilized egg) and the nucleus of the vegetative auxiliary cell [VoL. 2 334 ANNALS OF THE MISSOURI BOTANICAL GARDEN (Schmitz, ’83). Recent cytological work on the red algae has not confirmed this theory, but, on the other hand, has discred- ited it, since in the cases examined the diploid nucleus of the odblastema filament and the haploid nucleus of the auxiliary cell are said to repel each other and no fusion between them occurs. It should be emphasized that the fusion of the oöbla- stema filament and the auxiliary cell is a fusion of a diploid structure with a haploid one, that it is probably of a nutritive, or parasitic, nature comparable to the fusion of the moss sporogonium with the tissue of the gametophyte, a physiolog- ical, nutritive requirement in the absence of other means of nourishing the moss sporogonium. The fusions occurring be- tween cells of the same ascogenous hypha are fusions between cells of the same phase and serve to bring into association nuclei of more or less remote ancestry, but each endowed with the same number of chromosomes (probably the 1x number). Thus, while there are somewhat analogous variations in the splitting up of the ascogonium in the sac fungi, and of the car- pogonium in the red algae, with progress in the direction of increasing the output of spores, it seems fair to conclude, that, so far as the evidence at present in hand is concerned, the rela- tion between the fusion of odblastema filaments and auxiliary cells in the red algae, and those between the ultimate and an- tepenult cells of the ascus hook (of the ascogenous hyphae), however interesting it may be, has no phylogenetic signifi- cance, and is at best a rather strained parallel. Ascogenous hyphae, gonimoblasts, odblastema filaments, the several fertile cells of certain ascogonia which communicate by resorption of the intervening septa, the fused procarp, may be considered as morphological equivalents, as suggested by Dodge (714, p. 174), but there is no evidence of a phyletic rela- tion between the ascogenous hyphae and fusing ascogonial cells, and their morphological equivalents in the red algae. They illustrate different modes of increase of spore output by splitting up of the oögonium. NOTE II The fundamental difference in the method of development of ascospores and carpospores is one of the great barriers in the 1915] ATKINSON—PHYLOGENY IN THE ASCOMYCETES 335 way of the descent of the Ascomycetes from the Florideae. Some (Bessey, E., ’13, p. 151) have attempted to overcome this difficulty by suggesting the homology of the ascus and tetra- sporangium. But this effort leads to so many suppositions and supporting hypotheses because of the fundamental dif- ference between the process of spore formation in the ascus, and the processes of carpospore or tetraspore formation, that the descent of the ascus fungi from the red algae would re- quire a far more labyrinthian course than would be necessary in deriving them from the Phycomycetes. NOTE III IS NUCLEAR FUSION IN THE ASCUS OF A VEGETATIVE OR SEXUAL NATURE? It is unfortunate that there is such great divergence of opinion in the interpretation of the nuclear phenomena in the archicarp and ascogenous threads. These conflicting results are probably, in a large measure, due to the difficulties pre- sented in the minute size of the nuclei. The divergence of opinion relates primarily to the question as to whether the fusion nucleus of the ascus is the result of two successive nuclear fusions, the first taking place in the ascogonium and the second in the ascus, or whether the nuclear fusion in the ascus is the only one. The principle of a single nuclear fusion, that in the ascus, interprets this act as the final stage in the process of fertiliza- tion, by the fusion of two nuclei of more or less remote an- cestry. At some time prior to ascus formation these two nuclei may possibly become associated in pairs into a syn- karion and multiply in the ascogenous threads by conjugate division, or the synkarion and conjugate division may be post- poned to the ascus hook and the complicated series of fusions between the ultimate and antepenult cells of the erozier, or proliferations of the young ascus with accompanying con- jugate divisions of the synkarion. Dangeard (’94) first described the presence of two nuclei in the young ascus, and their fusion, in several species (Borrera ciliaris, Peziza vesiculosa, Helvella ephippium, Geoglossum [voL. 2 336 ANNALS OF THE MISSOURI BOTANICAL GARDEN hirsutum, Acetabula calyx, Exoascus deformans, and some lichens). The origin of the ascus was correctly described in a number of cases, but in the majority of cases at that time he thought the young ascus arose by the copulation of two unicel- lular gametes according to a method similar to the formation hy => `G Ac J STIRS SEIT De LOS >= F. DN PR ry IE > RE Fig. 8. Pyronema confluens: A, section ir were discocarp; B, group of archicarps copulating with antheridia by m of the slender prolonga- tion (tr richogyne) of the ascogonium which is separated as a zen. cell; C, pair of ee organs copulating by means of the trichogyne gonium at left, ann at right; D, showin multinueleste condition sexua a nication of antheridium and trich antheridium ; b or ; ¢, ascogonium. der — of a tutes group of sexua al Er en after the antheridial nuclei have ered asco- =: nuclei — a nerated; also showing early stage gr hyphae ascogonium; F, showing relation of ascogonia, ascogenous De asci, ar paraphyses in mature fruit body.—After Harper 1915] ATKINSON—PHYLOGENY IN THE ASCOMYCETES 337 of the ascus in Eremascus, so that the ascus appeared to be supported on two stalks. Frequently, however, in Peziza vesiculosa and Helvella ephippium he observed the origin of the ascus from a single hypha curving at the end in the form of a hook or crozier. The four nuclei resulting from the divi- sion of two were so situated in the crozier that after the forma- tion of two cross walls the ultimate and antepenult cells each contained one nucleus, while the penult cell contained two nuclei. The association of two nuclei in the young ascus and their fusion he interpreted as a sexual act, and the young ascus was looked upon as an oögonium. Later, Dangeard found that the crozier method of ascus formation was the usual one in the forms studied and that in no case in these higher forms did the ascus arise immediately from the conjugation of two dif- ferent hyphae. This important pioneer work by Dangeard was a great stimulus to further studies which has led to a more or less clear knowledge of the history of the nuclei from the archicarp through the ascogenous hyphae to the ascus, while the origin of the ascogenous hyphae from the fertile cells of the archi- carp was first described by Janczewski (’71) in Ascobolus, and later by Kihlman (’83) in Pyronema confluens. Harper first demonstrated the origin of the ancestral ascus nuclei in the archicarp of Sphaerotheca castagnei (’95") and Pyronema confluens (700) and their migration in the ascogenous hyphae, though he does not give the nuclear history in the ascogenous hyphae, except the later stages at the time of formation of the ascus. Their archicarp origin has been abundantly confirmed by several investigators in a number of different forms, both among the lichens and other Ascomycetes. The different opinions in regard to the significance of nuclear fusion in the ascus rest upon the interpretation by different investigators of the behavior of the nuclei in the archiearp, or ascogenous cells, before they begin to move into the ascogenous hyphae. Some maintain that there is a fusion, in pairs, of the sex nuclei (1) in the archicarp when fertilized [VoL. 2 338 ANNALS OF THE MISSOURI BOTANICAL GARDEN by an antheridium (Harper in Sphaerotheca castagnei,' ’95*, 96; Erysiphe, ’96; Pyronema confluens, ’00; Phyllactima, ’05; Blackman and Fraser in Sphaerotheca, ’05; Claussen in Bou- diera [=Ascodesmis], ’05); or (2) in the archicarp where the antheridium is functionless or absent (Blackman and Fraser, 06, in Humaria granulata; Fraser, ’07, in Lachnea stercorea). In Aspergillus herbariorum Miss Fraser (07, p. 420) finds that the antheridium often degenerates and did not observe disap- pearance of the intervening wall when fusion with the tricho- gyne took place. She nowhere describes or figures fusion in pairs of the ascogonial nuclei. She merely assumes it, for, in the summary (’07, p. 428) she says: ‘‘It seems probable that normal fertilization occurs in some cases, and that in others it is replaced by a fusion of ascogonial nuclei in pairs’’; Wels- ford (’07) in Ascobolus furfuraceus; Dale (’09) in Aspergillus repens; Cutting (’09) in Ascophanus carneus believe in the fusion of archicarp nuclei in pairs; or (3) of nuclei in vegeta- tive cells where the archicarp is wanting or functionless (Fraser, ’07, ’08, in Humaria rutilans, fusion of the nuclei said to take place soon after entering the ascogenous hyphae; Car- ruthers, ’11, in Helvella crispa; Blackman and Welsford, 712, merely found evidence of nuclear fusion in vegetative cells of Polystigma rubrum). 1Dangeard (’97) claims that the antheridium is functionless and that the single nucleus in the oögonium divides into two. After his study of Pyronema Claussen (’12) is inclined to question the fusion of the two sex nuclei in the oögonium of Sphaerotheca, Erysiphe, Phyllactinia, and Pyronema as described by Harper (’95a, ’96, ’00, 05), and by Blackman and Fraser (’05) in Sphaerotheca as well as in the case of Boudiera (= Ascodesmis) studied by him in 1905, I respect to his work on Boudiera he now says: “My own statements upon the nuclear fusion in the ascogone of Boudiera (Ascodesmis) are clearly wrong.” He points out that in none of these cases is the history of the nuclei in the ascogenous hyphae known, and thinks that a reinvestigation will show paired nuclei here. A question to be considered, says Strasburger (’05, p. 24), is whether the chromosomes of the nuclei united in the oögonium do not remain in separated groups in the ascogenous yphae, in order to fuse as individual nuclei in the ascus. Lotsy (’07) has ex- pressed a somewhat similar view in an attempt to harmonize the situation in the Ascomycetes and Basidiomycetes. The fusion nucleus in the oögonium remains for a time a 2x nucleus but some time prior to ascus formation the 2x nucleus karion. Conjugate division now takes place with ascus formation occurring immediately or after several successive conjugate divisions. 1915] ATKINSON—PHYLOGENY IN THE ASCOMYCETES 339 Others maintain with equal assurance that there is no fusion of the sexual nuclei in the archicarp. There is merely an asso- ciation of sex nuclei. 1). In forms with a func- tional antheridium and archicarp may be mentioned Monascus! (Schikorra, ’09) and Pyro- nema confluens (Claussen, ’07, ? (2). In forms where the an- theridium is absent or function- less may be mentioned Pyronema confluens (Brown, W. H., ’09, an- theridium functionless), Lachnea scutellata (Brown, W. H., ’11, antheridium absent). In both of these examples, cases of division g and 0, conjugate division Er of the nuclei in the ascogonium nuclear pairs in the ascogenous were observed which might be ascus hook; E, tips of branched mistaken for fusion. Since no 4scogenous hyphae with divisions of nuclei in the as- conjugation of the ultimate and cogonium have been described by antepenult cells of the ascus hooks; authors in the forms where they ultimate and antepenult cells of believe sexual fusions of nuclei 0° to take place, W. H. Brown (’11) nium; suggests that they may have had before them division stages. In Ascophanus carneus and Ascobolus immersus the anther- idium is absent, but association of the nuclei in several of the multinucleate ascogonial cells occurs after pore formation in the walls. Most of these nuclei become paired and remain paired as they migrate in the ascogenous hyphae to the ascus hooks, where conjugate division takes place. The only fusion of nuclei is that in the ascus, except in badly fixed prepara- tions or in degenerating nuclei in the ascogonium (Ramlow, 14). In Leotia (Brown, W. H., 10) the ascogenous hyphae 1 Barker (’03) ascribed his failure to find a fusion of nuclei in the ascogonium of Monascus to the absence of proper stages in his material, Fig. 9. Pyronema confluen =] =) ® B © = „ua m» ar "5 Er D © ; ase. with paired ‘ ‘sexual” nuclei.— After Claussen [VoL. 2 340 ANNALS OF THE MISSOURI BOTANICAL GARDEN are supposed to arise from an ascogonium in the base of the ascocarp, but the nuclei are believed to arise from a haploid nucleus. Conjugate division occurs in the ascus hooks, the majority of which are formed by proliferation of the binu- cleate penult cell and from fusions of the ultimate and ante- penult cells of croziers, so that many conjugate divisions of the haploid nuclei take place, and the first nuclear fusion is in the ascus. In Laboulbema chaetophora and L. Gyrinidarum (Faull, ’11, 712) fusion of nuclei does not occur in the as- cogonium, the mature binucleate ascogenic cell develops the asci by budding, each ascus bud being preceded by a con- jugate division of the nuclear pair. In Polystigma rubrum (Nienburg, 714) no fusion in the ascogonium occurs. In Collema pulposum (Bachmann, ’13) the nuclei in the ascogenic cells were often found in pairs, but no cases of fusion were observed. (3). Forms in which an archicarp is absent or functionless, and certain vegetative cells take on the function of ascogenic cells, in which the authors believe nuclear fusion does not take place except in the ascus: Gnomonia erythrostoma (Brooks, 10); Helvella elastica (McCubbin, ’10) in which the ‘‘as- cogenous hyphae’’ form an intricately interwoven subhy- menial layer of threads each with two nuclei in the end. The ends of these hyphae form croziers with conjugate division of the two nuclei followed by about six repeated proliferations of the young ascus and crozier formations, accompanied by fusions of the ultimate and antepenult cells and crozier formation, resulting in many successive conjugate divisions of the haploid nuclei, with fusion first in the ascus. In Xylaria tentaculata (Brown, H. B., ’13) the ascogenie cells which appear to be derived by the separation of the cells of «“Woronin’s hypha’’ are uninucleate and soon become multi- nucleate by nuclear division. The nuclei multiply also in the ascogenous hyphae. The theory of a vegetative fusion in the ascus arose from the belief on the part of some students that sexual fusion of the nuclei occurred in the ascogonium, that the nuclear fusion in the ascus must be a second fusion with no relation to the 1915] ATKINSON—PHYLOGENY IN THE ASCOMYCETES 341 sexual process, and, therefore, it must be of a vegetative na- ture. If a second fusion of the nuclei occurred it would call for a triple division of the fusion nucleus in order that the haploid condition should be again reached. The universal occurrence of the triple division in the ascus in the formation of the spores is by some ascribed to a ‘‘quad- rivalent character’’ of the chromosomes in the fusion nucleus, and rendered necessary in the return to the univalent condi- tion (Harper, ’05; Overton, ’06), and Overton states, ‘‘that all these divisions persist, no matter how many spores are to be produced, which shows their necessity in the process of reduction.”’! Eremascus controverts this statement since there is certainly but one fusion (Stoppel, ’07; Guilliermond, ’09) and yet triple division occurs in the ascus. The results of cytological investigations by different stu- dents in connection with the triple division show considerable variation. Thus Harper (’00, ’05) finds the same number of chromosomes in all three divisions (10 in Pyronema, 8 in Phyllactinia). The two ascus nuclei ‘‘fuse with all their cor- responding parts’’ (Harper, ’05, p. 67), so that the quadriva- lent nature of the chromosomes in the fusion nucleus is not to be seen, though he conceives it to exist. Synapsis occurs in the first division. Miss Fraser (’07, ’08) describes Humaria rutilans as having 16 chromosomes in the first division where synapsis occurs (heterotypic) which split transversely and the daughter nuclei have each 16 chromosomes which appear on the nuclear plate in the second division. ln the second division the chromo- somes split longitudinally (homöotypie) and 16 chromosomes pass to each daughter nucleus. In the third division the 16 chromosomes are supposed to separate et the nuclear plate without division, 8 going to each daughter nucleus. This divi- sion she terms ‘‘brachymeiotic’’. A similar situation is de- scribed by Fraser and Welsford (’08), Fraser and Brooks (’09), and Carruthers (711). Faull (’05) finds the same num- 1 Polysporous asci resulting from several to many nuclear divisions may be the retention of an ancestral character, the number of divisions being reduced to three in most forms. [VoL. 2 342 ANNALS OF THE MISSOURI BOTANICAL GARDEN ber of chromosomes in all three divisions, in some species 4 or 5 (Hydnobolites), in others 8 (Neotiella). More recently Claussen (’12) after a very thorough study of Pyronema confluens finds the same number of chromosomes (about 12) in all three divisions. The first division is hetero- typic accompanied by synapsis, diakinesis and a splitting of the chromosomes. The second is homöotypie, while the third is typic. Faull (’12) in a recent study on Laboulbenia also finds that the two first divisions in the ascus agree with the usual phenomena accompanying reduction in spore mother cells, the first being heterotypic, while the second follows ‘‘very swiftly on the heels of the first.’? He concludes that “probably the only nuclear fusion in the life cycle is that in the ascus,’’ and that conjugate divisions of nuclei are an im- portant phase in the sexual phenomena of the Ascomycetes. The evidence from recent investigations, therefore, supports more and more the interpretation of nuclear fusion in the ascus as a process of exactly the same significance as the nuclear fusion in the basidium of the Basidiomycetes, and in the teleutospore of the Uredinales, i. e., it is the fusion of a pair of nuclei of a longer or shorter history of conjugate divi- sions from a pair of ancestral nuclei of more or less remote association. This association of nuclei arises in a variety of ways and at different periods in the ontogeny just as it does in the Basidiomycetes (Maire, ’02; Ruhland, ’01; Harper, ’02; Nichols, S. P., ’04; Kniep, ’13), and Uredinales (Sappin- Trouffy, ’96; Maire, ’99, ’01; Blackman, ’04; Christman, ’05, 707; Blackman and Fraser, ’06; Olive, ’08; Hoffmann, 712; Werth und Ludwigs, 712). The association is accomplished in some cases through the copulation of two gametangia (Pyronema, Monascus, Gymnoascaceae, and the Erysipheae). Such an association represents nearly, if not quite exactly, the true type of sexuality. The other methods of association represent a variety of modified types of sexuality (see Note 1) where the archicarp is present and the antheridium absent, or functionless, or where the archicarp is absent and vegetative cells, either with or without the migration into them of nuclei 1915] ATKINSON— PHYLOGENY IN THE ASCOMYCETES 343 from adjacent vegetative cells, give rise to the ascogenous threads. The results of recent work tend more and more to show that there is no fusion of the associated nuclei in the as- cogonium, or ascogenic cells, whether certain of the nuclei have been derived from an antheridium (Pyronema, Claussen, 12; Monascus, Schikorra, ’09), or not. Conjugate division in the ascogenous threads has been abundantly proven, though in some cases it may occur only one or a few divisions prior to the formation of the ascus. What the peculiar features of nuclear fusion in the ascus are which characterize it as vegetative, seem to rest more on an ex parte judgment of a fusion of nuclei in the ascogonium than upon any well established idea of the nature of vegeta- tive nuclear fusion. Thus, Miss Fraser (’08, p. 37) states that in Humaria rutilans the two nuclei in the ascus enter in- dependently upon the prophases of the first division, fusing in the spirem stage. This she regards as evidence in disproof of the sexual nature of the fusion of nuclei in the ascus (08, p. 44). Harper (’05) raises a similar objection. On the other hand, it seems to me that it is excellent evidence that it is not of a vegetative nature. It is well known in a number of cases that the egg and sperm nuclei, lying side by side in the egg, undergo the prophase stages of division up to the formation of the chromosomes before fusion of the two takes place. I cite certain examples in the Abietineae: Pinus sylvestris (Blackman, ’98); P. strobus (Miss Ferguson, ’01, 04); Tsuga canadensis (Murrill, ’00). In support also of the supposed vegetative nature of the fusions in the ascus Miss Fraser (’13, p. 559) cites ‘‘vegeta- tive nuclear fusions’’ in the quadrinucleate ascus of Humaria rutilans and her work on this plant in 1908. But she no- where describes or figures the fusion of the four nuclei in such asci. She says (’08, p. 41) ‘‘trinucleate (Fig. 50) and quadrinucleate (Fig. 51) asci are sometimes formed; their fate could not be determined.’’ It is very likely that such tetranucleate young asci found by Miss Fraser in Humaria result from further conjugate division prior to the prolifera- (Von. 2 344 ANNALS OF THE MISSOURI BOTANICAL GARDEN tion of the young ascus to form branches and further eroziers resulting in an increase of asci as shown to take place in Pyronema confluens by Claussen (’12, p. 25, fig. 6, III). It has been suggested by some who regard the fusion in the ascus as a second fusion of nuclei (Harper, ’05; Overton, ’06) that if the synkaryophytie condition of the terminal portion of the ascogenous hyphae in Pyronema, and far back in those of Galactinia succosa (Maire, ’03, 0D), could ‘‘work back until the egg cell was reached,” an apogamous condition might result similar to that in the Hymenomycetes. Certainly those who have suggested this theory have not thought far enough ahead, for how would the univalent condition of the spore nucleus pass to the bivalent condition of each nucleus prior to the paired (= quad- rivalent) condition in the ascogenous hyphae of the next generation unless this were preceded by a nuclear fusion. Such a condition would not be apogamy. The quadrivalent character of the fusion nucleus of the ascus, or of the syn- karion in the ascogenous threads, demands two successive nuclear fusions, if the triple division in the ascus brings about the reduction of a quadrivalent nucleus to a univalent one as maintained by the adherents of this theory. As to such an apogamous condition being similar to that in the Hymenomycetes it must be remembered that there are only two divisions in the reduction process in the Hymenomycetes, so that when two univalent nuclei become associated in cells of the mycelium or basidiocarp the bivalent condition of these cells is attained. In a very interesting and scholarly argument Harper (’05) has attempted to explain the inclusion and fusion of two nuclei in the young ascus on the basis of the nucleo-eytoplasmie relation or balance in the cell. The abundance of food ma- terial in the tips of the ascogenous hyphae inhibits cell wall formation so that two nuclei are enclosed in one cell. Rapid growth of the ascus and eytoplasm follows in order to balance the relation of the latter with the nuclear mass. The fusion of the nuclei and growth of the fusion nucleus again over- balance the cytoplasm, which then by growth increases again 1915] ATKINSON—PHYLOGENY IN THE ASCOMYCETES 345 in mass. The process is thus a reversible one, and by a sort of see-saw growth of nucleus and cytoplasm the ascus cell is pushed up to the large size characteristic of spore mother cells. It is very true that the ‘‘regulative function is a reversible one,’’ that an active cell with a large amount of cytoplasm demands a correlative amount of nuclear substance, that the increase in one may result in the increase of the other. Also it is very true that the ascus belongs to the category of spore mother cells, which are characterized by relatively large nuclei and cytoplasmic mass compared with most vegetative cells, but this does not explain why, when ascus or spore mother cell formation is about to take place, cell division does not occur at a period when the food relation would permit the forma- tion of young uninucleate asci if these nuclei are bivalent in nature. The regulative functions accompanying growth and maturity of such a young gonotokont would assure sufficient size, sufficient food material, and the necessary equilibrium. The fact that asci in different species and groups vary so greatly in size shows this, and also that there is no general standard of mass in relation to surface area which would demand two nuclei at the origin of the ascus. In fact it is very clear, from the morphological processes which take place in the tip of the ascogenous hyphae of most of the forms studied, that cell division, or cell wall formation, is more likely governed by the last division of the two nuclei so that the cell walls are laid down between the daughter nuclei. If the inclusion and fusion of two nuclei in the young ascus were controlled entirely by nutritive and cyto-regula- tive processes, why are not sister nuclei included? Surely the purely cyto-regulative functions would be just as well satis- fied. It appears that in rare cases sister nuclei may be in- eluded in the ascus (Brown, W. H., ’10, in Leotia chloro- cephala). Of the four nuclei resulting from the two successive divi- sions of the zygote nucleus in Spirogyra, Chmielewski (’90) states that two fuse to form the nucleus of the single germling which is usually formed in the Zygnemaceae. Harper inter- [VoL. 2 346 ANNALS OF THE MISSOURI BOTANICAL GARDEN prets this as a vegetative fusion in support of his interpreta- tion of vegetative fusion in the ascus. Karsten (’08) de- scribes the divisions of the zygote nucleus into four nuclei in Spirogyra jugalis, but does not state the relation of the nuclei to the germling (second division sometimes omitted). Trön- dle (’07) interprets the process in Spirogyra Spréeiana as presenting but a single division of the zygote nucleus. Re- sults of this nature, so divergent from expectations based on the normal history in many other organisms in widely sepa- rated groups, are usually received with considerable reserve, particularly where they are pioneer investigations in a group not yet studied. Recently Kurssanow (711) in a thorough study of nuclear division and germination of the zygote in two species of Zygnema (Z. cruciatum and Z. stellinum) has shown that the process is normal, there being two successive divisions, three of the nuclei usually degenerating, while one becomes the nucleus for the single germling characteristic of the Zygnemaceae. Occasionally only two of the nuclei de- generated, but then two germlings were formed, an interest- ing case showing a tendeney to retain what is believed to be the ancestral eondition where four germlings are formed as in the Mesotaeniaceae, while in the desmids two germlings are regularly formed. Other cases cited as examples of vegetative nuclear fusion and classed with nuclear fusion in the ascus, are those of the endosperm nucleus with the second sperm nucleus in seed plants (Harper, ’05), and (Fraser, 713) nuclear fusions in paraphyses and in hairs of the excipulum of certain discomy- cetes. Such cases, however, cannot be legitimately compared to fusion in the ascus, since those nuclei are shut off from further participation in the line of successive ontogenies. The example cited by Harper of Boveri’s (’88) experiment in shaking sea urchin’s eggs after fertilization, resulting in the production of an abnormally large larva with 72 instead of 36 chromosomes, is in a different class from most of the other examples of vegetative fusion given. This is equiva- lent to a true double fertilization and it is quite within the bounds of possibility that among many such larvae some 1915] ATKINSON— PHYLOGENY IN THE ASCOMYCETES 347 might under favorable conditions be the starting point of a new ontogeny which would be similar to certain mutants. The case of Oenothera gigas (see De Vries, ’03, 713) a mutant from Oe. Lamarckiana with double the number of chromosomes is similar.t Other tetraploid mutants are known (see Gates, 13), the diploid gametophyte and tetraploid sporophyte of the mosses produced experimentally by Marchal (’09, ’11) is in- teresting in this connection. Now, the possibility of a similar double fertilization in an as- comycete is not, a priori, excluded. There might be an isolated example. But the normal expectation is that it would have afterward a nuclear history in its ontogeny similar to others with one nuclear fusion and one reduction from 2x tolx. But it is not likely that the entire group of sac fungi is founded on such a mutation, followed by a double reduction with triple division and then double fertilization again and so on. The several cases where it has been quite well established that there is no nuclear fusion prior to the ascus, together with the great uniformity of the ascus nuclear phenomena in the group, controverts the idea of any such origin for the sac fungi. All of these facts go to prove that the inclusion and fusion of two nuclei in the young ascus is of a very different and far greater significance than a vegetative one. The process of nuclear fusion in the ascus does not comprise in itself the entire series of events generally accepted as belonging to the process of fertilization, for in most organisms nuclear fusion occurs in the same cell where nuclear association takes place. It is generally conceded that before the haploid con- dition of the nucleus is again established important pro- cesses take place which we call reduction phenomena, the full significance of which we perhaps are as yet ignorant of. These processes, including synapsis, cannot take place unless nuclear fusion has occurred, and some students see in how the doubling arose in this instance is of course difficult to determine. Stomps (’12) suggested that it arose through the union of two unreduced diploid gametes, while Gates (’09, ’13) thinks it arose through “sus- pended mitosis of a megaspore mother cell” having (4x) 28 chromosomes, and its apogamous development. [Vou, 2 348 ANNALS OF THE MISSOURI BOTANICAL GARDEN them the real act of fertilization (Strasburger, ’00, ’04, ’05). Remarks on the origin of the specialized ascus.—In the direction of progression from the generalized ascus by split- ting up of the zygote, the diploid phase has been prolonged and the number of spores multiplied. The filamentous out- growths of the zygote, or its equivalent, provide numerous terminal cells of restricted size suitable for the production of a small number of spores in each, following the meiotic divi- sions of the fusion nucleus which terminate the diploid phase. The situation in species with polysporie asci, where the spores result from numerous divisions of the fusion nucleus, is interpreted by some as a germination phenomenon (Over- ton, ’06), but it seems to me more comprehensible to regard it as a retention of a primitive feature existing in certain phyco- mycetous ancestors, and characteristic also of primitive As- comycetes like Dipodascus. The formation of internal non-motile spores through free cell formation in the zygote, under conditions adapted for dis- persion by ejection from either the generalized or specialized ascus, may be sufficient to account for the distinctive processes of spore formation in the sac fungi. In the odgonium of Saprolegnia, functional nuclei in the oögonium are very simi- lar to the nuclei of the ascus preceding ascospore formation. The nucleus is provided with a prominent central body at its pointed end from which kinoplasmic radiations extend (Har- tog, 95; Claussen, ’08; Mücke, ’08). In most of the Ascomycetes the cytoplasm in the ascus is differentiated into epiplasm and spore plasm, the former as- sisting in the ejection of the ascospores. This separation of the plasm may have been one of the direct causes of the peculiar method of ascospore formation. NOTE IV THE PHYLOGENETIC RELATION OF THE TRICHOGYNE AND SEXUAL APPARATUS OF THE ASCOMYCETES AND THOSE OF THE RED ALGAE The sexual apparatus of the Ascomycetes, particularly the trichogyne and the so-called spermatia, is generally conceded to be the strongest evidence in support of their phylogenetic 1915] ATKINSON—PHYLOGENY IN THE ASCOMYCETES 349 relation to the red algae. The analogy at least between the trichogyne of the red algae and that of the Ascomycetes is very striking. The evidence brought forward by Stahl (’77) and others of the relation of the trichogyne to the ascogo- nium in the lichens, together with the fusion of spermatia to the trichogyne, followed by the gradual and peculiar degenera- tion of the latter and the subsequent development of the as- cogenous threads, was generally accepted as proof of fertil- ization in the ascogonium by a spermatium. Also the early studies of Polystigma rubrum (Fisch, ’82; Frank, ’83) and Gnomonia erythrostoma (Frank, ’86) in which similar struc- tures and phenomena were observed at that time, were gen- erally accepted as indicating a well developed condition of sexuality. These studies gave a great impetus to the theory suggested by Sachs (’96) that the Ascomycetes had their origin from the red algae, or that the two groups had an- cestors in common. This theory has taken very deep root and probably is accepted by a majority of botanists even at the present time, especially by those who are not special students of the fungi. It should be stated also that a number of our foremost students of the fungi, perhaps a majority of them, are firm disciples of this theory. Recent investigation, however, including a cytological study of several of the now classic types, including Collema (Bach- mann, Miss F. M, 712, ’13), Polystigma rubrum (Blackman and Welsford, 712; Nienburg, ’14), Gnomonia erythrostoma (Brooks, ’10) have failed to furnish any evidence of a real sexual function on the part of either the trichogyne or sper- matia in any of the species of fungi possessing these two structures. Pairing of nuclei in the odgonium, or the pairing of these with nuclei from adjacent cells of the ascogonial branch or archicarp, furnish the synkaria, or the synkaria are organized at different stages in the development of the as- cogenous hyphae (see Note m1). In some quarters these re- sults have led to a loss of confidence in the sexual significance of the trichogyne and spermatia of the Ascomycetes. Some have therefore attributed to the trichogyne a physiological significance of another kind, that of a respiratory organ for [VoL. 2 350 ANNALS OF THE MISSOURI BOTANICAL GARDEN example (Brooks, ’10), or a boring organ, a terebrator (Lindau, ’99). Zukal (’89) interpreted the trichogyne of Pyronema confluens as a haustorium to provide food for the large ascogonium with its numerous ascogenous threads. Recent investigations on Collema pulposum (Bachmann, F. M., 13) have revealed an interesting departure in the rela- tion of the trichogyne and spermatia from that thus far found in other lichens, and is in strong contrast with the condition found by Stahl in Collema. The ‘‘spermatia’’ are not free and are not formed in large numbers in superficial receptacles, but are imbedded in the thallus and remain attached to the supporting hypha. The trichogyne does not extend to the surface but migrates through the interior of the thallus, seeks the spermatia and fuses with one. Then the trichogyne un- dergoes the usual deterioration, but no evidence was obtained of the migration of the nucleus of a spermatium to the as- cogonium, although a nucleus supposed to be the sperm nucleus appears to have been observed in the terminal cell of the trichogyne. In the red algae the only variations and progression in the trichogyne is in variations in length to meet the requirements of thin or thick cortex, some more or less sinuous or spirally wound, and a few stout and blunt. It is universally a con- tinuous, enucleate,! prolongation of the odgone, i. e., not sep- tate nor a separate cell. So far as we know the sperm always functions in the red algae. In the sac fungi, there is great variation and marked morphological progression from an odgone without a trichogyne through short one-septate trichogynes to long, simple, several-celled ones, and also to profusely branched, multi-septate trichogynes. It is more comprehensible to regard this progression and variation in the light of evolution from the simple to the complex, in the as- comycete phylum, independent of the red algae, than to con- 1Davis (’96) describes the trichogyne of Batrachospermum as having a nucleus of its own, but it is not separated from the egg nucleus by a wall until just prior to the development of the gonimoblasts from the egg. He also states that the sperm nucleus never passes out of the trichogyne into the egg. How- ever, Schmidle (’99) and Osterhout (’00) find no trichogyne nucleus and describe a real fertilization by fusion of sperm and egg nucleus. 1915] ATKINSON— PHYLOGENY IN THE ASCOMYCETES 351 ceive the long septate trichogyne of the highly specialized Collema to be derived directly from the simple trichogyne of the red algae, and then degenerate to the simple gamete of lower more generalized Ascomycetes. NOTE V MODIFICATION OF SEXUAL PROCESS ALONG WITH STERILITY OR LOSS OF THE ANTHERIDIUM AND STERILIZATION OF THE ARCHICARP Sterility or loss of the antheridium.—Several species are known in which the antheridium, though present, does not function. In such cases sexuality is modified in such a way that sex differentiation occurs among the nuclei in the as- cogonium or in the ascogenous hyphae. Several examples may be cited as follows: In Pyronema confluens (Brown, W. H., ’09) the antheridium sometimes fuses with the trichogyne but there is no migration of its nuclei; in other cases it may not connect with the trichogyne. The antheridial nuclei degenerate. In still other cases the antheridium is ab- sent. In Lachnea stercorea the antheridium fuses with the terminal cell of the archicarp but its nuclei degenerate (Fraser, ’07). In Aspergillus herbariorum (Fraser and Chambers, ’07) and A. repens (Dale, ’09) a similar situation exists. In those numerous examples where spermatia (mostly free ‘‘antheridia’’) are present it is very likely that the sperm nuclei no longer play a röle in fecundation due to such exten- sive sterilization of the terminal segments of the archicarp, but the cytology of only a few species has been determined. They no longer perform the function of fecundation in Poly- stigma rubrum (Blackman and Welsford, ’12; Nienburg, ’14), Gnomonia erythrostoma (Brooks, 710), and in Collema pul- posum (Bachmann, ’13) the sperm nucleus has not been traced through the long succession of sterile segments of the archicarp, and it is very probable that it does not reach the ascogonial cells. The spermatia are entirely absent in a num- ber of species where archicarps are present, as in Laboul- benia chaetophora (Thaxter, ’96; Faull, ’12). Sterilization of the terminal portion of the archicarp and differentiation of sex nuclei in the ascogonium or ascogenous [VoL, 2 352 ANNALS OF THE MISSOURI BOTANICAL GARDEN hyphae.—A moderately large number of species, in which more or less extensive sterilization of the terminal portion of the archicarp has occurred, have been examined by cytological methods and in most cases a reduced or modified sexual con- dition has been found. In Pyronema confluens great variations occur in the sexual nature of the ascogonium. In what may be called normal cases, antheridial nuclei enter and become associated with the ascogonial nuclei (Harper, ’00; Claussen, ’07, 712). Under cultural conditions the antheridium may be normal, rudi- mentary or absent, but the ascogonium develops in a normal manner (van Tieghem, ’84). Different strains may also þe- have differently. In some the antheridium does not fuse with the trichogyne, while in others it does (Brown, W. H., ’09). In some cases even when the antheridium fuses with the trichogyne, its nuclei do not pass into the ascogonium (Dan- geard, ’07), but degenerate in situ (Brown, W. H., ’09). In these cases where the antheridium does not function the sex- uality of the ascogonium is modified in as much as its nuclei are differentiated sooner or later so that in pairs they per- form the function of sperm and egg nuclei. According to W. H. Brown (’09) in cases where the origin of the pair of nuclei in the ascus hook could be determined, they were sisters. After the one conjugate division in the hook the two nuclei in the ascus, or penult cell, are ‘‘cousin’’ nuclei. The archicarp of Lachnea scutellata (Woronin, ’66; Brown, W. H., ’11) consists of about nine cells. No antheridial struc- ture has been observed. The penultimate cell functions as the ascogonium (Brown, W. H., ’11). It is multinucleate and no fusion of nuclei in pairs takes place here. The nuclei are increased in numbers by division, not only in the ascogenous threads where they do not appear to be paired or show con- jugate division, but also in the ascus hook where conjugate division takes place. The numerous fusions of the terminal and basal cells of the ascus hook result in numerous succes- sive conjugate divisions. In Leotia, although the archicarp has not been clearly observed, it would appear from the ac- count (Brown, W. H., ’10) that the antheridium is absent (or 1915] ATKINSON—PHYLOGENY IN THE ASCOMYCETES 353 if present, funetionless) and that the ascogonium consists of a single coenocytic cell. Conjugate division takes place in the ascus hook, and the subsequently fusing cells, so that in most cases rather distantly related pairs of nuclei form the fusion nucleus in the ascus. In L. chlorocephala (Brown, W. H., ’10), it appears that the pair of ascus nuclei are some- times sisters. This would indicate an extreme case in the modification of sexuality, the distance of relationship between the sex nuclei being reduced to the minimum. It recalls the very close relationship of the sex nuclei in many of the lower algae, particularly in certain diatoms! (Oltmanns, ’04), and in the species of Spirogyra having buckle-joint conjugation (Chodat, 710). In the case of Spirogyra it is not known whether the pair of sex nuclei in this type of conjugation are cousins or sisters, or whether now one and then another of these possibilities exists. Such species of Spirogyra in which certain threads present scalariform as well as buckle- joint conjugation offer an interesting parallel to the variation in distant relationship of the fusing nuclei in the young ascus. In some other species where the antheridium is function- less or wanting, sex differentiation is said to take place among the nuclei in the ascogonium. This indicates a sex differentia- tion much earlier than that which is supposed to occur in the species just cited. This differentiation in sex nuclei has been described in Humaria granulata (Blackman and Fraser, ’06). Another species in which similar phenomena are described is Lachnea stercorea (Fraser, ’07). Here the archicarp con- sists of several coenocytic large cells and the terminal tricho- gyne of 4-6 smaller eoenocytie cells. The unicellular coenocytic antheridium fuses with the terminal cell of the trichogyne, but its nuclei do not reach the single-celled ascogonium, among whose nuclei sex differentiation is said to take place. For a number of years Polystigma rubrum, a parasite on cherry leaves, as the result of studies by Fisch (’82) was re- garded as an example of fertilization of an ascogone coil by 1In Achnanthes subsessilis, the protoplast divides into two parts along with nuclear division. The two uninucleate protoplasts now immediately unite in auxospore formation, [Vou 2 354 ANNALS OF THE MISSOURI BOTANICAL GARDEN sperm nuclei from spermatia after passing through a long succession of cells constituting the trichogyne or sterile por- tion of the archicarp. The trichogyne, or sterile portion of the archicarp, is very long and branches into two portions, one extending to either surface of the leaf. But according to Nienburg (’14) sex differentiation has occurred between the basal cells of the archicarp and a nucleus from the basal cell migrates into the adjacent cell, which becomes the ascogonium or ascogenic cell, but nuclear fusion does not take place here. Loss of function by the archicarp or its disappearance.— A number of examples are known in which the archicarp has either lost its function as a sexual organ or ascogone, or has disappeared. In such cases differentiation of sex occurs in special vegetative cells, sometimes by the migration of a nu- cleus from certain cells into adjacent ones. In Gnomonia erythrostoma, although Frank (’86) described coiled ascogone- like structures with trichogynes, and believed that the coils were fertilized through the agency of the spermatia, recent cytological work (Brooks, ’10) on this species appears to show that the tufts of hair-like structures emerging through the stomates of cherry leaves, on which this species of Gno- monia is parasitic, are not now connected with the coiled hyphae deeper in the tissue. It appears also from the same work that the ascogenous hyphae do not arise from the coils, but from one or more slightly differentiated hyphae in the center of each coil. A similar example is found in Xylaria polymorpha (Fisch, 82), where an extensively coiled hypha (‘‘ Woronin’s hypha’’) occurs in the early stages of the formation of the ascocarp, but later disappears and certain vegetative cells give rise to the ascogenous hyphae. In Humaria rutilans (Fraser, ’08) no archicarp or ascogone coil is discernible, but certain vegetative cells function as as- cogenic cells following the migration into them of nuclei from adjacent cells. MORPHOLOGY OF THE ARCHICARP If the history of the Ascomycetes is correctly read from the simpler and more generalized forms to the complex and 1915] | ATKINSON— PHYLOGENY IN THE ASCOMYCETES 355 highly specialized ones as Sachs (’74, ’96), de Bary (’81, 84), and many other students have advocated, the female organ or archicarp first appeared as a ‘‘unicellular’’ or con- tinuous organ, not differentiated into an oögonium or fertile portion, and a trichogyne. The presence of a ‘‘procarp,”’ whether consisting of one or several cells, which ultimately gave rise to the asci or ascogenous threads was the predomi- nant character which led Sachs in 1896 to believe in the phy- letic relation of the sac fungi and red algae, although earlier he had regarded the morphology of the ascocarp and cysto- carp of greater importance in showing relationship. No known red alga possesses a procarp simple enough to repre- sent the prototype of the two groups. Gymnoascus was se- lected by Sachs as representing the simplest Ascomycetes. The archicarp of Gymnoascus is a continuous structure more or less coiled around the antheridium from which it copulates directly without the intervention of a trichogyne. After copulation the ascogonium divides into several cells which give rise to the ascogenous hyphae. In some forms the splitting up of the ascogonium by transverse division occurs at an earlier period, before copulation. There is some evi- dence which indicates that the ‘‘trichogyne’’ in the Ascomy- cetes primarily was a prolongation of the ‘‘unicellular’’ oögone (or carpogone), and that when it was first separated as a distinct cell it was still a fertile part of the archicarp. In Aspergillus repens the terminal cell, or ‘‘trichogyne,’’ some- times gives rise to ascogenous hyphae (Fraser, ’08). The terminal cell became merely a trichogyne when it ceased to give rise to ascogenous hyphae, and acted as a transport tube for the sperm nuclei from the antheridium to the as- cogonium, as in Pyronema and Monascus. The septum be- tween the terminal cell and the functional ascogonium was an impediment to the passage of the sperm nuclei, as well as the fact that when they entered the terminal cell of the archi- carp they did not meet with functional egg nuclei. This situa- tion very likely favored the assumption of sperm and egg functions by the nuclei of the functional ascogonial cell. The variations in Pyronema where the antheridium may or may [VoL. 2 356 ANNALS OF THE MISSOURI BOTANICAL GARDEN not be present, and often when present and fused with the trichogyne its nuclei degenerate and the ascogonium is still functional producing ascogenous hyphae and asci, is in sup- port of this interpretation. Further sterilization of the terminal portion of the archi- carp proceeds as it becomes longer and more septate, the fer- tile ascogonial cell or cells being near the center or base. All of the sterile portion of the archicarp distal to the ascogonial cells is usually interpreted as the trichogyne. I believe it would be more in harmony with the historical origin of the archicarp, and with the real homologies, if only the terminal sterile receptive cell of the archicarp were called the trich- ogyne, the other portions to be regarded as sterile portions of the archicarp or ascogonium. This would be in harmony also with Thaxter’s (’96) interpretation of the archicarp of the Laboulbeniales.‘ In this group the inferior and superior sup- porting cells are sterile cells of the archicarp derived by a transverse splitting of the ascogonium. Even with this inter- pretation of the trichogyne of the Ascomycetes, it would be a different structure from that of all the red algae where it is merely a continuous prolongation of the egg cell. NOTE VI The coenocytic character of the mycelium of the Phycomy- cetes has been presented as an obstacle to the derivation of the sac fungi from the sporangium fungi (Bessey, E. A., 713) ; this character can, however, have very little or no significance, for many of the Ascomycetes are coenocytic. As in most of the fungi, cell wall formation is delayed so that new portions of filaments are often multinucleate, the cell walls being laid down subsequently, sometimes enclosing one nucleus, some- times several in a cell. There are the monoenergid and poly- energid species of sac fungi. In the Phycomycetes cell wall formation is usually longer delayed or does not occur except where reproductive cells are formed. In the Mucorales old mycelium frequently becomes multiseptate. It should be noted that in Basidiobolus (Eidam, ’86; Raciborski, ’96; Fair- 1 Except in the case of the multiseptate branched trichogynes. 1915] ATKINSON—PHYLOGENY IN THE ASCOMYCETES 357 child, ’97, and others) the cells are uninucleate. The varia- tion in coenocytic character of mycelium probably is due in some measure to the usually fundamental difference between cross wall formation in dividing cells, in the thallophytes and the higher groups of plants, where the fibers of the inner spindle play a part and the cell wall development is centrif- ugal, while in most thallophytes the spindle fibers do not play such a part, wall formation being centripetal, like a clos- -ing iris diaphragm. The strong plasma connections between the protoplasts of the Laboulbeniales (Thaxter, 96) present a very striking re- semblance to those in the red algae. This feature is regarded by some as very strong evidence of a phylogenetic relation between the Laboulbeniales and the red algae. But intercellu- lar plasma connections are a common feature in all groups of plants, though in many plants these connections are very minute. The single central pore in the wall of the Laboul- beniales is perhaps the result of incomplete closing of the ring- forming wall, and in the Laboulbeniales would seem to be of physiological rather than of phylogenetic significance. The firm cell walls which are characteristic of the members of this group bear a very definite relation to their habit as external parasites of insects. Standing out free from their bodies and thus having no other means of support than their own rigidity, thick cross walls would interfere with transport of food ma- terial, while the prominent plasma connections permit easy passage of nutrients. NOTE VII BRIEF OUTLINE OF SOME OF THE THEORIES AS TO THE PHYLOGENY OF THE ASCOMYCETES I. Descent from the Rhodophyceae—Sachs (’74, p. 287) regarded the resemblances between cystocarp and ascocarp as the most important character indicating a relationship be- tween the red algae and sac fungi, although the form of the sexual organs, particularly the carpogonial branch, was also believed to point in the same direction. In his ‘Lehrbuch der Botanik’ he did not even suggest that the Ascomycetes were derived from the Florideae. The relationships were based [VoL. 2 358 ANNALS OF THE MISSOURI BOTANICAL GARDEN on the principle of morphological homology, which he believed were great enough to justify their inclusion in the same class. To justify his arrangement in one large group of plants with such diverse aspects and habitats, he cites the inclusion of the Lemnaceae and palms in the great group of the monocots. We could not then interpret his inclusion of the sac fungi and red algae in one class, the Carposporeae, as indicating that the former were derived from the latter. Sachs says (’74, p. 288) that in order to find the relation- ships between plant divisions one must compare the simplest, not the highest forms. By this method he finds that the Coleochaetaceae and Characeae are linked, on one hand to the simplest Florideae, and on the other to the simplest As- comycetes. Each of these series, he says, has developed in its own peculiar manner to higher forms, so that if one com- pared the most complete Ascomycetes with the coleochaetes only very slight resemblances are to be found. From this it is very clear that Sachs, at that time, had no thought of the derivation of the Ascomycetes from the Florideae. There is nothing to indicate that he believed the Ascomycetes descended from the charas and simplest coleochaetes, to which he says the simplest Ascomycetes are most closely related. Nor would his theory require a common ancestor for the two groups. Because of the morphological resemblance between cystocarp and ascocarp, he would have united the Ascomy- cetes and Florideae into a higher group even had he believed that the former were derived from the Phycomycetes. It has been said by Sachs (’96, p. 204) that the fungi as a whole cannot be valued as an architype because, as apochlo- rates, they must be descended from green plants. The bacteria he would derive from the Cyanophyceae, the Phycomycetes from the Siphoneae, and the Ascomycetes (or at least the Discomycetes) from the Rhodophyceae. The predominant feature indicating the descent of the sac fungi from the red algae he now sees in the procarp of both groups (’96, p. 205). The chlorophylless seed plants have only a slight form-pro- ducing power or motive, as Sachs has pointed out (796, p. 205), since they occur mostly as small plant groups within certain 1915] ATKINSON—-PHYLOGENY IN THE ASCOMYCETES -~ 359 green leaved families and show very plainly the morpholog- ical characters of their antecedents. But he says it is quite otherwise with the fungi. The simplest primitive forms of the Ascomycetes, Phycomycetes and Basidiomycetes have given rise independently to an enormously high state of dif- ferentiation. Now Sachs in 1896 (and earlier, ’74, p. 310) recognized Gymnoascus as belonging to the simplest Ascomy- cetes, the sexual organs of which are a simple carpogone and pollinode. It is very clear then that Sachs would not derive the Ascomycetes from any primitive form at all like any known red algae, much less through such forms as the highly specialized Collema or Polystigma. This warrants us in con- cluding that Sachs had in mind a primitive hypothetical an- cestor of the sac fungi and red algae, which possessed simple copulating gametes. With the knowledge we possess to-day of such forms as Dipodascus, Eremascus, etc., where the zygote becomes the ascus (generalized or simple) I believe he would have recognized in the Phycomycetes, as we know them to-day, a situation very closely approximating an ‘‘Urform”’ for the Ascomycetes, particularly in view of the fundamental difference in the cytology of the red algae and sac fungi. But whether the fungi represent one or several architypes it by no means follows that, because of the absence of chloro- phyll, they must be derived from green plants, or that each great series must be derived separately from different groups of algae. The appearance of the higher fungi (Eumycetes) was, in the opinion of Vuillemin (’12, p. 223), contemporaneous with the emergence of sea-shore, which abandoned certain red algae to a terrestrial life. This new environment introduced the change, which, accompanied by loss of chlorophyll, gave rise first to the Pyrenomycetes, from which the other higher fungi (Uredinales, Basidiomycetes) have originated. The sapro- phytic forms represent the productive and progressive stock. Parasitic groups, like the Uredinales, Laboulbeniales, lichens, etc., are composed of highly specialized and uniform members, thelr progressive potentialities being suppressed, but they re- tain their hold on existence because of their specialized hab- [VoL. 2 360 ANNALS OF THE MISSOURI BOTANICAL GARDEN itat. The first Pyrenomycetes, according to his view, were some of these depatriated red algae, losing their pigments while preserving the structure, the sexual organs and the gen- eral evolution. But he recognized no known member of the red algae as a prototype of the Pyrenomycetes. Primitive trichogyne-bearing algae gave rise to the red algae on one hand, and to the Pyrenomycetes on the other, the now known colorless red algae (like Harveyella mirabilis, Choreocolax alba) being recently reduced forms having no significance in the origin of the sac fungi. But the Pyrenomycetes with well developed trichogyne and spermatia are chosen as the primi- tive forms, the simplest represented by Polystigma (in his “Polystigmales’’) the higher ones (his ‘‘Pyreniales’’) giving rise successively to the Hysteriales and Phacidiales. From the Polystigmales three other lines arose, their simplest forms being represented by first, Gymnoascus; second, Pyronema; and the third line represented by the Laboulbeniales (see Vuillemin, ’12, pp. 338-341). Bessey (714) regards the Discolichenes as the most primi- tive Ascomycetes. This theory is based on the supposed phyletic relation of the multiseptate trichogyne of the lichens (Collema, for example) to the trichogyne (a mere tubular, continuous, prolongation of the egg) of the red algae. Cer- tain of the red algae became parasitic on blue-green algae and on simple members of the green algae, forming a lichen thallus. It is supposed that this parasitism may have had its origin while both kinds of organisms still lived in the water, but finally the lichen assumed the land habit. The improba- bility of such a derivation of the sac fungi as suggested in the above theories has been fully discussed in the preceding pages. II. Descent from the Phycomycetes—De Bary (’81, ’84, ’87), as already stated in the first part of this paper, be- lieved the Ascomycetes were derived from the Phycomycetes, particularly through such forms as the Peronosporales. The — criterion for the relationship is the close homology and mor- phological resemblance of the sexual organs, though he sug- gested that Eremascus might have been derived from the Mucorales through some such form as Piptocephalus where 1915] ATKINSON— PHYLOGENY IN THE ASCOMYCETES 361 the zygote is the outgrowth from the fusion point of two equal gametangia. Brefeld (’89, ’91) also derived the Ascomycetes from the Phycomycetes but interpreted the ascus as the phyletic hom- ologue of the sporangium, the ascus representing a special- ized structure derived from the generalized sporangium in one direction, while the conidia were regarded as reduced one- spored sporangia. But the nuclear fusion and reduction phenomena in the ascus are so fundamentally different from any known cytological processes in the sporangium, that its phyletie relation to the sporangium is doubtful.* The con- jugation of the gametangia he interpreted as ordinary fusion of hyphae which occurs in numerous instances devoid of all sexual significance. Protomyces, Ascoidea and Thelebolus, with numerous spores in the ascus, were interpreted as rep- resenting an intermediate condition between the generalized sporangium of the Mucorales and the specialized ascus. In Thelebolus it has been found that the development of the ascus follows the type with crozier formation and that it is closely related to Ascobolus and Rhyparobius (see Ramlow, ’06; Dangeard, ’07). As for Protomyces and Ascoidea they prob- ably represent forms with reduced sexuality while retaining the ancestral character of many divisions of nuclei to form numerous spores. Zukal (’89), influenced by Brefeld, derived the hymenial Ascomycetes (like Ascobolus, Pezizales, ete.) through Thele- bolus and Monascus; the stromatic Ascomycetes (whether Pyrenomycetes or Discomycetes) from the Uredinales; the Gymnoascales and others with asci arising directly from the mycelium, from another ancestral type. Lotsy (’07, p. 469) sees no difficulty in deriving the polyener- gid forms like Pyronema from the Phycomycetes. The forms with spermatia, which are usually monoenergid, it would seem rational, he thinks, to derive from the red algae, and this raises the question as to whether the Ascomycetes are of poly- phyletic (or biphyletic) origin. The great uniformity of the 1 The nuclear phenomena in the “germ” sporangium (from the zygote) are not known. [VoL. 2 362 ANNALS OF THE MISSOURI BOTANICAL GARDEN ascus in the entire group is a great obstacle in the way of accepting a polyphyletic origin for the group. All things con- sidered he is inclined to accept de Bary’s view of their phy- comycetous origin. The origin of the Ascomycetes from the Phycomycetes is recognized by Dangeard (’07) through such forms in which there is still a union of gametangia. Dipodascus and Ere- mascus represent such forms in his scheme. The generalized ascus resulting from the union of the gametangia of Dipo- dascus he terms a ‘‘sporogone.’’ From Eremascus, by re- duction, forms like Endomyces arose, while the Ascomycetes with ascogenous hyphae were derived from such forms as Dipodascus by delayed nuclear fusion and the proliferation of the gametangium into what he terms ‘‘gametophores’’ (= as- cogenous hyphae). The gametes then are formed in the nuclear pair which fuses in the ascus. This terminology arises from his persistent belief that the ascus is the egg. Shorn of the change in terminology and his, perhaps, unfor- tunate insistence on homologizing the ascus with the egg, his interpretation of the relation which such a form as Dipodascus bears to the Ascomycetes, has much merit. Nienburg (714) suggests the origin of the Ascomycetes from the Phycomycetes through some such form as Monoblepharis. He would find the evidence for this in the homology of the archicarp of Polystigma rubrum with such forms of Monoble- pharis in which the stalk cell of the oögonium is an anther- idium, and where the odgonium is terminated by one or more sterile cells. The archicarp of Polystigma he interprets as having two fertile cells at the base and prolonged into a long sterile septate portion (so-called trichogyne) which forks, sending a branch to either surface of the leaf. The basal multinucleate cell is the antheridium. After pore formation one nucleus migrates into the unicellular egg. Interesting as this suggestion is, forms of Pythiwm (see de Bary, ’81, ’84; Atkinson, ’95) with interealary oögonia and stalk antheridia present a closer analogy to the archicarp of Polystigma as described by Nienburg, but it is extremely doubtful if the point of contact is to be sought through such structures. 1915] ATKINSON—-PHYLOGENY IN THE ASCOMYCETES 363 Brief comparative summary of the above views on the phylogeny of the Ascomycetes—The adherents to the doc- trine of the red algal origin of the Ascomycetes interpret the point of contact in three different ways: first, sac fungi with highly developed ‘‘trichogyne’’ (sterilized archicarp) of the Collema type with red algae like certain of the existing forms, Nemalion, or some of the higher forms in the vicinity of Har- veyella, ete.; second, sac fungi with highly developed ‘“trichogyne’’ (= sterilized archicarp) of the Polystigma type with hypothetical trichogyne algae representing the com- mon stock for the origin of both groups; third, sac fungi with simple generalized copulating gametes of the Gymnoascus type with hypothetical algae having a simple procarp repre- senting the stock from which both groups originated. According to the two first interpretations the sac fungi have been derived through highly developed and specialized forms from either quite highly developed and specialized red algae, or both groups from a common trichogyne algal stock, and then by degeneration have slid backward from complex and specialized structures to simple, generalized and primi- tive ones. The third view which recognizes a simple procarp, without regard to a trichogyne, as the important character of the hypothetical stock, is far more comprehensible. But if we must go back to some hypothetical ancestor, which cannot be represented by any known red alga, for the source of the sac fungi it is far more reasonable to search for one in another fungus line, where, in the light of present-day knowledge, there are known forms with sexual organs very much like the sexual organs of simple, known forms of the Ascomycetes. But we are not yet in a position to name any known phycomycete! as a probable ancestor of the Ascomy- cetes, though it appears very likely that the ancestral stock possessed phycomycetous characters. 1 Lotsy (’07) suggests Cystopus; Miss Dale (’03) in her study of Gymnoascus suggests Basidiobolus; Nienburg (’14), Monoblepharis; while Dangeard (’07) suggests Myzocytium vermicolum as the prototype of the higher fungi. [VoL, 2 364 ANNALS OF THE MISSOURI BOTANICAL GARDEN PROVISIONAL ARRANGEMENT OF MAIN LINES OF DEVELOPMENT IN ASCOMYCETES For those who are interested in the suggestions as to the phylogeny and relationships of the Ascomycetes presented in this paper, a diagrammatic arrangement of the principal series or lines which will illustrate the relationships tenta- tively held by the writer may be acceptable. It is with con- siderable hesitation that this arrangement is presented. The writer trusts that it will be accepted as provisional and in the nature of a working hypothesis which he hopes will fur- ther stimulate investigation, suggestions and criticisms on the ideas embodied in this paper, all of which, for or against, will be gladly welcomed. Dipodascus, a primitive form, cells of mycelium polyen- ergid, gametogenous branches large, unequal, polyenergid. Ascus is elongated, broadened zygospore, zygote germinating immediately forming a broad germ tube in which spores are formed. Since the process does not go on to the formation of a sporangium, a different mode of internal free cell-forma- tion then arose in connection with the precocious formation of spores in the zygote and retention of epiplasm which assists in discharge of spores. Dipodascus retains tendency of gamogenic branches to copulate early before they become strongly differentiated as gametangia, just as in Mucorales. I. Proroascomycerss are derived by descent and degenera- tion from some such primitive ascomycete form as Dipodascus. The ascus when of sexual origin is the zygote, except in Nad- sonia. Endomyces Magnusii is the nearest known form to the gen- eralized condition seen in Dipodascus. Cells of mycelium usually polyenergid, those of stout mycelium are polyenergid. Formation of ascus in Endomyces Magnusii repeats formation of zygospore in Zygorhynchus. Gamete branches in both are multinucleate, but when cell wall is laid down delimiting the gametangia all but one nucleus in each gametangium of E. Magnusu are excluded. After contact of the two sexual branches the male gametangium is formed by enlargement of its tip, into which protoplasm and the one nucleus migrates, 1915] ATKINSON— PHYLOGENY IN THE ASCOMYCETES 365 exactly as male gamete of Zygorhynchus is formed, except the latter is multinucleate. By disappearance of the separating wall, ascus is formed of the two gametes. Endomyces series, then, derived from Dipodascus-like an- cestors, with Endomyces Magnusii the lowest and most gen- eralized. Developmental tendencies from here in four, five, or six different directions: i bo S? j © Eremascus, both gamogenic branches uninucleate, ascus more definite and specialized in s ape. Loss of conidial formation. Endomyces fibuliger indicates step toward Eremascus (E. fer- tilis) in poni size of gametes . Endomyces diverging into die two series, one chiefly with sprout oe the other chiefly with oidia; the latter preserves the nusii character, the former takes on sprout conidia in addi- kun to oidia (E. fibulig er and E. capsularıs form both oidia and sprout conidia) ; oidia Formation the more primitive and gener- alized condition in Ascomycetes. Saccharomycetes. Still more specialized and reduced than in Endomyces fibuliger and in this same line. Schizosaccharomyces may have come from same line with dropping of sprout conidia, or may be descended from form near Endomyces Magnusii. Exoascaceae. From Endomyces-like ancestors. Nuclear phe- nomena not well known. Diploid young ascus may have arisen in connection with cell wall formation, two nuclei being retained in ascogone instead of one as in E. Magnusii, where all but one a excluded at time of wall formation, i. e., ascus fundament may have retained the polyenergid character of the most primi- tive forms like E. Magnusi. Tende ency to form hymenia may be controlled by host — asci in all except Taphrina laurencia, come to surface to matur . Ascocorticium, iis 25 on wood where food is not so rich, tendency to drop conidial formation (?), association of asci in hymenium, highest development of the Endomyces series, or of the Protoascomycetes. Series is terminated early, tendency i in Endomyces line to specialization of zygote into one ascus with reduced number of spores, and line soon terminated. . Ascoidea, Protomyces, Taphridium, etc., probably represent forms derived by reduction and loss o istinct sexual organs but preserving primitive feature of many divisions of nucleus in the generalized ascus. IJ. Evascomycetsts. Lowest forms with generalized archi- earp. Similar to Monascus, Gymnoascus, ete. (VoL. 2 366 ANNALS OF THE MISSOURI BOTANICAL GARDEN 1. Tendency to late copulation of gamogenie branches, so that archicarp becomes large and many-nucleate, or tendency to elongate, or both. 2. As it elongates tendency to septation, first a single terminal cell (“trichogyne”’), and later longer and multiseptate “trichogyne,” or rather sterilization of terminal portion of archicarp. One o the early tendencies in een ge ne... of the archi- carp may have been the origin of a receptive terminal portion under ae or aie stimulation; such a condition sug- gested in Cystopus. 3. This made the passage of antheridial nuclei increasingly difli- cult, and resulted in early tendency to sterilization of anther- idium or failure to function because of functionless condition of “trichogyne.” Led in many cases to modified sexuality by dif- ferentiation of sex among nuclei in ascogonium, ee Sa cells, or ascogenous threads. > . Progressive tendency to multiplication of spores by postpone- ment of nuclear fusion and spore formation; conjugate division of sex nuclei, and multiplication of the specialized structures (asci) in which spores are formed, so that spore formation wo distribution is extended over greater period of time. This m advantageously attained by sprouting of zygote fen. branching of threads, and terminal formation of specialized asci. Diverging lines from Gymnoascus and Monascus-like an- cestors or related prototypes in which asci are irregularly arranged but associated in groups with imperfect envelope. 1. A line with interwoven asci, Plectascales as a highly specialized lateral group, with Gymnoascaceae at base. Aspergillaceae a progressive line, with Perisporiales an offshoot, or Perisporiales direct from Monascus-like ancestors. > Elaphomycetaceae, asci interwoven in groups but separated by sterile walls w . Pezizales, asci remaining in groups not interwoven in mycelium, but spaced by sterile threads (paraphyses). Pyronema repre- sents one of the generalized, lower forms. The Helvellales, ete., are probably derived from the Pezizales. AN . The Microthyrialest have usually been placed among the Peri- sporiales with which they have little in common. I believe they 1 Recent studies by several authors, particularly by von Höhnel (’10) and by Theissen (’12, ’13, ’14) have greatly increased our knowledge of these interest- ing fungi, partly by the discovery of new forms but especially by uncovering many forms from the clouded situation in which they have been placed for lack of an adequate study of their structure. — 1915] ATKINSON— PHYLOGENY IN THE ASCOMYCETES 367 represent reduced forms derived on the one hand from the Pha- cidiales and perhaps on the other from the Sphaeriales and pos- sibly some from the Perisporiales. The formation of the char- acteristic shield has rendered superfluous the perithecial wall as a protective structure. The genus Diplocarpon, the sie and development of which was investigated by one of my for students (see Wolf, 712), I believe is an excellent ion of a form on the way (by reduction of the perithecial wall in con- junction with the formation of the shield) from the Phacidiales to the condition presented by many members of the Micro- thyriales. The above provisionally suggested relationships may be represented by the following five or six series, or lines of development, with the accompanying diagram (fig. 10): 1. Apocarp line from Dipodascus-like forms and by reduction. 2. Plectocarp line from Dzipodascus-like forms, perhaps similar to Monascus. 3. Perispore line arising from Monascus-like prototype, before splitting of archicarp, or from Aspergillaceae. 4. Pyrenocarp line arising near Monascus-like prototype. Laboul- beniales side line near base, and some of the Mycrothyriales as reduced from Sphaeriales. 5. Discocarp line from Dipodascus-like forms near Monascus, but lower (it is not improbable that some of the cetes showed considerable variation in the strength of the ascocarp envelope, also in its presence or absence in forms where it is more or less rudimentary!) ; and some of the Microthy- riales as reduced forms from Phacidiales. Or a 6th line also, Laboulbeniales from Monascus-like ancestor. ı This variation sometimes oceurs in existing forms. Zukal (’89) describes an abnormal case in Eurotium (Aspergillus) herbariorum where the anthe ridial branch and envelope are wanting, the mass of asci being exposed. nection it is worthy of note that Fraser and Chambers (’07) regard Aspergillus “as represen e a primitive ee type from which most othe n be derived.” This suggestion was based on the assumption that the red were the ancestors of the sac ec wn the basis of the counter theory (phyco- mycetous origin) Gymnoascus and Monascus-like forms are more comprehensible as primitive Huascomycetes. ANNALS OF THE MISSOURI BOTANICAL GARDEN [VoL. 2 w” _—_ u or v © S } 5 È Fi E- 5 d £ g % & / 5 /é es JS è /$ S 5% ar a a cog % gh F., į FR © SD % f a/ SS t+ Hyster aes Y ? I RYSA ai / \ I yn S % yj A Tuberales A x > ww. : ee EUASCOMYCETES i Y Ve a woe Saccharomycetes | Te E protonas PROTOASCOMYCETES ASCOMYCETES PHYCOMYCETES | Fig. 10. Chart showing suggested phylogeny of the Ascomycetes LITERATURE CITED came > F. (’95). Damping off. Cornell Univ. Agr. Exp. Sta., Bull. 94 231-2 pl. 1-6. 1895 Bachmann, Miss F. M. (’12). A new ace of oe and fertilization in Collema. Ann. Bot. 26: 747-760, pl. 69 The origin and development of the apothecium in Collema posum (Beri) Ach. Archiv f. Zellforsch, 10: 369-430. pl. 30-36. 1913 1915] ATKINSON—PHYLOGENY IN THE ASCOMYCETES 369 Barker, B. T. P. (03). The morphology and development of the ascocarp in Monascus. Ann, Bot. 17: 167-236. pl. 12-13. 1903. ——, (04). Further observations on the re ” Age : caters British Assoe. Adv. Sci., Cambridge, Rept. 1904: 825-826. de Bary, A. (’81). Untersuchungen iiber die Peronosporeen und Saprolegnieen und die nn eines natürlichen Systems der Pilze. In de Bary und Work Beitr. z. Morph. u. Physiol. d. Pilze 4: 1-145. pl. 1-6. 1881. ————, (’84). Vergleichende Morphologie und Biologie der Pilze, usw. Leipzig, 1884. ‚ (87). Comparative Morphology and Biology of the Fungi, Mycetozoa, and Bacteria. 87. Baur, E. (’98). 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Soc. London, Bot., Proc. 77: 354-368. pl. 13-15. 1906. — —— (06). Further studies on the sexuality of the Uredineae. Ibid. 20: 35-48. pl. 3-4. 1906. ————,, and Welsford, E, pos (712). The development of the en of Poly- Be rubrum DC, . Bot. 26: 761-767. pl. 70-71. 191 Bar Th. (’88). Zellen Studien II. an cabs und Zellteilung des Eies n Ascaris megalocephala. Jena, Brefeld, 2 (788). Basidiomyceten II. Protobasidiomyceten. ee aus dem Gesammtgebiete der BIS e 7:1-X and 1-178. pl. 1-11. 1888 — (’89). Basidiomyceten IH. ren page und die Begründung des natürlichen Systemes der Pilze. Ibid. 8: 1-274. pl. 1-11. 1889. ——, (91). Die Hemiasci und die Ascomyceten. Ibid. 9: 1-156. pl. 1-3B. 1891 , (’91). Ascomyceten II. Ibid. 10: 157-378. pl. 4-13. 1891. Brooks, F. T. (’10). The development of Gnomonia erythrostoma Pers, Ann. Bot. 24: 585-605. pl. 48-49. 1910. ; [VoL, 2 370 ANNALS OF THE MISSOURI BOTANICAL GARDEN Brown, H. B. (13). Studies in the development of Xylaria. Ann. 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Recherches sur le ii du périthèce chez les Ascomy- cetes. Ibid. 10: 1-385. pl. 1-91. 1907. Darbishire, O. V. (00). Über die STEN der “heer eo pulverulenta (Schreb.) Nyl. Jahrb. f. wiss. Bot. 34: 329-345. pl. 11. 1900. Davis, B. M. al The fertilization of Batrachospermum. Ann. 10: 49-76. pl. 6-7, ——,, (’03). ER in Saprolegnia. Bot. Gaz. 35:233-249, 320-349. pl. 9- 10. 1903 1915] ATKINSON— PHYLOGENY IN THE ASCOMYCETES ar Dodge, B. O. (’12). Artificial cultures of Ascobolus and Aleuria. Mycologia 4: 218-222. pl. 72-73. 1912. ——, (’12a). Methods of culture and the morphology of the archicarp in certain “ee x =: Ascobolaceae. Bull. Torr. Bot. Club 39; 139-197. pl. 10-15. f. 1 —, (’l4). The morphological ers of the Florideae and the Ascomycetes. Ibid. 41: 157-202, f. 1-13. Eidam, E. (’80). Beitrag zur Kenntniss der Gymnoasceen. Beitr, z. Biol, d. Pfl. 3: 267-305. pl. 12-15. 1880. — (33). en. der Entwicklung bei den Ascomyceten. Ibid. 3: 376-433. pl. a 1 ———, (’86). 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Further contributions to the cytology of the a iavestes. Ann. Bot. 22; 465-477. pl. 26-27. 1908. [VoL. 2 372 ANNALS OF THE MISSOURI BOTANICAL GARDEN Gates, R. R. (’09). The stature and a of Oenothera gigas De Vries. Archiv. f. Zellforsch. 3: 525-552. 909. ——, (’13). Tetraploid mutants and chromosome mechanisms. Biol. Cen- tralbl. 33: 92-150. f. 1-7. 1913. ares ge A. (08). La question de la sexualité chez les Ascomycetes. Rev. Gen ae 32-39, 85-89, 111-120, 178-182, 298-305, 333-334, 364-377. f. 1:86. 1908 ———, (09). Recherches eytologiques et taxonomiques sur les Endomycetées. Ibid. 21: 354-391, 401-419. pl. 13-19. 1909. ———, (712). Les levures. 1-565. f. 1-163. Paris, 1912. a R. A. (795). u zur Kenntniss der a oe er ir m Ascus, Ber. d, deut. bot. Ges. 13:(67)-(68). pl. 27. ‚(’95a). Die Entwickelung des Peritheciums bei Sphaerotheca Castagnei. Ibid. 13: 475-481. pl. 89. 1895. — (9 Ueber das Verhalten der Kerne bei der here a a einiger Ascomyceten, Jahrb. f. wiss. Bot. 29: 655-685. pl. 11-12. 189 ———, (99). Cell-division in sporangia and asci. Ann. Bot. 13; 467-525. pl. 24-26, 1899. ———,, (00). Sexual ee in Pyronema are and the morphology of the ascocarp. Ann. Bot. 14: 321-400. pl. 19-21. 1900. —, ( Binucleate cells in certain Hymenomycetes, Bot. Gaz, 33; 1-35. pl. 1. 1902. ——, (’05). Sexual reproduction and the organization of the nucleus in certain mildews. Carnegie Inst. Washington, Publ. 37: 1-104. pl. 1-7. Hartog, M. M. (’95). On the cytology of the vegetative and reproductive organs of the ye Roy. Irish Acad., Trans. 30: 649-708. pl. 28-29. 5 Hoffmann, A. W, (’12). Zur Entwicklungsgeschichte von a sem- pervivi. Cenitralbl. f. Bakt. II. 32: 137-158. pl. 1-2. f. 1-14. von ti F. (710). Fragmente zur Mykologie. X. Mitteilung. K. Akad. Wiss. Wien., Math.- naturw. K1., Sitzungsber. 119: 393-473 (1-81). f. 1. 1910. Janczewski, E. (’71). Morphologische Untersuchungen über Ascobolus furfur- aceus. Bot. Zeit. 29: 257-262, 271-278. pl. 4. 1871. Juel, H. O. (02). Taphridium ee & Juel. Eine neue Gattung der Protomy- cetaceen. Bihang K. Sv. Vet.- Akad. Handl. 27'*; Afd. III. 1-29. pl. 1. 1902. » (702). ge Trg RE und Sporenbildung bei Dipodascus. Flora 91: 47-55. a G. (708). Die Te der Zygoten von Spirogyra jugalis Ktzg, a 99: 1-11. pl. 1. 1908 Kihlman, O. (83). Zur rer aaa der Ascomyceten. Soc. Sei. Fen- nicae, Acta 13: 1-43, pl. 1-2. — ar Af 13). Beiträge zur Ba der Hymenomyceten, I, II. Zeitschr. f. t. 5: 593-637. pl. 2-5. f. 1. 1913 Kurssanow, L. (’11). Ueber Befruchtung, Reifung und Keimung bei Zygnema. Flora 104: 65-84. pl. 1-4. 1911. Lagerheim, G. de (92). Dipodascus albidus, eine neue, geschlechtliche Hemiascee, ahrb. f. wiss. Bot. 24; 549-565. pl. 24-26. 1892. 1915] ATKINSON—PHYLOGENY IN THE ASCOMYCETES 373 Lindau, G. (’88). Ueber die art und Entwicklung einiger Flechtenapothecien. Flora 71; 451-489. pl. 10. (799). Beiträge zur Kenntniss der Gattung Gyrophora, Festschrift fiir Schwendener. Berlin, 1899. Lotsy, J. P. (07). Vorträge über botanische Stammesgeschichte 1; I-IV and 1-828. f. 1-430. 1907. Maire, R. (’99). Sur les phénomènes cytologiques précédant et accompagnant la Sramana de la ee chez le Puccinia Liliacearum Duby. Compt. end. acad. Paris 129: 839-8 1899. ——, (’01). L’evolution nucléaire chez les Urédinées et la sexualité. Bull. Soc, "Mye. 17: 88-96. 1901. ————,, (02). Recherches cytologiques & taxonomiques sur les Basidiomycétes. Ibid. 18: 1-209. pl. 1-8. 1902. — —., (03). Recherches cylotogiques sur le Galactinia succosa. Compt. rend. acad. Paris 137: 769-771. 1903. ——, (705). zen. Asa aaa sur quelques Ascomycetes. Ann. Myc. 3: 123-154. pl. 3-5. 1905 aur El. et Em. (’09). Aposporie et sexualité chez les Mousses, II. Bull. d. Belg. (classes des Sciences) 1909: 1249-1288. 1909. A Ibid, III. Ibid. 1911: 750-778. f. 1-19. 1911. FERN W. A. (’10). Development of the Helvellineae. I. Helvella elastica. Bot, Gaz or 195-206. pl. 14-16. 1910. Mücke, M. (’08), Zur Kenntnis der Eientwicklung und Befruchtung von Achlya polyandra de Bary. Ber. d. deut. bot. Ges. 262: 367-378. pl. 6. 1908. Murrill, W. A. (’00). The development of the archegonium and Poe y the hemlock spruce (Tsuga canadensis Carr.). Ann. Bot. 14: 583-607. pl. 22. 1900. Nichols, M. A. (’96). The morphology and development of certain pyrenomycetous Bot. Gaz, 22: 301-328. pl. 14-16. 1896. Nichols, S. P. (’04). The nature and origin of we binucleated cells in some Basidiomycetes. Wis. Acad. Sci., Trans. 15: 30-70. pl. 4-6, 1904, ir W. (07). Pe zur Entwicklungsgeschichte einiger Flechtena- pothecien. Flora 98: 1-40. pl. 1-7. 1907. — (714). Zur Entwicklungsgeschichte von Polystigma rubrum DC. Zeitschr, f. Bot. 6: 369-400. f. 1-17. 1914. Olive, E. W. (705). The morphology of Monascus pupureus. Bot. Gaz. 39: 59-60. 1905. —-, (’07). Cell and nuclear division in Basidiobolus. Ann. Myc. 5:404- 418. pl. 10. 1907. ————, (08). Sexual cell fusions and vegetative nuclear divisions in the rusts. Ann. Bot. 22: 331-360. pl. 22. 1908. Oltmanns, F. (’98). Zur Entwicklungsgeschichte der Florideen. Bot, Zeit. 56: 99-140. pl. 4- 7. 1898. ——, (’04). Morphologie und Biologie der Algen 1: 1-733. f. 1-467. Jena, 1904. er W. J. V. (’00). Befruchtung bei Batrachospermum. Flora 87: 109- pl. 5. 1900. [VoL. 2 374 ANNALS OF THE MISSOURI BOTANICAL GARDEN Overton, J. B. (06). The morphology of the ascocarp and spore-formation in the many- spored asci of Thecotheus Pelletieri. Bot. Gaz. 42: 450-492, pl, 29-30. 1906. gi M. (’96). Studya oe hr ung Studien I. Karyo- ese bei Basidiobolus ranaru robus v. sp., Penicillium Poir- aultii nov. sp., Entyloma Nymphaea eae Ara d. d. Wiss., Krakau, Anz. 1896: 377-386. 1 pl. 19 f. 1896. Ramlow, G. (’06). Zur en a a i von Thelebolus stercoreus Tode. Bot, Zeit. 64: 85-99. ———, (14). Beiträge zur Entwicklungsgeschichte der Ascoboleen, Myc. Centralbl. 5: 177-198. pl. 1-2, f. 1-20. 1914. Ruhland, W. (’01). Zur Kenntnis der intracellularen Karyogamie bei den Basi- diomyceten, Bot. Zeit. 59: 187-206. pl. 7. 1901. Sachs, J. (’68). Lehrbuch der Botanik. 1-632. f. 1-465. Leipzig, 1868. , (74). Ibid. 1874. ———-, (96). Physiologische Notizen X, Phylogenetische Aphorismen = über innere Gestaltungsursachen oder Automorphen. Flora 82: 173-2 1896. Sappin-Trouffy, M. (’96). Recherches SR sur la famille des Urédinées. Botaniste 5:59-244. f. 1-70 N W. (’09). Ueber die r a NER von Monascus. Zeitschr. f. . 1: 379-410. pl. 2. f. 1-3 BEN W. (’99 Einiges erg die Befruchtung, Keimung und Haarinsertion n Batrachospermum. Bot. Zeit. 57: 125-135. pl. F. 1899 x Schmitz, F. (°79). Ueber die Fruchtbildung der Squamarieen. Niederrhein. Ges. f. Nat.- u. Heilkunde, Bonn, Sitzungsber. 36: 376-377. 1879. ————.,, (80). Ueber die Zellkerne der Thallophyten. Ibid. 37: 122-132. 1880, ————., (’83). Untersuchungen über die Befruchtung der Florideen. K. Preuss. Akad. Wiss., Berlin, Sitzungsber. 1883: 215-258. pl. 5. 1883. ————.,, und Hauptfleisch, P. (’97 Rhodophyceae. In Engler & Prantl, Nat. ae 1°: 298-544. # ir 288. Leipzig, 1897. Stahl, E. (’77). Beiträge zur Entwickelungsgeschichte der Flechten. 1-55. pl. 1-4. Leipzig, 1877. na F. L. (99). The ee oösphere of Albugo bliti. Bot. Gaz. 28: 149- , 225-245. pl. 11-15. 1899 —, (’01). es and fertilization in Albugo. Bot. Gaz. 32: 77-98, 157-169, 238-261. pl. 1-4. f. 1. 1901. aigi ae T. J. (12). Die Entstehung von Oenothera gigas deVries. Ber. d. deut. t. Ges. 30: 406-416. 1912. Stoppel, R. (°07). Eremascus fertilis nov. spec. Flora 97: 333-346. pl. 11-12. f. 1—6. 1907. Strasburger, E. (’00). Uber Reduktionsteilung, Ppindebidung, Centrosomen und Cilienbildner im Pflanzenreich. Histolog. Beitr. 6:1 1900. ———, (’04). Uber Reduktionstheilung. K. a Akad. Wiss. Berlin, phys.- math. K1., Sitzungsber. 18: 587-615. f. 1-9. 1915] ATKINSON— PHYLOGENY IN THE ASCOMYCETES 375 ———,, (05). Typische und ee a Ergebnisse und Erör- terungen. Jahrb, f. wiss. Bot. 42: 1-71. ——,, (09). Sexuelle und T ke. bei Urticaceen. Jahrb. f. wiss. Bot. 47: 245-288. pl, 7-10. ui! R. (796). Contribution toward a monograph of the Laboulbeniaceae. Am. Acad., Mem. 12: 189-429. pl. 1-26. 1896, ———., (08). Contribution ee a monograph of the Laboulbeniaceae. Il. Ibid. 13: 219-469. pl. 28-71, 1908 Theissen, F. (712). Die Gattung Clypeolela v. Höhn. Centralbl, f. Bakt. II. 34: 229-235. 1912, —, (712). en ee RM nebst ra über einige andere Asterina- Arten, Ann. Mye. 10: 2. f. 1-5. 1912 —, (’12). Fragmenta brasilica V nebst Besprechungen einiger palaeo- tropischer Microthyriaceen. Ann. Myc. 10: 159-204. 1912. , 13). Lembosia-Studien. Ann. Myc. 11: 425-467. pl. 20. 1913. ————, (’13). Hemisphaeriales. (Vorläugfige Mitteilung.) Ann. Mye. 11: 468-469, 1913. ————, (’13). Über einige Mikrothyriaceen. Ann. Mye. 11:493-511. pl. 21. f. 1-7. 1913. ———, (’13). Die Gattung Asterina in ee: ee K.K. zool.-bot. Ges., Wien, Abhandl. III. 7; 1-130. pl. 1-8. 1913 —, (113). ur Revision der Gattungen Mycrothyrium und Seynesia. Österr. bot. Zeitachr. 63: 121-131. 1913. _—, (’14). eres n. Frog a sae) Centralbl. f. Bakt. ER 39: 625-640. pl. 1. f. 1-7. i CI). Über Polystomella, Microcyclus, u. a. Ann. Mye. 12: 63-75. pl. 6-7. 1914. Treub, M. (’05). Agere: oe ee er Brogn. Ann. Jard. Bot. Buitenzorg II. 5: 141-152. pl. 4-11. 190 Tröndle, A. (707). Ueber die Kopulation und Keimung von Spirogyra. Bot. Zeit. 65':187-210. pl. 5. f. 1-13. 1907. Twiss, W. ©. (711). rn erua delesserioides J. Ag. Univ. Calif. Publ. Bot. 4: 159-176. pl. 21-24. 1911. van n Ph. (’84). Culture et re aa du Pyronema confluens. Soe. Bot. France, Bull. 31: 355-360. 1884 De ed H. (703). Die Mutations-Theorie 1; I- gerd m 1-752. pl. 1-2. f. 1-159. ; 2: I-XII and 1-648. pl. 1-8. f. 1-181. ————, (’13). Gruppenweise Artbildung unter spezieller Berücksichtigung der Gattung Oenothera I-VII and 1-365. pl. 1-22. f. 1-121. 1913. Vuillemin, P. (’12). Les champignons. Essai de classification, 1-425. Paris, 1912. EN E. J. (’°07). Fertilization in Ascobolus furfuraceus, New Phytol. 6: 61. pl. 4. 1907. Werth, E., and Ludwigs, K. (712). Zur ee bei Rost- und Brandpilzen. Ber .d. deut. bot. Ges. 30: 522-528. pl. 15. 1912 [VoL 2, 1915] 376 ANNALS OF THE MISSOURI BOTANICAL GARDEN Wolf, F. A. (712). The perfect stage of Actinonema Rosae. Bot. Gaz. 54: 218-234. pl. 13. 1912. Wolfe, J. J. (’04). Cytological studies on Nemalion. Ann. Bot. 18: 607-630. pl. 40-41. f. 51. 1904, Woronin, M. (’66). Zur Entwickelungsgeschichte des Ascobolus ag imus Cr. und einiger Pezizen. In deBary und Woronin, Beitr. z. Mor rph. u. Physiol. d. Pilze 2; 1-11. pl. 1-4. 1866. ———, (’70). Sphaeria Lemaneae, Sordaria coprophila, fimiseda, Arthrobotrys oligospora. Ibid. 3: 1-36, pl. 1-6, 1870. re Z. (’04). Einige neue S707 ek zur EEE von Basi- iobolus ranarum. Flora 93: 87-97. pl. 4. f. 1. 1904, Yamanouchi, S. (’06), The life history of Polysiphonia. Bot. Gaz. 42: 401-449. A 8. 1906. Zukal, H. (’89). a ee Untersuchungen aus dem Gebiete der scomyceten. . Akad. Wiss., Wien, Math.- naturw. Kl., Sitzungsber. 98: 520-603, pl. Er 1889. A CONSPECTUS OF BACTERIAL DISEASES OF PLANTS ERWIN F. SMITH U. 8. Department of Agriculture, Washington, D. C. All our knowledge of these diseases has come within a gen- eration. It began thirty-six years ago with the announcement _ of the bacterial origin of pear blight by Professor T. J. Bur- rill of the University of Illinois, who is with us to-day. During the first half of that period progress was slow and doubt uni- versal, especially in Europe. It is now eighteen years since I ventured the statement,! that ‘‘there are in all probability as many bacterial diseases of plants as of animals.’’ This statement was received with much skepticism, not to mention active opposition, but time has more than borne out my statement, and there is now no one left to dispute it. To-day I will venture another, and broader generalization, to wit: It appears likely that event- ually a bacterial disease will be found in every family of plants, from lowest to highest. This prediction is based on the fact that although the field is still a very new one, with no workers in most parts of the world, such diseases have been reported from every continent, and are already known to occur in plants of one hundred and forty genera distributed through - more than fifty families. DISTRIBUTION Following Engler’s arrangement, I will list these families that you may see how wide is the distribution of bacterial diseases in plants and how utterly wrong were those who said that there were no such diseases, and also those who conceded a little but said that they were very rare and restricted to the soft underground parts of a few bulbous and tuberous plants, and generally preceded by fungi. In this list, I have included only the flowering plants, but some of the eryptogams are also * Am. Nat. 30: p. 627. 1896. ANN. Mo. Bot. GARD., VoL. 2, 1915 (377) [vor. 2 378 ANNALS OF THE MISSOURI BOTANICAL GARDEN subject to bacterial attack. The number following the family name indicates the number of bacterial diseases known within the limits of the family. The total of the figures, however, will not give the number of bacterial parasites, because some of the diseases overlap. TABLE I SHOWING THE FAMILIES OF FLOWERING PLANTS ARRANGED SERIALLY FROM LOWEST TO HIGHEST. THOSE CONTAINING GENERA SUBJECT TO BACTERIAL DISEASES ARE UNDERSCORED, AND WHEN SEVERAL DISEASES HAVE BEEN RECOGNIZED THEIR NUMBER IS ALSO GIVEN 1. Cycadaceae 34, Juncaceae 68. Myzodendraceae 2. Ginkgoaceae 35. Stemonaceae 69. Santalaceae 3. Taxaceae 36. Melanthiaceae 70. Grubbiaceae 4. Pinaceae 2 37. Liliaceae 3 71. Opiliaceae 5. Gnetaceae 38. Convallariaceae 72. Olacaceae 6. Typhace 39. Smilacaceae 73, Balanophoraceae 7. Pandanaceae 36. 74. Aristolochiaceae 8. Sparganiaceae 37 Lilas 75. Rafflesiaceae 9. Potamogetonaceae 38. 76. Hydnorace 10, Naiadaceae 39. 77. Polygonaceae 2 11. Aponogetonaceae 40. Haemodoraceae 78. Chenopodiaceae 4 12. Scheuchzeriaceae 41. Amaryllidaceae 79. Amaranthaceae _ 12. Juncaginaceae 42. Velloziaceae 80. Nyctaginaceae 13. Alismaceae 43. Taccaceae 81. Batidaceae 14. Butomaceae 44. Dioscoreaceae 82. Theligonaceae 15. Vallisneriaceae 45. Iridaceae 8 Cynocrambaceae 15. ydrocharitaceae 46. Musaceae 83. Phytolaccacea 16. Triuridaceae 47. Zingiberaceae 84. Aizoaceae 17. Poaceae 48. Cannaceae 85. Portulacaceae 17. Gramineae 7 49, Marantaceae 86, Basellaceae 18. Cyperaceae 50. Burmanniaceae 87. Silenaceae 19, Phoenicaceae 51, Orchidace 87. Caryophyllaceae 19. Palmae E ere eA N 20 Cyclanthaceae 53. Saururaceae 89. Ceratophyllaceae 21. Araceae 54. Piperaceae 90. Trochodendraceae 22. naceae 55. Chloranthaceae 91. Ranunculaceae 23. Flagellariaceae 56. Salicaceae 2 92. Lardizabalaceae 24. Baloskionaceae 57. Myricaceae 93. Berberidaceae 24. Restionaceae 58. Balanopsidaceae 94. Menispermaceae 25. Centrolepidaceae 59. Leitneriaceae 95, Magnoliaceae 26. Mayacaceae 60. Juglandaceae 2 96. Calycanthaceae 27. Xyridaceae 61, Betulaceae 97. Lactoridaceae 28, Eriocaulaceae 62. Fagaceae 98. Annonaceae 29. Rapateaceae 63. Ulmaceae 99. Myristicaceae 30, Bromelia 64. Moraceae 100. Gomortegaceae 31. Commelinaceae 65. Urtieaceae 4 101. Monimiaceae 32. Pontederiaceae 66. Proteaceae 102. Lauraceae 33. Philydraceae 67. Loranthaceae 103. Hernandiaceae a t te H ko | + y SMITH— BACTERIAL DISEASES OF . Papaveraceae ; en uciferae 5 N oo . Penthoraceae y l Crassulaceae . Cephalotaceae . Grossulariaceae Saxifragaceae . Pittosporaceae rothamnaceae . Bruniaceae . Hamamelidaceae . Platanaceae . Crossosomataceae . Rosaceae . Malaceae . Amygdalaceae Rosaceae 6 . Connaraceae Mimosaceae . Caesalpiniaceae 6. Krameriaceae Fabaceae Leguminosae 5 . Geraniaceae 2 . Oxalidaceae 140. 141, 142. Tropaeolaceae 3 a . Malpighiaceae . Trigoniaceae . Vochyaceae aceae ; oc . Tremandraceae . Polygalaceae . Dichapetalaceae horbi uphorbiaceae . Callitrichaceae . Buxaceae . Coriariaceae ERBE . Hipp . Stackhousia k Staphyleaceae hanthaceae . Elaeocarpaceae . Schizolaenaceae . Chlaenaceae . Gonystylaceae . Tiliaceae PLANTS 184. {oo 186. Bomb 187. 188. 189. 190. 191. 192. 193. Ma 194. 195. Th . Hypericaceae . Clusiaceae 185 379 Malvaceae 2 mbacaceae Sterculiaceae Scytopetalaceae 7. Guttiferae : Dipterocarpaceae Fouquieriaceae . Cistaceae ixaceae . Cochlospermaceae . Koeberliniaceae . Canellaceae . Punicaceae [VoL, 2 380 ANNALS OF THE MISSOURI BOTANICAL GARDEN 230. Lecythidaceae 252. Primulaceae 275. Bignoniaceae 231. Rhizophoraceae 253. Plumbaginaceae 276, Pedaliaceae 232. Combretaceae 254. Sapotaceae 277. Martyniaceae 233. Myrtaceae 255. Diospyraceae 278. Orobanchaceae 234. Melastomataceae 255. Ebenaceae 279. Gesneria 235. Onagraceae 256. Styracaceae 280. Columelliaceae 236. Trapaceae 257. Symplocaceae 281. Pinguiculaceae 236. Hydrocaryaceae 258. Oleaceae 2 281. Lentibulariaceae 237. Haloragidaceae 59. Salvadoraceae 282. Globulariaceae 23 alorrhagidaceae 260. Loganiaceae 283. Acanthaceae 238. Cynomoriaceae 61. Gentianaceae 284, Myoporaceae 239. Araliaceae 2 262. Menyanthaceae 285. Phrymacea 240. Apiaceae 261. } eT Ere 286. Plantaginaceae 240 Umbelliferae 3 262. 287. Rubiaceae 241. Cornaceae 263. Apocynaceae 288, Caprifoliaceae 242. Clethraceae 264. Asclepiadaceae 289. Adoxaceae 243. Pyrolaceae 265. Convolvulaceae 290. Valerianaceae 244. Monotropaceae 266. Cuscutaceae 291. Dipsacaceae 243. 265, 292. Cucurbitaceae 3 244. } Pyrolaceae 266. } Convolvulaceae 293. Campanulaceae 245, Lennoaceae 267. Polemoniaceae 294. Goodeniaceae 246. Ericaceae 268. Hydrophyllaceae 295. Candolleaceae 247. Vacciniaceae 269. Boraginaceae 296, Calyceraceae 246. | Bricaceae 270. Verbenaceae 297. Cichoriaceae ot ieee ee 271. Menthaceae 298. Ambrosiaceae 248. Epacridaceae 271 Labiatae 299. Asteraceae 249. Diapensiaceae 272. Nolanaceae 297. 250. Theophrastaceae 273. Solanaceae 9 298. Compositae 3 251. Myrsinaceae 274. Scrophulariaceae 299. The widest gap, it will be observed, is between Cruciferae and Rosaceae, but I believe this represents nothing more than lack of knowledge. Also I should like to list the genera within the limits of which one or more species are now said to be subject to attack, because many of these genera contain plants of great economic importance. Where I have some personal knowledge of the subject I have italicized the genus name, and in what follows the reader will naturally expect me to draw illustrations prin- cipally from the diseases most familiar to me. TABLE II SHOWING GENERA OF FLOWERING PLANTS SUBJECT TO DISEASES OF BACTERIAL ORIGIN m Bromus Y Phleum Zea Saccharum se Andropogon Triticum Cocos 1915] SMITH—BACTERIAL DISEASES OF PLANTS 381 Oreodoxa Beta Prosopis (?) Syringa Richardia Amaranthus rythrina Olea Amorphophallus Dianthus Geranium Fraxinus Hyacinthus Delphinium elargonium Strychnos Allium Papaver Tropaeolum wu Lilium Brassica us Tectona Iris Raphanus Cedrela Verbena Ixia Cheiranthus Manihot Capsicum Gladiolus Matthiola Mangifera lanum usa Amelanchier Euonymus Lycopersicum Zingiber Sorbus Vitis Nicotiana Dendrobium Eryobotrya Gossypium Physalis Cattleya Pyrus Malva etuni ncidium Cydonia Sterculia Datura Odontoglossum Prun lod Calceolaria Cypripedium Rubus Begonia esamum Phalaenopsis Crataegus Opuntia Pavetta Vanilla Fragaria Eucalyptus Psycotria alia osa Oenothera Benincasa Populus Heteromeles Aralia ucu Juglans ; Hede Cucurbita Castanea Lathyrus Carota Citrullus Corylus Indigofera Pastinaca Sicyos us Kraunhia (?) Levisticum Echinocystis Pouzolzia Lupinus i Ageratum Cannab Mucuna Arbutus Chrysanthemum Acalypha Phaseolus Vaccinium Lactuca Humulus } Ardisia Blumea Ficus Crispardisia Synedrella Rheum Trifolium Amblyanthus Tragopogon Polygonum Medicago Amblyanthopsis Bellis Atriplex Arachis Diospyros Aster Spinacia i Ligustrum PERIOD OF GREATEST SUSCEPTIBILITY In certain diseases the brief seedling stage of the plant is the one most subject to attack, e. g., Stewart’s disease of maize due to Bacterium Stewarti, and brown rot of tomato and to- bacco due to Bacterium Solanacearum, but many bacterial diseases of older plants are also rather strictly time-limited. n both groups it is a question of abundant immature tissue. To the latter class belong the numerous leaf-spots, fruit-spots, and blights, e. g., black spot on the plum and peach, due to Bacterium Pruni, and fire-blight of the pear, apple, quince, etc., due to Bacillus amylovorus. In such cases, so far at least as they occur in temperate climates, the disease appears in [voL. 2 382 ANNALS OF THE MISSOURI BOTANICAL GARDEN the spring and the greater part of it occurs during a brief period in the early summer, in which growth of roots, leaves and shoots is proceeding rapidly and there are many young and succulent parts. The cause of the disease may and often does remain on the plant over winter in a latent or semi-latent condition (walnut blight, pear blight, plum canker), but the active period is limited to three months, more or less, of actively growing weather in which developing tissues, subject to infection, are abundant. With definitive growth and the hardening of the tissues in late summer and autumn, the disease is checked and disappears, or remains as a slow canker to appear again on other parts the following spring. It is a very instructive experiment to see, for example, inoculations of Bacillus amylovorus on ripening fruits and shoots of the pear wholly fail toward the end of July, which were eminently successful on the same trees at the beginning of June. The difference in this case is not due to lessened virulence on the part of the organism, but to changes in the host-plant, making it non-susceptible. Similar changes leading to non-suscepti- bility occur in the Japanese plum subject to Bacterium Pruni; the young fruits are very susceptible, the maturing fruits cannot be infected. Other parasites on the contrary are able to attack, disin- tegrate and destroy matured tissues, e. g., the pith of cabbage stems, turnip roots, the ripened tubers of the potato, well de- veloped roots of sugar beets, the bulbs of onions and hyacinths, full-grown melon and cucumber fruits. In both of these types the action of the parasite is expended chiefly on the parenchyma, although in some cases (the plum disease, Appel’s potato rot) there is more or less bacterial invasion of the local vessels. Vascular occupation is not a special characteristic. In the typical vascular diseases the case is reversed. Here parenchyma is also destroyed, more or less, but the most con- spicuous and destructive action is on the vascular bundles themselves, which are occupied for long distances, to the death, or great detriment, of the whole plant. In maize attacked by Bacterium Stewarti, it is not unusual, indeed one might rather 1915] SMITH—BACTERIAL DISEASES OF PLANTS 383 say it is customary, to find the vessels of the stem filled with the bacteria continuously for a distance of 3-6 feet from the point of infection, i. e., from the surface of the earth to the top of the full-grown plant. In cucurbits attacked by Bacillus tracheiphilus and in sugar-cane attacked by Bacterium vas- cularum the same thing occurs, and many of the vessels are filled solid with the bacterial slime to a distance of 8 or 10 feet from the place of infection. In such cases infection has taken place generally near the base of the plant, which continues to grow for some weeks or months. Transitions, of course, occur. Bacterium Stewarti, for ex- ample, is confined much more strictly to the vascular bundles of the maize stem than is Bacterium Solanacearum to those of the tomato, potato, or tobacco stem, although it also is a vascular parasite; that is, following infection of the vessels we do not find in the maize stems that extensive breaking down of the pith and phloem into vast cavities which is so common, for example, in tobacco and tomato stems. WHAT GOVERNS INFECTION Within the plant we may suppose, from certain indications, that abundant juiciness is the chief factor governing the in- fection of immature tissues. To this may be added an abun- dant well-adapted food supply and, in some cases, probably the absence of inhibiting substances, which may appear later. As the parts approach maturity the water content becomes less. Along with this, acids, sugars, amids, proteids, etc., are consumed and converted into substances less well adapted to the needs of the meristem-parasites, if not wholly inimical. In young shoots of potato and tomato, or of pear and apple, as contrasted with old ones, or in the roots of carrots as com- pared with the leaves, or in rapidly-growing cabbages, as compared with slow-growing ones, we know that there is an excess of water, and this alone appears to be sufficient to ex- plain the difference in behavior of their respective parasites in old versus young parts. When, however, we come to ripen- ing fruits, such as the pear and the plum, it would seem that they are still juicy enough to favor the growth of almost any [vor. 2 384 ANNALS OF THE MISSOURI BOTANICAL GARDEN bacterium, and we are forced to the hypothesis of chemical changes within the fruits to account for the failure of inocula- tions. As a rule (there are striking exceptions), parasitic micro-organisms are rather sensitive to changes in their en- vironment, e. g., to drying, exhaustion of food supplies, multi- plication of their own by-products, conversion of an easily assimilable substance into one less assimilable or actually harmful, appearance of esters, new acids, ete. But why speculate! Much additional experimenting must be under- taken before we shall have precise and full data. We are still largely in the observational stage. The parasites of ripened tissues do not require so much water, are able to convert starch into sugar, or have a special liking for some other element of the plant tissue. Externally, a number of factors favor infection. One of these is excessive shade, either of clouds or of foliage. An- other is high temperature. When these two factors are ac- companied by excessive rainfall, wet earth, and heavy dews, the conditions are ideal for the rapid dissemination and the destructive prevalence of a variety of bacterial diseases of cultivated plants. The bean spot due to Bacterium Phaseoli, the black spot of plum due to Bacterium Pruni, and the lark- spur disease due to Bacterium Delphini, are all favored by heavy dews and by shade. In hot, wet weather in July pear blight due to Bacillus amylovorus often bursts out like a con- flagration and sweeps over whole orchards. In warm, moist autumns bacterial diseases of the potato may destroy almost or quite the entire crop over extensive districts. HOW INFECTION OCCURS As I have already described elsewhere how infection oc- curs,! I will only dwell for a moment on it here, offering a few examples. The commonest way of infection is probably through wounds. 1 Smith, E. F. Bacteria in relation to plant diseases. Carnegie Inst. Wash- ington, Publ. 27°: pp. 51-64. 11. 1915] SMITH—BACTERIAL DISEASES OF PLANTS 385 In Italy, the olive tubercle due to Bacterium Savastanoi has been observed to begin very often in wounds made by hail- stones. In South Africa, crown-gall is said to be disseminated in the same way. In this country and also in Sumatra, Bac- terium Solanacearum enters the plant more often than other- wise through broken roots. A tomato or tobacco plant with unbroken roots will thrive in a soil deadly to one that has been root-pruned. I have myself observed this. We may suppose that substances attractive to the particular bacteria diffuse into the soil from the broken roots, following which they enter the plant. Resistant plants may be supposed to diffuse indifferent or repellant substances. All infections must be chemotactic. More interesting perhaps are those diseases which begin in natural openings, i. e., in places where the protective covering of the plant gives place to special organs such as nectaries, water-pores, and stomata. All the pome fruits subject to fire-blight are liable to blos- som infection. The bacteria multiply first in the nectaries of the flower, passing down into the stem by way of the ovary and pedicel. Blossom blight of the pear is a very conspicuous and common form of the disease as everybody knows. Thou- sands of blighted blossom clusters may be seen in any large orchard subject to this disease. In the black rot of the cabbage due to Bacterium campestre, the majority of the infections begin in the water-pores. These are grouped on the margins of the leaf at the tips of the ser- ratures. From this point the bacteria burrow into the vas- cular system of the leaf and so pass downward into the stem and upward into other leaves. In the black spot of the plum, almost or quite all of the infections are stomatal. A large proportion of them are also stomatal in the leaf-spot of cotton, and other leaf-spots. TIME BETWEEN INFECTION AND APPEARANCE OF THE DISEASE As in animal diseases, the period of latency may be very short or surprisingly long. Some time must be allowed the parasitic organism to multiply inside the plant before it does [vor. 2 386 ANNALS OF THE MISSOURI BOTANICAL GARDEN damage serious enough to be recognized externally as a dis- ease. This is the so-called ‘‘period of incubation,’’ during which the parasite is growing and its enzymes and toxins are becoming active. The microscope shows it to be present in the tissues, but the latter have yielded only a little in the immediate vicinity of the bacterial focus. This time is short or long depending on whether the parasite or the host has the first advantage. If the host is growing rapidly it may either en- tirely outstrip the parasite, or be only so much the more sub- ject to it. All depends on whether the parasite finds the initial conditions entirely suited to its needs, or by means of its secretions and excretions can quickly make them so, and con- sequently can from the start make a rapid growth, or must first slowly overcome obstacles of various sorts, such as in- hibiting acids and resistant tissues. The plant may show siens of infection within as short a time as one or two days after inoculation (various soft rots), or it may be as long a time as one to two months before they appear (Cobb’s disease of sugar-cane, Stewart’s disease of sweet-corn). In the latter, infection generally occurs in the seedling stage and the maize plant may be three months old and six feet tall before it finally succumbs. Of course, as in case of bacterial animal diseases, the greater the volume of infectious material the shorter the time. I have seen many instances of that law. In general, the period of latency may be said to vary from one to three weeks (yellow disease of hyacinth, black rot of cabbage, black spot of plum, cucurbit wilt, pear blight, angular leaf-spot of cotton, sorghum leaf-stripe, ete.). RECOVERY FROM DISEASE Mention has already been made of the self-limited spot diseases and blights. As the actively growing season draws to a close such diseases cease their activity. Also in some plants well developed signs of vascular dis- ease may be suppressed (squash, maize, sugar-cane) or re- main in abeyance for a longer or shorter period, according to the varying fortunes of the host and the capabilities of the parasite. The tomato plants inoculated with Bacterium Sol- 1915] SMITH— BACTERIAL DISEASES OF PLANTS 387 anacearum (Medan ur) and photographed for Volume mı of ‘Bacteria in Relation to Plant Diseases’ (plate 45 D), en- tirely outgrew the disease, as did also certain sugar-canes (series v1) inoculated with Bacterium vascularum.! Also, I have seen tomato plants recover only to develop a second and fatal attack of the vascular brown rot three months after the first attack, during which period they had made an extensive healthy-looking growth.? Recovery from disease may depend on loss of virulence on the part of the parasite. This often occurs when bacteria are grown for some time on culture-media, and it occurs also in nature, but its cause is obscure. AGENTS OF TRANSMISSION These may be organic or inorganic. In many cases the plant itself harbors the parasite indefinitely, carrying it over from year to year on some portion of its growth. Seeds, tubers, bulbs, grafts, or the whole plant may be re- sponsible for the appearance of the disease the following year in the old localities, and through the agency of seedsmen, nurserymen, or whoever disseminates plants, for outbreaks in regions hitherto exempt. There is good reason to believe that the black rot of cabbage and Stewart’s disease of sweet corn have been disseminated broadeast in the United States in recent years by ignorant and unscrupulous seedsmen. Both diseases are transmitted to seedling plants from the seed. The yellow disease of hya- cinths is carried in the bulb. Potato tubers from diseased fields may infect healthy fields. Apple grafts have transmit- ted crown-gall. Slightly infected trunks and limbs of trees (hold-over pear blight, walnut blight, canker of the plum) may infect shoots, leaves, blossoms, or fruits the following season. The soil around the infected plant may serve for years as a source of infection to other species (crown-gall), or to other individuals of the same kind (various leaf-spots). Occasion- ally, however, a parasite seems to die out of certain soils (Bac- 1 Smith, E. F. Bacteria in relation to plant diseases. Carnegie Inst, Wash- ington, Publ. 27°: p. 33. 1914. 2 Ibid. p. 179. [voL. 2 388 ANNALS OF THE MISSOURI BOTANICAL GARDEN terium Solanacearum). The pear blight organism probably dies out of soils quickly as it does in a majority of the blighted branches. Pear blight by soil infection is not known. Among extraneous agents, wind and water have been sus- pected. Ihave never seen any clear indications of wind-borne infection, not even when conditions seemed to invite it, but water often carries parasites and furnishes conditions favor- able to infection. Horne has shown that the olive tubercle in California is transmitted in this way. Honing, in the tobacco fields of Sumatra, has traced infection several times to the watering of plants from infected wells, and has cultivated the parasite from the water. I have discovered experi- mentally that to obtain several sorts of bacterial leaf-spots (bean, cotton, peach, plum, carnation, larkspur, sorghum, geranium) the surface of the leaves must be kept moist to the same extent they would be in case of prolonged dews or fre- quent light showers. Such conditions are necessary to enable the bacteria to penetrate the stomata and begin to grow. In case of water-pores, however, the plant itself furnishes the water necessary for infection, if the nights are cool enough, i. e., if the air remains near enough to saturation to prevent for some hours the evaporation of the excreted water from the leaf-serratures. Every plant with functioning water-pores awaits its appropriate bacterial parasite. The genus Im- patiens is a good example. I have looked on it for one in vain but I am sure it must occur. Man and the domestic animals, especially through the agency of the dung-heap, infallible repository of all sorts of discarded refuse, undoubtedly help to spread certain bacterial diseases of plants (potato rots, black rot of cabbage, ete.). Birds probably transmit some of these diseases on their feet or in other ways. In connection with the bud-rot of the coconut palm in the West Indies, I suspect the turkey-buzzard, but the evidence is not complete. Long since, Mr. Waite ob- tained (once in Florida, once in Maryland) the strongest kind of circumstantial evidence going to show that pear blight may be spread by birds. 1915] SMITH—-BACTERIAL DISEASES OF PLANTS 389 Respecting insects, molluscs, and worms, the evidence is complete. They often serve to carry these diseases. I have summarized our knowledge in another place! and will here content myself with a brief statement calling renewed atten- tion to the subject. We had very good evidence of the transmission of one bac- terial disease of plants by insects long before the animal pathologists awoke to the importance of the subject,” but it cannot be said that they have ever paid much attention to it, although it antedates by two years the work by Theobald Smith and Kilborne showing that Texas fever is transmitted by the cattle tick (Ixodes bovis). That discovery also belongs to the credit of the U. S. Department of Agriculture, and the two together may be said to have laid broad and deep the foun- dations of this most important branch of modern pathology. Waite isolated the pear blight organism, grew it in pure cul- tures, and proved its infectious nature by inoculations. With such proved cultures he sprayed clusters of pear flowers in places where the disease did not occur and obtained blossom- blight, and later saw this give rise to the blight of the sup- porting branch, found the organism multiplying in the nectar, and reisolated it from the blighting blossoms. On some trees he restricted the disease to the sprayed flowers by covering them with mosquito netting to keep away bees and other nectar-sipping insects. On other trees where the flowers were not covered he saw bees visit them, sip from the inoculated blossoms and afterwards visit blossoms on unsprayed parts of the tree which then blighted. Finally he captured bees that had visited such infected blossoms, excised their mouth parts, and from these, on agar-poured plates, obtained Bacillus amylovorus, with colonies of which he again produced the dis- ease. These experiments were done in several widely sepa- rated localities with identical results. I saw them and they made a great impression on me. 1 Smith, E. F. Bacteria in relation to plant diseases. Carnegie Inst. Wash- ington, Publ. 27°: p. 40. 1911. 2 Waite, M. B. Results from recent investigations in pear blight. Bot. Gaz, 16: 259; Am. Assoc. Adv. Sci., Proc. 40: 315. [VoL. 2 390 ANNALS OF THE MISSOURI BOTANICAL GARDEN The writer has since proved several diseases to be trans- mitted by insects, notably the wilt of cucurbits, and here the transmission is not purely accidental, but there appears to be an adaptation, the striped beetle (Diabrotica vittata), chiefly responsible for the spread of the disease, being fonder of the diseased parts of the plant than of the healthy parts. This acquired taste, for it must be that, works great harm to melons, squashes, and cucumbers. Whether the organism winters over in the beetles, as I suspect, remains to be determined. Certainly the disease appears in bitten places on the leaves very soon after the spring advent of the beetles. In 1897 I showed that molluses sometimes transmit brown rot of the cabbage, and last year I saw indications in Southern France which lead me to think that snails are responsible for the spread of the oleander tubercle, i. e., I saw them eating both sound and tubercular leaves, and found young tubercles developing in the eroded margins of bitten leaves. Parasitic nematodes break the root tissues and open the way for the entrance of Bacterium Solanacearum into tobacco and tomato, as was first observed by Hunger in Java and later by myself in the United States. One of the serious problems of plant pathology is how to control Heterodera radicicola, not only because of its wide distribution on a great variety of cultivated plants and the direct injury it works, but also on account of the often very much greater injury it causes through the introduction into the roots of the plant of bacterial and fungous parasites. The man who shall discover an effect- ive remedy will deserve a monument more enduring than brass. Our Southern States in particular are overrun with this parasite. Much remains to be done before we shall know to what ex- tent fungous parasites function as carriers of parasitic bac- teria. H. Marshall Ward sought to explain the presence of bacteria in diseased plants by supposing that they must enter the plant through the lumen of fungous hyphae. In this he was wrong, certainly if it be stated as a general proposi- tion, but it appears to be clear that in some cases the two types of parasites work together, the fungus invading first, and the 1915] SMITH—BACTERIAL DISEASES OF PLANTS 391 bacterium following hard after and often doing the major part of the damage. The reverse of this also occurs, the bacterium entering first and the fungus following. Parasitic bacteria are soon followed by saprophytic bacteria which complete the destruction of the tissues, and, if the dis- ease is somewhat advanced, cultures from the tissues may yield only the latter (potato rots). Also, as in animals, one parasitic disease may follow another and the second be more destructive than the first, e. g., fire-blight following crown-gall on the apple. EXTRA-VEGETAL HABITAT OF THE PARASITES Here is perhaps the place to say a few words about the non- parasitic life of the attacking organisms. All are able to grow saprophytically, i. e., on culture media of one sort or another, and probably all live or may live for a time in the soil. Very few, however, have been cultivated from it. The vast mixture of organisms present in a good earth rather discourages search. In some of the unsuccessful attempts failure may have been due to not having undertaken isolations at exactly the right time, or in just the right place, or on just the proper medium, but more often probably to the swamping tendency of rapidly growing saprophytes. How long a parasite is able to maintain its virulent life in a soil must depend largely on the kind of competitors it finds. I have used the term virulent, because it is conceivable that an organism might remain alive in a soil long after losing all power to infect plants, just as we know it can in culture media. Bacterium Solanacearum causing brown rot of Solanaceae, Bacillus phytophthorus causing basal stem rot and tuber rot of the potato, and Bacterium tumefaciens causing crown-gall, certainly live in the soil, and the soundest plants when set in such soils, especially if wounded, are liable to contract the disease, if they belong to susceptible species. The root-nodule organism of Leguminosae, which I have not considered here, also lives in many soils, as every one knows. [VoL, 2 392 ANNALS OF THE MISSOURI BOTANICAL GARDEN MORPHOLOGY AND CULTURAL CHARACTERS OF THE PARASITES Most of the plant bacteria are small or medium sized rod- shaped organisms. Very few parasitic coccus forms are known. In fact, none are very well established. Some of these bacteria are Gram positive, others are not. All take stains, especially the basic anilin dyes, but not all stain with the same dye or equally well. Most of the species are motile by means of flagella—polar or peritrichiate. A few are non- motile, genus Aplanobacter.‘ Some develop conspicuous cap- sules, others do not. Few, if any, produce endospores. Grown pure on culture media in mass, they are either yellow, pure white, or brownish or greenish from the liberation of pigments. Red or purple parasites are not known. We for- merly supposed that there were no green fluorescent species capable of parasitism, but now several are known, e. g., the organism causing the lilac blight of Holland, with pure cul- tures of which the writer obtained typical infections at Amsterdam in 1906, and afterwards in the United States (now first recorded). Some species produce gas, liquefy gelatin, consume asparagin, destroy starch, and reduce ni- trates; others do not. Their fondness for sugars and alcohols is quite variable. Some are extremely sensitive to sunlight and dry air (Bacillus carotovorus, Bacillus tracheiphilus). Others are remarkably resistant, remaining alive and infec- tious on dry seeds for a year (Bacterium campestre, Bac- terium Stewarti, Aplanobacter Rathayi). Some are strictly aerobic, others can grow in the absence of air, if proper foods are available. Some are very sensitive to acids, alkalies and sodium chlorid, others are not. Some have wide ranges of growth from 0°C. upwards. Some will not grow at or near 0°C., others will grow at or above 40°C. Very few, however, will grow at blood temperature, certain ones even in plants or on culture media are killed by summer temperatures, and none are known definitely to be animal parasites. mith, E. F, Bacteria in relation to plant diseases. Carnegie Inst, Wash- ington. Publ. 271: p. 171. 1905; Ibid. 27°: pp. 155, 161. 1914. 1915] SMITH—BACTERIAL DISEASES OF PLANTS 393 ACTION OF THE PARASITE ON THE PLANT In some cases it is hard to draw the line between parasitism and symbiosis or mutualism. Probably we shall find more and more of these transition states. I have included Ardisia in my list of genera and have excluded the genera of legumes subject only to root nodules. But a nodule on the root of a legume, so far as the local condition is concerned, is a disease as much as a leaf-spot, and, if Nobbe and Hiltner’s statements are to be credited, the general effect of the root-nodule or- ganism on the plant may be excessive and injurious and not to be distinguished from a disease. In the tropical East Indian Ardisia, which is one of the strangest cases of mutualism known to me, and on which Miehe has done such a beautiful piece of work, we perhaps have something akin to what occurs in the root nodules of legumes. Here the bacterial injury is local and internal. There are no superficial indications of disease. The bacteria are most abundant in the leaf-teeth where they form pockets or cavities and multiply enough to make the leaf serratures appear blanched or yellowish and slightly swollen, but never enough to kill them. In smaller numbers the bacteria occur in other parts of the plant including the inner parts of the seed from which they are transmitted to the seedling, whose leaf serratures, infected through their water-pores, in turn become the chief focus of the bacterial multiplication. Ap- parently the bacteria are always present, and we do not know what would happen to Ardisia plants grown without them, nor do we know how to obtain such plants. It would be an interesting experiment to see if they could be produced and to watch their behavior. The action of such organisms as I have mentioned differs probably from the behavior of active parasites in that they liberate much weaker toxins and enzymes, can attack only very actively growing parts, and also give off compensating nitrog- enous substances. Not yet proved for Ardisia. 1 Smith, E. F. Bacteria in relation to plant diseases. Carnegie Inst. Wash- ington, Publ. 27?: p. 131, last paragraph. 1911. [VoL. 2 394 ANNALS OF THE MISSOURI BOTANICAL GARDEN The active parasites produce toxins freely, poisoning the tissues, and enzymes converting starches into sugars, com- plex sugars into simpler ones, and so on, for their nutrition. They also neutralize and consume plant acids, and feed upon amido bodies and other nitrogenous elements of the host. As a result of their growth, many of them liberate both acids and alkalis to the detriment of the plant. The solvent action of their products on the middle lamellae separates cells and leads to the production of cavities in the bark, pith, phloem and xylem. There is also, or may be, a mechanical splitting, tear- ing or crushing due to the enormous multiplication of the bac- teria within confined spaces. The whole intercellular mech- anism may be honeycombed and flooded in this way, and if the cavities are near the surface the tissues may be lifted up or the bacteria may be forced to the surface through stomata in the form of tiny beads or threads (pear, plum, bean, maize, sugar-cane, etc.), or by a splitting process. The splitting in the black spot of plum fruits and peach fruits, however, results from local death of the attacked tissue with continued growth of the surrounding uninjured parts. A majority of the forms known to cause plant diseases are extra-cellular parasites occupying chiefly the vessels and inter- cellular spaces, causing vascular diseases, soft rots, spot dis- eases, etc. But intra-cellular parasites also occur, e. g., Bac- terium Leguminosarum causing root-nodules on legumes, and Bacterium tumefaciens causing crown-gall. The former mul- tiplies within the cell myriadfold, prevents its division, destroys its contents including the nucleus, and enormously stretches the cell wall so that the cell becomes much larger than its normal fellow cells and is packed full of the bacteria. The latter does not multiply abundantly within the cell, does not enlarge it, does not injure its viability, and would be a harmless messmate were it not for the fact that it exerts a stimulating effect on the cell nucleus, compelling the cell to divide again and again. THE REACTION OF THE PLANT We now come to the reaction of the plant. What response does it make to this rude invasion? Ten years ago we might 1915] SMITH—BACTERIAL DISEASES OF PLANTS 395 have said, ‘‘With rare exceptions, the plant is passive or nearly so,’’ but that would have been a superficial observation. In every disease we must suppose that the plant makes some effort to throw off the intruder, although often its forces are paralyzed and overcome very early in the progress of the disease. One of the most conspicuous results is lessened growth. In some of my plants recovering from brown rot due to Bac- terium Solanacearum,: a month after external signs of the disease had disappeared the check plants were twice the size of the inoculated ones, and there was still a very decided dif- ference after more than two months. I do not know how to explain this checked growth unless it be the response to ab- sorbed toxins. On potato plants attacked early by Bacterium Solanacearum the tubers remain small. On maize attacked by Bacterium Stewarti the ears are imperfect. Olive shoots inoculated and infected by Bacterium Savastanoi are always dwarfed, and the crown-gall dwarfings are frequently very conspicuous. The dwarfing of melon and squash plants attacked by Bacillus tracheiphilus is also conspicuous. Uninoculated sugar-cane stems soon surpass in height and vigor those successfully in- oculated with Bacterium vascularum. Changes in color are also conspicuous. The attacked parts may become greener than normal, or fade to yellow, red, brown or black. In tomato fruits there is often a retarded ripening on the attacked side with persistence of the chlorophyll. Crown-galls on daisy are greenish. In certain leaf-spots also the leaf green persists in the vicinity of the spot while the rest of the leaf becomes yellow (bean-leaf spot). The male inflorescence of maize attacked by Bacterium Stewarti ripens prematurely and becomes white. Distortions of various kinds appear (leaves of bean, lilac, larkspur, hyacinth, mulberry, Persian walnut). The leaves of tomato plants attacked by Bacterium Solanacearum are bent a gl so are the fronds of the coconut palm when 1 Smith, E. F. Bacteria in relation to plant diseases, Carnegie Inst. Wash- ington, Publ. A pl. 45-D. 1914. [VoL. 2 396 ANNALS OF THE MISSOURI BOTANICAL GARDEN attacked by the bacterial bud-rot. Knee-shaped curvatures of the culms appear on Dactylis attacked by Aplanobacter Rathayı, and in the buds of the sugar-cane attacked by Cobb’s disease. Organs may be developed in excessive number or out of place, as roots in hairy-root of the apple, witch-brooms on Pinus, and incipient roots on the stems of tomato, tobacco, chrysanthemum, nasturtium, ete. Hunger found a bud on a tomato leaflet which he attributed to the stimulus of Bac- terium Solanacearum. In various diseases the plant removes starch from the vicin- ity of the bacterial focus which it endeavors to wall off by the formation of a cork barrier, and in this effort it is sometimes successful if the parasite is growing slowly. The most conspicuous response of the plant is in the form of pathological overgrowths,—cankers, tubercles, and tumors. Some of these are very striking, e. g., those on the ash, olive, pine, oleander, and on a multitude of plants attacked by erown- gall. In some of these growths there is a great reduction of the vascular system, and a great multiplication and simplifica- tion of the parenchyma. There are also various other phe- nomena nearly related to what takes place in certain insect galls. In crown gall cell division under compulsion proceeds at such an abnormally rapid rate that the cells are forced to divide while still immature, and in this way masses of small- celled unripe (anaplastic) tissue arise. These develop tumor- strands on which secondary tumors arise. PREVALENCE AND GEOGRAPHICAL DISTRIBUTION Economically considered, bacterial diseases of plants may be classed as major or minor. Most of the leaf-spots would fall into the latter class. Various soft rots, blights and vascular diseases, being wide-spread and destructive to plants of great economic importance, may be classed as major diseases. Cankers and tumors would fall midway in such a grouping. Occasionally a minor disease, e. g., lettuce rot, celery rot, under favorable conditions may assume great importance. 1915] SMITH—BACTERIAL DISEASES OF PLANTS 397 It will be of interest to mention a few of these diseases with particular reference to their distribution and prevalence. Dutch East Indies—The tobacco disease of Sumatra and Java is probably the most destructive, if the Sereh of sugar- cane is not bacterial. Each of these diseases has caused enorraous losses. Each threatens an industry. The tobacco disease occurs also in the West Indies, in the United States, and probably also in South Africa. If Janse’s root disease of Erythrina, the coffee shade tree of Java, is also bacterial, as he supposed, then there is another great bacterial plague in that region, for hundreds of thousands of trees have died, and another species has been substituted as a shade tree. West Indies.—Here the most destructive disease is the bac- terial bud-rot of the coconut palm, which occurs all around the Caribbean, and threatens the entire destruction of a profitable industry in Cuba. There is also the bacterial disease of bananas and plantains, but the most wide-spread and de- structive Musa disease of the Western Hemisphere is the Panama disease, due to a Fusarium. Australia.—Cobb’s disease of sugar-cane has probably at- tracted more attention in Australia than any other bacterial trouble, although bacterial rots of the potato are also very destructive. The cane disease in both Queensland and New South Wales has in many cases destroyed the output of whole plantations and greatly discouraged planters. This disease occurs also in Fiji, and probably in South America. Japvan.—Probably the tobacco wilt, which has destroyed many fields, is the worst Japanese disease. This is believed to be identical with the tobacco wilt of Sumatra and of the United States. Several other bacterial blights have been reported, including one of the basket willow. India—The brown rot of Solanaceae is common and de- structive. Most of Asia is a terra incognita. South Africa—The mango disease in recent years has greatly reduced the exports. Potato and tomato wilts are common. There is a serious tobacco disease, probably bac- terial. Crown-gall is common and injurious on shade and orchard trees. Other diseases occur. (VoL, 2 398 ANNALS OF THE MISSOURI BOTANICAL GARDEN South America—There is a serious disease of sugar-cane in Brazil and another in Argentina, both of which I believe are of bacterial origin, and identical with Cobb’s disease. Bondar has reported a destructive manihot disease. The bud-rot of the coconut occurs in the north. United States and Canada.—Potato rots probably cause the greatest losses one year with another. Following these I should think pear and apple blight. Perhaps the latter should be placed first, for the destruction of an acre of potatoes would scarcely equal the value of a single fine pear tree, and thou- sands are destroyed every year. In California, which was free from pear blight until recently, the losses in the last fif- teen years have been enormous, amounting to about one-third of all the full-grown orchards and to a money-loss estimated at $10,000,000 for the five years preceding the efforts for its restriction begun in 1905 by the U. S. Department of Agri- culture. Very serious losses from this disease are experi- enced every year in the East, or were until growers became generally familiar with methods of control. In our southern states the tobacco and the tomato wilt have made it impossible to grow these crops on many fields. In the northern United States the cucurbit wilt is wide-spread and destructive, but cucurbits are of course a minor crop. The walnut blight has done much damage in California. This occurs also in New Zealand and Tasmania. The bacterial disease of alfalfa has been serious in parts of the West. It is most injurious early in the season, i. e., on the first cutting. Holland.—Here the yellow disease of hyacinths is always destructive and will eventually put an end to hyacinth-growing for export if means cannot be had for its control, since the land suited for hyacinths is limited in amount. Brown rot of cab- bage occurs in Holland and Denmark, and is common now also in many parts of the United States. It was probably imported into the United States from Denmark on cabbage seed. Some years in nurseries about Amsterdam the lilac blight has been troublesome. 1915] SMITH—BACTERIAL DISEASES OF PLANTS 399 Great Britain and Germany.—Potato rots are probably the most destructive bacterial diseases. France and Italy—Potato diseases are common. Olive tubercle, common also in California, and all around the Medi- terranean, is prevalent in spots. Vine diseases, especially Maladie d’Oleran and crown-gall, do considerable damage. Pear blight seems to be absent in France, but has been re- ported from several places in Italy. The destructive Italian rice disease, brusone, is not due to bacteria as reported, but to a fungus (Piricularia). METHODS OF CONTROL In conclusion, some words on prophylaxis will be in order. Until recently almost nothing was known. Unfortunately so far as regards most of these diseases, methods of control must still be worked out. But with rapidly increasing knowledge of the biological peculiarities of the parasites causing these diseases, and of the ways in which they are disseminated, light begins to dawn, so that before many years have passed we may confidently expect the more intelligent part of the public to be applying sound rules for the control of these diseases,— rules based on the individual peculiarities of the parasites and carefully worked out experimentally by the plant pathologist. The little that we now know may be summarized in part as follows: Waite has shown that pear blight winters over in excep- tional trees on trunk and limbs in the form of patches which ooze living bacteria the following spring and are visited by bees and other insects, and that if these ‘‘hold-over’’ spots are cut out thoroughly over regions several miles in diameter (wide as a bee flies), the disease does not appear on the blos- soms and shoots the following spring, except as it is intro- duced into the margins of this area from remoter uncontrolled districts. He has tried this method of control very success- fully, both in Georgia and California. Sometimes only one tree in many carries over the disease, but such is not always the case, and the success of this method involves the inspec- tion of every pome tree in a district with complete eradication [VoL. 2 400 ANNALS OF THE MISSOURI BOTANICAL GARDEN of every case of the hold-over blight, and this in great fruit regions requires a small army of trained inspectors. During the blighting period in late spring and early summer, if one would save his orchard, the trees must be cut over for removal of diseased material as often as every week, and in the worst weather oftener. The introduction of diseases transmitted by way of seeds, bulbs, and tubers may be avoided by obtaining these from plants not subject to the disease. As this freedom cannot always be known, bulbs and tubers should be inspected criti- cally before planting, and firm-coated seeds should be soaked for 15 minutes in 1:1000 mercuric chlorid water. In case of two plants (cabbage and maize) we know positively that the diseases are transmitted on the seed and this is probably true for several others—beans, sorghum, orchard grass. All shrivelled seeds should be screened out before planting. The seed bed in case of tobacco, tomato, cabbage, and trans- planted plants generally, should be made on steam-heated or fire-heated soil, or new earth which one has good reason to think free from the parasite in question. Nematode-infected soil should be avoided. Cuttings of carnations, chrysanthemums, roses, peaches, plums, apples, quinces, sugar-cane, etc., used for slips, buds, or grafts should be from sound plants. By following this practice, recommended in ease of sugar-cane by Cobb, the more intelligent cane planters in New South Wales have overcome the disease due to Bacterium vascularum. On badly infested soils a careful long rotation should be practised and the low places should be drained. Certain diseases may be held in check by germicidal sprays. Pierce reduced the number of infections in walnut blight fifty per cent by this method. Scott and Rorer combated leaf- spot of the peach in this way, the sprayed trees retaining their leaves, the unsprayed ones becoming defoliated. in Italy has recommended it and used it successfully on olive trees following hail-storms to keep out the olive tubercle. When diseases are transmitted by insects the destruction of the latter must receive prompt attention. 1915] SMITH—BACTERIAL DISEASES OF PLANTS 401 Great care should be taken to keep the manure heap free from infection. Diseased rubbish should be burned or buried deeply. It must not be thrown into a water supply or fed to stock or dumped into the barnyard. It has been found that some varieties of plants are less sub- ject to disease than others (pear, apple, plum, maize, potato, tomato, sugar-cane, banana, cabbage, etc.), and there are also individual variations within the variety. These phenomena lead us to hope that by selection, or hybridization, valuable re- sistant strains may be originated. Meanwhile the resistant sorts when they are of any value commercially should be sub- stituted for sensitive sorts in localities much subject to the disease. Unfortunately some of the resistant sorts have other less desirable qualities. A vast amount of experimental work must be done in this field before we shall have substantial re- sults, and at least a generation or two will be required to learn even the boundaries of the field. But the problem offered is so enticing and has such immediately practical bearings that in the near future we may suppose many pathologists will de- vote themselves to it, and that long before the whole field is worked over, many useful results will be forthcoming. The labor involved is enormous and exacting to discouragement at times, the results come so slowly, so much must be done to be certain of so little, all because the organisms dealt with are very small—how small, we seldom realize! Many a time in the past when downcast I have repeated to myself Seneca’s rolling words, Palma non sine pulvere per viam rectam, and have had more or less encouragement out of them. They are a good motto for any man, since nothing is more certain than this, that without plenty of well-directed hard work there can be no worthy success in any field of human endeavor. < a < € February-April, 19 | y 5 lon owering : oti Garden. of Oaxaca, S The Origin! ee Monocotyl < The Histor: ecent Investigations on the Protoplasm c Colloidal - ertie BT, y and Functions oF Botanic "Garden pied ; The Experimental “Modification ‘of Germ-Plasm. D. T. Sao ae Re ‚between Scientific ge and Eo ee ace of Temperature ‘Connected wih the stil -elass matier ‘at the Post- Omice “at. St aus, Misao: vader the Ast 5 TREA , 1870 ne, eS BIRLA “See Snes we a FSGS EASTER BE yee ae x e E tah u Bere ets z on Se RIA 5 3 ETF ae, eee ps et ne a Mi- Fa HERR i x x 3 cD ee i = y à x SS i A as \ € ` ty BY eee g RR inh wee Woda ea ye yids aa x er TR a BR

N AS Le kN RAR Cob US ee pie IT LII 4. Rhizoctonia Crocorum: a, from a section of a large sclerotium; Fig. b, extreme forms of cells isolated from a macerated sclerotium. SUGGESTIONS REGARDING THE PERFECT STAGE It has been noted that Du Hamel and other early observers stated that the affinities of the violet fungus were with the truffles. Persoon, Fries, and others placed the genus near Sclerotium. Tulasne considered the small sclerotia as prob- ably a stage in the development of an ascomycete (pyreno- mycete). This suggestion of Tulasne has apparently in- fluenced many mycologists, and a search in this direction for the perfect stage has continued practically until the present time. Fuckel suggested that Lanosa nivalis Fr. might be considered the first or conidial stage of this fungus and he believed that the minute sclerotia or penetration cushions gave rise during the latter part of the season to pycnidia. With the more complete disintegration of the af- fected tissues he reported the development of a perithecial stage, and this fungus he called Byssothecium circinans (Lep- tosphaeria circinans (Fckl.) Sace., Trematosphaeria circinans (Fekl.) Wint.). It will be noted that Winter regarded this [VoL 2 420 ANNALS OF THE MISSOURI BOTANICAL GARDEN view of the genetic relation to Rhizoctonia as improbable; and Saccardo, who at first accepted the relationship, subse- quently changed his opinion. Prunet (’93) states that he made certain inoculation experiments from which he was con- vinced that Fuckel was correct; but we possess no indica- tions as to how these experiments were conducted. The writer in 1899, at Leipzig, germinated the spores of Lepto- sphaeria circinans and obtained a mycelium bearing no re- semblance to the Rhizoctonia hyphae. The idea that Lepto- sphaeria constitutes a perfect stage of the Rhizoctonia has had no support recently, although Comes (’91) incorporates it in an extreme form in his treatment of the genus. Rostrup (’86) found in the spring on the old roots of af- ` fected plants a pyenidial stage which he considered to be con- nected with the Rhizoctonia hyphae; and on the old roots of Ligustrum he found reddish filaments and scattering peri- thecia; the latter he identified as a species of Trichosphaeria. His assumption, however, has received no encouragement. When Hartig (’80) discovered a Rosellinia as the perfect stage of his Rhizoctonia Quercina there was a temporary re- vival of interest in the quest for one of the Ascomycetes as the perfect stage of R. Crocorum. Frank (’97) reported observing the violet fungus on the grape, and associated with it he found a species of the The- lephoraceae. This he regarded as the perfect stage, and to the fungus he applied the name Thelephora Rhizoctoniae. This observation has failed of confirmation. Eriksson (’13) has recently presented an extension of his earlier account (’03%) of diseases produced by Rhizoctonia, and in this he records a new ‘‘Hypochnus,’’ H. violaceus (Tul.) Eriks. as the perfect stage of ‘Rhizoctonia violacea, Tul.” In this he was stimulated by the observations of Rolfs (703) and others in America, and Pethybridge (’11) in Ire- land, on the occurrence of the basidial stage (Corticium vagum B. & C. or Hypochnus Solani Prill. & Del.) of Rhizoc- tonia Solani Kühn, resulting in a reéxamination of some ma- terial of the violet fungus on roots and stems of certain wild plants. This material had been preserved in alcohol thirteen 1915] DUGGAR—RHIZOCTONIA CROCORUM AND R. SOLANI 421 years earlier. The result of his study is reported as follows: “D’après ces renseignements, il faut—du moins pour ce qui concerne les formes du champignon qui envahissent les Car- rottes—considérer comme résolue la question tant débattue de savoir à quel groupe rapporter le mycélium stérile connu sous le nom de Rhizoctonia violacea. Dans ce qui suit, je vais in- diquer le nom scientifique qu’il faut donner, ainsi que les car- acteres diagnostiques du champignon autant que j’aie pu en juger sur les documents conservés que j’avais 4 ma disposition.” On the basis of these observations he creates the Hypochnus mentioned. No adequate diagnosis is given, but the im- portant part of the account is as follows: “Ensuite le champignon forme autour des tiges de la méme plante ou d’autres especes de plantes immédiatement au-dessus du sol, une enveloppe annulaire, membraneuse, d’un rose tendre, qui, montant souvent sur les tiges jusquà une hauteur de 5 a 15 mm. et s’étalant parfois sur la surface du sol comme une feuille aves mince, produit des basidiospores. C’est le stade Hypochnu This apparently refers to material on Stellaria media, Myo- sotis arvensis, Galeopsis Tetrahit, Erysimum cheiranthoides, Urtica dioica, and Sonchus arvensis, which hosts he would regard as harboring the Hypochnus stage of that form of the violet fungus attacking the carrot, and for this reason the names just given appear in the list of hosts. In the writer’s opinion he properly considers it remarkable that the fructification stage should attack hosts other than those producing the sterile stage. In view of the character of the material, the incompleteness of the account, and the pos- sibility of confusion with Corticium vagum B. & C. it would appear necessary to await confirmation of the observation that a Corticium (Hypochnus) may represent the perfect stage of the fungus here discussed, although, reasoning from the apparent relationship of this species to R. Solani, a Corticium stage might well be assumed. The writer has been unable thus far to secure any of the material mentioned. In a footnote Eriksson expresses himself thus: ‘‘Quant à la Rhizoctone de la Luzerne, je suis porté à croire, d’après les (VoL. 2 422 ANNALS OF THE MISSOURI BOTANICAL GARDEN observations de cette année (1912), quelle doit étre rapportée à un groupe d’Ascomyeétes.’’ This suggestion is both in- teresting and surprising since Hriksson adopts the Tulasnes’ name for the Rhizoctonia on carrot and this would seem to concede the identity of the carrot and alfalfa forms. It is also in a measure inconsistent with his inoculation results, as reported later.! CROSS INOCULATION AND CULTURAL STUDIES The amount of cross inoculation work yet reported is not considerable, and for this, doubtless, the inability to cultivate the organism is largely responsible. Throughout the early literature numerous indications are offered showing that fol- lowing a severe outbreak of the disease on any crop, it may appear on susceptible plants grown in the affected area—ob- servations which tend to establish the identity of the fungus on different hosts. Among later observations may be men- tioned those of Giintz (’99) who records that in a field where alfalfa and red clover had been seriously affected, beans, potatoes, and tuberous artichokes were planted; the potatoes subsequently developed the disease in serious form, and the other plants showed indications of its presence. In England it is reported (Bd. of Agr., ’06) that potatoes are affected by the violet felt fungus, especially when following alfalfa; and under similar conditions the fungus appears upon clover, car- rots, beets, and mangolds. Eriksson (713) undertook some cross inoculation work em- loying, in zine cylinders, soil from diseased carrot fields (eight cylinders) in contrast with soil taken from areas free from the disease (two cylinders). At the same time, to the diseased soil he added pieces of carrots affected by the fungus. The cylinders were permitted to stand over winter eg proof of this paper I have received from Prof. Eriksson an Penn reprint of his paper, “Fortgesetze Studien über Rhizoctonia violacea DC.” Arkıv för Be Py 14 (Art 12) : 1-31. f. 1-13. 1915. It is impracticable to include here a full discussion of this s paper. It is necessary to state, however, that he irite. at length Rhizoctonia —n DC. and R. Asparagi Fekl., and includes inoculation experiments indicating form differences. After germinating the spores of Leptosphaeria circinans he comes to the conclusion that, in spite of his earlier work on Hypochnus violaceus, the yrenomycete mentioned is the enn stage of R. Medicaginis. Prof. Eriksson has also furnished material of Asparagi and of the Leptosphaeria 1915] DUGGAR—RHIZOCTONIA CROCORUM AND R. SOLANI 423 and the following spring were planted to several varieties of carrots, to beets, mangolds, red clover, and alfalfa. At the time of harvest, the carrots were all more or less severely affected, while the sugar beets and alfalfa showed very light attacks, and the clover none at all. Continuing the work in subsequent seasons he obtained evidence in one case—that of the sugar beet—pointing to an increased virulence of the fungus with adjustment to that host. On the contrary, in the second year the alfalfa exhibited greater resistance, thus rendering a decision as to the existence of physiological races hazardous. He also reported, that on placing diseased soil and diseased carrots in a box in which various weeds were permitted to grow, the fungus appeared on eight species of weeds (representing several families), apparently a consider- able proportion of those present. This also would seem to discourage the idea of marked host specialization. Attempts to cultivate the violet fungus on artificial media have been made by several investigators without success. While in Leipzig, 1900, I obtained particularly good material on alfalfa from Bavaria. Dilution cultures were attempted both on various kinds of agar and on gelatin, but no growth of the fungus was secured in any case. Further trials were made with material from France in 1902, and again upon re- ceiving comparatively fresh material from Kansas in 1911. Bailey (’15) reports an endeavor to cultivate the organism in Oregon, also without success. It is quite possible that special conditions are essential to its growth in artificial eul- ture, but we should not assume that it is incapable of growth in this way. It would appear that the presence of contami- nating organisms is not the sole cause of the difficulty, since isolated hyphae in the dilution cultures remain free from the growth of contaminating organisms, and yet themselves fail to develop a colony of growth. It will be recalled that At- kinson! found difficulty, but ultimate success, in growing Ozonium omnivorum (Lk.) Shear, the cause of the south- western root rot of cotton. The writer also found that this organism is not readily cultured, but obtained a satisfactory 1 Bot. Gaz. 18: 16-19. 1893. [vor. 2 424 ANNALS OF THE MISSOURI BOTANICAL GARDEN growth on cotton decoction starch paste in 1902. Since in general pathology and physiology the cotton Ozonium and the violet Rhizoctonia have much in common, a further careful investigation of their life relations would doubtless yield interesting results. PREVENTION AND CONTROL Relief measures respecting the violet fungus are very largely limited to the practices of good culture, good drain- age, and sanitation. The early pathologists have generally recommended pulling up diseased plants and burning them. It is well to point out, however, that after a careful examina- tion of the distribution of the fungus on the smallest fibrous roots, it has been found to invest these to a considerable depth in the case of alfalfa, and therefore a very small meas- ure of security may be expected unless one carries out this recommendation in a far more thorough manner than is practicable in the field. The further suggestion has been made that where the diseased areas are few, small, and clearly defined, trenches may be dug to prevent the further spread of the disease; but if this should prove feasible under any con- ditions, it would be advisable only in connection with a thor- ough disinfection of the isolated areas by formaldehyde or sulphuric acid—the former disappearing from soil in time, and the latter being easily neutralized by liming. The rota- tion of crops is undoubtedly desirable, but complete immun- ity from the disease cannot be expected if we may trust the statements of Du Hamel and other observers to the effect that the fungus may remain alive in the soil for periods of from three to twenty years. The fact that many hosts are affected also complicates the practice of rotation. Tue Common Rarzocronia, R. SoLanı KÜHN (Corticrum vacum B. & C.) EARLY STAGES In addition to his discussion of the violet Rhizoctonia on beets and carrots Kühn (’58) described a disease of potatoes, of which the causal organism was recognized as a species of r 5 0 ian o / A D E A $ 1915] DUGGAR—RHIZOCTONIA CROCORUM AND R. SOLANI 425 Rhizoctonia differing notably from the violet organism, and to this potato fungus he gave the name R. Solani. The life history of the fungus and the symptoms of the disease induced were very imperfectly known at the time, so that the deserip- tion could not be complete. As a result, those who subse- quently discussed the genus Rhizoctonia have sometimes recognized R. Solani, while others have referred the organism to R. Crocorum (R. violacea), and still others have assumed that R. Solanı Kühn was also the cause of another disease of beets and of carrots mentioned by Kühn without identifying the causal organisms. After a study of certain diseases in America induced by Rhizoctonia, I was keenly aware of this confusion, so when opportunity presented itself in the winter of 1899-1900 I conferred with Professor Kühn regarding those diseases, and also endeavored to obtain satisfactory specimens of the fungi. There has been no earlier oppor- tunity to utilize the information obtained in connection with a general discussion of the genus. Kühn laid special stress upon a scab (‘‘Schorf oder Grind,” later termed ‘‘Pockenkrankheit’’) of potatoes, sometimes followed by deeper seated injuries and decomposition (‘‘als Raude und Kratze bezeichnet’’). The symptoms are clearly those that we now know as one type (cf. McAlpine, 712) of the potato diseases ascribed to R. Solani Kühn (Corticium vagum B. & C.). It has been noted that the fungus was not so well described as might be wished, and the spores men- tioned were evidently those of contaminating organisms, or else the oval cells of the tufted stage of the fungus; but when we use in connection with this general description Kiihn’s comparison of this plant with the violet fungus (Kiihn, ’58, p. 248) it is convincing that the fungus on the potato which he had under consideration was not Rhizoctonia Crocorum. The sclerotia were also inadequately described and figured. With reference to that point, however, Professor Kühn stated that while a common form of the fungus on the tubers con- sisted of irregular superficial sclerotia, this form did not lead to serious consequences and therefore received less attention from him. Material of this superficial sclerotial stage was 7 m : QGTON UNIVE pN 4 PR iBRARY \ MA ý Pn —YICAL SCH AN je [VoL. 2 426 ANNALS OF THE MISSOURI BOTANICAL GARDEN furnished the writer by Professor Sorauer in 1900 (for a photograph see Duggar, ’09, p. 477, fig. 219), and, subsequently, from other points in Germany. It is clearly the ‘‘black speck”’ form of the disease now generally recognized. Professor Kühn also identified cultures of the American fungus on sugar beets (Duggar, ’99) as very close to, if not identical with, his R. Solan. In 1858 Kühn was obviously unaware of the fact that the violet fungus also occurs on potato in Germany; and, in fact, he told me in 1900 that it was subsequent to 1858 when he first collected specimens of the violet fungus on this host. “The violet fungus produces no serious epidemics of the potato in Germany,’’ he declared. Professor Kühn was un- able to locate type material of R. Solanı, and such material is doubtless unavailable. Before presenting still other indi- cations pointing unmistakably to their identity, I shall proceed on the basis that it is correct to refer the sterile stages of the commoner American Rhizoctonia on potato and other plants to R. Solani Kühn, and once studied comparatively there can be no confusion of this plant with R. Crocorum (Pers.) DC. A disease of carrots was also described by Kühn with which no fungus was positively associated. The indications are in- sufficient to determine whether this was a fungus or a bacterial disease. So far as the writer is aware no disease of carrots in Europe due to R. Solant has since been reported, though in 1900 Professor Kiihn stated as his opinion that carrots as well as beets in Germany were affected by a fungus similar to R. Solana. The violet root felt fungus was clearly distinguished by Kühn (’58, see pp. 235-237, 243-249) in its occurrence on both beets and carrots. It is not possible to mistake his statements in which the organism on these hosts is referred to Rhizoc- tonia Medicaginis DC.’’ Moreover, he nowhere suggests the combinations R. Dauct Kühn and R. Betae Kühn, which later crept into the literature of the subject. This fact makes it difficult to understand the nomenclature employed by Eidam (’87) and Comes (’91). In discussing a beet disease prevalent in Germany, Eidam refers the organism to Rhizoctonia Betae Kiihn. He gives a description of the disease and of the fun- 1915] DUGGAR—RHIZOCTONIA CROCORUM AND R. SOLANI 427 gus, including its growth on culture media. It is clearly the beet disease now well known in America, and of which the causal fungus is referred to R. Solani. Kühn did describe the symptoms of another disease of beets, and this last bears every indication of being the heart rot later known to be due to Phoma Betae (Phyllosticta tabifica), much discussed by Frank and others. Kühn’s discussion of this other beet disease has been interpreted, also, in the way I have indicated by Prillieux and Delacroix (’91) and others outside of Germany. In my conference with him, Professor Kühn stated that the only Rhizoctonia diseases of beets and carrots which he knew in the vicinity of Halle in 1858 and earlier were those due to the violet fungus, and of these he exhibited specimens having the usual characteristics. From the evidence at hand, therefore, the Rhizoctonia disease of beets described by Eidam was new on that host. It would seem, then, that Eidam is the authority for the combination R. Betae, which he attributes to Kühn. In any case it be- comes a synonym of R. Solani Kühn (Corticium vagum B. & C.). In discussing the Rhizoctonia disease of potatoes in Europe Sorauer (’86) describes unmistakably the ‘‘black speck’’ or sclerotial form of the fungus, and while he, like many others, assumed that it would be found to belong among the Ascomy- cetes, it is obvious that the characteristics of this stage of Kühn’s fungus were well recognized. Among the forms of Rhizoctonia which he enumerated and discussed Comes (’91) includes R. Dauci Kiihn, and R. Betae Kiihn. In his discussion of the first-named he reviews Kiihn’s account of the violet fungus on carrots, already mentioned; but in the account of R. Betae Kühn he evidently refers both to Kühn’s account of the heart rot of beets and to the Rhizoc- tonia disease of this host described by Eidam. Pammel (’91) was the first American pathologist to report in this country a disease now known to be caused by R. Solani. He, however, followed Comes and Eidam in referring to the fungus causing the beet rot as R. Betae Kühn. : Atkinson (’92, ’95) studied a ‘‘sterile’’ fungus causing sore (Vou. 2 428 ANNALS OF THE MISSOURI BOTANICAL GARDEN shin or damping off in cotton, and ascertained that the same fungus was commonly associated with, and, capable of, induc- ing damping off of various seedlings in the greenhouse. Duggar (’99) also referred to the beet rot fungus in Amer- ica as Rhizoctonia Betae Kühn, following Comes, and was able to determine that this beet fungus was identical morpho- logically (mycelium and sclerotia) with the damping off fun- gus found by Atkinson. The characteristics of the two organ- isms in culture were also identical, both forming on certain media a rich mycelium and finally numerous flaky or tufted centers of growth, some of which become irregular, often erust-like, sclerotia. Neither on affected seedlings nor on beets were sclerotia ordinarily produced (compare, however, Edson, 715, pl. 23). Subsequently, Duggar and Stewart (’01) reported that several types of disease, on a variety of hosts, including the potato, were induced by Rhizoctonia. The account given was intended to be merely preliminary, and for this reason a few words of explanation are necessary. The account referred to did not (perhaps unfortunately) explicitly indicate that, as far as the studies had progressed, there was evidence that the organism, or forms of the organism (except in the case of the form on rhubarb, referred to later) exhibited morpho- logically and in culture the characters of the beet rot and damping off fungus. The authors were likewise convinced, after a study of European material of Kühn’s fungus on the potato, of the identity of the American and European forms on this host. Cultural studies were being carried forward with Rhizoctonia Solani from many hosts, since there was the possibility of establishing definite forms or races, of find- ing the perfect stage, and of discovering other species. Again, specimens of the violet root felt fungus on various hosts had been obtained by one of us, and it was intended to include in a final paper a general account of the genus. This failure to designate the form with which we worked has doubtless led to some misunderstanding (see Prillieux 97, Eriksson 713, p. 17). However, in a more recent account (Duggar, ’09, pp. 477-478), it will be seen that the diseases 1915] ‘ DUGGAR—RHIZOCTONIA CROCORUM AND R. SOLANI 429 discussed are ascribed to R. Solani (Corticium vagum B. & C.). DISTRIBUTION Rhizoctonia Solan is distributed throughout the United States and Canada. There is every reason to believe that it exists as a saprophyte in most arable soils, and under certain conditions may attack many species of plants. It is perhaps most frequently noted as a damping off disease in green- houses and seed beds, but this occurrence may be explained by the fact that here the conditions are probably more con- ducive to the pathogenicity of the fungus. On the potato it is likewise wide-spread, although, as noted later, the eco- nomic importance of the diseases induced varies in different sections of the country, probably in accordance with climatic and soil conditions. In all potato-producing states and re- gions it is a well-known disease. On the sugar beet it has been observed in many states. The fact that it is an important disease of one crop or another in every section of the country is alone sufficient indication of its general occurrence. Rhiz- octonia has been mentioned in Brazil by Potel (’00), but it is not clear to which species he refers. It is rather surprising to find that R. Solani has received relatively little attention in Europe. Although recognized as inducing a disease of the potato widely distributed in central Europe, and occasionally reported on the beet, yet little care- ful work has been bestowed upon the fungus. Eriksson (’13), seems to be unfamiliar with the fungus in Sweden. On this account we can gain no incidental information regarding R. Solani as a result of his extensive studies of the related spe- cies in that country. The following will express his attitude regarding R. Solan: “TI paraît tres douteux, du moins si l’on en juge d’après les descriptions et les figures données, que les nouvelles formes de la Rhizoctone stérile signalées dans ces derniers temps par B. M. Duggar et F. C. Stewart sur une quantité de plantes différentes en Amérique (* * *) soient vraiment identiques aux formes du Rhizoctonia violacea qui ravage l’Europe. We have very little data regarding its occurrence in other sections of continental Europe, although from conference (VoL. 2 430 ANNALS OF THE MISSOURI BOTANICAL GARDEN with Prof. Delacroix in Paris (Nov. 28, 1901) and from an examination of material furnished by him I learned that it is not uncommon throughout France on the potato. It will be recalled that the perfect stage was described by Prillieux and Delacroix (’91). Judging from the amount of the black speck disease observed on the potato in the markets of various cities in southern Europe during 1905-’06 the writer would infer that it is of more frequent occurrence than is reported. Pethybridge (’11) finds the fungus (including the Corticium stage) well distributed in Ireland, and it is reported from other parts of Great Britain. McAlpine (’11) has reported this fungus on the potato from several points in Australia, and he states that it occurs upon a variety of economic plants. Since it has proved a serious disease in very few localities, it receives little attention, and is therefore freely disseminated by commercial intercourse. It is also known in New Zealand and Japan. The investigations of Shaw (’13) suggest that Rhizoctonia Solanı may be an important disease-inducing organism in some of the more humid regions of India. Reference is made later (pp. 448-450) to the fact that he has obviously misap- plied this name, however, and also that other confusion has resulted. In spite of this, it seems certain that he has ob- served all stages of the fungus. TYPES OF DISEASES INDUCED, SYMPTOMS It is not my purpose to attempt a complete description of the more important diseases caused by this species, yet suf- ficient will be included to indicate the main types of diseases thus far investigated, their general distribution, and their striking pathological relations. By types of disease, I have reference to general effects or symptoms. The effect of the fungus upon the stems may occasion a different appearance from its action upon the root, and thus there arise the differ- ent types referred to. With respect to penetration and action up- on the cell the behavior of the fungus may be the same in all cases. Moreover, as a result of the primary injury, second- ary effects may occur, and sometimes such secondary phe- 1915] DUGGAR—RHIZOCTONIA CROCORUM AND R. SOLANI 431 nomena may be so striking in appearance as to dominate the primary injuries or lesions. For convenience we may arrange the types of disease in the following categories: (1) damping off, (2) stem rot, (3) root rot, (4) leaf rot, (5) scab, and (6) such secondary effects as rosette, little potato, and leaf roll. Since more than one type of disease may occur upon a single host, and especially since one form of the disease may grade into another, it will be more practicable to discuss these under the following captions: (1) damping off, (2) potato diseases, (3) rot of fleshy roots, (4) stem and root rots of herbaceous plants, and (5) fruit and leaf injuries. DAMPING OFF It would appear that the first mention of a disease of seed- lings caused by Rhizoctonia is that of beets, recorded by Eidam (’87), although he gives no complete account of the evidence. It is preferable to date our knowledge of damping off diseases caused by Rhizoctonia from the work of Atkinson (792), who studied particularly sore shin of cotton, but he also found the ‘‘sterile’’ fungus to cause damping off of seedling beets, radish, lettuce, egg plants, cabbage, and other plants in the forcing house. The later identification of the fungus concerned (Duggar, ’99) and its association with the damping off of various plants (Duggar and Stewart, 01) was only the beginning of the observations which have now served to direct our attention to the vast importance of this fungous disease throughout the United States both in the greenhouse and in the outside seed bed. Among numerous instances in which damping off has been reported due (or in all probability due) to this fungus may be noted the following: (1). It has been found as a source of serious injury to ginseng in the seed bed (Van Hook, ’04; Whetzel and Rosenbaum, 712). (2). Tobacco seedlings are so frequently injured that soil treatment has received special consideration in the case of this crop (Selby, ’04; Cook and Horne, 05). (3). As a damping off disease of cotton (sore shin) it occurs not only in America but in Africa (Balls, ’05, ’06) and possibly in India (Shaw, ’13) as well. (4). Tomato [VoL 2 432 ANNALS OF THE MISSOURI BOTANICAL GARDEN seedlings seldom attacked by Pythium have been found to succumb to Rhizoctonia in Louisiana (Edgerton and More- land, 713). (5). Alfalfa seedlings have been reported sus- ceptible in one instance (Stewart, French, and Wilson, ’08). (6). Seedlings of various species of conifers from a few days to nine weeks old have been reported attacked in several in- stances (Hartley, ’12, Clinton, 713). The majority of the instances reported above were under normal seed bed or field conditions. Many other cases of the damping off of seedlings might be included where seeds are grown in crowded condition in moist greenhouses. Again, damping off of cuttings by Rhizoctonia is now a well-known phenomenon in the propagating house, and special precau- tions are taken with respect to drainage and moisture in order to reduce the injuries to a minimum. It is safe to assume— since the fungus seems to be found in practically all soils— that it is in general the worst enemy of seedling plants. In fact, it may be anticipated that under conditions favorable for the fungus the damping off of seedlings of numerous species may be anticipated. So far as the writer has been able to ascertain there has been no report of the damping off of mon- ocotyledonous plants under normal seed bed conditions. While Rhizoctonia Solanı may perhaps induce damping off in innumerable species regarding which observations are lacking, some of the host plants which have come to the writ- er’s attention as particularly susceptible are the following: lettuce (Lactuca sativa), celery (Apium graveolens), beet (Beta vulgaris), cress (Lepidium sativum), tobacco (Nico- tiana Tabacum), balsam (Impatiens balsamina), snapdragon (Antirrhinum majus), cotton (Gossypium spp.), cucumber (Cucumis sativus), squash (Cucurbita spp.), sunflower (Heli- anthus annuus), carrot (Daucus Carota), radish (Raphanus sativus), and phlox (Phlox Drummondit). Since the phycomycetous damping off fungus Pythium has been known to pathologists much longer, and prior to 1895 was practically the only fungus to which this type of disease was ascribed, it is probable that much damage due to Rhi- zoctonia has been ascribed to Pythium. Moreover, unless 1915] DUGGAR—RHIZOCTONIA CROCORUM AND R. SOLANI 433 examined microscopically, there are no symptomatic differ- ences between the effects of the two organisms. Seedlings affected exhibit symptoms somewhat different with age. The youngest seedlings of all delicate plants show what may be called the usual damping off characteristics. Near the base of the stem an hygrophorus or translucent appearance is quickly followed by shrinkage of the tissues and weakness of the stem. The plants topple over, the fungus invades all parts, and spreads rapidly to the neighboring individuals. The cells of the sap-perfused tissues are flaccid and injured, some showing this even before the entrance of the hyphae into the cells. Somewhat older plants and the more robust seed- lings of cotton, bean, etc., often exhibit characteristic lesions. Atkinson (’95) gives a description of its effect on cotton seedlings as follows: “The trouble is caused by the fungus growing first in the superficial tissues of the stem near the ground and disintegrat- ing them before it passes to the deeper tissues; in other words the fungus never seems to penetrate far in the living tissues, but ‘kills as it goes,’ and the tissues become brown, depressed, and present the appearance of the plant having a deep and ugly ulcer at the surface of the ground. The fungus does not spread into the tissues either above or below the ulcer to any extent, but literally eats away at that point until it has severed the stem at the affected place or the plant has recovered from its effects.” DISEASES OF POTATOES The potato is the most interesting of the host plants with respect to the parasitism of Rhizoctonia by reason of the many types of disease induced under diverse conditions. The conditions may be in part climatic and, in part perhaps, de- pendent upon the pathogenicity of the particular strain of the fungus or upon the stage and development of the host at the time of infection. It has been noted that when Kühn first de- scribed the disease of potatoes in Germany he laid emphasis up- on a scab which was often followed or accompanied by decay. This form of the disease was probably less prevalent in the country as a whole at that time, and the more recent accounts indicate that the ‘‘black speck scab’’ or ‘‘black speck,’’ prop- erly the sclerotial stage, is the feature by which the main type [Vor. 2 434 ANNALS OF THE MISSOURI BOTANICAL GARDEN of the disease is now generally known. At present the following main types of injury are recognized for the potato: (1) black speck scab or selerotial stage, (2) Rhizoctonia scab, (3) Rhizoe- tonia rot, (4) stem lesions and root rot, (5) rosette and leaf roll, and (6) little potato and aerial potato. Black speck is a form of the disease most widely distrib- uted and in itself scarcely merits consideration as a ‘‘dis- ease’’ at all, since the sclerotia are superficial on the tuber, and it is merely the appearance of the potato which is affected. The sclerotia may lead to other types of disease which are more serious. The black specks show up most clearly when the potatoes are wet and it is only at this time that they present the appearance of being black, for, as indicated later, the nor- mal color of the sclerotia is deep brown. It was this form of the disease which first gave evidence of the wide distribution of the fungus in America (Duggar and Stewart, ’01), and it has been shown to exist in practically all potato-producing sec- tions of the United States and Canada. It occurs throughout Europe, especially on the later varieties of potatoes. It is also reported from India, Africa, and Australia, so that it may be assumed to be world-wide in its distribution on this host. It is safe to say that this is the only form of the disease which does not result directly in serious injury and loss to the crop. In the United States, especially from Ohio westward, other forms of the potato disease assume a seriousness nowhere else attained. If all such forms of the disease mentioned below occur in the Atlantic states they are of little consequence. They are, more- over, far less frequent in Europe, India, and Australia. The Rhizoctonia scab is believed to occur as a result of the penetration of hyphae during the early stages of sclerotial development, and occasionally it may be induced by a late growth of new hyphae from old sclerotia. The writer has had an opportunity of examining only casually this form of the dis- ease. It is one of the types doubtless seen by Kiihn. Accord- ing to McAlpine (’11), when this disease occurs, practically every part of the tuber is affected, no normal skin remaining. In severe cases the scab areas may be thrown into folds or puckers and these rub off easily in the form of ‘‘cork dust.’’ Ean r a pih AES ‘ iby a ve ae Faas t ie 1915] DUGGAR—RHIZOCTONIA CROCORUM AND R. SOLANI 435 It is reported that the irritating hyphae are then found at the bases of such scab formations. This scab has been re- ported fairly common in Europe and in Australia. Giissow (’05) seems to refer to the same type in England, and Rolfs (’03) describes it from Colorado. Specific scabs of the potato have been clearly defined and related to particular organisms. The capacity of the tuber to respond with cork formation to varied injuries suggests that in certain modifications of Rhi- zoctonia scab this fungus may accompany other active scab inducing agents. The Rhizoctonia rot is a form of disease which appears rel- atively late in the season when certain conditions prevail, or possibly when the fungus has for one reason or another de- veloped unusual virulence. The disease is supposed to origi- nate either from stem infections, from sclerotia, or from scab areas. In any case penetration of the mycelium occurs to a considerable depth, and according to McAlpine (’11) there is produced in Tasmania a form of the disease known as brown rust, characterized in the early stages by dark spots in the tuber resembling certain symptoms of Phytophthora. It may also be associated more or less with the deeper form of the Rhizoctonia scab. During the latter part of the season a typi- cal stem rot may occur which is not characterized by the definite lesions described later. Instead, the affected cortex slips readily from the wood and about the bark a considerable web of the yellow-brown hyphae may be found superficially, below and just at the surface of the ground, and the pith may be fairly stuffed with the mycelium. Plants only slightly af- fected with this form of the disease, especially when growing on rich garden or muck soil, have been found to yield the collar or Corticium stage. It is not always easy to distinguish as separate forms of the disease, stem lesions, rosette, little potato, aerial potato, rolling, ete., for these types of injury are often associated. All of these types except stem lesions are properly secondary ef- fects, and there is abundant evidence that all represent responses of the plant to disturbed condition or nutrition, sometimes associated with native weakness. It would not be [VoL. 2 436 ANNALS OF THE MISSOURI BOTANICAL GARDEN strange, therefore, if somewhat similar effects should charac- terize, as they do, purely ‘‘physiological’’ disturbances. Stem lesions are generally dark, sunken areas, clearly different from black leg, occurring at the surface of the ground or on any of the underground stems, or tuber-forming stolons. These lesions may result in the early death of the affected plants. Selby (’02, ’03) maintains that generally the lesions upon young shoots are associated with stunted growth and the pro- duction of rosette-like clusters of the upper leaves, as well as with less marked modifications of habit, including slight leaf rolling. Drayton (’15) finds the hyphae in the lesions. If the tuber-bearing stolons are the seat of injury, the food supply is cut off from the young tubers and there may result ‘little potato,’’ a form of the disease which Rolfs (’04) has found to be an important cause of the potato failures in Colorado. Little potato in Australia is considered an evi- dence of underground injuries occurring late in the season. Injuries which effectually girdle the stem, especially if these occur during a moist season or when the crop is frequently irrigated, lead to the formation of aerial tubers. In the re- lation of Rhizoctonia to the various types of potato diseases much remains to be investigated, and Orton (’14) rightly suggests that inadequate attention has been bestowed upon the question of the predisposition of the tubers used as-seed, since it is quite possible that these may yield offspring with tendencies toward rosetting, leaf rolling, and other morpho- logical modifications. ROT OF FLESHY ROOTS The root rot of beet, apparently first described by Eidam (’87) in Germany, and shortly afterward found by Pammel (791) in Iowa, was observed in New York (Duggar, ’99) some years later. Since that time it has appeared epidemically in Nebraska (Lyon and Wianco, ’02) and other western states. The fungus is most virulent during midsummer or later. In- fection may take place at the bases of the leaves or on the fleshy root. The leaf bases blacken, the leaves become paler, and finally wilt. Pammel (’91) has drawn attention to the 1915] DUGGAR—RHIZOCTONIA CROCORUM AND R. SOLANI 437 fact that when fleshy root crops of this type are attacked by such fungi they die gradually, while herbaceous plants (cot- ton, alfalfa, ete.) wilt suddenly. This is probably closely re- lated to the effect of the fungus on the conducting tissues. In the beet root the invaded tissues are pale brown, and often cracks or rifts occur, though rotting may take place without such lesions. Sometimes there is partial recovery after the cracks are formed, and in this case callous tissue is developed. A soft crown rot of the radish induced by this fungus has apparently been reported only once (Duggar and Stewart, 01). A similar disease of the carrot was found in 1900 in New York and this is possibly the disease first reported by Kühn (’58, pp. 241-243), although he did not identify it as due to a Rhizoctonia. STEM AND ROOT ROTS OF HERBACEOUS PLANTS Rhizoctonia Solani produces serious stem and root rots of a number of economic herbaceous plants, among which the following are known to be important: carnation (Dianthus caryophyllus), Sweet William (Dianthus barbatus), bean (Phaseolus vulgaris), sweet-pea (Lathyrus odoratus), and violet (Viola odorata). The carnation stem rot is one of the most destructive dis- eases occurring on this host and is wide-spread in the United States. The general symptoms of the disease on carnation and Sweet William are much the same. The stem is affected at or just below the soil level. The fungus penetrates and kills the cortex which may be readily slipped from the wood. Through the medullary rays the hyphae also enter the pith, which likewise decays. In later stages of the disease the wood shreds, due to the complete penetration by the fungus of all parenchymatic tissues. Several important epidemics of Rhizoctonia on bean have been reported from different parts of the United States. In addition to the outbreak described by Duggar and Stewart (701), Hedgecock (’04), a few years later, found the bean dis- ease severe near St. Louis. The base of the stem and the larger roots bore characteristic ulcerations; pods were af- "y ‘ vt ag ý- on % w "7 [VoL. 2 438 ANNALS OF THE MISSOURI BOTANICAL GARDEN fected, and through the sunken areas of these the hyphae penetrated the seed and produced small sclerotia on the seed- coats. The fungus was cultivated and typical Rhizoctonia hyphae and sclerotia were obtained. Fulton (’08) observed the disease in Louisiana on stems and pods, with the char- acteristic ulcerations, especially at the surface of the soil or just below. He proved the causal relation of the organism through cultures, and inoculations yielded positive results with the damping off of seedlings. McCready (’10) reported the bean disease as new to Ontario, where it was also char- acterized by stem and pod ulcers. In New York Barrus (’10) observed an epidemic of this host in which as many as 30 per cent of the plants were affected. He determined the fungus by cultural studies and proved its pathogenicity by inoculation. On the sweet-pea the disease is mainly a root rot, yet the base of the stem may also be considerably affected before the plant succumbs. On the violet it is primarily a crown disease, but where the plants are succulent and the con- ditions are moist, the leaves are considerably invaded. FRUIT AND LEAF INJURIES In discussing stem diseases the occurrence of Rhizoctonia on bean pods has been mentioned. Another case of fruit injury is described by Wolf (714), who found a severe rot of egg plant fruits from which the fungus was obtained. The pathogenicity of the organism was determined by inocula- tions, and cross inoculation from tomato and potato led to the conviction that the organism was Rhizoctonia Solan. Direct attacks of leaves by Rhizoctonia Solami are infre- quent. From the habits of the fungus this would be expected. The one serious leaf disease reported is that of lettuce (Stone and Smith, ’00), in which the fungus spreads over the whole surface, causing a moist rot. Sclerotia are frequently formed in connection with this affection. It would be anticipated, perhaps, that diseases of a similar nature might be found on other plants with the rosette habit. Leaf stalks are fre- quently invaded, or may be the regions of first attack, in the case of the beet disease. The disease of leaf stalks of rhubarb 1915] DUGGAR—RHIZOCTONIA CROCORUM AND R. SOLANI 439 reported by Duggar and Stewart (’01) is not due to typical R. Solam. MYCELIUM AND SCLEROTIA The morphological characteristics of the hyphae and sclerotia have been adequately described by several writers, but it may be well to summarize some of the more important features. Upon such hosts as the potato, sugar beet, carnation, and others there is more or less de- velopment of an external web, but never over the general root system such a complete invest- ment of roots by a mantle of hyphae as characterizes the violet fungus. The external hyphae are somewhat colored, usually yellow- ish brown, and they are generally of two types. One type may be designated as purely vegetative and another as constituting the external tufts or masses when these occur. All hyphae are prac- tically colorless when young, vac- uolate, more or less irregular, septate with the septa at intervals of 100-2004. The diameter of TEI Es vegetative hyphae is 8-12 u. Fig. 5 5. Rhizoctonia Kolani (Cor- 3 . a m): vegetative Branches arise, and when young these are inclined in the direction of growth and are invariably somewhat constricted at the point of union with the main hyphae (fig. 5). As the hyphae mature and become more deeply colored they are more uni- form and rigid, the distances between cross walls are greater, the constrictions where branches arise less marked, and the branches are approximately at right angles to the main hypha. On certain affected plants a short tufted or mealy growth occurs and this is made up of hyphae of very different char- acteristics. In the young condition threads are profusely ypha and small rg from artificial aiias on pota [VoL. 2 440 ANNALS OF THE MISSOURI BOTANICAL GARDEN branched and lobed, sometimes botryoid, and they are ulti- mately divided into short, ovate cells, arranged in short chains, or elbowed, and producing branches in a more or less dichotomous fashion (figs. 7 and 8). In culture the denser F . Rhizoctonia Kolani : Fig. 7 Vordative hyphae. Rhizoctonia Solani: a, young hyphae from young sclero- tial tuft on lettuce; b, older cells from same source, masses give rise to sclerotia. With maturity these hyphae. be- come light brown in color, they break up readily into short hyphal lengths or single cells, the individuals of which bear some resemblance to conidia. However, they could not easily be mistaken for spores, although they may function as such inasmuch as most of them may germinate within a few hours when placed under suitable conditions. I have previously de-. seribed (’99) this process as follows: “So far as observed, germination is always by the protrusion of a tube through a septum. En several cells are connected, protoplasm, and from neighboring protoplasmic cells the germ 1915] DUGGAR—RHIZOCTONIA CROCORUM AND R. SOLANI 441 tubes seem to pass into such empty cells as readily as N into the nutrient solution. When the germ tube is from 10 u to 20 yw in length, it is invariably narrowed towards the outlet from the parent cell, and a septum forms at a short distance from this outlet.’ Fig. 8. Rhizoctonia Solani: En ag moniliform, and elbowed cells from tufted growth in artificial culture The hyphae which penetrate the tissues remain colorless so long as they are in active growth, and while generally less in diameter they present much tba same appearance as the young external hyphae. In the different strains which have been studied, originating from different hosts, certain minor modifications of the general habit of the fungus in culture have been observed. But these have not seemed to be suff- [vor. 2 442 ANNALS OF THE MISSOURI BOTANICAL GARDEN cient to be considered of specific importance, except in the case of the form on the rhubarb. In general, the differences referred to consist in a variable amount of the mealy or tufted growth, or of the amount of aerial growth; differences in the color of the colony are also observable; and the rapidity with which selerotia are formed are all minor distinguishing features. The subject needs further investigation, but in gen- eral it is felt that these differences are such as might be due to permanent differences in the pathological strains, on the one hand, or may be regarded as temporary differences due to the recent environment, on the other. It may be pointed out that the appearance of the mycelium of the beet fungus from the damping off seedlings is not exactly comparable with that of the mycelium derived from the beet rot. When the organisms from both sources are grown in culture they are found to be identical. Strains do occur, however, evidence of which may persist for some time in the general appearance of the cultures. The exact conditions under which sclerotia may occur on the various hosts affected have not been determined. It has been noted that affected potato tubers are the main seats of selerotia formation when the fungus attacks that host. Upon this plant they are typical, and the numerous illustrations published are sufficient evidence that the appearance is much the same under a variety of conditions. Special attention may be called to the illustrations of Duggar and Stewart (701), Rolfs (’02), Duggar (’09), McAlpine (’11), Pethy- bridge (711), and Morse and Shapovalow (714). On the majority of hosts, however, sclerotial formation is relatively rare. From the various illustrations referred to it will be seen that the sclerotia vary in size from those so minute as to be scarcely visible, to others which may be a centimeter or two in diameter. They are generally more or less flattened, irregular, deep chestnut-brown, and generally smooth on the surface (that is, free from a looser growth of investing hyphae). Smoothness of sclerotia, which has been regarded by Kiihn as of much diagnostic value, should not be considered 1915] DUGGAR—RHIZOCTONIA CROCORUM AND R. SOLANI 443 an important character except under natural conditions. Sclerotia which develop on fleshy organs in moist chambers as well as those which develop in culture show to a certain degree, a semi-persistent hyphal investment; but such invest- ing hyphae are readily worn away, whereas in the violet fungus they are truly persistent. Sections of the denser sclerotia exhibit a fairly homo- geneous structure (fig. 9), with the cells more uniform in size and appearance than in Rhizoctonia Crocorum. Fig. 9. Rhizoctonia Solani: a, from a section of sclerotium on potato; b, cells isolated by maceration of sclerotium. THE BASIDIOSPORE STAGE, SYNONOMY, AND MATERIAL EXAMINED Besides suggestions of a general nature no indications regarding the perfect stage of Rhizoctonia Solani were made prior to the discovery of the Corticium. Prillieux and Dela- croix (’91) described Hypochnus Solani from potato stems, and although at this time the Rhizoctonia diseases were known in Europe no connection with this Hypochnus stage was suspected. The characteristic collar of mycelium was found surrounding the stem just above the surface of the ground, but they found nothing to indicate that the fungus had injured particularly the plant affected. Rolfs (’03) found the collar fungus during his studies of potato diseases in Colorado. The material was determined by Prof. E. A. Burt as referable to the species Corticium vagum B. & ©. On account of the parasitic habit, however, [VoL. 2 444 ANNALS OF THE MISSOURI BOTANICAL GARDEN it was considered advisable to make the fungus a variety of the Berkeley and Curtis species, so that it was written Cor- ticium vagum B. & C. var. Solani Burt. Prof. Burt also recog- nized that it agreed closely with, and might be identical with, Hypochnus Solan Prill. & Del. This conclusion the writer accepts, but in view of the fact that Professor Burt is pre- paring a monograph of the Thelephoraceae, I shall not dis- cuss this point; for the same reason I need only express doubt regarding the validity of Shaw’s suggestion that Hypochnus ochroleucus Noack and Corticium vagum B. & C. are identical, although there is a certain similarity in the various stages. Rolfs (’04) was able to germinate the basidiospores and to develop characteristic Rhizoctonia hyphae from these. Riehm (’11) also reported germinating the basidiospores and producing a characteristic Rhizoctonia mycelium together with the formation of sclerotia. Pethybridge (’15) gives a more complete account of mycelial production from spores. The herbarium and fresh material which has been examined and found to agree with the authentic descriptions of Rhizoc- tonia Solani Kühn (Corticium vagum B. & C.) may be briefly enumerated : Exsiceati: Rhizoctonia Napaeae nov. sp., Westendorp and Wallays, Herb. Crypt. Fase. 5: 225. (On decaying turnips which had been stored in a cave.) American material: Hyphal stages on numerous hosts, many of which are mentioned in this paper, also others not included; sclerotia, on potatoes grown throughout the eastern and central United States, on potato stems (New York, 1900), on bean pods (New York, 1910), also on carnation stems, let- tuce leaves, ete. Corticium stage from Prof. F. H. Rolfs, Colorado, 1901, on potato stems; from Dr. I. C. Jagger, Rochester, New York, 1914, on potato stems and on crown of carrot; from herbarium of Prof. E. A Burt, material on moist soil and decayed wood, collected by Prof. Farlow, Mag- nolia, Mass., 1903; from Herb. Mo. Bot. Garden, Nos. 44679, 44681, and 44682; collected by Dr. Geo. L. Peltier, Urbana, Il., 1915. European material: Sclerotia on potato tubers from Prof. 1915] DUGGAR—RHIZOCTONIA CROCORUM AND R. SOLANI 445 Sorauer, Berlin, 1900; from Prof. Magnus, Berlin, 1901; from Prof. Delacroix, Paris, 1901; and material secured on the markets of various cities, 1905-06. As far as the writer has been able to determine, the fol- lowing synonomy may be listed for Corticium vagum B. & C.: Rhizoctonia Solanı Kühn (1858). Rhizoctonia Betae Eidam [non Kühn] (1887). Rhizoctonia Napaeae West. (1846). Rhizoctonia Rapae West. (1852). Hypochnus Solani Prill. & Del. (1891). PREVENTION AND CONTROL Much the same situation confronts us regarding the pre- vention and control of Rhizoctonia Solani as in the case of R. Crocorum. The presence of the fungus in practically all soils serves to emphasize the importance of cultural methods including drainage and sanitation. In this case, however, since the fungus is of so much importance in the seed bed and in the greenhouse special preventive measures may be prac- tised. Selby (’06) found that the treatment of the seed bed with formalin (1:160 to 1:200) proved satisfactory in most cases. In general, the best results have been obtained by steam sterilization, and where the facilities are at hand it is practicable to apply this to any type of greenhouse work, and, in certain cases, to seed beds outside. Liming has been recom- mended for the control of the disease in the field, but this has “not been uniformly successful, and cultural studies have shown that the fungus is able to withstand a high percentage of alkalinity. Nevertheless, when liming results in the im- provement of physical and sanitary conditions of the soil it undoubtedly assists in restraining the activity of the fungus in an indirect way, possibly by raising the resistance of the host. Even though the fungus may be widely distributed, it is advantageous to plant clean ‘‘seed.’’ This applies particu- larly to the case of the potato. The presence of the sclerotia upon the tuber makes possible the early spread of the fungus [voL. 2 446 ANNALS OF THE MISSOURI BOTANICAL GARDEN to the young shoots. It has been positively determined that the more effective tuber treatment is the standard corrosive sublimate solution, as for potato scab. In all cases, however, it would be better to employ seed which are not infected, if this is possible. CONCLUSIONS AND NOTES In the account already given of Rhizoctonia Crocorum per- haps sufficient discussion of the occurrence and the character- istics of this form has been entered upon, except in the way of a direct comparison between this species and R. Solani, subsequently included. Further work upon the first named species should consider especially the culture of this organ- ism, inoculation experiments, the development of the organ- ism as it occurs on several hosts, the formation of sclerotia and infection cushions, and the confirmation or more definite declination of Eriksson’s view that the fungus is referable to Corticium (Hypochnus). From the study of this organism thus far the following conclusions seem justified: 1. The views of L. and C. Tulasne that the forms of Rhi- zoctonia on crocus, alfalfa, and other hosts may be included in a single morphological species is confirmed. 2. The correct name of the violet root felt fungus, so long as a spore stage remains uncertain, is Rhizoctonia Crocorum (Pers.) DC. 3. This organism occurs throughout a considerable part of Europe and has been found in a few localities in America. 4. It attacks a variety of plants representing many fam- ilies, mostly dicotyledonous. 5. The mycelium and sclerotia exhibit no important dif- ferences in equivalent stages on the different hosts, but large sclerotia which form freely in contact with crocus, and often near the affected roots of alfalfa, are seldom observed in con- nection with the attacks upon beets, carrots, and some other hosts. 6. The existence of distinct forms or races of this species requires further extended study. eS ee ee e 1915] DUGGAR—RHIZOCTONIA CROCORUM AND R. SOLANI 447 7. The organism has not yet proved culturable with the usual laboratory methods. 8. At the present time there is insufficient evidence to de- termine what the perfect stage of this organism may be. Obviously much still remains to be done regarding the physiological, pathological, and taxonomic relationship of the culturable forms which in the vegetative stage may be re- ferred to the form-genus Rhizoctonia. The writer has grown in culture Rhizoctonia from twenty-three different American hosts, most of which are mentioned by Duggar and Stewart (701). Most of these were grown upon a variety of culture media including prune juice, beet, and potato agar; also beans, stems and pods, celery, sugar beet and potato cylinders, and corn meal mush. With one exception (the organism from rhubarb) the cultural characteristics have been sufficiently similar, especially after protracted culture in the laboratory, to suggest a single species, with characteristics of the beet and cotton fungus, already sufficiently described (Atkinson, 92, ’95; Duggar, ’99). Moreover, these cultural studies have confirmed in all cases the conclusions tentatively arrived at from the preliminary microscopic examination of the fungus on the different hosts. Reasons have already been given to indicate why this. species is properly R. Solani. It is recog- nized, however, that much culture and inoculation work is necessary to establish the point that the fungus on the various hosts is the same species, and to determine to what extent physiological forms may occur. The following brief summary of conclusions may be pre- sented with regard to Rhizoctonia Solani: 1. The common American species of Rhizoctonia is R. Solanı Kühn. 2. This fungus is widely distributed in America and else- where, and would seem to occur on the potato in most regions of the world where this crop is a staple product. 3. The host plants represent many families of dicot- yledons, Asparagus Sprengeri being the only monocotyle- donous host thus far reported. [VoL, 2 448 ANNALS OF THE MISSOURI BOTANICAL GARDEN 4. The types of disease induced are most diverse, damp- ing off and root and stem rots being the most important direct effects. Secondary effects have been studied only in a few localities. 5. The mycelium and the sclerotia, as well as the general appearance on the host, readily distinguish the fungus from Rhizoctonia Crocorum (Pers.) DC. 6. The organism is readily eulturable by the usual labora- tory methods. 7. The evidence seems clear that the perfect stage of this organism is Corticium vagum B. & ©. It is to be regretted that the fungus causing a disease of rhubarb (Duggar and Stewart, ’01) was lost before adequate study could be bestowed upon it. The fungus bore a close resemblance to Rhizoctonia, but the aerial hyphal cells were shorter and of greater diameter than those of R. Solam. No selerotia were found on the host, and they did not develop in culture. Shaw (’13) has contributed interesting notes on diseases of plants in India attributed to two species of Rhizoctonia. Unfortunately, however, he has added to the general con- fusion regarding this subject by a preliminary discussion which does not sufficiently designate the forms referred to, but more especially by the advancement of certain ideas re- garding species which are made, apparently, without adequate study of material from other countries. The conclusions ar- rived at are necessarily at variance with our present knowl- edge of the forms of Rhizoctonia. Of the organisms producing diseases in Indian crops he refers to Rhizoctonia Solani Kühn, a fungus which he found on jute, mulberry, cotton, groundnut, and cowpea. The mode of branching of young hyphae of his fungus is characteristic of R. Solani, but with this the resemblance apparently ceases. Basing an opinion wholly upon his descriptions and figures, the adult mycelium (Shaw, ’13, pl. 7 and 8) differs from R. Solani (1) in being usually much finer; (2) in the abundant development of short ‘‘barrel-shaped”’ cells in the ordinary ae i 1915] DUGGAR—RHIZOCTONIA CROCORUM AND R, SOLANI 449 vegetative mycelium, which would seem, from his figures, to have little in common with the chain-like, ovoidal, often branched or lobed cells (designated ‘bale shaped’’ by Balls) of R. Solani (see Atkinson, ’92, ’95; Balls, ’05, ’06; Duggar, ’99; Duggar and Stewart, ’01; and others); and (3) in the verrucose or warty, wall markings (Shaw, ’13, pl. 8, figs. 2-3), all of which indicate some other fungus. Again, the development of sclerotia (Shaw, ’13, pl. 8, fig. 4) discloses a type of hyphal cell not characteristic of R. Solani; and the small discrete sclerotia themselves (Shaw, ’13, pl. 2, fig. 3, pl. 8, fig. 1) convincingly indicate that another fungus was under consideration. I can find no record of a description of sclerotia resembling these in the literature of Rhizoctonia diseases. I am at a loss to understand how a fungus with such characteristics could be likened to Kühn’s fungus on the potato, even though depending upon Kühn’s imperfect de- scription. On the other hand, neither in general appearance nor in structure (as described and figured by Shaw) am I able to find any resemblance to the ‘‘small selerotia’’ or in- fection cushions of R. Crocorum (R. violacea). In moist situations the sclerotia of Rhizoctonia Solanı may occur on aerial organs (as on the pods of beans, Hedgecock, ’04, on lettuce leaves, Stone and Smith, ’00) but the frequent and apparently normal occurrence of minute sclerotia, fairly regularly arranged, on the dead tips of stems, as described by Shaw, finds no parallel in R. Solan. Again, in regard to the hyphae, it may be said that while there is a characteristic location of the septum when a branch is formed in a hypha of Rhizoctonia, this character alone is not sufficient to identify the fungus. It is necessary to take into consideration all of the mycelial characteristics which have been referred to, and if possible also the cultural characters. The writer finds that the ‘‘Rhizoctonia type’’ of branching is more or less similar to that found in the hyphae of certain species of Sclerotinia, Morchella, Pleospora, Rosellinia, and many others. It would be unwise to offer any definite suggestions regarding the fungus described by Shaw and referred to above. What rela- tion it may bear to the fungus of ‘‘bangle blight’’ (Cunning- (VoL. 2 450 ANNALS OF THE MISSOURI BOTANICAL GARDEN ham, ’97) must also remain, for the present, uncertain. It is possible that Shaw’s fungus is one of the Ascomycetes, at least this is suggested by the figures of the sclerotia. In my opinion Shaw has correctly referred to Corticium vagum B. & ©. (accordingly to Rhizoctonia Solam Kühn, representing the vegetative phases of that species) another fungus which he also found in India on the groundnut and cowpea. Both the mycelium and the sclerotia of this second organism as described by him agree with R. Solan as we know it on carnation, beet, bean, lettuce, potato, ete., in America and elsewhere, as far as reported. The descriptions and measurements of basidia and spores are also in sufficient accord. Shaw has even suggested that Rhizoctonia violacea Tul. is the vegetative stage of Corticium vagum B. & C. No such unfortunate confusion could result, however, had he been able to study that which is accepted as Kiihn’s organism on the potato together with the violet root felt fungus of Europe on any of its hosts. He has obviously failed to find material of the last named fungus in his studies thus far. Between Rhizoctonia Crocorum and R. Solanı in the vege- tative condition some of the important and easily observed contrasting features as usually found are presented in the following table: Rhizoctonia Crocorum Rhizoctonia Solani An external felt, or mantle, of External mycelium, if notice- investing h yphae, confined able, only a web, or some- almost exelusively to under- times with flaky tufts, the ground organs. formation of a “collar” oc- curring only at the time of fruiting. Color of mycelial felt pink-red Color of web, if evident, dirty or violet to violet-brown yellow to yellow-brown. with age. Protoplasm of young hyphal Young hyphal cells hyaline, cells soon develops a violet and even when flavous later, reddish pigment. pigment confined to walls. Infection cushions conspicu- Nothing comparable to infec- ous in the root-investing tion cushions, though on mycelium on most hosts. potato sclerotia may serve as points of infection. 1915] DUGGAR—RHIZOCTONIA CROCORUM AND R. SOLANI 451 Sclerotia, when present, Sclerotia normally free from ensely wooly with invest- any definite or permanent ing mycelium and filaments investment of mycelium, or of short, ovoidal or ellip- filaments of elbowed hyphal tical hyphal cells. Internal cells. Internal structure structure not truly plec- homogeneous in the larger, tenchymatic, cells variable denser sclerotia. n size, Cultures difficult—not yet ob- Cultures readily obtained on tained by usual methods. any nutrient medium. Typically a parasite, with per- Grows rapidly saprophytic- haps the possibility of con- ally on the invaded host, tinuing existence only for a and apparently on debris in time saprophytically. the soil when conditions are favorable. The following species may be excluded from Rhizoctonia as far as can be judged from reference to the descriptions and to the exsiccati material examined: Rhizoctonia Allii Graves, de Thuemen, Myc. Univ. Fase. 6: 600 (obviously not closely related to the forms here dis- cussed). R. bicolor Ell. N. Am. Fung. Fase. 10: 977 (with sclerotia like those of a Botrytis, e. g., B. cinerea). R. Bras- sicarum Lib., Libert, Pl. Crypt. Arduennae, Fase. 3 : 240 (no characteristics of Rhizoctonia). R. muscorum Fr. Ellis, N. Am. Fung. Fase. 13 : 1266; Libert, Pl. Crypt. Arduennae, Fase. 2 : 141. From the descriptions alone it would seem that the follow- ing species have insufficient affinities with Rhizoctonia to be included, but critical study of material is needed: Rhizoctonia aurantiaca Ell. & Ev. on decaying wood of Acer; R. Batatas Fr. on Ipomoea Batatas; R. placenta Schw., and R. radiciformis Schw., on decaying wood (the three last mentioned are distributed in Schweinitz’, Syn. N. Am. Fung., to which, however, the writer has not yet had access); R. destruens Tassi, reported parasitic on five species of Del- phinium, and on Lobelia laxiflora, and Hibiscus rosa-sinensis; R. moniliformis Ell. & Ev. on branches of Nyssa. Rhizoctonia Strobi Scholz (’97) on roots of Pinus strobus in Austria, is insufficiently described to warrant a suggestion; and R. subepigea Bertoni (’97) on coffee should be included in a further comparative study. [VoL. 2 452 ANNALS OF THE MISSOURI BOTANICAL GARDEN BIBLIOGRAPHY ~~ G. F. (92). 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(’11). Rhizoctonia rot, or potato collar fungus. Handbook of fungous diseases of the potato in Australia and their treatment. pp. 60-65. pl. 9-13, 19, 39-43. 1911. (cf. pp. 75-77.) a ri S. B. (710). Pod and stem rot of ers ae ath Ont. Agr. and Exp. Farms, Rept. 36 : 46-47. f. 6. Montagne ie ee ). Etude micrographique de la s, du safran, connue Tacon. Soc. de Biol., Paris, Mém. Pea e 1850. [Translation by Berkeley, Jour. Hort. Soc. London mg 21-25. 1850 mn W. J. and Shapovalow, M. (’14). The Rhizoctonia disease of potato. Agr. Exp. Sta., Bull. 230 : 193-216. f. 61-73. 1914 Nees von eg TORR Das System der Pilze und Schwämme. p. 148. Wurzburg, 1 [vor. 2 456 ANNALS OF THE MISSOURI BOTANICAL GARDEN sata? and related diseases. U. S. Dept. . Bull. 64: pp. 40-41. pl. u L. H. (’91). Fungous diseases of the sugar beet. Ia. Agr. Exp. Sta., Bull. 15: 234-254. pl. 1-7. 1891. [See Preliminary notes on a root-rot disease of sugar beets. pp. 243-251. pl. 3-6.] RN = (797). Il mal vinato della medica e delle barbietole. Bol. di Entom. Agron. e Patol. Veg. 4 : 367-369. Padua, 1897. Peltier, G. L. (’14). Rhizoctonia in America. Phytopath. 4; 406, 1914. Persoon, C. H. (1801). Synopsis methodica fungorum. pp. 119-120. 1801. Pethybridge, G. H. (’10). Potato —. in Ireland. Dept. Agr. and Tech. Instr. for Ireland, Jour. 10: 1-18. f. 1-8. 1910. —, (’l0a). A little known potato disease. Garden 74:560. f. 1. 1910. ———, (’11). Investigations on potato diseases (second oe Dept. Agr. and Tech. Instr. for Ireland, Jour. 11: 29-32. f. 11-14. 1 ——, (15). “Black speck” scab and “collar fungus.” Ibid. 15:513-517. 1915. Pierson, W. R. (702). Sterilized soil for stem rot. Gardening 10: 179-181. 1902. dae = (700). Molestias eryptogamicas da batata ingleza e seu tractamento. da Agr. Estado de Säo Paulo 1 :45-48. 1900. [See p. 4 wre E. (’83). Etude we ge maladies du safran. Ann. de l’Inst. Nat. 6: 17-31. pl. 3-4. ————, (91). Sur la pénétration de la Rhizoctone violette dans les racines de la betterave et de la luzerne. Compt. rend. acad. Paris 113: 1072-1074. 1891. _—, (’96). Ibid. Soc. Bot. de France, Bull. 43 :9-11. f. 1. 1896. , (97). Maladies des plantes agricoles 2 : 144-157. f. 282-287. 1897. ————., et Delacroix, G. ve Hypochnus Solani nov. sp. Soc. Myc. France, Bull. 7: 220—221. f. 1. 1891 Prunet, A, (’93). Sur le Rhizoctone de la lucerne. Compt. rend. acad. Paris 117: 252-255. 1893. nn . 11). Ueber den Zusammenhang zwischen Rhizoctonia Solani Kühn Hypochnus Solani Prill. u. Del. K. Biol. Anstalt f. Landw.- u. Forstw., Mitt. 6:p. 23. Berlin, 1911. Rolfs, F. M. ae Corticium vagum B. and C. var. Solani Burt. Science, N. S. 18: 729 on = 04). Potato failures. Colo. Agr. Exp. Sta., Bull. 70 : 1-20. pl. 1-12. 1902; 91: 1-33. pl. 1-5. 1904. ——, (05). (Tomato un en vagum (B. and C.). Fla. Agr. Exp. Sta., Rept. 1905 : 46-47. A E. (’86). Undersgelser a. Ar tele Rhizoctonia. K. . Vid. Sels. Forhandl. 1886 : 59-77. pl. 1-2. 1915] DUGGAR—RHIZOCTONIA CROCORUM AND R. SOLANI 457 wie) bo (796). Observations sur le rhizoctone de la pomme de terre. Compt. d. acad. Paris, 123: 1017-1019. 1896 —, (97). La maladie de la gale de la pomme de terre et ses rapports avec le Rhizoctonia Solani Kühn. Soc. Myc. de France, Bull. 13 : 23-28. 1897. Saccardo, P. A. (’99). Syll. Fung. 14: pp. 1175-1177. 1899. Salmon, E. S. (’08). Disease of seakale, Gardeners’ Chronicle 44 : 1-3. f. 1-3. 1908. , and Crompton, T. E. (’08). The Rhizoctonia disease of seakale. S. E. Agr. Coll., Wye, Jour. 17 : 348-353. pl. 21-25. 1908 Scholz, E. R. (’97). Rhizoctonia Strobi, ein neuer Parasit der een K. K. Zool.- Bot. Akad. Ges., Verhandl. 47 : 541-557. f. 1-6. 1897. Selby, A. D. (02). A disease of potato stems in Ohio, due to Rhizoctonia. Science, N. S. 16: 138. 1902. —, (0 2: A rosette disease of potatoes. Ohio Agr. Exp. Sta., Bull. 139 : 51-66. f. 1-5. 1903. —, (0 06). Studies in potato rosette II. Ibid. 145 : 13-28. f. 1-4. 1903. (et > Cire. 57 : 1-7. 1906; and 59 : 1-3. 1906.] —, (’04). Tobacco diseases, bed rot. Jbid. 156: pp. 97-99. pl. 1. 1904. Shaw, F. J. F. (13). The morphology 7 fee of Rhizoctonia. Dept. Agr. India, Mem. 6: 115-153. pl. 1-11. 1913. Sorauer, P. (’86). Pflanzenkrankheiten. pp. 354-361. 1886. (2d ed.) ———, (08). Ibid. 2:471-474. 1908. (3d ed., revised by Lindau 1908.) Stevens, F. L. (713). The fungi bar on plant disease. Rhizoctonia. pp. 406-408. f. 298-294; pp- 659-600. ——, and Hall, J. G. Hoel Hypochnose of pomaceous fruits. Ann. Myco- logici 7: 49-59. f. 1-8. ———, and Wilson, G. W. (’11). N.C. Agr. Exp. Sta., Rept. 1911 : 70-73. 1911. Stewart, F. C., French, D. T., and Wilson, J. K. (’08). Troubles of alfalfa in New York. N. Y. Agr. Exp. Sta., Bull. 305 : 330-416. pl. 1-11. 1908. [See Root-rot and damping off. pp. 392-393.] Stift, A. (700). Der Wurzeltödter oder die Rotfäule der Rüben oe hun. violacea Tul.). Die Krankheiten der Zuckerriibe. pp. 67-72. pl. 8-9. Wie 1900. (13). Zur — des Wurzeltödters oder der Rothfiiule e octonia violacea Tul.). Oesterr.-Ungar. Zeitschr. f. Zuckerind. u. Landw 42 : 445-461. 1913. Stoklasa, J. (°98). Wurzelbrand der Zuckerrübe. Centralbl. f. Bakt. II. 4: 687-694. 1898. Se: = E. and Smith, R. E. (’00). The rotting of greenhouse lettuce. Mass. . Exp. Sta., Bull. 69 : 1-40. f. 1-10. 1900. [See A Rhizoctonia disease of ie pp. 16-17. f. 8-10.] ———, ———-,, (02). Carnation stem rot. Mass. Agr. Exp. Sta., Rept. 14: 67-68. 1902. Tassi, F. (’00). Di una 2 mes Bul. de Lab. ed Orto Bot. Siena 3: 49-51. pl. 4, f. A-M. [VoL. 2, 1915] 458 ANNALS OF THE MISSOURI BOTANICAL GARDEN a E J. (714). The diseases of the sweet pea. Del. Agr. Exp. Sta., Bull. 1-93. f. 1-43. 1914. [See Root rot. pp. 18-27. f. 9-11.] Tubeuf, K. von (’97). [Trans. by W. G. a Diseases of plants induced by cryptogamic parasites. pp. 201-202, 1897 Tulasne, L. et C. (’62). Fungi Hypogaei. pp. 188-195. pl. 8, f. 4; pl. 9; pl. 20, f. 8-4. 1862. Van Hook, J. M. (’04). Diseases of ginseng. Cornell Univ. Agr. Exp. Sta., Bull. 219: non f. 18-42. 1904. [See Damping off by Rhizoctonia. pp. 289- 291. f. 32-33.] Webber, H. J. (’90). of the flora of Nebraska. Nebr. State Bd. Geol., Rept. 1889 : p. 216. 1890. pime. H. H. and a (712). The diseases of ginseng and their ontrol. U. S. De ept. Agr., Bur. Pl. ae Bull. 250 : 1-44. pl. 1-12. 1912. [See Damping-off of seedlings. pp. 22-23.] Westendorp, G. D. (52). Notice sur poe, Bul Cryptogames — ou nouvelles pour la flore belge. Acad. Roy. , Bull. 18? : p. 402. 1852. Winter, G. Die Pilze. [In Rabenhorst’s Cryptogamen-Flora von Deutschland, Oesterreich, u. d. Schweiz 1? : p. 277.] Wolf, F. A. (’14). Fruit rots of egg plant. Phytopath. 4:38. 1914. line ir H. W. (13). Pilzparasitäre Welkekrankheiten der te sa . d. deut. bot. Ges. 31: 17-33. 1913. [See Rhizoctonia. p. 30.] SOME RELATIONS OF PLANTS TO DISTILLED WATER AND CERTAIN DILUTE TOXIC SOLUTIONS M. C. MERRILL Formerly Research Assistant to the Missouri Botanical Garden I. INTRODUCTION In view of the extensive use of distilled water as a medium in which to grow control plants for comparative purposes in solution-culture work, there is well-grounded justification for the performance of considerable experimental work in order to determine more definitely the relations of plants to this medium. The subject is an important one, and it will require much experimentation for the ultimate solution of all phases of the problem involved. While the results herewith reported are only preliminary in their nature, the fact that they give positive indications along certain lines has been deemed sufficient warrant for their publication at this time. In addi- tion to determining the growth relations of plants in this and other media, consideration has also been given to the effect produced by growing plants in this medium as determined by means of electrical conductivity measurements. II. HISTORICAL ASPECTS or THE SUBJECT The relation of plants to distilled water is a matter that has been under more or less serious consideration at differ- ent periods for a long time. Woodward (1699), who first em- ployed the method of water eulture in 1691-1692 in his interest- ing experiments, found that plants grew better in river water than in either rain water, spring water, or distilled water. The difference was of course due to the quantity of plant food contained in the medium, and this idea, coupled also with the character of the nutrients, has been the basis for a vast amount of physiological work since that time. Coming down to more modern times, there has been a diversity of opinion among the investigators of the subjeet ANN. Mo. Bot. GARD., VOL. 2, 1915 (459) [VoL. 2 460 ANNALS OF THE MISSOURI BOTANICAL GARDEN in regard to the reason why plants and animals thrive so much better in natural water or aqueous media than in dis- tilled water. Considering the period from about 1860 on down to the present, the most important explanations offered may be summed up under the following three heads: 1. Lack of essential nutrients; 2. The presence of deleterious substances ; 3. Extraction of salts, or nutrient materials, from the organism immersed in the distilled water. Holding each of these views there has been a formidable array of scientists at different periods, each group contend- ing strongly to establish the correctness of its viewpoint. Among the earlier workers in the field may be mentioned Boehm (’75), Dehérain (’78), and others, who believed that the lack of essential nutrients in the distilled water was responsible for the resulting poor condition of the organism. Boehm, for example, believed that calcium played a funda- mental rôle in the metabolism of the plant, and that in its absence certain processes, notably that of starch formation, could not be carried on and that therefore deterioration re- sulted. He also believed that calcium was necessary for the transfer of the reserve materials from the cotyledons to the formative organs. Dehérain repeated Boehm’s experiments and confirmed his results. | Owing to the fact that even distilled water, which had been unquestioningly regarded as pure, produced effects simulat- ing toxieity, a great deal of attention has been given in the past to the chemical and other properties of water distilled from different kinds of apparatus and under various condi- tions. On the animal side, workers, among whom may be mentioned Kölliker (’56) and Nasse (’69), had early noticed the injurious effeets on tissues when the same were placed in distilled water. Nasse, for example, found the deleterious effeet of distilled water about equal to that of the following solutions: 2.5 per cent NaCl, 3.3 per cent NaBr, 3.7 per cent Na»SO,, and 5.0 per cent Nal. Nägeli (’93), in his classical work published twelve years after his death, found that very minute amounts of toxic sub- 1915] MERRILL—DISTILLED WATER 461 stances, notably copper, in solution produced injurious ef- fects on organisms (Spirogyra), and to this phenomenon he applied the term ‘‘oligodynamik”’ action. This line of work was extended to include other substances and other organisms, and claimed the attention at different times of Aschoff (’90), Loew (’91), Locke (’95), Ringer (’97), Copeland and Kahlen- berg (’99), Dehérain and Demoussy (’01), Lyon (04), Bokorny (’05), Hoyt (’13), and others. It is of particular interest to note that Ringer in some of his earlier work ascribed the injury to the extraction from the organism of necessary nutrient materials; but after the publication of Locke’s experiments (’95), which Ringer duplicated and con- firmed, the latter concluded that the injury done in the par- ticular case under consideration (Tubifex) was due to dele- terious materials in the distilled water. He says: ‘‘Copper in even infinitesimal quantities will disintegrate tubifex whilst water free from copper or other heavy metals and without any salts such as calcium salts can sustain the life of tubifex.’’ In regard to the third idea pertaining to the effects of dis- tilled water on organisms, early workers, both on the plant and animal side, found that salts were extracted from organisms placed in distilled water, even though their methods for determining the extraction were somewhat crude. Among the early investigators on the animal side may be mentioned Plateau (’83), Ringer and his school (’83, ’84, ’85, ’94, 94°, 94”, ’97), Loeb (’03), and others. The writer has another paper ready for publication in which is given a historical treat- ment of the subject of excretions from roots and other plant parts, so the discussion of certain phases of the plant work is reserved for that publication. Upon the perfection and the employment of conductivity apparatus by physical chemists, it soon began to be used also by the various workers in the fields of soil, plant, and animal investigations. In this connection distilled water came in for its share of consideration. The determination of the purity of water by ascertaining its electrical conductivity speedily came into vogue, and it should be said that as far as elec- [Vor. 2 462 ANNALS OF THE MISSOURI BOTANICAL GARDEN trolytes are concerned it is a very accurate and excellent method and has deservedly come into more and more general use for this purpose in the fields of chemistry, physics, and biology. Koeppe (’98), for instance, determined the electrical con- ductivity of water obtained from various sources and com- pared his results with those of other workers. He believed that distilled water has a deleterious effect which is partly due to a withdrawal of salts necessary to the organism, and partly to a swelling of the tissues. He was supported in his views by Oldham (’09), while Winckler (’04), Kobert (705), and others argued in favor of the harmlessness of dis- tilled water, especially in medical practice. Peters (’04) used the electrolytic conductivity method in his work on Stentor and found that there was an exosmosis of electrolytes when the organism was placed in distilled water, and he therefore concluded that the injurious effects noted were due to an extraction of salts. True and Bartlett (712, 715, ’15") considered, for certain salts, not only the excretion but also the absorption of electrolytes under balanced and unbalanced conditions of the medium. In a recent paper in which a historical discussion of the subject is also given, True (714) concludes that over and above any injurious effects caused by deleterious substances in the distilled water there is still a ‘‘residuum of harmful action due to no known type of impurity.’’ Because this harmful action seems to be most marked in water of least conductivity True believes that the withdrawal of electrolytes from the root tissues best accounts for the deleterious action, but that this withdrawal is ‘‘not due to the aggregate differ- ence in osmotic pressure between the cells of the roots and the external medium.” He chose lupine seedlings for his work because Frank (’88) had found them very sensitive to distilled water. Schulze (’91), however, after several years of experience with Lupinus luteus, claimed that distilled water produced no toxic effects upon those plants. Both before and after the appearance of the recent con- tribution by True just referred to, I carried on the investi- 1915] MERRILL—DISTILLED WATER 463 gations reported in this paper, which, as previously stated, are but preliminary in their nature, but which have given indications leading to the conception of an idea differing somewhat from the majority of those above mentioned re- garding the relation between plants and distilled water. This conception will be briefly mentioned here, while the evidence and a further discussion will be given later; it is that pure distilled water is not harmful or injurious per se, but that because of the static condition forced upon them as a con- sequence of the absence of plant food, the growing cells become disorganized and thus become easy prey to bacterial and fungous action. Excretion of electrolytes does occur but this should be considered merely as a concomitant condition, or resulting effect of the conditions under which the plants are placed, and should not be considered as a cause of degen- eration unless the electrolytes themselves be toxic. III. Merxops (GERMINATION, CULTURE, AND CONDUCTIVITY) Canada field peas (Pisum sativum) and horse beans (Vicia faba), the small variety, were the plants selected, as both were known to be well adapted for growth in solution cultures. Of the various methods of seed sterilization tried out, the one in which the seeds were treated with 1-600 formalin-water for 15 minutes after being soaked for 24 hours in running water gave best satisfaction. For germinating the seeds a modification of the method used by Boussingault (’74), and also by various investigators in the Bureau of Soils, was employed. This consisted in the use of ordinary enameled-ware pans about 12 inches in diameter and 3 inches in height, filled with tap water and covered with 6 X 6-mesh galvanized iron “hardware cloth,’’ on which the previously soaked and sterilized seeds were placed. The seeds were then covered with filter paper or paper towelling which was kept moist throughout the ger- mination process or until the radicles reached the water below. The germination was carried on in the greenhouse. In the [VoL. 2 464 ANNALS OF THE MISSOURI BOTANICAL GARDEN course of four or five days a splendid lot of vigorous, uniform seedlings which have serviceably straight radicles about 2 inches long with no laterals yet formed is obtained by this method; such seedlings are well adapted, both by their char- acter and their accommodation to an aqueous medium, for solution-eulture work. At this stage the plumules have grown to about one-half inch in length, and the plants are now ready for transfer to the culture medium, an operation which is easily and quickly done. This method of germination, which is shown in pl. 16 fig. 2, recommends itself both by reason of its simplieity and ease of operation and the certainty of secur- ing excellent results. In the transfer process from the ger- minating pan to the culture medium, the entire seedling was always immersed and carefully rinsed in once-distilled and again in twice-distilled water; by this means the roots became free of any adhering impurities. As containers for the cultures, ordinary glass tumblers. were used, the sides of which were covered with black paper to prevent algal growth and the top covered with perforated paraffin paper. (For a complete description and illustration of the method see the paper by McCool, 713.) Ten plants were grown in most cases in each tumbler; exceptions to that number will be noted in each case when the series are dis- cussed in detail. Galvanized iron wire supports were used to hold the plants upright when the seedlings had attained suffi- cient size to require them. In all cases doubly distilled water was used, the second dis- tillation being carried out in the laboratory with KMnO, added to the once-distilled water to oxidize any organic matter that might be present. Conductivity tests of this water showed it to possess a specific conductivity of 2.064 10%. The nutrient solution used was that of Pfeffer, redistilled water being the solvent for the necessary salts. Each tumbler was filled to a convenient level with either the water or the full nutrient solution as the case might be, approximately 250 cc. being required. To replace transpiration loss, doubly dis- tilled water was added as needed. In the early days of conductivity work on solutions, 1915] MERRILL—DISTILLED WATER 465 measurements could be made only by means of a continuous current. Because of the resulting polarization effects, how- ever, the resistance of the solution increased to such an ex- tent as to introduce serious errors into the results. But thanks to the classical work of Kohlrausch and others, the al- ternating current method was devised and perfected, whereby the determinations became practically independent of polar- ization effects. A vast amount of work has since been done in the realm of physical chemistry on conductivity measure- ments, a review of which, however, is outside the scope of this paper. For a clear and concise discussion of this sub- ject see Jones (’09), Walker (’10), or Findlay (710). In addition to the investigations already cited which deal with the practical applications of conductivity work, there might well be mentioned in this connection the work done by investigators in the Bureau of Soils of the U. S. Department of Agriculture: Whitney, Gardner, and Briggs (’97); Whit- ney and Briggs (’97); Whitney and Means (’97); and Gard- ner (’98). Heald (’02) used the Kohlrausch method for de- termining the conductivity of plant juices in order to get indications regarding the dissolved mineral substances in different parts of the plants under experimentation. Nicolosi- Roneati (’07), Bouyoucos (712), Dixon and Atkins (713, ’13°) and others have also carried on conductivity determina- tions with different plants and under various conditions. Sjöqvist (’95) was the first to use the conductivity method in enzyme investigations, which he did in his work on the action of pepsin on protein solutions. Similar work was done by Oker-Blom (’02), who also extended the applications of this method. Oker-Blom (’12) has recently given an account of his own and previous investigations in the field of bacteriology, wherein the electrical conductivity method was used. Various other investigators have also made use of it, among whom may be mentioned Bayliss (707). Stiles and Jörgensen (’14) give a partial review of some of the historical aspects of this subject as it pertains to plant work. [voL. 2 466 ANNALS OF THE MISSOURI BOTANICAL GARDEN For the conductivity work herein reported the following apparatus was used: Wheatstone bridge (Central Scientific Co., catalogue number, 2475 a box, 11 ‚110 ohms (Central Scientific Co., catalogue number, 2 Induction coil (Eimer and Amend, catalogue number, 4100) ; Dry battery cells (Eimer and Amend, catalogue numbe r, 592) ; Conductivity cell, Freas (Eimer and Amend, catalogue number, Telephone receiver (Central Scientific Co., catalogue number, 2355) ; Thermometer graduated to 1/10°C; Water tank holding 50 gallons, specially constructed for the purpose, pilot flame underneath; temperature regulated to Tiffany laboratory reie with which to operate a stirring apparatus in the ta In the method Ei for the work the procedure given below was consistently followed: the tumblers were always filled to approximately the 250 ec. level with either the solu- tion or redistilled water, depending on the culture. Before taking readings, doubly distilled water was added to bring the water or solution up to the original level, if the transpira- tion loss since the previous reading made the addition neces- sary. This was of course essential in order to keep the con- centration factor under control. Readings in all cases were taken at 25°C. The control of temperature exactly to within 1/10°C. was comparatively easy by the use of the pilot flame underneath and the stirring apparatus in the tank of water. For absolutely accurate and final quantitative determina- tions or ultimate values, as were required, for example, in the case of the standardization measurements for the cell constant with N/50 KCl, or the determination of the specific con- ductivity of the doubly distilled water, the greatest precau- tions possible were taken in regard to the conductivity cell and the concentration of its contents. But in making hun- dreds and even thousands of determinations, most of them as rapidly as accuracy permitted, due to the time factor involved, it was both impossible and unnecessary to dry the cell after each reading, since relative, and not absolute, values were 1915] MERRILL—-DISTILLED WATER 467 desired for the most part. The method employed, therefore, was to remove from the carefully stirred solution in the tumbler a 25 cc. sample with a pipette of the same capacity, the latter having previously been rinsed with the solution. Using exactly the same amount for each determination further reduced any possibility of error due to unequal dilution in the conductivity cell. Between readings the pipette was kept almost entirely immersed in redistilled water in a tall cylinder attached to a stand in the water bath. After carefully pour- ing the sample back into the tumbler, in case further readings were to be taken, the cell was rinsed twice with doubly dis- tilled water and rapidly drained before taking the next read- ing, whether of the same or of a different culture. Any minute amount of doubly distilled water that might be present to dilute the next sample was a constant factor throughout all the readings and was of course inconsequential. In using fresh batteries it was necessary to insert resis- tance coils between the battery and the induction coil in order to reduce the current. For this purpose German silver wire was used. While polarization phenomena may possibly be operative to a certain extent, such would be so small as to be practically negligible, especially in view of the fact that the effects from such a cause would be entirely relative and would therefore not affect the validity of the results. Some of the conductivity results given in this paper are shown in tabular form and others are plotted as curves. In some instances the data are calculated as specific conductivity ; in other cases the conductivity is represented by the value of x on the Wheatstone bridge. To make it clear what x actually represents, when the apparatus is set up as it was for the determinations, the following proportion is given: R:R’ :: x2 : 100 R is the resistance in ohms inserted in the resistance box; F’ is the resistance in ohms of the solution; and a is the num- ber on the bridge wire (graduated in millimeters from 0 to 100 centimeters). As the position of x on the bridge varies with R and R’, the R for each series of curves or tables will be given (though in the great majority of cases it was 9,110), (VoL, 2 468 ANNALS OF THE MISSOURI BOTANICAL GARDEN from which R’ can then be calculated. Having these values, the specific conductivity can be calculated for any determined cell constant (the value of the cell used being .4088). For a fuller discussion see Findlay (710). IV. Recovery oF PLANTS AFTER BEING IN DISTILLED WATER FOR VARYING PERIODS The first question studied pertained to the recovery of plants in full nutrient solution after being kept in doubly dis- tilled water for varying periods. To determine the com- parative condition for optimum recovery, the distilled water and the full nutrient medium were renewed every four days in some cultures and left unrenewed in others, in such a way that for either condition of the medium of each set in a cer- tain period the other medium would be both renewed and unrenewed so as to give all possible methods of combination. Examination of table 1 will make this clear. Thus, for example, with cultures 11, 12, 13, and 14 of the 10-day period in distilled water the doubly distilled water in Nos. 11 and 12 was unrenewed; but when these cultures were placed in full nutrient solution this medium was unchanged or unrenewed for No. 11 and was renewed for No. 12. The distilled water in Nos. 13 and 14 was renewed, and the full nutrient solution unrenewed and renewed respectively. In series 1 the small variety of horse beans (Vicia faba) was employed, 8 plants being used to a culture. The condi- tion of the media and duration of growth, the green weight of tops, and the dry weight of tops and roots of series 1 are given in table 1. On examining this table it is seen that even after the plants had remained for 20 days in distilled water, they recovered on being placed in the full nutrient solution, while those remaining for 10 days in distilled water produced practically as much growth when later placed in the full nutrient solution as did the plants which were in the latter medium during the entire period. Of course, as would be expected, the cultures wherein the full nutrient solution was renewed every four days gave much better growth than did those in the unrenewed medium, due, 1915] MERRILL—DISTILLED WATER 469 no doubt, to an increased amount of available nutrients. But an interesting comparison is manifest in connection with the effect of renewing the distilled water; the greater growth of both tops and roots may be noted in cultures 3 and 4, in which TABLE I (Series 1) EFFECT OF RENEWED VS. UNRENEWED MEDIA ON GROWTH OF HORSE BEANS Length of | Dist. HzO} Length of | Full nutr. | Green | Dry wt.| Dry wt. Culture | period in |renewed or| period in |renewed or| wt. of of of no. ist. H20 | unrenewed| full nutr. | unrenewed| tops tops | roots days days gms. gms. | gms. 1 4! Untenewedi. :. ee... 2, "1111 >:124 y 5 Uneeda e a bir .666 | .096 k 45 lee Biya. Sey E 4.40 .887 e212 4 45 SCHERER rate Ins inte 4.40 870.1. 235 £ Unrenewec 44 Unrenewed| 16.30 | 2.069 485 6 Unrenewec 4 Renewed | 27.00 | 3.069 | .707 s Unrenewec 4 Unrenewed { 994 429 8 Unrenewec 4 Renewed | 32.: 3.543 743 9 Unrenewec 40 Unrenewed .90 ais 463 0 Unrenewec 40 Renewed | 26.2: 895 .700 10 Unrenewec Unrenewed Hs, 623 382 y ) Unrenewec Renewed | 26.35 928 697 K ) Renewed Unrenewed| 14.90 887 463 4 ) Renewed Renewed | 22.90 | 2.376 .548 k Unrenewed ) Unrenewed| 14.00 719 388 6 Unrenewed 30 R 8.( 880 457 Renewed ) Unrenewed| 15.60 .807 .417 3 Renewed : Renewed | 21.20 | 2.403 .642 ) ) Unrenewed y Unrenewed)| 12.! „447 .300 ) ) Unrenewed ; Renewed „40 .247 .328 ) Renewed : Unrenewed 40 319 284 ) ) Renewed 5.50 670 454 AT ES | ae ane eee 4 Unrenewed| 14.60 975 419 7 el ae en DE) ee ange 4 Unrenewed| 14.70 | 2.044 443 miele era Fe RR re 4 Renewed | 27.8 2.925 .690 re ee 4 Renewed | 27.0 3.025 . 764 the distilled water was renewed every four days throughout the period, as compared with that of eultures 1 and 2, where the distilled water was not renewed, except, of course, for the occasional addition of water to replace the transpiration loss, which, however, was small. Furthermore, in noting the growth of cultures 11-22 inclusive, it is seen that in four of the six cases of comparison between the renewed and unre- newed distilled water, better growth of both tops and roots resulted where the distilled water was renewed. Considering eultures 1-4 and 11-22, inclusive, the total green weight of tops for the unrenewed distilled water as compared with the [vor. 2 470 ANNALS OF THE MISSOURI BOTANICAL GARDEN renewed distilled water, and the same conditions for the dry weight of tops and roots, gave the results to be seen in table 1. The total weight in all cases is therefore greater in the cul- tures in which the distilled water was renewed. TABLE II (Series 1) EFFECT OF RENEWED VS. UNRENEWED DISTILLED WATER ON GROWTH OF BEANS (Summarized Results of Part of Table I) 3 Green wt. of Dry wt. of Dry wt. of Medium tops in tops in roots in grams grams grams Water renewed... 110.30 13.219 3.315 Water unrenewed 99,56 12.287 2.712 These results therefore indicate that the so-called injury to plants in distilled water cannot be entirely or even satis- factorily explained on the basis of extraction of solutes from the plant tissues. If that were the case we should have the greatest injury and least recovery in those cultures in which the distilled water was renewed, the periodically renewed water effecting in toto a greater exosmosis of the salts than the water which is not renewed. This statement will receive verification under the section on conductivity measurements. It would therefore seem that we must seek other explanations for the phenomena observed when plants are placed in dis- tilled water. This phase of the subject will also be discussed later. The points noted will be clear from an examination of pl. 13 figs. 1 and 2. Plate 13 fig. 1 shows the various stages of recovery after varying periods in the distilled water. The better growth is to be noted of both tops and roots of No. 2, in which the distilled water was renewed, as contrasted with No. 1, in which it was not renewed. It is interesting to ob- serve how plants, even after 20 days in distilled water, will recover in full nutrient solution and then give even better growth than plants in unrenewed full nutrient solution the entire period, and that after 10 days in distilled water, plants will recover in renewed full nutrient solution and equal in 1915] MERRILL—DISTILLED WATER 471 growth, plants grown the entire period in renewed full nut- rient solution. Plate 13 fig. 2 shows first (Nos. 1 and 2) the contrasted effect of renewing and not renewing the full nutrient solution. The remaining 8 cultures of the plate show the effect of renewing and of not renewing both the distilled water and the full nutrient solution. In cultures 3-10 the comparison should, of course, be made between the alternating numbers for the distilled water effect (renewed or not renewed), and be- tween successive numbers for the effect of the renewal or the non-renewal of the full nutrient solution. While the culture represented by No. 7 of the plate gave greater growth than did No. 9, that excess was probably due to the individual hardi- hood of two plants. It is seen that a much more uniform and desirable growth was made by the plants of No. 9. An interesting point in connection with the horse beans is that 16 days after setting up the series the tips of those plants still in distilled water were more or less blackened, probably as a result of enzyme (oxydase) action, and many of them were considerably inrolled. Such conditions were entirely absent from the cultures in full nutrient solution at that time. When the affected plants were later placed in full nutrient solution there was a gradual recovery from the blackening of the leaves, and this recovery was greater in the case of those cultures in which the distilled water had been renewed than in those in which it had not been renewed. Twenty days later Nos. 3 and 4 were in very much better condition than Nos. 1 and 2. There was much less blacken- ing, some leaves not being blackened at all. The general height of the plants in Nos. 1 and 2 was 14-2} inches; and in Nos. 3 and 4 it was 24-4 inches. A very noticeable feature at the end of the experiment was the condition of the medium, that of Nos. 3 and 4 being of course clear while that of Nos. 1 and 2 was milky, turbid, and opaque, indicating abundant fungous and bacterial action, a condition further emphasized by the hyphal threads and gelatinous coating on the roots. The roots of the plants in Nos. 3 and 4 were also in much better condition at the end of the experiment than were those [VoL, 2 472 ANNALS OF THE MISSOURI BOTANICAL GARDEN of Nos. 1 and 2, especially as regards length and the amount of lateral root development. The root growth in No. 13 at the end of the experiment was also greater than that in No. 11; but in Nos. 12 and 14 it was about equal. The plants of No. 17 also showed greater root growth than did those of No. 15, and this difference was more marked than in the case of the tops. The lateral roots in No. 17 were produced all along the main roots, while in No. 15 they were practically confined to the upper or older portion of the main roots. An- other interesting difference observed was that in No. 17 the main root tips were not permanently injured in the distilled water and when placed in the full nutrient solution they con- tinued growth. This was not the case in No. 15. In general there was not much difference between the roots in Nos. 16 and 18; the plants in No. 18, however, had slightly greater growth of roots and showed less injury and some continua- tion of growth of the tips, whereas those in No. 16 did not. The same condition of the roots above noted for Nos. 15 and 17 held also in Nos. 19 and 21 respectively; but the difference in favor of the renewal of the distilled water though less marked was nevertheless evident. Likewise, Nos. 18 and 20 were similar to Nos. 16 and 18 respectively. Strong evidence was therefore afforded by the cultures of horse beans that renewing the distilled water has a favorable effect upon the plants. Series 2 is in every respect a duplicate of series 1 except that Canada field peas (Pisum sativum) were used instead of horse beans (Vicia faba), and that the dry weight of the tops was not determined; furthermore, the length of the ex- perimental period was different. The condition of the media and duration of growth, the green weight of tops, and the dry weight of roots are given in table m, seven plants being grown in each culture. An examination of this table reveals results similar in many cases to those contained in table 1; plants recovered even after 20 days in distilled water, but after 10 days in this medium the recovery was not so com- plete as in the case of the horse beans, for the plants so treated did not equal in growth similar ones which had remained in 1915] MERRILL—DISTILLED WATER 473 full nutrient solution the entire period. However, plants which had been in distilled water only 5 days before being transferred to full nutrient solution subsequently equalled in growth other plants which had been in the latter medium from TABLE III (Series 2) EFFECT OF RENEWED VS. UNRENEWED MEDIA ON GROWTH OF PEAS Length of | Dist. H20 | Length of | Full nutr. | Green wt. | Dry wt. Culture period in | renewed or | period in | renewed or of of no. | dist. HzO | unrenewed | full nutr. | unrenewed tops roots days ays gms. gms 1 47 Unrenewed |..........|. .80 073 2 47 Te Seen RE NT .60 .076 3 47 Renewerit.. ar [Soden re A .067 4 47 Renewed BEN, 1.30 .091 8 Unrenewec x Unrenewed 6. .400 6 Unrenewecd k Renewed 11.1 .500 Unrenewec k Unrenewed 5.50 .401 8 Unrenewec Renewed 12.90 . 568 9 : Unrenewed 2 Unrenewed 5.9 .263 0 h Unrenewed newed 1253 .467 10 Unrenewec Unrenewed 5.3 .254 2 ) Unrenewed 7.90 s21 E 0 Renewed Unrenewed 3.80 .160 4 ) Renewed 6. .202 s Unrenewed Unrenewed 3.30 .144 ( Unrenewed 4.40 .175 Renewed Unrenewed 4.30 .162 3 Renewed 4, .169 ) 20 Unrenewed Unrenewed 3.1 .124 ) 20 Unrenewed 2 .092 20 Renewed Unrenewed 4.0: .139 : 20 Renewed : ne 4.4 .141 BEER EANA EN 3: Unrenewed 5.50 .368 er RR EEE 3: Unrenewed 6.94 .420 re ee T 3: Renewe 9.60 .536 N et E FE ee 3: Renewed 13.45 .611 the start. The period between 5 and 10 days in distilled water is therefore a eritical one, and will be discussed later in other connections. Renewing the full nutrient solution again showed beneficial results, as might be expected. But the renewal of the distilled water did not produce such striking results in some respects as in the case of the horse beans; in other ways, however, the results were equally or even more striking. Where the plants remained in distilled water for 47 days the growth was better in one case and poorer in the other where the distilled water was renewed than where it was not renewed. The average [VoL. 2 474 ANNALS OF THE MISSOURI BOTANICAL GARDEN growth, however, of the two cultures in the renewed medium was better than that of the two in the unrenewed distilled water. In Nos. 11-22, there was better growth of tops and roots TABLE IV (Series 2) EFFECT OF RENEWED VS. UNRENEWED DISTILLED WATER ON GROWTH OF PEAS (Summarized Results of Part of Table III) Mediam Green wt. of tops Dry wt. of roots in grams in grams Distilled water renewed... 28.55 1.131 Distilled water unrenewed. 28.08 1.259 in four cases where the distilled water was renewed and better growth in four cases where it was not renewed. Considering eultures 1-4 and 11-22 the results given in table ıv were ob- tained, from which it is again evident that renewing the dis- tilled water exercises no injurious influence, and the conclu- sion is reinforced that an exosmosis of mineral nutrients is not the fundamental basis of the injury which plants suffer in distilled water. Furthermore, the difference between the renewed and the unrenewed distilled water cultures was very marked if the plants remained for 20 days in distilled water before being changed to the full nutrient solution, the differ- ence being greatly in favor of the cultures in which the medium was renewed. Figures 1 and 2 of pl. 14 illustrate the points above men- tioned. In pl. 14 fig. 2 should be noted the better growth of Nos. 9 and 10—which were in renewed distilled water for 20 days before transfer to full nutrient solution—as compared with Nos. 7 and 8, which had remained in unrenewed distilled water for the same length of time before transfer. The excess of growth in No. 4 over that in No. 6 is probably to be accounted for on the ground that since those cultures were in distilled water but 10 days neither the renewal nor the unrenewal of the medium exercised much effect. Hence the greater growth of No. 4 represents an individual variation. At the expiration of the experimental period the following conditions prevailed in series 2: while the top growth in 1915] MERRILL—DISTILLED WATER 475 cultures 1-4 was about the same in each case, the root growth in Nos. 3 and 4 was much better than that in Nos. 1 and 2, the roots of the former being whiter, cleaner, and having longer and more numerous lateral roots. In the case of those cul- tures grown in distilled water 10 days before removal to full nutrient solution, Nos. 11 and 12 were in somewhat better condition than Nos. 13 and 14, a difference which might readily be expected for the shorter periods in distilled water due to individual variation. After 15 days in distilled water and 18 days in full nutrient solution the benefits derived from renewing the former were markedly evident in the appear- ance of cultures 15-18, even though the actual weights did not show such difference. Nos. 17 and 18 were in better condi- tion than Nos. 15 and 16 respectively, especially as regards the root growth; similarly, Nos. 21 and 22 were in better condition than Nos. 19 and 20 respectively. Some special conditions which are of particular interest were observed when the cultures were examined carefully at the close of the experiment. The first point pertains to the method of recovery. After being in the distilled water only one or two days the top growth of such cultures when placed in full nutrient solution proceeds unhindered from the tips of the main stems, i. e., the tips of the stems remain unin- jured and resume growth. But 5 days in distilled water almost marks the limit at which growth can be resumed at the tip of the main axis of the stem when such cultures are subsequently placed in full nutrient solution. After 10 days in distilled water the tips of the stems become injured so that the later growth in full nutrient solution is made from new lateral branches. Hence the period from 5 to 10 days in distilled water before removal to full nutrient solution may be considered a crucial period as regards the recovery and growth of the main stems. Another point of interest is the delayed maturity which results in the case of the cultures which are grown for some time in distilled water and later are placed in full nutrient solution. Such plants remain in a green and growing condi- tion much longer than do those which have been in full [VoL. 2 476 ANNALS OF THE MISSOURI BOTANICAL GARDEN nutrient solution for the entire period, or those which re- mained in distilled water for a shorter period before being transferred to the full nutrient solution. The growing season of the former is thus prolonged and the date of maturity delayed. The foregoing series having given evidence of the recovery of plants in full nutrient solution after being in distilled water for 20 days, the question arose as to the maximum length of time plants might remain in distilled water without preventing recovery when subsequently transferred to full nutrient solution. Series 3 was therefore set up. This con- sisted of cultures of Canada field peas grown in distilled water for 10, 20, 30, 40, and 50-day periods before transfer to the full nutrient medium. The condition of the media and dura- tion in each and also the results of the series (as shown by the green weight of tops) are given in table v, Nos. 1-20 in- clusive. Renewals in this series also were made every four days. Nos. 21-28 under different conditions and concen- tration of nutrient solution are given for purposes of com- parison. The maximum time limit in distilled water above referred to is thus seen to be approximately 30 days, and this was practically attained only in case of the cultures in re- newed distilled water. After 40 days in distilled water, whether renewed or unrenewed, the recovery was almost nil, though somewhat better in the renewed, while after 50 days in either renewed or unrenewed distilled water all the cultures were dead. In the 10 cases furnishing comparisons between cultures in which the full nutrient solution was preceded on the one hand by renewed and on the other by unrenewed distilled water, greater growth was attained in 7 cases where the distilled water was renewed. The total weight of green tops is more nearly equal in the two sets of cultures, however, being 24.20 grams in the case of those in the unrenewed and 22.38 grams in the case of those in the renewed distilled water. We thus see that no injurious effects attend the renewal of the distilled water when compared with the non-renewal of the same; on the other hand, positive benefits are derived from such a 1915] MERRILL—DISTILLED WATER 477 renewal, especially in the case of plants approaching the maximum time limit of durability in distilled water—a period which enables the results of the two conditions to be more readily seen and compared. TABLE V (Series 3) et en en IN RENEWED AND UNRENEWED TE a ee i BER PTO. T To WATER AT DIF FER ENT IN TERVALS MEDIA UNDER VARIOUS CONDITIONS Length of . Length of Green Cute | feat | Dino | Teega | ut nue, | Sree no. dist. H,O full nutr tops ays unrenewed ays unrenewed gms. 1 0 Unrenewed 42 Unrenewed 4.50 2 0 Unrenewed 42 enewed 8.00 3 ) Renewed 42 Unrenewed 4.85 4 ) Renewed 42 Renewed 6.30 5 ) Unrenewed 32 Unrenewed 2.7 6 ) Unrenewed 32 Renewed 5.( 7 Renewed 32 Unrenewed E: 8 ) Renewed 2 Renewe 1.40 9 30 Unrenewed 22 Unrenewed i LO 30 Unrenewed 22 1.30 1 30 ewe 22 Unrenewed 1.90 2 30 Renewed 22 1.90 5 40 Unrenewed 12 Unrenewed .50 + 4 Unrenewed 12 5 40 enewe 12 Unrenewed -C 6 40 enewed 12 Renewed “a 7 k Unrenewed h eere nesnese eee .60 8 b Unrepewed 1. eau iiss sea] ects « eee 4 9 k Renewed © I. vases cosa a Ex 20 g Renewed ed eee ee an 21 Unrenewed full Ber ie days, dist. H0 added every 8 days 6.40 22 Unrenewed full n days, dist. H20 added every 4 day .00 23 Renewed full aie ir days, the sol’n. renewed every 8 days «59 24 Renewed full 2 days, the sol’n. renewed ry 4 days| 18.50 25 nrenewed 1/10 full nutr. 42 days, dist. H,Oaddedevery4 d’ys .90 26 nrenewed 1/5 full Eee 42 days, dist. HzO added every 8 days 95 27 Renewed 4 10 full nutr. 42 avs. sol’n. renewed every 4 days} 10.10 28 Renewed 1/5 full ae. 42 days, sol’n. renewed every 8 days .85 In pl. 15 fig. 1 some of the cultures are illustrated, the ones of special interest being Nos. 9-14. e exceptionally small or irregular growth of No. 8 is difficult to account for, because in the renewed full nutrient it should be greater than that of No. 7. Individual resistance is apparent, however. [VoL, 2 478 ANNALS OF THE MISSOURI BOTANICAL GARDEN V. Recovery or PLANTS AFTER BEING IN Toxic SOLUTIONS Having thus ascertained the maximum time plants may remain in distilled water and then recover on being placed in full nutrient solution, we may turn our attention to toxic solutions. If distilled water in itself is toxic then it should be interesting to get quantitative data on its effects as measured by the power of plants so treated to recover. This power should furnish a good index regarding the extent of any injury suffered. By comparing the ultimate time limits for various media after which recovery in full nutrient solu- tion is possible, we are able to get a basis on which to de- termine the relative toxicity of each medium. Almost simul- taneously with series 3, series 4 was set up. The plan of the series and the green weight of tops and dry weight of roots of the plants in series 4 are given in table vi, while pl. 15 fig. 2 shows the actual condition of the plants in some of the media. The results obtained indicate the following relative toxicities of the substances used, the time expressed in days having reference to the longest period in the toxic solution after which recovery is possible: Kein WE a Bien 2. yas gi 20 EEE Orr errr ry Pee re ern eee eee ee eee cee igi MEGS on ee ere Tere err errr er rere seers E about > are N/1000 bee ENO Miso oyes cc ea E A EE EEE about 16 days N/12 2 98 7 rrr err rer err errr Tre rere ree rere ee Tey RT about 20 days N/40 0 KOH ee E S Be ROHR er Rear a Face Rese ETC 1. SACHEN: about 20 days We thus see that as compared with the toxic solutions men- tioned distilled water, if it be considered as a toxic agent at all, is much less so than either of the others given above. In this connection it is interesting to note that Kahlenberg and True (’96) found that N/12800 H2SO.1 and N/400 KOH were approximately the critical concentrations for Lupinus roots. Hence, the fact that plants can remain much longer in distilled water than in these solutions and still recover would seem to indicate that as regards toxicity distilled water is only very slightly if at all deleterious. But the writer believes that it is entirely incorrect and misleading to speak of distilled water as being toxic. What is illustrated above for distilled water is not toxicity, therefore, but merely the length of time 1915] MERRILL—DISTILLED WATER 479 TABLE VI (Series 4) EFFECT ON GROWTH OF PLANTS OF VARIOUS PERIODS IN TOXIC SOLUTIONS Length off First |Length of} Green | D Culture First sol’n. period in) medium period in| wt. of | wt. of no. or medium first enewed /fullnutr.| tops | roots medium or ay gms. days |unrenewed 1 PBE EO er 32 Unrenewed]|......... 1019 116 2 Bist HA cere ee 32 Renewed 1,95 130 3 100 Met)... win... 32 Unrenewed|......... a 012 4 100 MeCh....,.... 32 enewed |......... .40 | .016 5 100 MeCh....... 1 Unrenewec 31 10.1 .428 6 Te ogre 2 Unrenewec 30 8.40 | .372 7 ID Rag lers. 3S. 54s 4 Unrenewec 28 S: .132 8 Te Saree 8 Unrenewec 24 ef .018 9 100 Math... 2.3... 12 Unrenewec 20 30 | .020 0 100 Melle... veces 16 Unrenewec 16 .40 | .016 1 S00 Mech... it 20 Unrenewed 12 ö .012 2 1000 MgCl......... 32 Inrenewed|......... 1.00 | .085 3 1000 MgCl:........ 32 Renewed |......... 1.00 | .038 4 1000 MgCl:........ 2 Unrenewec 30 8. . 385 5 1000 MgCl:........ 4 Unrenewec 28 9.70 | .384 6 N/1000 MgCh........ 8 Unrenewec 24 Ke: .305 7 N/1000 MgCh........ 12 Unrenewed 20 Si .192 18 ] hee MEC O TA 16 Unrenewed 16 2.0 421 19 N/1000 MgCh........ 20 Unrenewed 12 1.0 .093 20 I 1000 CaCl: and N/20 MgCl ee eee 32 Unrenewed|......... .75 | .092 21 N/1000 CaCl: and N/20 ee eee eee 32 Renewed .85 | .099 22 N/1000 CaCl: and N/20 | Rr are 1 Unrenewed 31 10.60 | .409 23 N1000 CaCl: and N/20 ee aek 2 Unrenewed 30 9,35 . 388 24 N/1000" CaCl: and N/20 A PEA 4 Unrenewed 28 10.35 | .384 25 N/1000 CaCl: and N/20 RE E N 8 Unrenewed 24 8.40 | .294 26 N/1000 CaCl: and N/20 ANSET 12 Unrenewed 20 3.00 | .144 27 N/1000 CaCl: and N/20 re 16 Unrenewed 16 15017 2117 28 BI CaCl: and N/20 JS ee Sere 20 |Unrenewed 12 7 . 103 29 PUN Rutt, a E 32 Unrenewed 32 8.95 | .411 30 Haate SOL Ils. 0.2 32 Renewed 32 tS- S0 .930 31 N/12800 H:SO........ 32 Unrenewed]......... oes 5450 32 N/12800 H:SO........ 32 Renewed |......... 1.25 .124 33 N/12800 H:SO........ 2 Unrenewed 30 7.05 | .318 34 N/12800 cayenne 8 Unrenewed 24 7.45 | .289 39 Fabio te AD 0 Ve Fee 16 Unrenewed 16 4.40 | .236 36 N/12800 H:SO........ 20 Unrenewed 12 LOS eA he 37 NAOKO AR NER 32 Unrenewed|......... 1.25 | .094 38 NAO KOH. nn. 32 Renewed |......... 1.50 .108 39 N/400 KOH re 2 Unrenewed 30 8.60 | .444 40 OOD OT er 8 Unrenewed 24 6.60 | .214 41 N/400 KOH... -on an. 16 Unrenewed 16 2.55 | .092 42 N/400 KOH.......... 20 Unrenewed 12 2200 1 EES [VoL. 2 480 ANNALS OF THE MISSOURI BOTANICAL GARDEN plants can survive in a medium without nutrient materials. That these plants could not survive for that length of time in the other media, however, shows that in those cases a real toxicity enters into consideration. In addition to the actual time limits for recovery just tabu- lated, as well as the method of recovery and delayed maturity mentioned in the preceding section, another interesting point, which was very noticeable in the cultures and which can also be seen in the plates, is the character of growth of the root- lets in the boundary cultures, by which is meant those cultures which have remained in the inimical media nearly as long as their endurance would permit, and whose recovery in full nutrient solution is slower or more difficult than the normal unaffected plants. In the latter case the roots are short and compact and usually extend down only to about one-half the distance to the bottom of the tumbler. In the case of the first mentioned cultures, however, when transferred to full nutrient solution the rootlets develop a long, slender growth easily extending to the bottom of the tumbler. VI. EFFECT or STERILIZING THE WATER DURING GROWTH OF PLANTS The foregoing series pointed, therefore, to factors other than extraction or loss of solute from the plant tissue as being responsible for the deteriorating phenomenon observed when growing plants are placed in distilled water. In the unre- newed water cultures in the previous series a brownish colora- tion developed and the roots appeared, in their gelatinized condition, to be covered by bacterial and fungous growths. Suspecting that these organisms played an important röle, it was decided to grow additional cultures to test this point. Four cultures, each containing ten plants of Pisum sativum, were set up in distilled water: in one the medium was not renewed; in a second the water was renewed every four days; and in the remaining two the medium was sterilized every four days by boiling in a return condenser one-half hour. The re- sults are given in table vir (series 5) and the cultures are shown in pl. 16 fig. 1. The full nutrient solution cultures. 1915] MERRILL—DISTILLED WATER 481 were grown for purposes of comparison. The duration of growth was 30 days. Whether the beneficial effect of the sterilization was due to the destruction of the bacterial and fungous floras of the TABLE VII (Series 5) EFFECT PRODUCED ON GROWTH OF PLANTS BY STERILIZING THE WATER IN WHICH THEY ARE GROWN tis Green wt. Dry wt Gene Medium Condition of of tops of roots 2 gms. ms 1 Dist. H,O Unrenewed eos .141 2 Dist. H,O enewed 1.65 .150 3 Dist. H,O Sterilized 2.40 225 4 ist. H,O Sterilized 3:05 .233 5 Full nutr. Unrenewed 10.30 .342 6 Full nutr. Renewed 17.65 .507 medium, to a decomposition of any contained toxic substances (thereby rendering them less toxic), or to incidental effects such as aération of the water by the boiling process, was not definitely determined. Neither was this effect compared with that produced by the addition of various bodies (tannic acid, pyrogallol, calcium carbonate, various hydrates, carbon black, and other substances mentioned by Livingston and his co- workers, ’05, ’07, Dachnowski, ’08, ’09, and others). In the last paper of Livingston and his co-workers referred to are given the results of boiling the aqueous extracts from soils containing toxic properties as determined by the growth of plants in the same. The boiling improved the extracts, but this effect was explained by ‘‘supposing the process of boiling to remove or change the toxic action of this extract, the toxic materials being perhaps partly volatile with steam.’’ But since in our sterilization process a return condenser was used the removal of toxic substances by volatilization would not occur. A breaking down of toxic compounds into less toxic constituents may possibly be a condition induced by the boiling, however. It will be recalled that Lyon (’04) found the toxicity of tap water reduced by boiling. While the oxidizing power of roots, due to enzymatic activity, may be an important factor in aiding in the decom- [VoL, 2 482 ANNALS OF THE MISSOURI BOTANICAL GARDEN position of vegetable matter in the soil, as pointed out by Schreiner and Reed (’07) and others, it is not believed that in the case under consideration the oxidizing power of the roots was altered to any appreciable degree by the boiling of the medium. Dachnowski (’12) mentions the effect of oxida- tion upon the toxic substances found in bog water. In the sterilization method by boiling under a return condenser, however, the aération or oxidation phenomenon would no doubt play only a subsidiary role. The stronger line of evi- dence seems to favor the destruction of injurious bacterial and fungous agencies as the chief factor in the beneficial effect of the sterilization. VII. CONDUCTIVITY MEASUREMENTS The excellence of the electrical conductivity method for determining any change in the electrolyte content of an aqueous medium naturally led to its adoption for the experi- mental work described below. This phase of the investiga- tion was especially concerned with determinations pertaining to the extraction of electrolytes — including the essential nutrient salts—from the roots of plants in distilled water. The generally beneficial results attendant upon a frequent renewal of the distilled water in which the plants were placed has already been noted, as well as the evidence in favor of the view that conditions other than extraction of essential salts constitute the underlying cause of the deterioration of plants in distilled water. The next point to be determined was the relative amount of the total exosmosis in the renewed distilled water as com- pared with that in the unrenewed. In placing roots in dis- tilled water it is pertinent to this subject to inquire whether all the exosmosis occurs during the first four days. If it does, we should have the same amount of extraction in both the unrenewed water and that renewed every four days. Or is there a renewal of the exosmosis of the electrolytes follow- ing the renewal of the water each time, thereby giving rise to a greater exosmosis than in the cultures in which the water was not renewed? If such a condition obtains and yet in 1915] MERRILL—DISTILLED WATER 483 spite of it the renewal of the water shows no baneful effects, or indeed produces beneficial results, then may we well con- clude, and with increasing assurance, that extraction of nutrient salts is in no way responsible for any injury plants undergo in distilled water. The results obtained strongly substantiate that conclusion. A series of cultures (series 6) was set up in which healthy plants of Canada field peas were grown in full nutrient solu- tion for about three weeks and then transferred, after care- fully rinsing the roots, to doubly distilled water. In half of the cultures the distilled water was renewed at certain definite intervals for each culture, while in the other half of the eul- tures the water was not renewed. Conductivity determina- tions were then made of the water under both conditions— renewal and non-renewal—at certain regular intervals, vary- ing for each set of cultures, for several days after the plants had been placed in this medium. By numerous readings it was ascertained that with a resis- tance of 9,110 ohms in the resistance box the average value of x on the Wheatstone bridge for the water in the vessel after being rinsed and before placing the roots therein was approxi- mately 6.0, rarely varying 1 em. either way. Considering that figure, then, as the basis or the starting point for the exos- mosis, and subtracting it from the different values found for the renewed, and from only the final value obtained for the unrenewed distilled water, we get the figures in the last column of table vr. The plan of the experiment with respect to renewal of the distilled water and the time of readings, the values of the individual readings, and the comparative amounts which represent the total exosmosis of the electrolytes under the various conditions of the experiment are all given in table vi. The numbers given are the values of w on the Wheat- stone bridge when the resistance inserted in the box was 9,110 ohms. It is thus seen that by far the greater exosmosis was ob- tained in the case of those cultures in which the distilled water was renewed. Another point of interest was the reabsorp- [vor. 2 484 ANNALS OF THE MISSOURI BOTANICAL GARDEN tion of electrolytes—as seen by the decrease in conductivity of the medium—in those cultures in which the distilled water was not renewed. The reabsorption of electrolytes has been observed to be a phenomenon characteristic of normal, healthy TABLE VIII (Series 6) COMPARATIVE EXOSMOSIS IN RENEWED AND UNRENEWED DISTILLED WATER CONDUCTIVITY READINGS Dura-| To tion |; Culture) Water of yar ary no. | renewal | Fre- | ist | and | 3rd | 4th | Sth | 6th | 7th | ©et: \conduc- quency ment | tivity days 1 Every Every day day | 32.91 10.4} 10.0] 8.9] 9.7} 9.4)10.2) 7 49.5 2 None Every day | 36.3} 22.8] 21.4) 17.8| 15.2|12.5| 11.4) 7 5.4 3 Every Every 2days| 2 days|10.8} 9.3} 9.6/10.7]....]....]..-- 8 16.4 4 | None Every 2 days | 25.0) 14.3] 13.6} 11.0). 8 5.0 5 Every Every 4days| 4days|12.9115.0116.1[16.1|....|....|....| 16 36.1 6 |None Every 4 days | 10.7] 12.4) 15.9] 19.5]....|..- .|....| 16 13.5 peas, when transferred from a full nutrient solution to dis- tilled water, after being in the latter medium one or two days. In order to obtain some additional information regarding the relations between the conductivity of the medium and the plants grown therein, series 7 containing 50 cultures was set up in full nutrient solution, ten Canada field pea plants to each culture. The nutrient solution was not renewed. At the end of each five-day period 5 of the cultures were taken down, the green weight of tops of the plants in each determined, and the conductivity of the solution measured; and from these results the average green weight of tops and the average conductivity of each set of 5 cultures were obtained. This was done throughout the entire period of 50 days. The re- sults obtained are given in table ıx and plotted as curves in fig. 1. In the latter the abscissa represents days, and the ordinate both specific conductivity and green weight of tops. The values given for conductivity should be multiplied by 10~° 1915] MERRILL—DISTILLED WATER 485 in order to get the specific conductivity values. In the case of the weights the numbers in the margin represent ten times the actual weight in grams, e.g., 40 in the margin = 4.0 grams. From the results it is seen that both the increase in green TABLE IX (Series 7) GROWTH OF PLANTS AND PORT 8 OR FULL NUTRIENT MEDIUM FOR Av. green wt. Specific conductivity * at end Cultures Ban gi period i of period nos. = rent | each culture = Minimum Average Maximum 1- 5 5 3.81 93.22 96.25 98.19 6-10 10 8.12 57.47 61.03 66.93 1-15 15 9.47 32.38 34.83 37.59 20 20 8.66 24.38 32.68 46.05 21-25 25 8.69 15.73 18.19 21.69 26-30 30 8.00 13.74 20.63 30.28 1-35 35 7.10 10.02 15.98 t.23 36-40 40 6.77 11.16 14.23 16.19 41-45 46 6.09 6.88 11.44 22.31 a 50 5.01 13.00 16.97 28.11 he numbers in the three columns are to be multiplied by 10° in order to arrive at the specific conductivity values. weight of tops and decrease in conductivity of the medium are most rapid and pronounced during the first 15 days. After that period both the green weight and the conductivity gradu- ally decline, but the latter more slowly than the former. ile the curves of the minimum, average, and maximum conductivity remain very close together during the first 15 days, they become more divergent after that time. The green- weight curve shows a gradual decline as the age of the plant increases, after a certain period, due to the drying of the ops and consequent loss of water. Different curves would, of course, have been obtained had the nutrient solution been renewed. In table x and fig. 2 are seen the results of series 8, a similar experiment with distilled water, the same units being used as in the previous case. The green weight of tops in- creased during the first 10 days and then gradually declined to the end of the experiment. The conductivity of the water was practically the same on the 10th as it had been on the 5th day. Evidence from other experiments, however, indicates a oO Specific conductivity and green weight of tops [VoL. 2 ANNALS OF THE MISSOURI BOTANICAL GARDEN TT 11 I NT Cl Wo NY 7 En EEE U A. Sees N Benen tt ttf N. a m IR EEE SER EEE ER IT / pp ee ee aa 4 Ob eb Nan BS ee Ji q See ee8258 ae SSRBEEnE COO q Ae p+ REECE EEEL b BERGER CREE ji N ji ji N | 1 i 4 1 ' iY = iY Pd it A ai z i ai ‘a y 111 7 N EET 4 LOA 4 za T 4 N, 1 I 4 N ‘A B bq IILN P. “um we z 4 | FT EERE ETTI CEFE BRES a 5 10 15 20 25 30 35 40 45 50 Days Fig The eo =e oe. elo for the full nutrient station T Pfeffer’s) in which 50 days, the medium being unrenewed, (For por i a, see the text.) 1915] MERRILL—DISTILLED WATER 487 that in the interim the curve might have risen and fallen. After the 10th day the curve inclined with fluctuations. Here again are seen evidences that the 10-day period for seedlings in the distilled water may properly be considered a crucial one for the plants. After that time the growth declines and the conductivity increases markedly. Suspecting that the question of injury to plants in distilled m Pu ° ~ ETEL u H ame © 30 HERSE) + DILU a BLL = cI Q $ 25 7 [=] 4 4 S 5, 20 A N E > P +3 10 Q "4 * 3 = = I 7 +A = 7 zSa =e Cow a 9 5 Land CEEI > S RT] TI © S08 TIT EDS E 1 Loo (i ei S = LI coor LLLI A. RD 5 10 15 20 25 30 35 40 45 50 Days Fig The conductivity and growth curves for the unrenewed redistilled water in which pea BR: were grown for 50 days. (For complete explanation see the text.) water might be intimately bound up with that of lack of reserve food materials, the writer carried out an experiment bearing upon this matter. The experiment consisted, first, in placing some Canada field pea seedlings directly into redis- tilled water and determining the specific conductivity of the water at intervals for 20 days; and, next, in transferring some pea plants which had been grown in full nutrient solution for 1, 5, 10, 20, 30, and 40 days respectively to redistilled water, and determining the specific conductivity of the water at in- tervals for 20 days. The results are plotted as curves in fig. 3, the conductivity values being represented in terms of x on the Wheatstone bridge with a resistance of 9,110 ohms in the box. Four cultures of 10 plants each (except in the case [VoL 2 488 ANNALS OF THE MISSOURI BOTANICAL GARDEN of the 20-day period in full nutrient solution in which 12 cul- tures were used) were grown under each of the specified con- ditions, and the curves represent the averages for the 4 (or 12) cultures under each condition. To determine how much TABLE X (Series 8) GROWTH OF PLANTS AND CONDUCTIVITY OF DISTILLED WATER MEDIUM FOR 50 DAYS : l Specific conductivity * at end Length of period| Av. green wt. : Cultures in dist. water of tops of period ` ays grams Minimum | Average | Maximum 1- 5 5 2.04 1.46 1.54 1.66 6-10 10 3.07 1.01 1.34 1.81 11-15 15 2.89 3.04 5.43 7.41 16-20 20 2.34 4.34 5.92 8.63 21-25 25 .89 5.66 8.15 19.77 26-30 30 55 2.60 8.26 15.82 31-35 35 .15 4.43 7. 17.95 40 sta 17.62 19.31 26.04 41-45 45 .68 6.87 13.32 17.95 46-50 50 .60 10.51 16.48 29.00 _ * The numbers in the three columns are to be multiplied by 107° in order to arrive at the specific conductivity values. increase in conductivity was contributed by the glass tumblers in which the cultures were grown, 4 such containers filled only with redistilled water, and containing no plants, were used and the conductivity of the water determined at intervals for 20 days. It is seen that from the seedlings which had not been in full nutrient solution at all (Nos. 5-8) the highest conduc- tivity resulted, while from those which were in the full nutrient solution longest before being placed in the distilled water (Nos. 37-40 and 33-36), the lowest conductivity was found at the end of 20 days. The other cultures at the end of 20 days were midway between the two extremes. It is also seen that, whereas the conductivity curve for Nos. 5-8 shows very little tendency to decline in the early stages, the curves for the cultures which had first been in full nutrient solution show that tendency to a considerable extent. And that ten- dency, as we have previously remarked, is a characteristic fea- ture of normal plants transferred from full nutrient solution to distilled water. Attention should be called to the difference in the character of the conductivity curves in fig. 2 and that of 5-8 in fig. 3. It 1915] MERRILL—DISTILLED WATER 489 will be noted that both represent the conductivity curve of dis- tilled water containing the roots of seedling peas. The differ- ence mentioned no doubt finds its explanation in the different conditions under which the two series were grown (the series 40 = rt ~ I am HO i bi 7 = tit 35 T ee coy” TILL? Bz -A = 30 — ‘4 DATH j ên i HHH = PEEHFEBERRALEH = x 2 ae coor 3 25 pann HEHH $ Z TH SEMS 7 7 ALL 3 H au Panne = m 208 z an eee = Ban 5 x tooth = a eum 8 “ea À 220 H an > E an 3 b = ILLILEB a qa wa 172 Fir a 5 ra 8 3145 et bo © = A SMEH n 15 1 Q 3 i = > 7 10 Bun» 5 H ao 2 4 6 8 10 12 14 16 18 20 Days Fig. 3. The conductivity curves for cultures in distilled water 20 days— ied hula = en nutrient solution for varying periods of time, as follo l day; > os. 13-16, 5 day Nos. ar 20, 10 a» Nos. 21- 32, 20 da ays; Nos. 33-36, 30 days; Nos. 37-40, 0 da ays. —8 were grown only in distilled water, while Nos. 1-4 were “without East, N ow only of distilled water. (Vou. 2 490 ANNALS OF THE MISSOURI BOTANICAL GARDEN for fig. 2 being run in the fall when the seeds were fresh, and that for fig. 3 in the winter), in the vigor of the seeds, and in the difference in the units used in plotting the curves. It must be said, however, that various factors of the problem of exosmosis from the roots of plants remain as yet unknown. The early drop in the curve of the conductivity of the con- trols (1-4) is an interesting feature which would seem to be explained by an adsorption of the electrolytes on the surface of the chemically clean glass tumblers. At the end of 20 days in distilled water the roots of the plants which had not been in full nutrient at all showed marked deterioration (being badly decomposed and covered with a gelatinous coating), while the roots of those which had previ- ously been in full nutrient solution for some time remained normal in every respect, even after 20 days in distilled water. These results seem plainly to indicate that injury which plants sustain in distilled water is very closely related either to the lack of available nutrients in the medium or of reserve food material in the tissues. A seedling is in an exceedingly plastic state of growth. If no food materials become avail- able the embryonic tissues which are in such an active condi- tion of growth soon become disorganized, possibly suffering partial autolysis and becoming the prey to bacterial and fungous action. We would expect, therefore, that the larger the seeds (and hence also the supply of stored materials), the longer the seedlings could remain in distilled water before deterioration. Comparison of True’s results on Lupinus with those here presented on Pisum sativum and Vicia faba seems to fulfill that expectation. We should also expect that the more nutrient materials the plant absorbed, the better it would be able later to withstand any deteriorating influences in the distilled water, and the experiment above noted seems to bear out that idea also. In the light of what has been said we are led to believe that the conductivity curve of Nos. 5-8 is not a pure representa- tion of exosmosis and that the products of bacterial and fungous action and cell decomposition account for at least a part of the conductivity. While the same condition may be 1915] MERRILL—DISTILLED WATER 491 true of the other cultures to a certain extent, it no doubt plays a lesser, and real exosmosis a greater, part. In connection with the above experiment it was thought desirable to determine whether a difference in the initial tem- perature of the water into which the roots were placed had any immediate or subsequent effect upon the exosmosis from the roots; plants which had been grown in full nutrient solu- tion for 20 days were used for this purpose. Four cultures were prepared with distilled water at a temperature of 6.5°C., four at 17.2°C., and four at 35.0°C., and conductivity readings were taken after exactly one-half hour, and then at various intervals for 20 days. No attempt was made to keep the water at the initial temperatures and it therefore gradually returned to the temperature of the room. After one-half hour, when the first readings were taken, the respective temperatures were 8.9°C., 16.6°C., and 27.4°C. The average conductivities of the water of these cultures are plotted for 20 days in fig. 4, the same units being used as in fig. 3. From these results it may be concluded that the initial differences of temperature can not be said to have exercised much, if any, effect. The results would probably have been different had the temperatures remained at the original point during the 20 days. Wächter (’05) has con- sidered the röle of the temperature factor in exosmosis. VIII. Discussion AND CONCLUSIONS It is believed that the evidence furnished is sufficient to support the conclusion that pure distilled water per se is not toxic or injurious to plants, and that various other factors enter in to cause the deterioration noted when plants are placed in that medium. Of course by qualifying the assertion to include pure dis- tilled water only, we have thus eliminated the effect that may be produced by toxic substances in the distilled water, no matter from whence derived. The abundance of work that has been done on the toxicity of various substances to plant tissues would of course lead us to expect injurious effects if such substances were present in any quantity in the distilled [VoL. 2 492 ANNALS OF THE MISSOURI BOTANICAL GARDEN water. With that phase of the question we are therefore not much concerned at present. With a distilled water prepared as indicated, and with a specific conductivity which is approxi- mately 210~-°, we have a water sufficiently pure for use in the consideration of other aspects of the question, and atten- tion is directed to these. The evidence presented has inclined us strongly to the view PrI EE LLI 2 30 4 a a = & AH I A A A þa A > = Pr/A Q a p KE £ 2 3 v Fa E at 4 = 20 aay g ° A 8 H S Ea Zannnn D aar B15 E £ ‚8 - 10 2 + 6 8 10 12 14 16 18 20 Days Fig. 4. The conductivity curves for cultures z distilled m. 20 days—after growth in full nutrient solution for 20 days. The initial temperatures of the distilled water into which the roots were ee were as follows: Nos, 21-24, 6.5°C.; Nos. 25-28, 17.2°C.; Nos. 29-32, 35°C. that the fundamental basis of the deterioration of plants in distilled water rests upon the food relations of such plants, but that, on the other hand, an exosmosis of food materials or nutrient salts is in no way responsible for the diffieulty. It is considered that the question of the food relation plays an important röle in the incipiency of the disorder, but that this is quickly followed by factors which have been initiated as a result of the inimical food or nutrient relation. 1915] MERRILL—DISTILLED WATER 493 A plant must assuredly have food in order to thrive. The more food it has stored up in its tissues, the longer it can survive in a medium devoid of it. But because of the absence of available food it is believed that the tissues of the plant begin to become disorganized and in that condition fall a ready prey to bacterial and fungous action, which may then set in and play a very important part in the subsequent de- composition of the tissues. While it may seem paradoxical to assert in one clause that absence of food is the fundamental basis of the injury which plants undergo in distilled water, and in the very next to say that exosmosis of nutrient salts plays no röle, yet the results obtained have substantiated that idea. Furthermore, it is essential to consider the various other factors attendant upon these two conditions in order to arrive at the proper conclu- sions respecting their operation. Among such factors may be mentioned the decrease in conductivity after a short period coincident with exosmosis from normal tissues, the relation of sterilization to bacterial and fungous action, the recovery of plants under different conditions, and the numerous other questions already considered in the body of the article, all of which lend weight to the conclusions arrived at. IX. Summary A brief historical review is given in this paper of the views held in regard to the cause of injury to plants in distilled water. The methods of work are outlined. The experimental work is given and the results discussed, especially with reference to the conclusions of other workers. A discussion is given of the results obtained in the experi- mental work and the conclusions derived therefrom are stated. Some of the results obtained from the experimental work may be summarized as follows: (a). Renewing the distilled water of the cultures every 4 days was in general beneficial, as shown by increased growth of both tops and roots. The plants were also able to survive longer in the renewed than in the unrenewed distilled water, x [VoL. 2 494 ANNALS OF THE MISSOURI BOTANICAL GARDEN and continued growth better after being placed in a full nutrient solution. (b). The period between 5 and 10 days in distilled water is a crucial one for plants; if they remain longer in this medium they are unable to recover normally or completely when subsequently placed in a full nutrient solution. (c). By keeping the plants in distilled water a certain period before transferring to full nutrient solution the ma- turity of the plants is delayed. (d). The longest period during which plants can be kept in distilled water and later recover on being placed in full nutrient solution was found to be 30-40 days. For certain dilute toxie solutions this period was much less, thus indicat- ing that the so-called toxieity of distilled water is, if it exists at all, very slight. (e). The lateral roots of ‘‘boundary cultures’’ were characteristically long and thread-like. (f). Sterilizing the distilled water by boiling one-half hour every 4 days exercised a beneficial effect upon the growth of plants in that medium as compared with the growth of those in unsterilized distilled water. (g). Greater total exosmosis was obtained in the renewed than in the unrenewed distilled water. (h). Normal plants which have been grown for some time in full nutrient medium and then transferred to distilled water exhibit at first greater excretion than absorption of elec- trolytes. After one or two days, however, there is greater absorption than excretion and the conductivity curve declines. This condition may be maintained for a considerable period. (i). The conductivity curve of the full nutrient solution in which plants were grown rapidly fell during the first 15 days or so; then it was more or less horizontal for a period, and finally began to incline after about 50 days. The growth curve was in general opposite in character to the conductivity curve. (j). The conductivity of the distilled water in one series in which the roots of pea seedlings were placed was practically the same on the 10th as on the 5th day. After the 10th day 1915] MERRILL—DISTILLED WATER 495 it rose considerably. The growth curve showed a rise the first ten days, then a decline. (k). Higher conductivity in the distilled water after 20 days was caused by plants which had not previously been in full nutrient solution than by plants grown for a time in full nutrient solution before transference to distilled water. The former cultures also failed to give the decline in conductivity characteristic of normal plants transferred from full nutrient solution to distilled water. (1). Greater deterioration of the roots in distilled water occurred if the plants had not previously been in full nutrient solution than in the case of plants which had been grown for a time in the latter medium. (m). Initial difference of temperature of the distilled water produced no effect on the exosmosis of electrolytes. The sincere thanks of the writer are cheerfully extended the following, who have generously aided in various ways in the preparation of this paper: Dr. B. M. Duggar, for his helpful suggestions and criticisms throughout the work; Dr. J. F. Merrill and Prof. Lindley Pyle, for suggestions in regard to some features of the conductivity apparatus; Mr. ©. H. 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Concerning the action of small ira of calcium, sodium, and potassium salts upon the vitality and fune of contractile tissue and the cuticular cells of fishes. Ibid. 6: Teri i885. ————., and Phear, A. G. (’94a). The influence of saline media on the tadpole. Ibid. 17: 423-432. 1894-1895. — —— (°94b). The influence of saline media on Tubifex Rivulorum. . Ibid. 17; XXIII-XXVII. 1894-1895. [See section, “Proc. of the Physiol. Soc.”] [vor. 2, 1915] 498 ANNALS OF THE MISSOURI BOTANICAL GARDEN and Sainsbury, H. (’94). The action of potassium, sodium, and calcium salts on Tubifex Rivulorum. Jbid. 16:1-9. 1894. Schreiner, O., and Reed, H. S. (’07). The röle of the pree power of roots in soil fertility. Jour. Biol. Chem. 3: XXIV-XXV. 1906-1907. Schulze, E. (’91). Ueber das zn der Lupinenkeimlinge gegen destillirtes Wasser. Landw. Jahrb. 20: p. 1891. er J. (’95.) Physiologisch-chemische Beobachtungen über Salzsäure. Skand. rchiv f. Physiol. 5: 277-376. pl. 7-8. Stiles, = ey Jörgensen, I. (’ 14). The measurement of electrical conductivity as a method of investigation in plant physiology. New Phytol. 13: 226-242. f. aii True, R. H. (’14). The harmful action of distilled water. Am. Jour. Bot. 1: 556-578. f.1. 1914. and Bartlett, H. H. (’12). Absorption and excretion of salts by roots, as influenced by concentration and composition of culture solutions. I. Concentration relations of dilute solutions of calcium and magnesium nitrates to pea roots. U. S. Dept. Agr., Bur. Pl. Ind., Bul. 231: 1-36. pl. 1 f. 1-21. 1912. —, ——+——,, (’15). The exchange of ions between the roots of Lupinus albus and culture get ee containing one nutrient salt. Am. Jour, Bot. 2: 255-278. f. 1-13. _— —— (15a). The exchange of ions between the roots of Lupinus albus -5 culture solutions containing two nutrient salts. Jbid. 2: 311-323. f. 1-3. 1915. Wächter, W. (’05). nee ger 7a über den Austritt von Zucker aus den Zellen der Speicherorgane von Allium Cepa und Beta vulgaris. Jahrb. f. wiss. Bot. 41: 165-220. 1 f. 1905. Walker, J. (’10). Introduction to physical chemistry. London, 1910. [See p. 237 ff.] Whitney, M., and Briggs, L. J. (’97). An electrical method of Serta ee, the te emperature of soils. U. S. Dept. Agr., Div. Soils, Bul. 7: 1-15. f. 1. 1897. —_——— , Gardner, F. D., and Briggs, L. J. (’97). An eae method of de- termining he se content of arable soils. Ibid. 6: 1-26. f. 1-6. 1897. ———_——, and Means, T. H. (’97). An electrical wae of determining the soluble salt content of soils, with some results of investigations on the effect of water and soluble salts on the disetz resistance of soils. Ibid. 8: 1-30. f. 1-6. 1897. Winckler, A. (°04). Ist gooi Wasser ein Gift? Zeitschr. diätet. u. phys. Therapie 8: 567-571. 1904-1905 TSN J. (1699). Some thoughts and experiments concerning vegetation. Roy. Soc. London, Phil. Trans. 21: 193-227. 1699. [vor. 2, 1915] 500 ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 13 igure Culture no. Conditions of growth. 1 (2 ae enewed distilled HO, De days. 2 (3) Renewed distilled H20, 45 days. 3 (6) 1 day dist. H20, 44 Er in renewed full nutr. 4 (8) 2 days dist. HO, 43 days in renewed full wires 5 (10) 5 days dist. HsO, 40 days in renewed full n 6 (14) 10 days dist. HO (renewed), 35 in A onal full nutr. 7 (18) 15 days ei H.O (renewed), 30 in renewed full nutr 8 (22) 20 ur t. H,O ee Ped in renewed full nutr. 9 (23) 45 days in run ewed fu 10 (25) 45 days in renewed full ey (9) 5 days in unrenewed dist. H,O, 40 days in rn pol . (10) 5 days in unrenewed dist. H»O, 40 days in renewed full n (11) 10 days in unrenewed aisi 150, 35 days in ee full ir SO SID D er Bee jet = Q =) Me w — [=] = B oO EE er 5 f: a) oo Zu S te „u m. B c z] = © B oO = © je? —, = A a = 5 4 10 (22) 20 days in renewed dist. HO, 25 days in renewed full nutr. * The numbers in parentheses correspond to the culture numbers of series 1. (See table I.) PLATE 13 om me 1915 ANN. Mo. Bor. GARD., VOL. 2, MERRILL—DISTILLED WATER COCKAYNE, BOSTON SCeONaurnrwnde _ CONQourP wrt e= 10 table [vor. 2, 1915] ANNALS OF THE MISSOURI BOTANICAL GARDEN Figure 1. Culture no. EXPLANATION OF PLATE PLATE 14 Conditions of growth aie days in unrenewed dist. H20 at time En. was taken. (3) 3 (6) (8) (10) 5 (14) (18) (22) (23) (26) Figure 2. (9) 3 days in renewed dist. H,O at time pictur ei 1 had in dist. H,O, 32 daya in renewed full n 2 days in dist. H20, 31 days in renewed full ae 5 days in unrene ewed rag Hs0, 28 days in renewed full nutr. 10 days in renewed dist. H20, 23 days in renewed full nutr. 15 days in renewed dis t. H,O, 18 days in renewed full nutr. 20 days in renewed dist. H20, 13 days in a full nutr. 33 days in unrenewed full nutr. 33 days in renewed full nutr. 5 days in unrenewed dist. H0, 28 days in unrenewed geo ... 5 days in unrenewed dist. H20, 28 days in renewed full n 10 days in unrenewed dist. H,O, 23 days in unrenewed full. nutr. 10 days in unrenewed dist, H30, 23 days in renewed full nutr. 10 days in renewed dist. H20, 23 days in unrenewed full nutr. 10 days in renewed dist. I 1,0, 23 days in renewed full nutr 3 da days in renewed dist. Hs O, 13 days in renewed full nutr. * The zn in ee correspond to the culture numbers of series 2. (See 1.) Ann. Mo. Bor. Garp., Vor. 2, 1915 PLaTE 14 Fig. 2 MERRILL—DISTILLED WATER COCKAYNE, BOSTON ee ee ed te u a aa 504 ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 15 Figur Culture no. Conditions of growth. 1 (1)*10 days in unrenewed dist, H,O, 42 days in unrenewed full nutr. 2 (2) 10 days in unrenewed rags H20, 42 days in renewed full nutr. 3 (3) 10 days in renewed dist. H,O, 42 days in ENS full nutr. 4 (4) 10 days in renewed dist. H50, 42 days in renewed full nutr 5 (5) 20 days in unrenewed dist. H,O, 32 days in unrenewed full nutr. 6 (6) 20 days in unrenewed dist. H0, 32 days in renewed full nutr. 7 (7) 20 days in renewed dist, 1,0, 32 days in unrenewed full ats 8 (8) 20 days in renewed dist. H>O, 32 days in renewed f tr. 9 (9) 30 days in unrenewed dist. H,O, 22 days in hd full nutr. 10 (10) 30 days in unrenewed rp H,O, 22 days i wed full n 11 (11) 30 days in renewed dist. H0, 22 days in ARED full we 12 (12) 30 days in renewed dist. HO, 22 days in renewed full nutr 13 (13) 40 days in unrenewed dist. H5O, 12 days in unrenewed full nutr 14 (15) 40 days in renewed dist. H20, 12 days in unrenewed full n Figure 2. l (1) 18 days in unrenewed Ps 2 (2) 32 days in renewed dist 3 (20) 32 days in oe N MgCl, & N/1000 CaCl. 4 (22) 1 day in unrenewed N/20 MgCl & N/1000 CaClg, 31 days unre- newed full ii 5 (23) 2 bine in unrenewed N/20 MgCly & N/1000 CaCle, 30 days un- wed full nutr. 6 (24) 4 days in unrenewed N/20 MgClo & N/1000 CaCl,, 28 days un- wed full nutr. T (25) 8 days in unrenewed N/20 MgCl & N/1000 CaClo, 24 days unre- wed full nutr. 8 (26) 12. an in unrenewed N/20 MgCly & N/1000 CaClg, 20 days un- ewe t: 9 (27) 16 days in unrenewed N/20 MgCl & N/1000 CaClo, 16 days unre- we utr. 10 (28) 20 days in unrenewed N/20 MgCl & N/1000 CaCl, 12 days unre- newed full nutr. 11 (29) 32 days in unrenewed full nutrient solution. *The numbers in parentheses correspond to the culture numbers of series 3. (See table v.) (See +The numbers in parentheses correspond to the culture numbers of series 4. table v1.) [vor. 2, 1915] Ann. Mo. Bor. GARD., Vor. 2, 1915 PLATE 15 ee r 5 PARA eu pes MANOR RIES Lan saa aaa terri ay Fig. 2 MERRILL—DISTILLED WATER COCKAYNE, BOSTON [vor. 2, 1915] 506 ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 16 Figure 1. Culture no. Conditions of growth. (1)*30 days in unrenewed distilled H30. (2) 30 days in renewed distilled H30. (3) 30 days in distilled H20, sterilized every four days. (4) a days in Sue H20, snare -= four days. (5) 30 days in unrenewed full nutrient solution, hpa 30 days in pps full actin tion. Figur Showing a method used for seed germination. *The numbers in parentheses also correspond to the culture numbers of series 5. (See table VII. ) OTAU Ann. Mo. Bor. Garp., VoL. 2, 1915 PLATE 16 Fig. 1 MERRILL DISTILLED WATER COCKAYNE, BOSTON ELECTROLYTIC DETERMINATION OF EXOSMOSIS FROM THE ROOTS OF PLANTS SUBJECTED TO THE ACTION OF VARIOUS AGENTS M. C. MERRILL Formerly Research Assistant to the Missouri Botanical Garden I. [INTRODUCTION In a previous paper the writer (’15) gave some results showing the exosmosis curves when normal growing plants are taken from a full nutrient medium and placed in redis- tilled water. Those results and the data herewith given show that exosmosis of electrolytes is a constant feature associated with the transfer of normal growing plants from a full nutri- ent solution to distilled water. In the paper above mentioned evidence was introduced indicating that such exosmosis was not a causal injury but that it was simply a concomitant con- dition or incidental effect and had but an indirect relation to the inimical condition of the plant in the distilled water. For convenience we might designate the agency or agencies caus- ing such exosmosis as passive in their effects. In this paper are given results on exosmosis in terms of the electrolytic conductivity of the medium when such excretion is caused, or at least is accelerated, by various factors or agen- cies which we may designate as active in their effects. Accord- ingly, plants have been treated by injurious agents or subjected to conditions of different kinds and the comparative effects on the exosmosis from the roots have been noted. By determin- ing the conductivity of the medium at various intervals sub- sequent to the treatment, data have been secured for plotting the exosmosis curves shown in this paper. It has also been the aim to determine in each case the approximate boundary between the normal and the abnormal exosmosis by varying either the duration of application or the concentration of the substance applied, or both. Hence in most cases there will be found the two extremes with any given substance—at the upper end of the scale the curve of excessive exosmosis due to ANN. Mo. Bor. GARD., Vou. 2, 1915 (507) [vor. 2 508 ANNALS OF THE MISSOURI BOTANICAL GARDEN cytolysis or death of the cells (though it should be noted here that excessive exosmosis from the roots may result even when those tissues are in an apparently normal condition), and at the lower end of the scale the curve of slight exosmosis that is in the region of the normal curve of exosmosis for un- treated plants placed from full nutrient solution into distilled water. Between these two extremes lie various gradations depending on conditions. II. Hisrorrcat Review The work that has been done on the problem of excretions from the roots of plants is very interesting from several stand- points and has been considered by various workers to be of great practical importance. Nearly a century ago De Candolle (732) advocated a theory of crop rotation on the basis of root excretions in which he claimed that certain plants excreted from their roots substances which are harmful to succeeding crops of closely related plants, but not so to plants less closely related. This theory was based partly on his own observations and partly on the statements of earlier workers. At De Candolle’s suggestion Macaire (’32) performed some experimental work pertaining to root excretions. He took plants from the soil, washed the roots carefully, and placed them in rain water. After several days, during which the water was frequently changed, the water was yellow and had odor, taste, and chemical reactions indicative of contained exuded materials. By placing one part of the roots of a plant in a vessel of pure water and another part in a second vessel containing a solution of lead acetate and later finding the salt in the pure water, he concluded that a plant can excrete a poison which it has absorbed. The results of Macaire’s ex- periments with water cultures led him to favor the theory of crop rotation on the basis of the excretions from the roots of plants, as advanced by De Candolle. Braconnot (’39) repeated many of Macaire’s experiments but was unable to convince himself that plants excrete toxic substances from their roots, and hence he did not look with favor upon De Candolle’s theory. Braconnot believed that 1915] MERRILL— ELECTROLYTIC DETERMINATION OF EXOSMOSIS 509 capillary action played a röle in Macaire’s experiments whereby he obtained an excretion of lead acetate into distilled water, as noted above. Boussingault (’45) considered that under ordinary conditions radicular excretion is doubtful, and that any excretion from the roots in water is caused by dis- ease. He also advanced various arguments opposing De Candolle’s theory. Gyde (’47) grew various agricultural plants in soil for a time and then, after carefully washing the roots, placed them in pure water. After 3-17 days, during which the plants con- tinued in good condition for the most part, the water was evaporated. The finding of a residue of yellowish or brown matter, part organic and part inorganic, caused him to con- clude that plants excrete both organic and inorganic substances in minute quantities, similar in composition to the sap. But he denied that root excretions have any injurious effect upon plants later grown in the same medium. An examination of the literature on the subject of root ex- cretions reveals the tendency among the workers of the par- ticular period at which we have now arrived in our review, to pay more attention to the morphological and chemical aspects of root excretions, and perhaps not so much to the purely agri- cultural phases. Hence we find from this period on, consider- able emphasis laid on the structure of the root and a more detailed account given regarding the chemical nature of the substances excreted from the roots, even though the experi- mental methods were somewhat crude in most cases. Further- more, it should be said that opinion was divided on the ques- tion of whether or not there is an actual excretion from the roots. Among those whose influence was felt in the development of the chemical aspects of the subject at this time Liebig should probably be mentioned first. In the American edition of his work (’41, p. 195) occurs the following statement: “It is evi- dent that plants, also, by producing carbonic acid during their decay, and by means of the acids which exude from their roots in the living state, contribute no less powerfully to destroy the coherence of rocks.’ An appended note by Dr. Webster in the [voL. 2 510 ANNALS OF THE MISSOURI BOTANICAL GARDEN same work (’41, p. 411) says that other chemists were unable to obtain results similar to those of Macaire. If they did, they were inclined to ascribe them to injury of the roots examined. Various workers were thus attacking different phases of the problem. Chatin (’47) mentioned the excretions from roots and especially considered the elimination of toxic substances by them. Link (’48) held that the slimy drops found on root tips should not be considered as actual excretion inasmuch as they arise from the cast-off cap cells of the root. Garreau and Brauwers (’58) maintained a similar view in regard to the gummy, nitrogenous substance they found given off by the roots to the water in which they were placed. The observa- tions of Liebig (’58) concerning the dissolving action of roots on limestone were later substantiated by the experimental work of Sachs (’60), which has been so much referred to since that time. Of the two possible explanations Sachs advanced —excretion of carbonic acid by the roots, and the liberation of acids by the decomposition of the cell walls of the roots—he inclined to favor the latter as being the best explanation for the marble etchings caused by the roots in his experiments. In his extensive series of experiments, Knop (’60, ’61, 62) studied, among other things, the character and amount of root excretions from certain plants placed in distilled water, and the conditions governing the same. His analyses indicated that, in addition to other substances in small amounts, potas- sium, calcium, phosphoric acid, and some organic matter were excreted. The studies of Cauvet (’61) resulted in his declar- ing that physiologically sound roots do not excrete any sub- stances, toxic or otherwise, and that all theories based on the ideas of root excretion advanced by De Candolle and Macaire were necessarily false. Sachs (’65) made further contribu- tions along his line of work indicated above, while Liebig (’65) says: “Wir haben allen Grund zu glauben, dass diese Absonderung an der ganzen Oberfläche stattfindet, wir beobachten sie nicht nur am Stamme, sondern auch an den kleinsten Zweigen, und wir müssen daraus schliessen, dass dieser Exeretionsprocess auch an den Wurzeln vor sich geht. . . . . Eine Aus- scheidung von Exerementen kann demnach bei den Pflanzen 1915] MERRILL—ELECTROLYTIC DETERMINATION OF EXOSMOSIS 511 nicht geleugnet werden, wiewohl es möglich ist, dass sie nicht bei allen Pflanzen in gleichem Grade stattfindet.” Molisch (’87) branched out in a new direction as regards the subject of root excretions; he held that such excretions exer- cise an influence on organic bodies in the soil which is even more important than that exercised upon the inorganic con- stituents of the same, for he considered the latter merely a dissolving action but the former a real chemical transforma- tion. His main work along this line pertained to a study of the ferments in the root excretions, and their reactions and properties. Johnson (’90), after considering Gyde’s results above noted, says that ‘‘we may well doubt whether agricul- tural plants in the healthy state excrete any solid or liquid matters whatever from their roots,’’ but that ‘‘under certain circumstances, small quantities of soluble salts or free acids may indeed diffuse out of the root-cells into the water of the soil. This is, however, no physiological action, but a purely physical process.’’ Goebel (’93) found that after the roots of Hordeum and Lepidium plants had been in distilled water for six days the medium gave the reaction for formie acid. We thus see that the early work on root excretions was characterized by contradictions and uncertainties. While the nature of the more recent work has been more exact and com- prehensive, the subject, as we shall see, is still beclouded by a considerable degree of confusion. A classic piece of experimental work was undertaken by Czapek (’96, ’96") to determine the exact chemical nature of the excreted substances from roots. In his report (’96*) he discussed the earlier work, especially with regard to the rela- tion between excretion from injured cells and actual exos- mosis. In his experimental work he found that root excretions are composed of soluble substances, partly organic and partly inorganic. Of the inorganic, he identified K, Ca, Mg, HCl, H>SO,, and H;PO,, only the first and last mentioned—in the form of the primary potassium phosphate—being excreted in any quantity. Of the organic substances he identified carbonic acid and also formie acid, the latter in the form of its potassium salt; oxalic acid was also isolated as a primary [Vou, 2 512 ANNALS OF THE MISSOURI BOTANICAL GARDEN potassium salt. Czapek believes the reddening of litmus paper by root excretions to be due ordinarily to the acid reaction of monopotassium phosphate, but in the case of hyacinth roots to the primary oxalate. The corrosion of marble he attributed to the dissolving effect of carbonic acid. While considering as possible the results obtained by Molisch (’87), who claimed that diastatic ferments were normally present in the root excretions, Czapek’s own work in repetition of Molisch’s experiments offered only negative results. Prianischnikov (’04) performed some experimental work dealing with the action of organic acids on phosphates. It will be remembered that because the roots did not attack aluminum phosphate Czapek concluded that organic acids were not ex- ereted by them, inasmuch as this substance is soluble in cer- tain organic acids. Prianischnikov found that phosphates derived from different sources were utilized by various plants but in different degree, and he suggested that this might be correlated with a different amount of CO2 excretion, in which case the presence of organic acids would not be necessary. Kunze (’06) found that free mineral acids are not excreted from the roots of higher plants and concluded that any acidity in the excretions is probably not due to the presence of acid salts of mineral acids, but to excreted organic acids. These, however, were present in such minute amounts as to be below the sensitiveness of litmus. He held that a greater effect is produced on the soil by fungi than by the roots of the higher plants. Lemmermann (’07) held views similar to those of Kunze. Stoklasa and Ernest (’08) disagree with the findings of both Czapek and Kunze. No potassium or phosphoric acid were ever found as a result of their determinations, and they main- tain that in the economy of the plant the excretion of such useful or necessary substances is unthinkable. Only COs was found to be excreted under conditions of normal aerobic res- piration of the root system; no other free inorganic or organic acids were detected. In aerobic respiration of the root system, they believe the organic acids in the living cells would be split up to give COs and He, the latter then being oxidized to H20. 1915 MERRILL—ELECTROLYTIC DETERMINATION OF EXOSMOSIS 513 They determined the amount of CO2 excretion per gram dry weight of roots of wheat, oats, rye, and barley. The amount varied for the different plants but a correlation was found between the amounts of P205, K, and Na contained in the dry roots of plants grown on gneiss and basalt and the amount of CO; excreted. We now come to the work of various soil investigators whose results have again focused attention during the past decade upon De Candolle’s original theory. The essential features of this work have become so well known that for our purpose it is not necessary to do much more than merely men- tion it here. Though not considering directly the phases of the subject with which we are dealing, yet the much-discussed paper by Whitney and Cameron (’03) is historically impor- tant and bears an intimate relation to the later work of the investigators in the Bureau of Soils of the U. S. Department of Agriculture, the results of which led to the so-called toxic- excretion theory. Among the workers most prominently con- nected with the early studies along this line may be mentioned Livingston, Britton, and Reid (’05); Livingston, Jensen, Breazeale, Pember, and Skinner (’07); Schreiner and Reed (’07); Schreiner, Reed, and Skinner (’07); Schreiner and Reed (’07*) ; and others. As is well known, opinion is much divided on the various phases of this subject, however. Among those opposing the ideas or theories advanced along this line by the investigators named above should be mentioned Hop- kins (710) ; Hall, Brenchley, and Underwood (’14) ; and others. That the question is one upon which investigations are still being pursued is shown by the publications from various quar- ters. As recent examples of these the work of Molliard (’13) and Prianischnikov (’14) may be cited. The former found that peas grown in water cultures in which previous crops of peas had grown produced a smaller growth than the original crops. This he attributed to the excretion of toxic substances in the medium by the earlier plants. The latter, from his own experimental work and from the results observed by him at the Rothamsted Experiment Station, is inclined to believe that the hypothesis of root excretion is not sufficiently demon- (Vou. 2 514 ANNALS OF THE MISSOURI BOTANICAL GARDEN strated. He says that other factors, as, for example, the physi- eal nature of the soil, decomposition of roots, change in re- action of soil, ete., might be supposed to accomplish the same results as toxic excretions from the roots. In pure distilled water he found no decrease in either the size or quality of the crops of the second and third plantings, either where wheat fol- lowed wheat or where wheat followed oats. Experiments in sand, however, showed great decrease in the amount of the harvest of the second and third crops, but this, he believes, might be explained by the operation of the above-named factors. So much for root excretions; we now come to a general con- sideration of exosmosis from living cells, both under natural conditions and under treatment of different kinds. While a great deal of attention has been given in the past to the in- take, or endosmosis, of substances by the cell from its sur- rounding medium, comparatively little has been done on the opposite effect—the outgo, or exosmosis, of substances from the cell. It should be said, however, that the latter process, both in extent and in importance, is no doubt of much less significance in the plant’s economy than the former. Sachs (’60") referred to the exosmosis of soluble material from germinating seeds when they remain for some time in distilled water. Knop (’64), in his studies on the absorption of salts by healthy seeds, also determined the quantities of the different salts which pass out of the seeds during the time they are swelling in distilled water. He found that both or- ganic and inorganic substances were excreted. Hofmeister (’67) ascertained that when fresh pieces of sugar-containing plants were placed in water, no sugar passed out of the tis- sues into the medium. The much-cited experiments of De Vries (’71) showed that pieces of red beet placed in water for 15 days gave no trace of sugar or of colored material to the water during that time. In a NaCl solution of sufficient con- centration, however, he obtained an exosmosis of both sugar and colored material. Turnips, beets, and the seedling roots of wheat, barley, and corn were used in the experiments of Boussingault (’74) but from none of them did he detect any 1915] MERRILL— ELECTROLYTIC DETERMINATION OF EXOSMOSIS 515 exosmosis of sugar into the water in which they were placed. Pfeffer (’76, ’”77) and Detmer (’79) also confirmed the re- sults above noted regarding the absence of sugar in the water in which roots or other plant parts had been exposed for some time. Wilson (’81) found that in some cases (Dionaea and Drosera) the excretions may be influenced by external factors, e. g., partly by irritation caused by nitrogenous substances and partly by osmotic action. In general, he believed that the excretion of nectar is caused by the osmotic action of a fluid on the surface of the nectary. Pfeffer (’86) studied the ef- fects of various organic acids (citric, picric, and tannic) and some inorganic compounds in causing the exosmosis of ab- sorbed methylene blue from Lemna, Trianea, Azolla, and Elodea. Wachter (’05) obtained considerable exosmosis of sugar, especially in the case of Allium Cepa; he found, however, that salts like NaCl and KCl tended to inhibit this exosmosis. He also investigated the effect of ether on this phenomenon. While he obtained greater exosmosis of sugar the first two days in a solution of ether alone than in one of ether and KCl, he at- tributed this increase to leaching from cells killed as a result of contact with ether, and believed that the ether itself has no effect on the actual process of exosmosis. Lepeschkin (’06), from his experimental work on sporangia of Pilobolus, concluded that the exosmosis of water was due to an alteration of the plasma membrane caused by the anes- thetics he used, provided the amounts employed were sufficient to be toxic. Small amounts of ether and chloroform, on the other hand, were found to decrease the exudation of water, and he believed this to be due to a decrease in permeability of the plasma membrane. An interesting line of investigation was undertaken by Czapek (’10, ’10°, 710", 711) a few years ago to determine the surface tension relations of the plasma membrane. That work is especially pertinent to our discussion here because of the prominent part exosmosis played in his experiments. He used for the most part species of Echeveria, Spirogyra, and Saxi- fraga, in the cells of which is found a tannoid substance, [VoL. 2 516 ANNALS OF THE MISSOURI BOTANICAL GARDEN anthocyan, which is precipitated by caffein, giving a loose compound of tannin and caffein, called a ‘‘myelin-formation.’’ Ammonia also gives this precipitate even in a solution as dilute as 1-15,000. Czapek investigated the effect produced by the application of a great variety of organic compounds and some inorganic acids in varying dilutions and for different periods of time, and determined the concentration at which exosmosis just occurred, i. e., the critical point. At the higher concen- trations exosmosis of the tannoid substance readily occurred, as shown by the absence of the ‘‘myelin-formation’’ when caffein or ammonia was subsequently added. At the lower concentrations exosmosis did not occur and a precipitate was obtained, while at the critical point the precipitate was barely visible and usually in the form of fine particles. By the use of his ‘‘capillar-manometer,’’ Czapek was able to measure the surface tension exerted by the various concen- trations, and found that, considering the surface tension of water as unity, that of the critical concentrations was approx- imately .68 in most cases. This lowering of the surface ten- sion he considered as essentially a physical phenomenon which is intimately connected with the osmotic activities of the plasma membrane and is to be differentiated from the toxic action of injurious substances, e. g., anesthetics, whose action is chemical in large part, since even in very dilute solutions these caused marked exosmosis. Czapek used both aqueous and colloidal solutions and found that in general the critical concentrations had a surface tension of .68 in terms of water as unity. Inversely, he therefore concluded that the surface tension of the plasma membrane was also approximately .68 for the plant cells investigated. In his study of acids he found results coincident with those of Kahlenberg and True (’96) in that N/6400 was the critical concentration for exosmosis of the tannin bodies, just as those workers had found it to be the critical concentration for growth of Lupinus seedlings in solution culture. In his later experimental work Lepeschkin (’11) obtained additional evidence tending to confirm and add to his previous results, as mentioned above. Thus he found that aniline dyes 1915 MERRILL—ELECTROLYTIC DETERMINATION OF EXOSMOSIS 517 penetrated cells of Spirogyra more slowly in the presence of one per cent chloroform than when the anesthetic was not used. If the cells were killed by the narcotic the rate was the same as for normal cells. He also used Tradescantia discolor and by the plasmolytic method found that the permeability to KNO; decreased during narcosis. This he explained on the assumption that the anesthetics (chloroform and ether) ac- cumulated in the disperse phase of the plasma membrane which thereby leads to a hindrance of the solubility of KNOs and aniline dyes in the same. He considered that his results therefore showed that Nathansohn’s hypothesis regarding the mosaic structure of the plasma membrane is not correct. Another important piece of work dealing with the phenom- enon of exosmosis from living tissue is that accomplished by Lillie (’09, ’10, 711, 712, 712%, 713, ’13°, 713") and discussed at length in his various papers. Among other things he worked on the larvae of Arenicola and the eggs of Arbacia, each of which contains a pigment, and found that on placing them in NaCl or KCl solution (.55m) isotonic with sea-water, there was a rapid exosmosis of the contained pigment into the surround- ing medium. When, however, the organisms were placed in the salt solutions to which had previously been added in a cer- tain concentration any one of several anesthetics belonging to various classes (alcohols, esters, hydrocarbons, and miscel- laneous compounds) a checking or possibly a complete preven- tion of exosmosis resulted. In general, all the anesthetics tried gave cytolysis in strong concentrations and therefore a rapid exosmosis of the pigment, while in weaker concentrations they showed a definite protective or anticytolytic action against the salt solution when used in conjunction with it. Lillie finds the explanation of the observed phenomenon in the relations of the plasma membrane, the salt solutions used having a permea- bility-increasing action which is offset or prevented by the tem- porary alteration of the membrane as the result of the action of the anesthetic. The alteration, he believes, is accompanied by an increase in the volume of the lipoid particles of the membrane. Tn connection with the general subject of exosmosis it might [vorL. 2 518 ANNALS OF THE MISSOURI BOTANICAL GARDEN be well briefly to mention the results obtained by some of the earlier investigators working on the products exereted by the leaves of plants. De Saussure (1804) found that leaves im- mersed in distilled water soon lose a considerable amount of substance, composed for the most part of alkaline salts. Treviranus (’38) mentioned the results of various workers who studied the inerustation of minerals on the surface of leaves and found it to consist of calcium and silicon salts, especially of calcium carbonate. Gaudichaud (’48) and Payen (’48) both found that there is an alkaline excretion on certain parts of the leaves of some plants, yet they disagreed as to the extent of this phenomenon in nature. Sachs (’62) ascer- tained that drops of water on the leaves of certain plants soon become alkaline, which he considered to be the result of an out- ward diffusion of alkaline salts in the leaf. Volkens (’84) studied the deposit of calcium carbonate found on the leaves of various plants. Dandeno (’02) made a comprehensive study of the different phases of the subject. Among other things, he determined that the alkaline substances extracted from leaves by distilled water are largely potassium and calcium carbo- nates and probably potassium oxalate. He further found that the residue from the evaporation of dew drops, guttation drops, and of water used in drenching the leaves is practi- cally the same, and is similar to the calcareous deposit found upon the leaves of certain plants. The above investigations may therefore be considered as tending to substantiate the idea of exosmosis from leaves. III. METHODS or EXPERIMENTATION The methods used for the electrolytic determination of exosmosis were the same as those described in the writer’s paper referred to above. In that contribution (Merrill, ’15) some of the curves were plotted on the basis of the specific conductivity. In the present paper, however, all curves are plotted on the basis of the values of x on the Wheatstone bridge when the resistance in the box is 9,110 ohms; as these values increase the specific conductivity also increases. In order to have a basis of comparison between the values of x MERRILL—ELECTROLYTIC DETERMINATION OF EXOSMOSIS 519 and the specific conductivity, the corresponding values of the latter for the values of v at 5, 10, 25, 50, 75, and 85 are given herewith: Corresponding values in terms of spe- Values of æ% on a... bridge for eifie conductivity (to be multiplied resistance u. 9,110 o by 10°) ee rl EDER .23 10 ERENTO ET .4 DE IRRE I ESEL E Te 1.49 RR SCREEN SE TER 4.48 (OSS CESS il Ee rene eer 13.46 es er 25.43 It is also advisable to have the conductivity values repre- sented in terms of the concentration of some salt. The follow- ing are the values of the specific conductivity of NaCl solu- tions at 25°C. which had been determined by the writer for the concentrations indicated : Concentration of NaCl Specific conductivity (to be multiplied by 10—*) tf. Brees een ven ne A EEE ELITE 686.13 ING Sree Sake TITS er EA ESO 353.94 LOB REIT. 181.93 NY V8) Wham stefan ee ee an EEA AA 93.25 ET AA E sane ae hele ie eke) E 47.79 Ve te CRORE OCU LCR aR PORTE eC 24.54 IN ODS ee ee ae exis nl 12.60 The correction for the specific conductivity of the water itself is not considered in the above values. Neither is that cor- rection applied in any of the work here reported, since it is always a constant factor and only relative values are desired for the most part. Plants of Pisum sativum were used. For the method of growing the seedlings, and other manipulations, see the writ- er’s paper referred to (Merrill, 15). The plants were grown in full nutrient solution until a vigorous or well-developed con- dition was attained and then they were transferred to redis- tilled water! after rinsing the roots carefully and thoroughly in once-distilled water. Ten plants were grown in each cul- ture. The treatment was always given when the plants were either in distilled water or in the solution, the effects of which on the plants were being studied. In all cases where the read- 1 Hereafter, en this paper, whenever “distilled water” is referred to it will be understood to mean redistilled water with a specific conductivity of ap- proximately 2 X os If the ordinary distilled water is referred to, it will be specially designated as “once-distilled water” or some such distinguishing term. [vor. 2 520 ANNALS OF THE MISSOURI BOTANICAL GARDEN ings were made in the distilled water, the resistance in the re- sistance box was 9,110 ohms. In some media other resistances were used; in such cases the values are given only in tables, and in terms of specific conductivity. TABLE I EFFECTS OF VARIOUSLY TREATED PLANTS ON THE DISTILLED WATER MEDIUM AS SHOWN BY GROWTH OF SECOND CROP Green wt. of u Kind and duration of treatment tops of 2ndcrop* grams 1 and 2 | Controls—no treatment; full nutrient to dist. H:O..... 2.90 3 Plant tops packed in ice 19 hrs.; dist. H:O unchanged 1) 2.80 4 Plant tops packed in ice 19 hrs.; dist. H:O changed . i 5 gas incuba 30.3 RR .60 6 as incubator at 50°C., 3. 2 hs Deo Segen vaste e eens oo 7 and 8 | Inoculated with la “agp Mey SEER EEE TERT .80 9 lum. gas under bell jar, 6 reg ; dist. H:O unchanged... AS 10 llum. gas under bell jar, 6 hrs.; dist. H:O changed..... = 11 and 12| N/1 MgCl. in full nutrient as the solvent, 7 hrs........ .20 13 and 14| .5% H:SO: in full nutrient as the solvent, 7 hrs........ 2:30 15 and 16| 1% KOH in full nutrient as the solvent, 7 hrs......... 8.45 17 and 18| Plants grown throughout in dist. H,O; replaced by ‚fresh seedlings in the unrenewe A." 0 eee 2.55 19 and 20) Same as es and 18, except that seh crop was EEE EURE O 6.85 21 and 22| Canada field peas in he dist. soa no second crop. 2.82 23 and 24| Horse beans in fres t. H:O; no second crop........ 8.15 * The 2nd crop was 27 days = at time of mane IV. PRELIMINARY EXPERIMENTS In order to determine in a preliminary way whether the exosmosis from the roots of plants seriously affected by in- jurious agencies was sufficient to noticeably influence a new crop of seedlings in that medium (distilled water plus the ex- creted substances) as compared with control cultures in pure distilled water, the following series was set up. Canada field pea seedlings were grown in full nutrient solution until they were 15 days old, at which time they were about 8 inches high, and were green, vigorous, healthy, and in good condition. They were then treated in accordance with the plan given in table 1. In some cases, depending on the nature of the agent applied, the treatment was given after the plants had been transferred to distilled water. This was the case with Nos. 3, 4, 5, 6,9 and 10. Cultures 7, 8, and 11-16 were treated while still in the 1915] MERRILL—ELECTROLYTIC DETERMINATION OF EXOSMOSIS 521 full nutrient medium, after which they were transferred to dis- tilled water. In all instances, however, the roots were care- fully rinsed before being placed in the water. To determine if any impurities had contaminated the dis- tilled water in the cultures treated with ice, the distilled water in No. 4 was renewed after the operation. The resulting crop, however, was practically the same in cultures 3 and 4 and hence it may be considered that no plant food had entered from the ice. The distilled water was renewed in No. 10 a few hours after the treatment. The result of so doing was to discard the plant foods already excreted during, and immediately after, the treatment. This fact was evident from the better growth of the plants in No. 9 as compared with those in No. 10. Later work also showed that exosmosis caused by treatment with illuminating gas and other agents is comparatively rapid and immediate. After the treatment the plants remained in the distilled water for 5-6 days, after which they were discarded. The dis- tilled water level was then raised to the original height by adding fresh distilled water, and into this medium fresh Can- ada field pea seedlings were placed and the resulting growth determined. Cultures Nos. 17-24 are given in table ı for comparison. After pea seedlings had been grown for 21 days in the unrenewed distilled water of cultures 17-20, the original plants were discarded and fresh seedlings of peas and horse beans were placed in the same distilled water. For compari- son, cultures of these plants (Nos. 21-24) were set up at the same time in fresh distilled water. Returning now to the effects of the treatments on the plants and noting the results given in table 1, we see marked differ- ences evident. Neither the ice nor the inoculation with Asco- chyta Pisi? produced any effect either on the plants or on the excretions from their roots, and hence these cultures are sim- 1The usual method of rinsing throughout this work was as follows: The solution to be discarded was thrown out, the tumbler filled twice with once-distilled water (the roots replaced and the whole thoroughly shaken each time), and then distilled water (redistilled) was added, the roots replaced, and the readings taken. *Cultures of Ascochyta Pisi were kindly supplied the writer by Dr. R. E. Vaughan. (Vou, 2 522 ANNALS OF THE MISSOURI BOTANICAL GARDEN ilar and comparable to the untreated controls. Marked injury resulted in the case of the heat, illuminating gas, MgCle, and H>SO,, all in characteristic manner. The injury from KOH was rather slow in manifesting itself, but the coloration of the roots was a noticeable feature. An interesting condition to be noted here, which holds true also in the later experiments, is in regard to the effect of the heat and the illuminating gas. It should be borne in mind that during these treatments the roots remained in water. The tops only were affected and died; the roots remained white, turgid, and normal in appear- ance even though the exosmosis from them had been exces- sive, thus indicating a transfer of some electrolytes from the tops and down into the medium through the roots. Later ex- periments also substantiated the fact that abundant exosmosis sometimes occurs from roots which remain normal in appear- ance. The other agents (MgCle, HeSOs, and KOH) caused more or less injury to both tops and roots. The exosmosis of nutrients into the water from the affected plants is evident by the greater growth of fresh pea seedlings placed in such water as compared with the controls. Both peas and horse beans grew somewhat better in fresh distilled water than in distilled water in which pea seedlings had already been grown for 21 days. Further preliminary experiments along this line gave sim- ilar results. Thus in another series, vigorous, thrifty plants of Canada field peas grown 10 days in full nutrient solution were transferred, after rinsing the roots, to distilled water, some being untreated and others treated. The treatment con- sisted in placing some of the cultures in an atmosphere of illuminating gas for 3 and 6 hours, and others in a gas-heated oven for 1 and 2 hours where it was not aimed to keep the temperature constant. For those cultures in the oven 1 hour the temperature at the outset was 53°C. and at the end 33°C., while for those remaining in the oven 2 hours the initial tem- perature was 60°C. and the final 33°C. The conductivity of the water was measured soon after the plants were placed in it but before the treatment, and again 5 days after treatment, 1915] MERRILL—ELECTROLYTIC DETERMINATION OF EXOSMOSIS 523 at which time the plants were discarded and a fresh lot of pea seedlings substituted. The average reading (value of x) of the water for the 4 untreated controls at the beginning was 14.6 on the Wheat- stone bridge, and with the same resistance in the box (9,110 ohms) it was 12.6 after 5 days. For the 4 cultures treated with illuminating gas (2 cultures for 3 hours and 2 cultures for 6 hours, the resultant effect being approximately the same for the two periods of exposure) the average initial reading was 17.0 for a resistance of 9,110 ohms, and at the end of 5 days it was 43.7 for a resistance of 1,000 ohms. In this case the increase in terms of specific conductivity was from 9.2 x 10 to 317.3 x 10°. In the 4 cultures placed in the oven at the temperature desig- nated there was no marked difference as regards variation in conductivity of the medium. The average initial reading was 17.2 and at the end (after 5 days) it was 12.4, the resistance in the box being 9,110 ohms in both readings. The rather high initial readings in the above cases are due to the fact that it was some hours after the roots were placed in the water be- fore the readings were taken. In later work it was found to be advantageous to have the interval between placing the roots in the water and taking the first reading reduced to exactly one-half hour in order to obtain comparative data on the initial rate of exosmosis under different conditions. We have, of course, no indication from the above regarding the exosmosis or conductivity curve during the 5-day interval. Subsequent work shows that it is very probable that the curve rose considerably in the case of the untreated and the oven- treated cultures and then fell, at the end of 5 days, to a posi- tion lower than that of the initial reading, due to the absorp- tion being greater than the excretion after the first 2 or 3 days. Let us turn now to the results obtained with the fresh seed- lings grown in the same water in which the first crop had re- mained for 5 days under the conditions indicated above. After the second crop had been growing in this medium for just 15 days the green weight of tops of the 4 cultures in each group [VoL. 2 524 ANNALS OF THE MISSOURI BOTANICAL GARDEN was determined, with the following results, the figures repre- senting the average green weight of tops in each culture: Previous crop untreated........cccesecesesersreccces 3.39 grams Previous crop treated with illuminating BAS. eee ee eee 4.38 grams Previous crop in oven 1 and 2 hrs. at 60-33°C.......... 3.35 grams V. EFFECTS or ANESTHETIC VAPORS For this work the method used was to place the cultures (in some cases the medium also, in which instances the roots were in the water during exposure, and in other cases only the plants themselves, thus exposing the roots directly to the vapor) under bell jars into which the anesthetics were subse- quently placed. In the case of ether and chloroform a meas- 20 CaF Ase ows ow ww a a Oa a A i iE ® ENENEENEEENERERERERERERENERENE| 111 is 2 38 ENENEREEERERERERRERERERENERNEN! co Corrs 3 HEHEH sss . j=] a'ž 15 COCO ee TI °2 TATS u ro m HH ag na =S === seeeneeae ar © 10 - te Foi nan CEFER ot u meesi TT ns coe COCO Coo} o 8 TERTI Coe ima Coo 38 5 PETER Coo Cr coor Sg CoCo Coo Cot Coy > COO COCO rH Co > Coot COC Cr Coo Coo Core as Coo | ME O BE TD EI STIL CL LL Le LLLI III I IT I I it 1 2 3 4 5 6 7 Days Fig. 1. Conductivity curves of cultures (series 11) er distilled water subsequent to treatment with anestheties, as follows: No. 21, ether i t No. 22, cont exposed under bell yA 1 minute; No. 23, ether vapor, 2 minutes, roots sn. T No. 24, ether vapor, 5 minutes, roots exposed ; = ed ether vapor, 10 setter roots exposed; No. 26, ether vapor, 1 nutes, — ‘exposed. The plants used were 39 days o old. The first govt in each case is of the distilled water before the roots were placed in it. ured amount of these agents was placed in an open evaporat- ing dish under the bell jar, and after the treatment the residue was measured to determine the amount which had evaporated; in the case of the illuminating gas, however, the agent was run in until the air in the bell jar was more or less completely replaced. Where the plants alone were placed under the bell jars they were carefully attached by cheese-cloth bands to the leg of an inverted tripod, over which the bell jar was then placed. 1915] MERRILL—ELECTROLYTIC DETERMINATION OF EXOSMOSIS 525 In figures 1 and 2 are shown the results of treatment with ether and illuminating gas for varying periods of time. The plants for this experiment were 39 days old at time of treat- ment and had roots in good condition and well developed. The i ER) | | | | | = > wn \ iT \ A © \ N an \ Values of æ on the Wheatstone bridge a [1 Cc] i i \ \ IY IH 1 1 | I 1 [ii I H Mil IT} I na II III Voy U | I | | mn it nee BEN. Seeks Days Fig. 2. Conductivity curves of cultures (series 11) in distilled water subsequent to treatment with gr ne follows: No. 27, illuminat- ing gas, 5 minutes, roots exposed; No. illuminating gas, 10 minutes roots exposed; No. 29, illuminating = 15 minutes, roots expos sed; No. 30, control—roots exposed under bell jar 15 minutes; No. 31, ether vapor, 3 hours, roots in tumbler; No. 32, control—under bell jar 3 "hours, roots in tumbler. The plants used were 39 da ays old. The first reading in each case is of the distilled water before the roots were placed in it. first conductivity readings of the water were taken before the plant roots were introduced. As seen from the plotted re- sults the ether had no effect on the exosmosis when the dura- tion of the exposure ranged from 1 to 15 minutes; after 3 [vor. 2 526 ANNALS OF THE MISSOURI BOTANICAL GARDEN hours exposure, however, the exosmosis was pronounced, even when the roots were not in direct contact with the vapor. An exposure of only 5 minutes te illuminating gas pro- duced no effect, but one of 10 or 15 minutes’ duration eaused considerable exosmosis. That the 15-minute exposure should result in less exosmosis than the 10-minute one is an inter- esting point which finds an analogy, we shall see, at different places throughout the work, where in isolated cases a briefer exposure or milder treatment results in greater conductivity of the medium than a somewhat more prolonged exposure or more severe treatment. Where such a condition exists it is usually found near the boundary line of noticeable effect, and not where the effect is either nil or very pronounced. At this critical point the individual hardihood of the plants them- selves seems the most plausible explanation of the difference. As the manipulation methods were exactly similar for any given series it is altogether unlikely that difference in tech- nique was responsible for the variation. The only plants to sustain any injury were those of cultures 28, 29, and 31. The tops of those in No. 31 drooped imme- diately after the treatment and soon died, though the leaves remained green; the roots, however, remained entirely normal to all appearances and retained their turgor. This is an in- teresting point and was referred to above. After 7 days Nos. 28 and 29 plainly showed some injury, but it was slight, and its visible effects were slow in making their appearance. At that time the tops of these cultures showed greater yellowing and drying than did those in the controls, No. 29 being somewhat more affected than No. 28; the roots of both, however, re- mained normal in appearance. The greatest contrast between the treated plants and the controls is seen in fig. 3. The effect on the treated cultures corresponds to the duration of treatment, the curves espe- cially showing the difference in the speed of initial exosmosis. It will be seen that the conductivity curves of the controls rise rather high during the first day. This is no doubt due to the effect of rather prolonged exposure of the roots to the air in the bell jar, even though it was saturated with water vapor. 1915] nN n © wn n an Values of æ on the Wheatstone bridge w an MERRILL— ELECTROLYTIC DETERMINATION OF EXOSMOSIS 527 Ep? FET T T Coo ER 2 | 1 = = Set 5 EA w IT E 5 1] = A -3 4 A Ai LA A 4 N it T WCIN \t NIT ~ = “= IT EFI ABM E A iS A E E E E E S A e Sa e a E za EENBRRBERENG. 17377 2 4 6 8 i0=212 14 Or ger 922724 Days Ber en; Pe of en En 11) in distilled water subsequent to treatm fol er inating gas mi n control—roots exposed under a: jar 30 min gas, 1 hour, roots exposed; . 38, control— water for the following ee No. 33, 35 m 1 hou minutes, par exposed ; 36, utes; No. 37, illuminating roots exposed under bell jar l hour. The plants were 22 FA ays ‘ade hen treated. The first readings were En in the various cultures after the roots had been in distilled a No. 34, 1 hour and 15 minutes; and 13 minutes; 36, ‘minutes; No. 37. 1 a aa 5 minutes; No. 38, 1 tie and 16 minutes. [Vou. 2 528 ANNALS OF THE MISSOURI BOTANICAL GARDEN The subsequent decline in the curve, however, is characteristic for normal root tissues. It is also seen here that the 15- minute exposure to illuminating gas resulted in a greater rise in the conductivity curve than did a similar exposure in the case of the cultures recorded in fig. 2. That this is due to the different ages of the plants in the two cultures was borne out by treatment of plants of different ages with other agents. The older the tissues the more resistant they become to the toxic substance. McCool (713) was the first to point this out, in his experiments with manganese chloride, and we see that it here holds for anesthetics as well. Figure 4 shows the effect of illuminating gas at different in- tervals when only the tops are exposed directly to the gas, the roots meanwhile remaining in distilled water. The plants were affected in proportion to the duration of treatment. The tops of No. 39 were only very slightly injured, so that there was practically no difference between them and the tops of the controls; No. 41 was affected more; and No. 43 still more, finally dying, after progressive drooping and yellowing. But here again the roots of the treated plants were in all respects similar to those of the controls and entirely unaffected, vis- ibly, even though exosmosis was considerable. In such cases it was also presumed that the excreted substances came in part from the tops and that here we had an illustration of the downward flow of food materials which occurs in plants under natural conditions. This presumption was considered experi- mentally as follows: Some cultures were placed under a bell jar and treated with illuminating gas as before, the roots meanwhile being in distilled water. The tops of one culture were not cut off, while those of another were removed just before treatment, and finally those of a third were removed just after treat- ment. The controls were not treated, but their tops were cut off immediately after the roots were placed in the distilled water. The treated plants all gave approximately the same exosmosis, which was considerably more than that from the controls. A point to be noted here is that even though the treated tops which were not cut off were very much affected, 19 Values of x on the Wheatstone bridge nN © MERRILL— ELECTROLYTIC DETERMINATION OF EXOSMOSIS 529 DA A p HH = 2 ro = IT E co = A To = = a = A < A | 4 A Z I TT Pr S39 u 4 _—, \ \ N, \ ~~ N 5 A = Za H m neu s at nu > M “ = sl ELI ] cH Coco Coo Coco Coo eH A ie se EERLGAR LLLELI 125 EE a R, 117 1.003 LET Coo Coo See FSB (BRS: a | 2 + 6 8 9. 22 4. WEB Fi 2 24 Days Fig. 4. Conductivity we of menge en ee in distilled water subsequent to treatment, follow 39, i inating gas, 15 minutes, roots in reir "No. 40, control under ‘bell | ar 15 minutes, roots in oe er; No. 41, illuminating gas, 30 minutes, roots in tu En No. 42, trol—under bell jar 30 minutes, roots in tumbler; No. illuminating gas, 1 hour, roots in tumbler; No. 44, pe or aa PN bell jar 1 hour, r oots in tumbler, The plants were 22 pve old when treated. The ee Pending was taken in the various cultures after the roots had n in the a water to the treatment for the following periods (but t e periods should be added the time the cultures were under the ae Aen for the Haie were in > distilled wer susie that interval also): No. 39, 2 hours and 12 minutes; No. 40, 2 hours and 23 minutes; No. = Ven an Sy 20 minutes; No. 42, 2 hours and 30 minutes; No. 43, 2 and 11 minutes; No. 44, 2 hours and 23 tes. [voL. 2 530 ANNALS OF THE MISSOURI BOTANICAL GARDEN the roots meanwhile remaining practically normal, transpira- tion no doubt still continued. It remains an open question, how- ever, whether such transpiration caused lower conductivity readings, due to the consequent absorption of electrolytes, than would have been the case had there been no, or only slight, transpiration, as in the cases where the tops were removed. The roots in all the cultures remained turgid and practically TABLE II EFFECTS OF ILLUMINATING GAS ON THE EXOSMOSIS FROM THE ROOTS OF PLANTS UNDER VARIOUS CONDITIONS Conductivity Readings * Interval in Increaset Re Treatment re n eo. After | After | over dist. 7 endl i 24 hrs. | 88 hrs. | H:O after ading |interval 1 and 2 page in dist. H:O, as t roots in dist. H:O. ...| 10 hrs. 33.6 37.9 | 41.81 35.8} 3 Illuminating gas 1 hr. roots in tumbler. ties 1 hr., nok CUE OR EE eases 17 min. 18.4 38.9 | 56.7 50.7 4 zuen gas 1 hr. ots mbler. Tops cut off Feen rt 2a 1 hr., after exposure....... 23 min 23.4 46.6 | 61.6 55.6 5 gnome pa gas 1 hr.; roots in tumbler. Tops cut off fo before ex- Uhr, posure Ca pa ee 3 28 min. 18.6 41.2 | 60.5 54.5 eadings aba the values of x on the Wheatstone bridge, resistance in box halog 9,110 ohm t After 99 hour 1 The average adi of the distilled water before roots were placed in it was approximately 6.0. normal. The higher readings of the treated cultures whose tops had been removed, over those of the untreated controls are to be considered as due to the effect of the illuminating gas, even though in one case only part of the plants was ex- posed to the agent. The results of this experiment are given in table nm. The results of ether vapor treatment for different periods are seen in fig. 5. An interesting point in this connection is the decline in the curves of the treated plants comparable in 1915] MERRILL—ELECTROLYTIC DETERMINATION OF EXOSMOSIS 531 85 80 TCE SN b 4 N 75 f N Ba 5 N T N 70 N RN N, > N, N N x 65 . iee A o &o N N N Let © N Ps ro 5 60 b A = u = z = h 55 x = N g N Vv << N = 50 > S I a S : 8 TH i 7 © 40 a 8 À A E S35 Hf / \ A 30 -H 25 = zeug) \ Sa > 20 = 15 N zu 10 2 4 6 8 10 12 14 16 18 20 22 Days ig. 5. Conductivity curves of — (series 11) in distilled water subsequent to treatment, as follow No. 45, ether vapor, 30 inutes, roots exposed; 46, Sabrata exposed under bell jar 30 minutes; . 47, ether vapor, i hour, roots se o 5 trol—roots ex posed under bell jar 1 hour; No. 4 her vapor, 2 hours, roots e Yo. 50, control—roots sed under bell jar 2 hours T lants were 25 days old at the time of treatment. In culture 49, 17cc. of the initial 50ce. o her remai a e end of the 2 rs The first reading plotted in each case was taken after the roots had been in the Pigs dapi ar isk the treatment be bag follow- ing periods: No. eae l minute; No, 46, 1 hour and 12 minutes; No. 47, 7 m ache No. 48, À a pa 38 minutes; No. 49, 1 “ie Sr 17 minutes; No. 50, l hour and 28 minutes [VoL. 2 532 ANNALS OF THE MISSOURI BOTANICAL GARDEN some respects to that in the curves obtained from normal plants. A distinction should be made here, however, from the causal agency in this decline in conductivity and the anes- thetic reversibility that Osterhout (’13) describes. The de- cline in the curve indicates that the absorption of electrolytes by roots occurs at a greater rate than they are excreted, for both processes, absorption and excretion, are undoubtedly going on and the curve represents the proportionate amounts of each for any given time. Thus if A represents the excre- tion and B represents the absorption, the curve declines when B is greater than A, and inclines when A is greater than B. Hence the curve may be represented as A — B = C, where C represents the number of ions or charge-carriers in the solu- tion. The tops of the treated plants showed no visible effects whatever when compared with the controls. The roots of No. 45 were very slightly affected, but those of Nos. 47 and 49 were considerably so and to about an equal degree, as shown by flaccidity, root coloration, and the colored and turbid ap- pearance of the medium; the tops, however, continued normal for 21 days after the treatment. Hence the metabolic proc- esses no doubt proceeded unimpaired in many respects, as did also transpiration. The decline of the conductivity curve therefore represents merely a partial return to normal condi- tions. But the higher conductivity of the medium shows greater exosmosis than from the normal plants. This is due to the unalterable and invariable (and not reversible) effect of the anesthetic upon certain cells. Culture 50 shows in the higher position of its curve, as compared with the other con- trols, an effect that is no doubt due to the 2-hour exposure of the roots to the air in the bell jar. As seen in fig. 6 no marked results followed the ether appli- cation for one-half to two hours when the roots were in the water during the treatment, though a slight rise is evident for the culture exposed 2 hours. No visible effects were pro- duced on either the tops or roots. Comparing the effects on plants of an ether vapor-saturated atmosphere with those produced by an illuminating gas-satu- rated atmosphere, it is thus seen that illuminating gas is much 1915] MERRILL—ELECTROLYTIC DETERMINATION OF EXOSMOSIS 533 more injurious than is ether vapor under the conditions of the experiment. Equal amounts of each might give different results, however. The gas used was a mixture of water- and coal-gas with a specific gravity of .62 as compared with air; © = TT 5 35 Cot = IH =) EB: i 2 30 Zone = NECN coo E * pa > 25 Er co 3 \ Zid Rar + MES b anya Pp B ane = IH St ae = S15 NE - HH 3 = TETEA: ETEL 3 « EEREEERENEN| ELEI = EEERENEREEEEEE PELI s 10 O Leer! | a ELI > 2 4 6 8 10 12 14 16 as 20 22 Days E Fig. 6. Conductivity curves > ae Nager: od in distilled water subsequent to ee wi follo No. ether vapor, 30 minutes, roots in tumbler; 52, ontrol under bell jar 30 minutes, roe bei tumbler; No. 53, er er, 1 hour, roots in tumbler; No. 54, trol—under bell jar 1 hour, roots in tumbler; No. = ether hie ot 2 tumbler. The plants were 25 days old at the time of treatment. In culture 55, 60cc. of the initial 100cc. ae ether remained at the end of the 2 hours. The following bee represent the time elapsing in the various cultures between the removal of the cultures from the bell jar and the taking of the first ose (to which period should be added the duration of treatment, for the roots were in the distilled water glo | that time also): No. 51, 1 hour and 24 minutes; No. 52, 1 hour and 46 minutes; No. 53, 1 hour and 51 minutes; No. 54, 2 2 hou urs er 6 arora No. 55,1 hour and 39 minutes; No. 56, 1 hour and 50 minutes. one of the daily samples analyzed by the gas company (the officials of which kindly supplied the writer with the data and informed him that they may be considered an approximately fair average) showed the following constituents : OOs were a a a a 3.0% Oe E re a es ee E wee eee .5% amiat (unsaturated hydrocarbons, e. g., ethylene and AA D EE E E E E E E S sera .0% Se A RE RN ET A ous 16.1% a a ee re ee 25.6% a Lee ee eee rn NEE 42.8% [VoL. 2 534 ANNALS OF THE MISSOURI BOTANICAL GARDEN Crocker and Knight (’08), in their work on the question of injury by illuminating gas and its constituents, concluded that ‘‘there is much evidence that indicates that the toxic limits of illuminating gas upon these flowers [carnations] is determined by the ethylene it contains.’’ They used a small greenhouse of 1.69 cubic meters’ capacity in which they placed potted plants for varying intervals, specified amounts of gas being introduced. The buds were easily injured but the vegetation was apparently not affected even after an exposure of about 72 hours, during which 10 liters of gas had been introduced, 2 or 4 liters at a time. The method was therefore somewhat different from the one employed by the author, in which the plants were placed in an atmosphere saturated with illuminat- ing gas, but for a much shorter period. The underlying cause of the effect in both cases, however, is probably the same. The etherization of plants as a practical process has been in operation for many decades, especially as a means of hasten- ing the activities of plants, particularly of bringing them into bloom earlier. Some experimental work has also been done, as we have seen, on the effect of such treatment (though in most cases only when the anesthetics were in solution) upon the exosmosis of non-electrolytes, as determined by various methods, from plant or animal cells. It is interesting, there- fore, to observe the exosmotic phenomena of electrolytes when the plants are anesthetized under various conditions. To determine whether the amount of substance excreted cor- responded to the conductivity readings, the water in the tum- blers was evaporated and the residue weighed. The following are the results: Total wt. of substance from illuminating er -treated cultures (Nos. 33, 35, 37, 39, 41, and 43)........ 0.1514 grams Total wt. of substance from ether- trented cultures (Nos. aD, Er 60, 91.58, 000-DB) «xk lone vara sone ee ook 0.0674 grams Total wt. of substance from the 12 controls............ 0.1077 grams Total wt. of substance from 6 controls, therefore...... 0.0538 grams We may obtain a rough basis for estimating this residue in terms of NaCl by comparing the figures just given with the data on a previous page which gave the corresponding spe- cific conductivity values for some values of x on the Wheat- 1915] > MERRILL— ELECTROLYTIC DETERMINATION OF EXOSMOSIS 535 stone bridge and also for various concentrations of NaCl. Thus 0.15 gram residue was obtained from 1500 ce. of the media from illuminating gas-treated cultures. This is equiv- alent to 0.10 gram in 1 liter, which in terms of NaCl would be 65 60 ber s 8, 3 pA = 55 = E - s s = E 50 nr. B 5 Š 45 = = = 7 = 40 + g 4 aT 8 35 va um ° g 30 f 3 T A IH = a +H am EBEN + === 20 HHHH RR $ cry IH 111 = HH EHH 18 O2 EEI BEAR! IH 1 2 3 4 5 Days Fig. 7. etz curves of a (series 14) in distilled water subsequent to treatm as follo No l, temperature of — 6 to — 2° hour, ee in ee No. 2. temperature of — 6 to —2°C., 2 hours, roots in tumbler; No. 3, temperature o to — 2°C., 3 hours, roots in tumbler; No. 4, control— om temperature, well in ‘tumbler ; ontrol—room temperature, ‘Toots in tumbler. The plants were 23 py ‘old at the time of treatm The first reading was taken in each case after the roots had war in nthe distilled water for the follow- ing respective HEE (cultures 1-3 were being treated during part o that time): No. l hour and 47 minutes; No. 2, 2 hours and 29 et No. 3, 3 hours and 45 minutes; No. 4, 3 hours and 6 minutes; ‚2 hours and 2 minutes. approximately N/500. The specific conductivity of N/500 NaCl is about 25 x 10°, the x value of which on the Wheat- stone bridge is 85. The average final reading of the 6 cul- tures treated with illuminating gas is 79.5. Hence the residue in terms of NaCl would be in the neighborhood of N/500. [VoL, 2 536 ANNALS OF THE MISSOURI BOTANICAL GARDEN VI. Errects or Hicem anp Low TEMPERATURES After the preliminary experiments noted above on the effect of heat had been carried out it was desired to study the ques- tion further and determine the resulting exosmosis curves at the extreme temperatures, high and low. The preliminary ex- periments had involved temperatures requiring a considerable time interval to produce positive results. The data now to be presented concern temperatures sufficient in themselves to ef- fect decided injury in a very short period. By varying the time factor, therefore, results could readily be obtained on both sides of the point of injury. For the experiment, the results of which are plotted in fig. 7, cultures were set out of doors for the time indicated, directly exposed to the winter temperature. The tops showed some signs of freezing after a few moments, but the effects did not become noticeably worse until the cultures were brought in- side, when all the plants in each culture immediately drooped over the wire supports and became entirely limp, and soon died. The tops did not yellow, but retained the green color after death. Except for the root tips of the plants in No. 3, which were slightly brown at the end of 5 days, all the roots of the treated plants remained turgid, white, normal, and in healthy condition. This is interesting in view of the fact that while no ice was formed in No. 1, there was a slight fringe of it between the water and the tumbler in No. 2, and a hollow eylinder of ice one-fourth inch thick formed next to the tumbler wall in No. 3. In the last-mentioned culture there was also a film of ice over the surface of the water and the roots were frozen to the ice mass so that on lifting the plants from the tumbler the mass of ice adhered to the roots. The first readings were taken only after the ice had melted. The tem- perature at first was —6°C. but by the end of the first hour it had risen to —2°C., where it remained practically con- stant for the balance of the interval. At a temperature of —6.5°C. it is seen by reference to fig. 8 that while for exposures of the plants alone (the roots being out of the water) of 2 and 3 minutes, marked exosmosis imme- 1915] MERRILL—ELECTROLYTIC DETERMINATION OF EXOSMOSIS 537 diately results, exposures of 1 minute or 4 minute produce no results. Culture 13 has rather high exosmosis for a control, an © cs an Values of x on the Wheatstone bridge N n I | | | il H IT {i 1 IH 1! ii i II ali cy mi i | | | 1 ji 1 17 {Ty im al B: EE: I fit IL! 1 2 3 4 Days Fig. 8. Conductivity curves of cultures (series 14) in distilled mep „Subsequent to treatment, as follows: No. 11, temperature of d laboratory temperature 30 minutes; No. W control— va exposed ia laboratory t rature 5 minutes; o. 1 emperature of — 6.5°C., 2 minutes, roots ne o. 16, temperature of minute, roots ar . 17, temperature of —6.5 one-half minute, roots exposed. a were p nás old at the time a wein ent. The first re itn was taken in each case after the had oa in the distilled water for pore 30 minutes ep T = treatment. VE ne I, ee a Ss a AA eg io Be [voL. 2 538 ANNALS OF THE MISSOURI BOTANICAL GARDEN but this is readily accounted for by the exposure of its roots to the atmosphere of the laboratory for 30 minutes, a condition noted in other cases above. Cultures 9 and 10 of this series, the results from which are not represented because both tops and roots were killed out- right, the resulting exosmosis therefore being immediate and high (a being about 88.0 cm.), were exposed for 15 and 33 minutes respectively to a temperature of — 6.5°C., the roots being out of the medium. In a very short time, on returning them to the laboratory, the tops wilted and drooped over the supporting wires and the roots became very flaccid. In the case of No. 11, however, an interesting gradation or interme- diate condition was observed between it and Nos. 9 and 10 on one hand and between it and the controls on the other. While the tops in No. 11 wilted and drooped somewhat soon after being returned to the higher temperature of the laboratory, they did not become entirely limp and the roots were only slightly less turgid than those of the controls. Even after 4 days the tops of No. 11 were not drooping much, though the tips of the branches and the upper leaves were dead; the lower part of the stems and the lower leaves remained green and normal. The lateral roots and the older part of the main roots remained nearly normal, but the tips of the latter were flaccid and shrunken for about 2 inches. Culture 15 showed a very slight flaceidity in the tops and roots soon after the treat- ment, and after 4 days some of the younger leaves and the tips of the older leaves were blackened, curled, and dried some- what, but the great part of the tops remained normal in ap- pearance; the roots were slightly flaccid at the tips, but were in general practically normal. Cultures 12, 13, 14, 16 and 17 were normal in respect to both roots and tops. The interval between 15 and 30 minutes is shown in fig. 9 to be the critical period for the pea plants exposed in a tumbler to a temperature of from — 2°C. to — 2.5°C., for an exposure of 30 minutes caused considerable exosmosis, while one of 15 minutes gave a curve approximately that for normal plants. To contrast the effects of low and high temperatures, Nos. 1915] MERRILL—ELECTROLYTIC DETERMINATION OF EXOSMOSIS 539 25-28 inclusive are plotted in the same figure with Nos. 18 and 23. With plants enveloped in a steam bath the injury, as expected, is very speedy and effective. Even one-half minute —.\ ‚en the roots are exposed—causes immediate and marked 85 mi 80 sz HH = co | 75 == am 4 maa 7 - "A + 70 5 <= & Pa A OS a 9 f=] | 2 60 f- B 2 ss HZ Pagasi BE N] = = o Be. z 4 1 A 4 — gr T == 8 45 parz A S = 8 40 HH an E X 35 an 4 BEE 25 =e T] CEI Crt BE 20 I I iH 1 2 5 6 7 Days = Fig. 9. Conductivity curves of cultures t gr 14) in distilled = _,water subsequent to treatment, as follows > hen mperature of —2.5°C., 30 minutes, roots in tumbler; a temperature of —2.0°C., 15 minutes, roots i in tumbler; No. 25, a one-half minute, roots exposed; No, 2 , steam, 2 minutes, roots in tumbler; No. 27, steam, 1 minute, roots in tumbler; No. 28, 10 minutes, roots in tumbler. The plants ge 29 days old’ at the time = treatment. The first reading was taken in each case after the had been in the distilled bag for the idomiai ag ardh pe eriods ir he u. wick roots were umblers during the treatment were likewise in the di m Water); No. 18, 4 hours et "49 Ru: No. 23, 4 a and nutes; No. 25, 4 hours and 43 minutes; No. 26, 4 hours and % minutes; No. 27, 4 hours and 31 minutes; No. 28, 4 hours and — 12 minutes (VoL, 2 540 ANNALS OF THE MISSOURI BOTANICAL GARDEN exosmosis which is greater than that caused by a 10-minute exposure when the roots are in distilled water meanwhile. The condition of the plants immediately after the treatment and again after 7 days is given in table m. Here again is illus- TABLE III CONDITION OF PLANTS AFTER EXPOSURE TO VARIOUS TEMPERATURES — Condition of Condition of roots Condition of plants i tely after the t 18 Considerably ‘flaccid and er TEE E en normal 23 Very slightly drooping, nearly on a ee Entirely normal rooping considerably................... 26 and 27 Drooping, green and damp............... mal f Drooping, green and damp............... Apparently practically normal Condition of plants 7 days after the treatment: - 18 About half dead and half alive; 3 live stems with green, normal leaves.............. Entirely normal 23 Almost normal ; 2 of a few stems killed injure ut some stems Et throughout; a few blackened leaves, but for the most part stems and leaves. green and normal............... Entirely normal“ —= 25 7 URN RER se E ir very slightly flac- ge nearly no appearance 26 and 27 1 17.2. T a PEEN RER ne a me T Potini normal in appearance 28 a T, SEE ERT EAE ENE Almost normal trated, therefore, the case where there is considerable exos- mosis without very marked visible effects resulting to the root tissues. The effects of moist heat, as graphically represented in fig. 9, having been considered, we may now turn our attention to fig. 10, where the results are plotted of an exposure of plants to dry heat for short intervals, both with the roots directly exposed and with the roots remaining in the tumbler of water- during the treatment. It is seen that definite and positive exosmosis is obtained after a 4-minute exposure of the unprotected roots. The de- cline of the curve of No. 29, roots exposed for 2 minutes, is probably best accounted for by assuming greater hardihood of the plants in that culture, or that some condition effected an increase in transpiration. A 1-minute exposure (No. 30) pro- 1915] MERRILL—ELECTROLYTIC DETERMINATION OF EXOSMOSIS 541 ooo 70 Cee ee eet EE EEE EEE HEE eee Tritt. Ch csp li a 65 CCE Ee EHH HEH et EREEREREERERERNBEHERER-- „nERHERERREHERHERRHEB> CCC CECE CCC eS Cee ee Coot ik Oe OS R PLCC LLC E E E a E E ASR CITITI TTT TEN TILL E 60 AARDSE Cee ee i 7 2 F CEET] PCC ert tery = $ AHH T Heer aa ee i 455 BEL tice ct Cotto) = teten rape ee 2 ETETETT EPI EP A A ELIITTI coo CCC S 50 Ty ESZ-«BERERENNEENNEEREEER| 7 7 Ea EEEENENEEERRERENENE + = PETTI i Coot 3 Cort t zs i HH H E45 H Im co Q i Bi 3 40 7 g i mi im 8 3 mm I z TEE TS HH N g H HH tet 5 30 Coot See 8 KENNEN yy > PELE LeeLee peel EEEFESHEHHEEESESRSH EEE, 25 AERERERERRENERERBER TooToo ooo = CoCo HEHE En 20 Coe CoC eet COC eet COC ee eect i | 15 FS Im Coot IT i 1 2 S 6 7 Days 10. Conductivity curves A cultures (series 14) in distilled water subsequent to treatment, as fo > ows: No. 29, temperature y 92°C es, roots expel N 30, temperature of 92°C, No. 31, temperature of 92°C., 4 minutes, or 2°C., 2m ee roots exposed; osed ; emperature of 9 minutes, roots in tumbler; 3, temperature of 92°C inutes, roots in tumbler; No, 34, control—roots in tumbler at laboratory temperature. The plants were 29 days old at the time of treatment. reading en each case after the roots had been in the distilled water for the follow- whose tumble time) l minutes; No. 31, 2 hours and 44 minutes; No. 32, 2 hours and 45 rn No. 33, 2 hours and 46 minutes; No. 34, 2 hours and 46 duced less exosmosis at the beginning than was the case in No. 29, but finally caused more. The same irregularity is also noticed in Nos. 32 and 33. These irregularities near the boundary line of endurance have been discussed above. The tops of Nos. 29-33 were killed by the treatment, but the roots of all remained practically normal in appearance except in No. [voL. 2 542 ANNALS OF THE MISSOURI BOTANICAL GARDEN 31, where the tips were slightly shrunken at first but became almost normal in the water after 7 days. In the temperature experiments we have thus used the ex- tremes of temperature and have reduced the interval of expo- sure in order to approach the point at which the effect is just evident. VII. EFFECTS or ANESTHETICS IN SOLUTION Having seen some of the effects of anesthetic vapors, we may turn our attention next to the results obtained with anes- thetics in solution. In the investigations of others pertaining to the effect of anesthetics, already cited, the result has been almost universally noted that small amounts of anesthetics decrease the exosmosis of coloring matters, ete., while toxic amounts increase it. In most cases this exosmosis was ex- plained on the basis of an alteration in the plasma membrane, small amounts of the anesthetics presumably reducing the permeability and large amounts increasing it. But a point worthy of note is that wherever such effects have been deter- mined the substance under observation was either a colored compound or one of complex organic nature. Thus Czapek (711) used the myelin-formation of a tannoid substance, anthocyan, as a basis of observation. From the standpoint of a physical phenomenon, i. e., the lowering of surface tension, his experiments beautifully illustrated the principle under consideration. But from the standpoint of exosmosis in the broader sense we must include electrolytes (salts, bases, and acids) as well as tannin compounds in any discussion dealing with agents affecting exosmosis, and while the critical concentrations which he determined are undoubt- edly characteristic of the plants and the compounds studied, the results given herewith show that they are not the limit- ing concentrations which effect the exosmosis of electrolytes from the roots of certain plants. The limiting concentrations which he found are given in table tv. Czapek believed the permeability of the plasma membrane was altered under the influence of alcohols, ethers, ete., so that abnormal exosmosis occurred. Whatever may be the expla- 1915] MERRILL—ELECTROLYTIC DETERMINATION OF EXOSMOSIS 543 ANIC COMPOUNDS AS DETERMINED B CRITICAL OS cnt one (THOSE JUST SUFFICIENT TO a EXOSMOSIS) CZAPEK FOR CERTAIN PLANTS : Surface Agent Plant Concentration of agent kenn? Methyl alcohol | Echeveria....... 18% aqueous solution (by volume) cri Ethyl alcohol te = m. aqueous solution axifrag volume): oeae a uon cee eee .65-.70 Ethyl ether ne pee ‘i 3 satu et Fe aan solution Viola acting tor Fi: ie .61- .71 Chlorofor Echeveria.. .... is ous solution f.. .98 Chloral a ER TEE 09% aqueous solution.......... 93 Ethyl acetate | Saxifraga hairs 30, its solution acting for ) ape va ariegated yh. ere Sheet ee leave p- lismenus imbe- + .69-.73 Ethyl acetate | Red beets....... 2% aguian solution acting for FA ET ER * In terms of water as unity. t+ After 24 hours the cells had lost all tannin. nation for the phenomena observed, it will be seen by compar- ing the results in the following experiments with the data just given that the limiting values found for the exosmosis of elec- trolytes do not at all correspond to the values found by Czapek for the exosmosis of the tannoid substance. Values of æ on the Wheatstone bridge 1 3 5 7 9 11 13 15 Days Fig. 11. Conductivity curves of roa Tee ava gl user water subsequent to tre ee as follow G minutes, eae exposed; No. 2, 1 per ane et in u 15 oo No. 15, eontrol—roots Bick under bel jar 15 minutes. The plants were 31 Sara old when treated. In culture od koas of the —— 15cc. of ether ee after the 15- a nute expos first readin s taken exactly 30 minutes, na in No. 15, 48 neue. after the Ne wo placed in fg distilled water [voL. 2 544 ANNALS OF THE MISSOURI BOTANICAL GARDEN In fig. 11 are shown the results with ether in distilled water and, for comparison, also with ether vapor for the same period. The curves for the ether-treated cultures are closely parallel for the entire period of observation of 15 days, and both are V EFFECTS OF VARIOUS ANESTHETICS ON THE EXOSMOSIS FROM THE ROOTS OF PLANTS (See curves in fig. 12) Vapor Treatment, Roots Exposed . . Resulting Anesthetic Time of exposure Culture no. Fe: 2S Ether 30 minutes 3 el git ias Illuminating gas 15 minutes 5 3 and 5 Eh er pt gas 30 minutes 7 Higher Chlorofor 30 minutes 9 Highest Treatment with Anesthetics Dissolved in Water pe el 10 30 minutes 4 igh Er 4% Throughout experiment 11 Highest 30 minutes 12 Higher et gas- 15 minutes 6 Medium low turated sol’n Illuminating gas- 30 minutes 8 Like control satur. sol’ Illuminating gas- Throughout experiment 13 Medium low turated sol’n. Illuminating gas- 30 minutes 14 Slightly above turat ‘1 control frequently re- rat Chloroform, 4% 30 minutes 10 Very high Controls Roots exposed to the air under a bell jar 30 minutes 16 ir = con- ro Roots exposed to the air under a bell jar 30 minutes 17 j aai con- ro Roots not exposed, but in water from first 18 Normal forcon- rol above that of the control. The excretion was nil during the first half hour and at the end of 5 days it was scarcely more, though it may have risen and fallen in the meantime, as no readings were taken in the interim. After 5 days a greater rise in the conductivity curve occurred with the ether-treated cultures than with the control; no apparent effects, however were produced on either the tops or roots. > 1915] MERRILL—ELECTROLYTIC DETERMINATION OF EXOSMOSIS 545 80 75 = eo 70 Pa g j- Ai A 65 L E = i — n — T as Lert +-+ >= 17 o 60 EEJ = ct A op a LLI LI Cl A a 7 [11 A 11-1 s” J 2 HER HH Toe £55 pn f = CEELI g f HL rm 3 = 1 F ti n 50 Ct tty E EHER HH = a a TEETE mezua b ! CLLI = 45 —-- — — = Wit pr © ial E ~ $ Ari H ; 0 Wie ce S bEELEE 41 © aamaan m A- t 8 ERSEM Pa 88 PERRE ZA wan ° PRAS YP 7 5 p a8 ve TITEL 20 EZE Hass = = TAT. 1] = > HGY | = 25 Amey, 7 I ry: f 71 HA 4 E 20 TH Ak 4 TII 7 A 15 =~ — oo << a z225 = H N ie N Pritt iz a a AB T S 10 ai tee = Ba | (PE 1 5004.72 Rr i im ERRB SERS Es HE wort SG $i) 5 ERER.: I I Ji (BREED KASTEN 1 2 3 4 5 6 7 8 9 Days Fig. 12. as curves e cultures (series 15) in distilled water subsequent to treatment, as follows: No. 3, ether vapor, 30 minutes, roots exposed; per so ether in pig 30 minutes; No. 5, illuminating ‚15 minutes arme exposed; No 6, distilled =e saturated with illum- inating gas nutes; No. 7, , 30 po a No. ’8, distilled water saturated with i Borir gas, 30 minutes; chlor oform bir > r, 30 minutes, roots exposed; No. 10, 4 per cent chloroform in water, 30 nemoe; No. 11, Se per cent ether in water, to the aad of he ace ent; No. 10 per ent ether i i No. 13, Be er ae with ee gas, end of experi- ment; No. 14, distilled water Pe with nn gas, 30 minutes ese saturated); ; No. ss co bie ts exposed 3 minutes under bell jar; No. 17, rol—roots expos ag nutes under bell jar; No. 18, control—roots a pees into distilled n will The ee A given above refer to volume-per cent. The plants were 37 d ays old when treated The first reading was m i o of ether remained at the end of the 30- 2 exposure; in culture 9, 23.5ce. of the initial 25cc. of chloroform rema (Vou, 2 546 ANNALS OF THE MISSOURI BOTANICAL GARDEN To show the comparative effects on the exosmosis from the roots of plants treated with ether, chloroform, and illuminat- ing gas—both when applied as vapor and when introduced into the water—the conductivity curves of fig. 12 were plotted. The results, somewhat classified, are also given in table v. It will be seen that the quantity of anesthetics used and the dura- tion of treatment varied in individual cases. The indications are, therefore, that for an equal exposure the vapors range in order of effectiveness as follows: ether, least; illuminating gas, more; and chloroform, most. The dif- ference in effectiveness between the ether and the chloroform is especially interesting, more so when we note that 8 cc. of ether were used and only 14 ce. of chloroform. This would seem to be in harmony with the findings of Graham (’14) ; he was able to produce liver necrosis by some aliphatic halogen substituted compounds, but not by ether or chloral hydrate. As regards the fact that No. 11 (4 per cent ether, remaining in the water) has a higher curve than No. 12 (10 per cent ether for 30 minutes) and especially at the beginning, it should be stated that in the case of Nos. 4, 6, 8, 10, 12, and 14, the treat- ment was given while the roots were in distilled water plus the anesthetics. Following the treatment the roots, after rins- ing, were placed in distilled water, and at the end of one- half hour the first reading was taken. In the case of Nos. 11 and 13 the water containing the anesthetic was not replaced by fresh water and the first reading was taken one-half hour after the treatment began. Since the exosmosis during the first half hour is unusually rapid as a result of anesthetic treatment, it will be seen that in replacing the medium at the end of that period, the excreted material was thus discarded for that interval. Hence, such curves represent a secondary exosmosis. The curve of No. 11, therefore, is for total exos- mosis, while that of No. 12 is for partial exosmosis. The condition of the cultures which furnished the results plotted in fig. 11 is given for various periods in table vr. In fig. 13 the secondary exosmosis after the first half hour is graphically represented for some organic compounds in con- siderable concentration. The purpose was, of course, to use a 1915] MERRILL—ELECTROLYTIC DETERMINATION OF EXOSMOSIS 547 concentration sufficiently effective to give results in a short interval of time. After the treatment the roots were rinsed and placed in distilled water. It is interesting to note that the alcohols used were only slowly effective at first, but that CONDITION OF PLANTS AFTER TREATMENT WITH ANESTHETICS Culture no. Condition of tops | Condition of roots Condition of plants 2 days after treatment: 3 and 4 Slightly subnormal, but almost normal | Somewhat flaccid 5 Practically same as in Nos. 3 and 4 . . .| Practically normal 6 Practically same as in Nos. 3 and 4 ...| Practically normal, but somewhat flacci 7 Burns dad REED Somewhat flaccid 8 RE RR 2 os en ern Slightly flaccid RE E87 a es a ks se ee Considerably flaccid 13 a RE eR, Sars EEE WEN Pri ee Tr 14 nn RE ees ee = 16, 17, and 18 hun RN RESET PERIL E Nor Condition of plants 9 days after er ed 3 Much dried and VEHOWER. «use Practically normal 4 Slightly worse than in N DSen ESEN Considerably flaccid 5 ete O08 a une cc cce es Less flaccid than in No. 4 6 POMEL CRE U Practically 7 Pils NING UP eek nn ae a meen Slightly 8 Eracucallyanormal: ane o sis x sea we Practically normal 9 Practicalvy Bormal. -es msee seme Considerably flaccid 10 a BOLMAN Ee e a AA Somewhat flacci 11 and 12 a atoian ri ME ae Somewhat flaccid 13 and 14 Normale meeste aa aar AE N, Practically normal 16 Slightly SOIR s ea aa ee Practically normal 17 and 18 TS SE” SECTS TEER TR TORTIE EET after 8 days the conductivity readings for those cultures were as high as those of the other cultures. Benzol and toluene produced almost identical effects. The effect produced by chloral hydrate remained constant after 1 day. Ethyl acetate and benzaldehyde were especially effective. The condition of the plants at the end of 8 days is given in table vu. In fig. 14 are shown the effects of smaller amounts of the same substances, the curves of which are exhibited in fig. 13. Here, however, concentrations only one-fourth as great as those previously employed were used, but the chemicals were allowed to remain in the water during the entire period (or until evaporated, as may have been the case with some). ile the alcohols gave a greater effect than the control, they gave no greater exosmosis than one of the controls in the Values of œ on the Wheatstone bridge [Vor. ANNALS OF THE MISSOURI BOTANICAL GARDEN | {I mal LLJ | i KLEI \ Vo LEI 1 LELLELI ELERI ELLLELLLI Pa eT = 1} : vum Days Fig. 13. Conductivity rg of cultures (series gl in distilled water subsequent to treatment, as follows: No. 19, 4 per cent ethyl alcohol in water, 30 minutes; No. 20, 4 per cent methyl aleohol in water i 21 o. 25, bosses 30 minutes; No. 26, control—placed directly into distilled No. 27, control—placed directly into distilled wa = The plants were 38 pe old at the time of treatment. The first reading was taken in all cases after the roots had in in distilled. yore exactly 30 minutes. In the case of the treated plants the roots — in distilled water containing the _. for the specified tim fter which they were transferred to the car water, the con- ductivity of which was subsequently pindia 2 á 1915] MERRILL—ELECTROLYTIC DETERMINATION OF EXOSMOSIS 549 previous figure. The other substances, however, even in the small concentration employed, produced a marked rise in the conductivity of the medium during the first day, after which it remained practically constant. The benzaldehyde and the TABLE VII CONDITION OF PLANTS EIGHT DAYS SUBSEQUENT TO TREATMENT WITH EFFECTIVE CONCENTRATIONS OF ANESTHETICS FOR A SHORT PERIOD Culture no. Condition of tops Condition of roots 19 Normal and in good condition........... Sons flaccid 20 ERBE ER ie oe flaccid than those 19 21 Somewhat subnormal................... Somewhat flaccid 22 me stems considerably affected, others | AR Erea E Considerably flaccid 23 en en, os eae Very fla 24 bout the same as in No. 22............ Considerably flaccid 25 Considerably subnormal................ Very flaccid 26 pe" = many green, vigorous, turgid N ee Practically normal 27 Practically ohn 39% 2.5 23 0 Ca Normal ethyl acetate, which themselves give a high conductivity in aqueous solution, should be considered apart from the other substances, which give no such increase. The two substances mentioned are given here merely for the purpose of compari- son with the others employed. A 1 per cent solution of ethyl acetate had a conductivity of 65.2 on the Wheatstone bridge, while that of a similar solution of benzaldehyde was 88.6. These corrections should therefore be applied to the curve values in order to obtain the true value of the exosmosis from the roots in those cultures. The condition of the plants after 8 days is given in table 8. VIII. EFFECTS or SUBSTANCES USED SINGLY AND COMBINED IN AIRS It is not the writer’s purpose here to go into the historical aspect of the increasingly voluminous work on toxic agents, antagonistic action, and balanced solutions, and the numerous related subjects. But since those subjects have assumed such great importance in the realm of physiology it was thought de- sirable to consider the effect of certain toxic and unbalanced Values of x on the Wheatstone bridge n on wm © > a A = | [voL. 2 IITFILLII III] DE TEE LLLIS#LII ICH LLELLE r. Peo / CeCe 4 CELLET LER PETTI AHHH TT ot tegen yi] ie HH a HW We HAH afin HFF q HHF al TH = 1 — PAs + IL Fe ji : u IT + = Iwan IH IH Pa Lh 17 = Y | ™ x p va beer á ime u Be a a z =. u i sur m = ERER == (LI = panan Coo EEI ETE TATT TH TITI ILL or itt TLLELI I uf I 1 2 3 4 5 6 7 8 Days g. 14. URN: Be of ur er 15) in distilled water subsequent to treatment, as follow , 1 per cent ethyl leohol in water, to of retinal, y 29, l per cent methyl alcohol in water, to end of experiment; No. 30, 1 per cent chloral hydrate in water, to end of experiment; A r cent benzol in water, to end of experiment; a yi = cent ethyl acetate in water, to end of Be o 1 per cent tol n — f experiment; 34, eh benzaldehyde in water, to end of ex- riment; No. 35, co ih directly into distilled re No. 36, 1 per cent methyl alcohol in water, to end of experim The plants ia 38 days old at the time of treatment. he first reading was taken n all cases after the roots had a in = distilled ee containing the anesthetic exactly 30 minutes. The rol, however, was exposed KA the distilled pir only, the Aei ec being taken after 30 nutes. 1915] MERRILL—ELECTROLYTIC DETERMINATION OF EXOSMOSIS 551 solutions on the exosmosis from plant roots in order to obtain a basis of comparison with the other agents used. In this connection it might be well to consider more in de- tail the work of Lillie already referred to in the historical TABLE VIII CONDITION OF PLANTS EIGHT DAYS SUBSEQUENT TO TREATMENT WITH LOW CONCENTRATIONS OF ANESTHETICS FOR THE ENTIRE PERIOD Culture no. Condition of tops Condition of roots 28 Somewhat subnormal................66. Slightly flaccid 29 TR oe ER Gousiceeaidier flaccid; ag less flaccid than in 28 but upper part more so See 5 3 aa eee Peer eee. Considerably flaccid 31 Alno RE ec e EENT Very flaccid 32-34 aina n A AT a 0 ow on ees Ve 35 PEA ODER: > > ana es baa Lees Practically normal 36 go eee ee eee ee Somewhat flaccid review. His work on Arenicola and the eggs of Arbacia per- tains largely to the exosmosis of the pigment and the man- ner in which they were affected by isotonic salt solutions alone and in the presence of various anesthetics. From the effect observed, he concluded that the salts have a permeability-in- creasing effect on the plasma membrane which is counteracted by the anesthetics. But in dealing with the question of per- meability it would seem that we must take into consideration the effect on the exosmosis, not only of any contained pig- ment, but of electrolytes as well. It would have been exceedingly interesting, and would have furnished a means of strengthening or shattering his hypoth- esis, as the case might be, had Lillie also measured the elec- trical conductivity of the medium in which the Arenicola larvae and the Arbacia eggs were placed and thus determined whether the electrolytes contained in these organisms behaved as did the pigment. It would seem that the work of Loeb (703), Peters (’04), and others might be considered as sug- gesting possibilities for electrolytic determinations along this line with marine organisms. Without such facts at hand any general conclusions in regard to permeability effects based on the coloring matter only must be considered imperfect. What (VoL. 2 552 ANNALS OF THE MISSOURI BOTANICAL GARDEN the reaction may be between the anesthetics and the larval pig- ment is another question which Lillie does not touch upon. In a recent article Miss Wheldale (’14), in discussing the natural and artificial extracts of plants, states that whereas artificial anthocyanin is soluble in ether the natural anthocyanins are not. May we not have a similar effect in the pigments con- cerned? Small amounts of the anesthetics may render those pigments insoluble and in that manner prevent their exosmosis rather than by bringing about any considerable alteration of the membrane; larger amounts of the anesthetics would act chemically on the membrane to a point of disintegration suffi- cient for the physical escape of the pigment. It will be seen from the following experiments that in the case of roots of Pisum sativum certain salts caused a marked exosmosis of electrolytes. In the presence of anesthetics this exosmosis was not decreased or prevented, as Lillie found in the case of the pigments referred to, but was even increased. Hence these results do not indicate any permeabilit y-decreas- ing action on the part of the anesthetics and are theróforei in harmony with the findings of Dixon and Atkins (’13) and others. Another interesting condition is seen in the exosmosis resulting from single and combined salts acting for different periods of time. It was expected that such results would cor- respond with those obtained on plant-growth studies of antag- onistic action between various nutrient and non-nutrient salts. That equally as high, or in some cases higher, exosmosis values were obtained from combined salts as from single salts is an unexpected and interesting result. As previously indicated, the method used was to place the plants in the various solutions for the period specified and then transfer them, after careful rinsing of the roots, to distilled water in which the conductivity readings were to be taken. It was ascertained that the rinsing was effective in removing electrolytes from the roots. Figures 15 and 16 show the re- sults for the briefer treatments with certain salts, and it is there seen that for a period of treatment less than 17 hours the N/20 MgCl: has no effect. While in the case of the cul- ture treated for one-half hour with the MgClo, the conductivity 1915] MERRILL—ELECTROLYTIC DETERMINATION OF EXOSMOSIS 553 reading was higher at the end of four days than in the other cultures (2-5) of that group, and continued higher through- out, this fact loses its significance, as far as comparative ef- fects are concerned, when the curve resulting from a 4-hour 40 vo on a = = & 35 s j Q A = = 3 30 4 PR 3 = g 2o = Vv r. = A. =a E- = = = 25 7 P] y a = ov J "s = 20 f=] 7 d d = = 2 ESIE A “u pnez = nt mm a auuu ° = A m ii g T THIN f F HHH B 10 Yrr | KELTI = ET PP tert Coe ee Peete ett SSEL jae aus! CELLS 2 4 6 8 -10 12 14 16 18 20 Days Fig. 15. Conductivity curves Sis ame sada 16) in ar water I sss nt to treatment, as follow nn fal 0 MgCls, minutes; No. 2, N/20 ‘CaCl, 30 “nieces No. N/20 MgCls = » N/ N/20 CaClg, 30 minutes; No. 4, control—placed directly into art water; No. 5, N/20 MgCl», 4 hours. The cultures were 17 days at the time of treatment. The first reading was taken in all cases en the roots had been in the distilled water exactly 30 minutes. treatment with MgClə is considered, and should no doubt be interpreted as an individual variation irrespective of treat- ment. In the case of No. 6, however, the curve for which rep- resents the results of a 17-hour treatment with N/20 MgClo, we no doubt have a real effect clearly distinguished from the controls. At the end of 20 days in distilled water following the treat- ment the tops of Nos. 1-10 were all in about the same con- dition, those of the treated plants showing no injury. Like- wise the roots of Nos. 1-5 and 8-10 were practically normal, with no, or only very slight, flaccidity ; those of No. 6, however, were brownish in color and somewhat flaccid, while those of [vor. 2 554 ANNALS OF THE MISSOURI BOTANICAL GARDEN No. 7 were brownish only in spots, but were of about the same flaccidity as those of No. 6. Having found that a treatment of 17 hours under the con- ditions indicated above was not sufficient to yield the most 55 A 50 Q &o 045 .- fa = J o w @ 40 PA A 8 A / 12 7 > at i £ g 35 7 R= J 5 = A A g 30 F ü p it od pe E Al: Tai A HH g J | i 4 © 25 IN 8 ICH X - fer CUE © “+ e 20 = Tr = Q t = Z ENAH > 15 „un: zemi 7 TII = TH HH HH 10 FREER EEE mr Pc eee eee CCC eee 5 LLL BIT TI TE I I IE IT 57 2 4 6 8 10 12 14 16 18 20 Days ig. 16. Conductivity curves of cultures (series En in distilled water subsequent to treatment, as follows: No. 6, N/20 MgCl, 17 N 0 hours; No. control—distilled water, not renewed. ne cultures were i7 dave a at the time of treatment. The first readings were = in all cases after the roots had been in the distilled water tly 30 minutes. No. 10 remained in the full nutrient solution until the ge ae ulture es were transferred (after the 17-hour period) from the respective solutions to distilled wa positive results, it was decided to try stronger concentrations and longer periods. Figure 17 shows the conductivity curves after a period of treatment extending 75 hours. Some interesting results were obtained. N/10 MgCl- gave the high- est readings, closely followed by N/10 MgCl- plus N/10 CaCle; the N/20 MgCle plus N/20 CaCle curve is very similar to that 1915] MERRILL—ELECTROLYTIC DETERMINATION OF EXOSMOSIS 555 obtained from N/10 MgCle plus N/20 CaCls, while the N/10 MgCle plus N/100 CaClə causes a rise higher than that in the two curves just mentioned after the fifth day. It was unexpected that N/20 CaCle should exceed N/20 MgCl: in its Er an © »> a | aus im ti ii i Ww a | Ww © \ Ji N on Values of ~ on the Wheatstone bridge > © 5 I I I I : I I 1 3 S 7 9 11 13 Days Fig. 17. Conductivity curves of cultures E re 16) in distilled water subsequent to treatment, as follows: No. 11, N/10 MgCl, 75 hours; No. 12, N/20 MgClo, 75 hours; No. 13, N/10 CaCl», 75 hours; 8: o. 16, N/20 j N/10 MgCl plus N/20 CaClo, 75 hours; No. 18, N/10 MgCla plus N/100 CaClo, 75 hours; No, 19, control—distilled water, rg after 75 hours; No. 20, control— distille d water, not renewed. e plants were 21 days old when ee The first reading was aban: alter the roots had been = distilled water exactly 30 minutes (in . 20, 75 hours). Nos. 19 and 20 were placed in distilled ee at ay same time omg the pans to be treated were placed in their respective solution [vor. 2 556 ANNALS OF THE MISSOURI BOTANICAL GARDEN effect on exosmosis and that the conductivity curve resulting from treatment with N/10 CaCl should rise so high at the end. TT 85 BS eae rc IH 80 aa co r | BE BE 7s EE = mann BR nan 70 - nu -amm yar, ae i } : - ~ are sa o 65 H -> = Ba rt a0 a = a = a u BEER = 60 - m waas © = = =] = — 3 a 55 8 Q r- = 50 a 3 g 45 = prag 8 r = ~ 7 ‘5 40 = = = 35 HH > H 30 N H x HHHH 25 H+ ~ rT HE H H 20 > | = ELI = — = =; - 15 fr = z H ST ss HH HH 10 OOI I I EN I 1 3 5 7 9 11 13 Days . 18. Conductivity curves of cultures ages 16) in distilled water subsequent to treatment, as follows 21, N/10 NaCl, 75 hours; No. 22, pr KCl, 75 hours; No. er 0 Nacl Lag N/10 KCl, 75 hours; No. 24, N/20 NaCl plus X/20, KCL 75 hou No. 25, N/10 NaCl plus N10 CaClo, 75 hours; No. N/10 KCL plus N/10 CaClo, 15 hours; No. 27, N/20 NaCl, 75 Hek a u e on 75 ho 29, control—distilled water, not ren No. on- trol—distilled wi i not renewed. The plants were 21 days old sl treated. The first reading was en after the roots had been in the distilled water 30 minutes (in 29, 75 hours). Culture 30 was placed in distilled water at the end ‘of the 75-hour period, having been in full nutrient solution up to that time. 1915] MERRILL—ELECTROLYTIC DETERMINATION OF EXOSMOSIS 557 At the end of 13 days in distilled water following the treat- ment, the tops of Nos. 11-20 were of the same appearance throughout, i. e., normal. The roots were also practically nor- mal in the case of Nos. 12-20, except for a brownish color on those of Nos. 12, 13, 15, and 16-18, being especially evident in the case of No. 15. In addition to being brown, however, the roots of No. 11 were considerably flaccid. Figure 18 shows similar relations for NaCl, KCl, and CaCle. It is seen that KCl is more effective than NaCl in causing exos- mosis. Far from ameliorating the exosmotie condition, the treatment with combined NaCl and KCl likewise yields high conductivity readings of the medium, the N/10 concentration of each combined giving the highest. It can not be argued that this effect is due solely to the osmotic pressures of the solutions of the agents in question, for if that were the case we should expect more comparable results on the basis of the osmotic effects of the various solutions at the concentrations used. There is a reduction in the effect when the NaCl and KCl used singly are reduced to concentrations of N/20. The condition of the plants 16 days after first applying the treatment, or 13 days after being in distilled water, is shown in table rx, from which it is evident that there was great exos- mosis with but little or no visible effect accompanying it. TABLE IX CONDITION OF PLANTS TREATED WITH VARIOUS SALTS FOR DIFFERENT PERIODS OF TIME Culture no. Condition of tops Condition of roots 21 and 22 Normal Slightly brown and very slightly flaccid 23 Dead—badly wilted at end of | Very limp and fla treatment and br i 24-28 Normal Very slightly brown- ish but practically orma 29 and 30 Normal Practically normal That osmotic effects play practically no part in the phe- nomenon under consideration is indicated from the results of Loeb (’03) on Gammarus and those of True (’14) on Lupinus seedlings. The writer also performed experimental work to [VoL. 2 558 ANNALS OF THE MISSOURI BOTANICAL GARDEN determine this point. Solutions of pure saccharose of vary- ing concentrations were used and the effects produced by the same during a period of 24 hours as compared with pure dis- tilled water, measured by the determined conductivity of the medium both during the 24 hours and after (when the plants which had been in the sugar solutions were also placed in dis- tilled water). These results are given in tavle x. As there seen, no differences were obtained from the different concen- trations. TABLE X EXOSMOSIS FROM THE ROOTS OF PLANTS IN SUGAR SOLUTIONS AND DISTILLED WATER * Culture 1 Culture 2 Culture 3 Culture 4 1.28% sac- | 2.56% sac- | 5.13% sac- control Time of readings cinean = charose sol’n.| charose sol’n. ist. ours |H:O throughout followed oy followed by | followed by changed dist. H,O dist. H:O dist. H:O conductivity Conductivity readings of the sugar solutions: Before .. Een in WABCO POR 45% 9.4 10.6 10.9 10.9 After 10 ug EEE 28.0 19.7 30.8 32.9 After 24 hrs... 6.665% 28.9 18.3 29.0 32.9 Increase over original sol’n. during 24 hrs 19.5 3 18.1 22.0 Coad uctivity readings of the distilled water: Alter Lea 9. 8.6 8.8 Aftor 23 Nros sen 10.5 11.5 11.0 10.4 After 48 hrs........... 10.9 11.3 10.2 10.0 Increase over dist. H2 the first half hour t 3.4 2.6 2.8 y Increase over dist. H:O during 48 hours t.. 4.9 5.3 4.2 4.0 * All readings represen values of x on the Wheatstone bridge, the resistance in the box a t The aver age lied a the distilled water before placing roots in it was ap- ee 6.0. In table xı are shown the effeets produced by salts alone as well as by salts plus anestheties in weak concentrations. It was desired to use approximately the same concentrations of anesthetics as indicated by the work of Lillie (’12), Oster- hout (’13), and others. The conductivity of the water con- taining the anestheties was not determined after the 53-hour treatment and hence the resulting exosmosis during that in- terval was not ascertained. But from other experiments on 1915] MERRILL—ELECTROLYTIC DETERMINATION OF EXOSMOSIS 559 the effect of anestheties in solution we have seen that the exosmosis is rapid and considerable during the first day or so and then remains stationary, i. e., the curve becomes hori- TABLE XI EFFECTS OF SALT tanz USED SINGLY AND COMBINED WITH ANESTHETICS UPON er XOSMOSIS FROM THE ROOTS OF PLANTS (PL NTS 40 DAYS OLD WHEN TREATED) Conductivitv * Culture 2 ee Readings Increase over dist. H:O ** no. After After 1st Next | Total in $ hr. | 42 hrs. 3 hr. | 414 hrs. | 42 hrs. 31 N/10 MgCh, 53 hrs..... 35.4 57.1 29.4 21.7 51.1 32 N/10 NaCl, 53 hrs...... 34.4 60.9 28.4 26.5 54.9 33 N/10 KCL $3 hen... ...... 40.8 63.0 34.8 22.2 57.0 34 0. 1% ether in H:O, 53 EV Pee EFF ee 10.0 11.6 4.0 1.6 5.6 35 0. TA CHCl; in H:O, E ee 42.1 36.3 6.1 24.2 30.3 36 0.7% benzol in H:O | ae oa ae 71:3 1747 5 6.4 11.7 37 N/10 MgCl: and 0.7% ether, 53 hrs......... 41.2 64.7 39.2 23.3 58.7 38 N/10 MgCl: m 0.7% Te ER 45.5 57.2 39.5 11.7 51.2 39 N/10 MgCl: aad 0.7% benzol, 53 hrs........ 44.0 49.4 38.0 5.4 43.4 40 N/10 NaCl and 0.7% ether, 53 hrs......... 37.4 59:5 31.4 22.1 53.5 41 N/10 NaCl and 0.7% CHCE 53 BM... 5 47.5 62.4 41.5 14.9 56.4 42 N/10 NaCl and 0.7% SS 49.5 56.0 43.5 6.5 50.0 43 N/10 KCl and 0.7% 3 ae 50.2 76.0 44.2 25.8 70.0 44 N/10 KCI and 0.7% CESS D.. $1.7 65.4 45.7 13.7 59.4 45 N/10 KCI and 0.7% enzol, 53 hrs........ 49,2 55.8 43.2 6.6 49.8 46 and 47| Control (dist. H:O re- newed after 53 hrs.)...| 10.9 1119 4,9 1.0 5.9 48 ne ag H:O not VE Sea TE 18.27 | 15:51 9.22 = 9.5] 49 and 50 ar (full nutr. until Nos. 31-45 were placed oe: eee 42.3 39,0 6.5 26.5 33.0 * All nee pent values of x on the Wheatstone bridge with a resistance in the box of far ms. *Th average reading of the distilled water before placing roots in it was approximately 6. t After 53 ene t After 95 hours. ĝ In 53 hours. || In 95 hours. zontal. We can therefore safely infer that such was the case here, except possibly in the ether-treated cultures in which the roots and tops showed no effect whatever from the treat- 560 [vor. 2 ANNALS OF THE MISSOURI BOTANICAL GARDEN ment. We can thus account for the fact that the increase in the conductivity of the medium in Nos. 34, 35, and 36, was so slight and so similar to that given by the controls. It will be seen that the anesthetics did not antagonize the salts so far as exosmosis of electrolytes is concerned. The condition of the plants after the 53-hour treatment is shown in table xu. TABLE XII CONDITION OF PLANTS FIFTY-THREE HOURS AFTER TREATMENT WITH SOLUTIONS OF SALTS AND ANESTHETICS Culture no. Condition of tops Condition of roots 31 Moral er rs re Yellowish brown, somewhat flaccid 32 Practically normal... eresse Slightly yellow, practically norma 33 Subnormal, drying considerably... . By ie practically 34 m ee ay ee es Practically normal 35 Sa N E EE N E EEA = considerably flaccid 36 aeg Son P SEEE EPET T Ver ae 37 Practically paesi OET EEA Yellowish er considerably 38 PEG normal rer enter Somewhat flaccid 39 Lin considerably..............- Very flacci MNO aaa AA a Almost normal 41 Drying NOTRE RE Taar Sete flaccid 42 Drying read EN zen EIN cid 43 Practically Benin Pr ey normal 44 Drying kee an r TEEN Conside ite flaccid 45 Ore A oy s ee ee ee flac 46—50 Normal rn EAEE TAN Dacia normal The concentration of the anesthetics used in the above ex- periments was near the boundary which would just produce exosmosis. To eliminate such action entirely when these sub- stances were used alone, therefore, the concentrations used were reduced to a point below that at which they cause exos- mosis to any appreciable extent, if at all. The results of that series are given in table xm, where we see again no indica- tions that there is any decreasing effect by the anesthetics on the exomosis induced by salts. On the contrary, the combined salt and anesthetic cause a greater exosmosis than the salt alone. As measured by the resulting growth of roots, Hibbard (713) found an antagonistic action between CuSO, and chloral hydrate. To determine if such action would also hold true in 1915] MERRILL— ELECTROLYTIC DETERMINATION OF EXOSMOSIS 561 the case of exosmosis, an experiment was set up, the results of which are given in table xıv. As there seen, there was no decrease in the exosmosis caused by either substance when the two were combined. TABLE XIII EFFECTS OF SALT SOLUTIONS USED SINGLY AND COMBINED WITH ANESTHETICS ON THE EXOSMOSIS FROM THE ROOTS OF TREATED PLANTS* Conductivity Culture EEE Readings Increase over dist. H.O]| no. After After ist Next Total 4hr. | 41 hrs. 3 hr. | 403 hrs. |in 41 hrs. 1 1/8 cer rene CHCl; in 2.311 “205 2:33 2.5 5.0 ee LIES. aaa 2 M/ 200 Ben hydrate, a vinta Reet 4 13.47 | 18.9 7.41 5-5 3 N/10 NaCl, 44 hrs...... 38.4} | 59.3 32.41 | 20.9 33.3 4 N/10 KCl, 46 hrs....... 53.6 76.2 47.6 22.6 3 yE Sen CHCl; i & N/10 NaCl, 44 EUe Oa Ts 46.2 61.6 40.2 15.4 55.6 6 ed rated CHCl: & N/10 KCI, 46 hrs..... 47.2 76.5 41.2 29.3 70.5 7 M/200 ech hydrate N/10 N hrs 49.1 79.3 43.1 30.2 13.3 8 M/200 chloral hydrate N/10 , 46 hrs 60.5 84.2 54.5 23.7 78.2 9 N/10 NaCl & N CEG Drs 50492; 70.7 82.7 64.7 12.0 76.7 10 nee So = N/20 Wy nen 41.8 Wa 35.8 29.9 65.7 11 Control Cee H:O re- wed every 2 days)..| 11.4 10.8 5.4 -.6 4.8 12 Dral (dist. H:O not Fenewed)............ 16.7 25.0 10.7 8.3 19.0 * All ae ve Rees values of x on the Wheatstone bridge with a resistance in the box of 9,11 t Reading en aie 50 minutes. ft Increase in first 50 minutes | The average reading of the distilled water before placing roots in it was approximately 6.0. Merely to get a basis of comparison between the effects pro- duced by the various agents above mentioned and acid and alkali in certain concentrations, plants were placed in solu- tions of KOH and H250, of approximately the limiting con- centrations for root growth, as found by Kahlenberg and True (’96). Instead of excretion being greater than absorption the reverse was found to be true during the period the plants re- mained in the solutions. The plants, to all external appear- ances, were not affected adversely in the least, and when later [vor. 2 562 ANNALS OF THE MISSOURI BOTANICAL GARDEN placed in distilled water gave practically no greater exosmosis than the control. With stronger concentrations a marked ef- fect would undoubtedly be produced. The results obtained are given in table xv. Another point worthy of note in this TABLE XIV EFFECTS OF COPPER SULPHATE AND CHLORAL HYDRATE USED SINGLY AND COMBINED UPON THE EXOSMOSIS FROM THE ROOTS OF PLANTS CIFIC CONDUCTIVITY OF THE SOL’NS. * VALUES OF x, f OR BRIDGE READINGS OF THE DISTILLED WATER Cult. 2 ge . 7 ++ iis. Treatment Ta E A P EPS E- o leyl gta FH a | RO] ak lemcOlmcols4 | SXhi shu oo | So | so |L Bm onma] Ove Pl BS] pS] En [ost 4er ges FRA tn ge | fo] BS [e228] S52) 858/" oe oF ed |< q = 1 | M/10,000 CuSO,, naa See 2.92| 16.68| 13.76| 19.7 | 55.4 | 13.7 | 35.7 | 49.4 2 | M/100 CuSO, Wr”, ere 142.40}/151.10) 8.70) 22.0 | 22.4 | 16.0 .4 | 16.4 3 | M/8,000 chloral te, re- an: toen * i OPED. crise 35| 1.16 81| 20.6] 13.1 | 14.632) -7.5 | 7.1 4 M/100 choral ar i 37| 3.04| 2.67| 14.8 | 16.8 | 8.8 | 2.0 | 10.8 5 M/i0, 000 CuSO and M/8,00 chloral a Lae ERTE 2.82| 19.77| 16.95| 15.0 | 53.3 | 9.0 | 38.3 | 47.3 6 M7100 CuSO. and M/100 chloral hydrate, 26 hrs.} 82.69) 95.25} 12.56] 17.2 | 20.2 | 11.2 | 3.0 | 14.2 7 | Control (dist. H:O, changed every 4 TT tt nn Peer iY .79 .47| 15.0|| 12.9 | 9.08) -2.1 | 6.9 8 Control (dist. H:O, not chandod) al. ee MP ees 14.8 | 10.7 | 8.8]-4.1] 4.7 = ” Gr values given are to be multiplied by 10° to obtain specific conductivity valu + Resistance i in box 9,110 ohms, j After 26 hours i = the solution. The first 26 hou || After 26 hours i 5 “distilled H.0. tł The average reading of the distilled water before placing roots in it was ap- proximately 6.0. * After 89 hours in the solution. tt After 89 hours in the water connection and seen in table xv is the additional verification of the fact that the rinsing method used throughout this in- vestigation was effective and that no electrolytes were carried 1915] MERRILL— ELECTROLYTIC DETERMINATION OF EXOSMOSIS 563 over on the roots from the full nutrient solutions, salt solu- tions, and other media to the distilled water, at least not in sufficient quantity to affect the validity of the results in any way. Although the conductivity of the acid and alkaline media was very high, it is seen by reference to table xv that after rinsing the roots in the usual manner and transferring the cultures to distilled water the readings were very low, thus showing that practically no electrolytes were carried over on the roots. TABLE XV CONDUCTIVITY READINGS OF THE CULTURE MEDIA OF PLANTS IN KOH, H:S0;, AND LATER IN DISTILLED WATER r VALUES OF x,t OR DR rg BRIDGE READINGS OF THE TILLED WATER Cult . a ooma ee d | © v a| SE oae Treatment Ep Ela F, EQ EQSEISE g É 2312 |n98 | Be | wn lo! of! gej gs pol "ot Fal ie „u |? o F Hia 3|.83 SE | 35 | 88 | 35 | 29 138] Sy] 52182 ass | <8 285758] et 1 | N/12,800 H:SO,, a, Serer 2.39} 1.50) 1.03) -1.36} 10.8] 10.5) 4.8) -.3} 4.5 2 | N/6,400 H:SO,, es Serre 5.62} 2.53} 1.17] —4.45 8.7| 9.61 2.71 .9| 3.6 3 | N/400 KOH, 47 I DT PEA PA 42.02| 20.09) 16.39|—-25.63| 11.5} 8.7| 5.5| -2.8| 2.7 4 | N/200 KOH, 47 Se ee 58.50] 27.98] 24.16/-34.34) 11.2) 10.5} 5.2) —.7| 4.5 5 | Control (dist. H:O, ** aa not changed). . 98) 2.56} 1.32) .34) 22.8t] 17.8/16.8ł| -5.0| 11.8 * The values given are to be sea 2 1075 t The resistance in the box was 9,11 t After being in distilled water 47 hou he — reading of the distilled water before placing roots in it was approximately 6. ** After 97 ao. IX. GENERAL Discussion In the foregoing experiments we have been able to note the exosmosis of electrolytes following different treatments. As compared with the controls we have seen marked excretions in some cases and slight or no exosmosis in excess of that in the controls in others. In the normal untreated cultures, or controls, we have seen that there is almost universally a slight exosmosis from the roots into the distilled water for about 24 [VoL. 2 564 ANNALS OF THE MISSOURI BOTANICAL GARDEN hours or so, and then in most cases there is a decline in the conductivity curve to a point approaching the original posi- tion, after which there may or may not be a gradual incline, depending, probably, on various factors. It might be well briefly to consider some theoretical aspects of the subject, especially in regard to the causal agencies ef- fecting the increased exosmosis of the treated cultures. The mere transfer of a culture from a full nutrient solution to dis- tilled water is not in itself sufficient to account for the effects produced, as we have seen that osmotic effects play little or no role in this connection, a conclusion in harmony with the find- ings of Loeb (’03) and of True (’14). To what then is the exosmosis due? Can it all be laid at the door of cell cytolysis? What influence has an alteration of the plasma membrane? In any case, we are dealing with the effect of physical and chemical factors upon the plant cell. For our purpose here it is not considered necessary to enter upon a discussion of the various ideas regarding the details of the structure of the cell and its limiting membrane, or the work and theories of the different investigators on both the animal and plant side con- cerning the permeability of the plasma membrane. Yet in passing, it may be well to mention Overton’s theory regard- ing the lipoid nature of the plasma membrane, Nathansohn’s idea of a mosaic structure of the same, Czapek’s experiments indicating the presence of neutral fats in the membrane, Lep- eschkin’s view that the plasma membrane is a continuous film (some of the work of the last two investigators being sum- marized by Blackman, 712), and Kite’s work on the structure of protoplasm, and also make note of the recent work of Cran- ner (’14) on the lipoid content of the cell wall. The effect of the two physical factors, heat and cold, may undoubtedly be considered as resulting in a complete or in- cipient disorganization of the cell, depending upon the dura- tion of exposure, and a consequent escape of some of the con- tents into the surrounding medium. In the case of the various chemical factors or agents used the matter is probably not so simple or so easily disposed of. However, a conception that would fulfill the requirements 1915] MERRILL— ELECTROLYTIC DETERMINATION OF EXOSMOSIS 565 theoretically and also accord with the experimental results would seem to be based on the specificity of chemical reaction. The cell, with its complex aggregation of chemical substances, may be considered as interacting with the substance employed, be it anesthetic, toxic agent, salt solution, or other chemical. It may be assumed that each substance has a greater affinity (if we may use that tabooed chemical term) for a particular component of the cell than for other constituents and hence reacts accordingly. This was exemplified by the striking com- parison between the effect produced by anesthetics in certain concentrations and that produced by the KCl or NaCl solution. The exosmosis, it is true, was considerable in both cases, but the resulting appearance of the roots was markedly different, the anesthetics causing indications of flaccidity, while the roots exhibiting quite as much exosmosis in the salt solutions, re- mained practically normal. If we assume that the anesthetic acted upon the colloidal matrix or gel portion of the cell and thus more or less destroyed its organization, while the salts re- acted with the substances in the sol condition and left the matrix more or less intact, we would seem to have a basis for explaining the differences observed. Anesthesia has been considered by Lillie, Osterhout, and others to be essentially a reversible process, provided that the concentration of the anesthetic was not sufficient to be toxic. The experimental work reported herewith, however, on the excretion of electrolytes induced by various anesthetics does not seem to substantiate that view. If the concentration of the anesthetics employed was below a certain point there was no observable effect whatsoever. By increasing the con- centration the critical point was attained when excretion be- gan, and as the concentration of the anesthetic was further in- creased, or as the period of application was lengthened, excre- tion likewise increased. The excretion process induced by anesthetics therefore conformed in every way to an irrever- sible chemical reaction. In Osterhout’s conductivity measure- ments of tissue, secondary agglutinization phenomena may possibly have entered in to give the observed effects, and thus have masked the real chemical reaction. Recovery of organ- [voL. 2 566 ANNALS OF THE MISSOURI BOTANICAL GARDEN isms after anesthetic treatment has also been considered by some as evidence indicating the reversibility of the anesthetic action. If such be viewed from the standpoint of chemical reactions, however, the mere fact of recovery of the organism to a normal condition following the application of anes- thetics would not seem to be sufficient justification for con- cluding that the chemical reaction which initiated the effect is a reversible one, especially when one considers the manifold activities of the cell and the wonderful recuperative powers possessed by organisms, these no doubt involving numerous reactions. Hence the writer is inclined to the belief that an irreversible chemical reaction was at the basis of the phe- nomena observed as a result of the treatment of the plant with anesthetics and the consequent exosmosis of substances contained in the cell, and that any alteration of the plasma membrane resulting in changed permeability finds its best explanation on the basis of actual chemical reactions. It is further believed that the results obtained by antagonis- tic pairs of salts and by single salts are also to be explained, as far as resulting exosmosis is concerned, in the specificity of the action of each. The method employed herein gives a deli- cate register of such action and is considered to be especially desirable because in it growth phenomena, with their result- ing complex nutritive relations, may be left out of considera- tion. That the high conductivity readings in the case of the salts and certain other electrolytes was not due to insufficiency of the washing before the roots were placed in the distilled water was abundantly proved in various ways. In regard to the method of experimentation employed in the work here reported, mention may well be made of its adapt- ability for delicate determinations pertaining to the relative toxicity of different substances. In the past such determina- tions have been made by means of growth measurements. It would seem that in this method we have, in some respects, a more rapid and satisfactory procedure for such work. X. SUMMARY AND CONCLUSIONS A brief historical review is given of the subject of excretion 1915] MERRILL—ELECTROLYTIC DETERMINATION OF EXOSMOSIS 567 from plant roots, exosmosis from living cells, and of excretion from leaves and other tissues. The methods of experimentation are described. A theoretical discussion is given of the various aspects of the subject. The following are some of the experimental results ob- tained: (a) Pea seedlings grew better in distilled water in which exosmosis from the previously treated plants of the first crop had occurred than in fresh distilled water, or in distilled water in which untreated plants had been grown. (b) Peas and horse beans did not do as well in distilled water in which pea seedlings had already grown for 21 days as in fresh distilled water. (c) Abundant exosmosis may occur from treated plants, even though the roots remain entirely normal in appearance. When the tops were badly affected and the roots remained normal, abundant exosmosis also occurred and the indications pointed in some cases to a downward flow of substances into the roots and out into the aqueous medium. No conclusive proof of this was obtained, however. (d) Anesthetic vapors cause marked exosmosis upon con- siderable exposure of the plants to them, but there is none if the exposure be short. The interval required to initiate exosmosis was accurately determined. The order of effec- tiveness of the vapors tried is, ether, least; illuminating gas, more; and chloroform, most. (e) The time limits for the exposure of plants to extremes of temperature in relation to exosmosis were determined. Comparison was also made between the effect of dry and moist heat. (f) The exosmosis curves for various organic compounds were found. In general, at the concentrations used, marked excretion was produced. (g) The effects of single salts, salts in pairs, and salts plus anesthetics in solution were ascertained as regards the exos- mosis produced upon the plants in such solutions. Antag- onistic relations in the sense of one substance decreasing the (VoL, 2 568 _ ANNALS OF THE MISSOURI BOTANICAL GARDEN exosmotic effect produced by another substance were found not to hold in the cases tried and under the conditions of the ex- periment. It is with pleasure that the writer acknowledges his indebt- edness to Dr. B. M. Duggar for numerous helpful suggestions in the prosecution of this work, and to Mrs. Amy Lyman rill for valuable assistance in the calculations involved and in the plotting of the curves; also to Dr. J. R. Schramm, who kindly aided in the tedious work of preparing the manuscript for the printer. LITERATURE CITED Blackman, F. F. (’12). The plasmatic membrane and its organisation. New Phytol. 11: 180-195. 1912. 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Bot. Gaz. 22: 81-124. 1896. Knop, W. (’60). Ueber die Ernährung der Pflanzen durch wässrige Lösungen bei Ausschluss des Bodens. Landw. Versuchs-Stat. 2: 65-99. 1860. [See pp. 86-87.] —, (61). Quantitativ-analytische Arbeiten über den Ernährungsprocess der Pflanzen, I. Ibid. 3: 295-324. 1861. —, (’62). Ibid, II. Ibid. 4: 173-187. 1862. — (64). Untersuchungen über ur on der Mineralsalze durch das Pflanzengewebe. Ibid. 6 : 81-107. 18 ag ` (06). Über ee bei hair und Pilzhyphen und ihre eutung. Jahrb. f. . Bot. 42: 357--393. temmerman; 0 i SA „Untersuchungen gl einige ee nee der dw mineen wahrscheinliche Ursache. Lan nn. Stat. = 207881. 1907. nn un 216-230.] (Vou. 2 570 ANNALS OF THE MISSOURI BOTANICAL GARDEN Lepeschkin, W. W. (’06). Zur Kenntnis des Mechanismus der aktiven Wasser- ausscheidung der Pflanzen. Beih. z. Bot. Centralbl. 19: 409-452. f. 1-3. 1906. , 11). Über die Einwirkung anästhesierender Stoffe auf die osmo- tischen Eigenschaften der Plasmamembran. Ber. d. deut. bot. 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The relation of stimulation and conduction in irritable tissues to changes in the permeability of the limiting membranes. Ibid. 28: 197-222. 1911. s Antagonism between salts and anesthetics. I. On the con- ditions of the anti-stimulating action of anesthetics with observations on their pecbactive or antitoxic action. Ibid. 29: 372-397. 1911-1912. , (12a). Ibid. II. Decrease by anesthetics in the rate of toxic action of pure isotonic salt solutions on unfertilized starfish and sea-urchin eggs. Ibid. 30: 1-17. 1912 (13). Ibid. III. Further observations arie: parallel decrease in ei stimulating, permeability-increasing, an actions of salt solutions n the presence of anesthetics. Ibid. 31: 055-287. “1912-1 913. , (13a). The physico-chemical conditions of anesthetic action. Correla- tion between the anti-stimulatin ng and the anti-cytolytie action of anesthetics. Science N. S. 37: 764-767. 1913. ————, (13>). The physico-chemical conditions of anesthetic action. Ibid. 37: 959-972. 1913. Link, (’48). Gelehrte Anstalten und Vereine. Ben nase ha rn naturforschender Freunde zu Berlin. Flora N. 8. 6: 590-59 Livingston, B. E., Britton, J. C., and Reid, F. R. (’05). Studies on the rg erties of an unproductive soil. U. S. Dept. Agr., Bur. Soils, Bul. 28 : 1-39. 1905. —, Jensen, C. A., Breazeale, J. F., Pember, F. R., and Skinner, J. J. (07). Further ar Ni on the properties of rn soils. Ibid. Bul. 36 : 1-71. pl. 1-7. Loeb, nn On the TE er = distilled water, sugar solution, and Are of the various constituen the sea-water for marine animals. Univ. Cal. Publ., Physiol. 1: 55-69. rn = 32). Mémoire pour servir a l’histoire des Assolemens. Soc. Phys. et t. nat., Genève, Mém. 5 : 287-302. 1832. . . Se Bee SE 1915] MERRILL—ELECTROLYTIC DETERMINATION OF EXOSMOSIS 571 McCool, M. M. (713). The action of certain nutrient and non-nutrient bases on plant growth. Cornell Univ. Agr. Exp. Sta., Mem. 2: 113-216. f. 1-15. 1913. Merrill, M. C. (715). 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Club, "Bul. 34: 279-303. f. 4:31907, — —— and Skinner, J. J. (707). Ocrtain organic constituents of soils in relation to soil fertility. U. S. Dept. Agr., Bur. Soils, Bul. 47 : 1-52. pl. 1-6. 1907. Stoklasa, J., and Ernest, A. (’08). Beitriige “mn Lösung der Frage der rer Natur des Wurzelsekretes. Jahrb. f. wiss. Bot. 46 : 55-102. pl. 1-4. Treviranus, L. C. (’38). Physiologie der Gewiichse. Bonn, 1838. [See 2: 100- 103.] True, R. H. A: HA The harmful action of distilled water. Am. Jour. Bot. 1: 255— 273. f. 1 : (Vou. 2, 1915] 572 ANNALS OF THE MISSOURI BOTANICAL GARDEN Volkens, G. (84). Die Kalkdrüsen der Plumbagineen. Ber. d. deut. bot. Ges. 2: 334-342. pl. 8. 1884. Wächter, W. (’05). Untersuchungen über den Austritt von Zucker aus den Zellen er gs ag von Allium Cepa und Beta vulgaris. Jahrb. f. wiss. Bot. 41 : 165-220. 1 f. 1 yi eret . (14). Our present knowledge of the chemistry of the agg ctors for flower-colour. Jour. Genet. 4: 109-129. pl. 7. 1914. [See p. 125.] et M., and Cameron, F. K. (’03). The chemistry of the soil as related crop production. U. S. Dept. Agr., Bur. Soils, Bul. 22: 1-71. 1903. Wilson, W. P. (’81). The cause of the excretion of water on the surface of i Bot. Inst. z. Tübingen, Untersuch. 1; 1-22. 1881-1885. MONOGRAPH OF THE NORTH AND CENTRAL AMERICAN SPECIES OF THE GENUS SENECIO—PART II! J. M. GREENMAN Curator of the Herbarium of the Missouri Botanical Garden Associate Professor in the Henry Shaw School of Botany of Washington University INTRODUCTION The study upon which this monograph is based was begun nearly twenty years ago, at which time the author was an Assistant at the Gray Herbarium of Harvard University. Nearly every collection of any considerable size which came to the Herbarium, particularly from western United States, Mexico, and Central America, contained specimens of Senecio, many of which were either undetermined or referred doubt- fully to some obscure or little known species. The identifica- tion of such material was often a laborious task, since all species recorded from a given region had to be listed and then specific identity established by a process of elimination. The available publications for such work were De Candolle’s ‘Prodromus,’ Gray’s ‘Synoptical Flora,’ and Hemsley’s splendid contribution to the systematic literature of the botany of Mexico and Central America in the ‘Biologia Centrali-Americana’; but the results obtained were often very unsatisfactory, because of the large number of new species published in scattered papers during the two decades following the appearance of the ‘Synoptical Flora’ and the ‘Biologia.’ It was felt, therefore, that a revision of the genus, in the light of recent and more complete collections, which have accumulated from the numerous botanical explorations in different parts of North America, would „be helpful to those concerned with this difficult group of plants and especially in the organization of material in different herbaria. A critical study of Senecio with the view of publishing eventually a 1 Issued October 8, 1915. ANN. Mo. Bot. GARD., VoL. 2, 1915 (573) [voL. 2 574 ANNALS OF THE MISSOURI BOTANICAL GARDEN monograph was suggested to me by Dr. B. L. Robinson, Curator of the Gray Herbarium, who very kindly offered to place at my disposal the entire representation of this genus in the Gray Herbarium, and who, moreover, willingly granted me the exceptional privilege of taking abroad the North American specimens, including all the types, for comparison and study in European herbaria. Accordingly nearly 2,000 mounted specimens were taken to Berlin; and through the courtesy of the authorities of the Royal Botanical Gardens and Museums of Berlin every facility in that institution, which is remarkably rich in Central and South American plants, was accorded me and work on the task was begun under the direction of Professor A. Engler. It was necessary first of all to acquire a detailed knowledge of the general morphology of the genus Senecio as a whole, and also of the closely allied genera. The results of these investigations are briefly recorded in the first part of this monograph, namely ‘Monographie der nord- und central- amerikanischen Arten der Gattung Senecio, I. Teil’ which is frequently referred to in the following text. This prelim- inary work and the rich eolleetions of the Gray and Berlin Herbaria form, therefore, the basis for the present systematic part of the monograph. After completing my studies in Berlin I went to London, taking the Gray Herbarium speeimens with me, and there spent several weeks, particularly in the examination of authentie and type speeimens at the Kew Herbarium and in the Linnean Herbarium. The opportunity at Berlin, Kew, and Paris to actually compare side by side and in detail, recent specimens, or series of specimens, with many of the older types, some of which are more or less incomplete, has been of very great advantage, and, in fact, has made it possible to establish beyond doubt the identity of many of our American species. In addition to those herbaria mentioned it also has been my good fortune to study this group of plants in several American institutions, notably the Herbarium of the Geo- logical Survey of Canada, the United States National Her- 1915] n GREENMAN—MONOGRAPH OF SENECIO 575 barium, the New York Botanical Garden Herbarium (includ- ing the Torrey Herbarium), the Herbarium of the Field Museum of Natural History, the Herbarium of the Phila- delphia Academy of Natural Sciences, the Missouri Botanical Garden Herbarium, and a number of private collections. To the directors and curators of all these, as well as the owners of the private herbaria, and correspondents who have facili- tated my work, I wish to express personal thanks; but I desire especially to extend most grateful acknowledgments to Dr. Benjamin Lincoln Robinson, Asa Gray Professor of Systematic Botany at Harvard University, and Geheimrath Professor Dr. Adolph Engler, Director of the Royal Botanical Gardens and Museum of Berlin, without whose cooperative interest and extreme liberality in the use of valuable scientific material under their charge, this work would have been im- possible. I am also grateful to Mr. W. Botting Hemsley, of the Kew Herbarium, through whose courtesy I secured type material of certain rare Mexican species and a number of excellent drawings, some of which are herein reproduced. I have cited exsiccatae rather freely, particularly such as occur in American herbaria, but by no means all that have been examined, and I have given even at the expense of much repetition detailed citation of specimens in different herbaria, hoping that this would be helpful in the interpretation of species and to future students of the genus. The few plates which it is possible to include are chosen to illustrate more especially the different sections as here defined. SENECIO [TOURN.] LINN. Senecio [Tourn. Inst. 456. pl. 260. 1700] L. Sp. Pl. 2 : 866. 1753; Gen. Pl, ed. 5, 373, n. 857. 1754; Hill, Hort. Kew. 25. 1768, and ed. 2, 1769; Juss. Gen. Pl. 181. 1789; Less. Syn. 391. 1832; DC. Prodr. 6: 340. 1837; Endl. Gen. 458, n. 2811. 1838; Hook. Fl. Bor. Am. 1: 331. 1840, in part; Torr. & Gray, Fl. N. Am. 2 : 436. 1843; Benth. & Hook. f. Gen. Pl. 2 : 446. 1873, in part; Pfeiffer, Nom. Bot. 2? : 1136. 1874; Hemsl. Biol. Cent.- Am. Bot. 2 : 235. 1881, excl. Cacalia; Gray, Syn. Fl. N. Am. 1°: 383. 1884, and ed. 2, 1888; Hoffmann in Engl. & Prantl, [VoL, 2 576 ANNALS OF THE MISSOURI BOTANICAL GARDEN Nat. Pflanzenf. IV. Abt. 5, 296. 1892, excl. Emilia; Greenm. Monogr. Senecio, I. Teil, 1901, and in Engl. Bot. Jahrb. 32 : 1-33. 1902; Dalla Torre & Harms, Gen. Siph. 563. 1900-1907, mainly. Jacobaea Thunb. Fl. Cap. Prodr. Praef. 1794. Obaejaca Cass. Diet. Sei. Nat. 35 : 270. 1825. Roldana LaLlave & Lex. Nov. Veg., fasc. 2, 13. 1825. Rugelia Schuttlew. in Chapm. Fl. Southern U. S. 246. 1860. Cacalia, Cineraria, and Gynoxis, in part, of authors. Heads heterogamous and radiate, or discoid. Involuere cylindrical campanulate, occasionally flask-shaped, usually subtended by calyculate bracteoles; bracts of the involucre uniseriate, or by overlapping subbiseriate, variable in number but tending to approach a definite series of numbers, namely 5-8-13-21. Ray-flowers when present disposed in a single row, fertile; rays sometimes more or less reduced. Disk- flowers perfect; corollas slenderly tubular to abruptly am- pliated above into a campanulate 5-toothed limb, teeth mostly short. Anthers obtuse or slightly sagittate at the base. Style-branches subterete, recurved-spreading, truncate, rounded-obtuse, occasionally terminated by a small penicillate tuft of hairs, or (in the subgenus Pseudogynowis) terminated by a triangular acute or acuminate appendage. Achenes sub- terete, usually ribbed, glabrous, or more or less hirtellous especially on the ribs. Pappus of numerous usually white setae.—Annual, biennial, or perennial herbs, shrubs, climbers, or even arboreous plants, with alternate or radical, very variable, pinnately or palmately veined, entire or variously divided leaves. Synopsis OF THE SuBGENERA and SECTIONS Subgenus I. Eusexecıo Hoffm. Style-branches truncate, rounded-obtuse or occasionally terminated by a penicillate tuft of hairs. A. Stems erect or ascending, not climbing. a. Stems not abruptly terminated by a fore shortening of the main axis; oil-tubes not richly developed in the peripheral portion of the stem. a. Leaves pinnately veined; lateral nerves not numerous or conspicuous. Te Annual Derbs: Sraa an assess aes § 1. Annui 1915] GREEN MAN—MONOGRAPH OF SENECIO II. Biennial or perennial herbs (rarely annual). t Stems herbaceous. * Heads usually radiate; — yellow, except in 8. Gree and 8. crocatus. ne leafy to the inflo ee leaves laciniately o. Nativ § 2. Eremophili 00: Introduced u ...§ 3. Jacobaeae es lower simple ided. Leaves pinnate or pin- era rarely undi- 00,2 owe, leaves Sohne ovate, s le and un- s divide a ee ...$ 5. Bolanderiani ttt Stem not a leafy to the inflorescence; leaves o lyra start ore or less per- eat ct ntose; pu- bescencee never of long duced on the stem..$ 6. Aurei vided .: ..:.:4. nase 7. Lobati ly reduced ........ 8. Tomentosi ttit Stem leafy to the inflo- escence (exce in §9); pubescence aria E of long ore airs. tem-leaves not am- digi tately divide a .§ 9. Columbiani eav Digitati vided 00. Stem - nn. amplexi- cau ò. Involuere ecaly- LATE 11. Cineraroidei 56. Seer calycu- late sis RE § 12. Amplectentes 577 Sanguisorboidei [vor. 2 578 ANNALS OF THE MISSOURI BOTANICAL GARDEN ** Heads discoid; flowers whitish or purplis + Heads 2 more high; corollas a 5-lobed..... § 13. Rugeliae +} Heads 1 em. high; corollas shortly 5-toothed ......... § 14. Mulgedifolu 2. Stems ligneous wi the bas *Involucre barely xe culate; plants igual white-tomentose thronpiont anne § 15. Incani ** Involuere calyculate; plants glabrous or pubescent......... § 16. Suffruticosi Shrubs or tree-like plants........ 817. Fruticosi ß. Leaves nn ot A san $ 18. Palmatinerves y. Leaves pinnately veined; lateral parallel-arcuate, numerous and ekas mh $ 19. Multinervii Stems abruptly iea by a fore- polio of the main axis and bearing at the top t J pound corym cym oil-tubes richly d veloped in the pores orn portion of the stem. "20. Terminales B. Fio OIDE ae usa § 21. Streptothamni Subgenus II. Psrupocynoxis Greenm. Style-branches ter- minated by triangular acute or acuminate dorsally hispidulous appendages..... eer ete eee eee + $22. Convolwuloidei Suggenvs I. Eusenecıo Hoffm. Subgenus I. Eusexecıo Hoffm. in Engl. & Prantl, Nat. Pflanzenf. IV. Abt. 5. 297. 1892; Greenm. Monogr. Senecio, I. Teil, 21, 30. 1901, and in Engl. Bot. Jahrb. 32 : 17, 26. 1902. Annuals, biennials or perennials; stems erect, scandent or climbing; leaves pinnately or palmately veined; heads radiate or discoid; style-branches truncate or rounded-obtuse, not infrequently bearing a penicillate tuft of hairs at the extreme tip. Sect. 1-21. Secr. 1. Annur Hoffm. $1. Annur Hoffm. in Engl. & Prantl, Nat. Pflanzenf. IV. Abt. 5, 297. 1892; Greenm. Monogr. Senecio, I. Teil, 21, 23. 1901, anid i in Engl. Bot. Jahrb. 32 : 17, 19. 1902. Giasisous DC. Prodr. 6 : 341. 1837. Annual herbs; heads radiate or discoid; involucre nar- rowly campanulate or subcylindric, usually calyculate; achenes pubescent or glabrous. Sp. 1-7 1915] GREENMAN—MONOGRAPH OF SENECIO 579 KEY To THE SPECIES A. Heads radiate or discoid; rays when present minute, barely surpassing the sae pas a.. Plants viscid-pubescent -sssr e rreran 000 1. 8. viscosus b. Plants glabrous or ene not vis a. Leaves coarsely dentate, Aaria rsa oan by 2. OR eer S. mohavensis B. Leaves hieng pinnatifid, not greatly expanded at the bas L Aueh black-tipped, heads discoid..... 8. vulgaris II. Bracteoles not black-tipped; heads a 1. Plants je ch Te RE aE 4, er ants. glabrous. -acses nanea 5. K. aphanacti B. Heads ap eta ‘ae hei much surpassing the involuer a. Plants ee or pubescent, not arachnoid- tomentose Case CR UND Een er 6. 8. californicus ß. Leaves thickish, r <<. Ss cca 6a. var. ammophilus bis Plante arachnoid-tomentose =... aaa 7. 8S. ampullaceus 1. Senecio viscosus L. Sp. Pl. 2: 868. 1753, and ed. 2, 1217. 1763; Sow. Eng. Bot. pl. 32. 1790; Willd. Sp. Pl. 3 : 1984. 1800; Oeder, Fl. Dan. pl. 1230. 1799; Schkuhr, Handb. pl. 267. 1808; DC. Prodr. 6 : 342. 1837; Gray, Syn. Fl. N. Am. 1? : 394. 1884; Greenm. Monogr. Senecio, I. Teil, 23. 1901, in Engl. Bot. Jahrb. 32:19. 1902, and in Gray, Manual, ed. 7, 853. 1907; Britton, Manual, ed. 2, 1029. 1905; Britton & Brown, Il. Fl, ed. 2, 3: 540. 1913. Obaejaca viscosa Cass. Dict. Sci. Nat. 35 : 270. 1825. A strong-scented annual, viscid-pubescent throughout; stem erect, 2 to 4 dm. high, usually branched from the base; leaves sessile, half-clasping, 3 to 6 em. long, two-thirds as broad, once or twice pinnatifid with angulate-sinuate lobes and rounded sinuses; heads radiate (rarely discoid); rays incon- spicuous; achenes glabrous. Distribution: eastern North America from Nova Scotia to Pennsylvania, near the coast. Specimens examined: Nova Scotia: Pictou, 1 Nov., 1874, Fowler (Field Mus. Herb.) ; Pictou Landing, 21 July, 1883, Macoun 14883 (Geol. Surv. Canada Herb.) ; Kentville, 22 Aug., 1902, Fernald (Gray Herb. and Geol. Surv. Canada Herb.). New Brunswick: Schediac, 11 Sept., 1874, Fowler (Geol. [Vor. 2 580 ANNALS OF THE MISSOURI BOTANICAL GARDEN Surv. Canada Herb. 14882 and Kew Herb. 872, in part); Painsee Junction, 8 Aug., 1901, Churchill (Gray Herb.). Massachusetts: along Boston and Albany Railroad, Sept., 1879, Boott (Gray Herb.); streets of Cambridge, 1 Sept., 1897, Robinson (Gray Herb.). Rhode Island: wharves at Providence, 4 Sept., 1874, Congdon (Gray Herb.); streets of Providence, coll. of 1876, Bailey (Gray Herb. and Field Mus. Herb.) ; East Providence, 20 July, 1890, Collins (Mo. Bot. Gard. Herb.). Pennsylvania: on ballast, Girard Point, July, 1877, Martin- dale (Gray Herb.) and Aug., 1877, Rothrock (Field Mus. Herb.). Introduced from Europe. 2. S. mohavensis Gray, Syn. Fl. N. Am. 1°: 446. 1884, and ed. 2, 454. 1886; Greenm. Monogr. Senecio, I. Teil, 23. 1901, and in Engl. Bot. Jahrb. 32:19. 1902. Plate 17. Glabrous throughout; stems erect or nearly so, 1.5 to 4 dm. high, freely branching; leaves membranous, ovate to oblong-ovate, 2 to 6 cm. long, 1 to 4 em. broad, apiculate-acute, irregularly toothed, or somewhat laciniate-dentate, the lower- most narrowed into a petiolate base, those of the stem sessile and amplexicaul; inflorescence a terminal corymbose cyme; heads 1 em. high on slender peduncles, discoid or with much reduced ligulate flowers; involucre calyculate with few short inconspicuous bracteoles, 18-20-flowered; bracts of the in- voluere about 13, linear, acute, slightly shorter than the flowers of the disk; achenes canescent pubescent. Distribution: southern California, Arizona, and northern Mexico. Specimens examined: California: Pleasant Canon, Panamint Mountains, alt. 900 m., 10 May, 1906, Hall & Chandler 6910 (Mo. Bot. Gard. Herb. and Field Mus. Herb.) ; Hall Canon, Panamint Moun- tains, 18 April, 1891, Coville & Funston 697 (U. S. Nat. Herb.) ; Panamint Valley, alt. 450 m., 5 May, 1897, Jones (Mo. Bot. Gard. Herb.) ; Mohave region, April-May, 1884, Lemmon 3129 (Gray Herb.), Tyre; Colorado Desert, April, 1889, C. R. Orcutt (U. S. Nat. Herb. and Gray Herb.). 1915] GREENMAN— MONOGRAPH OF SENECIO 581 Arizona: Tempe, 21 April, 1892, Ganong & Blaschka (Gray Herb.). Sonora: near the U. S. boundary line, 28 March, 1884, Pringle (Gray Herb. and U. S. Nat. Herb.). 3. 8. vulgaris L. Sp. Pl. 2 : 867. 1753, ed. 2, 1216. 1763; Fl. Dan. pl. 513. 1770; Willd. Sp. Pl. 3:1979. 1800; Sow. Eng. Bot. pl. 747. 1800; Pursh, Fl. 2:528. 1814; DC. Prodr. 6:341. 1837; Reichb. Ic. Fl. Germ. & Helv. 16:35. pl. 68 (CMLIX), fig. I, 1-9. 1854; Gray, Syn. Fl. N. Am. 12 : 394. 1886; Greenm. Monogr. Senecio, I. Teil, 23. 1901, in Engl. Bot. Jahrb. 32: 19. 1902, and in Gray, Manual, ed. 7, 853. 1907; Britton, Manual, ed. 2, 1029. 1905; Britton & Brown, Ill. Fl., ed. 2, 3: 539. 1913. Annual, 1 to 4 dm. high, glabrous or subfloecose pubescent especially in the axils of the upper leaves and in the inflo- rescence; leaves 2 to 8 cm. long, 0.5 to 2.5 em. broad, more or less lyrately pinnatifid and angulate-toothed, lower leaves narrowed into a margined petiole, the upper sessile and semi- amplexicaul; heads discoid; the rather numerous small calye- ulate bracteoles as well as the bracts of the involucre usually black-tipped; achenes hirtellous-puberulent along the angles or ribs. Distribution: Labrador, Newfoundland to North Carolina, west to Alaska, California, and New Mexico. Europe, Asia, and Africa. Specimens examined: Labrador: Hopedale, 4-6 Aug., 1897, Sornborger 162 (Gray Herb.). Newfoundland: rocky hills, St. John’s, 1 Aug., 1894, Robin- son & Schrenk (Gray Herb., U. S. Nat. Herb., Geol. Surv. Canada Herb., and Mo. Bot. Gard. Herb.) ; Funk Island, 23 July, 1887, Palmer (U. S. Nat. Herb.); rich soil, field near shore, Channel, 27 July-1 Aug., 1901, Howe & Lang 802 (Gray Herb.); Barred Island, 13 Aug., 1903, Sornborger (Gray Herb.). Nova Scotia: dry soil, roadsides, North Sydney, Cape Breton, 21-25 July, 1901, Howe & Lang 639 (Gray Herb.) ; [VoL. 2 582 ANNALS OF THE MISSOURI BOTANICAL GARDEN Boylston, July, 1890, Hamilton 22848 (Geol. Surv. Canada Herb.) ; Baddeck, Cape Breton Island, 25 July, 1898, Macoun 19721 (Geol. Surv. Canada Herb.). New Brunswick: along railroad, Conners, 22 July, 1908, Mackenzie 3646 (Mo. Bot. Gard. Herb.) ; Shediac, 11 Sept., 1874, Fowler 872 in part. (Kew Herb.). Quebec: shore of St. Lawrence, Gaspé, Matane Co., Forbes (Gray Herb.); Gaspé Basin, 24 July, 1882, Macoun 14889 (Geol. Surv. Canada Herb.). Ontario: Ottawa, 20 July, 1891, Scott 14885 (Geol. Surv. Canada Herb.) ; Belleville, 10 Aug., 1877, Macoun 14890 (Geol. Surv. Canada Herb.); northeast of Sarnia, Lambton Co., Wheatley (Mo. Bot. Gard. Herb.) ; Wingham, Aug., 1890, Mor- ton 14886 (Geol. Surv. Canada Herb.) ; Kingston, Sept., 1896, Fowler (Field Mus. Herb.); Sarnia, 18 June, 1901, Macoun 26677 (Geol. Surv. Canada Herb.). Saskatchewan: between Cumberland House and Hudson Bay, Richardson 14887 (Geol. Surv. Canada Herb.) ; Prince Albert, 13 July, 1896, Macoun 12174 (Geol. Surv. Canada Herb.). Alberta: waste ground, Prince’s Island, near Calgary, 21 Aug., 1913, Moodie 31 (Field Mus. Herb.). British Columbia: Burrard Inlet, 22 July, 1889, Macoun (Gray Herb. and Geol. Surv. Canada Herb.); vicinity of Victoria, 9 April, 1908, Macoun 78949 (Field Mus. Herb.) ; along railway embankment, Sicamous, 20 July, 1904, Macoun 62191 (Geol. Surv. Canada Herb.); Cedar Hill, Vancouver Island, 21 May, 1887, Macoun 14884 (Geol. Surv. Canada Herb.) ; near Victoria, 23 May, 1893, collector not indicated, 550 (Geol. Surv. Canada Herb.); Victoria, 10 June, 1875, Dawson 14888 (Geol. Surv. Canada Herb.). Alaska: vicinity of Sitka, July, 1891, Wright 1538 (Mo. Bot. Gard. Herb.); Sitka, July, 1881, McLean (U. S. Nat. Herb.); Skagway, 29 July, 1907, Cowles 889 (Field Mus. Herb. and Mo. Bot. Gard. Herb.). Maine: Baker’s Island, 19 July, 1883, Redfield (Mo. Bot. Gard. Herb.). 1915] GREENMAN—MONOGRAPH OF SENECIO 583 Vermont: waste ground, Rutland, 1 Sept., 1899, Eggleston 1383 (Gray Herb.). Massachusetts: Ipswich, Oakes (Gray Herb. and U. S. Nat. Herb.) ; Nahant, 6 July, 1878, Kellermann (Mo. Bot. Gard. Herb.); Revere Beach, 9 July, 1898, Greenman 515 (Gray Herb.); Cambridge, Chickering (U. S. Nat. Herb.); road- sides, West Cambridge, 29 Sept., 1894, local collection (Gray Herb.) ; Swampscott, 21 June, 1897, Weatherby (Gray Herb.) ; Ipswich, July, 1874, Morong (Field Mus. Herb.). Rhode Island: waste places, Providence, Sept., 1844, Thurber (Gray Herb.); Providence, 2 July, 1892, Collins & Bailey (U. S. Nat. Herb.) ; Cat Swamp, Providence, 23 June, 1895, Collins (U. S. Nat. Herb.) ; Providence, 16 Aug., 1873, Congdon (Field Mus. Herb.) ; Providence, July, 1878, Bailey (Mo. Bot. Gard. Herb.). New York: Syracuse, June, 1887, Overacker (Mo. Bot. Gard. Herb.); Troy, collector and date not indicated (Gray Herb.) ; Ithaca, 12 Oct., 1892, H. von Schrenk (Mo. Bot. Gard. Herb.) ; near Fiske mansion, Ithaca, 21 May, 1884 (U. S. Nat. Herb.) ; Hunter’s Point, Long Island, Sept., 1879, J. Schrenk (U. S. Nat. Herb.) ; Elmira City, 28 Aug., 1898, Lucy (Field Mus. Herb.) ; Troy, June, 1873, Jesup (Field Mus. Herb.). Pennsylvania: Girard Point, Philadelphia, Aug., 1877, Rothrock (Field Mus. Herb.). New Jersey: Camden, July, 1876, Martindale (U. S. Nat. Herb.) ; Kaighn’s Point, Camden, 16 July, 1865, Parker (Mo. Bot. Gard. Herb.). Maryland: vicinity of Oakland, 5 Sept., 1910, Steele (U. S. Nat. Herb.). District of Columbia: waste ground, Washington, 14 Sept., 1891, Blanchard (Mo. Bot. Gard. Herb.) ; above Uniontown, 27 May, 1883, Ward (U.S. Nat. Herb.). North Carolina: cultivated grounds, Biltmore, 4 May, 1897, Biltmore Herb. 883° (Gray Herb., Mo. Bot. Gard. Herb., and Field Mus. Herb.). Ohio: Oberlin, June, 1892 and 1895, Ricksecker (U. S. Nat. Herb.). [vor. 2 584 ANNALS OF THE MISSOURI BOTANICAL GARDEN Michigan: waste ground, Keweenaw Co., July, 1887, Far- well (Gray Herb.). Wisconsin: St. Croix Co., coll. of 1888, Matthews (U. S. Nat. Herb.); Preble, 20 May, 1888, Schuette (Field Mus. Herb.) ; Green Bay, 11 July, 1897 and 29 Sept., 1901, Schuette (Field Mus. Herb.). Nebraska: Valley Co., July, 1886, Webber (Field Mus. Herb.). Montana: Willow Creek, 14 June, 1883 Scribner 123° (Gray Herb.); Columbia Falls, 21 June, 1894, Williams 965 (Gray Herb. and U. S. Nat. Herb.). Wyoming: Sundance, 4 July, 1896, Nelson 2201 (Mo. Bot. Gard. Herb.). Colorado: valley near Empire, Sept., 1892, Patterson (Gray Herb.); along railroad at Georgetown, Aug.-Sept., 1892, Patterson (Field Mus. Herb.). New Mexico: Sante Fe, 14 Sept., 1895, Mulford 1301 (Mo. Bot. Gard. Herb.) ; 4 May, 1897, A. A. € E. G. Heller 3657 (Gray Herb., Field Mus. Herb., and Mo. Bot. Gard. Herb.). Idaho: frequent in moist places, Julietta, Latah Co., 8 June, 1892, Sandberg, McDougal & Heller 343 (Gray Herb., Field Mus. Herb., and U. S. Nat. Herb.) ; waste ground in the Palouse Country and about Lake Coeur d’Alene, June—July, 1892, Aiton (Field Mus. Herb. and Mo. Bot. Gard. Herb.). Washington: on mountains near the lower Cascades, 29 May, 1886, Suksdorf (Gray Herb.); Seattle, 6 March, 1889, Smith (Mo. Bot. Gard. Herb.); in fields, Pullman, 2 June, 1894, Piper (Mo. Bot. Gard. Herb.) ; Hoquiam, 5 June, 1897, Lamb 1146 (Field Mus. Herb. and Mo. Bot. Gard. Herb.) ; San Juan Island, July, 1914, Reynolds (Field Mus. Herb.) ; Index, Snohomish Co., July, 1898, Savage, Cameron & Lenocker (Field Mus. Herb.); Granddalles, 3 Sept., 1904, Westgate 3997 (U. S. Nat. Herb.) ; Klickitat Co., June, 1878, Suksdorf (Gray Herb.). Oregon: cultivated fields, Sauvie Island, June, 1880, Howell (Gray Herb.) ; Portland, 1 June, 1884, Henderson 555 (Mo. Bot. Gard. Herb.); Portland, Feb., 1900, Lunell, and without date Sargent (Gray Herb.) ; Bonneville, 6 Aug., 1895, 1915] GREENMAN—MONOGRAPH OF SENECIO 585 Canby (U. S. Nat Herb.); Catching Inlet, 10 May, 1911, Smith 3700 (Field Mus. Herb.) ; Charleston Bay, 6 May, 1911, Smith 3668 (Field Mus. Herb.) ; North Slough, 1 March, 1911, Smith 3487; Coos Co., 2 March, 1911, Smith 3494 (Field Mus. Herb.) ; Portland, March, 1889, Drake & Dickson (Field Mus. Herb.) ; without definite locality, coll. of 1868-69, Kellogg £ Harford 536 (U. S. Nat. Herb.).* California: Oakland, March, 1864, Bolander 2777 (Gray Herb. and Mo. Bot. Gard. Herb.) and May, 1865, Bolander 434 (Gray Herb.) ; without definite locality, coll. of 1880, Norton (Mo. Bot. Gard. Herb.) ; near Mendocino, May, 1898, Brown 758 (Mo. Bot. Gard. Herb.); Mendocino Co., June, 1898, Brown 458 (Field Mus. Herb.) ; Stanford University, 2 March, 1902, Baker 311 (Gray Herb., Field Mus. Herb., and Mo. Bot. Gard. Herb.) ; Russian River, near Trenton, 16 March, 1902, Heller € Brown 5072 (Gray Herb., Field Mus. Herb., and Mo. Bot. Gard. Herb.); Big River, Mendocino Co., July, 1903, McMurphy 374 (U. S. Nat. Herb.); near Saratoga, Santa Clara Co., 25 Feb., 1906, Pendleton 288 (U. S. Nat. Herb.). 4. S. sylvaticus L. Sp. Pl. 2:868. 1753, and ed. 2, 1217. 1763; Sow. Eng. Bot. pl. 748. 1800; Willd. Sp. Pl. 3:1985. 1800; Fl. Dan. pl. 869. 1782; DC. Prodr. 6 : 342. 1837; Gray, Syn. Fl. N. Am. 1° : 394. 1884; Greenm. Monogr. Senecio, I. Teil, 23. 1901, in Engl. Bot. Jahrb. 32:19. 1902, and in Gray, Manual, ed. 7, 853. 1907; Britton, Manual, ed. 2, 1029. 1905; Britton & Brown, Ill. FL, ed. 2, 3: 539. 1913. Obaejaca sylvatica Cass. Diet. Sei. Nat. 35 : 271. 1825. Stem erect, simple or branched, 1 to 4 dm. or more high, usually somewhat pubescent; leaves more or less pinnatifid with unequal lobes, 2 to 15 em. long, 1 to 8 cm. broad; the lower leaves petioled, the upper sessile, clasping and auricu- late-sagittate; inflorescence naked or nearly so; heads cylin- drical, sparingly calyculate, radiate; ligules barely surpassing the involucre, not infrequently much reduced; achenes can- escent-pubescent. Distribution: Newfoundland to Maine, Ohio, and on Pacific coast. [VoL. 2 586 ANNALS OF THE MISSOURI BOTANICAL GARDEN Specimens examined: Newfoundland: railway ballast, Whitbourne, 17 Aug., 1894, Robinson & Schrenk (Gray Herb., U. S. Nat. Herb., Mo. Bot. Gard. Herb., and Geol. Surv. Canada Herb.). Prince Edward Island: sand dunes, Tracadie Beach, 25 July, 1901, Churchill (Gray Herb. and Mo. Bot. Gard. Herb.) ; waste places, Brackley Point, 28 Aug., 1888; Macoun 14874 (Geol. Surv. Canada Herb.). Nova Scotia: clearings and open woods, Sydney, Cape Breton Island, 17 Aug., 1902, Fernald (Gray Herb.) ; Boylston, Aug., 1890, Hamilton 22847 (Geol. Surv. Canada Herb.) ; Truro, without date, Maccullock (Gray Herb.); Elizabeth- town, Cape Breton Island, 2 Aug., 1898, Macoun 19719 (Gray Herb. and Geol. Surv. Canada Herb.); Baddeck Bay, Cape Breton Island, 11 Aug., 1898, Macoun 19720 (Gray Herb. and Geol. Surv. Canada Herb.); sea cliffs, Black Hole, near Baxter’s Harbor, 24 Aug., 1902, Fernald (Gray Herb.) ; on pebbly beach, Purcell’s Cove, Halifax Harbor, 2-6 Sept., 1901, Howe & Lang 1512 (Gray Herb.) ; open woods, Starrs Point, Kings Co., 23 Aug., 1902, Fernald (Gray Herb.); MeNiels Harbor, Cape Breton Island, 4 Aug., 1898, sheet 19722 (Geol. Surv. Canada Herb.). New Brunswick: Grand Manan, 26 July, 1891, Churchill (Field Mus. Herb. and Mo. Bot. Gard. Herb.) ; Falls of the St. John River, St. John, 22 July, 1902, Williams & Fernald (Gray Herb.). Quebec: beach of Gaspé Bay, Gaspé Co., 24-27 Aug., 1904, Collins, Fernald & Pease (Gray Herb.). British Columbia: Vancouver Island, 6 Aug., 1909, Macoun 78950 and 78951 (Field Mus. Herb.). Maine: island in Penobscot Bay, Aug., 1896, F. L. & L. H. Harvey 554° (U. S. Nat. Herb.). Ohio: near Painsville, coll. of 1892, Hacker 123 (Gray Herb.). Washington: Seattle, Aug., 1909, Piper (U. S. Nat. Herb.) ; on old burn near farms, Port Crescent, Aug., 1911, Webster 19 (U. S. Nat. Herb.) ; old camps, Granite Falls, Snohomish Co., 31 Oct., 1911, Smith 4226 (Field Mus. Herb.) ; Iron Mountain, GREENMAN— MONOGRAPH OF SENECIO 587 Granite Falls, alt. 300 m., 28 Oct., 1911, Smith 4224 (Field Mus. erb.). Oregon: region of Coos Bay, 10 Sept., 1911, House 4848 (U. S. Nat. Herb.). California: Vance’s Camp, Humboldt Co., 5 June, 1911, Smith 3778 (Field Mus. Herb.) ; vicinity of Eureka, 20 June, 1907, J. P. Tracy 2571 (Univ. Calif. Herb. and Mo. Bot. Gard. Herb.). 5. S. aphanactis Greene, Pittonia, 1: 220. 1888, and Fl. Franciscana 464. 1897; Greenm. Monogr. Senecio, I. Teil, 23. 1901, and in Engl. Bot. Jahrb. 32:19. 1902. S. sylvaticus Gray, Bot. Calif. 1: 410. 1876, not L.; Jepson, Fl. West. Mid. Calif. 512. 1901. A slender annual, 1 to 3 dm. high, glabrous or somewhat tomentulose especially in the inflorescence; stem simple or branched; leaves linear to lanceolate, 1 to 4 em. long, 1 to 12 mm. broad, entire to coarsely dentate or even pinnately lobed, glabrous or nearly so; the lower leaves narrowed into a petio- late base, the upper sessile; inflorescence terminal, few to several-headed; heads somewhat flask-shaped, 6 to 7 mm. high, radiate; involucre sparingly bracteolate, glabrous to tomen- tulose at the base; rays small, scarcely exceeding the in- volucre; achenes appressed-canescent. Distribution: central California, northern Mexico and adjacent islands. Specimens examined: California: Mare Island, 30 March, 1874, Greene (Gray Herb. and Field Mus. Herb.), co-rypr; San Luis Obispo, Brewer 463 (Gray Herb. and Mo. Bot. Gard. Herb.); San Luis Obispo, coll. of 1886, Summis (U. S. Nat. Herb.) ; Avalon, Santa Catalina Island, March, 1901, Trask (U. S. Nat. Herb. and Mo. Bot. Gard. Herb.); edges of cañons and alkaline flats, San Diego, Brandegee 3414 (Gray Herb., Field Mus. Herb., and Mo. Bot. Gard. Herb.); San Diego, Geological Survey of California 1860-61, Cooper (Gray Herb.); North American Pacific coast flora, Parry 170 (Gray Herb.); San Diego, 5 Feb., 1884, Orcutt (Field Mus. Herb.). [VoL. 2 588 ANNALS OF THE MISSOURI BOTANICAL GARDEN Lower California: Cedros Island, April, 1897, Brandegee (Gray Herb. and U. S. Nat. Herb.) ; San Quentin Bay, Palmer 606 (Kew Herb.). 6. S. californicus DC. Prodr. 6: 426. 1837; Torr. & Gray, Fl. N. Am. 2 : 437. 1843; Gray, Bot. Calif. 1 : 410. 1876, Syn. Fl. N. Am. 1? : 393. 1884, and ed. 2, 454. 1886; Greene, Fl. Fran- ciscana, 465. 1897; Greenm. Monogr. Senecio, I. Teil, 23. 1901 and in Engl. Bot. Jahrb. 32:19. 1902; Abrams, Fl. Los Angeles and vicinity 439. 1904. S. californicus var. laxior DC. Prodr. 6 : 426. 1837; Torr. & Gray, Fl. N. Am. 2: 437. 1843. S. coronopus Nutt. Trans. Am. Phil. Soe. 7 : 413. 1841; Torr. & Gray, Fl. N. Am. 2 : 437. 1843. An herbaceous glabrous annual; stem erect simple or branched, 1 to 5 dm. high; leaves oblong-spatulate to lanceo- late, entire to subpinnatifid, 2.5 to 7 em. long, .2 to 2 em. broad, often reddish; the lower leaves often narrowed to a subpetiolate base, the upper sessile and auriculate-clasping at the base; heads radiate, few to several in a loose cyme; bracts of the involucre about 21, often brownish or black- tipped, much exceeded by the yellow conspicuous rays; achenes canescent-pubescent. Distribution: central California, vicinity of Monterey, south to northern Mexico. Specimens examined: California: sand hills, back of Seaside, Monterey Co., 3 April, 1903, Heller 6509 (Gray Herb., Field Mus. Herb., and Mo. Bot. Gard. Herb.) ; Gigling Station, east of Del Monte, in sand, 11 May, 1903, Heller 6710 (Gray Herb., Field Mus. Herb., and Mo. Bot. Gard. Herb.); Bardins, Monterey Co., April, 1903, Elmer 4893 (U. S. Nat. Herb. and Mo. Bot. Gard. Herb.) ; Del Monte, April, 1902, Elmer 3576 (Mo. Bot. Gard. Herb.) ; Arroja Grande, San Luis Obispo Co., 21 Feb., 1886, Summers (Gray Herb.) ; Cuyama, near the boundary between Santa Barbara and San Luis Obispo Counties, 6 May, 1896, Eastwood (Gray Herb.); hillsides, Los Angeles Co., 19 March, 1888, Hasse (U. S. Nat. Herb.); Los Angeles, May, 1915] GREENMAN—MONOGRAPH OF SENECIO 589 1888, Hasse (Field Mus. Herb.) ; copses and grassy slopes, Los Angeles Co., May, 1890, Hasse (U. S. Nat. Herb.); Santa Monica, coll. of 1885, A. Gray (Gray Herb.) ; hillsides, Los Angeles Co., Aug., 1890 and June, 1891, Hasse (Mo. Bot. Gard. Herb.) ; ‘‘Pueblo los Angeles,’’ Gambell (Gray Herb.) ; without definite locality, Coulter 335 (Gray Herb.), and coll. of Nov., 1846, Fremont (Gray Herb.) ; Los Angeles, 5 April, 1890, Fritchey (Mo. Bot. Gard. Herb.); San Bernardino, S. B. € W. F. Parish 198 (Field Mus. Herb. and Mo. Bot. Gard. Herb.); San Bernardino, coll. of 1880, Vasey 330 (Field Mus. Herb., U. S. Nat. Herb., and Gray Herb.) ; near San Bernardino, May, 1893, Parish (Mo. Bot. Gard. Herb.) ; mesas, San Bernardino Co., May, 1888, Parish (Mo. Bot. Gard. Herb.) and April, 1896, Parish (Field Mus. Herb.) ; San Bernardino Co., coll. of 1876, Parry & Lemmon 206 (Field Mus. Herb. and Mo. Bot. Gard. Herb.) ; Arrow Head Springs, 15 May, 1891, Fritchey 18 (Mo. Bot. Gard. Herb.) ; San Ber- nardino, Parish 7 (Gray Herb.); ‘‘Cocomurgo,’’ in sandy places, March, 1854, Bigelow (Gray Herb.); San Ber- nardino Co., Feb.-April, 1882, Parish 233 (Gray Herb.) ; without definite locality, coll. of 1833, Douglas 46 (Gray Herb.), co-TYpE of var. laxior; vicinity of Riverside, alt. 600 m., March, 1903, Hall 3721 (Gray Herb. and Field Mus. Herb.) ; San Diego, April, 1873, Bolander & Kellock (Gray Herb.) ; San Luis Rey, Parry (Gray Herb.) ; vicinity of Riv- erside, 26 March, 1907, Reed 1252 (Field Mus. Herb.) ; vicinity of San Bernardino, 13 April, 1903, Parish 5188 (Field Mus. Herb.) ; without definite locality, Nuttall (Gray Herb.) ; San Diego, April, 1905, Brandegee (U. S. Nat. Herb.) and April, 1902, Brandegee 1647 (Gray Herb., Field Mus. Herb., and Mo. Bot. Gard. Herb.) ; San Diego, April, 1882, Jones (U. S. Nat. Herb.) ; hills, San Diego, 25 April, 1882, Pringle (U. S. Nat. Herb. and Field Mus. Herb.); San Diego, 4 May, 1882, Orcutt 328 (Mo. Bot. Gard. Herb.) ; Potrero, 6 April, 1889, Orcutt (U. S. Nat. Herb. and Mo. Bot. Gard. Herb.) ; Moro hills, near Fallbrook, 28 April, 1903, Abrams 3332 (U. S. Nat. Herb. and Mo. Bot. Gard. Herb.) ; Fallbrook, 27 March, 1882, Jones 3118 (U. S. Nat. Herb.) ; San Diego, Cleveland (Field [VoL, 2 590 ANNALS OF THE MISSOURI BOTANICAL GARDEN Mus. Herb. and Mo. Bot. Gard. Herb.); San Diego, coll. of June, 1906, K. Brandegee (U.S. Nat. Herb.), and coll. of 1875, Palmer 200 (Field Mus. Herb. and Mo. Bot. Gard. Herb.) ; side hill, Del Mar, Oct., 1894 and 22 March, 1895, Angier 14 and 97 (Mo. Bot. Gard. Herb.); Mesa, April, 1895, Angier (Field Mus. Herb.) ; La Jolla, San Diego Co., 17 Feb., 1899, Snyder (Field Mus. Herb.) ; Las Paderes Ranch, San Diego Co., 26 Feb., 1888, Deane (Field Mus. Herb.). Lower California: Todos Santos Bay, July, 1883, Orcutt 708 (Gray Herb.) ; All Saints Bay, May, 1882, Fish (Gray Herb.) ; Punta Bauda, 25 Jan., 1883, Orcutt 708 (Mo. Bot. Gard. Herb.) ; Nachoguero Valley, Schoenfeldt 3401 (U. S. Nat. Herb.). Var. ammophilus (Greene) Greenm. comb. n Senecio ammophilus Greene, Bull. Cal. rey 1: 193. 1886. Leaves thickish, somewhat succulent, 2 to 4 em. long, .2 to 1.5 em. broad, the lower oblanceolate subentire, those of the stem auriculate-clasping, pinnately lobed into oblong or linear obtuse lobes. Lower California: Cape San Quentin, 10 May, 1885, Greene (Gray Herb.), co-TYPE. The thick leaves of this variety give the plant a somewhat different appearance from typical forms of the species; but an examination of a large suite of specimens shows numerous transitional forms such as those secured by Fritchey, Pringle, Bigelow, Palmer 200, Orcutt 708, and K. Brandegee. 7. S. ampullaceus Hook. Bot. Mag. pl. 3487. 1836; DC. Prodr. 6: 428. 1836; Torr. & Gray, Fl. N. Am. 2: 440. 1843; Engelm. & Gray, Boston Jour. Nat. Hist. 5: 250. 1845 (Pl. Lindh. 1: 42. 1845) ; Gray Syn. Fl. N. Am. 1? : 393. 1884, and ed. 2, 1886; Coulter, Contr. U. S. Nat. Herb. 2: 241. 1892; Greenm. Monogr. Senecio, I. Teil, 23. 1901, and in Engl. Bot. Jahrb. 32 : 19. 1902. S. ampullaceus var. glaberrimus Engelm. & Gray, Boston Jour. Nat. Hist. 5 : 250. 1845 (Pl. Lindh. 1: 42. 1845). S. ampullaceus var. floccosus Engelm. & Gray, Boston Jour. Nat. Hist. 5 : 250. 1845 (Pl. Lindh. 1: 42. 1845). 1915] GREENMAN—MONOGRAPH OF SENECIO 591 Annual, or occasionally becoming biennial, more or less floccose-tomentose throughout, somewhat glabrate; leaves ob- long-obovate, acute to lanceolate and acuminate, 5 to 18 cm. long, 1 to 7 em. broad, entire to coarsely and irregularly den- tate; the lower leaves narrowed below into a winged petiole, those of the stem sessile, semiamplexicaul, gradually smaller towards the few to many headed cymose inflorescence; heads 10 to 12 mm. high, radiate, including the rays 1.5 to 3 cm. in diameter; involucre setaceous-calyculate; bracts of the in- volucre glabrous; achenes pubescent. Distribution: eastern Texas. Specimens examined: Texas: San Felipe, Austin Co., Drummond (Kew Herb. and Gray Herb.), TYPE; Corsicana, Reverchon (Mo. Bot. Gard. Herb.) ; near Richland Station, 13 March, 1880, Joor (Mo. Bot. Gard. Herb.) ; Dawson, 16 April, 1903, Reverchon 3965 and 5965 (Mo. Bot. Gard. Herb.) ; Llano, May, 1885, Reverchon 1545 (U. S. Nat. Herb., Field Mus. Herb., and Mo. Bot. Gard. Herb.); banks of Pecan Bayou, April, 1882, Reverchon 81 (Gray Herb. and Mo. Bot. Gard. Herb.) ; sandy soils, Lampa- sas Co., May, 1884, Reverchon 1321 (Mo. Bot. Gard. Herb.) ; Crabapple, Gillespie Co., Jermy (Mo. Bot. Gard. Herb.) ; Hockley, Harris Co., coll. of 1890, Thurrow (Field Mus. Herb.) ; banks of Colorado River, 4 April, 1914, Young (Mo. Bot. Gard. Herb. and Univ. of Texas Herb.) ; on dry ground, Hempstead, 24 April, 1872, Hall 369 (Gray Herb., U. S. Nat. Herb., and Mo. Bot. Gard Herb.) ; without locality, coll. of 1848, Wright (Gray Herb.); Industry, Austin Co., coll. of 1890, Wurzlow (Field Mus. Herb.); banks of railroad near Rosenberg, Fort Bend Co., 13 April, 1900, Eggert (Mo. Bot. Gard. Herb.) ; common on prairies, Columbia, 10 April, 1899, Bush 95 (Gray Herb. and Mo. Bot. Gard. Herb.) ; Columbia, 23 April, 1900, Bush 122 (Mo. Bot. Gard. Herb.) ; Columbia, 25 March, 1900, Canby, Sargent & Trelease 153 (U. S. Nat. Herb.) ; on moist prairie between the Brazos and the Colorado Rivers, April, 1844, Lindheimer 268, 269 (Mo. Bot. Gard. Herb.), co-Typzs of var. glaberrimus and floccosus. [VoL. 2 592 ANNALS OF THE MISSOURI BOTANICAL GARDEN Sror. 2. EREMOPHILI Greenm. § 2. Eremorpnını Greenm. Monogr. Senecio, I. Teil, 21, 23. 1901, and in Engl. Bot. Jahrb. 32:17, 19. 1902. Annual or biennial herbs, not infrequently becoming peren- nial by the development of a ligneous base; stems leafy; leaves laciniately pinnatifid; inflorescence a terminal corymbose or paniculate cyme; heads radiate, rays conspicuous; achenes glabrous or pubescent. Sp. 8-13 KEY TO THE SPECIES A. Pag Lee — = or slightly ee on to high; involucral bra 5 to fone, ea conspicuously black: Et a. ee Te 3 to 5 mm. in diameter, 20-35- NOWATGR ee ee 8. 8. MacDougalii B. — 5 to 6 mm. in diameter, 35-50- POO care ches EEE TREUE . SS. ambrosioides b. Heads 10 to 12 mm. high; involucral pi 7 to . long, not conspicuously black-ti a. Apae species (Canada and the U. Ber eee 10. 8. eremophilus B. Southern species (Mexico)..,...........-+05- 11. 8. Townsendii B. Plants zn or less tomentose; achenes canescent- pubescent. a. ee at first tomentulose, later glabrate........ 12. 8. chihuahuensis b. Leaves permanently tomentulose..............000 3. 8. durangensis 8. S. MacDougalii Heller, Bull. Torr. Bot. Club 26 : 592. 1899; Greenm. Monogr. Senecio, I. Teil, 23. 1901, and in Engl. Bot. Jahrb. 32 : 19. 1902, in part; Rydb. in Fl. Colo. 397. 1906, in part; Wooton & Standley, Contr. U. S. Nat. Herb. 19: 745. 1915. S. eremophilus Gray, Syn. Fl. N. Am. 17: 392. 1884, and ed. 2, 1886, in part, not Richards. S. eremophilus var. attenuatus Greenm. Monogr. Senecio, I. Teil, 23. 1901, and in Engl. Bot. Jahrb. 32 : 19. 1902. Glabrous throughout or slightly puberulent above; stem simple or branched, 5 to 8 dm. high, leafy to the inflorescence ; leaves more or less laciniately pinnatifid, 3 to 10 em. long, 1.5 to 5 em. broad, segments linear to lanceolate, entire to coarsely and unequally dentate; inflorescence terminating the stem and branches in corymbose cymes; heads 7 to 10 mm. high, radi- ate; involucre narrowly campanulate, calyculate, 3 to 5 mm. in diameter; bracts of the involucre usually 13 (8-13), linear- 1915] GREENMAN— MONOGRAPH OF SENECIO 593 lanceolate, 4 to 5 mm. long, commonly black-tipped; ray-flow- ers 0 to 8, light yellow; disk-flowers 14 to 30; achenes glab- rous or slightly puberulent. Distribution: New Mexico and Arizona. Specimens examined: New Mexico: Santa Fe Cafion, Aug., 1880, Snow (Mo. Bot. Gard. Herb.); Santa Fe Creek, 9 Sept., 1881, Engelmann (Mo. Bot. Gard. Herb.) ; Santa Fe, 14 Aug., 1895, Mulford 1292 (Mo. Bot. Gard. Herb.) ; near Pecos, alt. 2040 m., 25 Aug., 1908, Standley 5311 (Mo. Bot. Gard. Herb.) ; Pecos River National Forest, alt. 2560 m., 10 Aug., 1908, Standley 4873 (U. S. Nat. Herb.) ; White Mountains, alt. 2130 m., 6 Aug., 1897, Wooton 290 (Gray Herb. and Mo. Bot. Gard. Herb.) ; White Moun- tains, alt. 2255 m., 25 Aug., 1907, Wooton & Standley 3672 (U. S. Nat. Herb.) ; head of Bear Creek, coll. of 1903, Plummer (U. S. Nat. Herb.) ; Gilmore’s Ranch, White Mountains, alt. 2280 m., 23 Sept., 1906, Standley (Mo. Bot. Gard. Herb.) ; G. O. S. Ranch, Grant Co., 27 Aug.-12 Sept., 1911, Holzinger (U.S. Nat. Herb.). Arizona: Walnut Cañon, alt. 2130 m., MacDougal 342 (Gray Herb. and Field Mus. Herb.), co-rypr; near Flagstaff, May-Oct., 1900, Purpus (U. S. Nat. Herb. and Mo. Bot. Gard. Herb.) ; Mt. Agassiz, alt. 3050 m., 10 Sept., 1909, Pearson 315 (U. S. Nat. Herb.) ; Humphrey Peak, July, 1883, Rusby 337 (Gray Herb. and Field Mus. Herb.); Barfoot Park, Chiri- cahua Mountains, 24 Oct., 1906, Blumer 1484 (U. S. Nat. Herb. and Field Mus. Herb.); Huachuca Mountains, Sept., 1882, Lemmon 2785 (Gray Herb., U. S. Nat. Herb., and Field Mus. Herb.) ; Huachuca Mountains, 17 Oct., 1903, Mearns 2581 (U. S. Nat. Herb.). 9. S. ambrosioides Rydb. Bull. Torr. Bot. Club 37: 467. 1910; Wooton & Standley, Contr. U. S. Nat. Herb. 19 : 745. 1915. S. eremophilus Gray, Pl. Fendl. 108. 1849, as to plant of Fendler; Pac. Rail. Rept. 4: 111. 1856, as to plant of Bigelow; Syn. Fl. N. Am. 1°: 392. 1884, and ed. 2. 1886, in part, not [VoL. 2 594 ANNALS OF THE MISSOURI BOTANICAL GARDEN Richards.; Nelson, in Coulter & Nelson, Manual Cent. Rocky Mountains, 583. 1909, in part, not Richards. S. MacDougalii Greenm. Monogr. Senecio, I. Teil, 23. 1901, and in Engl. Bot. Jahrb. 32:19. 1902, in part, not Heller; Rydb. Fl. Colo. 397. 1906, in part, not Heller. Herbaceous perennial, glabrous or essentially so through- out; stems one to several from a ligneous base, 3 to 5 dm. high; leaves oblanceolate to ovate-lanceolate in general out- line, 3 to 13 em. long, 1 to 5 em. wide, more or less laciniately pinnatifid into linear to lanceolate, entire to coarsely and un- equally dentate divisions; inflorescence a terminal corymbose eyme; heads usually numerous, 7 to 10 mm. high, radiate; in- volucre subeampanulate, 5 to 7 mm. in diameter, calyculate ; bracts of the involucre usually 13, linear-lanceolate, 5 to 7 mm. long, commonly black-tipped; ray-flowers 5 to 8; disk-flowers 30 to 45; achenes hirtellous-puberulent. Distribution: Wyoming to New Mexico, Idaho, and Arizona. Specimens examined : Wyoming: gravelly banks, Centennial Mountain, Albany Co., 2 Aug., 1902, Nelson 8773 (Gray Herb., Field Mus. Herb., and Mo. Bot. Gard. Herb.); moist ground in open woods, Centennial, 27 July, 1900, Nelson 7717 (Gray Herb. and Mo. Bot. Gard. Herb.) ; Bridger Peak, Carbon Co., 22 Aug., 1903, Goodding 1942 (U. S. Nat. Herb. and Mo. Bot. Gard. Herb.). Colorado: Chamber’s Lake, alt. 2895 m., 13 Sept., 1896, Baker (Mo. Bot. Gard. Herb.) ; cañon west of Palmer Lake, alt. 2435 m., 12 Aug., 1896, Crandall (Mo. Bot. Gard. Herb.) ; Steamboat Springs, 20 July, 1903, Goodding 1617 (U.S. Nat. Herb. and Mo. Bot. Gard. Herb.) ; Boulder, coll. of 1908, Pace (Mo. Bot. Gard. Herb.) ; Denver, 8 Sept., 1905, Moffat (Mo. Bot. Gard. Herb.); Georgetown, 19 Aug., 1895, Shear 4720 (U. S. Nat. Herb.), coll. of 19 July, 1886, Trelease, and coll. of 26 July, 1886, Letterman (Mo. Bot. Gard. Herb.) ; Rocky Mountains, Powell’s Colorado Exploring Expedition 1868, Vasey 337 (Gray Herb.) ; Golden City, 18 Aug., 1870, Greene 230 (Gray Herb.); Silver Plume, 21 Aug., 1895, Shear 4999 (U. S. Nat. Herb.) ; Manitou, Aug., 1881, Fritchey 14, in part, and coll. of 16 Aug., 1884, Letterman (Mo. Bot. Gard. Herb.) ; 1915] GREENMAN—MONOGRAPH OF SENECIO 595 Ruxton Park, alt. 2700 m., 21 Aug., 1901, F. E.G E. S. Clements 152 (Gray Herb., U. S. Nat. Herb., and Mo. Bot. Gard. Herb.) ; Breckenridge, coll. of 1887, Bereman (Mo. Bot. Gard. Herb.) ; Breckenridge, coll. of 1892, Wislizenus 1063 (Mo. Bot. Gard. Herb.) ; Oro City, 23 July, 1873, Hayden’s U. S. Geol. Survey, Coulter, in part (U. S. Nat. Herb.); Green Mountain Falls, alt. 2560 m., 2 Aug., 1892, Sheldon 485 (U. S. Nat. Herb.) ; Hotchkiss, alt. 1585 m., 30 June, 1892, Cowen 287 (U. S. Nat. Herb.) ; Oak Creek, Fremont Co., Aug., 1873, Brandegee 716 (Mo. Bot. Gard. Herb.) ; Gunnison, 25 July, 1901, alt. 2300 m., Baker 596 (Gray Herb. and Mo. Bot. Gard. Herb.) ; vicinity of Mount Carbon, Gunnison Co., alt. 2730-2800 m., 4 July and 10 Aug., 1910, Eggleston 5835 and 6159 (U. S. Nat. Herb.) ; Pandora, 10 Aug., 1901, Baker 748 (Gray Herb. and Mo. Bot. Gard. Herb.); Taylor River, 15 Aug., 1873, Hayden’s U. S. Geol. Survey, Coulter (U. S. Nat. Herb.) ; Telluride, alt. 2740- 3600 m., Aug., 1894, Tweedy 354 (U. S. Nat. Herb.) ; Ute Pass, 2 July, 1896, Shear 3695 (U. S. Nat. Herb.); near Pagosa Peak, alt. 3050 m., 8 Aug., 1899, Baker 706 (Gray Herb., U. S. Nat. Herb., Field Mus. Herb., and Mo. Bot. Gard. Herb.) ; Saguache Creek, Sept., 1873, Wheeler Expedition, Wolf 1086 (U. S. Nat. Herb.) ; Parrott City, alt. 2740 m., Baker, Earle & Tracy 475 (Gray Herb., Field Mus. Herb., and Mo. Bot. Gard. Herb.) ; northeast corner of North Park, 3 Aug., 1874, Barber (U. S. Nat. Herb.); Twin Lakes, Wheeler Expedition, 1873, Wolf & Rothrock 562 (Gray Herb., and Field Mus. Herb.) ; Rocky Mountains, coll. of 1862, Hall & Harbour 827 (Gray Herb. and Field Mus. Herb.), also coll. of 1861-62, Parry 26 (Gray Herb. and Mo. Bot. Gard. Herb.) ; mouth of Bear Creek Canon, 23 Aug., 1915, Drushel € Dougan (Drushel Herb.) ; upper Clear Creek Valley, alt. 3050 m., 10 Aug., 1874, Engel- mann (Mo. Bot. Gard. Herb.) ; Leadville, 8 July, 1886, Tre- lease (Mo. Bot. Gard. Herb.) ; Tolland, alt. 2895 m., 29 July, 1913, Overholts (Mo. Bot. Gard. Herb.) ; near Breckenridge, alt. 2950 m., Aug., 1901, Mackenzie 208 (Mo. Bot. Gard. Herb.) ; Penn’s Gulch, near Sunset, 30 July, 1886, Letterman (Mo. Bot. Gard. Herb.). New Mexico: pine forest, Jicarilla Apache Reservation, [VoL. 2 . 596 ANNALS OF THE MISSOURI BOTANICAL GARDEN near Dulce, alt. 2150-2470 m., 20 Aug., 1911, Standley 8183 (U. S. Nat. Herb.) ; Chama, 8 Sept., 1899, Baker (U. S. Nat. Herb. and Mo. Bot. Gard. Herb.); Santa Fe Canon, 3 Oct., 2380-2850 m., 8 July, 1911, Standley 6564 (U. S. Nat. Herb.) ; Navajo Indian Reservation in the Tunitcha Mountains, 8 Aug., 1911, Standley 7591 (U. S. Nat. Herb.) ; mountains near Las Vegas, July, 1881, Vasey (U. S. Nat. Herb.) ; Santa Fe Canon, 7 July, 1897, alt. 2440 m., A. A. € E. G. Heller 3819 (Gray Herb. and Mo. Bot. Gard. Herb.); Sante Fe Canon, 3 Oct., 1913, Rose, Fitch & Parkhurst 17714 (U. S. Nat. Herb.) ; Canoncinto, Santa Fe Co., coll. of 1879, Brandegee 12078 (Mo. Bot. Gard. Herb.) ; creek bottom, Santa Fe, 20 Oct., 1846, Fendler 475 (Gray Herb. and Mo. Bot. Gard. Herb.) ; Balsam Park, Sandia Mountains, alt. 2500 m., Aug.-Sept., 1914, Ellis 281 (Mo. Bot. Gard. Herb.) ; Pecos River Indian Reservation, 6 Aug., 1898, Coghill 144 (Mo. Bot. Gard. Herb.) ; Mineral Creek, Sierra Co., alt. 2130 m., 26 Sept., 1904, Metcalfe 1415 (U. S. Nat. Herb.); Santa Antonita, Whipple’s Exploration 1853-54, Bigelow (U. S. Nat. Herb. and Gray Herb.) ; Organ Mountains, alt. 2130 m., 23 Sept., 1906, Wooton & Standley (U. S. Nat. Herb.). Utah: Big Cottonwood Canon, Salt Lake Co., alt. 2774 m., 10 Aug., 1905, Garrett 1591 (U. S. Nat. Herb.); Tate Mine, Marysvale, alt. 2740 m., 22 Aug., 1894, Jones 5858 (Mo. Bot. Gard. Herb.) ; Bromide Pass, Henry Mountains, alt. 3050 m., 27 July, 1894, Jones 56957 (U. S. Nat. Herb.); slope of Aquarius Plateau, alt. 2750 m., 2 Aug., 1875, Ward 499 (U.S. Nat. Herb.). Arizona: Navajo Indian Reservation, about the north end of the Carrizo Mountains, 29 July, 1911, Standley 7876 (U.S. Nat. Herb.). Among the specimens here cited, a few, particularly Parry’s 26, Overholts’, Mackenzie’s 208, and Engelmann’s plant from Upper Clear Creek Valley, might be almost equally well re- ferred to the preceding species, S. MacDougalu, to which S. ambrosioides is very closely related; but in general the latter may be distinguished by the slightly larger and more numer- 1915] GREENMAN—MONOGRAPH OF SENECIO 597 ously flowered heads and usually, but not always, less pin- natisect leaves. 10, 8. eremophilus Richards. in App. Frankl. 1st Journ. 31. 1823; Hook. Fl. Bor. Am. 1: 334. 1840; Torr. & Gray, Fl. N. Am. 2: 444, 1843; Eaton, Bot. King Exp. 191. 1871, in part; Gray, Syn. Fl. N. Am. 17: 392. 1884, and ed. 2, 1886; Greenm. Monogr. Senecio, I. Teil, 23. 1901, and in Engl. Bot. Jahrb. 32 : 19. 1902; Nelson in Coulter & Nelson, Manual Cent. Rocky Mountains 583. 1909, in part. S. pembrinensis Greenm. Monogr. Senecio, I. Teil, 23. 1901, and in Engl. Bot. Jahrb. 32 : 19. 1902. An herbaceous perennial, glabrous or slightly puberulent in the inflorescence; stems erect, 3 to 8 dm. high, striate; leaves more or less laciniately pinnatifid with linear, lanceolate or oblong, entire or coarsely and unequally dentate divisions; the lower leaves petiolate, the upper subsessile; inflorescence terminating the stem in a somewhat leafy corymbose or pan- iculate cyme; heads rather large, 10 to 12 mm. high, radiate; involucre campanulate conspicuously calyculate; bracts of the involucre usually 13, linear-lanceolate, acute, 7 to 9 mm. long, glabrous, minutely brownish- or black-tipped; ray-flowers 8 to 10; disk-flowers 40 to 60; achenes ribbed, glabrous, or slightly hirtellous-puberulent. Distribution: northwestern Canada to Nebraska, Colorado, and Utah. Specimens examined: Saskatchewan: Lipton, 11 Aug., 1911, Clokey 1844 (Mo. Bot. Gard. Herb.) ; Qu’Appelle River, Assiniboia, Aug., 1883, Macoun 14839 (Geol. Surv. Canada Herb. and U. S. Nat. Herb.); near Prince Albert, 10 July, 1896, Macoun 12171 (Geol. Surv. Canada Herb.) ; in damp thickets north of Sas- katchewan River, 22 Aug., 1872, Macoun 14841 (Geol. Surv. Canada Herb.); Saskatchewan Plains, Macoun 868 (Kew Herb.). Alberta: ‘‘on gravelly banks of Cedar Lake, Lat. 54°,” Richardson (Kew Herb.), Tyre; Pembina, coll. of 1873, Coues (Gray Herb.) ; on damp banks, Bow River at Morley, 6 Sept., [voL. 2 598 ANNALS OF THE MISSOURI BOTANICAL GARDEN 1879, Macoun 14840 (Geol. Surv. Canada Herb.) ; Dunvegan, Peace River, 17 Aug., 1879, Dawson 26686 (Geol. Surv. Canada Herb.) ; Athabasca Plains, 14 Sept., 1872, Macoun 1040 (Gray Herb. and Kew Herb.). South Dakota: Sylvan Lake, 27 Aug., 1897, Griffiths (Mo. Bot. Gard. Herb.). Nebraska: mountain range, south of White Clay Creek, 23 Aug., 1859, Lieut. F. T. Bryan’s Expedition, 1856, H. Engel- mann (Mo. Bot. Gard. Herb.). Wyoming: on the summits of Big Horn Mountains, Aug., 1859, Reynolds’ Expedition to the headwaters of the Missouri and Yellowstone Rivers, Hayden (Mo. Bot. Gard. Herb.) ; Laramie Mountains, Hayden (Gray Herb. and Mo. Bot. Gard. Herb.) ; Laramie Mountains, 17 Aug., 1899, Schuehut (U. S. Nat. Herb.). Colorado: Cascade Canon, July, 1880, Eurney (Mo. Bot. Gard. Herb.) ; Rocky Mountains, Hall & Harbour 327, in part (U. S. Nat. Herb. and Mo. Bot. Gard. Herb.); Pike’s Peak, alt. 3050 m., 25 Aug., 1915, Drushel € Dougan (Drushel Herb.) ; Manitou, Aug., 1881, Fritchey 14 in part (Mo. Bot. Gard. Herb.), form. var. Kingii (Rydb.) Greenm. comb. nov. Senecio Kingü Rydb. Bull. Torr. Bot. Club 37: 468. 1910. S. eremophilus Eaton, Bot. King Exp. 191. 1871, as to plant of Watson. S. Watson. Greenm. Monogr. ai I. Teil, 23. 1901, and in Engl. Bot. Jahrb. 32 : 19. 1902 Leaves oblanceolate to oblong- lnneaclades coarsely dentate to pinnatisect with relatively broad divisions; but through several specimens connecting directly with the above species. Specimen examined: Utah: Cottonwood Cañon, alt. 2590 m., Aug., 1869, Watson 676 (Columbia Univ. Herb. and Gray Herb.), TYPE. 11. S. Townsendii Greenm.! Herbaceous perennial, glabrous throughout; stem 6 to 10 dm. high, striate, often purplish; leaves coarsely, unequally 1 Senecio Townsendii Greenm. sp. nov., herbaceus perennis ubique glabrus; caule 6-10 dm. alto, striato saepe purpurascenti; foliis inaequaliter et remote 1915] GREENMAN—MONOGRAPH OF SENECIO 599 and remotely dentate to laciniately pinnatifid, oblanceolate to oblong-lanceolate in general outline, 3 to 10 em. long, 1 to 4 em. broad, divisions linear and entire to dentate, acute or obtuse; lower leaves petiolate, the upper sessile; inflorescence a loose several to many-headed corymbose cyme; heads 10 to 13 mm. high, radiate; involucre narrowly campanulate, caly- culate, glabrous; bracts of the involucre commonly 13, linear- lanceolate, 8 to 10 mm. long, terminated by a small black or brownish penicillate tip; flowers pale yellow; ray-flowers 5 to 8, occasionally much reduced; disk-flowers 35 to 50; achenes glabrous. Distribution: northern Mexico. Chihuahua: near Colonia San Garcia in the Sierra Madre, alt. 2285 m., 9 Sept., 1899, Townsend & Barber 317 (Mo. Bot. Gard. Herb., Gray Herb., and U. S. Nat. Herb.), Tyee; Mound Valley, Sierra Madre Mountains, alt. 2130 m., 18 Sept., 1903, Jones (U. S. Nat. Herb.). The Townsend and Barber specimens have been distributed s “Senecio Chihuahuanus Wats.” and the Jones plant was distributed as ‘‘Senecio eremophilus’’ under which names they may be looked for in herbaria. 12. S. chihuahuensis Watson, Proc. Am. Acad. 23: 280. 1888; Greenm. Monogr. Senecio, I. Teil, 23. 1901, and in Engl. Bot. Jahrb. 32 : 19. 1902. An herbacous perennial; stem erect, 4 to 5 dm. high from a rather slender rootstock, striate-angulate, somewhat purplish; early leaves oblanceolate, 3 to 5 em. long, 1 em. broad, lacini- ately dentate, arachnoid-tomentulose on both surfaces; later stem-leaves short-petiolate, or subsessile, oblong-ovate in general outline, about 8 cm. long, one-half to two-thirds as grosse-dentatis vel a -pinnatis, oblanceolatis vel or ger er in circumscriptione, 3-10 em. longis, 1-4 cm. latis; laciniis linearibus et integris vel dentatis acutis vel ae: foliis inferioribus petiolatis, superioribus So oa bus; inflorescentibus laxe corymboso-eymosis multicapitatis ; capitulis s 10-1 altis, radiatis; involucris anguste campanulatis calyculatis glabris; ss involucri 13 lineari-lanceolatis 8-10 mm. longis minute atro- vel fulvo penicillatis; floribus ger aurantiabus; floribus femineis 5-8 nonnunquam multo ed s disci 35-50 achaeniis striatis glabris—Near Colonia San Garcia in the Sierra Made, State of Chihuahua, has alt, 2285 m., < Se = Ae 1899, Townsend & Barber 31 7 (Mo. Bot. Gard. Herb., Gray Herb., and U. Herb. ), TYPE; Mound Aj ek Sierra Madre wo alt. 2135 m., is Sept, 1903, Jones (U. S. Nat. Herb.). [Vou 2 600 ANNALS OF THE MISSOURI BOTANICAL GARDEN broad, subbipinnate, at first tomentulose, later becoming glabrous or essentially so, divisions narrow, unequal, carti- laginous-apiculate; inflorescence a terminal corymbose cyme; heads 10 to 12 mm. high, radiate; involucre cylindric- campanulate, calyculate with short linear subulate bracteoles; bracts of the involucre 7 to 9 mm. long, brownish- or black- tipped, shorter than the numerous flowers of the disk; ray- flowers about 8; achenes canescent-pubescent. Distribution: northern Mexico. Specimens examined: Chihuahua: ledges of the Sierra Madre, alt. 2955 m., 7 Oct., 1887, Pringle 1318 (Gray Herb., Kew Herb., and Mo. Bot. Gard. Herb.), TYPE. 13. S. durangensis Greenm. Field Col. Mus. Bot. Ser. 2: 275. 1907. Plate 18. S. ctenophyllus Greenm. Proc. Am. Acad. 43 : 20. 1907, not Phil. An herbaceous annual, or becoming perennial by the de- velopment of a ligneous base; stem simple or branched, erect, 3 to 4 dm. high, arachnoid-tomentose; leaves lanceolate, 2 to 9 em. long, 1 to 2.5 em. wide, more or less pinnately divided, permanently arachnoid-tomentulose on both surfaces, lower leaves petiolate, upper sessile; inflorescence a terminal tomen- tulose corymbose cyme; heads numerous, 8 to 10 mm. high, radiate, calyculate; involucre campanulate, glabrous or nearly so; bracts of the involucre 13, linear-lanceolate, 5 to 6 mm. long, minutely black-tipped, penicillate; ray-flowers 5 to 8, ligules pale yellow; disk-flowers 20 to 30; achenes canous- hirtellous. Distribution: northern Mexico. Specimen examined: Durango: barranca, below Sandia Station, alt. 2135 m., 15 Oct., 1905, Pringle 10105 (Gray Herb., Field Mus. Herb., and Mo. Bot. Gard. Herb.), TYPE. Sect. 3. JACOBAEAE DC. §3. JacoBAEAE DC. Prodr. 6: 348. 1837; Hoffm. in Engl. & Prantl, Nat. Pflanzenf. IV, Abt. 5, 297. 1892; Greenm. 1915] GREENMAN— MONOGRAPH OF SENECIO 601 Monogr. Senecio, I. Teil, 21, 23. 1901, and in Engl. Bot. Jahrb. 32 : 17, 19. 1902. Biennial or perennial herbs with lyrate or 2-3-pinnatisect leaves and radiate heads. Sp. 14-16. KEY TO THE SPECIES A. Stem and leaves glabrous or nearly so; invol- ucral bracts narrow, about 1 mm. broad; men usually black- WOO EN 14. 8. rupestris . Stems and leaves more or less permanently red -tomentulose; involucral bracts 1.5 to 2 mm. broad; bracteoles not black-tipped. a. Upper stem-leaves once pinnate............ 15. 8. erucifolius b. Upper item: leaves 2-3- Serie Haat OE i Se 16. 8. Jacobaea w 14. S. rupestris Waldst. & Kit. Descer. et Ic. Pl. Rar. Hung. 2 : 136. pl. 128. 1805; Reichb. Ic. Crit. 4:28. pl. 334. fig. 514. 1826; Strobl, Fl. Admont. 1:57. 1881, and in Flora 65: 478, 479. 1882; von Hayek, Fl. Stierm. 2 : 564. 1913. S. laciniatus Bert. in Desv. Jour. Bot. 2:76. 1813; Amoen. Ital. 102, 408. 1819. Senecio nebrodensis var. glabratus DC. Prodr. 6 : 350. 1837. Annual or biennial, sometimes becoming perennial, glabrous throughout or slightly pubescent; stem erect, 3 to 6 dm. high, simple or branched, striate; leaves lanceolate to obovate- lanceolate in general outline, 3 to 10 cm. long, 1 to 4 cm. broad, laciniately lobed or subpinnatiscent, thin in texture, the lobes again sharply dentate; the lower leaves narrowed into a sub- petiolate base, the upper sessile and semiamplexicaul; in- florescence a terminal corymbose cyme; heads 8 to 10 mm. high, radiate; involucre calyculate with black-tipped brac- teoles; bracts of the involucre about 21, linear-lanceolate, acute, 6 to 7 mm. long; ray-flowers about 13; disk-flowers numerous; achenes glabrous or slightly hirtellous. Distribution: on ballast near Philadelphia. Introduced from Europe. Specimen examined : Pennsylvania: on ballast, Philadelphia, July, 1880, Martin- dale (Gray Herb.). 15. S. erucifolius L. Fl. Suecica, ed. 2, 291. 1755; Huds. Fl. Ang. 366. 1798; DC. Prodr. 6: 351. 1837; Reichb. Ic. Fl. [Vou 2 602 ANNALS OF THE MISSOURI BOTANICAL GARDEN Germ. & Helv. 16: 38. pl. 75 (CMLXVI). fig. 1. 1854; Cosson & Saint-Pierre, Fl. Paris, ed. 10, 518. 1861. Beck von Man- nagetta, Fl. Nieder-Oesterr. 1221. 1893. An herbaceous biennial or perennial, more or less floccose- tomentulose throughout and on the stem and lower leaf- surface often intermixed with hirsute hairs; stems erect, 3 to 10 dm. high, simple or branched; leaves lyrately pinnatifid to pinnatisect, 2 to 10 em. long, 1 to 6 em. broad, the lobes subentire, blunt, and submucronate to sharply dentate; lower- most leaves narrowed into a subpetiolate base, the upper ses- sile and semiamplexicaul; inflorescence a terminal few- to many-headed corymbose cyme; heads about 1 em. high, radiate; involucre campanulate, calyculate; bracts of the in- volucre usually 13, lanceolate-oblong, 4 to 5 mm. long, glabrous or slightly floccose-tomentulose, with rather broad scarious margins; ray-flowers about 13; disk-flowers numer- ous, 50 to 60; achenes hirtellous. Distribution: on ballast near Philadelphia. Introduced from Europe. Specimens examined: Pennsylvania: on ballast, Philadelphia, 30 Aug., 1879, Parker (Gray Herb.). New Jersey: on ballast, Kaighn’s Point, Burk (Field Mus. Herb.). 16. S. Jacobaea L. Sp. Pl. 2: 870. 1753; Willd. Sp. Pl. 3: 1997. 1800; DC. Prodr. 6 : 350. 1837; Sm. & Sow. Eng. Bot. 16: pl. 1130. 1803; Schkuhr, Handb. pl. 267. 1808; Reichb. Ic. Fl. Germ. & Helv. 16: 38. pl. 73 (CMLXIV). figs. II. 3, 4. 1854; Gray, Syn. Fl. N. Am. 1°: 383. 1884, and ed. 2, 1886; Britton, Manual, ed. 2, 1029. 1905; Gray, Manual, ed. 7, 853. 1907; Brit- ton & Brown, Ill. Fl., ed. 2, 3 : 542. 1913. Jacobaea vulgaris Vahl in Fl. Dan. 6: pl. 944. 1787; Gaertn. Fruct. 2 : 445. pl. 170. fig. 1.1791. An erect, biennial or peren- nial herb, 3 dm. or more high, at first usually arachnoid- tomentulose, more or less glabrate; basal leaves petiolate, somewhat lyrate; stem leaves sessile, semiamplexicaul, ovate- oblong in general outline, 3 to 15 cm. long, 1.5 to 7 cm. 1915] GREENMAN—MONOGRAPH OF SENECIO 603 broad, 2-3-pinnatisect; inflorescence a terminal corymbose cyme; heads numerous, radiate; achenes pubescent. Distribution: Newfoundland to New Jersey, occurring along roadsides, in pastures, and on ballast. Introduced from Europe. Speeimens examined: Newfoundland: roadsides, St. John’s, 7-19 Aug., Robin- son & Schrenk (Gray Herb., U. S. Nat. Herb., Geol. Surv. Canada Herb., and Mo. Bot. Gard. Herb.). Nova Scotia: L’Ardoire, Cape Breton Island, Aug., 1892, Faxon (Gray Herb.); Sydney and Mira Bay, Cape Breton Island, 17 Aug., 1898, Macoun 19723 (Geol. Surv. Canada Herb.) ; eastern Nova Scotia, 16 Aug., 1890, Chickering (U. S. Nat. Herb.) ; Boylston, Hamilton 22844 (Geol. Surv. Canada Herb.); Pictou, 1 Nov., 1874, Fowler (Field Mus. Herb.) ; Pictou Landing, 24 July, 1883, Macoun 14859 (Geol. Surv. Canada Herb.); pasture, Windsor Junction, 11 July, 1901, Howe & Lang 427 (Gray Herb.) ; pasture, near Pictou, 12-18 July, 1901, Howe & Lang 540 (Gray Herb.). Prince Edward Island: Tignish, 26 July, 1888, Macoun, 14858 (Geol. Surv. Canada Herb.); Tracadie Beach, 27 July, 1901, Churchill (Gray Herb. and Mo. Bot. Gard. Herb.). New Brunswick: Miramichi, Fowler (Gray Herb. and U. S. Nat. Herb.) ; near railroad station, Anagance, 19 July, 1901, Churchill (Gray Herb. and Mo. Bot. Gard. Herb.). Quebec: on ballast-filling about fish houses, York, Gaspé Co., 25 Aug., 1904, Collins, Fernald € Pease (Gray Herb.). Ontario: Burlington, 23 Aug., 1883, Burgess 14857 (Geol. Surv. Canada Herb.). Pennsylvania: on ballast, July, 1876, Martindale (Mo. Bot. Gard. Herb.). New Jersey: on ballast, Camden, coll. of 1878, Martindale (Gray Herb., U. S. Nat. Herb., Field Mus. Herb., and Mo. Bot. Gard. Herb.); on ballast, Kaighn’s Point, Burk (Field Mus. Herb.). Sect. 4. SANGUISORBOIDEI Greenm. §4. SANGUISORBOIDEI Greenm. Monogr. Senecio, I. Teil, 22, [vor. 2 604 ANNALS OF THE MISSOURI BOTANICAL GARDEN 23, 1901, and in Engl. Bot. Jahrb. 32:18, 19. 1902. Lobati Rydb. Bull. Torr. Bot. Club 27 : 169. 1900, in part. Annuals, biennials, or perennials, often forming merely a rosette of leaves during the first year; stems erect, 1.5 to 10 dm. high from a distinetly annual root or from a rather stout rootstock; leaves once, twice, or thrice pinnately divided; heads numerous; achenes glabrous or pubescent. Sp. 17-29. KEY To THE SPECIES A. Annuals or biennials. a. Bracts of the involucre usually 13, linear- lanceolate, 1 to 2.5 mm. broad .......... 17. 8, sanguisorboides b. Bracts of the in nvolucre beige in = linear or linear-lanceolate, 0.5 t broad. a. Lateral leaf- esi: oa not “abruptly con- tracted into a narrow bas I. Plants of southeastern United States..18. 8. glabellus II. Plants of a Texas northern pr nen 19. 8. Greggiü ET kaka paw sine aes . 8. imparipinnatus B. Perennials; ari ate ee gine a horizontal, or suberect rootstock. a. gie es 2—3-pinnatisect; segments narrow.....21. 8. Millefolium b. pps = ar! “segments narrowly obo- rate subreni a. "Heath bee at 5 to 10 mm. high. I. Involucral bracts usually 21. l. Dara glabrous; achenes hirtellous.22. 8, tampicanus 2. Leaves pubescent beneath; achenes WIADTOUS is ote ee en 23. K. hypotrichus LI: Involueral Bons usually 1 1 ral leaf-divisions oe than ans ad. * Midrib glabrous ............ 24. 8. Sanguisorbae ** Midrib floccose-tomentulose....25. 8. pinnatisectus 2. Lateral leaf-divisions as broad as ONG ya T ns T, 6. 8. coahuilensis 8. Heads fewer and larger, 10 to 14 mm. high. I. Leaves er) divided nearly to the i. Leaf- drtato few, cuneate to reni- for a leonensis 2. Leaf-divisions many, cuneate to x a montereyana Il. si er divided slightly more half-way margin to en ee 8. zimapanicus 17. S. sanguisorboides Rydb. Bull. Torr. Bot. Club 27: 170. 1900; Wooton & Standley, Contr. U. S. Nat. Herb. 19: 745. 1915. 1915] GREENMAN—MONOGRAPH OF SENECIO 605 Annual or biennial, glabrous or slightly white tomentulose in the axils of the leaves; stem 1.5 to 5 dm. high, striate; leaves usually pinnately divided into cuneate to reniform dentate or crenate-dentate divisions, the terminal division ovate-reni- form, 1 to 5 cm. broad; basal and lower stem-leaves petio- late and occasionally undivided; upper stem-leaves sessile and amplexicaul; inflorescence a terminal few to several-headed corymbose cyme; heads radiate; involucre campanulate, barely calyculate; bracts of the involucre usually 13 (rarely 16), lanceolate, 6 to 6.5 mm. long, glabrous; ray-flowers 8 to 10; disk-flowers 30 to 50; achenes ribbed, glabrous. Distribution: mountains of New Mexico. Specimens examined: New Mexico: Willow Gulch, Colfax Co., alt. 3050 m., Aug., 1896, St. John 115 (Gray Herb.); Santa Fe Canon, 7 July, 1897, alt. 2440 m., A. A. £ E. G. Heller 3820 (Mo. Bot. Gard. Herb.), co-rype; Santa Fe Creek, 22 June, 1847, Fendler 438 (Mo. Bot. Gard. Herb.) ; White Mountains, Lincoln Co., alt. 3048 m., 16 Aug., 1897, Wooton 494 (Mo. Bot. Gard. Herb.) ; mouth of Pouchuelo Creek, Pecos River National Forest, alt. 2590 m., 30 June, 1908, Standley 4093 (Mo. Bot. Gard. Herb.) ; mouth of Mora River, Pecos River National Forest, alt. 2470 m., 7 July, 1908, Standley 4250 (Mo. Bot. Gard. Herb.) ; Pecos River Indian Reservation, 17 July, 1898, Coghill 71 (Mo. Bot. Gard. Herb.). 18. S. glabellus Poir, Dict. 7: 102. 1806; Greenm. Monogr. Senecio, I. Teil, 23. 1901, and in Engl. Bot. Jahrb. 32:19. 1902; Gray, Manual, 853, ed. 7, 1907; Britton & Brown, Ill. Fl. 3:540, ed. 2, 1913. S. lyratus Michx. Fl. Bor. Am. 2 : 120. 1803, not L. S. lobatus Pers. Syn. 2 : 436. 1807; Nutt. Gen. 2: 165. 1818; Elliot, Sk. 2: 332. 1824; Torr. & Gray, Fl. N. Am. 2: 4387. 1843; Gray, Syn. Fl. N. Am. 17: 394. 1884, and ed. 2, 1886, mainly; Chapman, Fl. Southern U. S. 266, ed. 3, 1897; Britton & Brown, Ill. Fl. 3: 481, ed. 1, 1898; Small, Fl. Southeastern U. S.. 1303. 1903, and ed. 2, 1913; Mohr, Contr. U. S. Nat. Herb. 6 : 815. 1901. [VoL. 2 606 ANNALS OF THE MISSOURI BOTANICAL GARDEN S. carolinianus Spreng. Syst. 3 : 559. 1826. S. densiflorus Martens, Bull. Acad. Roy. Soc. Brux. 8: 66. 1841. S. Schweinitzianus Nutt. Trans. Am. Phil. Soc. 7: 413. 1841. Annual or biennial, glabrous or slightly tomentulose in the axils of the leaves; stems erect 1 to 10 dm. high, striate; radical leaves petiolate, lyrate, occasionally undivided; those of the stem petiolate or sessile and semiamplexicaul, pin- nately divided into rather remote, narrowly cuneate to sub- reniform unequal divisions; inflorescence a terminal corym- bose cyme; heads 6 to 8 mm. high, radiate; ray-flowers 8 to 12; disk-flowers about 50; achenes usually hirtellous- puberulent. Distribution: North Carolina west to Illinois, Missouri, and South Dakota, south to Florida and eastern Texas. Com- mon on river bottoms and flood-plains. Specimens examined : North Carolina: near Wilmington, April, 1888, McCarthy (U. S. Nat. Herb.) ; without locality, Curtis (Gray Herb.). South Carolina: Goose Creek, 19 May, 1885, A. C. € F. W. Maier (Gray Herb.); swamps, Summerville, April, 1890, Taylor (Field Mus. Herb.). Georgia: Macon, coll. of 1875, Curtiss (U. S. Nat. Herb.) ; central Georgia, coll. of 1846, Porter (Gray Herb.); Butler Island, McIntosh Co., 27 May, 1909, Smith 2185 (Field Mus. Herb.). Florida: without locality, Chapman (Gray Herb., U. S. Nat. Herb., and Kew Herb.); Fort Orange, 10 April, 1895, Straub 108 (Gray Herb.); near Chattahooche, Curtis 1565 (Gray Herb., U. S. Nat. Herb., Kew Herb., and Field Mus. Herb.) ; River Junction, 19 April, 1898, Curtis 6370 (U. S. Nat. Herb. and Mo. Bot. Gard. Herb.) ; Sand Point, 8 April, 1874, Palmer 301 (Gray Herb., U. S. Nat. Herb., and Mo. Bot. Gard. Herb.) ; near St. Marks, coll. of 1843, Rugel (Mo. Bot. Gard. Herb., and Kew Herb.); Losman’s Key, May, 1891, Simpson 154 (U. S. Nat. Herb. and Field Mus. Herb.) ; New 1915] GREENMAN—MONOGRAPH OF SENECIO 607 Smyrna, Burgess 563 (Field Mus. Herb.); Gulf Hammock, April, 1876, Garber (Field Mus. Herb.). Illinois: in a damp meadow near Peoria, coll. of 1903, McDonald (Field Mus. Herb.) ; river bottom opposite Decatur, April, 1864, Stewart (Field Mus. Herb.) ; Eldred, Green Co., 9 May, 1891, Andrews (Mo. Bot. Gard. Herb.); opposite St. Louis, July, 1839, and May, 1845, Engelmann (Mo. Bot. Gard. Herb. and Kew Herb.); Mississippi Valley, St. Clair Co., colls. of 1874, 1875, and 1879, Eggert (Mo. Bot. Gard. Herb.) ; near Falling Spring, 1 June, 1890, Glatfelter (Mo. Bot. Gard. Herb.) ; East St. Louis, 11 June, 1890, Hitchcock (Mo. Bot. Gard. Herb.). Kentucky: Muhlenberg, 5 June, 1901, Price (Mo. Bot. Gard. Herb.) ; without locality, Short (Kew Herb.). Tennessee: in swamps, Rutherford Co., July, 1892, Bain (U. S. Nat. Herb.). Alabama: Tuscaloosa, April, 1892, Ward (U. S. Nat. Herb.); Greensboro, coll. of 1857, Watson (Gray Herb.) ; Auburn, Lee Co., 9 April, 1898, Earle & Baker (Field Mus. Herb.). Mississippi: damp fields, North Carrollton, 21 April, 1899, Clute 24 (Field Mus. Herb.); without locality, coll. of 1843, Holton (Kew Herb.). South Dakota: Fort Pierre, July, 1853, Hayden (Mo. Bot. Gard. Herb.). Missouri: Courtney, 15 May, 1896, Bush 701 (U. S. Nat. Herb. and Mo. Bot. Gard. Herb.) ; vicinity of St. Louis, coll. of about 1840, Duerinck (Mo. Bot. Gard. Herb.) ; Creve Coeur Lake, 8 May, 1859, Glatfelter (Mo. Bot. Gard. Herb.) ; near St. Louis, Hus 4007 (Mo. Bot. Gard. Herb.); St. Louis Co., 24 May, 1896, Shannon 250 (Mo. Bot. Gard. Herb.) ; St. Louis Co., 20 May, 1879, Eggert (Mo. Bot. Gard. Herb.) ; Jefferson Barracks, 6 May, 1890, Hitchcock (Mo. Bot. Gard. Herb.) ; Jefferson Co., 5 May, 1896, Eggert (Mo. Bot. Gard. Herb.) ; Kimmswick, 20 May, 1860, Engelmann (Mo. Bot. Gard. Herb.) ; Kimmswick, 23 May, 1885, Wislizenus (Mo. Bot. Gard. Herb.) ; Sulphur Springs, 14 Aug., 1910, Sherff 1062 (Field Mus. Herb.) ; Osage, 13 May, 1901, Norton (Mo. Bot. Gard. Herb.) ; [VoL. 2 608 ANNALS OF THE MISSOURI BOTANICAL GARDEN Batesville, Butler Co., 21 May, 1908, Smith 534 (Field Mus. Herb.) ; St. Louis, coll. of 1832, Drummond (Kew Herb.) ; St. Louis, Riehl 382 (Kew Herb.). Arkansas: Fulton, 17 April, 1905, Bush 2354 (Mo. Bot. Gard. Herb.) ; Fulton, 24 April, 1914, Palmer 5381 (Mo. Bot. Gard. Herb.) ; Arkansas Post, 20 March, 1909, Kellogg (Mo. Bot. Gard. Herb.) ; Little Rock, 22 April, 1909, McNair (U. S. Nat. Herb.) ; Little Rock, June, 1886, Hasse (Field Mus. Herb.). Louisiana: without locality, Hale (Gray Herb. and Kew Herb.) ; Gretna, 28 April, 1899, Ball 315 (Gray Herb., U. S. Nat. Herb., Field Mus. Herb., and Mo. Bot. Gard. Herb.) ; between New Orleans and Balize, May, 1829, Berlandier 556, 1946 (Gray Herb. and Mo. Bot. Gard. Herb.) ; Balize, April, 1839, Lindheimer (Mo. Bot. Gard. Herb.); Baton Rouge, 22 Jan., 1874, Joor (Mo. Bot. Gard. Herb.) ; Holly Ridge, West Carroll Parish, July, 1910, Mosely (Field Mus. Herb.) ; swampy woods, Natchitoches, 16 April, 1915, Palmer 7253 (Mo. Bot. Gard. Herb.); New Orleans, Drummond 176, 626 (Kew Herb.) ; New Orleans, coll. of 26 March, 1847, Brom- field (Kew Herb.). Texas: low ground, San Augustine, 31 March, 1915, Pal- mer 7114 (Mo. Bot. Gard. Herb.). Forma robustior, forma nova. Stout herb; upper stem-leaves 1.5 to 2 dm. long, 8 to 10 cm. wide; the large lateral obovate leaf-lobes alternating with smaller wedge-shaped divisions of the leaf. Georgia: ditch banks, near Savannah, 21 March, 1882, J. D. Smith (Gray Herb.), type. This plant appears to be a giant form with rather marked foliage. 19. S. Greggii Rydb. Bull. Torr. Bot. Club 27: 170. 1900. S. tampicanus Gray, Pl. Fendl. 109. 1849 (in Mem. Am. Acad. N. 8. 4), not DC. S. lobatus Gray, Pl. Wright., part 2, 99. 1853 (in Smithson. Contr. 5), not Pers. Annual or biennial, glabrous or with a slight tomentum in the leaf-axils and on the upper side of the leaf along the mid- 1915] GREENMAN—MONOGRAPH OF SENECIO 609 rib; stems one to several from a common base, 1.5 to 4 dm. high, striate; leaves lyrate to pinnately divided into cuneate to subrotund divisions; inflorescence a terminal corymbose cyme; heads 5 to 8 mm. high, radiate; involucre campanulate, slightly calyculate; bracts of the involucre about 21, linear-lanceolate, 3 to 5 mm. long, glabrous; ray-flowers 8 to 12; disk-flowers 45 to 60; achenes hispidulous. Distribution: southern New Mexico, western Texas, and northern Mexico. Specimens examined: New Mexico: ‘banks of the Rio Grande near El Paso, Wright 1413 (Gray Herb.). Texas: valley of the Rio Grande, below Dofiana, Mexican Boundary Survey, Parry 659 (U. S. Nat. Herb.); El Paso, May, 1881, Vasey (U. S. Nat. Herb.); southeastern Texas, Sept., 1879 to Oct., 1880, Palmer 754 (Gray Herb.). Chihuahua: valley of Rio Parral, near Santa Rosalia, 21 April, 1847, Gregg 11, (Gray Herb.) co-rypn; valley near Ortiz, 11 April, 1887, Pringle (Field Mus. Herb.). 20. S. imparipinnatus Klatt, Natur. Gesell. Halle 15 : 333. 1881; Greenm. Monogr. Senecio, I. Teil, 23. 1901, and in Engl. Bot. Jahrb. 32 : 19. 1902. S. lobatus Gray, Syn. Fl. N. Am. 1? : 394. 1884, and ed. 2. 1886, in part, not Pers.; Coulter, Contr. U. S. Nat. Herb. 2: 241. 1892, in part, not Pers. Annual or biennial, glabrous or slightly floceose-tomen- tulose in the axils of the leaves; stems slender, 1.5 to 4 dm. high, simple or branched from the base; leaves 2 to 10 em. long, 1 to 3 em. broad, lyrate to pinnately divided or the lower- most occasionally undivided; the upper stem-leaves remote, sessile, and pinnately divided into small linear and entire to abruptly cuneate and unequally toothed lateral divisions; in- florescence a terminal few-headed corymbose cyme; heads 6 to 8 mm. high, radiate; involucre campanulate, glabrous, min- utely calyculate; bracts of the involucre usually 21, linear- lanceolate, 3 to 5 mm. long, acute; ray-flowers 8 to 12; disk- flowers commonly 50 to 60; achenes hirtellous-puberulent. [vor. 2 610 ANNALS OF THE MISSOURI BOTANICAL GARDEN Distribution: western Louisiana, Oklahoma, and Texas. Specimens examined: Louisiana: without locality, Leavenworth (Gray Herb. and Kew Herb.). Oklahoma: Rock Creek, coll. of 1884, Tufts (U. S. Nat. Herb.) ; between Fort Cobb and Fort Arbuckle, coll. of 1868, Palmer 462 (U. S. Nat. Herb.) ; near Indianola, Pope (Gray Herb.) ; Muskogee, May, 1894, Schenck (Field Mus. Herb.) ; near Paul’s Valley, Garvin County, 19 April, 1913, Stevens 108 (Mo. Bot. Gard. Herb.). Texas: Dallas, 16 April, 1901, Reverchon 558 (Mo. Bot. Gard. Herb.); in waste ground, Tarrant Co., 5 May, 1912, Ruth 367 (Mo. Bot. Gard. Herb.) ; Waco, Pace 122 (Mo. Bot. Gard. Herb.); Navarro Co., 22 May, 1880, Joor (Mo. Bot. Gard. Herb.); wet ground, Houston, May, 1872, Hall 368 (U. S. Nat. Herb. and Field Mus. Herb.); Harrisburg, 24 April, 1899, Eggert (Mo. Bot. Gard. Herb.) ; Harris Co., 13 and 22 May, 1876, Joor (Mo. Bot. Gard. Herb.) ; vieinity of Huntsville, 6-12 May, 1910, Dixon 516 (Field Mus. Herb.) ; Columbia, 6 April, 1899, Bush 56 (Mo. Bot. Gard. Herb.); Columbia, 31 March, 1902, Bush 1263 (Mo. Bot. Gard. Herb.) ; along Corpus Christi Bay, 21 March, 1894, Heller 1476 (Gray Herb., U. S. Nat. Herb., Field Mus. Herb., and Mo. Bot. Gard. Herb.) ; Corpus Christi, 7 April, 1905, Tracy 8927 (Field Mus. Herb. and Mo. Bot. Gard. Herb.) ; low prairies near Rosen- berg, 5 April, 1900, Eggert (Mo. Bot. Gard. Herb.) ; Rich- mond, 15 March, 1914, Palmer 4954 (Mo. Bot. Gard. Herb.) ; Hungerford, 4 March, 1914, Palmer 4844 (Mo. Bot. Gard. Herb.) ; Austin, March, 1870, Bodin 52 (U. S. Nat. Herb.) ; “Bejar a la villa de Austin,’’ Berlandier 1741, 421 (Gray Herb.), co-rypr; near Belknap, 20 April, 1858, Sutton Hays 515 (Field Mus. Herb.) ; Brazos, coll. of 1889, Nealley 91, 280 (Field Mus. Herb. and Mo. Bot. Gard. Herb.) ; Brazos, April, 1859, Lindheimer (Mo. Bot. Gard. Herb.) ; bottom land be- tween Laredo and Palafox, Schott (Field Mus. Herb.). 21. $S. Millefolium Torr. & Gray, Fl. N. Am. 2: 444. 1843; Gray, Syn. Fl. N. Am. 1?:392. 1884, and ed. 2, 1886; Chap- 1915] GREENMAN—MONOGRAPH OF SENECIO 611 man, Fl. Southern U. S., ed. 3, 266. 1897; Small, Fl. South- eastern U. S. 1305. 1903, and ed. 2, 1913. An herbaceous perennial, glabrous or with a white floccose- tomentum at the base of the stem and in the axils of the leaves; stems 3 to 7 dm. high, striate; leaves bi-tri-pinnately dissected into linear segments; basal and lower stem-leaves petiolate, 1 to 2.5 dm. long, 1.5 to 6 em. wide, the upper ones sessile; inflorescence terminating the stem in a corymbose cyme; heads 8 to 10 mm. high, radiate; involuere campanulate, sparingly calyculate, glabrous; bracts of the involucre 4 to 6 mm. long; ray-flowers 8 to 12; disk-flowers numerous, usual- ly 50 to 60; achenes hirtellous-puberulent. Distribution: mountains of North Carolina and South Carolina. Specimens examined: North Carolina: slope of Caesar’s Head, 3 Sept., 1876, Engelmann (Mo. Bot. Gard. Herb.) ; without locality, coll. of 1888, Boynton (U. S. Nat. Herb.) ; dry, rocky places on White Oak Mountains, Polk Co., alt. 850 m., 4 May, 1897, Biltmore Herb. 1301” (Gray Herb., Field Mus. Herb., and Mo. Bot. Gard. Herb.); Skyuka Mountains, Polk Co., 25 May, 1899, Churchill (Gray Herb.). South Carolina: Table Rock, coll. of 1842, Buckley (Gray Herb. and Mo. Bot. Gard. Herb.) ; ‘‘Carolina,’’ Fraser (Gray Herb.), part of Tyre; Caesar’s Head, Aug., 1876, Canby (U. S. Nat. Herb.). 22. 8. tampicanus DC. Prodr. 6: 427. 1837; Hemsl. Biol. Cent.-Am. Bot. 2: 248. 1881, excl. plant of Wright. S. Ervendbergu Greenm. Monogr. Senecio, I. Teil, 23. 1901, and in Engl. Bot. Jahrb. 32:19. 1902; Field Col. Mus. Bot. Ser. 2 : 275. 1907. Glabrous throughout; stem 4 dm. or more high, terete, striate, leafy; leaves thin, pinnately divided into cuneate to obovate, unequally dentate divisions; lower leaves petiolate, 1 to 3 dm. long, the upper ones sessile and amplexicaul by a large stipular-like base; inflorescence a terminal compound corymbose many-headed cyme; heads small, 5 to 7 mm. high, [vor. 2 612 ANNALS OF THE MISSOURI BOTANICAL GARDEN radiate; involucre campanulate, glabrous, minutely calyculate ; bracts of the involucre 21, linear-lanceolate, 3 to 4 mm. long; ray-flowers about 13; disk-flowers numerous, 70 to 90; achenes hirtellous along the ribs. Distribution: eastern Mexico. Specimens examined: Tamaulipas: Tampico, coll. of 1827, Berlandier 186 (Ber- lin Herb., tracing and fragments in Gray Herb.), co-TYPr. Vera Cruz: Wartemberg, near Tantoyuca, coll. of 1858, Ervendberg 90 (Gray Herb.); without locality, Liebmann 172 (Copenhagen Herb., tracing and fragments in Gray Herb.). Puebla: near Metaltoyuca, alt. 240 m., 27 Feb., 1898, Gold- man 74 (U. S. Nat. Herb. and Gray Herb.). San Luis Potosi: without definite locality, Parry & Palmer 533 (Gray Herb.). 23. S. hypotrichus Greenm.' S. Sanguisorbae Hemsl. Biol. Cent.-Am. Bot. 2: 246. 1881, in part, not DC. An herbaceous perennial; stem 7 dm. high, aad striate, glabrous, somewhat purplish, branched above; leaves pin- nately divided into cuneate to rhomboic-ovate dentate unequal divisions, glabrous above, crisp-hirsute beneath; lower leaves including the petiole 2 to 3 dm. long, 4 to 9 em. broad, the upper stem-leaves sessile, semiamplexicaul and gradually reduced towards the terminal corymbose eyme; heads 8 to 10 mm. high, radiate; involucre campanulate, sparingly calye- ulate; bracts of the involucre usually 21, linear-lanceolate, u hypotrichus Greenm. sp. nov. herbaceus he caule erecto cir- citer 7 dm ahs tereti striato stramineo vel plus minusve purpurascenti glabro, superne ramoso; foliis pinnatifidis, inferioribus ne usque ad 3 dm. longis, m. latis, ins oa rioribus sessilibus et s semiamplexicaulibus gradatim reductis, laciniis anguste cuneatis vel oba vatis vel rhombo-ov ond es renato- — supra glabris subtus erispo-hi rsutis; inflorescentiis terminalibus corym capitulis 8-10 mm. altis r tiation involucri squamis plerun ıque 21 Ina lanceo- latis = mm. lon ngis Bigot flosculis liguliferis — 13, ligulis oblongis, 6 -7 m ge ngis, 2.5 mm. latis, 4-5-nerviis; floribus disci 60-70; achaeniis — —Re er of San Pig Potosi, eect alt. 1830-24 40° m., coll. of 1878, Parry & Palmer "533 (U A Nat. Herb.), TY The Gray Herbarium rew en of dah j and Palmer’s No. 533 differ .. the United States National Herbarium speci men above cited in having er s leaves, smaller and more numerously flower ed heads and hirtellous achenes ; it tas been referred to 8. anna DC. 1915] GREEN MAN—MONOGRAPH OF SENECIO 613 5 to 6 mm. long, glabrous; ray-flowers 13, rays oblong, 6 to 7 mm. long, 2.5 mm. broad, 4—5-nerved; disk-flowers 60 to 70; achenes glabrous. Distribution: central Mexico. San Luis Potosi: ‘‘region of San Luis Potosi,’’ alt. 1830- 2440 m., coll. of 1878, Parry & Palmer 533 (U.S. Nat. Herb.), TYPE, 24, S. Sanguisorbae DC. Prodr. 6: 427. 1837; Hemsl. Biol. Cent.-Am. Bot. 2 : 246, 1881, in part; Greenm. Monogr. Sene- cio, I. Teil, 23. 1901, and in Engl. Bot. Jahrb. 32 : 19. 1902. An herbaceous perennial; stem erect, 3 to 10 dm. high, striate, glabrous, simple or branched; leaves pinnately divided, the radical and lower stem-leaves petiolate including the petiole 1 to 4 dm. long, 3 to 13 em. broad, glabrous on both surfaces or slightly subarachnoid beneath, the upper stem- leaves sessile and more or less amplexicaul; lateral leaf-seg- ments oblong-cuneate to oblong-ovate, 1 to 7 em. long, .3 to 5.5 em. broad, rather coarsely dentate, the terminal segment usually broadly obovate; inflorescence a terminal many-headed corymbose cyme; heads 6 to 8 mm. high, radiate; involucre narrowly campanulate, sparingly calyculate; bracts of the involucre 8 to 13, linear-lanceolate 4.5 to 6 mm. long, glabrous; ray-flowers 5 to 8; disk-flowers 15 to 25; achenes glabrous. Distribution: southern Mexico. Specimens examined: Hidalgo: by brooks, Sierra de Pachuca, alt. 3050 m., Aug., 1902, Pringle 9959 (Gray Herb. and Mo. Bot. Gard. Herb.) ; Sierra de Pachuca, 1 Sept., 1903, Rose & Painter 6739 (Gray Herb.). Mexico: Toluca, coll. of 1854, Schaffner (Gray Herb. and Berlin Herb.); Valley of Mexico, Sante Fe, Bourgeau 832 (Gray Herb., U. S. Nat. Herb., Berlin Herb., and Kew Herb.) ; without locality, Gregg 691 (Mo. Bot. Gard. Herb.); Cima, 24 Aug., 1910, Orcutt 3767 (Mo. Bot. Gard. Herb.) ; in moist soil along brooks, Mt. Ixtaccihuatl, alt. 3050-3350 m., Nov., 1905, Purpus 1514 (U. S. Nat. Herb. and Mo. Bot. Gard. Herb.) ; in moist soil, Mt. Popocatepetl, Sept., 1908, Purpus [Vou 2 614 ANNALS OF THE MISSOURI BOTANICAL GARDEN 3044 (Field Mus. Herb. and Mo. Bot. Gard. Herb.) ; Mt. Popo- catepetl, 7 and 8 Aug., 1901, Rose & Hay 6069 (U. S. Nat. Herb.) ; without locality, Uhde 582, 602, 603, 609, 624 (Berlin Herb.) ; without locality, coll. of 1848-49, Gregg 673 (Gray Herb.). Michoacan: Angangueo, Hartweg 313 (Berlin Herb.) ; cool summits of mountains near Patzeuaro, 2 Aug., 1892, Pringle 4129 (Gray Herb., U. S. Nat. Herb. and Mo. Bot. Gard. Herb.). 25. S. pinnatisectus DC. Prodr. 6 : 427. 1837; Hemsl. Biol. Cent.-Am. Bot. 2: 245. 1881; Greenm. Monogr. Senecio, I. Teil, 23. 1901, and in Engl. Bot. Jahrb. 32 : 19. 1902. Cineraria pinnata La Llav. & Lex. Nov. Veg. Descr. fase. 1, 26. 1824. An herbaceous perennial; stem erect, 4 dm. or more high, striate, glabrous or slightly tomentulose; leaves pinnately divided, the lower petiolate, including the petiole 1 to 3 dm. long, 3 to 8 em. broad, the upper sessile and amplexicaul, at first white floccose-tomentulose, later glabrate except for the persistent tomentum along both sides of the rhachis; lateral divisions of the leaf narrowly oblong, sharply serrate-dentate, terminal division obovate-cuneate; inflorescence a terminal compound compact corymbose cyme; heads numerous, 6 to 7 mm. high, radiate; involucre calyculate, glabrous; bracts of the involucre usually 13; ray-flowers commonly 6 to 8; disk- flowers 15 to 20; achenes glabrous. Distribution: southern Mexico. Specimens examined: Hildalgo: Real del Monte, Ehrenberg 386 (Berlin Herb. and Gray Herb.), also 386*, 386” (Berlin Herb.); Real del Monte, coll. of 1830, Graham (Gray Herb. and Kew Herb.). Michoacan (?): Angangueo, Chrismar (Berlin Herb.); ‘‘ Cuesta de las papao Angangueo,’’ Schiede (Berlin Herb.). Mexico, without definite locality: Bates, Mackenzie, and also Parkinson (Kew Herb.). This species is elosely related to the preceding, but differs in the narrower lateral leaf-segments, slightly smaller heads, 1915] GREENMAN— MONOGRAPH OF SENECIO 615 and persistent floccose tomentum along the rhachis or midrib. 26. S. coahuilensis Greenm. Monogr. Senecio, I. Teil, 23. 1901, and in Engl. Bot. Jahrb. 32 : 19. 1902; Field Col. Mus. Bot. Ser. 2 : 275. 1907. Plate 19, fig. 2. An herbaceous perennial, glabrous or essentially so through- out; stem erect, 3 to 8 dm. high, branched, striate ; leaves pin- nately divided into obovate to subreniform cuneate-dentate divisions, thickish and firm in texture, glabrous on both sur- faces or slightly pubescent on the veins beneath; lower leaves including the petiole 1 to 3 dm. long, 2 to 5 em. broad, the upper stem-leaves sessile and amplexicaul; inflorescence ter- minating the stem and branches in a compound corymbose cyme; heads 7 to 10 mm. high, radiate; involucre campanulate, calyculate with a few small bracteoles, glabrous; bracts of the involucre 13 to 18, linear-lanceolate, 4 to 6 mm. long, thick- ish; ray-flowers 8 to 10, rays oblong, 3 to 5 mm. long, 4-nerved; disk-flowers 35 to 45; achenes ribbed, glabrous. Distribution: northern Mexico. Coahuila: Lerios, Feb. to Oct., 1880, Palmer 755 (Gray Herb., Kew Herb., and U. S. Nat. Herb.), rypz; without local- ity, coll. of 1848-49, Gregg 403 (Gray Herb. and Mo. Bot. Gard. Herb.). 27. 8. leonensis Greenm. Monogr. Senecio, I. Teil, 23. 1901, and in Engl. Bot. Jahrb. 32: 19. 1902; Field Col. Mus. Bot. Ser. 2 : 276. 1907. Plate 19, fig. 1. An herbaceous perennial, more or less lanate-tomentose throughout, somewhat glabrate in age; stem 2 to 3 dm. high, leafy at the base, essentially naked above; leaves petiolate, pinnately divided, including the petiole 8 to 12 cm. long, about 3 cm. broad, at first lanate-tomentulose on both surfaces, later glabrate; divisions of the leaf rather coarsely, somewhat unequally and sharply dentate, the terminal segment subren- iform, the lateral ones (3 to 6 on either side) obovate-cuneate; heads few, about 1 cm. high, radiate; involucre campanulate, slightly calyculate and, as well as the bracteate peduncle, tomentulose; bracts of the involucre about 13; disk-flowers numerous, 50 to 60; achenes pubescent. [voL. 2 616 ANNALS OF THE MISSOURI BOTANICAL GARDEN Distribution: northern Mexico. Specimen examined: Nuevo Leon: Sierra Madre, near Monterey, 1 June, 1889, Pringle 2894 (Gray Herb.), TYPE. 28. S. montereyana Wats. Proc. Am. Acad. 25 : 155. 1890; Greenm. Monogr. Senecio, I. Teil, 23. 1901, and in Engl. Bot. Jahrb. 32 : 19. 1902. An herbaceous perennial, more or less white-tomentose throughout; stems one to several, 2.5 to 4 dm. high, from a rather stout ascending rootstock; leaves mostly radical, in- cluding the petiole 1 to 2 dm. long, 1.5 to 3 em. broad, pinnately divided into narrow, oblong, cuneate to sublinear, entire or few-toothed divisions, at first white-floccose-tomentose on both surfaces, somewhat glabrate above; heads few, 10 to 12 mm. high, radiate, on long naked peduncles; involucre campanulate, calyeulate with minute bracteoles, tomentose; bracts of the in- volucre slightly shorter than the numerous flowers of the disk; ray-flowers about 12; achenes hirtellous-pubescent. Distribution: northern Mexico. Specimens examined: Nuevo Leon: dry shaded ledges of the Sierra Madre, near Monterey, 27 June, 1888, Pringle 1922 (Gray Herb., U. 8. Nat. Herb., Kew Herb., and Mo. Bot. Gard. Herb.), TYPE. 29. S. zimapanicus Hemsl. Biol. Cent.-Am. Bot. 2: 248. 1881; Greenm. Monogr. Senecio, I. Teil, 23. 1901, and in Engl. Bot. Jahrb. 32 :19. 1902. An herbaceous perennial; stems 3 to 4.5 dm. high, simple, leafy below, nearly naked above, striate, more or less pubes- cent with flaceid-hirsute, jointed, and somewhat matted hairs; leaves mostly basal, sessile or essentially so, 3 to 18 em. long, 1 to 3 em. broad, pinnately lobed or divided into oblong-ovate dentate divisions, flaccid-hirsute or subarachnoid-pubescent on both surfaces, more densely so beneath; inflorescence a terminal corymbose few-headed cyme; heads large, 10 to 14 mm. high, conspicuously calyculate, radiate; bracts of the in- volucre commonly 21 (15-21) linear-lanceolate, 7 to 9 mm. long, thickish, glabrous except at the penicillate tip; ray- 1915] GREENMAN—MONOGRAPH OF SENECIO 617 flowers 12 to 15, rays oblong, 10 to 12 mm. long; disk-flowers numerous; achenes about 3 mm. long, ribbed, slightly pubes- cent on the ribs. Distribution: eastern Mexico. Specimens examined: Hildago: Zimapan, Coulter 423 (Kew Herb.), TYPE. Tamaulipas: near Miquihuana, alt. 2140 to 2740 m., 10 June, 1898, Nelson 4492 (Gray Herb. and U. S. Nat. Herb.). Sect. 5. BoLANDERIANI Greenm. $5. BoLanperianı Greenm. Monogr. Senecio, I. Teil, 22, 23. 1901, and in Engl. Bot. Jahrb. 32 : 18, 19. 1902. Slender, herbaceous perennials; stems erect or nearly so, 1 to 5 dm. high, from a slender more or less horizontal root- stock; leaves undivided and orbicular-ovate to pinnatifid; heads of medium size, about 1 cm. high, radiate; achenes glab- rous. Sp. 30-32. A. Stems 1.5 to 5 dm. high, leafy to the inflorescence. eaves ani pubescent beneath; bracts of the involucre 6 to 9 mm. long, more or as hairy.. 30. 8. Bolanderi of the involucre 5 to mm. long, glabrous ........ 31. 8. ee B. Stems 1 to 2 dm. high, leafy only at the base...... 32. 8. Fle 30. 8. Bolanderi Gray, Proc. Am. Acad. 7 : 362. 1868; Bot. Calif. 1:411. 1876, in part; Syn. Fl. N. Am. 12; 392. 1884, and ed. 2, 1886, in part ; Howell, Fl. N. W. Am. 1: 379. 1900, in part; Greenm. Monogr. Senecio, I. Teil, 23. 1901, and in Engl. Bot. Jahrb. 32:19. 1902. A slender herbaceous perennial; stems ascending or erect, from a creeping rootstock, 1.5 to 5 dm. high, striate, often somewhat purplish; radical and lower stem-leaves undivided and crenately lobed-dentate to pinnately divided into oblong, obovate to subrotund, crenate to sharply dentate divisions, glabrous above, usually pubescent beneath, including the petiole .5 to 1.5 dm. long, 1 to 3 em. broad; the upper stem- leaves sessile; inflorescence terminating the stem in a few- headed subcorymbose cyme; heads 10 to 12 mm. high, radiate; involucre campanulate, calyculate, usually tawny pubescent; bracts of the involucre about 13, linear-lanceolate, 6 to 9 mm. [voL. 2 618 ANNALS OF THE MISSOURI BOTANICAL GARDEN long; ray-flowers 5 to 8; disk-flowers rather numerous, 25 to 45; achenes glabrous. Distribution: California and Oregon, near the coast. Specimens examined: California: on sand-stone bluffs at the mouth of the river below Mendocino City, May, 1866, Bolander 4816 (Gray Herb., Field Mus. Herb., and Mo. Bot. Gard. Herb.), TYPE; Humboldt, coll. of 1868-69, Kellogg & Harford 539 (U. S. Nat. Herb. and Mo. Bot. Gard. Herb); Humboldt, coll. of 1866, Kellogg 539 (Gray Herb.); Redwoods, Eel River, coll. of 1878, Rattan 33 (Gray Herb.); near Crescent City, Del Monte Co., June, 1892, Burt-Davy & Blasdale 1072 (Field Mus. Herb.). Oregon: Coast Mountains, Lat. 42°, June, 1884, Howell 162 (Gray Herb.) ; Newport, June, 1892, Mulford (Mo. Bot. Gard. Herb.). 31. S. Harfordii Greenm. Contr. U. S. Nat. Herb. 11: 597. 1906. S. Bolanderi vər. oregonensis Greenm. Monogr. Senecio, I. Teil, 23. 1901, and in Engl. Bot. Jahrb. 32 : 19. 1902. A slender herbaceous perennial, glabrous throughout; stem erect or ascending from a creeping rootstock, 2 to 5 dm. high, usually leafy; leaves mostly pinnately divided into cuneate to subrotund crenate to laciniate-dentate divisions; the radical and lower stem-leaves petiolate, including the petiole 4 to 14 em. long, 1 to 5 em. broad, occasionally undivided, subro- tund and crenately lobed and the lobes again crenate-dentate, thin in texture, pale green in the dried state; the upper stem- leaves sessile; inflorescence a few-headed corymbose cyme; heads 8 to 10 mm. high, radiate, including the conspicuous yellow rays 1.5 to 2 cm. in diameter; bracts of the involucre usually 13, narrowly lanceolate, 5 to 6 cm. long, acuminate, acute, glabrous; ray-flowers usually 5 (-8); disk-flowers 15 to 25; mature achenes 2.5 to 3.5 mm. long, glabrous. Distribution: mountains of Washington and Oregon. Specimens examined : Washington: on mountains near the Lower Cascades, Skamania Co., 29 May, 1886, Suksdorf (Gray Herb.); in 1915] GREENMAN—MONOGRAPH OF SENECIO 619 woods, Lower Cascades, 29 May, 1887, Suksdorf 872 (Mo. Bot. Gard. Herb.) ; summit of Mt. Adams, 4 Aug., 1899, Flett 1087 (Piper Herb.). Oregon: Rooster Rock, June, 1877, Howell (Gray Herb.) ; Cascade Mountains, 31 May, 1868-69, “Kellogg & Harford,” namely Harford & Dunn 540 (Gray Herb.), TYPE; near Bonneville, Multnomah Co., 11 July, 1885, Suksdorf 572 (Gray Herb.); Multnomah Falls, 25 June, 1904, Piper 6212 (Gray Herb.) ; Bonneville, 24 June, 1905, Palmer (U. S. Nat. Herb.). 32. S. Flettii Wiegand, Bull. Torr. Bot. Club 26: 137, pl. 355. 1899; Greenm. Monogr. Senecio, I. Teil, 23. 1901, and in Engl. Bot. Jahrb. 32:19. 1902; Piper, Contr. U. S. Nat. Herb. 11 : 597. 1906. An herbaceous perennial, 1 to 2 dm. high, glabrous through- out; leaves mostly basal, petiolate, including the petiole 4 to 12 em. long, 1.5 to 2 em. broad, undivided, ovate-orbicular and crenate-dentate to pinnately parted, upper stem-leaves few, 1 to 3, incisely pinnate to linear and bractiform; inflorescence terminating the stem in a few-headed corymbose cyme; heads about 1 em. high, radiate; involucre narrowly campanulate, sparingly calyculate; bracts of the involucre 8 to 13, linear- lanceolate, 5 to 6 mm. long, thickish, glabrous; ray-flowers commonly 5; disk-flowers about 20; achenes glabrous. Distribution: Washington. Specimens examined : Washington: loose rocks, Olympic Mountains, alt. 1830 m., 27 Aug., 1898, Flett 801 (Piper Herb.), co-rypz; Olympic Mountains, Clallam Co., Aug., 1900, Elmer 2620 (Mo. Bot. Gard. Herb.); Angeles, Clallam Co., 29 June, 1908, Flett. 3351 (U. S. Nat. Herb.) ; in volcanic sands, Olympic Moun- tains, alt. 1525 m., Sept., 1890, Piper 929 (Gray Herb., Mo. Bot. Gard. Herb., and U. S. Nat. Herb.) ; crevices of volcanic rock, Olympic Mountains, alt. 2135 m., Aug., 1895, Piper 2196 (U.S. Nat. Herb., Gray Herb., and Piper Herb.) ; Yakima Region, coll. of 1882, Brandegee 176 (Mo. Bot. Gard. Herb.). (To be continued.) 620 [Vor. 2, 1915] ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 17 Senecio mohavensis Gray California From the type specimen, Lemmon No. 3129, in the Gray Herbarium of Harvard University. Ann, Mo. Bor. GARD., Vor. 2, 1915 PLATE 17 GREENMAN—MONOGRAPH OF SENECIO COCKAYNE, BOSTON 622 [Vor. 2, 1915] ANNALS OF THE MISSOURI BOTANICAL GARDEN ExPLANATION OF PLATE PLATE 18 Senecio durangensis Greenm. Mexi From the type specimen, Pringle No. 10105, in the Gray Herbarium of Harvard University. Ann. Mo. Bor. GARD., Vor. 2, 1915 PLATE 18 GREENMAN—MONOGRAPH OF SENECIO COCKAYNE, BOSTON [Vor. 2, 1915] 624 ANNALS OF THE MISSOURI BOTANICAL GARDEN ExPLANATION OF PLATE PLATE 19 Fig. 1. Senecio leonensis Greenm. Mexico From the type specimen, Pringle No. 2894, in the Gray Herbarium of Harvard University. Fig. 2. Senecio coahuilensis Greenm. Mexico From the type specimen, Palmer No. 755, in the Gray Herbarium of Harvard University. Ann. Mo. Bor. GARD., Vor. 2, 1915 PLATE 19 GREENMAN—MONOGRAPH OF SENECIO COCKAYNE, BOSTON 626 [vor, 2, 19151 ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 20 Fig. 1. Senecio Burkei Greenm. Canada From Macoun’s No. 69359 in the Gray Herbarium of Harvard University. Fig. 2. Senecio sawosus Klatt United States From Baker’s No. 770 in the Herbarium of the Missouri Botanical Garden. w Ann. Mo. Bor. GARD., Vor. 2, 1915 GREENMAN—MONOGRAPH OF SENECIO COCKAYNE, BOSTON PLATE 20 THE THELEPHORACEAE OF NORTH AMERICA. IV! ExoBAsIDIUM EDWARD ANGUS BURT Mycologist and Librarian to the Missouri Botanical Garden Associate Professor in the Henry Shaw School of Botany of : Washington University EXOBASIDIUM Exobasidium :Woronin, Naturforsch. Ges. Freiburg Ver- handl. 4: 397-416. pl. 1-3. 1867.—Saccardo, Syll. Fung. 6: 664. 1888.—Hennings, in Engl. & Prantl, Nat. Pflanzenfam. (I.1**) : 103. 1897. The type species of the genus is Exobasidium Vaccinii Fuck. ex Wor. Fungi parasitic in leaves, shoots, and flowers, which they deform more or less, producing on the surface of these organs an effused hymenium, rarely composed of basidia alone and more usually felt-like and composed chiefly of interwoven hyphae bearing basidia and conidiophores; basidia simple; spores white, simple or septate. Exobasidium resembles so closely in the thinness of its fructifications such species of Corticium and Peniophora as Corticium byssinum, Peniophora asperipilata, P. pilosa, and P. subalutacea that I follow Saccardo and include it with the above genera in the Thelephoraceae. Hennings in Engler & Prantl’s ‘Die Natürlichen Pflanzenfamilien,’ has raised Ez- obasidium to ordinal rank but this is not justified by the structure of the many fructifications of Exobasidium which I have sectioned; the illustrations in text-books of the structure in section of the fructification are decidedly diagrammatic and simplified. . In his work already cited, Woronin gives a detailed account of the morphology and life history of Exobasidium Vaccinii and illustrates this account with three double plates. The interest in this fungus which Woronin’s work aroused has 1 Issued October 8, 1915. NoTE.—Explanation in regard to the citation of specimens studied is given in Part I, Ann. Mo. Bot. Gard. 1: 202, footnote. ANN. Mo. Bor. GARD., VoL. 2, 1915 (627) [vor. 2 628 ANNALS OF THE MISSOURI BOTANICAL GARDEN resulted in the publication of other species by various authors, whose descriptions contrast sharply with that of Woronin in giving little weight to the morphological characters of the fungus under consideration, but extended description of the form and color of the gall of a particular collection, with pass- ing reference to the occurrence of the fungus upon a hitherto unpublished host. In case of the galls, the descriptions usually fail to state what other forms besides the one mentioned the galls may have on other organs of the new host and likewise omit mention of the different forms they may have at other times in the year than the particular time at which the type collection was made. Woronin’s description of E. Vaccini was based upon field observations extended through two sea- sons, during which more than a thousand specimens were col- lected. He gives one double page colored plate to show the various types of galls produced by the different organs of Vaccinium vitis-idaea. Plate 21 is a photographic reproduction, reduced one-fifth, of Woronin’s colored plate; it shows the forms of galls as determined by the particular organ of the host, Vaccinium vitis-idaea, which makes hypertrophic response to local stimu- lation by the parasitic fungus. A local change of color from green to some shade of red is common in plant portions in- fested with Exobasidium. In the photographic reproduction of Woronin’s plate the reddened areas of the original appear light colored. In fig. 1, the left side of the uppermost leaf was attacked by the fungus, producing what I term a leaf spot gall. The affected region of the leaf is reddened on the upper side and bears the fructification which may be felty or seurfy on the under side; this leaf is not distorted much in form and thickness. Figures 2-9 present leaf galls, reddened on the upper side of the leaf and distorted and thickened by hypertrophic growth so as to become more or less concave with respect to the upper surface. I designate this form of gall as leaf concavity. Figures 10-17 illustrate shoot galls, in the production of which, stems of the current season’s growth have been greatly 1915] BURT—-THELEPHORACEAE OF NORTH AMERICA. IV 629 enlarged and have turned pale and slightly pink under the stimulus of the infecting fungus. In figs. 10-15 the lateral axillary buds along the infected stem have abnormally en- larged by the stimulation of the fungus and have developed in several instances short, delicate, wax-like or coralloid branchlets of carmine color. Such branchlet shoot galls are beautiful objects in their vegetative condition; they consti- tute a noteworthy type of gall which is quite different in ap- pearance from the more common leaf galls, produced in re- sponse to local infection of leaves. Nevertheless, the common cause of these different gall forms is well brought out by Woronin’s illustrations, especially by figs. 11, 12, 13, and 15. Upon shoot galls similar to the above, there have been pub- lished Exobasidium Andromedae Karst. non Peck for the shoot galls of Andromeda polifolia, E. cassiopes Peck for the shoot gall of Cassiope Mertensiana, and E. Oxycocci Rostrup for that of Oxycoccus palustris. Figures 16-18 show the flower type of gall of Vaccinium vitis-idaea, that is, the abnormal growth form made by indi- vidual flowers in response to the stimulation of their tissues by the fungus. That both the flower gall and the leaf gall have a common cause has been brought out well by the selec- tion of the specimens used for figs. 16 and 17. In fig. 18 there is presented local infection of a single flower. This is import- ant because isolated flower galls upon a new host have in some cases been regarded as prima facie evidence that they have been caused by a new species of Exobasidium. Other host plants produce some types of galls, when in- fected with Exobasidium, which were not figured by Woronin for Vaccinium vitis-idaea but which are more or less common. Such gall types are: (a) Leaf type in which scattered whole leaves of the host are infected. These leaves redden more or less on the upper side and bear on the whole under side the scurfy or felty fructification but are not notably thickened or deformed. This gall differs from the leaf spot gall of Woronin’s fig. 1 merely in having the whole of the leaf infected. (b) Shoot gall with all the leaves toward the tip of the [Vou. 2 630 ANNALS OF THE MISSOURI BOTANICAL GARDEN i shoot infected but not deformed. These leaves may be almost normally green on the upper side or they may be more or less reddened, sometimes to carmine red; on the under side they become clothed with the felty fructification of the fungus but the leaves are not deformed. This is merely a more gen- eral infection than the leaf type a, described above, and is often associated with it on the same plant as well as with the leaf spot and leaf concavity forms. (c) Bag gall of Andromeda ligustrina. This is the ex- treme in gall production. This gall finally becomes a hol- low bag which attains a maximum size of 10-15 cm. in length by 5-10 cm. in diameter. These bag galls are either terminal or lateral on leafy shoots of the current season’s growth. When lateral, such a gall has the morphological position of a leaf. (d) Bud gall of Symplocos tinctoria. The expanding leaf buds are deformed into a subglobose mass which may be 3-34 cm. in diameter. In this gall, the undeveloped stem of the bud is greatly enlarged and the individual leaves of the bud are greatly thickened and deformed. In North America, we have a large number of species of Ericaceae which produce galls when infected by Exobasidium. The specimens which have accumulated under Exobasidium in herbaria show that none of the gall forms which I have designated under distinctive names in the preceding para- graph are isolated forms. Favorable hosts show a connection and gradation between the various gall forms as intimate as that presented by Woronin for Vaccinium vitis-idaea. How- ever, the terms which I employ are useful for contrasting and comparing the data presented by the specimens which I have studied. These data are later given in tabular form. The microscopic examination of an Exobasidium gall shows that it is composed principally of the tissues of the host plant. Hyphae of the fungus ramify about between the cells of the host and, in the galls in which deformation has taken place, the presence of the fungous hyphae has caused the host both to multiply and enlarge its cells in the infected region. The gall is, therefore, a direct product of the host plant, which 1915] BURT—-THELEPHORACEAE OF NORTH AMERICA, IV 631 is stimulated to growth by the presence of the parasitic vegeta- tive hyphae, by absorption of organic products from the host, and, undoubtedly, by excreta from the hyphae. We may see from Woronin’s figures that the various organs of a given host produce different galls when infected by the same fungus; from which we may conclude that the several organs of the host make different growth responses to the same stimulating cause. We have in the host itself, in its several organs, and also in the age of tissues of these organs, as I shall point out later, factors not only able to produce, but actually producing, diversity in gall form even though but a single species of Exobasidium is the parasitic stimulant. Of what value, then, is the form of the gall as a taxonomic char- acter for species of Exobasidium? The different organs of the host differ in the resistance which they offer to infection by Exobasidiwm. Woronin notes in his work cited that out of more than a thousand specimens of Exobasidium Vaccinii, only twelve showed flower galls. Hence the flowers of Vaccinium vitis-idaea are much less sub- ject to infection than the leaves. In only the one case, which he illustrates by fig. 18, did he observe local infection of a flower. In figs. 16 and 17, the infected flowers are borne on infected shoots and may have become infected through these shoots. We may therefore conclude that in a given host a high resistance of certain organs to infection by Exobasidium restricts the galls for that host to fewer organs and to a smaller number of forms than in some other host with a lesser resistance. That the age of the organs, or their cells, of a host is an important factor in the determination of gall form is appar- ent if one observes throughout a season the succession of galls produced by a favorable host. In this connection Richards! has stated, ‘‘and also on Gaylussacia resinosa in the earliest formed distortions, whole shoots are transformed. Later in the season the Exobasidium forms only slight local distortions on the leaves, and still later one finds forms which do not dis- tort the tissues of the host plant at all, but simply form a 1Bot. Gaz. 21 : 107. 1896. [VoL, 2 632 ANNALS OF THE MISSOURI BOTANICAL GARDEN scurf on the lower side of the leaves. The same succession is found in the forms on Andromeda down to the last men- tioned.” Richards determined by culture experiments that the remarkable bag galls of Andromeda ligustrina are merely early (June in Massachusetts) productions under the same speeifie fungous stimulus which later in the season induces leaf concavities on this host. The account of his experiments? may be summarized as follows: During July, Exobasidium spores were removed with suitable precautions from fresh mature bag galls of Andromeda ligustrina and were im- mediately transferred to buds and young leaves of experi- mental plants of the same species, which were isolated in a moist chamber. In about ten days faint discolorations of the leaves were noticed, at first yellowish and then pink. About five days later, the spots which had considerably enlarged, began to show unmistakable signs of thickening, forming the peculiar concavities in the leaves seen in other Exobasidia. In external form, and also in the matter of basidia and spores, this distortion resembled precisely the leaf form on Andro- meda ligustrina, and indicates that the Exobasidium which produces the bag galls of the young buds is identical with the fungus which produces the leaf form found later in the season. The foregoing presentation of the Exobasidium gall as a growth response of the host under stimulation by the fungus shows that very different forms of galls and differences in regard to abundance of each form on a host may result— (a) From the different organs making the response. (b) From differences in resistance of the several organs, which, in many cases, may undoubtedly be so great as to give complete immunity for certain organs. (c) From the age of the organ attacked. Since the host produces a great variety of gall forms as growth responses to attack by a single species of Exobasidium, how are we to decide whether a given gall form is ever suffi- ciently distinct to entitle its causative organism to separate specific rank? Gall forms are host products to so large an 1 loc. cit., p. 105. 1915] BURT—THELEPHORACEAE OF NORTH AMERICA, IV 633 extent that they can have little, if any, value for discrimin- ating between species of Exobasidium. Into the formation of such galls so many other factors besides the Exobasidium hyphae enter that it is impossible to consider galls as homol- ogous with the fructification of an ascomycete or that of a toadstool, and they should not be used therefore in the way these true fungous fructifications are used for affording in their form specific characters. As a matter of fact, the layer of basidia and conidia-bearing hyphae at the outside of the gall comprise the whole fructification of the parasitic fungus; this layer alone is morphologous with a toadstool. The mere form of the foreign substratum covered by the resupinate fructification of Exobasidium should have no greater tax- onomic weight than it has in the closely related genus Corticium. We should now consider the distribution of Exobasidium Vaccinü as a parasite upon various genera and species of the Ericaceae. Woronin limited his investigation of E. Vaccini to what he observed on Vaccinium vitis-idaea and left the matter there for other investigators to go on with, if they were so disposed. As the collections which are made on this host nearly always show the fungus occurring in leaf spot galls and leaf concavity galls, and since these forms of galls are the only ones on this host common enough for distribution in published exsiccati, the species Exobasidium Vaccinu seems to have become altogether too closely associated with, and limited in mycological practice to, merely the very com- monest gall forms which are produced under stimulation by E. Vaccinit. For example, Shear! states, ‘‘The typical form of Exobasidium Vaccinii occurs on Vaccinium vitis- idaea, producing hypertrophied spots on the leaves. No record has been found of the occurrence of hypertrophied shoots on this host similar to those found on cranberry plants. Rostrup! seems to have been the first to describe this form. In 1883 he reported it as occurring on Oxycoccus palustris in Denmark.’’ 1 Cranberry Diseases. U. S. Dept. Agr., Bur. Pl. Ind., Bul. 110: 36. 1907. [VoL. 2 634 ANNALS OF THE MISSOURI BOTANICAL GARDEN Without doubt, this misapprehension of the galls produced by Vaccinium vitis-idaea is due to the searcity of copies of Woronin’s original account of Exobasidium Vaccinia, for Woronin is at great pains to show that to E. Vaccinü are due both shoot galls and flower galls. That the erroneous tendency of limiting to E. Vaccinw the production of only the commonest leaf galls is potent, is ap- parent from inspection of the table towards the close of this paper where under the heading, ‘‘Exobasidium Vaceinii (Fuck.) Wor. The following have been referred here invari- ably’’ there are grouped all Exobasidium galls produced by Vaccinium vitis-idaea, V. vacıllans, V. arboreum, V. penn- sylvanicum, V. stamineum, Gaylussacia frondosa, G. resinosa, Arctostaphylos uva-ursi, A. nevadensis, Arbutus Menziesti, Rhododendron canadense, R. maximum, and Lyonia jamai- censis. Our Gaylussacia frondosa and G. resinosa of this list merit some detailed consideration for they compare very favorably with Vaccinium vitis-idaea as hosts for Exobasidium Vaccinit. The galls of these two species of Gaylussacia include during the season two shoot forms, leaf concavity type, leaf spot type, and the flower type. The flower type of gall is probably very rare; I have seen a dried herbarium specimen of it collected by Dr. Farlow, at Brewster, Massachusetts, and two others, preserved in alcohol in Seymour Herbarium, one of which was collected by A. B. Seymour, at Woods Hole, Massachusetts, and the other by Mrs. Pier, at Biddeford, Maine. These flower galls have a diameter of 10-12 mm.; all the floral organs are enlarged as in case of the flower galls illustrated by Woronin. Bartholomew collected and distributed in his ‘Fungi Columbiani,’ 3429, the shoot gall of the wax-like or coralloid type such as is produced by Vaccinium vitis-idaea. Gaylussacia resinosa very frequently produces as its earliest galls the other form of shoot gall with all the leaves felty on the whole under surface, more or less reddened above, and not deformed. Such a shoot gall is produced by Vaccinium Myrtillus in Europe; it has usually been regarded by Euro- pean mycologists as due to Exobasidium Vaccinii. Its regular 1915] BURT—THELEPHORACEAE OF NORTH AMERICA. IV 635 occurrence in North America in a series of E. Vaccinii forms confirms the correctness of the reference. As we take up the consideration of North American species of Exobasidium which have been published since 1867, we find that in nearly all cases peculiarities of galls have furnished the distinctive portion of the description. These odd or strik- ing forms of galls have been discovered upon new hosts, as was to be expected, for a new host species would without doubt have composition and properties at least slightly different from those of Vaccinium vitis-idaea—so different that the growth response, i. e., the gall of this new host, might differ somewhat, perhaps differ notably, from that of V. vitis-idaea, even though the stimulus should be given by the same fungus. Two of the specific names to be considered are based entirely upon the occurrence of Exobasidium on a new host, and the other eight are founded upon more or less noteworthy galls. Reference to the second division of my table shows that gall form rather than host has caused the publication of specific names in Exobasidium. Exobasidium Pecku, for example, was published as the cause of flower galls produced by Andromeda Mariana. Its flower galls are produced so frequently that they attracted attention; leaf concavity galls are common here also. The morphological characters of the fungous cause of these galls agree closely with those of Exobasidium Vaccinii, and the galls themselves are of types that Vaccinium vitis-idaea pro- duces under stimulation by Exobasidium Vaccinii. No evi- dence of any nature has been offered tending to show that E. Peckt is not E. Vaccinu in all respects. The frequent pro- duction of flower galls by Andromeda Mariana can be simply accounted for as due to the susceptibility of the young flower to infection by the fungus, that is, to a special property of this host. I regard Exobasidium Peckii as a synonym of E. Vaccinit. In connection with the discussion of E. Peckii, attention should be called to occasional flower galls produced by Lyonia (Andromeda) ferruginea. I have seen only four specimens of these galls, two from Georgia and two from Florida. All [Vou. 2 636 ANNALS OF THE MISSOURI BOTANICAL GARDEN resemble monstrous flowers—up to 5 em. long in the dried state—with all floral organs enlarged proportionally, as in the flower galls of Andromeda Mariana, Gaylussacia resinosa, and Vaccinium vitis-idaea. Only flower galls are as yet known to me for Lyonia ferruginea, but as the morphological char- acters of the fungus found on the galls are those of Exo- basidium Vaccinii, I regard these galls as similar to those of Andromeda Mariana but much larger and due to Exobasidium Vaccinii. The large size of these Lyonia galls is the expres- sion of the growth response of the flower tissue of this host. It will be interesting if further collections of this host show that only the flowers are susceptible to infection by Exo- basidium. Exobasidium Oxycocci was proposed as a name for the fungus causing the shoot galls of wax-like or coralloid habit which are produced by Oxycoccus palustris. Similar galls are produced in the United States by Vaccinium macrocarpon and V. intermedium. Shoot galls of V. macrocarpon are illus- trated in color by Shear! and also the leaf spot and leaf con- cavity galls which this host produces. The morphological characters of the fungus producing the shoot galls on the cranberry species of Vaccinium are the same as those of Exobasidium Vaccinii; the galls produced by cranberry plants are such as E. Vaccini produces. As there is no evi- dence of any kind that E. Vaccinii, common throughout the same region, does not cause the cranberry galls, the name E. Oxycocct seems quite unnecessary. Exobasidium Cassiopes and E. Karstenii have been pub- lished as causes of the shoot galls produced by Cassiope Mer- tensiana and Andromeda polifolia respectively. These shoot galls are of the wax-like or coralloid type such as Vaccinium vitis-idaea produces under stimulation by Exobasidium Vac- cinii. As the morphological characters of the so-called Æ. Cassiopes and E. Karstenü are those of E. Vaccini, and as no evidence has ever been presented that E. Vaccinit does not cause the galls referred to, E. Cassiopes and E. Karsten should also be regarded as synonyms of E. Vaccini. 1 loc. cit., pl. 8. 1915] BURT—-THELEPHORACEAE OF NORTH AMERICA. IV 637 Exobasidium Andromedae Peck is based on the bag gall produced by Andromeda ligustrina. This gall described in detail on a preceding page, is so very large and remarkable in structure that it did seem that here, if anywhere, must be the anomaly for higher fungi of a fungous cause, specifically dif- ferent from Exobasidium Vaccini, yet having the same mor- phological characters. From this point of view, Richards’ experiment,! already described, of growing on the leaves of Andromeda ligustrina a July crop of leaf concavity galls from spores produced by a bag gall which had matured at the be- ginning of July, was very illuminating. It showed that such a bag gall is noteworthy only because it shows peculiar prop- erties inherent early in the season in shoots and leaves of Andromeda ligustrina, that this bag gall belongs in the series with, and is caused by, the same fungus as the leaf concavity galls such as Exobasidium Vaccini produces. Richards made other experiments tending to show that E. Vaccinii produces the bag galls on Andromeda ligustrina. He demonstrated that the latter species is not immune to un- doubted Exobasidium Vaccinii, that it is as susceptible to such spores as to those produced by its own bag galls. In July, spores of E. Vaccinii gathered from leaf concavity galls of Gaylussacia resinosa were transferred to buds and young leaves of Andromeda ligustrina. After about the same lapse of time as when spores from the bag galls were used, there appeared on the Andromeda leaves infected with Exobasidium Vaccinii distortions very similar to those produced by spores from the bag galls. As the large bag gall was the only occa- sion for the name E. Andromedae Peck, I agree with Richards that this name is a synomym of E. Vaccinu. In confirmation from the herbarium side of the correctness of the above conclusion, I have a specimen collected in Idaho by Professor Piper, 772, on Menziesia glabella, which has a small terminal bag gall such as is produced by Andromeda ligustrina, and also a leaf concavity gall. In the light of what we now know about bag galls the names Exobasidium Azaleae, E. discoideum, and E. Rhododendri 1 loc. cit. [Vou, 2 638 ANNALS OF THE MISSOURI BOTANICAL GARDEN appear superfluous, for their galls pass through the concavity stage and the morphological characters of the fungi concerned differ in no respect from those of E. Vaccinit. Exobasidium Cassandrae was based on a leaf concavity of Cassandra calyculata. The new host was the sole basis for this new name and its author closed his description with the comment, ‘‘perhaps this is only a form of E. Vaceinii.’’ Since we now regard E. Vaccini as able to infect many species of the Ericaceae, the host alone in this case (with the morpho- logical characters of the fungus agreeing with those of E. Vaccinii) does not afford sufficient justification for regarding E. Cassandrae as distinct from E. Vaccinit. Exobasidium Arctostaphyli was founded on a leaf spot on Arctostaphylos pungens. As in the case of Exobasidium Cas- sandrae, there is no evidence whatever that the fungus con- cerned is not E. Vaccini, the characters of the fungus and its work being quite those of the latter species. The usual errors in connection with the preceding series of synonyms which are grouped together in the second division of my table are due, it seems to me, to attaching to a strange gall form—a host product—the same weight which one would give to a toadstool, and to ignoring the true fructifications of the Exobasidium concerned. In the taxonomy of the Hy- menomycetes, species are based upon differences in morpho- logical characters. It is so remarkable an innovation in our taxonomic usage in this group of plants to propose a new species which has precisely the same morphological characters as a well-known and established one that it makes it incum- bent upon, and an unusual opportunity for, an author so establishing a species to show conclusively the truth of the paradox that actually good and distinct species of Hymenomy- cetes have the same morphological characters. In all the cases which have been considered, no evidence tending toward such proof has been offered. In the above, I but express the views of many of the best mycologists, who have consistently regarded the above-mentioned Exobasidium names as synonyms of E. Vaccinia. 1915] i BURT—THELEPHORACEAE OF NORTH AMERICA. IV 639 Winter! wrote of Exobasidium Vaccinii in Europe where there is a similar confusion as to species, ‘‘der Pilz erzeugt ausnahmslos Formänderungen der verschiedensten Art an den von ihm bewohnten Pflanzentheilen . . . . . Ich finde zwischen den einzelnen verschiedene Nährpflanzen bewohnen- den Formen keine wesentlichen Unterschiede.’ The specimens which I have studied show that we have in North America perhaps three species of Exobasidium, two of which are rare and are present in herbaria in so few speci- mens that present conclusions concerning them are somewhat tentative. These species are as follows: 1. E. Vaccini (Fuck.) Wor. This species is common and wide-spread and is parasitic on many ericaceous host plants. There is as yet no evidence of which I am aware tending to show that so-called physio- logical races or forms with parasitism limited to a particular host exist in this species. This fungus attacks leaves develop- ing leafy shoots, and flowers of susceptible plants, making its most successful infections when these organs are very young. The vegetative hyphae live in the infected organs between the cells, which are stimulated by the presence and activities of the parasitic hyphae to make a more or less marked hyper- trophic growth response, termed a gall. The galls are of varied and sometimes strange form according to the host, the organ, and its age. The distribution of the galls upon the host is dependent upon the susceptibility of its various organs to infection. In fruiting, the hyphae push through the epidermis to the surface and produce there a resupinate fructification which is amphigenous in the case of galls from tissues so young that they form galls of wax-like or coralloid structure, and hypophyllous on the more common leaf galls. The fructification is variable in thickness, consisting sometimes of scattered clusters of basidia but usually with hyphae present in vari- able quantity between the basidia so that the fructification may attain a maximum thickness of 60-70 y, as in the case of col- 1 In Rabenhorst, Krypt. Flora 1*: 322. 1884. [voL. 2 640 ANNALS OF THE MISSOURI BOTANICAL GARDEN lections on Vaccinium vitis-idaea. As shown by Richards,* these hyphae bear simple, acicular, conidia about 6-91-14 a. Conidia are nearly always present in the preparations but have been entered only occasionally in my table. The basidia are generally 4-spored. The basidiospores from herbarium specimens are colorless, simple or with some uniseptate, 10-20 24-5 a, but are usually about 12-18X 3-34 u. They are sometimes a little shorter, or a little longer, or a little thinner, or a little thicker, but are so variable within the extremes stated for different collections on the same host within the same regions or distant regions—as will be seen by reference to my table—that a moderate latitude in spore dimensions seems evident. 2. E. Vaccini uliginosi Boud. The European specimen of this species distributed from Norway in Briosi and Cavara, ‘Funghi Paras.,’ 261, has a resupinate, hypophyllous felty fructification, 30-45 y thick, which is composed almost wholly of large basidia, standing close together and presenting in sections the appearance of a distinct palisade layer. This fructification begins below the epidermis and tears the cells of the latter loose and apart from each other and carries them outward between the basidia. The hymenium is abundantly fruited with basidio- spores, borne two to a basidium. The spores are simple, colorless, even, curved towards the base, 18-206-7 u. No conidial hyphae could be found between the basidia in this specimen. The specimen distributed in Eriksson, ‘Fungi Par. Scand.,’ 286a, has similar spores 16-208 yp. This specimen is in poorer condition and does not show basidia clearly. In some — places the fructification is composed of very fine, short-celled hyphae, which are not bearing conidia. Both the above speci- mens are shoot galls with leaves felty below and reddened above. Professor Piper, 443, collected on Vaccinium membran- aceum, at Mt. Ranier, Washington, in August, a shoot gall similar to the European specimens and having a well fruited 1 loc, cit. 1915] BURT—THELEPHORACEAE OF NORTH AMERICA, IV 641 Exobasidium with 2-spored basidia and spores 16-208 u. The fungus agrees in all respects with the specimen in Briosi and Cavara, 261. Several other collections on Vaccinium membranaceum of buff colored leaf concavity and leaf spot galls appear to bear Exobasidium Vaccini. The very thick spores, borne two to a basidium, distinguish E. Vaccini uliginosi from E. Vaccinit. 3. E. Symploci Ell. & Mart. This fungus attacks the developing leaf buds of Symplocos tinctoria and deforms them into a lobed mass. In fruiting, the hyphae protrude on the surface of the mass and bear acicular, simple, colorless, slightly curved conidia, ranging from about 7X1 „ upward. The largest spores are 24X2 p, acicular, curved, and of the same form as those of inter- mediate size and so on down to attached conidia. I have not found any of the largest spores attached, nor have I found basidia. In the original description the reference to spore characters is ‘‘conidia hyaline, cylindric, nearly straight, 15-212 u.” I conclude that basidia have yet to be demonstrated for this fungus. As I have had an opportunity to examine a large number of Exobasidium specimens, collected in widely separated localities, on many hosts and at various times in the growing season, it has seemed that a concise summary of the data ob- tained in regard to each specimen might prove useful for comparison purposes to others who study our specimens of this genus in the future. Pains have been taken to give the hosts accurately. I am indebted to Dr. J. M. Greenman for aid in host determinations in several cases. In the matter of spores the stated dimensions are those of the preparations which were studied. No effort was made to study preparation after preparation from the same collec- tion in order to find spores possibly larger or smaller than those of the first preparation which showed the spores well. The dimensions stated are those obtained by treating all speci- mens in exactly the same way and give such results as her- barium specimens afford. (Vou. 2 ANNALS OF THE MISSOURI BOTANICAL GARDEN 642 »Aoge 166% “qH I'd “OW ‘AN | sunf | pas pue ynq ‘moyaq Ayımas ‘gods yea] 1 $¢-€XST-ZIT t46% “4H '9 “d “OW ‘ely | AeWw “Surpaceid se aures 1 EXZI wnaurwegs "A 916b “GH DA ON ey | mdy "Burpacaid se aures 1 ER-EXSI-ZI i ‘aAoqe “AS “pig “FIV ety | mdy | pas qep ‘moq Aymos “Jods yea] " €XSI-ZI 91FF7 “qH “OD ‘d “OW UUW [>i ‘Surpaoeid se aures "EXET »Aoge Sth? “GH ‘Dg “OW | sung 'N | UCC ysıppsı *‘mojaq Ajınas ‘jods jeaT s3ınyewuw] i PIPPP “qH “Dd ON ‘sty | “3ny |'aaoqe ysrppas ‘mojaq 4413} ‘0ds year] SPETTA d Ce eA »Aoge S86F “4H I'd “OW "SIM | ystpper ‘mojq Ajinos ‘zods jea'] H EXET S46} "AH I'd ON ‘ey | Indy ‘Surpooaid se ates 1 €XSI-ZI . “OP Ling Torg “ely ‘ely | Indy |’aaogeysıppa1‘mojoq Ajınas‘Jods yea] n §-EXST eee ae 6t6t “qH DA “OW ‘OW | APIA "3uıpass1d se sures 1 1 X6-9 eIpruo) TEZE “100 ‘Bung “"yueg pm | APN "Surpaoa1d se aures 1 $-€X8I-ZI SZLI “109 *Buny “yyeg a| ACW *Burpaooid se sweg a EXST ‘aaoqe zeg “JOD ‘Suny "yueg ‘PIN | unf | ystppes ‘sapun Ajay ‘saavay Aue n £6-¢XC]-ZT FERN OE] “Bung ‘oq ‘apeg 9 ‘Aas ssew | Amf| ‘adoqe pas ‘mojaq Ajay ‘ods year] i EXSI-ZI iit A “aAoqe qet "ung ‘oq ‘Seg X “Aas “SSRI Amf | ysıppsı ‘mojq Ajinos ‘yods jea7y n ¢-27XZT “2aoqge ere, “Zuna ‘oq ‘eg X A ‘sse | əunf | ystppor ‘səpun 44} ‘saavay LUEN 1 ¢XFI-ZI yang uspoms | ‘Sny *Sutpoooid se auleG rl £6_¢XCT-ZI Ipwoy uspaaS | "Iny ['əaoqe pə ‘moq £49} ‘AIA PIUOD Jea’T r EXSI-ZI ‘2A0qe ysıpp3ı pue mojaq Ajay eaepr-snra 79 “Xes “Suny ‘sadaııy | Luew Ájnf | 10 Apınas—AyıAe9uo9 zea] ‘zods Jeo i EE-EXSI-ZI iat 20 3das "e3 Jomoy ‘pres J00ys SEARA o} |—ə4oqe ysıpp3ı pue mojpq Alay ƏPIHEL Ss UTUOIO M eissny | Avy | 10 Ayanos—A}AvIUOD Jeo] ‘zods yeaT | (0M) 7 8'7TX8'91-P1T AIAVIAVANI AAAH CAAYAAAA NAAI AAVH ONIMOTION AHL “WOM CAONA IINIIIVA WNICISVAOXA "quay 10 "[]0> Anje | sed | We) | aınseaw sods 3soH4 GAHINIWNYXAI WNIAISVIOXY JO SNIWIIAIS DONINAHINOD VIVA AO ATAVL YALLVYAVdWOD I ATaVL 1915] 643 BURT—-THELEPHORACEAE OF NORTH AMERICA. 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[je—projfe1oa ‘jež J00US iex IIT UOU ISAD avpamospuy ‘H E'HOLI Q "IS NUASADY MUNIPISDQOXT 6£ “diq 'ysem | aunt 'ad4} PIo]fe109 Jo je 300yS SXPL] Ip ur "A "3AOge pol AZIEZ “A “WY CN "AIR II “SSRI ‘Sny | ‘Ajanos ‘Ayıaesuo9 ea] ‘Jods year n ¢XZI 'qH ‘9'E “Oy ‘aseaqary, sse | "das *Surpadeid ay} 31] [fe 4004S 1 EXSI-ZT | vodıesonew 1 $1-1X6-9 eIpruoD WINTUTIOeA, ‘dH "dA pioj “By jo daq 'S 'n ur suur Ended a A Eee "| -Te109 PIEPI-SIHA "A 947 JO Jfe3 JOOYS 7 $€-€XST GNÄSON 1990IKXO WNIPISDIOKT TLL “did 'YSEM "any "Zuipa9a1d ay} ayı] [fe3 J00yS 1¢X€I-ZI L 4 P ar Li ra. Per 25 TOS ‘popsang "yseM | “Bn | -T2109 BILpI-SHIA *A IYI JO [eB OOYS 7 €XeI-ZI werd 4994 sadoisspy wnrpispgoxy [voL. 2 ANNALS OF THE MISSOURI BOTANICAL GARDEN 648 "dH ‘9 I ON ‘sepoyy ‘puy | pdy *ZuIpaceid se ales aınyewur i , PIPTUOS aTe [je sdey l 6967 “4H DI OWN ‘ey | mdy ‘Surpaoaid se aues | -Jəd ‘d 7-$] Xp7-8 J | POU 896fF “qH I I ON ei] | yore *SuIpacaid se aures aaoge sy sosoJdusAg eıpruos are jje sdey } 9697 “A “WY "N “AJ X NA "eg | YEN "wo 7xg sseur ‘ed png eag | -sod A 7-F1XPI-L J "LAVW 3 SITIA IOOTAWAS WNICISVEOXa p310ds-7 eıpıseg epp ‘did ‘ysem | ‘Sny *Buipaseid ay} Əy! [[e3 4004S 1 9XQ7-OT TENSIEHBIQUISUN "A eggz “seg “Suny ‘uossyug | Aemıon |" *Surpadeid ay} ƏŞ! [je3 JOOYS n 9-1 XL I-ST SNITHÄN "A 197 "3Aoge pal ‘MOJ pa10ds-z eıpıseg | | winsoursiyn “yeg ‘Suny ‘eese 9 oug Aemion | ‘Sny | -3q Ajay saavay je yyım [Tes 3004S "i 1-9 X07-81 wnmumoeA‘ ‘ano ISONIÐITA IINIOOVA WNOICISVdOxa 'q19H 10 “JOD | Áe | əqd | I9 | ainsvaul 310dg | 3soH (panuruo)) | AIAVL 1915] BURT—THELEPHORACEAE OF NORTH AMERICA, IV 649 SYSTEMATIC SUMMARY 1. Exobasidium Vaccinii Fuck. ex. Wor. Naturforsch. Ges. Freiburg Verhandl. 4: 397-416. pl. 1-3. 1867. Plate 21. Fusidium Vaccinü Fuck. Bot. Zeit. 19: 251. 1861.—Exo- basidium Andromedae Peck, Buffalo Soc. Nat. Hist. Bul. 1: 63. 1873; N. Y. State Mus. Rept. 26: 73. 1874.—E. Azaleae Peck, Buffalo Soc. Nat. Hist. Bul. 1: 63. 1873; N. Y. State Mus. Bul. 26: 72. 1874.—E. discoideum Ellis, Torr. Bot. Club Bul. 5: 46. 1874.—E. Rhododendri Cramer in Rabenh. Fung. Eur. 1910. 1875.—E. Andromedae Karst. in De Thuemen, Myc. Univ. 1110. 1878; Finland Natur och Folk Bidrag 37: 153. 1882.—E. Karstenit Sace. & Trott. in Sace. Syll. Fung. 21: 420. 1912.—E. Cassandrae Peck, N. Y. State Mus. Bul. 29: 46. 1874.—E. Arctostaphyli Harkn. Calif. Acad. Sci. Bul. 1: 30. 1884.—E. Myrtilli (Thuem.) Karst. Finlands Natur och Folk Bidrag 37: 152. 1882.—E. Vaccinu Myrtilli (Fuck.) Juel, Svensk. Bot. Tids. 6: 364. 1912.—E. Oxycocci Rostr. Bot. Tidsskr. 14: 243. 1885.—E. Cassiopes Peck, N. Y. State Mus. Rept. 45: 24. 1893.—E. Peckii Halst. Torr. Bot. Club Bul. 20: 437. 1893. Illustrations: Woronin. loc. cit—Richards, Bot. Gaz. 21: pl. 6. f. 1-20.—Petri, Ann. Myc. 5: 342-346.—Brefeld, Unter- such. Myk. 8: pl. 1. f. 17-22.—Duggar, Fung. Dis. f. 215, 216.— Shear, U. S. Dept. Agr., Bur. Pl. Ind. Bul. 110: pl. 7. f. A-D.— Juel, Svensk. Bot. Tids. 6: 353-372. f. A-C.—Engl. & Prantl, Nat. Pflanzenfam. (I. 1**): 104. f. 65.—Other illustrations in many text-books. References to other illustrations in Sacce. Syll. Fung. 19: 694. Fructifications hypophyllous or amphigenous, resupinate, effused, scurfy or felty and compact, grayish, consisting of somewhat scattered clusters of basidia or of basidia and fine, suberect, more or less interwoven and branched hyphae which bear conidia and give to the fructification a maximum thick- ness ranging up to 60-70 u; basidia with 4 sterigmata usually; basidiospores colorless, simple or with some 1-septate, 10-20 21-5 yp, but usually about 12-183-34 u, becoming 3-septate in germinating; conidia simple, 6-91-14 u. [voL. 2 650 ANNALS OF THE MISSOURI BOTANICAL GARDEN Parasitic in leaves, young shoots, and flowers of various ericaceous hosts, and stimulating the infected parts to the production of leaf, shoot, or flower galls which bear the fructifications on their surface. Leaf galls are usually some- what reddish on the upper side and bear the fructification on the lower side. From Newfoundland to Florida and westward to California and Washington, also in Jamaica. I have referred here, with some doubt, the Exobasidium causing yellow-buff leaf spot galls on Rhododendron albi- florum, collected on mountains in Washington by W. N. Suks- dorf. The basidia are 20-306 u, with 4 prominent sterig- mata; the basidiospores are mostly 18-2143-6 u, and are nearly all 3-septate. Some of these spores are germinating, hence the septation of the spores may possibly be due to their over maturity when collected, combined with weather condi- tions at that time favorable to germination. Other collections which show the full series of gall forms on this host are desir- able and should give the needed information in regard to sep- tation of the spores. Specimens examined: Exsiceati: Ellis, N. Am. Fung., 107, 722; Ell. & Ev., N. Am. Fung., 1586a, 1586b, 1718, 2312a, 2312b; Ell. & Ev., Fung. Col., 220, 1210; Bartholomew, Fung. Col., 1728, 2729, 3231, 3232, 3323, 3324, 3429, 3430, 3523; Seymour & Earle, Econ. Fung., 137a, 137b, 137c, 487, 488, 489; Shear, N. Y. Fung., 117; De Thuemen, Myc. Univ., 115, 210, 1110, 1808; Eriksson, Fung. Par., 286b; Jaczewski, Komarov & Tranz- schel, Fung. Rossiae Ex., 72; Kunze, Fung. Sel. Ex., 302; Krieger, Fung. Sax., 62, 665, 768; Rabenhorst, Fung. Eur., 1910; Romell, Fung. Scand., 38. Austria: On Rhododendron ferrugineum, Tyrol, P. Magnus (in Mo. Bot. Gard. Herb., 4988). Germany: On Vaccinium vitis-idaea, Königstein, Krieger, Krieger, Fung. Sax., 62; Bavaria, De Thuemen, Myc. Univ., 910; on Rhododendron ferrugineum, P. Magnus; on Vac- cinium Myrtillus, Leipzig, G. Winter, De Thuemen, Myc. 1915] BURT—THELEPHORACEAE OF NORTH AMERICA. IV 651 Univ., 115; Königstein, Krieger, Fung. Sax., 665; on V. uliginosum, Altenberg, Krieger, Fung. Sax., 768. Russia: On Cassandra calyculata, Novgorod, Jaczewski, Fung. Rossiae Ex., 72. Finland: On Vaccinium uliginosum, Mustiala, P. A. Karsten; on Andromeda polifolia, Mustiala, P. A. Karsten; and also in De Thuemen, Myc. Univ., 1110. Sweden: On Vaccinium vitis-idaea, Femsjé, L. Romell; Up- sala, E. A. Burt; on Andromeda polifolia, L. Romell, Romell, Fung. Scand., 38; on Vaccinium uliginosum, Eriksson, Fung. Par. Scand, 286b. Switzerland: On Rhododendron ferrugineum, Luzern, G. Winter in Kunze, Fung. Sel. Ex., 302; same host, Mader- aner Thal, Cramer, Rabenhorst, Fung. Eur., 1910. Canada: on Cassandra calyculata, London, J. Dearness, El. & Ev., N. Am. Fung., 2312a. Newfoundland: on Cassandra calyculata, Pennie’s River, B. L. Robinson & H. von Schrenk (in Mo. Bot. Gard. Herb., 4779); on Rhododendron canadense, Bluff Head, A. C. Waghorne, 940 (in Mo. Bot. Gard. Herb., 42608) ; Virginia Water, B. L. Robinson & H. von Schrenk (in Mo. Bot. Gard. Herb., 4981). New Brunswick: on Vaccinium pennsylvanicum, Hays, 16 (in Mo. Bot. Gard. Herb., 44415). Maine: on Gaylussacia baccata, Biddeford, Mrs. A. M. Pier (in Seymour Herb., T55). New Hampshire: on Andromeda polifolia, Shelburne, H. von Schrenk (in Mo. Bot. Gard. Herb., 4778). Massachusetts: on Vaccinium vacillans, Arlington, Magnolia, and Medford, A. B. Seymour, Sey. & Earle, Econ. Fung., 137a, 137b, 137c respectively; Plymouth, E. Bartholomew, Fung. Col., 3324; Weston, A. B. Seymour, T56 (in Seymour Herb.) ; Rafes Chasm, A. B. Seymour, T58 (in Seymour Herb.) ; Middlesex Falls, J. @. Jack (in Seymour Herb.) ; on V. macrocarpon, Woods Hole, W. Trelease (in Mo. Bot. Gard. Herb., 4982) ; Chatham, Miss Minns, and also(in U. S. Dept. Agr. Herb.) ; Harwich, B.D.Halsted, Ell.& Ev.,N.Am. Fung., 2312b; Waverly, A. B. Seymour, T60 (in Seymour 652 [VoL. 2 ANNALS OF THE MISSOURI BOTANICAL GARDEN Herb.); on V. pennsylvanicum, Rafes Chasm, A. B. Sey- mour T59 (in Seymour Herb.) ; on Gaylussacia frondosa, Woods Hole, W. Trelease (in Mo. Bot. Gard. Herb., 4948) ; Plymouth, E. Bartholomew, Fung. Col., 3323; on G. resi- nosa, Manchester, W. C. Sturgis, Sey. & Earle, Econ. Fung., 488; Falmouth, A. B. Seymour, T53 (in Seymour Herb.) ; Woods Hole, A. B. Seymour, T54 (in Seymour Herb.) ; Dartmouth, W. G. Farlow (in Seymour Herb.) ; Brewster, W. G. Farlow (in Seymour Herb.) ; on Andromeda lig- ustrina, Cambridge, Mr. Rush; Dedham, H. L. Jones, and also B. M. Duggar (in Mo. Bot. Gard. Herb., 44411) ; Woods Hole, W. Trelease (in Mo. Bot. Gard. Herb., 44410) ; Hamp- den, A. B. Seymour, T51 (in Seymour Herb.) ; Granville, A. B. Seymour (in Seymour Herb.); on Rhododendron cult. sp., Brookline, A. B. Seymour, Sey. & Earle, Econ. Fung., 489; on R. nudiflorum, Granville, A. B. Seymour (in Seymour Herb.) ; on R. viscosum, Woods Hole, W. Tre- lease (in Mo. Bot. Gard. Herb., 44405, 44408). New York: on Vaccinium stamineum, Ithaca, W. Trelease (in Mo. Bot. Gard. Herb., 4991); on Gaylussacia frondosa, Eastport, J. Schrenk (in Mo. Bot. Gard. Herb., 4953) ; East- port, H. von Schrenk (in Mo. Bot. Gard. Herb. 4957); on G. resinosa, Deer Park, H. von Schrenk (in Mo. Bot. Gard. Herb., 4781); on Andromeda ligustrina, Alcove, C. L. Shear, N. Y. Fung., 117; on A. Mariana, Westbury, F.C. Stewart, Sey. & Earle, Econ. Fung., 487; on Cassandra calyculata, Adirondack Mts., C. H. Peck, Ells, N. Am. Fung., 722; Buffalo, G. W. Clinton. New Jersey: on Andromeda ligustrina, Ellis, N. Am. Fung., 107; on A. Mariana, Newfield, Ellis, Ell. & Ev., Fung. Col., 1210; on Rhododendron viscosum, Newfield, Ells, Ell. & Ev., N. Am. Fung., 1718; and (in Mo. Bot. Gard. Herb., 4959). Maryland: on Vaccinium vacillans, Rosecraft, Bartholomew, Fung. Col., 3231; on Gaylussacia resinosa, Lanham, E. Bartholomew, Fung. Col., 3429, 3430; Bartholomew & Swingle, Fung. Col., 3523. 1915] BURT—THELEPHORACEAE OF NORTH AMERICA. IV 653 District of Columbia: on Vaccinium vacillans, Takoma Park, C. L. Shear, Fung. Col., 1728. Virginia: on Gaylussacia resinosa, Vienna, E. Bartholomew, Fung. Col., 3232. North Carolina: on Rhododendron maximum, H. von Schrenk (in Mo. Bot. Gard. Herb., 4951) ; on R. nudiflorum, H. von Schrenk (in Mo. Bot. Gard. Herb., 4950). Georgia: on Lyonia ferruginea, Brunswick, comm. by U. S. Dept. Agr. Herb.; W. Trelease (in Mo. Bot. Gard. Herb., 4955). Florida: on Gaylussacia frondosa, Dunedin, S. M. Tracy, 6649 (in Mo. Bot. Gard. Herb., 44404); on Andromeda ligustrina, St. Leo, Rev. Jerome (in Mo. Bot. Gard. Herb., 44326); on A. Mariana, White Springs, H. H. Hume, 88 (in Mo. Bot. Gard. Herb., 4966), and also (in aaa Herb.) ; Chapman (in Mo. Bot. Gard. Herb., 4954) ; Lyoma ferruginea, Chapman (in Mo. Bot. ad Herb, 44409). Alabama: on een arboreum, Auburn, Ala. Biol. Surv., and also (in Mo. Bot. Gard Herb., 4975) ; on V. stamineum, Auburn, Ala. Biol. Surv., and also (in Mo. Bot. Gard. Herb., 4976); Auburn, F. S. Earle & L. M. Underwood (in Mo. Bot. Gard. Herb., 4971); on Rhododendron nudiflorum, Auburn, Ala. Biol. Surv., and also (in Mo. Bot. Gard. Herb., 4964, 4963). Mississippi: on Rhododendron viscosum, Ocean Springs, F. S. Earle (in Mo. Bot. Gard. Herb., 4970) ; and S. M. Tracy (in Mo. Bot. Gard. Herb., 4960). Michigan: on Galylussacia frondosa, Lansing, M. B. Waite, 118 (in U. S. Dept. Agr. Herb.) ; on G. resinosa, Agricul- tural College, G. H. Hicks (in Seymour Herb.); on Cas- sandra -calyculata, Republic, W. Trelease (in Mo. Bot. Gard. Herb., 4983); Agricultural College, G. H. Hicks (in Seymour Herb.). Minnesota: on Vaccinium pennsylvanicum, Hokal, L. H. Pammel (in Mo. Bot. Gard. Herb., 44416). Wisconsin: on V. pennsylvanicum, i Crosse, L. H. Pammel (in Mo. Bot. Gard. Herb., 44414) ; Kir (in Mo. Bot. x [vorL. 2 654 ANNALS OF THE MISSOURI BOTANICAL GARDEN Gard. Herb., 4985) ; on Gaylussacia resinosa, Kirkland (in Mo. Bot. Gard. Herb., 4961). Missouri: on Vaccinium vacillans, Crystal City, (in Mo. Bot. Gard. Herb., 4949). Wyoming: on V. membranaceum, Teton Mts., A. Nelson, E. Nelson, 6525 (in Mo. Bot. Gard. Herb., 44413). Idaho: on V. membranaceum, Forest, Nez Perces Co., A. A. & E. @. Heller, 8465 (in Mo. Bot. Gard. Herb., 4989); on Menziesia glabella, Bitter Root Mt., C. V. Piper, 772. Colorado: on Arctostaphylous uva ursi, Glacier Lake, Bar- tholomew & Bethel, Fung. Col., 2729. Washington: on Vaccinium deliciosum, Mt. Rainier, C. V. Piper, 842; on V. membranaceum, Mt. Paddo, W. N. Suks- dorf, 448; Chiquash Mts., W. N. Suksdorf, 504; on Vac- cinium sp., probably V. membranaceum, Mt. Paddo, W. N. Suksdorf, 447; on V. intermedium, Seattle, C. V. Piper, 39; on Arctostaphylos uva ursi, Orchard Point, C. V. Piper, 434; on A. nevadensis, Mt. Paddo, W. N. Suksdorf, 840; Longwire Springs, C. V. Piper, 428; on Cassiope Merten- siana, Chiquash Mts., Skamania Co., W. N. Suksdorf, 501; Olympic Mts., C. V. Piper, 771; on Rhododendron albi- florum, Chiquash Mts., Skamania Co., W. N. Suksdorf, 841; Mt. Paddo, W. N. Suksdorf, 449. California: on Arctostaphylos pungens, H. W. Harkness (in Mo. Bot. Gard. Herb., 4972); and also Ell. & Ev., N. Am. Fung., 1586a; on A. manganita, Sisson’s, Siskiyou Co., W. C. Blasdale (in Seymour Herb.) ; on Arbutus Menziesu, H. W. Harkness, Ell. & Ev., N. Am. Fung., 1586b. Jamaica: on Lyonia jamaicensis, Cinchona, H. von Schrenk (in Mo. Bot. Gard. Herb., 44403). 2. E. Vaccinii uliginosi Boud. Soc. Bot. Fr. Bul. 41: CCXLIV. 1894. Illustrations: Juel, Svensk. Bot. Tids. 6: 353-372. pl. 7. f. 5. text. f. D. Fructification hypophyllous, resupinate on the whole lower surface of the leaves, felty, 30-45 „ thick, composed of large basidia arranged side by side in a compact hymenium; basidia 1915] BURT—THELEPHORACEAE OF NORTH AMERICA. IV 655 with 2 sterigmata; spores colorless, even, curved towards the base, 16-20 x 7-8 u. Parasitic on Vaccinium membranaceum, which produces shoot galls with all the later leaves of the gall red on the upper side, felty below, and but slightly, if at all, deformed. Mt. Rainier, Washington. August. In the original description of this species, the spore dimen- sions are stated as 25-32 X 8-12 a. The European specimens in the exsiccati cited below, which European authors refer here, have spores of the dimensions of the American collection. Shoot galls of the type stated are the only form known to be caused by this species, but other forms may yet be found. Specimens examined: Exsiccati: Briosi & Cavara, Fung. Par., 261; Eriksson, Fung. Par. Scand., 286a under the name Exobasidium Vaccinii. Norway: on Vaccinium Myrtillus, Eriksson, Fung. Par. Scand., 286a; on V. uliginosum, G. von. Lagerheim, Briosi & Cavara, Fung. Par., 261. Washington: on Vaccinium membranaceum, Mt. Rainier, C. V. Piper, 443. 3. E. Symploci Ell. & Mart. Am. Nat. 18: 1147. 1884. Fructification amphigenous, resupinate, effused, consisting of lax, slender, colorless hyphae which bear solitary conidia at the tips of very short, lateral, ascending branches; conidia colorless, even slightly curved, acicular, 7-24x1-2 u; basidia and basidiospores unknown. Parasitic on Symplocos tinctoria which produces bud galls 3-34 cm. in diameter, lemon yellow, subglobose and sublobate. Florida, Alabama, and Indiana. March and April. In the original description it is stated that the galls are dis- torted flower buds. In a specimen collected in Indiana, the gall is a partially developed leaf bud. Specimens examined: Exsiceati: Ell. & Ev., N. Am. Fung., 1696. Florida: on Symplocos tinctoria, Green Cove Springs, G. Martin (in Mo. Bot. Gard. Herb., 4968) ; and in Ell. & Ev., N. Am. Fung., 1696. (Vou. 2, 1915] 656 ANNALS OF THE MISSOURI BOTANICAL GARDEN Alabama: on Symplocos tinctoria, Auburn, Ala. Biol. Surv. (in. Mo. Bot. Gard. Herb., 4969). Indiana: on Symplocos tinctoria, Robertsdale, A. M. Rhodes (in Mo. Bot. Gard. Herb., 741178). SPECIES IMPERFECTLY KNOWN E. decolorans Harkness, Cal. Acad. Sci. Bul. 1: 31. 1884. “Receptaculum effused, producing conspicuous yellowish- white, orbicular spots, 1-2 em. in diameter, not at all distort- ing the leaf; spores appearing upon the under surface, hyaline, straight, p 7-8 X 4-5. “On living leaves of Rhododendron occidentale. Tamalpais [Cal.]. Autumn. 2887.’’ The above is the original description. I have seen no speci- mens referable here nor on the host stated. EXCLUDED SPECIES E. mycetophilum Peck ex Burt, Torr. Bot. Club Bul. 28: 285-287. pl. 23. 1901. Tremella mycetophila Peck, N. Y. State Mus., Bul. 28: 53. pl. 1. f.4. 1879. This curious structure on Collybia dryophila, I no longer regard as parasitic but, rather, as a teratological production of C. dryophila, induced by protracted wet weather during development of the fructification. (To be continued. ) (Vou, 2, 1915] 658 ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 21. is plate is a photographic reproduction, XH» of Plate 1 by colo and with all figures natural size; “red colors of the orig- inal cn photographed light colored. g. Leaf spot gall, on left side of uppermost leaf; the leaf is reddish on the apere side in the infested area, not deformed, and was felty or scurfy on the lower s Fi Se Karin galls. More or less deformation of the infected region is present he her Figs. 10-15. Shoot galls of the wax-like or coralloid type. Extended por tions of peer shoots are infected. Figure 11 shows whole branchlets cothiplitaly hypertrophied. Figs. 16-17. Flower galls borne on, and a part of, shoot galls. Fig. 18. Flower gall. Local infection of a single flower, noted as the only such instance observed. 1 loc. cit. PLATE 21 Ann. Mo. Bor. GARD., Vor. 2, 1915 Hhßeronin del. BURT—THELEPHORACEAE OF NORTH AMERICA Jab / € lane lich. COCKAYNE, BOSTON Annals of the Missouri Botanical Garden Vou. 2 NOVEMBER, 1915 No. 4 TOXICITY OF GALACTOSE FOR CERTAIN OF THE HIGHER PLANTS! LEWIS KNUDSON Assistant Professor of Plant Physiology, Cornell University In the course of investigations upon the effect of sugars on the growth of certain higher plants, the sugar galactose was employed. In experiments with vetch (Vicia villosa) the plants grown in the presence of 2 per cent galactose showed very marked injury, the injury being especially manifest by a killing of the roots and accompanied by a reduction in the growth of tops. The results secured were the more surpris- ing in view of the fact that lactose sugar employed coin- cidently influenced beneficially the growth of the same plant. Certain experiments were therefore made to determine whether or not the effect of the galactose was consistent. Method of experimentation.—The plants were grown under sterile conditions on agar media containing Pfeffer’s nutrient solution? of one-half its normal strength. This solution is neutral in its reaction. The solution contained varying amounts of galactose sugar, the source of which is indicated in each case. he writer acknowledges gladly his indebtedness to the officers of the Mis- souri Botanical Garden for facilities and courtesies extended to him during his stay in St. Louis. N Ass A eee CER eT EME SCROTAL ER TOR A ee eeeee 2 s EN Ope ee en TE a nto nan 0.5 grams KOR ne a a coe ee a Se 0.25 grams E HRO a bs eae Ee ESS REET 0.50 grams RS een tees e a E NAE RO er eae 0.50 grams PC E nern. ee a ee E ame + milligrams PN 558 00 ae eel Seca eee eee ee On 6 8 ANN. Mo. Bor. GARD., VoL. 2, 1915 (659) [VoL. 2 660 ANNALS OF THE MISSOURI BOTANICAL GARDEN The seed employed were sterilized by means of a method devised in the Laboratory of Plant Physiology of Cornell University by Dr. J. K. Wilson.’ In brief it is as follows: 10 grams of chloride of lime are shaken up with 150 ec. tap water and after standing for ten minutes the supernatant liquid is filtered. The filtrate is used as the sterilizing agent. The seeds are placed in a test-tube covered with about five times their volume of the filtrate and the tube then tightly stoppered. The seeds are treated for from 4 to 24 hours, depending upon the character of the seed. In the experiments here mentioned the vetch seeds were exposed to this treatment for 12 hours and the peas for 4 hours. The seeds are directly transferred to the culture vessels from the chloride of lime solution, care being observed to drain off all of the chloride of lime solu- tion. In transferring the seed the usual bacteriological pre- cautions are observed. Experiment with vetch (Vicia villosa).—The plants were grown in large glass cylinders 60 em. high and 10 em. in diameter, having a volume of approximately 4 liters. In each of the cylinders were placed 250 ec. of the nutrient solution plus 1 per cent washed agar and galactose sugar. The cylinders were then fitted with cotton plugs and sterilized for one hour in an autoclave at a pressure of 15 pounds. The cultures were made in triplicate and the galactose was tested at 2 per cent and at 0.2 per cent concentration. After a growth period of 30 days the cultures showed the injurious action of the galactose, in each case the roots being markedly injured. The primary root tip coming in contact with the agar medium was killed and the lateral root produced met with the same injury, so that ultimately a multi-branched root system was produced after the manner of the pea roots shown in pl. 22 fig. 5. Whatever portions of the roots re- mained in contact with the agar medium were ultimately killed. It should be mentioned in this connection that the vetch grown in the presence of glucose, saccharose, lactose or maltose at concentrations of 2 per cent was greatly benefited. These sugars are absorbed and assimilated. 1 Am. Jour. Bot. 2: 420-427. 1915. 1915] KNUDSON—TOXICITY OF GALACTOSE 661 Experiment with Canada field pea (Pisum sativum) —In the first experiment with the pea the large cylinders were again employed and to each were added 200 ce. of the nutrient solution plus 1 per cent agar and the sugar whose effect was to be tested. Cultures were made with raffinose, saccharose, lactose, glucose, and galactose (‘‘Merck’s Highest Purity’’), the concentration of the sugar employed in each case being 2 per cent. The cylinders were fitted with cotton plugs as in the previous experiment and then sterilized for a period of one hour. In each cylinder were sown four peas which had TABLE I DATA ON CANADA FIELD PEA (Duration 25 days. Taken February 13) ies Height| Total ey Dry | Dry |Total | Av. | Gain à f | green tw wt. | dry | dry per plants} wt. |jedons| roots | tops | wt. | wt. | plant cm. | grams] or grams | grams | grams | grams | grams Culture o plants 4 Glucose 3 40 |6.250] .155 | .170 | .364 | .689 | .229 | +.085 Lactose 4 40 16.700! .169 | .105 | .355 | .629 | .157 |+.007 *Raffinose 4 | 33 |6.500| .192 | .130 | .328 | .650 | .162 | +.012 Saccharose 4 = 7.600| .160 | .144 | .430 | .734 | .183 | +.036 5 2 Check 3 23 |4.450} .150 | .075 | .190 | .415 | .138 | —.012 4 Maltose 4 20 16.600] .222 | .142 | .386 | .750 | .187 + .034 Galactose Plants small and roots injured. (See pl. 22, figs. la and 5.) * Reducing sugar formed in medium probably as a result of secretion of invertase and raffinase from roots. Acidity of entire medium at time of examination equiva- lent to 0.7 cc. N/10 KOH. [Vou, 2 662 ANNALS OF THE MISSOURI BOTANICAL GARDEN been sterilized by the method described. The plants were grown for a period of twenty-five days and then data taken on the various cultures. The various cultures are shown in pl. 22 fig. 1. The galactose plants are separately shown in pl. 22 fig. 5 and the detailed data are given in table 1. An examination of the table reveals the fact that every sugar acted beneficially except galactose. If the plants had been examined a month later (as was the case with other cul- tures), much greater differences would have been secured between the check cultures and the sugar-containing cultures. Lactose is undoubtedly utilized by Canada field pea as well as by vetch and probably before assimilation is converted into glucose and galactose. Raffinose, which is also utilized, yields on hydrolysis first levulose and melibiose, and the latter is further transformed to galactose and dextrose. In the light of the foregoing, it would appear from the results secured with lactose and raffinose that levulose and glucose must exert some protective action against the injurious action of galactose. Influence of concentration of galactose.—In all of the pre- vious experiments the galactose sugar was employed at only two concentrations, namely, 0.2 per cent and 2 per cent. In the following ee a series of cultures was made con- taining galactose at the following concentrations: 0.125 per cent, 0.25 per cent, 0.50 per cent, 1.0 per cent, 2.0 per cent, and control cultures lacking galactose. The plants were grown in large test-tubes 30 cm. X 4 cm., containing 50 ce. of the nutrient medium plus 1 per cent agar. The galactose sugar employed in this experiment was provided by Dr. ©. S. Hud- sont, Chief of the Carbohydrate Laboratory, U. S. Bureau of Chemistry. The galactose sugar provided had been reerystal- lized and was stated by Dr. Hudson to be of a very high degree of purity and probably purer than any which could be secured upon the market. The tubes were plugged with eotton and sterilized in an autoclave at 15 pounds pressure for a period of 20 minutes. One pea was sown in each tube and the cul- * The writer gratefully acknowledges his indebtedness to Dr. Hudson for the galactose furnished. 1915] KNUDSON—TOXICITY OF GALACTOSE 663 tures made in triplicate. The seeds germinated in four days and even by this time in the higher concentrations of galac- tose, browning of the cotyledons was becoming evident. This browning of the cotyledons intensified with time and at the end of 20 days the peas in the 1 per cent and 2 per cent galactose cultures showed marked discoloration, and death of roots soon occurred. The height of tops was also markedly affected in the presence of galactose of a concentration of 1 per cent or over. (See pl. 22 fig. 4.) The above experiments were repeated with wheat and corn and the results secured were similar. Antagonistic action of glucose toward toxicity of galactose. —It was noted previously that raffinose and lactose are utilized by Canada field pea, and this has been verified by other experiments. The use of lactose by vetch has also been decidedly shown by experiments not yet reported. Since both lactose and raffinose are assimilated by pea and vetch, and since it is highly probable, as previously suggested, that these sugars are hydrolyzed before assimilation, it is possible that the glucose and levulose exercise a protective action against the galactose. An experiment was made to test the hypothesis with respect to glucose. Test-tube cultures were prepared as in the pre- vious experiment, but in this case were made in quadrupli- cate. One series contained 1 per cent glucose plus 1 per cent galactose and the second series contained 1 per cent galactose alone. The plants were grown for 25 days in the greenhouse and the general results are clearly evident in pl. 22 figs. 2 and 3. In the case of the 1 per cent galactose culture the pri- mary roots were killed, but with the 1 per cent glucose added, the primary root tip was killed and the epidermis and part of the cortex, but the inner part of the root was not appar- ently injured, for secondary roots developed which seemed to be more resistant to the toxic action of the galactose, for these root tips suffered no injury and not even a browning of the root was secured as was the case with the primary root (pl. 22 fig. 3). The experiment was repeated a second time and the results secured are concordant with the first. [Vou. 2, 1915] 664 ANNALS OF THE MISSOURI BOTANICAL GARDEN Discussion. —So far as the writer has been able to discover, no previous mention has been made of the toxic nature of galactose for plants. Molliard!, however, intimates that galactose is toxic for radish, for unlike other sugars, the galactose permitted no development beyond a 5 per cent con- centration and with 2 per cent galactose the plants are very small. He concludes that galactose is not utilized by radish. That galactose is injurious to the green plants employed is definitely shown. It does not appear to be toxic to fungi since Aspergillus niger, several species of Penicillium, a species of Fusarium, and a species of Mucor were all found growing in cultures which became contaminated. It is defin- itely known also that certain yeasts are able to ferment galac- tose. The character of the injury effected by the galactose in the above experiment and the method of action have not yet been determined. Incidental observations indicate that the galactose on penetrating kills the cells in its path. In the case of peas grown on 1 per cent galactose the peripheral layers of the cotyledon showed the original starch reserve undigested. In the presence of glucose it was only the epi- dermis and part of the cortex which suffered injury. It would appear that the outer layers of cells were injured before sufli- cient glucose had accumulated to render them resistant to the toxic action of galactose, or perhaps the penetrability of the inner cells for galactose was altered by the presence of glu- cose. In what manner the glucose antidotes the toxicity of galactose cannot yet be stated. It may be possible that it is the oxidation products of galactose that are the injurious agents and that the glucose prevents the formation, or modifies the character, of the oxidation products and that the toxicity is thereby overcome. Investigation into other phases is in progress. 1 Molliard, Marin. — Sea ng er de ie rg substances organiques sur les végétaux supérieurs. . Gén. de Bot. 19: p. . 1907. [VoL. 2, 1915] ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 22 Fig. 1. a, ou. 2 = cent; b, lactose 2 per gee c, check—no sugar; d, raffinose 2 per cent; e, saccharose 2 per cent; f, glucose 2 per cent. (Coven Dinge were removed at the me of photo- Fig. 2. The two outside tubes contain 1 per cent galactose while the en middle ones contain 1 per cent galactose plus 1 per cent gluco Fig. 3. The small plant was grown on 1 per cent er the larger one on 1 per cent galactose plus 1 per cent gluc Fig. 4. a and b, 2 per cent galactose; c, 1 per cent galactose; d, 0.500 per cent galactose; e, 0.250 a cent alae: f, 9.125 per cent galactose; g, check—no galactos Fig. 5. Peas (shown in fig. la), showing character of root grow when tips alone come in contact with 2 per cent galactose-containing medium. Ann. Mo. Bor. GARD., Vor. 2, 1915 PLATE 22 KNUDSON—TOXICITY OF GALACTOSE COCKAYNE, BOSTON COMPARATIVE STUDIES IN THE POLYPORACEAE L. O. OVERHOLTS Formerly Rufus J. Lackland Fellow in the Henry Shaw School of Botany of Washington University The subclass Basidiomycetes of the class Fungi contains a natural group of plants sharply separated from related groups in that the hymenium (basidia, paraphyses, ete.) forms the lining of hollow tubes on the ventral surface of the fruit body. This group of plants constitutes the tribe Polyporeae. It is divided into two families, the Boletaceae and the Polyp- oraceae. The Boletaceae are separated from the Polyp- oraceae in that they are fleshy and soon decay and the tubes are easily separated from the pileus, while the Polyporaceae vary in texture from coriaceous to hard and woody, and the tubes are inseparable from the pileus. These characters are susceptible of some variation, as there are a very few fleshy species in the latter family, and in two or three cases the hymenium is waxy and the tubes separable. In this article we are concerned only with the Polyporaceae. HISTORICAL Accurate knowledge of the classification of the Polyporeae dates back only to the last few years of the eighteenth or the beginning of the nineteenth century. The first attempt worthy of consideration was that of Persoon in 1801, although we still have occasion to refer to articles by earlier writers, especially Bulliard (Herbier de la France, 1780-1793), Schaeffer (Fung. Bav. 1780), and Sowerby (Eng. Fung. 1797-1809). These three, while contributing considerable in the way of illustra- tions of the species known at that time, knew very little about the correct classification of the species they illustrated. The binomial method of naming species had come into general use following its introduction by Linnaeus (Species Plan- tarum) in 1753, and many new species were described in the succeeding years, but the descriptions were inadequate and ANN. Mo. Bor. GARD., VOL. 2, 1915 (667) [VoL. 2 668 ANNALS OF THE MISSOURI BOTANICAL GARDEN the type specimens not preserved, so that it is impossible to tell to what plants the descriptions refer. By the beginning of the nineteenth century those interested in this line of study had begun to feel the need of permanent herbaria containing specimens of all the species described. The appreciation of this need augmented the demand for a more systematic and a more natural arrangement of the genera and species of fungi. It thus came about that while Linnaeus in 1753 had listed but one genus, Boletus, and 12 species of pore fungi (Bole- taceae and Polyporaceae), the number of genera had in- creased to 3 and the number of species to 93 when Persoon published his ‘Synopsis Fungorum,’ in 1801. This was fol- lowed by the work of Albertini and Schweinitz (Conspectus Fungorum) in 1805, which was modeled after the work of Persoon and contributes nothing to the systematic arrange- ment of the Polyporeae. It must not be supposed, however, that there was any extraordinary change from the incomplete descriptions of the earlier writers to a more or less perfect standard of description that should include all the facts neces- sary for the identification of the species. The descriptions in Persoon’s ‘Synopsis’ were still far from what could be de- sired, and it is only where these are supplemented by her- barium specimens or by accurate illustrations or by both that the species can be identified beyond all doubt. But the fact remains that the beginning of the nineteenth century wit- nessed a growing inclination on the part of mycological systematists toward a form of record for the species that would be more concrete in its conception and thus give an added impetus to the study of the fungi. Among the vast array of mycologists produced in the nine- teenth century by far the most prominent was Elias Fries. His first work of importance was the ‘Systema Mycologicum,’ published in 1821-1832, in which the known fungi were mar- shalled in order. To the genera of the Polyporeae listed by Persoon he added the genus Polyporus (first proposed by Micheli in the eighteenth century) and thus made the first attempt to separate the Boletaceae from the Polyporaceae. 1915] OVERHOLTS—STUDIES IN THE POLYPORACEAE 669 The genera treated by him contained 164 species in all, of which probably two-thirds were in the single genus Polyporus. This genus was divided into 3 sections, Favolus, Microporus, and Polystictus, the first named being later raised to generic rank. The section Microporus contained by far the largest number of species. It was divided into 5 subgenera: Mesopus, Pleuropus, Merisma, Apus, and Resupinatus. This arrange- ment was continued in his ‘Epicrisis Systema Mycologicum,’ published in 1836-38. In the meanwhile the genera Trametes, Cyclomyces, Hexagona, Favolus, Laschia, and Porothelium had been carved from the old genus Polyporus, and the num- ber of species described had increased to 361 (entirely exclu- sive of the genus Boletus). Of these, 280 were included in the genus Polyporus. The same disposition of the pore fungi was followed by Fries in his last publication, ‘Hy cetes Europaei,’ in 1874, and, indeed, that system has er been followed in its entirety since or has served as a founda- tion for all other systems of classification that have been pro- posed from time to time by others. Correlated with the increase in the number of described species there is manifest a tendency on the part of some later writers toward a change in the conception of what should constitute a genus. There has been a tendency away from the old idea of large genera containing a heterogeneous col- lection of species, and toward the breaking up of genera into smaller units consisting of closely related individuals. This tendency finds its best expression in the work of Karsten, Quelet, and Murrill, each of whom has published papers deal- ing with the classification of the Polyporaceae. IMPORTANT MICROSCOPIC CHARACTERS USED BY EARLIER WORKERS Having glanced at the beginnings of the various classifica- tions that have been proposed, we may now turn our atten- tion to an analysis of the characters used in separating genera and species. For the most part the generic characters were macroscopic ones, such as presence or absence of a stipe, con- sistency of the sporophore, nature of the hymenium, ete.,— characters that arrested the attention of the collector without [Vou 2 670 ANNALS OF THE MISSOURI BOTANICAL GARDEN recourse to the microscope, for the microscope was unknown when the foundations of this study were laid. In the separa- tion of species other macroscopic characters of minor import- ance were used. Color, pubescence, habitat, form, size, etc., were characters that were largely drawn upon in fixing the limits of species. It was unfortunate, however, that though the characters named are the most conspicuous ones, yet they are more sub- ject to modification and variation than are certain internal characters that require the use of the microscope for their detection. Perhaps the desideratum in systematic botany would be a classification in which genera are well defined and sharply separated from each other by gross morphological characters, and in which the microscope would be necessary only in determining specific characters. Perhaps this demand is more nearly filled in the family Agaricaceae than in any other group of the fungi. There the genera are divided into sections on the color of the spores, and the genera in these sections are more or less well differentiated on gross morpho- logical characters. In those groups of the fungi that have been most carefully studied, e. æ., the Myxomycetes, considerable attention has been paid to the minute anatomical structure of the plant. Spore markings that are scarcely visible, except with an oil- immersion lens, have been used as points of separation in closely related forms, and in certain of the Discomycetes the spore markings and the nature of the paraphyses have been largely drawn upon to furnish specific characters. Durand? has gone somewhat farther, and in his studies in the fleshy Pezizineae has taken into account the structure of the apothecium in fixing the limits of the families. Burt? has recently set new limits to some of the genera of the Thele- phoraceae, in keeping with their inner anatomical structure. In the Polyporaceae, Miss Ames? has recently attempted to outline a scheme of classification of the genera based largely on the structure of the sporophores, but only a few forms 1 Bul. Tor. Bot. Club 27: .. en 2 Ann. Mo. Bot. Gard. 1: —196. š Ann. Myc. 11: 211-253. nn 1915] OVERHOLTS—STUDIES IN THE POLYPORACEAE 671 were investigated and the results not as satisfactory as could be desired. It is a significant fact, however, that no attempt has been made to classify the Polyporaceae on the basis of spore or other hymenial characters, although it is recognized that, out- side of the algae, the organs concerned in reproduction are usually subject to less variation than are external morpho- logical characters. That no such attempt has been made is due to two causes: first, the dislike on the part of students of the careful and painstaking observations that must often be made to determine those characters; and second, to the wide- spread belief that the pore fungi are spore-bearing only for a short interval of time during the year, and that they must be examined at the right moment or the spores will have dis- appeared. When it has been shown that the second objection is invalid and that hymenial characters are usually not hard to make out, the first objection will largely disappear. In the course of the last year the writer has spent a con- siderable portion of his time in searching for these char- acters, not only in the Polyporaceae but in other related families as well. The methods employed are given on a fol- lowing page, and suffice it to say here that probably 75 per cent of the collections examined contained spores, and a large percentage afforded other microscopic characters that played a considerable part in distinguishing one species from another. The characters that may be obtained by the use of the micro- scope are here enumerated and some indication given as to their possible value. Discussion or Microscopic CHARACTERS Now AVAILABLE FOR USE as Generic AND SPECIFIC CHARACTERS The characters that may be obtained by the methods out- lined on a following page are as follows: spore characters, presence or absence of cystidia, setae and other sterile organs in the hymenium, basidial characters, hyphal characters, and the presence or absence of sterile structures in the sub- hymenial tissue. [VoL, 2 672 ANNALS OF THE MISSOURI BOTANICAL GARDEN Spore characters. —Spore characters are probably worthy of a great deal more consideration than they have yet received in the greater part of the mycological work that has been done up to the present time. As previously stated, in the A gari- caceae the primary divisions of the family are made on the basis of spore colors. This distinction was made as early as 1821 by Fries in his ‘Systema Mycologicum.’ The fact that this character was so early recognized was not because spores are more abundant or their colors more striking in the gill fungi, but because the period of spore production more closely coincides with the period of maximum development of the plants. Unfavorable conditions, i. e., drought, super- abundance of moisture, cold, ete., result in the disorganiza- tion of the tissue in a fleshy fungus, and consequently the duration of the period of spore liberation is permanently shortened. In the coriaceous or woody forms these same con- ditions result only in a temporary suspension of the act of spore liberation and with the return of normal conditions the suspended function again becomes active. In this way the period during which spores are present in the hymenium of a pore fungus is greatly lengthened, and it is safe to assume that the number of mature spores present at a given time in the hymenium of one of the more durable pore fungi is less than the number of mature spores on an equal hymenial sur- face of a gill fungus. Contrary to the condition in the Agaricaceae, the introduction of spore colors as generic char- acteristics would mean an entire revision of all the genera, and it may well be doubted whether the advantage obtained from such a limitation of genera would compensate for the confusion that would be sure to arise. On this basis, how- ever, the species could easily be grouped into sections under the genera, but even were that done the white-spored species so far outnumber those with colored spores that the adoption of the idea would delimit only a small group of species that perhaps could be better separated in other ways. Very little exact evidence bearing on the variation in size in the spores of a given species is obtainable. The work of 1915] OVERHOLTS—STUDIES IN THE POLYPORACEAE 673 Falck! showed that the mature spores of certain species of Lenzites were very constant in the length of their short axes, the variations being only a fraction of one micron, while the length of the long axis varied considerably, although in that case the variation rarely went beyond 3 u in different spores from different fruit bodies. Cotton? investigated variations in the spores of Stropharia semiglobata and found that when the pileus was cut from the stem and a series of spore prints obtained from the former, the spores shed during the first hour measured 18 X 10 u, while those shed during the twenty- third hour measured 15 X 9 yw, and those shed during the eighty-third hour measured only 12 X 7 u. The diminution in size was ascribed to the artificial conditions, i. e., the pileus be- ing severed from the stipe, under which the spores were pro- duced. Experiments carried on with sporophores collected and placed in large test-tubes and supplied with water, showed that the spores shed the first day did not differ in size from those shed during the fifth or sixth day. The first experiment suggests the possibility that in plants growing in nature the size of the spores might be reduced if the fungus was grow- ing on a substratum in which the required amount of food substances was not present. No comparative studies along this line have yet been reported and the question of the amount of variation in size of spores is still an open one. However, spore measurements have been very successfully used in separating species of fungi and no doubt the limit of their usefulness has not yet been reached in systematic mycology. Inaccurate spore measurements may creep into the litera- ture through a misdetermination of species quite as easily as species may be misdetermined because of inaccurate spore measurements. The former condition is especially liable to be pronounced in the literature of a fungous flora as little known as is that of this country, and where species are not determined on microscopic characters, but these same char- acters are entered in the literature when the species is re- * Moeller’s Hausschwamm-forschungen, Heft 3, pp. 79-96. 1909. 2 Trans. Brit. Myc. Soc. 4; 298-300. 1914. [VoL. 2 674 ANNALS OF THE MISSOURI BOTANICAL GARDEN corded. This latter procedure is entirely commendable, but it has been so much abused that the spore characters carried in the literature are far from being reliable in a large num- ber of cases. However, allowances must be made for some variation in measurement by different individuals as no two persons will report exactly the same measurements for one species. The shape of the spores is probably subject to somewhat less variation with age than is the size. Spores begin to take their characteristic shape while they are yet comparatively immature and from seeing such a spore one can judge of its mature form more accurately than of its mature size. Often the spores of two or more species are so similar in shape that it is perhaps best not to try to distinguish between them, al- though the distinction may be perfectly apparent to one who has before him the spores of all the species in question. The terms used to describe spore forms are not as rigidly defined as we could wish, and it does not add to the clearness of dis- tinction between two species to describe the spores of one as “elongate-ellipsoid’’ and of the other as ‘‘narrowly fusoid”’ and expect the users of the manual to distinguish the species on that basis. There are many cases, however, where the form of the spores may be used to good advantage. Spore markings are so universally absent in the Polyp- oraceae that the subject requires very little comment here. There are probably not more than a dozen species that are characterized in this way and they are so widely separated that the character is given an added value. In some groups of the fungi, especially among the Ascomycetes, not only the presence or absence of markings on the spore wall but also the nature of these markings is taken into account. Cystidia.—Cystidia may be defined as more or less con- spicuous sterile organs found either in the hymenium or in the subhymenial tissue of various basidiomycetous fungi. They are usually unicellular and they may be smooth or they may have a more or less incrusted surface, the incrusting substance probably always being calcium oxalate. The name ‘¢setae’’ has been given to these bodies when they are colored 1915] OVERHOLTS—STUDIES IN THE POLYPORACEAE 675 (usually brown) and sharp-pointed, and that distinction is maintained in this paper, although there may be some doubt as to the advantage that accrues from its use. The presence or absence of setae has been made a generic character in some groups of the Basidiomycetes, and even in the Polyporaceae the genus Mucronoporus was founded by Ellis and Everhart on the presence of the setae in the hymenium. The genus probably has not received the acceptance that it has deserved at the hands of mycologists. It is difficult to say at times whether a given structure should be designated as a cystidium or not, but the writer is of the opinion that the term should be used in its broadest sense, except that it should not be ap- plied to those structures usually referred to as paraphyses. These latter can usually be distinguished by the frequency of their occurrence as they usually alternate with the basidia, while cystidia or setae are scattered irregularly through the hymenium. In by far the largest number of cases the cystidia are very conspicuous on account of their size, coloration, in- crustation, or other characters. In a few cases the presence or absence of setae is a variable character, in some specimens being abundant and in others very scarce. In such cases the writer has found it advisable to make longitudinal sections of the tubes, as the setae are sometimes more abundant in one part of the tubes than in another. A cross-section of the tubes of Fomes igniarius will sometimes fail to show a single seta, but in only one specimen has the writer failed to locate them in longitudinal sections from the hymenium of the same plant. They are also almost entirely lacking in some speci- mens of Polyporus dryophilus. Basidia.—It is very seldom that the basidia offer char- acters that can be used in separating species. They are al- most universally 4-spored in the Polyporaceae and in those few species where 2- and 3-spored basidia do occur there are always a goodly number of 4-spored ones present also. In a very few cases the basidia are conspicuous on account of their large size. This is true of Trametes Peckw where they are 8-10 » broad, while usually they vary from 3 to 6 u broad. [VoL 2 676 ANNALS OF THE MISSOURI BOTANICAL GARDEN Hyphal characters—The characters of the hyphae that make up the subhymenial tissue and the tissue of the trama of the pileus have never been used in the classification of the Polyporaceae. While the size of the hyphae may depend to a considerable degree on the food supply of the plant, yet in examining a large number of species the writer has found that some are characterized by hyphae two to three times as large as in most species. These cases have been thoroughly investi- gated as far as herbarium material would permit and as all specimens have showed the character about equally well, it has been taken as a means of identifying the species in which it has occurred. The writer knows of no factor or combina- tion of factors that would be operative on a large number of individuals from widely separated localities and in the case of but a limited number of species. If it be dependent on nutrition, then the species possessing this character are so constantly associated with that kind of nutrition that the character is as constant a one as can be obtained. The same is true of the unbranched hyphae of the context of Polyporus albellus. Incrustation of the hyphae has never been observed in the pileate Polyporaceae, though it is a well-marked character in the species of certain groups of resupinate fungi. METHODS EMPLOYED A few words may not be amiss here concerning the methods employed by the writer in obtaining these microscopic char- acters. In general the method is that already described in a previous number of this journal.! Obtaining spore prints.—In the case of fresh specimens just brought into the laboratory from the field, spore prints are very easily obtained by placing the specimens on a glass slide in such a manner that the tubes are in a perpendicular position so that the spores do not lodge on the sides of the tubes when they are liberated from the basidia. The slide with the fungus in position should be either wrapped in waxed paper or left over night or for several hours in the collecting 1 Burt, E. A. loc. cit. 1915] OVERHOLTS—STUDIES IN THE POLYPORACEAE 677 basket or other receptacle in which a fairly high humidity will be maintained, so that the liberation of the spores will not be prematurely stopped by the drying-out of the tissues of the fungus. If the specimens are dry when brought into the laboratory they may be moistened thoroughly with water and then treated as described above. One unaccustomed to this procedure will be surprised to find how large a percentage of the collections so treated will produce a good spore print. Specimens collected on the warm days that frequently come in January and February have often been treated in the above manner with gratifying and surprising results. When desic- cation takes place by exposure to the air the vitality of many species is not destroyed. Buller! was able to restore normal vitality to such plants by placing wet cotton-wool on their upper surfaces. He was even able to revive the fruit bodies of Daedalea unicolor after they had been exposed to ordi- nary air at room temperatures for eight years and three months, and of Schizophyllum commune after an exposure of six years and three months. In most species, e. g., Polyporus versicolor, P. hirsutus, and Lenzites betulina the vitality was retained for a period of but two to three years. Sectional preparations.—In case one is working with ma- terial that has been in the herbarium for several years the above method will not answer. Neither does it furnish any evidence as to the other microscopie characters of the plants. One must then resort to sectional preparations. These are eut free-hand with a very sharp sectioning razor. Free-hand sections are quickly made and the results from them are usually better than from microtome sections. It is impossible for the spores to retain their position on the basidia when subjected to the different processes involved in preparing ma- terial for microtome sectioning. The first requisite in suc- cessful free-hand sectioning is material in good condition; the second is avery sharp razor (preferably flooded with alcohol) ; the third is some little skill and experience. The hymenium of the specimen is first moistened with alcohol, then with water, 1 Researches on fungi, pp. 105-111. 1909; and in Trans. Brit. Myc. Soc. 4: 106-112. 1913. (VoL, 2 678 ANNALS OF THE MISSOURI BOTANICAL GARDEN and a piece about 2 mm. square on the hymenial surface is cut out with a scalpel. If material is abundant the process may be reversed and a larger piece than needed may be cut out with the scalpel, trimmed to the requisite size, immersed in 95 per cent alcohol for a few seconds and then transferred to water. In the writer’s experience the latter method is the more pref- erable and has probably been the one most used. The ma- terial does not soften while in alcohol, but that reagent is used only to facilitate the absorption of water by the tissue. Any rigidity that may be imparted to the tissue by the alcohol is probably overcome when the material is transferred to water. In some cases when this transfer is made the tissue either becomes very soft or very friable so that no razor, how- ever keen, will cut a clean section through it. It is here that the latter method obtains preference over the former, for after some experience one can judge of the probable effect the water will have and by shortening the period that the material remains in the water the tissue is in better condition for sec- tioning. The most instructive preparations are often those contain- ing both longitudinal and cross-sections of the tubes. Such sections are easily obtained in one mount by cutting out the piece of material somewhat longer in one direction than stated above—say about 2 X 4 mm. on the surface. Several longi- tudinal sections may be cut from this and the position of the remaining bit of tissue so changed that cross-sections may be obtained. For sectioning, the tissue is placed in the proper position in a piece of pith and as the sections are cut they may either be transferred directly to the slide by means of a camel’s- hair brush dipped in alcohol, or they may be allowed to ac- cumulate in the alcohol on the razor and then flooded off into a watch-glass containing alcohol. By the last method one can pick out with more accuracy the thinner sections by observing them under the lens of a low-power dissecting microscope. The writer has found it to be sufficient in most cases to trans- fer the sections directly to the slide, disregarding the thicker sections that are cut, or brushing them off the edge of the 1915] OVERHOLTS—-STUDIES IN THE POLYPORACEAE 679 razor with an outward stroke of the finger. The sections are placed in a drop of 7 per cent KOH solution on the slide. This immediately expands the hyphae of the tissue to their normal size. The KOH solution is then drained off and a drop of stain added. Staining and mounting.—I have tested a considerable num- ber of the more common stains and so far I have failed to find one that gives universally good results if the sections are to be made into permanent mounts. For temporary mounts there is nothing superior to a 1 per cent water solution of eosin, but when sections so stained are mounted in glycerin the color soon completely disappears. The same strength solution of alcohol eosin (in 95 per cent alcohol) often gave a good permanent stain but quite as often it, too, faded out in the course of several weeks, and when used it gives a pre- cipitate that must be washed off with water before the cover glass is applied. Why this stain should remain permanent in some cases and not in others is a question that has not been answered. It may be due to the KOH that remains on the slide and in the sections, but flooding the sections with water after draining off the KOH solution did not seem to have any beneficial effect. Different strengths of alcohol were used in preparing the stain, but with alcohols weaker than 95 per cent the stain disappeared even more quickly and the precipitation obtained was so great that such stains were of no value. From the facts observed it seems more reasonable to suppose that the difference may be in the tissue of the fungus rather than in the stain or the glycerin. A solution con- taining equal parts of a 1 per cent water solution and a 1 per cent alcoholic solution of eosin gave no better results. Magdala red, Congo red, neutral red, acid fuchsin, methylen blue, and saffranin T were used, and of these, only the last one gave a permanent stain and it has been used in a large part of the work. It is a rapid stain, though probably not quite so rapid as alcoholic eosin, and it is well to leave the stain on the sections for about one minute. A 1 per cent alcoholic solution was used, the stain being dissolved in 95 per cent alcohol. When a drop of this stain is added and drained [VoL. 2 680 ANNALS OF THE MISSOURI BOTANICAL GARDEN off, the sections must not be allowed to become dry or an orange precipitate is obtained that necessitates the addition of alcohol to dissolve it. This also dissolves the stain from the tissues and the sections must be restained. This pre- cipitate is not formed if a little water is added to the stain after it is made up. This stain imparts a uniform dull red color to the tissue but the color brightens when glycerin is drawn under the cover glass. Since it is not a differential stain its use is not advised where only temporary mounts are desired. It gives best results with very thin sections or with sections in which the hyphae are loosely arranged. After the cover glass is applied the sections are ready to be examined under the microscope, but if the saffranin T stain is used, it is better to place a drop of glycerin at one side of the cover glass, at the same time drawing off some of the surplus water from the opposite side by means of filter paper. Several slides of each species are retained and mounted in 66 per cent glycerin. After a week or more all traces of the glycerin are removed from near the outer edge of the cover glass by means of a soft eloth dipped in 95 per cent alcohol. The slides are then ringed with some suitable cement—gold-size being most often used—labeled, and filed away in order. It will usually facilitate subsequent examina- tion of the slides if the spore characters for each species are written on a slip of gummed paper and glued to one end of the slide. It is sometimes quite impossible to find spores in the sec- tions treated in the manner outlined above, since they are often easily removed from the sterigmata and washed away before the cover glass is applied. To overcome this difficulty the writer sometimes finds it advisable to distribute between two slides the sections obtained, one slide to be treated as outlined above, the other to be mounted for temporary ob- servation only. This last one should be stained with a water solution of 1 per cent eosin, a drop of the solution being added to the drop of KOH containing the sections. Sometimes the staining is unnecessary, especially if one is dealing with species which have colored hyphae and colored spores. A 1915] 5 OVERHOLTS—STUDIES IN THE POLYPORACEAE 681 preparation made in this manner will often show spores when other methods of demonstrating them have failed. Even with the most careful manipulation one will some- times fail to find the spores, and, indeed, some species seem to be almost always sterile. In the case of Fomes fomentarius I cut sections of all the specimens available, and only when as a last resort, I sectioned a small and very unpromising specimen did I find the spores. I have been able to locate them in but one of the few speeimens of Polyporus graveolens that were available for examination. As stated above, the literature dealing with American Poly- poraceae contains many inaccurate observations concerning spores. This is due mostly to a lack of care in making sure that a given body in the hymenium is really the spore of the fungus in question. The writer is of the opinion that spores should not be recorded for a collection unless they are ob- tained from a spore print or are seen attached to basidia. The spores found on basidia are usually somewhat immature, at least as regards size, but from their shape one can judge whether the spores found free-floating in the mount have any relation to the species under consideration. Where such free spores alone are present there is always the possibility that they belong to some other fungus and they should not be taken into consideration unless present in large numbers. One must also guard against the fact that the cut ends of hyphae may be in such a position as to appear globose in form and such may be mistaken for spores. Examining the context hyphae.—In obtaining the char- acters of the hyphae of the context a bit of tissue is picked out with the forceps and mounted on a slide in a drop of KOH solution. In the case of some of the species of the genus Fomes where the context is hard and woody, it is usually better to boil a bit of the context in a KOH solution for a few minutes. In this way the tissue is softened and when teased apart on the slide with needles, a cover glass added, and pressure applied, the hyphae will generally separate out so that their characters may be obtained. In all cases the [vVoL. 2 682 ANNALS OF THE MISSOURI BOTANICAL GARDEN hyphal measurements given are for the hyphae in the con- text of the plant and not for those in the subhymenial tissue. STATEMENT OF PROBLEM The writer presents in this paper the results obtained by carefully investigating some of the more common species of pore fungi, using the methods outlined on the preceding pages. There are certain groups of species in the Polyporaceae that are very much in need of just such treatment, and it is to these groups that the writer has turned his attention. The groups consist of closely related species that have been separated heretofore largely on external characters and in a great many cases the results have only led to confusion. The problem, as the writer saw it, was one involving a contribu- tion toward a more exact characterization of these species and their separation, wherever possible or feasible, on some con- stant internal microscopic character. Some species are well enough marked by external characters so that such distine- tions should be used only as supplementary characters, while in other cases the characters obtained by this study should displace those hitherto used. The results obtained were not as gratifying as was expected when the work was undertaken. Only a small beginning has been made, for it is a laborious task involving the cutting and examination of many sections for each species in order to be sure that the characters shown by the first sections are con- stant for all collections of the same species. The work should be carried on although several years would be required for its completion. Permanent mounts of the sections have been made for each species and these are available for future reference. Criticisms and suggestions, both of methods em- ployed and results obtained, are invited and will be given careful consideration. ACKNOWLEDGMENTS . Acknowledgments and thanks are due to Dr. E. A. Burt of the Missouri Botanical Garden for aid, criticism, and ad- vice in the preparation of the paper and for free access to 19157 TER OVERHOLTS—STUDIES IN THE POLYPORACEAE 683 his valuable herbarium; to Dr. B. M. Duggar for his interest in the work and for aid in various ways; to the Missouri Botanical Garden for the use of the herbarium material and library facilities; to Mr. C. G. Lloyd of Cincinnati, Ohio, for aid in determining some of the species. PoLyPoRUS ABIETINUS Dicks. Ex Fries and P. PARGAMENUS FRIES P. abietinus was first described by Dickson,! in 1793, and appears to be almost cosmopolitan in its distribution. In the United States it is found wherever coniferous forests abound, fraza Canada to the Gulf of Mexico, and from the Atlantic to the Pacific Ocean. It is never fowad on the wood of decidu- ous trees, and as will be pointed out later, this fact affords almost the only constant character by means of which it can be separated from its near relative, P. pargamenus. P. pargamenus was described by Fries,? in 1838, from plants collested on pine wood in Arctic America by the Franklin Ex- -pedition. The plant has not been reported from the western coast of the United States, but has been found in practically every state east of the Mississippi River, ranging west as far as Wisconsin, Kansas, Arkansas, and Colorado. It is also found in Europe. Most of the collections in this country under the name P. laceratus Berk., P. xalapensis Berk., or P. ilicincola Berk. and Curt., belong to this species. An ex- —auination of P. pseudopargamenus, as distributed by de- Thuemen,? shows it to be identical with P. pargamenus. The writer has not seen authentic specimens of the other species named above, but they are given as synonyms by Murrill. By some writers the two species have been confused, due to the fact that the type specimens of P. pargamenus were __sieotted as growing on the wood of coniferous trees, while in the United States the plant that has gone under die name P. pargamenus is confined entirely to the wood of deciduous trees. This has led some authors to regard the original P. 1 P]. Crypt. Brit. 3: p. 21. 1793. 2 Epicr. Syst. Myc. p. 480. 1838. ® Myc. Univ. 1102. [VoL. 2 684 ANNALS OF THE MISSOURI BOTANICAL GARDEN pargamenus as probably a synonym for P. abietinus. In that event, the species on the wood of deeiduous trees would have to be given another name. This point can be settled only by a study of the type specimens of P. pargamenus, if they are still preserved. Nearly all the exsiccati material has been dis- tributed under the name P. pargamenus, and the plant is so common and the name so well established that it is the writer’s opinion it should not be changed without recourse to the types. The two species under discussion are very closely related and they are connected by intermediate forms to such an ex- tent that it is difficult to refer some collections to their proper species. However, the usual form of the fructification is dis- tinct enough. P. abietinus is usually much smaller, is fre- quently effused-reflexed with a narrow and often laterally con- tinuous pileus, rarely more than 2 cm. in length, and the tubes sometimes break up into lamellae-like plates—a condition I have never found in P. pargamenus. That species often grows much larger than P. abietinus, sometimes attaining a length of 6-7 cm., and is often fan-shaped or cuneate in out- line and attached by a narrow, attenuate, sometimes stem- like base, so that the form and size of the fruiting body will usually separate it from P. abietinus. The color, zonation, and pubescence of the pileus is similar in both species, though the pubescence is often inclined to be strigose in the latter plant and more velvety in the former. Both species often have a violaceous or lavender tint to the hymenium or on the margin of the pileus. The microscopic appearance of the hymenium of the two species does not furnish additional characters for their sepa- ration. The spores are similar in size and shape, being eylin- drie or sometimes allantoid, hyaline, smooth, and measuring 5-7 x 1.5-2.5 u (not globose, 4.5-5.5 „ as stated by Murrill). Murrill states that no cystidia are present in the hymenium of P. abietinus and to the writer’s knowledge their presence has never been recorded. I have examined several collections of both P. abietinus and P. pargamenus and I find that the plants vary as regards this character. I am of the opinion that cystidia are probably always present, but at times are so rare 1915] OVERHOLTS—STUDIES IN THE POLYPORACEAE 685 or sO inconspicuous that close observation is necessary to detect them and I have often examined whole sections without being able to locate them. A similar section taken elsewhere in the hymenium may show an abundance of them. The ac- companying illustrations (figs. 1 and 2) show the different forms they may assume, but perhaps the most common form is as shown in a of fig. 1. They are often scarcely larger in size than the basidia, but are differ- ent a shape, usually Fig. 1. Section of the hymenium of P. abiet- with the appearance of inus showing cystidia and spores. slender pegs tapering to a rather blunt point. Rarely they are somewhat fusiform in shape and reach a length of 20 „ and a thickness of 6 u. These sizes are unusual, however. They are colorless or almost so, sometimes scarcely extending beyond the basidia, but sometimes projecting enough that one can easily pick them out with the low power of the microscope. They are usually unincrusted, but sometimes their tips are somewhat capitate with small erystals (see fig. 2). They are then much more conspicuous, and in some collections this appears to be the 7 predominating eondition. Fig. 2. Section of the Before the writer had seen this hymenium of P. abietinus A . showing cystidia inerusted more conspicuous type it was thought at the apes. these sterile, inconspicuous structures might be basidia that had discharged their spores and had thus been rendered hyaline, as it is frequently found in other species that the mature spore-bearing basidia project somewhat be- yond those that have not reached maturity. The shape of these bodies and the fact that they often assume a capitate apex, as do cystidia of many other species, make this view untenable. If more proof were needed it might be pointed out that these bodies are present in young specimens and in [VoL 2 686 ANNALS OF THE MISSOURI BOTANICAL GARDEN the growing margins of mature specimens where it is evident that no mature basidia have yet been formed. Neither can these structures be regarded as paraphyses that have become elongated and, therefore, more conspicuous. While there may be no ground for the belief that paraphyses can not assume such a form, yet there is no evidence to indi- cate that conspicuous sterile structures ever have arisen in such a manner. Moreover, the distribution of these struc- tures under consideration makes impossible any such idea, as they are scattered promiscuously and do not alternate with the basidia. These two species then are to be distinguished only by their habitat, and the size and shape of the pileus. In my own col- lecting experience the former character alone is enough to separate them, but when once the two plants are learned, the matter of form and size will usually be sufficient for the identification of the specimens, even if the habitat be unknown. As stated above, the hymenium of P. abietinus may at times be lamellate. This statement is made only after a careful study of the facts in the case. They are as follows: There is a plant with apparently the same distribution as P. abietinus, in which the hymenium is entirely lamellate. No exactly intermediate conditions have ever been seen by the writer, though he has collected both forms in Colorado. In all other characters the two plants are precisely similar. The host is always the wood of coniferous trees; the pubescence and color- ation of the pileus is the same; the spores and cystidia are similar; and the hymenium often has the violaceous tint char- acteristic of P. abietinus. Irpex fuscoviolaceus is in all prob- ability only another form of the same plant, although I have never seen specimens of that species with the well-marked lamellate hymenium of this form. The illustration (pl. 23 fig. 1) is from specimens communicated by Prof. C. R. Orton, of State College, Pennsylvania. He writes that the rot pro- duced by this fungus is almost identical with the one produced by P. abietinus. Patouillard! represents the eystidia of Irpex 1 Hym. Eur. pl. 3. f. 23. 1887. 1915] OVERHOLTS—-STUDIES IN THE POLYPORACEAE 687 fuscoviolaceus as incrusted at the apex in the same manner as shown in the accompanying illustration of P. abietinus. I have also found this condition to be predominant in the lamel- late form of our species. The following comparative synopsis of the two species dis- cussed in this section is appended here: 1. Polyporus abietinus Dicks. ex. Fries. Plate 23, figs. 1, 2. Pileus coriaceous, sessile or effused-reflexed, 0.5-5 X 0.5-5 x 0.1-0.2 cm., white, cinereous, or blackish with age, villous, zonate; context not more than 1 mm. thick; tubes not more than 3 mm. long, the mouths white, bay, or violaceous, averag- ing 2-3 to a mm. in poroid forms, but sometimes entirely lamellate; spores cylindric or allantoid, hyaline, 5-7 x 1.5- 2.5 u; cystidia present or inconspicuous, hyaline, rarely in- crusted at the apex, 3-6 u in diameter, projecting 5-15 p; hyphae of context hyaline, 3-4 a in diameter. On wood of coniferous trees, especially of Pinus. Illustrations: Dicks. Pl. Crypt. Brit. 3: pl. 9. f. 9 —Fl. Dan. pl. 1298, 2079. f. 2.—Gill. Champ. Fr. pl. 463.—Swant. Brit. Fung. pl. 33. f. 2-3. Specimens examined: Barth. Fung. Col. 3108.—Cooke, Brit. Fung. 512, 605.—Thuem. Myc. Univ. 6, 706.—EIll. N. Am. Fung. 8.—Hll. & Ev. Fung. Col. 303.—Krieg. Fung. Sax. 1205. —Rab.-Wint. Fung. Eur. 3235 (as Irpex fuscoviolaceus).— Rav. Fung. Am. 422; Fung. Car. I, 12.—Shear, N. Y. Fung. 307.—Mo. Bot. Gard. Herb. 4726, 4727, 4728 (Newfoundland), 3854, 4213 (New York), 4214 (Labrador), 4220 (Alabama), 4074 (Colorado).—Burt Herb. (collections from Vermont and Washington).—Overholts Herb. 2001 (Colorado), 2465 (Penn- sylvania), 2472 (Maine). 2. Polyporus pargamenus Fries. Plate 23, fig. 9. Pileus coriaceous, sessile, often narrowed at the base, 1-7 x 1-7 x 0.1-0.4 cm., whitish, cinereous, or brownish with age, villous or velvety-pubescent, zonate; context less than 1 mm. thick; tubes not more than 3 mm. long, the mouths white, bay, or violaceous, averaging 2-3 to a mm. in poroid [vor. 2 688 ANNALS OF THE MISSOURI BOTANICAL GARDEN forms but usually soon irpiciform; spores cylindric or allan- toid, hyaline, 5-6 X 1.5-2.5 u; eystidia present or inconspicu- ous, hyaline, rarely incrusted at the apex, 4-5 „ in diameter, projecting 5-15 4; hyphae of context hyaline, 4-5 u in diameter. On wood of deciduous trees. Illustrations: Freeman, Pl. Dis. f. 36—Hard, Mushrooms, f. 345. Speeimens examined!: Barth. Fung. Col. 2825, 2924 (as Coriolus prolificans) —Ell. N. Am. Fung. 312.—Ell. & Ev. Fung. Col. 302.—Rav. Fung. Am. 423, 108 (as Irpex fusco- violaceus).—Rav. Fung. Car. I, 13.—Rab.-Wint. Fung. Eur. 3331.—Shear, N. Y. Fung. 38.—Thuem. Mye. Univ. 1102 (as P. pseudopargamenus).—Mo. Bot. Gard. Herb. 4086 (Mis- souri), 4431 (Arkansas), 3855 (New York), 4443 (Indiana), 4439 (Kentucky), 4433 (Illinois), 4436 (Alabama), 4559 (Georgia), 4557 (Florida), 42875 (New Hampshire).—Burt Herb. (collections from Pennsylvania, Vermont, Kansas, and Massachusetts). —Overholts Herb. 476, 269, and others (Ohio), 1756 (Colorado). PoLYPoRUS ADUSTUS WILLD. Ex Fries, P. rumosus PERS. EX Fries, P. FRAGRANS PECK, AND RELATED SPECIES Perhaps no species have been more confused in American mycology than these three, together with a few other closely related forms both of Europe and America. They all agree in the one character of having a hymenium that usually be- comes more or less smoke-colored at maturity. In P. adustus and its closest relatives, P. crispus Fries and P. Burtü Peck, the hymenium is usually black or grayish black from the first, while in P. fumosus and P. fragrans it frequently becomes 1 Ell. & Ev. Fung. Col. 804, distributed as P. pargamenus, is P. hirsutus | not P. pubescens as stated by Lloyd, Letter No. 52, p. 20). Ell. & Ev. N. Am. Fung. 1934, distributed as P. pargamenus, is not this species. The a pearance of the plant suggests a form of Irpex tulipifera. I have made a micro- scopie study of the hymenium of the specimen and I find it has the larger in- menus, Mycological literature contains several names for plants closely related to, if not identical with, Irpex tulipifera and until the limits of the species are better known the writer hesitates to refer the above specimen with certainty. 1915] OVERHOLTS—STUDIES IN THE POLYPORACEAE 689 darker in mature plants but often remains white, sometimes assuming an ochraceous tint in herbarium specimens. Of the above-named species, the first three have been re- ferred to P. adustus by Murrill. P. adustus was described by Willdenow! in 1787. P. crispus was first described as a species by Persoon,? in 1799, and was later (1815) accepted by Fries? and so maintained by him in his ‘Hymenomycetes Europaei.’ P. Burtii was described from Vermont by Peck,‘ in 1897, and has not since been reported. P. fumosus was first described by Persoon,? in 1801, and P. fragrans by Peck,® in 1878. There are several other names for plants closely re- lated to, if not identical with, these species but the writer has had no opportunity to study them. One of these, P. sub- cinereus, described by Berkeley, in 1839, is said to have been repudiated by its author and the plants referred to P. adustus. P. Halesiae Berk. & Curt.” is probably distinct, and P. Lind- heimeri Berk. & Curt.® is not at all related to P. adustus, as stated by Murrill, but is a large-pored species with a brown context. In working over the collections referred to P. adustus in the herbarium of the Missouri Botanical Garden, the her- barium of Dr. E. A. Burt, and the writer’s herbarium, it be- came evident that we are here concerned with a species that has been used as a sort of dumping-ground for all plants with a black hymenium and a rather thin context, while plants of thicker context and lighter-colored hymenium have been re- ferred to P. fragrans or to P. fumosus, according to whether a pleasant odor was or was not noticed in the plants. Such procedure has resulted in the bringing together of a heter- ogeneous mass of material under the name P. adustus. This material was very readily separated into three fairly distinct sections besides the collections that properly belonged under ı Fl. Berol. p. 392. 1787. 2 Persoon, C. H. Obs. Myc. 2: p. 8. 1799. * Fries, E. Obs. Myc. 1: p. 127. 1815. ‘Bul. Tor. Bot. Club 24: : P. 146. 1897. ë Syn. Fung. p. 530. * Rept. N. Y. oa Mus, a p- 45. 1878. T Grev. 1: p. 52. 8 Ibid. p. 50. 1872, (Vou, 2 690 ANNALS OF THE MISSOURI BOTANICAL GARDEN P. fumosus. After considerable study the writer has decided that to P. adustus should be referred those collections with a thin, finely tomentose pileus, a thin, even margin, and minute black pores. The species does not grow densely imbricate as in P. crispus (see pl. 23 fig. 7) and does not have the crisped margin of that species. The illustration of P. adustus given by Patouillard! represents our plant very well. From P. Burtü it is to be distinguished by the smaller and more equal pores, the thinner, sterile margin of the pileus, and the firmer context. It is much more abundant than the other three species and frequently grows semi-resupinate. According to Fries, P. crispus differs from P. adustus in having a thin, crisped, margin and large unequal pores. One lot of segregates from my P. adustus material possesses just those distinguishing characters, and I have, therefore, revived the Friesian name and applied it to my plants. They are certainly distinct from the specimens referred to P. adustus though connected by intergrading forms to some extent. The illustrations (pl. 23 figs. 7 and 8) show typical specimens of the two species. I have seen no specimens other than the types that could be referred to P. Burtu. The type specimens differ from the above conception of P. adustus in having a somewhat thicker context, a thicker margin that is fertile below, and larger and more unequal pores. The hymenium is black, as in that species, and the surface of the pileus is finely tomentose. The flesh of the pileus is also very soft and almost floccose in texture. It has been held by some that the mouths of the tubes in P. adustus become larger and more irregular in ma- ture plants, and if such a character stood alone in the differ- entiation of these forms it probably should not be considered a specific character. But it is the writer’s opinion that in P. adustus they do not become much larger in old plants, and since P. Burti differs also from that species in the other char- acters mentioned above, we must consider it a valid species, at least until other collections throw more light on the subject. From P. crispus it may be separated by the fact that the 1 Tab. Anal. Fung. f. 142. 1915] OVERHOLTS—-STUDIES IN THE POLYPORACEAE 691 margin is not crisped, sterile, and thin, that the pubescence of the pileus is not nearly so prominent, and that the context is soft and floccose. The type specimens are not densely im- bricate as in P. crispus but more nearly approach the condi- tion found in P. adustus. The microscopic characters of these three species are identical and do not afford additional means of separating them. The tramal tissue of the pores is decidedly brown in color, the hyphae are small, and a large percentage of them are cut transversely in a cross-section of the hymenium. The spores in all three species are oblong or oblong-ellipsoid, and measure 3.5-4.5 X 1.5-2.5 u. There are no cystidia or other sterile bodies in the hymenium. In endeavoring to find characters on which to separate the three above-named species (and especially P. adustus) from specimens heretofore referred to P. fumosus and P. fragrans, recourse was had to microscopic sections of the hymenium. It was at once apparent that when longitudinal sections were prepared, according to directions given on page 678 of this paper, the tramal tissue of the tubes of P. adustus, P. crispus, and P. Burtit were decidedly brown in color, while those of P. fumosus and P. fragrans were entirely hyaline, except for the eosin stain. This character has been tested out thoroughly and is believed to be a satisfactory and constant one on which to differentiate these two groups of species. By obscuring the labels on the slides containing the sections of the different species it was found possible to easily separate the sections of the species of the one group from those of the other group by this character, and then verify the separation by uncovering the labels. Since suitable sections can be readily prepared in a very few minutes, the task of deciding between the two groups is an easy one when they cannot be readily separated on the general appearance of the specimens. Some such method of procedure is especially desirable in separating P. adustus from P. fumosus, since thin or young specimens of the latter are easily confused with the former species. How- ever, care must be taken not to confuse the dark color some- times obtained in thick sections of P. fumosus with the truly [VoL. 2 692 ANNALS OF THE MISSOURI BOTANICAL GARDEN brown color of the hyphae in P. adustus. In the hyphae of the latter species the color is brown, whether the sections are thick or thin. This test will usually apply to cross-sections of the tubes as well as to longitudinal sections, except that when the hymenium of a growing specimen is bruised, dried, and then sectioned, the mouths of the tubes and the hyphae at the ends of the tubes often show a brownish discoloration that may be confusing. P. crispus and P. Burtii usually are easily dis- tinguished without this test, but the results are even more marked in the case of those two species than in P. adustus. When Peck first described P. fragrans he stated that it was closely related to P. fumosus, but differed in having unequal pores and an agreeable odor. In a later report he remarked that it should perhaps be considered a variety of that species. Microscopically the two plants are the same. There are no cystidia and the spores are oblong-ellipsoid, and measure 4.5-6 X 2-3 u, thus being slightly larger than the spores of the three species discussed above. The spore characters given for both species in the ‘North American Flora’ are erroneous. From our present knowledge of the variability of odors in the fungi! we are not warranted in laying much stress on the fragrant odor ascribed to P. fragrans. Bresadola? discusses P. fumosus under the name P. imberbis and states that the plant at times has a subanise odor. I have never obtained such an odor from plants heretofore referred to that species, but frequently the plants do have an odor that I would not describe as pleasant. In the face of such evidence, it seems reasonable to conclude that the odor alone should not separate the two species in question. As to the size and regularity of the pores of the two species, I find collections of P. fumosus in which the younger specimens have minute pores and the older ones have large and irregular pores, and collections of P. fragrans with both large and small pores. I conclude, te, g., Polyporus graveolens Schw. I have collected this species several times and have had growing plants under observation for three seasons and at no time have I been able to obtain the slightest trace of an odor that would warrant the application of “sweet knot” to that species. Similar results have been reported by others. There is good authority, however, for stating that it is at times very fragrant. 2 Fung. Trid. p. 29. 1915] OVERHOLTS—STUDIES IN THE POLYPORACEAE 693 therefore, that we are here dealing with a character that varies witli the age of the plants or even varies in different plants of approximately the same age. In other characters the two species are identical. Bearing in mind then the fol- lowing points: (1) Peck’s admission concerning his species, (2) the little reliance that is to be placed on odors in at least some of the fungi, (3) the evidence that P. fumosus is some- times fragrant as it grows in Europe, and (4) the variability in the size of the pores in a single collection, we can only con- clude that P. fragrans is at most only a form of P. fumosus and not worthy of a distinct name. There are a few other names that need to be mentioned be- fore dismissing this group of species. P. salignus Pers. ex Fries is generally held to be P. fumosus, and Fries’ illustra- tion! certainly agrees with the species as it grows in this country. P. Holmiensis Fries, as distributed by Romell,? is surely our plant and it is so regarded by Bresadola. P. im- berbis Bull. ex Fries, as represented by Bresadola, is the same plant, but the name was not recognized by Fries in his ‘Systema Mycologici’ and so cannot be used for our plant. The following key will aid in distinguishing the four species presented here: Jogok oa er pe menium black or smoky black; tramal hyphae distinctly ee thicker; “hymenium ipag to somewhat smoky; tramal hyphae y apam arly so ection. o eena lee ee ee Mik rool cre 4. P. fumosus Pileus er poe margin thin, even, sterile below; context firm when dry; pores minute; plants slightly, if at all, imbricate. .1. P. adustus Pileus sd bscasodiy can age 3 on ae margin, pesay Be toward the — . base; margin thin, crisped or wavy, sterile below; context firm when dry; pores es and tt) aik Me sel imbri- EN EEE Fer ER en 2. P. crispus Pileus finely tomentose; ei acute but thicker than in the pre- ceding species, even, fer zn; context soft and floccose; > unequal; plants scarcely ae Pe E see 3. P. Burtü 1. Polyporus adustus Willd. ex Fries. Plate 23, fig. 8. Pilei not much imbricate though somewhat so at times, 1-6 X 3-8 x 0.1-0.6 em., white to smoky white or pale tan, rarely with reddish blotches or zones, finely tomentose to short villous-tomentose, zonate or azonate; margin thin, even, tIe. Hym. 2: pl. 181. 2 Fung. Scand. 11 [VoL 2 694 ANNALS OF THE MISSOURI BOTANICAL GARDEN often black in dried specimens, sterile below; context white or pallid, firm and corky when dry, 1-4 mm. thick, in large specimens separated from the hymenium by a narrow dark line; tubes less than 2 mm. long, the mouths grayish black to black, scarcely visible to the naked eye, averaging about 6 to a mm.; tramal tissue decidedly brown in color under the microscope; spores oblong or oblong-ellipsoid, rarely slightly curved, smooth, hyaline, 3.5-5 X 1.5-2.5 u; eystidia none. On dead wood of deciduous trees. Illustrations: Pat. Tab. Anal. Fung. f. 142.—Rostk. in Sturm’s Deutsch. Fl. 3: fase. 16. pl. 38. Specimens examined: Cooke, Fung. Brit. 2.—Ell. N. Am. Fung. 6.—Ell. & Ev. Fung. Col. 206.—Krieg. Fung. Sax. 1319. —Rabenh. Herb. Mye. 412.—Rav. Fung. Am. 421.—Shear, N. Y. Fung. 32.—Mo. Bot. Gard. Herb. 4222 (Newfoundland), 4223 (New York), 3851 (Missouri).—Burt Herb. (collections from Vermont, Ohio, Massachusetts, and New York).—Over- holts Herb. 284 (Ohio), 572 (Missouri), 2239 (New York), 1780 (Colorado), and others. 2. Polyporus crispus Pers. ex Fries. Plate 23, fig. 7. Pilei more or less densely imbricate and overlapping, 2-7 X 1-5 x 0.1-0.4 cm., gray to avellaneous, sometimes cin- namon to clay-colored in herbarium specimens, adpressedly fibrillose toward the margin, usually strigose toward the base, zonate or azonate; margin very thin, radiate-lineate, crisped or wavy, often becoming black, sterile below; context white or pallid, often brownish in herbarium specimens, soft and fibrous to corky, 1-3 mm. thick, usually separated from the hymenium by a narrow dark line; tubes 1-3 mm. long, the mouths grayish black to black, unequal, irregular, averaging 3-6 to a mm.; tramal tissue decidedly brown in color under the microscope; spores oblong or oblong-ellipsoid, smooth, hyaline, 3.5-4.5 X 1.5-2.5 u; cystidia none. On dead wood of deciduous trees. Illustrations: Fl. Dan. pl. 1850. Specimens examined: Romell, Fung. Sax. 8 (as P. adus- tus).—Thuem. Myc. Univ. 604 (as P. fumosus).—Mo. Bot. 1915] OVERHOLTS—STUDIES IN THE POLYPORACEAE 695 Gard. Herb. 42868, 42848 (Arkansas), 4180 (Missouri).—Over- holts Herb. 386 (Indiana), 105 (Ohio). 3. Polyporus Burtii Peck. Plate 23, fig. 4. Pilei not closely imbricate, 1-2.5 X 2-5 x 0.3-0.5 cm., gray or pinkish buff, finely tomentose, azonate; margin acute but rather thick, deflexed, even, concolorous, fertile below; con- text soft and sub-floccose in dried plants, 2-4 mm. thick; tubes 1-2 mm. long, the mouths grayish black to smoky black, un- equal, irregular, averaging 2-4 to a mm.; tramal tissue de- cidedly brown in color under the microscope; spores oblong- ellipsoid, smooth, hyaline, 44.5 X 1.5-2 u; cystidia none. On stump of yellow birch. Known only from the type locality, Middlebury, Vermont. Specimens examined: Burt Herb. (type collection). 4, Polyporus fumosus Pers. ex Fries. Plate 23, fig. 3. Pilei simple or imbricate, 2-10 x 3-15 X 0.5-2 cm., white to ochraceous or smoky white, sometimes stained with red- dish, finely tomentose to glabrous, sometimes with a rather broad, marginal furrow; context white or pallid, soft corky to woody when dry, 2.5-10 mm. thick, usually zonate, always separated from the hymenium by a narrow dark line, anise- scented or with a disagreeable odor; tubes 1.5-4 mm. long, the mouths white to grayish black, usually becoming black when bruised, averaging 3-4 to a mm.; tramal tissue hyaline or nearly so under the microscope; spores oblong-ellipsoid, smooth, hyaline, 4.5-6 X 2-3 u; cystidia none. On dead wood of deciduous trees, especially elm. Illustrations: Fries, Ic. Hym. pl. 181 (as P. salignus).— Bres. Fung. Trid. pl. 135 (as P. wmberbis).—Massee, Brit. Fung. Fl. f. 14-15.—Rostk. in Sturm’s Deutsch. Fl. 3: fase. 16. pl. 42. Specimens examined: Ell. & Ev. N. Am. Fung. 2902.— Shear, N. Y. Fung. 31.—Thuem. Mye. Univ. 5.—Mo. Bot. Gard. Herb. 43648 (Missouri), 4277 (Kansas).—Overholts Herb. 455, 527 (Ohio), 436 (Canada), 370 (Indiana), and others. [Vou, 2 696 ANNALS OF THE MISSOURI BOTANICAL GARDEN THe WHITE SPEcIES or Potyporus— THosE WATERY AND Fursuy-TouGH WHEN FRESH AND WITH WHITE CONTEXT AND SPORES This group of plants has probably been the source of more trouble and exasperation to those collecting them than any other group in the Polyporaceae. Collectors have sent them to various mycologists for determination, and quite often no two will agree on the name that should be applied to any one form. The group of species with which we are here concerned has been divided into two genera by Murrill, namely, the genus Tyromyces and the genus Spongipellis. Since the characters that separate the latter from the former genus are not always well defined, it would seem better had they been united into one genus. The group includes those species found only dur- ing the summer and fall, growing on logs or on living trees, and further characterized by being white or whitish through- out, and having a more or less watery and soft fibrous con- text. Some of the species have characteristic odors that will usually aid in their identification. When dry the context of some of these is soft and friable, sometimes more solid, and sometimes differentiated into an upper soft portion and a lower firm portion. We cannot include here all of the species referred by Murrill to the two above-named genera, partly because there has been no opportunity to study all of them and partly because many of them are limited in their distribution and are only infrequently found by collectors. Those that are of common occurrence in the Ohio and the upper Mississippi River valleys have been studied and the results here pre- sented. The series thus limited includes the following species: P. albellus Peck, P. caesius Schrad. ex Fries, P. chioneus Fries, P. delectans Peck, P. fumidiceps Atk., P. galactinus Berk., P. lacteus Fries, and P. spumeus Sow. ex Hornemann. These are not all closely related and most of them are not difficult to determine but they have been more or less confused in this country, and their distinguishing characters are here pointed out. 1915] OVERHOLTS—STUDIES IN THE POLYPORACEAE 697 P. chioneus, P. albellus, and P. lacteus.—P. chioneus was described by Fries,’ in 1815. In his ‘Hy ycetes Europaei,’ published in 187: (p. 546), he described it some- what more fully as follows ‘‘Albus, pileo carnoso, molli, laevigato, azono, postice siepe porrecto, margine inflexo; poris curtis, exiguis, rotundi , aequalibus, integerrimis...... Ad truncos v. c. Betulae...... unciam latus, odore acido.’’ In 1878 Peck? described P. al sellus from New York, also grow- ing on birch. Peck eviden ly was not acquainted with P. chioneus, but he regarded his species as probably more closely related to P. parado wus Fries and P. betulinus Bull. ex Fries. The only points o: difference in the descriptions of P. albellus and P. chioneus are: (a) in size, Peck’s species being described as ‘‘two to {>ur inches broad, one to one and a half thick,’’ and (b) in pub scence, the pileus being ‘‘smooth or sometimes slightly rough med by a slight strigose tomen- tum.” Both descriptions :ention the soft context, white color, and ‘‘acid’’ odor. S:ccardo? has listed P. albellus as a synonym for P. betulinus, and while the general form and size of the two species is at times somewhat similar, it does not require close observatio: . to distinguish them. The same cannot be said of P. albellu: and P. chioneus. Maurrillt has listed them as synonyms a: d the writer has expressed the same opinion in a recent pa er.” P. lacteus may well be bi ught into the discussion at this point. It was described in 1321. The description and figure® call for a plant similar in ; ize and habit to P. chioneus but differing from that species a from P. albellus in having a decidedly pubescent pileus a id a lacerated and labyrinthiform hymenium. These characte s should be sufficient to separate at once P. lacteus from the = two species, and the writer can neither accept nor undeı stand the determinations of those who would refer our commo | plant with a glabrous pileus and 1 Obs. Myc. 1: p. 125. 1815. 2 Rept. N. Y. State Mus. 30: p. t5. 1878. 3 Syll. Fung. 6: p. 139. dan “N. Am. Fl. 9; p. 35. 8. 5 Ann. Mo. Bot. Gard. r p. 97. 1914. ° Fries, E. Ic. Hym. 2: pl. 182. f. 1. (Vou. 2 698 ANNALS OF THE MISSOURI BOTANICAL GARDEN even hymenium to P. lacteus. Romell,! after a short descrip- tion of P. lacteus as he understands it, says: “This species seems to be identical with one known in America as Polyporus chioneus. . . . My specimens agree with the authentic specimens of P. lacteus at Kew. In Fries’ herbarium neither P. lacteus nor P. chioneus is represented by authentic specimens as far as I know. There is, however, a col- lection referred to P. chioneus by Robert Fries, and this collec- tion differs from my plant not only by the glabrous surface of the pileus but also 2 having the hyphae us parallel s.) and simple. ..... ” (Italics are the writer It is unfortunate if, with the easy access to Fries’ de- scription, American mycol- ogists of repute have sent specimens of a pubescent Polyporus to Europe under the name, P. chioneus. On the other hand, if the de- termination were that of an amateur it should not have been seriously considered by Mr. Romell. Whichever may have been the case, it is the writer’s opinion that such determinations are the exceptional ones and not the rule, for the plant that is usually referred to P. chioneus (including P. albellus) is usually, if not always, entirely glabrous and has even tube mouths. In fact, it is the writer’s opinion that P. lacteus and P. chioneus have been less confused in this country than in Europe. If there has been a tendency to confuse P. lacteus with anything it is with P. galactinus, as I have found several collections so mis- determined. The important point of the extract from Romell’s paper is, however, that the collection to which reference is there made as having a glabrous pileus and simple hyphae in the context, in all probability represents the species that is in- terpreted in this paper as P. albellus. Having fixed upon the distinguishing characters of P. A Hym. Lapp. p. 15. A Fig. 3. Hyphae of P. chioneus. 1915] OVERHOLTS—STUDIES IN THE POLYPORACEAE 699 chioneus and accepting Fries’ idea of P. lacteus, it becomes an easy matter to differentiate between P. chioneus and P. albel- lus. As stated above, and as will be seen in the accompanying illustration (fig. 4), the hyphae in the context of P. albellus are unbranched or at most very infrequently branched, while those of P. chioneus (fig. 3) are branched to a very great de- gree, and they vary considerably in size, some being narrow (5-6 „) and others twice as thick. This is not the only dis- tinguishing character, nor the one that was first hit upon by the writer, al- though it is probably the most reliable. The rela- tive thinness of the pileus in proportion to its length is a distinguishing char- acter of P. chioneus. In other words, the pileus is usually thin and spreading in P. chioneus, while in P. Fig. 4. Hyphae of P. albellus. albellus it is thicker, con- vex or ungulate, and triangular in section. This is only a general statement of a character that varies considerably. An additional character is found in an examination of a cross-section of the hymenium, though the sections must be cut very thin to see it at its best. In sections of P. albellus the hyphae in the trama of the pores appear to run in all directions and give a peculiar, ever-changing ap- pearance as they are viewed at changing foci. They are also all of one size. In P. chioneus the hyphae in the trama of the pores all run in one direction and practically all are cut trans- versely in a cross-section of the hymenium. The trama is seen to be made up of a background of a pseudocellular structure, with minute openings that indicate the cavities of the closely compacted hyphae. Interspersed over this background one sees cross-sections of hyphae two to three times larger, and standing out much more plainly than the sections of the com- pact hyphae in the background. It was at first thought these [VoL. 2 700 ANNALS OF THE MISSOURI BOTANICAL GARDEN larger hyphae might belong to some other fungus living within the tissues of this species. This supposition is ren- dered improbable, however, by the fact that they are in- variably present in all collections, and that while other fungi frequently attack all of these white species, their hyphae are invariably much smaller than those of the fungi attacked. The evidence seems very clear, however, that these two species should be considered as distinct. When once differ- entiated they can usually be separated on the basis of their general habit, without recourse to the character of the branched or unbranched hyphae in the context, though that character can always be relied upon in establishing beyond a doubt the identity of the species. In other characters the two species are very similar. Both are glabrous or practically so; are covered with a thin grayish or yellowish pellicle that be- comes more evident when the plants are dried; have a sweet acid odor when fresh, a soft and friable context when dry; and the spores are the same, being cylindric, often slightly curved, and measuring 3-40.7-1.5 u. There are no cystidia. There is considerable doubt in the writer’s mind as to whether the true P. lacteus occurs in this country. There is a collection in the herbarium of the Missouri Botanical Garden and another in the writer’s herbarium that should perhaps be referred to that species, but the hymenium has been dis- organized by the growth upon it of another fungus, so that no spores are present. If future collections should show that the spores are similar to those of P. chioneus, the plants should in all probability be referred to P. lacteus. The pileus is somewhat strigose or fibrillose-pubescent, though the mouths of the tubes are not labyrinthiform. The pileus is too pubescent for either P. chioneus or P. albellus to which latter species the plants were once referred by Lloyd. It is possible that they represent P. lacteus as more recently defined by Lloyd.! I have seen no specimens so referred by him and his description of the plant as ‘‘a common white species’’ and again as ‘a frequent plant’ throws some doubt on my opinion, for the plant is a rare one. 1 Letter No. 49, p. 14. 1915] OVERHOLTS—STUDIES IN THE POLYPORACEAE 701 According to the writer’s notes on specimens of P. lacteus in the herbarium of the New York Botanical Garden, that species, as it appeared in the ‘North American Flora,’ is P. albellus as here defined, at least in part. Neither can the writer accept Romell’s interpretation of P. lacteus, but if such a plant exists it must agree in the main with Fries’ descrip- tion and figure, and neither of the above interpretations do so agree. I do not know what Bresadola’s latest ideas on the subject are, but at one time he regarded P. lacteus and P. chioneus as synonyms—a position just as untenable as that taken by Murrill and Romell. According to the above interpretation of P. chioneus and P. albellus, the presentation of the two species in a recent paper! by the writer should be modified, and those collections that show simple hyphae in the context should be referred to P. albellus and those with branched hyphae should be referred to P. chioneus. P. delectans and P. spumeus. —The first one of these species was described by Peck,? in 1884, from specimens collected in Ohio by Morgan. It is a large or medium-sized plant and was described as having a fleshy-fibrous context, a glabrous or floccose-tomentose pileus, and long tubes with large unequal mouths. By this last character and by the large size of the plant and the ellipsoid or subglobose spores it is easily dis- tinguished from the species discussed above. In size of pores and length of tubes it is intermediate between the above species and P. obtusus Berk. A much more closely related species, however, is P. spumeus. The original notes of Sowerby on this species are very meager. The plant is de- scribed as ‘‘oozes from decaying elms in a very soft frothy mass, hardening in a day or two; and if it dries favorably, the pileus becomes hispid. The pores are small and nearly round; the tubes not long.’’ In Sowerby’s text? this species is followed by P. betulinus. Plates 211 and 212 are cited as representing the two species, respectively. Plate 211 shows a 1 loc. cit. p. 97. * Bul. Tor. Bot. Club 11: p. 26. 1884 3 Colored Figs. Eng. Fung. pl. 211-212. 1797-1803. [VoL. 2 702 ANNALS OF THE MISSOURI BOTANICAL GARDEN plant with a substipitate base, an incurved margin, and short tubes. One figure shows the plant from a front-underneath view, the other shows half of the plant with the cut surface outward and the hymenium upward. Plate 212 shows prac- tically the same thing but with a little more detail, and it is a fair representation of P. betulinus. All later descriptions of P. spumeus are either based entirely on pl. 211, or else on plants that have no resemblance to the one that has since been referred to P. spumeus. Fries’ description! says: ‘‘basi stipitiformi, margine incurvato.”’ This gives us but two alternatives from which to choose. Either Sowerby confused his illustrations of P. spumeus and P. betulinus and inserted two plates of the same species (P. betulinus), or else there existed at that time a plant closely related to P. betulinus but growing on elm and thought by Sowerby to be distinct. Since the mutual resemblance of Sowerby’s two plates is so great, it is the writer’s opinion that he had drawn two plates of P. betulinus and by mistake inserted both of them instead of one of that species and one of P. spumeus. This theory is borne out by the fact that he makes no mention of a stipe-like base nor an incurved margin to the plant. We may also conclude that Fries’ description was drawn, in part at least, from pl. 211, for it is incon- ceivable that with access to Sowerby’s figure he would have referred to that species a plant that departs so widely from the authentic illustration, unless he was also of the opinion that pl. 211 was a mistake. This mistake (for so it seems we must regard it) has caused some little confusion in the literature. Fries’ idea of P. spumeus was evidently gained, in part at least, from Sower- by’s plate, for he refers as a synonym for P. spumeus, Boletus suberosus of Wahlenberg?. But Wahlenberg was aware of the existence of a Boletus suberosus of Linnaeus? and ex- pressed the doubt that his species was the same as that one. Boletus suberosus of Linnaeus has always been regarded as 1 Hym. Eur. p. 552. 1874. 2 Fl. Upsal. p. 457. 1820. °Sp. Plant. p. 1176. 1753. 1915] OVERHOLTS—STUDIES IN THE POLYPORACEAE 703 a synonym for P. betulinus. In 1823 Hornemann: published a figure of P. spumeus entirely different from Sowerby’s original figure, but in all probability a better representation of his original species. It was not, however, so accepted at the time. In the text accompanying the plates in ‘Flora Danica,’ Hornemann refers to Sowerby’s original figure as a variety (var. stipitatus) of P. spumeus. This was evidently only a makeshift to dispose of a troublesome figure, and since the figure itself was evidently an error, Hornemann’s disposi- tion of it need have no weight. Subsequent writers did not concur in his opinion, however, and the confusion was only made worse, for now some regarded that there were two dis- tinct plants passing under the name of P. spumeus. In Hooker’s ‘English Flora,’? in which the fungi were written up by Berkeley, both Hornemann’s and Sowerby’s illustra- tions are cited as representing P. spumeus, and Hornemann’s figure is given priority in the order of citation. Again the plant is described as possessing an obsolete stipe and an in- curved margin—characters either taken from Sowerby’s il- lustration or copied from Fries. That Berkeley was in doubt as to the correctness of Sowerby’s plate is evidenced by the statement: ‘‘ According to Fries, the figure of Sowerby repre- sents the species in an imperfect state...... ’ In 1874 Fries* accepted Sowerby’s figure as representing P. spumeus and referred Hornemann’s figure to P. epileucus. This refer- ence was evidently followed by Saccardo. Berkeley* pub- lished an illustration of P. spumeus that corresponds well with Hornemann’s figure and agrees with the plants since referred to that species. Thus there has arisen an interesting situation in which, according to the writer’s interpretation, a well-known species is referred to an erroneous illustration that cannot possibly represent it, while the authentic illustra- tration is referred to another species. Of course it is possible that Hornemann may have misinterpreted Sowerby’s P. ı Fl. Dan. pl. 1794. 1823. 2 Eng. Fl. 5°: p. 139. 1836. ®Hym. Eur. p. 552. 1874. *Outl. Brit. Fung. pl. 16. f. 4. 1860. [Vou. 2 704 ANNALS OF THE MISSOURI BOTANICAL GARDEN spumeus, in which case the name should be written P. spumeus Hornemann, Fl. Dan. pl. 1794. 1823, since there is no doubt that Hornemann’s figure represents P. spumeus as it is known in Europe to-day. But the writer prefers to accept Horne- mann’s plate as a correct interpretation of Sowerby’s species (disregarding pl. 211) and write the name as P. spumeus Sow. ex Hornemann. If the writer’s theory is correct, there never existed a plant, the name of which could be written as P. spumeus Sow. ex. Fries, Syst. Myc. 1: 358. 1821,' since Fries never illustrated the plant, and his descriptions, several times repeated, were based, in part at least, on the erroneous pl. 211 of Sowerby. In the American literature the plant was first described by the writer in a recent paper.” The relation of Sowerby’s figure to the species was not then understood and the state- ment was there made that ‘‘the plants so referred do not agree with the figure given by Sowerby, nor with Fries’ de- seription.”’ There are but few references to its occurrence in this country, although it is a fairly common species. Lloyd reports receiving it from several widely separated localities. Whether others may agree with the writer or not, the evi- dence here presented should at least have the effect of doing away with the inconsistency of citing both Sowerby’s illustra- tion and that of Hornemann as representing the same species. P. spumeus is not likely to be confused with any species except P. delectans. These two intergrade to some extent. The former species has a strigose-tomentose surface to the pileus while the latter is glabrous or only slightly tomentose. Heavy rains or a little handling of the plant may cause the pubescence on P. spumeus to become matted and appressed, but when specimens are found growing imbricated so that the lower pilei are protected by the ones above, the character is very marked. The tubes in both species are long and slender, but in P. delectans the mouths are larger and more sinuous, usually measuring 0.5-1 mm. in diameter, while those of P. spumeus are smaller, measuring about 3-4 to a mm., and col- lef, Ann. Mo, Bot. Gard. 1: p. 99. 1914. 2 loc. cit. 1915] OVERHOLTS—STUDIES IN THE POLYPORACEAE 705 lapse when dry. This collapsing is due to the thinness of the dissepiments—a character easily made out in transverse sec- tions of the hymenium. The illustration (pl. 24 fig. 14) shows the larger tubes of P. delectans. The spores of the two species are practically the same, varying from ellipsoid to ovoid or subglobose, and measuring 5-64-5 u. They are frequently guttulate in both species. There are no cystidia in the hymenium. P. galactinus.—This species is a fairly well-marked one and only its distinguishing features will be pointed out here. It was originally described by Berkeley from specimens collected in Ohio by Lea. It is eastern in its range in the United States, occurring from Maine to Missouri and probably no farther south than West Virginia. There are but three common plants in this section of Polyporus that possess characteristic odors when fresh and growing. P. galactinus is one of them. The odor is usually described as ‘‘acid,’’ but to the writer it is a very pleasant and fragrant odor, but not persisting in the dried plants. Characters are not wanting to separate this species from the group just discussed in this section. The pileus is strigose-pubescent, as shown in the illustration (pl. 24 fig. 15), the tubes are very small, and the spores are minute, ellipsoid or subglobose, uninucleate, and measure 3-4 X 2-3 p. From P. delectans and P. spumeus it may be separated by the minute pores and the smaller spores. From P. fumidiceps Atk. it differs in the decidedly pubescent pileus and larger size. From P. caesius, which it resembles in its hairy covering, it differs in its larger size and ellipsoid spores. There are no eystidia. P. caesius.—This species has long been recognized as a well- marked one, characterized by the villous-strigose pubescence on the pileus, the bluish or grayish blue tint often present on the hymenium, and the minute, eylindrie, curved spores. From P. galactinus it is separated by its small size and dif- ferent spores; from P. chioneus and P. albellus by the pubes- cent pileus; from P. lacteus by the more strigose pileus and the unbranched hyphae of the context. [Vou, 2 706 ANNALS OF THE MISSOURI BOTANICAL GARDEN P. fumidiceps.—This species was described by Atkinson! in 1908, and has not since been reported. Since the writer finds it to be a rather common species in Missouri, and since a description has not appeared in the American literature, a few notes will be appended and the plant described on a fol- lowing page. In size and shape the species corresponds most closely to P. chioneus, but it is of a different color and the spores are ellip- soid to subglobose. From P. galactinus and P. caesius it is separated by the almost or quite glabrous pileus and from the latter also by the spores. The writer finds it most often on dead willow logs in willow thickets along river bottoms. The types were described from similar locations. Fresh plants have the same peculiar fragrant odor that is found in P. galactinus. The following key will aid in the determination of the species here discussed : Spores cylindric-oblong, often allantoid..........-.... 000s eeee sneer e eee 1 Spores ellipsoid to globose. ... 1... 6... cece eee tenet eee e eter teens Spe villous- ae je se; a. zn us or grayish blue..5. P. caesius Pileus glabrous or very slightly pubescent. ...... 2... nennen rere 2 Hyphae of context simple or rey pni | pileus usually tri- angular in section; tubes usually 4-9 mm. long..........- 2. P. albellus Hyphae of on much ae pileus asually more BEI tubes 1-3 mm. lo . P. chioneus Spores 5-6 a in longest direction; plants not fragrant when faa ars 4 Spores 2-4 u in longest direction; plants fragrant when fr ORR isdn kead 5 Pileus strigose-tomentose or strigose-hispid, especially on the ee tubes collapsing on drying, the mouths equal, small, vs E 3—4 oa mm. 3. spumeus Pileus glabrous or floccose-tomentose; tubes scarcely collapsing on Er the mouths usually somewhat sinuous, averaging g a ee Oe EERE EES STE PRERE A CE TE SOE ETE ES Oe HOSS — D eee u IE Te Bar a To Tor er ar or Ye Er Var Dr Ma ME BE Soc Dar Dec oc vu: De Sor DS BEL Der DE eee eee ee = > ing, to aloalii Pileus gisbrona or nearly 8O....... cee cece eee ee ener encees P, fumidiceps Pileus conspicuously pubescent, often strigose-tomentose at in m PERECA TEET oe be ee ES Se Swe OS 8H O48 ee TER ET RUE WR 1. Polyporus chioneus Fries. Plate 24, Se 13, 16b Pileus soft and watery when fresh, rigid when dry, 2-7 X 1-6 X 0.5-1.5 cm., white, often grayish or yellowish when dry, glabrous or nearly so, covered with a thin continu- ous gray or yellowish pellicle that becomes more evident when the plants are dried; context white, usually with a fragrant 1 Ann. Myc. 6: p. 61. 1908. on . ga la ctinus 1915] OVERHOLTS—STUDIES IN THE POLYPORACEAE 707 odor when fresh, soft and friable when dry, 2-7 mm. thick; tubes 1.5-3 mm. long, the mouths white or yellowish, averag- ing 3-4 to a mm.; spores cylindric or allantoid, minute, hya- line, 3-4 X 0.7-1.5 u; eystidia none; hyphae of context hya- line, much branched. l On dead wood of deciduous trees. Specimens examined: Mo. Bot. Herb. 4311 (Missouri).— Burt Herb. (collections from Vermont and New York).— Overholts Herb. 2325, 2261, 2277, 2276 (New York), 2326 (Ohio). 2. Polyporus albellus Peck. Plate 23, fig. 5, Plate 24, fig. 16a. Pileus soft and watery when fresh, rigid when dry, more or less triangular in section, 1-8 X 1-7 X 1-4 cm., white or yel- lowish, glabrous or nearly so, covered with a thin yellowish pellicle that is more evident in dried plants, but often disap- pears in patches; context white, soft and friable when dry, 0.5-3 cm. thick; tubes 4-9 mm. long, the mouths white or yel- lowish, averaging 3-4 to a mm. ; spores cylindric or allantoid, minute, hyaline, 3-4 X 0.7-1.5 „; eystidia none; hyphae of context hyaline, unbranched or nearly so. On dead wood of deciduous trees. Specimens examined: Mo. Bot. Gard. Herb. 48756 (Idaho). —Burt Herb. (collection from Pennsylvania).—Overholts Herb. 591 (Vermont), 408, 149, 207 (Ohio), 2243, 2270 (New York), 440 (Missouri). 3. Polyporus spumeus Sow. ex Hornemann. Plate 24, figs. 10, 11, 14a. Pileus soft and watery when fresh, rigid on drying, 5-20 X 6-20 X 2-6 cm. (much thinner when dried), white or some- what yellowish, villous-strigose or matted strigose-tomentose; context white, rigid on drying, 1-3 em. thick; tubes 0.5-1.5 cm. long, collapsing when dried, the mouths white or yellowish, averaging 2-4 to a mm.; spores ellipsoid to subglobose, hya- line, smooth, often once guttulate, 5-6 X 4-5 u; cystidia none. Illustrations: Hornemann, in Fl. Dan. pl. 1794.—Berk. Outl. Brit. Fung. pl. 16, f. 4. [VoL, 2 708 ANNALS OF THE MISSOURI BOTANICAL GARDEN Specimens examined: Cooke, Fung. Brit. 5111—Thuem. Mye. Univ. 7091.—Mo. Bot. Gard. Herb. 43719 (Missouri).— Overholts Herb. 101 (Ohio), 526, 625 (Missouri). 4. Polyporus delectans Peck. Plate 24, fig. 14b. Pileus soft and watery when fresh, 3-15 X 5-20 X 1.5-5 cm., white, yellowish, or grayish, glabrous to finely tomentose; context white, often with a soft upper layer and a more firm lower layer, firm when dry, 0.5-2 em. thick; tubes 0.5-1.5 em. long, the mouths white or yellowish, averaging 1-2 to a mm.; spores ellipsoid to subglobose, often uninucleate, hyaline, smooth, 4-5 X 5-6 u; cystidia none. Growing from wounds of living trees and on old logs. Illustrations: Jour. Cine. Soc. Nat. Hist. 8: pl. 1. Specimens examined: Overholts Herb. 145, 519, 250, 415, 659, 93, 258, 255 (all from Ohio and Missouri). 5. Polyporus caesius Schrad. ex Fries. Pileus more or less triangular in outline, rather soft and watery when fresh, 1-5 x 1-4 x 0.5-2 cm., white or grayish, rarely bluish gray, villous-pubescent or strigose; context white, 3-10 mm. thick; tubes 3-5 mm. long, white or grayish blue, large, unequal, averaging 1-3 to a mm., the dissepiments thin, torn and lacerated; spores cylindric or allantoid, smooth, hyaline, 3-4 X 0.7-1.5 u; cystidia none. On dead wood of deciduous trees. Illustrations: Sow. Col. Fig. Eng. Fung. pl. 226 (as Boletus albidus).—Gill. Champ. Fr. pl. 458. Specimens examined: Krieg. Fung. Sax. 1913.—Mo. Bot. Gard. Herb. 43650 (Missouri).—Burt Herb. (collections from Canada and New York).—Overholts Herb. 627 (Missouri), 2271 (New York). 1 These specimens or sections of specimens are not well preserved. They contain no spores, and while the general appearance, i. e., shape of pileus, size of pores, length of tubes in comparison with thickness of context, etc., are very much the same, the context appears to be more woody and zonate than in our specimens. Ellis N. Am. Fung. 1103 is referred to P. spumeus Fries. It is the same as distributed by Cooke, Fung. Brit. 603, under the name P. spumosus Fries. There is no such species listed by Saccardo. Lloyd (Letter No. 52, p. 25) refers the Ellis specimen to Fomes geotropus Cooke. 1915] OVERHOLTS—STUDIES IN THE POLYPORACEAE 709 6. Polyporus galactinus Berk. Plate 24, figs. 12, 15, 17. Pileus more or less triangular in sections, sometimes gib- bous behind, rather firm but watery, 3-8 x 5-10 x 1-3 cm., white or yellowish, strigose-tomentose at the base, short tomentose on the margin; context fibrous when fresh, hard and sometimes resinous when dry, white, 0.3-2 em. thick, strongly zonate, with a strong fragrant odor in fresh spect- mens; tubes 5-10 mm. long, the mouths white or yellowish, minute, averaging 4-6 to a mm.; spores ellipsoid, smooth, hyaline, once guttulate, minute, 3-4 X 2-3 u; eystidia none. On old logs in woods, especially in overflow river bottoms. Specimens examined: Mo. Bot. Gard. Herb. 4092, 43636 (Missouri), 4138.—Overholts Herb. 42, 489, 382, 134, 252, 2178, 511, 611, 583 (mostly from Ohio and Missouri). 7. Polyporus fumidiceps Atkinson. Plate 23, fig. 6. Pileus thin, soft and watery when fresh, 1-4 X2-5 X 0.5-1 cm., vinaceous buff to avellaneous or wood-brown, minutely pubescent or glabrous; context white, watery, with a strong fragrant odor, 2-5 mm. thick; tubes 2-5 mm. long, sometimes olive-green within on drying, the mouths concolorous, averag- ing 4-5 to a mm.; spores ellipsoid to subglobose, smooth, hya- line, 2.5-3.5 X 1.5-2.5 u; cystidia none. On dead wood of deciduous trees, especially willows, in woods and along overflow river bottoms. Specimens examined: Mo. Bot. Gard. Herb. 43712 (Mis- souri).—Burt Herb. (part of type collection, from New York). —Overholts Herb. 552, 2305, 2318 (Missouri). PoLyporus Lucipus Leyss. ex Fries, P. Tsucaz Murer., P. URTISII BERK., AND CLOSELY RELATED SPECIES These species form a rather natural group of plants pos- sessing the common character of a laccate or varnished pileus. P. lucidus was described in 1780 by Leysser (as Boletus) from plants collected in England. The description calls for a plant with a lateral stipe and it is so figured by English mycolo- [VoL. 2 710 ANNALS OF THE MISSOURI BOTANICAL GARDEN gists. P. Curtisit was described by Berkeley, in 1849,! from plants collected in South Carolina by Curtis. P. Tsugae was more recently described by Murrill? from plants collected in New York City on decaying trunks and stumps of Tsuga canadensis. Ganoderma sessile was described at the same time and by the same author. In Murrill’s first treatment of this section? Polyporus lucidus was reported as a synonym for P. pseudoboletus, the latter name being used for the plant. The species was re- ported as occurring in most of the states east of the Missis- sippi River with the exception of the New England states. P. Curtisu was there listed as a synonym for P. pseudoboletus with the remark that specimens referred to P. Curtisii were only variations of the other species, due to age, rapidity of growth, and perhaps to differences in the host. The next species described was Ganoderma sessile and that was de- scribed as differing from G. pseudoboletus in being annual and sessile, with a very acute margin and a more rugose sur- face. It was reported as occurring in Indiana, New York, Ohio, Alabama, Louisiana, and Kentucky. In the ‘North American Flora,’* six years later, the names Ganoderma pseudoboletus and Polyporus lucidus were both entirely omitted and P. Curtisii was restored as a specific name. No comment was made as to why this was done, nor as to what disposition was made of the numerous collections previously referred to Ganoderma pseudoboletus. The writer has seen material referred to G. sessile by Murrill, and the supposition is that all collections, except those belonging under Polyporus Curtisii, were referred to his new species Ganoderma sessile. This supposition is borne out by the fact that the description of that species is there so amended as to include stipitate forms also, while the species as originally described was limited to sessile forms. We must also conclude that G. sessile was regarded by its author as distinct from Polyporus lucidus of Europe, else that name or an older one would have 1 Lond. Jour. Bot. and Kew Gard. Misc. 1: p. 101. 1849. ® Bul. Tor. Bot. Club 29: p. 601. 1902. 8 loc. cit. “N. Am. Fl. 9: p. 120. 1908. 1915] OVERHOLTS—STUDIES IN THE POLYPORACEAE 711 been used. Mr. Murrill remarks concerning Ganoderma ses- silet: “Very similar in its stipitate forms to Polyporus lucidus of Europe.’? The American plants are usually re- ferred to P. lucidus by European mycologists, and taking into account the general agreement with the European descrip- tions and illustrations, and the fact that Murrill has consist- ently failed to cite any distinguishing characters upon which the legitimacy of his species might be established, we must conclude that there is no such distinction to be made between the European and the American plants. The American plant is variable in respect to the presence or absence of a stipe, and that cannot enter into the discussion. There is a tendeney among mycologists? to disregard the Ganoderma Tsugae deseribed by Murrill. To the writer this species appears to be a perfectly good one, although it cannot be differentiated on host character alone. A further discus- sion of this species is reserved for a following paragraph. In 1908 Atkinson? described a species of Ganoderma which he called G. subperforatum. After an examination of the type specimens the writer referred‘ this species to Polyporus lucidus. This leaves us three species of this section of Polyp- orus that are found in the central states. There are no spore characters of sufficient importance or constancy that can be used in separating them. There is a color difference but it probably cannot always be relied upon. The pileus of P. Tsugae is shining and mahogany-colored or darker; that of P. lucidus is of a lighter red color; and that of P. Curtisii is yellowish, at least in mature plants. Moreover, P. Curtisii is southern in its distribution, not being found north of the Ohio River; P. Tsugae is not reported south of Virginia; and P. lucidus is not limited in its north and south distribution in the United States. 1 Northern Polypores, p. 55. 1914. ®cf. Atkinson, Bot. Gaz. 46: p. 335. 1908. @. Tsugae is here listed as a synonym for G. pseudoboletus (=P. lucidus). Later on the same page it is given varietal rank; also Lloyd (Letter No. 52, p. 27) eites it as a synonym for Fomes lucidus. ® Bot. Gaz. 46: p. 337. 1908. * loc. cit. p. 123. [VoL. 2 712 ANNALS OF THE MISSOURI BOTANICAL GARDEN A more constant difference that serves to separate P. Tsugae is the color of the context. In P. lucidus and P. Curtisu the context is never pure white, but is usually sep- arated into an upper light-colored and a lower brown layer. This lower layer is more firm than the upper one and often contains horny fibers. In P. Tsugae the context is uniform in texture and almost pure white throughout, but often with a very slight tinge of brown next the tubes. Under the microscope this effect is magnified. There are no brown hyphae in the context of P. Tsugae, — while in the other two species brown hyphae are very pronounced, especially in the layer of con- text next the tubes. A compari- son of the size of the hyphae in the three species is interesting but does not always give conclusive evidence : 7 as to the identity of the species. The hyphae of P. Curtisu vary from 4 to 6 » in diameter. Those of P. Fig. 5. a, hyphae of P. > ‘ Curtisii; b, hyphae of P. luc. lucidus are more variable. In some ae width of hyphae of P. cases they cannot be differentiated from those of P. Curtis in point of size, but in some specimens they attain a diameter of 10 u. Those of P. Tsugae often attain a diameter of 15 a. The dif- ference in the branching of the hyphae of these three species is very striking and is shown in figs. 5 and 6, all drawn to the same scale. Figure 5a represents the hyphae of P. Curtisü, which are not extremely branched but can by no means be said to be unbranched. Figure 5b shows the hyphae of P. lucidus, and the branching does not differ materially from that of P. Curtis. In both species the large hyphae may extend more than across the field of the high-power microscope and not branch at all in that distance. This condition is never found in the hyphae of P. Tsugae. There the hyphae are ex- tremely branched, as shown in fig. 6. The large hyaline 1915] OVERHOLTS—STUDIES IN THE POLYPORACEAE 713 hyphae are not continuous for any distance but break up into numerous smaller branches that are often rapidly narrowed to fine thread-like hyphae. This condition must be seen to be best appreciated. It affords, however, another character on which the species can be separated from those closely allied. The following brief diagnoses of these species is appended: 1. Polyporus Curtisii Berk. Plants perhaps al- D ways stipitate; pileus | reniform or flabelliform, S 3-12 Xx 3-20 x 0.7-2 cm., covered with a thin crust that is at least in ! part ochraceous in ma- \ ture plants, zonate ; con- text soft and nearly white above, brown and firmer next the tubes, 0.5-1.5 cm. thick; tubes 0.3-1.2 cm. long, the mouths white to brown- Fig. 6. Hyphae of P. Tsugae. ish, averaging 3-5 to a mm.; stipe lateral, with color and context as in the pileus; spores light brown, ovoid with a truncate base, apparently echinulate, 8.5-11.5 X 4.5-7 u; eystidia none; hyphae of con- text hyaline or brown, 4-6 u in diameter. On and about trunks of deciduous trees. Illustrations: Bot. Gaz. 46: f. 1-3. Specimens examined: Ell. N. Am. Fung. 802.—Rab.-Wint. Fung. Eur. 3430.—Mo. Bot. Gard. Herb. 1438 (Louisiana), 4746 (Alabama).—Overholts Herb. 305 (Florida), 962, 518 (Missouri), 2235 (New York). Also reported from most of the other states east of the Mississippi and south of the Ohio Rivers. 2. Polyporus lucidus Leyss. ex Fries. Plants sessile or stipitate; pileus dimidiate or reniform, 3-12 x 3.5-20 x 0.5-2.5 cm., covered with a thin reddish or [VoL. 2 714 ANNALS OF THE MISSOURI BOTANICAL GARDEN chestnut crust, zonate; context white to light brown, usually separated into an upper light-colored layer and a lower brown layer, never entirely white, 0.2-1.5 em. thick; tubes 0.3-1.5 em. long, the mouths white to umber, averaging 3-5 to a mm.; stipe lateral or excentric when present, with color and con- text as in the pileus; spores light brown, ovoid with a trun- cate base, smooth or appearing echinulate, 9.5-11 X 5-6.5 u; cystidia none; hyphae of context hyaline or brown, branched, 4-10 u in diameter. On and about stumps and trunks of deciduous trees. Illustrations: Bot. Gaz. 46:f. 5—Dufour, Atlas Champ. pl. 49. f. 116.—Gill. Champ. Fr. pl. 457.—Hard, Mushrooms, f. 332—Krombh. Abbild. u. Beschr. pl. 4. f. 22-24.—Rostk. in Sturm’s Deutsch. Fl. 3: fase. 5. pl. 13. Specimens examined: Ell. N. Am. Fung. 5.—Ell. & Ev. Fung. Col. 202 (Delaware).—Krieg. Fung. Sax. 1116.—Rav. Fung. Am. 5.—Thuem. Myc. Univ. 104.—Mo. Bot. Gard. Herb. 43149, 4095, 4024, 4144 (Missouri), 43939 (Illinois).—Burt Herb. (collection from Vermont). —Overholts Herb. (collec- tions from New York, Florida, Ohio, Illinois, and Missouri). 3. Polyporus Tsugae Murrill ex Overholts n. comb. Plants stipitate; pileus flabelliform or reniform, 5-15 X 7-20 x 1-4 cm., with a mahogany-colored or almost black, shining, incrusted surface, suleate; context white or nearly so throughout, 0.5-2 cm. thick; tubes 0.5-1 em. long, the mouths white to brown, averaging 4-6 to a mm.; stipe present, with color and context as in the pileus; spores light brown, ovoid with a truncate base, apparently echinulate, 9-11 X 6-7 u; eystidia none; hyphae of context very irregular and much branched, up to 15 win diameter. On or about stumps and trunks of hemlock and pine. Specimens examined: Burt Herb. (collection from Ver- mont).—Overholts Herb. 2338 (Vermont). Fomes Exuistanus AND. AND F. FRAXINOPHILUS PECK Fomes fraxinophilus was described by Peck from New York in 1882. It was first described as a Polyporus and later trans- 1915] OVERHOLTS—STUDIES IN THE POLYPORACEAE 715 ferred to the genus Fomes. F. Ellisianus was described from Montana by Anderson in 1891, and redescribed as Polyporus circumstans by Morgan from South Dakota in 1895. The former species is abundant in the central and eastern United States, growing only on the trunks of ash trees. The latter species is found occasionally in the western United States, growing only on trunks of Shepherdia. Lloyd has recently expressed the opinion that these two species are identical, except for host, and he has so treated them in his recent synopsis of the genus Fomes. The plants are much alike in their old stages but I cannot agree with him that Fomes Ellisianus is ‘‘exactly the same plant” as our eastern species on the ash. First, there is the distinction in host, but that of itself would not be important. Second, plants of F. Ellisianus that are fairly mature have a decidedly corrugated or radiate-rugose surface and a reddish tinge of color. I have seen no indication of either of these characters in F. fraxinophilus though I have been familiar with that species for a number of years and have observed it in all stages of growth. When the plants are several years old they become similar in appearance and it would be an easy matter to mistake the one for the other if the host were unknown. But the characters pointed out here are believed to be amply sufficient for retaining the two plants as distinct species. The following brief descriptions are appended: 1. Fomes Ellisianus Anderson. Pileus convex to ungulate, 3-10 x 3-8 x 1.5-4 cm., pallid to brown, radiate-rugose and with a reddish tinge when young, black and usually somewhat rimose with age, sulcate; context pallid to wood-colored, punky to corky, 0.5-2 em. thick; tubes 2-6 mm. long each season,! not distinctly stratified, the mouths white or yellowish, averaging 2-3 per mm.; spores oblong- ellipsoid to broadly ellipsoid, 6-8 X 4-5 y; cystidia none; hyphae hyaline, 3-5 up. On Shepherdia in the west-central states. 1 The tubes in this plant are sometimes continuous to a length of 1.5 cm., but I do not believe that such lengths are attained in a single year’s growth. [VoL, 2 716 ANNALS OF THE MISSOURI BOTANICAL GARDEN Illustrations: Bot. Gaz. 16: pl. 12.—Jour. Cine. Soc. Nat. Hist. 18: pl. 1. f. 4 (as P. circumstans Morg.). Specimens examined: Anderson, Paras. Fung. Mont. 537 (as P. fraxinophilus) —Baker, Pl. N. N. Mex. 55.—Mo. Bot. Gard. Herb. 4272 (New Mexico). —Burt Herb. (collections from Montana and New Mexico). Also reported from North Dakota and Colorado. 2. Fomes fraxinophilus Peck. Pileus convex to somewhat ungulate, 2-25 X 3.5-40 x 1.5- 10 em., at first white, soon grayish black or black, not rugose, somewhat rimose with age, sometimes sulcate; context woody, 0.5-1.5 em. thick; tubes 2-4 mm. long each season, indistinctly stratified, the mouths white to brownish, averaging 2-3 to a mm.; spores ellipsoid to ovoid, 5-6 X 6-7 u; cystidia none; hyphae 3-5 u. On living or dead ash trees. Illustrations: U. S. Dept. Agr., Bur. Pl. Ind. Bul. 32: pl. 2.— Hard, Mushrooms, f. 350. Specimens examined: Ell. & Ev. N. Am. Fung. 3302 (Kan- sas); Fung. Col. 909 (Kansas).—Mo. Bot. Gard. Herb. 4780, 1437, 4826 (Missouri).—Burt Herb. (collections from Kan- sas).—Overholts Herb. 46, 157, 159, 122, ete. (Ohio), 559, 624 (Missouri), 626 (Iowa). Also reported from Kentucky, Ne- braska, Pennsylvania, Indiana, and New York. FOMES IGNIARIUS LINN. EX GILLET AND F. nigricans FRIES Much confusion has existed concerning the limits of these two species, and many different ideas are stated in the litera- ture. Murrill has referred Fomes nigricans as a synonym for F. igniarius. Lloyd has kept them apart, though recognizing a close relationship between them. Others have concluded with Bresadola that we are here dealing with two species that can be easily separated on the presence or absence of setae in the hymenium. Romell has held that such is not the case, but that setae may be present or rare in either species, and has stated that they are usually most abundant near the bot- tom of the tubes. This would account for the fact that some 1915] OVERHOLTS—STUDIES IN THE POLYPORACEAE 717 observers have stated that they have been unable to find setae in the hymenium of F. nigricans. The original illustration of F. nigricans does not agree with any present-day conception of what the species really was. The manner in which the plates for Fries’ ‘Icones’ were gotten together does not at all preclude the existence of grave errors regarding the identity of the species there illustrated. Hence the original illustration of F. nigricans has been discounted by careful European workers, they preferring to base the species rather on specimens authenticated by Fries himself. Of these, there appear to be specimens both at Upsala and at Kew. The F. nigricans of my ‘Ohio Polyporaceae’ proves to be F. Bakeri Murrill. The specimens referred by me to F. igniarius are of two types. One of these has the pileus convex or ungulate, the surface sometimes becoming rimose, and setae not at all abundant. The second type is most commonly found on birch trees. The pileus is plane or slightly convex, sometimes shining black in color, and the surface often cracks in both directions but does not become roughly rimose. The setae are often more abundant. Of this second form, Lloyd recently wrote as follows concerning a collection sent to him by me: “It agrees with his (Fries’) specimens (of F. nigri- cans) both at Upsala and at Kew...... It is usually thinner than typical F. igniarius and the setae are more abundant than in the type form.’’ On the strength of this information I am now able to separate my collections of these forms into what I am con- vinced are the two species, F. igniarius and F. nigricans, re- spectively. I have examined all available material of the two species and have thoroughly confirmed Romell’s observation on the presence of the setae. In but one collection was I un- able to find setae and I do not doubt that further attempts would show their presence in that instance. It is advisable, however, as stated on a previous page of this article, to cut longitudinal sections of the hymenium, since by so doing one will be more likely to strike the setae if there is any variation in their abundance at particular places in the tubes. [VoL. 2 718 ANNALS OF THE MISSOURI BOTANICAL GARDEN The characters cited above do not appear to the writer to be sufficient to warrant the complete separation of the two species. They are sufficiently distinct, however, to enable one to refer to one form or the other all the specimens collected. It has been thought best to refer F. nigricans as a variety of F. igniarius. The following diagnosis of the species and its variety is appended: 1. Fomes igniarius Linn. ex Fries. Typical form: Pileus convex or ungulate, 3-10 X 5-20 X 2-10 em., grayish black or black, rarely roughly rimose with age, not incrusted; context hard and woody, brown, 0.5-1 cm. thick; tubes 2-5 mm. long each season, the older layers con- spicuously white-stuffed or incrusted, the mouths brown, averaging 4-5 per mm.; spores globose or subglobose, smooth, hyaline, 4-6 a; setae present though sometimes rare, sharp- pointed, 16-25 X 6-8 u; hyphae 3-4 u. Var. nigricans Fries: Pileus plane to convex, 3-10 X 3-15 X 2-7 em., black, sometimes shining black, the surface often cracked in both directions but never roughly rimose; context and tubes as in the typical form, decidedly white in- crusted; spores, setae, and hyphae as above, the setae often abundant. On trunks of living deciduous trees. Illustrations: Published illustrations passing under the name of this species and its variety are abundant, but typical representations of my plants so referred are scarce. The type form intergrades into the variety to such an extent that some illustrations are hard to refer. The typical form is repre- sented by Hard, Mushrooms, f. 349, and in pl. 25. f. 18. of this paper. The variety is well represented by Lloyd, Mye. Notes 29: f. 193; Rostkovius in Sturm’s Deutsch. Fl. 3:fase. 17. pl. 51. Specimens examined!: Ell. & Ev. N. Am. Fung. 915 (Ken- tucky).—Krieg. Fung. Sax. 526.—Thuem. Mye. Univ. 105.— Mo. Bot. Gard. Herb. 4037* (New York), 4043* (New York), ı Collections assigned to var. nigricans are marked with an asterisk. 1915] OVERHOLTS—STUDIES IN THE POLYPORACEAE 719 43627" (Vermont), 42958 (Florida).—Burt Herb. (collections from Vermont and Canada).—Overholts Herb. 378* (Indi- ana), 423 (Ohio), 2460* (Vermont), 2256 (New York), 450 (Missouri). FoMES SCUTELLATUS ScHw. EX CookEe AND F. ontensis BERK. EX MURRILL These two species are closely related and have on more than one occasion been treated as a single species. Fomes scutellatus was first collected by Schweinitz on dead Syringa in Pennsylvania. It has since been reported on a few other hosts, namely, alder, witch- hazel, and sweet-gum. F. ohiensis was origin- ally described from Ohio by Berkeley and is a very common species in that state. It is especially abundant on dead limbs on the ground in woods in September and October. Quite frequently it grows on fence posts, pickets, and a variety of other structural timbers. Both species were formerly fre- quently referred to the genus Trametes, but it seems best to restrict that genus to annual forms only. Besides the host distinction, other char- acters may be used to distinguish between the a ar two species. In typical specimens of F. scutellatus the pileus is entirely black and attached dor- sally to the under side of branches. F. ohiensis is rarely found so attached, and the whole plant is at first white, the upper or basal part of the pileus becoming blackish with age, as in many species of Fomes, but the margin remaining white, even in perennial forms. F. scutellatus is rarely ungulate in form, while old specimens of F. ohiensis become steep in front, much as in F. fomentarius. The spores of F. scutellatus have never been recorded and Lloyd has recently stated! that he has failed to find them even in freshly collected material. Murrill records them as 1 Syn. Fomes, p. 218. 1915. [voL. 2 720 ANNALS OF THE MISSOURI BOTANICAL GARDEN ‘smooth, hyaline,” but that conclusion is reached only from inference. I find them to be cylindric, hyaline, smooth, 8-9 X 2.5-3.5 u. They thus differ from those of F. ohiensis, which are ovoid with a truncate base, hyaline, smooth, 10-12 X 6-7 u. It is apparent then that the spores of F. ohiensis are similar in shape to those found in all species recently segre- gated into the genus Ganoderma, while those of F. scutellatus point to an alliance of that species with the genus Trametes, they being typical trametoid spores. It is only in rare cases that the branching of the hyphae of the con- text can be used as a distinguishing character. The hyphae of F. scutel- latus are much branched, while those of F. ohiensis are practically simple. These differences are shown in figs. 7 and 8. It is thus apparent that these closely related species are separated by rather wide differences, and their determination need no longer be considered difficult. The following descriptions are appended: 1. Fomes scutellatus Schw. ex Cooke. Pileus convex, sometimes attached by the vertex and cir- cular in outline, 0.5-1.5 X 0.5-2 X 0.1-0.5 cm., entirely dark brown or black, at least when mature, slightly sulcate ; context corky, about 2 mm. thick; tubes 1-2 mm. long, the mouths white or pallid, averaging 4-5 per mm., thick-walled; spores cylindric, 8-9 X 2.5-3.5 u; eystidia none; hyphae hyaline to light brown, much branched, 2-4 u; basidia 6-9 u broad. Usually growing on alder and witch-hazel. Specimens examined: Ell. & Ev. N. Am. Fung. 1597 (Penn- sylvania) ; Fung. Col. 1010 (Vermont).—Mo. Bot. Gard. Herb. 4469 (New Jersey).—Burt Herb. (collections from New York and Vermont).—Overholts Herb. 337 (Ohio), 2394 (Florida). Also reported from Maine, Delaware, and Ala- bama. Fig. 8. Hyphae of F. scutel- latus. 1915] OVERHOLTS—STUDIES IN THE POLYPORACEAE 721 2. Fomes ohiensis Berk. ex Murrill. Pileus convex to ungulate, sometimes attached by the vertex and circular in outline, 0.5-2.5 X 0.5-3 X 0.2-1 cm., pure white, then black at the base, the margin remaining white, often zonate or sulcate; context corky or woody, 1-3 mm. thick; tubes 1-4 mm. long, the mouths white, averaging 3-5 per mm., thick-walled; spores! ovoid with a truncate base, 10-12 X 6-7 u; eystidia none; hyphae hyaline, unbranched, 3—4 u; basidia 8-11 u broad. On dead wood and on structural timbers. Specimens examined: Ell. N. Am. Fung. 923 (as Trametes) (Ohio).—Burt Herb. (collection from South America, ex Herb. Romell).—Overholts Herb. 38, 39, 131, and others (Ohio), 479 (Missouri), 503 (Illinois). Also reported from Kansas, Michi- gan, and New York. TramMEeTES Pint Tumor. Ex Fries, T. Aswrts Karst., ano T. PICEINUS PECK. Trametes Pini dates from the year 1803, when it was de- scribed by Thore,? and again in the following year, 1804, by Broteri.? The typical form of the perennial plant is rather large, has a more or less ungulate pileus, and in age becomes blackish and rimose. At times, however, the first year’s growth is thin and applanate and thus differs markedly in form from the typical plant. This condition was observed by Peck and the name Polyporus (later changed to Trametes) piceinus was proposed by him for the form that he collected on Picea about 1889.4 Karsten had already? described the same plant in Europe, in 1882, as Fomes Abietis, and the two names have been used interchangeably in this country for several years. In 1889 Karsten® referred to his species as 1 According to Murrill (N. Am. Flora 9; p. 96. 1908) the spores of the size and form given here are conidial, but they represent the only type of spore I have been able to find in the hymenium of this species. ® Chlor. Land. p. 487. 1803. °F]. Lusit 2: p. 468. 1804. ‘Rept. N. Y. State Mus. 42: p. 121. 1889. 5 Bidrag Finl. Nat. Folk. 37: p. 242. 1882. °Finl. Basidav, p. 336. 1889. [VoL, 2 722 ANNALS OF THE MISSOURI BOTANICAL GARDEN Trametes Pim var. Abietis, and that name has also appeared in the American literature. The writer has not seen Karsten’s types and his opinion as to the synonymy of the species of Peck and Karsten is based entirely on the use of the names in this country and on the fact that T. Pini var. Abietis, as distributed by Romell,! is certainly to be referred to Peck’s species. In the case of Polyporus piceinus and Trametes Pini, however, the evidence is not so clear, and there are yet mycologists who distinguish between the two species. Peck has stated? that the pileus of T. piceinus is persist- ently tomentose, while that of T. Pini is not tomentose, and on this ground and also in view of the fact that the former is thin and applanate while the latter is thick and ungulate, the two have been kept apart to some extent, though Murrill, in 1908, declared them to be not specifically distinct. During the sum- mer of 1913 and again in 1914 the writer had the privilege of collecting in the almost unexplored (mycologically) region of the Rocky Mountains in central Colorado. Here the forests are principally composed of the lodge-pole pine (Pinus Mur- rayana) and the Engelmann spruce (Picea Engelmannit), the former genus being the typical host of T. Pini and the latter the same for T. piceinus. No extensive field observations had been previously reported as to the intermingling of these sup- posed species of fungi, and the opportunity was taken to pro- cure some notes on the subject. In that region the species is more abundant on the spruce than on the pine, probably because the best spruce forests follow the courses of the streams, while the pine often represents the only tree growth on the mountain sides and in the higher parts of the mountain parks where the soil often contains a higher percentage of sand. Such forests are not dense and quickly become dry, unless kept moist by daily rainfalls. Hence the statement that T. Pini is more often found on spruce in that locality is not surprising. In one instance in an area of no more than four square feet on a spruce snag the writer counted 18 sporo- phores, and of these about half were the 7. Pini form and 1 Fung. Scand. 7. 2? Rept. N. Y. State Mus. 54: p. 170. 1901. 1915] OVERHOLTS—STUDIES IN THE POLYPORACEAE 723 the rest were good specimens of the thin form known as T. piceinus. There is no doubt in the writer’s mind that all these sporophores came from a common mycelium. In 1914 a similar find was made, the substratum being an old spruce log. Portions of these two collections are preserved in the writer’s herbarium. Attempts were later made to sepa- rate the specimens in these collections by means of micro- scopic characters, but it was found to be impossible. In view of these observations it is seen that the recently expressed opinion of Meinecke! that the variation in shape is due to the host, is not true for the fungus, as it sometimes occurs in Colorado. In some localities it may be more convenient to consider the thin form as a variety of T. Pini, for it must be admitted that the two forms do not always grow in such close associa- tion as described above. Yet the evidence is clear that they cannot be regarded as distinct species. The writer believes that it will add to the clearness of the general situation in the Polyporaceae to include in the genus Fomes all perennial plants of whatever structure. This not only simplifies the definition of the genus Fomes, but also gives a clearer idea of the genus Trametes. As it has been commonly understood, the genus Trametes is a very poorly defined one, and any attempt to make its limits clearer is a step in the right direction. The transfer of this species to Fomes has already been made by Lloyd’. The species is here described under that name. 1. Fomes Pini Thor. ex Lloyd. Sporophores very variable, the variations grouping them- selves as follows: Typical form: Sporophore perennial, often ungulate, 6-15 X 4-20 X 1-15 cm., at first tawny and with elevated zones of appressed tomentum, becoming blackish and glabrous, the surface cracking or becoming rough and irregular; context not more than 5 mm. thick, tawny or ochraceous tawny, woody; tubes 2-6 mm. long each season, the mouths ochraceous to * Forest tree diseases common in California and Nevada, p. 43. 1914. 2 Syn. Fomes, p. 275. 1915. [Vor. 2, 1915] 724 ANNALS OF THE MISSOURI BOTANICAL GARDEN brown; spores globose or subglobose, hyaline, 4-5 y broad; setae abundant, sharp-pointed, brown, extending 20-30 u be- yond the basidia; hyphae 3-5 a. Var. Abietis Karsten: Sporophores usually annual, rather thin and applanate, 1-5 X 1-7 X 0.8-1 cm., tawny or russet- tawny toward the margin, the immediate margin sometimes brighter-colored, zonate with elevated ridges of tomentum, grayish black or brownish black toward the base; context colored as in the typical form, 1-3 mm. thick; tubes usually in a single layer; spores, setae, and hyphae, as in the typical form. On wood of coniferous trees, both living and dead. Illustrations: Boudier, Ic. Myc. pl. 161.—Delacroix, Atlas Path. Veget. pl. 19. f. 10-12.—Meinecke, For. Tree Dis. Calif. and Nev. pl. 4-5.—Rostk. in Sturm’s Deutsch. Fl. 3: fase. 17. pl. 50. Specimens examined: Ell. N. Am. Fung. 602 (New Jersey). —EIl. & Ev. N. Am. Fung. 2507 (as T. Abietis) (Canada).— Linh. Fung. Hung. 348.—Rabenh. Crypt. Samm. Schule & Haus 8; Herb. Myc. 118.—Romell, Fung. Scand. 7 (as T. Pina var. Abietis—Seym. & Earle, Econ. Fung. 11:549.—Mo. Bot. Gard. Herb. 42958 (Washington), 4609 (Newfoundland), 42970 (Maine), 42954 (Michigan), 42956 (Vermont), 4618 (Colorado), 43810 (Missouri), and others.—Overholts Herb. 154 (Ohio), 630, 2033, 642, and 2391 (Colorado), 2458 (Mon- tana), and others. Graduate Laboratory, Missourt Botanical Garden. Saree SS [Vor. 2, 1915] ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 23 Specimens of P. abietinus with lamellate hymenium. Surface view of typical am of P. abietinus. Typical sporophores of P. fum P. Burtii. Photograph ie type aak Upper surface of P. albe P. ers, showing on surface and section through a sporopho crispus, Ton the densely imbricate mode of growth and the pubescent pileus . adustus. View of MUSTER of a. and hymenium. 9. Typical sporophores of P. pargamenus ANN. MO. BOT. GARD., VOL. 2, 1915 OVERHOLTS—POLY PORACEAE COCKAYNE, BOSTON. Se) eee ae ee PLATE 23 728 [Vor. 2, 1915] ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 24 View of hymenium of P. spumeus. The pores of P. spumeus somewhat enlarged. Section through a sporophore er P. galactinus. Note the prominent zonation of the contex Upper surface of P. chio nd = size = = tubes in (a) P. spumeus and (b) P. delec Upper Mut of P. galactinus. Note the prominent pubescen > ons came the relative DE Re of the pilei in (a) P. albellus and (b) P. chioneu Hymenium of P. EEE OVERHOLTS—POLY PORACEAE COCKAYNE, BOSTON. 730 [VoL, 2, 1915] ANNALS OF THE MISSOURI BOTANICAL GARDEN . 18. . 19. EXPLANATION OF PLATE PLATE 25 Sporophore of F. igniarius growing on beech trunk. View of upper surface and section through a sporophore of F. fraxinophilus nl ae a ionic sporophore of F. igniarius var. e the strongly white incrusted layers of a ie and Eare A inot view of the same specimen of F. igniarius var. nigrica Sporaphores of F. ohiensis. isianus, showing _ upper surface and section of hymenium with long t Sporophores of F. os on limbs of alder. a OVERHOLTS--POLYPORACEAE 4 p COCKAYNE, BOSTON. THE THELEPHORACEAE OF NORTH AMERICA V! TREMELLODENDRON, EICHLERIELLA, AND SEBACINA EDWARD ANGUS BURT Mycologist and Librarian to the Missouri Botanical Garden Associate Professor in the Henry Shaw School of Botany of Washington University The group of fungi comprising the present part probably attains its greatest development both in form and numbers in the western continent where it culminates in the erect Tremellodendron, apparently confined to North America. This continent has five of the seven species of Eichleriella; it has twenty-six species of Sebacina against fifteen for the Old World. The better-known species of these genera were originally described in Thelephora, Stereum, and Corticium, with which they conform so closely in general habit of growth and con- sistency of the fructification that it is impossible to separate them from the latter except by microscopic examination of preparations which show the mature basidia to be longitudin- ally cruciately septate. Collectors invariably roughly grade their findings of Sebacina as Corticium. The recognition of longitudinally septate basidia is not always easy with the aid of the microscope; for example, the fungus originally de- scribed as Stereum Leveillianum B. & C. has been studied critically at several times by experts without their observing the true structure of the basidia. I regret that the present account of our species and their range in North America does not include all the material at hand. The Missouri Botanical Garden herbarium contains several hundred undetermined specimens of possible Cor- ticiums which have been received during the last two years. NoTE.—Explanation in regard to the citation of specimens studied is given in Part I, Ann. Mo. Bot. Gard. 1 : 202. 1914, footnote. The technical color terms used in this work a of Ridgway, Color Standards and Nomenclature. Washington, D. C., 191 1 Issued December 20, 1915 ANN. Mo. Bor. GARD., Vou. 2, 1915 (731) [VoL. 2 132 ANNALS OF THE MISSOURI BOTANICAL GARDEN I have looked through these colleetions very carefully to sort out, without examination now of everything by microscopic methods, just those specimens which ought to be studied at once for citation in this part, but some of the specimens most desirable for citation have undoubtedly been deferred for the present as probable Corticiums. As it is really a nice microscopical task to recognize longi- tudinally septate basidia when they are not at their best, some notes, based on my experience, may be helpful. Species of Tremellodendron are the most easily recognized, for a little of the moistened and softened hymenium may be picked out with a scalpel, placed in a drop of water, stained with aqueous solution of eosin, 7 per cent potassium hydrate solu- tion added, and then crushed down by pressure on the cover glass. In the detection of species of Eichleriella and Sebacina, thin vertical sections of the fructification are necessary. After the sections have been made turgid and clear by potassium hydrate solution, the latter should be drained off and the sec- tions stained by merely a sufficient amount of solution of Gruebler’s eosin soluble in alcohol, and mounted in water for temporary examination. It may be necessary to spread apart the tissues of the preparation somewhat by pressure upon the cover glass. If the preparation is to be preserved per- manently in glycerin, a drop of dilute solution of sodium chloride should be run under the cover glass before the glycerin is added to insure a permanent stain by the Gruebler eosin. Longitudinally cruciately septate basidia are simple and pyriform or subglobose when young, but so are the pro- basidia of Septobasidium, the possible storage organs of Corticium polygonium, and the basidia of some species of Corticium. The basidia of the latter are likely to form a layer at the surface of the fructification and are certainly simple if any can be detected bearing sterigmata and perhaps spores while still non-septate. Ina fructification having longi- tudinally septate basidia, the hymenial surface is usually com- posed of paraphyses and of long, slender sterigmata arranged side by side; in this surface layer—but sometimes at a con- 1915] BURT—THELEPHORACEAE OF NORTH AMERICA. V 733 siderable distance from the surface, as in Thelephora Helvel- loides Schw.—is situated the layer of basidia. Only very rarely do the basidia of Sebacina or Eichleriella constitute the surface of the fructification. If a fructification contains a palisade layer of deeply stain- ing, pyriform bodies among or underneath the paraphyses and with no simple basidia in the surface layer, more or less prolonged examination of the pyriform bodies is likely to show longitudinal septa in some of them. The three genera which comprise the present part of this monograph, are treated here by the writer, because their gen- eral habit and consistency conform so closely with Thele- phoraceae having simple basidia, that they may be regarded as a connecting group, although belonging with the Tremel- laceae by the structure of their basidia. Such of the species as were described in the past were described as Thele- phoraceae or by authors with special knowledge of the Thele- phoraceae; the taxonomic recognition of fungi of these genera seems likely to continue to fall in the future to students of the Thelephoraceae, for other mycologists will hardly care to glean for material of so few species among the many Thele- phoraceae of similar aspect. TREMELLODENDRON Tremellodendron Atkinson, Jour. Myc. 8: 106. 1902; Sac- cardo, Syll. Fung. 17: 208. 1905. The type species is Merisma candidum Schw. Fructifications coriaceous, erect, pileate, branched or rarely simple; hymenium amphigenous or inferior; basidia longi- tudinally cruciately septate; spores white, even. The species of Tremellodendron are indigenous to North America; none have been reported for other regions, so far as I am aware. The fructifications spring up on the ground in deep woods during wet weather in summer and early autumn, and have the general habit of Thelephora vialis, of branched Clavarias, or, very rarely, of simple clubs. In active vegetative condition the fructifications may be distinguished from species of Clavaria of similar habit by coriaceous and [vor. 2 734 ANNALS OF THE MISSOURI BOTANICAL GARDEN tough consistency and by lack of brittleness. The longitudin- ally septate basidia afford a decisive character in all doubtful cases. The specific distinctions between the more common species of this genus are based largely upon the form of mature and well-developed fructifications; very young, deformed, or frag- mentary specimens can not be referred very confidently to their species. KEY To THE SPECIES ee branched when well developed. Simple forms may be when very young or in the same colony with normal branched ee nee Er DER DE DAE SUES LER ER E TREES Re age | 44a rere er nee es Ei 4 . Fructifications es cespitose, more or less grown together.........- 2 . Fructifications solitary or scattered... 0... 66. e eee eee ee eee eee ees 2. bahar pileate divisions flattened, 3% de at many points of ontact, ey rosette- like masses 2— in Be = T. pallidum 2. With the stems grown together i inte a nae stem 2-10 mm. thic pileate divisions cylindric, spreading, grown tonetar a ‘only few points of contact; the smaller divisions about 1% mm. thick...... EEE EEE ELTERN aOR A ET WERE ERT FS 2. T. candidum 2. Sometimes with both stems and pileate divisions grown together into compact bundles, usually merely closely cespitose and with the branches intricately eier al much slenderer rae preceding species and with the habit of Pterula..............- 5. T. merismatoides . Stem about 1144 mm. thick, palmately ia branched; branches once or twice similarly branched, cylindric or subcylindric, often channelled on the u side; basi idia 15X9 u; spores 9-15 X 414-6 p, pointed at the base DIY ics 558s eek PR RADE CORE ESS EEE EEE EBEE E S 3. T. Cladonia . Stem about 14-1 mm. thick, sometimes with occasional, scattered, divergent branches from its > dilated at the ne r end, divided into a few, short, finger-shaped branches; basidia 20-24xX12-14 u; spores x6- e at both e d Known from Jamaica only.....ssssssssss . T, tenue ee er orange, probably with medullary tissue pale as in the preceding species; basidia subglobose, 10-12 mw in diameter a en ee a ae 6. T. au rantium 4. Fructification black with the exception of the hymenium; hymenium olive-ocher, amphigenous on the lower third of the fractifieation; basidia 11X7 u. Known from Porto Rico only..........- 7. T. simplex — ji ie) = 1. Tremellodendron pallidum (Schw.) Burt, n. comb. Plate 26, fig. 6. Thelephora (Merisma) pallida Schw. Am. Phil. Soc. Trans. N. S. 4: 166. 1834.—T. Schweinitzii Peck, N. Y. State Mus. Rept. 29: 67. 1878; Saccardo, Syll. Fung. 6: 534. 1888.— Tremellodendron Schweinitzü (Peck) Atk. Jour. Mye. 8: 106. 1902. Illustrations: Hard, Mushrooms f. 381.—Moffatt, Chicago Acad. Sci. Bul. 7: pl. 22. f. 1. 1909. 1915] BURT—THELEPHORACEAE OF NORTH AMERICA. V 735 Type: in Herb. Schweinitz and a portion in Curtis Herb. Fructification cespitose, erect, white or pallid, drying warm buff, stipitate by one to several or many stems which may be distinct below or arise from a common, swollen, basal mass; above, the stems branch into flattened, more or less furrowed, pileate divisions which grow together at surfaces of contact to form a somewhat cup-shaped or rosette-like mass; divisions in center of mass somewhat subulate at the apex, those at margin dilated and sometimes fimbriate, splitting when dry into sharp fibers or spicules; hymenium inferior, warm buff, best developed towards the base of the pileate divisions; basidia pyriform, longitudinally cruciately septate, 12-15 x 9 u; spores from a spore collection, white, simple, 10-12 x 44- 54 u, and 9-12 x 44 u from an herbarium specimen. Fructifications 2-10 em. high, 2-15 em. broad. On the ground in deep woods. Canada to South Carolina and westward to Missouri. June to October. Common. Full-grown and well-developed specimens are rosette-like and resemble Thelephora vialis when viewed from above but may have the pileate mass supported by many stems; small specimens with only a single stem do oceur. The large speci- mens are apparently due to the concrescence of many small fructifications. In the large specimens the pileate divisions on the outside of the mass become broader and more flattened than those in the interior. The flattened form of the divisions of the pileus and their growing together at numerous points of contact are characters separating Tremellodendron palli- dum from T. candidum. The small specimens, distributed as T. pallidum in published exsiccati, are often so immature and fragmentary that they cannot be distinguished from T. candidum. Forms of T. pallidum which have the tips of pileate divi- sions split into sharp fibers or spicules are the Thelephora cristata and T. serrata of Schweinitz, ‘Syn. N. Am. Fungi,’ Nos. 621 and 623. Specimens examined: Exsiccati: Ravenel, Fungi Car. II, 29; Ellis, N. Am. Fungi, 510; Ell. & Ev., Fungi Col., 1208; Shear, N. Y. Fungi, 50. [voL. 2 736 ANNALS OF THE MISSOURI BOTANICAL GARDEN Canada, Ontario: London, J. Dearness, and also in Ell. & Ev., Fungi Col., 1208; Belleville, J. Macoun, 174, 230 (both in Can. Geol. Surv. Herb.). Maine: N. Parsonfield, R. G. Leavitt. Vermont: near Burlington, L. R. Jones, two collections; Middlebury, E. A. Burt, two collections. Massachusetts: Sprague, 773 (in Curtis Herb. under the name Thelephora vialis) ; Brookline, S. Davis. Connecticut: East Hartford, C. C. Hanmer; and also No. 1567 (in Hanmer Herb.). New York: Alcove, C. L. Shear, N. Y. Fungi, 50; Floodwood, E. A. Burt; Taughannock, H. H. Whetzel, Cornell Univ. Herb., 13600; Buffalo, G. W. Clinton (in U. S. Dept. Agr. Herb.); Tarrytown, H. von Schrenk (in Mo. Bot. Gard. Herb., 42800). New Jersey: Laning (in Mo. Bot. Gard. Herb., 701330, 701331, 701333); Newfield, J. B. Ellis (in Mo. Bot. Gard. Herb., 5162), and also N. Am. Fungi, 510. Pennsylvania: Bethlehem, Schweinitz, type (in Herb. Schweinitz, and a portion in Curtis Herb. and also the Nos. 621 and 623 of Schweinitz, ‘Syn. N. Am. Fungi,’ under the names Thelephora cristata and T. serrata, respectively) ; Trexlertown, W. Herbst (in Lloyd Herb.); Kittanning, D. R. Sumstine. Delaware: Newark, H. S. Jackson, B10. District of Columbia: Washington, O. F. Cook, 2, comm. by P. L. Ricker. Virginia: Great Falls, C. L. Shear, 1044. North Carolina: Blowing Rock, @. F. Atkinson, Cornell Univ. Herb., 10666, 10667, 10669, 10664 (of which the first two numbers and part of the third are in Cornell Univ. Herb. and part of the third and the last in Mo. Bot. Gard. Herb.). South Carolina: Ravenel, Fungi Car. IT, 29. Ohio: C. G. Lloyd, 2346 (in Lloyd Herb.) ; Loveland, D. L. James (in U. S. Dept. Agr. Herb.). West Virginia: Eglon, C. G. Lloyd, 02601. Kentucky: S. M. Price (in Mo. Bot. Gard. Herb., 5141, 5144, 701332, 712372) ; Mammoth Cave, C. @. Lloyd, 1071. 1915] BURT—-THELEPHORACEAE OF NORTH AMERICA. V vor Illinois: H. C. Beardslee (in Lloyd Herb., 2175); Newton’s Ferry, E.T.& S. A. Harper, 441; Riverside, E. T. & 8. A. Harper, 696. Wisconsin: Blanchardville, Univ. of Wis. Herb., 52; Madison, E. T. & S. A. Harper, 881; C. J. Humphrey, 948 (in Mo. Bot. Gard. Herb., 44783). Iowa: T. J. Fitzpatrick (in Lloyd Herb.). Missouri: St. Louis, N. M. Glatfelter (in Mo. Bot. Gard. Herb., 701335, 701370, 701371) ; Cliff Cave, J. B. S. Norton (in Mo. Bot. Gard. Herb., 5126); Columbia, B. M. Duggar, 140; Creve Coeur, Miss E. M. Briggs (in Mo. Bot. Gard. Herb., 44756). 2. T. candidum Schw. ex Atkinson, Jour. Myc. 8: 106. 1902. Plate 26, fig. 3. Merisma candidum Schweinitz, Naturforsch. Ges. Leipzig Schrift. 1:110. 1822—Thelephora candida Fries, Elenchus Fung. 168. 1828; Schweinitz, Am. Phil. Soc. Trans. N. S. 4: 166. 1834. Type: in Herb. Schweinitz, Acad. Nat. Sci. Phila. Fructifications cespitose, erect, coriaceous-soft, white, dry- ing warm buff, stipitate; stem thick, palmately branched, with branches spreading, branching, cylindric or subcylindric; hymenium inferior on the main branches, often amphigenous on secondary branches; basidia longitudinally septate, 10- 12 x 74-9 u; spores colorless, simple, even, 74-10 44-04 u. Fructifications 24-5 em. high, 2-5 em. broad; stem 2-10 mm. thick; smaller pileate branches about 14 mm. thick. On ground in open woods. Vermont to North Carolina and westward to Missouri. July to September. Infrequent. The type of T. candidum has the dimensions given above for recent collections. In the original description Schweinitz noted that fructifications may attain a breadth of 15 cm.; at that time he had not given specific recognition to the large and common T. pallidum and it may be that the large speci- mens to which he referred were of the latter species. T. candi- dum is closely related to T. pallidum but contrasts with the latter in having consolidation between adjacent fructifications [vor. 2 738 ANNALS OF THE MISSOURI BOTANICAL GARDEN confined to the main stems from the base upward to about the region of branching; from here the branches spread so that they grow together only rarely; furthermore, the branches are distinctly cylindric or subeylindric. The spores average a little shorter than those of related species. Specimens examined: Vermont: Lake Dunmore, E. A. Burt; Newfane, C. D. Howe. Massachusetts: Woods Hole, G. T. Moore. New York: Alcove, C. L. Shear, 1218; Fishers Island, C. C. Hanmer, 192, 193, 194 (all in Hanmer Herb.). North Carolina: Schweinitz, type (in Herb. Schweinitz) ; Blowing Rock, G. F. Atkinson, Cornell Univ. Herb., 10662, 10668 (in Mo. Bot. Gard. Herb., 44775, 44776) and (in Cornell Univ. Herb., 10663). Ohio: Granville, 7. L. Jones. Missouri: near St. Louis, N. M. Glatfelter (in Mo. Bot. Gard. Herb., 701336). 3. T. Cladonia (Schw.) Burt, n. comb. Plate 26, figs. 1, 2. Merisma Cladonia Schweinitz, Naturforsch. Ges. Leipzig Schrift. 1:110. 1822.—Thelephora Cladonia Fries, Elenchus Fung. 168. 1828; Epier. 537. 1836-1838; Schweinitz, Am. Phil. Soc. Trans. N. S. 4:166. 1834; Saccardo, Syll. Fung. 6: 535. 1888.—Thelephora gracilis Peck, Torr. Bot. Club Bul. 25: 371. 1898. Type: in Herb. Schweinitz. Fructifications solitary or gregarious, erect, coriaceous-soft, pallid, drying warm buff, sometimes with the older portions pale olive-gray, stipitate; stem cylindric, palmately branched into a few— often three—cylindrie branches, each or some of which occasionally branch again in similar manner; branches arranged in a plane from flattened end of stem or branch or in a circle about the cylindric end of the stem which is then sometimes perforate and the branches often channelled; hymenium amphigenous, or inferior when the branch is chan- nelled; basidia longitudinally septate, pyriform, 15 X 9 a; spores colorless, simple, even, curved, 9-15 X 44-6 u. 1915] BURT—-THELEPHORACEAE OF NORTH AMERICA. V 739 Fructifications 24-5 em. high, 7 mm.-2 em. broad; stem about 14 mm. thick. On ground in woods. Canada to Mississippi and westward to Missouri. August and September. The fructification of this species is smaller than that of T. candidum and has but few branches, which are often arranged in a circle about the end of the stem so as to appear some- what proliferous on the margin of an imperfect cup as in some species of the lichen, Cladonia—hence the specific name—or with the branches standing up side by side from the com- pressed apex of the main stem. Both forms of branching have been found so associated in the same collection as to preclude the possibility of regarding this difference as a basis for two species. The branches are so frequently in threes that ‘‘tri- faria’’ was contemplated as a name for the species by one author. Specimens examined: Canada: J. Macoun, 78. Vermont: Smugglers Notch, L. R. Jones; Middlebury, E. A. Burt; Brattleboro, C. C. Frost (in Univ. Vermont Herb.). Massachusetts: Sprague, 871 (in Curtis Herb., 5762). New York: Hague, C. H. Peck, 7; Ithaca, G. F. Atkinson, Cornell Univ. Herb., 7708. Pennsylvania: Trexlertown, W. Herbst (in Lloyd Herb.). District of Columbia: Takoma Park, P. L. Ricker, 822 (in Ricker Herb.). North Carolina: Schweinitz, type (Herb. Schweinitz and a portion in Curtis Herb.) ; Blowing Rock, @. F. Atkinson (in Cornell Univ. Herb., 10665, 10008. A part of the latter num- ber is in Mo. Bot. Gard. Herb., 44774). Georgia: Tallulah Falls, A. B. Seymour, Farlow Herb., O, P, Q, R, U, W (in Mo. Bot. Gard. Herb., 44619, 44623-44625, 44628, 44630). Alabama: F. S. Earle, 13, type of Thelephora gracilis (in Coll. N. Y. State). Mississippi: Biloxi, Mrs. F. S. Earle, 324. Ohio: Cincinnati, A. P. Morgan (in Lloyd Herb., 32) ; Love- land, D. L. James. [VoL. 2 740 ANNALS OF THE MISSOURI BOTANICAL GARDEN West Virginia: Eglon, C. G. Lloyd, 02634. Missouri: Creve Coeur, E. A. Burt (in Mo. Bot. Gard. Herb., 44755). 4. T. tenue Burt, n. sp. Plate 26, fig. 7. Type: in Burt Herb. and in N. Y. Bot. Gard. Herb. Fructifications scattered, erect, very slender, coriaceous- soft, drying warm buff, stipitate; stem equal, flexuous, drying somewhat twisted and flattened, becoming fibrillose, some- times giving off two or three scattered, divergent, small branches, dilated above and divided in a few palmately ar- ranged, finger-shaped branches; hymenium inferior on the dilated portion and branches; basidia longitudinally septate, 20-24 X 12-14 p; spores colorless, simple, even, curved, pointed at both ends, 14-16 X 6-7 u. Fructifications 2-34 em. high, 3 mm. broad; stem 13-23 cm. long, about 4-1 mm. thick. On the ground in wet mountainous region, altitude 3000- 5200 ft. Jamaica. December and January. This species is characterized by its long and slender stem, few branches, and the largest basidia and spores of any species of the genus. The spores differ from those of the other species in being pointed at the apex. Specimens examined: Jamaica: Chester Vale, W. A. & E. L. Murrill, N. Y. Bot. Gard., Fungi of Jamaica, 400, type; Cinchona, W. A. & E. L. Murrill, N. Y. Bot. Gard., Fungi of Jamaica, 614. 5. T. merismatoides (Schw.) Burt, n. comb. Plate 26, fig. 4. Clavaria merismatoides Schweinitz, Am. Phil. Soc. Trans. N. S. 4:182. 1834.—Merisma Schweiniteti Leveille, Ann. Sci. Nat. Bot. IV. 5:157. 1846.—Lachnocladium merismatoides (Schw.) Morgan, Cincinnati Soc. Nat. Hist. Jour. 10: 193. 1888.—Pterula merismatoides (Schw.) Saccardo, Syll. Fung. 6: 742. 1888.—Thelephora merismatoides Lloyd, Letter No. 26: 2. 1909. Nomen nudum.—Tremellodendron merismatoides Lloyd, Letter No. 40:2. 1912. Nomen nudum.—Thelephora pteruloides Berk. & Curt., Hooker’s Jour. Bot. 1:238. 1849; Grevillea 1: 148. 1873. 1915] ‘ BURT—THELEPHORACEAE OF NORTH AMERICA. V 741 Type: In Herb. Schweinitz, Acad. Nat. Sci. Phila. Fructifications erect, cespitose or fasciculate, and sometimes with stems grown together, coriaceous, branched, pallid, dry- ing with stems warm buff and branches tawny; branches few, rather straight, filiform, angular-terete; branchlets many, dilated and fimbriate at the apex, then splitting into spreading branchlets; hymenium glabrous, amphigenous; basidia longi- tudinally septate, pyriform, 12-15 X 8-9 yu; spores in prepara- tions from herbarium specimens hyaline, even, simple, 8- 10 X 4-5 u. Cluster of fructifications 2-5 em. high, 2-3 em. broad. In- dividual from cluster has stem 5-10 mm. long, 4-1 mm. thick; branches about 4-4 mm. thick. On the ground in open woods. Massachusetts and New York to South Carolina and westward to Missouri. June to August. This is a small species with the habit of a Pterula but with coriaceous structure and longitudinally septate basidia. The fructifications of a cluster may have their stems distant from one another by spaces equal to the diameter of the stems, but the branches interlock above; in other cases the fructifications are crowded closely together and united throughout their whole length. T. merismatoides may be distinguished from the preceding species by the smaller diameter of the stems and branches and from all the following species by its cespitose to fasciculate habit. The collection from West Virginia, distributed as Thele- phora pteruloides in Ell. & Ev., ‘N. Am. Fungi,’ 3415 and ‘Fungi Col.,’ 1117, has the hymenium composed of basidia standing side by side in a distinct palisade layer and the basidia not longitudinally septate in my opinion. Specimens examined: Massachusetts: near Boston, Murray, comm. by Sprague, 250 (in Curtis Herb. under the name Thelephora pteruloides B. & C.); Woods Hole, G. T. Moore, 58. New York: Ithaca, G. F. Atkinson, 37; Fishers Island, C. C. Hanmer, 1478 (in Hanmer Herb.). New Jersey: Haddonfield, T. J. Collins comm. by C. G. Lloyd. Pennsylvania: Bethlehem, Schweinitz, type (in Herb. [Vor. 2 742 ANNALS OF THE MISSOURI BOTANICAL GARDEN Schweinitz); York County, N. M. Glatfelter (in Mo. Bot. Gard. Herb., 44742) ; Kittanning, D. G. Sumstine. South Carolina: M. A. Curtis, 1745 (the type and cotype of Thelephora pteruloides in Kew Herb. and Curtis Herb. respectively). Ohio: Cineinnati, A. P. Morgan, Lloyd Herb., 2589 (deter- mined by Morgan as Thelephora filamentosa). Wisconsin: Lake Geneva, E. T. € S. A. Harper, 842. Missouri: Meramec Highlands, N. M. Glatfelter (in Mo. Bot. Gard. Herb., 44743). 6. T. aurantium Atkinson, Ann. Myc. 6:59. 1908. Type: in Cornell Univ. Herb. but cannot be found at present. “Plants simple, slender, 1-3 em. long, 2-3 mm. stout, dark orange, tough. Basidia subglobose, 10-12 p, longitudinally divided; sterigmata 4, long, slender, flexuous. Spores oboval- subelliptical, granular, then with an oil drop, 7-10 X 5-6 p, white, hyaline.—C. U. herb., No. 10684, ground, woods, along small stream crossing Boone Road, Blowing Rock, Blue Ridge Mts., N. C. Q. F. Atkinson, Aug. 19-Sept. 22, 1901.” —Original description. T. aurantium differs from the preceding species of Tre- mellodendron by its simple fructifications. I have seen no specimens referable here. Professor Atkinson had intended to make a negative from his type so that I could include a figure of the species, but, upon going to the envelopes labelled T. aurantium, he found that they contained—by error of a helper—T. merismatoides instead. The specimens of T. aurantium have not been found. 7. T. simplex Burt, n. sp. Plate 26, fig. 5. Type: in Mo. Bot. Gard. Herb. and in Farlow Herb. Fructifications scattered, erect or suberect, drying hard, brittle, somewhat longitudinally wrinkled and sometimes com- pressed, black above, olive-ocher with the hymenium towards the base; hymenium amphigenous on the lower third of the fructification, olive-ocher, hyaline under the microscope, with surface consisting of colorless clavate paraphyses 5 a thick, 1915] BURT—THELEPHORACEAE OF NORTH AMERICA. V 743 and with basidia and spores at base of the paraphyses; basidia longitudinally septate; 11 X 7 p; spores colorless, even, 74- 9 X 5-6 u. Fructifications about 2 em. long, about 2 mm. thick. In cane field. Porto Rico. T. simplex is noteworthy by the column composed of longi- tudinally arranged, black hyphae, which extends the whole length of fructification and constitutes the whole, upper, sterile two-thirds of the fructification and is clothed by the ochraceous hymenium on the lower third. The specimens are broken off at the base, hence I cannot be sure that a stem was not originally present, but if present it would doubtless have been included in the packet. The general habit is that of a small Geoglossum or cylindric Xylaria. Specimens examined: Porto Rico: J. R. Johnston, comm. by W. G. Farlow, type (in Mo. Bot. Gard. Herb., 5119). EICHLERIELLA Eichleriella Bresadola, Ann. Myc. 1:115. 1903.—Hirneolina as a section of Sebacina Patouillard, Essai Taxon. 24. 1900.— Hirneolina (Pat.) Saccardo, Syll. Fung. 17: 208. 1905. Fructifications coriaceous, waxy or membranaceous, sub- gelatinous, cup-shaped or plano-concave, rarely pendulous, hymenium typically superior, discoid, inferior in pendulous forms, even or somewhat rugulose; basidia globose-ovoid, eruciately divided, with 2-4 sterigmata; spores hyaline, cylindric, somewhat curved. It is a Stereum or Cyphella with tremellaceous hymenium. The type species of the genus is Eichleriella incarnata Bres. The original definition of Eichleriella, which is translated above, should be broadened to accurately describe our North American species, which are as coriaceous as Stereum spadiceum. All have the hymenium inferior. Eichleriella gelatinosa is our only species with subgelatinous hymenium. But few species of this genus are known. Five species of Eichleriella have been recognized up to the present time in North America, three in Europe, and two in South America; [VoL, 2 744 ANNALS OF THE MISSOURI BOTANICAL GARDEN of our five, only one species, Eichleriella Leveilliana, ranges through the eastern United States; E. spinulosa occurs in both Europe and North America. Key TO THE SPECIES Fructifications gray, small, 13-2 mm. long, %,-1 mm. broad, en habit of Oyphella . E. Sec ER EIER ees a ETE OE DOARA ER ENT hrenkit Pructitcations the gee yi Eh ae and cream, and peltate a first, 1-5 . long, %-1% e Dalaran 2. E. Leveilliana Fructifications ica a 200-300 u thick; hymenium even; Bee fro uba EEE BER. su dh bes aes oesaneeeb sega ess eeseenne 3. E mae Fructifications wood-brown, with whitish margin; hymenium dry, with tubercules like: Radulum.:: ».:..:0. 00.000 as 200 0a 4. E. spinulosa rigen white at first, then clay-color, tomentose, soft and spongy, ne ick; hymenium gelatinous; known from Jamaica only........ MN ER whee Gila a BWA E Slag Mace ean 8 T 5. E. gelatinosa 1. Eichleriella Schrenkii Burt, n. sp. Plate 27, fig. 8. Type: in Mo. Bot. Gard. Herb. and in Farlow Herb. Fructifications gregarious, coriaceous, sessile, pezizoid, oblong or rotund, margin free and strongly inrolled, pubes- cent, smoke-gray; hymenium concave, pale smoke-gray to pallid neutral gray; basidia longitudinally septate, pyriform, 22 X 11 a; spores white in collection on slide, simple, curved, pointed at base, 12-19 X 6-74 u. Fructifications 4-2 mm. long, 4-1 mm. broad, 4 mm. thick. On bark of dead limbs of Prosopis (mesquite). San Antonio, Texas. February. The general habit of this fungus resembles that of very small specimens of Corticium Oakesii, of large species of Cen- angium, or of a sessile Cyphella; from all of which Eichleri- ella Schrenki is easily separated by its longitudinally septate basidia which show clearly in sectional preparations. The fructifications are much smaller than those of any other species of this genus heretofore described. Specimens examined: Texas: San Antonio, H. von Schrenk, type (in Mo. Bot. Gard. Herb., 42579), and also (in Mo. Bot. Gard. Herb., 42580). 2. E. Leveilliana (Berk. & Curtis) Burt, n. comb. Plate 27, fig. 9. Corticium Leveillianum Berk. & Curtis, Hooker’s Jour. 1915] BURT—THELEPHORACEAE OF NORTH AMERICA. V 745 Bot. 1:238. 1849.—Stereum Leveillianum Berk. & Curtis, Grevillea 1: 163. 1873. Type: type and cotype in Kew Herb. and in Curtis Herb., respectively. Fructification coriaceous, soft, dry, rather thick, vinaceous fawn at first, whitening with age, resupinate-effused, with the margin free, sometimes narrowly reflexed, concolorous, minutely tomentose; hymenium composed of a surface layer about 30 » thick of paraphyses 14-2 u in diameter and of a layer of basidia under this; basidia longitudinally septate, 10-18 x 6-12 u; spores in spore collection, white, simple, curved, pointed at base, 12-16 X 5-6 u. Fructifications often 5 mm. in diameter at first, finally up to 1-5 em. long, 3-1} cm. broad, about 4 mm. thick. On dead limbs of several species. New York to Texas, Cuba, Jamaica, Central America, and Brazil. November to May. This is a well-marked species upon which Berkeley made the following excellent graphie comment: “At first forming little peltate orbieular spots, which, as they dilate, become closely attached to the matrix, with the exception of the margin, which is often free, soon confluent, soft, rather thick; of the colour of raspberries and cream. Hymenium often minutely pitted. Old specimens lose in great measure their ruddy hue, and are of a dead white.’’ I have seen no specimens having the hymenium minutely pitted. Specimens examined. Exsiccati: Ravenel, Fungi Car. II, 35. New York: Hudson Falls, S. H. Burnham (in Mo. Bot. Gard. Herb., 44009, 44170, 44194) ; Buffalo, G. W. Clinton. South Carolina: M. A. Curtis, 1220, 92 (types and cotypes in Kew Herb. and Curtis Herb., respectively); Ravenel, Ravenel, Fungi Car. II, 36. Georgia: Tallulah Falls, A. B. Seymour, Farlow Herb., © (in Mo. Bot. Gard. Herb., 44608). Texas: Austin, W. H. Long, 570, Cornell Univ. Herb.; San [VoL, 2 746 ANNALS OF THE MISSOURI BOTANICAL GARDEN Antonio, A. B. Langlois, bd; same locality, H. von Schrenk (in Mo. Bot. Gard. Herb., 42576). Cuba: San Diego de los Baños, Earle & Murrill, 296, 356 in part, N. Y. Bot. Gard. Herb. Jamaica: Cinchona, W. A. £ E. L. Murrill, N. Y. Bot. Gard., Fungi of Jamaica, 493. Brazil: Blumenau, A. Möller, comm. by G. Bresadola; Matto Grosso Cuyaba, G. Malme, 599, comm. by L. Romell. 3. E. alliciens (Berk. & Cooke) Burt, n. comb. Plate 27, fig. 10. Stereum alliciens Berk. & Cooke, Linn. Soc. Bot. Jour. 15: 389. 1876; Massee, Linn. Soc. Bot. Jour. 17 : 201. 1891. Type: in Kew Herb. Fructification coriaceous, resupinate, sometimes narrowly reflexed, separable, ochraceous buff, the margin slightly paler, the reflexed portion tomentose; structure in section, 200-300 u thick, (1) with hyphae next to substratum ochraceous, loosely interwoven and protruded, 3 p in diameter, similar to those on outer surface of reflexed portion, (2) with inter- mediate layer 100-180 a thick, composed of longitudinally arranged hyphae 2 a in diameter, (3) with hymenium com- posed of basidia 10 » below the surface, imbedded in jelly through which rise a few filiform paraphyses or hyphae to the surface; hymenium even, ochraceous buff; basidia longi- tudinally cruciately septate, pyriform, 12-15 X 9-10 yw; spores colorless, simple, even, curved, 10-13 X 34-5 p. Fructifications of type described as several inches long, originally orbicular; Cuban specimen 1 em. long, 1 em. broad, reflexed side 1-2 mm. long, 1 em. broad. On dead wood in virgin forest. Cuba and Brazil. March. The fructification resembles in habit and coloration that of a resupinate specimen of Stereum hirsutum with a very nar- rowly reflexed margin. The Cuban collection, of which but a single fructification was communicated to me, is much smaller than the Brazilian type and has the hyphae of the intermediate layer with gelatinously modified wall. Specimens examined: 1915] BURT—THELEPHORACEAE OF NORTH AMERICA. V 747 Brazil: San Antonio da boa vista, Rio Javary, Traill, 1, type (in Kew Herb.). Cuba: San Diego de los Baños, Pinar del Rio Province, Earle & Murrill, 405, N. Y. Bot. Gard. Herb. 4. E. spinulosa (Berk. & Curtis) Burt, n. comb. Plate 27, fig. 11. Radulum spinulosum Berk. & Curtis, Grevillea 1: 146. 1873. —Radulum deglubens Berk. & Broome, Ann. and Mag. Nat. Hist. IV. 15: 32. 1875.—Eichleriella deglubens (Berk. & Br.) Lloyd, Letter No. 45:7. 1893; Wakefield, Brit. Myc. Soe. Trans. 4: 305. 1914.—Stereum rufum of English authors but not S. rufum Fries—Radulum Kmetii Bresadola, I. R. Accad. degli Agiati Rovereto Atti III. 3: 102. 1897.—Kichleri- ella Kmetii Bresadola, Soc. Myc. France 25: 30. 1910. Type: in Kew Herb. Fructifications longitudinally and broadly effused, wood- brown, coriaceous-soft, separable, with the margin whitish, finally narrowly reflexed on the upper side and tomentose, or with margin everywhere free and curved outward; hymenium wood-brown, dry, usually bearing tubercules singly or in small clusters, with pale tips; basidia longitudinally septate, clavate, 25-369 u, arranged between paraphyses with brown tips; spores simple, colorless, cylindric, curved, 15-16 X 6 p. Fructifications range up to 6 cm. long by 1-2 em. wide and may be larger by confluence, about 700 u thick; tubercules about 4-1 mm. long. Alabama. On bark of dead Populus trichocarpa, Idaho, and Oregon. July to September. This species is distinguished by having a hymenium with configuration of a Radulum and cruciate basidia. The tuber- cules are often simple and cylindric, sometimes deformed and multifid. The wide distribution and yet the extremely local occurrence of this species together with the absence, until recently, of observations on its basidia have resulted in a very interesting synonymy. It is remarkable that this species, which occurs on Fraxinus, Populus, ete., in several countries of Europe, should have been collected in the United [VoL. 2 748 ANNALS OF THE MISSOURI BOTANICAL GARDEN States in Alabama, Idaho, and Oregon only. I am greatly in- debted to Mr. L. Romell for a preparation from the type of Radulum spinulosum which makes possible the reference to this species. Specimens examined : Sweden: Stockholm, L. Romell, 327, and three unnumbered collections. Alabama: Peters, Curtis Herb., 4543, preparation from type (in Kew Herb.). Idaho: Kaniksu National Forest, Priest River, J. R. Weir, 55. Oregon: Eugene, C. J. Humphrey, 1103. 5. E. gelatinosa Murrill, n. sp. Plate 27, fig. 12. Type: in N. Y. Bot. Gard. Herb. and in Burt Herb. Fructification eoriaceous, effuso-reflexed, white when young, finally clay-colored, tomentose, soft to the touch, margin obtuse; context soft, spongy, zonate; hymenium tough, gela- tinous, drying Hay’s brown, even; basidia longitudinally septate, 13 X 11 p; spores simple, colorless, even, flattened on one side, 8-10 X 6 a. Reflexed portion of fructification 14-2 em. long, 2} em. wide, 4 em. thick. On rotting wood in wet, wooded regions. Jamaica. Decem- ber and January. Only two collections of one fructification each were made. That of December 17 is a white, young specimen, with no basidia developed, which shows the general habit and early characters of the species, but would not have been determin- able except for the later collection of January 12-14, which shows the darker coloration assumed at maturity. The thick, spongy, soft pileus of the mature fructification distinguishes this species from others known at present. Specimens examined : Jamaica: Troy and Tyre, Cockpit country, W. A. Murrill & W. Harris, N. Y. Bot. Gard., Fungi of Jamaica, 1087, type (in N. Y. Bot. Gard. Herb.), a portion in Burt Herb.; Blue 1915] BURT—THELEPHORACEAE OF NORTH AMERICA. V 749 Hole, Priestman’s River region, W A. Murrill, N. Y. Bot. Gard., Fungi of Jamaica, 180, immature specimen. SEBACINA Sebacina Tulasne, L. R. and C., Ann. Sci. Nat. V. 15: 223- 226. pl. 10. f. 6-10. 1872; Linn. Soc. Bot. Jour. 13:35. 1873; Brefeld, Untersuch. Myk. 7:102-106. pl. 6. f. 22-26. 1888; Patouillard, Essai Taxon. Hym. 24, 25. 1900 (with the exclu- sion of section Hirneolina) —Exidiopsis Brefeld, Untersuch. Myk. 7:94. pl. 5. f. 20-22. 1888.—Stypella Möller, A., Bot. Mitth. a. d. Tropfen. 8, Protobasidiomyceten 166. pl. 4. f. 6, 7. 1895. Fructification coriaceous, membranaceous or floccose, gela- tinous, waxy or pulverulent, resupinate, with habit of Corti- cium; basidia longitudinally septate, close together or scat- tered, sometimes between bushy conidiophores; spores color- less, producing in germination a similar spore or a cluster of conidia. The type species of the genus is Corticium incrustans Pers. Sebacina incrustans occurs sometimes on the ground and incrusting herbaceous stems and various erect objects but is often on decaying wood; S. Helvelloides occurs on the ground and incrusting erect objects; S. chlorascens has been observed incrusting the mossy bases of living trees; the other species have been recorded only on dead wood and bark. A few mem- bers of this genus are thick and spongy and were originally included in Thelephora; usually the species are thin and Corticium-like in general habit and were in several instances published under Corticium. In the dried conditions some species of Sebacina may be tentatively recognized as such by having the hymenial surface glassy or resembling dried carti- lage; but such a separation from Corticium is very uncertain, for some species of Sebacina dry with a dull, soft surface and some true Corticiums assume the appearance of dried carti- lage in drying. It seems probable that it will always be difficult to deter- mine resupinate species of Hymenomycetes; it is not possible to do so from the descriptions alone of the earlier botanists. European authors have recently been enlarging such descrip- [VoL. 2 750 ANNALS OF THE MISSOURI BOTANICAL GARDEN tions by giving spore characters, dimensions of basidia, eystidia, and hyphae, and the presence or absence of clamp connections. Such additional characters may often be ob- tained quickly by microscopic examination of a portion of the fructification which has been teased out and crushed down in dilute potassium hydrate solution; by these helpful addi- tional characters, some species may be recognized with reason- able accuracy, but there are comparatively few such species. Structure in section of the fructification affords important characters for the identification of resupinate species. In practical work with these species, a microscopical mount of a sectional preparation of a type specimen is the next best thing for purposes of comparison to having the type itself. My method of determining a resupinate specimen is to ob- serve closely its general habit and characters, such as con- sistency, adnation, thickness, surface, margin, substratum, and color. Color is an important character when given in terms of an adequate color standard. The color which the specimens retain in drying is often the only color character available; it is more constant, fortunately, than is com- monly appreciated, for it has to be the color factor in the comparison of herbarium specimens. The preliminary ob- servation may suggest that the species is one of several of somewhat similar habit which may be of the same genus or of various genera. The sectional preparations, which are now made, may present (a) a uniform, homogeneous arrange- ment of similar hyphae from substratum to hymenium, (b) dissimilar hyphae or organs distributed uniformly through- out the whole fructification, (c) a layered, heterogeneous ar- rangement of various types with the layers more or less sharply differentiated from one another, (d) a stratose ar- rangement having the first stratum extend from the sub- stratum to the upper surface of the first hymenium, the second stratum a repetition of the first and borne on the first, and so on. Under a there are characteristic varieties of struc- ture, constant for each species, such as all the hyphae in erect position extending from substratum to hymenial surface, or all interwoven, or all procumbent, and there are also constant 1915] BURT—THELEPHORACEAE OF NORTH AMERICA. V 751 differences in regard to whether the hyphae are crowded close together or are loosely arranged. Under c, a conspicuous example would be one in which the layer next to the sub- stratum is composed of longitudinally arranged hyphae (that is, parallel with the substratum) crowded closely together; ratte this layer, a few branches might extend outward at right angles to the first layer and form a layer of loosely arranged, erect hyphae — the second layer; the hyphae of the second layer might branch abruptly at its outer surface and bear a compact hymenial layer. Some species invariably form a loosely interwoven layer next to the substratum, and on the surface of this layer form a dense hymenial layer, as, for ex- ample, Sebacina incrustans, S. chlorascens, and S. Helvel- loides. Sterile fructifications may frequently be determined by their general characters and structure in section. The preparations which reveal structure in section, give also spores, basidia, paraphyses, and other organs. From the combination of general characters, structure in section, and details of spores and noteworthy organs, the species becomes manifest. Our species of Sebacina are described in accord- ance with this method in the following pages. KEY TO THE SPECIES en 2 the ee running up and incrusting the bases of living runks well as dead objets: |: ee EEE . merustans . Pileate branches = eng cream-color with a glaucous tint, ws mg apices spiculose or fimbriate; hymenial layer drying vinaceous brow =) it ° zE oO gt PE oO Dm ES D m A = R 5 5 yet rd a A E E EEE ETDS 3. 8. emio l. Not fo hr free branches or flaps; hymenial T 200-300 u thick; paraphyses straight and rod-like; basidia 20-25 X15 w..... 48. Helvelloides 2. Fructifications white or whitish Wate er eee. 3 B, RUM MOE WEE eo). soso ss ne ann nenn, 4 3. Hymenium composed of unbranched, flexuous, even-walled, deeply staining, clavate organs 40-456 u, in addition to few- branched paraphyses and WORDS Fae ET ya ci PORT REVERSE. a. 8. ka 3. Hymenium composed of paraphyses and basidia; gegen > 300-400 thick; margin thick, not closely adnate to substratum...... 6. S.m silatoepsie 3. Hymenium composed of basidia and paraphyses; ia tification ‘age thick, shining hit at first; margin very thin and closely adnate..7. 8. ER 3. Hymenium composed of basidia and paraphyses; fructification 300-3 300 thick, dirty whitish; hyphae er in upper two-thirds of fructifica- ion; margin thin and closely adnate. s -asnes ce 8. 8. monticola [vor. 2 752 ANNALS OF THE MISSOURI BOTANICAL GARDEN crystalline matter about 100 u below surface. On Alnus, South EI TER ER eT 8 scariosa 4, a some variety Of bDrOWN........ ce cece ee eter nennen nenn 4. Drying fuscous to black........... ses seeeee eee rece nennen nn 6 5. Drying cacao-brown (testaceous of Saccardo’s ‘Chromotaxia’) ; separable from substratum; resembling S. incrustans but with margin soon de- tached and spores 6-7 X4%4-5 m. On juniper, Alabama...... 2. 8. deglubens 5. Blue-purple when — — = awny olive to Saccardo’s umber where directly on the wood; ser e to substratum; 30-45 a thick; basidia 7-10X6-8 u; he B-T KBB Miceeccccscnscccceenveanens 10. 8. podlachica 5. Drying cinnamon-brown ; dna | to substratum; 100-140 a thick; scattered paraphyses with Bun “branched, brown tops rise 45-60 p ‘above the basidia. On Magnolia, Delaware. .....esssssererrrsreto 1. 8. cinnamomea 6. Hay’s brown when moist, eo. fuscous, the margin pale cartridge- uff; separable from substratum; 500-600 u thick. On ur [Pe rrr errr rere rer rer rr kr eee re . 8. adusta 6. hag blackish plumbeous; adnate to E oa p thick, the margin indeterminate. On Populus, Was ington ness 13. K. plumbea 6. Grayish when moist, drying dark mouse-gray and shining; adnate to substratum ; 50-160 H e i the margin indeterminate. On very rotten wood, New England. ......sssssssrssssesererrereto 14. K. atrata 1. Sebacina incrustans Pers. ex Tulasne, Ann. Sci. Nat. Bot. V. 15:225. pl. 10. f. 6-10. 1872; Linn. Soc. Bot. Jour. 13: 36. 1873. Plate 27, fig. 13. Corticium incrustans Persoon, Obs. Myc. 1:39. 1796.— Thelephora incrustans Persoon, Syn. Fung. 573. 1801; Fries, Syst. Myc. 1:448. 1821.—Thelephora sebacea Persoon, Myc. Eur. 1:155. 1822; Fries, Elench. Fung. 1:214. 1828; Hym. Eur. 637. 1874; Saccardo, Syll. Fung. 6: 540. 1888.—Corticium sebaceum Massee, Linn. Soc. Bot. Jour. 27:127. 1891.— Merisma cristatum Persoon, Syn. Fung. 583. 1801.—Thele- phora cristata Fries, Syst. Myc. 1: 434. 1821; Hym. Eur. 637. 1874; Saccardo, Syll. Fung. 6: 539. 1888.—Sebacina incrustans Tul. ex Bresadola, in part (Hym. Hung. Kmet.), I. R. Acad. Sei. Agiati III. 3: 117. 1897. Illustrations: Tulasne, loc. cit—Persoon, Com. Fung. Clav. pl. 4. f. 4; Berkeley, Outlines Brit. Fung. pl. 17. f. 6; Brefeld, Untersuch. Myk. 7: pl. 6. f. 22-26. Hennings in Engl. & Prantl, Nat. Pflanzenfam. (I. 1 **): 91. f. 59 C, D; Nees, System pl. 34. f. 256 B; Patouillard, Tab. Anal. Fung. f. 155; and Essai Tax. Hym. 25. f. 17 a, b; Soc. Mye. Fr. Bul. 5: pl. 7. f. 11—See Saccardo, Syll. Fung. 20: 945 for references to some additional illustrations which I have not seen. 1915] BURT—THELEPHORACEAE OF NORTH AMERICA. V 753 Type: authentic specimens of Thelephora incrustans and Merisma cristatum from Persoon in Kew Herb. Fructifications coriaceous-fleshy, varied in form, creeping on the ground and ascending and incrusting small erect objects and forming little columns and free branches, the apices somewhat awl-shaped or fringed, or effused and resup- inate on bark, whitish, drying warm buff; structure in section, 250-400 a thick, (1) with a broad layer of very loosely inter- woven rather stiff hyphae, 2-24 p in diameter, which divide above into fine branches and form (2) a densely interwoven layer about 60-150 „ thick with the basidia in the upper 40-90 u among the very fine (14 „a in diameter), densely crowded, somewhat interwoven filaments from the subhymenium; basidia longitudinally septate, ovoid or pyriform, 12-20 x 9- 14 »; spores colorless, simple, even, flattened on one side or curved, 12-14 X 6-8 u. Fructifications 5-6 em. long, 2-5 em. wide, ascending ob- jects 2-5 em.; pileate flaps, when present, 4-1 cm. long. On the ground in woods and incrusting objects, and resup- inate on logs. Canada to Louisiana and westward to Mis- souri. June to October. Common. S. incrustans is the common incrusting Sebacina of Eastern North America. It may usually be recognized at sight by coriaceous-fleshy consistency, occurrence on earth and run- ning up and incrusting living objects, and pallid color. The thinner hymenial layer, paraphyses less rod-like in form, and finer and thinner-walled hyphae of layer next to the sub- stratum afford structural characters separating specimens of this species from those of S. chlorascens and S. Helvelloides. I exclude from the synonymy of S. cristata, Clavaria laciniata of Bulliard’s ‘Hist. Champ.’ 1:208. pl. 415. f. 1, because in the absence of authentic specimens and observa- tions in regard to spores and basidia, it is not certain that C. laciniata Bull. is Merisma cristatum. Bulliard’s figures repre- sent quite as well an incrusting European fungus communi- cated to me by Bresadola under the name Thelephora fas- tidiosa (Pers.) Fr., which has simple basidia and colorless echinulate spores. This species is the Thelephora cristata [Vou, 2 754 ANNALS OF THE MISSOURI BOTANICAL GARDEN of Patouillard’s ‘Tab. Anal. Fung.’ No. 559, and Cristella cristata of his ‘Essai Taxon. Hym.’ f. 28. Patouillard notes that Clavaria laciniata is a synonym of the species which he figures. Because of the uncertainty as to whether figures of Thelephora cristata by European authors represent the true Merisma [Sebacina] cristatum Pers. or the echinulate- spored T. fastidiosa (Pers.), I have refrained from citing any illustrations except that of Persoon, of whose species I have studied an authentic specimen. Specimens examined : Exsiceati: Ellis, N. Am. Fungi, 513. The specimen in Thuemen, Mye. Univ. 2009, under the name Thelephora sebacea, collected in France, is Thelephora mollissima Pers. Europe: authentic specimens of Thelephora incrustans and Merisma cristatum from Persoon in Kew Herb. Sweden: sterile specimen determined as Thelephora cristata by E. Fries (in Fries Herb.) ; Stockholm, L. Romell, 54. Canada: J. Macoun, 5, 10. Quebec: Hull, J. Macoun, 203, 313. Ontario: near Ottawa, J. Macoun, 40 (in Can. Geol. Surv. Herb.) ; London, J. Dearness. Maine: Portage, L. W. Riddle. New Hampshire: Shelburne, W. G. Farlow (in Farlow Herb.). Vermont: Middlebury, E. A. Burt, two collections. Massachusetts: Williamstown, W. G. Farlow (in Farlow Herb.). New York: Hudson Falls, S. H. Burnham, 2 (in Mo. Bot. Gard. Herb., 43995). Pennsylvania: Michener, 5821 (in Curtis Herb.); Trexler- town, W. Herbst. District of Columbia: Rock Creek, C. L. Shear, 793. North Carolina: Asheville, H. C. Beardslee, 03126. South Carolina: Ravenel, 1619 (in Curtis Herb.). Louisiana: St. Martinville, A. B. Langlois, F, 2015; the same locality and collector, (3022 in Lloyd Herb.) ; Baton Rouge, Edgerton & Humphrey, 667. Ohio: A. P. Morgan (in Lloyd Herb., 2655, 2656) ; Cincinnati, 1915] BURT—-THELEPHORACEAE OF NORTH AMERICA. V 755 C. G. Lloyd, 4198; Loveland, D. L. James (in U. S. Dept. Agr. Herb.). Wisconsin: Blue Mounds, E. T. and S. A. Harper, 864, 879, 880; Madison, W. Trelease (in Mo. Bot. Gard. Herb., 5145, 44779); C. J. Humphrey, 2146 (in Mo. Bot. Gard. Herb., 44784). Illinois: Riverside, E. T. and S. A. Harper, 698. Missouri: Creve Coeur, E. A. Burt (in Mo. Bot. Gard. Herb., 44763). 2. 8. deglubens (Berk. & Curtis) Burt, n. comb. Corticium deglubens Berk. & Curtis, Grevillea 1: 166. 1873. Type: type and cotype in Kew Herb. and Curtis Herb. Fructification resupinate, effused, coriaceous, separable, white beneath, drying about cacao-brown, the margin very narrow, white, byssoid, soon. detached; structure in section 250-300 y thick, (1) with a very loosely interwoven layer 180- 200 » thick, having hyphae 14-2 „ in diameter which branch and form (2) a very densely interwoven layer 80 „ thick with the basidia in the upper 30 a, not quite reaching to the sur- face, among the very fine, densely interwoven filaments from the subhymenium; basidia longitudinally septate, 15 x 10-12 u; spores colorless, simple, even, flattened on one side, 6-7 x 44-5 u. On juniper, Alabama. This fungus has the same type of structure which is found in resupinate specimens of Sebacina incrustans. It differs from the latter in having the hymenium darker, all the spores found in a sectional preparation a little smaller, and the hyphae of the layer next to the substratum a little smaller and more flaccid than those of S. incrustans, and the margin was described as soon detached. These differences may be merely the variation from specific type of a single collection, or they may be those of a subspecies of S. incrustans which has taken on the saprophytic life on dead wood, prevalent for most species of Sebacina. Until other collections, referable to S. deglubens are made, the former view appears the more probable. [VoL. 2 756 ANNALS OF THE MISSOURI BOTANICAL GARDEN Specimens examined: Alabama: Peters, Curtis Herb., 4557, type (in Kew Herb.). 3. S, chlorascens Burt, n. sp. Plate 27, fig. 15. Type: in Mo. Bot. Gard. Herb. and in Farlow Herb. Fructification coriaceous, drying cream-color with glaucous tint, effused, ascending and incrusting the mossy bases of trees and forming imbricated, free, pileate, sterile branches, the apices spiculose or fim- briate; hymenium gelatinous, drying vinaceous brown, occurring in somewhat scattered spots on the lower portions of the fructification; structure in section 800 a thick, with (1) a broad, spongy layer next to the substratum of loosely interwoven, rather rigid, even-walled hyphae 23-3 p in diameter, which bear (2) a sharply differentiated hymenial layer 140-240 u thick, composed of rod-like paraphyses 2 „ in diameter, between which occur basidia through- out the outer 60 u of the layer; basidia longi- tudinally septate, pyriform, 15-18X12 u; spores Fig. 1 simple, colorless, flattened on one side, 10-103 X S. chlorascens Par: ; 6-7 u. ia AU, Ascending objeets 2-4 em., 1-2 em. broad; free branches up to 5 mm. long. On mossy bases of living trees. Florida. Autumn. As shown by the figures in pl. 27, the pileate branches of S. chlorascens do not resemble those of S. incrustans. The structure in section is different in every detail from that of specimens of the latter species and approaches more closely that of S. Helvelloides, but the fructification is thinner than that of the latter, has smaller basidia and spores, and the basidia distributed from the surface to about 60 u below the surface, and forms free pileate branches. Specimens examined: Florida: Cocoanut Grove, R. Thaxter, 98, type (in Mo. Bot. Gard. Herb., 43923, and in Farlow Herb.). 4, S. Helvelloides (Schw.) Burt, n. comb. Plate 27, fig. 14. 1915] BURT—THELEPHORACEAE OF NORTH AMERICA. V 157 Thelephora Helvelloides Schweinitz, Naturforsch. Ges. Leipzig Schrift. 1: 108. 1822; Am. Phil. Soc. Trans. N. S. 4: 168. 1834; Fries, Elenchus Fung. 1:193. 1828; Epier. 541. 1836-1838.—Corticium Helvelloides Massee, Linn. Soc. Bot. Jour. 27: 153. 1891.—Corticium basale Peck, N. Y. State Mus. Rept. 43:69 (23). 1890. Type: in Herb. Schw. and portions in Curtis Herb. and in Kew Herb. Fructification coriaceous, spongy, effused, convex, closely adnate and incrusting, on ground in mosses and on bark at bases of living trees, at first whitish, drying honey-color to warm buff; structure in section, with (1) a very thick spongy layer next the substratum, of loosely interwoven, branched, rather rigid, even-walled, brownish hyphae, 3-34 p in diameter, which bear (2) a fertile layer 200-300 » thick made up of great numbers of erect, straight, cylindric paraphyses 2 u in diameter, between which occur the basidia at about 40-50 u below the surface; basidia longitudinally septate, pyriform, 20-25 X 15 u; spores colorless, simple, flattened or slightly curved on one side, 12-13 X 6 u. Fructifications 3-15 em. long and wide, drying about 4-2 mm. thick to 9 mm. thick in type which covers a cushion of moss plants. On ground and bark at bases of living trees. New York to North Carolina. July and August. Specimens of this species have somewhat the habit of thick specimens of Coniophora puteana but are of very different structure. The abundant, erect, unbranched, cylindric para- physes often 200 » long which compose the greater part of the hymenium, and the large basidia are reliable characters for identifying Sebacina Helvelloides when sections are studied; the coarser and colored hyphae of the species give an additional character separating it from S. incrustans when the latter occurs strictly resupinate. The type specimen is abnormal in thickness and ridged surface by running over and incrusting a bed of moss. The hanging rootlets referred to in the original description are [VoL. 2 758 ANNALS OF THE MISSOURI BOTANICAL GARDEN moss stems. The specific name is rather fanciful and mis- leading. Specimens examined New York: Whitehall, c. H. Peck, type of Corticium basale (in Coll. N. Y. State); Alcove, C. L. Shear, 1221. North Carolina: Salem, Schweinitz, type in Herb. Schw., in Curtis Herb., and in Kew Herb.). 5. S. Shearii Burt, n. sp. Plate 27, fig. 16. Type: in Burt. Herb., and in Shear Herb. Fructification coriaceous, effused, dull white, drying pale olive-buff, cracked, the margin determinate, entire; structure in section, 140-200 u thick, with (1) a broad and dense layer next to the substratum of longitudin- ally arranged, slightly brownish, even-walled hyphae 14-2 „ in diameter, which branch and curve outward at a right angle and form (2) a fertile, less compact layer 60-75 a thick of sub- erect, few-branched paraphyses 3 p in diameter, of basidia at about 15-20 a below the surface, and of scattered, even-walled, flexuous, cylindric- l clavate organs—perhaps gloeocystidia—40-45 X it aa 6 u, not emergent above the surface; basidia longi- Paraphysis tudinally septate, pyriform, 15 X 9 a, with sterig- Bde mata 18 X 3 a; spores colorless, simple, curved, 9-15 X 44-6 u. Fructifications in crevices of bark at first, 2 X 1 mm., at length, by confluence, 7 cm. long, 1 em. broad. On dead Berberis vulgaris. District of Columbia. October. This species is well characterized by the presence in the hymenial layer of flexuous, even-walled organs, which are either latex or gloeocystidia, and by the broad layer of longi- tudinally arranged hyphae which shows relationship to Eichleriella, although the margin is not distinctly free. A few small granules are present on the hymenial surface but I do not know that they are a constant character. Specimens examined: District of Columbia: grounds U. S. Dept. Agr., Washington, C. L. Shear, 1238, type. 1915] BURT—THELEPHORACEAE OF NORTH AMERICA. V 759 6. S. macrospora (E. & E.) Burt, n. comb. Corticium macrosporum Ell. & Ev., Torr. Bot. Club Bul. 27:49. 1900. Type: in N. Y. Bot. Gard. Herb. ; specimens from type col- lection in Lloyd Herb., and in Burt Herb. Fructification coriaceous, appressed, thin, dull white, cracked, the narrow, white, cottony margin sometimes nar- rowly involute; structure in section, 300-400 a thick, with (1) a very broad layer of longitudinally arranged and somewhat obliquely ascending crowded hyphae 14 a in diameter, color- less next to substratum but brownish in upper part of layer, which pass into (2) the hymenial layer 60-100 „ thick, con- sisting of erect, bushy paraphyses and of basidia; basidia longitudinally septate, pyriform to subglobose, 15 X 9-12 u; spores colorless, simple, flattened on one side or curved, 104 x 44-6 u. Appearing at first in orbicular patches 3-5 mm. in diameter, at length confluent and up to 4 em. long, 14 em. broad. On pine (Pinus Strobus) limbs. Ohio. September. This species is near Sebacina calcea, but the single collec- tion which has been studied seems distinct from the latter by the thick, determinate margin, sometimes free and slightly upturned, by the greater thickness of the fructifications, by the brown hyphae of the middle region, and by walls of hyphae not gelatinously modified as in S. calcea. A relation- ship to Eichleriella is manifest in the broad layer of longi- tudinally arranged hyphae and in the tendency of the margin to be slightly free. The original description gives this species as on ‘‘Fraaxinus?’’, but the limbs are Pinus strobus. The spores are not exceptionally large; the specific name was probably based on immature basidia. Specimens examined. Ohio: Linwood, C. G. Lloyd, 3113, type collection. 7. S. calcea (Pers.) Bresadola, Fungi Tridentini 2: 64. pl. 175. 1892. Plate 27, fig. 17. Thelephora calcea Persoon, Syn. Fung. 581. 1801; Myc. Eur. 1:153. 1822.—Thelephora calcea c. albido-fuscescens [voL. 2 760 ANNALS OF THE MISSOURI BOTANICAL GARDEN Fries, Elenchus Fung. 1:215. 1828.—Thelephora acerina forma Abietis Fries, Syst. Myc. 1:453. 1821.—Corticium Abietis (Fr.) Romell, Bot. Not. 1895: 72. 1895.—Xerocarpus farmellus Karsten, Finska Vet.-Soc. Bidrag 37: 139. 1882. Illustrations: Bresadola, loc. cit.; Patouillard, Essai Taxon. Hym. 25. f. 17 Fructification effused, closely adnate, crustaceous, slightly pulverulent, shining white at first, at length darkening in the central portion from cartridge-buff to pale drab-gray, cracked, the margin much thinner and farinaceous; structure in section, 50-150 a thick, (1) with hyphae next the substratum interwoven, 2 a thick, the wall gelatinously modified, (2) hy- menial layer 40-60 a thick, composed of basidia and of paraphyses branched at the apex into very fine branches loaded with Fig. 3 minute granules; basidia more abundant in ea T the lower portion of the hymenial layer, longitudinally septate, 14 X 9 yw; spores colorless, simple, cylindric, curved, 8-12 x 4-5 u. Fructifications 3-9 em. long, 1-3 em. broad. On bark and wood of dead branches of spruce, pine, hem- lock, white cedar, oak, ash, elm, maple, and elder. Canada, northern New England, and New York to Georgia, and in Washington. March to January—perhaps throughout the year. As good distinctive macroscopic characters this species has: chalky white color with central portions ashy; powdery sur- face under a lens; thinness on drying and margin still thinner, so that it appears mealy under a lens rather than mem- branous. The fine branches and granules at the tips of the paraphyses show best in lactic acid preparations; potassium hydrate solution has a solvent action here. I have not been able to study an authentic specimen of Thelephora calcea Pers. and accept Bresadola’s conclusion on this point. Specimens examined: Exsiccati: Romell, Fungi Exs. Scand. 129. Austria: G. Bresadola. Sweden: L. Romell, 58, 59; Stockholm, L. Romell, Fungi Exs. 1915] BURT—THELEPHORACEAE OF NORTH AMERICA. V 761 Scand. These specimens are under the name Corticium Abietis. Norway: Christiania, M. N. Blytt (in Herb. Fries and deter- mined by Fries as Corticium calceum). Finland: Mustiala, P. A. Karsten, under the name Xero- carpus farinellus. Canada: J. Macoun, 30, 33. New Hampshire: Chocorua, W. G. Farlow, two collections. Vermont: Middlebury, E. A. Burt, two collections; Ripton, E. A. Burt; Little Notch, Bristol, E. A. Burt. New York: Alcove, C. L. Shear, 1134, 1208; Hague, C. H. Peck, 10; Clear Water, G. F. Atkinson, Cornell Univ. Herb., 5049. Georgia: Tipton, C. J. Humphrey, 177; Savannah, C. J. Humphrey, 5106 (in Mo. Bot. Gard. Herb., 15081). Washington: Bingen, W. N. Suksdorf, 695, 711, 763, 765, 864. 8. 8. monticola Burt, n. sp. Type: in Mo. Bot. Gard. Herb. Fructification coriaceous, resupinate, cracked, dirty whitish approaching pale smoke-gray, the margin closely adnate; structure in section 200-300 yw thick, with hyphae colorless, 3-4 u in diameter, ascending obliquely from substratum to surface, densely crowded together, more interwoven and little inerusted in the lower third of the fructification, but more loosely arranged and heavily incrusted in the whole upper two-thirds, terminating in incrusted paraphyses which are either simple or 2-4-branched and with the hyphal body about 24 p in diameter under the incrustation; basidia about 40 y below the surface of the hymenium, longitudinally septate, 15-20 x 9-12 u; spores simple, colorless, even, cylindric, straight or curved, 9-104 X 5-54 u. The portion of the fructification described is 5 cm. long, about 14 cm. wide. On bark of log of Picea Engelmanni, altitude 8,500 ft., Pike’s Peak, Colorado. August. This species belongs in the group with Sebacina calcea and S. macrospora; it is distinguished from both of these by the [VoL. 2 762 ANNALS OF THE MISSOURI BOTANICAL GARDEN incrustation of its hyphae and by simpler paraphyses, which are either unbranched or with only about 2-4 branches not branching repeatedly and becoming so attenuated as to be nearly invisible except for the granules which they bear. Specimens examined: Colorado: Pike’s Peak, G. G. Hedgcock, comm. by C. J. Humphrey, 2571, type (in Mo. Bot. Gard. Herb., 15157). 9. 8. scariosa (Berk. & Curtis) Burt, n. comb. Corticium scariosum Berk. & Curtis, Grevillea 2:3. July, 1873.—Corticium secedens Saccardo, Syll. Fung. 6: 635. 1888. Type: type and cotype in Kew Herb. and Curtis Herb., respectively. ‘“Forming a thin, oblong, membranous stratum, without any distinct border; hymenium pulverulent ochroleucous.’’ —Original description. Structure in section 300-600 a thick, with hyphae 2 a in diameter, branched, very loosely interwoven, extending from substratum to basidia, with walls gelatinously modified, im- bedded in jelly, much crystalline matter about 90-120 a below the hymenial surface; basidia at or near the surface, longi- tudinally septate, pyriform to subglobose, 12-15 X 9-12 u; no spores found. On alder, South Carolina. The type specimens of this species have the general habit of Peniophora gigantea, which they also resemble in being separable and in cracking and peeling up from the substratum, but they are more lemon-yellow in color than specimens of the latter species. The structure in section is distinetive and suggestive of that of Eichleriella alliciens. Authors have sometimes confused Corticium scariosum B. & C. with Cor- ticium scariosum B. and Br., published from Ceylon a few months later in the same year. The types of these fungi are not of the same genus, the American specimens having longi- tudinally septate basidia. Specimens examined: South Carolina:-Society Hill, M. A. Curtis, 4916 (type and cotype in Kew Herb. and Curtis Herb.). 1915] BURT—THELEPHORACEAE OF NORTH AMERICA. V 763 10. 8. podlachica Bresadola, Ann. Mye. 1:117. 1903. Type: in Bresadola Herb. and a portion in Burt Herb. Fructification effused, closely adnate, described as ‘‘e pallido-caerulea caesio-hyalina,’’ drying tawny olive to Sac- cardo’s umber where directly on the wood; structure in sec- tion 30-45 a thick, with hyphae 2 „ in diameter closely crowded together and rising obliquely from substratum to the surface; basidia in upper 15 yu of fructification among the hyphal fila- ments, longitudinally septate, pyriform, 7-10 X 6-8 u; spores colorless, simple, even, curved, 6-7 X 3-5 u. Covering areas 5 em. long, 2 em. broad. On decaying coniferous wood, Massachusetts; on decaying beech wood, Russian Poland. The Massachusetts collection was noted as blue-purple when fresh; in some places algae coating the wood have been covered by the fructification and the modified color of this algal layer is seen through the dried fructification; where the fungus coats the wood directly, the color of specimens which have been several years in the herbarium is the tawny olive. The American collection agrees closely with that communi- cated by Bresadola. Specimens examined: Russian Poland: on beech wood, Eichler, comm. by Bresadola, part of type. Massachusetts: on coniferous wood, W. G. Farlow. 11. S. cinnamomea Burt, n. sp. Plate 27, fig. 18. Type: in Burt Herb. Fructification effused, coriaceous, dry, close- ly adnate, drying cinnamon-brown, the margin determinate, thick, entire; structure in section 100-140 „ thick, with (1) a layer 10-30 a thick next to the substratum of longitudinally ar- ranged, densely interwoven hyphae 2-24 a in diameter, which bear (2) the hymenial layer g sinnam composed of basidia at the lower side of Paraphysisx540. the layer, and of loosely arranged, highly branched, bush- Fig. 4 (VoL, 2 764 ANNALS OF THE MISSOURI BOTANICAL GARDEN shaped paraphyses with brown branches of zigzag form, which rise 45-60 u above the basidia and give the characteristic color of the hymenium; basidia 15-20 x 9-11 a, longitudinal septa- tion not positively made out; no spores found; paraphyses 75 u long, trunk 14-2 u in diameter, sweep of branched top about Fructification 4 em. long, 1 em. broad. On limbs of dead Magnolia glauca. Maryland. December. It is not certain that this fungus is a Sebacina, for none of its basidium-like organs show longitudinal septa, although in a very few there is arrangement of the protoplasm suggestive of such septation. The specimen is a little too immature for generic reference but is probably a young Sebacina in my opinion. The species is distinct from others in possible genera by cinnamon-brown color, paraphyses scattered as to trunks but with such brown, bushy-branched tops as to form a compact surface of the color stated. Specimens examined: Maryland: Takoma Park, C. L. Shear, 1339, type. 12, S., adusta Burt, n. sp. Plate 27, fig. 19. Type: in Burt Herb. Fructification broadly effused, coriaceous, separable from the substratum, Hay’s brown when moist, drying fuscous, the margin pale cartridge- buff, fibrillose-fimbriate; structure in section, 500-600 u thick, composed of densely inter- woven and obliquely ascending hyphae 3 „in diameter, the walls not gelatinously modified, which bear the basidia at the surface of the hymenium; basidia longitudinally septate, pyriform, 12-16 x 8-10 pu; spores colorless, simple, curved, 10-12 X 4-5 u. Fructifieation 12 em. long, 4 em. broad. On decortieated trunk of Populus trichocarpa. Idaho. July to September. In the single eolleetion of this species which has been re- ceived the margin is everywhere closely applied to the sub- stratum and shows no tendency towards becoming free or ig. S. adusta. hyphae X 540. 1915] BURT—THELEPHORACEAE OF NORTH AMERICA. V 765 reflexed, hence the species must be included in Sebacina. The distinguishing specific characters are easy separation as an unbroken membrane of the moist fructification from the sub- stratum, thickness of fructification, and position of the basidia at the surface of the hymenium. Specimens examined: Idaho: Kaniksu National Forest, Priest River, J. R. Weir, 12, type. 13. S. plumbea Burt, n. sp. Plate 27, fig. 20. Type: in Burt Herb. Fructification effused, closely adnate, drying blackish plumbeous, pruinose, the margin indeterminate; structure in section, 150-200 u thick, with (1) a broad layer next to the substratum containing much crystalline matter in the interspaces between the interwoven suberect hyphae 14-2 „ in diameter, the wall gelatinously modified, and (2) a hymenial layer about 60 a thick consist- ing of basidia, and of hyphae which branch and Fig. 6 S. plumbea. : : Basidia and form a densely interwoven hymenial surface; hyphaex540. basidia about 30 u below the surface of hymenium, longitudinally septate, pyriform, 15-18 x 10-13 a; spores colorless, simple, even, curved, 13-15 X 43-6 u. Fructification 4-8 em. long, 4-1 em. broad. On blackened wood of Populus trichocarpa. Washington. November. The coloration and habit of specimens of this species agree closely with those of the European Corticium plumbeum Fr. which have been received from Karsten, but the internal struc- ture is wholly different from that of the latter. Specimens examined: Washington: Bingen, W. N. Suksdorf, 862, type. 14, S. atrata Burt, n. sp. Plate 27, fig. 21. Type: in Burt Herb. and in Farlow Herb. Fructification effused, somewhat gelatinous, closely adnate, grayish when moist, drying dark mouse-gray and shining, the margin thinning out and indeterminate; structure in section, [Vor. 2, 1915] 766 ANNALS OF THE MISSOURI BOTANICAL GARDEN 50-160 a thick, with even-walled hyphae 3 y in diameter, densely interwoven next to the substratum, then curving out- ward to form a hymenial layer 50-90 a thick, consisting of basidiax 540. erect, parallel, rod-like paraphyses 2 a in diam- eter and of basidia about 30 u below the surface of the hymenium; basidia longitudinally septate, pyriform, about 18 X 12 p; spores colorless, simple, somewhat flattened on one side, 8-10 X 6-7 u. Fructifications 24 em. long, 14 em. broad. On very rotten coniferous and frondose wood. New Hampshire and Massachusetts. May. When bits of dried specimens of this species are moistened, they become softer and more gela- tinous than is usual with those of other species of the genus, but walls of the hyphae do not show gelatinous modification in sectional preparations. The paraphyses are as noteworthy as those of Sebacına Helvelloides, being arranged close to- gether side by side in a palisade layer. They are sometimes simple rods, sometimes divided into equal branches which rise side by side to the surface of the hymenium. Specimens examined: New Hampshire: Chocorua, W. G. Farlow, two collections (of which No. ais in Mo. Bot. Gard. Herb., 44782). Massachusetts: Magnolia, W. G. Farlow, type. (To be continued. ) 768 [Vor. 2, 1915] ANNALS OF THE MISSOURI BOTANICAL GARDEN EXPLANATION OF PLATE PLATE 26 The figures of this plate have been reproduced natural size obi i of dried her- barium specim Fig. 1. Trem —: Cladonia. a, from specimen collected in Canada by J. Macoun, 78; b, collected at "Hague, New York, by C. H. Peck, 7; ¢, pervert at Cincinnati, Ohio, by A. P. Morgan, Lloyd Herb., 32. Fig. 2. T. Cladonia, Be the type of Thelephora gracilis, collected in an pis F. S. Earle, 13. Fig. 3. candidum. Collected at .. Vermont, by C. Howe. a i closely with the type and is my standard for co parison; b could be Rn without fracture into three portions, each having seu of a matoides. a, from specimen collected at York Co ounty, Pennsylvania, by = M. Glatfelter; b, single fructification from the clus c, from a very fascicula te s specimen having stems grown ei, act Saidiy still en at apex, collected at Had- re ei Sapien Fig. 5. mplex. From type coll ected in a. Rico, by J. R. Johnston. The gener ent hig on the right is i Fig. En ie pali dg m. a, from specimen eat er eek Verm yE. a. b, from speeimen in Mo. Bot. Ga rd. Her 712370, Collet at "st. uis, Missouri, by N. M. Glatfelter. Bath wth no of the flattened pileate divisio Fig. 7. a tenue. a, from types eateries at Chester aa. aan by . and E. L. Murrill, b, from specimens collected at Cinchona, Jamaica, by the same Pe en 614. oS eae: eee ae ee ee eee jar e PE a > ee TE = r en 2 s " i ng i__ANN. MO. BOT: GARD., VOL. 2,1915 ] ‘ PLATE 26 Oe ai en II EURT-TEELEFHORATEAE OF NORTH AMERICA 1 AND 2. a ay iam ewe ante CLADONIA.— CANDIDUM.—4 T. MERISMATOIDES.— . T. SIMPLEX.—. T. PALLI eg —7. T. TENUE. COCKAYNE, BOSTON. 770 [Vor. 2, 1915] ANNALS OF THE MISSOURI BOTANICAL GARDEN EXXPLANATION OF PLATE PLATE 27 The figures of this plate have been reproduced natural size from oh a ar of dried her- barium a cimens, except in the cases noted otherw Fig. 8. Eichleriella cg nkit. From the type collected at San Antonio, Texas, by H. von Schrenk. a, photograph of a piece of limb bearing many Intita nos, and b, drawing of median longitudinal section z single fructification, x 16. Fig. E. Leveil dira From specimens collected at San Antonio, Texas, N H. von Schre Fig. 10. E. alliciens. "Fro are rar at San Diego de los Beton Cuba, by Earle and "Murrill, 4 art. Fig. 11. E. ge From. siber i ‘eae at Priest River, Idaho, 4 J. R. Weir, Fig. 12. E. a From sen collected in Jamaica by W. A. Murrill and W. Harris. a, upper surface of No. 180; b, type specimen, 1087, split longitudinally to oe thickness of pileus and tructure. Fig. 13. Sebacina incrustans. a, from specimen collected at Middl ebury, Vermont, by E. A. Burt; b, from specimen with pileate es collected at Asheville, North Carolina, by H. C. Beardslee, 0312 Fig. 1. S. Helvelloides. From feom collected at Alcove, New York, by C. L. Shear, 1221. shows upper su a. . is a vertical section from the same riesig to show thickne Fig. 15. 8. chlorascens. From type specimen collected a cane nut Grove, en by R. Thaxter, 98. Fig. 16. 8. arii. From type eg collected at Washing- ton, Distriet ee Columbia, by C. L. Shear Fig. 17. 8S. calcea. From nn on nn. cedar bark, collected at Middlebury, Vermont, by E. A. Burt. ig. 18. S. cinnamomea. ze type specimen collected at Takoma Park, Maryland, by C. L. Shear, 1339. ig. 19. 8. adusta. P iiin: ri specimen collected at Priest River, Idaho, 2 J. R. Weir, 1 Fig S. ee From ‚pe specimen collected at Bingen, Washington, by W. N. Suksdorf, 862 Fig. 21. 8. atrata. From \ specimen collected at Chocorua, New Hampshire, by W. G. Farlow ee ea ye a Da. Be ? 5 ws ANN. Mo. BOT. GARD., VOL. 2, 1915 PLATE 27 BURT—THELEPHORACEAE OF NORTH AMERICA 8. rege —9. E. LEVEILLIANA.—10. E. a papap 3. SEBACINA INCRUSTANS.—14. ee sA SHEARII.—17. S. CALCE 18. 8. en. —19. S. ADUS —20. S. PLUMBEA.—21. COCKAYNE, BOSTON. ALLICIENS.—11. E. ca T S. er A ATRATA. ENZYME ACTION IN THE MARINE ALGAE A. R. DAVIS search Assistant to the Missouri Botanical Garden, Formerly aa J. Lackland Fellow in the Henry Shaw School of Botany of Washington University In a previous contribution from this laboratory! attention has been called to the difficulties experienced in demonstrating enzyme action in Fucus vesiculosus. Because of the negative results there obtained it was deemed worth while to extend the study to certain representative forms of the three great groups of marine algae, the ‘‘greens,’’ the ‘‘browns,’’ and the ‘‘reds’’; first, to ascertain whether this apparent inac- tivity were generally characteristic of the algae, and second, because of the light such an investigation might shed upon the general metabolism of the group. HISTORICAL Knowledge concerning enzyme activity and the distribution of enzymes in the algae is extremely meagre. The few papers that have found their way into the literature have been, for the most part, by-products of other studies and as such have dealt merely with isolated phases of the subject. From time to time, previous to actual demonstration, the presence of enzymes has been suggested by the work of various investi- gators. Arber (’01), attacking the problem of carbon as- similation in Ulva latissima, found that the accumulation of starch in the tissue disappeared very slowly when the plant was subjected to darkness. This would suggest the presence of a diastase acting slowly. Spargo (’13) observed that Chlamydomonas began growth more slowly when the medium contained sucrose as a source of carbon than when dextrose was supplied. She suggests that the sugar is probably assim- ggar, B. M. and Davis, A. R. Enzyme action in Fucus vesiculosus. Ann. Mo. Bot. Gard. 1:419-426. 1914. ? The binomials used throughout the historical review are those employed by the original investigators, no attempt being made to have them conform to any different existing nomenclature. ANN. Mo. Bor. GARD., Vou. 2, 1915 (771) [VoL. 2 772 ANNALS OF THE MISSOURI BOTANICAL GARDEN ilated in the hexose form and that sucrose must be split by invertase before becoming available. It is a well-known fact that diverse fresh-water algae can be grown in pure culture on media where asparagin and peptone are sources of nitrogen. It is hardly conceivable that the large protein molecule is assimilated directly and, a priori, this would argue for the presence of both an ereptase and a desamidizing enzyme. ENZYMES FOUND IN THE MARINE ALGAE Few workers have demonstrated enzymes present in either the fresh- or salt-water algae. Fischer (’05), working on the storage carbohydrates of Anabaena and Oscillatoria, found that the specific carbohydrate involved, which he named ana- baenin, disappeared when the algal tissue was autolysed at 40°C. Microchemical tests showed glycogen split off. The action here, if it be due to ferments of the alga, is interest- ing in that the action was inhibited by .1 per cent acetic acid, by 1 per cent carbolie acid, and still more strangely, by con- centrations of ethyl alcohol as low as 5 per cent. One per cent carbolic acid is quite often used as an antiseptic in enzyme experimentation, and the resistance of enzymes to even high concentrations of alcohol is common knowledge. No attempt was made to isolate the enzyme or to carry on experiments outside the cell. Teodoresco (’12) found that Chlamydomonas in pure cul- ture gave rise to an extracellular enzyme that decomposed sodium nucleate with the liberation of phosphorus. Later, (712°) he demonstrated nucleases present in certain ‘‘blue- greens,’’ ‘‘browns,’’ and ‘‘reds.’’ Unfortunately, differences in methods do not permit a true comparison of activity with that of the nuclease isolated by Dox (710) from Penicillium camemberti, nor with that determined by Zaleski (’07) in the growing tips of Vicia faba, yet even a crude comparison is in- teresting. Dox added 2 grams of mold powder to 100 ce. of a 2 per cent solution of yeast nucleic acid, and maintaining his flasks at a temperature of 35-37°C. for forty-five days, found 51 milligrams of phosphorus (caleulated as phosphoric acid) liberated. Teodoresco used a .5 per cent solution of sodium 1915] DAVIS—ENZYME ACTION IN MARINE ALGAE 773 nucleate with an unstated amount of crushed seaweed. The temperature during the incubation period varied from 21 to 26°C. for the different forms used. The following are his results for 100 ce. of substrate: TABLE I Phosphorus as P205 Alga Days | mgms. Cladophora frusta............ 57 54.3 Ceramium rubrum...... ee 51 76.6 Griffithsia setacea............ 37 62.5 Phormidium 8P..............» 15 90.0 Zaleski crushed growing tips of Vicia faba, added water and an antiseptic, and allowed this material to autolyse at 34°C. for 4 days. At the end of that time the control flask showed a free phosphorus content of 13.6 milligrams and the one containing the active enzyme 51.2 milligrams. We have no means of knowing even the relative amount of enzyme present in any of these experiments and yet it seems that the algal nuclease compares very favorably with that isolated from the fungi and the higher plants. The classes in plant physiology at the Marine Biological Laboratory, Woods Hole, for several years past have quali- tatively determined diastase in Ulva lactuca. Bartholemew (’14), working on the question of starch in the Florideae, conclusively demonstrated diastase present in such ‘‘reds’’ as Polysiphonia variegata, Dasya elegans, Agardhiella tenera, and Ceramium sp. In order to isolate the enzyme, he used the ordinary method of precipitation by alcohol from an aqueous extract of crushed tissue. Starch as paste was hydrolysed rather slowly to an undetermined reducing sugar, presumably dextrose, 5 ce. of .25 per cent starch paste with a relatively large amount of the enzyme material requiring from 6 to 9 days for the completion of hydrolysis. Micro- scopic observation of the attacked starch grain showed corro- sion similar to that caused by the translocation diastase of the barley. Torup (Krefting and Torup, ’09) had previously isolated an enzyme from fresh Laminaria that hydrolysed the characteristic storage carbohydrate of that alga, laminarin, to dextrose. [VoL, 2 774 ANNALS OF THE MISSOURI BOTANICAL GARDEN Atkins (’14) investigated the oxidases and peroxidases of twenty-nine diverse algae. Using guaiacum as a reagent, oxidases were demonstrated in but one—Furcellaria fasti- giata—while peroxidases were shown present in seven. Alpha naphthol gave negative reactions for all the forms studied, while with it peroxidases could be determined in but two— Delesseria sanguinea and Furcellaria fastigiata. He calls at- tention to the redueing power of the tissues of certain algae and suggests that such agents may be responsible for the failure to obtain positive tests in the other forms. Reed (’15, 715"), on the other hand, holds that many of these algae may show a specific oxidative ability. Like Atkins, he found that the ordinary reagents, such as gum guaiac, alpha naphthol, and aloin, gave negative results in all but one or two instances. When, however, alpha naphthol and para-pheny- lenediamine, para-phenylenediamine alone, or the hydro- chlorides of these two were used in the presence of peroxide, positive tests were very generally obtained. As earlier indicated, the results obtained by Duggar and Davis (’14) for Fucus vesiculosus were very generally nega- tive. This was true even though a great variety of substrates were used under varying conditions, and only vigorously growing plants, fresh crushed, or dried and powdered, were employed for enzyme action. The results are exceedingly difficult to explain. It might well be that the enzymes were present but in such small amounts as to escape detection by the ordinary methods. Methods of enzyme isolation are still crude and they undoubtedly involve some loss of the ferments. Another factor suggested in the preliminary paper, was that the death of the cell might liberate certain substances which would then be free to unite with the enzyme complex, throw- ing it out of the sphere of action. SOME STORAGE PRODUCTS OF THE ALGAE It is often assumed that the presence of storage products in the plant is generally linked with the presence of specific enzymes—starch with diastase, inulin with inulase, fats with lipase, hemicelluloses with eytase, ete. These enzymes may be present at all times, as the diastase of the potato tuber and 1915] DAVIS—ENZYME ACTION IN MARINE ALGAE 775 the diastase and maltase of the barley grain, or they may only arise when there is food transformation and translocation, as in germinating seeds. However, in the light of such possi- bilities of association, it is worth while to call attention briefly to some of the work that has been done on the chief storage products of the algae. The carbohydrates have been more worked over in this respect than has any other chemical group, but much confusion still exists regarding their exact status in assimilation. Much of the study has been on the cleavage products, obtained by acid hydrolysis, of undetermined carbohydrates. These, how- ever, are not a true index of the distribution and more re- stricted chemical nature of assimilable carbohydrates in the living plant; one must look rather to the work of those who have limited themselves to the isolation and determination of unaltered carbohydrates. CHLOROPHYCEAE Polysaccharides.—Nigeli (’63) reported ‘‘sphirokristalle”’ in Acetabularia which Leitgeb (’87) later showed were inulin. The former worker also demonstrated the presence of this carbohydrate in various members of the Dasycladaceae. Küster (’99) has more recently found characteristic crystal formations in Derbesia and Bryopsis which, from the many reactions they gave, appear to have been inulin. Famintzin (67) and Krause (’70) worked on the effect of light on starch formation in Spirogyra, and within recent years, Tim- berlake (’01) has contributed observations on the starch of Hydrodictyon. Oltmanns (’05, p. 147) speaks of starch ac- cumulation in the Conjugales, Volvocales, Ulotrichales, Charales, Siphonocladiales, and some of the Siphonales. He considers it the first visible product of assimilation, but thinks that it may also function as a reserve. Starch in the marine forms seems to be quite widely distributed. In the work of Arber (’01), to which reference has already been made, starch accumulation in the tissues of Ulva, Cladophora, and Enteromorpha was easily demonstrated by means of iodine. Swartz (’11) isolated starch from Ulva but was unable to prove its presence in Enteromorpha, a closely re- [VoL. 2 776 ANNALS OF THE MISSOURI BOTANICAL GARDEN lated genus. She concluded that the carbohydrates existed in the form of hemicelluloses, probably as pentosans. Glycogen, although frequently found in the ‘‘blue-greens,”’ where, as held by some authors (Fischer, ’05), it functions as the chief reserve carbohydrate, has been demonstrated in but one case, as far as is known, in the Chlorophyceae, and that by Beyerinck (’04) in Chlorella variegata. Simple sugars—The nature of the simple sugars in the group is indefinite. Klebs (’96) reported a substance in the cells of certain Heterokonteae that reduced Fehling’s solu- tion, but this means little since most algae contain non- carbohydrate reducing substances made up chiefly of tannins and tannoidal bodies. Tihomirov (’10) used the phenyl- hydrazine method as modified by Senft (’04) for the detection of osozone-forming sugars in algal tissues in this group, chiefly those of Codium bursa and C. tomentosum. After a period of thirty days, for these two forms, yellow amorphous deposits appeared in the cells indicating a sugar reaction. The definite sugars these osozones represented could not be determined, but he suggests the possibility of dextrose and d-galactose. It seems evident that they must be present in very small quantities in the tissues investigated. PHAEOPHYCEAE Polysaccharides.—Starch is conspicuously absent from the great group of ‘‘browns,’’ but there are, however, certain less highly condensed polysaccharides present. Schmiedeberg (’85) speaks of a dextrin-like compound which he isolated from Laminaria. He gave to it the name ‘‘laminarin’’ and the general formula, 10(Cs01005)-9H20. There seems, how- ever, to be some confusion regarding his method of arriving at these figures. Torup (’09) was able to extract a dextrin from Laminaria sp. with warm water, that gave dextrose on hydrolysis. This could be isolated only during the winter months. He called it ‘‘kreftin.’? Kylin (’13), extracting crushed Laminaria saccharina, Fucus vesiculosus, and Asco- phyllum nodosum, obtained a dextrin-like compound similar to that described by Schmiedeberg and he retained Schmiede- 1915] DAVIS—ENZYME ACTION IN MARINE ALGAE 777 berg’s name, ‘‘laminarin.’?’ He showed also that Torup’s ‘‘kreftin’’ was without doubt a modification of ‘‘laminarin.’’ Kylin ascribes to ‘‘laminarin’’ the same physiological func- tion that starch performs in the higher plants, i. e., that of a reserve product. In a more recent paper (’15) he shows that there is an accumulation of the ‘‘laminarin’’ in the tissues of the algae during the summer months, while during the winter and spring this reserve is drawn upon by the young fronds until by the end of March very little of it is demonstrable. Kylin was also able to clear up much of the confusion that has attended observation of the light-refracting granules present in the cells of many members of the group. They had been variously considered as of fatty nature, protein- aceous, tannin-like, and glucosidal. Reinke (’76) demon- strated fat-like bodies in the cells of Fucus that he looked upon as the first visible products of assimilation, a point of view later supported by Hansen (’93). Schmitz (’83) claimed two distinct bodies present, one of which, although it did not react with iodine, he called ‘‘phaeophyceenstirke,’’ the other giving the ordinary reactions for fats. Hansteen (’92) had observed bodies in the same plant which he maintained were of carbohydrate composition and to which he applied the term, ‘‘fucosankorner.’’ Crato (’92, ’93), the same year, in- vestigating the fat globules observed by Schmitz, suggested that they were either phloroglucin or a derivative of it, since they colored red with vanillin-hydrochloric acid. This con- ception was held by Bruns (’94) as well. In a later paper, Hansteen (’00) observed that the ‘‘fucosankérner’’ were formed in the presence of light, and this to his mind indicated that they function as the first assimilable products. Hunger’s (702) work two years later pointed to Hansteen’s ‘‘fucosan- körner’’ as being glucosidal in nature, the carbohydrate at- tached being bound up with phloroglucin, or at times, with tannic acid. Some of the larger ‘‘korner’’ gave fat reactions, some protein. Kylin found three definite bodies in the cell, the nature of which had been confused by earlier workers— fat globules, proteinaceous particles, and tannin-like bodies— these latter probably representing the ‘‘fucosankörner’’ of [voL. 2 778 ANNALS OF THE MISSOURI BOTANICAL GARDEN Hansteen. He holds that none of these are to be considered the first visible products of assimilation, and suggests that here, as in most phanerogams, carbohydrates function in that role. Simple sugars.—As far as is known, Tihomirov (710) was the first to definitely demonstrate simple sugars in these plants. He used the same phenylhydrazine method employed with the‘‘greens,’’ but as was the case there, was unable to con- nect the osozones with definite sugars. The osozones took con- siderable periods of time to form, in some cases as long as five months, evidence pointing to the low concentration of sugars in the cell. It is a question, too, whether during this long period of incubation some of the more highly condensed carbo- hydrates in the cell were not hydrolysed far enough to give the sugar tests. Using the same method, Kylin (713) was unable to substantiate these results. However, by using 40 per cent alcohol as an extracting agent, precipitating the in- organic material with lead acetate, and then purifying with alcohol, he was able to obtain reducing sugars from several of the Fucoideae, particularly Laminaria digitata, L. sac- charina, Ascophyllum nodosum, and Fucus vesiculosus. In all cases Seliwanoff’s test for fructose was positive, while dextrose was demonstrated by its osozone. These sugars he considers the first products of assimilation referred to above. RHODOPHYCEAE Polysaccharides.—The so-called Florideae-starch has been the source of many investigations, from the time of Nageli (758) and Van Tieghem (’65) to the present day. Although not identical perhaps, it is very similar to the starch of the higher plants, and as very generally held, it undoubtedly func- tions in the same manner. Meyer (’95), Kolkwitz (’00), and Bartholemew (714) hold the opinion that it repre- sents a combination between true starch and dextrin, while Biitschli (’03) suggests the possibility of its being a transitional stage between amyloporphyrin and amyloery- thrin. Kylin (’13) considers it as standing midway between starch and dextrin. This investigator succeeded in isolating 1915] DAVIS—ENZYME ACTION IN MARINE ALGAE 779 Florideae-starch from Furcellaria fastigiata, that was readily hydrolysed to dextrose by malt diastase, and it will be re- membered that Bartholemew (’14) isolated diastase from several of the ‘‘reds’’ that split phanerogamic starch to re- ducing sugars. Simple sugars.—Very little work has been done on the di- and monosaccharides of the ‘‘reds.’? Tihomirov (’10) suc- ceeded in obtaining the same yellow amorphous osozone de- posits in the tissues of Sphaerococcus crispus and Gigartina mamillosa that he had in certain members of the ‘‘greens’’ and ‘“browns,’’ but here, as in the other groups, the specific osozone involved could not be determined. FATS AS STORAGE PRODUCTS Many observations have made it evident that fats in some form or other are generally present in the algae, their peculiar röle, however, having been very little investigated. In some of the siphonaceous forms, particularly Vaucheria, they seem to replace carbohydrates. Whether fats are to be regarded as the first visible products of assimilation in these forms is disputed. Some workers hold them to be reserve products, some by-products of metabolism. If they are utilized as a reserve or storage product in any of the forms, one might expect to find evidences of lipolytic action, yet none has been reported so far. As stated by Czapek (’13, p. 761), Loew and Bokorny find that Spirogyra and other filamentous forms contain 6 to 9 per cent of the dry weight as fat. This probably includes lecithin. The same authority gives the following results as obtained by Sestini, the figures being percentages of the dry weight: POOROTIG DENE 55 oo ck a eee 2.94 EE ee RE En 21 IESE WORIOUTOSUE. | o.oo ne esses ck ans ea cae .67 Valomia aegagropia ::...-.00H00n00 0000 .15 Gracilaria confervoides ......cerecccveces mal König and Bettels (’05) made a large number of analyses of the dry tissues of a variety of marine algae and found a fat [Vou 2 780 ANNALS OF THE MISSOURI BOTANICAL GARDEN content ranging from .20 per cent in Enteromorpha to .98 per cent in Porphyra. RELATION OF THE ALGAE TO NITROGEN Some of the recent work on pure culture methods with fresh-water algae, such as that of Beyerinck (’90), Charpen- tier (’03, ’03*), Chick (’03), Artari (713), Spargo (’13), and Schramm (714) have conclusively proved that these forms can utilize organic nitrogen. Furthermore, the work of Letts and Hawthorne (711), and Foster (’14) point to the fact that the marine forms may have this capacity as well. Letts and Haw- thorne and also Letts and Richards (’11) showed that Ulva latissima grew better in sewage-contaminated sea-water than in water from the open sea. Foster placed strips of Ulva lactuca in normal and artificial sea-water, containing in addi- tion compounds of nitrogen in varying concentrations. When urea or ammonium sulphate was added to either solution an accelerated growth took place. The current conception concerning the assimilation of organic nitrogen by the animal organism is that the protein and amino acid molecule must be completely desamidized be- fore the building-up process can begin. In the absence of definite information to the contrary, we can conceive of a par- allel situation existing in the plant. The question at once arises in regard to the algae, whether this be due to the agency of amidases formed by the tissue, or to the activity of desamid- izing bacteria, the presence of which Brandt (’99), Gran (’02), Baur (’02), Reinke (’03), Benecke and Keutner (’03), and others have shown to exist abundantly in harbor waters. Neither Letts and Hawthorne nor Foster worked with pure cultures, and these bacteria may have been the agency in their experiments to render the amino-nitrogen assimilable. CARBOHYDRATES AND CARBOHYDRATE CLEAVAGE PRODUCTS OF ALGAL SLIME Besides the carbohydrates that may be directly assimilable, we find those whose function in metabolism is more or less disputed. The so-called algal slime is made up chiefly of such products. 1915] DAVIS—ENZYME ACTION IN MARINE ALGAE 781 Chemical composition—Greenish (’81) found agar from Fucus amylaceus to consist of 37.21 per cent gelose (probably galactan since it passed to galactose on hydrolysis) and that from Sphaerococcus crispus of 60 per cent of the same carbo- hydrate. Konig and Bettels (’05) give the carbohydrate com- position of agar-agar from Gelidium as 33 per cent galactans and 3.1 per cent pentosans; by hydrolysis, d-galactose and levulinie acid were split off. Günther and Tollens (’90) found fucosan in Fucus from which the methyl-pentose, fucose, was split off. Galactose was also demonstrated. Sebor (’00) obtained galactose, glucose, and fructose from the slime of Chondrus crispus by acid hydrolysis. He held that the slime is a very complex carbohydrate of high molecular weight, made up chiefly of galactosan, glucosan, and fructosan. The cleavage products of Porphyra laciniata, as investi- gated by Oshima and Tollens (’01), were found to consist chiefly of l-galactose and mannose, but glucose, fucose, and other pentoses were also obtained. Miither and Tollens (’04) found methyl-pentosans in several of the Fucaceae. Konig and Bettels (’05), working on the carbohydrate hydrolytic products of various species of Porphyra, Gelidium, Laminaria, Cystophyllum, and Enteromorpha, found them to consist of such hexoses as galactose, dextrose, and fructose, as well as several pentoses, chiefly methyl-pentoses. Enteromorpha yielded a pentose—rhamnose. The results of Swartz (711) agree with those above, namely, that for all forms studied, representatives of the ‘‘greens,’’ ‘‘browns,’’ and ‘‘reds,”’ pentosans were always present, and galactans frequently so. Kylin (713), by direct extraction with warm water of crushed Ceramium, Furcellaria, and Dumotia, obtained substances that gave the mucie acid test for galactose, as well as the phloroglucin test for pentosans. Substances giving pen- tosan reactions alone were isolated from the slime of Asco- phyllum nodosum, Fucus vesiculosus, and Laminaria sp. He was apparently unable to substantiate the finding of galactan in Fucus by Ginther and Tollens, and this negative result also conflicts with the statement of Swartz, who says that the gelatinization in the algae is due to the galactan groups. [VoL. 2 782 ANNALS OF THE MISSOURI BOTANICAL GARDEN Kylin (714) and others have also demonstrated pectin-like compounds forming the middle lamella in various members of the Fucaceae. These exist as the calcium salts of pectic-like acids which Kylin designates ‘‘Fucinsiiure’’ and “Algin- saure.”’ PHYSIOLOGICAL SIGNIFICANCE OF ALGAL SLIME It is seen that algal slime is made up chiefly of the an- hydrides of hexoses and pentoses—carbohydrates that must be broken down to simpler form before assimilation by the plant would be possible. Two questions naturally arise: (1) Do the algae concerned form enzymes that will hydrolyse these highly condensed carbohydrates to assimilable form? (2) Does the slime itself arise through the breaking down of the hemicelluloses of the cell wall through enzymic or other causes, or does it represent a final stage in the condensation of those hemicelluloses? Algal slime as a reserve product.—Galactanases and man- nases have been demonstrated in the phanerogams and in the fungi by Bourquelot and Hérissey (’99), Griiss (’02), and Hérissey (’03). The last worker especially has clearly shown the distinct röle that galactans and mannans may play as re- serve products in the tubers of the Orchidaceae and in many of the Leguminoseae. It is significant that Gran (’02") was able to isolate a marine bacillus, B. gelaticus, that acted on part of the constituents of agar-agar to give a reducing sugar. From the standpoint of a possible symbiosis it would be in- teresting to know if this organism has the ability to fix free nitrogen. Saiki (’06) experimented with a number of algal and lichen preparations containing a large proportion of carbohydrates as galactans and pentosans, and concluded that the latter could not be transformed into sugars readily by carbohydrate digesting enzymes of animal origin and scarcely more so by the vegetable enzymes, either of the higher plants or of bacteria. Still less is known of the digestion of pentosans by the higher plants. Schöne and Tollens (’92) found no decrease in the amount of pentosans during germination and conclude 1915] DAVIS— ENZYME ACTION IN MARINE ALGAE 783 that they cannot function as reserves. Cross, Bevan, and Smith (’95) consider the pentosans as by-products of metab- olism and once formed remain unalterable. Ravenna and Cereser (’09), on the other hand, in some very interesting ex- periments, found that when dextrose was supplied as the sole nutrient to the leaves, pentosans increased greatly, especially in the light. If, however, the function of chlorophyll is in- hibited, a decrease in the amount of pentosans takes place. These results form the basis for their conclusion that pento- sans may sometimes function as reserves. The origin of algal slime—The question concerning the origin of the slimy and gummy constituents of cells, whether they arise through enzyme action or through other causes, has provoked much discussion. There is considerable doubt whether such gums can arise directly from true cellulose or whether they are, at least in the case of the plant mucilages, laid down as such. One might roughly group the plant gums into those arising as a result of some external excitant, such as, for example, cherry gum, acacia gum, gums of citrus, etc., and those which seem to be normal constituents of the plant, as the mucilages found in the epidermis of many seeds and plant organs. The former arise as a result of a pathological condition; the latter, as far as we know, are normal physiological products and as such are more nearly comparable to the algal slime. Klebs (’84), investigating slime formation in some of the lower algae, particularly some of the Desmidiaceae, held that it was not a conversion product of cellulose. Hauptfleisch (’88) substantiated the conclusion of Klebs, and going further, states that it arises in this particular case through the activity of the protoplasm, being excreted through pores. Oltmanns (704, p. 76) illustrates very clearly the arrangement of these pores. Tschirsch (’89) differentiates these slimes or muci- lages into those giving a cellulose reaction and those not doing so, the former having some relation perhaps to the cellulose, but the latter being laid down on the cell wall as such by the protoplasm. He holds the epidermal slime of Spirogyra to be of this latter type, which he calls ‘‘echter Schleim.” In [VoL. 2 784 ANNALS OF THE MISSOURI BOTANICAL GARDEN the same work the author concludes the slime of the Fucaceae and of the Florideae to be of the ‘‘echter’’ type, occurring here, however, not as a layer laid down on the inner cell wall, but as an intercellular substance. Guignard (’93) held much the same view, and in an excellent histological investigation, clearly demonstrated the presence of slime or mucilage ducts in the Laminariaceae. Mucilages very similar in nature and origin to the algal slimes occur in the higher plants, and much more work has been done with them than with those occurring in the algae. It is hardly necessary to go into the historical aspect of this phase of the work. The current conception of its origin is voiced by Walliezek (’93), who, investigating rather fully the location of different types of normal mucilages by means of suitable stains, found that in almost all cases they were laid down as such. According to him, the slime forms secondary layers on the cell wall which he designates ‘‘ Membranverdic- kungsschichten’’—layers that in many instances almost com- pletely fill the cell. Where the epidermal layer of seeds be- comes gelatinous, as, for example, in those of flax, mistletoe, various Cruciferae, ete., it is this inner cell wall which Wal- liczek holds to be the seat of slime formation. Upon contact with water the slime swells remarkably, filling the cell and at times even bursting it. There may or may not be an actual hydrolysis of the true cellulose, but if there is it seems rarely to enter into mucilage formation. EXPERIMENTAL Forms used.—The algae to be used for enzyme investiga- tion were collected in the vicinity of Woods Hole, Massa- chusetts, during the summers of 1913-14, at which time the plants were also dried for winter work at the Missouri Botanical Garden. Work with the fresh tissue was carried on at the Marine Biological Laboratory, Woods Hole, during the latter summer. The selection of forms with which to work was limited to those relatively abundant in the neighboring waters, a further limiting factor in selection being relative freedom from adhering marine organisms. Only those plants 1915] DAVIS—ENZYME ACTION IN MARINE ALGAE 785 were selected that were ‘‘clean.’? This was an important pre- caution, since many adhering organisms have been found to be quite active enzymatically, and the presence of even a few might well lead to serious errors in the final results. The fol- lowing forms lent themselves most readily to the work:! Chlorophyceae Ulva lactuca (L.) Le Jolis Enteromorpha intestinalis (L.) Link Phaeophyceae Laminaria Agardhi Kjellm. Ascophyllum nodosum (L.) Le Jolis Mesogloea divaricata (Ag.) Kutz Rhodophyceae Ceramium rubrum (Huds.) Ag. Agardhiella tenera (J. Ag.) Schmitz Rhodymenia palmata (L.) Grev. Chondrus crispus (L.) Stack. Preparation of algal material.—In addition to the question of cleanliness, great care was taken to select only plants that were in a young, vigorously growing condition. These were brought into the laboratory, placed in large aquarium jars containing salt water, picked over, and all detectable foreign matter removed. A thorough washing in running salt water for two hours was then given, after which, with the exception of one or two forms that rapidly gelatinized, the plants were placed in running fresh water for 10 or 15 minutes. This fresh water treatment was very efficacious in causing small snails and other minute marine organisms to loosen their hold. The plants so washed were either crushed and used at once with the substrate for enzyme action, or they were dried for future use. In either case, two general ways of using the ma- 1 With the exception of Laminaria Agardhii and Agardhiella tenera, these binomials conform to the nomenclature as given by Farlow (Marine algae of New England, pp. 1-210. pl. 1-14. 1881); these two forms are as given by De Toni (Sylloge Algarum 3: p. 349. 1895) and Engler and Prantl (Nat. Pflanzenfam. 17:371. 1896), respectively. [Vou, 2 786 ANNALS OF THE MISSOURI BOTANICAL GARDEN terial for such action were employed. The tissue was added directly to the substrate, or it was extracted with water by the method to be described later and a water-diffusion used of the alcohol precipitate. If the fresh tissue were to be used directly, it was ground in a meat chopper two or three times, then pounded in a large mortar with an equal amount of fine, clean, quartz sand. This treatment gave a very homo- geneous pulp, one in which a large number of the cells were broken down. If desired for future use, the plants were either dried at room temperature or dehydrated by the following modified Buchner ‘‘dauerhefe’’ process: 3 volumes 95 per cent alcohol for 15 minutes. 3 volumes acetone for 15 minutes. 3 volumes 95 per cent alcohol for 10 minutes. 3 volumes acetone for 5 minutes. 2 volumes absolute alcohol or ether for 5 minutes. After each treatment, the dehydrating liquid was pressed out through two thicknesses of cheese cloth by making a tourniquet. Upon the removal of the absolute alcohol or ether, the tissue was spread out on adsorbent paper, either filter paper or paper toweling, until all the dehydrating agent had evaporated. A uniformly dry, brittle, easily crushed material usually resulted that was roughly broken up and stored in tightly stoppered bottles for future use. Those plants that were dried at room temperature were simply wrapped in paper or placed in paper bags until needed. The crushing of the dry material was accomplished in the same manner as was the fresh. Usually it was ground twice or more in an ordinary meal mill, then pounded in a mortar with an equal weight of quartz sand until a very fine powder was obtained. The sand was dispensed with if the tissue were easily crushed. Methods of isolating the enzymes.—As indicated above, there were two general methods of using the material for enzyme action: first, adding the crushed tissue directly to the substrate, either as fresh pulp or as ‘‘dauerhefe’’ powder; 1915] DAVIS—ENZYME ACTION IN MARINE ALGAE 787 second, by extracting the tissue with water and precipitating the protein-enzyme complex with several volumes of 95 per cent alcohol. Wherever possible the first method was used, since it was thought that in this way the maximum enzymic activity would be obtained. However, the fresh pulp and the powdered material contained a substance, or substances (probably tannoidal bodies), that reduced copper from Fehl- ing’s solution, and so in all experiments where sugar deter- minations were involved, it was found necessary to use the extraction and precipitation method; by this means all the unknown reducing substances were avoided. The method was as follows: To a known amount of the crushed, fresh algal material, 3-5 volumes by weight of distilled water were added; to the powdered tissue, 8-10 volumes. The amounts varied owing to the differences in viscosity produced by the different algae. In some forms a relatively large amount of water was neces- sary in order to overcome difficulties in handling due to this high viscosity. Two per cent toluene was generally added as an antiseptic, or in some cases, 1 per cent chloroform-thymol mixture was used (5 per cent thymol dissolved in chloroform), and the extraction allowed to go for 12 hours at room tempera- ture, or for 4 hours at 35° C. The water extract, if at all viscous, was then filtered off through two thicknesses of cheese cloth and the algal tissue pressed out as completely as possible by making a tourniquet of the cloth. Filtering through cotton was tried at first, both with pressure and without, but the method had the disadvantage of slowness and also that of adsorption by the cotton. Neither did filter paper lend itself efficiently to the filtration of such viscous liquids, a drier residue being obtainable in a shorter time by the cheese cloth-tourniquet method. A press would have been desirable but none was at hand. If the medium were not viscous, it was filtered with pressure through a thin layer of cotton or a coarse filter paper in the bottom of a Buchner funnel. The protein-enzyme complex was precipitated with 3 volumes of 95 per cent alcohol. After a few moments the [voL. 2 788 ANNALS OF THE MISSOURI BOTANICAL GARDEN coagulum either came to the top or settled to the bottom of the vessel—if to the top, it was usually very much aggregated and little difficulty was experienced in the filtering, if to the bottom, it was generally in a very finely divided condition and unless care was exercised in the decantation of the super- natant liquid the pores of the filter soon became clogged, re- sulting in extremely slow filtration. Time was therefore given for a complete settling out (15 minutes to half an hour sufficed) and all the clear fluid filtered off before the coagulum reached the filter paper. A homogeneous diffusion of the precipitate was made by placing the filter paper with the attached coagulum in a known volume of distilled water. The paper could soon be removed without loss of material, and the weight of the original fresh or dry tissue represented by an aliquot portion of the solu- tion easily reckoned. If the precipitate were not required im- mediately, it was dried on a filter paper at room temperature and stored in stoppered jars. In none of the experiments was the enzyme material purified further. When dissolved in water, the precipitates behaved differ- ently. Some, especially those where much slime had been noticed in the extraction, gave an extremely viscous suspen- sion, others a suspension of low viscosity. In Laminaria and Chondrus, where the extract had been quite viscous and slimy, the protein was caught up in the precipitated slime in such a way as to make the freeing of it practically impossible. The precipitate in these cases was very large and when diffused in water gave a suspension difficult to handle. Rhodymenia, Ceramium, and Enteromorpha, on the other hand, gave a finely divided precipitate that produced no viscosity. Glassware, antiseptics, solutions, ete.—With few exceptions, the various experiments were set up in 125 cc. Erlenmeyer flasks. All glassware was thoroughly cleaned with strong soap and then with chromic-sulphuric cleaning mixture, after which it was rinsed several times with tap and distilled water. Solutions were made up from either Merck’s or Kahlbaum’s ‘‘ouarantiert’’ chemicals. 1915] DAVIS—ENZYME ACTION IN MARINE ALGAE 789 Three general antiseptics were used—toluene, alcohol to 20 per cent, and 5 per cent thymol in chloroform. Toluene was, in general, the most satisfactory. Usually it was used to 2 per cent concentration, but where large surfaces were exposed, as high as 4 per cent was found necessary. The chloroform- thymol was also very efficacious, but in the carbohydrate ex- periments chloroform could not be used because of its power of reducing copper. In the lipase work the substrate was made up to 20 per cent alcohol since the action seemed to proceed best in the presence of this antiseptic. In all cases where the experiments were maintained over a considerable period of time, it was necessary to add additional antiseptic from time to time. Checks were set up in all experiments—on the substrate, on the material used to demonstrate enzyme action, and on the substrate plus such enzyme material boiled to destroy any ferments that might be present. CARBOHYDRASES OF THE ALGAE In these experiments the alcohol precipitate from an aqueous extract of crushed, fresh or dried, algal tissue was employed as an enzyme source, this precipitate being diffused in such a volume of distilled water that one gram of the original material was represented by 5 cc. of the diffusion. Thus one can more closely compare the amounts of enzyme present in definite amounts of different algal tissue. The number of cubic centimeters of diffusion will be noted in con- nection with each set of experiments. Substrates.—Starch, dextrin, inulin, sucrose, maltose, lac- tose, glycogen, and in one or two cases, laminarin isolated from Laminaria Agardhü, were used as substrates. These were made up in 1 per cent concentrations with the ex- ceptions of maltose and glycogen, where .25 per cent, and laminarin, where .5 per cent concentrations were employed. Of the many suggested methods for making up starch paste, the following one used by Clark (’11) was found to give the best satisfaction. Ten grams of potato starch were weighed out and placed in a beaker with 250-300 ce. of distilled water. [VoL. 2 790 ANNALS OF THE MISSOURI BOTANICAL GARDEN This was brought to a boil with constant stirring, and when an opalescent solution resulted the paste was transferred with rinsing to a 2-liter flask containing about 500 cc. of boiling water. The lot was boiled under a reflux condenser for two hours, cooled, and made up to a liter. Although, as is stated by Clark, this treatment is very effective in breaking down the starch grain physically, no detectable hydrolysis takes place, and the additional advantage is gained in obtaining a paste that will not settle out, even after long standing. Two per cent toluene was employed as an antiseptic if the starch were not to be used immediately. Since all dextrin obtainable contained some reducing sugar, it was found necessary to purify it by making a concentrated solution in hot distilled water, and then precipitating out with several volumes of 95 per cent alcohol. The dextrin was caught on a filter paper and dried at a low constant tem- perature. Laminarin, a dextrin-like carbohydrate found in many of the Fucaceae, was isolated from Laminaria Agardhuw accord- ing to the method employed by Kylin (’13), with some few slight modifications. Freshly collected Laminaria was crushed in the usual way and 1,680 grams of the pulp were boiled with 7 liters of water for 24 hours, water being added from time to time to replace that lost through evaporation. The extract was then filtered off through a double thickness of cheese cloth, and the residue pressed out with a tourniquet. About 3,000 ce. of a dirty brown filtrate were obtained which was divided into three lots of 1,000 ec. each. To the first of these was added a concentrated Ba(OH)z solution until the precipitation of the inorganic matter was complete. The pre- cipitate was caught on a cotton filter in a Buchner funnel, the filtrate being a clear, golden-colored liquid. The inorganic material in the other two lots was precipitated with basic lead acetate, the liquid filtered off through cotton, and the excess of lead removed with H:S. The solutions were filtered while hot through double filter paper to remove the lead sulphide, and then the excess of H:S was driven off with heat. The three portions were first evaporated to about one- 1915] DAVIS— ENZYME ACTION IN MARINE ALGAE 791 third their volume, when the scum that formed was filtered off; this filtrate was then further evaporated to about one- fifth the original volume on the water bath. At this point the two lead acetate portions were placed together. Ninety- five per cent alcohol was added to each of the lots to about 80 per cent concentration when a flocculent precipitate came down rather slowly. With the Ba(OH)» portion this was copious, with the lead acetate, slight. After two hours the precipitates were filtered off, washed with absolute alcohol, redissolved in a small amount of distilled water, and then reprecipitated with 4 volumes of absolute alcohol, the result- ing precipitate being dried over CaCle. From the Ba(OH); portion, 4.2 grams of a creamy white powder were obtained that gave a very slightly reddish tinge with iodine, did not reduce Fehling’s, and was easily soluble in water, giving a clear solution. Upon hydrolysis with weak H>SO, a reducing sugar was split off. The lead acetate portion gave but two grams of the same material. This powder was taken to be the laminarin described by Kylin. The determination of reducing sugars.—The reduction of copper, or in the case of maltose and lactose, the increase in the reducing value of the substrate plus the enzyme over that of the checks, was taken as the measure of carbohydrate hydrolysis. In this determination the permanganate titra- tion method, as modified and described by Shaffer (’14), was used, it being possible with it to determine amounts of sugars as low as 2 milligrams! very accurately and quickly. Shaffer’s description may not be generally available to plant workers who may desire to use this really splendid method, and so the various steps in the process as used here are set down in some detail. Ten cc. of the carbohydrate-enzyme substrate were placed in a large test-tube containing 5 cc. of water, and just brought to a boil. At this point a drop of 50 per cent acetic acid was added. When the slight protein precipitate formed, 5 cc. of 1 Shaffer determines values below two milligrams, but as used here, con- sistent results could not be obtained where less than that amount was involved. Below this point the relative increase in the experimental error is large. [voL. 2 792 ANNALS OF THE MISSOURI BOTANICAL GARDEN colloidal iron (Iron dialysed, Merck) were pipetted in and the tube well shaken, the iron then being flocked out with .25 gram of NazSOu. Upon the addition of this latter the mixture was again thoroughly shaken and the iron precipitate thrown down by centrifuging, the resulting clear, supernatant liquid then being decanted off through a small filter. This filtrate was entirely free of proteins or other substances which, through oxidation later, would lead to errors in the perman- ganate values. Ten cc. of this filtrate were placed in a 50 ec. lipped centrifuge tube, and standard Fehling’s solu- tion added, the copper content of which was in excess of that reducible by the sugar present.! The tube was then placed in a boiling water bath for 10 minutes, at the end of which time it was centrifuged at a moderate speed for 2 minutes, the supernatant unreduced Fehling’s carefully decanted off, a like volume of distilled water added, and the euprous oxide again thrown down by a 2-minute centrifuging. All but 1 or 2 cc. of this wash water was carefully decanted off, and the copper dissolved in the smallest amount necessary of a mix- ture of equal parts of 10 per cent ammonium ferric sulphate and 50 per cent sulphuric acid. It was found that if the cop- per were stirred up with a glass rod just before dissolving, it went into solution more readily. The dissolved copper was titrated directly in the centrifuge tube against N/50 KMnO:.? By calculation it is found that 1 ce. of N/50 KMnO; is equivalent to 1.27 milligrams of copper, and for the conver- sion of this into glucose use was made of the table prepared by Shaffer.? As stated by Shaffer, care must be observed on the three following points: (1) to eliminate all oxidizable substances other than sugar, (2) to titrate the cuprous oxide immedi- ately after dissolving, (3) to use poor conductors of heat as containers of the centrifuge tubes in the water bath, else many broken tubes will result. As employed here, circular wire 1 In the determinations made here this amount never exceeded 10 ce. ? It is necessary to titrate immediately after dissolving because of the danger of oxidation of the cuprous oxide. If larger amounts of sugar are concerned, N/10 KMnO, may be used. ” DAVIS— ENZYME ACTION IN MARINE ALGAE 793 baskets having wooden bottoms and tops were used, the tops containing holes large enough for the free insertion of the cen- trifuge tubes, and the bottoms, slight depressions into which the tubes might rest. It is always necessary to run blanks with Fehling’s solution since some reduction always takes place. The cuprous oxide solvent must be free from ferrous iron, and this can be assured by the addition of a trace of per- manganate. Method of setting up experiments.—Fifty ec. of the sub- strate to be used were placed in 125 ce. Erlenmeyer flasks with 2 per cent toluene as an antiseptic. If the series were main- tained longer than six weeks, another 2 per cent toluene was added. As previously noted, in these carbohydrate experi- ments the material used for enzyme action was an alcohol precipitate from a water extract of algal powder or pulp. This was diffused in water so that 10 ce. of the diffusion represented 2 grams of the original tissue. Usually this amount was added to the substrate to be tested. Duplicates and checks were set up in accordance with the following model series for starch: 50 ce. starch, 10 ec. enzyme diffusion. 50 ce. starch, 10 ec. enzyme diffusion. 50 cc. starch, 10 cc. boiled enzyme diffusion. . starch, 10 ec. boiled enzyme diffusion. 50 ec. starch, 10 ce. distilled water. 50 ce. starch, 10 ec. distilled water. DIPYN a oO Q O : To make this table more generally available, it is printed here in full. Shaffer’s table of copper-glucose equivalents mgms. mgms. mgms. mgms. mgms. copper glucose copper glucose copper glucose 0.7 47 6.0 2.74 20. 1 1.0 .62 7.0 3.21 25.0 12.25 1.5 .88 8.0 3.68 30.0 14.80 2.0 1.11 9.0 4.15 35.0 17.40 2.5 1.32 10.0 4.65 40.0 20.00 3.0 1.50 12.0 5.61 50.0 25.00 3.5 1.67 14.0 6.61 60.0 30.10 4.0 1.82 16.0 7.61 80.0 40.40 5.0 227 18.0 8.65 100.0 50.70 [VoL. 2 794 ANNALS OF THE MISSOURI BOTANICAL GARDEN In addition, at the end of a complete carbohydrate series there were included for each alga the following checks: 1. 50 ce. distilled water, 10 ce. enzyme diffusion. 2. 50 ce. distilled water, 10 ce. enzyme diffusion. 3. 50 ce. distilled water, 10 ce. boiled enzyme diffusion. 4. 50 ce. distilled water, 10 ec. boiled enzyme diffusion. Where the enzyme diffusion referred to above actually con- tained carbohydrases, it was extremely difficult to render them inactive by heating—10 minutes at the boiling point not being sufficient in most cases to more than slow down the action. This was probably due to the impurities contained, the rela- tively large amounts of protein and slime present tending to protect the enzymes. Those extracts relatively richer in such constituents proved the more difficult to render inactive. The expedient was finally adopted of placing the enzyme material in the autoclave and bringing the pressure in the latter up to 15 pounds. This proved quite effective. THE CARBOHYDRASES OF ULVA LACTUCA The effect of an extract of Ulva lactuca on different starches. —Since starches were to be used in many of the following E Il THE ACTION OF ULVA LACTUCA “DIFFUSION-EXTRACT"* UPON CERTAIN STARCHES 15 days 30 days ee. Sugar as Sugar as 1 per cent Ar Iodine test glucose Iodine test gms. mgms 2.1 o PER EIER 10.1 Blue, trace red 17.4 Complete hydrolysis Arrowroot.......+» 8 Blue, trace red 16.9 Complete hydrolysis ee eres 9.9 Blue, trace red 37.1 Complete hydrolysis a 2 VE 6.3 Blue 10.8 eddish Soluble, Iesnaassar 8.7 Blue : Traces dextrin Jouin. ee ee ere eee eee TAGE BE TESTEN * Wherever the term “diffusion-extract” is employed, it refers to a diffusion in water of the alcohol precipitate from an aqueous extract of the alga under discussion. sugar values in ee and the following tables are net, i. e., sugar values for all checks have been deducted. tIn all the an eam an amount of sugar below 2 mgms. is designated a “trace 1915] DAVIS—-ENZYME ACTION IN MARINE ALGAE 795 experiments, it was desired to know which, if any, were the most favorable substrates for the diastases of the algae. The action of diastase from Ulva lactuca was taken as an index. Potato, arrowroot, wheat, corn, and soluble starch, as well as inulin, were made up in 1 per cent concentrations in the man- ner previously described. To 50 ce. of each of these sub- strates were added 10 cc. of a diffusion of an alcohol precipi- tate from a water extract of dehydrated Ulva lactuca. Two per cent toluene was added as an antiseptic, and the flasks maintained at a temperature of 35°C. for 30 days. The re- sults of the experiments are given in table m. The data show but slight differences in the rate of digestion of the starches with the exception of corn starch, and the reason for this is not clear. One would expect it to be due to some impurity in the starch rather than to an inherent dif- ference in the granule. The action on inulin was so slight as not to warrant the assumption of hydrolysis due to inulase. The action of an extract of Ulva lactuca upon various carbo- hydrates.—A series was arranged using a ‘‘diffusion- extract” from Ulva lactuca with the following substrates: potato starch, dextrin, glycogen, sucrose, maltose, and lactose. Ten ce. of the ‘‘diffusion-extract’’ were added to each flask with 50 ce. of substrate, 2 per cent toluene used as an anti- septic, and the flasks maintained at a temperature of 35°C. for 30 days. The data are given in table mı. TABLE III THE ACTION OF AN EXTRACT OF ULVA LACTUCA UPON VARIOUS CARBOHYDRATES Sugar as glucose in 5 ce. Substrate mama, 15 days 30 days EEE 10.20 15.50 a5 i gt tla. een na 30 9.95 oka, See ee eee 2.25 3.50 unse | une Trace ry OO ee Pe Cn: BE 22 E Matrose taken at —- |. waxed Trace The two polysaccharides, starch and dextrin, are very readily attacked even though the action is slow. Glycogen, which is hydrolysed by most diastatic enzymes with about the [VoL. 2 796 ANNALS OF THE MISSOURI BOTANICAL GARDEN same ease as starch, seems very slightly acted upon by the carbohydrases of Ulva. The failure of action on sucrose and lactose is not so surprising as is that on maltose, for one would expect the action on polysaccharides to continue to what is generally held to be directly assimilable sugars, i. e., the hexoses. THE CARBOHYDRASES OF ENTEROMORPHA INTESTINALIS This series (table rv) was run under exactly the same con- ditions as the one preceding. The ‘‘diffusion-extract’’ was from dehydrated tissue about two months old. Ten ce. of this were used with each 50 ce. of substrate, toluene added as an antiseptic, and the flasks kept at a temperature of 35°C. for 30 days. TABLE IV THE ACTION OF A “DIFFUSION-EXTRACT” FROM AIR-DRIED ENTEROMORPHA TISSUE UPON CERTAIN CARBOHYDRATES Sugar as glucose in 5 cc. Substrate mgms. 15 days 30 days AT ae e hov05 2 coves Se da ee 9.7 13.1 Dextrin.. :...::..,30504% 5.1 9.8 Glycogen, eee 8 3.9 Bin; cosas GES ee es Trace Trace SUCTORC) 050020556005 008% a Trace Datas. 5:00 Bu Maltose.......222 22222. % The results for this closely related form are consistent with those obtained for Ulva, the action in the present case, how- ever, being somewhat slower. The more common polysac- charides are acted upon while the disaccharides are not attacked. THE CARBOHYDRASES OF LAMINARIA AGARDHII The water extract from air-dried Laminaria tissue was extremely viscous and upon addition of alcohol, a very heavy precipitate was thrown down that contained a large amount of algal slime. When water was added to this precipitate in the usual ratio a very viscous diffusion was obtained. Ten ce. of the ‘‘diffusion-extract’’ were used with 50 cc. of the sub- strate and 2 per cent toluene added as an antiseptic. The flasks were kept at a temperature of 20-22°C. for 100 days, 1915] DAVIS—ENZYME ACTION IN MARINE ALGAE 797 portions being removed and sugar determinations made at the definite intervals noted in table v. The carbohydrases in this form appear to be limited to those acting on starch and dextrin, and with these the hydrolysis proceeds much more slowly than was true with either of the preceding ‘‘greens.’’ The lower temperature at which the hydrolysis occurred does not explain completely the lessened action. Inhibiting substances or else an actually smaller con- centration of the enzyme seem to be important factors. TABLE V THE ACTION OF A “DIFFUSION-EXTRACT” FROM AIR-DRIED LAMINARIA TISSUE UPON CERTAIN CARBOHYDRATES Sugar as glucose in 5 cc. Substrate — 15 days 45 days 75 days 100 days Fg ol | ane ace Seen Sr Trace 3.25 4.7 6.5 aara I PE cen RE Trace : 6.8 8. Pe RO eee Trace Trace Trace Trace TUL oe ess rer en Be Trace Trace SICLOBE sete ee S Trace Trace BOtOse en aks ase Re Be Maltose....... ee Trace Trace Another series (table vı) of flasks was set up with the same form, using a ‘‘diffusion-extract’’ from the fresh tissue. Ten ce. of this diffusion represented 6 grams of the Laminaria pulp. In addition to the usual substrates, .5 per cent lamin- arin was used. Toluene was added and the flasks maintained for 60 days at room temperature (22-23°C.). TABLE VI THE ACTION OF > EXTRACT” FROM FRESH LAMINARIA TISSUE UPON RTAIN CARBOHYDRATES Sugar as glucose in 5 cc. Substrate mgms. 7 days 15 days 30 days 45 days 60 days Athena T aa Trace 27 2 5.35 Eo ans E EEE E Trace Trace 3.6 ber 7.40 CAMINEM Zune. Trace 2: 3.9 5.4 5.65 Elaa Ta 0 ren Trace Trace Trace Trace Trace HUN Ense er, Be ait Vanes Trace Trace SUCrOBe A Gael el eee Trace Trace Trace Lactose. ¢.cc cc bcee case's | See |) mass | Dee sot cot meres ec IMaltoser xc bccn ate ae cates Fee Trace Trace Trace [vor. 2 798 ANNALS OF THE MISSOURI BOTANICAL GARDEN The diastases of fresh Laminaria seem slightly more active than those isolated from the dried tissue; however, no other carbohydrases were evident than those shown in the previous table. THE CARBOHYDRASES OF ASCOPHYLLUM NODOSUM AND MESOGLOEA DIVARICATA The Mesogloea material was dehydrated as soon as brought into the laboratory, the preliminary fresh-water washing being omitted because of the rapid gelatinization of the tissue. The erushed dried tissue, extracted in the usual way, gave a very heavy, stringy precipitate with alcohol, consisting, as did that from Laminaria, mostly of slime. This, when diffused in the usual volume of water, gave a very viscous mixture. Crushed fresh Ascophyllum was extracted directly. The viscosity of the extract was high, but the alcohol precipitate from it came down in a flocculent mass that gave only a slightly viscous diffusion with water. Experiments were set up with the various carbohydrates heretofore employed, including laminarin, and in the different series, amounts of the ‘‘diffusion-extract’’ were used varying from 5-15 ec. As was true with the Fucus reported in the previous study, in no case were there evidences of hydrolysis even after 60 days at room temperature. THE CARBOHYDRASES OF RHODYMENIA PALMATA The air-dried Rhodymenia tissue proved to give rise to one of the most viscous extracts encountered in the algae, 20 volumes of water being necessary to make handling possible. With alcohol, a very rubbery, white precipitate came down that was made up of a large proportion of algal slime. This diffused very slowly, giving an extremely viscous mixture. Ten cc. of the ‘‘diffusion-extract’’ were used with the sub- strate to determine action, and toluene was added. The flasks were kept at a temperature of 21-22°C. for 100 days, sugar determinations being made from time to time, the results of which are given in table vir. The results here are quite comparable to those obtained with Ulva and Enteromorpha, the same carbohydrates being 1915] DAVIS—ENZYME ACTION IN MARINE ALGAE 799 acted upon, although perhaps a little more slowly. This action is definitely progressive with starch, dextrin, and laminarin, but with glycogen it takes a sudden jump during the 15-45- day period, then remains practically stationary for the rest of the time the series is being maintained. As was true of the results shown in the previous tables, this carbohydrate was less favorable as a substrate than any of the other polysac- charides employed. TABLE VII THE ACTION OF an “DIFFUSION-EXTRACT” FROM tg RHODYMENIA TISSUE UPON VARIOUS CARBOHYDRATES Sugar as glucose in 5 cc. Substrate gms. 15 days 45 days 75 days 100 days SEATCH er et Bs lea 9.2 12.2 14.8 18.2 Bestim nn ee ? 9.7 19:3 11.1 RE Trace 6.1 6 Laminatnaa.a. Sees 4.7 7.3 9.6 10.5 rT tpg Sear A er ee Trace Trace SUCHORTE Trace Trace Trace Lacto La on et a ese en 2 er Trace Maltosen aan Trace Trace THE CARBOHYDRASES OF AGARDHIELLA TENERA The very succulent nature of the freshly collected material compelled its partial dehydration immediately, Two 15- minute treatments with 95 per cent alcohol were used, then the tissue spread out on paper toweling to dry at room tem- perature. After drying, it was very easily powdered without the aid of quartz sand. The alcohol precipitate from a water extract of this powder was quite fine and floceulent, differing much from that of Rhodymenia, both in amount and in nature. TABLE VIII THE ACTION OF A “DIFFUSION-EXTRACT” FROM DEHYDRATED AGARDHIELLA TISSUE UPON CERTAIN CARBOHYDRATES Sugar as glucose in 5 cc. Substrate mgms. 15 days 45 days 75 days 100 days Starch ts Ses i eas 6.35 9, 20.9 Dextrin.. ae ee 7.00 10.5 15.95 19.7 CHYCOMOR occa cessed au 2.35 6.15 6.65 6.85 Paare 8. 11.6 13.2 Tour ehe Trace Trace Trace DÜECHOSE ee Trace Trace Trace Trace Lactose. ee sa Trace Maltose................. ee Trace Trace [VoL. 2 800 ANNALS OF THE MISSOURI BOTANICAL GARDEN It diffused readily in water with no resulting viscosity. Ten ce. of the ‘‘diffusion-extract’’ were used for enzyme action, toluene added, and the flasks kept at a temperature of 21-23°C. for 100 days. The data here obtained are given in table vim. Dextrin here more nearly approaches starch as a favorable substrate, differing from the action evidenced by the other algae with the exception of Laminaria, where all action was slow. There is also a slightly increased action over that evi- denced by Rhodymenia, for all the carbohydrates hydrolysed. THE CARBOHYDRASES OF CERAMIUM RUBRUM As was the case with Rhodymenia, it was necessary here to use 20 volumes of the water-extracting medium, not, how- ever, because of the great viscosity, but on account of the great adsorption of water by the tissue particles. The alcohol precipitate was copious and finely floceulent. It diffused in water rather slowly, giving a mixture that was only slightly viscous. Ten ce. of the ‘‘diffusion-extract’’ were used for action, the usual percentage of toluene added, and the flasks maintained at a temperature of 21-23°C. for 100 days. The data are given in table ıx. TABLE IX THE ACTION OF A‘ MEN EXTRACT” FROM FRESH CERAMIUM TISSUE UPON RTAIN CARBOHYDRATES Sugar as glucose in 5 cc. Substrate g E 15 days 45 days 75 days 100 days oE y ol t ARAR essen 6.85 8.1 11.75 16.9 Dextrin.............005. 11.5 15.0 17.5 19.6 GIVEREN eseo raris Trace 6.2 7.3 8.85 CA CeT: PEE 7.2 9.4 12.1 12.2 MIN = che RETTEN eae ER Trace Trace SUCKOSE 6x15 64.4 ae sees Trace Trace Trace Lactöse.. 20000 aia : Trace Maltose............00005 Trace Trace Trace Dextrin proved the most favorable substrate for the carbo- hydrate enzymes of this alga, the hydrolysis being about the same as that evidenced by Agardhiella. With the exception of glycogen, the other carbohydrates showed a decreased hydrolysis when compared with this latter form, and when 1915] DAVIS—ENZYME ACTION IN MARINE ALGAE 801 compared with Ulva the difference is quite marked. As in the other algae, Ceramium showed no ability to hydrolyse the disaccharides used. A COMPARISON OF THE DIASTATIC ACTIVITY OF ULVA LACTUCA WITH THAT OF LEAF TISSUE FROM SOLANUM TUBEROSUM One of the very evident facts brought out by the data in the preceding tables was the relative slowness with which hydrolysis was carried on. This point made it seem worth while to compare, in a general way, the activity of such a form as Ulva with the starch-forming leaf tissue of a higher plant, one from which diastase could be isolated rather easily. The potato (Solanum tuberosum) was chosen. The Ulva tissue was from an air-dried lot that had been tried out earlier and had been found quite active. Fresh potato tops were brought into the laboratory, and both these and the Ulva given the ‘‘dauerhefe’’ treatment. After de- hydrating and drying at room temperature, both lots were ground in a mill, then reduced to a fine powder in a mortar. Exactly 18.5 grams of each were extracted with 250 ce. of water for 12 hours at room temperature with toluene added as an antiseptic, and then the protein-enzyme complex precipi- tated with 2.5 volumes of 95 per cent alcohol. The Ulva pre- cipitate was the characteristic heavy white mass to which attention has been called before, while that of the potato was finely divided and dark. The entire amount of each precipitate was diffused in 60 ce. of water. The Ulva precipitate gave a rather viscous diffu- sion, due to the adsorption of water by the protein particles; that from the potato did not all go into solution, making it necessary to shake the flask so that a true sample might be obtained. Five ce. of the ‘‘diffusion-extract’’ represented 1.84 grams of the original dehydrated tissue, and this volume was used with 50 cc. of a starch and dextrin substrate. Toluene was added as an antiseptic, and the flasks kept at a tempera- ture of 31°C. for 42 days. Portions of the substrate were removed from time to time and sugar determinations made, the results of which are shown in table x. [voL. 2 802 ANNALS OF THE MISSOURI BOTANICAL GARDEN The action of the potato extraet upon starch was about two and one-half times that of Ulva, and its action on dextrin about twice in all of the determinations made. For some un- known reason the hydrolysis of dextrin by the diastase from Ulva ceased after the twenty-eighth day. TABLE X A COMPARISON OF THE DIASTATIC ACTIVITY OF ULVA WITH THAT OF POTATO LEAF TISSUE Sugar as glucose in 5 cc. Substrate MEMS, 50 cc 14 days 21 days 28 days 35 days 42 days Ulva | Potato Ulva | Potato Ulva | Potato Ulva | Potato Ulva | Potato Starch....| 8.7] 18.1 9.6] 26.3 |11.8| 28.1 12.9 | 33.5. 113.8] 35.5 Dextrin...]10.5| 17.3 |11.5| 25.1 |17.8| 27.5 [17.9| 30.3 |17.9| 31.9 ACTION OF VARIOUS ALGAL a. u THE CARBOHYDRATE CONSTITUENTS OF AGAR-AGA D OF VARIOUS GUMS, AS WELL AS EXPERIMEN’ TS UPON THE AUTOLYSIS OF ALGAL SLIME Because of the large amounts of carbohydrate-containing slime formed by many algae, and because of the röle this might play as a reserve product, it was deemed advisable to try out the various algae for enzymes capable of hydrolysing such complex carbohydrates to assimilable sugars. It was assumed on the basis of the work done by König and Bettels (’05) and others, that such hydrolytic products would be re- dueing sugars, in all probability galactoses and pentoses. A series was set up with each of the several algae, using 50 ce. of .25 per cent agar as a substrate and varying amounts of a ‘‘diffusion-extract’’ from fresh tissue. The agar sub- strate was slightly viscous in the cold, but when kept at a temperature of 40°C., the optimum temperature for diastase, this was not noticeable. Toluene was used as an antiseptic. The flasks were shaken at regular intervals during a 30-day period and at the end of that time aliquot portions were re- moved and tested for reducing sugars. There was no reduc- tion in any case. As a parallel series, thin strips of agar were placed in test- tubes and 20 cc. of ‘‘diffusion-extract’’ added. Toluene was used as an antiseptic and the tubes kept at a temperature of 40°C. for two months. At the end of that time no hydrolytic 1915] DAVIS—ENZYME ACTION IN MARINE ALGAE 803 action was observable, either by reduction of Fehling’s or by mieroscopical examination. In the experiments on the hydrolysis of various poly- and disaccharides, checks were set up in which the usual amount of ‘‘diffusion-extract’’ was placed in distilled water. This was to determine the reduction of copper, if any, due to the ‘‘diffusion-extract’’ itself. In no case was there more than a very slight trace that might have been due to other causes than enzymic. However, it was thought that a self-digestion series would more definitely determine whether the hydrolysis of the carbohydrates of the slime could be brought about by specific algal enzymes. With this in mind, a series was arranged in which the flasks contained 50 cc. of a water extract from each of the forms investigated. Checks were set up in which the ‘‘diffusion-extract’’ was inactivated in the autoclave. Toluene was used as an antiseptic and the flasks maintained at a temperature of 22-23°C. for two months. Aliquot portions removed from time to time failed to show the slightest trace of hydrolysis. It will be remembered that Tihomirov (’10) had found osozone-forming sugars in the conceptacles of Ascophyllum and Fucus that he thought might be dextrose and d-galactose, possibly also fucose and arabinose. Thinking that these might possibly have arisen from their corresponding an- hydrides contained in the conceptacle slime, a self-digestion series was set up with an extract from the abscised, crushed conceptacles of those two forms. The Fucus was in a fruit- ing state. The series were set up in duplicate, one kept at room temperature and the other at 32-383°C. Fehling’s test showed no hydrolysis after a month. Pentosans alone were then used as substrates. Two series of flasks for each of the algae investigated were set up, each containing a .5 per cent solution of gum arabie.! To one series was added 10 cc., to the other 20 ce. of ‘‘diffusion- extract,’’ and the flasks placed at room temperature with toluene as an antiseptic. No hydrolysis was apparent either "The gum arabic was dissolved in water, then precipitated with several volumes of 95 per cent alcohol to get rid of reducing sugars. [VoL. 2 804 ANNALS OF THE MISSOURI BOTANICAL GARDEN by the phloroglucin test or by sugar determinations, even after 60 days. THE ACTION OF ALGAL “DIFFUSION-EXTRACTS” UPON CELLULOSE AND HEMICELLULOSE Experiments were carried out to determine the presence or absence of cellulose hydrolysing enzymes in the algae, and to this end several methods were employed. First, strips of filter paper were placed in test-tubes and entirely covered with 20 ce. of ‘‘diffusion-extract.’? Checks were maintained with distilled water and also with the ‘‘diffusion-extract’’ alone. The series were set up in duplicate—one kept at room tem- perature and the other at 35°C., both with toluene as an anti- septic. After definite intervals during a 60-day period, the contents of the tubes were tested for reduction. None was observable in any case, and microscopic examination of the filter paper failed to reveal any decomposition whatsoever. A double series was then set up in a similar way, except that 2 grams of fresh, crushed algal tissue were added to the tubes instead of the ‘‘diffusion-extract,’’ together with 20 ce. of distilled water. At the periods noted above, microscopic examination revealed no attack. It was thought an inherent difference between algal and filter paper cellulose might be responsible for this absence of action. Accordingly, cellulose was prepared from the tissue of Ascophyllum after the method described by Fowler (’11, p. 159) and used by Cooley (714). Fifty grams of air-dried tissue were placed in a liter flask, 500 ce. of distilled water added, and the lot placed in the autoclave at 15 pounds for 15 minutes to destroy any cellulase that might be present, and also to extract as much as possible of the water-soluble substances. The water was filtered from the tissue, fresh water added, and the flask placed in an incubator at 35°C. It was kept at this tempera- ture with daily changes of water for 10 days, at which time the water-soluble constituents seemed to be almost entirely removed. The treatment from here on was the same as that described by Cooley. To the tissue was added a liter of potassium-chlorate-nitric-acid solution made up in the pro- portion of 30 grams of potassium chlorate to 520 ce. of nitric 1915] DAVIS—ENZYME ACTION IN MARINE ALGAE 805 acid (sp. gr. 1.1). The flask was kept in the ice-box for two weeks, when the oxidizing mixture was changed and the new lot allowed to remain another fortnight. At the end of this time a yellowish white tissue was obtained, representing fairly pure algal cellulose. This was filtered off, washed well with distilled water, and dried in the oven at 75-80°C. The final product weighed 19.7 grams. This cellulose was used in a way similar to the filter paper in the first series. One gram was placed in each flask and well shaken up with 50 cc. of distilled water. A concentrated ‘‘diffusion-extract’’ was prepared from Ascophyllum, Lamin- aria, Ulva, and Chondrus, 10 ec. of which represented 5 grams of the original dried tissue. This volume was added to the flasks, and the series set away at 30°C. with toluene as an antiseptic. At the end of two months no reduction of Fehl- ing’s was observable and under the microscope there seemed to be no decomposition of the cellulose particles. Action on hemicelluloses——Hemicellulose was used from two sources—from date seeds, and from the seeds of the wild persimmon, Diospyros virginiana. In both cases the experi- ments were essentially the same. The horny coats were broken and the embryos removed. Small pieces of the hemi- cellulose were then taken, placed in a flask with water, and heated in the autoclave at 15 pounds for 15 minutes to kill the cytase present. Upon removal from the autoclave the pieces were washed several times in distilled water, being left in the last wash water for several days with toluene as an antiseptic—this to get rid of any reducing sugars present. Two of these washed pieces were placed in test-tubes with 10 ce. of the concentrated ‘‘diffusion-extract’’ used in the ex- periments with cellulose. Another lot was covered with 10 ce. of distilled water and 2 grams of the dried algal powder added. In a third series shavings of the hemicelluloses were mounted in a Van Tieghem cell with a drop of enzyme solu- tion. All the algae under investigation were tried out, but in no case was there the slightest trace of decomposition, either microscopically or by the reduction of copper. [Vou 2 806 ANNALS OF THE MISSOURI BOTANICAL GARDEN Results—These negative results do not necessarily argue against the production of slime through the agency of enzymes. It is impossible to exactly reproduce the conditions of the cell in vitro, and enzymes which might act upon cellu- lose in the living tissue to produce slime might easily be in- hibited from action on cellulose or hemicellulose under the conditions of the experiments. Grüss (’10) found that fresh cherry gum contained cytase, but that none was demonstrable in the older gum. He also found that malt diastase would not act upon such gum until the tannins had been removed. It is known that the algae do contain tannins or ‘‘tannoidal’’ bodies, the writer having demonstrated a ‘‘tannoid’’ content in Ascophyllum of 1.1 per cent of the dry weight. These, or other agents, could be involved in the partial or complete in- hibition of cytolytic action. On the other hand, indirect evi- dence, at least, points to the presence of the galactan and pentosan groups as due to their being laid down as such, that is, they do not arise as the direct result of hydrolytic enzyme action, but probably represent the final step in the condensa- tion of those particular hemicelluloses. Tschirsch (’89) and his students have shown that the algal slime exists as an in- tracellular substance, and they hold that in most instances, at least, it does not arise from the cellulose. This seems to be the logical view, and we in turn seem justified in looking upon the galactan and pentosan groups in the algae as normal products of the plant’s metabolism, present at all stages in the plant’s growth, and capable of giving rise to gelatiniza- tion at any time upon the adsorption of water. If one ex- amines, for instance, such forms as Fucus, Mesogloea, and Chondrus, the slime is hardly detectable when the plants are growing under normal conditions, but when brought into the laboratory and placed in fresh water, a rapid adsorption begins at once. The dissolved salts in sea-water are un- doubtedly the inhibiting factors in such adsorption under normal conditions. That this inhibition is not bound up with the living cell may be shown by the simple experiment of killing two fronds of Chondrus, for example, and placing one in fresh, the other 1915] DAVIS— ENZYME ACTION IN MARINE ALGAE 807 in salt water, with toluene to keep down bacterial action. Very slight, if indeed any, gelatinization is evident with the frond placed in salt water, while that in fresh water begins to gelatinize immediately. It is also a well-known fact that in histological or cytological work with these forms, the kill- ing fluids must be made up in sea-water or water containing a high percentage of salts, else gelatinization interferes. These facts, together with the apparent absence of cellulase and cytase, tend to show that the galactan and pentosan groups are always present as final condensation forms of their particular ‘‘generic’’ carbohydrate line, and that sliming in the marine algae, at least, is the result of the adsorption of water by these already existing carbohydrate groups. DISCUSSION OF RESULTS OF CARBOHYDRASE EXPERIMENTS It is seen from the data presented in the foregoing tables that carbohydrases in the algae, at least those that can be isolated by standard methods, are very few. Furthermore, in all cases where such carbohydrase action is evident, it is limited to the polysaccharides—starch, dextrin, laminarin, and glycogen. In no case were the disaccharides hydrolysed. As groups, the ‘‘greens’’ are more active than the ‘‘reds,”’ while of the ‘‘browns,’’ Laminaria is the only form in which carbohydrate action is demonstrable. Moreover, the action here is extremely slow and is limited to starch, dextrin, and laminarin. Mesogloea and Ascophyllum are similar to Fucus in failing to show the presence of carbohydrases. Within the groups there is little difference in the rate of carbohydrase action. This is especially true in the ‘‘greens.’? Of the ‘‘reds,’’ Agardhiella is a little more active than the other forms investigated, while Ceramium is slightly the slowest. Bartholemew (’14), in the work already referred to, also found that Ceramium was less active than the other ‘‘reds”’ with which he worked. The various polysaccharides, with two exceptions, prove favorable as substrates for the various algae in the same order, viz., starch, dextrin, laminarin, and glycogen. The carbohydrases of Ceramium act more rapidly upon dextrin than upon starch and this is also true of Laminaria, although [voL. 2 808 ANNALS OF THE MISSOURI BOTANICAL GARDEN to a lesser extent. Glycogen, which is very generally hydrolysed by diastase, is here decidedly less readily attacked than the other polysaccharides. This would seem to indicate that we are dealing with a distinct enzyme, one that might be placed in the same category with dextrinases. These latter always occur with the diastases but are held by many workers to be distinct. | Some of the substrates tested for hydrolysis do not, as far as we know, oceur in the plants investigated. This is true of sucrose, lactose, and inulin. However, although this might reconcile us to the failure to find their specific enzymes, it does not argue conclusively against such enzymes being formed. It is well known that tissues do form ferments that have no de- tectable substrates upon which to act—the rennen of the bird’s stomach and the urease of the Soja bean being notable examples. Inulin, as pointed out previously, does occur in certain ‘‘greens,’’ as in Acetabularia and members of the Dasycladaceae. Unfortunately, none of these forms were available for investigation. The absence of lactase and sucrase is not so significant as is that of maltase. It is very generally considered that in the plant, as well as in the animal organism, poly- and disac- charides must be hydrolysed to simple sugars before assimila- tion can take place. It is hardly possible that the algae are an exception to this general rule and yet it is difficult to account for this important negative result. It is known that inhibit- ing agents do not affect all enzymes alike, and it may be here that if such agents are liberated on the death of the cell, the maltase might prove more sensitive to them than the other carbohydrate enzymes. According to the findings of Kylin (713), both dextrose and fructose have been demonstrated in the tissues of Ascophyllum, Fucus, and Laminaria, but in ex- tremely small quantities. These results would tend to con- vince one that an enzyme giving rise to them is probably present in the algal cell. Such carbohydrates as galactans, pentosans, and mannans, are very frequently met with in the algae and are potentially capable of being split to assimilable sugars. That they are 1915] DAVIS—ENZYME ACTION IN MARINE ALGAE 809 not so split, however, seems evident, at least not through the activity of demonstrable algal enzymes, and in the face of the negative evidence obtained, we would consider them as by- products of metabolism rather than as playing the role of reserves. As such, they would not be so comparable to the reserve carbohydrates of the date as they would be perhaps to the mucilaginous constituents of various seeds, as those of flax, mistletoe, etc. These latter adsorb water readily with gelatinization, and as far as is known, never function as re- serves but act in a purely mechanical way (Czapek, ’13, p. 705). LIPASES IN THE ALGAE The almost universal presence of fats in the marine algae led to the question of their assimilation. Accordingly, experi- ments were set up to determine the lipolytic activity upon emulsions of neutral fats as well as upon certain esters of the lower fatty acids. For the neutral fats olive oil was chosen as a substrate, and two general methods were employed in forming the emulsion. The first, an olive oil-casein emulsion was made up after a method described by Bloor (714). Four grams of casein were placed in a warm mortar on a water bath and water added until the whole formed a paste of medium viscosity. A drop of phenylphthalein was added, then N/1 NaOH poured in and stirred with the casein until the latter had been dissolved, this point being indicated by a permanent pink tinge of the mix- ture. Hight ce. of olive oil were stirred into the hot solution and then ground with a pestle until all the oil globules had disappeared. At this point the mortar was removed from the bath and the emulsion cooled. During the cooling it was found necessary to stir the mixture occasionally. The thick, creamy mass resulting was diluted up to the required concen- tration by the careful addition of water. If this dilution is too great, the oil globules tend to rise to the surface. The second method was also suggested by Doctor Bloor, but, as far as is known, has not been described. Hight ce. of olive oil were dissolved in the smallest amount of absolute alcohol necessary. This solution was run through a hot fun- [VoL. 2 810 ANNALS OF THE MISSOURI BOTANICAL GARDEN nel to which a drawn-out piece of glass tubing had been at- tached, into about 100 ce. of cold distilled water, the water being stirred constantly while the olive oil was being run in. A milk-white emulsion made up of extremely small suspended globules of oil resulted. In an emulsion carefully made, most of these globules are small enough to show Brownian move- ment. The alcohol was driven off finally by heating and the emulsion made up to the desired concentration. Both emulsions stand up well. In the latter, however, there is a tendency toward flocking out by some of the smaller par- ticles upon the addition of any salt-containing substance, such as, for instance, algal powder; but, on the other hand, it has the advantage of being more easily checked up Ba of its simpler composition. TABLE XI LIPOLYTIC ACTION OF THE SEVERAL ALGAE UPON OLIVE OIL-CASEIN EMULSION Number cc. of N/10 NaOH to neutralize 10 cc. of substrate 4 days 10 days 15 days ei g 8 agoj g v ed] e y 3#1 512% 1551| 5]|1%5 T 22 3/25 oO E+] €/Stloolet+!] g | s+) oS o/Et] E |] s+] o's a la ls zla ja ls laa (ale |z*® TT ere rer i 1.3 1.29) -1 1-95 RR OEA Enteromorpha ee a 1,25] .2 Be eee ees SEE ree Mesogloea......... .6| .00) .1 5 |1.00} .1 11.8 21 .6 | .075 11.525 Ascophyllum....... Be PER DER i BO. 1 Up 11. lis ı 0 00 TE es cleus catacvad meer tee clear] she 31500 0 0 ondrus.......... 1.2} .00} .05 11.15[1.85| .00| .0511.8 [2.3 2.200 Agardhiella EEE BEE ERA ES EAU, RR. PO) SOL sae S ES ices thecaad Ceramium......... RESEN VER: reer eer: | ew wet Du Aap ı Gee, E Pee Parana S Rhodymenia....... Bi 1 ck PSL del oO Rak bo I. | Sol E S ETE E Champia.......... 21 d | OB) OS) 351.3 05| 00). cal cancfessasdiaees In all the lipolytic experiments, algal powder or fresh algal tissue crushed with fine quartz sand, was used as a source of enzyme action. In some of the original series the olive oil- casein emulsion was employed, but on account of the danger arising from a possible hydrolysis of the casein with a re- sulting increase in acidity, the aleohol emulsion was used in the later work. Lipolytie action of the several algae upon olive oil-casein emulsion.—In this experimental series (table x1) flasks were set up containing 50 ce. of olive oil-casein emulsion as a sub- 1915] DAVIS—ENZYME ACTION IN MARINE ALGAE 811 strate, 5 grams of crushed algal tissue for enzyme action, and 10 ce. of 95 per cent alcohol as an antiseptic. Checks were employed wherein the flasks in one case contained the emul- sion alone, and in another case, the same weight of algal pulp in distilled water. The flasks were maintained for 15 days except for the forms especially noted. At intervals 10 ce. por- tions were removed and titrated against N/10 NaOH with phenylphthalein as an indicator. Lipolytie action on alcohol-water-olive oil emulsion.—Be- cause of the possibility of the hydrolysis of the casein in the emulsion used in the preceding experiments, a series (table x11) employing the alcohol-water emulsion was set up as a check. This emulsion alone was practically neutral but a TABLE XII LIPOLYTIC ACTION OF THE SEVERAL ALGAE UPON ALCOHOL-WATER-OLIVE-OIL EMULSION Number cc. N/10 NaOH to —. 10 cc. substrate after 10 day Alga Emulsion | Emulsion | Water+ |Water+| Net + tissue alone boiled tissue | tissue | acidity E AAA reese ey ea 9 .00 .05 .05 85 Bike ERTL tay a oa 8 .00 .025 1 N EEE .65 .00 .05 075 575 Ascophylium.............. = .00 3 15 nn RE eee EEE 1,15 00 .02 3 85 EEEE le a ceiets 1.45 00 a 35 1.10 A gordbiella re th -25 00 .05 05 20 Ceramium.... ennauaneenn .85 00 1 20 Rhodymenia............:.. ‚525 .00 05 15 375 EEE 125 .00 15 15 slight acidity was produced by the addition of the algal powder. A negligible amount of the oil globules ran together and collected at the surface of the liquid after some days, but the bulk of the emulsion stood up well. As in the preceding series, 5 grams of the fresh tissue were used as a source of lipolytic activity, and the alcohol in which the olive oil had been dissolved served as an antiseptic. Fifty ce. of the emul- sion were used as a substrate, and the flasks maintained at a temperature of 22-23°C. for 10 days. Lipolytic action on triacetin.—The lipolytic activity of dry tissue powder of Ulva, Mesogloea, and Chondrus was tested, using a .5 per cent solution of triacetin as a substrate. Two [VoL. 2 812 ANNALS OF THE MISSOURI BOTANICAL GARDEN grams of the tissue powder were used, otherwise the series (table x) was arranged exactly as the preceding and kept at room temperature for 25 days. Action on other esters.—A series was set up with methyl acetate, ethyl acetate, and ethyl butyrate in .25 per cent solu- tion, using 2 grams of algal powder with 50 cc. of the sub- TABLE XIII THE ACTION OF POWDERED TISSUE FROM CERTAIN ALGAE UPON TRIACETIN Number cc. of N/10 NaOH to neutralize 10 cc. substrate aft Alga 10 days 25 days Tri- Tri- Tri- Tri- :_ {Water+ . Net . , Water + . Net acetin : acetin ege jacetin+] |. acetin . 4; +tissue| TSU | alone acidity tissue | HSSUe | alone acidity Ulva.......... 3 .025 | .175 > 15 15 2 Mesogloea...... .25 .05 1 1 4 2 .15 .05 Chondrus...... .55 .35 .1 4 8 4 .15 25 strate in 20 per cent aleohol. Titrations were made from time to time against N/10 NaOH with phenylphthalein as an indicator. Even after 60 days at room temperature no in- crease in acidity was observable over the checks. General results for experiments with lipases—The results serve to show that, although slight, there is distinet lipolytie activity in most of the forms investigated. The various groups of algae are not so distinet regarding this activity as was the case with the carbohydrases, nor does the activity of the individual alga in this case relate itself partieularly to the activity shown by the form in its carbohydrase action. Agard- hiella hydrolyses the polysaccharides more rapidly than any other alga, yet its lipolytie activity is very low. Likewise, Laminaria, so inactive in the previous group of enzymes, is among the most active on fats. Fucus, on the other hand, was found in previous work to have no action on either carbo- hydrates or fats. The action is especially evidenced by use of the olive oil- casein emulsion. In general, the increases were less where the aleohol-water emulsion was used—a difference probably ex- 1915] DAVIS—ENZYME ACTION IN MARINE ALGAE 813 plainable on the ground that the casein gave rise to a slight acidity. Specificity of action might explain the failure to obtain action on most of the esters. Euler (’12) differentiates the lipases into true lipases and esterases, the former acting on neutral fats particularly, the latter on the methyl and ethyl esters of the lower fatty acids. Even in this latter restricted field, great specificity may be shown. Reed (’12) found that ethyl acetate was quite rapidly acted upon by an esterase isolated from Glomerella rufomaculans, while ethyl butyrate was only slightly hydrolysed. THE PROTEINASES The proteolytic activity of the various algae was tested on albumin, casein, legumin, peptone, gelatin, and in certain cases, on proteins isolated from the algal tissue—most of these under acid, alkaline, and neutral conditions. The first four were made up in 1 per cent concentrations. Albumin and peptone went into solution quite readily; legumin and casein, being insoluble in water, were either weighed out directly, or dissolved in N/10 NaOH. The albumin and gelatin were also tested in the form of Mett’s tubes, and the gelatin alone in test-tubes where it was held at a temperature high enough to keep it in a liquid state while in contact with the algal powder. In all cases, algal tissue was used directly, either fresh crushed, or dry powdered—usually 2 grams of the powder or 5 grams of the fresh tissue to each 50 ce. of substrate. Determination of hydrolysis—Proteolytie action was de- termined in several ways, each acting as a check on the others. The biuret test was used for the demonstration of tryptic action, the proteins being precipitated by (NH4)2SO,4 in saturated solution and the test applied in the usual way. The tryptophane test was employed for ereptic action and this also furnished a check on the action of trypsin. In this, 1 ee. of the protein solution was placed in a small evaporating dish, a drop of glacial acetic acid added, and then a few drops of strong chlorine water. The hydrolysis to the amino acid stage [VoL. 2 814 ANNALS OF THE MISSOURI BOTANICAL GARDEN was also demonstrated in two other ways—by the formal- dehyde-titration method of Sörenson (’08), and the determina- tion of the amino-nitrogen by the micro-Kjeldahl method of Folin (713). The Sörenson method consisted in adding 2 ce. of formalin, made alkaline to a faint pink tinge with N/20 NaOH, to 10 ce. of the filtered protein solution, made alkaline to the same color. Upon mixing, the color disappeared and the acidity resulting was titrated against N/50 NaOH, using phenylphthalein as an indicator. In the determination of the amino-nitrogen by the ‘‘micro”’ method of Folin, the protein in a 5 ee. filtered portion of the solution was precipitated with 2 ec. of a 25 per cent solution of phosphotungstie acid in 5 per cent H:S04. The precipitate was filtered off and a 2 ce. portion of the filtrate removed for the determination of the nitrogen. Duplicate determinations were made in all cases. These portions were placed in Jena test-tubes, 20200 mm., 1 ce. concentrated H250; added, then 1 gram of K2SO4, and a drop of 5 per cent CuSO4. The digestion was carried on over the flame from a micro burner, the fumes being carried away by the fume adsorbers described by Folin. Usually 20 minutes sufficed for the completion of the digestion, although in a few instances 25 minutes were required. After cooling slightly, 6 cc. of distilled water were carefully added. The tubes were then transferred to the distilling apparatus where concentrated NaOH was added to alkalinity, and the tube contents distilled over for three minutes, the NH; being collected in a known volume of N/10 HCl. The acid in the collection flask was titrated against N/10 NaOH with alizarin red (alizarin sulfonsäure Natrium, Merck), and the amount of nitrogen represented by the acid neutralized, determined. In the method originally described by Folin, the NH; was not distilled but was forced over from an alkaline solution by a strong air current. However, students in his laboratory have made use of a micro distilling apparatus, and the sug- gestion for the ones employed here owes its origin to one of Folin’s assistants. Distillation has the advantage of quick- ness, and from the writer’s experience, of accuracy as well, 1915] DAVIS—ENZYME ACTION IN MARINE ALGAE 815 at least where suction instead of compressed air is employed in the air method. The results with the air current were very often below the theoretical. The distilling tubes used were made in the laboratory from glass tubing, the outer jacket measuring 402 cm., and the inner being 5 mm. in diameter. The lower end of this latter, where it dipped into the collec- tion acid, was fitted with a larger tube 14 mm. in diameter— this to prevent a back flow of the acid; to the upper end of this inner tube was attached a safety bulb made from a 10 ce. pipette, and this in turn fitted into the Jena tube containing the distilling mixture, by means of a two-hole rubber stopper. Through the second hole in this stopper was a small piece of glass tubing closed at the upper end with a bit of rubber tubing and a pinch clamp; it was through this that the alkali was added after the apparatus was connected up for dis- tillation. Considerable trouble was experienced at first with bump- ing, especially after the digestion mixture had become con- centrated. Neither bits of glass nor pebbles would overcome it. Finally the expedient was adopted of using short pieces of glass tubing sealed at one end and this end placed upper- most. These were of such a diameter that after digestion, the digestion mixture drawn up into them by the cooling of the contained air, would easily drain out when the boiling tube was forced up on the side of the test-tube by a quick down- ward motion. The action of Enteromorpha, Mesogloea, and Chondrus powder upon various proteins.—Fifty ec. lots of casein, legumin, albumin, and peptone were used as substrates in this series—all in 1 per cent concentrations. The albumin and peptone were dissolved directly in distilled water, the legumin and casein in N/10 NaOH. Two grams of air-dried tissue powder were used for proteolytic action, with the exception, however, of Mesogloea, which, as before stated, was partially dehydrated before being air-dried. The various substrates were made neutral by the addition of N/10 alkali and then acid or alkaline by further addition of 2.5 ce. of N/10 HCl or NaOH. In the formaldehyde titrations 10 ce. of the sub- (VoL. 2 ANNALS OF THE MISSOURI BOTANICAL GARDEN 816 “HORN 10 JOH OT/N yH% suegre 10 poe 007/N epeul 2M sorensans oy], — sg = o£’ se = = Sz 07 T 1M PE OF = 06 I Os I — pis So'z orz | Sey” + S 06°C => STZ S6 I F + OTL se A = u ss I = SLT co'T — = sot 09T | PPe |" = K OLI en 91 ort F +F 09€ 00 [4 unundsa] = T ss T — 00°C 06'T Fa = c6 T OS tt a = K 017 Zn: OL T SS'I F T S6 P Or T — — E orz = 07T Of °C — — Svc Ot | Aer TF T ‘OT | 0€'S un SLG 09°C + T Of ET 9 T I — T 08°7 == Svc Orc aa — 047 097! pre |" = == T SL'T = set OTe — == OF’ a T upse) — = T 07°C — ss T OSZ z — 047 OFC | mau |" zu + L or € zu SIT OET + + OS TT | SLE A Se EEE 1 eg | —- poce ort | ort es - are 071 oo | am ee + .....%. s. ZI oss see ee ee 061 cep + s.s.s,’ Of’ 6 os’ 7 E reaa 0 | = pees c6 03° EEE, PER) coz | etd | poe het + . 10... Fi 62T |||... Cf" F os ı + corres TE co? Z əuozdəd Ei rere J 0 | been 08° SL: Re ER aiT 6 | mau pete + or 0... ZT <6F er... or. OL°Z En .eor ee... Or "AK 7 = SE SL Fr ss sr — = Og” oS |; epee + t b | orz + 08°F | STZ + + I|s6# | SL T ‘2 = so. a OF: OF: a — | oe op | ppe frets — = T s9'I 2 06° 0L == T 06 I 08° T unungy _ — OF a OL: oc’ u — | oF: og: | camau | + + 'S | OL°2 + oe 7 | OFT + + (oze | ozz z sep OF |sAep SI sAep 08 |SAep ST sAep 0¢ |sAep CT wo | g w 25 w 2 F| sus ES 5 suoyenm S suogenn Bog = }uonenn Z o & | Jepmod ‘99 OS 25 u jowo 13 joumo a5 = jow40 4 a "5'| ee areigsqns i + 5 |g o ~|,| t4°PM SnApUoyD D20180S2 J] DYGLOMOLNUT SNIGLOUd SNOINVA NOdN AVOTV NIVLYAO NOAAI AAGMOd ANSSIL AO NOILOV AHL AIX ATAVL 1915] DAVIS—ENZYME ACTION IN MARINE ALGAE 817 strate were titrated against N/50 NaOH. One per cent chloro- form-thymol was used as an antiseptic, and the flasks were kept at a temperature of 22-23°C. for 30 days. The forms used in table xıv show a general ability to hydrolyse proteins. All four proteins employed were acted upon by one alga or another, but peptone and casein in neutral and alkaline solution were the most readily attacked. Enteromorpha split albumin and legumin but poorly; Chon- drus acted upon legumin only in alkaline solution, and then slightly; Mesogloea failed to hydrolyse casein and legumin, and its action on albumin and peptone was very slow. The action of Ulva, Laminaria, and Agardhiella powder on peptone and casein in alkaline and neutral solution.—In- dications in the preceding experiment seemed to point to the fact that peptone and casein were more easily acted upon than the other proteins—and these more especially in neutral and alkaline solution. Accordingly, a series was set up with these two substrates, similar in all respects to the preceding one, except that the acid substrate was omitted and that Ulwa, Laminaria, and Agardhiella were used for proteolytic action. Five grams of air-dried tissue were employed with 100 ce. of substrate. One per cent chloroform-thymol served as an anti- septic, and the flasks were kept at 35°C. for 30 days. Formal- dehyde titrations were made after 15 and 30 days and trypto- phane tests and amino-nitrogen determinations after 30 days. In the titrations 10 cc. of substrate were titrated against N/50 NaOH, and the amino-nitrogen represents that in 2 ce. of the filtrate from phosphotungstic precipitated protein. The data in table xv tend to substantiate that of table xıv concerning the hydrolysis of peptone and casein. The higher temperature at which the flasks were maintained undoubtedly had something to do with the larger amounts of amino acids split off from these two proteins than was the case in the pre- ceding series, yet if we can judge by the action on carbohy- drates and fats, we are dealing here with the more active members, enzymatically, of their respective groups. On the whole, peptone and casein seem to be the most favor- able substrates of those used for proteolytic activity, and [Vou, 2 *a}eIJSGNS [CUISIIO ay} JO ‘90 FT syuasaıdaı urajoid payeydrioaid o13s3unjoydsoyd wor 372174 Jo "9 z ur uadonu-ourwe oY] $ ANNALS OF THE MISSOURI BOTANICAL GARDEN ‘azeijsqns Jo ‘09 QT ƏZHLIMƏU 0} HORN OS/N ‘99 Jo Jaquinu ay} Juasaıdas sane, , T or I so'l IT’ — $o'T 06° 80° — s6 OP ia S 137EM EA OLS 072 Ss | SE = OL'S Oz S | SE = OLS oz S mye joe" + OS #1 | OLS | OTT + se 97 | S9 | 9S I E sTez | 098 ALe c = ot 9 SS S | OF T Or'9 sss | 8F — 0r 9 ao) eee ee bid aad + 09'ST | S6S | 76 + 0L 21 | 079 | OFT T O0O'ZZ | OL'8 | "mau - = sg oo. 00 = $8 gs 00 rg $8 ss ATE ee + StF 087 | Or = se 8 SO'S | #81 T 00'8T | 0€°S ae S — 00 I OL: 20° — 00°T 04° 20° wee 00°T 04° hee pee auojdag viet + ao S$ So'z | 901 F SL'9 087 | 89T + 00°ST | sep | mau A ase gg hore Oe EP St 5P] on sAep 0f |sAep SI = 5 > SPP OF APP ST) o z 53 ke) >53 |, 53 25 £ "sw d 325333 | suonenn [SPF] FES] sonen 1898 |EES| suonem | FoF zapmod | 35 got | an. =) Jowo fai eo jowo ETU °o Jowo Be [esye saıensqan z Z z ; 35 | aaam | TS DIADUIMD'T | pan D11214 p408 V 818 NIGSVO GNV ANOLddd NOdN AVOTV NIVLYAD WOU ANSSIL AAAAAMOd AO NOILOV JHL AX YIdVL 1915] DAVIS—ENZYME ACTION IN MARINE ALGAE 819 these in neutral and slightly alkaline solution. This was shown in the formol titrations! and in the determination of the nitrogen in the amino acids split off. Albumin was slowly acted upon by Enteromorpha, Mesogloea, and Chondrus. The first and last also hydrolysed the vegetable protein, legumin, to a slight extent—an action that was not shared by Mesogloea. The action of algal powder on the proteolysis of gelatin and albumin in Mett’s tubes.—T wo lots of Mett’s tubes were made up, one containing coagulated egg-white, and the other, 15 per cent gelatin. In each of a series of flasks containing 50 ce. of distilled water, N/200 NaOH and N/200 HCl respectively, were placed one tube each of egg-white and gelatin. Two grams of the powdered tissue from each of the several forms under investigation were added for enzyme action and the usual percentage of toluene used as an antiseptic. The sev- eral series were kept for two months at room temperature. At the end of that time the albumin tubes in the alkaline solu- tion containing the algal powder of Ulva, Enteromorpha, Chondrus, and Agardhiella showed a slight digestion. The checks in the alkaline solution alone showed swelling. How- ever, although this was indicative of action, it was not definite, since the great length of time the protein was in contact with the complex constituents of the tissue may have been a factor in either causing a slight hydrolysis or a contraction of the albumin. On the other hand, Laminaria, Ascophyllum, Meso- gloea, and Ceramium caused no such action. The gelatin tubes showed no evidences of action even after 60 days. The effect of proteinases on the hardening of gelatin —Dox (710) describes a method for testing the hydrolysis of gelatin which consists in keeping the protein in a liquid state during contact with the material being tested for proteolytic activity, then at the end of a stated period noting whether the gelatin congeals when placed in cold water. This method was used in the following way: Five ce. of 20 per cent gelatin were placed in each of a series of test-tubes, and 5 ee. of the standard t The formaldehyde titrations, as used here, were satisfactory only in a general way, i. e., to show relative rather than exact differences in the amounts of amino acids split off. The differences brought out by the amino-nitrogen determinations are much more exact. [VoL. 2 820 ANNALS OF THE MISSOURI BOTANICAL GARDEN ‘““diffusion-extract,’’ described under ‘‘carbohydrases,’’ used for action. The contents of the tubes were made neutral, and acid and alkaline to N/200, as was done in the other pro- teolytic experiments. Five drops of chloroform-thymol were added as an antiseptic. Checks were set up containing the gelatin together with 5 ec. of boiled ‘‘diffusion-extract.’’ The tubes were placed in an incubator at 35°C. for a week, at which time they were removed and cooled in running water. All tubes hardened in a short time, showing that no hydrolysis had taken place. General results for experiments on proteolysis —The pro- teolytic activity, although slow, as was the case with the other enzymes investigated, is definite enough to warrant the statement that proteinases and peptases are very gen- erally present in the algae. When present, such enzymes act best under neutral and alkaline conditions. This last finding is interesting in the light of the existing differ- ences of opinion regarding the relative value of acid and alkaline substrates for vegetable proteinases. It will be re- called that Vines (’97) found that acidity favored the pro- teinase contained in the leaf pitchers of Nepenthes, and in a later paper, he states that peptase (hydrolysing albumoses and peptones to amino acids) always act best under faintly acid conditions. Emmerling (’02), on the other hand, demon- strated that the papain of Carica papaya acted more rapidly when the substrate was alkaline. Euler (’12) states in a general way that peptases require a neutral or faintly alka- line substrate, and proteinases (tryptases) an acid one. Of the proteins employed, solutions of casein and peptone prove the most favorable substrates. Albumin in solution is acted upon slowly, but when employed in the form of Mett’s tubes, doubt exists regarding its digestion. Legumin appears to be slowly hydrolysed by Enteromorpha and Chondrus, but not by Mesogloea. Gelatin, either in the liquid state or in the form of Mett’s tubes, is not attacked. As groups, the ‘‘reds’’? -appear more active in proteolysis than do the ‘‘ereens,’’ while, as was true for carbohydrases, the ‘‘browns”’ show the least activity. 1915] DAVIS—ENZYME ACTION IN MARINE ALGAE 821 THE AMIDASES The tissues from the several algae were tested for their ability to split NH; from such amino and amido compounds as urea, acetamid, asparagin, and methyl amine. These com- pounds were used in 1 per cent concentrations. Series were set up in which 50 ce. of the substrate to be tested were placed in flasks together with 2 grams of the powdered tissue and chloroform-thymol as an antiseptic. Checks were used with the nitrogen compounds alone and with the algal tissue in distilled water. The flasks of duplicate series were kept at room temperature and at 35°C. respectively, for 30 days, at the end of which time Folin’s method was employed for the determination of any NH; that might have been split off. In the collection of the NHs, Friedrich’s improved gas washing bottles containing 250 ce. of N/50 HCl were used. Air was bubbled through by means of a suction pump for two hours, then 25 ce. portions of the collection acid were removed and titrated against N/50 NaOH, with alizarin red as an indicator. In no case was there any action over that evidenced by the checks. These results are extremely interesting, in the case of Ulva especially. This form, as has been shown, thrives in waters where the organic nitrogen content is high. The question would at once arise whether this increased growth were due to the ability of the Ulva to break down the protein molecule and thus obtain an increased supply of nitrogen as NH3, or whether it were due to the activities of the denitrifying bac- teria rendering available a larger assimilable supply. That such bacteria are relatively abundant in sewage-contaminated water has been shown in the review of literature. We can conceive of another factor entering in—that of selective for- mation of enzymes. It might well be that with plenty of the amino-nitrogen available through the activity of bacteria, no amidases would be formed. The possibility of shedding some light on this point led to the experiments following. Experiments on amidase formation by Chlamydomonas.— Chlamydomonas was grown in pure culture upon two differ- ent media; one (with one or two modifications, that used by [VoL. 2 822 ANNALS OF THE MISSOURI BOTANICAL GARDEN Schramm (’14) ), containing (NH;)>2SO, as a source of nitro- gen, the other with nitrogen supplied as peptone and aspar- agin. These media complete were as follows: A. B. DAGIT usa 10.00 grams. AZAT 6 isssagscusdansedy 10.00 grams ) PEN .25 grams Peptone .............6. 4.00 HEBT ED ua am AsSparagin ia 1.00 grams Perr eT Teer ee 10 grams MgS0O4.7 Hs0 .......... .10 grams DOG. cade oes aaa race 1 © 0 VE EEE .10 grams NOONE sioe raran cee 10.00 grams . Ter ee Te Te trace Distilled HAD eerie Se 500.00 ce. BHUCDBS fc ca cus eseds asa’ 10.00 grams Distilled AP oaue nates 500.00 e These media, designated ‘‘A’’ and ‘‘B,’’ were placed in 125 ce. Erlenmeyers, about 25 ec. to each flask, and the flasks placed horizontally until the agar hardened. A relatively large surface was obtained in this way and the harvesting of the alga later was facilitated. In inoculation, the alga was smeared over the surface of the agar to give an even growth. After a growth of thirty days, it was harvested by scraping from the agar surface with a stiff, platinum needle. The cells were then dehydrated with alcohol, acetone and ether, dried, and ground with an equal weight of fine quartz sand. Flasks were set up in duplicate in Wollf wash bottles, using 1 per cent asparagin as a substrate, with an amount representing .35 grams of sand-free algal powder. Checks were run on both the asparagin and the algal powder alone. One-half the series was taken down at the end of 7 days, the other half at the end of 15 days, and the NH; split off determined by the Folin method previously employed. The flasks were kept at a temperature of 35°C. The results are given in table xvı. TABL I THE ACTION OF DEHYDRATED CHLAMYDOMONAS CELLS UPON ASPARAGIN Be Weight Nitrogen as NH; in 50 cc. substrate i algal mgms. 1 per cent powder 7 days Net N 15 days Net N .35 grams “A” .36 .16 45 21 Asparagin .35 grams “B” 1.17 .96 1.84 1.60 eS ee eT re .18 Eee .22 EEE Water .35 grams “A” Me titews enna Oe RE ree .35 grams “B” WS ncecancaele . OR. ee ee The amount of nitrogen in the checks is so small as to be well within experimental error. The NH; split off by powder 1915] DAVIS—ENZYME ACTION IN MARINE ALGAE 823 “B” would be almost negligible were the findings not so con- sistent. There is a definiteness about the increase over the checks that can hardly be ignored. In order to get further evidence on this point, however, another series (table xvr) was set up, using urea and asparagin in 1 per cent concentra- tions as substrates. The flasks were maintained at a tempera- ture of 35°C. for 30 days. TABLE XVII THE ACTION OF DEHYDRATED CHLAMYDOMONAS CELLS UPON ASPARAGIN AND UREA Weight Nitrogen as NH; split Substrate algal i s Net nitrogen powder mgms. .5 grams “A” 1.15 .20 Asparagin .5 grams B” 3.10 2.20 PO ee A .65 ir .5 grams “A” 1.45 Urea .5 grams B” 3.70 2.47 ar ee N .98 EEE Water .5 grams ‘‘A” S50 a | ea en s5 grams > AI T EN In this, as in table xvi, the evidence goes to show that al- though the desamidization is practically negligible where the alga is grown with (NH3)2SOx4 as a source of nitrogen, it is definite where the nitrogen is supplied in the amino and amido form. The actual splitting is small in any case. On the basis of the above, we can simply reason by analogy, and yet this analogy points to the fact that the probable reason for the failure to demonstrate amidase in Ulva lies in the failure to form that enzyme. This in turn would indi- cate that the great growth of Ulva in sewage-contaminated waters is probably due to the abundance of desamidizing bac- teria which those waters maintain—bacteria which break down the protein molecule with the ultimate setting free of NH;. Nitrogen, as such, becomes directly available to the plant. NUCLEASES The presence of nucleases in the algae has already been reported by Teodoresco (’12), but since he investigated only [VoL. 2 824 ANNALS OF THE MISSOURI BOTANICAL GARDEN one of the forms falling within the scope of this study, experi- ments were carried on to determine the presence or absence of nucleases in one representative of each group, Ulva, Ce- ramium, and Ascophyllum. One-half per cent nuclein was dissolved in N/10 NaOH, the complete solution of the compound being shown by a drop of phenylphthalein. One hundred cc. of this neutral solution were added to each flask together with 3 grams of air-dried algal powder. Toluene was added as an antiseptic. Checks were set up by adding autoclaved algal powder to the nuclein and also by using nuclein solution alone. The flasks were placed at 35-36°C. for 38 days, at the end of which time the phosphoric acid split off was determined as P205 by the uranium-acetate method.’ Five ce. of a sodium acetate solu- tion? were added to 25 ec. of the nuclein substrate, this brought to a boil and titrated while hot. Potassium ferrocyanide was used as an indicator—a drop of the titration mixture being removed from time to time and brought into contact with a drop of the indicator on a porcelain plate. The results ob- tained are given in table xvi. TABLE XVIII THE ACTION OF POWDERED TISSUE FROM CERTAIN ALGAE UPON NUCLEIN Substrate ; Free HPO; as P:O5 | Net amount Weight algal powder Jin 100 cc. in 38 days| P:Os in 100 cc. 1 per cent mgms. mgms. 3 gms. Ceramium 70.00 56.25 3 gms. Ceramium boiled ite «<€ «sives i 3 gms. Ulva 56.70 39.20 Nuclein 3 gm a boiled 17:50 nn, ien 3 gms. Ascophyllum 17.25 35 3 gms. Ascophyllum bld. 1090 O ears These findings substantiate those of Teodoresco (’12) re- garding the general presence of nucleases in the algae. The values for Ceramium agree very well with those he obtained for the same form, i. e., 56.25 milligrams in 38 days at 35°C., as compared with 76.6 oin im 91 days at 22-26°C. Ulva * The standard solution of the uranium acetate contained 8.8652 grams of the = in 250 ce. of water, and each cc. by calculation was equivalent to 5 milligrams 0 ? The sodium acetate solution N 25 grams of sodium acetate and 25 ce. of 30 per cent acetic acid in 250 c 1915] DAVIS—ENZYME ACTION IN MARINE ALGAE 825 shows less nuclease activity than does Ceramium, while Asco- phyllum, true to its reputation for inactivity, gives a value so small as to be negligible. An interesting point is brought out by the use of nuclein— one that proves a check on some of the previous proteinase experiments. Nuclein is composed of nucleic acid bound up with some protein (according to Abderhalden, ’11, this is albumin) which must be split off by a proteinase before the nuclein residue is exposed to the attack of the nuclease. That unmistakable nuclease activity was evident, only serves to show again the presence of proteolytic enzymes. OXIDASES AND CATALASES Oxidases.—Direct and indirect tests for oxidase action, that is, for the oxidases and the so-called peroxidases, were carried out in all cases with fresh tissue. The general method de- scribed by Clark (’10) was employed, using guaiacum, alpha naphthol, and phenylphthalin as reagents. Five grams of the fresh tissue, crushed with an equal weight of fine quartz sand, were extracted for half an hour with 25 cc. of distilled water. The extracting fluid was then filtered off, the tissue residue pressed out, and the filtrate made up to 50 ce. Five ce.- portions were placed in test-tubes, and for the direct test, ten drops of the reagent were added; for the indirect test, this amount plus 1 cc. of fresh 3 per cent hydrogen peroxide. In only two cases was direct oxidization observable—with Agardhiella and Ulva. With the former, direct action was strong with all three reagents, and when peroxide was added an immediate deepening of the color occurred, showing the presence of peroxidases as well. With Ulva, however, both direct and indirect tests were only weakly positive. Atkins (714), it will be remembered, obtained direct tests with but one of twenty-nine diverse algae investigated and indirect tests with but seven. He thought that reducing substances prevent the demonstration of oxidases in other forms. As brought out in the review of literature, Reed (’15*) has since demonstrated indirect oxidation of the alpha naphthol-para- phenylenediamine group of compounds by many of these [VoL. 2 826 ANNALS OF THE MISSOURI BOTANICAL GARDEN forms. In the filamentous forms he showed the presence of oxidases by the formation of colored granules within the cells surrounded by these reagents. Reed concludes that oxidases of specific oxidative ability are very generally present in the algae, and where negative results are obtained, either the necessary specific compound is not present or other factors enter in, such as the destruction of the oxidase equilibrium of the cell upon crushing. Catalases.—Both fresh and air-dried tissue were used for catalase demonstration. In a preliminary series, the addition of 5 cc. of 3 per cent hydrogen peroxide to about a gram of fresh crushed algal tissue showed evolution of oxygen in all cases except one, that of Mesogloea. Later, a series (table xıx) BLE XIX CATALASE ACTIVITY OF CERTAIN ALGAE Al Number cc. O: evolved at 21.5°C. > 2 minutes 5 minutes 10 minutes Ascophyllum..........:.. 3 9 9 ANMAVID. . ccc cca ccecas 1.4 2.3 2.3 Mesogloed i. ssc acces 0.0 0.0 0.0 WS cay aia a) alae ean 2 4 5 Agardhiella............»- 3.3 4.6 5.6 Chondrus.... ccc cece A 2.0 2.5 Rhodymenia 9 1.4 2.0 MIUM een 3.1 5.9 8.2 Potato leaf tissue......... 22.6 > er was set up in which 1 gram of powder was placed in 125 ce. Erlenmeyer flasks, 10 cc. of 3 per cent hydrogen peroxide added, and the oxygen evolved collected in a gas burette over water. The flask in which the action was taking place was shaken every 15 seconds, and the volume of oxygen evolved read at the end of 2, 5, and 10 minutes. The temperature of the room was practically constant during the experiments and no especial precautions were taken to control the temperature of the flask other than keeping the hands away from it dur- ing the action. The results are not meant to be quantitatively exact, but they do give the relative catalase activity of the several forms. In addition, air-dried potato leaf tissue that had been in the laboratory about the same length of time as the algal tissue was tested for comparison. 1915] DAVIS—ENZYME ACTION IN MARINE ALGAE 827 Catalase, so wide-spread in all plant tissues, is found here in all the forms investigated except Mesogloea. The ‘‘reds’’ prove more active than the ‘‘browns,’’ and these latter slightly more active than the ‘‘greens.’’ No alga is strikingly active, however, when compared with potato leaf tissue. Strangely, Ulva, most active in regard to the other enzyme groups, is one of the least so here. GENERAL DISCUSSION AND CoNCLUSIONS The data obtained in the foregoing investigation serve to show that the number of enzymes in the algae that can be iso- lated, by standard methods at least, is quite limited. This is especially true of the ‘‘browns,’’ in two forms of which, Ascophyllum, and the Fucus of the earlier study, such action is limited to catalase alone. In this group the demonstrable carbohydrases are restricted to very slowly acting diastases in Laminaria; in neither Ascophyllum nor Mesogloea is there the slightest trace of what might be termed carbohydrate hydrolysis. Moreover, negative results are obtained in these forms for most of the other enzymes sought. Laminaria shows lipases and catalases (it was not tested for proteolytic or nuclease activity), and action in Mesogloea is restricted to lipases and proteinases, both tryptic and ereptic. On the other hand, very general enzymic activity is demonstrable in the ‘‘greens’’ and the ‘‘reds’’—diastases, dextrinases, lipases, proteinases (tryptic and ereptic), nuclease, and catalase being isolated from the crushed tissue. Oxidase is shown present in one ‘‘red,’’ Agardhiella, and in one ‘‘green,’’ Ulva. Such action, as a whole, appears a little more rapid in the ‘‘reds’’ than in the ‘‘greens,’’ but no enzyme stands out as being specific for either a group or an alga within a group. The carbohydrases demonstrated are restricted in their action to those hydrolysing starch, dextrin, glycogen, and laminarin of the polysaccharides used as substrates, and in Laminaria, such action was further limited by a failure to act upon glycogen. In no case, in any member of the three groups was there evidence of disaccharides being attacked. While [vor. 2 828 ANNALS OF THE MISSOURI BOTANICAL GARDEN this is not so surprising perhaps for sucrose and lactose, it is difficult to understand the failure of enzymic hydrolysis of maltose. The results obtained by Kylin (’13) indicate that both dextrose and fructose are found in algal tissues, and reasoning from results found for plant and animal tissues in general, it seems, as is true in those cases, that in the algae, maltose must be broken down to glucose before assimilation can take place. The failure to isolate this enzyme points to the possible presence of some inhibiting factor, rather than to the non-formation of the ferment. Lipases, acting very slowly, appear wide-spread in algae, being demonstrable in all the forms used in this study except- ing Ascophyllum. Along with the fact that fats are very generally found in the algae, these results are significant in that they indicate the importance of the röle these compounds may play as assimilatory products. It is not thought, as was advanced by Reinke (’76), Hansen (’93), and others, that these fats function as the first products of assimilation, but rather, that they act as storage products of more or less importance. The algae, in general, show the presence of enzymes capable of hydrolysing certain proteins. Casein and peptone in alka- line and neutral solution prove the most favorable substrates of those tested, although legumin and albumin are also slightly attacked. The ‘‘greens’’ and the ‘‘reds’’ are about equally active in this way, the ‘‘browns,’’ as usual, acting more slowly. The fact that both native proteins and peptones were hydrolysed, points to the presence of both tryptic and ereptic enzymes. Still further evidence of the presence of the first of these was the splitting of the protein molecule from nuclein preceding the action of nuclease. Amidases seem not to be formed by any of these algae. The results obtained with Chlamydomonas, from which the amidases were isolated when the alga was grown on a medium containing asparagin and peptone as a source of nitrogen but not when the nitrogen was in the form of ammonium sulphate, indicate that such amidase formation may depend upon the nature of the supply of assimilable organic nitrogen. This has a distinct bearing upon the reason for the increased growth of 1915] DAVIS—ENZYME ACTION IN MARINE ALGAE 829 Ulva in sewage-contaminated waters. In order to break down the proteins present in the surrounding waters and even those in close contact with the plant itself, it would be necessary for the Ulva to secrete an extracellular enzyme, since the large protein molecule is not diffusible into the cell. If so secreted, the enzyme would be quickly dissipated in the large volume of surrounding water. Desamidizing bacteria, on the other hand, have been demonstrated in harbor and shore waters where such algae abound. They can come into much more intimate contact with the protein than can the plant, and un- doubtedly play an important röle in rendering available at all times an abundant supply of organic nitrogen. The demonstration of nucleases acting upon the previously split nuclein molecule, substantiates the findings of Teodoresco for this enzyme. Both Ulva and Ceramium showed the presence of the ferment, while Ascophyllum, the only repre- sentative of the ‘‘browns”’ investigated, gave negative re- sults. Where such enzymes were formed, they compared more favorably with enzymes of fungi and higher plants than do any of the other algal ferments. None of the ‘‘browns’’ studied showed the presence of oxidative enzymes, while in the ‘‘reds’’ and the ‘‘greens’’ but one form gave the characteristic reactions. It is interesting to note that these algae, Agardhiella and Ulva, were the most enzymatically active forms studied. The oxidase reactions with guaiacum, alpha naphthol, and phenylphthalin were very positive, both directly, and indirectly with hydrogen peroxide. In all cases where enzymes were demonstrated, the action was very slow, being with the exception of nuclease, much less rapid than in the higher plants. The reason for this is not clear, but it cannot in all instances be due to inhibiting sub- stances set free upon the death of the cell. Arber (’01), as has been mentioned before, found that Ulva, Cladophora, and Enteromorpha, placed in the dark but under otherwise pre- sumably normal conditions, required from two weeks with Ulva, to two months and more in the case of Enteromorpha for destarching. This indicates the presence of a very slowly acting diastase in the cells of these algae. The metabolism [voL. 2 830 ANNALS OF THE MISSOURI BOTANICAL GARDEN of the algae is also probably slower than that of the higher plants and one might expect, a priort, the enzymes also to be less rapid in their action. Although the algal enzymes may be inherently slow, it seems that there may also be substances set free on the death of the cell which either partially or en- tirely inhibit enzyme action. The writer has found evidence in some preliminary experiments, that the action of taka diastase upon starch is directly proportional to the amount of free tannin present. In connection with this, it was also found that Ascophyllum had a ‘‘tannoidal’’ content of 1.1 per cent of the dry weight. It is possible that such tannoids, if in an uncombined state, may after the death of the cell unite with an enzyme to throw it out of the sphere of action. That diastases are demonstrable in tissues having a high tannin content may perhaps be explained on the basis that they are bound up in such a way as to render them incapable of uniting with the ferments. Still other organic inhibiting compounds may be present, and the point opens up a very interesting problem concerning inhibition, not only in algal tissues, but in those of many higher plants as well. SUMMARY 1. Using standard methods of enzyme isolation and de- termination, the following enzymes have been found in fresh or dried algal tissue: Carbohydrases hydrolysing the polysaccharides, starch, dextrin, glycogen, and laminarin, but not those hydrolysing the several disaccharides employed as substrates. . Lipases acting upon neutral fats but not upon the esters of the lower fatty acids. Proteinases (tryptic and ereptic) acting best under neutral and alkaline conditions. P oO’ © oF) . Nucleases. . Oxidases and peroxidases (in but two forms—Agard- hiella and Ulva). f. Catalases. (ee) 1915] DAVIS—ENZYME ACTION IN MARINE ALGAE 831 2. Negative results were obtained for cellulase, cytase, maltase, lactase, sucrase, amidase, and esterase. 3. The action of all the enzymes isolated was very slow. It is a pleasant duty to acknowledge indebtedness to those who in various ways have lent support to this investigation. Such acknowledgments are due Dr. George T. Moore for the privileges of the Missouri Botanical Garden, especially its library and laboratories; to Dr. Benjamin M. Duggar, at whose suggestion the problem was undertaken, and under whose direction and constant kindly interest it has been pur- sued; to Dr. Phillip A. Shaffer, of the Biochemical Labora- tories of the Washington University Medical School, for sug- gestions and advice concerning biochemical methods, and for the courtesy of his laboratory from time to time; and finally, to the Woods Hole Biological Laboratory, for the privileges extended during the summers of 1913-14. 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Ges. 28:2604-2609. 1895. Crato, E. (’92). Die ein Organ des Zellenleibes. Ber. d. deut. bot. Ges. 10:295-302. f. 1-8. ————, (’93). Ueber die Hansteen’chen Fueosankörner. Ibid. 11:235-241. 1892. ————, (93a). Morphologische und mikrochemische Untersuchungen ueber die Physoden, Bot. Zeit. 511:157-195. 1893. Czapek, F. (’13). Biochemie der Pflanzen. 1:pp. 760-761. 1913. Dox, A. W. (’10). The intracellular enzymes of Zn and Aspergillus. U. S. Dept. Agr., Bur. Animal Ind., Bul. 120: 1-70. 19 Duggar, B. M., and Davis, A. R. (’14). Enzyme action in Fucus vesiculosus. Ann. Mo. Bot. Gard. 1:419-426. 1914. Emmerling, O. (°02). Über die Eiweissspaltung durch Papayotin. Ber. d. deut. chem. Ges. 35:695-699. 1902. Euler, H. (712). General chemistry of the enzymes. 1912. nn... —— 1915] DAVIS—ENZYME ACTION IN MARINE ALGAE 833 Famintzin, A. (67). Die Wirkungs des Lichtes auf Spirogyra. Acad. Imp. d. Sei. de St. Petersbourg, Mélanges biol. 6:277. 1867. [cited from Tay on ar ; 65 Die Zelle der Cyanophyceen. Bot. Zeit. 63:51-129. pl. 4-5, 1-64. 1905 en = and Farmer, C. J. (712). A new method for sr N a of total trogen in urine. Jour. Biol. Chem. 11:493-501. Foster, G. L. (’14). Indications regarding the een = combined nitrogen for Ulva lactuca. Ann. Mo. Bot. Gard. 1:229-235. Fowler, G. J. (’11). An introduction to bacteriological and enzyme chemistry. Gran, H. H. (’02). Studien ueber Meeresbakterien I. Bergens Mus. Aarbog 1901:°:1-23. 1902. (7022). Die Hydrolyse des Agars durch ein Enzyme. Centralbl. f. Bakt. _—, (’028). Il. 9:562-563. 1902. Green, J. R. (’99). The soluble ferments and fermentation. 1899. Greenish, H. (’81). Untersuchung von Fucus amylaceus. Pharm. Zeitschr. f. Russl. 20:501-507. 1881. [Ber. d. deut. chem. Ges. 14:2253. 1881.] —, (’82). Die Kohlenhydrate des Fucus amylaceus. Archiv Pharm. 17: 241-257, 321-335. 1882. [Ber. d. deut. chem. Ges. 15:2243-2244. 1882.] Griiss, J. (702). Ueber den Umsatz bei der Keimung der Dattel. Ber. d. deut. bot. Ges. 20:36-44. 1902. ——, (’10). Ueber das Verhalten von Cytase und apa ar bei der Gummibildung. Jahrb. f. wiss. Bot. 47:393-429. pl. 13, f. 1-3. ming hy L. (792). Observations sur ry ara mucifére des Laminariacées. . d. Sci. Nat., Bot. VII. 15:1-46. f. 1 1892. Günther, A., und Tollens, B. (°90). Über die Fucose, einen der Rhamnose isomeren Zucker aus Seetang (Fucus-arten). Ber. d. deut. chem. Ges. 23:2585-2586. 1890. Hansen, A. (°93). Ueber Stoffbildung bei den Meeresalgen. Mitth. aus d. Zool. ta. zu Neapel 11. Berlin, 1893. [cited from Kylin ’13.] Hansteen, B. (°92). Studien zur Anatomie und or ast der Fucoideen. Jahrb. f. wiss. Bot. 24:317-362. pl. 7-10, f. 1-30. 1892 ——, (’00). Ueber das Fucosan als erstes scheinbares Product der Kohlen- saüreassimilation bei den Fucoideen. Ibid. 35:611-625. pl. 14, f. 1-11. 1900. ake reg (788). Zellmembran und Hüllgallerte der Desmidaceen. Disserta- on. Greifswald, 1888. [cited from Walliczek ’93.] ger a a (703). Recherches are ger et tape sur le digestion des nes et des galactanes par la séminase, chez les végétaux. Rev. Gen Bot. "18! 345-368, 369-392, 406-417, 444-404, 1903. Hunger, F. W. T. (’02). ae iy ee ee der Dictyotaceen. Jahrb. f. wiss. Bot. 38:70-82. rT = TE Ueber die Organisation der Gallerte bei einigen Algen und Flagel- . Inst. Tübingen, Arb. 2:p. 333. 1884 —, (’85). Ueber gr a und Schleimbildung der Desmidaceen. Biol. Centralbl. 5:353-367. 188 [VoL. 2 834 ANNALS OF THE MISSOURI BOTANICAL GARDEN ———-, (96). Die Bedingungen der Fortpflanzen bei einigen Algen und Pilzen. Jena, 1896. Koenig, J. und Bettels, J. (705). Die Kohlenhydraten der Meeresalgen und daraus hergestellter a Zeitschr. f. Untersuch. d. Nahrungs- und Genuss- mittel 10:457-473. 1905 Kolkwitz, R. (’00). Beiträge zur Biologie der Florideen. by Meeresunters. N. F. Abt. Helgoland, Kiel, und Leipzig 4:31-62. f. 1-7. 1900. Krause, G. (°70). Einige Beobachtungen ueber den Einfluss des ee und der Wärme auf der Stärke ee im Chlorophyll. Jahrb. f. . Bot. 7: 511-531. pl. 24, f. 1-4. 1870. Krefting, A. (97). Ueber wictige organische oe aus Tang. Chem. Ind. 1897. No. 20. [Just’s bot. Jahresber. 25°:76. 1897.] r S. (09). Et nyt Katha i Laminariaarterne. Tid- skrift a ia Farmaci, og Terapi, Aarg. 6, Kristiania 1909. [cited from Kylin, °15.] Küster, E. (’89). Ueber Derbesia und Bryopsis. Ber. d. deut. bot. Ges. 17:77-84. pl. 6, f. 1-8. 1889 Kylin, H. (712). Ueber die Inhaltskorper der Fucoideen. Arkiv f. Bot. 115:1-26. 1912. ———, (’13). Zur Biochemie der Meeresalgen. Zeitschr. f. physiol. chem. 83: 171-197. 1913. ————, (15). Untersuchungen über die Biochemie der Meeresalgen. Ibid. 94: 337-425. 1915. aii a T LRA Die Inkrustation der Membran von Acetabularia. Sitzungsber. ad. d. Wiss., Wien, math.- naturw. Kl. 1887:p. 96. 1887. Letts, E. A., and Hawthorne, J. (00). The seaweed Ulva mien and its rela- tion to the pollution of seawater by sewage. Brit. Assoc. Adv. Sci., Rept. 1900:935-936. 1900. —, ———,, (01). On the absorption of ammonia from polluted sea water by the Ulva latissima. Ibid. 1901:831-833. 1901. , and Richards, E. H. (°11). On green riban Rey ge Ulva latis- sima) in relation a pollution of waters in w hey occur, Seventh Report Roy. Com n Sewage Disposal, Anei 3: 72-100. 1911 Meyer, A. (’95). Untersuchungen ueber die Stärkekörner. Jena, 1895. Müther, A., und Tollens, B. (’04). Ueber die zo. der Hydrolyse von Seetang Fucu I d 37: Laminaria, und Caragheen Moos . deut. chem. Ges. 298-305. 1904. Nägeli, C. (63). Sphärokrystalle in Acetabularia. Nägeli’s Bot. Mitth. 1:206- 213. pl. 1. 1863. Oshima, K., und Tollens, B. (’01). Ueber das Nori aus Japan. Ber. d. deut. chem. Ges. 34:1422-1424. 1901. Oltmanns, F, (’04). Morphologie und Biologie der Algen 1:p. 76. 1904. , (C05). Ibid. 2:pp. 147-164. 1905. Ravenna and Cereser, (’09). Origin and physiological function of pentosans in plants. Jour. Lond. Chem. Soc. 96:1946. 1909. [cited from Swartz ’11.] Reed, G. C “ae oo in plant oxidases (Preliminary report). Science N. S. a. 175. — (15 Evidences for the general distribution of oxidases in plants. Bot. Gaz. 59:407-409. 1915 + a > i 1915] DAVIS— ENZYME ACTION IN MARINE ALGAE 835 ET 23 S. (12). The enzyme activities involved in certain fruit diseases. Va. r. Exp. Sta., Ann. Rept. 1911-1912:51-77. 1912. Reinke, J. (76). Beiträge zur Kenntniss der Tange. Jahrb. f. wiss. Bot. 10:317- 381. pl. 25-27, f. 1-18. 1876. ————, (’03). Die zur Ernährung der Meeresorganismen disponiblen Quellen an Stickstoff. Ber. d. deut. bot. Ges. 21:371-380. 1903. Saiki, T. (’06). The digestibility and utilization of some polysaccharide carbo- hydr rates derived from lichens and marine algae. Jour. Biol. Chem. 2:251- 265. 1906. Schmiedeberg, (’85). Ueber der Bestandtheile der Laminaria. Tageblatt der 58th. Ver rsammlung ‘lee a Naturforscher und Arzte in Strassburg. pe [cited from Swartz ’11.] re =i (°83). Die Chromatophoren der Algen. Verhandl. d. naturh. Ver. d. preuss. Rheinlander u. Westfalens 40: . [cited from Kylin.] Schöne und Tollens, B. (’92). Untersuchungen ueber Kohlenhydrate. Landw. Versuchssta. 40:377- —. 1892. Schramm, J. R. (°14). Some pure culture methods in the algae. Ann. Mo. Bot. Gard. 1:23-45. 1914 ———, (’l4a). A contribution to our knowledge of the perky of grass-green algae to elementary nitrogen. Ibid. 1:157-184. pl. 3. f. 1. 1914. Sebor, J. (’00). Ueber die Kohlenhydrate des Caragheen-Moos. Oestereichische Chemiker- -Zeitung. Jahrgang III: p. 441. 1900. [ Bot. Centralbl. 86: p- 70. 01.] En E. (’04). Ueber den mikrochemischen Zuckernachweis durch er azin. Sitzungsber. d. k. Akad. d. Wiss., Wien, math.- naturw. Kl. 113: 3-27. mi 1-2. 1904 Shaffer, P. A. (14). On the determination of sugar in blood. Jour. Biol. Chem. 19:285-295. 1914. Sörenson, S. P. L. (’08). Enzymestudien. Biochem. Zeitschr. 7:45-101. 1908. Spargo, M. W. (’13). The genus Chlamydomonas. Washington Univ. Stud. 1: 65-88. pl. 1, f. 1-17. 1913. Er, Re (84). Ueber das Vorkommen von Mannit in Laminaria saccharina und einigen anderen Seegrassern. Liebig’s Annalen der Chemie 1884:51. [cited om Swartz ’11 Swartz, M. D. (711). Nutrition investigations on the carbohydrates of lichens, algae, and related substances. Conn. Acad. Arts and Sci., Trans. 16:247-382. 11. Teodoresco, E. ©. (’12). Assimilation de l’azot6e et du phosphore nucléique par s algues inférieures, Compt. Rend. Acad. Paris 155:300-303. 1912. —-, (’122). Sur la presence d’une nucléase chez les algues. Ibid. 464-466. 1912. Tihomirov, W. A. (’10). Sur la valeur de la réaction mierochemique de la phenyl- hydra: azine pour la constatation du sucre dans les een des plantes. Ann. ard. bot. Buitenzorg, Suppl. 3°:536-582. pl. 13-15. 910. Timberlake, H. G. (01). Starch formation in Hydrodietyon iaa Ann. Bot. 15:619-634. pl. 84, f. 1-31. 1901. [Vou. 2, 1915] 836 ANNALS OF THE MISSOURI BOTANICAL GARDEN Torup, S. (09). Ein neues Kohlenhydrate bei den Laminariaceen, Tidskrift f. emi, Farmaci, og Terapi, Christiania. 1909. [Biochem. Centralbl. 8:770. 09.] Tschirsch, A. (’89). Angewandte Pflanzenanatomie. pp. 193-217. 1889. Vines, S. H. (’97). The proteolytic enzyme of Nepenthes. Ann. Bot. 11:563-584. 1897. Walliczek, H. (93). Studien ueber die membranschleime vegetativen Organe. Jahrb. f. wiss. Bot. 25:209-277. pl. 11-13, f. 1-22. 1893. Zaleski, Buln (07). Ueber den Umsatz der Nucleinsäure in keimenden Samen. Ber. d. . bot. Ges. 25:349-356. 1907. GENERAL INDEX New scientific names of plants and the final members of new combinations are printed in bold face type; synonyms and page num bers having reference to figures or plates, in italic; and previously published scientific names and all other matter, in ordinary type. A Abietis (Corticium), 760 acerina forma Abietis nen: 760 adusta ren, 764, pans action of al- 1.8 en tenera: carbo ohydrases of, 799; ca atalases of, 826; lipases of, 809; vi e: Enzyme action in the, aw of temperature con- ected with the distribution of, 287 alliciens (Eichleriella), 746, 77 allici nn Fer m), 74 midases in the marine algae pe (Exobasidium), =. 637, 646, 647, Anesthetic vapors, effect of, on exos- mosis, 524 Anesthetics in solution, effect of, on exosmosis, Appel, eech delivered E ne twenty- -fifth anniversary banque Translation of the speech a A the banquet, 13; The relations be- tween scientifie botany and phyto- pathology, 275 Arctostaphyli (Exobasidium), 638, 646, Ascomycetes, oo and relation- ships in the, 2 Ascophyllum “n sum: carbohydrases , 798; sel of, 826; lipases of, 800 Atkinson, = nr Phylogeny and rela- tionships the Ascomycetes, 315 atrata ira), 765, 770 aurantium (Tremell odendron), 742 Azaleae Heobasidium), 637, 645, 649 Bacterial diseases of plants, A conspec- tus of, Banquet, speeches at twenty-fifth anni- ary, Pera (Corticium), 7 Peny: ; e phylogenetic taxon- my of ein plants, 109 erie eier 708; suberosus, 702 Botanic gar ATY , The history and func- ps: of, 1 Botanical Garden of Oaxaca, The, 165, Britton, N. L. The vegetation of Mona Isla nd, 33 Burt, E. A. The ooo of North America, IV, 627; V, 731 C calcea (Sebacina), 759, 770 calcea c. albido- -fuscescens (Thelephora), 759 calcea (Thelephora), 759 candida (Thelephora), A Ae candidum (Merisma), T candidum er Aa i "988, 737, 768 Carbohydrases in the marine algae, 789 Cassandrae (Exobasidium), 638, 646, Cassiopes (Exobasidium ), 629, 636, 647, Catalases in the marine algae, 8 Cellulose and hem ge ra of le trans es of, 0; catalases of, 826; lipases of, 809 ee diff ffusa, 41; nulata, 41 9 e action in, 821 756, 770 s of, 826; lipases of, 809; proteinases = rg cinnamome a (Sebacina an 763, 7 Cladonia er; Cladonia (Thelephor A a: 738, 768 ls, laciniata, 753; merismatoides, i (Sebacina ) 5 oO = J o = w on O © = ‚® Collybia dryophila, 656 ee ar electrical: ap- used i ; measurements of Kerr ytes trom roots of plants, determined by, 4 Conzatti, C. The Botanical Garden of , 165 Coriolus prolificans, 688 (837) 838 ren Abietis, 760; basale, 757; deglubens, 755; Helvelloi ides, 757; in- rte et ns, "752; Leveillianum, : macrosporum cariosum, ra 752; secedens, 762; vagum, Coryphanta nivosa, 45 . The origin of mono- cristata (Cristella), eristata (Thelephora), 5735, 752 cristatum re Cristella nn 754 Czapek, Recent investigations on the nn of plant cells and its colloidal properties, 241 D avis, A. R. Enzyme action in the marine algae rn (Exobasidium), 656 eglubens (C re 755 iella), 747 discoideum (Exobasidium), 637, 645, 649 Distilled water and certain dilute toxie solutions, Some relations of plants to, 459, 500, 502, 504, 506 dryophila (Collybia), 6 656 Duggar, M. Rhizoetonia Crocorum ( Pers. ) “DC. R. Solani Kühn an (Corticium vagum notes on other species, “40 E 743; .), wit Eichleriella, 731, alliciens, 746, Leveilliana, 744, 770; rn 744, : osa, a ei ek aga of e from the r of Beer subjected = the Pre or various agents, 507 intestinalis: carbohy- 96; lipases of, 809; pro- 815 n in the marine algae, 771 on exosmosis, 524, 530 646, 647, 649; Arelostaphyl, 646, 649; Azaleae, 64 5, 649; Cassandrae, 646, 64 647, 64 9; decolorans, oideum, 645, 649; Kar arstenti, mycetophilum, eta Andromedae, Myr , Pecki, Rhododendri, a 649; Symploci, 641, 648, 655; Vaceinii, ANNALS OF THE MISSOURI BOTANICAL GARDEN 627, 639, 642, 649, 658; Vaccinii myr tilli, oA, 649; Vaccinii uliginosi, 640, 648 Exosmosis, Electr nn determination rb from the roots of plants, subjecte ed o the action of various agents, 507 F farinellus (Xerocarpus), 760 Farlow, W. Speech ae livered at twenty-fifth on gi ci 20 einig an ora), nes, Abie A Bakeri, 717; Eli , 730; EN er 14, 716, 730 on 708; igniar- ius, 716, 717, "718, 730, var. nigricans, oo: ne 718, 730; nigricans, 716; ohiensis, 719, 720, 721, 730; Pini, 723, var. Abietis, 724; scutellatus, 719, 720, 730 Fusidium Vaccinii, 649 G Galactose, Toxicity of, for certain of the higher plants, Ganoderma pseudoboletus, 710; 710; subperforatu gelatinosa Peer hey. Gerard’s in nn = "title page of, Germ- plasm, ‚The experimental modifica- sessile, i Teugao, 711 228 Glucose, antagonistic —— of, toward toxicity o of galac janie ( Thele ape "785, 768 Greenman, Mo ograpl e North and Central American species of the genus Senecio—Part Guilandina divergens, 41; melano- p H Helvelloides (Corticium), 757 Helvelloides ciate es 756, 770 Helvelloides (Thelephora), 757 Hill, A. W. The e history and functions of botanic es 185 Hirneolina, 743 Hypochnus Solani, 445; violaceus, 408 I atone age gs effect of, on exosmo- , 524 in ncarmata, (Eichteriella), = ineru s (Cor m), 7 inerustans soe 150, 110 inerustans (Thelephora), 752 Innen, Hee ie et 688; tulipifera, GENERAL INDEX J Jacobaea vulgaris, 602 K Karstenti (Exobasidium), 636, 647, 649 otanie Ga T : Rhododendron Dell, 238 enry. Speech 1 delivered at -fifth anniversary banquet, 15 Kmetii (Eichleriella), 747 Kmetu (Radulum), 74 Knudson, Lewis. Toxicity of galactose for certain of the Mah plants, 659 L Lachnocladium merismatoides, 740 laciniata (Clavaria), 753 Laminaria Agardhii: carbohydrases of, 79 6; catalases of, T lipases of, 809; p arenan es 0 of, 8 Leveilliana (here), a 770 Leveillianum (Corticiu Leveillianum (Stereu a Be Lipases in the marine algae, 809 ee ugal, a a sm, 253 macrosporum (Corticium), 759 Mallotonia gnaphalodes, 47 Merisma candidum, 733, 737; Cladonia 7 cristatum, 752; Schweinitzii, 740 merismatoides (Clavaria), 740 merismatoides (Lacknocladium), 740 meer ri la), m (T 2), 7 toides halos Sanasto san oh); 740, 768 rg M. nn Electrolytie determina- mosis from the roots of ne subjecte ed to the action of vari- ous agents, 507; Some relations of plants to distilled water and certain dilute toxic solutions, Mesogloea divaricata : ci sE ES of, 798; catalases of, 826; proteinases of, 15 Mona Island, The vegetation of, 33, 56, Monocotyledony, The origin of, 175 monticola (Sebacina), 761 Moore, George T. ddress at twenty- fifth re banquet, 25; Ad- dress of welcome at the twenty- -fifth anniversary ee 29 839 ee (Tremella), 656 ycetophilum (Exobasidium ), Muyrtilli (Hobel 649 N Nagel, Charles. Speech delivered at twenty-fifth anniversary banquet, 23 ur The flora of, and its immigra- 656 ee in the marine algae, 823 ee The Botanical Garden of, 165, Oba NA viscosa, = sylvatica, 585 an lts, L. O. mparative studies es a , 667 Oxford Botanic Garden, plan = Sg Oxidases in the marine algae Oxycocci (Exobasidium), 629, 637, 647, 649 ig Padua Botanic Garden, 224 pallida (Thelephora [Merism al), 7 pallidum (Tremellodendron), 734, er. 76 Parkinson’s ‘Paradisi in sole Paradisus an photograph of title page os Beobasi idium), 635, 645, 649 s, 42 nomy of flowering pla “he, 109 Phytopathology: in the tropics, 307; The relations between scientific bot- any and, 275 Pisa Pokai age n, 226 Pisum sativum: effect of galactose on, 660; use of in distilled water experi- ments, 463, in exosmosis experiments, plumbea (Sebacina), 765, 770 een k (Sebacina), 63 olyporaceae, Comparative studies in the, Polyporus, 668; abietinus, 683, 684, 685, 696, 7 fraxinophilus, taille 606, 706, 709, 726; fumo- 840 sus, 688, 689, 691, 692, 695, 726; galactinus, 696, 705, 709, 728 ; Hal- ag kand irsutus, 688; Holm ien- sis, 33 ’ imber bis, 692, 693, 695; P Bas 696, 697, 698, 70l; Lind- heimeri, 689 eae 709, a? 712, 713; parg , 683, 687, 726; ae se a 2 ee ‘i Er 8; salignus, 693, ; spu- meus, eae. 701, 702, 703, 70% 707, 72 sp » 708; subcinereus, ; Tsugae, 709, 711, 712, 714 689; Proteinases in the ma. chil rag Recent investigations on, josey it 4 eblloidal properti es, 241 merismatoides, 740 pi ili et (Thelephora), 740, 741 Radulum deglubens, 747; Kmetü, 747; spinulosum, 747 Rhizoctonia, 403; Asparagi, ira Betae, ge 404, 408, cross inocu- iat a: “as ral ates of, 422, rien of, 409, host plants and general s symptoms of, 411, and en = 413, sen ps the perfect aeae, 445; Rapae, 445; Rubiae, 408 Solani, 424, 445, ee Er of, 443, dis tribution of, 429, mycelium and sclerotia e = ag of -oii induced by, 4 iolacea Rhizoctonia eae rum (P no De. and R. Solani Kühn Corkins vagum B. & C.), with notes on other species, 403 Rhododendri (Exobasidium), 637, 645, Rhodymenia palmata: carbohydrases 0 Aus catalases pe 826; lipases of, Ricci Brittonii, 50; ali 51 m (Stereum), 747 er (Stereum), 747 S scariosa (Sebacina), 762 scariosum (Corticium), 762 u. ern, Schw iù (Merisma), 740 Sohaoeiniteti (Thelephora), 734 Schweinitzii (T nen, roti 408 744, 770 734 ane "(Thelephora), 752 sebaceum (0 m), 752 Sebacina 731, te. adusta, 764, 770; ANNALS OF THE MISSOURI BOTANICAL GARDEN atrata, 765, 770; calcea, 759, 770; chlorascens, 756, 770; cl 763, 770; deglubens, 755; loides, 756, 770; incrustans, 752, 770; macrospora ; monticola, 761; plumbea, 765, 770; podlachica, 763; scariosa, 762; Shearii, 758, 770. m. (Corticium m), Se , Monograph of the North and species of the < LSPS nal American 3 mbrosioides, 593; am- pullaceus, 590, var. landeri v regonens 626 5 pee lk ie 588, var. ammo- m 590; californicus, var. laxior, 8; carolinian us, 606; chi hua- coahuilensis, 615, 588; hilus, ar. Kingii, 598; eremophilus, 592, 593, s, 592; erucifolius, 601; Ervendbe he “ll: Flettii, 619; gl abellus, 605, forma robustior, 608; Greggii, 608; Harfordii, 618; hypo- 612; imparipinnatus, en 602; Kingit, 598; lacinia- s, 601; leonensis, 615, 624; lobatus, lyratus, 605; ac- M f 609; ; Dougalii, 592; acDougalii, 594; Millefolium, 610; mohavensis, 580, 20; montereyana, 616; nebrodensis var. glabratus, 60 pembrinensis 5 597; pinnatisectus, 614; rupestris, 601; Sanguisorbae, 613; Sangui- sorbae, 612; sanguisor s, 604; saxosus, 626; Schweinitzianus, 606; sylvaticus, 585; she s, 587; tam- 611 mpicanus, nn i To nd 598; viscosus, 579; garis, 581; Watsoni, 598; a ‚616 Ben (Thelephora), 735 Setchell, W. A. The law of temper ee connected with the disina f the marine re ae, 287 iography of. See Ed- s Whitaker’s eee at twenty- filth ey bar Shearii (Sebacina), 758 770 simplex A en). 742, 76 th, E. A sh of bacterial diseases > plants, 377 Solanum tuberosum: comparison of dia- static rn of Ulva lactuca with that of, catalase action in, 826 on 696. spinulosa (Bichleriella), 747, 770 sp Libr peat (Radulum), 747 GENERAL INDEX Stereum, alliciens, 746; Leveillianum, 731, hoe rufum, 747; rufum, 747 Stypella, Symploci (Exobasiatum), 641, 648, 655 > Tabebuia heterophylla, 48; Temperature: e lucida, 48 exosmosis, w of, connecte with the giatetbation of the marine algae, tenue Seirus ae ne 768 fo orma, N 760; 738, 768; Helvelloides, 157; incrustans, 7 2; ee oides, en pallida, 734; pteruloides, be) five NG: ae Ag 734; sebacea, 752; serrata, 735 Thelephoraceae of North America, The, IV, 627; V, 731 Toxic solutions: effect of, on en, 49; Some relations of plants to dis- tilled water rn rtain dilate 459, 500, 502, 504, 5 Toxicity of galactose for certain of the higher plants, 659 Trametes Abietia 724; piceinus, 721, 122; Pini, 721, 722, var. Abietis, 722, 724 en 731, 733; aurantium, 7 42; candidum, 737, 768; Cladonia, 738 268: merismatoides, 740, 768; pallidum, 734, 768 ; Schweinitzii, 734; simplex 42, 768; ’ tenue, 740, 76 ) T 4 ci, 408 Ten nty-fifth anniversary ein un Pu ee u = oz 841 ge 1; delegates and Fai at- tending, 1; program for ee 696 U Ulva lactuca: Sr genial = poe catalases of, 826; comparison of t diastatie activity ‘of, with that of loaf tissue from Solanum tuberosu pi 801; lipases of, 809; proteinases of, 8 y Vaccinii (Exobasidium), 627, 639, 642, 6 K 49, Vaccinii myrtilli (Exobasidium), 647, 649 sre oo (Exobasidium), 640, 648, Vicia, faba, u use of, in distilled water experiments, 463; villosa, effect of galactose on, 660 distilled, and certain dilute Some relations of , 006; ect x sterilizing, on growth of 2 nts, 480 Westerdijk, Johanna: nic opathology the tropics, 307; Speech delivered a ae twenty- fifth anniversary ban- 0 Water: xi que Whitaker, Edwards: Address at the 9; Speech delivered at the ne "Eth anniversary banquet, X Xerocarpus farinellus, Xylophylla Epiphyllanthus, 42 5 = ie 0g ler} © BR EN (Pers) DC. a a Ri Astas Ki üha. Cortichin. aie eee al goa aih We io ee Shea abs M. Fr Ü tesked, to, ‘the Action of Shee comic ae tM: €. Merrill RN e of P: Nov Gs merican. Species of the ‘Genus fi ‚Senecior-Part Mr SER nt a Ei ee Oe Er M, Greenman ` Ray The Thelephoraceae of North Avior mw. INES RN, “EB. at Bure PUBLASHED UARTERLY BY. TER BOARD ol TRUSTEES € OFT missoni {Pr Enact gy a ST. LO ee MIBHOURE ua ur fy y 2 Rai Ý Ast ‚of neha a. Bievotric Diaen nation ‘of, cok ae the Roots ‘of Plants ERSTEN. | 07-572 Entered as secondée ‘matter at ths ER, om ice! hat: "st. Tau, ‘Missoni, ‘ander the if ATA 403-458 459.306