MISSOURI BOTANICAL GARDEN TWENTY-FIRST ANNUAL REPORT ST. LOUIS, MO. PUBLISHED BY THE BOARD OF TRUSTEES 1910 JOHN GREEN, M.D. BOARD OF TRUSTEES OF THE MISSOURI BOTANICAL GARDEN. President, EDWARDS WHITAKER,! Vice-President, DAVID S. H. SMITH 2 Epwarp C. Exior, Davin F. Houston, Chancellor of Washington University.* CHRISTOPHER W. JoHNson,! President of the Board of Public Schools GEORGE C, HircHcock. of St. Louis.* FREDERICK H. KREISMANN, LEONARD MATTHEWS. Mayor of the City of St. Louis. * SAUNDERS NORVELL.? WILLIAM TRELEASE, President of the Academy of Science of WILLIAM H. H. Pertus. St. Louis.* DANIEL S. TUTTLE, JOHN F, SHEPLEY. Bishop of the Diocese of Missouri.* A. D. CUNNINGHAM, Secretary. * Rx-Officio. 1 Elected President of the Board in November, 1910, to sueceed David F. Kaime, who had succeeded Rufus J. Lackland in that office early in the year, and occupied the office at the time of his death. 2 Elected Vice-President of the Board in November, 1910, to fill a vacancy caused by the election of Mr. Kaime to the presidency on the resignation of this office by Mr. Lackland very shortly before his own death. 3 Elected to membership in November, 1910, to fill a vacancy caused by the death of Rufus J. Lackland, one of the Trustees designated by Mr. Shaw. 4 Elected President of the School Board in October, 1910, to succeed Robert. Moore, who had held that office for one year. (2) ee ee 6 Oa -) | dae Y OPS 6 a 4 may BS ee ae oe | . 1 ee e Se age a ON ets, Es Ee ae ae era STAFF OF THE MISSOURI BOTANICAL GARDEN. Director, WILLIAM TRELEASE. GEORGE T. MOORE, Henry C. IRIsu, Plant Physiologist. Superintendent. HERMANN VON SCHRENK, Plant Pathologist. * JAMES GURNEY, Head Gardener.t REGINALD R. GATES, Research Assistant. JOHN BANNES, Cora J. HOGAN, Foreman. t Curator of Library. Moses CRAIG, OTTO BoGuLA, Curator of Herbarium. Foreman. HARRY W- ANDERSON, CHARLES O. CHAMBERS, SrockTon M. McMURRAN, CAROLINE RUMBOLD, JACOB SCHRAMM, Rufus J. Lackland Research Fellows. * Honorary. + Emeritus, (3) PREFACE. Under direction of the Board of Trustees, the twenty-first annual report of the Missouri Botanical Garden i is presented to the public. The twentieth report, of 1909, was not issued until Jan- uary 14, 1910, because of unavoidable delays in press and bindery; but separates of Dr. Widmann’s paper had been issued early in the season, those of Dr. Griffiths’ paper on March 22d, and those of the other scientific papers on December 31st. These reports are sent to scientific institutions and journals in exchange for publications and specimens desirable for the library, herbarium, laboratories or living collections of the Garden. So far as is possible, reprints of the botanical articles they contain are sent to botanists occupied with a study of the subjects to which they pertain. Any of the Garden publications not out of print may be purchased, at approximately the cost of publication, from Messrs. R. Friedlander & Sohn, Berlin, Germany; W. Wesley & Son, London, England; or the undersigned. WILLIAM TRELEASE. St. Louis, Mo., November 15, 1910. (4) CONTENTS. Pace 1. Reports ror THE YEAR 1909:— a. Report of the Officers of the Board................-006- b. Twenty-first Annual Report of the Director............. 11 2. Screntiric PAPERs: — a. The Algal flora of the Missouri Botanical Garden.— BR AC GIO ooo nose 0:54:3 00 bo ye mee Ae 25 b. Periodicity in Spirogyra.— Beg... Danforth «cues 3256-04 ees eh es 49 c. The fungous root tubercles of Ceanothus americanus, Elaeag- nus argentea, and Myrica cerifera.— By B. GG. AraBergee ie eG GE os tak MES d. Development and nutrition of the embryo, seed and carpel in the date, Phoenix dactylifera L.— By Francs 2. DIO sa haere ce nes ees e. Illustrated studies in the genus Opuntia-III.— By David. Griftthax. 50k Soren clk j. Abnormalities in Oenothera.— By I. Fe. Gated, oi isa oe ae ee eke kce cans Ba g. Botrytis as a Parasite upon Chrysanthemums and Poin- settias.— By Perley Spaulding i... 0k. Piven ves Cacteecekts h. Fungi of Clay Mines.— By Parley SUGUMGg isi hn sok a4 ee cen an sas she (5) 60 103 165 175 185 189 a 7 LIST OF ILLUSTRATIONS. Frontispiece: The library and laboratories. Vanilla flowers and fruit ‘ A Sauromatum . Garden-grown vanilla . On an “open Sunday” . The graduate lecture room . ; Distribution map for Garden algae Plates 1-5 Plates 6-14 . Plates 15-18 Plates 19-28 Plates 29-31 Plate 32 . (6) Facing ce p. “ec VANILLA FLOWERS anv FRUIT. THE LIBRARY anp LABORATORIES. SR Ly Fe PTE oe ee ea PL ee Om ee ees * Oe Sy et a i REPORTS FOR THE YEAR 1909. REPORT OF THE OFFICERS OF THE BOARD. SUBMITTED TO THE TRUSTEES JANUARY 12, 1910. To the Board of Trustees of the Missouri Botanical Garden: We submit for your consideration the financial results for the year ending December 31, 1909. Our receipts from rentals were $2,322.32 in excess of those for the year 1908, in addition to which our taxes were | $3,534.02 less than the year previous, that amount having been assumed by the lessee of one piece of property as part of the rental, so that the actual increase was $5,866.34. Our loss through vacancies during the year amounted to $2,620.68, and $282.50 was charged off as uncollectable,— about one-fifth of 1% on the total earnings. We have several warehouses vacant at this time, but we do not anticipate any diminution in our income during the ensuing year. No improvements to the income property have been made other than necessary repairs. We have disposed of 178 feet on Flora boulevard, for the sum of $13,650.00; and 210 feet on McRee avenue between Grand and Spring avenues, for $7,316.87. Demands for street improvements have not been so heavy as in recent years, but the following expenditures have been made and charged to real estate as a betterment: Lawrence street . . . . Roadway andsidewalks . . $ 2,885 79 Thurman avenue... . Sidewalks. ..... .- 882 64 Tower Grove avenue. . . Sidewalks. ..... . 957 76 Spring avenue. . . . . Sidewalks. ..... .- 1,033 96 Sewer between McRee and Lafayette avenues ... .- 7,440 27 $12,700 42 Several streets just completed pr under contract will call for additional expenditures during the year 1910, as follows: (7) g MISSOURI BOTANICAL GARDEN, some of which were anticipated in our last report, but com- pletion of the improvements was delayed: Shawavenue .... . 8,112frontfeet . .. . . $18,672 00 Klemm street .... . 2,000frontfeet... . . 10,000 00 Vandeventer avenue. . . 2,000frontfeet.... . 8,500 00 Peewee 65. 6 < -, E000 test hee... ve. 8,000 00 $40,172 00 The library and laboratory building at the Garden, men- tioned in our last report, has been completed at a total cost of $54,593.82 and the sum of $6,793.80 has been expended for metal book and herbarium cases, etc. No other expendi- tures have been made at the Garden other than for its main- tenance, at a total cost of $54,145.28. The following amounts have been credited to the Stock Account for the year: ee Oe ee ee aaa ee ee aa Herbarium . . a el Se ok See ee 5,543 85 Library building and ‘Siinisbings Pe rer Ge ae me $72,400 20 The annual bequests provided for in Mr. Shaw’s will, with the exception of the Trustees’ Banquet, have been met by the expenditure of $766.50, but the full amounts authorized, $2,100.00 have been charged against the year’s income, to be used at some future time. RECEIPTS. Receipts on accountof rentals . . . . $139,591 36 Interest and dividends . ...... 1,370 10 Garden handbook sales...) we es 166 00 Pence saiek oo eas Be 4 50 Beerret CON 68a ak ae | ee 19 20 Total income receipts . . .. . $141,151 16 Sales of real estate under decree . . . 20,966 87 Shaw School of Botany, rent ... . 3,900 00 PE PORRIVEDIO <5 hie we ae hue 4,360 00 29,226 87 Total receipts. . . $170, 378 03 Cash on hand December 31, 1908 . 4,070 34 $174,448 37 * on Ls Sy REET, ee Tee aie ey RE a OP ee ae ee Ow eee ee Ais ee Meteo, er ee cing rvs ONG PSC Chogd © BS a ul at A Ae eh REPORT OF THE OFFICERS OF THE BOARD. a DISBURSEMENTS. Garden Account, Labor pay-roll. . . . . $22,966 80 Students’ pay-roll .. . 2,152 32 Office assistance . . .. 1,256 25 $26,375 37 MO ae ks te ee 1,904 80 VORUOE ester. ence wits a oi 557 00 Repairs andsupplies. . ...-.+ . 2,693 56 Stableandimplements . .... . 364 54 Plants and seeds = of; sc. Se es 759 78 $32,655 05 Herbarium Account, MI 9 fg ns cg eae ks, 8 _ 1,080 00 NE ik ee ees at a ie ee 419 99 Coieent Chena ne os eee 1,833 92 3,333 91 Library Account, MalAMICR ery Per wh che + oo line Paina 2,220 57 Pui Oo es: oy eee 518 73 Current expenditures ...... 2,872 30 5,611 60 Office Account, eG oo ee te Re ae ee 5,796 51 ONS Sa ee ae” 278 03 Current expenditure. ..... . 470 30 6,539 84 Research and instruction, Salaries << oe. se neh eee ee 4,087 42 . Current expense’ .... °.° 42 te ee 172 18 Furnishing laboratories. . ... . 1,745 28 6,004 88 Total maintenance «2-62 ee $54,145 28 Garden Improvements, Library building, balance for comple- $i0n: bo. ee ak i et Ste 14,253 62 Renovating old library building . . . 457 33 Metal cases for library and herbarium 6,793 80 21,504 75 Total expended on the Garden . . $75,650 03 Publication Account, Twentieth annual report .... . 1,905 00 Seventh report, reprint. . . .. -» 380 00 2,285 00 Carried forward... . $77,935 03 10 MISSOURI BOTANICAL GARDEN. Brought forward $77,935 08 Property Account, State, school, city and sprinkling taxes $33,701 50 Streets, sidewalks and sewers ... 15,158 32 en ee ae 8,974 59 Ronin er kA ig a eS a 7,608 27 a ee eee ger 300 00 60,742 68 Bequests, Annual Flower Sermon. . .... 200 00 Annual Flower Show ...... 396 00 Annual Gardeners’ Banquet . .. . 170 50 766 50 Sundries, A SU es ck ie ak Oe 6,680 37 Legal and professional expenses . . 268 70 es sae i 2,341 25 9,290 32 Bonds, stocks and certificates 14,747 11 Shaw School of Botany, rents 8,936 75 Total Disbursements ee $167,418 39 Cash on hand December 31, 1909 . 7,029 98 Respectfully submitted, $174,448 37 R. J. LACKLAND, President. Attest: A. D. CUNNINGHAM, Secretary. A SAUROMATUM. my” at) ig A F eee See ee Fo ee ee ee ne ee ae ea a) as ea ps a : ea i aS ee ne ae aie wwe Dis i - heh ire OE ay ER a ee Se Ee he ee gt ye ey her * 2 ; : ma | s TWENTY-FIRST ANNUAL REPORT OF THE DIRECTOR. SUBMITTED TO THE TRUSTEES JANUARY 12, 1910. To the Board of Trustees of the Missouri Botanical Garden: The following report on the Missouri Botanical Garden and the School of Botany therewith connected is respectfully submitted, in compliance with your rules. GARDENING. Decorative gardening in 1909 followed essentially the same lines as for several years past, the most observable changes consisting in modification of the borders flanking the entrance, in the substitution of foliage for flowering plants in the sunken garden, and in the use of more than the usual number of succulents in attractive and skillful mosaic designs. In the early spring the sunken garden and its sur- roundings were devoted to tulips, as has been the case for several years past, and the display of these flowers was ex- tended by planting additional beds with the brilliant, late- blooming “parrot” varieties. Aside from the tulips, of which 28,800 plants representing 222 varieties were used, the bed- ding plants placed during the season numbered 33,000. Chrysanthemums were again grown in numbers, and 3,511 plants, of 520 varieties, were shown under canvas through the fortnight beginning with November 15th. Though the season had been less favorable than usual for the growth of these plants, and the specimen plants were not as large as in some earlier years, a very creditable display was made, and in variety of instructive forms this collection has not before been equaled. : About 2,500 Oenotheras, grown from pedigree-seed, added much to the attractiveness of the Garden in the early even- ing during the summer, because of their profuse production of large and fragrant flowers, though they were intended primarily for research use. Plant and seed accessions, apart from those propagated or collected at the Garden, number 326, and comprise 16,433 (11) 12 MISSOURI BOTANICAL GARDEN. plants or packets of seeds of which 9,960, corresponding to .31 accession entries, were purchased at a cost, including transportation charges, of $759.78; 6,473, representing 295 of the accessions, valued at $694.82, were presented; and 3,271, or 39 accessions, valued at $194.03, were collected by Garden employees—in addition to a large number of seeds for exchange purposes. Garden propagations amounted to 23,618 plants, valued at $1,653.26 ; and 13,250 seedlings, valued at $662.50, were also raised by the gardeners. The exchange seed list issued by the Garden last winter included 2,369 species or named varieties; and 8,159 packets of seeds, valued at $407.95, selected from the list have been issued to correspondents. Living plants to the number of 169, valued at $16.30, have been similarly distributed. In addition to these exchanges 207 duplicate plants, valued at $26.20, have been presented to schools and colleges for edu- cational use, and 1,133 surplus plants, removed from the grounds on the approach of winter or remaining after the spring planting, were given to schools and playgrounds. Chrysanthemum plants which were still usable, numbering 208, and many cut flowers from others, were distributed to hospitals and similar charities after the November exhibition. PLANTS CULTIVATED. During the year 822 species or varieties were added to the collection, and 520 lost or discarded, leaving a net recorded gain of 302, and bringing the total at the end of the year to 11,764, as compared with the 11,464 noted for 1908: These forms represent 1,777 genera, belonging to the follow- ing 197 families of plants: FAMILIES GENERA SPECIES FAMILIES GENERA SPECIES Acanthaceae........ 14 35 Anacardiaceae...... 8 32 Aceraceae.......... 2 36 Anonaceae. ........ 3 6 Agaricaceae........ 1 5 Apocynaceae....... 17 34 Alismaceae......... 3 10 Aponogetonaceae... 1 1 Aizoaceae.......... 4 16 Aquifoliaceae....... 2 10 Amarantaceae...... 14 45 Araceae............ 32 254 Amaryllidaceae. .... 25 366 Araliaceae ......... 9 36 1 Rept. Mo. Bot. Gard. 20:15. TWENTY-FIRST ANNUAL REPORT OF THE DIRECTOR. FAMILIES Aristolochiaceae.... Asclepiadaceae..... Balsaminaceae ..... Begoniaceae........ Berberidaceae...... Betulaceae......... Bignoniaceae....... Bixacede ? i, +3 Bombaceae......... Borraginaceae...... Bromeliaceae....... Brunelliaceae....... Burseraceae........ Butomaceae........ Buxac@ae:... . is +5.50% Cactaceae.i. iodsiy Calycanthaceae..... Campanulaceae..... Cannaceae.......... Capparidaceae...... Caprifoliaceae...... Caricaceae ......... Caryophyllaceae.... Casuarinaceae...... Celastraceae........ Chenopodiaceae. .... Chloranthaceae..... Cistaceae: 0%... s. Clethraceae.......:. Cneoraceae......... Cochlospermaceae .. Combretaceae...... Commelinaceae..... Compositae......... Convolvulaceae..... Coriariaceae........ Cornaceac.......... Corynocarpaceae... Crassulaceae ....... Cruciferae.......... Cucurbitaceae...... Cycadaceae......... Cyclanthaceae...... GENERA SPECIES - Dap al NR Aa aot nee rac ME Belk Di oh re woo) Sa Be a ie aa hie Tae = ete dk 16 114 8 51 33 4] 55 13 FAMILIES GENERA SPECIES Dilleniaceae’........ A 4 Dioscoreaceae...... 1 8 Dipsacaceae........ 4 29 Droseraceae........ 2 4 Ebenaceae.......... 3 i Elaeagnaceae....... 3 12 Empetraceae....... 1 1 Equisetaceae....... 1 3 Ericaceae .......... 13 52 Eriocaulaceae...... 1 1 Euphorbiaceae...... 23 158 Fagucemé.. 5.5.40 <3 3 50 Filicinese <.. <3... 44 187 Flacourtiaceae...... 4 5 Fouquieriaceae ..... 1 2 Gentianaceae....... 3 5 Geraniaceae........ 3 94 Gesneriaceae....... 15 108 Ginkgoaceae........ 1 1 Gnetaceae.......... 1 2 Gramineae......... 84 301 Guttiferae; . 6.05... 8 35 Haemodoraceae..... 2 2 Halorrhagidaceae . 2 2 Hamamelidaceae... 3 3 Hippocastanaceae.. 1 7 Hydrocharitaceae... 3 4 Hydrophyllaceae.... 5 11 Teacinaceae......... 1 1 PriGnGene co. ss 18 232 Juglandaceae....... 3 16 Juncaceae.....-:..). 3 22 Juncaginaceae...... 1 2 TREO’, ob ois wc 3% 43 173 Lardizabalaceae.... 2 2 Leuracege, .:..., 2. 10 12 Lecythidaceae...... 3 3 Leguminosae....... 117 = «537 ‘Leitneriaceae....... 1 1 Lemnaceae......... 1 1 Lentibulariaceae.... 1 1 PANBRONG, oA sc 3s « at 7192 THUOCORG S20 cs 5. ose 2 18 Loasaceae.......... 3 3 Loganiaceae........ 5 13 Loranthaceae....... a 1 14 MISSOURI BOTANICAL GARDEN. FAMILIES GENERA SPECIES Lycopodiaceae...... 1 1 Lythraceae......... 6 21 Magnoliaceae....... 5 23 Malpighiaceae...... 6 8 Malvaceae.......... 22 96 Marantaceae....... 4 24 Marchantiaceae..... 6 10 Marsileaceae....... 2 3 Martyniaceae....... 2 8 Melastomaceae..... 6 8 Meliaceae.......... 5 7 Melianthaceae...... 1 2 Menispermaceae.... 6 9 Moraceae........... 16 ~=— «64 Moringaceae........ 1 1 Musacese.....05.:::. 3 14 Myricaceae......... 1 4 Myrsinaceae........ 3 12 NEYTIACORE. 5155 55s 18 98 Nepenthaceae...... 1 82 Nolanaceae......... 1 4 Nyctaginaceae...... 3 10 Nymphaeaceae..... 6 61 Ochnaceae.......... 1 1 Canoes: kX: 10 135 Onagraceae ........ 9 61 Orchidaceae........ 99 640 Oxalidaceae........ 3 45 PUIG, 6 cee es 39 86 Pandanaceae....... 11 Papaveraceae...... 13 80 Passifloraceae...... 1 7 Pedaliaceae.. ...... if a Phytolaccaceae..... 5 12 PIRGCGRO «oid sis 16 101 Piperaceae......... 2 12 Pittosporaceae...... 3 19 Plantaginaceae..... | 23 Platanaceae........ 1 4 Plumbaginaceae.... 4 29 Polemoniaceae...... 3 35 Polygalaceae....... 1 3 Polygonaceae....... 12 63 Pontederiaceae..... 3 4 Portulacaceae...... 5 25 Potamogetonaceae.. 1 3 Primulaceae........ 7 57 FAMILIES GENERA SPECIES Proteaceae......... 3 6 Punicaceae......... 1 2 Ranunculaceae ..... 20 285 Resedaceae......... 3 13 Rhamnaceae........ 1} 87 Rosaceae........... 34 876 Rubiaceae.......... 31 66 Rutaceae........... 16 82 Salicaceae.......... 2 81 Salvadoraceae...... i 1 Salviniaceae........ 1 1 Santalaceae........ 2 2 Sapindaceae........ 14 20 Sapotaceae......... 7 12 Sarraceniaceae..... 1 5 Saururaceae........ 2 2 Saxifragaceae...... 16 96 Scrophulariaceae... 24 114 Selaginellaceae.... 1 7 Simarubaceae....... 1 1 Solanaceae.......+.. 22 262 Sparganiaceae...... 1 1 Staphyleaceae...... 1 4 Sterculiaceae....... 8 19 Styracaceae........ 2 7 Symplocaceae ...... 1 1 Taccaceae.......... i 3 Tamaricaceae....... 1 3 Taxaceae. . iis sects 2 8 TREACORC. . isi retes 5 14 Theophrastaceae... 1 1 Thymelaeaceae..... 3 3 Tiliaceae...... tes 6 20 Tropaeolaceae...... 1 6 Typhaceae......... 1 4 Ci macesew enw 140% 4 12 Umbelliferae....... 38 111 Urticaceae ....... ee Et 25 Valerianaceae ...... 3 5 Verbenaceae........ 12 80. Violacease.. . 25. 6s66: 1 82 Vitacene.. i cescee ws 8 66 Zingiberaceae....... 9 23 Zygophyllaceae..... 2 2 Totals e: < ces 197 2 1,777... 11, 764 GARDEN-GROWN VANILLA. TWENTY-FIRST ANNUAL REPORT OF THE DIRECTOR. 15 DIAGRAMS A AND B. ee Bass +1+-25—F- rt nat | poor oe 100--} pa uh? ~. +-50—F= og ~. -— 50-47 LMEF at a ay ae eae apne woe Peel ~~] 25°F — ae = ee = = eed Pn es A \ E VANS Lp r—MAR= ADR APA? JUNE JUL —AUG? -SEPT —OCT= NOV DEC? = = | A. — TEMPERATURE AND HUMIDITY, 1909. mE, care Zz Il IN; KS x we | ra ate end We ee vl oo ~ ae i i » O-4 = Tw ; ut Zz : = VAN res MAR APR MAY. J JULY. , 28 oe. | ee by ¢ x eee + ee pio ace 8 eo ee Pare Sy em ee epee re eae el 16 MISSOURI BOTANICAL GARDEN. THE WEATHER. Though January, February and November, and to a less extent August, were this year markedly warmer, and Decem- ber decidedly colder, than usual, the mean temperature for the season (Diagram A) closely follows the average, except for the fall and winter months. The precipitation for the year (Diagram B) has been rather abundant (47.5 in.) ; but the rainfall in June was considerably below the average and less than an inch of rain fell in August, which, with the rather high temperature, increased the difficulties of garden- ing and especially affected the blue-grass lawns. The dia- grams are derived from the local Weather Bureau records. VISITORS. , A gratifying number of visitors is to be reported. As may be seen from the accompanying diagram, they closely agreed DIAGRAM C, ‘ 6000) ; eh pT Liye ty ake Bee. - bo tf Ft FG : i = Beck Ba Sy 7 +909— e aN ape SUNDAY VISITORS, 1890 To 1909. with the average for previous records in January, February, March and May, but considerably exceeded this average in the other months except December. In November, however, they fell far below the average for the past few years, though the fact that over 16,000 were recorded justifies the special chrysanthemum display by which the larger part of these were attracted—the courtesy of Captain Robert McCulloch having again made possible the lighting of the tent so that it might be opened in the evening. oe ON AN “OPEN SUNDAY.” TWENTY-FIRST ANNUAL REPORT OF THE DIRECTOR. 17 The number of visitors for 1909 is 120,748, of whom 18,- 379 were recorded on the first Sunday afternoon in June and 17,572 on the first Sunday afternoon in September. The relation of these 35,951 Sunday visitors to those of other years, and to the total for this year (29.7%), is indicated in Diagram C. The seasonal distribution of the 84,797 week- day visitors, and its comparison with the average for earlier years, are graphically presented in Diagram D. The total recorded for 1909 has been exceeded (apart from the Exposi- ae at ie i SEL ee Fee eae eee ee, | DIAGRAM D. ae ! L /\\ 120000 ! \ ‘Be -1-5000- ! / \ [/\\\ call Y_A\\ ++-0000 A a Fay ae Ds ee as \ \ va be ere | r“ VERE \ \ pH OO0- - via Via \ “7 A\ tT [AS —FEB—WAR__APR___ MAY-JUNE JULY AUG SEPT OCT NOV. DEC, WEEK-DAY VISITORS, 1909. tion year) only in 1907, when the unusually large number of persons attracted by the chrysanthemums brought the number for the year to 135,497. THE HERBARIUM. Rather few large purchases of specimens have been made, but the herbarium has been enlarged by the customary cur- rent collections offered for sale. Advantage has been taken of the lessened purchases to have the unincorporated accumu- lations of recent years mounted; and relatively little un- mounted material now remains. Eight hundred duplicate 2 leet etek ha eo 18 MISSOURI BOTANICAL GARDEN. specimens, valued at $40.00, were distributed to correspond- ents during the year. The additions to the mounted collection in 1909 number 36,959 sheets of specimens, of which 6,344, valued (un- mounted) at $317.20, were presented; 2,332, valued at $116.60, were collected by employees; and 28,283 were pur- chased, the Secretary’s books showing an expenditure of $1,508.69 for specimens and material during the year. The herbarium, so far as now mounted, consists of : The Engelmann Herbarium (all groups) . . . . 97,859 specimens. The General Herbarium: — Higher plants. The J. J. Bernhardi Herbarium . 62,507 The Henry Eggert Herbarium? . 26,703 The J. H. Redfield Herbarium . 16,447 The Sturtevant and Smith Her- NS Geet oat ae ee ae ee The Gustav Jermy Herbarium ._ 5,118 The A. W. Chapman Herbarium? 3,536 The Julien Reverchon Herbarium? 17,210 The Nicholas Riehl] Herbarium . 3,359 Other specimens. . . . . . 862,099 504,425 ne Thallophytes. The J. J. Bernhardi Herbarium? 3,191 The Gustav Jermy Herbarium ._ 1,659 The S. M. Tracy Herbarium? . 4,334 The Wm. Trelease Herbarium . 11,000 Other specimens . . . . . . 33,857 53,541 as Making atotalof ... . 655, 825 dd VWEINt he.) ae $98,373. 75 Supplementing the herbarium, and the shelved or incor- porated exsiccatae, which are here counted as a part of it,* the Garden possesses specimens of economic plant-products, woods, seeds, etc., valued at $280.00, and 1,851 preparations for microscopic study, valued at $410.00, which have not been added to since their enumeration in my last report.° 2 So far as yet incorporated. 3 This valuation at the rate of $15.00 per hundred mounted sheets. 4 Rept. Mo. Bot. Gard. 16:21. * See Rept. Mo. Bot. Gard. 20:26. TWENTY-FIRST ANNUAL REPORT OF THE DIRECTOR. 19 The addition to the herbarium and library building reported last year has made it possible for the herbarium to be united under one roof again; and one floor of the new building has been equipped with steel cases, as nearly dust and insect tight as such cases can be made, the unit size adopted being a ground plan of 40% 5514 inches and a height of 90 inches. Advantage was taken of the neces- sary handling of specimens to rearrange them in the now accepted phylogenetic sequence. “THE LIBRARY. The increase in the library during 1909 has been closely comparable with that for a number of years preceding; the more essential current publications have been procured, in addition to a number of works rounding out the library in the fields of bacteriology and phycology, though unfortu- nately means have not been found to add more than a very few of the earlier books which now and then appear in the market and are becoming more difficult of purchase and more costly with the passage of each year. Additions number 883 books, valued at $1,886.45, 1,631 pamphlets, valued at $285.15, and 6 manuscripts, valued at $114.00, presented or received in exchange for Garden pub- lications; and 485 books and 219 pamphlets bought, the expenditure for purchases and binding amounting — to $2,727.80. The card index has been increased by the addition of 45,533 cards, of which 35,301 were written by employees and the remainder purchased. As for several years past, in addition to indexing the plant illustrations of the library, the attendants have given special attention to references to germination and seedlings. The serial publications now received number 1,464, of which 107 are bought and 1,357, issued by 970 institutions or publishers, are received in exchange for the Reports of the Garden. This number is twelve more than that reported last year. It is to be observed that to this exchange use of the Garden publications a large part of the yearly accessions to the library is directly attributable. 20 MISSOURI BOTANICAL GARDEN. As now constituted, the library contains: i teal te i eee SS eee: 1 61,654, valued at . .- $96, 182.79 Manuscripts: Engelmann, Notes and Sketches ... . . . 60, valued at . . $600.00 Engelmann, Thesis ... 1 Ae ee Roetter, Sketches . ... 1 < ™ fy ree ae re | 7 4 ae Sturtevant, Index Rerum . 11 66 At eo Price, Bird and Insect Sketches en + eae to 250.00 Leconte, Insect Sketches 8 So ee Bay, Bibliotheca Riviniana . 1 CO Va ee Lindheimer, Miscellaneous 1 ocaaial gl 10.00 Theses by Garden Pupils . 10 ‘“ ‘* 10.00 Theses by Graduate Students 8 ‘ hg 8.00 Se ee ery 1,538.00 ee. eer Sk oon... TO Ie eee 7,198.77 Total value of library Prk. woe Though it has not been possible to provide for the entire library under one roof, as was done for the herbarium, the addition to the library building has made it possible to bring the most-used classes of books once more together, those left in the old Museum building, though less quickly acces- sible, being safely housed. ‘Two floors of the new building have been equipped with steel cases corresponding in style, length, and height with those used in the herbarium, so that books referring to a given group of plants are appropriately shelved near the specimens; both the herbarium and library are capable of rearrangements and replacements at will, as they increase and are fitted into new quarters from time to time. To correspond with the changed sequence of families in the herbarium, the portion of the library that deals directly with taxonomy has been readjusted so as to follow the’same classification. THE GRADUATE LECTURE ROOM. ey omeey ’ ‘ie a de oy ee Me GARY Shere, ee ee Pek te ee ee Ree ee oe TWENTY-FIRST ANNUAL REPORT OF THE DIRECTOR. 2] RESEARCH AND THE USE OF FACILITIES. The long-established policy of making the facilities of the garden available to investigators has been taken advantage of by a number of visiting botanists. During the year 19,771 herbarium specimens have also been loaned to 26 investi- gators who were unable to visit Saint Louis or who could not complete their use of material at the Garden; and 360 books or pamphlets have been loaned similarly to 73 persons. As in previous years, such part of the time of capable employees as could be given to original study has been so used, and a number of papers have been published embody- ing its results. THE HENRY SHAW SCHOOL OF BOTANY. One of the most cherished purposes of the founder of the Garden was the provision of means and appliances for in- struction in botany—referred to by him not merely as a specific science but “in its application to horticulture, arbori- culture, medicine and the arts,”* and with special mention of “vegetable physiology, the diseases of plants, the study of the forms of vegetable life and of animal life injurious to vegetation, experimental investigations in horticulture, arboriculture, etc.” 7 Inaugurated before Mr. Shaw’s death by the endowment of a School of Botany as a special depart- ment of Washington University® with provision for the closest affiliation between this department and the Garden® and with authorization for the Trustees of the latter “to allot, if they think it expedient, from time to time, any of the income not needed for the development and maintenance of the said Garden to the augmentation to the means and appliances of instruction,”?° the realization of this purpose has necessarily rested, until the present year, upon the single professorship established in 1885. Though still burdened by the necessary cost of holding the large tracts of unimproved and unproductive real estate ®° Rept. Mo. Bot. Gard. 1:86. TL. c. 1:87. 8 1. c. 1:56-59, ® 1. c. 1: 36-87. 10 [. c. 1:87. 29, MISSOURI BOTANICAL GARDEN. in which a large part of the endowment of the Garden con- sists, the Trustees, after careful consideration, have decided that the time has now arrived for availing themselves of the authorization given for augmenting means and appliances for instruction, and as a first step toward this they have built and equipped laboratories suited to graduate work in certain lines of botany, and have established the post of plant physi- ologist at the Garden. To this post, Dr. George T. Moore has been called by the Trustees, and he has been elected by the Directors of Washington University to a newly estab- lished professorship of plant physiology and applied botany in the School of Botany. In connection with this professor- ship, two research fellowships in botany have been established which are this year held by Mr. E. G. Arzberger and Miss Ada Hayden. The recently published Catalogue of the University enum- erates the following regular courses now offered by the School of Botany in addition to special work suited to the individual needs of advanced students who, when possible, are given such work in preference to less elastic courses: CouRSES OF INSTRUCTION. — A. Primarily for undergraduates: 1, 2. General Botany.—2a, Field Ecology. —3, 4. Plant Physiology. — 5. Bacteriological Methods. —6. Histological Methods. — B. For wn- dergraduates and graduates: 7. Bacteriology. —9. Morphology and Taxonomy of Fungi. — 10. Morphology and Taxonomy of Algae. — 11. Morphology and Taxonomy of Bryophytes. — 12. Morphology and Taxonomy of Pteridophytes. —13. Morphology and Taxonomy of Spermatophytes. —16. Plant Ecology. —18. Advanced Physiology. — 19. Water and Sewage Purification. RESEARCH UNDER SUPERVISION. — Taxonomy. — Applied Mycol- ogy. — Physiology. : INDEPENDENT RESEARCH, in any field for which the necessary equipment is available, is open to persons qualified by training and experience to carry it on without instruction. To such advanced in- vestigators the utmost freedom is allowed, and all the facilities of the School of Botany and the Botanical Garden are placed at their dis- posal free of expense, unless their work is done in candidacy for a degree, in which case the prescribed fees are chargeable. The electives offered to undergraduates and jointly to undergraduate and graduate students, are intended in a gen- eral way to indicate the breadth of botanical preparation ex- 0 Sat oe ee ~h é eee ee oe oe ee a SP ea a hw! or oon a. a ak 2 i" P h TiteIN RRME ERit Pa al ae pa cy ts gh oe Go “a . TWENTY-FIRST ANNUAL REPORT OF THE DIRECTOR. 23 pected of successful candidates for the Master’s degree in bot- any; while the research courses indicate the field in which work leading to the Doctor’s degree may now be undertaken. Undergraduate work, in the year just closed, has differed little from that of several seasons past. From the opening of the current college year, Mr. C. D. Learn, as Teaching Fellow in Botany, has continued the assistant’s work per- formed last year by Mr. Nehrling. The undergraduate en- rollment for the first term of 1909-10 was: Biology, eight, Botany 1, twenty; Botany 3, seven; Botany 5, five—a total of forty students, of whom the eight first noted give equal time to botany and zoology and the others take one full botanical course each. On the completion of the new building at the Garden, one three-story pavilion, about fifty feet square, was reserved for use in connection with the graduate needs of the School of Botany and independent research, and an excellent equip- ment has been provided for work in bacteriology and other branches of mycology, phycology, and certain lines of plant physiology. In these directions research work, under the immediate direction of Professor Moore, is being carried out by four candidates for advanced degrees; while another ap- plicant for the Master’s degree is giving a part of his time to less advanced graduate study. GARDEN PUPILS. In March, Mr. Arno H. Nehrling and Mr. Henry Ochs, who had completed the prescribed course of study and passed an examination satisfactory to the Garden Commit- tee, were given the customary certificates; and in June, Miss Herta A. Toeppen, who had also completed the required course and passed the requisite examination, was granted a similar certificate. On the results of competitive examina- tion, duly announced, the scholarships released by Mr. Nehr- ling and Mr. Ochs were awarded to Mr. Clark Craig, of Rush Lake, Wisconsin, and Mr. Carl Haltenhoff, of Gotha, Florida. In June Mr. Raymond B. Wilcox, for family reasons, gave up the scholarship which he had held since 1907, and it was given to Mr. Homer E. Reed, of Louisiana, Missouri, ” 24 MISSOURI BOTANICAL GARDEN. who had ranked highest among the unsuccessful competitors for the two scholarships awarded in the spring. At the end of December, the scholarship held by Mr. Jesse B. Tuggle was relinquished, and is still free. No changes have been made during the year in the teach- ing staff or the prescribed course of study; but several of the Garden pupils have found time for additional work, of col- lege grade, as special students in Washington University. THE GARDEN STAFF. Aside from a few changes among library assistants en- gaged temporarily in indexing and cataloguing, no changes are to be reported further than the addition of Professor Moore, already referred to. As in previous years, I can not too warmly commend the cheerful and efficient assistance that has been rendered by my associates, in caring for the important collections to which the Garden owes its chief value as an establishment for research, and I am especially indebted to Miss Hogan and Miss Brown for important contributions to the last Garden Report. SPECIAL TESTAMENTARY PROVISIONS. Three of the annual events provided for in the will of Mr. Shaw have taken place in 1909. The flower sermon was preached in Christ Church Ca- thedral, St. Louis, on the morning of May twenty-third, by Rt. Reverend F. K. Brooke, Bishop of Oklahoma. The sum set apart for floral premiums was once more en- trusted to the.St. Louis Horticultural Society, for use in con- nection with an exhibition held between November ninth and twelfth, but no award was made of the Shaw Medal. The twentieth Gardeners’ banquet, in the form of an open- air collation, was given in the grounds of the Director’s resi- dence at the Garden on July first; some 150 persons being present, of whom many were in attendance at the summer meeting of the State Board of Horticulture. Very respectfully, WILLIAM TRELEASE, Director. ALGAL FLORA OF THE MISSOURI BOTANICAL GARDEN. i} AU GARDE The 1 Doane 1 550U su d 72% ; ay: fi. {. 7 HABITATS. (E 6-7). (E 6-7). (D 4). 1) 3). (E 8). ( Ms (B ties. i Vegetable Garden. Mint Bed. Nelumbium Pools M. Nymphaea Pool. Earth. Local Fountain Pond Beds. Arboretum Stream. L. N. (A-D 3-9). (B 9). (D 1-2). (C-D 1). (B 1-3). (E 7-8). (D 1). Waterfall Stream. Lagoon. Arboretum Stream. J. Arboretum Pond. Crescent Pool K. Fountain Pond. Typha Pool. F. G. H. I Waterfalls all numbered westwards, from F. to H. SCIENTIFIC PAPERS. THE ALGAL FLORA OF THE MISSOURI BOTANICAL GARDEN.! BY ADA HAYDEN. INTRODUCTION. While holding a research fellowship in the Henry Shaw School of Botany at the Missouri Botanical Garden an oppor- tunity was afforded to investigate the algae found in the pools, ponds and streams of that place. The work was done under the direction of Professor G. T. Moore and the deter- mination of the species in the systematic list, based on stand- ard taxonomic works and exiccatae, has been corroborated by Professor Moore. The photographs of the habitats were made by Mr. Emil G. Arzberger and the species marked by an asterisk are taken from a manuscript list prepared by Dr. Henri Hus some years ago. Any habitat study of plants involves ecological principles. In the present consideration of the garden algae none of the main habitat factors 7. e., light, heat, temperature and water vary from what may be considered typical for this region. While the water is artificially conducted to ponds and pools its source is the Mississippi and though passed through the purification process its chemical value as regards mineral content (U.S. Bull. Bur. Pl. Ind. No. 64) is not essentially different from that to be found in any chance location. It is seldom, however, that such a variety of types are found in such a limited area. This may be accounted for by the fact 1 Abstracted from athesis presented to the Faculty of Washington University, in candidacy for the degree of Master of Science, June, 1910. (25) 26 MISSOURI BOTANICAL GARDEN. that the artificial arrangement of pools, ponds and streams brings within a small space a varied number of natural con- ditions such as small bodies of quiet, shallow, constantly re- newed water with or without outlet, swiftly and slowly run- ning streams, ete. So far as the sources of a fresh-water algal flora are con- cerned, there are present the possibilities of dissemination by (1) currents of air bearing spores through short distances; (2) transportation by birds, animals and insects. It has been observed in a study of the dissemination of Lemna, by Mr. C. H. Thompson, that Belostoma americanum, commonly found flying about electric light globes on the street, carried Lemna attached to its body. This insect stays in the water during the day and flies about at night. If this is true of Lemna it might easily be true of algae which grow in the same habitats. (3) Introduced plants in ponds and pools, many of which are of tropical origin, thus having been de- rived from widely differing habitats. There is but slight pos- sibility of dissemination through the agency of water cur: rents directly except within the garden itself, for only two small streams enter it, and the boundaries due to street or grading are higher than the surrounding territory. Of the two small streams, which enter, the one from the south drains | the grassy, sparsely wooded portion of Tower Grove Park and during the period under observation has been quite free from algal growth large enough to be noted without microscopic inspection. The stream which comes from an ordinary city block on the west contains few or no algae and is often dry. Tt is evident from the chart that the main bodies of water within the garden are connected and it is to be noted that the city water at its entrance is free from all vegetation. Having considered these deviations from the probable typical environmental characters and those characters bear- ing on the source of the algal flora, it is evident that this large number of forms present, offers a good opportunity for consideration of their relative grouping and adaptation to habitat conditions which in range and variety express in miniature the probabilities of a much larger area. Sue ROLY Ree tere oe Oe poh eee ee Ee en See ee a ee ee Ui es, Yan eee a Bea >. : i ate Rage e ; * < : \ ea ALGAL FLORA OF THE MISSOURI BOTANICAL GARDEN. 27 HABITATS. The habitats observed in this study are indicated by letters on the phyto-geographic map. For purposes of discussion they may be resolved into the following types: I. Moist earth. II. Water. Running. a. Waterfalls. b. Rocky stream beds, e. g., Arboretum stream. c. Smooth stream beds, e. g., Arboretum stream in part. - Waterfall stream in part. Quiet. a. Ponds or pools with no outlet, e. g., Crescent pool. Arboretum pool and Nelumbium pools. b. Ponds or pools with outlet, e. g., Fountain pond lagoon. Having viewed the general problem of habitat characters it is in order to consider the particular conditions in the loca- tions studied which for convenience may be designated as Fountain pond, Waterfall stream, Lagoon, Arboretum stream, Arboretum pond, Nelumbium pools, Nymphaea pool, Crescent pool. Their location in the garden may be seen in the phyto-geographic map. The first four in the series are connected in the order named and as may be seen lie respect- ively in the lowest part of the tract between rolling elevations which drain into them. The surrounding territory is grassy with the exception of the Arboretum stream where the herb- age is sparse due to the shade and more or less of loose soil. The fact that these bodies of water are connected makes quite probable the dissemination to succeeding connected bodies of water of anything which grows in the first of the series. Some of the more obvious habitat characters in con- formation to which plants arrange themselves in groups may be mentioned. The amount of moisture in the earth depends on precipitation, air currents and heat, of which the two latter affect evaporation from the earth; also level of water table and type of soil whose greater or less porosity affects the water holding and retaining power. ee Malls tae St a %, . SS 2 re ie “e 28 MISSOURI BOTANICAL GARDEN. In water habitats are encountered such factors as depth, motion, rapidity of running water; attachment surfaces, i. ¢., vertical faces of rocks subject to direct force of water; con- cave or protected surfaces under waterfalls where the atmos- phere may be saturated and surfaces moist though plants are not submerged; soft, smooth, muddy floors of streams from which thalli may be easily swept by variation of currents de- pending on variation in volume of water which fluctuates periodically with rains; rocky, brick or cinder covered beds; the better aeration of water rapidly running over rocky beds; turbidity of water; presence or absence of sewage or decaying vegetation; wave beaten rock surfaces subject to variable water level; stagnant pools with no outlet; and little aeration with tendency to accumulate vegetation. STABILITY OF HABITAT CHARACTERS, These particular habitat characters are naturally affected by the more general meteorological conditions and granting that the physical characters of a habitat are fairly constant, the percentage of plants that might be able to live under those physical conditions at that time may become necessarily di- minished in that some may become so prolific as to monopo- lize so large a proportion of light, moisture, space or nutri- ment, 2. ¢., life necessities, as to crowd weaker ones out. Thesa stronger dominant plants, however, may serve as a protec- tion to another type which might not have found the original conditions favorable for existence, hence the equilibrium is subject to continuous variation. It is evident that physio- logical, physical and biotic factors in the ecological problem are closely related. While physiological factors are of vital importance in the study of associations it will hardly be pos- sible to give them specific consideration though an attempt will be made to note as far as possible the influence of physi- cal and biotic factors. ANALYsIS or Haprrars, The Fountain pond is at an average 100 ft. in diam. and 4-5 ft. deep in the center, gradually sloping to the edge. The od ey ee, Re 2 ele Pay tie., SP ee ee ee a. Tee ee Ne ALGAL FLORA OF THE MISSOURI BOTANICAL GARDEN. 29 water (city water derived from the Mississippi) enters at two points, the fountain in the center of the pond and a pipe at the south side. The level is fairly constant except in rainy weather, when it is variable. The water as it enters the pond maintains a fairly constant temperature, varying slowly through the year, the temperature being practically the same as that of the earth at four feet. It is covered in the winter with a coat of ice for about two months. The algae here group themselves into free swimming, those attached to water plants such as Nymphaea and Potamogeton (sta- tionary) or attached to Azolla (floating). The free floating groups are swept across the surface of the pond by winds and in overflows after rains are swept down the stream. Of the algae listed for this habitat, Bulbochaete, the Spiro- gyras, Mougeotia, Palmodictyon, Cladophora, and Oedogo- nium were at some stage attached. The others were floating or free swimming. Pleodorina was always found in warm, shallow water near the edge of the pond in a tangle of fila- mentous algae and small water plants as was generally true of the Volvocaceae in this pond. This piece of water con- tains several Spirogyras, only three of which have been de- termined, since conjugation had not taken place. All the filamentous forms are more or less closely associated in masses, Spirogyra and Oedogonium being predominant. Typha angustifolia pool. This pool at the edge of the Fountain pond is about 4X6 ft. in size and seldom contains more than five inches of water, usually about two inches, and is often muddy without standing water. It contains a thick growth of Typha angustifolia. Observations were not taken here until March when an interesting association was found. Mougeotia scalaris, Spirogyra tenuissima and Gonatonema sp. formed a closely interwoven mass in the spaces of which were several species of Cosmarium, Closterium and one of: Micrasterias. Waterfall stream. This stream connecting the Fountain pond and Lagoon contains fourteen waterfalls, all of which have limestone surfaces with the exception of X and XII, whose surfaces are cement covered. 30 MISSOURI BOTANICAL GARDEN. Cladophora sp., Pleurococcus vulgaris, and Stigeoclonium tenue are the only forms found on the rocks themselves. Pleurococcus grew on all the rocks presenting a saturated surface, except the cement falls and Cladophora grew below the water-level of the stream at the base af the waterfalls X, XI, XII, and XIII. Some of this Cladophora was the same species as that which grew on the rocks of the Lagoon. Stig- eoclonium was present on all the falls in the parts where the water ran rapidly or struck breaking into spray; especially where it fell perpendicularly on a horizontal surface, these being the points of greatest aeration. A few small plants sparsely scattered were found on the perpendicular surfaces of the two cement waterfalls X and XII over which the wa- ter ran slowly in an unbroken stream. Whether the unde- sirability indicated by the absence of vegetation here was due to the cement or to the flow of the water is difficult to say. So far as water is concerned conditions were similar to those where Cladophora and Pleurococcus grew on the other falls but none appeared here. Stigeoclonium tenue grew abund- antly on waterfall I all winter, but by the 16th of May was quite degenerate, the fall becoming covered with small leeches. S. tenwe was noted on all of the falls but X, whose face was shaded by cat-tails and IX, over which the water ran sluggishly with little fall. All the other forms found in the intervals of the stream between the falls have been noted in the Fountain pond with the exception of Hydrodictyon, which probably originated there. It grew in a tangle of fila- mentous forms among the stems of cat-tails. The Lagoon is a long, irregular body of water 180540 ft. and approximately 12 ft. max. depth. It is deepest at the west side and slopes gradually toward the east. This larger body of water yields slowly to temperature changes, freezing later than the upper pond. On cold mornings when the small pond had a fringe or thin coating of ice the lagoon had none. The greater size makes the force of the wind of more import- ance here as waves starting from one side on reaching the other beat the floating fronds against the earth. Oedogonium is abundant on the shallow east side of the lagoon. The aa Me may eae as oe» ss ra ie es Bie oa aie oo ok sod ve vet UN Ne Sb ee hi Cg a te ei Rie ALGAL FLORA OF THE MISSOURI BOTANICAL GARDEN. 31 vegetative state which lived through the winter in the lagoon compared with that of the upper pond and stream was found to be badly beaten by the wave motion. The pond on the south and west side becomes abruptly deep. The prevailing. winds during the growing season are in a southerly. direc- tion. These combined factors seem to make the shallow east and north side a more valuable field for algal forms which are practically absent from the west side except at the northwest rocky dam which separates the lagoon from the overflow stream. Here on the rocks is a group which must adapt itself to the wave motion and rise and fall of the water level which is variable within a range of 6-8 inches. The lagoon, probably due to the fact that it is a larger body of water with more variability of habitat shows less connection between groups. On the deep south and west shores only a few strands of Oedogoniwm were noted. On the shallow east side, abundant Oedogonium, apparently the same species as that in the Fountain pond and some Spiro- gyra were present. On the mud at the north end, in March for a brief period, a thick film of Chlamydomonas was seen. The rock of the dam at the west was the most favorable situ- ation. There a slender Cladophora sp. grew all the year. Spirogyra, Phormidium, Oscillatorias, Desmids, Diatoms and, Tetraspora were prominent during parts of the year. The most conspicuous association and the only well-marked one was that of Anabaena Flos-aquae, Clathrocystis aeruginosa var. major, and Oscillatoria Agardhii floating on the water often mixed with quantities of soot when the wind blew the smoke in that direction. (See Anabaena description.) Conju- gatae and Cyanophyceae are predominant. Arboretum stream. The water from the lagoon after it enters the pasture to the northwest contains only some frag- ments of Spirogyra or Oedogonium until it enters the Arbo- retum. Near the center of the Arboretum the incline be- comes more abrupt than above, the bed is here narrower and rough with bricks and stones. Here Cladophora flourishes. Toward the east side of the Arboretum the bed widens and the slope is slight so that the current is sluggish. In this i: 32 MISSOURI BOTANICAL GARDEN. region a sewage pipe enters. Here Oscillatoria is dominant. The floor of this stream is smooth and muddy. The surface of the water is never frozen in winter. It varies in volume with precipitation and becomes very turbid at these times. Cladophora canalicularis during the winter was covered with diatoms which as the spring advanced diminished until by the first of May they had disappeared. The plants looked ragged and unhealthy at this time, but soon began to branch and regain their bright green color. On the stones near Cladophora, Stigeoclonium glomerata suddenly appeared in the spring and within a month disappeared. In the east end of the stream where the water was sluggish the Blue Greens were the principal forms. On the mud at the edge of the water in this region, a small form of Vaucheria grew,—a different species from the one in the Arboretum pond. Here the dominant forms were present during the whole period of study. Arboretum pond. This is a small crescent shaped body of water from a few inches to two feet deep. It has no outlet so that the water is stagnant. It contains much decaying as well as living vegetation, consisting of Typha, Juncus, Iris, Acorus and Nymphaea, which makes conditions favorable for a rich growth in algal forms. This pond freezes earlier and remains frozen longer than the Fountain pond. This body of water has more characteristic forms than any other. In the west wing planted with Iris is about % in. of water. Here Vaucheria forms a thick mat with several Clos- teriums (see list) Oscillatorias and a Lyngbya scattered among its filaments. In the center and east wing in deeper water the other algae were attached to the stems of Acorus or Nymphaea or floating in a tangle of decaying vegetation. Oedogonium was very rare here, though common in the habitats which had Cladophora. Tetraspora was first attached then floating over the whole water surface. Scenedesmaceae were practically absent. Chaetophoraceae are predominant. This is the only location in which Ulothricaceae were found. Nelumbium pools. These are two cement basins with no outlet. The water is 2-3 in. deep. They are covered in win- Ba A ae ee og See ee eee RCS eso ak. ee Ge Set te Te OE he ee eae Enel ge Se ea ey ALGAL FLORA OF THE MISSOURI BOTANICAL GARDEN. 33 ter by mulching, freeze early and remain frozen late. The Nelumbium speciosum which grows in them is a tropical plant native to Eur-Asia. There are two well marked seasonal groups here. None of the fall (Oct.-Dec. incl.) were observed in the spring (Mar.- May incl.) and vice versa with the exception of the diatoms which have been found in all the stations in greater or less, abundance. The four species which were particularly char- acteristic in the fall are Anabaena Flos-aquae, Nostoc, Spiro- gyra setiformis and Pithophora Mooreana (see syst. list) a new species. These genera by history are well adapted to tropical life. Oedogonium though represented in some de- gree in most of the stations was rare here. The spring group is free swimming, free floating including Volvocaceae, Flag- ellatae, Scenedesmaceae,—Chlamydomonas gloeocystiformis, Euglena proxima and Gonium pectorale being very abund- ant for a short time. This is the only location in the garden in which Spirogyra setiformis and Pithophora Mooreana were found. This with the fact that the Nelumbium speciosum is a tropical plant is of interest here. The genera Scenedes- mus and Phacus are the best represented of the spring group with regard to species though the relative representation of ~ each species is not great. Nymphaea pool is a large cement basin 1530 ft., situated between the two Nelumbium pools. The desoriptian of the Nelumbium pools applies to this one except that the water is from 6 in. to 1 ft. in depth. Here are found Nymphaea Marliacea var. chromatella, N. Marliacea var. carnea, N. Glad- - stoniana and N. Robinsoniana. The algae in this pond were in the fall casually observed, not studied by weekly microscopic examinations, as those from the other locations. It was noted, however, that Spziro- gyra setiformis and Pithophora Mooreana were not present in the Nymphaea pool though the Nelumbium basins were but 4 ft. away. The most conspicuous thing here was a very abund- ant growth of Spirogyra dubia which was first attached to bricks in the pool becoming free as it rapidly developed. ‘The filaments simultaneously went into conjugating state April 8 34 MISSOURI BOTANICAL GARDEN. 15, after which it sank to the floor of the pool. The free floating or swimming forms of which the other representa- tives consist seem scattered before this time, but when small masses of the conjugating Spirogyra were examined spaces between the filaments were found to be very abundantly filled with these small algae. Here Conjugatae, Volvocaceae, Scene- desmaceae and Flagellatae were represented. During the early part of May just after the Spirogyra had disappeared a large number of tadpoles made their appearance in the pool and the remaining small algal forms rapidly approached the vanishing point, the more abundant ones becoming rare. Crescent pool. This is a small pool of the shape of its name with earth sides and floor and has no outlet. It con- tains Acorus, Nymphaea, several species, and Nelumbium. Observations were here taken during the spring, in the early part of which one form was found in this place only, i. e., Stigeoclonium glomerata. This pool contains more Oscillatorias than the other pools. In the latter part of April it was cleaned preliminary to the spring planting and the only forms which endured were the group of Oscillatorias. Earth habitats. Those in which algae have been studied are: 1. Vegetable garden, Botrydium Wallrothii and Proto- siphon; 2. The mint beds, Botrydium Wallrothii; 3. Flower beds near Fountain pond, Protosiphon botrioides, Chlorococ- cum humicola, Oscillatoria animalis and Stichococcus sub- tilis; 4. Edge of Arboretum stream, Vaucheria sp. The soil of the first three locations is loess, moderately moist ordinarily, occasionally becoming quite dry on the surface. The fourth location is usually saturated, simply mud. SYSTEMATIC ENUMERATION. Schizophyta. SCHIZOMYCETES. BEGGIATOACEAE, Beggiatoa alba (Vauch.) Trev. Stagnant water contain- ing sewage or decaying vegetation. Arboretum stream. ALGAL FLORA OF THE MISSOURI BOTANICAL GARDEN. 35 ScHIZOPHYCEAE. CHROOCOCCACEAE. Chroococcus limneticus var. sub-salsus Lemm. Appeared end of May in Nelumbium pool. Coelosphaerium Kiitzingianum Nig. In quiet water with larger algae. Fountain pond. Coelosphaerium confertum W. and G. S. West. Nelum- bium pools. Nymphea pond. Clathrocystis aeruginosa var. major Wittr. This species was noted near the end of September, associated with Oscilla- toria Agardhii and Anabaena Flos-aquae forming a conspicu- ous green scum on the surface of the water of the Lagoon. Clathrocystis was seen as late as the end of November, but Anabaena and Oscillatoria had disappeared by that time. This association has been observed by Mébius in the Botani- cal Garden at Frankfort with the substitution of C. aerugi- nosa for C. aeruginosa var. major. Merismopedia tenuissima Lemm. Nymphaea pool March to May. Merismopedia elegans A. Braun. Waterfall stream. La- goon. Sept. to Nov. Merismopedia convoluta Bréb. Arboretum stream. Foun- tain pond. Sept., Oct., Nov., June. OSCILLATORIACEAE. » Oscillatoria Agardhii Gomont. Lagoon on surface of wa- ter associated with Clathrocystis aeruginosa var. major and Anabaena Flos-aquae. End of Sept. to end of Oct. (See Clathrocystis. ) — Oscillatoria animalis Agardh. Mingled with Stigeo- clonium tenue on the perpendicular surface of the first water- fall at the outlet of the Fountain pond. April. Noted on earth near Protosiphon in May. (K,) Oscillatoria amphibia Ag. Arboretum pond among Oedo- gonium, Some trichomes were noted 3.24 wide, which is slightly larger than the type measurements. March, April. Rather commen. 36 MISSOURI BOTANICAL GARDEN. _ Oscillatoria tenuis Ag. Crescent pool, forming a dark green stratum on the floor of the pond,—closely associated with 0. anvmalis and O. limosa. Mar., Apr. Abundant. Oscillatoria chalybea Mertens. Arboretum stream. As- sociated with 0. limosa. Sept., May. Common. Oscillatoria formosa Bory. Arboretum pond. Nelum- bium pool. Apr., May. Rare. Oscillatoria limosa Ag. Lagoon. Crescent pool. Arbore- tum stream. LKarth. This species occurs in greater abund- ance and in more habitats than any of the others. At the lower end of the Arboretum stream it is found in the greatest quantity. In the upper part of the stream very little is present. The upper half of the stream flows rapidly over rocks, while the lower half, into which sewage enters, flows sluggishly. Here in the sluggish part of the stream O. limosa covers the floor in a thick stratum, which now and then | breaks up into tufts and floats away or is forcibly all carried away by freshets, after which, in a short time, the floor of the stream is recovered. Sept. to May. Oscillatoria splendida Greville. Crescent pool. Associated with O. limosa and O. tenuis. Common. Oscillatoria limnetica Lemm. Arboretum stream. Rare. Lyngbya Digueti Gomont. Attached to Stigeoclonium tenue on Ist Waterfall and to Vaucheria in Arboretum pond. Rare. Lyngbya Lagerheimii (Méb.) Gomont.* Microcoleus vaginatus (Vauch.) Gomont.* Phormidium uncinatum (Ag.) Gomont. On rocks West Lagoon. Very abundant, forming a thick dark green stratum. Sept. to Dec., Apr., through May. Spirulina major Kiitz. West Lagoon, associated with Os- cillatoria limosa. Sept. to Dec. Common. NOSTOCACEAE. Anabaena circinalis (Kiitz.) Raben. Quiet water. La- goon. Rare. Oct. Anabaena Flos-aquae (Lyngb.) Bréb. Nelumbium pool. Abundant in Lagoon. (See Clathrocystis.) Sept. to Oct. S oe, Te x ss ee a Sek dele ee hom.» es ae pte ter Ss 24 4 A "wird ~~ a ae ee ee ee oo Th ~~ = age eee ean wet ea < ‘d bere. ated Ca Es ela Fe of = al lh kode 7 pete he a ae = So ee - eas Sis Ry « 4 f 2 } : , ‘ WRT ae ese ‘ Balan i ‘ ALGAL FLORA OF THE MISSOURI BOTANICAL GARDEN. 37 Nostoc sp. Nelumbium pools. Sept. to Nov. Common. Cylindrospermum minutissimum Collins. Nelumbium pools. May and June. Common. SCYTONEMACEAE. Scytonema Hofmanni Agardh.* RIVULARIACEAE. Calothrix sp. Nymphaea pool on edge of cement basin. Common. May, June. Associated with Stigeoclonium aestivale, Chroococcus and Hydrodictyon. Flagellata. RHIZOMASTIGACEAE. Mastigamoeba aspera Schultze.* HYMENOMONADACEAE. Synura uvella Ehrenb. Arboretum pool. Sept., Oct. Rare. OCHROMONADACEAE. Dinobryon sertularia Ehrenb. Arboretum pond. Rare. Oct. Uroglena volvox Ehrbg.* CRYPTOMONADINEAE. Chilomonas sp.* EUGLENACEAE. Euglena acutissima Lemm. Nymphaea pool. Rare. Euglena acus Ehrbg.* Euglena oxyuris Schmarda.* Euglena prozima Dangeard. Wesan ieee pool. Arbore- tum stream. Abundant. April, May, June. This species was noted thickly covering the surface of the Nymphaea pool, with a green scum. Many of the individuals were in motion, though the scum consisted largely of those in resting state, more or less enveloped in mucus and globular in form. At intervals this scum appears on the mud at the edge of the Arboretum stream. 38 MISSOURI BOTANICAL GARDEN. Euglena spirogyra Ehrbg. Arboretum pool. Large Ne- lumbium pool. Fission observed Apr. 12. Euglena viridis Ehrbg.* Phacus longicauda Dujardin. Arboretum pool. Rare. Apr., May. Phacus pyrum (Ehrbg.) Stein. Garden stream. Nym- phaea pool. Sept., Oct., Mar., Apr., May. Rare. Phacus pleuronectes Nitzsch. Arboretum pool. Waterfall stream. Nelumbium pool. Fountain pond. Common. Sept., Oct., Mar. to May. Phacus triqueter Ehrbg.* ASTASIACEAE. Astasia sp.* Bacillariaceae. Navicula sp.* Gomphonema acuminatum Ehrbg.* Gomphonema constrictum Ehrbg.* Heterokontae. CONFERVALES CONFERVACEAE. Ophiocytium sp. Arboretum pond on Microspora. Rare. April. BOTRYDIACEAE. Botrydium Wallrothii Kiitz. (E3) Cabbage patch. Earth. Associated with Protosiphon botryoides (Kiitz.) Klebs. Nov. Botrydium granulatum (L.) Greville. Mint beds on damp loess soil (D4). Abundant. In the first part of March on the damp soil of a flower bed (soil had not been stirred since the previous summer) was noted what proved to be Botry- dium aplanospores in such abundance that the earth looked light green. No Botrydium plants were observed at this time. At the end of March in another bed in a different location were found Botrydiwm plants in abundance forming dark green masses among the more conspicuous brighter green ALGAL FLORA OF THE MISSOURI BOTANICAL GARDEN. 39 aplanospores. In location I. at this time the earth had dried out, the aplanospores had disappeared and no plants were observed in this place. At the end of March most of the plants were producing aplanospores which immediately began to grow into plants. The aplanospores found earlier in the season on the earth were studied directly from the earth and in drop cultures, but showed no indication of developing into plants at that time. Usually, as soon as these aplanospores taken from the earth were placed in water, they began to produce zoospores. Others of these same aplanospores, when mature, apparently divided into another generation of aplanospores within their walls, which broke, setting the young ones free. They usu- ally remained agglutinized in a spherical group for a time, then broke apart and grew to normal size. All of the young aplanospore cells contained clearly defined, sub-angular chro- matophores, which lost their outline, becoming diffuse granu- lar as the cells increased in size. The aplanospores, then, may develop into plants, remain resting for a time, produce zoospores or divide into other aplanospores, depending on environmental conditions, water probably being the most important. The normal size of the aplanospores up to the time of maturity as indicated by send- ing out a rhizoid process or internal re-organization, 1s 16—38.4n. Chlorophyceae. DESMIDIACEAE. Closterium acerosum Ehrenb. Arboretum pond. Nym- phaea pool. Nelumbium pools. Crescent pool. Mar., May. Common. Closterium acerosum var. elongatum Bréb. Arboretum pond. Nymphaea pool. Nelumbium pools. Crescent pool. Mar., May. Common. ' Closterium Lunula var. intermedium Gutw. Lagoon. Fountain pond. Arboretum pond. Sept., through Nov. Mar. to June. Rather common. Closterium moniliferum Ehrenb. Typha angustifolia pool. Lagoon. Oct., May. Rare. 40 MISSOURI BOTANICAL GARDEN, Closterium strigosum Bréb. Arboretum pond. Mar., May. Rather common. Cosmarium Botrytis Menegh * Cosmarium Broomei Thwaite.* Cosmarium granatum Bréb. Nymphaea pool. Apr., May. Cosmarium margaritiferum Menegh.* Cosmarium Phaseolus var. minor Boldt. Nymphaea pool. Mar., May. . Micrasterias americana (Ehrenb.) Ralfs. Typha angusti- folia pool, with filamentous algae. Rare. May. Pleurotaenium Trabecula (Ehrenb.) Nig. West side of Lagoon. Rocks. Rather common. . Pleurotaenum Trabecula var. granulata West. West La- goon. Rather common. Penium margaritaceum (Ehrenb.) Bréb. Nymphaea pool. Rare. May. Staurastrum sp. ZYGNEMACEAE. Spirogyra dubia Kitz. Nymphaea pool. Abundant. Con- jugating Mar. 15. Spirogyra Grevilleana (Hass.) Kiitz. Fountain pond. Waterfall stream. Rather common. Sept. and through May. Conjugating early in Mar. eS Spirogyra longata (Vauch.) Kiitz. Waterfall stream. Fountain pond. September and through May. The mate- rial in the garden does not exactly conform to the type de- scription. There is, however, observed a variation in the measurements given for the vegetative cells as described by Collins, who gives 20-364 as diameter; Petit 25-30u; De Toni, 24-304. There is also more or less variation in regard to the length of the cells in descriptions. Collins de- scribes the spore as broadly ovoid; Wolle, twice as long as broad, though all state that the spore completely fills the diameter of the cell without swelling, which is true in this case, whatever may be the width of the filament. The mea- _ surements of the garden specimens are: veg. fil. 21.4-28.8y: chromatophore up to 6.4 with as many as 5 coils. The vegetative cells are up to 10 diameters long, usually less; BS ah ae ee, oi gee a ae ania Seid re Sc -—s . .- Ain io 5." ye a ali Le : ALGAL FLORA OF THE MISSOURI BOTANICAL GARDEN. 41 spores 21.4-28.8 48-804, up to 3 diameters in length, ellip- soid with rounded tips, conjugating the middle of May. Spirogyra porticalis (Miiller) Cleve. Fountain pond. Waterfall stream. Sept., through May. In an instance where 3 filaments lay parallel, the two outer filaments contained _ zygospores, the result of conjugation of aplanogametes from the central strand. In two cells of the central strand were two zygospores, the cells of which showed connection with ‘cells of adjacent strands mentioned, indicative that the whole strands were not of one sex, but that the distinction of sex applies to the individual cell. Conjugation observed at end of March. Spirogyra setiformis (Roth) Kiitz. Nelumbium pools. Fairly abundant. Conjugating in November. This is the only species which conjugated in the fall (Dec.) Spirogyra tenuissima (Hass.) Kiitz. Arboretum pond. Associated with Mougeotia scalaris and Gonatonema sp. Sept. to June. Conjugating through May. Conjugation is both lateral and scalariform. The receptive cell swells before dis- sociation of the chromatophore. Chromatophore of male cell is usually dissociated and ready to pass out of its cell before the chromotaphore of the receptive cell has lost its spiral form. In some cases two normal appearing zygospores (aplanospores?) were found in one cell. These were some- what smaller than the ordinary spores. MESOCARPACEAE. Gonatonema sp. Typha angustifolia pool. Abundant. Mougeotia scalaris Hassall. Arboretum pond, closely as- sociated with Spirogyra, Microspora, Stichococcus. Quiet wa- ters. Conjugation observed Apr. 5. In one instance 2 gametes from one filament were conjugating with one from another adjacent filament to form a zygospore. VOLVOCALES. CHLAMYDOMONADACEAE. Chlamydomonas glococystiformis Dill. First noted forming a thick, bright green coat on the mud of the north branch 42 MISSOURI BOTANICAL GARDEN. of the Lagoon, later forming a green scum on the surface of the Nymphaea pool. The cell remains motile long, finally becoming motionless, assuming a spherical form and lying imbedded in a gela- tinous secretion. The first division in the formation of young zoospores is longitudinal, the next transverse. Dimensions, 9.6-174X6.4-11.24, while active; 6.4-12.8, resting. VOLVOCACEAE. Eudorina elegans Ehrenb. Fountain pond. Nymphaea pool. Waterfall stream. Quiet water. Sept. to Dec. Colo- nies were breaking up the early part of Dec. Gonium pectorale Miiller. Nelumbium pools. Nymphaea pool. Arboretum pond. Producing auto-colonies Apr. 10. Pandorina Morum (Miill.) Bory. Fountain pond. Ne- lumbium pools. Arboretum stream. Waterfall stream. Nymphaea pool. Sept., Dec., Mar., through June. Produc- ing auto-colonies abundantly in April and March. Pleodorina californica Shaw. Edge of Fountain pond. Shallow water. Colonies were observed in perfect state the last of September. During the first part of November they lost their vegetative cells and the number of colonies decreased. The colonies at this stage resembled Eudorina. Sept., Nov. . TETRASPORACEAE. Tetraspora gelatinosa (Vauch.) Desvaux. Arboretum pond. Rocks, west Lagoon. Ineffigiata neglecta W. and G. S. West. Fountain pond. Rare. PROTOCOCCALES. PROTOCOCCACEAE. Chlorococcum infusionwm (Schrank) Menegh. Lagoon. PROTOSIPHONACEAE. Protosiphon botryoides (Kiitz.) Klebs. Cabbage patch (E3). Associated with Botrydium Wallrothit in Nov. in abundance. In March, scarce on a flower bed near Foun- tain pond. May, abundant. In loess soil. ALGAL FLORA OF THE MISSOURI BOTANICAL GARDEN. 43 SCENEDESMACEAE. Actinastrum Hantzschii Lagerh. Nymphaea pool. Mar., June. Multiplication takes place by longitudinal division of cells. Observed Apr. 6. The size of the individual found here differs from that given by De Toni, Hansgirg, West and Chodat, 7. e., 3.64 10-24u. The dimensions of those found in the Garden are 2.4-3.2u49-16u4.: Colony 19.2-32y diam. Ankistrodesmus falcatus (Corda) Ralfs. Fountain pond. Waterfall stream. Nelumbium pools. Nymphaea pool. Abundant. Sept. to Dec., Mar. through June. Ankistrodesmus falcatus var. spiralis (Turn.) West. Nym-. phaea pool. April-June. Raphidium polymorphum var. contortum (Thur.) Wolle, and Ankistrodesmus contortus Thur., according to the descriptions given in Wolle (I resh- water Algae of U. 8. 198. pl. CLX., seem to be synonymous with West’s Ankistrodesmus falcatus var. spiralis. Thuret’s description antedates the others here mentioned. Ankistrodesmus falcatus var. mirabilis West. Nelumbium pools. Waterfall stream. Nymphaea pool. Sept. to Dec., Apr., June. Ankistrodesmus falcatus var. tumidus West. Nelumbium pools. Fountain pond. Waterfall stream. Nymphaea pool. Oct., May. Rare. Coelastrum cubicum Nig. Fountain pond. Common. Sept. and Oct. Rare. The form found in the garden cor- responds closely with Lemaire’s figure and descriptions of C. cornutum, which Senn discusses and declares is not suf- ficiently distinguished from C. cubicum to exist as a species. Coelastrum microsporum Nig. Fountain pond. Quiet water of Waterfall stream. Common. Sept. to Dec. Dictyosphaerium Ehrenbergianum Nig. Nymphaea pool. Most abundant near the floor of the pond. Apr., May. Colo- nies observed here consist of 4-16 cells. Diam. of col. 16-41.6 wscells rather uniform in size, measuring about 3.2-6.4y. The cup-shaped chromatophore has a red eye spot. This form corresponds closely in size to the Dictyosphacrium Se Ya MISSOURI BOTANICAL GARDEN. - which Bernard describes (1909 Algues Unicellulaires) from Singapore. Though smaller than the type as generally de- scribed, he considers this variable character not sufficient to establish a new species. Kirchneriella lunaris (Kirchner) Mobius. Fountain pond. Sept. to Dec. Nymphaea pool. Mar., June. Rather com- mon. Kirchneriella obesa (West.) Schmidle. Nymphaea pool. Mar. to June. Producing auto-colonies in April. Scenedesmus bijuga (Turp.) Wittr. Nelumbium ponds. Nymphaea pool. Fountain pond. Waterfall stream. Sept. to Dec., Mar., June. Common. Scenedesmus obliquus (Turp.) Kiitz. Nelumbium pools. Fountain pond. Waterfall stream. Nymphaea pool. Rather common. Sept. to Dec., Mar. to June. Scenedesmus obliquus var. dimorphus (Turp.) Hansg. Fountain pond. Nymphaea pool. Nelumbium pool. Wa- terfall stream. Sept., Dec., Mar., June. Rather common. Scenedesmus quadricauda (Turp.) Bréb. Fountain pond. Nelumbium pool. Waterfall stream. Nymphaea pool. Sept., Dec., Mar. to June. Common. Yas ad No loss. At 43°C 1. Normal. .25 g. 217 g. -033 g. 2. With 5 ce. HCl. (1%). ies 115" 185 ‘* 3. With 5 cc. NasCOs. at ang ** -O01 ‘* 4. Heated to 80° C. 25 ** we “Sf No loss. Experiment V. With extract from root tissue. At 20° C. Extract. - Original wt. After 24 hrs. Loss. 1. Normal. .25 g. .25 g. None. 2. With 5 cc. 1% HCl. Bet 2% None. 3. With 5 cc. 1% NazCOs. 20 ** ap % None. 4. Heated to 80° C. ie ap" None. Similar experiments were carried on with root extract at 43° C., but no loss of weight was obtained. 94 MISSOURI BOTANICAL GARDEN. Experiment VI. With aqueous extract from Ceanothus tubercles, .25 gram of fibrin was placed in 50 cc. of extract which was kept at 33° C. A drop of chloroform was added as an antiseptic. Extract. At end of 7 hrs. After 24 hrs. 1. Normal. Slight action—liquid Some digested. becoming turbid. 2. With 5 cc. 1% HCl. Apparent digestive Fibrin becoming filled action. with bubbles. 8. With 5 cc. NasCOs. Liquid dark, fibrin No action on fibrin. contracted. 4. Heated to 70° C. No action. A slight precipitate. The same extract. At end of 48 hrs. After 72 hrs. 1. Normal. Fibrin digested, Half of fibrin digested. liquid turbid. 2. With 5 ec. 1% HCl. One-half of fibrin Only a few small pieces digested. left. 8. With 5cc.1%NazCOs. No digestion. No digestion. 4. Heated to 70° C. No action. No action. After 48 hours of digestion some filtered liquid from each flask was tested for proteid. The solutions from 1 and 2 gave good biuret tests, indicating further that normal and acid extract will digest fibrin. No proteid reaction could be obtained from 3 and 4, showing that the enzyme was de- stroyed under the conditions or else its action was inhibited. After six days all the fibrin in 1 and 2 was digested. Experiment VII. To 50 cece. of dilute glycerin extract of root tissue of Ceanothus .25 gram of fibrin was added. A drop of chloro- form was added as an antiseptic. Temperature was 23° C. Extract. After 7 hrs. 24 hrs. 72 hrs. 1. Normal. Liquid clear. Nodigestion. No digestion. 2. With 5 cc. 1% HCl. No digestion. No digestion. No digestion. 3. With 5ec. 1% NasCOs. No digestion. No digestion. No digestion. 4. Heated to 80° C. No digestion. No digestion. No digestion. Judging from these results no enzyme which will digest fibrin is present in the root tissue of Ceanothus. FUNGOUS ROOT-TUBERCLES. 95 Experiment VIII. To 50 ce. of dilute glycerin extract of Ceanothus tubercles .25 gram of fibrin was added. A drop of chloroform was added to each flask as an antiseptic. The temperature was 22°-23° C. Extract. After 14 hrs. After 24 hrs. 1. Normal. No action, fibrin No digestion. very loose. 2. With 5cc. 1% HCl. No digestion. Slight digestion. 8. With5cc. 1% NazCOs. No digestion. No change of fibrin. 4, Heated to 80° C. A slight precipitate. No digestion. Extract. After 48 hrs. After 72 hrs. 1. Normal. Some digestion. Considerable digestion. 2. With 5 cc. 1% HCl. Liquid turbid. All digested. 3. With5cc. 1% NasCOs. No change of fibrin. No reaction. 4. Heated to 80° C. No digestion. No reaction. Extract. After 96 hours. 1. Normal. Only a few pieces undigested. 2. With5 cc. 1% HCl. All digested. 3. With 5 cc. NasCOs. No reaction. 4. Heated to 80° C. No reaction. Two other experiments with a similar extract were carried on at 33° and 43° C. At 33° C. the results were similar to those which were obtained at 23° C., whereas at 43° C. the digestive activity was much slower. Hence the optimum temperature for the enzyme is lower than 43° C. At 28° C. there is no perceptible digestive action during the first thirty- six hours, but following this period, the process goes on very rapidly and in the normal and acid extract the fibrin is readily digested. No fibrin was digested in the alkaline ex- tract or in that which was heated to 80° C. Even 60° will stop the action of the enzyme. If my interpretation of the preceding data be correct there is present, in the tubercles of the alder and Ceanothus, an enzyme capable of digesting fibrin. The enzyme obtained from the Ceanothus tubercles is more active at a lower tem- perature than that from the alder which digests more readily at a higher temperature. The enzyme, however, is found Fy ge, tee eS te mr eo a a oe ¥ oe ae 96 MISSOURI BOTANICAL GARDEN. only in the tubercles, for experiments with root tissues show that.it is not present in the normal root. The question still presents itself whether the enzyme is produced by the host cell or by the fungus. J udging from cytological data, the digesting of the fungus is an indication that the host cell produces an enzyme. The dissolving of the cell walls in the tubercles of Ceanothus presents a fact which indicates that the fungus also produces an enzyme. Thus there may be two enzymes present, one produced by the host cell and another by the fungus, for it is hardly probable that the host cell forms an enzyme which dissolves its own walls. Until a sufficient amount of pure culture of the fungus can be grown, it is impossible to decide whether the fungus secretes an enzyme or not, but the conditions are probably quite similar to those which Marshall Ward found in Botrytis, which produces cytase capable of dissolving the walls of the host cell. SUMMARY OF RESULTS. The Tubercles of Ceanothus. 1. Judging from the common occurrence of the tubercles, the infee- tion of this plant by the fungus is quite universal. 2. External in- fection probably takes place through a root hair or an epidermal cell from which the subsequent tubercle is formed. 3. The tubercle con- sists of three systems of tissues: the outer or corky layer; the inner, the vascular cylinder; and the middle or cortex, which contains the infected cells. 4. Internal infection occurs in the growing region and takes place by the fungus passing from cell to cell. 5. Three distinct stages of fungal development may be noted: the mycelia stage found in the host cell; the stage with the sporanges, which initiates the conditions for the digestive cell; and the last stage, where all but the walls of the mycelium are absorbed. 6. Because of infection, hypertrophied cells and nuclei are formed. The fungus dissolves the walls of the host cell. 7. The host nucleus increases in volume; with it, there is an increase of the nucleole and in the amount of chromatin. 8. Following the vesicular stage the cytoplasm and nucleus of the host cell are absorbed. Subsequent to this, the cell content of the fungus disappears. 9. Both the host cell and the fungus finally die and undissolved portions of the fungus remain in the cell. 10. Symbiosis exists, which is quite apparent in the early stage. Mitta ere Sh. 2 <)> | ha tase ele Tig Ee a Ee Ce PTE OS ok ee NE eee en ee ar aes eae 9 , St FUNGOUS ROOT-TUBERCLES. 97 Elaeagnus. 11. The tubercles are not found as abundantly as on Ceanothus. Regarding the form and structure, several resemblances can be noted. 12. External and internal infection takes place as in Ceanothus. 13. The fungal mycelium differs from that of Ceanothus in being very narrow. It branches profusely, forms the vesicles, the content of which breaks up into several segments. The infected cell passes through various stages. The fungus is not entirely absorbed by the digestive cell. 14. The walls of the host cell are not broken down as a result of the fungal infection. 15. Hypertrophied cells and nuclei are formed, but the nucleo-cytoplasmic relationship is maintained in the infected cells. 16. No ‘‘Exkretkérperchen,’’ such as Zach reports, can be found in the digested cells. 17. Both the host cells and the fungus die as a result of their previous relationship. Myrica. 18. The tubercles and fungus of Myrica differ in many respects from those of Ceanothus and Elaeagnus. All species of Myrica possess tubercles. 19. The fungus confines itself to one or two layers of cells and internal infection takes place acropetally. No hypertrophy or symbiotic relationship exists. The fungus is best regarded as a parasite. 20. The unicellular hyphae of the fungus form branches which change to club-shaped structures in which no further differenti- ation takes place. 21. The fate of the host cell and fungus is similar to that in Ceanothus. 22. The form, structure and behavior of the ' fungus indicate that it belongs to the genus Actinomyces. Enzymes. 23. In the tubercles of the alder and Ceanothus enzymes are pres- ent capable of digesting fibrin. Whether two enzymes are present, one produced by the host and another by the fungus, could not be determined without a pure culture of the fungus. INDEX OF LITERATURE. 1. Atkinson, G. F. ’92. The genus Frankia in the United States, (Bull. Torrey Bot. Club. 19: 171-177). 2. Bjérenheim, C. G. ’04. Beitriige zur Kenntnis des Pilzes in den Wurzelanschwellungen von Alnus incana. (Zeitschrift fur Pflanzenkrankheiten. 14:3 128-134). 8. Bourquelot, M. E. et H. Hérissey. ’95. Les ferments solubles de Polyporus sulphureus. (Bull. Soc. Myc. de France. ~ 11: 235-240). 4. Brefeld, C. ’02. Versuche iiber die Stickstoffaufnahme bei den Pflanzen. (Centrb. fiir Bakt. u. Parasitenkunde. 28 : 24-25). 98 5. 16. at: MISSOURI BOTANICAL GARDEN. Brown, Escombe. ’98. -On the depletion of the endosperm of Hordeum vulgare during germination. (Proc. Roy. Soc. Lond. 68: 3). . Brunchorst, J. ’86. Uber einige Wurzelanchwellungen, beson- ders diejenigen von Alnus and Elaeagnaceen. (Unters. bot. Inst. Tiibingen. 2151-177). Burgeff, H. ’09. Die Wurzelpilze der Orchideen und ihr Leben in der Pflanze. Jena. . Déobner, E. P. 753. Lehrbuch der Botanik fiir Forstmanner. 3 ed. 71. . Frank, B. ’80. Die Krankheiten der Pflanzen. 648. Breslau. ’87. Sind die Wurzelanschwellungen der Erlen und Elaeagnaceen Pilzgallen? (Ber. deutsch. bot. Ges. 5:50). . Gerassimoff, J. J. 01. Uber den Einfluss des Kerns auf das Wachstum der Zelle. (Bull. Soc. Imp. des Nat. de Moscou. n. 8. 153 185-220). . Gravis, A. ’79. Le Schinzia alni Woronine. (Mém. Soc. Roy. de Bot. de Belgique. 18: 50-60). . Green, R. ’99. The soluble ferments and fermentation. (Cam- bridge Natural Science Manuals). . Griiss, J. ’93. Uber den Eintritt von Diastase in das Endosperm. (Ber. deutsch. bot. Gesell. 2: 286-292). . Harshberger, J. W. ’03. The form and structure of the myco- domatia of Myrica cerifera. (Proc. Acad. Nat. Sci. of Phila. 55 : 352-361). Hiltner, L. ’96. Uber die Bedeutung der Wurzelknéllchen von Alnus glutinosa fiir die Stickstoffernahrung dieser Pflanze. (Landw. Versuchsst. 46: 153-161). 99. Uber die Assimilation des freien atmosphar- ischen Stickstoffs durch in oberirdischen Pflanzenteilen le- bende Mycelien. (Centrb. fiir. Bakt. u. Parasitenkunde. Abt. 2. 5: 831-837). . Jager, G. 60. Uber eine krankhafte Veriinderung der Bliithen- Organe der Weintraube. (Flora. 43: 49-54). . Kolle, W. and A. Wassermann. ’03. Handbuch der pathogenen Mikroorganismen. 2: 861-918. . Life, A. C. ’01. The Tubercle-like rootlets of Cycas revoluta. (Bot. Gaz. 31: 265-71). . Magnus, P. ’79. Sitzungsberichte bot. Ver. Prov. Brandenburg. 91:119. . Magnus, W. ’01. Studien an der endotrophen Mykorrhiza von Neottia Nidus avis L. (Jahrb. fiir wiss. Bot. 35: 205-272). 36. 37. FUNGOUS ROOT-TUBERCLES. 99 . Meyen, J. ’29. Uber das Hervorwachsen parasitischer gewaschse an den Wurzeln anderer Pflanzen. (Flora. 12: 49-64). . Miller, H. ’85. Plasmodiophora alni. (Ber. der deutsch. bot. Gesel. 3: 102-105). 90. Beitrag zur Kenntniss der Frankia subtilis Brunchorst. (Ber. deutsch. bot. Gesel. 8: 215-224). 791. Beitrage zur Kenntniss der Frankia subtilis. (Bot. Centrb. 45: 60-61). - Nobbe, F. und L. Hiltner. 795. Vermdégen auch nichtlegumi- nosen freien Stickstoff aufzunehmen? (Landw. Versuchsst. 45: 155-9). . Nobbe, F. und L. Hiltner. ’99. Die endotrophe Mycorrhiza von Podocarpus und ihre physiologische Bedeutung. (Landw. Versuchsst, 51: 241-5). . Pecklo, J. ’09. Beitrage zur Lésung des Mykorrhizaproblems. (Ber. deutsch. bot. Gesel. 27: 239-247). . Ratzeburg, J. T. C. ’68. Die Waldverderbniss. 2:90, 239-242. Rossmassler, E. A. ’62. Der Wald. 3 ed. 460-461. Schacht, H. ’53. Beitrage zur Entwickelungsgeschichte der Wurzel. (Flora. 36: 257-266). ——_—— ’53. Die Pflanzenphysiologie und Herr Dr. G. Walpers in Berlin. (Flora. 86: 10-11). D4. Beitrage zur Anatomie und Physiologie der Gewichse. 160. ’b9. Lehrbuch der Anatomie und Physiologie der Gewichse. 2: 147-148. 59. Grundriss der Anatomie und Physiologie der Gewichse. 121. — ’60. Der Baum. 172-174. . Shibata, K. ’02. Cytologische Studien iiber die endotrophen Mykorrhizen. (Jahrb. fiir wiss. Bot. 37 : 643-684). . Sorauer, P. ’85. Pflanzenkrankheiten. 2 Aufl. 1: 747. 40. Stanl, E. ’00. Der Sinn der Mykorrhizabildung. (Jahrb. f. wiss. Bot. 34: 539-668). . Ternetz, Charlotte. ’04. Assimilation des atmosphirischen Stick- stoffs durch einen torfbewohnenden Pilz. (Ber. deutsch. bot. Ges. 22: 267). - Von Tubeuf, K. ’95. Pflanzenkrankheiten durch kryptogame Parasiten verursacht. 117. Berlin.—Mykodomatien der Erlen, Elaeagnaceen und Myricaceen, veranlasst durch Frankia- Arten. . Van Tiegham, Ph. ’79. Sur la fermentation de la cellulose. (Bull. Soc. Bot. de France 263 25-81). ata eel ial aa 100 MISSOURI BOTANICAL GARDEN. 44. 45, 46. 47. 48. 49. 50. 51. 52. Vines, S. H. ’97. Proteolytic enzymes of Nepenthes. (Annals of Botany. 11: 563-584). —— ’02. Tryptophane in proteolysis. (Annals of Botany, 16: 1-22). 03. Proteolytic enzymes in plants—I. (Annals of Botany. 17: 237-264). 703. Proteolytic enzymes—II. (Annals of Botany. 17 : 597-616). —__———— ’04. Proteases of plants. (Annals of Botany. 18: 289-317). ; —____—— ’09. Proteases of plants. (Annals of Botany. 23: 1-18). —___——— ’10. Proteases of plants. (Annals of Botany. 24: 213-222). Ward, H. Marshall. ’88. A lily disease. (Annals of Botany. 2: 319-382). Warming, E. ’76. Smaa biologiske og morfologiske Bidrag. (Botanisk Tidsskrift. iii. 1: 84-110). . 53. Wilson, Robt. W. ’09. Research on proteolytic enzymes in fungi and bacteria, (Notes fr. Roy. Bot. Gard. Edin. 21: 27-37). 54. Wolpert, J. ’09. Die Mycorrhizen von Alnus. (Flora. 100: 55. 56. 57. 58. 59. 60-67). Woodhead, F. W. 700. On the structure of the root nodules of Alnus glutinosa. (Rep. British Assoc. Adv. Sci. 1900: 931). Woronin, M. ’66. Uber die bei der Schwarzerle (Alnus gluti- mosa) und der gewodhnlichen Garten-Lupine auftretenden Wurzelanschwellungen. (Mém. Acad. Imp. de St. Peters- bourg. vii. 10°: 1-13). 85. Bemerkung zu dem Aufsatze von Herrn H. Miller iiber Plasmodiophora alni. (Ber. deutsch. bot. Gesel. 3: 177-178). Zach, F. ’08. Uber den in den Wurzelknéllchen von Elaeagnus angustifolia und Alnus glutinosa lebenden Fadenpilz. (Sitz. d. kais. Ak. Wiss. zu. Wien. Math. u. Naturw. KI. 117: 973-983). 709. Untersuchungen iiber die Kurzwurzel von Sem- pervivum und die daselbst auftretende endotrophe Mykor- rhiza. (Sitzber. der kais. Akad. d. Wiss. zu Wien. Math. u. Natur. Kl. 118: 185-200). 10. Studie iiber Phagocytose in den Wurzelkndéllchen der Cycadeen. (Bot. Zeitschrift. 60: 49-55). ee ee ecg Se TL ~ . oe eM ite ‘4 ee eee eS Pe OS SE oe a eee ees ee oe 4 sib Shy eee ae : FUNGOUS ROOT-TUBERCLES. 101 EXPLANATION OF PLATES. All of the figures were drawn with the aid of a camera lucida. A Leitz 1/12 oil immersion objective and ocular no. 4, giving a magnification of 1350 diameters, were used for all except figures 1, 14, and 29, for which use was made of a no. 3 objective and no. 1 ocular, giving a magnification of 85 diameters. Plate 6.—A portion of a Ceanothus root with many young tubercles on the small lateral roots, above. Part of a Ceanothus root on which older tubercles are formed into loose clusters, below. Plate 7.—A large Elaeagnus root showing the dense mass of tuber- cles attached to it by a shortbranch, above. A portion of a root with large clusters, below. Plate 8.—Part of a stem of Myrica cerifera showing the masses formed at the ends of short adventitious roots. Plates 9-10.—Ceanothus americanus. 1, Transverse section of a root tubercle of Ceanothus indicating the infected region and some of the fungal stages. 2, Cells of the meristematic region showing early stages of infection. One cell shows where a hypha is just entering. The cell wall between some of the cells is being dissolved. 8, An embryonic cell just infected showing the branched mycelium. 4, An older hypertrophied host cell with enlarged nucleus. The mycelium is much branched and entwined. No vesicles have yet been formed; portions of the cell walls are being dissolved. 5, 6, Nuclei of the fungus set forth by the haematoxylin stain. The hyphal walls are difficult to differentiate. 7, A stage where the sporanges are formed at the end of the hyphal branches. Infection of adjoining cells is also shown. 8, Hypertrophied nuclei of the host cell differing from the following. 9, Nuclei of digestive cells similar to the one shown in f. 7. 10, Young sporanges showing their content. 11, Older and mature vesicles with a single nucleus. 12, Sporanges burst open, the content has disappeared. 13, A cell showing the last stage of the fungus where all but the walls of the mycelium is absorbed. Plates 10-12.—Elaeagnus argentea. 14, Cross section showing the infected region of the tubercle and its various tissues. The large dense cells contain the fungus in the vesicular stage,—other cells show younger stages. 15, Two uninfected cells showing nucleus, large starch grains and fine granular cytoplasm. 16, An infected cell showing hypertrophied nucleus, the mass of mycelial threads and the mode of infection. 17, 18, Infected cells where the hyphae pass through the cell wall into adjoining cells. 19, A large hypertrophied cell with an amoeboid nucleus. The fungus has the sporanges, in 102 MISSOURI BOTANICAL GARDEN. which the content is broken up into parts. 20, 21, The same vesicles drawn on a larger scale. 22, A stage where the fungus is partially destroyed, the walls of the vesicle and hyphae remaining. 23, A host cell which has collapsed in which the remains of the fungus are still present. 24, Nuclei of the host cell before they become amoeboid inform. 25, Stages of nuclei found in the digestive cells. 26, Very late stages of degeneration of nuclei just before their final disappear- ance, Plates 13, 14.—Myrica cerifera. 28, Life-sized tubercles as they are found in the clusters. 29, A longitudinal section of a tubercle showing the infected region and the various tissues of the tubercle. 80, Several uninfected cells showing the cell content. 31, Cells indi- cating the method by which internal infection takes place. 32, Cells showing the large number of hyphae which pass through the walls to infect the cell. 33, Several cells of the infected region showing young and old stages of the fungus where the branches of the hyphae have enlarged into club-shaped structures. 34, A portion of mycelial thread showing the nuclei. 35, The club-shaped ends of the hyphae. 86, The same but older structures where the nuclei have passed into them from the mycelium. 387, A stage where the host cell and the nucleus begin to disintegrate. The fungus also shows similar stages. 38, Several degenerating nuclei found in host cells. . REPT. Mo. Bot. GARD., VOL. 21. PLATE 6, CEANOTHUS. Rept. Mo. Bor. GARD., VOL. 21. PLATE 7. ELAEAGNUS. Rept. Mo. Bot. GARD., VOL. 21. PLATE 8. MYRICA. PLATE 9. ReEpT. Mo. Bot. GARD., VOL. 21. WSK WAS ie CEANOTHUS., REPT. CEANOTHUS AND ELAEAGNUS. PLATE 10. ee es a es PLATE 11. Rept. Mo. Bot. GARD., VoL. 21. ELAEAGNUS. Rept. Mo. Bot. GARD., VOL. 21. ELAEAGNUS. PLATE 12. bie Bait | eo PLATE 13. Rept. Mo. Bot. GARD., VOL. 21. 0G @. oe saan a IIT PEAT a orgs EEE URE SN SD EO UO Be RS , ae ; io spa Fate CES aay ry ‘ ep Oem * ES fe Gt a oS > c “ 7 a ) AAT Tet tae yi PE ES ASI ES 5 eee wa Seta es ee SK SD ape B tro aoe ‘ Pat egnts aes es oe eh ponte LIP SOOO NYA LIED Z Pas, ia ts ES = MYRICA. Bini Bi bey Ach agli A as Ee eT te a one eyes ' : : Rept. Mo. Bot. GarpD., VOL. 21. MYRICA. ey ee Toe ee ee PLATE 14, q Tg ae 1 a * me fer ”, Wag ee yf nbieed DEVELOPMENT AND NUTRITION OF THE EMBRYO, SEED AND CARPEL IN THE DATE, PHOENIX DACTYLIFERA L. BY FRANCIS E. LLOYD. PURPOSE AND SCOPE OF THE STUDY. The work here reported was begun in 1907 upon my appointment as Cytologist to the Agricultural Experiment Station, Tucson, Ariz., in connection with the special investi- gations on the date which have there been carried forward during recent years, more especially by Dr. A. E. Vinson. The material was collected, through the co-operation of Dr. Vinson, at the Station Date Orchard, at Tempe, Ariz. At the inception of the work it was my purpose to study exhaustively the whole period of embryogeny with reference to the rdle of the various foods and other materials in the seed and carpel, and for this purpose, it would have been necessary to make use of both preserved and fresh material. This object was defeated by my removal to Mexico, and I was therefore compelled to make use exclusively of preserved material, with the exception of some of the earlier stages which I studied before leaving Tucson. In consequence, the sugars have, I regret, been left out of account. This is of less consequence as regards the carpel, as they have been studied, by the methods of the chemical laboratory however, by Dr. Vinson, who has embodied his results in various papers to be later referred to. The present paper records my studies therefore of the anatomy and histology of both seed and carpel from the developmental point of view, and of the roles of tannin, starch, oil and reserve cellulose. Mate- rial of two well marked races, Rhars and Deglet Noor, invert and cane sugar types,’ respectively, has been examined, but it has developed that such differences as exist are, from the present point of view, negligible. When desirable, I have. noted such differences. 1 Vinson, 1906. (103) 104 MISSOURI BOTANICAL GARDEN. The date seed has long furnished material for studies begun with Malpighi, later continued by Sachs, whose work is fundamental, and followed by several others whose work will be mentioned beyond. This has been due in part to the ease of obtaining material and the conspicuousness of the phenomena during germination. In the development of our knowledge of enzymes it has played an important part, not the least important contributions in this field having been made by Americans. The positive evidence thus gained is of value not only in interpreting what goes forward dur- ing germination, but also during embryogeny. Working backward from the resting period, at which point the studies available up to the present time have begun, we are able to understand many details for which otherwise only specu- lative explanation could be advanced. Methods. Material was gathered on alternate weeks, from the time of pollination till maturity, from one of the two races and preserved in three series, one in a watery solution of copper acetate, as recommended by Strasburger, one in chrom-acetic fluid, followed by alcohol, the third in alcohol-acetic, (2:1). For examination, free-hand sections were used, except in earlier stages, in which it was necessary to use microtome methods for the determination of minutiae of structure. Free-hand sections were treated with iron salts, (ferric acetate or chlorid). After long standing in copper acetate, the material con- tained, in many cases, a considerable amount of metallic copper, either within the tissues, on its surface or in the fluid. This was evidently due to the reduction of the copper salt, chiefly, it seems probable, by the invert sugars present. The possible source of error, due to the presence of tannin, was excluded by the use of ethyl nitrite, on a small but satisfactory series of material prepared for me during the spring of 1910 by Dr. Vinson at my request. This check material showed that my observations on the copper acetate preparations were correct. This is of considerable impor- . ae >; i ET + Ad - Pye 7 ba Fg Soe -. Satta eit tk * “ j “4 ae et oe . “et ¢ EMBRYO, SEED AND CARPEL IN THE DATE. 105 tance during the development of the endosperm, where the fixation seemed to lead to misinterpretation. Similarly the long exposure to copper acetate of material containing oil results in the formation of a white, apparently amorphous deposit in certain situations. In a few prepara- tions, under conditions which I am unable to determine ex- actly, beautiful dendritic masses of minute crystals have appeared. In others, large numbers of pale green sphaero- crystals have been found on cutting a section, which had the appearance of being an oil-copper compound. Their insolubility in suitable solvents seems to preclude this inter- pretation. Nevertheless, these should be understood in order to exclude completely sources of error, as it is not at all unlikely that where oil is exposed to copper salts, the oil would be reduced in amount. In the present case, the alco- holic material was used as control. I am obliged to Professor R. H. Forbes and Dr. A. E. Vinson for much assistance in the obtaining and preserva- tion of material, and to my colleagues, Professor B. B. Ross and Professor C. L. Hare, for criticisms from the chemical point of view of certain interpretations. This study, begun at the Arizona Agricultural Experiment Station, has been largely prosecuted at the Alabama Agricultural Experiment Station. STRUCTURE AND DEVELOPMENT.. Organogeny of the fruit. Some account of the develop- ment of the parts of the fruit will be necessary in order to make evident the anatomical and histological changes which take place during the time between pollination and the final maturation of the fruit. The materials have been examined, not with the object of studying the cell-to-cell minutiae of the embryology, but rather to follow the main outlines of development of the embryo, seed and fruit, to- gether with their nutritive inter-relations discoverable with the methods at hand. The whole extent of the development of the date fruit falls rather naturally into three periods: a, that extending from the time of pollination (Stage I, f. 2) until the endo- 106 MISSOURI BOTANICAL GARDEN. sperm ends its pavement phase of development (Stage IT, f. 7); b, that between this time, and the final closure of the endosperm cavity (Stage III, f. 14); and ¢, the period following until maturation (Stage IV). It will be convenient for cross reference to speak of these stages and periods. Period I occupies about eight weeks. The young seed is then 4 mm. long by 2 mm. in diameter. The whole fruit meas- ures 7.5 by 7 mm. (broad). Period II occupies about three to four weeks. The fruit is 9.5 mm. broad by 10 mm. long; the seed 7 mm. long by 2.5-3 mm. broad. It is relatively a very short period and one of rapid change, characterized by the development of the endosperm, and marked topo- graphic changes in the ovule in general. The third period occupies a period, following the closure of the endosperm cavity, covering about 15 weeks, and is characterized by the development of the embryo, which, until the end of period II, remains very small. Stage I. Beginning of the First Period. The pistil (f. A), with the exception of the stigma, is en- closed in the involucre. Three pistils are present, only one of which persists normally. Carpel. The whole pistil at the time of pollination meas- ures about 3 mm. in length by 1.8 mm. broad. The dorsi- ventral diameter (1.2-1.4 mm.) is somewhat less. The locule is small, and completely filled by the anatropous, central basal ovule. The short style is traversed by a canal lined by secretory cells—pollen tube guiding tissue. The canal is continuous with a groove, which at the upper part of the ovule is single, but bifurcates as it passes downward, one groove passing on each side of the funicle (f. 2-2e). In front of the micropyle the glandular tissue spreads out to form a cap over the exostome (f. 2). These grooves per- sist and enlarge, and may be traced (f. 11) throughout the whole development of the fruit. At this time, with the exception of the epidermis of colum- nar cells, external and internal (endocarp), there is to be recognized only the tanniferous layer of idioplasts. Within rn pe Ta op cant Tile ge EE Se aR in te EMBRYO, SEED AND CARPEL IN THE DATE. 107 a week after pollination, active differentiation of tissues has commenced at the apex of the carpel, which begins to pro- ject beyond the bracts. The epidermis is thus more or less exposed and is becoming strongly cuticularized. Hypodermal tannin cells are evident here and there, and the layer of stone cells is rapidly forming. Within the mesocarp, numer- ous raphide bearing cells have been formed, and the tannin-idioplasts are more strongly developed. Ovule. The ovule at the time of pollination pre- sents several matters of in- terest from the present point of view. Of the nucellus nothing remains but a cap of a single layer of cells, in a state of rapid disintegration (f. 1). The bulk of the ovule is of nucellar origin (f. 2, 5), and, after the disappear- ance of the nucellus, may be regarded as a chalazal tissue. It thus comes about A. PHOENIX DACTYLIFERA. that only the upper end Longitudinal section through pistil of the embryo-sac is sur- at approximately the time of polli- rounded by the integu- nation. The distribution of starch is ments. which form a shown by stippling.—t, Tannin idio- : plasts. s, Stonecells. 1, Rhaphide idioplasts. The stylar canal is seen. A fia, crown resting on the top of it (f. 3). The micro- pyle is evident, and is lined by actively glandular cells, the inner superficial cells of the inner integument. As seen by the figure, the endostome juts forward into the exostome, which is open. The anatomy of this region suggests the explanation that the glandular tissue of the carpel facing the exostome is a center of attraction for the pollen tube till it reaches this point. The secretion, which is doubtless 108 MISSOURI BOTANICAL GARDEN. thrown out by the glandular cells of the endostome, then exerts a superior attraction perhaps quantitatively only, or, it may be, qualitatively, and this leads the pollen tube to enter the exostome, which is its normal course. I have else- where dealt with the problem of the direction of the pollen tube (Lloyd, 1902) suggesting that the pollen tube is guided in its course by the differential distribution of a stimulant arising from the egg-apparatus, chiefly the synergidae, and that the whole phenomenon is chiefly a chemical one. The evidence here before us prompts a modification of the above suggestion, which however does no violence to its funda- mental feature, to the effect that the stimulant may be handled in a system of relays, the pollen tubes being guided from one relay to the other. In the date the relay stations, so to speak, are in the carpellary guiding tissue, the glandular endostome tissue and the egg-apparatus and egg cell itself. There is no objection to the assumption that these offer either a renewed stimulus of the same kind, or even a dif- ferent kind of stimulus each time, in view of the work of Lidforss, who showed that positive curvatures are shown by pollen tubes toward nineteen proteins of various groups.” It appears not improbable that refined methods may discover that where different guiding tissues occur, each one involved produces its specific secretion which restimulates the pollen tube from time to time on its course. The innermost layer of cells of the inner integument, already mentioned, also presents a special degree of activity. The cells become deeply columnar and distended at their free extremities where they touch the embryo-sac. This layer of cells is clearly a tapetum, and is analogous to that described for the Compositae* and for a number of other plants by various later authors. This tapetum is contributed to, to some extent, by the adjacent chalazal cells, so that it extends some distance down the embryo-sac, and further down on the funicular aspect, where it reaches as far as the antipodal region. Here it has a distinctly pronounced development. 2 Lidforss, 1909. 5 Goldfluss, 1898-9. EMBRYO, SEED AND CARPEL IN THE DATE. 109 At this moment, namely at pollination, or very soon after, the antipodal end of the embryo-sac shows a remarkable amount of activity. This is seen especially in the very irreg- ular and rapid backward extension of the endosperm, pre- vious to the division of the endosperm nucleus, leaving the antipodal portion of the embryo-sac in its original position (f. 3, 5). There is formed in this way a curious several- armed chalazal extension which, in view of the digestion of the tissues in its patht must be regarded as a haustorium. Its function is the same, I believe, as that of analogous haustorial structures formed by the endosperm in Plantago,® in which, however, they are more highly specialized in form. Its total activity, when followed through the whole course of events, is relatively very great, as will appear from its position and volume in the ripened seed. The observed phenomena relating to this activity are recorded beyond. At this point it is sufficient to say that, in a week after pollination, the amount of development is quite marked, there being several cul-de-sacs penetrating deeply into a tissue heavily loaded with tannin. At the same time there is evident the beginning of that torsion which ends finally in the complete displacement of the embryo (f. 5). Stage II. Close of the First Period. Carpel. The epidermis is strongly cutinized. The hypo- dermal parenchyma cells are still cubical or rounded, but show the accumulation of tannin. The layer of stone-cells is completely developed, as also the tannin-idioplast layer. Tannin-idioplasts occur also throughout the mesocarp in the sutural sector (f. A, 7, 11), and in all parts of the basal region. They are especially numerous near the funicle. Morphologically, this is placental tissue, and is constantly 4 At first similar in appearance to the irregular cavity formed in the pine nucellus by the growing pollen tubes. 5 Balicka-Ivanovska, 1899. 6 It is evident that the chalaza in the mature date seed is not prim- ary. Its developmental continuity, however, is clear and so we may properly call it the chalaza. Pee Ce ee pcre ee en Lee a ee ne eet a ae Ne NL OE Lge, gM Oe ee OP MIE en gm Ne Bae ann % “ om ri : * t 110 MISSOURI BOTANICAL GARDEN. characterized by scattered tannin cells. Below the insertion of the funicle, there are two longitudinal grooves, one on each side of the ovule, which may be called sutural sulci. These arise as the continuations of the stylar canal, and are earlier functional as pollen-tube guiding grooves already described. Above the funicle the sulci unite and have a common meatus, finally becoming single. The epidermal (endocarp) lin- ing of the sulcus has the appearance of glandular tissue, and simulates the nectar grooves found in certain plants (e. ¢., Liliaceae). There is a greater amount of tannin in them, in common with the tissues of the placental region than elsewhere in the endocarp. There is, however, no indica- tion of glandular activity. The sulci, as a result of growth pressures, are secondarily more or less irregular in their dis- position, as is seen from the examination of a series of sections. Raphide cells are now relatively much less numerous, though they occur in scattered positions throughout the mesocarp. The endocarpal epidermis is so far differentiated that the cells are considerably elongated and irregular in tangential contour. Seed (f. 7). The torsion begun during the first week (f. 5) has progressed so far that the embryo is now one- third of the entire length of the ovule distant from the orig- inal position. A corresponding amount of torsion has been experienced by the upper end of the ovule, so that the back- ward extension of the endosperm, beyond the chalaza, is well started. The endosperm is parietal, and of a single layer of cells. The tanniferous tissue about the chalaza is very pronounced but is not readily distinguishable on account of the general tannin reaction. The chalazal cul-de-sac has enlarged, and continues to do so beyond this period, so that in the definitive seed its size is marked. The torsion which the integuments undergo is a differen- tial one. The epidermal cells move relatively less, while the greatest movement is found in the innermost layer of cells. Thus it happens that the exostome of the micropyle does not EMBRYO, SEED AND CARPEL IN THE DATE. 111 change position as much as the endostome, which retains its topographic relation to the embryo. As a condition of further growth, there is yet but little differentiation in the integuments. There is growth in thickness and extent, but the cells retain their undifferentiated character. Stage III. Close of the Second Period. Carpel. Aside from the extension of the tissues by growth, there is little to record, except only that, as a result of the mutual pressure of tissues, the inner zone of the carpel becomes slightly compressed. A small amount of disorgan- ization is apparent here and there, foreshadowing the shin- ing fibrous threads regarded as a part of the endocarp. The crushing is more apparent at the placental region than else- where, and it is especially noticeable in its effects upon the placental sulci, which become distorted. The contingent cells within the sulcus become denser in character and there appears as it were a mucilaginous thickening of some of the cell walls, especially near the meatus. There are numerous tannin cells of idioplastic nature, though the tannin is not wholly confined to these. Seed. In the seed the greatest change is in the inward growth of the endosperm, and its extension backward beyond the secondary chalaza. The centripetal growth proceeds at first at the chalaza (f. 10) filling the cul-de-sac; the inwardly moving walls then meet at the upper end, the fusion pro- gressing toward the opposite (micropylar) pole of the em- bryo sac. All nutritive changes in the endosperm progress, similarly, from the chalaza toward the micropylar pole. When the space is entirely filled, and before secondary changes set in, the endosperm cells are very thin walled, with the nucleus suspended in the middle of the cell by numer- ous radiating protoplasmic threads. They are isodiametric, but their radial measurements begin to increase rapidly, con- currently with the later growth of the seed. The growth of the integuments is very rapid, in view of the rapid increase in volume of the seed, and their definitive characters are still absent. 112 MISSOURI BOTANICAL GARDEN. The embryo at this time is spherical in form, 60-75 microns in diameter; at the time when the cavity is just obliterated, the seed measures 2.5 mm. in diameter in the transverse plane in which the embryo lies. The increase from this time, to a transverse diameter of 8.5 mm., in the ripe seed; and from a length of 8-9 mm. to 28-30 mm., is the period of maturation, anatomically speaking, during which the greatest changes are to be found in the endosperm and embryo. There is usually still more torsion, which brings the embryo into the ultimate position, about midway the seed. The position is, however, variable and may occa- sionally be quite abnormal. From the circular embryonic area, visible on the outside of the seed, a slender line may be traced downward. This is the evidence of the torsion, and is the anatomical indication of the micropyle. Stage IV. Close of the Third Period. Carpel (f. 32, 33). The definitive epidermis, 15 microns deep, is heavily cutinized, the cuticle 4 microns thick. The cells are nearly isodiametric and straight walled. There are occasional stomata, each supported by four or five acces- sory cells. Beneath the epidermis is a thin layer of paren- chyma 45 microns thick, of four to five layers of tannin cells, compressed radially. Within this layer again is the zone of stone cells, occupying a very irregular thickness (60-120 microns) according to the position of the individual cells. The longer ones are placed radially. It will be con- venient to regard these tissues as constituting the exocarp. The mesocarp is composed of very thin-walled parenchyma, with here and there a raphide, or tannin idioplast, and penetrated longitudinally by vascular strands. The midrib of the carpel is marked by a median vascular strand, and the suture by a dipping in of the layer of stone cells (f. 11). The inner zone of the mesocarp is composed of more or less crushed and disorganized parenchyma, which, in the ripe fruit is conspicuous as loose, shining, fibrous masses (the “rag’’). These are only loosely attached to the endo- carp, and constitute an irregular, poorly delimited zone which MMR Se UE ae aii, | soi a Sept EP Oe hE Ee ee ee ee "pee o ' EMBRYO, SEED AND CARPEL IN THE DATE. 113 must properly be regarded as part of the mesocarp. The endocarp is a definite, rather tough membrane (f. 34), com- posed of the inner epidermis and more or less underlying tissue of compact cells. This membrane adheres somewhat firmly to the seed but splits readily longitudinally on removal. Along the side of the placental ridge there are two parallel strands of endocarp epidermis which are especially obvious in chromic acid and ethyl nitrite material. They appear to be differentiated on account of their anatomical relation to the underlying placental tissue which is rich in tannin. It is possible, therefore, that they represent transfusion areas, where the tannin passes from the carpel into the ovule. Seed. The outer integument (f. 31). The epidermis is completely sclerosed, having pitted walls. The cells are, on the whole, elongated parallel to the axis of the seed, but at the same time they show en face, a very great deal of torsion and irregularity. There is an irregular hypoderm of more or less similarly sclerosed cells, beneath which are thin- walled tannin cells. It may be an expression of physiological correlation that tannin is not to be found in the sclerosed cells. The inner integument is somewhat difficult to delimit sharply; it consists of at least two layers of cells, the outer and inner epidermis, with usually one or two additional layers of compressed cells between. The entire integumental covering measures about 30 microns in thickness, but thick- ens toward the raphe and above the embryo. Directly impinging upon this, and within it, is the thick- walled endosperm. The outermost cells are very frequently isodiametric, are thinner walled than those within, and have few pores or none. The longer ones have pores at their inner ends, or on the sides when unusually long. All the cells radiate toward the morphological middle of the endos- perm, which is marked by a mass of isodiametric, roughly spherical cells. | The embryo is short and cylindrical in form, but is fre- quently distorted longitudinally. It is placed at right angles to its original position. The end toward the axis of the seed 114 MISSOURI BOTANICAL GARDEN, is the haustorial cotyledon, with which numerous students, beginning with Malpighi and Sachs, have familiarized us, and may be somewhat enlarged, when seen in a longitudinal section of the seed. In transverse section, this end of the embryo is usually smaller than the radicular end. The whole embryo measures about 2 mm. in length, of which one-fifth is the hypocotyl. The radicle proper is scarcely .o mm. long. The form of the seed, as seen in transverse section, varies with the race. In Deglet Noor, the outline is nearly circular, but may be disturbed by a low, lateral ridge. This is very marked in Rhars, so that the outline is lobular. OCCURRENCE AND DISTRIBUTION OF STARCH, TANNIN AND OIL. STARCH. Starch is to be observed, in the form of transitory grains, only during a relatively brief period of the earlier develop- mental stages. Its appearance at all seems to be connected with the slower rate of growth prior and immediately suc- ceeding pollination. During the later period of rapid de- velopment of the fruit, none appears at any time. Observations. Nondescript. May 16, unpollinated. Starch in the upper zone of pedicel, just beneath the receptacle, in large grains. Also in the inner (ventral) part of the parenchyma of the outer bracts, and in all the parenchyma of the inner bracts toward their bases, and in the ventral moiety in the basal half. In the receptacle below the insertion of the pistils, but much less in a zone above the pedicel. Pistil: Rather abundant in the basal part, becoming reduced in quantity in the upper half, where it is of very minute grains. Sim- ilarly minute grains in the ovule, in the funicle, outer and especially in the inner integument. The amount of starch is greater near the nucellus, becoming reduced to none toward the periphery. There is no starch in the tannin idioplasts in the carpel or elsewhere. EMBRYO, SEED AND CARPEL IN THE DATE. 115 The same material one day after pollination showed the same distribution of starch. Tronja. May 16. One day after pollination gave similar results qualitatively but much less marked. Deglet Noor. Pollinated, April 6; fixed, May 16. Starch in a few scattered grains in the pedicel in the paren- chyma adjacent to the bundles, in the parenchyma at the base of the carpel, and in the inner integument between the funicle and micropyle. Nondescript. Pollinated, Apr. 11; fixed, May 28. Starch in the ovule restricted to the basal part of the funicle and to inner zone of outer integument. Deglet Noor. Pollinated, April 6; fixed, May 16. Starch restricted to the mass of tissue between the funicle and micropyle and to a small volume of tissue along the funicle nearby. More in the base of the carpel itself. Up to this time the growth of the ovule has involved chiefly the chalazal half, from which concurrently the starch has disappeared, and in which it never reappears. It is thus seen that this form of carbohydrates plays only a brief réle during the embryological period. The deposition of starch appears to be inconsistent with a very rapid develop- ment such as characterizes the date fruits after the seventh week following pollination. The course of the disappearance of starch seems to be connected, in part, with the general growth of the ovule, as well as with its removal into the embryo-sac.7. The latter undoubtedly accounts for a part of it, but that which remains in the outer integument is probably consumed during the earlier phases of growth in this structure. Embryo. Starch appears in the embryo at first at a com- paratively last stage of development. At 17 weeks (f. 17) a very small amount is seen in the root cap and in the apex of the cotyledon. During the embryological history starch is unimportant quantitatively; during germination as de- scribed by Sachs (1862) its importance increases. 7 Ikeda, 1902. ee grr 116 MISSOURI BOTANICAL GARDEN TANNIN. One who now-a-days makes use of the blanket term tannin throws himself open to criticism. Nevertheless, it is not always possible to avoid it, especially when blazing the way into material necessitating the use of histological methods. A distinction has been drawn between “plastic” and “aplas- tic’ tannins, and objection has been raised’ to the use of these terms. They explain themselves, but it is obvious that we can use them only to describe tannins in a particular structure when they have been shown, in the end, to remain definitively, or to disappear during metabolism. ‘Aplastic’ may, despite that objection, be used merely as a matter of temporary convenience to designate that tannin which ap- pears in a particular situation to remain permanently there- after. Conversely, plastic tannin is that which appears only to disappear. Such tannin is analogous, in appearance at least, to transitory starch, and the evidence to be offered indicates that the comparison is justified by more than ap- pearance alone. If the attempt is not made in what follows, to distinguish chemically the tannins with which I have con- cerned myself, this is due to the conditions and, not less, to the point of view. The history of the study of tannins is extensive, and the materials which have been studied profuse. Happily, Dek- ker (1906), in a most painstaking way, has brought the available data into the compass of a small but rich work, and for this the thanks of the botanical fraternity are due him. A perusal of the historical survey in Dekker’s paper shows that there are two general views held as to the physio- logical réle of tannin. These are, obviously, that tannin is a waste product, and, opposed to this, that it is related to glucose and is of use in forming more complex carbohydrates than itself. Doubtless both are true, but, doubtless also, the same substances are not meant in all cases. Thus, Sachs (1862) regarded the tannin in the germinating date seed- 8 Pfeffer, Physiology of Plants. p. 493. EMBRYO, SEED AND CARPEL IN THE DATE. 117 ling as an excrete, and the evidence supports his view. Con- trariwise, Servettaz (1909) has observed that tannin occurs in certain situations in the young, rapidly developing ovule (in the Eleagnaceae) and argues, with equal right, that here this substance is concerned with nutrition. In view of these observations, it would seem the rational procedure to regard the tannin which accumulates, and remains permanently, in certain cells of the germinating date as different chemically from that in the young ovule of the Eleagnaceae. The chemist may tell us later that, indeed, the former is tannin, and the latter not. These two instances only are cited, because as we shall presently see, they are pertinent to the matter in hand. For the present purpose, we must be con- tent to call those substances tannins which afford us the visible reactions which are usually resorted to, and rely upon observation to tell us whether a particular tannin is an excrete or a nutrient. Aplastic tannin is known to occur in the date. Thorn- ber® studied a series of date fruits from the “size of a pea until full ripeness” and came to the conclusion that there is “no general distribution of tannin” but that it is “strongly segregated in a layer of very large cells near the surface of the fruit and, especially in the younger stages, near the seed.” Avoiding hypercriticism as to the meaning of “near the seed” these facts have been further mentioned and illus- trated by Vinson (1910) for the purpose of demonstrating the value of ethyl nitrite vapor in the study of tannin in plant tissues. Vinson believes that ‘‘a green date may also be easily divided into astringent and non-astringent portions with a pocket knife” and has said to me personally that, before ripening, the portion (mesocarp) lying beneath the layer of idioplasts (f. 32) is not astringent, but that the contents of the idioplasts may be expelled by gentle crush- ing and appear mucilaginous. The inference is at hand that the tannin is confined wholly to the idioplasts, but, for reasons which will be given beyond, I think this is not quite 9 1906. Not over his personal signature. 118 MISSOURI BOTANICAL GARDEN. true. Howard (1906) on the other hand concluded from his study of the ripening persimmon that before ripening the tannin is distributed in the cells of the “loose parenchyma where the tannin cells are located” as well as in the idio- plasts. During ripening the tannin, according to Howard, “becomes condensed in certain specialized cells’ (Bigelow, Gore and Howard, 1906, p. 702), and there becomes insol- uble. The observed facts in the date (Lloyd, 1907),*° as we shall see, do not accord with this view, though in the date as in the persimmon, either during natural or artificial (Vinson, 1909) ripening, the tannin becomes insoluble," and hence the lack of astringency in the fully ripe fruit, in these as in other cases, e. g., the sapodilla, according to Geerlig (1909). What is true of the specialized tannin cells, in this regard, is true also of the remaining tannin cells, those namely in the inner mesocarp and endocarp, and, also, in the integuments, since they are alike tasteless in the ripe fruit. The only remaining observations on the date which touch on the following account are the following: Pond (1907) reported finding tannin in the membranes (endocarp) about the seed, and cautioned students of digestion in the germinat- ing date against the introduction of tannin from the integu- ments into the fluids to be tested for reducing sugars. Sachs (1862) discovered tannin diffused through the tissues of the resting embryo, and described the final accumulation of such material in certain parenchyma cells of the seedling. Reference has been made to Servattaz’ observations on the Eleagnaceae. The tannin, “substances tannoides,” he says, occurs in the ovule, in the elongated cells of the nucellus at the base of the embryo-sac and connecting this with the vascular tissue of the raphe. It occurs also in the mid- layer of cells in the external integument. Continuing, he says, “Dans l’ovule, ces substances ne prennant pas naissance i la suite de la destruction de l’amidon, car il ne s’y forme jamais de réserves amylacées. Le glucose abonde dan toutes 10 Not over his signature. 11 Mylius (see Dekker, 1906), appears to have made observations to the same effect, but I have not seen his paper. EMBRYO, SEED AND CARPEL IN THE DATE. 119 les parties de ’ovule . . .” (p. 353). Servettaz, fur- ther, inclines to the belief that the tannin may be employed in the nutrition of the embryo-sac, and advances the interest- ing view that antipodal cells play an active réle in the process: “. . . les tannins amenés & la base du nucelle seraient transformés en glucose par la ‘cellule antipode’ qui occupe l’extrémité du sac embryonnaire, car cet organe ne renferme pas de tannins . . .” (p. 411). He concludes that, though tannins occur in such situations that they must - be considered a waste, he also takes the position that they are in other situations useful, and cites the finding of Gerber (1897), who has shown that tannins give rise to glucose dur- ing the maturation of some fruits. In what follows it is hoped to show that (1) in the date tannin occurs in special cells in such a state that we are precluded from supposing that it serves any but secondary functions, such as protection, as shown by Vinson (1909). This tannin is aplastic. (2) That there is no condensation of tannin just prior to ripening, meaning by this a move- ment of tannin from the surrounding parenchyma to the special cells or idioplasts. (38) that tannin (plastic) occurs - and disappears at such times and in such situations as to warrant the conclusion that it is a nutrient, and in this is analogous to starch, reserve cellulose, oil, etc. Carpel. Wypodermal tanniferous zone (f. 33). The parenchyma cells lying between the epidermis and the zone of stone cells are more or less flattened radially, and take up, with the epidermis, a thickness of about 60 microns, of which the epidermis accounts for 15. In the Rhars, the majority of these cells, usually excluding the epidermis, con- tain tannin, and they constitute a zone one to three cells in thickness, immediately beneath the epidermis. Occasional cells among them are sclerosed and may also contain tannin. In Deglet Noor there is a similar zone, but usually of only one cell in thickness. The hypodermis of commercial dates displays the same tanniferous layer, the cells containing tan- nin “vacuoles,’”’ or masses lying in the vacuoles. In material fixed with a chromic acid mixture, the tannin does not ap- 120 MISSOURI BOTANICAL GARDEN. pear in the vacuole in all cases, but in or against the proto- plasmic lining. In addition to the tannin in the more conspicuous tan- niferous cells lying immediately beneath the epidermis, a small amount of tannin occurs in all the parenchyma to a depth of 180 to 200 microns inside the zone of stereids (f. 33). This tannin is always in the form of minute drop- lets scarcely ever more than 6 microns in diameters and for the most part smaller than this. The cells themselves show no peculiarities, and in no way are differentiated from the remaining parenchyma within, except in containing these droplets of tannin. Thinking, upon discovery, that they might be oil, I tested them with alkanet, with negative re- sults. Upon allowing the section to lie over night in very weak methyl blue, they became very deeply stained. Their color reactions are identical with the more abundant tannin of the hypodermal or idioplast layers, but they are not con- spicuous on account of their small size. Their occurrence does not extend as far as the idioplast layer, nor do any of the parenchyma cells near to this layer show the pres- ence of tannin droplets. Origin—Indications of this tannin zone are to be seen after pollination. One week thereafter, distinctly differentiated hypodermal columnar or cubical tanniferous cells may be seen, though as yet the zone is not continuous. At the time of pollination, these cells are not yet differentiated, but the epidermis shows a diffused tannin reaction. With the dying back of the stigma and very short style, the tannin cells ap- pear rapidly. These cells, constituting the hypodermal tan- nin zone, are sharply differentiated once for all, and differ from the idioplasts only in their lack of special structural character. They are distinguishable only by their contents. Sub-hypodermal tanniferous zone (f. 82). This zone is composed of large idioplasts (giant cells'*) and is of vary- ing thickness (1 mm. more or less), lying about 1 mm. be- low the surface of the carpel.!* The layer is four to six cells 12 So called by Swingle, according to Howard ‘l. ce. 13 The character of the zone varies considerably with the race. A few of the difference are illustrated by Vinson, 1910. in ee AEE Oe pm ong eats me heey cae oC Ae Un ale St MCS te ane EMBRYO, SEED AND CARPEL IN THE DATE. 121 deep, roughly speaking, in Rhars and Deglet Noor, the num- ber of the cells depending on the size of the elements as well as upon the number which may be counted in radial direc- tion. Tannin similar to that in these cells occurs also in the integuments of the seed, and in the mass of tissue, partly of integumental, of chalazal and of funicular origin, which fills the deep prominent groove characteristic of the date seed. Origin of the layer of tannin idioplasts. This layer makes its appearance at a very early stage of development, some time before pollination. For the present purpose it is suffi- cient to sustain my contention to show its condition prior to pollination and its development thereafter. Both fresh and preserved materials including nitrous ether preparations, were studied. At this time the layer in question is continuous from the base of the carpel, upwards, overarching the ovule. At the base the component elements are nearly or quite spherical, and about 15 microns in diameter. Toward the base of the style, where the elements are the largest, they reach a size of 45-60 microns. The arch is interrupted at the apex by the stylar canal and its immediately surrounding tissues, and here also the thickness of the layer of four to six cells is the greatest. Toward the base of the carpel, it dwindles to the width of a single cell. The layer is homologous with the similar tissues in the bracts surrounding the three pistils, and appears the same physiologically. Precisely the same condition is found in Chamaerops humilis. Tracing them through the succeeding stages of development, there is no change in the constituent elements, except an increase in the amount of their tannin content. Other cells are added to the tissue, being intercalated between those already present, or in a radial direction. The contents of the cells either lie in a layer about the wall, or partially fill the lumen, and are highly vacuolated, this, as we shall presently see, in contrast to the same cells later on. In material which has not been prepared with a 4 Due perhaps to the liberation of gas during the reaction. 122 MISSOURI BOTANICAL GARDEN, view to the detection of tannin, they offer no points of strik- ing contrast so that they may be distinguished from their neighboring parenchyma only by their size, and in this re- spect resemble the primordia of the stereids, which lie, also in a layer, between the tannin cells and the epidermis. Both the former and the stereid primordia are paren- chyma cells which become secondarily specialized, and both have this in common also, that once their peculiar content is laid down, it remains unchanged: namely, in the tannin cells, tannin of the fixed variety, and 1 in the stereids, cellulose and lignin. What appears however in this condition as a simple peti: pheral layer of protoplasm, must be in a living condition, as shown by the proper reagents, protoplasm imprisoning a solution of tannin. Alcohol acetic material, even after pro- longed treatment, does not seem to be depleted of the tannin, for upon the addition of methyl blue, a fine, floceulent pre- cipitate is discoverable, occupying the whole of the interior vacuole. Material treated with copper acetate displayed a variety of curious forms of solid tannin-copper compound, and these are, I believe, to be regarded as artefact. Thus, sometimes a coarser or finer network of tannin forms a sponge-like mass, filling the interior; or into this may ex- tend, from a solid, continuous lining, arms of the same; or the whole interior may be filled with a vacuolated mass; or finally it may appear as a complete, homogeneous lump filling the whole of the cell as seen in ethyl nitrite material, as well as in that prepared by other reagents. In these con- ditions, one may hardly discover the protoplasm which is responsible for the secretion from this, and for this purpose copper acetate, with subsequent treatment with other metal salts for color effect, is really inadequate. Much better is methyl blue in this regard. Not however to do the copper acetate method too great injustice, I may add that sometimes a quite fine precipitate is to be seen, though the protoplasm is still not to be success- fully differentiated to the eye. To return to the anatomy of the tannin idioplast layer. EMBRYO, SEED AND CARPEL IN THE DATE. 123 While generally continuous, and forming a zone of fairly uniform thickness there is, along the line of carpellary fusion, a scattering of the cells. Single cells, or groups of these occur in all the tissue between the inner limit of the carpel, and the outer limit of the zone in question (f. 11). In the condition before pollination, this dipping inward of the tan- nin tissue is not obvious, for the reason that there is little thickness of the carpel here. But with this secondary thick- ening following pollination, the number of tannin cells in- creases within the region of carpellary fusion, as within the idioplast zone proper. At maturity, the number of tannin idioplasts has increased enormously, while, also, the size of the elements is also much greater. The largest are upwards of .2 mm. in radial measurement, and over .1 mm. in diameter, though these measurements must be taken as indicative of the gen- erally large size, rather than a specific statement of size, which varies with the race. I measured the tannin cells in a fruit of Deglet Noor. | Little further needs to be said by way of description of these cells. One may remark, however, that an examination of a full series of material suitably preserved, extending over the whole period of maturation from April to October, taken at intervals of two weeks, shows no evidence of the secretion of tannin within the parenchyma adjacent to the idioplasts indicating that these latter act merely as receivers of tannin or tannin-like material. If such is the case the evidence would be discoverable since it has been obtained elsewhere in the fruit and seed. Intercellular Tannin.?*—A peculiar condition is not infre- quently to be observed, namely, the injection, as it would appear to be, of the intercellular spaces of the idioplasts and adjacent tissues with tannin.” I have been unable to fix upon the explanation of this though there is some evidence 8 According to Winckler, extracellular tannin pockets occur, but I have been unable to see this paper. 16 Howard (1906, p. 570); speaks of this condition, in the persimmon; I have recently confirmed his observation. 124 MISSOURI BOTANICAL GARDEN. that it is due to bruising of the fruit. The masses of tissue showing this condition are irregular, but always associated with the idioplast layer. Aside from this, they are not at all constant in position. Wherever the surface of the fruit has been slightly damaged by pressure, as shown by brown- ish coloration, one may generally find more extensive injec- tion of the intercellular spaces beneath than elsewhere. Where actual wounding and tearing of the tissues have inter- vened, there may be observed a still more pronounced ap- pearance of the tannin in question. Whether however the injection of the intercellular spaces is due to an excretion or to rupture of the tannin-containing cells there is no evi- dence to decide. Vinson has observed that mature dates rupture on being placed in certain fluids, and this occurred in some of my own material, as well as some shrinkage at times. The unequal pressures that are set up would probably be sufficient to bring about the results described. Similar injection of the intercellular spaces frequently occurs along the sutural tract, in contact with the idioplasts occurring there (f. 11). Mesocarp; endocarp. The line of delimitation between the mesocarp and endocarp is not definite. It will, however, make a clear enough anatomical distinction to regard the toughish membrane which frequently adheres somewhat to the seed as the endocarp. This may vary in structure in be- ing of from one to several cell-layers in thickness. The cells are somewhat elongated with square, oblique or irregular ends, occasionally sclerosed, and not infrequently, as Pond (1907) discovered by chemical methods, contain tannin which is similar to that found in the idioplasts. The mesocarp embraces two zonal regions, the outer and inner. The outer mesocarp shows no peculiar anatomical changes in the later period of development. That sector of it which includes the tissues along the line of fusion contains a good many scattered idioplasts, similar in all respects to those of the periclinal zone, except that they are more or less elongated. The closer they lie to the seed, the greater their length; so great, indeed, that they are only EMBRYO, SEED AND CARPEL IN THE DATE. 125 with difficulty recognizable as of similar origin to the iso- diametric idioplasts themselves. The inner mesocarp may, for our present purpose, be de- scribed as a mixture of collapsed, chiefly elongated, thin- walled elements penetrated by a few displaced and distorted vascular strands. Here and there, isolated or in small groups, are very long cells rich in tannin. Sclerosed cells of similar form, having dimensions upward of 10 mm. in length and .2 mm. in diameter, their walls penetrated by simple oval or circular pores, and vaguely obliquely striated, are also to be found, more especially opposite each lateral angle of the seed, where a group readily visible to the naked eye, may occur. In transverse section, these stereids are circular or oval and sometimes contain tannin (f. 34). Additional tannin is occasionally observable also through- out the mesocarp elements and bundles, in both hadrome and leptome. Frequently vessels filled with tannin have been seen. I am unable, however, to make a sufficiently detailed account of this phase of the matter to warrant any definite ‘conclusions. This would require a more particular study of the vascular tissues than I have been able to give them. There is some evidence that plastic tannin occurs in small amounts in the parenchyma of the mesocarp in certain re- gions. Copper acetate and ethyl nitrite material frequently displays a copper-red or orange coloration respectively, which blackens with iron salts. As maturity is approached, this behavior is not to be observed. Fresh material was not studied, and the facts just stated may be misleading, but it would seem to be the case that plastic tannin, similar to that to be described in the endosperm, occurs in small amounts in the mesocarp during development. The reaction upon — which this conclusion is based is more marked in the sutural sector than elsewhere, and in the inner than outer mesocarp. In the latter, however, the amount, as previously stated, is exceedingly small at any time. For some time previous to ripening, as attested by Thornber, Vinson and myself, no _ tannin occurs in the major portion of the mesocarp. There is, therefore no evidence in this instance that tannin gives 126 MISSOURI BOTANICAL GARDEN. rise to any of the abundant sugar, one of the alternative views advanced (Bigelow, Gore, and Howard, 1906). Ovule. Stage I. At the time of pollination there is tannin distributed throughout the whole of the ovule, as shown by a diffuse reaction. There is, however, a much greater quantity in the embryo-sac, inner integument, chal- aza, raphe and pedicel (f. 2). The endocarpal pollen-tube guiding tissue shows a similar amount of tannin. Regarding, for a moment, the anatomical facts recited above, it is difficult to avoid the conclusion that there is special nutritive activity connected with tannin both at the antipodal end of the embryo-sac and in the tapetum. In the Eleagnaceae (Servettaz, J. c.), while tannoid substances occur in the nucellar cells beneath the antipodal cells, and do not occur in the embryo-sae, it is argued that the anti- podel cells play an active réle. The conception of the physio- logical importance of these cells, in spite of their frequently small size and lack of histological peculiarities, is one to which we have become accustomed through the work of Westermaier, Goldfluss, Lloyd, and others,’* but it still re- mains to determine in detail what their method of operation may be. Servettaz’ suggestion should prove fruitful. In the date, although the antipodal cells are small, their anatomical relations are peculiar. In the definitive embryo- sac, they are placed at the end of a slender pit, surrounded by elongated cells with thickened walls (f. 4). The thicken- ing appears to be due to swelling, and this must, I think, be of significance since it is only the walls forming the anti- podal pit which are swollen (f. 1, 3). Whether this is the same sort of thing observed by Servettaz (J. ¢., p. 354) or not, I cannot say. He appears to regard the thickening and gelatinization of the antipodal cell walls observed by him as an accompaniment of disintegration. In the case before us, the swelling of the cell walls is not due to digestion, since their persistence (f. 5) shows the contrary. They appear, ~ however, to be full of tannin, as they show a definite reaction 7 Coulter and Chamberlain, Morphology of Angiosperms. 2 EMBRYO, SEED AND CARPEL IN THE DATE. 127 at once on the addition of reagents.’® A careful study of the development and nutrition of the embryo-sac previous to pol- lination will, I believe, show that they behave in some such way as suspected by Servettaz in, e. g., Hippophae rham- noides. Unfortunately my material has not been suitable for this. I have been able, however, to follow the behavior of the antipodal cells after pollination. It appears that disintegra- tion proceeds from the basal cell, operating successively on the adjacent and the innermost (f. 4, 4a). The disintegra- tion is preceded by the reduction of the nucleolus, and the enlargement of those of the adjoining cells. The order of disintegration is the reverse of that followed in Hippophae and other Eleagnacee (Servettaz, 1. c. p. 354). Whatever the explanation of the difference, their position at the end of the elongated cells which represent the end of the vascu- lar supply to the embryo-sac, as in Hippophae, the presence and evident nutritive importance of tannin, the peculiar thickening of the contiguous cell walls, and their ultimate behavior, all offer positive evidence of their physiological importance. It would similarly appear that the tapetum”® is also impor- tant. At the early stage of development both tannin and starch occur in the inner integument and_ tapetum. This tissue does not break down—is not digested by the embryo-sac. The character of the tapetal cells indicates that its nutrients are transferred to the embryo-sac, following the decomposition of the thin nucellus. Just before, or, at any rate, immediately following polli- nation, the endosperm begins a rapid chalazal growth, pre- vious to the division of the secondary nucleus. In doing so, it digests the tissue at the base of the embryo-sae quite irregularly, and forms several cul-de-sacs which extend back- ward beyond the antipodal pit. This is, for some time, left projecting into the utriculum of the embryo-sac (f. 5). 18 This is not the secondary staining of the wall spoken of by Stras- burger in his Practical Botany. 19 Goldfluss, 1899. 128 MISSOURI BOTANICAL GARDEN. Finally the tissue beneath it is undermined, and so a single antipodal cul-de-sac is formed, which continues its growth _ upward and toward the raphe. During this time the tissue about the cul-de-sac shows a reducing action on Fehling’s solution. The cell walls are swollen, and stain deeply with Bismarck brown. Thus we may infer that active digestive processes are in progress; but we are not, of course, able to say whether the reaction is one to tannin alone, or to a reduc- ing sugar, or both. The cul-de-sac is therefore a haustorium, for a prolonged period constantly encroaching upon the sur- rounding tissues. Its size increases as the seed enlarges till its dimensions are sufficient to make it a prominent feature of the mature seed (figs. 5, 6, 7, 10, 18, 12). It is impor- tant to notice in this connection that, resulting from the stimulus of the growing cul-de-sac, the surrounding chalazal tissue increasing concomitantly by cell division, its cells become radially disposed. These always contain tannin, the least densely filled cells being those next to the endosperm. A part, at any rate, of the food material supplied to the endosperm is a tannoid substance and this passes over into the endosperm chiefly at the cul-de-sac. As we shall see, a tannin is also present in the endosperm during some part of its development. That the chalazal cul-de-sac is the active center for the distribution of nutrients is indicated also by the circumstance that it is the center from which progress the physiological as well as anatomical changes which over- take the developing endosperm; and by the further fact that the remaining tissues contingent on the embryo-sac grow, pari passu, with the endosperm. Why the endosperm digests the tissue touching it at one point and not at another is an interesting and important question, which has received little attention. The same question applies to the embryo-sac, during its development in many instances; and similarly to the developing embryo. I have elsewhere”? shown that the embryo-sac may develop in a quite abnormal manner in this regard and behave much as a pollen tube, but in a more aimless fashion, so to speak. ” Lloyd, 1902. Ba ek Sel i iy tele core amis . Er pe CRT 8 Gitte 0 ee is rn an, eer eran ea ce gees ee FR oe hs ; 5% EMBRYO, SEED AND CARPEL IN THE DATE. 129 The facts observable with the microscope seem to force the conclusion that the mechanical conditions determine the be- havior. Thus, in certain Rubiaceae, the embryo-sac extends forward along the micropyle, during the development of the single thick integument. If, as may happen, instead of remaining within normal bounds, it continues to grow until it extends beyond the exostome, it then pushes its way in any direction—the easiest mechanically. But if, instead of moving forwards, it develops backwards into the chalazal tissue, its path is indirect, and appears to be wholly indeter- minate. In the date, however, the backward growth of the endosperm is direct and determinate. To be sure, it is pointed in the right direction to begin with. Though it digests the nucellus—a very thin layer—and to this extent moves forward, it is resisted by the integumental tapetum. So far the same phenomenon is seen in other plants (Elaeagnus, Servettaz, 1. c. Cucurbitaceae, Kirkwood, 1904) but to a more marked degree. This resistance may, probably, be explained by a chemical response, or by a resistant layer, say a cuticle, as observed in T'ricyrtis by _ Ikeda (l. c.). But how shall we explain the direction of the antipodal haustorium, unless we assume, as a working hypothesis, that the response is chemotactic and that, there- fore, many facts about the embryo-sac are to be explained in the same way that we explain the behavior of the pollen tube! To assume a local enzymatic activity would be, I think, to assume too much, though this also is not impossible. Later stages (the young seed). During the whole period of development until maturity the raphe and integuments contain iron-blue tannin. For some time—no exact period - can be determined— the reaction in the integuments is diffuse, as it is in the bulk of the raphe, in which, however, are groups of cells showing a superior content. The appear- ance of these cells suggests that the tannin in them is aplastic, while their position, which is removed some distance from the endosperm, also indicates this to be the case. Similarly, as stage III is approached, there is evidence below the level of the embryo, that the tannin is accumulating in permanent 130 MISSOURI BOTANICAL GARDEN. form in some cells. At this time (f. 14) the endosperm cells in contact with the raphe and the integument contain more tannin than the inner ones, and it would seem that the integuments serve in some measure to distribute the tannin. The markedly greater activity in this regard of the chalazal tissue has already been noted. As maturity is approached, many of the cells of the outer integument lose their tannin. These are chiefly the epi- dermis and an irregular zone of underlying cells, in ‘all of which not the least trace of tannin is to be found (f. 31). The cells of the inner integument are all tanniferous. What becomes of the tannin in the cells which definitively do not contain it is not easy to say. There is no evidence that it is removed to adjoining cells; on the other hand, there is none that it is transformed, beyond the circumstance that there is a considerable sclerosis of the epidermal cells. It has been noted that of cells of similar origin, apparently, in the endocarp, some become sclerosed and do not contain tannin (or occasionally, very little), while others remain thin walled, and are tanniferous. This lends some probability to the view that the tannin is used in physiological changes in the individual cell. If, however, the removal of tannin from the epidermis and underlying cells is to be explained by lateral movement into those cells which are finally tan- niferous, this happens long before the period of ripening, and is not the rapid segregation described by Howard (l. c.) in the pericarp of the persimmon. After ripening, the cells of the integument and raphe, with the exception noted, are heavily loaded with insoluble aplas- tic tannin. To this the deep brown color of the seed is due. To sum up briefly, it appears that the tissues of the ovule, setting aside the endosperm for the present, are, during the period of growth, supplied with plastic tannin which is passed over to the developing endosperm. During the same period, tannin cells containing aplastic tannin appear, few at first in the raphe, but at length throughout both raphe and integuments. During the latter part of the interval be- EMBRYO, SEED AND CARPEL IN THE DATE. 131 tween stages III and IV, tannin disappears from the outer part of the outer integument. All the tannin finally be- comes insoluble; it is found to be so in the ripe fruit. There is evidence that during development tannin moves from the integuments and raphe into the endosperm, as well as from the chalaza, where the activity is especially evident (f. 27a). Endosperm. Stage I. A strong tannin reaction is seen in the embryo-sac. Just at the time of pollination the endosperm nucleus and its protoplasm become blackened by iron following copper. The same evidence is had by the use of ethyl nitrite, which gives a brown reaction. Alcohol material gives a less pronounced, but readily observable reac- tion. For a period of eight weeks, or somewhat longer, the endosperm retains a syncytial character, and remains as a thin protoplasmic lining. All the material collected during this period (Rhars and Deglet Noor— in alternate weeks) shows that during the whole of this time, the endosperm is always replete with tannin. Copper acetate and ethyl! nitrite material invariably show also a thick precipitation mem- brane lining the endosperm utriculum, from which it must be argued that the fluid within was rich in soluble tannin. It is also to be inferred from the facts of distribution, that the tannin is diffusible, and this is supported by a study of the endosperm in a later stage. Stage II. From this time on the endosperm develops centripetally (f. 10), but this more rapidly in the chalazal cul-de-sac than at the micropylar end. During the periods of growth between stages I and ITI, there is a general distor- tion, producing a displacement of the embryo, so that this comes to lie definitely in the familiar midway position. (f. 12). The filling of the utriculum occupies about four weeks, in which time the seed attains a length of 9 mm. with a diameter of 3 mm. (Deglet Noor). The lack of any pronounced tannin reaction (more marked in the peri- pheral cells than elsewhere) at this time, indicates that there has been a substitution of one material for another. As a matter of fact for the first time there is to be noted a general 132 MISSOURI BOTANICAL GARDEN. distribution of oil in small droplets throughout the endo- sperm, but whether there is any causal relation to be traced is not to be decided off-hand. Stage III. Very soon the thickening of the cell walls, _ to form reserve cellulose, begins, (f. 26 a). Concomitantly, there is a reappearance of tannin, so that, in material two weeks older, (3 months old) there is tannin throughout*! the endosperm in large amount (f. 27). The amount is how- ever much greater in the central portion, namely in those cells, strictly speaking, which are undergoing secondary thickening of the cell walls, looking toward the definitive condition of the seed. The reaction at this time is very pro- nounced, and the tannin-bearing portion of the endosperm forms the most conspicuous feature of the seed macroscopi- cally (f. 27). As I had some doubt of the facts here presented, another lot of material was obtained for me by Professor R. H. Forbes, and treated by Dr. A. E. Vinson with ethyl nitrite. The material, Deglet Noor, included material pollinated March 9, and fixed June 9, 1910. Upon examining this, the whole of the endosperm and the embryo were perfectly white, a quite unexpected result. The area (shown by stippling in f. 28) which should have been deeply stained, was a more _ opaque white than the surrounding zone. But upon the ad- dition of ferric chlorid, a prompt reaction followed, in quite the expected manner. Upon prolonged treatment with very dilute ferric chloride, the central part of the endosperm was stained dense blue-black, with the outer zone obviously less so. Re-exposure to vapor of nitrous ether produced the reaction also, from which we may infer that the period of original exposure was insufficiently prolonged. | During another two weeks the seed grows rapidly, attain- ing a transverse diameter of 7 mm, (f. 29), and the tannin tissue has extended toward the periphery, but is less pro- nounced in its response to reagents. After this, the density of the reaction is quickly reduced, but extends quite to the 21 See also p. 134. CN ge POO ee Rg ES Em NEE Re ae Se ee OR meer | a eee a wee, Mae cee ea PRT Ce ere Nt ae ee : “ a2 Sak anil EMBRYO, SEED AND CARPEL IN THE DATE. 133 periphery. I have found a relatively small amount of tannin present even as late as October 8, in Deglet Noor material pollinated on March 28. Frequently the disappearance of the tannin is even in rate throughout the whole mass of the endosperm, but one more often observes that it is at different — rates in different parts. In some instances (f. 28, 30), and these are particularly instructive, the tannin remains longer in certain sectors, having their vertices at the line of fusion marking the obliteration of the endosperm utriculum, than in others. The wedge-shaped areas (f. 28, 30) thus made conspicuous, reach quite to the outermost layer of the endo- sperm, and their superior tannin content is quite evident to the unaided eye. In other cases, similar but irregular areas occur, sometimes centrally placed and showing a radial dis- ' position, or it may be less definite relations. On the whole, the tannin is retained rather longer in the general region of the raphe than elsewhere, thus suggesting a movement of the material concerned from this part of the ovule into the endo- sperm. The last evidences of tannin are to be seen in the outermost, peripheral endosperm cells, which are the last to take on their definitive character. These are at this time passing from an obliquely distorted condition, due to shearing pressures exerted by the endosperm on the one side and by the integuments on the other. With further growth they finally attain a radial disposition, and the tan- nin content entirely disappears, save in isolated positions. The mature endosperm shows very little tannin, and this is confined to irregular sulci, caused by uneven rates of growth and the consequent tangential crushing of peri- pheral portions of endosperm which are unable to thicken their walls, and to adjust themselves finally to the sur- rounding tissues. The minute quantity of tannin found by Pond (1906, p. 74) is probably to be explained by this circumstance, though a partial explanation may be had in the incomplete eradication of tannin elsewhere. From the circumstance that there are no special tannin cells in the endosperm at maturity, we must conclude either that it disappears as the results of chemical change or that 134 MISSOURI BOTANICAL GARDEN. it migrates into the surrounding integuments. The facts described above do not consist, I believe, with the latter alter- native, but are quite applicable to the former. The appearance of tannin simultaneously with the begin- ning of secondary thickening of the endosperm cell walls has a good deal about it to warrant the belief that the tannin plays some role during the formation of reserve cellulose. The reaction is not in the cell lumen, but definitely in the cell wall, and, in the more deeply reacting cells, in the primary membranes. This appears not to result from displacement of tannin®® as the reaction appears at first in the cell wall. In copper acetate material, in which we may imagine dis- placement of tannin from the lumen into the wall to have oc- curred, the tannin reactions in other tissues (e. g. the raphe) are in the lumina. Ethyl nitrite material gave identical results in the clearest and most convincing fashion. The endosperm, as stated elsewhere, did not show coloration, but, upon the application of iron acetate, gave a prompt, iron- blue reaction in the cell-walls, but not in the lumina. In the integuments and raphe, a similar reaction was given in the lumina of the tannin cells. This parallelism between the distribution of tannin and the thickening of the cell walls, and the ultimate disappear- ance of the tannin when the endosperm is fully developed, indicate an analogy between the tannin in question and other nutrients in transitory form. For this view speaks the fact that the tannin in the endosperm is throughout diffusible, since, in contrast to the tannin in specialized cells, it is found permeating the cell walls. This conclusion is justified from another point of view. Strecker, according to J. Reynolds Green** has shown that a “tannin” is a glucoside, yielding gallic acid and glucose by hydrolysis. The fer- mentation of infusions of certain galls, yielding, with the dis- appearance of tannin, gallic and ellagic acids, (also pointed 2 See above, p. 132. *% The Soluble Ferments and Fermentation, p. 160. See also Dekker, 1906. * Hin Ma gestation ts Ge, Ge a en ee ee as Se Ae eM ae eg ee EMBRYO, SEED AND CARPEL IN THE DATE. 135 out in Green’s work) taken together with the hydrolysis just mentioned, indicate that the comparison of the plastic tannin in the date endosperm with transitory starch is sub- stantiated. Confirmatory evidence is had further in the fact that fungi can assimilate tannin, among other aromatic substances,** though anything beyond a very low food value is questioned. Such tannin as is here described for the date may, however, not be a food of direct availability, but may be a link in a chain of metastates, or may afford energy in a more direct way, as by oxidation. The Embryo. An account of the occurrence of tannins in the embryo requires only the general statement that dur- ing the earlier stages of development, from pollination on, the embryo, together with the embryo-sac structures, and after fertilization, with the developing endosperm, is richly supplied with tannin. A pronounced general reaction is ex- hibited by the embryo as late as thirteen weeks after pollina- tion, (Deglet Noor,) when the embryo measures 50 by .42 mm. When full sized, the embryo measures 1.1 mm. by 2.1 mm., an increase which is accomplished in a period (18 weeks) equal to that required to reach the smaller dimen- sions. During this second period the tannin content of the embryo becomes less and less marked. Nevertheless, until a short time before maturation, (f. 18) a distinct reaction is discoverable, and, though it is diffuse throughout the tissues of the embryo, there is evident a more marked amount of tan- nin(1)beneath the epidermis of the cotyledon, (2)along the vascular tracts, (3) in the plumule and the tissue beneath it, and (4) in the active zone of cells between the hypocotyl and radicle, which, of course, are both very short and not readily distinguishable. These peculiarities of distribution are clear- ly connected with the physiological function of the tannin. At maturity, and during the resting condition tannin is quite absent, as stated by Sachs. There are, so far as I have ob- % Nageli and Reinke, quoted by Pfeffer, Physiology of Plants, p. 492. M. T. Cook has shown recently that tannin is a poison. It is, of course, obvious that different substances may be meant. 136 MISSOURI BOTANICAL GARDEN. served, no special tannin cells in the embryo during the em- bryonic or the resting period. In early germination there is according to Sachs’ account, a reappearance of tannin, which soon becomes localized. When the extra-seminal portion of the seedling has reached a length of a few cm. the constricted zone of the cotyledon, connecting it with the haustorium, shows a definite tannin reaction. I studied fresh material of the ordinary dates of commerce. Iron chlorid shows, in com- mon with methyl blue, (1) a general reaction throughout all the tissue of the isthmus and (2) a considerable number of special tanniferous cells in the parenchyma in the same region. These react as strongly as the idioplasts. The diam- eter of the neck is one-half that of the cotyledon just outside the seed, so that the areas of the cross-sections are as 1:4. The reduction of the area does not, however, affect the trans- porting capacity of this part of the cotyledon, since there is no constriction of the vascular tissues. It would appear therefore that these tannin cells are not to be explained by any nutritive relation. It is aplastic tannin, and, as Sachs believed, plays no active role. In the further nutritive phe- nomena seen in the growth of the haustorium or in the diges- tion of the endosperm, there is no evidence of tannin. In the cortex of the seedling, chiefly near the growing end, in the region therefore of the hypocotyl, there is observable a slight tannin reaction, but the facts are such as not to lend themselves readily to any explanation. At the close of ger- mination, there are tannin cells distributed throughout the cotyledon, but chiefly in the ventral moiety and in the parenchyma immediately adjacent to the vascular strands. The ventral epidermis contains also a goodly number of tannin cells. The cotyledon is of course moribund at this stage. In the first foliage leaf a similar behavior is to be seen save that the tannin appears more especially in the paren- chyma of the dorsal moiety. In the second foliage leaf, before it has yet emerged from the encasing, cylindrical first leaf, the tannin appears at first in the parenchyma, in scattered cells lying in the middle zone. There is no diffuse eS ey Pe Sone awe y ‘ we tattoo. Fe NS a Sl el ae a in Oo ih Ce ie EMBRYO, SEED AND CARPEL IN THE DATE. 137 reaction in any case, the tannin being completely confined to the cells in which the secretion at first takes place. The genesis of the tannin may be observed very exactly in the second foliage leaf, at the age indicated. It first appears as minute granules which become larger. These finally coalesce to form a continuous layer about the cell, or, at length, a mass with vacuoles. There is no evidence of a transitory tan- nin, so that it must be regarded, in the absence of definite evidence to the contrary, as aplastic. OIL. No oil appears in the carpel at any time. In the endosperm oil appears for the first time after about the seventh week following pollination, before the endosperm utriculum is obliterated (f. 8-10). There is a gradual in- crease, in the form of minute droplets, till about the tenth week, about the time, approximately, when the thickening of the cell walls begins in the endosperm. As already pointed out, this is the time when the tannin appears in con- nection with the thickening walls. For a short period (f. 26, 26a) at about the time of this phase of development, the oil content is reduced, during which time the seed reaches its definitive form. The oil content then increases again till, in the mature seed, the endosperm cells contain two carbo- hydrate foods, reserve cellulose and oil. According to du Sablon (1897) the oil constitutes 7-9% of the whole endo- sperm in the resting seed. I find no evidence of tannin within the resting endosperm save in certain cases due to irregularity of development already noted. The disappearance of oil and the beginning of cellulose accumulation are without much difficulty explained on the same grounds upon which we may also explain the accu- mulation of oil as the reserve cellulose approaches a definitive condition. The relation of tannin to these is more obscure, but there is sufficient evidence, already advanced, to regard it as related in some way to the changes which go on during the thickening of the cell walls. 138 MISSOURI BOTANICAL GARDEN. NUTRITIVE RELATIONS OF THE EMBRYO AND ENDOSPERM. The nutritive relations existing between the germinating seedling and the endosperm have been so frequently investi- gated that the date has become classic. Under the above caption, it is my purpose, so far as possible, to complete the account by detailing the corresponding relations during the embryonic period. Especial interest attaches to this phase because the growing embryo opposes itself to a growing endo- sperm, while during germination, the latter is wholly pas- sive.** This opposition of two growing and anatomically independent bodies must result in adjustments which may be mechanical merely; or they may be chemical with resulting secondary mechanical adjustments. There must also be a zone where the counter-influences meet each other, which may be termed the tension zone. Periods I and II. During the first and second periods, as I have already pointed out, the growth of the embryo is slow. A week after pollination it measures about 25 microns in diameter, and attains at stage III a diameter of only 60-75 microns, It is evident that, in view of the loose anatomical character of the endosperm, and the slow increase of the linear di- mensions of the embryo, there is but little mechanical re- adjustment involved. During the first period, I have found no optical evidence of digestive activity peculiar to the em- bryo, although, of course, this may obtain. This is ap- parent from the fact that, at 11 weeks, all the endosperm cells near the embryo are intact (f. 14a), and show no evi- dence of digestive action in the cell walls. It seems prob- able therefore, that the embryo and endosperm act as a physi- ological unit so far as nutrition is concerned. At any rate, the reactions appear to be quite the same in both, and show, e. g., that tannin is abundant in them throughout the first 2 Pond, 1906. EMBRYO, SEED AND CARPEL IN THE DATE. 139 period. Toward the close of the second period, although there is pressure upon the endosperm surrounding the embryo, there is still no digestion of the cell walls to be noted. There is therefore no breaking down of tissue. During the early part of this period, however, there is to be seen the independent action of the embryo in the unequal distribution of oil in the surrounding endosperm (f. 14). At this moment the accumulation of oil throughout the latter is marked, but much less so in a spherical mass surrounding the embryo. This sphere measures fully 250 microns, so that the embryo exerts an influence to a distance of some 100 microns, more or less, from it. In addition to the small amount of oil in this region, I have noted that my prepara- tions of material of this age all show an accumulation of tannin in the form of droplets (f. 26, 26a). I have not seen it in the same or corresponding region in this form at any subsequent time. The preparations in question were fixed in copper acetate, treated with ferric chlorid and with alka- net, before and after absolute alcohol and chloroform, so that there can be little doubt of the accuracy of the observa- tion. This appearance of tannin is synchronous with the beginning of reserve cellulose formation, when, as already stated, tannin appears in that portion of the endosperm where the thickening of the walls occurs. It may therefore very well be that this tannin is concerned in the nutrition of the embryo, but my material was not sufficient to settle the question and it must in consequence be left open. To complete the evidence that oil is a nutrient for the em- bryo at this time it should be noted that it occurs plentifully in the embryo as well as in the endosperm. The appear- ance of the oil in the embryo differs from that in the endosperm. In this, when the oil first appears, it occurs in extremely minute droplets, which collect in a zone about the nucleus. This is especially well shown by the pavement en- dosperm cells lining the cavity (f. 9). In the embryo, however, this relation is not to be seen. Here the oil occurs anywhere in the cytoplasm, as depicted by Sachs in the epi- thelium of the. growing haustorium. 140 MISSOURI BOTANICAL GARDEN. How the distribution of oil at this time is to be under-— stood is not immediately clear. There appear to be two al- ternatives. The comparative absence of oil in the vicinity of the embryo may be due to the immediate absorption by the embryo of most of the material which would, in this region, be turned into oil; or to the movement of the oil, after it has been laid down, toward the embryo. In the latter case, we may assume a lipase to account for digestion, or we may as- sume that the fatty material moves in the form of minute globules, as Sachs believed. The occurrence of droplets of oil between the embryo and the endosperm was regarded by Sachs as evidence that during germination the immediate entrance of this substance into the embryo is accomplished without change of its molecule. I myself have observed the occurrence of large droplets of oil in the same situation dur- ing both the embryonic period and germination. Figure 15 is an example of the former, which, however, is explainable otherwise. Assuming that the oil actually occurs here nor- mally, it seems probable that it is due to the accumulation of small droplets from the disintegration of the cells in which it occurs. (zone a, f. 15). This would readily be brought about if the digestion of the oil at this time did not proceed as rapidly as the breaking down of the cell walls. The large masses of oil in the space between the haustorium and the compressed endosperm during germination on the other hand, I believe to be purely accidental, and is due to the transposition of the oil by the knife-blade during section- ing. There is, furthermore, convincing evidence that dur- ing the major part of the third period of embryogeny, as well as during germination, that there is an actual digestion of the oil, resulting in water-soluble substances, and it is therefore entirely probable that the same is true of the still earlier periods during which oil plays a part in the nutri- tion of the embryo. Period ITI. The final period of development is of peculiar interest in that it leads to the articulation of the two distinet physio- - ae i i ok: Teen See ? EMBRYO, SEED AND CARPEL IN THE DATE. 141 logical periods, that immediately before and that following, germination. Of much information regarding the latter we are already in possession, chiefly from the work of New- combe and of Pond, following that of Sachs, Reiss, du Sablon, and others. The period in question involves a marked mechanical ad- justment of the embryo and endosperm. At first the embryo is spherical in form (f. 14) becoming, at 13 weeks (f. 16) broadly ovate-conical, with the base against the micropyle. Scarcely any change in shape is now to be noted for two weeks, after which, however, the growth is rapid. Within another month, the definitive form and almost full size are attained. (f.18). This, especially, is the period of mechan- ical adjustment, for it is now that the endosperm is also growing rapidly, and that the secondary thickening of the cell walls, to form the reserve cellulose, takes place. But during this time approximately, the radial growth of the endosperm is relatively much more rapid than that of the embryo, and there arises as a result a cavity in the endo- sperm, opposite the cotyledonary end of the embryo, which is at length nearly obliterated (f. 15-19). For the purpose of enabling the reader to follow the changes which ensue dur- ing this period, I have chosen six stages of development, and represented these in figures 14a-19, inclusive, to which the following descriptive notes may be applied. Figure 14a.—Zone a. Cells show no disintegration, but are large, with a relatively small amount of protoplasm, and reduced oil content. No blue reaction in the cell-walls with iodine.—Zone a becomes dif- ferentiated into zones a and a’.—Zone b. Small endosperm cells with divisions taking place approximately parallel to the integument. -_ This characterizes also the peripheral endosperm, beneath the inner integument.—Zone b’. Thin-walled endosperm cells, larger than in zone b. Figure 15.—Zone a. Cells with collapsed walls, empty or with minute protoplasmic content and a droplet of oil. The walls react blue to iodine. In the space between a and the embryo, oil accumulates. — Zone a’. Uncollapsed cells within the digestion zone. The outermost show no change. The innermost show a more or less disintegrated ‘ Tet we ee sr a eee ot re ee oe 142 MISSOURI BOTANICAL GARDEN, condition in the protoplasm. The walls react blue to iodine. The small quantity of oil becomes more evident in the inner cells as drop- lets removed from the protoplasm.—Zone b-b’. Thin-walled endos- perm cells in which cell divisions occur, more especially in the layer of cells between b and b’. Tension zone, contributing cells to zone a’ and to zone c.—Zone c. Endosperm in which the thickening of the cell walls has progressed. The limit between zone b and zone ¢ is not asharp one. (Zone c’ is not visible, but has begun in the interior of the endosperm. cf. fig. 26a.)—Zone b’ now becomes compressed between the advancing thickening endosperm, zone ec. Figs. 16, 17 and 18.—Zone a. Cell walls entirely collapsed, reacting blue with iodine. All the contents except minute droplets of oil have entirely disappeared.—Zone a’. Cell walls not collapsed, reacting blue with iodine. The cells nearer the embryo have no other content that each a single large droplet of oil. The outermost have more or less protein content.—Zone b. Unaltered endosperm cells, the inner of which are subject to digestion. The outermost cells of this layer are represented by zone b’.—The tension zone becomes narrowed down to zone 6’, of cells undergoing division, and shearing between the grow- ing endosperm and zone b.—Zone c. Endosperm with thickening cell walls, giving a tannin reaction onlyin the lumen. Zonec'. Definitively thickened endosperm cell walls. Deep tannin reaction in the cel- walls in the younger stages but disappearing in the older ones.—The zones now become compressed and the distinctions less marked or nearly obliterated. Figure 19.—Zone a. Crushed and compressed cells reacting blue walls and contents. This zone is separable into two zones by the successful application of iodine and sulfuric acid. This treatment produces a deep blue reaction which quickly fades away, and causes swelling and dissolution, These proceed differently however in the two layers a and a’ figure 20. The compressed cell walls of layer a swell and dissolve as a whole; those of a’ first expand and assume their original form previous to swelling, indicating that they are not so far disorganized by digestion as those of a. The compressed cells contain numerous minute oil droplets; the less compressed (a’, f. 20) large droplets.—Zone b’. Tension zone. The cells are partially thick- ened, less toward the embryo, more toward the outside. They are secondarily divided by transverse walls. They contain protoplasm and oil, the latter in large drops, the protoplasm more or less dis- organized.—Zone c. Definitively thickened and characteristic endos- perm cells. It may here be noted that the distribution of oil in the compressed zones is not as figured by Sachs (his figure 4, pl. 9, 1862), who repre sented large drops as occurring anywhere within the compressed layer. The treatment with sulfuric acid demonstrates this very beautifully, as the droplets are displayed in their relative positions as the wall material swells. This is true of the whole period of germination. EMBRYO, SEED AND CARPEL IN THE DATE. 143 We are now in a position to present in summary form the changes which occur. Zone of Digestion. At first a spherical mass of intact cells (a, figure 14a) this becomes differentiated into two zones (a, a’, figure 15) due to the collapsing of the walls in zone a accompanied by the digestion of their contents oil and pro- tein. We have therefore to note (1) the cause for the col- lapsing of the cells which is found in the digestion of the primary walls; (2) the disappearance of the contents, due to the digestion of the oil and protein. Digestion of primary cell walls. Sachs (1862) believed that the primary membrane is not digested during germina- tion, but that the growing haustorium pushes the exhausted and crushed cells before it. Reiss (1889) extended Sachs’ observation of the endosperm of Chamaerops humilis. I have found no evidence to the contrary after a stage of germina- tion roughly indicated by an embryo length of 2 cm., at which stage the haustorium has attained the form of a sphere (f. 24), so that there exists no lack of harmony be- tween my results on germination and those of Sachs and Reiss, which have been generally accepted. Newcombe (1899) extracted a ferment capable of digest- ing the whole of the endosperm cell wall. This result is of interest in that there is yet advanced no optical evidence that the primary membrane is actually digested in the date. Newcombe’s results appear to indicate that there are two ferments extractable together,?* one capable of acting on the primary wall, the other on the reserve cellulose. The former may be present during germination in such small amounts that the result of its action on the primary wall is inappre- ciable to the eye, but that, during the prolonged period of experimentation made use of by Newcombe, it had time to act. Probability is lent to this interpretation by the work of Green. % The contrary view that a single ferment isolated by Newcombe is capable of digesting both primary walls and the reserve cellulose is negatived by the positive evidence that such digestion does not take place during germination; though the logical possibility remains that it takes too long or that it is inhibited in some way. 144 MISSOURI BOTANICAL GARDEN. Green (1899, p. 97) in 1887 studied the progress of diges- tion in the germinating seedling of Livistona humilis, which is quite similar structurally to the date. After two months germination at which time the endosperm was about half absorbed, Green found that the “inner zone”? of the digested portion of the endosperm gave a blue reaction with iodine, but failed to react with chlor-zinc-iodine. Green concluded that under the action of a cytatic ferment the cell wall (primary membrane) was changed. This form of digestion in the primary membrane Sachs did not see in the date, and, as far as microchemical methods may tell us, it appears that he was right. During the embryonic period, however, it is otherwise. My observations show that soon after a stage rep- resented by figures 14a, 26, when the adjustments between the embryo and the cell wall of the endosperm are purely me- chanical, a substance is secreted, presumably by the embryo, which acts upon the nearest cells, attacking the primary membrane and changing it into an amyloid, as I may call it for the present. The reaction indicative of this conclusion is to be seen clearly at a stage represented by figure 15, in which a column of cells (a, in the figure) opposite the apex of the embryo all show a distinct and characteristie blue coloring. The walls themselves in zone a appear wavy and more or less crumpled by lateral pressure of the cells of zone a’, and show every appearance of undergoing some sort of change. The blue reaction is shown also by the walls of the uncrushed cells (a’, f. 15) flanking the column of crushed cells (a, f. 15). This behavior in the presence of iodine indicates clearly that the membranes are undergoing a chemical change, hydrolytic in nature, similar to that observed by Green during germination in Livistona. That this digestion is such in fact is shown also by the behavior of the crushed cell walls (a, f. 20) nearer the embryo as compared with those in a layer further removed (a, f. 20). The former are directly dissolved by sulfuric acid while the latter spread out and take their normal form before dissolution. The exceedingly tenuous character of 27 I understand this to mean that next the haustorium. EMBRYO, SEED AND CARPEL IN THE DATE. 145 the crushed walls next the embryo, as compared with the thicker condition further away also points to the same con- clusion. It may finally be noted that the earliest mechanical disruption of cells is not due to the pressure of the embryo upon the contiguous endosperm cells, but of the cells of zone a’ upon those of a (f. 15). It takes place previous to the secondary thickening of the endosperm walls by the deposition of reserve cellulose. As the embryo grows, the amount of tissue thus affected increases markedly (f. 16, 17) until a time of maximum activity is reached, which happens about eighteen weeks after pollination, at a stage represented approximately by figure 18. The reaction obtained at this time indicates that zone a’ (f. 18) may reach quite near to the outer limit of zone b. My preparations show further that the blue reaction is not always confined to the cell wall, but may frequently fill the lumen, indicating the presence of a soluble or colloidal substance here.”* On approach to maturity, the relative thickness of the layer of cells affected becomes reduced, and at the time of ripening, occupies the zone a shown in figure 19. I have verified this by control with commercial dates, which give the same reaction. During the early stages of germination I have found only a partial reaction and this is confined to the endosperm in contact with the older part of the growing haustorium (f. 24, 25). In a stage corresponding to figure 25, the blue reac- tion appeared along the flanks and around the end of the cotyledon, but in the latter position it was very thin and discontinuous. In a somewhat older condition, as in figure 24, the reaction was to be seen only along the sides of the baustorium, corresponding to the extent of the sides of the cotyledon seen in figure 25. It seems probable therefore that the change of the primary membranes into an amyloid is chiefly an embryonic phenomenon, and ceases during or 28 T have found some evidence that a small amount of starch in the form of minute granules occurs. These are scattered between the crushed cell walls. The amount seen however is very small, and I have not always been certain of its presence. This uncertainty may be due to its disappearance ana reappearance. 146 MISSOURI BOTANICAL GARDEN, perhaps previous to, the early stages of germination. It isa fact of very great interest however that this form of digestion, which in Livistona takes place during the course of germina- tion, occurs in the date only during the embryonic period. I therefore conclude that there is secreted by the growing embryo during the period from the age of about 10 weeks till maturity an enzyme, probably a cytase, which causes the hydrolysis of the primary walls of the developing endosperm in the region of the embryo. It is to be further inferred that this digestion leads to the formation of a sugar, in which form it is absorbed by the embryo. Digestion of oil and protein—(a) During embryogeny.— The separation of the zone of digestion into two sub-zones, a and a’ in the figures, of collapsed cells within, next the embryo, and uncollapsed cells further out enables us to de- scribe the course of events relative to oil and protein with reference to the mechanical condition of the cell walls. At the beginning of the period the zone (a, f. 14a) is undifferentiated within itself. The cells contain only small amounts of oil (f. 14). Before the digestion of the cell walls, as already described, sets in, the amount of oil increases. The digestion of a column of cells in front of the embryo (a, f. 15) is accompanied by the disappearance of the proto- plasm and the isolation of the oil so that small droplets are seen within the strand of crushed and empty cells. Assum- ing for the moment that there is an actual digestion of this oil, it must be slow, since there collect large droplets in the space next the embryo. That the breaking down of the cells at this time should proceed more rapidly than the undoubtedly slow digestion of oil may be understood by the fact that the volume of these cells is relatively much greater than if their compression were due merely to the pressure exerted by the embryo. In point of fact, this pressure is exerted by the adjacent undigested cells in the direction indicated by the arrow points in the figure. Now begins the rapid growth of the embryo, accompanied by the com- pression of the cells adjacent to its surface (f. 16), but the growth of the whole endosperm in radial direction, as indi- ES eae re ae Ser t ; : i ? “if day Wintel RRM A ia hae bd iS a eek i 8 I) Nee eg a i i ae i EMBRYO, SEED AND CARPEL IN THE DATE. 147 cated by the arrow point at the bottom of the figure, results in the formation of a pit in front of the cotyledon. This is obliterated only with complete maturation (f. 19). The crushed layer of cells lines this pit, their crowding, which does not amount to compression, still caused by the adjacent tissues. The later progress of events consists in the amplifi- cation of these relations till a stage represented by figure 18, is reached, followed by a reduction during maturation. Observation during the whole of the period shows that the distribution of protein and oil in these tissues may be briefly summarized. The outermost of the uncollapsed cells (zone a’) contain both their oil and protoplasm in practically an unchanged condition. Passing toward the embryo, the protoplasm shows evidence of disintegration and obvious reduction in amount till the inner layer of uncollapsed cells is reached, where only the oil remains usually as a single drop in each cell. Within the crushed layer, these oil drops are quickly reduced in size (f. 18a and 20). (b) During germination.—The zones*® of digestion sur- rounding the growing haustorium are as follows, beginning at the plane of contact with the embryo (f. 24; text figure B). The thickness of the zones depends upon the position relative to the haustorium, being thickest opposite the rap- idly advancing edges: a. Crushed cellsderived from embryonic period, reacting blue with iodine. This zone is discontinuous and is derived from the resting condition. b. A zone of compressed cells, of primary membranes only, about 15 microns thick. The oil is in minute droplets. (Sachs’ layer 1). ce. A zone in which the oil is ina large single drop in each cell; the protoplasm has disappeared. The reserve cellulose is less in amount proceeding toward the embryo and the cells are more compressed (text figure b). (Inner part of Sachs’ . middle layer.) *? The reader will not infer that these zones are sharply delimited, any more so than the others which I have described. Nevertheless, they represent an objective reality. * ater AS wi. Le 148 MISSOURI BOTANICAL GARDEN. d. A zone from which the protoplasm is disappearing, and in con- sequence, the oil becomes agglomerated. The reserve cellu- lose begins to undergo change. This zone takes up Bismarck Brown more readily than elsewhere. (Outer part of Sachs’ middle zone.) e. Unchanged endosperm. (Sachs’ corresponding layer.) This somewhat sharper analysis than that of Sachs has for its purpose to show that the agglomeration of the oil is coincident with the disappearance of the protoplasm, and that the disappearance of oil begins when the cell-walls are reduced to the primary membranes. This harmonizes with’ du Sablon’s conclusion that the amount of oil in the undi- gested endosperm remains the same (7-9%) during germ- ination, and that “on peut done dire que la matiére grasse de l’albumen n’est absorbée par le cotyledon qu’au fur et & mesure de la digestion des parois mémes des cellules de Valbumen” (1899, p. 396). It may be observed however that the relative amount of oil in zone ¢ is greater than else- where, because of the reduction in the amount of both the protein and reserve cellulose. The comparison of the be- havior of oil and protein during embryogeny and during germination shows further that the facts are identical, while the cessation of growth in the endosperm and the presence of reserve cellulose remove one condition, the presence of the tension zone, and impose another, the necessity of digesting the reserve cellulose, a different material from any attacked by the embryo previous to the resting stage. The conclusion seems obvious, that during the embryonic period the oil is digested and, before absorption by the em- bryo, changed into water-soluble substances. This conclusion must be extended to the period of germination. Sachs be- lieved to the contrary, for the reason, before stated, that oil occurs in large quantities in the compressed layer and in the space between this and the epithelium of the haustorium, and because the immediately underlying layer of epithelium also contains oil-droplets®® (1862, p. 250). The presence of 80 Reed (1904) in his studies of the cytology of the epithelium of the date appears to have overlooked the important modifications in the appearance of the cytoplasm, and possibly of the nucleus, which ee ee ee ee SS ee =" Lien i, AUS Wb ee ee i eS te ee EMBRYO, SEED AND CARPEL IN THE DATE. 149 only a small amount of oil in the embryo was noted by du Sablon, but he observed also notable quantities of sugar (l. c. p. 398). I have already explained the presence of oil in the space between the embryo and endosperm as due to accident in the cutting of freehand sections. It would, I think, be appar- ent to anyone at a glance that this explanation is the true one. The presence of oil in the embryo is explainable on the ground that the materials derived from the endosperm which are not immediately used by the growing embryo are reformed into oil and starch. This explanation was applied by Sachs to starch. It is evident that the presence of a “notable proportion of sugar” was regarded by du Sablon as the part equivalent of the oil. My own contention is that the oil found in the embryo is derived from the water-soluble materials, derived from digestion of oil, and which, upon entrance into the epithelium of the embryo, are not removed by use, either there or elsewhere. In support of this view the following additional evidence is advanced: In a germinating seedling, the extra-seminal portion of which was two millimeters long (figure 25) the digestion of oil in the endosperm in front of the end of the cotyledon, which alone at this stage is haustorial in nature, was pro- ceeding. There was, however, no trace of oil to be found in the epithelium of the cotyledon in this region, but only in that flanking the cotyledon, opposite endosperm cells in which no digestion is going on. The distribution of oil is shown in figure 25 by the stippling. In the same general region, but in the parenchyma beneath the epithelium there is starch to be found. In a somewhat older seedling (f. 24) there was no oil to be found in the epithelium at all, although must ensue upon the accumulation and reduction of oil, which doubt- less occur during relatively short periods of time. In examining liv- ing material, Reed further speaks of numerous large granules all of which react to protein reagents, but does not indicate that other re- agents were employed. The subject of Reed’s study is admittedly peculiarly difficult, and for this reason it is all the more important that the appearances of the cells in question should be explained only - after taking all the substances occurring in them into consideration. 150 MISSOURI BOTANICAL GARDEN. the region of digestion had by this time spread along the flanks of the cotyledon. In two seedlings in which the first foliage leaf was 3.5 em. long, in which therefore the haus- torium was quite large and nearly fully developed, I found the oil very unevenly distributed. In most of the epithelium cells there was none. A comparatively few cells in isolated groups showed a rich oil content. In no case was there any oil in the parenchyma below. In a similar seedling after a week’s exposure to ethyl nitrite, there was a very large oil content, the epithelial cells being quite replete,** so that one could hardly observe the remaining contents. Scarcely a cell varied from this condition. Similar results were ob- tained in a younger stage when the first foliage leaf was just emerging. The conclusion is therefore 7 ie that the disappearance of oil during the development of the embryo and during germination is due to the same cause, namely, digestion. This digestion results in the formation of water-soluble sub- stances, and not of an emulsion. The reappearance of oil in the embryo is due to its reformation from these sub- stances in consequence of the failure of the embryo to con- sume them as rapidly as formed. The particular behavior of the protein I have not espe- cially considered, beyond to note the time of its disappear- ance from the cell relative to that of the oil. Sachs believed that “albumen” enters the embryo “as such.” It is appar- ent that this material must undergo proteolysis if we accept evidence of the same character as that advanced with regard to oil and cellulose. The Tension Zone.—The limits of the various zones are not sharp, so that, in making diagrammatic representations there is necessary some compromise as a sacrifice to con- fusing detail. In the first stage considered (f. 14a) the tension zone is little more than a vague layer of cells (6) somewhat crowded between the digestion zone a and the thin 31 A large increase in the starch content of the parenchyma beneath the epithelium was also noted. RE ee TIS EMM Cg the EMO, Beg Raped RAN Gene ae gy. eae EMBRYO, SEED AND CARPEL IN THE DATE. 151 walled but growing endosperm, b’. With a slight advance in age, it becomes evident that the inner part of zone 6 is giving cells to be digested, and these then become part of zone a. At the same time, zone b’ becomes narrowed down by the advancing zone c (f. 16), endosperm in which re- serve cellulose is being laid down. This results in crowding the cells in which divisions are taking place into a narrower compass, and they occupy a region whose position is approx- imately represented by the narrow cross-hatched line between b and 0’. It will be observed that the actual histological results of tension are seen at first in zone b while in later stages they pass over into zone 0’, as represented in the dia- grams. That is to say, thin-walled endosperm cells entirely disappear, but are represented by cells which are secondarily transversely divided and at the same time somewhat second- arily thickened. Zone c’ disappears because tannin does so. This fact is represented in the diagrams by the line of de- markation overtaking, and becoming coincident with, the inner limit of zone ¢’. In endeavoring to understand clearly the course of events it is necessary to keep in mind the directions of growth (1) of the endospermic plug tissue overlying the base of the embryo, and which thickens independently of the main mass of endosperm; (2) of the embryo, which is growing in thick- ness and length, on the one hand; and (3) of the main body of endosperm, expanding radially in all directions toward the inner integument as a limit. The enlarging endosperm cells, namely those in the interior, push against the dividing endosperm cells, which are peripherally placed. These oc- cupy a position, therefore, between the inner integument and the inner part of the endosperm, the cells of which are enlorigating radially. These thin walled, dividing cells constitute a tension zone. But in the region of the embryo, the digestive zone takes the place of the inner integument, so that the tension zone, where the secondary division there- fore are taking place, dips inwardly. The plug tissue how- ever is also in division, and this independent disc of ten- sion tissue meets and merges with the other at the place 152 MISSOURI BOTANICAL GARDEN. where it dips inwardly. The place of juncture becomes more and more obvious with age till the completion of secondary cell-wall thickening, when it remains visible on account of the irregularly matched cells. This is a plane of weakness where, during the initial stage of germination, the endosperm plug is released. The constant advance of the definitive endosperm opposed to the growing embryo results in the reduction of the zone in question. I have already pointed out that the secondary divisions in the endosperm cells which characterize it are always parallel to the integuments. They are therefore at right angles to the axis of the embryo. If these are taken as the criterion of the zone, it is seen that it is at last reduced to a thin layer of cells (b’, f. 19, 20), which are able neither to take on the definitive character of endosperm cells, nor entirely to resist the influence of the embryo. This is shown by the partial thickening of the walls, and in the partially digested condition of the contents. The shearing caused by the opposed directions of growth is also to be seen in the oblique and more or less distorted forms of the cells (f. 20, 21). Tannin in the zones of digestion.—It has been shown that during the third period of development tannin figures prom- inently in the endosperm in connection with the laying down of reserve cellulose. It has been pointed out, however, that the reaction is not confined to the cells alone which are active in this regard, that, in a word, it occurs everywhere in the endosperm, the statement being intended to include the zones of digestion. For example, ethyl nitrite material thir- teen weeks after pollination showed that the tannin in the tension tissue and in the digestion zones proper is in marked amount, as it is also in the embryo. This is true of younger stages, and of more advanced ones until the condition is reached represented by figure 18, when, along with the gen- eral reduction of tannin throughout the endosperm, the amount in the digestive zones is also reduced. This disap- pearance is not synchronous with that in the embryo, in which tannin is discoverable for a longer period. It must ef ae Se el ke el ~ st . = a ee ah Y Ai ete _ | A a ere eae oe. ee EMBRYO, SEED AND CARPEL IN THE DATE. 153 also be added that the tannin in the cells in these zones, is not confined to the cell walls as it is elsewhere in the endo- sperm. This, with the earlier disappearance of tannin in them than in the embryo, may be taken to indicate that the tannin here is concerned chiefly with the embryo. Digestion of Reserve Cellulose.—As reserve cellulose plays, if any, a very inconsiderable part in the nutrition of the embryo and because it has been studied, first by Sachs, and very carefully by Reiss (1889), both histologically and chemically, it has been no part of my purpose to concern myself with it. I can find during embryogeny no evidence that there is any digestion of reserve cellulose, even during the short period when this may be suspected to occur. On the contrary, as I have already pointed out, the process of cell wall thickening proceeds inwardly toward the embryo until the resting condition is entered. One point of dis- agreement, however, as between Sachs and Reiss has been looked into by me, namely, the presence or absence of the optical evidence of the primary membrane in the defini- tive endosperm cells. Sachs says: “. . . die primiiren Zellhiiute sind leicht als doppelte conturicte Lamellen zwischen den Verdickungschichten zu erkennen’’*? (1862, p. 242). This Reiss denies categorically, thus: “Die Winde lassen keinerlei Schichtung, auch keine Mittellamelle sichtbar hervor.” The fact appears to be that the observa- tion is not as easy as Sachs’ wording would lead one to expect. While not everywhere visible with equal ease, how- ever, there is no very great difficulty in determining the substantial correctness of his statement. As for the manner of digestion of the reverse cellulose, I am able to verify Reiss’ account. Origin of the Ferments. Sachs believed that the digestion of the endosperm in the date is due to a ferment secreted by the endosperm. Were this not so it would be difficult to explain the remarkable coincidence that the softening of the endosperm exactly cor- 32 Quoted by Reiss. 154 MISSOURI BOTANICAL GARDEN. responds to the growth of the haustorium. “Dieser Umstand macht es eher wahrscheinlich, dass die Epithel einen Stoff an die nichsten Endospermzellen abgiebt, der die Lésung des Zellstoffs bewirkt.” B. PHOENIX DACTYLIFERA. Transverse section through a germinating seed in which the embryo has formed in an abnormal position.—h, Haustorium. 7, Integuments. p, Endosperm plug which caps the embryo during the resting stage. 7, Raphe, partly torn out. «x, y, Portions of endosperm the cells of which are represented on a larger scale (the same for both) at the left. The relative position of this abnor- mally placed embryo and of a normally placed one is shown at m and a. Nevertheless, Sachs’ conclusion was not accepted without demur. Griiss (1896) especially thought he had proved to the contrary, though his evidence is not convincing. New- combe (1899) showed that a cytohydrolytic enzyme could ey vice % oe ec ae "S “— acti Spells 2 tak @ oo) FR es eee ey! ee es? “Re eee Ae ee i tala th Pes calm Ma OR ne ata aS Se RRS Kha fhe Sy 22 +c . ye 4? Pes ¥ EMBRYO, SEED AND CARPEL IN THE DATE. 155 be extracted from the endosperm, and he thought that it was present “in the softened layer of endosperm against the cotyledon” but it is clear from the context that he did not believe the enzyme to originate in the endosperm but in the cotyledon. The point however being moot, Pond (1906) showed conclusively that cytase does not originate in the endosperm. _ Sachs’ reasoning is thus justified. It is a well-known fact, however, that the outline of the zone of digestion is not parallel to the surface of the growing haustorium, and while this does not vitiate the reasoning it calls for explana- tion. Figure 25 shows that the digestion begins at the end of the cotyledon, where the endosperm cells are isodiametric. It is clear that there is a localization of enzyme secretion in this region of the cotyledon. The movement of the fer- ments into the endosperm from this on must be due either to the anatomical conditions in the endosperm, or to a con- tinuation of the localization of more active ferment secre- tion. If one follows the movement of the haustorium, one sees that it is not related to the shape or position of the cells of the endosperm (text f. B). Its more rapid growth may be parallel to or athwart the longitudinal axes of the cells, and is not always the same in amount and direction in the regions where the cells are approximately isodiametric. There is no evidence, moreover, that there is any more usable passage-way through the endosperm by virtue of thin- ner walls or more pores. We must conclude, therefore, that the shape of the digestion cavity is in large part determined by the localization of greater activity of ferment secretion in the haustorium. We are urged to the conclusion, fur- ther, that the greater secretion of ferments is at first at the end of the cotyledon, but that it becomes relatively less active, the scene of greater activity passing over to the sides of the haustorium. In this connection, the finding of a seed in which the embryo had suffered abnormal displacement, so that it lay in a position almost antipodal to the normal one. is of interest. It was discovered only after it had been germinating for some time (f. B). 156 MISSOURI BOTANICAL GARDEN, The endosperm was normal in respect to its histological characters, its cells being arranged in the usual way. Pre- sumably there was some adjustment quite near the embryo, and there is evidence for this in the configuration of the cells in the plug. Both the region of greatest growth in the haustorium and the area of most active secretion are opposite to the greatest thickness of endosperm to be digested. We cannot in this case say that this part of the haustorium is the end or the side, but it appears conclusive that the char- acter of the endosperm cells has nothing to do with the direc- tion of growth of the haustorium. We may, indeed, say that the behavior of the haustorium is adaptive, and that this organ adjusts its behavior adaptively when the normal conditions are disturbed, until a more satisfactory explana- tion is to be had. SUMMARY AND CONCLUSIONS. The more important features of the foregoing account may be summarized as follows: 1. The development of the embryo, seed and pericarp with reference to nutrition have been followed from the anatomical-physiologica} point of view. The embryological history has been articulated with the period of germination, and the continuity or discontinuity of the various pro. cesses involved in the digestion of the endosperm has been deter- mined. 2. Before the time of pollination, the antipodal cells, and for some time thereafter, a tapetum are active agents in the nutrition of the embryo-sac. This first digests the small nucellus; it then becomes active at the antipodal pole, and forms a group of digestive pockets around the degenerating antipodal apparatus which ultimately form a single cul-de-sac, the function of which is to digest ovular (chalazal) tissue and to receive nutrients from the raphe. The chalaza in the mature seed is not determined by the configuration of the integu- ments (as is frequently the case), but by the manner of development of the endosperm cul-de-sac, which may in turn be related to the main path along which food materials pass in the raphe. 8. The earlier phase of development of the seed is marked by a longitudinal distortion which results in altering the position of the embryo, swinging it through an arc of 90°. This feature is common among the Palmae. EMBRYO, SEED AND CARPEL IN THE DATE. 157 4, During the first three months following pollination the growth of the embryois very slow. This period is devoted to the growth of the tissues of theovule. The following period is marked especially by the development of the embryo and the laying down of reserve cellulose by the thickening of the walls of the endosperm cells. 5. Starch has been found to play only a brief réle in the basal por- tion of the carpel and in the ovule. It is found in small and con- tinually reduced quantities until the embryo is 6-7 weeks old. Atthis time traces only are found in the integument between the micropyle and the funicle. Subsequently none is found, till the embryo is about 17 weeks old, when it may appear in the radicle and in the cotyledon. Its appearance in any position may be taken as indicating a temporary reduction of growth activity in that place. This appears also to be true both of starch and of oil in the early stages following germina- tion. 6. Certain tannins are important quantitatively and in their nutri- tive relations. I have distinguished for convenience between aplastic and plastic tannin. Aplastic tannin appears in particular cells and remains there permanently. Plastic tannin undergoes translocation, is consumed and disappears. This is taken as evidence of its nutri- tive rdle. (a) Aplastic tannin in the carpel occurs in the hypodermis and in the idioplasts. The latter form a well marked sub-hypodermal zone _which begins to be laid down at about the time of pollination. Tannin as such does not migrate into these cells, nor into the hypodermal tannin cells. There is therefore no evidence that during ripening there is a segregation of tannin, as reported for certain other fruits. During ripening the tannin in these cells, as indeed all the aplastic tannin, becomes insoluble, and hence tasteless. There is no evidence forthcoming that this tannin is a glucoside and is oxidized, as tenta- tively held by Slade* and for which a slight amount of evidence has been mentioned by Vinson. Slade’s view, however, is possibly applic- able to the tannin described in the endosperm, and it would thus be a source of energy (vide infra). The idioplasts form a morphological layer of cells which bends toward the ovule in the sutural region, but is here discontinuous. There is evidence here however of the presence of a small amount of tannin of translocation which becomes more and more marked in quantity toward the base of the carpel and in the tissues subjacent to the ovule, or young seed. Aplastic tannin also occurs in a comparatively few, usually elon- gated, elements scattered throughout the inner mesocarp, and ina few cells also of the endocarp. 33 See Vinson (1907). 158 MISSOURI BOTANICAL GARDEN. (b) In the ovule all the tannin is at first plastic. Its distribution is such as to indicate clearly that it is concerned in the nutrition of the embryo-sac. The period following fertilization is characterized, on the anatomical side, by the torsion of the ovule, and, on the physi- ological side, by the rapid growth of the chalazal end of the embryo- sac into the tanniferous tissue of the raphe. The whole of the ovular tissues, properly speaking, now contain tannin, and there seems to be little doubt that it is given over to the growing endosperm. However, there begins the individualization of cells as tannin-cells, which appear at first in the raphe and about the outer limits of the chalazal] tissue. Later they appear also in the integuments, with the exception of the epidermis and an irregular hypodermal layer. This tannin is like that in the carpel, as it remains permanently in the special cells. Definitively, the integuments and raphe are completely loaded with insoluble tannin, with the exceptions noted. The precise relation between the plastic tannin and that which gradually appears in the manner described is not clear. They may be quite distinct, or the aplastic tannin may represent unused or unusable tannin side- tracked to accumulate as waste. (c) In the endosperm there is from the time of fertilization till about the 19th week a very large amount of tannin. Toward the period between the obliteration of the endosperm utriculum and the secretion of reserve cellulose, there is a reduction of tannin, but when the reserve cellulose begins to be laid down there is at once a large increase in the amount of tannin within the same area. This tannin is to be found in the walls as well as in the lumen, and not as artefact. With the maturation of the endosperm, the tannin disappears in a manner to preclude the explanation that it is thrown out of the endosperm as waste. Whether this disappearance is caused by oxidation, as is known to occur in the apple*, or by its incorporation into a substance (possibly the reserve cellulose) with a more complex molecule, the evidence does not help us to decide. (d) Tannin appears in the embryo in larger or smaller quantity throughout the whole of the time of development. It seems to be a principal nutrient during the earlier phase, for which there is the positive evidence that tannin is to be found in the sphere of embry- onic influence as droplets, when it is seen nowhere else in similar quantity or appearance. None is found in the resting embryo. Aplastic tannin occurs in certain situations in the seedling, as observed by Sachs, and rightly interpreted by him, so far as we can yet see, as waste. 4 Lindet, cited by Kastle, 1910. SSG a art BOR GE wee gee PE ke) aR tea, RRMA RE LEE fic RR, Yee ene Mee Ree ar ec ee Sema, bab: Py . bY PY EMBRYO, SEED AND CARPEL IN THE DATE. 159 7. Oil. There is no oil in the carpel, integuments or raphe at any time. Oil first appears in the endosperm during the process of ingrowth leading to the obliteration of the utriculum. The amount increases steadily until the utriculum is obliterated, or shortly thereafter. There is then a reduction in amount until the secretion of reserve cellulose has advanced somewhat, at a time approximately between that represented by figure 26a and that by figure 27. It then accum- ulates till the resting condition is reached. During germination it is digested before passing into the embryo. In the embryo, oil has been found soon after it appears in the endosperm. There continues throughout the embryonic period a digestion of oil in the endosperm, which is carried on in the same manner as during germination. Its appearance in any part of the embryo is correlated with the relative cessation of activity in that part. 8. Digestion of the primary cell walls near the embryo occurs. This begins approximately between the stages represented by figures l4a and15. The evidence for this conclusion is to be found in the change, effected in the cell walls, in a column of tissue opposite the cotyle- donary pole of the embryo. Here the walls react to rather strong iodine (KI-I) by becoming blue. This material appears to afford but a relatively small amount of food material. The same form of diges- tion has been described by Green in Livistona during germination. In the date, however, it ceases at the entrance of the embryo upon the resting stage, or, at the latest, very soon after germination be- gins. During the embryonic period therefore, the primary membranes are digested, and this, as recorded by Sachs, does not occur during germination. The middle lamella appears to persist. 9. As shown by Pond, the digestive ferments are secreted by the embryo entirely. It is here shown that the secretion is localized, and is not equally active throughout the superficies of the embryo ‘or haustorium. But this does not wholly explain the behavior of the movement of the haustorium through the endosperm. TEE ee ae, ee 14 z : " i 4 a 160 MISSOURI BOTANICAL GARDEN, LITERATURE CITED. 1899. Balicka-Ivanovska, G. P. Contribution a l’étude du sac embry- onnaire chez certaines Gamopétales. (Flora 86: 3-10). 1906. Bigelow, W. D., Gore, H. C., and Howard, B. T. Growth and ripening of persimmons. (Jour. Am. Chem. Soc. 38: 688- 703. June). 1906. Dekker, J. De looistoffen. Botanisch-chemische monographie der tanniden. I. Bibliographie, Botanie, Physiologie. (Bull. Kol. Mus. Haarlem, no. 35. December.)—An ex- tensive bibliography, summary of observations and methods and an outline of the more important trends of thought. Important to students in this field. 1908. Geerligs, H. C. Prinsen. Rapid transformation of starch into sucrose during the ripening of some tropical fruits. (In- tern. Sugar Journal. Manchester. 10: 372-380. Aug.). 1898-9. Goldfluss, M. Sur la structure etc. (Jour. de Bot. 12: 374, 1898; 18:9, 47, 87. 1899). 1899. Green, J. R. The soluble ferments and fermentation. Cam- bridge. 1896. Griiss, J. Beitrige zur Physiologie der sigs (Landw. Jahrb. 25: 385-452. pl. 2-3). 1897. Griiss, J. Studien iiber Reservecellulose. (Bot. Centralb. 70: 242-260). 1906. Howard, B. J. Tannin cells of persimmons. (Bull. Torr. Bot. Club. 83: 567.—(See also Bigelow, Gore and Howard). 1902. lkeda, T. Studies in the physiological functions of antipodals and related phenomena of fertilization in Liliaceae. 1. Tricyrtis hirta. (Bull. Coll. Agri. Imp. Univ. Tokyo. 5: 41-72). 1910. Kastle, J. H. The oxidases and other oxygen-catalysts con- cerned in biological oxidations. (Pub. Health Mar. Hosp. Serv. U. S. A. Hyg. Lab. Bull. 59). 1904. Kirkwood, J. E. The comparative embryology of the Cucur- bitaceae. (Bull. N. Y. Bot. Gard. 3: 313-402. 7 Oct.). 1909. Lidfforss, B. Untersuchungen iiber die Reizbewegungen der Pollenschlauche. 1. Der Chemotropismus. (Zeitschr. f. Bot. 1: 448. 1909).—Abs. in Bot. Gaz. 49: 230. March. 1899. 1902. 1907. 1899. 1906. 1904. 1889. 1899. - 1862. 1909. 1906. 1907. 1907. 1909. 1910. 1892. EMBRYO, SEED AND CARPEL IN THE DATE, 161 Lloyd, F. E. The comparative embryology of the Rubiaceae. (Mem. Torr. Bot. Club. 8: 1-26. Aug. 1899). Lloyd, F. E. The same, part 2. 27-112. Feb. [Lloyd, F. E.] 18th Annual Rep. Ariz. Exp. Sta. June 30, 1907. Statement, on p. 236, embodied in the report of the Associate Chemist, Dr. A. E. Vinson. Newcombe, F. C. Cellulose-enzymes (Ann. Bot. 18:49. Mar.). Pond, R. H. The incapacity of the date endosperm for self- digestion. (Ann. Bot. 20:61-78. Jan.) Reed, H. S. A study of the enzyme-secreting cells in the seed- lings of Zea mais and Phoenix dactylifera. (Ann. Bot. 183 267-287. April. Reiss, R. Ueber die Natur der Reserve-cellulose und iiber ihre Auflésungsweise bei der Keimung der Samen. (Land. Jahrbiich. 18; 711-765. pl. 14. du Sablon, Leclerc. Sur la digestion de l’albumen du dattier. (Rev. Gén. de Bot. 9: 395-398. 1897). Sachs, Julius. Zur Keimungsgeschickte der Dattel. (Bot. Zeit. 203 241-6; 249-252. pl. 9. 1 Aug.). Servettaz, C. Monographie des Eléagnacées. Thesis (Paris). Dresden. [Thornber, J. J.] Statement made in the report of the Asso- ciate Chemist, Dr. A. E. Vinson, p. 164, 17th Ann. Rep. Ariz. Agri. Expt. Sta. Vinson, A. E. The function of invertase in the formation of cane and invert sugar dates. (Bot. Gaz. 48:393. June). Vinson, A. E. Some observations on the date. (Plant World, 103: 259-262. Nov.). Vinson, A. E. The influence of chemicals in stimulating the ripening of fruits. (Science. n. s. 803604. 29 Oct.). Vinson, A. E. Fixing and staining tannin in plant tissues with nitrous ether. (Bot. Gaz. 493 222-224. March). Westermaier, M. Zur Embryologie der Phanerogamen insbe- sondere tiber die sogenannten Antipoden. (Verhandl. der K. L.-C. D. Acad. d. Nat. 56: 1-39). 162 MISSOURI BOTANICAL GARDEN, EXPLANATION OF PLATES. Plate 15.—Phoenix dactylifera. 1, Portion of an ovule at about the time of pollination, showing the embryo-sac, tapetum and nucel- lus, just prior to the disintegration of the last named. 2, Ovule with the contiguous carpellary tissue to show the stylar canal leading to the pollen-tube guiding tissue. The occurrence of tannin in specia- quantity in this tissue in front of the micropyle is indicated by stip- pling, as also in the embryo-sac, tapetum and inner integument, and chalazal tissue. ‘2a, 2b, Transverse section through the ovule to show the pollen tube canal, single above and double at the funicle. 2 c-e, Transverse sections to show the stylar canal at different levels. The dots in the hypoderm represent tannin; stone cells cross- hatched. Raphide cells are seen in 2d. 38, Embryo-sac and tape- tum at this time. The remains of the nucellus are seen as a cap over the end of the embryo-sac. Digestion of the chalaza is proceeding rapidly. 4, Pit formed of contiguous thick walls of adjacent cells; the antipodal cells within this pit before degeneration of the basal cell is apparent. 4a, The basal and middle cells degenerated. 5, Ovule with one-celled embryo. Multinucleate parietal endosperm. Its activity beyond the antipodal apparatus is evident. 6, Ovule 7-8 weeks after pollination. Distribution of tannin (the more densely reacting tissues) shown by the dots; starch by cross-hatching. Deg- let Noor. 7, Ovule, Deglet Noor, 8-9 weeks after pollination. The post-chalazal growth of the endosperm is seen, together with the general torsion of the ovule in the direction indicated by the arrow points. ‘Tannin indicated by the dots and conventionalized cells. 8. Endosperm cells at ten weeks, when the oil is first seen. 9, The nuclei of the flattened endosperm cells of the superficial layer bordering the utriculum. The manner in which the oil appears with reference to the nucleus is evident. Plate 16.—Phoenix dactylifera.—10, (Cf. f. 8-9) Ovule, Deglet Noor, 11.5 weeks. Endosperm growing inwardly to obliterate the utriculum. Beginning of oil secretion. 11, Portion of transverse section of car- pel and ovule to show the sutural sector in which a diffuse tannin reaction appears. 12, Longitudinal section of mature seed indicating the topography. Ch. chalaza. End. endosperm. 13, Transverse sec- tion through young seed through the chalaza. The chalazal tannin tissue proper is indicated by dots. Tannin is present however, else- where in the raphe and integuments. Same ovule as inf. 14. 14, Young seed at the time when the closure of the endosperm is com- plete. The distribution of oil about the embryo is indicated by the size of the dots, 11 weeks after pollination. 14a, The region about the embryo at about this time, in which no digestive action on the cell walls is observable; a, Digestive zone; b, Tension zone, where cell divisions occur; b’, Thin-walled growing endosperm. 15, Ten EMBRYO, SEED AND CARPEL IN THE DATE. 163 weeks after pollination. Area about the embryo as seen in transverse section soon after the digestion of the cell walls is begun in @ and a‘; a, Crushed cells empty save of oil drops; a’, Uncrushed cells like those in a; 6, Cells in which the protoplasm is being attacked and the oil segregates; b’, Thin-walled endosperm between the growing endos- perm cells of 6 and those of c. 15a, The integuments and edge of endosperm at about this time. As yet no special tannin cells have appeared (cf. f. 31); 0. 7. Outer integument. 7. 7. Inner integument, 16, Thirteen weeks after pollination. For full explanation see p.—. 16a, Single endosperm cell in the course of secondary thickening taken from the position in f. 16 indicated by the black square at x. Plate 17.—Phoenizx dactylifera. 17, About (17 weeks after pollina? tion) and 18, For full explanations of these figures see p. 142. The tannin in the embryo at the age of figure 18 is shown by the dotting, . which is intended to indicate the areas of denser reaction only. About 18-19 weeks after pollination. 18a, Camera lucida drawing to show the distribution of oil globules in zones a and a’ of figure 18, in which the same is shown diagrammatically. The irregular particles are protein. 19, Nearly mature embryo; a, Crushed cells (a, a’, f. 20); b’, Partially thickened cells (b’, inf. 20); ¢, Definitive endosperm. 20, Detail of f. 19 through the endosperm next the embryo; a, Crushed cells with minute oil droplets; a’, Partially crushed cells with larger oil droplets; b’, Partially thickened cells with transverse secondary walls, with large drops and the remains of the protoplasm. Definitive endosperm to the right of 6’. Deglet Noor. 21, Cells of definitive endosperm which have arisen by secondary division of elongated cells which have resisted digestion. 22, The same as 20 but in front of the cotyledon, showing the final divisions of the endosperm cells and the digestion of their contents. 23, Detail to show the position of the secondary division walls in the endosperm which resists digestion. a. Crushed cells. 6” partly thickened cells with their walls at right angles to the axes of the endosperm cells from which they arose. 24, Haustorium and adjacent endosperm of seedling with extraseminal portion 2 cm. long; a, Crushed walls, blue with iodine; b, Crushed walls, not blue with iodine; ¢, Cytatic digestion advanced, oil agglo- merated; d, Cytatic digestion beginning, oil agglomerating, protein digesting; e, unchanged endosperm. 25, A younger stage than 24, showing the embryonic layer blue-reacting with iodine still intact. Cytatie digestion has begun in front of the cotyledon. Dots in the embryo indicate tannin; cross-hatching, starch. Plate 18.—Phoenix dactylifera. 26, Embryo and adjacent endo- sperm showing the distribution of tannin. 11 weeks after pollina- tion. 26a, Transverse section of the same seed as 26, showing the areas in which the secondary thickening of the endosperm cells has begun. Here tannin is abundant, as shown by the dots. 27-29, Successively older stages showing the spread of the endosperm tissue oe Ae ee ee 164 MISSOURI BOTANICAL GARDEN. with thickened walls and large tannin content: 27 shows the tannin in and near the embryo; 28 shows the tannin in the embryo; 27a. Large tannin content of the endosperm cells of the chalazal cul-de- sac where the secondary thickening of the cells has gone on as in the main mass of endosperm. 30. Transverse section of seed near ma- turity in the endosperm of which tannin still is present in certain sectors. 31, The integuments in their definitive condition. All the cross-hatched cells contain tannin. 32, Exocarp. Diagrammatic rep- resentation of the various tissues; e, Epidermis; s, Stone cells; p, Parenchyma, of which a few cells are drawn here and there to show their relative sizes; id. tannin idioplasts. 33, Outer exocarp to show the tannin cells in the hypodermal layer, and the tannin globules in the parenchyma adjacent to the stone cells, st. (Rhars). 34, Endo- carp in transverse section, t, elongated tannin element. Rept. Mo. Bot. GARD., VOL. 21. PLATE 15. PHOENIX DACTYLIFERA. Rept. Mo. Bot. GARD., VOL. 21. PLATE: 16; PHOENIX DACTYLIFERA. PLATE TT. Rept. Mo. Bot. GARD., VOL. 21. PHOENIX DACTYLIFERA. Rept, Mo. Bor. GARD., VOL. 21. PLATE 18. eres PES LD Lig HT; PHOENIX DACTYLIFERA. so TL OW ee COR, ee ey. ERD RE TEA, RE ear VERE ere oe sia aia ek ea ine Be , " : ILLUSTRATED STUDIES IN THE GENUS OPUNTIA —III. BY DAVID GRIFFITHS. Studies in field and cultivated plantations during the past five years have brought together sufficient data to warrant the addition of the following species to the genus Opuntia. Opuntia alta sp. nov. A strictly arborescent species with distinct, short, cylindrical trunk, 3 to 3; dm. in diameter and huge, spreading branches 14 to 2 dm. in diameter, early becoming bare, brown-gray and scaly-cracked, 24 to 34 meters high in large specimens, mostly lower than this and often with a spread of 3 meters, mostly about 18 dm. high; joints sub-circu- lar, ovate to obovate, quite uniform in size, about 18 by 25 cm. or possibly more often 17 by 21, blue-green, thin, with surface only slightly raised at areoles, turning gray-green and sealy-cracked in age; areoles obovate, about 3 to 5 mm. long and 3 cm. apart, slightly raised, closer on edges where they are also larger, rendering a some- what congested appearance to spines and spicules, enlarging with age to sub-circular and often 1 cm. in diameter, tawny when young, be- coming dirty black in age; spicules yellow, abundant, 8 mm. long, mostly scattered throughout the entire areole but more numerous above, increasing with age and filling and crowding the entire areole, the tissues of which proliferate slightly into a raised hemispherical structure, the outer spicules becoming dirty yellow and the central newer ones a brighter color; spines yellow, typically, one 15 to 20 mm. long, erect, and one 10 to 12 mm. long, and sloping down on sides of joints and two or even 3 long ones on edges, not increasing with age to any appreciable degree, at about 5 years of age and older the trunks becoming comparatively bare of spines but covered with the scattered, formidable, bunches of spicules only, slightly flattened and the largest ones faintly annular; flowers yellow, with broadly-rounded, wavy-margined, obovate petals, with abrupt cuspidate point, filaments yellow, greenish at very base, style white, stigma yellowish tinged, 10 to 12-parted; ovary broadly obovate to conical, about 2 by 3 cm. having small sub-circular areoles bearing spreading, unequal, yellow, fugacious spicules about 5 mm. long. This species is distinctly arborescent in habit, one of the tallest and largest of our United States forms. It is very different indeed from Opuntia cacanapa, although one or (165) 166 MISSOURI BOTANICAL GARDEN. two of its characters seem to indicate a relationship. The type is yellow flowered, but there are forms which have flowers greenish-yellow, even lighter in color than the flowers of Opuntia leptocaulis. Owing to a constant tendency to segregation of species, it is considered preferable to recognize the yellow flowered form as typical, but I have little ques- tion but that the two forms must be considered specifically the same. The greenish-yellow flowers remain the same color throughout the day, but in the typical form where the flowers are lemon yellow they turn to orange in the aft- ernoon, and when dried or closed upon the plant are dis- tinctly reddish tinged, which is true of the type specimen. Corresponding changes in color take place in a large pro- portion of the species of the flat-jointed opuntias. The description is a compilation of a description and two sets of notes made in the type locality when specimens were collected. The type is No. 9914 D. G., collected March 13, 1910, near Brownsville, Texas, the flowers being collected from the same plant April 20, 1910.—Plates 19 and 20, upper figure. Opuntia xanthoglochia sp. nov. An erect to ascending, spreading, tuberous rooted species with ra- diating arms frequently resting on their edges on the ground, seldom over 3dm. high, but often 8 or 9 dm. in diameter; joints usually wid- est near middle, but sometimes obovate, mostly more or less pointed above and below, about 10 by 15 cm., dark green, glaucous when young, wrinkled and decidedly raised-tubercular at the areoles; areoles about 4 mm. long, obovate, about 2 cm. apart, enlarging and becoming sub-circular in age, sometimes 1 cm. in diameter; leaves subulate, cuspidate, slightly flattened; spicules light brown when young, but soon turning light yellow, conspicuous and formidable, in compact, 4 to 5 mm. long tufts in upper portion of areole, increasing with age, the new ones coming from center of areole and longer from year to year, the annular growths being in concentric circles, but brown color only appears on young joints, the change in color beginning to take place early in May; spines delicate, flattened, often twisted, usually 1 or 2, the longest about 2 cm., erect, or when 2 or 8, one sloping downward; flowers lemon yellow, brownish red within, lax, 7 cm. in diameter, turning very light orange to pinkish, filaments greenish, style white, stigma white or very light yellowish, 5 to 6 parted; ovary long, obovate or obconical, somewhat tubercular with raised areoles which are small and sub-circular, about 12 mm. by 6 cm. ILLUSTRATED STUDIES IN THE GENUS OPUNTIA—IlI. 167 The plant belongs to the Opuntia macrorrhiza group, but, as will be noticed from the description of the plant body, it is very different from that or any other of the related species. The flowers are exceedingly variable in character. At times they are pure lemon yellow throughout with no indication of red at base of the petals. At other times the lower one-third may be red and all degrees of variations between these two extremes may be found. Sometimes the red is confined to the mid-rib of the petal, being uniformly distributed throughout its length. The description is drawn from a cultivated plant grown from a single joint cutting set July 11, 1908. It was col- lected on that date near Milano, Texas. The description of the flowers and the notes on the flowers were made in the type locality from the type plants May 15, 1910. The type specimen is No. 9355 D. G., prepared May, 1910, from cultivated specimens bearing the same number.—Plate 20, lower figure. Opuntia Gomei sp. nov. Plant ascending with main branches commonly on edge and second- ary ones erect from them, mostly about 1 meter or less high and often 3 or more meters in diameter, the old centers containing much dead material when plants are large; joints sub-circular to ovate, rarely blue-green, varying to sometimes slightly yellowish green, 30 to 40 cm. in diameter, or often in last year’s joints only 20 cm. in diameter and then almost invariably sub-circular, more or less raised at areoles and larger joints always more or less wavy; areoles obovate, 6 to 10 mm. in diameter, bright brown when young, changing to dirty brown and finally to gray-black, mostly raised and surrounded by an irregular, dry, brown-gray, slightly cracked rim or area, varying from 2} to 5 cm. apart; spicules yellow, abundant, very prominent, about 1 cm. long, more abundant above, but often filling entire areole; spines yellow, somewhat flattened, often faintly annular, not twisted, 8 to 5 cm. long, erect, divergent, sometimes increasing irregularly with age, 1 to 5 in number, mostly 2 to 4; flowers yellow, stigma large, bright deep green, 9 to 10 parted; fruits reddish purple. This species belongs to the O. Lindheimeri group. It inhabits the lower edges of the slight elevations in the delta of the Rio Grande River and often extends both into the huisache flats and the mesquite areas of higher elevations. 168 MISSOURI BOTANICAL GARDEN. It is, therefore, in all probability, able to thrive upon land containing considerable soluble salts. It is often found growing scatteringly upon land entirely devoid of brush and which periodically overflows. In these areas, however, it frequently gets killed out on account of the overflows which occur at irregular intervals, and again becomes estab- lished thinly before another period of overflow occurs. The description was drawn in the field when the type specimen was collected, notes on the flowers being subse- quently secured. The type specimen is No. 9913 D. G., col- lected near Brownsville, Texas, March 13, 1910. It is named in honor of Mr. William Gome, whose assistance it is a pleasure to acknowledge.—Plates 21 and 22, lower figure. Opuntia pachona sp. nov. Plant tall, arborescent, widely branching but not as divergent as O. streptacantha, with distinct cylindrical trunk 1 to 1.5 meters long, the whole plant normally 3 to 5 meters high; joints about 20 by 32 em., obovate, deep dark green, with often a white bloom, similar to O. streptacantha in color but on the whole a little lighter; areoles 2} to 3 ecm. apart, ovate to sub-circular; spicules bright dark reddish brown, often formidable, in compact tufts in upper part of areoles, increasing in numbers and length with age, about 2 mm. long; spines white with bonelike tips, flattened or triangular, often slightly twisted, mostly slightly but never tightly recurved, 2 to 5 or 6 in number, mostly 2 to 4 on last year’s joints, increasing on old trunks to 6 or 8 and becoming larger and stouter than on young joints, diverging in all directions upon old trunks although upon young joints they are slightly recurved or sloping downward; fruits about 40 to 45 by 50 to 55 mm. bright, glossy, purplish-red when mature, beset with rather large cir- cular areoles containing formidable reddish brown spicules surrounded by the blackened ends of the dark tawny wool. Opuntia pachona belongs to an important group of prickly pears of the highland of Mexico in which the expressed juice contains a large amount of solids in suspension. It is, therefore, one of the forms used in the manufacture of queso. Although similar in color to O. streptacantha, it can hardly be considered as closely related to this species, but should be looked upon as representing the Opalillo- Lionero group none of which, so far as I am aware, have satisfactory descriptions. The description is a compilation from two sets of char- ILLUSTRATED STUDIES IN THE GENUS OPUNTIA—III. 169 acterizations, modified by several miscellaneous notes from various localities, one -description having been written in the type locality and the other drawn from a three year old seedling. The type specimen is No. 8141 D. G., collected near Zacatecas, Mexico, September 19, 1905.—Plate 22, upper figure. Opuntia lubrica sp. nov. A low ascending, spreading species very similar in habit to O. micro- dasys, frequently 44 dm. high and when well developed 10 dm. or more in diameter; joints sub-circular to obovate, about 15 by 20 cm., or in case of last joints of previous year about 12 by 15 cm., bright, glossy, leaf-green, very evidently papillate but scarcely pubescent under a lens; leaves subulate, cuspidate-pointed, 6 to 9 mm. in length; areoles 15 to 22 mm. apart, 4 to 6 mm in diameter, sub-circular, prom- inent; spicules prominent, 4 to 5mm. in length, erect, bushy, in cres- centic tufts in upper portion of areoles, becoming much more numerous ‘in age, and at 2 to 4years completely filling the areole, and, like O. rufida and some other species, becoming very abundant and conspicu- ous by proliferation of areolar tissue into short raised or columnar structures; spines exceedingly variable, sometimes {nearly absent, again quite abundant and irregularly distributed, none too many, mostly 1 to 8, becoming more numerous with age and in scattering areoles to as high as 16, mostly about 12 mm. long, but sometimes 2} em., yellowish, translucent, bonelike, sometimes darker at base; fruits decidedly acid, light red without with yellowish green rind and red pulp; seed small, thin shelled, about 3 mm. in diameter. The habit of this species resembles that of O. microdasys, but it is a more robust plant with heavier glossy joints smooth to the touch and with color of entirely different character. The description is a compilation of partial descriptions made in the type locality and notes upon cultivated, nearly mature plants. The type is No. 8439 D. G., collected near Alonzo, Mexico, August 23, 1906, backed up by several specimens put up from cultivated material. The cultivated plants have not yet bloomed, although one of them is now in the third year’s growth from a single joint cutting.— Plate 23. ; Opuntia nigrita sp. nov. An erect, open-branching, stout, arborescent plant with distinct cylindrical trunk and spread of branch about like O. pachona, com- — 170 MISSOURI BOTANICAL GARDEN. monly 3 to 4 or more meters high, in cultivation plants have made a 12-dm. growth in 2 years from single joint cuttings; joints obovate, broadly rounded above, about 17 by 25 em., papillate-pubescent under a lens, this scarcely detected by touch except in current season’s growth, deepdark green, with young growth of course lighter in color; areoles at first brown turning dirty black, broadly oval to sub-circular or even obovate, about 5 mm. in longest diameter, 2 to 24 cm. apart; leaves short, subulate, cuspidate, mostly only 8 mm. in length; spic- ules brown, in hemispherical bunches in upper part of areole, 2 to 3 mm. long at first, but developing anew from lower central portion of spicular area to often 1 cm. in length, this new development continuing for a couple of years; spines white, turning to a dirty gray, not very stout, but resembling those of O. chavefa, but longer, numerous, erect, spreading in all directions, 4 to 6 on last year’s joints, but be- coming very numerous in places on old trunks, even 25 to 30, varying in some areoles from 15 to 25 mm. in length; besides spines and spic- ules, current year’s joints bear two delicate, hairlike, fugacious spines, about 5 mm. long; fruit small, about 3 3.5 cm. purplish red, with pulp deeper in color and seeds easily separable. The species is one of the tree forms of the highland of Mexico of the southern Zacatecas and Aguas Calientes region. It is very different from any other species with which I am familiar, especially in size of fruit. My field notes indicate that when it was collected it was thought to belong to the O. chavefia group. Its brownish red spicules, however, make it appear quite different and its fruits are entirely different. It has been seen in cultivation in sev- eral localities and apparently native to the vicinity of Aguas Calientes. The description was drawn in the main from a cultivated specimen in the third year of its growth from a single joint . cutting, amended by notes from the type and other localities in Mexico. No flowers of it have been seen. The type specimen is one bearing my collection No. 8138, prepared from a cultivated specimen which was collected under the same number near Aguas Calientes, Mexico, September 16, 1905.—Plate 24. Opuntia Ellisiana sp. nov. Plant spreading, ascending, laxly branched, 10 to 14 cm. high and 14 to 20 dm. in diameter, depending upon moisture and fertility con- ditions; joints light glaucous blue-green, obovate or ovate, about 20 ILLUSTRATED STUDIES IN THE GENUS OPUNTIA—III. 171 by 24 cm., slightly elevated at areoles when young; areoles at first white, almost cottony, turning gray and finally black, small, 2 or 3 mm. in diameter; leaves long, prominent, circular in section, subu- late, cuspidate, soon recurved, 12 to 15 mm. in length; spicules yellow, but never prominent except on fruit where there are only a few in upper areoles, all but absent from joints; spines entirely absent; flowers deep yellow, changing to orange, reddish when closed, some of the outer perianth segments tinged with dull greenish red in bud, about 6 cm. in diameter when fully open, filaments and style white. stigma very light greenish yellow, 7 parted; fruit pyriform, deep reddish purple. The species is known only in cultivation and only from southern Texas. It is rather common in gardens at Corpus Christi and Brownsville, especially. It was received first through Mr. James Anderson, Jr., and Professor J. Cos- well Ellis, from Corpus Christi, and afterwards collected there by myself. It is only remotely related to any other spineless opuntia described. It is about as smooth as any species, much more hardy than the O. ficus-indica group, and is said never to be injured by cold weather at Corpus Christi and is apparently hardy at San Antonio. The description is a compilation of two sets of notes taken upon a cultivated plant. The type is a specimen bearing No. 8626 D. G., prepared from a cultivated plant, the cut- tings for which were secured by myself in Mexican gardens at Corpus Christi, Texas, on 1907.—Plate 25. Opuntia Wootonii sp. nov. A very open spreading, ascending species, about 6 dm. high (3 years old) and 14 to 2 meters inspread of branch, the main limbs horizontal, ascending or resting on edge on ground, the secondary ones erect from them; joints widest near the middle, pointed at either end, about 18 by 32 cm., glaucous, light blue-green becoming more yellow in age; areoles broadly oval, about 5 to 7 mm. in length and 3} to 4 cm. apart, increasing in size with age, at first tawny and then gray, leaves large, slightly flattened, erect, but recurved at tip in age, subulate, cuspidate-pointed, 15 to 20 mm. long; spicules long, formidable, in an unequal, hemispherical tuft in upper portion of areole, often 14 cm. long above and on edges of joints, increasing with age and often fill- ing the entire areole; spines very long, formidable, erect-spreading, flattened, twisted, faintly annular, the longest often 9 cm. in length and shortest about 1 cm., but the long ones greatly predominating and more commonly about 7 cm., 4 to 6on last year’s joints and increasing 172 MISSOURI BOTANICAL GARDEN. on old trunks to 8 or 10, the longest ones sometimes 11 cm. long, tips bonelike, changing abruptly to white or often yellowish or even trans- lucent which in turn fades into various degrees of reddish brown or even nearly black at base; flowers yellow to deep orange-red; fruit reddish-purple. This species has been cultivated several years by Pro- fessor E. O. Wooton in whose honor it is named. It is one of the most striking of our native opuntias and is easily recognized by its large joints, pointed at both ends like O. occidentalis, and exceedingly formidable, showy spines which resemble those of Opuntia tricolor more closely than any other species with which I am familiar. The description given above is taken from a cultivated plant in the third year of its development, supplemented by notes upon flowers and fruits grown at Messilla Park, N. M., by Professor Wooton, who collected the species orig- inally in the Organ Mountains of New Mexico. The type bears my collection No. 9171 which was prepared May 4, 1910, from a cultivated plant the cutting for which was secured in April, 1908, from Professor Wooton’s collection. The species was originally collected under Professor Woo- ton’s No. 3030. The plants from which the above descrip- tion was drawn bore but one flower bud this year, the third season from planting.—Plate 26, upper figure, and Plate 27. Opuntia atrispina sp. nov. Plant 7 to 10 dm. high and 12 to 15 dm. in diameter or often smaller, the main branches spreading with edges on the ground or ascending, the secondary branches mostly erect; joints obovate, rounded above to sub-circular, mostly slightly raised at areoles, about 11 by 15 em. and 1 em. thick, yellowish green; areoles obovate, or on young joints mostly elongated and raised below, about 5 mm. long and 25 mm. apart; spicules yellow, prominent, unequal, occupying a large triangular area in upper part of the areole, but finally scattered throughout its entire area but more numerous above, 6 to 12 mm. long, increasing with age; spines jet black to reddish brown at base with yellow tips, the transition between the two colors being quite abrupt, but the proportion of the two colors varying tremendously in different individuals, mostly one large, sub-erect one, 25 mm. long and one smaller, recurved one about 13 mm. long immediately below it and 2 shorter beside and a little below the latter about 6 mm. long; scattered among the spines are a few fugacious, reddish brown spic- ILLUSTRATED STUDIES IN THE GENUS OPUNTIA—III. 173 ules with yellow tips, all finally fading to a dull dirty gray or brown; flowers yellow changing to orange, about 4 or 5 cm. in diameter when fully opened, greenish within with filaments yellowish above and greenish below, style white, stigma yellowish, small, 7 parted; fruit small, pyriform, slightly to quite deep pitted above, reddish purple without and greenish yellow within, rind quite succulent and pulp small in amount having a slight aroma when first cut, covered with small, sub-circular areoles not over 1 mm. in diameter and 5 or 6 mm. apart. The species is more closely related to O. phaeacantha than any other species, but differs decidedly in many character- istics as indicated above. Well matured plants are quite different in habit. Although always greenish yellow within, the fruits differ decidedly in size. The description was drawn in the type locality and has been amended by subsequent notes secured near Devil’s River, Texas. The type is No. 9411 D. G., collected near Devil’s River, Texas, July 20, 1908.—Plate 26, lower figure. Opuntia Sinclairii sp. nov. An erect or ascending, open branching species making a shrub 12 dm. (high and 14 to 2 meters in diameter; joints broadly obovate and broadly rounded above, commonly 20 by 22 em., although often larger and smaller, blue-green, mostly somewhat glaucous, especially when young; areoles sub-circular to obovate, 5 to 6 mm. in length, at first brown, becoming dirty black; leaves 6 to 8 mm. long, sub-circular in section, subulate, cuspidate-pointed, mostly recurved; spicules reddish brown, numerous, scattered, unequal, 5 to 6 mm. long, the tips often fading to yellow and even dirty grayish; spines yellowish, bonelike to chalky white with light brown bases, mostly 3 or 4, often 2 to 5, erect, divergent, increasing with age to often about 8, the longest 4 to 44 cm. long, flattened, usually not twisted, faintly when at all annular; flowers yellow, fading to orange, filaments yellow, style white, stigma bright green, 8 to 9 parted; fruit reddish purple through- out; seed small. This species is rare in the type locality and is rather closely related to Opuntia Lindheimeri, from which it dif- fers in having reddish-brown spicules and spines colored at base, these characters being constant and distinct. It has been in cultivation with us now for the past 6 years and usually produces flower and fruit in abundance the third year from single joint cuttings. 174 MISSOURI BOTANICAL GARDEN, The description is a compilation of several drawn from a_ number of cultivated plants, together with subsequent notes upon the flowers. It is named in honor of Mr. Wm. Sin- clair to whom I am greatly indebted for assistance. The type is No. 9003 D. G., prepared from a cultivated specimen May 2, 1910. The original cuttings were secured near San Antonio, Texas.—Plate 28. EXPLANATION OF PLATES. Plate 19.—Opuntia alta, from type plant. Plate 20.—Above, Opuntia alta, type plant. Below, O. xantho- glochia, from cultivated plant. Plate 21.—Opuntia Gomei, from type plant. Plate 22.—Above, Opuntia pachona, showing a diseased spot, from a cultivated seedling. Below, O. Gomei, type plant. Plate 23.—Opuntia lubrica, from cultivated plant grown from cutting. Plate 24.—Opuntia nigrita, from a nearly mature plant cultivated from a cutting. Plate 25.—Opuntia Ellisii, from a cultivated plant grown from a cutting secured in cultivation at Corpus Christi, Texas. Plate 26.—Above, Opuntia Wootonii, in third year’s growth from a cutting from Professor Wooton’s plantation. Below, O. atrispina, from Devil’s River, Texas. A small plant. Plate 27.—Opuntia Wootonii. See upper figure in plate 26. Plate 28.— Opuntia Sinclairii, from a plant cultivated in the type locality. Rept. Mo. Bor. GARD., VOL. 21. OPUNTIA ALTA, PLATE 19. fate | Rept. Mo. Bor.!}GArRD., VOL. 21. PLATE 20. OPUNTIA ALTA anv O. XANTHOGLOCHIA. Rept. Mo. Bot. GARD., VOL. 21. OPUNTIA GOMEI. PLATE 21; REPT. Mo. Bor. GARP., VOL. 21. PLATE 22. OPUNTIA PACHONA anp O. GOMEI. Rept. Mo. Bot. GARD., VOL. 21. OPUNTIA LUBRICA. PLATE 23. Rept. Mo. Bor. GARD., VOL. 21. PLATE 24, OPUNTIA NIGRITA. Rept. Mo. Bot. GARD., VOL. 21. OPUNTIA ELLISIANA. PLATE 25. Rept Mo Bor. GARD., VOL. 21. PLATE 26. OPUNTIA WOOTONII anp O. ATRISPINA. Rept. Mo. Bot. GARD., VOL, 21. PLATE 27, OPUNTIA WOOTONILI. Rept. Mo. Bor. Garp., VOL. 21. PLATE 28. OPUNTIA SINCLAIRII. _ eae OT ee ee ee ee ABNORMALITIES IN OENOTHERA. BY R. R. GATES. In connection with my Oenothera cultures, particularly among plants grown during the past two seasons at the Missouri Botanical Garden, I have had occasion to observe several interesting ‘abnormalities’ of structure. These include virescence or frondescence and polymery of the flowers, tricotyly and variegation of leaves. I have thought it worth while to devote a short paper to a description of some of these cases which have an evident bearing on problems of variation and inheritance. Virescence.—In my experimental garden of 1909 four plants exhibited virescence of the flowers. These were all descendants in the second’ generation from plants which were derived from the English coast near Liverpool, the first generation having been grown at Woods Hole, except in the case of one (No. 47), which was grown in the tropical greenhouse at the University of Chicago. These four plants were therefore all from cultures of closely related forms, and in some of their characters were intermediates between O. grandiflora and O. Lamarckiana. The summer tempera- ture at St. Louis in 1909 ranged exceptionally high, read- ing 100° F. in the shade in one instance. The change in climate which the plants experienced was therefore very con- siderable, and one of the cultures had been subjected to such high temperatures for two successive seasons. ‘This may per- haps have had something to do with the appearance of these cases of virescence, the alteration in the conditions acting as a stimulus to the production of the abnormality. That the tendency to produce virescent individuals is inherited, is shown by the reappearance of virescent plants in one race in successive generations, and their failure to appear in many other races, ¢. g., 0. Lamarckiana and its mutants. (175) 176 MISSOURI BOTANICAL GARDEN. Two of the cases of virescence in 1909 occurred in a race which I have called O. multiflora, the description of which will be published at another time. This race is descended from a single individual grown at Woods Hole in 1908. A total of 376 first-generation offspring of this individual have been grown in the two following years, and also (in 1910) 50 plants of the sécond generation from the self-pollination of one individual of the first generation. The plants of the F, included a total of 15 virescent individuals, or very nearly 4%. The 50 plants of the F, contained one showing virescence. In a culture of 36 plants from seeds received from the Botan- ical Garden at Karlsruhe under the name Q. chilensis, which proved to contain two very distinct types, one plant was virescent. This abnormality has not appeared in any others of the many races of which I have grown cultures. All the plants showing virescence were affected in exactly the same way, although in some the early flowers were nor- mal and produced fruits, only the later flowers showing the peculiarity. I have not compared the offspring from such capsules with those of normal plants, though if this were done it might be found that the virescent tendency was inher- ited more strongly in the former case. In one plant a side shoot produced flowers which were quite normal while the main stem produced only flowers of the virescent type. One plant of O. multiflora, in which all the flowers but the earliest were virescent, is illustrated in plate 29. The peculiarities of structure exhibited by these flowers may now be described. Plate 30, f. 1, shows a group of the flowers, natural size. The sepals are green inside and outside, large and bag-like and more or less crinkled or curled. They are tapering at the end, terminating in long, slender sepal tips. Perhaps frondescence or phyllody would be a more suitable term than virescence to apply to this condition, for the sepals have become quite leaf-like. Plate 30, f. 2, shows several flowers opened and photographed to show the other organs of the flower. The petals retain a greenish yellow color, but are in all cases very small (usually about half an inch in length, though sometimes larger) and blunt at the tip. The ABNORMALITIES IN OENOTHERA. 177 anthers are small, with very short filaments, empty and sterile. The style is frequently markedly pubescent almost to the top. It tapers strongly and gradually to the top which is very slender, and the stigma lobes are reduced to four delicate prongs. A remarkable peculiarity of all these flowers is the com-. plete, or almost complete, suppression of the hypanthium. I have remarked elsewhere (Gates, 1910, footnote, p. 208) that the attacks of a certain insect also lead to suppression of this organ. Its wide variability, which Shull (1907) has proved statistically, and its suppression under various abnor- mal conditions, as I have shown, are probably significant facts, related to its recent phylogenetic development as sug- gested by MacDougal. A marked feature of this type of virescence is that the flowers do not drop off but remain per- manently attached to the stem. In many cases an elongation occurs below the ovary. This is more slender than the ovary and is hard and woody, tough, and strongly attached to the stem. In the meantime (see plate 30, f. 2, flowers to the left) leaves grow out from the interior of the flower and in this way the flower becomes transformed into a short side branch. The ovary in the meantime almost completely disap- pears, possibly becoming transformed into a portion of the woody branch by an alteration in its structure. This stem is always more slender than was the original ovary. A whole group of young leaves of abnormal shape (long and narrow) may grow out of the flower in this manner. The elongation to form a side branch is sometimes partly above and partly below the ovary, as may be seen from plate 29. It may also be seen from this figure, though not clearly, that the lower flowers on the main stem were normal and have . dropped off leaving the growing ovaries behind. Some of these afterward developed into large capsules. The plant in 1909 which produced only virescent flowers, wilted and died about August 10th for no assignable cause, while the other plants continued to bloom long afterwards. It seemed as though the production of virescent flowers was equivalent to seed production in the physiology of the plant, 178 ‘MISSOURI BOTANICAL GARDEN. and was therefore followed by drying up and death such as occurs with normal plants later in the season. The plant in the offspring of No. 47, showing virescence, was not ob- served to have virescent flowers until September 29, when the blooming season was nearly past. On this plant the ends of all the branches bore only virescent flowers, while farther down the branches normal seed capsules had been produced. Virescence therefore developd in all the flowers simulta- neously but only appeared at the end of the season. The virescent flowers on this plant became fairly stout branches, in some cases even possessing internodes. In the virescent flowers there was no departure from the normal number of parts, but when leaves developed within these they exhibited no regularity in number or arrange- ment, though always growing out from just within the cycle of the petals. DeVries refers to what appears to be a similar case in Oenothera subovata (1909, p. 423), but does not describe it. He has also described a different type of virescence which is pathological in nature, due to the attacks of certain para- sites. Masters (1869) gives a list of cases of virescence (p. 388) in which he includes Oenothera, with the suggestion that it might perhaps better be called frondescence or phyl- lody. On p. 252 of the work referred to, cases of frondes- cence or virescence of petals in Oenothera striata are cited. PoLyYMERY OF THE FLowers.—A number of cases of flow- ers with an increased number of parts were observed in the cultures of 1909 and 1910. No special effort was made to find them all, but they were recorded as they happened to be observed by myself or my assistant. Masters, on p. 44 of the work above cited, refers to species of Oenothera as exhibiting synanthy. Many of the cases of polyphylly in flowers of Oenothera, to be described shortly, are due to synanthy, as I shall show. Certain other cases will require a different explanation. DeVries (1909, pp. 472, 482) has recorded a number of cases of polymery from his cultures and in the field at ABNORMALITIES IN OENOTHERA. 179 Hilversum. I will add some observations which extend the range of variation in number of parts, and shall then suggest what appears to me a probable explanation of the phenome- non as it occurs in most of the races of Oenothera. All the cases in 1909, with the exception of two, occurred in hybrid O. Lamarckiana from various sources, and these two were O. brevistylis plants from a cross with O. Lamarckiana. Whether this is of significance as indicating greater variabil- ity in plants derived from a cross, I cannot say, but it seems not improbable that this is the case. Using signs for the flower parts I shall now give the formule for the flowers recorded in 1909, in which K=sepal, C=petal, S—stamen, N=stigma, lobe. y Re ee MS aT eee K7C?SisNis G... sick Coa ee KsCsSioN ) Seer pene KeCeSuxNu y Te ae ee a KeCeSizNo Me ee ss teens K7C7SieNis S$... a Saree K7C7SuNo Drew ered wav heise ss RyslpuNig. 9... cic. cease K7C7SieN17+5 ee oh pee ee KsCsSeN Wiikey ee oe KsCsSeN eee eres se a aes KsCsSeN This list of flowers, while shorter than that of DeVries, extends in both directions the range of variations observed by him. The highest number of stamens recorded by De- Vries in a flower is 14, while I observed one remarkable flower with 16 stamens and two styles which were separate at the top, in contact below, but terete and easily pulled apart. The hypanthium was also considerably flattened and much thicker than usual and even had a longitudinal groove. down its center. This flower gave me the clue to the explana- tion of these cases of polymery. DeVries states (1909, p. 483) in regard to his cultures of O. Lamarckiana forms, that “trimerous flowers are certainly not present,” but he has observed them, though very rarely, in O. biennis and in hybrid cultures. In the season of 1909 I observed three such cases, in O. Lamarckiana of various descent. The flowers were normal in every way except that the parts were in threes, which made the flowers smaller, though the individual organs were not reduced in size. 1This flower had two independent stigmas, and styles which were merely in contact in the calyx tube. 180 MISSOURI BOTANICAL GARDEN. Regarding the explanation of these cases, I soon found that every polymerous flower had two bracts at its base instead of being in the axil of a single bract. These bracts may be entirely independent or they may be more or less coalesced at the base, but they project in opposite directions. (See photograph by DeVries, 1909, p. 472.) In all these cases the ovary and hypanthium are more or less flattened. An examination of the stems which bore these polymerous flow- — ers, disclosed the fact that they exhibited irregularity in the placing of the flowers on the stem, or in other words, varia- tions in phyllotaxy. The flowers and their bracts were not uniformly distributed on the stem, but certain flowers were very close together and others long distances apart. It seems clear that this is the explanation of the phenomenon, which is therefore one of synanthy rather than of polymery. The Anlagen of the flowers are of course laid down and their position determined when the terminal rosette of the stem is very small. Anlagen of successive flowers therefore arise very close together, and if anything leads to variation in their position they will sometimes occur partly in contact or overlapping, giving a flower in which the parts are more or less completely doubled in number. The flower having two independent styles, and the fact that two bracts are always found at the base of polymerous flowers, shows that it must be due to a partial coalescence of primordia, such as I have mentioned. It is interesting to note that flower No. 5, hav- ing its parts in threes, was immediately below No. 4, which is heptamerous, and on the same side of the stem. Similarly, the plant bearing flower No. 9 also bore at the same time (Aug. 20) the two trimerous flowers, Nos. 10 and 11. It may also be noticed that in all the polymerous flowers the number of stigma lobes is less than the number of stamens. The same is true of DeVries’ records. These polymerous flowers are much larger than the normal owing to the larger number of parts, the parts themselves retaining their usual size, except that the hypanthium and style are stouter, as might be expected, and the filaments are sometimes thicker. In the season of 1910 a number of additional observations a ABNORMALITIES IN OENOTHERA. 181 were made on this subject. The records of these were kept by my assistant, Mr. V. Follenius, during my absence, but I had the opportunity of examining the most interesting cases before the end of the season. Cases of polyphylly or synanthy were found in a much wider range of Oenothera forms than in the previous year. The following is the list: FORMULA | RACE REMARKS 1..K7C7SuNu |O. multiflora Two bracts at base of flower. 2..KeCeSiNs |O. multiflora Two bracts partly coalesced. 3..KeCeSizNs |O. multiflora Two bracts at base. 4,.K4CaSsNa t Race No. 25, from |Two perfect tetramerous flowers, K4CsSgNs near Liverpool, with their hypanthia in contact England throughout their length and . ° partly fused. Ovaries in contact and partly fused. Two bracts. 5..KsCsSeNe |O. biennis >< Lamarc-|One bract. kiana 6..KsCaSeN4 rf he One bract. 7..KsCaSaNs te ‘“(same/One bract. plant as No. 6) 8..KeCeSuNs |O. grandiflora from |Hypanthium and ovary flattened. Alabama One bract. 9..K;CsS2N? /Race 54x 40 One bract. ~ 10..KsCsSsNs |O. biennis, Chelsea |Only one bract at base of each ; Physic Garden flower in this race. In one’ 11,..KsCsSsNs 7 case the bract had two tips, as 12.. KsCsSsNs = ss though resulting from the in- 13..KsCsSsN7 oe eS complete coalescence of two 14..KsCsSsNs af St bracts. 15.. KsCsSsNs nf uh 16.. KsCsSsN5 “ f 17..KsCsSsN5 ay ‘i 18..KsCsSsN5 ef a 19... Ks'CaSoN, et pee 1Two sepals of normal width, 83 narrower, Occupying about the width of the other two. The case of No. 4, in which two tetramerous flowers were found, each with its bract, and only partly fused by their ovaries and hypanthia, is particularly instructive and con- firmatory of my explanation. One hexamerous (No. 8) and one pentamerous (No. 9) flower, however, had each but one bract at its base, as well as the three trimerous flowers in the race O. biennis * O, Lamarckiana. It is therefore prob- 182 MISSOURI BOTANICAL GARDEN, able that the latter are real cases of polyphylly and not of synanthy, in which, instead of the partial coalescence of two primordia or their failure to separate, there is a variation in the division of the Anlagen of the various cycles of organs in the flower, resulting in a flower having a larger or smaller number of parts than normal. When, as in flowers 10-18, the androecium is normal while the calyx and corolla show an increase of parts, this may be considered to be due to polyphylly rather than synanthy. The ten pentamerous flowers in O. biennis from the Chelsea Physic Garden were found in a culture of 33 plants, and careful search would doubtless have revealed further cases. Evidently the phenomenon is rela- tively common in this race. The characters of the race are very constant and are remarkably different from those of any other race of O. biennis Ihave seen. They will be de- scribed elsewhere. These pentamerous flowers had invaria- bly but one bract at their base, which would indicate that the phenomenon of pentamery is here due to polyphylly rather than to synanthy, although the fact that one of these basal bracts had a double tip might be considered to favor the interpretation of this also as due to synanthy. It would seem therefore that while most of these are cases of synanthy, or coalescence of two primordia, the trimerous flowers and also evidently some at least of the other cases with only one bract at base, are real instances of polyphylly, due to variations in the divisions which the primordia of a flower normally undergo. My conception of the process of synanthy is that, owing to variations in phyllotaxy, two independent flower primordia become so closely approximated that they partly coalesce, and develop harmoniously into a single flower in a some- what similar fashion to the growth of a plant chimera (sec- torial chimera) as described by Baur and by Winkler. TricoryLy.—A number of cases of tricotyly and other abnormalities of the cotyledons have been observed in my 1 Penzig (1890) states that in O. biennis pentamerous flowers are common, the number of ‘‘carpels’’ often running up to 9. ABNORMALITIES IN OENOTHERA. 183 germinating pots each year. They are particularly common in O. gigas, but no record of them has been kept. VARIEGATION OF LeEAves.—Yellowish areas not infre- quently appear on the rosette leaves, particularly in the Eng- lish Oenotheras.. One striking case of what was evidently a sectorial chimera according to Baur’s (1909) terminology, occurred in a culture of 55 plants very closely resembling O. Lamarckiana, but having larger rosettes with rather broader leaves. They constituted the second pure generation from seeds of a plant near Liverpool, England. The green areas on the leaves in this plant are contrasted with areas which are yellowish white, showing a complete absence of chloroplasts. It will be seen that in several leaves the line be- tween white and green tissue passes down the midrib, while one or two leaves exhibit patches of white adjoining the mid- rib. Plate 31, from a photograph taken June 30, 1909, shows the partly developed rosette. The leaves arising from one side of the stem are wholly white, those on the opposite side are mostly pure green, while several others are green on one- half and white on the other. A few areas of pale green, owing to partial absence of chlorophyll, were also observed. The white areas were of course unable to nourish themselves, and continually died away. The plant never formed a shoot, but died before the end of summer, notwithstanding the most careful treatment. The plants of the previous generation gave no indication of such a condition, which therefore ap- peared suddenly in this individual, and appeared, moreover, from a seed of a plant which was normal green throughout. Presumably one side of the young growing point was without chloroplasts, but just how this condition came about is at present a matter of conjecture. 184 MISSOURI BOTANICAL GARDEN LITERATURE CITED. 1869. Masters, Maxwell, T. Vegetable Teratology. London. 1890. Penzig,O. Pflanzen-Teratologie. Genua. . 1907. Shull, Geo. H. In Mutations, variations and relationships of the Oenotheras, by MacDougal, Vail and Shull. Carnegie Institution Pub. No. 81. Washington. 1909. Baur, E. Das Wesen und die Erblichkeitsverhaltnisse der ‘‘Varietates Albomarginatz Hort.’’ von Pelargonium zonale. Zeitschr. f. ind. Abst.- u. Vererbungslehre 1:330-351. 1909. DeVries, Hugo. The Mutation Theory. Translated by J. B. Farmer and A. D. Darbishire. Vol. I. Chicago. 1910. Gates, R. R. The material basis of Mendelian phenomena, Amer. Naturalist 44: 203-213. EXPLAN/.TION OF PLATES. Plate 29.— Plant belonging to 2 race known as Oenothera multiflora, _ originally derived from the English coast near Liverpool. All but the earliest flowers are virescent or frondescent. : Plate 30.—1, Virescent buds photographed natural size, showing the peculiar baggy appearance of the calyx. 2, Virescent buds natural size, opened to show the small petals, tapering pubescent style and (buds on the left) leaves growing out from the interior. Plate 31.— Sectorial chimera, in which the leaves on one side of the rosette are lacking in chloroplasts. In certain cases half the leaf is white and half green. : Rept. Mo. Bot. GARD., VOL. 21 PLATE 29. VIRIESCENCE or OFNOTHERA. Rept. Mo. Bot. GARD., VOL. 21. PLATE 30. VIRESCENCE or OENOTHERA. Rept. Mo. Bot. GARD., VOL. 21. PLATE 31. CHIMERA or OENOTHERA. Rept. Mo. Bot. GARD., VOL. 21. PLATE 32, a BOTRYTIS on CHRYSANTHEMUMS. Sa e ee eh e . e ee oe Ge eS a a en ee ee ime BOTRYTIS AS A PARASITE UPON. CHRYSANTHEMUMS AND POINSETTIAS.* BY PERLEY SPAULDING. During the month of November, 1904, one of the smaller greenhouses at the Missouri Botanical Garden was devoted entirely to chrysanthemum plants which were just beginning to come into bloom. Because of lack of room they were badly crowded together, but especial pains were taken to have the conditions as favorable as possible for the’ plants. The taller ones, located in the middle of the house, came into bloom first, and it was noted almost at once that the flowers were affected by some disease which attacked the petals. The trouble soon spread to the lower plants as they came into bloom,.and the disease was very shortly scattered over the house. So far as observed, the trouble always first appeared on the petals. The diseased flowers were picked as soon as they were seen to be affected, so that the fungus did not have a chance to infest the other portions of the plants; for this reason it cannot be said that the fungus might not have attacked other parts of the plants if it had been left to run its course. The disease first appeared as tiny, watery, discolored spots, looking as if the petals had been pricked with a needle; this, of course, showed much plainer on the white flowers than on the colored ones (pl. 32, above). These spots were gen- erally more or less numerous on single heads, and even on individual petals; they were often located only on one side of the head, showing that the infection had come from some point on that side of the plant. They very rap- idly grew larger, and by the time the affected spot extended over a fourth of the petal, the diseased tissues wilted and dried up. Naturally the tips of the petals were first attacked, * Published by permission of the Secretary of Agriculture. (185) 186 MISSOURI BOTANICAL GARDEN. and they died down to the base as the disease progressed in its course. Very soon after they began to dry, a grayish brown velvety growth appeared upon them (pl. 32, below). This was at once perceived to be composed of the fruiting bod- ies belonging to Botrytis vulgaris. These fruiting bodies were formed within about two days after the beginning of the at- tack. If allowed to run its course, the disease invariably at- tacked every ray of the affected head, and in the last stages of disease the affected head was simply a mass of dried rays hanging from the stem, and permeated with the mycelium of the fungus, while all over the outside were the fruiting bodies crowded thickly together. After the first few flowers became affected, the disease spread very rapidly and caused considerable damage. Many of the finest blooms had to be removed as soon as they opened, thus defeating the purpose for which they were raised. The fungus, so far as could be determined, seemed to exhibit no partiality toward any one variety and counts showed that no one color of blossom was more often attacked than an- other. During the next two years, at about the same period, in the growth of chrysanthemum plants exhibited by the Botanical Garden, the disease has occurred in varying inten- sity. The first season it started earlier in the development of the flowers than it has since done, and for that reason, caused a greater amount of damage than at any other time. During the months of November and December, 1906, a Botrytis disease of “poinsettias’” (Luphorbia pulcherrima) was noted in the same house in which two years before had occurred the above mentioned Botrytis rot of chrysanthe- mum petals. In this case the fungus attacked the slightly projecting angles on either side of the leaves. It did not attack the red, bright colored leaves at the top of the plants, but only the green, broad leaves which grow along the stem below the red ones. The disease first appeared as a slightly deadened area at the very tip of the angles, which showed beneath tiny white drops of the hardened juice along the larger veins. The fungus seems to attack the tissues in such a way that the white juice of the plant finds exits and exudes BOTRYTIS UPON CHRYSANTHEMUMS AND POINSETTIAS. 187 through these weakened spots, hardening quickly upon being exposed to the air. These small, hardened drops of juice seem to be very characteristic of this disease upon this par- ticular plant, it being found to occur constantly only in con- nection with this trouble; the very earliest stages of the dis- ease could be found only by looking at the under sides of the leaves, and noting the presence of this dried juice at the angles of the leaves. In the earliest stages the leaf tissues upon the upper side gave little or no indication of being dis- eased, although there usually was a slight yellowish discolora- tion. As the disease progresses the affected area becomes larger and the extreme tips of the affected angles wither and become discolored; this dying of the tissues progresses toward the mid rib of the leaf as the fungus extends its field of action. In no case, however, did the Botrytis disease alone seem to extend over the whole area of the leaf. On the other hand, after the disease had progressed until it involved about one-fourth the area of the leaf, the effect seemed to be communicated to the petiole, and the leaf was prematurely shed. In this way considerable damage was done by the fungus, since the badly affected plants consisted only of a bare stem surmounted by the broad whorl of red leaves at the top. The absence of the green leaves greatly marred the plants for exhibition purposes. About two days after a leaf is first attacked, the characteristic fruiting bodies of Botrytis are formed in thick groups, clothing the under sur- face of the affected area. In the same greenhouse a number of plants of Primula obconica grandiflora were found to have the lower leaves also diseased by Botrytis. These plants were in very poor condi- tion, being very short-stemmed, so that the lower leaves lay flat on the surface of the soil, thus giving the fungus the best of opportunities for attack. In this case the affected leaves finally entirely succumbed to the disease, and it even spread from the diseased ones on to the adjacent healthy ones. These instances indicate the parasitism of Botrytis upon the leaves and petals of the above mentioned plants. No ex- my hal | Pee ed ee Pee ee a 188 MISSOURI BOTANICAL GARDEN. periments were made to prove the parasitism of the fungus, but considering the present state of our knowledge of this very point, we may well consider this fungus one of the very worst, under certain conditions, with which the gardener has to contend. The writer has seen little or no mention of dis- eases caused by Botrytis in this country, although this class of trouble seems to be well known upon the Continent. Be- cause of the very slight mention which has been made of diseases of this kind, it has seemed advisable to give a rather -extended account of the above cases as noted by the writer. EXPLANATION OF PLATE. Plate 32.—Chrysanthemums affected by Botrytis. Above, an early stage of the disease in which some flowers are spotted and a very few bear the fruiting fungus. Below, a later stage, in which the fungus is in full fruit. FUNGI OF CLAY MINES.* BY PERLEY SPAULDING. During the fall of 1906 and the spring of 1907, a num- ber of clay mines in the western part of the city of St. Louis were visited for the purpose of determining the species of fungi occurring upon the timbers. These mines are entered by vertical shafts descending from 75 to 125 feet from the surface. From this entrance shaft galleries run horizontally, or nearly so, for distances varying from a half mile to nearly a mile, and numerous doors are usually located at various places along these galleries. Considerable water seeps into the tunnels from overhead, the amount varying at different seasons of the year, but usually being greater during the winter and spring. Immediately after leaving the foot of the vertical shaft one could detect no light whatever. Along the galleries of these mines many timbers are used _ to prevent the caving-in of the roof. The timbers in deep coal mines are placed with considerable care, and are fitted together carefully, because of the great weight which they bear and the permanent nature of many of the galleries. In coal mines the tops of the braces standing each side of the tunnel are notched in such a way that the weight cannot pos- sibly cause the cross piece to slide, or the notches may be cut in the end of the cross pieces. In either case the two tim- bers are fitted quite accurately together. At this point most of the decay occurs, and the set of timbers has to be removed because of a comparatively small defect. In the clay mines, on the other hand, the timbers are intended only to prevent the caving-in of the earth imme- diately above the galleries. The timbers never prevent the settling of the large body of soil over the mine for one or two feet. In fact, a mined area could be detected by an * Published by permission of the Secretary of Agriculture. (189) i a 190 MISSOURI BOTANICAL GARDEN. expert by the dead trees standing on the surface which were killed by the settling earth pulling the roots in two. These timbers were light compared with those used in coal mining, and were rather carelessly placed in position. The top cross pieces were simple flat pieces of planking which were wedged in place on the supports. The rotting usually oc- curred in the oak supports. Because the weight sustained is not excessive the timbers rot practically throughout their diameter and for a considerable distance in length before, they must be replaced. The clay mines are worked for a number of years, and the timbers must be renewed several times, as they last only about two to three years ordinarily. The timbers used were oaks of various species, and southern pine. The oak pieces were used as braces at the sides, while the cross pieces overhead were of pine. The oak pieces were usually from 6 to 9 inches in diameter and did not have the bark removed, while the pine pieces were sawn mostly from heartwood, and were usually 2 to 3 inches in thickness, and about a foot in width. The pine was used for the overhead timbering because of its greater resistance to rot, it being in direct contact with the soil. All of the timber used was wholly untreated, with the exception of being cut in the proper sizes and shapes for use. It is shipped from outside the city, and is usually somewhat seasoned by the time it is placed in the mine, although little or no attention is given to this detail. The fungi growing upon the timbers in these mines were situated under very abnormal conditions. There was abso- lutely no light, and the air of the mines was noticeably moister than that out of doors. The temperature never goes down to freezing, and is very uniform, ordinarily being cool enough so that one may wear a coat with comfort even in - the hottest weather. In most of the galleries the sides and top were fairly moist, but not dripping, while in others the moisture was very noticeable, there even being small rivu- lets of water running along the tramway to the lowest parts of the mine. In all cases the water seeping into the mine had to be pumped out to prevent its filling the galleries. FUNGI, OF CLAY MINES. 191 Quite a number of wood-inhabiting fungi were found to- be fruiting in a normal manner, but there were evidently others which were unable to form any recognizable sporo- phores. Of these latter, one was very abundant, forming dense rounded masses of white mycelium which were so full of water that they collapsed when taken in the hand. Every place where touched immediately assumed a water- soaked appearance. Still another formed black mats of coarse mycelium upon the outer surface of the timbers. Somewhat to the writer’s surprise, the fungi found upon the timbers in these mines were not those which are seen most commonly in the forests of the immediate vicinity of the mines but in a number of cases at least, were compara- tively rare ones. In one of the mines visited by far the most abundant fungus noted was a bright red one, which usually grew in the shape of an inverted cone, attached upon one side or at its apex. It was also present in the other mines, but in lesser numbers. This was identified as Merulius rubellus Peck. It was present on over one half of the upright oak timbers, and must be quite destructive, as the timbers last ordinarily but two to three years. Sometimes there were dozens of the sporophores on a single timber, and sometimes but one or two. Usually they were scattered, but they also occurred in large imbricated masses. The young stages were simply rounded knobs of reddish mycelium, of greater or less regularity. The presence of this fungus as the most abundant one in the mine is somewhat anomalous in view of its evident rarity in the forests of the vicinity. It has never been found by the writer anywhere in the vicinity, although he has collected quite carefully for several years in that locality. Glatfelter,) however, mentions finding it once, about one hundred miles away. A single fungus was found occurring upon the pine cross pieces in considerable abundance; it grew in a resupinate form, and was identified as Fomes annosus Fr. This is known to be especially destructive to coniferous timber in 1 Glatfelter, N. M. Trans. Acad. Sci. St. Louis. 16381. 1906. 192 MISSOURI BOTANICAL GARDEN. — Europe, and is not unknown in America. In most locali- ties, however, it is not common. Its natural habitat is upon the roots of various coniferous trees, upon which it produces the so-called “root-rot”. The conditions in the mine would seem to be very favorable for this fungus. It was practi- cally the only one which was found occurring upon the pine timbers. : A considerable number of different species were found growing upon the oak timbers. A single sporophore of Fomes applanatus (Pers.) Wallr. was seen which came from the mine. It was perfectly normal in every way. Lenzites betulina (L.) Fr. was found somewhat sparingly upon the oak timbers, and in most cases was normal in ap- pearance, Polystictus versicolor (L.) Fr. was also quite plentiful: upon the oak timbers. It was very white in color, and did not exhibit the glistening zones on the upper surface at all distinctly, but otherwise seemed to be normal. One of the more common fungi upon the oak was Merul- ius lacrymans (Jacq.) Fr. var. verucifer Quel. This was noted as being quite frequent in some of the mines, but was not seen in others; it is one of the most destructive fungi occurring in the mines, as a large proportion of the timbers which had been removed were affected with a dry rot such as is caused by the action of this fungus. A rotted stick six inches in diameter when struck sharply upon the ground would break in two very easily, thus showing the extreme weakening effect of this fungus upon the wood tissues. Several other species of fungi were found occurring rather sparingly upon the oak timbers. Stereum spadiceum Fr. was found in a few cases. Bulgaria inquinans (Pers.) Fr. was found upon several of the timbers. It occurred in con- siderable number upon those timbers where it was found at all. Hydnum Erinaceus Bull. of a rather peculiar type was found hanging from the oak timbers. It grew in the form ‘ of a rounded mass, hanging pendant from the lower end of a stout stem several inches in length, and about an inch FUNGI OF CLAY MINES. , 193 in thickness (f. 1). Only two or three specimens of this fungus were found and they were all of the same type. Hydnum coralloides Scop. was found growing about nor- mally upon a number of the timbers. Hydnum artocreas Berk. was found somewhat sparingly upon the bark of a 1. SPOROPHORE OF HYDNUM ERINACEUS FROM CLAY MINE, x %. few of the oak timbers in a single mine. This fungus is apparently rare in this country. A number of edible fungi were also found growing in the mines, Several groups of sporophores of Coprinus atramen- tarius (Bull.) Fr. were found growing upon the clay at the sik op, Sy 194 MISSOURI BOTANICAL GARDEN. side of the galleries, and also overhead. They were normal, except that the stems were somewhat longer than usual. Mules are used quite extensively in the mines to pull the cars along the tramways, and a considerable amount of ma- nure accumulates, which is thrown to one side in some aban- doned gallery. In all of the mines entered edible mush- rooms of the Agaricus type were found growing upon this manure. One found in several mines has been provisionally identified as Agaricus placomyces Peck. The men were very well aware that these were indeed “mushrooms”, and it was difficult to find enough mature specimens for the purpose of identification, because the men kept them picked closely and took them home to be cooked. The peculiar feature of the growth of these fungi under such abnormal conditions was that so many of them grew in a nearly normal manner. As above mentioned, masses of mycelium of a number of other forms were very common, but they seemed to be unable to form perfect fruiting bodies. Experiments of the writer in cultivating the wood-rotting fungi for several years past have resulted in the production of perfectly normal fruiting bodies with but a single species, Schizophyllum commune Fr, Its absence in the mines was especially conspicuous, not a fruiting body of it being seen, except in one mine which seemed to contain more moisture than some of the others. This fungus seems to tolerate a higher degree of humidity in the air during the formation of perfect fruiting bodies than do most of the wood-inhabit- ing forms. In this mine the sporophores were numerous, but were somewhat abnormal. They were large, very pu- bescent, the gills were very wavy in outline, and many were suspended, being pendant at the center from a sort of stalk similar to the specimens of Hydnum Erinaceus. In some cases the entire fruiting body was composed of branched Clavaria-like growths, showing that the moisture was almost too great for normal sporophore formation. Polyporus gil- vus Schwein. is exceeedingly common in the forests of that locality, but not a normal fruiting body was found in the mines. A single mass of brown mycelium, however, was FUNGI OF CLAY MINES. 195 found which very probably was an abortive sporophore of this species. From its known parasitism of roots of living trees one would expect to find Armillaria mellea Vahl. in the mines, but no specimens of it were noted, although it is not uncommon in the vicinity upon rotting roots and stumps. The writer is indebted to Prof. Chas. H. Peck for identi- fying many of the fungi mentioned above.