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No a ww eid epee Ww. rave . lit) Ww evo secu = vtywby driyyettY Worry Wiican racer" iv v 1 aa NeW 4 vee ty viytvey We yw ivyutytyl | POSING bk TR ww™ Wy ww wheats taka ey We Lys WAY Anat bt A Wily east WIM, | set eet Ce vegt we Why we, WWW as - ihn Ve 0 ae, © ee yrr JU IU by y "UL Le CHIC ais ‘ Nyaa lcrewcee THI WAIL CA Wein com Ad Vv We Ae cane $s Stele PAM ad bY Moy yl Ft ere : wee és te] AAMT | MM Meer aM RA tl be bd ; MALE ny Wry, wae ae iy juvett eeuL eel *\ ici se © I Wiyvuvuve v Wy vgs ssdeldihdy ace fe adel J eee Ly “Ww Aan ene Syl Voy" SoS. eter MS asaay We foc uae Mut Vv tiyeY yyy, 00s Wey owt Le i eee “Veeeueeely: PIV Wecevetriey SCO TE Go” fy q wy wW ‘yu Ysa ‘ \d yy = eerie an == es ™ MAT pd tt yoruce” | | WEB r Y a Tila ' ge “Vows ifr [ atl od : A , A h re | we ! =i tad ee ‘ ) | i \ ad WS \ ‘ee | j atte A | i . x, =| | j = f t i i r4 } \ad : } rs , on a ‘ | Z ; 3 tee, : ; el he f pe ibe SP Sag Mad ; , { ‘\ f ‘3 F » 4 x. i J > : : 4 -i % : co \ : : ~ + VEU \e S oe ‘e/ ' ae 1 | . ewes: 4 Jv ts \ . nt oe whi ‘iyi | cp eueen SUSY vit sae Uta ns evades Lace = Wy ie wisianelooe mare L vec | ' w wn M4 iy Cawgte coun eect Pe vere mt ieee — | ; = ~. LA AS : f i~ We ef ow 1 ae, fe _ Vout if wy < PEL Hrrnyggneel Wt eae — HI ” aut 3 wh ¥ et j x} py Nae + we A Pee x y ? Ws 4 P =k ; y 5 \ A , sy x ‘ i . = 1 ar 1 -— } i) ‘ “ri ; 1 ' - eG = aed iis 2 i j e ' ; \ a 4 rn = Ma ¥ l ‘ . J iH 1a e} Y ‘ . oe F i 4 es ae ¢ Loans . . \ A “ . Es! i E di } : a ' \ = B ‘ > \ ; v as: Wee, 1 be ‘ ‘ : Le * ie ; 4 Hd ' > ‘ ia ’ fi y By os ‘ ed / ‘ - > , . “) ; - iy : j 4 ¥ . =F ‘ i 1 } ; 4 zy ~ . ~ i} +} HY ; “ 4 ay Fs f y - —_ c u ° a t y . 18 ri = D ’ “ cI * : ? rips i {> 5 : 3 eh ' ° t . ’ = 5 ? ‘ ' : : H & j ips * 5 ‘ 1 + 4 i ' y ' é %) 7 ~ ‘=f 7 ‘ ‘ 4 5 ¥ ‘ os ) 4 oS 4 . s FS = a . 1. eS a at ay / Be 3 m a / \ : 5 : + * |e if ba i I i ‘ H ; 4 q f b ' i ‘ o . : ‘ pags 7 ) taetc. 3 : ane . 6 if : ‘ 4 A % ‘ { i oe AMERICAN JOURNAL OF BOTANY OFFICIAL PUBLICATION OF THE BOTANICAL SOCIETY OF AMERICA EDITORIAL COMMITTEE ; 6 F. C. NEWCOMBE, Editor-in-Chief, A University of Michigan 4? C. STUART GAGER, Business Manager Avis: HiImcHCOck Brooklyn Botanic Garden Bureau of Plant Industry IRVING W. BAILEY, | L. R. JONES, Bussey Institution University of Wisconsin Heb BARTLETT, EDGAR W. OLIVE, University of Michigan Brooklyn Botanic Garden VOLEU MEE He-1915 WITH EIGHTEEN PLATES AND NINETY TEXT FIGURES PUBLISHED IN COOPERATION WITH THE BOTANICAL SOCIETY OF AMERICA BY THE BROOKLYN BOTANIC GARDEN @ At 41 Nortu Queen Street, Lancaster, Pa. PRESS OF THE NEW ERA PRINTING COMPANY LANCASTER, PA. ABE Or CONTENTS, VOLUME T1015 No: £, JANUARY PAGE Investigations on the phylogeny of the Angiosperms. 5. Foliar evidence as to the ancestry and early climatic environment of the Angiosperms (with plates I-IV). EpMUND W. SINNOTT AND IRVING W. BAILEY I The growth-forms of the flora of New York and vicinity. NORMAN TAYLOR 23 The temperature of leaves of Pinus in winter (with four text TITIES ae Ck Bea ae a Re eR Ge ere JoHn H. EHLERS 32 No. 2, FEBRUARY Some effects of the brown-rot fungus upon the composition of (LNG [OEIC as ie tira, ee Me eg ats 1 2 Lon A. HAWKINS 7I Negative heliotropism of urediniospore germ tubes (with two (RESETS TIE SY ou. aR esc arte aera F. D. FROMME 82 A woody stem in Merremia gemella induced by high warm water Gunilinian@stext fe UneS)ie ep mallee me ew, FRANK C. GATES 86 Picro-nigrosin, a combination fixative and stain for algae. Otis F. CurTIS AND REGINALD H. COLLEY 89 Normal and abnormal permeability...... W. J. V. OSTERHOUT 93 A simplified precision auxanometer (with three text figures). W\io elit Ss OWE ae The mutations of Oenothera stenomeres (with four text figures). FeoE. BARTERI: 100 No. 3, Marcu Morphology as a factor in determining relationships. J. M. GREENMAN III The genetic relationship of parasites....... FRANK DUNN KERN II6 The experimental study of genetic relationships. Me hee BARTER 122 ili 1V TABLE OF CONTENTS No. 4, APRIL A three-salt nutrient solution for plants....-... Joun W. SHIVE Preliminary note on the morphology of Gnetum. W. P. THOMPSON The persistence of viable pycnospores of the chestnut blight fungus on normal bark below lesions. R. A. STUDHALTER AND F. D. HEALD A taxonomic study of Setaria ttalica and its immediate allies. F. Tracy HUBBARD No. 5, May The morphology and systematic position of Podomitrium (with seven text figures)........ DouGLas HOUGHTON CAMPBELL On the relation of root growth and development to the temperature and aeration of the soil (with five text figures). W. A. CANNON The anatomy of a hybrid Equisetum (with plates V—VITI). | RutH HOLDEN Some features in the anatomy of the Malvales (with plates ESE aie te uh, Oe ee a C. C. ForsalrH The absorption of ions by living and dead roots. H. V. JOHNSON No. 6, JUNE The exchange of ions between the roots of Lupinus albus and culture solutions containing one nutrient salt (with thirteen text figures). RopNEY H. TRUE AND HARLEY HARRIS BARTLETT Notes on the forms of Castela Galapageia (with ten text figures). ALBAN STEWART The development of Pyronema confluens var. inigneum. WILLIAM H. BROWN : New or noteworthy grassés.o.......%.......A. 5. El@enegem Na. 77, JUEY The exchange of ions between the roots of Lupinus albus and culture solutions containing two nutrient salts (with three text figures). | RopNEY H. TRUE AND HARLEY HARRIS BARTLETT 157 161 162 169 199 201 225 238 250 hi: TABLE OF CONTENTS The probable non-validity of the genera Botryodiplodia, Diplodi- ella, Chaetodiplodia, and Lasiodiplodia (with plates XII-— DOING ee a J. J. TAUBENHAUS - Factors influencing flower size in Nicotiana with special reference to questions of inheritance (with four text figures). T. H. GooDSPEED AND R. E. CLAUSEN No. 8, OCTOBER ' The utilization of certain pentoses and compounds of pentoses by ClGmiereli CoMlala.. ates eos ek Lon A. HAWKINS The question of the toxicity of distilled water...R. P. HIBBARD An anatomical study of Gymnosporangium galls (with one text metre, and plates XV and XVI)...... 2.5. ALBAN STEWART An extension to 5.99° of tables to determine the osmotic pressure of expressed vegetable saps from the depression of the MMe ZEMOO UNG. Sect soe 6 iA. ke eee J. ARTHUR HARRIS Calcium hypochlorite as a seed sterilizer....JAMES K. WILSON No. 9, NOVEMBER Seasonal duration of ascospore expulsion of Endothia parasitica (with six text figures)... F. D. HEALD AND R. A. STUDHALTER Dimorphism in Contothyvrium pirinum Sheldon (with fifteen text INU OS Oe tac? ds Gs AU eetierersa near eee, Core CRABILE The genus Espeletia (with six text figures and plate XVII) PAE cO =.) SRANDI EY A study of the relation of transpiration to the size and number of stomata (with one text figure). WALTER L. C. MUENSCHER No. 10, DECEMBER A brief sketch of the life and work of Charles Edwin Bessey (with plate XVIII)... Rye Mohd on emer .RAYMOND J. PooL Heredity and mutation as cell Shenetie na. R. RUGGLES GATES The relation between vegetative vigor ney reproduction in some Saprolegniaceae (with two text figures)... ADRIAN J. PIETERS Prraemto. WOLUING: E13 ck ses * abate ty aie eee pnels te ite aa See 389 402 418 420 429 449 468 486 595 519 929 Di (Dates of publication, No. 1, Feb. 18; No. 2, Apr. 3; No. 3, Apr. 2a NO. 4, May 13; No:.5, June 16; Nos6, July 17- No. 7, Aug. No. 8, Nov. 4; No. 9, Dec.; No. 10, Dec.) ite} ERRATA ERRATA, VOLUME II. mS: Page 51, Fig. 3, for constantine, read constantan. ‘216, fourtia line from bottom, for mwmbers, read members. ‘ 318, line 16, transpose comma to follow days. 319, line 26, for mixture, read mixture. — . 310, fifth line from bottom, for supports, read supported. 6c a “CONTENTS, Losey on: the eee of the Angiosperms. 5 Rohan evi-- dence as to the ancestry and early climatic environment of the ~ Angiosperms. i Epmunp. W. SINNOTT AND. IRVING Ww. BAILEY ¥ | The growth-forms of the flora of New York and vieinity.. ei ‘Norman TAYLOR, 33 he, td ) ea MO i ere mh ; SH eA al Wehr ae weet” » x - i Erg yeni men } A B ooklyn Botanic Carden € N e Journal is published aaa except durin ne an ae ere price ce $3.00 « a hia ne members of the 1 | iS Aa — Are d be: made poe = A for mising nan shod | ow i gape ene m phe ron Neweambe, 9 : eae) eee fepitaee yA ne ‘ 3 j ( $ a j ~‘ f AMERICAN JOURNAL OF BOTANY ——— VOL. al January, 1915 ING ee ON, THE PHYLOGENY OF THE ANGIOSPERMS 5. FoLiAR EVIDENCE AS TO THE ANCESTRY AND EARLY CLIMATIC ENVIRONMENT OF THE ANGIOSPERMS EpMUND W. SINNOTT AND [RVING W. BAILEY The leaf of the Angiosperms is so variable in its shape and vena- tion among closely related species and so easily modified by environ- mental influences that gross foliar characters have been largely neglected by the taxonomist and the phylogenist except within small groups of plants. A careful survey of evidence obtained from the various botanical fields, however, apparently makes possible a recon- struction of the primitive Angiosperm leaf with a reasonable degree of certainty, and a determination of some of the factors which have modified it; and thus suggests not alone the probable ancestry of the Angiosperms but also the climatic conditions under which they first appeared. ‘The presentation of evidence on which such an hypothesis may be built is the purpose of the present paper. It is with the more ancient of the two Angiosperm classes, the Dicotyledons, that the problem necessarily rests. The two main types of leaf venation in this group are the palmate and the pinnate, between which intermediate conditions frequently occur. Leaf shape is, of course, generally correlated with venation although there are numerous instances where broad leaves are pinnate and narrow ones palmate. The main leaf types, having reference both to venation and shape, may be roughly designated as the palmate simple (fig. 2), [The Journal for December (1 : 499-550) was issued 29 Dec. 1914.] I 2 EDMUND W. SINNOTT AND IRVING W. BAILEY palmate lobed (fig. 3), palmate compound! (fig. 7), pinnate simple (fig. 1), pinnate lobed (fig. 17) and pinnate compound (fig. 10). A brief survey of the distribution of these types throughout the three main growth forms of Dicotyledons (trees, shrubs and herbs) in various regions of the earth is set forth in the following table.? It will be observed that in these modern floras the pinnate type is markedly predominant everywhere and that the simple pinnate condition alone constitutes from 60 per cent to 70 per cent of the species. Other noteworthy facts are the abundance of compound leaves in warm regions, the almost complete absence there of palmately lobed woody plants, and the practical confinement of the pinnately lobed type to herbaceous forms. ; In view of the preponderating evidence that woody Angiosperms are more ancient than herbacous ones,*® it seems probable that leaf — 1 Trifoliate leaves with stalked terminal leaflets (frequently called pinnately trifoliate) will be regarded in this paper as palmately compound. 2 Analyses have been made of the following floras as to types of leaf or types of node or both: North America: Flora of the Northern United States and Canada, Britton and Brown; Flora of the Rocky Mountains, Coulter; Flora of the Florida Keys, Small; Flora of the British West Indian Islands, Grisebach; Flora Nicaraguensis, Goyena. South America: Flora Brasiliensis, Martius and others; Historia de Chili: Botanica, Gay; Report of the Princeton Expedition to Patagonia: Botany, Macloskie. Europe: English Botany, Sowerby; Flora des Necrdostdeutschen Flachlands, Ascherson and Graebner; Flora Rossica, Ledebour; Flora des Alpes, Bouvier; Flora Espafiola, Lazara; Flora Italica, Fiori and Paoletti. Asia: Flora Orientalis, Boissier; Flora of Syria, Palestine and Sinai, Post; Flora Simlensis, Collett; Flora of the Upper Gangetic Plain, Duthie; Flora of Bom- bay, Cooke; Handbook of the Flora of Ceylon, Trimen; Maylayan Flora, King; Flora van Nederlandesch Indié, Miquel; Flora Hongkongensis, Bentham; Flora of Manila, Merrill. Africa: Manual of the Flora of Egypt, Muschler; Flora of Tropical Africa, Oliver, Thiselton-Dyer and others; Flora Capensis, Harvey and Sonder, and others; Forests and Forest Flora of Cape Colony, Sims; Natal Plants, Wood; Plantes de Madagascar, Baillon and Castillo; Flora of Mauritius and the Seychelles, Baker. Australasia: Flora Australiensis, Bentham; New Zealand Flora, Cheeseman. Oceana: Flora of the Hawaiian Islands, Hillebrand; Indigenous Trees of the Hawaiian Islands, Rock. Analyses were also made of the genera of woody Dicotyledons enumerated in Engler and Prantl’s Natiirliche Pflanzenfamilien; and of the species of trees and shrubs from China and Japan in the herbarium of the Arnold Arboretum. ’The Origin and Dispersal of Herbaceous Angiosperms. Sinnott, E. W .and Bailey, I. W., Annals of Botany, 28: Oct. 1914. INVESTIGATIONS ON THE PHYLOGENY OF THE ANGIOSPERMS 3 TABLE I Palm. | Palm, |)>PRalm. | Pinn, Pinn, Pinn, Se L. 5 Na ero alte OMAN 470R es 70rule 70 Engler Tropical Regions RSE MEL i 1,326 genera| 6 2 4 62 — 26 SIRS rie ek as des 1257 14 I 3 69 = 12 Temperate Regions MGCES Hk es ld ke B73 eb et 8 6 5 60 I 19 Silene ee TeOu eo Gi 2 5 76 8 mew giicd States... 2... ... 2,821 sps. SOAS SSic, = ie ae ae eee BOQ eee hx. 7 6 3 57 8 19 SIMMS Hosier cc Salen. S ess Be Orcne 5 9 8 72 — a |S Oe ele el aa Dea Ow e's II 4 10 58 6 10 GreGh BVMAIN. 2 8. oe I,310 sps. IS282C% (a5 eae ee Vee bret. 7 5 — 78 4 6 ‘SLU S Se eee eee E2G.- 3 oo I 7 15 63 — 14 SGI OSS cy, eran ae ElOles 3. 8 7h 8 56 9 12 HIEI0. 0.3 C/O eee 3,079 sps. LESS Se re Gy eg ar a ESe HcLe 5 48 6 13 SMUG ss ROOM A. 4 9 I2 55 I 19 PMU Peso e Pee PRT ON mite 3 5 5 I 56 II 10 idee ee MO ees Fe, 2,075 sps. LES ee tanta aan Oller ae 14 10 3 56 — 17, Samii ete ers ce tn PE TA We 4: 10 6 II 62 — | II LCA CASAS OGD Nt a er 542 sps. Mircesyte eta en. oS a (aes 120) | at! 2 60 = Le STEMS sass ew sian BATE. eee Gf 7 1a 66 == 7 PMG 2ONW VOUCWn. vc. sss 2 187 sps SWECeS mt ee rw 1 Fae: OAT. ae. cit 3 5 0 66 == 24 SUE UO Sera ees sass TOGS =. oe tes. 19 5 6 65 = 9 lise¥e| OS, se te el eee Lie tans Mae 25 5 8 56 2 3 NUON ORCH AOR 10} . 2. .-.<. 2,811| 85 15 69 31 58 42 Rocky, Mountains: 2... 1 sete oe 15405) 290 10 84 16 62 38 GreatiBuitain tine a oe ye ee T,2e0)\ (On 6 81 19 56 44 Cernran yasers cee caercec ee tae is 1, b20|mor 3 81 19 54 46 Russian Empire Wena. ee eee ee 3,676) 95 5 70 30 58 42 JEN TDCESs 5 wiki cations hist Sb Beso gba oe 1,735| 96 4 87 ia 57 43 SPA acne. cee ender os cone 2,901) Or 9 43 57 53 A7 talline cs Caotin seen ae oe *3,069| 92 8 67 23 60 40 MlorayOrnientalis™.-b4.9r5.gser er 9,848} 71 29 59 AI 63 37 Oh VR SRR ene oh cis ca. EL Canes ee 2,570) 84 16 54 46 68 20 BENNO Girt Aen 6.5 6 BE osu o> ao aaa 1,138)" 82 17 53 47 63 27, Ghia 515-o actatees Soe he eats 2,075 76 24 67 oe ETOP GA Gedismaadtho9 16 sooo od 9 ares 542, 87 13 63 37 atagoniawy.:.. ..ssvereeey uae ee 1,405| 84 16 66 34 67 aa Ghia sey ce ite a err tee See ke oe: 2,160) 76 24 67 33 67 ling INewsZealandie! ries ise Q74| 72 28 A7 53 61a. 30 Plongkong wines nares see tee 681} 69 31 AI 59 52 48 Srl a ee Pe ie eee ie ape eee ee 987| 76 24 61 39 56 44 Gangetic lainey. eee ene ea 996) 60 40 60 40 54 46 Florida Weys iis Geiger a,. sad 466) 56 44 50 50 47 53 Australia (extratropical). ) 5. =: 5,874) 51 49 67 Ze) 63 + 37 SOUtHAATTI Calas ey tice nee eo 7,284| 60 40 58 42 45 55 Amazon, Valllegoithanecets acres seca 2A TAN 57 43 30 70 38. 62 British West Indies; 4... 1... .. 2. 2,191; 46 54 46 54 50 50 Nicaragua: en nee ea eee 1,496} 66 34 54 46 58 42 TropicalpAfncac.c.. ) rer yt: 110,520) 54 46 32 68 39 61 Matinitits:: tact uy rae ote 555) 45 55 42 58 56 44 Ceylon ons ee ee sete eg ene 1,752} 50 50 43 57 36 64. Bombay, Uplands (eens. cteie eae: 996| 61 39 50 50 50 50 Bombay, Wowlandsw. 2.) eae. 1,229} 50 50 48 52 46 54 Dutch Bastsladiess cs veers. 6,246| 49 51 40 60 44 56 Malay heninsulat ey tocwua str 3,049| 49 51 2 68 26 74 Manila BE in Ss re ee ae. OR te A 232900 34 47 53 44. 56 Hawailcc.e. eee eee eee 508} 50 50 41 | 59 51 49 from 70 percent or 80 per cent in the former to 30 per cent or 40 per cent in the latter. Trees, however, which are probably the most ancient type of all, show the widest extremes and among them the relative proportion of the two nodal types is very closely correlated with climate. In the great land area of the north temperate zone INVESTIGATIONS ON THE PHYLOGENY OF THE ANGIOSPERMS 17 from 85 per cent to 95 per cent (averaging 90 per cent or more) of the arborescent species have three or more gaps at the node. This type is also dominant in the temperate non-arid regions of the Southern Hemisphere, such as Chili, Patagonia and New Zealand; and, to a less extent, in upland areas in or near the tropics, as Nicaragua, Simla and the Bombay uplands. In the tropics, however, there is a strong contrast for here 50 per cent or more of the tree species are unilacunar. Almost all the unilacunar arborescent genera are typi- cally tropical in their distribution. Subtropical regions, such as the Florida Keys, the Gangetic Plain and Hongkong, show a percentage intermediate between the two extremes. In South Africa and extra- tropical Australia, the great arid areas of the South Temperate zone, the unilacunar type is only slightly less common than in the tropics. In connection with this concentration of the presumably primitive trilacunar woody type in generally temperate and mesophytic habitats, it should also be borne in mind that most of the genera and families regarded as being particularly ancient are best developed in temperate or warm-temperate regions. These facts, which are in agreement with a large body of evidence gathered by the writers from other sources, all point strongly to the conclusion that tropical plants are specialized in almost every par- ticular and that the Angiosperms therefore could not have arisen under a tropical environment, as has generally been taken for granted, but must have had their origin in a climate which, though doubtless very equable and devoid of extremes of temperature, was essentially a temperate one. Suchaclimate, as far as we are able to judge of condi- tionsin the Mesozoic, could only obtain, asa general rule,in upland or mountainous regions. Onsucha supposition the Mesozoic Angiosperms which we know are to be regarded as plants which had migrated down into the tropical lowlands and swampy areas, where they could readily be preserved as fossils. If the earliest Dicotyledons were indeed largely confined to temperate upland regions (where fossilization could almost never take place), does not this explain why we know of so very few Mesozoic Angiosperms and why these are so diverse and often so highly specialized and far from primitive? And does it not strongly suggest the possibility that on these ancient mountains, about the vegetation of which we know so little, Angio- sperms may have been developing in extremely remote times, con- siderably earlier than the Lower Cretaceous? 18 EDMUND W. SINNOTT AND IRVING W. BAILEY PHYLOGENETIC CONCLUSIONS If the hypothesis which has here been presented is a sound one and the trilacunar palmate leaf type is indeed the most ancient among Angiosperms, the theory which derives these dominant seed plants from Cycad-like Gymnosperms meets with grave difficulty. The Mesozoic Bennettitales, from which many phylogenists believe the Angiosperms to have descended, were unilacunar and possessed, like all Cycads, pinnately compound leaves. The difficulty of deriv- ing from such a leaf the foliar types which prevail among living Angio- sperms has been noted by many writers. Wieland” suggests that the gap may have been bridged by a fusion of the leaflets of the com- pound leaf, with the eventual production of a simple pinnate one. But if a palmate leaf was indeed the primitive Angiosperm con- dition it is difficult to see how it could have arisen from any cycadaceous type. The Conifers, on the other hand, are invariably palmate in their venation. Although the common leaf type is a narrow one with but one or two strands, there are num- erous cases in the Araucarineae and Podocarpineae where the leaf becomes broader. In accommodation to this change the vascular tissue increases in amount, but always by a basal multiplication of the strands in palmate fashion rather than by the origin of branches from a midrib (figs. 74 and 75). It is possible to imagine how such a broad-leaved form, developing a trilacunar type of insertion (Agathis is occasionally bilacunar) might easily give rise to the palmate leaf which we have regarded as ancient. Evidence from the leaf certainly favors a coniferous rather than a cycadean stock as ancestral for the Angiosperms. Information from this source is also important in connection with the ancestry of the Monocotyledons. These plants are almost invariably palmate in their venation, and in many cases are three- veined. In the broad-leaved members of the Potamogetonaceae, Alismaceae, Araceae, Liliaceae, Dioscoreaceae and others we often meet with leaves which are essentially like those of the Piperaceae, Melastomaceae and other palmate Dicotyledons (figs. 76 and 77). The nodal conditions in such forms often suggest the dicotyledonous type, three traces departing to each leaf (fig. 78), and the petiole is 10Was the Pterophyllum Foliage Transformed into the Leafy Blades of Dicotyls? Wieland, G. R., Am. Jour. Science 38: 451-460. I914. INVESTIGATIONS ON THE PHYLOGENY OF THE ANGIOSPERMS I9 also frequently trifasciculate. The cotyledon and early leaves in several families of Monocotyledons have been shown by Chrysler and Coulter and Land” to have three bundles (fig. 79), apparently a vestige of the more ancient condition. The conspicuous tendency for the cotyledonary veins in the seed-leaves of many Dicotyledons to unite at the tip of the lamina (figs. 21, 27, 28, 38, and 44) producing a closed venation like that of the Monocotyledons, suggests either that the Monocotyledons are the more ancient of the two classes, a con- clusion opposed by too much evidence from other sources; or that the leaf in these plants has been developed by a process of reduction much like that gone through by the cotyledons of Dicotyledons and by the mature leaves of certain members of that class. The Mono- cotyledons apparently had their origin from a palmate dicotyledonous stock, very likely at an early period when the palmate leaf was pre- dominant among all Angiosperms. SUMMARY 1. The primitive Angiosperm leaf was palmate in type, probably lobed, and was provided with three main bundles which arose separ- ately at the node. 2. This conclusion is based on evidence from palaeobotany, that the palmate leaf was more frequent in the Cretaceous and Tertiary than at present; from morphology, that there is a correlation between the palmate leaf and the multilacunar node and between the pinnate leaf and the unilacunar node and that the former nodal type is the more ancient, that cotyledons and floral leaves are much more fre- quently palmate than are vegetative ones, and that vigorous growth emphasizes the palmate type; and from phylogeny, that palmate leaves are most frequent in relatively primitive groups and pinnate leaves in more advanced ones. 3. Transitions from the palmate lobed type to all other leaf forms may readily be traced. 4. The chief factor in the evolution of the now dominant pinnate leaf seems to have been the development of the petiole, in which 11 The Development of the Central Cylinder of the Araceae and Liliaceae, Chrysler, M. A., Bot. Gaz. 38: 161-184. 1904. 22 The Origin of Monocotyledony, Coulter, J. M. and Land, W. J. G., Bot. Gaz. 57: 509-519. I914. 20 EDMUND W. SINNOTT AND IRVING W. BAILEY the three originally distinct leaf bundles were pulled together into a single strand, the midrib. 5. Among woody plants, the multilacunar (more ancient) nodal type predominates in temperate regions and the unilacunar (more recent) in the tropics. The palmate lobed leaf among such plants is also almost entirely confined to temperate regions. These facts, in company with others, indicate that the Angiosperms first appeared under a climate more temperate than tropical, a climate in the Meso- zoic probably found only in the uplands. It is suggested that the absence of the earliest Angiosperms as fossils may be due to their confinement to such upland regions, where preservation would be very difficult. 6. Evidence from the leaf strongly favors the view that the Angio- sperms sprang from a coniferous (palmate) rather than from a cyca- dean (pinnate) stock. 7. The Monocotyledons were derived from some ancient palmate group of Dicotyledons. The writers are much indebted to the authorities of the Gray Herbarium and of the Arnold Arboretum for the use of their libraries and herbaria. BussEY INSTITUTION, HARVARD UNIVERSITY. DESCRIPTION OF FIGURES OF PLATES I-IV Fic. 1. Acer carpinifolium Sieb. & Zucc. (pinnate simple). Fic. 2. Acer tataricum L. (palmate simple). Fic. 3. Acer ginnale Maxim., vigorous leaf (palmate lobed). Fics. 4 AND 5. Acer ginnale Maxim. leaves of less vigorous growth. Fics, 6 AND 7. Acer glabrum Torr., two leaves from the same twig showing origin of palmately compound type. Fics. 8,9, IO ANDII. Acer Negundo L., series of leaves showing transitions from trifoliate to ternately compound. FIGS. 12 AND 13. Rubus negroensis Elm. and Rubus benguitensis Elm., tropical species reduced from primitive type. Fic. 14. Rubus odoratus L., presumably primitive type for the genus. Fic. 15. Rubus neglectus Peck, leaf showing the transition from palmately to pinnately compound. Fics. 16 AND 17. Solanum nigrum L. and Solanum carolinense L. two closely related species showing transition from pinnate simple to pinnate lobed. Fics. 18 AND 19. Ampelopsis tricuspidata Sieb. & Zucc., a leaf of vigorous and one of weaker growth. | INVESTIGATIONS ON THE PHYLOGENY OF THE ANGIOSPERMS) 2I Fics. 20 AND 21. Viburnum acerifolium L., a leaf of vigorous and one of weaker growth. FIGS. 22, 23 AND 24. Cercis occidentalis Torr., Bauhinia sp. and Hymenostegia sp., series showing origin of the pinnately compound type among certain of the Leguminosae. FIG. FIG. FIG. Fic. Fic. Fic. Fic. FIG. FIG. Fic. FIG. Fic. Fic. FIG. FIG. FIG. Fic. FIG. FIG. FIc. Fic. FIG. Fic. simple). FIG. FIG compound). Fic. Fic. Fic. Fic. Fic. FIG. s imple). Fic. FIG. FIG. Fic. FIG. simple). Fic. EG: 25. 26. 27, 28. 29. 30. a: 22) 33- 34- 35: 36. 37: 38. 39- 40. Al. 42. 43. 44. 45: 46. 47- 48. . 49. 50. 5I. 52s 53: 54. 55: 56. o7: 58. 59- 60. 61. 62. Engelhardtia spicata Blume, floral bract (leaf pinnate compound). Betula nigra L., floral bract (leaf pinnate simple). Carpinus caroliniana Walt., floral bract (leaf pinnate simple). Bocconia frutescens L., floral bract (leaf pinnate lobed). Dalechampia Passiflora C. &. H., floral bract (leaf palmate simple). Adhatodia vasica Nees, floral bract (leaf pinnate simple). Berberis Bealeit Curt., petal (leaf pinnate simple). Schizophragma hydrangeotides Sieb. & Zucc., petal (leaf pinnate simple). Guichenotia macrantha Turcz., petal (leaf pinnate simple). Anitsopiera glabra Kurz, calyx lobe (leaf pinnate simple). Calycopteris floribunda L., calyx lobe (leaf pinnate simple). Mussaenda tenuiflora Benth., petal (leaf pinnate simple). Casuarina stricta Ait., cotyledon (leaf greatly reduced). Pterocarya fraxinifolia Spach, cotyledon (leaf pinnate compound) Betula nigra L., cotyledon (leaf pinnate simple). Celtis occidentalis L., cotyledon (leaf palmate simple). Grevillea robusta A. Cunn., cotyledon (leaf pinnate compound). Antigonon leptopus Hook., cotyledon (leaf pinnate simple). Mirabilis jalapa L., cotyledon (leaf pinnate simple). Rivinia aurantiaca Warsz., cotyledon (leaf pinnate simple). Delphinium formosum Boiss. & Hook., cotyledon (leaf palmate lobed). Brassica olearacea L., cotyledon (leaf pinnate simple). Crataegus oxyacantha L., cotyledon, after Lubbock (leaf pinnate Mimosa pudica Mill., cotyledon (leaf pinnate compound). Canarium strictum Roxb., cotyledon, after Lubbock (leaf pinnate Euphorbia pulcherrima Willd., cotyledon (leaf pinnate simple). Rhus typhina L., cotyledon (leaf pinnate compound). Acer saccharum Marsh, cotyledon (leaf palmate lobed). Rhamnus cathartica L., cotyledon (leaf pinnate simple). Ambelopsis tricuspidata Sieb. & Zucc., cotyledon (leaf palmate lobed). Elaeocarpus oblongus Smith, cotyledon, after Lubbock (leaf pinnate Tilia americana L., cotyledon (leaf palmate simple). Althaea rosea Cav., cotyledon (leaf palmate lobed). Passiflora alaia Ait., cotyledon (leaf pinnate simple). Lecythis Ollaria L., cotyledon, after Lubbock (leaf pinnate simple). Aucuba japonica Thunb., cotyledon, after Lubbock (leaf pinnate Eucalyptus calophylia R. Br., cotyledon (leaf pinnate simple). Argania sideroxylon Roem. & Schult., cotyledon (leaf pinnate simple). De EDMUND W. SINNOTT AND IRVING W. BAILEY Fic. 63. Diospyros Lotus L., cotyledon (leaf pinnate simple). Fic. 64. Ipomaea purpurea Roth, cotyledon (leaf palmate simple). Fic. 65. Eranthemum leuconeurum Fisch., cotyledon, after Lubbock (leaf pinnate simple). Fic. 66. Coffea arabica L., cotyledon, after Lubbock (leaf pinnate simple). Fic. 67. Viburnum Opulus L., cotyledon (leaf palmate-lobed). Fic. 68. Cucumis Melo L., cotyledon (leaf palmate-lobed). Fic. 69. Senecio multiflorus DC., cotyledon (leaf pinnate simple). Fic. 70. Tropaeolum majus L., young leaf, near the growing poini. Fic. 71. Tropaeolum majus L., mature leaf. Fic. 72. Pterospermum Heyneanum Wall., leaf from coppice shoot. Fic. 73. Pterospermum Heyneanum Wall., leaf from mature branch (both after Brandis). : Fic. 74. Podocarpus Nagi Pilger, many-veined (palmate) leaf. Fic. 75. Podocarpus Halli Kirk, primitive type of leaf, with single bundle. Fic. 76. Sagittaria latifolia Willd., mature leaf. Fic. 77. Trillium cernuum L., mature leaf. Both this and the last are essen- tially like many dicotyledonous leaves in venation. Fic. 78. Trillium cernuum L., diagrammatic cross section through the node, showing the similarity to the dicotyledonous type. Fic. 79. Agapanthus umbellatus L’Her., diagrammatic cross section through the seedling, showing the three bundles in the cotyledon and in the first leaf (after Coulter and Land). | Fic. 80. Viburnum acertfolium L., cross section through the petiole. Fic. 81. Rzbes rubrum L., cross section through the petiole. Fic. 82. Ulmus americana L., cross section through the petiole. Fic. 83. Reconstruction of the leaf (with cross section of the node) supposedly primitive for the Angiosperms. The node is trilacunar and the veins depart directly into the lamina without approximation in a petiole. Fic. 84. A typical modern palmate lobed leaf. The node is trilacunar but the traces are pulled closely together in a narrow petiole before separating again in the lamina. Fic. 85. A pinnate simple leaf. The node has become unilacunar and the single trace continues as a strong midrib. AMERICAN JOURNAL OF BOTANY. VOLUME II, PLATE I. 5 SINNOTT AND BAILEY: FOLIAR EVIDENCE AS TO ANCESTRY OF ANGIOSPERMS. » ~# =) re VOLUME II, PLATE II. AMERICAN JOURNAL OF BOTANY. 15 L9 24 Hn en 21 SINNOTT AND BAILEY: FOLIAR EVIDENCE AS TO ANCESTRY OF ANGIOSPERMS. AMERICAN JOURNAL OF BOTANY. VOLUME II, PLATE IV. ens 78 EMS. SINNOTT AND BAILEY: FOLIAR EVIDENCE AS TO ANCESTRY OF ANGIOSPERMS. + ry b Fi 4 ‘ eas ‘ ’ ‘ Y . é = i ” THE GROWTH-FORMS OF THE FLORA OF NEW YORK AND VICINITY! NORMAN TAYLOR There has recently appeared in a work on the flora of New York,? an account of the relation between climate and the vegetation in which the length of the growing season was used as the chief tem- perature factor. This was done for the reason that it seemed to account for the distribution of the flora more closely than any other ascertainable temperature factor. While it is difficult to conceive of any climatic agency, such as the number of frostless days, the maxi- mum or minimum temperatures, or the accumulated heat units, as actually controlling the distribution of the flora, yet it is a matter of common observation that temperature does affect vegetation and its distribution. How, then, are we to measure the effect of climate, and particularly temperature, on plant life? All of the older methods, including the one used in the flora of the vicinity of New York, have studied climate as a rather distinct entity, and then imposed a somewhat rigid, usually instrumentally correct scheme, on a com- plex aggregate like a local flora. All such schemes require con- siderable wrenching of the purely climatic factors on the one hand, as they certainly do of the assumed vegetative response on the other. Until recently, and with the possible exception of Merriam’s ‘Life Zones,’ all studies of the effect of temperature on plants were of this type. They were essentially attempts to explain the facts of plant distribution by measured temperatures or heat units or frostless days or by some combination of these methods. Raunkiaer? has studied temperature factors from an entirely different viewpoint. His idea, briefly, is that we must study climate, -1 Brooklyn Botanic Garden Contributions, No. 9. *Taylor, N. Flora of the vicinity of New York: A contribution to plant geography. Mem. N. Y. Bot. Garden 5: 1-683. 1915. See pp. 33-36. All the statistics and figures used in this paper may be verified in that book. The nomen- clature of the present paper is essentially that of the Gray’s Manual. 3 A fairly complete account of Raunkiaer’s system, with a bibliography, may be found in Journ. Ecol. 1: 16-26. 1913. 23 24 NORMAN TAYLOR not as such, but as it is reflected in the vegetation which we all know it to have controlled. Such a method is not at all in line with older studies, it is really an ex post facto method of determining climate by the character of the vegetative response. From the standpoint of the ecologist and plant geographer, what could be more logical and reasonable? Temperature factors are not what one makes them out to be with an elaborate instrumental or niathematical method; they are rather what one finds them as reflected in the vegetation itself. Of course, in order to get the quality and kind of this implied vegetative response, we must study plants in a slightly new light, and Raunkiaer has devised, after Warming and a few earlier writers, a scheme for such a study. His theory is that plants react to climate by the kind and amount of protection exhibited by the perennating growth points during the winter or critical season. Upon this assumption he divides all vegeta- tion into several different groups of growth-forms sometimes called life-forms, depending on the kind and amount of protection to their growing buds exhibited by each. In the following account, I have included only those of his growth-forms that are found in eastern North America, which may be characterized as follows: Phanerophytes. Woody plants of all types, both evergreen and deciduous and exhibiting the least amount of protection from the cold, as showing the greatest amount of exposure. The group may be divided into Megaphanerophytes, trees over 30 m.; Mesaphan- erophytes, trees 8-30 m.; Microphanerophytes, shrubs or trees 2-8 m.; Nanophanerophytes. shrubs under 2 m. Examples of all these are too common to need citing. Chamaephytes. Perennial by virtue of the fact that the buds are just above the ground, or on the surface, and are thus often protected by the snow blanket. Among local species Arctostaphylos Uva-Ursi, Epigaea, Convolvulus, etc., are good examples. It includes, also, cushion-plants. Hemicryptophytes. With dormant buds in the upper crust of the soil, the top of the plant dying down in the winter. Common examples suggest themselves, as all our shallow-rooted herbaceous perennials belong here. Geophytes. Perennial by bulbs, rhizomes, tubers or by root- buds. Examples among our native plants: most Orchidaceae, Lili- aceae, Sanguinaria, Hydrastis, etc. GROWTH-FORMS OF FLORA OF NEW YORK AND VICINITY 25 Helophytes and Hydrophytes. The former has buds at the bottom of the water. They are mostly marsh species such as Typha, Spar- ganium, Acorus, etc. Hydrophytes have perennating rhizomes or winter-buds and are truly aquatic, such as Castalia, Elodea and Potamogeion. Therophytes. Annuals. A tabular view of these different growth forms, with the abbrevi- ations as used in this paper follows: MG = Megaphanerophytes CH = Chamaephytes MS = Mesaphanerophytes H = Hemicryptophytes MC = Microphanerophytes G = Geophytes N = Nanophanerophytes HH = Helophytes and Hydrophytes T = Therophytes Raunkiaer’s method of adapting the study of these growth-forms as related to temperature is to estimate the number of species char- acterized by these different forms, and to get the percentages of the different growth-forms in the flora. For the purpose of comparison he established a “normal spectrum”’ which is constructed on purely hypothetical lines. It consists of 400 species carefully chosen from 1,000 representative species. The analysis of these 400 species into their different growth-forms gives, theoretically, the ideal phyto- climatic spectrum of the whole earth. According to Raunkiaer the percentage of species belonging to each growth-form, in the ideal spectrum of 400 species, is as follows: PERCENTAGE OF GROWTH-FORMS IN NORMAL SPECTRUM? divine Of srowtn-fOrmM. .. <5... 0.62 o MEGre MS; MC N CH H GG HH T Percentage of growth-forms........ 6 Wp 2 Oy Oo) 27, 3 I 13 These percentages are supposed to reflect the average condition as to the growth-forms of the whole earth. Of course they may need future revision; it would be strange if they did not as our knowledge of the habits of various species increases. The method of comparing the climate of different parts of the earth’s surface, on this conception, involves working out the per- centages of the different growth-forms exhibited in the areas and a 4T have omitted epiphytes and stem-succulents, as being two of Raunkiaer’s — groups hardly applicable to our area. 26 NORMAN TAYLOR comparison of the figures thus obtained. The following table gives a few of the percentages as determined by Raunkiaer for widely separated areas. The percentages of the normal spectrum are given for comparison. PERCENTAGES OF GROWTH FORMS IN BIOLOGICAL SPECTRA, AFTER RAUNKAIER Type of Growth-form Le MC N CEH H G RH aE Normal spectrum......... 6 Wf 20 9 27, a I 13 Batinisdeands. eae en ae I 30 51 1h) 3 2 Georgia ce see toe ae Sel ae 16r 4 55 4 6 8 Denmark ac 3 een I 3 3 3 50 II II 18 Seychelles ie 2 'Jan = wee tee 10 22 24 6 12 3 2 16 Libyan: Desert. 1.352 328. ao 9 21 20 4 I 42 These figures give some idea of the variation of climate implied by the different growth-forms predominating in the different areas. They also make more cogent the terms phanerophytic, hemicrypto- phytic and chamaephytic as applied to climate. Raunkiaer has indicated three types of climate, as shown by ie study of growth-forms, namely a tropical area, an area of decreasing warmth correlated with an increasing difference between winter and summer temperatures and with a favorable distribution of preci- pitation, and lastly a region with decidedly decreasing temperature, or also, very commonly, with an unfavorable distribution of precipi- tation, such asin deserts. The tropics are typical of the first of these conditions, the eastern sides of North America and Asia are typical of the second, and the arctic region and some deserts are characteristic of the third type of climate, which may be seen also in our own South- west. Many refinements of these rather gross outlines of climate have been worked out, based on the so-called biochore, which is a line with the same percentages of a definite growth-form, as found in different parts of the continent. Such a study of the flora of North America would be extremely interesting. It can be rightly based only on complete percentages of growth-forms for different parts of the country, and it is with the idea of supplying this for the local flora area that the present paper has been written. In attempting to apply Raunkiaer’s principles described above and to get the biological spectrum of the flora near New York, I have thrown out of the calculation all the 615 species of introduced weeds, GROWTH-FORMS OF FLORA OF NEW YORK AND VICINITY P47 the 85 ferns and their allies, and 24 parasites. This leaves 1,907 native species that are found, roughly speaking, within 100 miles of New York City. Each species has been put in one or other of the categories mentioned above with the following result. BIOLOGICAL SPECTRUM OF THE FLORA OF NEW YORK AND VICINITY® Growth-form MG MS MC N CH H G HH 40 Gymnosperms...... 15 2 2 Monocotyledons.... 5 53 LQ7, 2), 202 164) 57 Dieotyledons: . 2. ... 10 62 130 | 65 AS | 43804). 105 60 | I9I “TCE Ss a 10 Vii neta 67 TOI | 635 | 397'| 224 | 248 %o Zo % % %o %o % % % Percentages of growth-forms..... SO IEO SM aor |i 3.50 le5<29) | 33620.|.20:23 | 11e74.| 13 The most remarkable figure in this list is the high percentage of gecphytes, 20.23 per cent. For no region in the world has there been published such a large percentage of these plants with bulbs, rhizomes, corms and other subterranean methods of winter protection. Among the 692 native monocotyledons in the area, over 29 per cent are geophytes, while for 1,200 native dicotyledons there are only 16 per cent of the same growth-form. Undoubtedly the high percentage of monocotyiedons in our flora, the pine-barrens of New Jersey are especially rich in them, has much to do with the large percentage of geophytes. Most of the regions with high geophyte percentages are arctic or sub-arctic; and, from this point of view, the high geophyte percentage in our area is misleading and it may be a response to quite other factors than climatic ones. As compared to the percentages of the normal spectrum those for the local flora are higher in the case of the aquatics, geophytes, and hemicryptophytes, lower in the case of chamaephytes and all the phanerophytes, and the same in the percentage of annuals. Figures for other countries, mostly northern, show for hemi- cryptophytes averages ranging from 50 per cent to 60 per cent, the local flora percentage is only 33.29 per cent, well illustrating the condition that prevails in our area, where only a moderately large number of species are of northern origin. The percentage of all phan- erophytes in our area is about 14.88, in the normal spectrum it is 43 5 Not counting .57 per cent of stem-succulents. 28 NORMAN TAYLOR per cent, in the Seychelles it is 57 per cent. Upon Raunkiaer’s assumption that phanerophytes are typical of warm and _ tropical regions the figures of the local flora are somewhere near what one would expect. In the flora of New York and vicinity 13 per cent of the wild species are southern plants reaching their northern distribution out- posts in the area within 100 miles of the city. We should expect to find these southern plants exhibiting a greater percentage of growth- forms characteristic of the warmer parts of the earth, than of those characteristic of the north. In this same area, also, 8 per cent of the native plants are typically northern and reach their southern distri- bution outposts in the region within about one hundred miles of the city. We should expect to find the percentages of growth-forms characteristic of the north predominating in these northern species. The following table gives the percentages of growth-forms in the southern species, the northern species, and, for comparison, the percentages in the whole native flora. The normal spectrum is in- cluded again, for comparison. PERCENTAGES OF GROWTH-FORMS IN THE NORTHERN AND SOUTHERN SPECIES OF THE FLORA OF N. Y. AND VICINITY THAT REACH THEIR DISTRIBUTION OUTPOSTS IN THE AREA mG | ms | Mc | N CE eet G |] Ceres Normal spectrum... 6 17 20 9 27, 2 I 13 Whole native flora..| *.52 | 4.03) 7.18 |—3:51 | 5:20 | 33-20 |: 203) | ik 74a Southern species.... 5.62 | 7.08 7.83, | 30.07 | 20.53) = 7.834) raso2 Northern species.... 1.31 | 3:904)) “8.55 '/ 3825594) 20.4 a eaeeaa 2s 3-94. There are, of course, hundreds of northern and southern species that range north or south of the local flora area,® but it seemed best to ignore these, and consider only those that find either their southerly or northerly distribution outposts within the region. An analysis of these percentages of northern and southern species shows that the main lines of Raunkiaer’s scheme are admirably illus- 6 The area included is as follows: All of the State of Connecticut; in New York the counties bordering the Hudson River up to and including Columbia and Greene also Sullivan and Delaware counties, and all of Long Island; all of New Jersey; and in Pennsylvania, Pike, Wayne, Monroe, Lackawanna, Luzerne, Northampton, Lehigh, Carbon, Berks, Bucks, Schuylkill, Montgomery, Philadelphia, Delaware and Chester counties. GROWTH-FORMS OF FLORA OF NEW YORK AND VICINITY 29 trated in the local flora. We find in the southern species about 12 per cent shrubs or trees, in the northern species only 5 per cent, well illustrating the tendency for the woody plants to increase in size and profusion as we travel southward. Among the small undershrubs there appear to be none in the southern group, but over 8 per cent in the northern, which is considerably over the percentage for this growth- form in the whole flora. One of the most notable elements is the large percentage of HH in the northern species (23 per cent) and the large percentage of geophytes in the same group (24 percent). Both these figures are so much above those published for any other region, that they may be open to the suspicion that other than climatic factors have influenced them. This region in eastern North America has never had this particular criterion of temperature response applied to it, in fact I know of no region where such a large number of species as our 1907 native plants have been used in making up the percentages as published by Raunkiaer, Vahl, and Paulsen for other countries. On the basis of the large numbers of species considered, and the obviously temperate nature of our climate, it may be that the normal spectrum as now understood is in need of revision. Certainly it seems to be much too low in the case of geophytes and the aquatics, and too high in the microphanerophytes. There are 19 northern species in the area, reaching their southerly distribution outposts here, but found only at elevations in excess of 1,000 ft., and most closely approximating an alpine habitat of any of our native plants. Of course, percentages of growth-forms based _on such a small number of species are apt to be misleading, but as ‘illustrating a tendency they prove interesting. The following is a list of such species: GROWTH-FORMS OF NORTHERN SPECIES REACHING THEIR SOUTHERLY DISTRIBUTION OUTPOSTS IN THE AREA, BUT FOUND ONLY AT ELEVATIONS IN EXCESS OF 1000 FT. Xyris montana = H Viola renifolia = G Juncus filiformis = G Moneses uniflora = G Spiranthes Romanzoffana = G Ledum groenlandicum = N Mitella nuda = H Vaccinium Brittonii = N Fragaria canadensis = CH Adoxa Moschatellina = G Fragaria terra-novae = CH Valeriana uliginosa = G Rubus pergratus = H Solidago macrophylla = H Pyrus sitchensis = MS Aster junceus = H Viola nephrophylla = G Petasites palmatus = H Viola Selkirkii = G 30 NORMAN TAYLOR PERCENTAGE OF GROWTH-FORMS IN ABOVE LIST (The percentages of the whole region included for comparison) Growth-form | mc | Ms | iC) ONS crs ae G | Serene Wiholeresionie.s. ae) 752.4 41.02 | 7.18 |\ (3251 |! -5.20)1123)20 |'20.23 ils meee Northern mountain SPECIES s.eeoruaa el 25-20 10.52 | 10.52 | 31.57 | 42.1 In the case of mesophanerophytes the percentage is obviously misleading, but the notable figure here, as in all those for the local flora area, 1s the high percentage of geophytes. It is often difficult to decide whether or no any given species belongs to the geophytes or hemicryptophytes, but errors of assignment to one or other of these groups should about equalize each other. It cannot be, then, that this abnormally high geophyte percentage is even partially explain- able upon the assumption that many plants are incorrectly credited to the group, as there is no more reason why they should have been incorrectly assigned to this group than to any other. The conclusion as to the climate of our area, as reflected in the spectrum of the whole flora is that the conditions seem more favorable here for the production of deep-rooted perennials of the bulb-bearing or rootstock type than any region as yet studied, and that the pro- duction of aquatics is relatively great. Figures for eastern Asia in this connection would be of interest. Such generalizations must mean very little as yet, because all such schemes of correlating climate and plant distribution deal with species rather than individuals. Biological spectra based on a census of individuals would very greatly alter the result. The chief value of this scheme of Raunkiaer’s, and it is the most suggestive of all schemes yet devised for the purpose, is the opportunity it gives for comparison of one flora with another, for comparing certain elements of the same flora, and it has been used in studying even smaller categories of vegetation. To the ecologist and phytogeographer it opens up a wide field of investigation. Its value to the practical agriculturist and horticulturist must be apparent, as it can be applied as a criterion of hardiness and suitability of plants from one region for another. A study of the scheme from this stand- point would surely reveal much information of value to growers. In the following four families, as illustrating the method, the species have been assigned to their respective growth-forms. In many cases it is not easy to assign the species, and much valuable work can be GROWTH-FORMS OF FLORA OF NEW YORK AND VICINITY Si done along this line. It would be of the greatest service to know all the growth-forms as shown in certain areas, formations, associations and so forth. IRIDACEAE Iris versicolor = G Iris primsatica = G Sisyrinchium angustifolium = H Sisyrinchium mucronatum = H Sisyrinchium arenicola = H Sisyrinchium gramineum = H Sisyrinchium atlanticum = H ALSINACEAE Stellaria uliginosa = T Stellaria pubera = H Stellaria longifolia = T Stellaria borealis = T Cerastium nutans = T Cerastium arvense = H Cerastium arvense oblongifolium = CH Sagina procumbens = T or biennial? Arenaria caroliniana = CH Arenaria stricta = CH Arenaria groenlandica = CH Arenaria lateriflora = H Arenaria peploides } é = stem succulents Spergularia marina ROSACEAE Physocarpus opulifolius = MC Spiraea latifolia = N Spiraea alba = N Spiraea tomentosa = CH? Gillenia trifoliata = G Potentilla pumila = H Potentilla canadensis = H Potentilla canadensis simplex = G Potentilla monspeliensis = T Potentilla arguta = H Potentilla palustris = CH Potentilla tridentata = CH Potentilla fruticosa = N Fragaria vesca americana = CH Fragaria virginiana = CH Fragaria canadensis = CH Fragaria terrae-novae = CH Sanguisorba canadensis = H Agrimonia gryposepala = H Agrimonia rostellata = H Agrimonia mollis = H Agrimonia Bicknellii = H Agrimonia striata = H Agrimonia parviflora = H Geum vernum = H Geum virginianum = H Geum canadense = H Geum flavum = H Geum strictum = H Geum rivale = H Waldsteinia fragarioides = H Rubus 28 species all H Rosa 7 species all N HYPERICACEAE Ascyrum stans = CH Ascyrum hypericoides = CH Hypericum Ascyron = H Hypericum densiflorum = N Hypericum adpressum = CH Hypericum Bissellii = G Hypericum ellipticum = G Hypericum virgatum = G Hypericum perforatum = G Hypericum punctatum = G Hypericum mutilum = T Hypericum gymnanthum = T Hypericum majus aay ; perhaps Hypericum canadense T? ) biennial Hypericum gentianoides = T Hypericum virginicum = T THE TEMPERATURE OF LEAVES OF PINUS IN WINTERS JoHn H. EHLERS I. INTRODUCTION There is evidence to show that the food reserve of decidous trees is at a maximum in autumn at the fall of the leaves, and that there is a gradual decrease in reserve material during the winter months to a minimum in the spring. For trees with persistent leaves, on the contrary, there is evidence to show that there is a gradual increase in reserve food during the winter, and that the maximum is not reached until just before the swelling of the buds in spring. Accepting the evidence as true leads to the conclusion that, while deciduous trees maintain their existence during the winter at the expense of the reserve food, trees with persistent leaves produce, by photosynthesis during the same season, food material not only for their maintenance, but in quantities sufficient for an accumulation of reserve. How they are able to do this in cold climates where the temperature rarely rises far above 0° C., and where the mean temperature for the winter months is several degrees below o° C. is a problem that has not been solved. | It has been shown by investigators that broad leaves, 7. e., leaves of deciduous trees, exposed to tropical insolation and leaves exposed to summer insolation in temperate regions, may attain in still air a temperature 16° C. above the shade temperature of the air. Assuming that a similar condition holds for trees with persistent leaves, such as the conifers, during the cold days of winter, may not this offer a solution to the problem suggested above? While a number of investigators have definitely determined the effect of solar radiation upon broad leaves for tropical regions, and for summer conditions in temperate regions, no such work has been done to find its effect upon persistent leaves under winter conditions. It was to fill this gap, 2. e., to determine the effect of solar radiation upon the temperature of persistent leaves under winter conditions, 1 Contribution No. 145 from the Botanical Department of the University of Michigan. 32 THE TEMPERATURE OF LEAVES OF PINUS IN WINTER 33 and its possible bearing upon photosynthesis and the accumulation of reserve food material during the same season, that this investigation was undertaken. The work was carried on in the Botanical Depart- ment of the University of Michigan during the winter months of 1912-1913 and 1913-1914. The problem was suggested to the author by Professor F. C. Newcombe, and was carried to completion under his supervision. To him my sincere thanks are due for helpful sug- gestions and kindly criticism. Grateful acknowledgment is also made to Professor H. M. Randall of the department of physics for valuable suggestions and help in setting up the apparatus, and for apparatus loaned by him; and to Professor N. H. Williams of the department of physics for the use of his laboratory and for helpful advice given. II. HISTORICAL The papers of particular interest for the consideration of the problem under discussion may be grouped under three heads: (1) the internal temperature of foliage leaves; (2) photosynthesis at low temperatures; (3) accumulation of reserve food material by evergreen trees in winter. 1. The Internal Temperature of Foliage Leaves Of interest here, merely because it is the first record of an investi- gation to determine leaf temperatures that could be found in botanical literature, is a paper by Rameaux (21) published in 1843. This in- vestigator placed neighboring leaves, attached to the stems, one upon another until the layer was sufficiently thick to prevent any light from passing through, and then bent the layer around a mercury thermometer. No record of leaf temperatures is given, however, since the investigation was chiefly concerned with finding the tempera- ture of stems. Schumacher (27) was the next to attempt a determination of leaf temperatures. His method consisted in placing a thermometer against the lower side of a leaf exposed to solar illumination. At least two serious objections may be raised to this method: (1) Assum- ing a difference in temperature between the leaf and the surrounding air, only the small portion of the mercury bulb in contact with the leaf would be influenced by the leaf temperature; (2) the rays passing through the leaf would tend to warm the mercury regardless of the leaf temperature. 34 JOHN H. EHLERS Askenasy (1) in 1874 made observations on the temperature attained by succulent plants when exposed to solar radiation. Sem- pervivum and Opuntia were used for this purpose. A mercury thermo- meter was laid against the upper surface of the leaf, or inserted in a cut made for that purpose. With the thermometer in the shade registering 31° C., the leaves of Sempervivum attained a temperature of 43.7° to 51.2° C., an excess of 20.2° C. over the air temperature. With leaves of Opuntia he obtained a temperature of 15° C. above the shade temperature. Thin leaves (Aubrietia and Gentiana) on the other hand reached a temperature of only 7° C. above that of the air. The difference in temperature attained by succulent and by thin leaves he ascribes to two causes: (1) the lower rate of transpiration of the succulent leaves; (2) their massive structure, exposing less surface in proportion to mass than thin leaves expose. Worthy of mention because of the method employed is an investi- gation by Stahl (29) to determine the difference in temperature between the red and non-red parts of variegated leaves. Stahl seems to have been the first to apply a thermo-electric method to determine leaf temperatures. His apparatus consisted of a thermo-couple of German silver and copper, and a mirror galvanometer read by means of scale and telescope. The junctions were made spatulate in form and were pushed into the leaf lamina. Variegated succulent leaves were used. The source of light was a gas flame. Since no absolute temperatures are given his work needs no further mention here. Under tropica! insolation, Ewart (5) obtained with leaves of Vanilla and Hoya temperatures of 45° and 50° C. respectively. The leaves were suddenly bent around and pressed against a delicate mercury thermometer originally a little below the expected temperature. The results obtained compare fairly well with those obtained by Askenasy. Ursprung (31) in the fall of 1901 made observations on the temper- ature of both succulent and thin leaves under natural illumination. The observations were made in the Basel Botanical Garden at temper- atures ranging from 14° to 28° C. Ursprung rejected the thermo- electric method on the ground that the direct reading of temperatures with a mercury thermometer was more simple, and because of the difficulty of getting an instrument sufficiently sensitive over a wide range of temperatures. Thermometers with small cylindrical bulbs and graduated to .5 degrees over a range of 10° to 50° C. were used. THE TEMPERATURE OF LEAVES OF PINUS IN WINTER 35 For finding the temperature of thin leaves, the thermometer was placed on the upper surface of the leaf. The sides of the leaf were folded over with the midrib as an axis, and fastened with a light wooden clamp. In handling, the leaves were kept from contact with the fingers by using a pad of wadding. In the case of succulent leaves a small hole, the exact diameter of the thermometer bulb, was bored and the bulb inserted. The results obtained for succulent leaves under strong solar illumination confirmed the findings of previous investigators, 7. e., for Opuntia 43.3° C., Mamillaria 43.5° C., and for Sempervivum 49.6° C. These were the maximal temperatures. ie ait’ temperatures were 27.5°, 28°, and 27.1° C. respectively. The temperatures found for thin leaves were much lower, Ulmus montana, Betula alba, and Saxtfraga crassifolia, showing maxima of 0.7°, 2.3°, and 8.1° C. respectively, above the air temperature. Ur- sprung ascribes the lower temperature of thin leaves, as did Askenasy, to the very large surface exposed in proportion to the total mass of the leaf. In 1905 four important papers appeared, important because more refined methods were used in the determination of leaf temperatures than had been employed by previous investigators. Of these, one, prepared by Miss Matthaei (14), was communicated to the Royal Society of London by F. Darwin in 1903. Miss Matthaei, in the course of her researches on vegetable assimilation and respiration, found it necessary to determine the internal temperature of the leaves used. For this purpose detached leaves of cherry laurel (Prunus Laurocerasus) held in a special leaf chamber and placed in a constant temperature bath, were exposed to light of varying intensity from an incandescent gas lamp. For strong illumination, a powerful Keith burner was used. The leaf was protected from direct heating effect by a system of circulating water. The temperatures were determined by means of a very fine thermo-couple of copper and constantan. One junction was inserted in the midrib of the leaf, the other, insulated by a rubber tube, was placed in a water bath the temperature of which could be varied at will. The electromotive force produced by the difference in temperature at the two junctions was read by means of a Thomson mirror-galvanometer, about 4 ohm in resistance. The deflections were read directly on the scale; no refinements, such as the use of a telescope, were considered necessary. This apparatus was found by calibration to be sensitive to0.1°C. The temperatures were 36 JOHN H. EHLERS not read directly by means of the galvanometer, but by a zero method which was to adjust the temperature of the bath containing the control junction until the galvanometer showed no deflections. The temperature of the bath then indicated the leaf temperature. This method was easily accurate to within .5° C., which was considered sufficient for the purpose. Under strong illumination from the powerful Keith burner, leaves of cherry laurel reached a temperature of 10° C. or more above the temperature of the bath. | The second of the above mentioned papers was presented before the Royal Society of London in March, 1905, by Brown and Escombe (4). The paper deals with investigations on the physiological pro- cesses of green leaves. It is interesting in this connection because of the method employed to determine leaf temperatures. The tempera- tures were arrived at by complicated calculations involving (1) the coefficient of absorption of radiation, (2) the specific heat of the leaf, (3) the energy expended in photosynthesis and respiration, (4) the thermal emissivity of the leaf, and (5) the effect of the wind velocity. Both attached and detached leaves of Helianthus annuus and Senecio grandtfolius were used. Under full solar illumination, a maximum temperature difference between leaf and air of only 2° C. was found by this method. This work was done at air temperatures of 15° to 27° C. In strong contrast to the results of Brown and Escombe are those obtained by Blackman and Matthaei (2) in their investigations of vegetable assimilation and respiration. Determinations of leaf temperatures under natural illumination, both with the leaf in open air and enclosed in a glass case, were made thermo-electrically. The apparatus and method used were identical with those used by Matthaei (14). For expériments in the open air detached leaves of cherry laurel were stretched on a frame, the leaf stalk dipping in a well of water. The thermo-junction was inserted in the midrib. Under brilliant insolation, temperatures varying from 7° to 16° C. above the shade temperature of the air were obtained. In diffuse light the leaves were found’ to be from 1° to 3° C. above the air temperature. Of the two methods—the complicated calculations of Brown and Escombe and the thermo-electric method of Blackman and Matthaei—the latter seems by far the more trustworthy. The sources of error are very much reduced. In confirmation of the results obtained by Blackman and Matthaei THE TEMPERATURE OF LEAVES OF PINUS IN WINTER 37 are those obtained by Smith (28) in Ceylon. The apparatus and methods employed by Smith were practically the same as those used by Blackman and Matthaei and, therefore, need no further description. His results may be summarized in part as follows: (1) Thermo-iunctions give the same result whether inserted in the lamina or in ‘the midrib of the leaf; (2) ‘‘in still air, with the black bulb in vacuum thermometer at from 55° to 62° C., the air temperature in the shade being 25° to 28° C., leaves, whether thick and fleshy, or thin and pliable, when placed normal to the sun’s rays, may reach a temperature of 15° C. above that of the surrounding air;’’ (3) ‘“‘in the shade such leaves have an internal temperature varying from 1.5° C. below to 4° C. above that of the surrounding air;’’ (4) thickness and texture of leaf have little effect, but thick leaves require more time to reach the final temperature and cool off more slowly; (5) air currents and transpiration are important factors in reducing leaf temperatures. 2. Photosynthesis at Low Temperatures While it has long been recognized that photosynthesis is influenced by the temperature to which the plant is exposed, comparatively little work has been done to determine the relationship between them. This is especially true of photosynthesis at low temperatures, such as prevail during the winter season in cold temperate regions. Until within recent years only a few more or less isolated determinations of the lowest temperature at which photosynthesis begins have been made. | Among the first investigators of photosynthesis at low temperatures was Boussingault (3) who observed an evolution of oxygen by Pinus laricio at a temperature of 0.5° to 2.5° C., and by meadow grasses at 1.5° to 3.5° C. It is interesting to note that Boussingault took into account the effect of solar radiation upon the leaf temperature. The plants with which he experimented were placed on the north side of a wall to protect them from the sun’s rays during the experiment. Heinrich (8) found that in Hottonia palustris carbon-dioxide assimi- lation began at 2.2° R., while Kreusler (11), experimenting with Phaseolus vulgaris, Ricinus communis, Prunus Laurocerasus, and Rubus, observed an assimilation of COs. at temperatures of — 0.9°, — 0.6°, — 2.2°, and — 2.4° C. respectively. Jumelle (9), as a result of experiments with Evernia Prunastri, Picea excelsa, and Juniperus communis, makes the assertion that 38 JOHN H. EHLERS these plants, when exposed to sunlight at temperatures as low as — 30° to — 40° C., absorb CO, and evolve oxygen, while respiration ceases at — 10° C. He suggests that the absorption of heat along with the light rays by the exposed leaves may prevent the cell sap from being entirely frozen at even these low temperatures and that, in consequence, assimilation may proceed. Pfeffer (20) offers in criticism of Jumelle’s results the following: “Since, however, all respiration ceases at — 10° C. to — 12° C., it is manifestly impossible that any assimilation of carbon dioxide can take place at — 40° C., for CO: assimilation is a vital process involving protoplasmic activity.’ Jumelle’s methods are generally considered faulty by later investigators, and not much credence is placed in his results. For criticism of his methods the reader is referred to Matthaei (is) sand tosewanta.)e Miyaké (18) has found that the leaves of a large number of ever- green plants, including trees, shrubs, and herbacous plants growing in the vicinity of Tokyo, contain more or less starch during the winter. To determine whether this starch was the product of photosynthesis in winter, or whether it was stored there as suggested by Sachs (25), he excluded light from several plants (Thea japonica, Fetsia japonica, Cinnamomum Camphora, Pinus Thunbergu, and Abies firma), some in the open, others in a dark chamber the temperature of which varied between 1° and 7° C., until the starch had disappeared, and then exposed them to sunlight. Microscopical tests showed the reappearance of starch within five hours after exposure. The tem- peratures were: for Thea, minimum 2.6° C., maximum 8.6° C., mean 2.1° Ci; for: Feista, minimum 0.8° C;; maximum 7: Co mean sence. He concludes that starch is formed by photosynthesis in winter and that its translocation occurs in the same season. This is further evidence, not only of photosynthesis in winter, but also of the accumu- lation of reserve material. The temperatures given were taken from the records of the Meteorological Observatory of Tokyo, and, pre- sumably, are shade temperatures and, therefore, do not represent the true temperature of the leaf. As will be shown later, the absorption of the sun’s radiations by the leaf will increase the internal temperature of the leaf from 3 to 10 degrees Centigrade on bright winter days. The next contribution of importance was by Ewart (6). In a paper on assimilatory inhibition he devotes a part to experiments on assimilation at low temperatures. He used the bacterium method. THE TEMPERATURE OF LEAVES OF PINUS IN WINTER 39 With this method the lowest temperatures at which determinations can be made is approximately 1° C. Parts of plants that were grow- ing in the open and had been exposed for several weeks to temperatures often reaching — 15° C. were thawed and examined at 1° C. The following plants among others, although most of their leaves remained alive, showed no power of assimilation at 1° C.: Ilex Aquifolium, Buxus sempervivens var. arborescens, Pinus montana, Taxus baccata, Thuya occidentalis, and Juniperus Sabina. Experiments were also made with tropical, subtropical and water plants. His results are sum- marized as follows: ‘‘It appears that all evolution of oxygen ceases in tropical plants between 4° C. and 8° C.; in warm temperate, sub- tropical and water plants between o° C. and 2° C., whilst in cool temperate, arctic, and alpine plants assimilation only ceases when the plants are frozen, 7. ¢., at a few degrees below o CC.” ‘The last case is, presumably, an inference, since he offers no experimental proof to substantiate it. He ascribes the cessation of assimilation in cool temperate, arctic, and alpine plants to physical causes, 7. e., the withdrawal of water from the protoplasm to form ice crystals and the consequent desiccated condition of the tissue. The most important contribution to the question under considera- tion was made by Matthaei (14). Determinations of the rate of assimilation through a range of temperatures from — 6° C. to 45° C. were made. The leaves of cherry laurel, Prunus Laurocerasus var. rotundifolia, were used for this purpose. Her work differs in two respects from that of all previous investigators: (1) In that careful attention was given to keeping the material under uniform conditions before the experiment; (2) in that precautions were adopted for obtaining the true internal temperature of the leaf. The leaf temper- atures obtained and the method of determining them have already been briefly stated. Of the results obtained those which are of particular interest here are: 1. The minimum at which assimilation could be observed. This was — 6° C. Disregarding Jumelle’s finding as untrustworthy, this is the lowest temperature at which photosynthesis has been observed. The author further suggests that, for such cold-enduring plants as the conifers, assimilation may take place at temperatures considerably lower. 2. The assimilation curve. This curve shows the maximal amount of assimilation at all temperatures from — 6° C. to 43° C. From 4.0 JOHN H. EHLERS — 6° C. to 38° C. the curve is convex to the temperature abscissa, the maximal amounts increasing rapidly as the temperature increases. For example, the maximal amounts increase from 0.2 mmg. of COs, per hour for 50 sq. cm. of leaf surface at — 6° C. to 3.8 mmg. at 9° C. The conclusion is that, other conditions being favorable, assimilation increases directly with the temperature; that for each temperature there is a definite amount of assimilation beyond which a further increase in light intensity produces no effect except in so far as it increases the internal temperature of the leaf. A greater assimilation can be obtained only by increasing the temperature. The author considers temperature, therefore, as the fundamental condition governing assimilation, intensity of light as well as percentage of CO., since they are always present in sufficient quantities, being of secondary importance. 3. Accumulation of Reserve Food Material by evergreen Trees in Winter In 1904 Sablon (23) published the results of his investigations on the reserve material of deciduous trees. He found that the reserve carbohydrates of these trees reached a maximum in autumn at the fall of the leaves at the end of the period of active assimilation. Dur- ing the winter the reserve decreased a little, while in the spring during the formation of new shoots there was a decided diminution—a fall to the minimum. Two years later (24) he published the results of an investigation of the reserve materials of evergreen trees (les arbres a feuilles persistantes). The species used were Quercus Ilex, Pinus laricio, Larix europoea, and Evonymus japonicus. The determinations in both investigations were made by chemical! analysis. The results obtained in this investigation were strikingly different from those obtained with deciduous trees. Instead of the maximum appearing in autumn, there is a constant increase in reserve material during the winter, and a maximum is reached only at the beginning of spring. To quote directly: ‘Pendent l’hiver, en effet, la végétation est sus- pendu, par conséquent la dépense de réserve est faible; d’autre part . l’assimilation du carbone se poursuit et l’on sait que l’abaissement de la température affaiblet beaucoup mains l’assimilation que la respir- ation. Il est donc naturel que l’hiver soit pour les arbres a feuilles persistantes une périod de formation de réserve.’’ In other words, in trees with persistent leaves photosynthesis is not only sufficient THE TEMPERATURE OF LEAVES OF PINUS IN WINTER 41 for the maintenance of the tree, but there is actually an increase in the reserve material during the ‘“‘rest period’’ so called. Mer (17) thirty years earlier working with the leaves of Hedera came to a similar conclusion. The experiments of both of these investigators were made with material growing in the open—Mer at Paris, Sablon at Toulouse. Unfortunately neither gives any data as to temperatures. How far their results may be applied in a given locality depends upon the factors governing photosynthesis in that locality. That they hold for the greater part of Japan has been shown conclusively by Miyaké (18) in the investigations cited above. III. MrtTHODs A. Preliminary Work in 1912-13 1. Material and Location.—Since the purpose of this investiagtion was to find the internal temperature of pine leaves in winter under as nearly natural conditions as possible, cut branches were avoided and leaves 1m situ used. As best suited for the purpose, the species Pinus laricio austriaca Endl. was selected. The leaves of this species are, in cross section, the largest of the conifer leaves available in this locality. The tree used during the preliminary work is about 2.5 meters high and is located in the university arboretum about a mile from the university campus. A small shed was built near the tree to house the apparatus. 2. Method.—The character of the leaves and the conditions under which the work was to be carried out made a thermo-electric method the only method possible. In the preliminary work an attempt was made to interpret directly into temperature differences, by means of a d’Arsonval galvanometer, telescope, and scale, the electromotive force produced by the difference in temperature of two thermo- junctions, one embedded in the leaf tissue, the other in a thermos- bottle kept at a known temperature—usually at 0° C. This method was, however, found unsatisfactory. The changes in the resistance of the lead wires and the wires connecting the leads with the galvan- ometer in the shed, as well as the changes in resistance in the galvan- ometer itself, due to changes in atmospheric conditions, made new calibrations necessary not only each day, but sometimes several times aday. After a thorough test this method was abandoned. 42 JOHN H. EHLERS B. Final Experiments 1. Material and Location.When the work was resumed in the autumn of 1913 the station in the arboretum was abandoned. An. Austrian pine growing on the university campus was selected for the final experiments. This tree is about 4o years old and about 15 meters high. It is protected from direct winds on the north and west by the library, and on the south by the physics laboratory. To give the operator access to the leaves and to hold the outdoor part of the apparatus, a wooden platform 4 meters high was constructed. Upon this platform a wooden screen was set up to protect the apparatus from the sun’s rays. Three pairs of heavy insulated copper wires connected the platform with the ground floor of the physics laboratory where the measuring apparatus was kept. 2. Method.—The first two months were spent in trying out various methods. A potentiometer method was finally adopted as best suited for the purpose. The principle of the method consists in opposing over the slide wire of a Wheatstone bridge the fall of potential due to a suitable current from a cell against the electromotive force produced by the difference in temperature between the two junctions, one of which is in the leaf. By moving the sliding contact, the portion of the bridge wire included in the galvanometer circuit (A to B, figure 1) may be changed until the fall of potential over this portion is equal and opposite to the resultant E.M.F. due to difference in temperature of the junctions. This condition is indicated by a zero deflection of the galvanometer. The displacement along the wire per degree temperature difference is determined empirically by placing the junctions in baths of known temperature. The great advantage of this method over the first one employed is that, since it is a zero method, no current flows in the galvanometer circuit and variations in resistance due to temperature changes and other causes may be neglected. Furthermore, its accuracy is inde- pendent of any assumption of proportionality between galvanometer deflection and current. 3. Apparatus —The apparatus in its essential parts consisted of a slide wire Wheatstone bridge, galvanometer, storage cell, resistance boxes, rheostat, and voltmeter. Figure 1 shows the arrangement. The galvanometer used was of the d’Arsonval type with a resistance of 30.6 ohms, and was made by the Eberbach and Son Co., of Ann Arbor, Michigan. The Wheatstone bridge was made by the same THE TEMPERATURE OF LEAVES OF PINUS IN WINTER 43 firm. The cell used to furnish the current for the potentiometer circuit (II, figure 1) was an ordinary storage cell with an E.M.F. of 2 volts. This cell proved entirely satisfactory, giving a uniform current throughout the experiments. As a check upon the cell, a Weston voltmeter was connected across the terminals of the potentiometer Vigure 1. L Galvanometler-Giroutl. Tr Polentiometer Girowr\, B Sliding Gowlact. G Golvanometler Jd, uy Thermo- junctions V_ Voltmeter RR Resistance Boxes. circuit. By throwing a knife switch, the voltage applied to this circuit of constant resistance could be measured, and any variations or weakening of the cell detected. Variations could be corrected by varying the resistance of a small rheostat which was placed between the cell and the potentiometer. It was not found necessary to use it however. 4. Thermo-Junctions—The thermo-junctions were made of advance (a trade name for constantan, which is an alloy of copper and nickel) and copper wire obtained from the Driver, Harris Co., of Harrison, N.J. The advance wire was 0.07 mm. in diameter, the copper slightly 44 JOHN H. EHLERS larger — 0.09 mm. The wires were carefully soldered, and all junc- tions having rough or thick joints rejected. A thin coating of shellac was given the junctions to protect them against oxidation and any possible action of the leaf juices. 5. Method of Insertion into the Leaf.—The leaves of Pinus laricio austriaca grow in bundles of two. The leaf in cross section represents © roughly a semi-circle in outline about 1.5 mm. in its shortest diameter. To insert the junctions into the leaf at temperatures ranging from o° to — 17° C. was attended with considerable difficulty and many junctions were broken, especially in the beginning of the experiments. The procedure was to thread the lead wire through a very fine steel needle and draw the wire through the leaf until one of the junctions was embedded in the tissue. To prevent too great a loss of heat by conduction along the lead wires, as much of the wire as possible should be embedded in the leaf tissue. Drawing the wire through at right angles to the long axis of the leaf would not, therefore, give the best results. Drawing the wire through the leaf lengthwise was found impracticable because of the firmness of the tissue. Attempts to do this usually resulted either in the splitting of the epidermis, or the breaking of the leaf. Placing the junction and lead wires between two leaves fastened together flat surface to flat surface did not give good results. The method finally adopted was to fasten the two leaves together by means of two single turns of very fine wire placed about I cm. apart, the wire covered with white insulation. The leads were drawn through both leaves at an angle of 45° with their long axis. Care was observed to leave the junction embedded in the leaf toward the sun and as near its center as possible. This gave a contact surface of about I mm. for the advance lead wire and from 3 to 4 mm. for the copper—the better conductor of the two. The second junction was left exposed to the air and shaded from the sun’s rays by a wooden screen. ‘The junctions, therefore, registered directly the differential temperature between leaf and surrounding air. 6. Position of the Leaf.—The leaves of the pines assume no definite position with relation to the sun’s rays. In all experiments, unless otherwise stated, a leaf normal to the sun’s rays, or as nearly normal as could be found, was selected. Two sets of junctions, each in a different leaf, were constantly in use—the one a check upon the other. A double-pole double-throw switch on the table beside the operator made it possible to read the temperatures of the two leaves in rapid THE TEMPERATURE OF LEAVES OF PINUS IN WINTER 45 CALIBRATION OF APPARATUS (RABLE. | Temperature Junction A —9.82° —9.82 —9.81 SO) —7.18 — 4.87 Junction B |Temp. Diff. —15.40°} 5.58° SS ILO 5.28 — 15.00 5.19 — 14.85 1°99 —14.70 7.52 —10.40 5-53 — 10.00 5.06 — 9.92 4.92 SONOS | O47 = 9-65 4.52 — 6.52 6.52 — 6.58 6.58 — 6.60 6.60 — 6.60 6.60 5-05 5.65 — 5.60 5.60 — 5.50 5-50 ar 9°45 5-45 O 5.00 o) 5.00 O 5.00 O 5.07 O 5.28 O 5.48 O 5.62 0) 7.70 e) 3.00 10.00 3.25 10.00 3.52 10.00 4.60 10.00 4.61 10.04 4.63 10.14 4.66 9-75 3.58 18.61 7.59 18.62 7.58 18.62 7.58 18.64 7.56 18.64 7.56 18.68 7.94 18.68 8.59 §6.71 8.55 Tos72 9.23 18.72 9.18 Bridge Reading in Cm. 44.6 AD a 41.5 60.7 60.4 44.7 41.0 39.8 By.t 36.7 53.6 54.0 54.3 54.2 46.5 46.0 45-4 45.0 41.6 41.6 41.5 42.0 43.8 45-4 46.5 64.2 25.0 27.6 29.9 39.2 39-3 39-4 39.6 39.3 65.8 65.6 65.6 65.5 65.5 68.8 74-3 74-0 80.0 79:5 Displacement per Degree C. Temp. Diff. 7-99 7-97 7-99 8.04 8.03 8.08 8.10 AO ee eee OO COs OO) CO Oo 0,00 is DADADAAADAAAAR hu ANARWTADAR AE Average Dis- placement per Degree Temp. Difi. in Cm, 8.00 8.10 8.22 8.30 8.50 8.65 Degrees C per Cm. Displace- ment 0.1250° 0.1234 0.1216 0.1205 0.1176 0.1156 46 JOHN H. EHLERS TABLE I.—Continued ai Bridge Reading) Displacement | placement per | Resress C pet A Oa piel ya Temp. Diff.) “Bétincme | ment 23.57 20.00 3.57 SITE 8.71 23.54 20.00 3.54 30.9 8.73 23.49 20.05 3.44 30.0 8.72 23.40 20.05 3.35 29.2 8.72 8.71 0.1148 22°26 20.05 B.31 28.9 8.73 25.15 20.15 5.00 43.4 8.68 26.10 20.15 5.95 51.8 8.70 26.13 20.18 5-95 51.9 8272 26.22 20.20 6.02 52.3 &.69 succession. By comparing fresh leaves with others in which the junction had remained embedded for several days, it was found that the same leaf could be used 3 or 4 days in succession without any appreciable effect upon the results. 7. Calibration of Apparatus——In calibrating the apparatus the junctions were tied to the bulbs of the thermometers, the leads separ- ated from one another by small glass tubes. Junctions and thermom- eters were then thrust into glass tubes containing kerosene oil and the tubes immersed in constant temperature baths. The leads of the junctions were connected with the apparatus by means of copper wires and copper binding posts. By means of resistance boxes the current from the cell was reduced until a displacement on the bridge of 100 cm. was equivalent to a temperature difference at the junctions of approximately 12°C. Readings were taken with the colder junction at temperatures varying from 18° to 20° C., while the warmer junction ranged from 0.5° to 10° C. higher. More than 50 readings gave an average displacement on the bridge of 8.66 cm. per degree centigrade temperature difference, or 0:1155° C. per centimeter displacement. But experiments showed that this factor could not be used for tempera- ture differences at 0° C. and below, the error varying from 0.05° to 0.5° C. Calibrations were therefore made at various points through a temperature range of 35 degrees, with the colder junction at — 15°, — 10°, — 5°, 0°, 1ro°, 18° and 20° C., the warmer junction varying from three to eight degrees higher in each case. Standard thermom- eters, one graduated to 0.2° C. the other to 0.1° C. were used for temperatures from 0° to 20° C. The low temperatures were obtained by means of two Beckmann thermometers. With these the tempera- tures could be read without lifting the bulb in the freezing mixture. The results are given in Table I. THE TEMPERATURE OF LEAVES OF PINUS IN WINTER 47 If these values are plotted against the temperatures as ordinates, a curve is obtained which, for all practical purposes is a straight line, the deviation at — 5° C. being negligible. From this curve (figure 2), by interpolation, values can be obtained for any temperature between — 20° and 20° C. The two columns of figures at the right of figure 2 contain these empirical numbers with the computed numbers inter- polated. 8. Sensitiveness of Apparatus—The galvanometer was found readily sensitive to a displacement on the bridge wire of two milli- meters. Taking the factor obtained for 0° C. as an example, the apparatus is sensitive to .2 + 8.30 = 0.024° C., which is amply sufficient for the purpose of this investigation. The apparatus may be made still more sensitive by decreasing further the current from the cell and increasing the sensitiveness of the galvanometer corre- spondingly. 9. Accuracy of the Method.—To determine whether the theoretical sensitiveness of the apparatus would hold in practice, tests were made in December and January, and again in March at the close of the experiments. The method consisted in placing the junctions in baths of known temperatures, finding the corresponding displacement on the bridge wire, computing the temperature by applying the proper factor obtained from the curve, and then comparing the calculated with the actual temperature difference. The results are given in Table II. Table II shows representative readings taken from a large number. The average error of the December and January readings is only 0.0155°; the greatest for any one reading 0.055°. For March the average error is 0.0233°; the greatest 0.056°. 10. Untformity of the Junctions.—Table III gives the results of an experiment to test the uniformity of the junctions used. Two sets of junctions were placed in the same baths. By throwing a switch, changes from one set to the other could be made in rapid succession. A number of other tests gave results similar to those in the table. The greatest difference in displacement is 2 mm. This, at the temperature difference given (5.04°) represents an error of 0.023° C., Of 4 per cent. . 11. Sources of Error.—The possible sources of error in this method are: (1) Variation in the current due to weakening of the cell; (2) the 48 JOHN H. EHLERS CONT CCCRE PrN D PTT Tt 2ABSBTELE2S AN EH NY) N TTT TART YE Fic. 2 Curve showing displacement in centimeters on wheatstone bridge. The tem- peratures from zero down the column are all minus. THE TEMPERATURE OF LEAVES OF PINUS IN WINTER 49 TABLE II Temperature Displacemen alculated Temp. Pie - F. pactual Ga as td CD RISGA DY eee 1merence Dece27 | 24.53° 18.38° 6.15° 53.2 6.149° 0.001° 24.52 18.39 6.13 52.9 6.115 0.015 26.25 18.40 7.85 68.3 7.895 0.045 24.65 18.85 5.80 50.2 5.803 0.003 24.62 18.85 Bai 49.8 5.757 0.013 23.35 18.90 4.45 38.6 4.462 0.012 22532 18.93 4.39 38.0 4.392 0.002 Dec. 28 ..| 20.21 | 18.04 PAN Coe 18.8 2172 0.003 21.86 18.02 3.84 BGr2 3.838 0.002 19.19 18.16 1.03 8.9 1.029 0.001 19.13 18.15 .98 8.5 .983 0.003 Jan. 5 8.00 0.00 8.00 66.7 - 8.037 0.037 5.00 0.00 5.00 41.6 5.013 0.013 2.97 0.00 2.077, 24.7 2.976 0.006 | Jan. 11 | —7.40 | —14.68 7.28 58.4 7.203 0.013 7 ahits) —14.70 Tee 60.4 7.543 0.023 —7.05 —14.00 6.95 55.6 6.950 0.000 —9.25 — 16.67 Fhe. © 58.7 7.365 0.055 —9g.80 -|-—17.03 7122 ie 7.198 0.032 —5.05 — 10.00 4.95 39.9 4.924 0.026 Average error | 0.0155 Mar. 22 4.15 e) 4.15 34.2 4.118 | 0.032 4.18 O | 4.18 34.8 4.190 0.010 4.28 Oo - 4.28 35-7 4.298 0.018 4.38 O 4.38 36.4 4.383 | 0.003 4.45 O 4.45 36.6 4.406 | 0.056 4.56 O 4.56 37-9 4.563 0.003 4-77 O 4.77 39-7 4.789 0.019 5.18 fe) 5.18 42.8 5.153 | 0.027 5-34 O 5.34 44.2 5-322 | 0.018 5:45 0) 5:45 45.0 5.418 0.032 5.50 O 5.50 46.0 5.538 | 0.038 Average error | 0.0233 formation of secondary couples; (3) heat due to wounding of the leaf tissue; (4) loss of heat by conduction along the lead wires. (1) Variation of the Current from the Cell—To guard against any variation of the current from the cell, a Weston voltmeter and a rheostat were connected across the terminals of the potentiometer circuit as already described. The simple throwing of a switch enabled the operator to detect any change in the voltage applied to the poten- tiometer circuit. Corrections could then be made by varying the resistance of the rheostat. 50 JOHN H. EHLERS TABLE III COMPARISON OF JUNCTIONS © ‘Temperature Displacement in Cm. Date rales Diff. in Mm. A B Diff. Junct. No.1 Junct. No. 2 Jan. 3 22.85. 20:42 - Praag 210 20.9 I 22.90 20.42 2.48 21.5 21.4 I 23.04 20.48 2.56 22.2 220 I 24.10 20:53° |. "3.57 31.0 | 31.0 re) 24.10 20.53 | 3.57 31.0 31.0 (a) 24.09 20.55 3.54 30.8 30.7, I 25.60 20.56 5.04 Aae7 43.5 2 25-57 20.57 9-00 43-4 43-4 e) 26.58 20.56 6.02 52.3 52.3 O 26.55 | 20.60 5.95 51.9 51.8 i Jan. 4 20.28 19.24 1.04 9.0 9.9 e) 19.60 19.21 <39 3.4 Gia) I 19.45 19.15 .30 Died, 2.6 I 19.50 1O-17, 123 2.9 2.9 e) 29.80 19.04 10.76 93.2 93.2 O 29.78 | 19.04 LORTA 93.1 93.0 I (2) The Formation of Secondary Couples.——Any difference in the composition of the metals at the connections, or any slight difference in the composition of the metal in the wires themselves may, if there is a difference in temperature at those points, cause the formation of secondary couples. For the purpose of this investigation the latter case may be neglected, as the error arising from this source would be exceedingly small. To guard against secondary couples at the connection of the lead wires with the heavy copper wires, copper binding posts were used and the joints placed as closely together as practicable to insure the same temperature for both. The connections with the apparatus in the laboratory were all under uniform tempera- ture conditions throughout the experiments and, consequently, the danger from secondary couples at those points was reduced to a minimum. (3) Heat Due to the Wounding of the Leaf Tissue—The develop- ment of heat in wounded tissue is due to an increased respiration of the injured part. Tiessen (30) found that the rise in temperature due to wounding increases with the extent of the wound; that its duration varies from one half to three days; and that its absolute value varies from 0.02° to 0.08° C. with an average of 0.04° C. Richards (22) has shown that a curve plotted for the heat developed in wounded tissue corresponds in the main to that of the respiration intensity under the THE TEMPERATURE OF LEAVES OF PINUS IN WINTER Sy! same conditions. Now since respiration is but feeble at 0° C. and decreases rapidly as the temperature falls [Maximow (16), Matthaei (14)], and since nearly all the readings were taken at 0° C. and below, and since the wound made in the leaf was very small, any increase in temperature due to wounding would not be appreciable. (4) Loss of Heat by Conduction Along the Lead Wires.—This was, in all probability, the source of greatest error and could not be entirely overcome. As already stated, two leaves were fastened together and the lead wires drawn through them at an angle in order to give as large a contact surface as possible. To determine what the loss from this source might be, the following experiment was made. A small glass tube 15 cm. long with a bore of 5 mm. was furnished with two side arms and two minute openings. Three thermo-junctions, such as were used in taking the leaf temperatures, were inserted in _ this tube as shown in figure 3. Through this tube kerosene oil was siphoned from a bath kept at a temperature of 10° C. above the room temperature. The leads of junction I were in contact with the heated oil the entire length of the tube, and this junction should therefore record accu- rately the temperature of the flowing oil. The leads of junction 2, on the other hand,_ were in contact with the oil for a distance of -5 mm. to I mm. for the advance lead and 4 mm. for the copper—a condition similar to that of the junction in the leaf. By connect- ing junctions 1 and 2 with the potentiometer, any difference in temperature between these junctions due to loss of heat by conduction from junction 2 could be measured. By con- necting junctions 1 and 3 in the same way the temperature gradient between the flowing oil and the surrounding air could be deter- mined. A three-way switch enabled the oper- Fic. 3. Showing the ator to measure both in rapid succession. Re- details of the three thermo- peated trials gave an average displacement on JUNCHONS. the bridge for junctions 1 and 3 of 84.5 cm. which at the room tem- perature was equivalent to a temperature difference of 9.7° C. Junc- tions I and 2 gave an average displacement of .36 cm. This repre- 52 JOHN H. EHLERS sents a temperature difference between these junctions of 0.04° C., 1. e., enough heat is lost by conduction from junction 2 to reduce its temperature 0.04° C. below the true temperature of the oil. When it is borne in mind that the temperature gradient between air and oil in this experiment was 9.7° C., and that the maximum temperature — gradient between leaf and air under natural conditions was 8.83° C. and the average considerably below this, it is evident that the error arising from loss of heat by conduction along the lead wires is also neg- ligible. Furthermore it is compensated for in part by the very small increase in temperature due to wounding. (5) Absorption of Radiation by the Junction in the Leaf—It may have occurred to the reader that the rise in temperature indicated by a junction placed in a leaf exposed to direct sunlight may be due in part to the absorption of radiation by the junction itself. Smith (28) has shown that thisis not true. By alternately shading a junction by means of a small piece of pith and exposing it to direct sunlight, he found that direct sunlight had no effect upon the junction whatever. He concludes from his experiment that ‘the rise of temperature of a junction registered when it is placed in a leaf exposed to direct sun- light must all be due to the absorption of radiation by the leaf, and no part of it to the absorption of radiation by the junction itself.” Smith’s result was accepted by the author and no experiments were made to verify it. 12. Meteorological Data. (1) Atr Temperature—lIn the earlier experiments the air temperature was obtained with a mercury thermo- meter graduated to degrees Centigrade. Later a registered thermo- meter graduated to .1° C. was used. No attempt was made to read the air temperature beyond one tenth degree. The thermometers were hung behind the screen upon the platform. (2) Solar Radiation——For purposes of comparison, the intensity of the solar radiation was also taken. For this a black bulb in vacuum thermometer graduated to the Fahrenheit scale was employed. It was placed on the platform near the leaf. For the sake of uniformity the readings were converted into degrees Centigrade. (3) Velocity of the Wind.—For this a self-registering anemometer making contact every one tenth mile was used. The miles of wind per five minute periods could be determined. The anemometer was placed upon the platform near the leaf; the registering device in the laboratory. THE TEMPERATURE OF LEAVES OF PINUS IN WINTER 53 (4) Relative Humidity.—The relative humidity was obtained from the records of the university observatory situated about one half mile from the campus. The values given represent the average from 7a. tO 7 p.1. IV. EXPERIMENTAL DATA A. Leaf Temperature under Winter. Conditions The collection of data began early in January. Of the mass of data obtained only a part can be given. The results shown by the tables following in the text may be taken as representative. The readings, as a rule, were taken from one half to three minutes apart for a period of five to ten minutes. Then the air temperature, radiation, and the wind velocity were recorded for that period. To save space, the average of the readings for the period is given, as a rule. In a few cases all the readings are given to show the rapidity of temperature changes within the leaf. The readings of the black bulb in vacuum thermometer are the maxima for the period unless otherwise indicated. The wind velocity given in column 9 is the average for the period as recorded by the anemometer on the platform and has only general value. Since the tree, partly sheltered from direct winds by the neighboring buildings, was subject to sudden gusts and eddy currents, some of which were vertical and did not, therefore, affect the anemo- meter, the velocity given does not necessarily represent the true velocity at the moment the leaf temperatures were taken. Column 4 in the tables gives the differential temperature between the leaf and the shade temperature of the air. The values are obtained by multiplying the bridge readings by the factor (see figure 2) indicated by the shade temperature given in column 5. Column 8 gives the actual temperature of the leaf. The values are obtained by adding the differential temperature to the air temperature. Column 7 gives the difference between the shade temperature and that recorded by the black bulb in vacuum thermometer—a better standard for com- parison than the values in column 6. In general, the differential temperature between leaf and air increases as the radiation increases. When this is not the case, an explanation can usually be found by referring to the wind velocity recorded in column 9. The data presented here are given to show: (1) the effect of full 54 JOHN H. EHLERS sunlight; (2) the effect of one fourth to one half full sunlight; (3) the effect of diffuse light; (4) the leaf temperature at night; (5) the effect of strong air currents; (6) the temperature of leaves at different angles to the sun’s rays. (1) The Effect of Full Sunlight—Unfortunately no data on the effect of full sunlight in still air could be obtained. Low temperature, still air, and full sunlight are a combination of conditions that rarely obtains. The nearest approach to these conditions was found on February 24. The data obtained are given in Table IV. TABLE IV February 24, 1914 Clear. Light breeze. Relative humidity 87% 1 2 3 4 Fe py 7 8 9 Aver. | Black : Wind Time of Obser- | No. of mide Calculated | Shade Bulb in | Diff. Col- Actual Velocitt vation Obser- | Reading Temp. Temp. | Vacu- umns5 |Leaf Temp.| Miles vations iniC@m. Diff. es and 6 per Hr. 9.00— 9.02 4 26.1 3.26° |—15.0°| 5.67) 20:65." ere 9.21I— 9.24 4 48.4 0,045) TTA eLAna 28.5 17200 1.2 9.28— 9.33 7. 44.4 5.54 |—14.0 | 20.0 34.0 — 8.46 2.4. 10.02—10.04 4 60.4. 75 j\=-20 22056 33.6 — 5.49 4 10.50-10.55 5h 44.0 5:46 | 2.27 92556 37.8 — 6.74 8 11.00 I 56.0 6.96 | — 5.54 11-005) 21 272 4.52 | | — 7.98 11.01 I 50.8 ove oi | — 6.19 II.O1I5| I 38.2 A758 — 7.75 11.02 I 64.0 7-05 Ge 125 20-5 40.8 — 4.55 2 TRO25 | an 59.6 7 ALi — 5.09 11.03 I 46.5 eo. ual — 6.72 11.04 I 40.0 4:07.) 9) — 7.53 11.05 I 54.0 6.71 | — 5.79 11.08-I1.12 4 58.1 7,24, Gi 12:07) 28.9 40.9 — 4.79 LZ EI.25-11.32 8 56.5 7.00) TET a e2826 40.0 — 4.10 9 I1I.2Q-12.35 a, 57.6 Fall) | Ore eZ Os4: 39.6 — 3.09 5 2.35- 2.40 8 41.5 55s i LOO ait yy, 21.7 — 4.87 3 a oe pe Averages) 6.045) |— 12:4 2258 — 6.36 1.0 | With the shade temperature of the air varying from — 15° to — 10°C. the black-bulb thermometer registering from 20.6° to 40.9° C. higher, and the wind velocity varying from 0.3 to 2.4 miles per hour, the average differential temperature between leaf and air for the entire time during which the readings were taken was 6.04° C., 1. e., = THE TEMPERATURE OF LEAVES OF PINUS IN WINTER 55 the leaf maintained, under the conditions given, a temperature of 6.04° C. higher than the air. This is shown by the averages of columns 4, 5, and 8. The maximum temperature difference for the day was Rom. C. The following Table V shows a maximum leaf temperature of 8.83° above that of surrounding air, the air temperature in this series of observations being considerably higher than that shown in Table IV. TABLE V February 27, 1914 Clear. Light breeze. Relative humidity 66.5% I 2 3 } 4 5 6 7 8 a4 9 Aver. : Wind Time of Ob-| No. of Bridge Calculated | Shade | Black Bulb Diff. Actual Neleere servation | Obser-| Reading Temp. Temp. in Vacuum | Columns Leaf Miles vations in Cm. Diff. 5 and 6 Temp. per Hr. 8.00? 5 5.7 .69° -|— 2.0° Fae 9.2° —T.31°) 1.2 9.15 4. 18.6 2.24 O 18.9 18.9 PAG |p V2 9.25 5 16.4 1.97 0.8 20.6 19.8 eis faa RN 9.33 8 32.5 3.90 1.5 23.9 22:4 5.40 | 1.0 10.05 5 46.9 5.60 3.0 24.4 21.4 8.607 | 1.2 10.54 I 50.5 6.02 10.02 10.55 I 66.5 7.93 11.93 10.555 I 73.0 Or7 5 12770 10.56 i 74.6 8.83 12.83 10.565 I 58.5 6.98 ace 29:3 25-3 10.98 ia 10.57 I 61.2 7.30 11.30 10.58 I 63.0 7.52 11.52 10.59 I 57.0 6.80 ‘ 10.80 11.05 5 28.1 2.25 4.2 Bie, 27.5 7.55 | 2.4 12.34 6 18.5 2.20 5.1 32.2 27.1 FEZO. |e 2e4 Average 3.82 2.66 6.48 The conditions under which the results in Table VI were obtained differ somewhat from those of the preceding Table IV. The air temperature averaged 3.85° C. lower and the average wind velocity was .7 miles per hour greater. Also the difference between the shade temperature and that recorded by the black-bulb thermometer was 2.7° C. lower. Under these conditions the average differential temperature between leaf and air was 3.56° C. and the maximum 5.19° C. (2) The Effect of One Fourth to One Half Full Sunlight——That a decrease in the intensity of solar radiation, other conditions remaining 2 Not included in the averages. 56 7 2 Time of Obser- vation "_ Ba 19.55 10.20 10.48 10.58 11.05 11.46 12.26 12.36 eye) | 1.47 | 1.56 i 1.36 2.50 2.54° ENB ORBN OF DUP HH No. of Obser- vations Clear. 3 JOHN H. EHLERS TABLE VI Moderate breeze. 4 Aver. Bridge Reading in Cm. Average Calculated Temp. Diff. ° 4.64 3.56° 5 Shade Temp. —I18.1 —17.2 —17.0 — 16.0 — 16.0 —15.8 —16.2 —15.5 —15.0 February 12, 1914 Relative humidity 86.5% 6 Black Bulb in Vacuum E2toe 12.8 16.1 7 Difference Columns 5 and 6 ; —19.0° 21.0- 30.9 33-3 37-0 2822 36.0 36.4 RIE) 29.4 2750 26.9 20.6 18.5 18.5 BRAT 8 Actual Leaf Temp. —14.32 —13.71 —12.70 —10.81 —12.24 —11.82 —I11.56 — 12.96 —I1I.16 —12.79 — 13.07 — 13.89 — 14.39 | —12.68 —13.88° sey ie constant, is followed by a decrease in the differential temperature between leaf and air needs, of course, no proof. tables given are of interest since they show the actual temperatures of the leaf under the conditions existing. Results are given in Tables VII, VIII and IX where the column headings are the same for all Nevertheless the tables. TABLE VII January 16, 1914 Hazy. Light breeze. Relative humidity 93% I 2 3 4 5 6 7 8 9 Time of| No. of Brace Calculated Grade Black Bulb | bilterence Actual Velocity Obser- | Obser- Reading Temp. Temp in Vacuum)! Comms Leaf Miles vation | vations in Cm. Diff. 5 and 6 Temp. per Hour II.10 5 oie3 2e5 AR 5.0° 25.6° 20.6° 7.54° 11.40 6 15.9 1.90 5.2 25.6 20.4 7210 11.58 2 15.9 1.90 5.5 25.6 20.1 7.40 a 12.30 5 Lo.2 217, 5.7 25.6 19.9 1.Ou = 12.40 6 20.4 2.42 5.8 27.0 21,2 8.22 ov 12.50 Ue 26.8 2.8 6.0 27.8 PRs) 9.18 mA 1.10 3 18.9 2.25 6.5 27.5 21.0 8.75 1.23) af 18.2 2. v7 5.3 20.7. 21.4 FAT E5642 20 8.2 507 5.2 26-1 20.9 6,57; | Average 27, 5.58 20.8 7.74 3 Leaf in shadow; not included in the averages. 4 Leaf in shadow. THE TEMPERATURE OF LEAVES OF PINUS IN WINTER S/ TABLE, VIID January 25, 1914 Approximately one half full sunshine. Light breeze. Relative humidity 78.5% I 2 3 4 | 5 6 7 | 8 9 9.45 I 22.5 277. 573. 9.47 I 21.5 2.65 —5.85 9.48 I 20.3 2.50 —8.5° 16.7° 25.2° } |—6.00 re 9.49 I 20.0 2.46 — 6302! 9.50 I 25-7 3-17 | | —oroo" | | 9.53 I 26.8 3.30 — 5.20 II.10 I 26.9 eu — 4.69 elie i 2a57) 4.14 3.00 PIE? oT 36.3 4.46 —8.0 20.0 28.0 | |—3.54 0.8 II.15 I 30°3 4.46 — 3.54 1.20 I 22.5 3.99 —4.01 11,22 I 30.7 4.51 — 3.59 F Average| 3.47 —8.25 26.6 —4.78 | TABLE IX February 2, 1914 Approximately one fourth full sunshine. Breeze moderately strong. Relative humidity 83.5% I 2 3 4 a5 6 7 8 9 Time of | No. of ata Calculated Gitade Plack Bulb: | Ditterence Actual Velouny Observa- | Obser- | Reading Temp. | Temp. in Vacuum | Columns Leaf Miles tion vations in Cm. Diff. 5 and 6 Temp. per Hr. 10.34 I 24.8 2800 —1.0° 20.0° 21.0" 2.00° 10.40 5 14.5 1.75 —0.8 22.2 23:0 95 4.8 11.00 4 14.0 - 1.69 —0.5 222 2207, 1.19 4.8 11.15 5 11.5 1.39 O 17.8 L729 1.39 4.8 11.46 5 13.5 1.63 fe) 18.3 18.3 Ge 4.8 11.55 3 LO: 1.94 0.2 19.2 19.0 2A 47. 12.36 3 10.9 1.31 1.0 stein, 15.7 2 oil a2 1.25 3 18.9 B27, io EOQu7 17.9 4.07 2.6 iol 4 2155 2.58 2.0 2252 20.2 4.58 3.6 2.34 3 11.0 1.39 2.5 19.4 16.9 | 3.89 3.6 Average} 1.89 0.52 19.25 | 2.41 With approximately one fourth of full sunlight, and with a light breeze blowing, the maximum differential temperature obtained was 3.18° C. and the average 2.17° C. (Table VII); with approximately the same radiation (compare column 7, Tables VII and IX) and a moderate breeze blowing, the maximum is 3.0° C. and the average only 1.89° C.; with approximately one half of full sunlight (Table VII) 58 JOHN H. EHLERS and a light breeze, the maximum is 4. St C. and the average 3. Bue CG; as compared with a maximum of 7.95° C. and an average of 6.09° C. under full sunlight (Table IV). (3) The Effect of Diffuse Light—Even on dark, cloudy, winter days, pine leaves absorb sufficient radiant energy to maintain a temperature slightly above that of the air. Ona few very dark days the potenti- ometer gave zero displacement, indicating that the leaf temperature was the same as that of the air, but, as a rule, the leaf was found to be from 0.34° C. to a little more than 2° C. higher than the air tempera- ture, depending upon the brightness of the diffuse light. In only one case was the leaf temperature found to be lower than that of the air during the hours of daylight. This occurred on January 29, when the air temperature was comparatively high. The average for the days given in Table X is 0.95° C. TABLE X January 18, 1914 Cloudy. Light breeze. Relative humidity 77% I 2 3 4 5 6 7 8 9 Time of | No. of ake Calculated Shade Biackebulb Diff. Actual Velocity Obser- | Obser- Reading emp Temp. in Vacuum | Columns Leaf Miles vation | vations Gm Diff. 5 and 6 Temp per Hr. 12.30 9 9.6 1.16° —2.2 Or4s 11-67 —1.04 12.48 43 8.2 99 —2.0 Ghee 9.2 —I1.01 Average! 1.08 —2.1 —1.02 January 23, 1914 Cloudy. Breeze moderate. Relative humidity 100% I 2 3 4 5 6 7 8 9 10.15 5 L120 Toa 4.0° Tepe} 12eS% aa 12 10.33 6 4.9 58 5.1 14.4 9.3 5.68 2.4 1.15 5 5.1 61 6.0 15.6 9.6 6.61 2.4 Average 83 5.03 5.86 February 19, 1914 Cloudy. Breeze moderate. Relative humidity 89.5% I 2 B 4 5 6 7 8 9 12.38 5 12.9 1.58° —6.0 T228~ 18.8° —4.42 3.0 1.30 Bi 17.8 2.17 —5.5 1722 222 — 3.33 2 Average! 1.88 —5.75 — 3.87 THE TEMPERATURE OF LEAVES OF PINUS IN WINTER 59 March 22, 1914 Cloudy. Light breeze I 2 3 4 5 6 7 8 9 8.40 6 2.8 3h —0.8° ye ae 8.0° —0.46° 12.30 7 5.6 67 1.4 9.4 Stu 2:07 1.30 I ) 0) 1.0 2.0 1.0 1.00 Average 34 53 .87 g (4) The Leaf Temperature at Night—Throughout February and a part of March readings were taken at six and ten p.m. The results obtained are represented by the values given in Table XI. A few readings show no difference in temperature between the leaf and the air, but, as a rule, the leaf is from 0.1° to 0.7° C. colder than the air. TABLE XI Rel. Bridge Read. Ta De Air Temp. Humid- Date 1ty | Atmospheric Conditions 6 PM. | 10 PM.| 6 PM. | 10 PM. 6 PM. TOMEI. ing Mi. Feb. 7 | —2.0°} —2.5*) 0.25° | 0.32° | —10.0° | —11.0°| 80%|Clear; wind light. as 8 Oo | —2.0]| 0 0.2 —18.5 | —20.0 67 |Clear; wind strong. 9 | —I.0/ —2.5| 0.13 |0.31 | —10.0 | — 9.5 74 |Clear; wind strong. * 10 | —2.8 OF AO835. 0 —12.0 | —14.0 55 |Wind light 6 P.M.; Calm 10 P.M. Bs Il | —2.5| —2.5| 0.31 | 0.32 | —16.0 | —19.5 77 |Wind moderate. i 12 | —2.0| —2.5| 0.25 | 0.32 | —17.0 | —19.0 73 |Wind moderate. % 13 Oo | —1.5/0 0.19 | —13.0 | —I4.0 75 |Cloudy; wind moderate. x 16 | —1.5| —1I.2| 0.19 | 0.15 | —10.0 | —I2.0 81 |Cloudy; wind | moderate. oa 16") -—5.0.| —3.6| 0.62 | 0:45 — 9.0 | —12.0 88 |Clear; calm. Ee 25 | —3.5| —6.0] 0.45 73 — 7.0 | —I10.0 44 |Clear; calm. Mar. 5 | —1.0| —1.5| 0.12 | 0.18 O — 2.0 78 (Cloudy; wind light. 7 6 | —0.5 Oo 10:06, (0 ee O5 re) 73 |Cloudy; wind light. oa 7 | —0.3 oO; | 0:04 50 — 1.5 — 4.0 | 100 |Snowing; wind light. Average] 0.21 | 0.25 (5) Effect of the Wind in Reducing the Leaf Temperature-—The effect of the wind in reducing the leaf temperature is well illustrated in Tables XII and XIII. The average differential temperature between leaf and air for the two tables is 2.93° C. as compared with 6.04° C.in Table IV. A part of this reduction in differential tempera- 5 Negative sign under bridge reading indicates that the current was reversed and that the leaf was colder than the air. 60 JOHN H. EHLERS ture is due, no doubt, to a difference in intensity of insolation, column 7 of Table IV showing slightly higher values than the corresponding columns in Tables VI, XII and XIII. But the difference in the intensity of insolation is slight and can account for only a small part of the reduction. The wind, therefore, is the important factor. TABLE XII February I, 1914 Clear. Strong wind.6 Relative humidity 71% I ape 3 4 5 6 i 8 9 Time of | No. of Bodes Computed Shade Black Bulb| _, Diff, Actual Velocity Obser- Obser Readings Temp. Temp. in Vacuum | Columns Leaf Miles vation | vations| in Cm. Diff. 5 and 6 Temp. | per Hr, 12.35 | 2 yey 2 Sit > —3.0° 28.6" Sule —0.89° | 6.0 12.45 6 22.4 2.68 —3.1 29.0 32.1 —0.42 6.0 1.50 ;3 BI 27.0 2.28 —3.5 29.2 227 —0.22 6.0 1.58 5 23.8 2.89 —3.2 20.0 2202 —0.31 4.8 Average! 2.74 —3.2 29.9 —0.46 March 2, 1914 Relative humidity of 90% I 2 2) 4 5 ir 6 7 8 | 9 8.52 7 15.4 1.90, —12.2° 9.4° 276° | —10:292 |e 9.36 4 1751 212 —I1.3 251 32.4 — 9.18 4.8 10.06 8 23.0 3.83 — 8.5 Q7ee 25.7 — 4.67 5.0 11.00 I 20.0 2.46 it i * — 6.04 | 3.6 I 10.5 1.29 ie i mg — 7.21 oi if 75 92 6c 6c oe a is 7.58 bc I 22.6 279 . se ‘ — 5.72 g I 26.5 3.26 r a . — 5.24 ore: I 122 1.62 s s - — 6.88 2 I 19.0 23d vy s e — 6.16 me I 7 86 ce oc bc mate 7.64 bc rp aes fag |e I 39-3 4.83 Pet nO 11.05 I 28.0 3.44 = ot ¥ — 5.06 ey 12.31 8 16.8 2.06 = 6:5 26.1 32.6 = 4.44 | 4.8 Average| 2.61 — 9.4 31.6 — 6.93 The effect of gusts of wind is shown in the rapid fluctuations in the differential temperature for the period I1.00 to 11.05, Tables IV and XIII. Fluctuations of 2° and 3° C. occur within one half minute. 6 Wind velocity, University Observatory record, Feb. I, 15 miles per hour; March 2, 26 miles per hour. ee ee THE TEMPERATURE OF LEAVES OF PINUS IN WINTER 61 TABLE XIII . February 8, 1914 Clear. Strong wind.’ Relative humidity 71% I 2 3 4 5 6 if 8 9 Time of | No. of ee Calculated Shade Blackebalb Diff. Actual Velocity Observa- | Obser- | Reading Temp. Temp. | in Vacuum | Columns Leaf Miles tion vations in Cu. Diff. 5 and 6 Temp. per Hr. 9.45 2 16.7 2.09° —15.5° 10.0° Poe oMnle ESAT |*, 6.0% 10.15 I 20.2 2.48 Ae II.1 25.6 12202 6.0 10.37 7 24.0 3.00 —14.5 15.6 30.1 —II.50 6.0 10.47 Gi 23.1 2237. —I4.5 T7c2 OTe, —I11.63 6.0 10.56 5 30.1 3.75 —14.3 18.2 32.5 —10.55 4.8 11.04 6 34.5 4.30 —14.0 19.8 33.8 =) (87 KO) 6.0 11.45 Ff 32.8 4.09 =A 19.9 34.1 —I0.1I 6.0 12.17 12 28:7 4.83 —13.8 22:2 36.0 — 8.97 6.0 qeaee 8 28:7, 3.58 —13.8 222 36.0 — 10.22 6.0 Average| 3.44 —14.34 | 3107 — 10.90 These fluctuations by no means represent all the changes in the leaf temperature. In fact, the changes were so rapid that it was not possible to adjust the sliding contact quickly enough to record them all. A somewhat nearer approach toward recording all the fluctu- ations was made in the following way: The galvanometer was cali- brated at the existing temperature in terms of displacement along the bridge wire. The sliding contact was held at a fixed point, and the galvanometer deflections read every five seconds for a period of two minutes, an assistant indicating the periods and recording the deflec- tions as read. To insure that the deflections should be due solely to changes in the leaf temperature, the second junction was enclosed in a thermos-bottle and kept at a constant temperature. The results are graphically represented by figure 4. One series of observations and measurements was made on a detached branch with cut end immersed in water, and the preparation protected from the wind by a thin cloth screen except on the south side. The results, given in the following table, show that in quiet air the temperature of the leaf may rise to 10° above that of the sur- rounding air. In making up the averages the average readings for the periods are taken. 7 Wind velocity, university observatory record, 18 miles per hour. 8 Leaf in shadow for a part of the period. 62 JOHN H. EHLERS 44.0 rade. » . t vees Cent? | in de » & pgvature y Tem ~ Time in Seconds. Fic. 4. Graph showing galvanometer deflections due to changes in leaf temperature. THE TEMPERATURE OF LEAVES OF PINUS IN WINTER Clear. TABLE XIV | March 10, 1914 Relative humidity 76% Detached branch partially protected against air currents by cheese cloth screen. Breeze light. 63 at Aver. : Time of No. of Bridge Calculated Shade Black Bulb Diff i Actual re ee | ape {Teme je Vaom | Ste | Tom. 10.45 I 64.0 Ow 9.69° I 50.5 6.07 8.07 10.46 I 63.8 7.67 eS : 9.67 10.47 I 72.0 8.77 2.0 Bae? BO" 10.77 10.48 I 85.8 10.31 12.31 10.49 it 82.0 9.86 11.86 10.50 I 75.0 9.02 11.02 11.47 i 74.0 8.83 £3.02 I 71.5 8.53 273 11.48 I 61.0 728 11.48 T 77-9 9.17 13.37 I 74.2 8.85 Ane 35.0 30.8 13.05 11.50 I 79.4 9.47 13.67 I 79.4 9.47 13.67 I 73:0 Sal 12.91 11.51 I 74.4 8.88 13.08 12.40 I 75.5 8.98 14.48 I 80.0 9.52 15.02 12.41 I 63-5 7.56 13.06 I 66.0 7.85 13335 12.42 I 71.5 8.51 14.01 I 69.5 8.27 5.5 36.7 B12 lie ard 12.43 I 70:4 9.09 14.59 I 65.0 Figo 13.23 12.44 I 75.0 8.93 14.43 I 61.8 7.35 12.85 12.45 I 76.5 9.10 14.60 12.5 82.0 9.76 15.26 eres 85.2 LO; L2 \ 5-5 36.7 3) 2 15.62 1.05 I 83.0 9.88 14.88 I 64.5 7.08 | & 5.50 35.0 30.0 12.68 1.06 I 79.5 9.46 14.46 1.07 I 72.5 8.63 13.63 ag Average 8.91 4.44 13.25 2 3 4 6 (6) The Temperature of Leaves at Different Angles to the Sun’s Rays.—Finally an experiment was made to determine the difference in temperature between two leaves at different angles to the sun’s 64. JOHN H. EHLERS rays—one at approximately 45°, the other at 90°. For this purpose a cut branch, partially protected from the wind by a screen of white cheese cloth, was exposed to solar illumination. The screen was open at the top to prevent the enclosed air from becoming heated. Doubtless there was some reflected radiation, but, since the leaves were side by side within the enclosure and therefore under the same conditions, this did not affect the results. These are given in Table XV. Other attempts in which the differential temperatures of the two leaves were read alternately in rapid succession gave similar results. TABLE XV March 5, 1914 Temperature of leaves at different angles Time of Aver. Bridge Calculated _Black Bulb in Observation | ReadinginCm.| Temp. Diff. Shade Temp. Vacuum 2.15 45.4 5.29” 49.8 5.80 46.3 5:39 Angle 45° 48.0 5.59 14.0° 20.1 38.6 4.50 43.0 5.01 31.0 3.61 2.20 35.5 4.14 i Average 42.2 4.92 2.25 58.9 6.86° 64.2 TAZ 64.5 7.51 | ed 6.65 ile) 36.07 Angle 90° 49.0 5.71 47.0 5-48 54-5 6.35 50.0 5.83 2.30 54.6 5.36 Average 55.5 6.36 B. Direct Evidence of Photosynthesis 1n Winter An attempt was made to obtain direct evidence of photosynthesis under winter conditions by examining for starch content the leaves of the various conifers growing in the university arboretum. The leaves were collected in early morning and again in late afternoon of clear days. They were kept in 75 per cent alcohol and later examined microscopically. Thin sections were placed on a slide and treated THE TEMPERATURE OF LEAVES OF PINUS IN WINTER 65 with absolute alcohol and ether to remove the fats, and then with a solution of chloral-iodine and potassium iodide. During the autumn and through December the leaves of Pinus laricio austriaca Endl. only were examined. Later other species were added. From five to ten leaves were examined in each case. In the leaves of Pinus laricio austriaca starch remained abundant in mesophyll, endodermis, and transfusion tissue throughout October and November. But leaves collected December 2, after a period of seven dark, rainy days—maximum temperature for the entire period 14° C. and the mean 6.7° C.—showed, when examined, no starch in the mesophyll and only a small amount in the endodermis. Leaves collected from the same branch December 4—maximum temperature 11.6° C. and the mean 6.7° C.—showed a small amount of starch in the mesophyll, endodermis, and transfusion tissue. On December I1, the temperature, in the meantime, having dropped to a minimum of — 7° C. and a maximum of 3° C., the starch had entirely disap- peared from all the tissues. Starch was again found on December 12 in the mesophyll, endodermis, and transfusion tissue, and in greater abundance on the following day, the temperature having risen to a maximum of 12° C. Since the temperature on December 4 was colder by several degrees than that of the preceding days, the reappearance of starch could not be due to a regeneration from sugar already present in the leaf, but must have been the result of photosynthesis. On December 13, however, the maximum temperature had risen 9° C. above that of December 11, and the reappearance of starch may have been the result, in part at least, of a regeneration of starch from the sugar present in the leaf, due to an increase in temperature. When next examined, December 20, starch had entirely disappeared from the mesophyll, though a small amount still remained in the endodermis and in the transfusion tissue. By January 15 the leaves were entirely free from starch with the exception of an occasional group of hyaline mesophyll cells, where it was evidently stored. Throughout January, February, and early March leaves of a number of conifers were collected and examined, but no further conclusive evidence of photosynthesis was obtained. Some of the species examined retained starch in the mesophyll in greater or less abundance throughout the winter, but no increase in quantity, even after a comparatively warm bright day, could be .detected micro- 66 JOHN H. EHLERS scopically. In other species groups of hyaline cells filled with large starch granules were observed more or less regularly. These groups usually appeared immediately below the epidermis. No explanation is attempted. The author hopes to make this a subject for further investigation. While no conclusive evidence of starch formation was observed after December 13, it does not follow that all photosynthetic activity had ceased. Carbohydrates may have been formed and used or trans- located as fast as formed. It merely indicates that photosynthesis was not sufficiently active under the conditions that obtained during January and February to result in the production of starch in the leaf. The species examined and the results obtained are as given below. No starch: Pinus strobus L. More or less starch: Juniperus communis L. Thuya occidentalis L. Juniperus virginiana L. Picea Abies (L.) Karst. Tsuga canadensis (L.) Carr. Abies balsamea (L.) Mill. Starch in groups of hyaline cells: Pinus laricio austriaca Endl. Pinus ponderosa Laws. Pinus sylvestris L. Pinus Banksiana Lamb. These results do not agree with those of Schulz (26) and Lidforss (13). The former maintains that the leaves of all gymnosperms except the Gnetaceae contain no starch in winter; the latter states as a universal rule that all green plant cells are free from starch during the winter months. Evidently their results do not hold for southern Michigan just as they do not hold for the warmer portions of Japan as was shown by Miyaké (18). This problem should be given more extended investigation. V. SUMMARY The data presented above show that evergreen conifer leaves, even under winter conditions, through the absorption of radiant energy, maintain temperatures from 2° to 10° C. higher than the surrounding air. The maximum obtained under brilliant illumination and with a light breeze blowing was 8.83° C. (Table V). In still air the temperature difference is considerably higher. This was shown Se | THE “TEMPERATURE OF LEAVES OF PINUS IN WINTER 67 by an experiment in which the leaf was partially protected against air currents. Under these conditions a differential temperature of 10.31° C. was obtained (Table XIV). Under less brilliant illumination and stronger air currents the differential temperature is correspond- ingly less. Even diffuse light, according to its brightness, will increase the leaf temperature from 0.5° to 2° C. For February, the coldest month of the year, the average differential temperature between leaf and air—650 readings in all, taken between the hours of 8 a.m. and 3 p.m. and under all kinds of weather conditions, cloudy days as well as days of sunshine—was 3.06° C. , The differential temperatures in the winter season, as was to be expected, are considerably less than those obtained by previous investigators for broad leaves under summer and tropical insolation, namely, from 7° to 16° C. The cause of the difference is to be sought in the lower rate of respiratory changes (Molisch (19)), the lower intensity of solar radiation, and in the greater loss of heat by con- vection, air currents being, as a rule, more constant and stronger during the winter season. Though smaller in winter, the differences are, nevertheless, of sufficient magnitude to become an important factor in photosynthesis. 7 It should also be remembered that the differential temperatures as found in this investigation are those of the leaf as a whole, and not necessarily those of the chloroplasts. It is the chloroplasts that absorb the most of the radiant energy; the temperature of the !eaf is due to radiation and conduction from the chloroplasts. It is entirely probable, therefore, that the chloroplasts have a temperature con- siderably higher than that of the leaf as a whole. Recalling briefly the more important facts of the evidence at our disposal concerning photosynthesis at low temperatures, we have: (1) Evidence of an accumulation of reserve food material through the winter in trees with persistent leaves as found by Sablon; (2) con- firmatory evidence both of photosynthesis and of an accumulation of reserve food in winter by evergreen leaves as found by Miyaké; (3) evidence of photosynthesis at — 6° C. as the result of very careful work by Matthaei; (4) the finding of Ewart that a number of ever- green shrubs and trees including conifers, after having been exposed for some time to a temperature often falling below — 15° C. had no power of assimilation when tested at 1° C. The results obtained by Ewart appear to contradict those of 68 JOHN H. EHLERS Miyaké and Matthaei. In making comparisons it should be borne in mind, however, that Matthaei’s material had previously been kept at a uniform temperature from 10° to 16° C., and that Miyaké’s experiments were made at Tokyo where the mean temperature for the three winter months is 3.8° C. and the minimum — 6.5 (Kusano (12)), while the plants from which Ewart obtained his material had been exposed for three weeks to temperatures falling below — 15°C. Ewart ascribes the inability of these plants to assimilate CO, at 1° C. to the inhibiting effect of the extreme temperature to which they had been exposed. The same plant material, when brought to a temper- ature of 15° C., showed weak assimilation within a few hours and quite active assimilation within eight hours to one day. The long latent period of recovery, according to Ewart, indicates that the exposure had nearly reached the plant’s limit of resistance. Un- fortunately Ewart does not state how long the tests for assimilation at 1° C. lasted. One gains the impression that the tests were of short duration. It is entirely possible that, had the plants been exposed to a temperature of 1° C. for a longer time, the inhibiting effect of the previous extreme temperature would have been overcome and assimi- lation begun. The contradiction between Ewart’s results on the one hand and those of Matthaei and Miyaké on the other is, therefore, more apparent than real. Bearing in mind the assimilation curve obtained by Matthaei, the application of the results of the present investigation becomes at once apparent. Kanitz (10) has shown that the law of van’t Hoff (32), that for every rise of 10° C. the rate of reaction is doubled or trebled, holds for the results obtained by Matthaei for temperatures between 0° and 37° C. assuming other conditions favorable. For temperatures from — 6° to o° C. the rate of increase is very much greater. The assimilation curve in Matthaei’s work shows an increase in assimilation from 2 mmg. of COs per 50 sq. cm. per hour at — 6°C., to 18 mmg. at o° C.,—an increase of 900 per cent. Since an increase of 6° C. in the temperature of the leaf due to the absorption of radiant energy is not at all uncommon and higher temperature differences are often found, the increase in assimilation resulting therefrom is of very great importance and goes far toward explaining the accumu- lation of reserve food material by evergreen trees. UNIVERSITY OF MICHIGAN, ANN ARBOR. IO. Il. 7. 18. 19. 20. ZN. Zaye 22. THE TEMPERATURE OF LEAVES OF PINUS IN WINTER 69 BIBLIOGRAPHY . Askenasy, E. Ueber die Temperatur, welche Pflanzen im Sonnenlicht annehmen. Bot. Zeit. 33: 441. 1875. . Blackman, F. F. and Matthaei, G. L.C. Experimental Researches in Vegetable Assimilation and Respiration. IV.—A Quantitative Study of Carbon-Dioxide Assimilation and Leaf-Temperature in Natural Illumination. Proc. Roy. Soc. London, Series B 76: 402. 1905. ~ . Boussingault, J. Sur les fonctions des feuilles. Agronomie 5: 16. . Brown, H. T. and Escombe, F. Researches on some of the Physiological Pro- cesses of Green Leaves, with special Reference to the Interchange of Energy between the Leaf and its Surroundings. Proc. Roy. Soc. London, Series B76: 29. 1905. : . Ewart, A. J. The Effects of Tropical Insolation.. Ann. Bot. 11: 444. 1897. Assimilatory Inhibition in Plants. Journ. Linn. Soc. Bot. 31: 389. 1895. Ibid., 402. . Heinrich. Beitrage zur Kenntniss des Temperatur-und Lichteinflusses auf die Sauerstoffabscheidung bei Wasserpflanzen. Landwirth. Versuchs-Sta- tionen 13: 148. 1871. . Jumelle, H. Recherches physiologiques sur les lichens. Rev. Gén. Bot. 4: 305. 1892. Kanitz, A. Ueber den Einfluss der Temperatur auf die Kohlendioxyd-Assimila- tion. Zeitschrift Elektrochemie 11: 689. 1905. Kreusler, U. Beobachtungen iiber die Kohlensaure-Aufnahme und -Ausgabe der Pflanzen. Landwirth. Jahrb. 1887. Abstract Bot. Centralbl. 34: 199. 1888. 2. Kusano, S. Transpiration of Evergreen Trees in Winter. Journ. Coll. Sci. Univ. Tokyo 15: 319. Igor. . Lidforss, B. Zur Physiologie und Biologie der wintergriinen Flora. Bot. Centralbl. 68: 33. 18096. . Matthaei, Miss G. L. C. Experimental Researches on Vegetable Assimilation and Respiration. Phil. Trans. Roy. Soc. Series B 197: 47. 1905. Ibid., p. 54. . Maximow, N. Ueber die Atmung der Pflanzen bei Temperaturen unter Null. Journ. Sect. Bot. éd. Soc: Imp. Natur. St. Pétersbourg 1908. Abstract Bot. Centralbl. 110: 535. - 1909. Mer, E. De la constitution et des fonctions des feuilles hivernales. Bull. Soc. Bot. France 23: 237. 1876. . Miyaké, K. On the Starch of Evergreen Leaves and its Relation to Photo- synthesis during the Winter. Bot. Gaz. 33: 32I. 1902. Molisch, H. Ueber hochgradige Selbsterwarmung lebender Laubblatter. Bot. Zeit. 66: 211. ‘1908. Pfeffer, W. Physiology of Plants 1: 338. 1900. Rameaux. Des températures végétales. Ann. Sci. nat. II. Bot. 19: 10. 1843. Richards, H. M. The Evolution of Heat by Wounded Plants. Ann. Bot. 11: 20), 1897. Sablon, Leclerc du. Recherches physiologiques sur les matiéres de réserves des arbres. Rev. Gén. Bot. 16: 341. 1904. 70 24. 25. 26. 27. 28. 20; 30. Bie BOR JOHN H. EHLERS Recherches physiologiques sur les matiéres de réserves des arbres. Rev. Gén Bot. 18: 82. 1906. Sachs, J. Microchemische Untersuchungen. Flora 20: 300. 1862. Schulz, E. Ueber Reservestoffe im immergriinen Blattern. Flora 46: 223. 1888. Schumacher. Physik der Pflanze. Berlin, 1867. As cited by Ursprung. Bibliotheca Botanica 12: 69. 1903-4. Smith, A. M. On the Internal Temperature of Leaves in Tropical Insolation. Ann. Roy. Bot. Garden Peradeniya 4: 229. Stahl, E. Ueber bunte Laubblatter. Ann. Jard. Buitenzorg 13: 153. 18096. Tiessen, H. Ueber die im Pflanzengewebe nach Verletzungen auftretende Wundwiarme. Beitr. Biol. Pflanzen 11: 53. 1912. Ursprung, A. Die physikalischen Eigenschaften der Laubblatter. Bibliotheca Botanica 12 (Heft 60): 68. 1903-1904. | van’t Hoff, J. H. Vorlesungen tiber theoretische und physikalische Chemie. Heft 1: 224. : oe . 1907, ‘ie itaiad for Me. Ruthertord Trowbides. b ' (OMA as Wb aes Haven, Conn this one compartment: house,,. 23 feet wide vei es SSS cor Seeds alclgg eta sl 33 feet long, “Last year, two more compartments were ae : bred as shown, abov Let us Plan some such plan for you. ‘ Z Daw why not nin mat eae a build, ¥ vie i to i the next thing cee ve "ti thm ceuly Ir now—not after ok have spent |) ONE a AVE, NEWYORK, CANADIAN OFFICE. 10 PHILLIPS PLACE. EsMONTREAS | "OFFICIAL PUBLICATION. OF THE BOTANICAL ‘SOCIETY OF ‘AMERICA STL AS G , i 4 t Vy ahs . e 74 3 % cas . 3 ao i } a ee CONTENTS Some effects of the brown-rot fungus upon the composition of the coe: : Lon A. HAWKINS ‘Negative heliotropism. of urediniospore germ tubes? y...-.F.D. FROMME ok woody stem in Merremia gemella induced by high warm water. FRANK C. Gates : S - Pito-nigrosi, a combination fixative aad stain for algae. y Oris F. Curtis, and REGINALD H, COLLEY’ “Normal and ‘abnormal permeability nF Ne wie te W. 4 V. OSTERHOUT | A singled precision auxanometer....-.- vevetee cose. WT. Bovie * cub Ay 4 j ie ( t 5 “ag 4 § - aes SNE Ae PUBLISHED 1 ra | f a oS : - neuen BY THE pad "BROOKLYN BOTANIC GARDEN Ar 41 oars Queen Sramer, Lancaster, Pa. 7 82 89 93 95 “The mutations of P¢aanibasien'e stenomeres Jel See Bi i et i. H. BARTtLeTt 100.— UN COOPERATION WITH THE BOTANICAL SOCIETY OF AMERICA. Se Sruant Gacer Business a Mage 4) ie “i 5 fone as nae a ee MY i | "i ‘oi ee es 00. ie "Blige copies 50 ceaeanh id all pat Stnean: Secept copa ce Port VTi tten, yi? will in genera i be limited to one full Me + : _ Separate should a ordered when pr : | cover and additional copies at ett os Remittances ston syle made aber tO, Am y Wigs 4 fa ae d be made on 30 as ly. missing r , 2 concerning editorial matters, shoul t ab S e eights, A ing g August and Septer : doy rats lowing (ea gp ene ENE = AMERICAN JOURNAL OF BOTANY Vou. I] FEBRUARY, 1915 ' No. 2 SOME EFFECTS OF THE BROWN-ROT FUNGUS UPON THE COMPOSITION OF THE PEACH! Lon A. HAWKINS The morphology and life-history of Sclerotinia cinerea (Bon.) Schréter, the fungus causing brown-rot of the peach, have been in- vestigated with considerable care. The fact that this fungus is, parasitic upon a number of other hosts is also well established. Not so much attention, however, has been paid to the effect of the fungus upon the composition of the peach; and it was to obtain some informa- tion upon this subject that the experiments described in this paper were carried out.” In these experiments most of the peaches were inoculated in the laboratory, though one series of analyses was made using peaches which had become infected under orchard conditions. The inocula- tions were made from stock cultures of the fungus grown on potato agar. The fungus was originally isolated from fruit in the early stages of brown-rot by removing portions of the deeper-lying tissue and transferring them to tubes of sterile potato agar. Pure cultures were obtained by this method and the fungus maintained in stock culture throughout the investigation. The method for studying the effect of the fungus upon the tissue of the peach was somewhat similar to that used by Behrens (1) in 1 From the Bureau of Plant Industry, U. S. Department of Agriculture, Office of Plant Physiological and Fermentation Investigations. Published by the per- mission of the Secretary of Agriculture. * The writer’s thanks are due to Mr. J. W. Kelly, laboratory technician in this office, for assistance in the experimental part of this work. [The Journal for January (2: I-70) was issued 18 Feb., 1915.| 71 APRIG 1915 ¢ RAL insta # X 72 LON A. HAWKINS studying the effects of Sclerotinia fructigena (Pers.) Nort. upon apples. This writer made a single inoculation upon the side of each apple. When the fruit was half ratted he split the rotten half from the sound portion and analyzed the two parts separately. In the present investigation this method was considerably modified. The peaches were first divided into quarters and the opposite quarters were combined. One of the two samples thus obtained was inoculated and the other was used asacontrol. Before the peaches were sampled; however, they were thoroughly scrubbed with a solution of bichlorid of mercury I to 1,000. They were quartered and separated from the stone with a sterile knife and immediately placed in sterile, glass- stoppered weighing bottles which had been tared, and then weighed. One set of these samples consisting of one sample from each peach was inoculated from the stock cultures of the fungus by shaking up spores in sterile water and pouring them over the cut surfaces of the fruit. The other set, the controls, were treated with a like quantity of sterile water. The two sets of samples were kept side by side under the same temperature and moisture conditions for two weeks or more. At the end of this time the samples were examined and any of the control samples found _-to be infected with fungi were discarded together with the corresponding inoculated halves. This procedure was also followed with any of the inoculated portions found to be infected with fungi other than Sclerotinia. As a further precaution cultures on beef agar were made from the interior of the remaining samples and if any of these cultures indicated the presence of contaminating fungi or bacteria in the fruit the samples were discarded. After these inoculations were made the samples were prepared for analysis. In the analyses, determinations were made of the pentosan, -acid, and sugar content of the peach samples. The amount of alcohol- insoluble material which reduced Fehling’s solution when hydiolyzed with dilute hydrochloric acid, was also determined. The data thus obtained were calculated on the basis of wet weight of fruit at the time of inoculation and it was then possible to compare the content of the different compounds in the sound and rotten portions of the same peach. In the pentosan and acid determinations it was found advisable to use an entire sample for each determination. In the case of the sugars, however, a sample served for the two sugar determinations and for the determination of alcohol-insoluble substance which reduced EFFECTS OF BROWN-ROT FUNGUS UPON PEACH 73 Fehling’s solution upon hydrolysis with dilute hydrochloric acid. The samples were prepared for analysis by slicing as thinly as possible with a sharp knife and then chopping up the slices. Inasmuch as all determinations were related to original wet weight, care was taken that none of the pulp or juice was lost. For pentosan determinations the chopped-up peach pulp was washed directly into half liter Erlenmeyer flasks and distilled immediately. Pentosan determinations were made according to Tollens’s phloroglucid method (8) and the phloroglucid was calculated as pentosan according to Krober’s (8) tables. Methyl-pentosan determinations made on portions of a number of peaches according to Ellett and Tollens’s (9) method, showed the presence in the peach-pulp of substances yielding methyl-furfurol when boiled with hydrochloric acid. The amount, however, was so small that it was not considered worth while to de- termine it in a]l the samples. The samples for acid determination were washed into 250 cc. Erlenmeyer flasks with 100 cc. of water and heated nearly to boiling for one hour. The mixture was then transferred to 250 cc. volumetric flasks which were filled to volume with water and allowed to stand with frequent shaking for one week. Toluol was added to prevent the action of micro-organisms. At the end of a week the solutions in the flasks were again made up to volume, filtered, and two 50 cc. portions of the filtrate titrated against standard potassium hydrox- ide. The acidity of the samples was calculated from these data in cc. normal acid per 100 g. original wet weight. The samples in which the sugar determinations were made weie washed into 250 cc. volumetric flasks with 70 per cent alcohol. About a gram of calcium carbonate was added to neutralize the acidity. The flasks were filled up to volume with alcohol and allowed to stand with frequent shakings for one week. At the end of this time the solutions were again made up to volume, filtered, and 200 cc. of the filtrate pipetted into beakers. The alcohol was driven off and the residue washed into volumetric flasks with water. The solution was cleared with neutral lead acetate, made up to the original volume and filtered. The excess lead was precipitated as oxalate, by adding sodium oxalate. The solution was again filtered and the amount of reducing sugar determined in the filtrate, using Allihn’s modification of Fehling’s solution (8). The copper was determined by direct weigh- ing of the cuprous oxide and the dextrose calculated according to 7A LON A. HAWKINS Allihn’s tables. The method used for sugars was similar to that given by Bryan, Given and Straughn (10). The total sugars were determined in the solution used for reducing sugars by inverting the sucrose in 50 cc. with hydrochloric acid, making the solution up to I00 c.c. and neutralizing with anhydrous sodium carbonate. The sugar was determined as in the case of the reducing sugar. The sucrose content was calculated from the difference in total and invert sugar in the sample, as is the usual procedure in investigations of this kind. Kulisch (7), Girard (5), Bigelow and Gore (2) aud others have considered this difference between the total and reducing sugars in peaches to be due to sucrose. It was, however, considered worth while, in view of the work of Davis and Daish (4), to obtain more evidence upon this point. Accordingly, a number of peaches were sliced up, a little calcium carbonate added, and the mixture extracted with 70 per cent alcohol, for one week. The solution was then filtered and three samples of the filtrate measured out. These solutions were prepared for analysis in the same way as the solutions from samples of peaches in the inoculation experiments and the reducing sugars determined. For total sugars duplicate samples were pipetted from each of the three so'utions. The one set of these was treated with acid in the usual way, while the sugar in the other set was inverted with invertase. The percentage of reducing sugar in the solutions and the percentage of total sugar as found by both methods are given below: Per Cent Sugar as Glucose after Inversion (Total Sugar) Per Cent Reducing Sugar Acid Inversion Invertase Inversion ! 0.77 3-34 3-37 0.77 3.36 3.48 0.77 3-47 3.46 From these results it would seem probable that the increase in reducing substance after treatment with acids is due to the inversion of the cane-sugar in the solution. At any rate, according to the work of Hudson (6), the increase in reducing power is not due to the hydro- lysis of starch, dextrins or pentosans or to the inversion of maltose or lactose. The alcoholic sugar solution, from which the original samples were taken, was later cleared with lead acetate, filtered, and the excess lead removed as sulphide. The filtrate was evaporated and a sugar 3 The writer’s thanks are due Dr. C. S. Hudson, of the Bureau of Chemistry, for the invertase solution used in these experiments. EFFECTS OF BROWN-ROT FUNGUS UPON PEACH 75 crystallized out. After several recrystallizations this sugar was iden- tified as sucrose by its melting point, by the fact that it did not reduce Fehling’s solution until after inversion with acid or invertase and by its specific rotation. As has been said, the alcoholic sugar solution was filtered off the peach sample and the amount of sugar determined in the filtrate. The residue on the filter was carefully removed to a porcelain extrac- tion thimble and extracted for one day continuously in a Soxhlet extractor. It was then dried, ground up and treated according to the usual routine for the determination of starch. The determinations were made by the direct acid hydrolysis method (8), as in the case of the sugars. Whether any considerable amount of these substances, which aie insoluble in alcohol and reduce Fehling’s solution after hydrolysis with dilute hydrochloric acid, are starch, is an open ques- tion. Bigelow and Gore (2) found starch grains in young peeches located only in a thin layer just under the epidermis. Determined quantitatively they found only one tenth of one per cent. The present writer examined portions of the ground pulp microscopically and, while bodies were present which gave the blue color with iodine, no definitely formed starch grains were discovered. To obtain more evidence upon this point, six twenty-five gram samples of peach pulp were weighed cut and extracted. After drying, three of the samples were treated by the direct acid hydrolysis method and the reducing substances determined in the usual way. The other three samples were carefully ground with fine sand and then digested with malt diastase and treated as in the diastase method for the determination of starch (8). The reducing substance was determined as in the other three samples. A comparison of the percentage of reducing substance in the two sets of samples, calculated as starch, is given below. Acid Hydrolysis Diastase Method 1.24 O32 1.33 0.38 1.34 0.37 From the results of these determinations it seems that a portion of alcohol-insoluble substance which reduces Fehling’s solution after hydrolysis with dilute hydrochloric acid, is either starch or some compound, such as dextrin, which is liquefied by diastase. The present work and the investigations of Bigelow and Gore would seem 76 LON A. HAWKINS to indicate that in the peach fruit starch is not a common form of reserve for carbohydrates translocated from other parts of the plant. The method of studying the effect of the fungus upon the peach in this work was to compare the percentage of the compounds as de- termined in the two samples taken from the same peach, one of which had been inoculated with the fungus, while the other remained sound. Any considerable variation between the two halves in the percentage of the substances determined was considered to have been caused by the fungus. This method is based on the assumption that the content of any of the substances determined is the same in the two samples of the same peach. It seemed then, of some importance to determine the extent of the variation between the two halves of the peach sampled in this way. Accordingly, a series of experiments were carried out using sound peaches and following the same method of sampling as in the inoculation experiments, except that the halves were pre- . pared for analysis immediately after sampling. The same variety of peaches, namely Champion, was used in these determinations as in the inoculation experiments described later. The results of these analyses are given in Tables I and II, which follow: TABLE I[ PENTOSAN AND ACID CONTENT, AND THE AMOUNT OF ALCOHOL-INSOLUBLE SUBSTANCE WHICH REDUCES FEHLING’S SOLUTION WHEN HYDROLYZED WITH DILUTE HCl, In SouND PEACHES, EACH SUBSTANCE DETERMINED IN THE Two HALVES OF THE SAME PEACH Per Cent of : : N Per Cent Alc.-Insol. Substance (as Starch) Re- Pentosans, rae era ae Hore | ducing Fehling’s Sol. when Hydrolyzed with Wet Weight ; = | Dil. HCl, Wet Weight Half a! Half é Half a Half 6 | Half a Half 6 1 | wor 28 13655 13.21 a 1.92 1.76 Oe Omit an. oijiaa| 11.57 12.50 | 1.54 1.48 2) LOR f LOS 14.67 15.64 | L7S | 1.64 AO. O77 0.07; | £292 2.08 5 | 2.01 1.88 From the results in the foregoing tables it seems that there is occasionally some variation in the composition of the two samples. This variation, however, is not as great as between the individual peaches. Two series of experiments were carried out in which the peaches were inoculated in the laboratory. In the first series of experiments EFFECTS OF BROWN-ROT FUNGUS UPON PEACH “NI “I TABLE II REDUCING SUGAR, TOTAL SUGAR, AND SUCROSE CONTENT IN THE HALVES OF THE SouND PEACHES, STATED AS PERCENTAGE OF WET WEIGHT ~ | Reducing Sugar as Glucose | Total Sugar as Glucose | Sucrose he Half a | Half 4 Half a | Half & Half a Half — (oe ae fem nee en bteonE e Ss ———- I 4.23 4.33 7.14 | 6.63 | 2.57 Daly 2 4.01 4.24 6.00 | 6.28 | 1.98 1.91 3 3.50 3.54 Seca ee OTA AUISO2 fh. 19? twenty peaches were picked, sampled and inoculated July 23. They were prepared and inoculated as already described and were aJlowed to remain in the glass-stoppered weighing bottles which were placed in a large covered glass dish until August 11. They were then ex- amined and prepared for analysis. | The second series of experiments was set up August 14; the same number of peaches were used as in the first series and they were treated in the same way. The samples were prepared for analysis in the usual way August 30. The results of the determinations in the two series of inoculation experiments are given in tables 3-6, which follow: TABLE III PENTOSAN AND ACID CONTENT AND THE CONTENT OF ALCOHOL-INSOLUBLE SUBSTANCE WuicH REDUCES FEHLING’S SOLUTION WHEN HYDROLYZED WITH DILUTE HCl, IN THE SOUND AND ROTTEN HALVES OF PEACHES, EACH SUBSTANCE DETER- MINED IN SOUND AND ROTTEN HALVES OF THE SAME PEACH Pentosans, Acid per 100 g., Wet Weight Reducing Fehling’s Sol. when Hydrolyzed with Per Cent of iAcidi Gontentisnece-eNiotmal | Per Cent Alc.-Insol. Substance (as Starch) Wet Weight | Dilute HCI], (Wet Weight) Sound | Rotten | Sound Half | Rotten Half | - Sound Half Rotten Half Fe 202) | 0.92 4.75 5.70 1.54 1.39 Dee We Weki7 < | ls 1-7 7.54 8.85 te 12 1.05 3 | 0.88 | 0.94 4.28 5-32 1.30 | 0.95 Api TV REDUCING SUGAR, TOTAL SUGAR, AND SUCROSE CONTENT IN THE SOUND AND ROTTEN HALVES OF PEACHES, STATED AS PERCENTAGE OF WET WEIGHT Reducing Sugar, as Glucose Total Sugar, as Glucose Sucrose Sound Half Rotten Half Sound Half Rotten Half Sound Half | Rotten Half = 2.41 2.83 | 4.43 | 2.836 | 1.92 | 0.004 2 ZOO ET IBIO8” a Nee Aes 0 Lo) 324 eee or | “0:14 78 LON A. HAWKINS TABLE V PENTOSAN AND ACID CONTENT, AND THE AMOUNT OF ALCOHOL-INSOLUBLE SUBSTANCE WHIcH REDUCES FEHLING’S SOLUTION WHEN HyDROLYZED WITH DiLuTE HCl, IN THE SOUND AND ROTTEN HALVES OF PEACHES. .EACH SUBSTANCE DETER- MINED IN SOUND AND ROTTEN HALVES OF THE SAME PEACH. Per Cent Aieeteeon Substance Per Cent Pentosans Acid Content in cc. Normal (as Starch) Reducing Fehling’s Wet Weight Acid per 100 g., Wet Weight Sol. When Hydrolyzed With Dil. HCI, Wet Weight | Sound Peach Rotten Peach | Sound Peach | Rotten Peach | Sound Peach | Rotten Peach I 0.57 | 0.59 2.45 | 7.40 | ©0883 0.80 2 0.67 | 0.69 4.75 7-30 O72 0.64 3 0.62 | 0.66 | 4.25 6.55 | 0.71 0.67 A | | 0.85 0.59 TABLE VI REDUCING SUGAR, TOTAL SUGAR, AND SUCROSE CONTENT IN THE SOUND AND ROTTEN HALVES OF PEACHES, STATED AS PERCENTAGE OF WET WEIGHT. | Reducing Sugar as Glucose | Total Sugar as Glucose | Sucrose Sound Half | Rotten Half Sound Half | Rotten Half Sound Half | Rotten Half | | | | > ae I | 2.47, | Ne Al 5-95 | An2O 2. ai | 0.12 28 T.35 1.68 3.93 | 1.88 2.44 0.19 2 1.14 by 2108) |e ese | 2.82 Mi7® | 0.18 A third series of analyses was begun August 16, using fruit that had become infected under orchard conditions. A quantity of peaches were picked, some of which bore typical brown-rot spots upon the surface. A number of the peaches which were apparently sound were selected, the pulp removed from the stones and cut up into thin slices. The whole mass was then chopped up into small pieces, well mixed and sixteen twenty-five gram samples weighed out, four for the determination of each of the various substances. In addition to these, three ten-gram samples were weighed out for the determination of the total dry matter. These last were covered with 95 per cent alcohol in glass-stoppered weighing bottles and the samples allowed to stand for several days. Most of the alcohol and water was then driven off at about 60° C. The residue was dried to constant weight in an oven at a temperature of 100° C. and the amount of dry matter calculated. The infected peaches were allowed to remain under warm humid conditions for two days after the sound peaches were sampled. At the end of this time they were rotten and in most cases covered with tufts EFFECTS OF BROWN-ROT FUNGUS UPON PEACH 79 of conidiophores of the fungus. They were sliced up and samp!ed and the samples treated as in the case of the sound peaches. The results of the determinations are given in Table VII, which follows. TABLE VII PENTOSAN, ACID AND SUGAR CONTENT AND AMOUNT OF ALCOHOL-INSOLUBLE SUB- STANCE WHICH REDUCES FEHLING’S SOLUTION ON HYDROLYSIS WITH DILUTE HCl, in SOUND AND ROTTEN PEACHES FROM SAME TREE | Wet Weight | Dry Weight | Sound | Rotten | Sound | Rotten PeCERCCTIMGIayATINAELEK 3). ari clay sutieie woe she Gels Gao Daveicge ss 14.40 | 14.40 PemmceitercduUCimorSUSat css scape ceS,c eres oe Bs Re Ses | 4.28 | 8.50 | 29.75 | 59.05 eimeent TOtAal Sugars ts shots. deck hi cule | 9.32 | 8.59 | 64.69 | 59.65 a MRCCIMGRSUICHOSC ts pia, oP ico ctn Pulte el GHA ede gases os AcSOni@:08123.22 1° 90.57, Per cent alc. insol. substance (as starch) which reduces) Fehling’s sol. when hydrolyzed with dil. HCl.... | IAZO HN TeO7 19205) 7-44. eMmGeIME PENLOSATIS wl. Ni vcas oe See ee cone ees Os Gr O73) 25.30), 5.08 CSwesronmeal acid’ per 100 C20. Fs es i ce ews 10.53 pLgsOO! 87.2512) |1 90-26 From the results in the foregoing tables it is evident that some of the compounds studied are much more readily available for the metabolism of the fungus than others. The pentosan content of the sound and rotten halves of the peaches was usually about the same, the differences being no greater than the variations in pentosan content of the two halves of a sound peach. Moreover, the pentosan content was sometimes higher in the rotten half of the peach than in the corresponding sound half. It seems probable then, that the pentosans were not utilized by the fungus. Cooley (3) has recently shown that the hyphae of this fungus are not found to any considerable extent in the middle lamellae of the cells and it does not apparently digest the pectin of plum fruits. It is possible, though not probable, that part of the pentosan, or furfurol- yielding material, might be used by the fungus and a like amount of pentosans laid down. The acid content was always higher in the rotten half of the peach than in the sound portion and this difference was greater than the variation in acid content between the two halves of a sound peach. It would seem then, that the fungus forms some acid or causes it to be formed by the peach. No attempt was made in the present in- vestigation to identify the acids of either the sound or 10tten peach. This work would, however, seem to corroborate the investigations 80 LON A. HAWKINS of Cooley (3) who found that oxalic acid was formed when this fungus was grown upon peach-juice. Behrens (1), on the other hand, found in his work with apples that the acid content of the rotten half of the apple was considerably less than that of the sound portion. In this case the fungus apparently used the acid. The percentage of alcohol-insoluble substance which reduces Feh- ling’s solution when hydrolyzed with dilute hydrochloric acid was somewhat less in all the rotten samples than in the corresponding sound halves. It is, therefore, to be concluded that the action of the fungus tends to decrease the amount of this substance in the peach, although the variation between the sound and rotten halves of the same peach is occasionally less than that found between the two halves of a sound peach, as shown in Table I, columns 6 and 7. The substance may be utilized by the fungus or changed so that it is soluble in alcohol. In the Jatter case it would have been extracted with the filtrate used in the determination of the sugars. In sugar content the sound and rotten halves of the peaches varied considerably. There was more reducing sugar in the rotten halves than in the corresponding sound portions, while in the total sugar content the order was reversed. There was very little cane-sugar in the rotten portions, much less than in the sound samples. The fungus then uses the sugar and causes the inversion of the sucrose. That the sucrose is inverted much more rapidly than the invert sugar is used by the fungus is evident from the fact that the reducing sugar content is higher in the rotten samples. This is especially evident in the case of the peaches rotted after they had become infected under orchard con- ditions in which only a small amount of the sugar had been used yet nearly all the cane-sugar had been inverted. Behrens (1) found that Sclerotinia fructigena used the sugar in apples; he, however, measured only the total sugars. In conclusion, it may be said that in peaches rotted by the brown- rot fungus, Sclerotinia cinerea, the pentosan content remains prac- tically the same, the acid content is increased, the amount of alcohol- insoluble substance which reduces Fehling’s solution when hydrolyzed with dilute hydrochloric acid decreases, the total sugar content de- creases, while the cane-sugar practically disappears. LITERATURE CITED 1. Behrens, J. Beitrage zur Kenntnis der Obstfaulnis. Centralbl. Bakter. Parasit. 4: 700-706. 1898. Q. EFFECTS OF BROWN-ROT FUNGUS UPON PEACH 8I . Bigelow, W. D. and Gore, H. C. Studies on Peaches. U.S. Dept. Agr. Bur. Chem. Bull. 97. 1905. . Cooley, J. S. A Study of the Physiological Relations of Sclerotinia cinerea (Bon.) Schréter. Ann. Mo. Bot. Gard. 1: 291-326. 1914. . Davis, W. A., and Daish, A. J. A study of the methods of estimation of carbo- hydrates, especially in plant-extracts. Journ. Agr. Sci. 5: 437-468. 1913. . Girard, Aimé. Recherches sur la composition des fruits frais. Bull. Ministre Agr. 17: 1523-1528. 1808. . Hudson, C. S. The quantitative determination of cane sugar by the use of invertase. U.S. Dept. Agr. Bur. Chem. Circ. 50. 1910. . Kulisch, P. Obstanalysen. Zeitschr. Angew. Chemie, 1894: 148-153. . Wiley, H. W., et al. Official and Provisional Methods of Analysis, Association of Official Agricultural Chemists. U.S. Dept. Agr. Bur. Chem. Bull. 107. Ellett, W. B., and Tollens, B. Ueber die Bestimmung der Methyl-pentosane neben den Pentosanen. Ber. Deutsch. Chem. Ges. 38: 492-499. 1905. 10. Bryan, A. H., Given, A. and Straughn, M. N. Extraction of grains and cattle foods for the determination cf sugars: a comparison of the alcohol and the sodium carbonate digestions. U.S. Dept. Agr. Bur. Chem. Circ. 71. 1911. NEGATIVE HELIOTROPISM OF UREDINIOSPORE GERM- TWBES: F. D. FROMME During the course of some germination tests with urediniospores of Puccinia Rhamni (Pers.) Wettst. in the spring of 1913 an apparent negative heliotropic reaction by the germ-tubes was seen. The spores had been sown in a drop culture exposed to a unilateral illum- ination on a window sill and a high per cent. of the tubes had grown directly away from the light. Subsequent tests with controls in darkness substantiated the first observations. A search of the liter- ature brought forth a single mention of a heliotropic reaction in ger- minating rust spores. Ward,? referring to a series of germination studies with urediniospores of Puccinia dispersa, has written: “‘My reasons for varying the direction of incidence of the light in certain cases were based on some results (as yet inconclusive) that the germ-tubes exhibit heliotropic curvatures.”’ That the sporidial germ-tubes of Puccinia malvacearum react negatively to daylight has been shown recently by Robinson,’ but aeciospore germ-tubes of Puccinia Poarum were found to be indifferent. Germ-tubes of conidia of Botrytis sp. also grew away from light but those of other non-rust fungi tested Alternaria sp., Penicillium glaucum aud Peronospora parasitica, were indifferent according to Robinson. During the past fall, 1914, the study of the effect of light on germ- inating urediniospores of Puccinia Rhamni was again taken up. Urediniospores were obtained at first from the field and stored in gelatin capsules and later from cultures on oat plants grown in the greenhouse. The urediniospores of this species are especially suitable to daylight tests on account of their quick germination and the rapid growth of their germ-tubes. In the tests made the germ-tubes aver- aged in growth once to twice the spore length in as many hours and four to six times in three to four hours. 1 Read before the American Phytopathological Society at the Philadelphia meeting, January I, 1915. 2Ward, TH. M.) Ann. Bot1216:) 267.9 1002: § Robinson, W. Ann. Bot. 28: 331-340. I914. | 82 NEGATIVE HELIOTROPISM OF UREDINIOSPORE GERM-TUBES 83 The following method of germination was chiefly used. Dry urediniospores were dusted over the surface of a drop of a 5 per cent non-nutrient gelatin placed on a glass slide in a Petri dish containing a moist filter paper. The Petri dish was then placed in a dark box on a window ledge with an aperture, 2.5 cm. in diameter, towards the window. All tests were made with diffused daylight. Controls were maintained in darkness. In all of the ten or more tests that were made with an exposure of four or five hours to 2 unilateral illumination more than four-fifths of the germ-tubes responded negatively to the light stimulus. A count of some 200 germ-tubes in three tests gave an average of 86 per cent that had grown away from the light (fig. 1a). Asmall part of the remainder Ss Orb FIG. I. Germinating urediniospores of Puccinia Rhamnz; a and b, exposed for three hours to a unilateral illumination; c, germinated in darkness. The arrows indicate the direction from which light was admitted. had grown towards the light and a large part were recorded as transverse to the incidence of light. The germ-tubes of the controls in darkness grew in al] directions (fig. Ic). Urediniospores of Puccinia Rhamni have from six to eight germ- pores that are distributed at approximately equal distances apart over the spore surface. A study of the germ-tubes that had grown away from the light showed that the large majority of them has issued from pores located on the part of the spore wall farthest from the light (fig. 2). Others that had issued from pores on the part of the wall towards the light or transverse to it had soon changed their direction 84 , F. D. FROMME of growth by a turn of 90° or 180° and had then grown away from the light (fig. 10). The incidence of light, therefore, not only had a pro- nounced effect in determining the direction of growth of the germ- tubes, but also determined to a considerable degree the approximate part of the spore walJl at which the germ-tube issued, 7. e., the part farthest from the light. The other pores on the shaded part of the spores were likewise stimulated to some degree and a noticeable swelling of their gelatinous contents was often apparent. Sometimes Fic. 2. Germinating urediniospores of Puccinia Rhamni after an exposure of three hours to a unilateral illumination. The arrow indicates the direction from which light was admitted. two germ-tubes issued from the shaded side but one of them soon outgrew the other. When the spores were germinated in darkness the tube issued from any one of the pores. No theory to account for the presence or significance of more than one germ-pore in urediniospores has been advanced. If a possible explanation in terms of advantage is permissible, it seems reasonable to assume that a germ-tube in nature arising from the shaded side of the spore, adjacent to the leaf surface, would be in a more advantageous position to effect an entrance into a stoma than one arising from the non-shaded side, away from the leaf. A urediniospore with several pores should, therefore, have a better chance of producing an infection than one with a single pore. The importance of these light reactions of germinating uredinio- spores from a pathological viewpoint may be found in the possible explanation of certain phases of infection that they suggest. The NEGATIVE HELIOTROPISM OF UREDINIOSPORE GERM-TUBES 85 nature of the stimulus, or combination of stimuli, that is responsible for the stomatal entrance of germ-tubes of rust spores and of other parasitic fungi has not been definitely ascertained. The more general belief is that the host exerts a positive chemotropic influence on the germ-tube, but the failure of attempts to prove that chemotropism plays an important part in this process has been practically universal. If a chemotropic attraction by the host is to,be assumed in the rusts it must be considered a very general property of plants and not a specific property of the hosts alone, since urediniospore germ-tubes ot a number of rusts have been shown by Gibson‘ to enter the stomata of non-hosts as well as those of their hosts. That positive hydro- tropism may partially explain stomatal entrance is suggested by the work of Balls® and of Fulton® but it is doubtful that this can be a factor of primary importance. What part the action of light may play in bringing about the stomatal entrance of the urediniospore germ-tube is as yet a matter of conjecture. It seems quite probable, however, that a continued turning away from light may serve to bring the germ-tube into close contact with the surface of the host and be chiefly responsible for its entrance into the stomatal opening. This is, of course, only a preliminary stage of infection. The success or failure of the attack on the host tissue from the substomatal chamber cannot be directly influenced by light. ; PURDUE UNIVERSITY, LAFAYETTE, INDIANA 4Gibson, C. M. New Phytol. 3: 184-191. 1904. 5 Balls, W. L. New Phytol. 4: 18-19. 1905. $ Fulton, H. R. Bot. Gaz. 41: 81-108. 1906. A WOODY STEM IN MERREMIA GEMELLA INDUCED BY HIGH WARM WATER FRANK C. GATES In the large swamp a little west of Los Banos, Laguna, Philippine Islands, Merremia gemella (Burm.) Hallier f. (Convolvulaceae) is a common vine which sometimes grows over Phragmites vulgaris (Lam.) Trin. and Sesbania (nearest S. sesban (L.) Merr.). As both of these species grow in water the greater part of the year and Merremia is neither a parasite, like Cuscuta, nor an epiphyte, its roots must also be under water. This is not a normal condition for Merremia but it does adapt itself and lives. Consequently, even near the center of the swamp, one finds festoons of Merremia draped over the Phragmites and Sesbania, even when they are growing in water a meter deep. Usually the stem of Merremia gemella in dryland thickets and as a weed in cultivated land is herbaceous and dies down each year. Under the conditions obtaining in the swamp, the first and perhaps most striking thing seen is the prominent woody stem which may be as much as 20 mm. in diameter. As a seedling Merremia must start in the ground and is continually dependent upon the ground for certain mineral salts. The seedling develops into a vine, rapidly making its way to the upper story of vegetation and spreading out over whatever happens to be there. To do this the normal herbaceous stem appears to be sufficient under ordinary conditions. Most of the swamp area is submerged during the rainy season, so that the roots of Merremia are under water. In ordinary years they are not generally far under water—less than 25 cm. rather than more. The sources of the water in the region are bubbling hot springs, whose temperature is usually between 70 and go° C. The water soon becomes cooled to a temperature between 30 and 40° C., which several plants withstand. The high water of 1914 was very high,—more than 1.5 meters over considerable area. The water covered the hot springs, which however continued to heat up the water in their vicinity. In the immediate vicinity of the hot springs the plants were killed outright 7 86 A WOODY STEM IN MERREMIA GEMELLA 87 and deleterious effects spread considerably further. Six weeks later, at the end of October, the temperature of the water a kilometer away from the hot springs was 27° C., whereas the surface water over them was 37 to 42° C. All of the Merremia in the vicinity of the hot springs was killed. Further away where the temperature did not go much above 30° C. the effect was to stimulate the growth of a woody stem. Thesecondary thickening was very irregular, forming what is known as anomalous structure. Beneath the warm water level, there was only a very little secondary thickening present, of the same general type as that above Fic. 1. The edge of the creek in the center of a swamp, showing Merremia gemella climbing on Sesbania. Sesbania in fruit is nearly leafless. Los Bafios, P. I. October 31, 1914. the water but much poorer in amount and diversity of anomalous structure. The xylem vessels were noticeably larger in the part of the stem above the water. The bark above the water was rugged, but beneath the water it was quite smooth and compact. It was frequently covered with putrifying bacteria, but did not produce aerenchyma. When the stem was submerged during only the highest part of the high water and especially when it was twining around a stem of Sesbania, at the places where aerenchyma was produced by the Sesbania, short (less than 25 mm.), horizontal, clinging roots were developed from the stem of the Merremia. Entirely above the water 88 FRANK C. GATES where no aerenchyma was produced by Sesbania, no clinging roots were present on the Merremia. These roots closely covered the aerenchymatous surface but did not seem to penetrate it. Lack of sufficient air in the lower part of the stem and in the roots was obvious. The smaller size and number of the vessels in the lower part, the simpler structure as well as the smaller amount of Fic. 2. authors should es aaa as soon as fe: to American Journal of Bot Ne: ‘American J ournal of I ts must Do wn on New York City.t ba | Claims on waning 3 numbers bo be made within 30 days followi is 2 mbers free 0. on _poncerning. pion colt be addresse , Garden N.Y. or gt age sane Soe va AMERICAN 3 tional th JOURNAL OF BOTANY Wows Ul Marcu, 1915 No. B MORPHOLOGY AS A FACTOR IN DETERMINING RELATIONSHIPS! J. M. GREENMAN The remarkable advance in our knowledge of relationship of organisms during the past quarter of a century is due primarily to extended studies in morphology in one phase or another. In this field. data have been gathered from various sources, and there is at present a vast array of recorded observations many of which have been interpreted in terms of relationship and phylogeny. In fact among morphologists and taxonomists relationship and phylogeny have been leading questions of the day. So important is morphology in these matters that it may seem almost axiomatic to speak of morphology as a factor in determining relationships. Nevertheless, the methods of attack have been from such different standpoints and the significance of the results is such that I trust a general statement at this time may not be entirely out of place. Naturally, I must speak mainly from the standpoint of a taxon- - omist, and furthermore I shall confine my remarks to a consideration of the higher plants. The general situation as to the morphology of the higher plants,, both the gymnosperms and angiosperms, has been so carefully summarized and so admirably presented by Coulter and Chamberlain that no further words are needed to emphasize the importance of modern morphology to a sound taxonomy. Notwithstanding the amazing progress in modern morphology and the fact that our knowledge of the life-history of individual organisms 1 Invitation paper read at the symposium on “The Genetic Relationship of Organisms”’ before the Botanical Society of America at Philadelphia, December 30, 1914. ind [The Journal for February (2: 71-110) was issued 3 Ap 1915.] III mace, APR 24 1915 —— | neiity, ee Wi A hf aye7 112 J. M. GREENMAN and genetic relationship of groups of organisms has been materially broadened there has been thus far at least little application of the data obtained to affect, except to amplify, our present system of clas- sification. The basis of classification of the higher plants today is still the old morphology, namely that of Caesalpini, John Ray, Linnaeus, Wolff, Goethe, De Candolle, Eichler, Gray, Bentham and Hooker, and Engler and Prantl; and it is expressed in epitomized form in Engler’s Syllabus der Pflanzenfamilien (1912), and, so far as the angiosperms are concerned, in a somewhat ampliated form by Wern- ham in recent numbers of The New Phytologist. Our present system of classification has resulted from a very gradual development of the science, and along with it there has also developed an elaborate terminology which is more or less arbitrary and to a certain extent fixed. Furthermore, it expresses, as has often been said, the best interpretation of correlated knowledge of relationships up to the time of its publication. Since the publication of Engler and Prantl’s Die Natdrlichen Pflan- zenfamilien morphological research has been directed to various groups of plants a more detailed study of which might throw light upon their genetic relationship. Nowhere has this been more striking than in the Gymnosperms and nowhere I think may it safely be said to have been more effective from a taxonomic standpoint than in this group. Investigations in plant anatomy have shown conclusively the importance of morphological anatomy in determining genetic relation- ship or phylogeny. Modern methods, through the work of Scott, Oliver, Arber, Jeffrey, Bailey, Eames, Sinnott and others, have made it possible and profitable to carry on researches along these lines. Mor- phological considerations of structural elements hitherto unused, or at least used to a very slight extent, have been demonstrated to be of value in determining relationship. How far such characters can be made use of in a general system of classification must remain until a correlation of all facts is effected and possibly for a future generation to decide. Personally, I am inclined to believe that these less obvious features must remain subsidiary to the more evident characters which are relatively easy of access. There has also been a revival in the study of botanical embryology in its broader aspect, which just at present seems likely to be of the utmost importance in determining finally the relation of monocotyle- donous to dicotyledonous plants. In fact morphological evidence is MORPHOLOGY AS A FACTOR IN DETERMINING RELATIONSHIPS I13 rapidly accumulating to show that the former group has been derived from the latter. Several writers have expressed this belief, but they have reached their conclusions from rather different points of view. I refer particularly to the recent contributions by Henslow and by Ethel Sargent. Miss Sargent’s paper entitled ‘A Theory of the Origin of Monocotyledons, founded on the Structure of their Seed- lings’? points out that “the cotyledon of Anemarrhena, a liliaceous genus, contains two. massive bundles which together form a tetrarch stele in the primary root’’; and she interpreted this as probably due to ‘‘a fusion of two seed-leaves in some remote ancestor to form the single cotyledon of Anemarrhena.”’ Seedlings of other Liliaceous genera were also examined, but this one she regards as representing a rather primitive type. Miss Sargent contrasts this condition with the condition shown by certain dicotyledonous plants, e. g., species of Anemone, Delphinium, Ranunculus, Trollius, Eranthis, etc., ‘‘pos- sessing seed leaves which are partially united, sometimes by one _margin only, but more often by both.’ The conclusion therefore being that the monocotyledons have been derived from the dicotyledons through a fusion of the two cotyledons into one. In this connection I may call attention to a very suggestive paper by Coulter and Land in a recent number of The Botanical Gazette; they record the results of a morphological study of seedlings of Aga- panthus umbellatus, a South American amaryllidaceous plant, seedlings of which were found to possess more or less indifferently one or two cotyledons. This study has been continued, and a paper by the same authors was read by Professor Coulter at the recent celebration of the 25th Anniversary of the Missouri Botanical Garden. Although this second paper is not yet published Professor Coulter stated, ‘The general conclusion is that monocotyledony is simply one expression of a process common to all cotyledony, gradually derived from dico- tyledony, and involving no abrupt transfer of a lateral structure to a terminal origin.” In other words morphological evidence has been presented by Miss Sargent that monocotyledony has resulted by a fusion of two coty- ledons and according to Coulter and Land by the reduction and disap- pearance of one cotyledon. In either case the phenomenon is inter- preted as indicating the origin of the monocotyledons from the dicotyledons. While recent studies have been concerned mainly in determining 114 J. M. GREENMAN the genetic relationship of the larger groups, yet, at the same time, attention has been given to the life history of individual species and groups of species, and to a discussion of the relationship and phylogeny of certain orders, such for example as the Salicales, a group recently investigated from an anatomical standpoint by Ruth Holden. This author takes objection to the relatively low position assigned the Salicales in the Engler and Prantl system; and she presents anatomical evidence, particularly concerning the primitive condition of the wood — —namely vasicentric parenchyma and multiseriate rays—to show that this simple structure is due to reduction. It is interesting to note that in this case at least the anatomical evidence in stem-structure is supplemented by reduction in floral structure and also by entomo- philous pollenation and porogamy. Furthermore, it has been maintained, through comparative mor- phological studies, that the Fagaceae have been derived from ancestors allied to epigynous types of Rosales; and it is not unlikely, moreover, that a similar genetic relationship may be the case among the Jug- landales. Several families of plants, for instance the Araceae in all probability represent lines of reduction and in this case possibly from some ancestral liliaceous type. It is not inconceivable also that the Gramineae may have developed from an ancestral stock allied to the present bamboo tribe. Among the dicotyledonous plants the Euphor- biaceae is generally regarded as a polymorphous family in which the floral structure in several genera has been very much reduced, as for example in Acalypha and Euphorbia. In such groups as these a more thorough knowledge of the embryol- ogy and seedling anatomy would undoubtedly throw light upon gene- tic relationship as well as generic affinity. I think the objection taken to the relatively low position assigned to several groups in the Engler and Prantl sequence is well founded, but we must wait for additional morphological evidence and its proper correlation before satisfactory change in their position can be made. Important as these embryological and anatomical studies are in determining genetic relationships, especially of the greater alliances, yet when it comes to a consideration of the relationship of different families and to generic affinity, taxonomy can be materially advanced by a more thorough knowledge of the floras of the different parts of the world. We need also to know more of the life-history of plants already recorded and to have our floras more fully represented in MORPHOLOGY AS A FACTOR IN DETERMINING RELATIONSHIPS II5 herbaria and garden. It is my belief that this can be done best through a co-operation of the larger botanical institutions. It is manifestly impossible for any one institution to cover the whole field with any degree of satisfaction, but it is possible for each institution either to direct its energies to a particular territory or to a particular group of plants. Thus it would be possible to place in the hands of the research student an abundance of the necessary kind of material for comparative morphological study and afford the opportunity to de- termine the range of variability. Our conception of the origin and limitation of species has been so modified by recent investigations in plant breeding, mutation, hy- bridization, etc., that relationship in a line of evolutionary sequence is an extremely complex problem. Nevertheless the new morphology, the intensive study of cell-phenomena, and experimental work have done much to advance our knowledge of the relationship of organisms; and it is not improbable that within a comparatively short time we shall have a more complete and satisfactory knowledge of the main phases of phylogeny and the chief lines of evolution of our higher plants. MissouRI BOTANICAL GARDEN, ST. LOUIS THE GENETIC. RELATIONSHIP” OF PARASI RES. FRANK DUNN KERN Parasitism is a mode of life having many forms and degrees. Nu- tritional dependence may be partial, temporary, or complete. All possible intergradations exist. It has been estimated that a large percentage of the total number of seed plants may use complex food derived from other organisms and yet only a comparatively few as- sume a mode of life which has been regarded as parasitic. Gradations also connect the organism, which is dependent upon other living or- ganisms for nourishment, with those which are dependent upon or- ganic, but non-living, food. 2 Organisms may be classified physiologically as parasites, sapro- phytes, and autophytes (or holophytes) but such a classification does not coincide with a taxonomic one. Upon morphological grounds parasites are variously distributed in the taxonomic series with inde- pendent or saprophytic forms. If our taxonomic classifications can be regarded as representing phylogenetic relationships their arrange- ments not only suggest the evolution of parasites from free-living an- cestors but also indicate many separate points of origin. The presence of free stages in the life-cycle of many parasites; the frequent occur- rence of temporary parasitism; and the close morphological resem- blances between some independent and some dependent forms afford abundant evidence to justify such a view. Organic dependence may have begun gradually or it is possible in some instances that the time element may not have been a factor and that parasitism may have come about as a sudden mutation. Plaus- ible explanations of the origin of the parasitic habit in animals do not seem so difficult asin plants. There is the possibility that the seeking of shelter may have been the starting point for some animal forms, others may have found protection accidentally having been swallowed in an embryonic or even adult condition. It has been suggested that lt Invitation paper read at the symposium on “The Genetic Relationship of Organisms”’ before the Botanical Society of America at Philadelphia, December 30, 1914. Contributions from the Department of Botany, Pennsylvania State College, No. 3. 116 THE GENETIC RELATIONSHIP OF PARASITES I17 in many cases the habit may have begun “with the naturally more sluggish females, prompted not by hunger, but by the impulse to seek some conveniently sheltered place for the birth of the young.” It may be possible eventually to gain some usable hints from the past ages but parasitic conditions are so dependent upon adaptations in the physiology and soft structures that the record in the rocks is exceeding- ly fragmentary and difficult to interpret. When parasitism is suggested the one feature concerning the para- site which presents itself most strongly is that of degeneration. The dogma of degeneration is so thoroughly drilled into the minds of the students of biology everywhere that parasitism and degeneration seem unalterably linked together. We are prepared for the expressions that parasites ‘‘are more or less degenerate according to the extent of their parasitism’’ or that parasitism ‘‘unfailingly involves degener- ation.’ Although we may expect old and popular beliefs to be over- thrown at any time there seems to be much in favor of the correctness of this one. So far as the seed plants are concerned there is evidence of somatic degeneration, consisting chiefly of reductions or atrophies of the shoot and root, even in those forms which are only partially parasitic; while those which are fixed parasites may be still further re- duced, the root failing to develop, the shoot remaining unbranched, the leaves lacking chlorophyll and frequently appearing as colorless bracts. With regard to the spore plants it is not so easy to form judg- ments concerning possible somatic modifications since there are likely to be long genetic series of heterophytic forms and no clearly related autophytic groups to form a basis for comparison. As regards sexuality it has been a widely accepted doctrine that the parasitic mode of life leads to its disappearance. Investigations by Ernst and Schmidt on the root parasite Rafflesia have caused them to draw the general theoretic conclusion that so far as seed plants are concerned parasitism does not tend to the disappearance of sexuality. Other studies have, however, shown that the fruits and seeds may show various specializations. In the case of the fungi we are not sur- prised to find in an authentic text-book, now only fifteen years old, the statement that sexual reproduction is common in the Phycomy- cetes, occasionally occurs in the Ascomycetes, and is not certainly known to exist in the Basidiomycetes, with the added explanation that probably certain of the higher forms of fungi that have lost their sexual methods of reproduction have been derived from the lower 118 FRANK DUNN KERN fungi. Recent researches have shown that the old belief in the dis- appearance of sexuality in the parasitic fungi is no longer tenable. Harper has summed up the situation for the fungi in the statement that “the evidence is now generally accepted that either a typical conjugation of normally differentiated gametes and their nuclei or some form of substitute for it is everywhere present.’’ The very fact, however, that there is here interpolated the phrase ‘or some form of substitute for it”? suggests strongly that although there may not be a disappearance of sexuality there may be a degeneration. In fact the author just quoted states further that the fungi exhibit some funda- mental modifications of the process of sexual fusion as found else- where. The question whether these variations are to be regarded as degenerations may be debatable but inasmuch as they are considered substitutions for sexual fusions that would appear to be implied. It should be noted here that while sexual reproduction can be shown not to be entirely impossible for certain forms yet its occurrence may be exceedingly rare. Long patient researches with some forms such as the late blight fungus of potatoes have led to an almost inevitable conclusion that there is not a disappearance of sexuality but that there is a loss of sexual vigor. It seems reasonable to conclude that the presence of both somatic and sexual modifications in parasites warrant the continued association of degeneration with parasitism. In connection with this view that parasitism is a degenerate adap- tation it would be interesting to know whether there are any cases where the parasitic habit has been conquered and a return made to a normal independent existence. MacDougal in speaking of the higher plants says that it is usual to consider them ‘‘as passing down an in- clined plane of atrophies, which would ultimately lead to their ex- tinction, without reference to the abundance of development of the host forms.” He has not in his investigations obtained the slightest hint which would indicate the possibility of a retracement by which a parasite might regain its standing as an independent. Nor is there any evidence from the bacteria or fungi to suggest that the parasitic habit when once established ever can be overcome. Some investi- gators hold the view that certain groups of obligate fungous parasites have made morphological advances of a sporophytic nature but this is questionable and even if true could not be taken as an indication of a possible step toward abandonment of parasitism. Some evidence which seems to point in this direction comes from the animal kingdom THE GENETIC RELATIONSHIP OF PARASITES II9 and is furnished by a paleontologist. The case referred to is that of the parasitism of the gastropods on the crinoids and has been very clearly set forth by Clarke. Gastropods of the limpet type were parasitic on crinoids from the Silurian on to the latter part of the Paleozoic when all traces of such a condition ceased. The evidence points toward the cessation of this parasitic condition during Car- boniferous ages. Clarke believes ‘“‘that all the positive and all the negative evidence we can now adduce on this deeply important sub- ject favors the presumption that the habit was abandoned or at least, to speak in terms of simple casuistry, was lost.’’ There are no para- sitic snails on crinoids today, but close allies of the crinoids are para- sitized by gastropods of the limpet type. This, however, is held to be entirely independent of the former association between these groups. Much has been written concerning the nature and origination of parasitism but much remains for further experimentation before the factors involved can be understood. In order to form proper concep- tions it is necessary not only to consider the parasite and its weapons, but also the host with its defense, so that the question of the origin and nature of parasites is inseparably associated with those phenomena generally grouped under the term immunity. We must take into account also the effects of the environment upon both parasite and host. The available evidence indicates such a difference in the genetic physiology of parasites belonging to the higher and lower plants, re- spectively, that it may be best to discuss the groups separately. The parasitic seed plants form a series of isolated groups each of which bears a more or less evident genetic relationship to some inde- pendent group. Any theory of origination must take into account the undoubted existence of a number of disconnected points or levels oforigin. MacDougal has extensively investigated the conditions under which two plants may enter into the relation of host and para- site by growing one plant upon another and has concluded that ‘‘the ruling factor was in all cases the osmotic ratio between the sap of the two plants,” a greater osmotic pressure being necessary in the incipient parasite. Of course many other factors are involved for one plant is not able to parasitize another simply because it possesses a higher osmotic pressure. It seems likely that parasites among the seed plants arose directly from independent ancestors without any inter- vening saprophytic condition such as probably existed in the evolution of the fungi. The first step intervening between independence and TZ20 FRANK DUNN KERN dependence in the seed plants may have been the absorption of water and food materials, the taking up of complex foods being a later de- velopment. This is in accord with a theory hazarded by Cowles who adds that as a final development there comes the loss by the seeds of the power to renew their growth, except when in contact with the host, and the development of a univorous habit. As intimated in the foregoing paragraph there is evidence indi- cating that the parasitic lower plants (bacteria and fungi) have de- veloped from independent plants not directly but through intermed- iate saprophytism. There are numerous transitional stages from autophytes to saprophytes and on to facultative parasites, ending finally in obligate parasitism. The development of the capacity to take in organic foods in a saprophytic manner seems to be a simpler first step for these forms, rather than the absorption of water and food materials in a parasitic way, and at the same time the existing forms grade into one another along such a probable course of development. Even after parasitism is attained there are still degrees of differentia- tion. The forms which can continue without sufficient detriment to the host to cause its death, and hence their own, are to be regarded as on a higher plane of development than those whose aggressive growth soon results in death for both host and parasite. Another condition which is agreed by many to indicate a high parasitic evolution is the physiological dependence upon a very restricted set of hosts. The greater the restriction, which may be a single host species, or even a variety, the higher the stage of parasitic development. Concerning the independent forms which may be looked upon as most nearly representing the probable ancestors of such forms as the parasitic bacteria and fungi there is general agreement in a broad way but naturally differences of opinion upon many important points. The probable connection between some dependent and independent forms has received definite recognition in the classifications used by some taxonomists. Bessey no longer maintains the Phycomycetes co-ordinate with the groups of higher fungi but places them in the phylum with the tube algae upon morphological grounds. In like manner the Synchitriaceae are grouped with the unicellular green algae. The series classed as higher fungi are more isolated and retain standing as a separate phylum with debate still going on as to their relationships and interrelationships. In a recent paper Jones has emphasized the fact that the funda- THE GENETIC RELATIONSHIP OF PARASITES 121 mental problems of parasitism remain unsolved, that ‘we have scarce- ly begun the study of the intimate relations of parasite and host, the conditions and results of parasitism.’’ He comments further that the very simplicity of the plant’s organization makes these relations more difficult to investigate than with the animal, the cell being the unit, not the organism, the study of chemical interrelations is rendered highly difficult. ‘‘Such problems,” he says, ‘“‘call for the combined skill of pathologist, physiologist, cytologist, and chemist.’ These forces, however, have not been mobilized adequately as yet. Chemotropism is frequently put forward as a factor involved in the origin of parasitism in the lower plants. Massee goes so far as to assert infection is due to positive chemotaxis. If it is the pulling power of some substance within a host which causes fungi to enter, it is difficult to explain highly restricted parasites. Either very few hosts contain the proper substance or all others must contain some- thing to neutralize this attraction or something of a more powerful repellant nature. Neither view seems adequate to give us a concep- tion of a large percentage of the world of plants with parasites more or less specialized to themselves. It must be kept in mind also that mere entrance of a fungus is only preliminary and not necessarily in- dicative of parasitism. The theories of immunity come to mind, pass in review, and leave us unsatisfied. One investigator states that “immunity depends chiefly (perhaps entirely) upon the ability of the cytoplasm of the host-cells to resist infection by secreting anti-toxins which will kill the mycelium of the fungus.’’ Tannin, vegetable acids, and oxidizing enzymes have been named as possible toxic substances. Others would not place the burden of keeping invaders out upon the cell-contents of the host, but explain the failure of parasites to grow promiscuously upon their lack of development of cytolytic enzymes sufficient to break down the barrier offered by the cell wall. It makes little difference which of the theories has the most supporters or which one appears most plausible; not one of them, or any combination of them, gets at the real intimate relation which must exist between host and parasite. Chemical, food, or structural features, as far as we are able to appreciate them, seem unable to explain why one parasite can attack only one variety of a single host species while another similar parasite may attack all the varieties, other species of the genus, and perhaps even extend to other closely related genera. Our problem would be sufficiently baffling if we were left to con- [22 FRANK DUNN KERN sider only the relation between a parasite and its host, or hosts when it passes to several allied forms, as indicated in the foregoing paragraph. No matter how difficult this may appear to be we are obliged to desig- nate this as simple parasitism. To contrast with this there are the cases of parasites which are known to inhabit wholly different and unlike hosts in different stages of their life histories. Such a surprising and, as it seems, unnecessarily complex parasitism exists in, both ani- mal and vegetable kingdoms. In those species which change hosts, and on that account are termed heteroecious, there is frequently more than one method of propagation. Protozoan forms which come under this category are said to multiply by schizogony, a kind of multiple fission, and by sporogony, a process of spore-formation preceded by conjugation. Sporogony usually either makes possible the invasion of a new host or takes place only in connection with the second or in- termediate host. The evident food relations between the hosts of many of the heteroecious animal parasites, such as the mosquito and man and various mammals in the case of malarial forms, renders the association of the unlike hosts not so difficult to appreciate. In the higher fungi, especially in the group popularly known as the rusts (Uredinales), there are many instances of heteroecious parasi- tism and in none of them is there the slightest hint of a relationship between the two hosts. There are many forms inhabiting coniferous trees and from these they may pass over in their alternate stage to plants widely separated in taxonomic classification. Thus we may find a form alternating between a fir and.a fern, a cedar and an apple, or a pine and a composite. It seems impossible to escape the con- viction that some extraordinary relationship must exist between these two sets of hosts occupied by a heteroecious form and yet in the half- century since we have had definite proof of heteroecism in the rusts no satisfactory explanation has been forthcoming. In North America we have today almost exactly one hundred demonstrated cases of heteroecious life-cycles in this group. The rusts are of especial interest from our present point of view not only because of heteroecism but also because of the existence of autoecism within the same group, and the consequent opportunity to compare these two types of adap- tation. Heteroecious forms always possess what may be termed a long life-cycle divided necessarily into two phases, on the one host consisting of telia, either alone or accompanied by uredinia, and on the other host of aecia, preceded by pycnia. Telia, uredinia, and aecia THE GENETIC RELATIONSHIP OF PARASITES 123 produce spores usually unlike one another and are all for purposes of propagation. Pycnia produce simple spores unlike any of the others and of unknown function. Autoecious forms may exhibit the same number of spore-forms and in the same sequence as just de- scribed for heteroecious forms, or they may have the aecia and ure- dinia lacking. In this case the pycnia, when present, are followed at once by telia and we have what may well be designated as a short life-cycle. The heteroecious forms always show well developed pleomorphy whereas the autoecious forms may or may not. The presence or absence of urediniospores, which simply repeat themselves over and over, is held to be of no fundamental importance in the forma- tion of a life-cycle. It is understood that telia are always present and that pycnia usually (but not always) follow from telial infection. Ifa heteromorphic spore-stage (aecia) follows the pycnia the form is thrown into the long-cycle series without regard to the presence or absence of a second heteromorphic stage (uredinia). Ever since heteroecism has been definitely known to exist in this group, or for fifty years, naturally there have been speculations concerning its origination. Within the last ten years since sexuality and an alternation of generations have been demonstrated the char- acter of the speculations has been somewhat modified. The probable evolutionary tendencies within such a remarkable group of obligate parasites must have an important bearing upon our general conception of the genetic physiology of parasitism. If we accept a view, which has found much favor, that the complex heteroecious types have been derived by amplification from the simple, autoecious, short-cycle forms, we then find it necessary to explain how parasites, in which degeneration might be expected, have made un- precedented advances. To be sure it seems most logical to derive the complex from the simple and the amplification, since it is in the sporo- phytic stage, is in accord with the conditions found in independent plants. But the questions immediately arise whether we can expect such obligate parasites to parallel the development of higher plants and whether we need to make such assumptions in order to explain present conditions. In the light of our observations on the behavior of parasites it seems that we ask a good deal of both evidence and probability in seeking to align these parasites with the independent higher plants on the basis of the development of the alternating generations. 124 FRANK DUNN KERN Both short- and long-cycle forms agree in the gametophytic genera- tion which is initiated by the reducing germination of the teliospore and ended by a doubling conjugation previous to the production of the first spore-structure (the pycnia being disregarded). In the short- cycle condition the first spore-structure is another telial stage hence the sporophyte is represented only by the teliospores themselves and a small amount of growth accessory to their production. In the long-cycle condition the first spore-structure following the conjugation may be known as the aecial stage without regard to its form or struc- ture. In all cases the aeciospores give rise to a plant body (mycelium) which is sporophytic. In some forms it produces repeating spores, either like or unlike the aeciospores, but ultimately forms the telial stage, the end of the sporophytic phase. In heteroecious forms the two phases are produced upon the unlike hosts. Reasoning by analogy we are justified in regarding the gametophyte as the primitive genera- tion and it would seem to follow that the forms with the least developed sporophyte should be nearest the ancestral conditions. By a progres- sive development of the sporophyte, a prolongation of the double nucleated phase, we have an evolution which is comparable with the course of development of the higher plants. Some investigators have become so confident that this is a reasonable presumption that they have ceased to argue this phase of the problem but have gone on to discuss the origin of heteroecism, how the jump from autoecism to a new host could have come about, and which was the original gameto- phytic host and which the secondary one. If the rusts have made such advances in their sporophytic genera- tion we have a condition which may force us to modify our ideas of parasitism. The whole question is inseparably connected with the large problem of the genetic relationship of parasites. The question whether this group with all its complexity conforms to the nature and development of other parasites or stands as an exception is one which interests us today. Those who have regarded the rusts, in spite of their complexity, as a degenerate group of parasites also have advanced theories as to the probable course of development. It may be idle to speculate further with such theories but perhaps another viewpoint may permit new relations to be perceived. According to one view pleomorphy and even heteroecism are held to be the primitive condition and the autoecious short-cycle forms are regarded as the latest in evolution THE GENETIC RELATIONSHIP OF PARASITES 25 having arisen by a process of degeneration and reduction. It is not denied that there must have been amplification some time in order to attain the long-cycle condition but it is believed that this did not take place after those organisms became parasites. Undoubtedly the long-cycle forms represent the highest development but probably not the latest. As suggested in a foregoing paragraph there is much in favor of the parasitic fungi having passed through an intermediate saprophytic stage. Ultimately, of course, they must have come from independent ancestors. The existence of independent thallophytes and saprophytes showing well developed pleomorphy indicates that this feature is not one which can be considered as having any peculiar connection with the parasitic mode of life. The transition of complex pleomorphic forms into heteroecious parasites may not seem an easy step but is not more difficult to appreciate than the interpolation of one or two new stages, upon an entirely foreign host, into the life-cycle of a simple parasite. Since our present interpretation of the evolution of the higher plants is the rise of the sporophyte and the decline of the gametophyte much has been made of an analogical application to these parasites. It has been pointed out that those forms with the longest sporophytic stages must have arisen in a progressive way. While this is doubtless the case there is much doubt whether they have progressed as parasites and whether the present short-cycle forms can be looked upon as representing their ancestors. It should be pointed out that there has been no decline of the gametophyte unless it be in the loss of pycnia in some species. However, the only forms in which pycnia are known to be absent are short-cycle forms, which are according to the theory of ancestral nature. The pycnia are without doubt functionless today. It would be odd indeed if the gametophyte of the latest type in evolu- tionary history should always develop a useless structure not univer- sally known in its ancestral forms. Surely this is not in conformity with the higher plants. There has been much discussion over the interpretation of the pycniain the rusts. The view that they represent vestigial male organs is highly plausible. Without much doubt they have not been functional since the parasitic adaptation. A former sexuality in which an Aecidium-like structure bore the female organs is now replaced by a somatic doubling conjugation in the mycelial mass below the spore-structure. The eventual disappearance of the pycnial stage might be anticipated in the latest forms. If this has 136 FRANK DUNN KERN any bearing it points toward the short-cycle autoecious type as the most recent for here the pycnia may be omitted although in ontogeny there elapses a time for their development. In a parasitic group where there is such a close relation between parasite and host we might gain some idea of genetic relationship from a study of the distribution of the different types of rusts upon the series of hosts. Although the phylogeny of the hosts may not be regarded as settled the arrangement will permit this sort of a general comparison. It seems logical to assume that the lowest hosts would be parasitized by the more primitive forms while the higher host orders would be expected to have upon them the more advanced types. As already indicated we recognize but three types of parasites, heteroe- cious long-cycle, autoecious long-cycle, and autoecious short-cycle. The ferns are the lowest hosts for these parasites and their forms are pleomorphic and heteroecious. In order to have some numbers available a careful survey of North American forms having a definite known life-cycle has been made. Out of fifty species on Coniferales forty-seven are heteroecious. Glumaceous rusts are heteroecious with a single possible exception. In the Lily order while only seven are heteroecious, twenty-three are autoecious long-cycle forms, with a single short-cycle form. The Ranales have the three types almost equally distributed. In the large rose order the long-cycle forms greatly predominate but autoecism is three times more prevalent than heteroecism. Upon the final great alliance, Campanulales, we find heteroecious phases but so far as present known life-cycles are con- cerned they are only half as numerous as short-cycle forms, the figures being thirty-two and sixty. If we make comparisons within the short- cycle series we find that they are almost unknown upon the lower orders whereas sixty out of a total of one hundred forty-eight known forms are found upon the highest order. If this sort of evidence is ‘competent its indication is certainly plain. In a recent paper Olive has taken it for granted that autoecism is the original condition and has brought forward arguments bearing on the question as to which. of the heteroecious hosts is the primary one. With his premises as a basis he comes to the very logical conclusion that the present aecial host was the original host of the hypothetical autoe- cious ancestor. Part of the argument is that the gametophytic gener- ation being the more primitive should be on the primary host. To evolve the heteroecious condition the gametophyte has remained un- THE GENETIC RELATIONSHIP OF PARASITES 127. changed but the sexual fusions have furnished the invigorating in- fluences which are responsible for pleomorphy and the pleophagic habit. It cannot be denied that sexual fusions should furnish in- vigoration but those fusions found here which appear to be a degenerate substitution for a former fertilization do not seem adequate to account for such a profound change in the life of a parasite. If we are to take the view that the present aecial host is the original and primary one, then it would seem reasonable to expect the aecial stages to be on the more primitive hosts as a rule. The condition found in our North American heteroecious forms does not bear out this expectation for we find out of ninety-nine species that seventy-eight have aecial hosts standing higher in classification than their telial hosts. To support the argument that the gametophytic host was the original autoecious one,it has been urged that there are instances where short-cycle forms are known on the aecial hosts of heteroecious species, the teliospores of the two showing structural parallelisms so striking that some kind of relationship is strongly suggested. Puccinia mesniertana a short-cycle form occurring on Rhamnus and having teliospores peculiarly like Puccinia coronata, a heteroecious grass form having its gametophytic stage on Rhamnus, has been cited. There are numerous other cases. Puccinia Xanthw is an autoecious form on Xanthium, which harbors the aecial stage of a sedge rust, Puccinia Cypert. Polythzlis fusca,ashort-cycle formon Anemonz, has teliospores identical with Tvanzschelia punctata which hasits telia on Prunusand its aecia on Anemone. Numerous other cases could be mentioned. May the evident genetic relationship best be explained by amplification or reduction? Not only does the latter more nearly conform to the physiology of parasitism but also agrees with certain conceptions of differentiation which have been formed from experimental genetics. Based upon many considerations we find Bateson saying that he feels no reasonable doubt that we may have to forego a claim to variations by addition of factors, yet variation by loss and fractionation of factors is a genuine phenomenon of contemporary nature. If there is a possi- bility that we may have to dispense with additions from without in independent forms how much more strongly does such a situation press its claim for recognition in parasites. To return briefly to the associated long- and short-cycle forms above mentioned we find that no startling variation has to be assumed in order to derive the latter from the former. If on the gametophytic host, following the sexual 128 FRANK DUNN KERN fusion, there should be produced at once teliospores instead of an inter- calation of one or more polymorphic spore-stages, the proper con-— ditions for a short-cycle autoecious form would be at once established. -” In heteroecious forms only the reduced basidiospores following telio-*~ spore germination can infect the aecial host. In the abbreviated con- ” dition just outlined we have the necessary requirements for reinfection °, and maintenance of a short-cycle telial form on what was the original. : aecial host. Believing that the only fundamental stages are those of sexual fusion and teliospore formation neither is disturbed by such a shortening process. Teliospore formation which was an inherent capacity simply takes place sooner than formerly without the inter- vention of a somatic stage of such extent between sexual fusion and reduction. If teliospore production is to take the place of the former aeciospore formation there might be two expectations concerning their nature. They might be exactly like the former teliospores in which case nothing happens except the pulling of the stage backward to the other host, or they might be expected to have the morphology of the aecidiospores but behave physiologically like teliospores. The so- called genus Endophyllum fits the latter hypothesis and may have arisen in some such manner. ‘The cases cited and numerous others fit the former. Such a theory does not fit badly with the present distribution of the types of rusts upon the phylogenetic series of host plants. Re- calling the fact that the tendency for heteroecious species is to have the gametophytic hosts higher in classification than the telial hosts we must expect the short-cycle forms which are to occupy the former gameto- phytic hosts to show an upward movement which would result in their occupation of the higher orders. I have already shown that is in accord with the facts based upon a survey of North American forms. Although it may be objected that these considerations do not explain the real origin of the group it may be claimed that they do express a trend of evolution, since a time when the group attained a parasitic mode of life, which accords with the course of development everywhere bound up with parasitic adaptations. Someone has said that nature produces individuals while man has created species. This seems hardly a proper observation for higher plants and animals but it applies well to the lower parasites. The fact that we hear of “physiological’’ species, ‘“biologic’’ races, and ‘““formae specialis,’’ in parasites is but an admission that man is the inventor of ‘‘species’’ of some groups and in order not to be misinter- THE GENETIC RELATIONSHIP OF PARASITES 129 preted finds it necessary to qualify the term. Cook and Swingle have pointed out that ‘normal and long-sustained evolutionary progress is not accomplished on single or narrow lines of descent, but is possible only for large companies of interbreeding individuals; that is to say, for species.’’ Certainly in all organisms where we have interbreeding and interweaving of distinct lines of descent there is such a thing asa real specific group but in the parasitic fungi all traces of the coherence of such a group has been lost. With the exception of the mucors, where the interweaving of strains is definitely known, and the possible occurrence of sexual strains in some downy mildews and anthracnoses, there is no suggestion of groups of interbreeding individuals. The individuals continue their existence without relation to other individ- uals. It is clear that man makes the species here solely for his con- venience and he must decide whether he will make morphology or physiology his standard. If it is true that progress cannot be main- tained along narrow lines of descent this will assist in understanding why advances are not to be found in the parasitic fungi for here the lines are the narrowest possible, simply the continuations of individual existences. If we are given an individual spore we can start a life-cycle which will bring us back to more spores of the same sort and we may raise generation after generation but they are descendants of a pure line in no way influenced by individuals of different origins. The factors which may influence them are their hosts and the environment. Through morphology we form our judgment concerning the possible similar origin of individuals which come to us to classify. In the parasitic fungi as a rule it is only the reproductive features that receive morphological consideration. If we considered only dead specimens there would be no difficulty, but with the advent of experimental culture work, perplexing questions have arisen. The work of Eriksson, Klebahn, and others served to emphasize this matter but it is clear from DeBary’s writings that he was aware of the specialization of a parasite to a host although he did not use the form of expression now prevalent. Morphological equivalents growing upon the closely allied — hosts may not have the same behavior. The powdery mildews, an- thracnoses and rusts furnish many familiar instances. This means that the plant body is coming tn for attention and that the consideration must be of a physiological nature. The question whether forms struc- turally alike but reacting physiologically differently should be regarded as belonging to the same species or a race, etc., may be an interesting 130 FRANK DUNN KERN one but is not the most important one. The bacteria are notable organisms which raise such questions. But the genetic relationship is of greater significance. It is a common view that the forms mor- phologically alike must have had a common origin and that physio- logically specialized forms have resulted by progressive variability on a special host. Nutritional adjustment and other interacting factors have been suggested as associated with the variations but no demon- stration up to the present has made the matter atallclear. It has been assumed that continual specialization will lead eventually to structural differentiations. There seems to be nothing in the methods of descent which would prevent the carrying forward of any variation once es- tablished. It is rather a question of how the physiological relations between host and parasite or the environment might operate to bring about a diversity of form and structure. Very little is known of the possible effect of the ordinary factors of environment upon the development of a parasite. There is some evidence regarding effects upon distribution. After a detailed com- parison of the geographical distribution of hosts and parasites in one group of fungi, the writer came to the conclusion that the environment had little if any influence at all, but such apparently is not true for all kinds of parasites. Ward has shown that external influences may undoubtedly exert important effects. Too high a temperature during incubation, starving the host of carbon dioxide or salts, or heating or cooling the roots may ruin a fungus and stop infection. Salmon has also carried out investigations to show that external as well as internal features are concerned in determining whether parasites may attack and continue development. As a conclusion we may consider, with brevity, parasites as an aid in determining genetic relationships in their hosts. Cobb has written on this subject in a very enthusiastic way, pointing out that parasites may be used (1) as an aid in discovering specific and generic relation- ships, (2) in following metamorphoses, (3) in physiology, (4) in chemistry. JI am particularly interested in the first of these. I trust I may be pardoned for drawing my examples from a group in which I have long had a special interest. A recently published result brings this phase of the subject to mind. The members of the genus Gym- nosporangium were long supposed to alternate solely between the juniper and apple families. Upon the morphology of a spore-form found upon a member of the rose family I suspected its connection to a THE GENETIC RELATIONSHIP OF PARASITES I3I cedar rust and assisted in proving the relationship. Later a similar case was discovered in the hydrangea family and it, too, was established. The Malaceae, Rosaceae, and Hydrangeaceae have always been regarded as close relatives, being included in the order Rosales. Re- cently Fromme has reported a fourth ramily, Myricaceae, as furnishing a host for a Gymnosporangium. Since the other three families are evidently related, there is a strong suggestion that this one should stand nearby in the scale of classification. In Engler and Prantl’s system it is, however, in the fourth order of the Dicotyledoneae while the Rosales is the twenty-first order. The Myricales order has a similar low position in the latest manuals of Gray and Britton. It is interesting to note that Bessey, on the other hand, places the family Myricaceae in the order Sapindales which he derives from the Rosales through the Celastrales. The latter arrangement is much more strongly supported by the parasites than the former. In this same group of fungi there is some evidence regarding the relationship of the other series of hosts. It is generally accepted that the § Oxycedrus, of the genus Juniperus, with its subulate leaves, is of earlier origin and probably ancestral to the § Sabina, which has its leaves subulate when young but scale-like when mature. The species of the § Oxycedrus are distributed in both hemispheres and appear to be identical. Species of the § Sabina are widely distributed but certain species are indigenous to one hemisphere and certain ones to the other, there is not one that is common to both. The parasites of the genus Gym- nosporangium parallel the condition of the hosts exactly. We do not find the same species indigenous to both hemispheres on the § Sabina but we do on the commonly distributed section. The distribution of the hosts would suggest separate origins of the derived section in North America and the Old World and this theory is strongly supported by the condition of the parasites. These examples will suffice to illustrate the matter. Our knowledge is too incomplete to determine the real value of this phase of the subject and yet we may conclude that a knowledge of the parasites is worthy of consideration in a study of the hosts. PENNSYLVANIA STATE COLLEGE, STATE (COLLEGE? PA, THE EXPERIMENTAL STUDY OF GENETIC REUATION. SEES: H. H. BARTLETT The earliest experiment designed to determine the genetic relation- ships of a plant was carried out by Linnaeus and recorded in his famous disquisition on the sex of plants. He tells us that late in the autumn of 1757 he stumbled upon some plants of Tvagopogon hybridus in the botanic garden at Upsala, growing in a bed where only Tragopogon pratensis and Tragopogon porrifolius had been planted. Before this time, as we know from Hartmann’s dissertation on hybrid plants, Linnaeus had thought it possible that Tvagopogon hybridus was a cross between Tragopogon porrifolius and Lapsana stellata—species of two quite unrelated genera. Now, however, he had a clue to its true relationship, which he set about to prove. The next year he removed the pollen from some heads of the yellow-flowered Tragopogon pratensis and sprinkled the styles with pollen from the purple-flowered T. por- rifolius. The hybrid seeds thus obtained were planted in the fall and gave plants which were found to be identical with Tragopogon hybridus. The flowers were not completely purple, as in the staminate parent, but showed the influence of the pistillate parent in their yellow bases. After this experiment, Linnaeus tells us, it was impossible to doubt that new species might come into being by hybridization. His con- ception had changed greatly since the publication in Fundamenta Botanica (1735) of the oft-quoted dictum, ‘Species tot numeramus, quot diversae formae in principio sunt creatae.’’ (Every species which we can enumerate was created in the beginning a distinct form.) The more mature views of Linnaeus, however, were in advance of his time. Philosophia Botanica (1737), in which the doctrine of special creation was set forth, continued, long after his death, to be the vade mecum of botanists. The conception of the hybrid origin of species was so completely disregarded that Sir James Edward Smith, an ardent Linnaean, the possessor of the Linnaean herbarium and 1Tnvitation paper read at the symposium on ‘‘The Genetic Relationship of Organisms”’ before the Botanical Society of America at Philadelphia, December 30, Igi4. 132 THE EXPERIMENTAL STUDY OF GENETIC RELATIONSHIPS 133 founder of the Linnaean Society, wrote in 1807, “‘By species are understood so many individuals, or, among the generality of animals, sO many pairs, as are presumed to have been formed at the creation, and have been perpetuated ever since; for though some animals appear to have been exterminated, we have no reason to suspect any new species has been produced; neither have we any cause to suppose any species of plant has been lost, or any new one permanently established, since their first formation, notwithstanding the speculations of some philosophers.” Although Linnaeus had reached the remarkable conclusion that species were, in some way, genetically interrelated, the second edition of the ‘Species Plantarum’’ and the inaugural dissertation of his pupil, Hartmann, contain so many preposterous guesses as to the parentage of certain supposed hybrid species, that he could certainly have had no conception of the limits of hybridization, or of the segre- gation which so often follows it. Obviously, he supposed that species originated as stable first generation hybrids—not a surprising theory, considering that, in his experiments with Tvagopogon, he had chanced upon the one family in which parthenogenesis is so common that hybrids are often constant from generation to generation. Linnaeus, knowing nothing about the geological history of organ- isms, had to construct a scheme of evolution which involved only the Mosaic ‘‘days of creation’’ and the few thousand years of recorded history. Having assumed the hybrid origin of species, it was clearly necessary for him to hypothecate the creation in the beginning of a considerable number of distinct forms representing the natural orders, for otherwise there could have been no material for hybridization to work upon. Moreover, since these originally created forms were so different that one could hardly believe in their crossing, he invoked miraculous intervention to account forit. Finally, in order to preserve intact the limits of the orders, he seems to have implied that the more fundamental characters were transmitted by the mother; the super- ficial ones by the father. His philosophical difficulties and the way he got around them now seem very amusing; but, as we shall see later, the same difficulties still confront certain modern geneticists, who have either not been able to circumvent them at all or are beginning to whisper a hypothesis more fantastic than that of Linnaeus. Although Sir James Edward Smith’s views were those which pre- vailed, for the most part, until the time of Darwin, there were occasional 134 H. H. BARTLETT outcroppings of evolutionary ideas. On the botanical side, the views of Dean Herbert and of Naudin were based largely upon experimenta- tion rather than speculation and were expressed with especial clearness. The former (1822 and 1837) said that “horticultural experiments have _ established beyond the possibility of refutation, that botanical species are only a higher and more permanent class of varieties.’’ The latter (1852) said that the methods by which the gardener produces new varieties coincide with the processes by which new species originate in nature. It remained for Darwin, however, to refute finally the doctrine of special creation. | Darwin ascribed the origin of new species in nature to the natural selection of favorable variations. With this view, in its most general aspect, nearly everyone is in accord. But the great problem of evolution still remains,—what is the source of the variations which are selected? Darwin could not answer this problem satisfactorily because no one had yet discovered the distinction between fluctuating varia- tions, which are not inherited, and mutations, or germinal variations, which are inherited. He also went astray, as we now think, in be- lieving that the effect of use or disuse of an organ could in some way impress itself upon the germ-plasm and become hereditary. The few who still hold that the selection of continuous variations would suffice to bring about specific differentiation can bring forward little or no evidence to support their view. The evidence all points to the utmost fixity of organisms, aside from mutations. In order not to perpetuate a misrepresentation of Darwin’s views which he himself particularly resented, it should be said that, after the publication of the Origin of Species, Darwin came to believe that he had formerly underrated the value of mutations (‘‘spontaneous variations’’) in bringing about diversity. Even in the first edition of his great work he stated his belief that the selection of insensible derivations had not been the exclusive means of modification. Darwin’s caution, however, was not shared by all of his followers. Exaggeration and misrepresentation of his views led to an almost universal conviction that modification was too slow a process to be made the subject of experimental inquiry. Thus it came about that the Origin of Species was followed by a period of stagnation, as far as experimentation was concerned. During this period there were indeed a few experimenters, with the courage of their convictions, who carried on genetical studies. One of them was Mendel, whose investigations aroused no interest among THE EXPERIMENTAL STUDY OF GENETIC RELATIONSHIPS 135 his contemporaries, although they were stated with unsurpassed clear- » ness, and would have changed the current of biological thought if anyone had realized their bearing on the theory of evolution. Another was Focke, who laid emphasis upon the importance of hybridization in species formation. Even then, as now, he believed that, in such large and polymorphic genera as Rubus and Rosa, many of the species have originated by hybridization, followed by the sorting out of stable forms. From Darwin’s time until very recently, however, it has been the prevailing view that the selection of extremes from a continuously varying population would result in a continuous, gradual modification of the entire population, and that such selection had brought about the formation of varieties in cultivation and of species in nature by the simultaneous transformation of masses of individuals. It need only be said that carefully planned and executed experiments lend no support to this view. All the evidence, on the contrary, seems to show that no amount of selection will suffice to modify the range of fluctuating variation of an organism. On the botanical side there is little evidence of the efficacy of selection as a factor in evolution; on the zodlogical side there are certain selection experiments of Castle’s, carried out with characteristic care and accuracy, but surely capable of a different explanation from that which he gives. Even if correctly interpreted they have at best a dubious bearing on the problem of species formation. His experiments deal with the inheritance of a certain color pattern in rats, which, in a presumably homozygous race, may be modified in either direction by selection. The changes from gen- eration to generation are very slight, however, and we cannot conceive of any agency in nature which would bring about assortive mating among such slightly dissimilar individuals. While it is becoming increasingly clear that the old selection theory is untenable, we are becoming more and more convinced that evolution does take place with measurable rapidity, and that the factors concerned with it are mutation and hybridization. | The new point of view we owe primarily to de Vries, who has deter- mined the distinction between non-heritable fluctuating variations and inheritable germinal variations, or mutations, and has developed the mutation theory. It should appeal especially strongly to systematists, most of whom have really never been convinced of the adequacy of the discarded selection hypothesis. Why, if species had come about by 136 H. H. BARTLETT such a gradual process, were not intergradations morecommon? Why did most of the supposed intergradations prove to be rare and partially sterile hybrids? Why, if evolution were still going on, could one recognize speciation at allin some of the groups? To the theoretical evolutionist, who knew as little as possible about species, such questions indicated merely the perversity of the systematist. The latter, accepting the general truth of evolution, but influenced hardly at all in his attitude by the manner in which it was supposed to have taken _ place, continued to describe species and then still more species, just as he had always done, and just as he will doubtless continue to do. Some- times he told what he thought about their relationships, oftener he did not, but he seldom failed to add to his description some variation of the formula “entirely distinct from its nearest ally.’’ He has been anathematized; some of his colleagues have even threatened to cast him into outer darkness. Nevertheless, his work has certainly been as truthful and as serviceable as the work of those who deplore his “raking together of straws and sticks and even antique dust.” It will require the combined efforts of morphologist, systematist, and geneticist to arrive at the whole truth in regard to genetic relation- ships. In one way the geneticist has a great advantage over the other workers, for his methods are inductive, whereas theirs are deductive. In another way he is at a great disadvantage, for he can deal only with the lesser categories, the variety, the species and perhaps the genus. The relationship of the larger groups must be determined, if at all, by the deductive methods of the morphologist, and, I may add, of the biochemist; for, as the years go on, biochemistry will come to be applied more and more to the elucidation of genetical problems. The immediate aims of the geneticist are (1) to observe the origin of new and distinct forms, the genetic relationship of which must, therefore, of necessity be known, (2) to determine the conditions which brought these forms about, and (3) to study their hereditary behavior and their morphological and chemical characteristics in order to provide a basis for sound deduction in regard to multitudes of types which we can never hope to know except as facts of nature. All three aims have already been realized in some measure as a result of the recent activity | in genetics. | Most of the new forms of which the origin has been. observed, belong to one class of organisms—namely, recessive Mendelian varieties. Such varieties have been observed to originate in two ways, THE EXPERIMENTAL STUDY OF GENETIC RELATIONSHIPS 137 by mutation, and by segregation from hybrids. Certain ultra- Mendelians maintain that only the latter method is operative, but their argument, which will be discussed after certain examples have been called to mind, can be supported by little or no evidence. As examples of recessive mutations, one naturally thinks first of the remarkable series of 150 which Morgan has observed to originate from the fruit fly, Drosophila ampelophila. All of them are recessive to the typical form. On the botanical side there is no correspondingly complex series of recessives known, of which all the members have originated under scientifically controlled conditions, but there is hardly the least doubt that the cases of the sweet pea (Lathyrus odoratus), of Primula obconica and of Primula sinensis are quite comparable with that of Drosophila. Some members of each of these large series of forms are known to have come about as mutations, and probably all did. Bateson, who has devoted especial attention to the sweet pea, is sure that all the varieties are recessive to the wild prototype, Lathyrus odoratus. It seems sufficiently well proved that recent hybridization has not modified Lathyrus odoratus, for no other plant has been found which will cross with it. In nature there are many examples of species and varieties which bear simple Mendelian relationship to one another. Such a case has been discovered by Trow in the series of elementary species compre- hended under the name Senecio vulgaris. It also appears that in Antirrhinum there are species which seem to be very unlike, neverthe- less all of the differences between them can be determined by Mendelian analysis. A large number of stable forms segregate from a hybrid between two such species, and these forms are themselves indistin- guishable from species. Their fertility is unimpaired. None of them contains a single character which is not identical with, or recessive to, a corresponding character in one or the other of the parents. Baur and Lotsy have studied the hybrids of Antirrhinum molle and A. majus from a Mendelian standpoint. The latter has been so impressed by the results that he has come to believe that there is no source of vari- ation in nature except hybridization followed by segregation; it seems to him the sole method of species formation. He explains the so-called recessive mutations, no matter how rare they may be, by the assump- tion of previous hybridization and a sufficiently large number of multiple factors. It seems fair to ask the holder of this view how the forms originate which supply the characters to be assembled and 138 H. H. BARTLETT reassorted by hybridization. This question brings him face to face with the philosophical difficulties of Linnaeus. Either’ he must deny evolution, or, with Bateson, confess that the studies carried on by Mendelians have thrown no light on the problems of evolution... Bate- son suggests that we should seriously consider the possibility that evolution has not taken place from the simple to the complex, but rather from the complex to the simple; that the original forms of life were heterozygous with regard to all the characters which have ever appeared in geological history; that for each character there was a corresponding inhibitor, and that the characters have come successively to light by the segregation of recessives from which the inhibitors have fallen away. We cannot believe, from the tentative way in which Bateson proposes this fantastic hypothesis, that he really places much faith in it. But the mere fact that he should whisper it shows the extreme pessimism of the ultra-Mendelian attitude in regard. to the problem of evolution. Other experimental workers, however, are more optimistic. The Mendelians have been so firmly convinced that differences between species were all capable of Mendel- ian analysis that they have disregarded facts which did not fit their formulations. Professor de Vries’ work is set aside with the statement that the chief reason why factorial analysis has been declared to be inapplicable to the Oenothera mutations is because no one (except Heribert-Nilsson) has set about such an analysis in the right way. Even those of us who doubt the universality of the Mendelian phe- nomena, see no reason to deny that species formation by hybridization and subsequent segregation has taken place on a large scale in many groups. One may, however, admit the great prevalence of hybridiza- tion, without believing that all variation which may take place sub- sequently to hybridization is a result of that hybridization. In other words, there is no reason why true mutations should not occur in hybrids as well as in pure lines. In this connection we may examine a little more closely the view that recessives always originate by segregation rather than by muta- tion, a hypothesis ancillary to the multiple factor hypothesis. In only one case among wild plants has it been satisfactorily shown that a recessive mutation differs from the parent form in the lack of duplicate factors. This is the case of Capsella Heegert and Capsella Bursa- pastoris, which has recently been studied by Shull. He finds that there is a difference of two duplicate factors between the derivative THE EXPERIMENTAL STUDY OF GENETIC RELATIONSHIPS 139 and the parent form. ‘The presence of either factor suffices to give the capsule the triangular shape typical of Capsella. C. Heegeri, the recessive, has a capsule shaped like that of Camelina, so that it really departs from the parent form in a generic character. As Solms- Laubach has shown, no systematist who did not know of its mutative origin or of its relationship as determined by crossing would place it in the genus Capsella. All the plants in existence have descended from one individual which was found by Professor Heeger in a colony of Capsella Bursa-pastoris in the market-place at Landau, Germany. When the two species are crossed the first hybrid generation is, of course, uniform and like Capsella Bursa-pastoris. In the second generation there is one plant of C. Heeger1 in 16. The 15 sister plants all look alike, but are in reality of three kinds. In the third generation seven will remain constant, four will give one plant of C. Heegeri in 4, and four will give one plant of C. Heegertin 16. As far as experimental evidence can be applied it shows that a plant into which the character of C. Heegert has been introduced by hybridization must either give rise to C. Heeger not less than once in 16 times, or not at all. Most recessives, if crossed with the parent form, reappear in the second hybrid generation in a typical 1:3 ratio. If they originated ac- cording to the multiple factor hypothesis we should have to assume that factors which had been independent for countless generations could suddenly become indissolubly associated in the generation in which the pure recessive appeared. There is not the least evidence that this takes place. Now that we have cases in which segregation in the second hybrid generation occurs in the ratios 15:1 and 63:1, we naturally expect that some of the recessives which are supposed to depend upon the concurrent absence of so many characters would not reappear at all when recrossed with their parents. With as few as six independent identical factors there would be only one recessive in a second hybrid generation consisting of 4,096 individuals. In a case like that of Capsella Heegert it would be necessary to postulate more characters than six to account for a non-mutative appearance so seldom that only one individual has ever been observed. On the whole there seems no reason to doubt that the sporadic appearance of recessives in supposedly pure lines is really due to mutation. The imaginary in- fluence of past hybridization is a bogey that need not bother us. Although recessive mutations and recombinations following hybridization may contribute much to specific and varietal diversity, 140 H. H. BARTLETT it is obvious that they cannot bring about the origin of any fundament- ally new type. We cannot, therefore, have any comprehensive knowledge of the genetic relationships of organisms until we know something about progressive mutation. Mendelian researches have shown us how to verify supposed relationships in the simplest cases, those in which characters have been lost. But can we always be sure that the recessive is the derived form? Is it not sometimes true that the dominant is the derived form, and that a new character has ap- peared? On this point the evidence is very unsatisfactory. Cases of progressive evolution have indeed been observed, but for the most part they have occurred out of the beaten track of the Mendelian and have been largely discounted, for Mendelism is the present fashion in genetics. In Primula sinensis, however, Professor Keeble has been fortunate enough to observe the origin of a Mendelian dominant. There are two types of gigantism in Primula. In one type the chromosome number is the same as in the typical forms, and the giant acts as a simple Mendelian dominant when crossed with the parent. The other type is of a very different nature, and will be referred to later. A number of giants of the simple Mendelian type have been known among the cultivated varieties of Primula, but no one knew how they had originated until the ‘Giant White Queen Star’’ appeared as a mutation in the third guarded generation of a pure line of the ‘‘White Queen Star’’ variety. It came true from seed and has remained uniform through five successive generations, which included several hundred individuals. Although we cannot doubt in this case that a new dominant has arisen, there is unfortunately one flaw in the evidence. The new form has proved sterile when crossed with the parent form and with all other non-giant forms, so that its dominance is only inferred from its perfect analogy with other giants of the same type. The latter have been found by Gregory to show simple dominance over the non-giant forms. Our argument is based upon evidence fitted together from two sources. Other examples might be brought forward, but they are not as striking or as well authenticated as this one. There is a type of non-Mendelian pro- gressive evolution, however, which has been far more satisfactorily studied. I refer to mutations in which new characters may be de- finitely associated’ with mutative changes in the chromosome number. It appears that, in general, each species is characterized by a constant number of chromosomes. Within a genus, different species THE EXPERIMENTAL STUDY OF GENETIC RELATIONSHIPS I4I are frequently characterized by different chromosome numbers; and, exceptionally, within a species, well-characterized nominal varieties may differ in this respect. Lists of genera in which there is variation in chromosome number have been published by Gates and by Strass- burger. Among the genera included in these lists, or concerning which information has been more recently published, we find the flowering plants Alchemilla, Antennaria, Crepis, Dahlia, Daphne, Drosera, Funckia, Hieracitum, Humulus, Houttuynia, Musa, Oenothera, Pri- mula, Rosa, Rumex, Spiranthes, Taraxacum, Thalictrum, and Wik- stroemia, the ferns Athyrium, Lastrea and Nephrodium, the mosses Amblystegium, Bryum, Mnium and Phascum. The numbers range from 3 (x) and 6 (2x) in Canna to 48 (x) and 96 (2x) in Castalea. The processes of species formation must have been frequently attended by changes in the number of chromosomes. We cannot escape this con- clusion when we take into consideration the two striking facts that within a species the number is constant, but that from species to species and from genus to genus it shows the greatest diversity. Recent discoveries are making it very clear that mutative changes in the chromosome number occur frequently, and that such changes are always associated with a modificdtion in the morphological characters of the plant. In other words, certain mutations are probably de- pendent upon, or, at any rate, closely associated with, visible changes in the nuclear mechanism. We have every reason to believe, therefore, that the different chromosome numbers of different species were ac- quired simultaneously with the acquisition of other specific characters. Among the mutations of Oenothera Lamarckiana there is just one which de Vries regards as unquestionably progressive. It is the re- markable Oenothera gigas, which differs from its parent not only in numerous external characters but also in having twice as many chro- mosomes. The gametophytic and sporophytic numbers are 14 and 28 in Oe. gigas, 7 and 14 in Oe. Lamarckiana. Gates has shown that the double chromosome number of the former is correlated with a larger cell size in all corresponding tissues and that many of the gross _ characters of the plant are in turn dependent upon the difference in the cells. Hybrids between Oe. gigas and Oe. Lamarckiana show no semblance of Mendelian inheritance. In general they are very sterile, but in one case a fertile strain was obtained which remained constant through five generations. The hybrids are intermediate whichever way the cross is made, and, if back crossed with either parent, the 142 H. H. BARTLETT secondary hybrids are again intermediate. Clearly we are not con- cerned here with a new dominant, in the Mendelian sense, but rather with the simultaneous origin of a whole group of non-Mendelizing characters, each of which is correlated with the increase in the number of chromosomes. Among the flowering plants we do not know of any way in which the number of chromosomes may be experimentally modified. Among the mosses however, the brilliant work of the Belgian investigators Elie and Emile Marchal has shown that tetraploid races may be produced at will. Their results are of the greatest importance because of the light which they throw upon such spontaneous mutations as Oenothera gigas. It has long been known that moss protonemata might be regener- ated from bits of the seta or young capsule; in the past both Pringsheim and Correns have obtained such aposporous protonemata. ‘The investigations of the Marchals were undertaken to determine (1) whether such protonemata would or would not give rise to moss plants with antheridia and archegonia, (2) whether, in case such plants were obtained, the gametes would have the 2x chromosome number of the normal sporophyte and give rise to a new sporophyte with the tetraploid chromosome number, and (3) whether the tetraploid races would be like the typical form of the species, or different. It was found that diploid gametophytes were obtained in which the vegetative cells were larger than in normal gametophytes and the generative cells were twice as large. The diploid gametophytes of dioecious mosses were absolutely sterile, and synoecious. They could be main- tained in culture only by regeneration from pieces of the axis. In the case of the monoecious mosses the results were far more interesting, ‘for the diploid gametophytes gave rise to tetraploid sporophytes, which produced good spores, and in turn reproduced the diploid gametophyte. The fertile races thus experimentally obtained were named Amblystegium serpens bivalens and Amblystegium subtile b1- valuens. Cytological studies showed that the reproduction of these new races was by normal fusion of the diploid gametes. The cytolog- ical relations were worked out for Mnium, Bryum, Amblystegium and Phascum. In the case of Amblystegium it was found possible to double the chromosome number again by regeneration from the tetraploid sporophyte, but the tetraploid gametophytes thus obtained © were completely sterile. The new bivalent race obtained from THE EXPERIMENTAL STUDY OF GENETIC RELATIONSHIPS 143 Phascum was anomalous in that it was dwarf, rather than giant. It was sterile, but reproduced itself by propagula, which do not usually occur in the parent form, Phascum cuspidatum. Many mosses re- produce almost exclusively by vegetative means, and this experiment is in the highest degree suggestive of the manner in which this habit has come about. All of the other bivalent races differed from their parents in characters which obviously depended upon the greater size of the individual cells. The experiments of the Marchals give us the strongest reason to believe that the visible differences between Oenothera Lamarckiana and Oe. gigas have been correctly interpreted as due to the doubling in th> latter of the chromosome number. ‘This explanation, first proposed by Gates, has been assented to by de Vries. The latter points out, however, that the genetical and physiological qualities of Oe. gigas are entirely different from those of Oe. Lamarckiana. ‘The seeds of Oe. gigas, for example, are much more viable than those of the parent species, and germinate more quickly. Oe. gigas does not give the laeta and velutina twin hybrids when crossed with unrelated species, as Oe. Lamarckiana does. Obviously, then, the process of mutation has changed the hereditary qualities of the germ plasm even more than the morphological characters of the sporophyte. Oe. gigas gives rise to certain secondary derivatives which have no counterparts among the variations of Oe. Lamarckiana. Are we too optimistic if we view the former as a newly evolved center about which an entirely new series of specific variations may spring up? It is natural to ask at this point how commonly mutative changes in the chromosome number occur. It is known that in Oe. lata, one of the most characteristic of the mutations from Oe. Lamarckiana, the unreduced number is I5 instead of 14. In semigigas mutations from Oe. Lamarckiana and Oe. biennis the number is 21. I have recently investigated the mutability of several species of Oenothera which belong to the small-flowered, self-pollinating series of forms which are generally lumped together under the name “Oe. biennis.”” Two of them have given rise to mutations characterized by an increased chromosome number. The cytological investigation of these new mutations has been undertaken by Mr. E. G. Arzberger, through whose kindness I am able to announce that Oe. stenomeres mut. gigas has 28 chromosomes, and that Oe. pratincola mut. gigas also has this num- ber. The former seems in every way analogous to Oe. gigas de Vries, 144 H. H. BARTLETT and will make it possible to determine the hereditary qualities of the latter much more satisfactorily than has thus far been possible. The method, of course, will be to compare the hybrids between the parent forms with those between the gzgas-mutations. The crosses in both cases will be between gametes with the same number of chromosomes, so we need not expect the high degree of sterility which prevents the study of hybrids between the forms characterized respectively by 14 and 28 chromosomes. It would give increased significance to the discovery of the new gigas-mutations if tetraploid species of Oenothera should be found in nature. I mention this fact in the hope that every- one who is interested in the problem of mutation may be on the look- out for them. I already have one wild species in my garden which appears to possess certain traits of the gigas-forms but Mr. Arzberger was unable to get his preparations ready in time to make a chromo- some count before this meeting took place. It has already been mentioned that in the genus Primula two types of gigantism occur. One type is characterized by a doubling of the chromosome number and seems to represent the same type of mutation as that which in Oenothera gives rise to the gigas-form. Gregory has found that not only are the chromosomes doubled in the tetraploid Primulas, but also the Mendelian factors for numerous characters. The hereditary behavior of these mutations is, therefore, entirely different from that of the diploid races. The differences in chromosome number which occur among species of the same genus represent changes which must necessarily have taken place abruptly. We can not imagine the origin of a gigas-race by gradual selection or by Mendelian segregation. Heribert-Nilsson has indeed offered a Mendelian explanation of Oe. gigas, but he has wholly neglected the cytological facts in the case. In Oe. stenomeres mut. gigas the chromosome count was made in the generation following the first appearance of the mutation. In the corresponding mutation from Oe. pratincola the count was made in the original mutation. There is the best of evidence, then, that the new chromosome number is acquired simultaneously with the new morphological characters. It is more reasonable to believe in a causal relationship between the cytologi- cal and morphological changes than to believe that the latter result from the hypothetical influence of hypothetical crossing in the indefinite past. It cannot be assumed that the modification of the chromosome number is itself due to Mendelian segregation, for there are too many facts THE EXPERIMENTAL STUDY OF GENETIC RELATIONSHIPS T45 which go to show that the chromosomes are themselves the mechanism by which Mendelian characters are transmitted. It is only necessary to call to mind Gregory’s discovery that the Mendelian factors are doubled in tetraploid plants of Primula. If.the mechanism is altered, the genetic qualities of the plant: must of necessity be altered. For this reason I am disposed to lay especial emphasis upon those mutations in which a cytological change in the cell can be demonstrated, not, however, without stating my belief that mutations for which there is no visible basis are quite as independent of ancestral hybridization as the gigas-mutations. In certain groups, as the Coniferae for example, there is great uniformity in the chromosome number, showing that evolution has taken place through invisible modifications of the germ plasm. Probably most of the mutations which take place in any group of plants, even Cknothera, have the same chromosome number as the parent. My Oenothera cultures of the last three years have given many mutations, aside from the gigas forms, which confirm in all essential points de Vries’s experience with Oe. Larmarckiana. Some of them I regard as progressive, although it has not been possible yet to demon- strate that they are dominant in a Mendelian sense. In fact, Mendel- ian inheritance seems to play so small a part in Oenothera that in general we cannot expect to apply the test of dominance in judging of the progressiveness of mutations. We must consider a mutation as progressive when it shows characters which are not present in the parent. It has been shown by de Vries that reciprocal hybrids between Oenothera species are frequently very unlike one another. Both de Vries and Davis have encountered cases in which the hybrids are strongly patroclinic. I have just the opposite experience with some of my interspecific hybrids, which are strikingly matroclinic. It is clear that in this genus reciprocal hybrids may be either matroclinic or patroclinic. In either case it is impossible to say that the characters of one parent or the other are dominant in the ordinary sense. With this explanation I shall proceed to a very brief discussion of two of my new mutations. In Oenothera pratincola the seedling leaves are ovate. Seven different strains of this species have given rise to a mutation with round seedling leaves, which I have called mut. nummularia. In three strains the mutation has appeared in three successive genera- 146 H. H. BARTLETT tions, with a frequency of approximately one mutation for every 400 seeds sown. Correlated with the shape of the leaves are other char- acters, involving the size and branching of the plant and the pubescence and dehiscence of the calyx. The entire group of characters are co- herent; it may be predicted from the shape of the seedling leaves alone that the other characters will appear in the mature plant. Never- theless it is quite impossible to imagine any necessary inter-dependence between the characters which cohere in this mutation. Nosystematist who did not know the parentage of mut. nuwmmularia could possibly decide which of two dozen elementary species in my garden had given rise to it. Reciprocal hybrids between the parent species and the mutation appear to be strictly matroclinic, but the plants are still very young seedlings. The progeny of the cross mut. nummularia X f. typica are all mut. nummularia, conversely, the progeny of the reciprocal cross are all f. typica, except for the fact that mut. nummularia appears with its usual frequency of one plant in several hundred. It seems that only female gametes bear the group of characters which distinguish mut. nummularia from f. typica. The other mutation which I wish to mention is Oe. stenomeres mut. lastopetala. It differs from its parent species in a group of coherent characters, one of which is the hairiness of the petals. The solitary primary mutation when self-pollinated gave rise to a progeny consisting of typical Oe. stenomeres and mut. lasiopetala in a ratio suggesting I : 1, although the former was in excess. It is highly improbable that a Mendelian explanation will apply to this case, either, but I do wish to’point out that such an explanation would necessitate viewing mut. lasiopetala as a dominant. A recessive could not have thrown the dominant parent. As my experience with this highly interesting group of plants in- creases I am more and more convinced that de Vries’ conception of the origin of species is the true one. He believes that new species, differing from the old ones in a coherent group of characters, may come into existance at one step, by mutation. The evidence for this special view of mutation has been doubted by several critics, who have brought forward several destructive arguments. I believe that all of these arguments can be met. There is first the argument that Oenothera Lamarckiana is known only in horticulture, and may be a garden product; consequently that THE EXPERIMENTAL STUDY OF GENETIC RELATIONSHIPS 147 its behavior cannot be held to throw light upon the behavior of wild plants. It has now been shown that wild species show absolutely parallel phenomena. It has also been said that the non-Mendelian behavior of Oenothera hybrids shows that the genus is so exceptional in its genetic behavior that it provides no basis for generalization. In reply we may say that the Mendelian school have in general confined their crosses to varieties of cultivated plants. If they had ventured into the field of inter- specific hybridization they would have found plenty of parallels to the behavior of Oenothera. As a case in point we may cite the experience of W. Neilson Jones, who obtained matroclinic reciprocal hybrids between species of Digitalis. He did not think the results quite com- parable to those of Oenothera for the reason that the Oenothera hybrids reported up to that time had been patroclinic. Now, however, this argument is removed by the discovery of matroclinic hybrids in Oeno- thera. The enormous literature of orchid hybridization contains frequent allusions to unlike reciprocal hybrids. In this largest family of the monocotyledons may be found numerous examples of both patroclinic and matroclinic hybrids. Many of them may prove to be cases of parthenogenesis, but the situation demands a much more thorough study than has yet been given it. The so-called “false hybrids’’ of Fragaria, as well as other Rosaceae, should be carefully investigated both cytologically and genetically. This much is sure; it is not yet time to speak of the universality of Mendelian phenomena, or of the exceptional nature of Oenothera. I am inclined to believe that such groups as the Orchidaceae may even provide parallels for the mutability of Oenothera. For example, Miss Pace has recently studied the cytology of Spiranthes cernua and S. gracilis from material collected near Chicago. Nine other species are interpolated between these two by Oakes Ames, in his Monograph of the American Species of Spiranthes. Yet they differ from one another in somewhat the same way that Oenothera Lamarckiana differs from Oe. gigas. Miss Pace finds 15 and 30 chromosomes in Spiranthes gracilis, as the reduced and unreduced numbers, but 30 and 60 in S. cernua. The latter may well be, as Miss Pace intimates, a tetraploid form of the former. Here isa fertile field for the experimentalist. Jeffrey has lately argued that all plants which have any defective pollen grains are in a state of genetical impurity, and that any con- clusions drawn from their genetical behavior, in connection with the 148 H. H. eBARTEBET vexed problem of the origin of species, must be subject to a large degree of reserve. Since all of the mutant Oenotheras are character- ized by more or less defective pollen, he thinks that the mutations are segregates from hybrids, and that ‘‘the mutation theory of de Vries appears accordingly to lag useless on the biological stage, and may apparently be now relegated to the limbo of discarded hypotheses.’’ Fortunately for the mutation theory, Professor Jeffrey’s argument is not sound. In the first place it must be insisted that there can. be no such thing as a morphological test of genetic impurity. We can only recognize genetical impurity by genetical tests. There is a certain sterile variety of the sweet pea, which, according to Jeffrey’s pollen test, would be adjudged a hybrid. When crossed with forms with normal pollen it acts as a simple recessive, and like the other recessive varieties of this plant it has doubtless arisen by mutation. Bateson, who has critically studied this series of varieties, writes of, : . the sweet pea, a form which is beyond suspicion of having been crossed with anything else, and has certainly produced all the multitude of types which we now possess by variations from one wild species.”’ Again, he states that “in spite of repeated trials, no one has yet suc- ceeded in crossing the sweet pea with any other leguminous species.” In the sweet pea, then, we find pollen conditions identical with those which Jeffrey believes are found only in hybrids; nevertheless there is no reason to believe that hybridization has ever occurred in the species. We may turn to another case. Humulus Lupulus, the hop, is normally dioecious, but monoecious individuals occur now and then which can hardly be considered as other than mutations. Winge has recently studied the pollen of one of these monoecious plants, found wild and transplanted from the woods into his garden at Carlsberg, Denmark. It bore staminate inflorescences at the base of otherwise hop-bearing branches. Cytological study showed that pollen mother cells were formed which divided normally but thereafter shrivelled up without making the tetrad division. Winge himself points out the similarity between this case and that of Oenothera lata. Of all the mutations from Oenothera Lamarckiana, Oe. lata is the most sterile. It is gener- ally completely so, but two or three strains are now known which yield a small amount of good pollen. Monoecious hop plants likewise vary greatly in pollen fertility, and Winge has lately made pollinations with apparently quite normal pollen from a monoecious plant. Winge worked with the wild hop of northern Europe. Aside from geographic THE EXPERIMENTAL STUDY OF GENETIC RELATIONSHIPS I49 races of Humulus Lupulus, and the variations of it which have arisen in cultivation, there is no other form in the genus except H. japonicus. The former has 20 chromosomes in somatic cells, the latter 16. The cross H. japonicusXH. Lupulus cannot be made; the reciprocal cross yielded variously malformed embryos, none of which were capable of further growth. So in this case again there can be no sus- picion that the pollen sterility has come about through hybridization. All plants which show transition from hermaphroditism to dioecism or monoecism present conditions parallel to those in Humulus. The case of Plantago lanceolata happens to be especially familiar tome. In most strains of this species the flowers are all perfect and the pollen is good. There are other strains, however, which are gynodioecious. Half of the plants are normal hermaphrodites and the other half functionally pistillate. I say functionally pistillate because in many of these strains the anthers develop, and contain pollen, but the grains are much smaller than normal pollen and are not liberated by the dehiscence of the anthers. Probably they are non-functional. From this condition there are various gradations through strains in which the anthers of the functionally pistillate form contain no pollen to strains in which the stamens are replaced by staminodia. In the latter form the gynodioecious state is not only functionally but also structurally attained. Plantago lanceolata is an introduced weed in the United States. In the Old World the well-marked subgenus to which it belongs contains six other species, but all are of comparatively restricted distribution. In northern Europe, as in the United States, where the sex forms have been studied, there is no allied species with which it could hybridize. We have, therefore, no reason to suspect that anther sterility in Plantago lanceolata has any relationship to hybridization. On the contrary, we may assume that the dioecious states of the species have been attained by a series of mutations, and that pollen conditions simulating those in hybrids may come about by mutation as well as by hybridization. At the risk of being tiresome there is one more type of anther sterility which I wish to touch upon. Bateson says: ‘‘ Without much more critical data I suppose no one would nowadays be inclined to foliow Darwin in instituting a comparison between the sterility of hybrids and that of illegitimately raised plants of heterostyle species. It is even difficult to imagine any essential resemblance between these two phenomena, nor has evidence ever been produced to show that 150 H. H. BARTLETT illegitimately raised plants have bad pollen grains, which is the usual symptom of sterility in hybrid plants. ...’ This statement does not do justice to Darwin’s evidence, which is quite convincing, es- pecially in the case Lythrum salicaria. This species is trimorphic, 1. e., its flowers are long-styled, mid-styled or short-styled. Each flower type has two sets of stamens, coinciding in length with the styles of the two other types of flowers. Thus there are three kinds of stamens in the species and each bears pollen which is morphologically and physiologically different from that of the others. A pollination is legitimate when it takes place between a style and stamens of the same length. There are 18 possible combinations between the 3 forms, of which 6 are legitimate and 12 illegitimate. Darwin made all the different pollinations and found that only the legitimate ones were fully fertile. In regard to pollen sterility we will quote his own words. An illegitimate progeny from the long-styled form, pol- linated from the longer or shorter stamens of the same form, consisted of 56 plants, belonging to three lots. ‘‘In several plants of all three lots, many of the anthers were either shrivelled or contained brown and tough, or pulpy matter, without any good pollen grains, and they never shed their contents; they were in the state designated by Gaertner as contabescent. .. . In one flower all the anthers were contabescent excepting two, which appeared to the naked eye sound; but under the microscope about two thirds of the pollen grains were seen to be small and shrivelled. In another plant, in which all the anthers appeared sound, many of the pollen-grains were shrivelled and of unequal sizes.”’ An illegitimate progeny of nine plants resulted from the pollination of the short-styled form with pollen from the shorter stamens of the same form. ‘‘The anthers in many of the flowers on several plants were contabescent.” Of 25 illegitimate plants from the mid-styled form, pollinated from the shorter stamens of the long-styled form, the pollen of 4 plants was examined ‘during the highly favorable season of 1866 . ; in one mid-styled plant, some of the anthers of the longer sta- mens were contabescent, but in the other anthers the pollen grains were mostly sound, as they were in all the anthers of the shorter sta- mens; in two other mid-styled and in one long-styled plant many of the pollen grains were small and shrivelled; and in the latter plant as many as a fifth or sixth appeared to be in this state.” Darwin also expressly states that contabescent anthers occurred as a resu** of il- legitimate pollination in Primula sinensis and Primula veris. THE EXPERIMENTAL STUDY OF GENETIC RELATIONSHIPS ESA We can hardly fail to agree that Darwin was right when he said that defective pollen could arise within a single well-circumscribed species as a result of illegitimate pollination. Wenow know, thanks to the investigations of Bateson and Gregory, that heterostylism in Primula is inherited as a simple Mendelian character. Legitimate pollination maintains heterozygosis: illegitimate pollination, on the contrary, leads to the extraction of dominants which, in a sense, are genetically ‘“‘purer’’ than the heterozygotes. Nevertheless, they may show, apparently as a result of their increased “‘purity,’’ the very character of the pollen which Bateson and Jeffrey consider a sign of hybridity. A further interesting fact relating to Primula is as follows: strains of P. sinensis sometimes throw a mutation in which style and anthers are of the same length. This form, called equal-styled by Darwin, has been shown to be recessive to the long-styled form, which, in turn, is recessive to the short-styled form. Darwin cultivated several equal-styled races. He tells us that “‘my son, Mr. W. E. Darwin, . . . examined pollen from two equal-styled plants which he procured at Southampton; and in both the grains differed extremely in size, a large number being small and shrivelled, whilst many were fully as large as those of the short-styled form and rather more glo- bular . . . The vast number of the small and shrivelled grains in the above two cases explains the fact that though equal styled plants are usually fertile in a high degree, yet some yield few seeds.’”’ Darwin tells us that his equal-styled races came true from seed, as, being extreme recessives, they must of course have done. Again we have a case of great genetic purity in association with defective pollen. There is no evidence, according to Bateson, that Primula sinensis has ever been hybridized. It seems to be one of the few cultivated plants in which great diversity has come about without any admixture with other species, although its purity is not as well attested as that of the sweet pea. 3 Examples might be multiplied indefinitely which show that de- fective pollen is as likely to indicate mutation as hybridization. In fact, I believe that it may be laid down as a rule that both processes are generally characterized by pollen sterility. With this conclusion in mind we may judge the mutation theory with a better chance of arriving at an unbiased decision. Why is it that the polymorphic groups in which mutation is taking place, or supposed to be taking place, all show such undoubted evi- 152 H. H. BARTLETT dence of the prevalence of hybridization? The answer is simple. The mutations, as a rule, are closely enough related to their parent species so that they hybridize readily with them. By hybridization the effect of a single mutation may be widely extended, for there is no experimental evidence that mutations can be ‘‘swamped out” by hybridization if otherwise fitted to survive. In groups which have perhaps long since passed their zenith and are now represented by a few very unlike species hybridization cannot readily take place be- cause the species which remain are too unlike to hybridize. Mutation and hybridization are usually associated with one another, and I do not see how we can escape the conclusion that hybridization is sub- sidiary to mutation rather than mutation to hybridization. Both processes are simultaneously concerned in the evolution of such poly- morphic genera as Oenothera, Rubus, Crataegus and Viola. In the beginning of this paper three aims of the experimental geneticist were stated. The first, as we have seen, has been attained with fair success. The origin of many spontaneous variations has been observed, and their genetic and systematic characters have been studied. The second aim was to determine the causes of mutability, so as to be able to produce the condition at will. Mutations have indeed been produced experimentally in the case of the bivalent moss varieties which have already been referred to, but other work along this line has been unsuccessful. It is impossible to view as conclusive the experiments of MacDougal in which genetic variation is supposed to have been induced by the injection of various solutions into the ovaries of Oenothera (Raimannia) odorata and a species called *“‘ Oeno- thera biennis.’” Since MacDougal’s views have had such wide pub- licity it may be well to summarize briefly the experimental evidence which he has brought forward. One mutation is said to have been induced in the strain called ‘‘Oe. biennts.’”’ This strain was started from presumably unguarded seeds of one individual mother plant, of which the first generation progeny were grown in 1904. We are no- where given any idea how large this progeny was. Four plants were self-pollinated, and the progenies were grown from each in 1905. We are told that one progeny included 669 individuals, and that the rest were not counted. With this generation of 1905, the first to be grown from guarded seed, the injection experiments were made. An ovary was injected with I : 500 zinc sulphate solution. The seeds obtained gave a progeny, the size of which we are not told, in which the solitary THE EXPERIMENTAL STUDY OF GENETIC RELATIONSHIPS 153 mutation occurred. We are not told of any check cultures of sister plants from untreated ovaries, or of any further test of the strain for mutability. MacDougal referred to this experiment in 1911 as the most conclusive which he had yet performed, and stated that the mutation had come true from seed for five generations. There is no doubt that it is a very interesting mutation, especially in view of the fact that its behavior on crossing with the parent strain resembles that of Oe. gigas. The evidence that it was induced by the zinc sulphate solution is, however, quite insufficient. In the case of Raiwmannia odorata 13 mutations are said to have been obtained from ovaries which were injected in 1905. We are not told whether the strain had been previously purified by self-pollination or not. One ovary, injected with Io per cent. sugar solution, gave two mutations; another, injected with 1 : 1,000 calcium nitrate solution, gave 10 mutations; a third, after exposure to radium, gave one mutation. We are not told whether the injected ovaries were all on one plant or not. There s no record of any check experiments with untreated ovaries, nor any record of the size of any of the cultures. More injections were made in 1906, with the contradictory result that calcium nitrate, which had been so potent the year before, induced no mutations at all. Three mutations were found in the progeny from “‘capsules’’ treated with I : 2,000 zinc sulphate, and also “other combinations”’ which were not followed in subsequent years. We are told, moreover, that the effect of the first injections with calcium nitrate persisted in following gener- ations, for normal plants belonging to the strain from the ovary which was treated in 1905 with calcium nitrate gave rise to the same mutation which was supposed to have been induced by the chemical treatment. It seems to me that these facts can only mean that the strain used by MacDougal was in a highly mutable condition and that the experiments were not properly checked. It is especially noteworthy that Compton, also working with Raimannia odorata, has been unable to induce any mutations by the use of MacDougal’s method. His strain was doubt- less a non-mutable one. It is much to be hoped that more decisive results will attend future work along this line, and that whoever under- takes such experiments will adequately check them. The third aim of the experimentalist was to provide a basis from which the systematist might determine deductively the genetic re- lationship of organisms. It must be admitted that except in the case of Mendelian varieties little progress has been made. Nevertheless 154 H. H. BARTLETT certain negative conclusions which may be helpful may be made. In the first place, we must conclude that the degree of sterility which follows hybridization can not be used as an index of relationship. The swarm of 200 elementary species which are included under Erophila verna all differ from one another in relatively trivial characters. Rosen has shown that some of the hybrids among them are fully sterile and only one pair of the species which he tried gave fully fertile hybrids. Yet we can not doubt that all are closely related genetically. In the Onagra group of Oenothera there are many species which cross more readily with Oenothera Lamarckiana than the latter does with its mutation Oe. gigas. On the whole, we may say that among closely related forms neither interspecific sterility nor the lack of it is a true guide to the degree of relationship. - The origin of a form by hybridization should not be inferred from likenesses to both of the supposed parents, nor should a high degree of sterility be interpreted as a sign of hybridization. On the other hand, true interspecific hybrids in some cases show almost no influence of one or the other parent, and are as fertile as, or more fertile than, either parent. The extreme morphological dissimilarity between some mutations and their parent species must teach us that little reliance can be placed upon the guesses of systematists regarding relationships in polymorphic groups. The herbarium botanist should fully realize the fact that his schemes of classification, in Rubus and Crataegus for example, are probably entirely artificial and do not represent natural relationships at all. Very important systematic characters may originate repeatedly and independently in unrelated lines of descent. There is no test of what constitutes a species, except that it shall reproduce itself from generation to generation. Systematists should have a pragmatic attitude in describing species. Subdivision should extend as far as any one finds necessary. The geneticist needs to have definite designations for much smaller groups than the ecologist or morphologist is likely to be interested in. The makers of manuals should therefore endeavor to produce books which will supply the needs of either class of workers without misrepresenting facts. If it is necessary to have a more simple treatment of Crataegus than that of Sargent, for example, it may be done in such a way as not to discredit a large amount of careful work. The synonymy which is so conspicu- ous a feature of systematic work should be given a different significance. THE EXPERIMENTAL STUDY OF GENETIC RELATIONSHIPS 155 There should be some discrimination between true synonyms, which are names applicable to the same identical organism, and the names of distinct units which it may not seem desirable to differentiate in a popular manual. The problem of genetic relationships is the greatest problem of biology. Only repeated attacks, from every side, will solve it. It is no reason for pessimism that results come slowly. Centuries may pass before the greater trends of evolution will be understood. In the meantime we must not scorn our advances, even though they seem slight. BUREAU OF PLANT INDUSTRY, WASHINGTON, D. C. : AS it ever ocurred to. ‘you au Gua greenhouse constructions differ | - greatly, and that a large share of the ie “success. of vote flowers depends: on. a i. construction 2s fa | Haven’ t you 1 aodabe. that prace : ealty the. only difference in’ green- a hoes aside. from design, was the - : “difference i in price? Oe a ) - With ‘everything else, i isn’ " “there.” Sie some one kind that ‘is gener- Cay, coriceded to be better than the enc - yest, and taken as a standard for c com-_ a he 1 _parbons? ie pe 3 ee ee ie Then i isn 't it tonly logical it should 1 Wee: thee zreenhouse builders - t aah their houses’ : are “as. light. as. the U-Bar’ s” it’s. significant: that the _U-Bar i is s the Henne of them a all. : er) iia dient RR STN LMT LD FETE, Ce TRE GE wk 0 7 fs pti ‘ ny i ITORIAL f eG \ ing Koni oy BARTLETT € Bureau of Plant Industry, fs DB; ie AMERICAN JOURNAL OF BOTANY VoL. II APRIL, 1915 No. 4 eine bone NUTRIENT SOLUTION FOR PLANTS JouHn W. SHIVE From a survey of the literature bearing on the growth of plants in nutrient solutions, it appears that the experimental work most nearly approaching logical completeness is that recently carried out by Tottingham.! This writer employed 84 different solutions, all of approximately the same total osmotic concentration but each solution differing from all the others in its proportions of the four nutrient salts, mono-potassium phosphate, potassium nitrate, calcium nitrate, and magnesium sulphate. Besides these salts each solution contained the usual trace of iron, as ferric phosphate. Tests of the 84 different proportions of the nutrient salts, with total osmotic concentration (2.50 atmospheres diffusion tension) about the optimum for young wheat plants, showed that the best solution for the growth of tops (during the first four weeks after germination) contained the four salts in the following volume-molecular concentrations: KNO;, .co49m.; KH2PO,, .0130m.; Ca(NOs)2, .ol44m.; MgSQ,, .o145m. The solu- tion giving the greatest dry weight of tops showed an improvement over Knop’s solution, of the same osmotic concentration, of II percent on the basis of dry weight of tops grown in Knop’s solution. The solution above described is Tottingham’s best solution for tops, and may be regarded as the most reliable standard nutrient solution for wheat during its first four weeks of growth, so far worked out. The present writer has repeated the test of Tottingham’s optimal series of solutions and of Knop’s solution (all with osmotic concentration of 2.50 atmospheres) with wheat, and the results are in very satis- factory agreement with those obtained by Tottingham. For tops, 1Tottingham, W. E. A Quantitative Chemical and Physiological Study of Nutrient Solutions for Plant Cultures. Physiol. Researches 1: 133-345. 1914. [The Journal for March (2: 111-156) was issued 23 Ap 1915.| 157 158 JOHN W. SHIVE the best growth (measured by dry weight) occurred with the same proportions of salts as those giving the best growth in the earlier work. While Tottingham reports an improvement of II percent over Knop’s proportions, the corresponding improvement here shown was I2 percent. This agreement between the two results becomes still more pronounced when it is noted that the two experiments were carried out in different years and at different seasons. Since the general problem of the salt requirements of plants largely remains to be studied, and since nutrient solutions will surely have to be employed in experiments bearing on this problem, it is quite essential that the standard solution used as a basis of comparison be as simple as possible and that it produce excellent growth of the plants. That distilled water is not suitable for use as such a standard is obvious from the fact: (1) that this medium removes salts from the plants, and (2) that the plants in pure water become visibly unhealthy after a few days and soon cease to grow at all. One phase of this matter has been dealt with by True and Bartlett? and by True.* It therefore seems highly desirable to devise a simpler solution than the four-salt mixture used by Knop and Tottingham, if this can be accomplished. An attempt was made in this direction, and the present announcement aims to state the main results in a preliminary way. A more complete account of this work will appear later. Combinations of three nutrient salts, which contain all the essential elements required for plant growth besides iron, and which do not precipitate when mixed in solution of the needed concentration, are comparatively few. The solution here employed contained mono- potassium phosphate, calcium nitrate, and magnesium sulphate; potassium nitrate is thus omitted from the Knop formula. These three salts contain all the essential elements except iron, and they dissociate in solution to form all of the ions present in the Knop- Tottingham four-salt solution. They do not readily precipitate when mixed in solution, and permit total concentrations suitable for plant growth. Thus the three-salt solution appears to be chemically adapted for the purpose in view. Growth tests are needed to show its suitability for supporting plant growth. 2 True, R. H., and Bartlett, H. H. Absorption and Excretion of Salts by Roots, as Influenced by Concentration and Composition of Culture Solutions. U.S. Dep. Agr: Bur, Pind Bull2ar.. sone: §True, R. H. Harmful Action of Distilled Water. Amer. Journ: Bote a- 255-273. I9I4. A THREE-SALT NUTRIENT SOLUTION FOR PLANTS 159 A method similar to that used by Tottingham was here employed, to give a series of solutions differing in the proportions of the three salts, but all having approximately the same total osmotic concentra- tion as measured by the lowering of the freezing point. A trace of ferric phosphate was added to each solution. Each series contained 36 different solutions, instead of the 84 necessitated by the four-salt solution used by Tottingham. The plants here used were wheat (Triticum vulgare) (from the same lot of seed as was used by Totting- ham) and buckwheat (Fagopyrum esculentum Moench.), and the seed- lings were germinated and mounted in a manner similar to that employed by the earlier writer. All the three-salt solutions used in this comparison, including that with Tottingham’s best proportions for wheat tops and the one with the regular Knop proportions, had approximately the same total osmotic concentration, but this was much lower than the concentration employed by Tottingham. They had a diffusion tension of 1.75 atmospheres (as measured by the lower- ing of the freezing point), while Tottingham’s corresponding series showed 2.50 atmospheres. Both these concentrations lie within the optimum range for wheat. Tottingham’s best solution for tops and the regular Knop’s solution were included in this series for purposes of comparison. The cultures lasted 24 days, with change of solution every three days, the containers holding 250 cc. The solution giving the best growth of wheat tops contained the three salts in the following volume-molecular partial concentrations: KH2POu,, .o180m.; Ca(NQOs3)s, .0052m.; MgSQO,, .o150m. This showed an improvement over Knop’s solution, of the same total osmotic concentration, of 27 percent, while Tottingham’s best solution here showed a corresponding improvement of but 16 percent. In the solution yielding the greatest dry weight of buckwheat tops the volume- molecular partial concentrations of the three salts were: KH,PO,, .0144m.; Ca(NOs)2, .0052m.; MgSO., .o200om. The improvement produced by this medium, in dry weight of buckwheat tops, over Knop’s solution of the same total osmotic concentration, was 61 percent. The increase shown by Tottingham’s best solution was 21 percent. The data here given represent averages from two series, one conducted in August and the other in October. The two corre- sponding series agreed, both for wheat and for buckwheat, in the 4Shive, J. W. The Freezing Points of Tottingham’s Nutrient Solutions. PI. World 17: 345-353. I915. 160 JOHN wW. SHIVE proportions of the three salts giving the greatest dry weight of tops. These results are expressed in the tabulated form below, with those of Tottingham’s tests added for comparison. RELATIVE Dry WEIGHTS OF Tops OF WHEAT AND BUCKWHEAT GROWN IN VARIOUS SOLUTIONS Experimenter and Plant Tottingham (wheat). . ShivexGvheat): 2... Shive diwheat esse Shive (buckwheat)... Four-salt Solution Knop’s (2.50 Atm.) | (1.75 Atm.) 1.00 1.00 T.00 1.00 Tottingham’s Best for Three-salt Solution Shive’s Best (1.75 Atm.) Wheat Tops For Wheat | For Buck- (2.50 Atm.) | (z.75 Atm.) Tops wheat Tops Dae Sa ace rete Mg I = I.1 L627 — L227 1.61 From these results it is clear that the three-salt mixture, with proper proportions, is not only eminently suitable for plant growth but that it gives a markedly better growth of tops than does either Tottingham’s or Knop’s four-salt solution, at least with a total osmotic concentration of 1.75 atmospheres, which is a suitable con- centration for general water-culture work. THE LABORATORY OF PLANT PHYSIOLOGY, Jouns Hopkins UNIVERSITY. eles fe Pk ae PRELIMINARY NOTE ON THE MORPHOLOGY OF GNETUM W. P. THOMPSON The writer has collected and examined abundant embryological material of several species of Gnetum in the hope of securing informa- tion concerning the affinities of the Gnetales, and of throwing light on the origin of the gametophytic conditions and endosperm of the Angiosperms. Some of the results of the study are enumerated below. The detailed information will be published shortly. 1. The pollen grains sometimes germinate well up in the so-called ‘style,’ the pollen tubes then growing down to the nucellus as in the Angiosperms. Usually, however, germination takes place on the tip of the nucellus. 2. In the male gametophyte no prothallial cells are produced. Distinct male cells are formed. 3. Only free nuclet (never cells) are formed in the embryo-sac before the pollen tube enters, though cells are formed before fertiliza- tion takes place. 4. One or more eggs are definitely organized just before the pollen tube enters, usually while it is pressed against the side of the sac. 5. Before fertilization takes place the female gametophyte becomes divided into a large number of multinucleate compartments. All the nuclei in each compartment then unite to form a fusion nucleus. Fertilization is delayed until the fusion nuclei are formed. 6. The endosperm is formed by the divisions of the fusion nuclei in the lowermost compartments. 7. Coenocytic pro-embryonal tubes are formed which grow down into the endosperm and branch freely. The bearing of conclusions five and six on the morphology of the endosperm of Angiosperms is obvious. The whole study indicates a close relationship between Gnetum and the Angiosperms. UNIVERSITY OF SASKATCHEWAN. THE’ PERSISTENCE ‘OF VIABLE PYCNOSPORES{@r ao CHESTNUT BLIGHT FUNGUS*® ON NORMAL BARK BELOW LESIONS R. A. STUDHALTER AND F. D, HEALD INTRODUCTION A study of the part taken by birds and insects in the dissemination » of the chestnut blight fungus, Endothia parasitica (Murr.) And., has shown that both birds (6, 7) and insects (9, 10) can carry high numbers of pycnospores of this fungus. There are a number of possible sources from which these pycnospores may have been obtained, but it was thought that by far the greater number of them were brushed from both diseased and healthy chestnut bark during the normal movements of the birds and insects over these surfaces. This sup- position was based primarily upon the fact, previously reported (4, 5), that pycnospores of the chestnut blight fungus, generally called the summer spores, are produced in large numbers even during the winter months, when only an occasional spore-horn can be found, and that they are washed down the trunks of trees with every rain. It is natural to suppose that a large number of the pycnospores thus carried down the tree trunks would lodge on healthy bark below lesions. The work herein reported was undertaken in order to obtain some definite information on the numbers of pycnospores which find lodg- ment on normal bark, and the length of time they may remain viable after a rain. ; A preliminary test of seven pieces of normal chestnut bark was made by Mr. M. W. Gardner in February, 1913, in which he obtained from 130 to 4,700 viable pycnospores per square centimeter of bark surface on the day following a rain. METHOD Pieces of smooth chestnut bark about 4 X 4 cm. in area were cut out with a flamed scalpel at varying distances below blight lesions. Without touching the bark it was dropped into an envelope which had previously been folded crosswise, placed in another envelope and 162 CHESTNUT BLIGHT FUNGUS 163 sterilized. By this means the bark was transported to the laboratory where the tests were made on the following day. In the laboratory 100 cc. of sterile tap water was poured into a shallow sterile dish. The piece of bark to be tested was trimmed down to 2 X 3 cm., stood on end in the dish of water, held by the corners and the outer surface scraped thoroughly with a cutting needle. The scalpels and needles were flamed before using, the hands and arms washed in mercuric chloride, and the entire operation carried on inaculture room. The dish was shaken at intervals for several hours, after which time I cc. of its contents was transferred to a flask con- taining 100 cc. of sterile tap water. This flask was also permitted to stand for at least an hour, and was shaken at intervals. From this flask I cc. and fractions were transferred to sterile Petri dishes; and it was also found advantageous in many instances to pour several plates using fractions of I cc. from the wash water. Chestnut bark agar was used in all the cultures. Incubation took place at laboratory temperatures and the plates were carefully watched for at least ten days. This was necessary on account of the fact that many of the spores of the blight fungus, although viable, would not begin to germinate at once, and colonies of Endothia parasitica frequently made their appearance 3 to 5 days after they were due under normal conditions. DISCUSSION OF RESULTS Six series of tests, representing 36 pieces of healthy chestnut bark, were made between December 22, 1913, and June 4, I914. Of these 36 pieces only five failed to yield positive results. The remaining 31 showed the presence of viable pycnospores of the chestnut blight fungus in numbers varying from 33 to 172,222 per square centimeter of bark surface (Table I). None of the colonies appeared in cultures at the time at which ascospore colonies usually show (3), but it should be stated that it was impossible to tell from the time of appearance of the colonies that all originated from pycnospores, on account of the frequency of delayed germination. It is a known fact, however, that the ascospores are forcibly ejected into the air, and that none are washed down the tree trunks as is the case with pycnospores (4, 5). We therefore feel certain that the colonies appearing in the cultures originated from pycnospores. Positive results were obtained almost uniformly during December 164 R. A. STUDHALTER AND F. D. HEALD TABLE I Results Obtained from the Tests of Healthy Chestnut Bark Below Blight Lesions : Series I Series 2 Collected at Martic Forge, Pa., in afternoon of 12/22/13. Rainfall night of 12/21/13, 0.13 inch. Date of cultures, 12/23/13. Collected at Martic Forge in after- noon of 1/5/14. Rainfall 1/3-4/14 and snow (melted). 1/4-5/14, 2.11 inches. Date of cultures, 1/6/14. No. of Viable | Viabl No. of Viable : Distance Be- | Pycnospores | No. of Viable Distance Be- | Pycnospores No. of Viable Number) Jow Blight | ot E.para- | Spores of |Number| jow Blight | of Z. para- | Spores of _ of Test | iesinn sitica per Other Fungi | of Test Tecion sitica per Other Fungi Sq. Cm, per Sq. Cm. Sq. Cm. per Sq. Cm. \ ; = I 30 cm 15,556 HEUTE 8 25 cm 3,889 I,J 01 2 TOvre 21,667 2,778 9 65° 566 o) 3 43 ie [Symi ti 4II 10 38 “ e) 4,167 4 Ole cs 4,688 7,813 II 32 25336 2778 5 a C7 7a 2,604 12 45 i, 28,125 2,604 6 395 4 833 833 13 Oho) ta 2,500 6,667 7 34 123778 23,333 14 30 24,242 10,101 Series 3 Series 4 Collected at West Chester, Pa., in afternoon of 1/26/14. Rainfall, 1/24/14, 1.35 inches. Date of cultures, 1/27/14. Collected at Martic Forge in after- noon of 1/26/14. Rainfall, 1/24/14, 1.21 inches. Date of cultures, 1/27/14. 15 2 cm. 4,611 1,854 22 10 cm. 633 649 TOun oer 667 1,222 23 TQees 256 51 iy fc Wet Ga ee 708 0) 24 15% 1,975 814 18 230s 3,764 3,667 19 ais 6,500 5,729 20 Live 1,458 5,302 21 eae 1,431 7,500 Series 5 Series 6 Collected at West Chester, in afternoon of 6/5/14. Rainfall, 6/4/14, 0.30 inches. Date of cultures, 6/6/14. Collected at West Chester, in afternoon of 5/26/14. Rainfall, 5/12/14, 0.56 inches. Date of cultures, 5/27/14. 25 0.5 cm fe) ©) 34 Tem. 17,593 O 26 toeean a8 167 35 at edge 35,185 0) oF, I x 139 641 36 2cm. | 172,222 5,556 28 O85 aK 67 aa | 29 Brita 67 100 | 30 122 eee O O | a1 A 1,181 694 22 7 O O a3 at edge fe) 417 CHESTNUT BLIGHT FUNGUS 165 TABLE II Summary of Results Obtained from the Tests of Healthy Chestnut Bark below Lesions eel nceot Collection: Last Rain Before Test SS aes No, of No. of GE io per a a = __| and Time of Made Date Am’t., in. Collection Max. Min. Average 1 | Martic Forge | 12/21/13 0.13 I 7 21,667 833 0;772 2 i: . 1/3-5/14. | 2.11 0) 7 28,125 0} 8,951 2 ee os 1/24/14 1.21 2 7 6,500 667 2734 4 | West Chester) 1/24/14 1.35 2 B 1,975 256 955 5 % ne 5/12/14 0.56 14 9 1,181 O 165 Sle i 6/4/14 0.30 I Bee 727222| 175593) |) 75,000 and January. Snow was on the ground during one collection, and not a single spore horn was found during these two months. That many pycnospores washed down by the winter rains (4, 5) remain clinging to healthy bark below lesions is definitely shown by the first four series in Table I, and the averages in Table II. It is to be expected, of course, that much higher results would be obtained during the summer, when pycnospores are formed in much larger numbers and spore-horns are plentiful. The single series tested in June, collected on the day following a rain of 0.30 of an inch yielded from 17,593 to 172,222 viable spores per square centimeter. The average for the June series was 75,000 per square centimeter (483,900 per square inch), while the average for the four series in December and January was 6,378 per square centimeter (41,151 per square inch). The distances below lesions at which the pieces of bark were taken varied from 0 to 70 cm., several pieces being cut at the very edge of the lesion. It is impossible to make any definite comparison between the number of viable spores obtained at the edge of a lesion and from points further down, for no two pieces of bark were cut below the same lesion at the same time, and no two lesions can readily be compared. From the data on hand, however, it would appear that the distance below the lesion has very little influence on the number of viable pycnospores present. Since pycnospores were very plentiful at the maximum distance tested, it seems certain that positive results could have been obtained at much greater distances. The majority of tests showed that spores of fungi other than Endothia parasitica were also present on the bark (Table I). It is interesting to note, however, that in the majority of the cultures a 166 R. A. STUDHALTER AND F. D. HEALD larger number of colonies of Endothia parasitica appeared than of all © other fungi combined, and that all of the plates from four pieces of bark developed only pure cultures of the chestnut blight fungus. RELATION TO RAINFALL With the exception of Series 5, all of the collections were made not more than two days after a rain. Series 5 was collected at West Chester on May 26. The last preceding rain, 0.56 in., fell on May 12. During this interval of 14 days the weather was generally fair and warm, offering very good opportunities for the desiccation of the spores. Of the nine pieces of bark tested in this series, five yielded viable pycnospores, ranging in number from 33 to II81 per square centimeter (Table I). Assuming that the average number of pycno- spores on the day following the rain was about 75,000 per square centimeter, as was the case in Series 6 (Table II), the number which remained viable at the end of 14 days of desiccation was only a frac- tion of one percent of those present at the start. The significant fact, however, is that some pycnospores could withstand two wecks of desiccation in five out of nine pieces of bark tested. After some rains the bark remains wet or moist in sheltered ples: for several days. Some of the bark tested was still wet at the time of collection. This was true for all of Series 2 and part of Series I. On such areas of bark practically all of the pycnospores present would no doubt remain viable until the bark begins to dry. When the bark once begins to dry the number of viable pycnospores is probably greatly reduced by the mere act of drying, after which the further reduction will be more gradual. This statement is based upon the results obtained under certain artificial conditions (8) where the mere act of drying was found to reduce very greatly the number of viable pycnospores; once dry, the decrease was quite gradual. It is brought out in the last mentioned work (8) that the decrease in the percentage of viable pycnospores is not as great when only a part of the mucilaginous coating is washed off, as when aJl the mucilage has been removed. Under natural conditions in the field it is very probable that only a small part of the mucilage is washed away by rains, for it may frequently happen that fragments of spore-horns may remain intact with nearly all of the mucilage still surrounding the spores. This condition leads us to believe that the number of pycnospores to resist desiccation is greater on the bark below lesions CHESTNUT BLIGHT FUNGUS 167 than under the artificial conditions referred to, where practically no mucilage was left on the spores. In this connection it might be stated that disease organisms have been found to resist desiccation on the seed of the host plant longer than on other material. Such is the case with the bacteria causing the black rot of cabbage (2). Spores of Glomerella gossypu, the cause of cotton anthracnose, retain their vitality much longer when dried on cotton seeds than on cover slips (1). Although little evidence is at hand to confirm the statement, it is not impossible that spores of Endothia parasitica will resist desiccation on chestnut bark much longer than on some other materials. PRACTICAL BEARING OF THE RESULTS ON THE DISSEMINATION BY BIRDS AND INSECTS The conclusion has been reached that birds, and especially migra- tory birds (6, 7), are capable of carrying large numbers of viable pycnospores of the blight fungus for considerable distances; and that insects (9, 10) may be important agents in the local dissemination of the blight. Birds were shown to be carrying pycnospores only; the same was true of nearly all of the insects from which positive results were obtained, although a very small number of ascospores were found on several beetles. The question of the source of these pycnospores has already been touched upon in the introduction. The results herein reported bring out the following reasons in support of the belief that these pycnospores were obtained in the main, if not wholly, as a result of the brushing of birds and insects over diseased and normal chestnut bark: 1. Viable pycnospores are present in varying numbers on healthy bark below lesions after both summer and winter rains. 2. Large numbers of pycnospores can be obtained immediately after a rain, but they may be present in smaller numbers for at least two weeks. 3. The largest numbers of spores were invariably obtained from birds and insects from about two to four days after a rain, correspond- ing rather closely with the time at which the largest numbers of viable pycnospores are present on normal bark below lesions. 168 R. A... STUDHALTER? AND ©... D. HEALD SUMMARY Viable pycnospores of the chestnut blight fungus were found to be present on normal bark below lesions in numbers varying from 0 to 172,222 per square centimeter of bark surface (1,111,176 per square inch). Of the 36 pieces of bark tested, only five failed to yield positive results, and four of these five were collected 14 days after a rain. Viable pycnospores were obtained in all but one of 24 tests made during December and January, when no spore-horns were present in the field. An abundance of viable pycnospores was obtained at as great a distance below a lesion as 70 cm., and it appears very likely that they could be obtained at much greater distances below cankers. Most of the tests were made one or two days after a rain; in one series, however, tested 14 days after a rain of 0.56 inch, positive results were obtained from five of the nine pieces of bark from which cultures were made. LITERATURE CITED 1. Edgerton, C. W. The rots of the cotton boll. La. Agr. Exp. Sta. Bull. 137: I-88. pl. 1-13. 1912. 2. Harding, H. A., Stewart, F. C. and Prucha, M. J. Vitality of the cabbage black rot germ on ‘cabbage seed. N. Y. State Exp. Sta. (Geneva) Bull. 251: 177-194. pl. I. 1904. 3. Heald, F. D. A method of determining in analytic work whether colonies of the chestnut blight fungus originate from pycnospores or ascospores. Mycologia 5: 274-277. pl. g8-IOI. 1913. 4. Heald, F. D. and Gardner, M. W. Preliminary note on the relative prevalence of pycnospores and ascospores of the chestnut blight fungus during the © winter. Science n. ser. 37: 916-917. 1913. 5. Heald, F. D. and Gardner, M. W. The relative prevalence of pycnospores and ascospores of the chestnut blight fungus during the winter. Phyto- pathology 3: 296-305. pl. 26-28. 1913. 6. Heald, F. D. and Studhalter, R. A. Preliminary note on birds as carriers of the chestnut blight fungus. Science n. ser. 38: 278-280. I913. 7. Heald, F. D. and Studhalter, R. A. Birds as carriers of the chestnut blight fungus. Journ. Agr. Research 2: 405-422. fig. 1-2, pl. 38-39. I914. 8. Heald, F. D. and Studhalter, R. A. Longevity of pycnospores and ascospores of Endothia parasitica under artificial conditions. Phytopathology 5: 35-44. Pl2: “1OLs. g. Studhalter, R. A. Insects as carriers of the chestnut blight fungus. Abstract in Phytopathology 4: 52. 1914. 3 10. Studhalter, R. A. and Ruggles, A.G. Insects as carriers of the chestnut blight fungus. Pa. Dept. Forestry Bull. [In press]. 4) Sa Pert Pa CONOVIG STUDY OF SETARIA ITALICA AND ITS IMMEDIATE ALLIES F. Tracy HUBBARD This paper deals with those species of Setaria grouped by Ascherson and Graebner, Syn. Mitteleur. F!. 2!: 74 (1899) under their super- species Panicum viride (that is S. verticillata, S. viridis including S. ambigua and S. italica) and joined by Kuntze, Rev. Gen. 2: 767, 768 (1891) to form his Chamaeraphis ttalica. The study was undertaken with a view to determine what S. italica var. germanica really was, as material of various forms was passing under that name and it gradually involved a careful examination of the whole group,—especially of S. viridis and S. ttalica since the old line of separation of these species did not hold good. SS. verticillata separated itself readily when con- fined to those specimens with retrorsely barbed setae. Some recent authors have reduced S. ztalica to a variety or sub- species of S. viridis,—for example Ascherson and Graebner, Syn. Mitteleur. Fl. 2!: 77 (1899), as Panicum viride B. P. italicum and Briquet, Prodr. Fl. Corse 1: 68 (1910), as Setarta viridis subsp. ttalica. This is readily understandable when we consider that length of panicle and size of spikelet, the key characters commonly employed to separate the two species, entirely fail to do so as these characters frequently overlap very strongly, though they hold good in the average specimens. Generally, the panicle of S. viridis is said to range up to 8.5 cm. in length while the length of spikelet is given as about 2mm. Careful study of the material at hand with measurements of every specimen examined, shows a range of panicle dimensions from I to 15 cm. long Dy 4 to 14 mm. in diameter. The spikelets range from 1.8 to 2.7, commonly 2.2 to 2.5 mm. long. SS. ialica, on the other hand, is said to have a panicle from I0 to 20 cm. long and spikelets about 3 mm. long. My measurements show that in the denser, more spike-like forms,—grouped by Alefeld, Landw. FI. 315 (1866), in his Sect. Moharium and best represented by subvar. germanica of this paper,— the panicle may be as short as I cm. and not more than 7 mm. in diameter while the spikelets of the whole species vary in length from 2 to 3.2, more commonly 2.6 to 3 mm. 169 170 F. TRACY HUBBARD In testing the stability of the various characters I found that ordinarily the second glume of S. viridis equaled the sterile lemma in length whereas in S. ztalica it was for the most part noticeably shorter. Furthermore the spikelets in S. viridis, as soon as they become at all mature, shell out the whole spikelet—quite readily—leaving a cup- like receptacle; whereas those of S. ttalica shell out the fruit! only leaving the first and second glume and sterile lemma behind. This appears to me to be a good specific character and the use of it as a key character in separating the specimens gave homogeneous groups. The name germanica was first used by C. Bauhin, Theatr. Bot. 518, fig. (1658) as Panicum Germanicum sive panicula minore. Exactly what form of S. ttalica Bauhin had is impossible to state though he undoubtedly had one of the larger lobulate forms as he states that the panicle is nine inches rarely a foot long. It may be yellow, purple or black, but he fails to say anything about the length of the setae. The plate is rather indefinite, but represents panicles with short setae. While the name originated with Bauhin the first use of it after 1753 is Panicum germanicum Miller, Gard. Dict., ed. 8, no. I (1768). Miller describes the species as follows ‘Spica simplici cernua, setis brevioribus, pedunculo hirsuto,’ basing his name on C. Bauhin, Pinax 27 (1671). In the discussion of his species of Panicum he makes the following statements regarding P. germanicum, “‘The stalks are terminated by compact spikes, which are about the thickness of a man’s finger at their base, growing taper toward their points, and are eight or nine inches long,? ... .’’ The phrase “setis brevioribus’’ has undoubtedly led to the recent interpretation of S. ttalica var. germanica as a form with setae shorter than the spikelets, but does this phrase compare the length of the setae with the spikelets; why may it not be a term comparative with the following species, Panicum ttalacum, which according to Bauhin’s plate in Theatr. Bot. 519 is a plant with long setae? I believe that this interpretation is correct as I have a photograph and spikelets of the Miller material in the British Museum which were kindly sent me by Mr. A. B. Rendle. This material was collected in Chelsea Garden in 1760 and is labeled ‘Panicum Germanicum sive panicula minore’’ and consists of a panicle and two leaves. As a centimeter scale has been placed on the 1 The term fruit is consistently used in this paper to mean the seed inclosed in the fertile lemma and palea. 2 These measurements are too long for subvar. germanica and also do not agree with Miller’s specimen. SETARIA ITALICA AND ITS IMMEDIATE ALLIES Liel sheet the measurements can be easily obtained. The leaves are respectively 30 cm. long by 14 mm. broad and 24 cm. long by 14 mm. broad; the panicle is dense, about 11.5 cm. long by 13 mm. in diameter, with the lobes scarcely noticeable except at the very base. The setae are quite visible in the photograph, decidedly exceeding the spikelets and the fragment sent me shows them to be green and more than twice the length of the spikelet. The fruit is quite immature, but un- questionably belongs in the straw-colored group. These characters enable me to identify the form as that named Panicum ttalicum var. praecox by Alefeld, Landw. FI. 315 (1866) and taken up by Kornicke in Kérn. & Wern. Handb. Getreideb. 1: 276 (1885) which I have classed as subvar. germanica. Early in the study of the subject I realized that a second form of the species was causing some of the confusion as some authors referred back to Panicum germanicum Mill., others to P. germanicum Willd. Willdenow, Sp. Pl. 1: 336 (1797), described a plant as P. germanicum, ‘“P. spica composita coarctata, spiculis glomeratis, involucellis setaceis flore longioribus, rachi hirsuta.’ As will be noticed, at once, the setae are described as longer than the spikelets as opposed to the variety 8 ‘‘involucellis flore brevioribus.” Also the fact that the spike is described as composite should be noted. Willdenow refers to Roth, Tent. Fl. Germ., but his description clashes with Roth’s in the length of the setae. Through the courtesy of Prof. Engler, Dr. Pilger wrote me as follows about the Willdenow Herbarium speci- men ‘“‘Was im Herbarium Willdenow unter Panicum germanicum liegt ist eine kraftige Form von Setaria italica. Obere Halmblatter (die letzten unter der Rispe) 20 cm. und dartiber lang, 15-17 mm. breit. Rispe 13 cm. lang, im oberen Teil sehr dicht, 1144 cm. in Durchmesser, an der Basis locker unterbrochen. Spelzen und Granen + braunlich violett gefarbt. Granen ziemlich kurz. Deckspelze oval, glanzend, griinlich-gelb, fast garnicht gerunzelt, nur hier und da schwache Runzelung.’’ Ata later date Dr. Pilger sent me a few spikelets of the Willdenow material which have purplish setae noticeably longer than the spikelets, and have straw-colored fruit. These facts place P. _ germanicum Willd. in the form called P. ttalicum var. macrochaetum by Ko6rnicke, in Kérn. & Wern. Handb. Getreideb. 1: 273 (1885) which I have renamed var. Hosiii as K6rnicke derived his name from Pennisetum macrochaetum Jacq. Eclog. Gram. 3: t. 25 (1815-20), a species pictured and described as having green setae: P. macro- chaetum Jacq. is referable to subvar. germanica. 12 F. TRACY HUBBARD Besides the two above mentioned there are other applications of the name germanica and a chronological table of some of these uses may be of interest. (1768) Miller, Gard. Dict., ed. 8, no. 1 (1768) previously discussed. (1788) Roth, Tent. Fl.Germ.1: 27 (1788); 2: 71 (1789) as Panicum germanicum. A comparison with P. italicum is given in volume two and four points of difference noted: of these, ‘3 Spiculis conglobatis nec elongatis. 4 Involucellis floribus brevioribus: nec triplo longi- oribus.”’ The exact form is indeterminable, one of the short-setaed variations of Setarza italica. (1797) Willdenow, Sp. Pl. 1: 336 (1797), previously discussed. (1802) Koeler, Descr. Gram. 16 (1802) as Panicum italicum Var. I Germanicum. The main point of his description is ‘“‘setis spiculas non superantibus.’’ I cannot place this further than to say it is a form of the Moharium group with very short setae. (1802) Host, Ic. Gram. Austr. 2: 12, t. 15° (1802) as: Pama germanicum. The description covers the plate which is of a plant of the Moharium group—dense panicled—with rather long purple setae and straw-colored fruit and is readily determined as subvar. Metzger1. The description is broad enough to include other forms. (1805) Lamarck, in Lam.’&'DC) Fl. Pr. .edi’3, 33 imGiees aa. Panicum italicum B Germanicum. Lamarck says “... les fleurs sont entourées de barbes trés-coutres dans le variété B, ....’’ The form is not determinable, but more stress is laid on the length of the setae than on the size or shape of panicle. Probably a form of the lobed group with short setae. (1812) Beauvois, Agrost. 51, 169, 178 (1812) as Setaria germanica. Transfer of name, only, based on Panicum germanicum Willd. cf. p. 169. I prefer to leave the form unstated though as far as the name is concerned it would belong under var.’ Hostit. (1817) Roemer and Schultes, Syst. Veg. 2: 492 (1817) as Setarta germanica. The description is copied from Willd. Sp. Pl., but their remarks lead me to believe they had one of the other variations, which one I cannot decide. (1827) Link, Hort. Berol. 1: 219 (1827) as Setaria germanica. Part of the description reads ‘‘Setae sursum scabrae spiculis 6-8 plo longiores interdum brevissimae aut deficientes’’ showing that he had several variants. (1829) Trinius, Spec. Gram. 2: t. 199 (1829) as Panicum ttalicum SETARIA ITALICA AND ITS IMMEDIATE ALLIES 173 var. germanicum. The plate shows a long, slender, cylindrical panicle with long setae. The form is indeterminable, but of the Moharium group or quite possibly a variation of S. viridis as suggested by the cup-like receptacles shown in one of the detail drawings. (1829) Eaton, Man., ed. 5, 322 (1829) as Pennisetum germanicum based on Panicum germanicum Willd. What Eaton really had is difficult to say without seeing his specimen, but it certainly is not var. Hostii to which the Willdenow reference is referable as Eaton’s description reads ‘‘spike compound compact: spikelets glomerate: rachis hirsute: bristles short.”’ (1838) Schrader, in Linnaea 12: 430 (1838) as Setaria ttalica 6 germanica. Based on Panicum germanicum Roth, a form with short setae which I cannot place. The other references given are Host Gram. 2: t. 17 [probably should be 15] which equals subvar. Metzger1 and Trin. Icon. t. [probably 199 is meant] which as before stated is indeterminable. There is no description given. (1816-46) Baumgarten, Enum. Stirp. Magno. Transs. 3: 277 (1816-46). References to the same page of this work are given for Paspalum germanicum and Pennisetum germanicum. I have not seen the work and do not know which one is correct or if both ccm- binations are given nor have I any idea to what form the citation is referable. (1847) Wood, Class-book Bot., ed. 2, 607 (1847) as Setaria ger- manica. There is nothing in the description to determine what form it applies to. The other editions of Wood are practically the same. (1848) Parlatore, Fl. Ital. 1: 114 (1848) as Setaria germanica. There is nothing to determine the form. (1853) Ledebour, Fl. Ross. 4: 471 (1853) as Setaria italica B germanica. All that can be gathered from the description and syn- onymy is that it treats of a form with the setae shorter than the spikelets. (1877) Doell, in Mart. Fl. Bras. 2?: 165 (1877) as Panicum italicum var. germanicum. Based on P. germanicum Willd., but the other citations refer to other forms. The description commences ‘‘Setae longiores . . .”’ so that he certainly had a long-setaed form. (1890) Richter, Pl. Eur. 1: 28 (1890) as Setaria itdlica b) ger- manica. Only a transfer of name based on Panicum germanicum Willd. and consequently referable to var. Hostit. (1890) Beck, Fl. Nieder-Oesterr. 1: 46 (1890) as Setaria italica 174 F, TRACY HUBBARD a) germanica. Based on Panicum germanicum Willd. and hence referable to var. Hostit as to name and probably as to description. Exclude the synonym P. italica var. brevisetum Doell which is another form. | 45 (1891) Kuntze, Rev. Gen. Pl. 2: 767 (1891) as Chamaeraphis italica 6 germanica. The synonymy given is Panicum germanicum L. non Roth; P. marittimum Lam. To what Kuntze refers as P. germanicum L. I am not certain as the following is the nearest approach to such a combination that I can find in Linnaeus’ works and the only reference given, in Linnaeus Codex. Linnaeus, Mant. 2: 323 (1771) ‘‘atalicum. Panicum germanicum muito minus est, spica ovata, nec elongata.’ This is in the section of the Mantissa headed Observationes in Species Plantarum cum Emendationibus et Animad- versionibus, and is certainly only a comment on the German form of S. italica. Panicum maritimum Lam. is indeterminable as to form. (1897) Scribner, U.S. Div. Agrost. Bull. 6: 32 (1897) as Chaetochloa italica germanica. Transfer, only, based on Panicum germanicum Mill. and consequently referable to subvar. germanica. (1899) Ascherson and Graebner, Syn. Mitteleur. Fl. 2!: 77 (1899) as Panicum viride |subsp.| B. P. ttalicum B. germanicum. This refers to some form that has the setae a little longer than the spikelets. (1900) Scribner and Merrill, U.S. Div. Agrost. Bull. 21: 21 (1900) as Chaetochloa italica germanica. This is based on Panicum ger- manicum Mill. and is referable to subvar. germanica as to name and possibly to subvar. Metzgeri as to plant. (1902) Hitchcock, in Bail. Cycl. Am. Hort. 4: 1662 %(1ree2) ae Setaria ttalica var. germanica. Based on Panicum germanicum Mill., but the authority given for the combination is Richter who based his combination on P. germanicum Willd. which is a different form. Prof. Hitchcock describes the plant as follows: ‘‘Asmaller form more nearly approaching the wild S. viridis: bristles much longer than the spikelets which would apply to subvar. germanica very well.” (1906) Dalla Torre and Sarntheim, FI. Tirol. 6!: 158 (1906) as Panicum italicum stirps germanica. Based on P. germanicum Mill. and referable to subvar. germanica [exclude synonym Setaria germanica Beauv. which is uncertain as to form, cf. (1812) Beauvois]. (1908) Hitchcock, in Gray Man., ed. 7, 119 (1908) as Setaria ttalica var. germanica. For discussion see (1902) Hitchcock. The descrip- tion and form referred to are not the same as in the Cyclopedia as SETARIA ITALICA AND ITS IMMEDIATE ALLIES — 75 Prof. Hitchcock in the Manual describes the variety as follows, ‘“GOLDEN-WONDER MILLET, which is more slender and has bristles shorter than the spikelets.”” This description would apply to several of the variations mentioned in this paper. The common name Golden Wonder Millet applies to forma breviseta, a very large, lobulate- panicled form. KEY TO SPECIES TREATED. a. Setae retrorsely barbed. S. verticillata a. Setae antrorsely barbed. 0. b. Spikelet articulate below the glumes; complete spike- let shells out leaving a cup-like receptacle. S. viridis and varieties b. Spikelet articulate above the glumes; fruit only shells out leaving the glumes and sterile lemma behind. S. ztalica and variations SeTARTA VERTICILLATA (1L.) Beauv. Agrost. 51, 171, 178 (1812). I have not attempted to go into the involved synonymy of this species as it is readily distinguished from S. viridis var. ambigua,—which it otherwise resembles,—by the retrorsely barbed setae. From the material I have seen I am inclined to agree with Sir. J. D. Hooker, Fl. Brit. Ind. 7: 80 (1896) in considering S. Rottleri Spreng. synonym- ous. RANGE.—Sparingly introduced in Canada, in the United States, mainly eastwards, in Mexico and Central America. In Venezuela? and Brazil fide Doell. Common in Europe, occurring in several sections of Africa, in Asia Minor, British India, Philippines, Australia and Hawaii. | SETARIA VIRIDIS (L.) Beauv. Agrost. 51, 171, 178, t. 13, f. 3 (1812): ima. syst..Ver, 2: 486 (1807) Link, Hort. Berol. 1: 218 (1827): Kunth, Rev. Gram. 1: 46 (1829) [excl. syn. in part]: Reichb. Fl. Germ. Pecurs. 2: 20 (1630): Kunth, Enuny, Pl..r: 151 (1833) [excl. 6 which refers to var. Weinmanni|: Reichb. Ic. Fl. Germ. 1: 68, t. 47 [188] heed) ed. 2 Ga (1650): Koch, Syn, Fly Germ., ed. 1, 773 (1837) [excl. portion refering to S. Weinmanni]: Parl. Fl. Ital. 1: 111 (1848): Gray, Man., ed. 1, 615 (1848); Hitche. in ed. 7, 118 (1908): Coss. & Dur. Expl. Sci. Alger. 2: 36 (1854): Miq. Fl. Ind. Bat. 3: 467 (1855- 60): Griseb. Fl. Brit. W. Ind. 554 (1864): Lowe, Nat. Hist. Brit. Grasses I01, t. 31, f. B (1865): Schur, Enum. Pl. Transs. 723 (1866); eemnov, |. co (1885): oyme, in Sowerby, Engel..Bot., ed. 3, II: 13, t. 1693 (1873) [as to name, descr. and plate questionable]: Benth. FI. Austral. 7: 494 (1878): Boiss. Fl. Orient. 5: 443 (1884): Hook. f. FI. 176 F. TRACY HUBBARD Brit. Ind. 7: 80 (1896): Wildem. & Dur. Prodr. Fl. Belg. 3: 80 (1900), PRELINNAEAN REFERENCES: C. Bauhin, Theatr. Bot. 138, fig. (1658), _ Gramen paniceum, sive panicum sylvestre simplict spica: C. Bauhin, Pinax 8 (1671) Gramen paniceum spica simplici . . .: Scheuchzer, Agrost. 46 (1719) Gramen paniceum, seu panicum sylvestre simplica spica [the Bauhin reference only]. Panicum glaucum B L. Sp. PI., ed: I, I: 56 (1753). WP. viride L. Syst. Nat., ed: 10, 2: 870 (1750) » Beene, FI. Herborn. 13; t. 2, f. 2 (1775): @urt. Fl Lond. fase. 4:0. 445 em [also cited as fasc. 4: t. 5] (1782?): All. Fl. Pedem. 2: 240 (1785): Vill. Hist. Pl. Dauph. 2: 64 (1787): Roth, Teut. Fl. Germ. 1: 27 (1788)n2- 69 (1789): Lam. Tabl. Ill. 1: 169 (1791): Hoffm. Deutschl, Fit aon (1791): Willd. Sp. Pl. 1: 335 4797):Lam. Encycl..4: 727 ernie 737] (1798): Sm. in Engl. Bot. 13: t. 875 (1801) [the plate seems to me to be Sefarta. tialica|: Host, Ic. Gram. Asin 2-1, t. 4 se2)- Koel. Descr. Gram. 10 (1802): Knapp, Gram. Brit. t. 10 (ees): Schrad. Fl. Germ. 1: 240 (1806): Gaudin, Agrost. Helv. 1: 17 (1819) fexcl. syn. Host, Gram. Austr. ‘2: t. 15]: Hornem. Hort. Matmaeen (1813):. Eaton,. Man., ed. 2, 339 (1818): Mert. & Koch, in Roh Deutschl. Fl., ed. 3, 1: 469 (1823): Trin. Gram. Panic 163 (18260): Gaudin, Fl. Helv. 1: 152 (1828): Trin. Spec. Gram. 2: t. 203 €1829): Bertol. Fl. Ital. 1: 420 (1833) [excl. syn. P. verticillatum b ambiguum Guss.]: Doell, Fl. Grossherz. Bad. 1: 233 (1857): Lehm. in inch: Naturk. Livl., ser. 2, 11: 137 [Fl. Poln.-Livl. 137] (1895): Dalla Torre & Sarnth.. Fl. Tirol. 6!: 156 (1906). PP. laevigatum Lam. Fle amas 578 (1778). P. crusgalla O. F. Mill..non L. m Fl. Dan sa4acess. t. 852 (1782) [as to plate, but not as to descr. or syn.]. P. reclinatum Vill. Hist. Pl. Dauph. 2: 64 (1787) [this is considered by some authors to be a synonym of var. Weinmann, but I can see no evidence to show that it really is the same]. PP. viride var. reclinata (Vill.) Hoffm. Deutschl. Fl. 1: 21 (1791). ‘Panicum, quale Linnae ?”’ Krock. FI. Siles. 1: 88 (1793) [The date may be 1787 cf. Pritzel Thesaurus. It is questionable if Krocker intended to make more than a comment, but various authors have treated it as though it were a combination]. P. bicolor Moench, Meth. 206 (1794). Pennisetum viride R. Br. Prodr. 1: 195 in obs. (1810) by implication only: combination first made by R. & S. Syst. Veg. 2: 489.(1817) in syn.: Nutt. Gems B55 (1818): Eaton, Man. ed. 4, 389 (1824): Torr. Fl. N. Y. 2: 430 (1843). Panicum viride var. majus Gaudin, Agrost. Helv. 1: 18 (1811) as ‘“8 Panicum viride majus N.”’: Gaudin, Fl. Helv. 1: 152 (1828) as ee Te SETARIA ITALICA AND ITS IMMEDIATE ALLIES bye Go mows >) Lehn. in Arch. Naturk. Livl.,:ser. 2, 11: 137 [Fl. Poln.- Livl. 137] (1895) as ‘‘Var. b major Koch”’ [Koch did not make the combination! Walla” Torre & Sarnth. FI.: Tirol. 6%: 157 (1906). Setaria villosa Beauv. Agrost. 51, 171, 178 (1812) fide Hook. f. FI. Brit. Ind. S. nana Dum. Obs. Gram. Belg. 139 (1823) fide Hook. f. 1. c. and Aschers. & Graebn. Syn. Mitteleur. Fl. S. affinis Schultes, Mant. 2: 276 (1824) questionable synonym fide Hook. f. 1. c. Panicum viride var. humifusum Lej. Rev. Fl. Spa 13 (1824) nomen: Lej. & Gourt. Comp. Fl.-Bele. 1: 52 (1828). -P. humile Thunb. ex Trin. Gram. Panic. 164° (1826) in syn: sub. P. viride. Setaria viridis var. nana Dum. Florula Belg. 150 (1827): Wildem. & Dur. Prodr. FI. Belg. 3:80 (1900). Panicum viride var. nanum (Dum.) Lej. & Court. Comp. Fl. Belg. 1: 52 (1828). P. viride var. longisetum Doell, Rhein. F1.128 (1843): Doell, Fl. Grossherz. Bad. 1: 234 (1857). Setaria penicillata Nees ex Wall. Cat. n. 8640D (1848) nomen, fide Hook. f. FI. Brit. Ind. Panicum viridescens Steud. Syn. Pl. Gram. 51 (1854) fide Mig. FI. Ind. Bat.- P. muticum Hort. Lips. ex Steud. I. c. 51 (1854) in-syn. sub. P. viride. Setaria viridis var. latifolia Ambr. Fl. Tirol. Merid. 1: 42 (1854). S. panis Jessen, Deutschl. Graser 248, 249, in part, figs. 391, 392 (1863). S. viridis var. nodiflora Saccardo, in Atti Ist. Ven., ser. 3, 92: 865 (1864). S. arvensis subsp. viridis (L.) Bruhin, in Bericht. Naturw. St. Gallen 1865-66: 215 (1866): S. arvensis subsp. viridis var. fallax Bruhin, |. c. 1865-56: 215 (1866). S. chlorantha Schur, Enum. Pl. Transs. 723 (1866); ed. nov., |. c. (1885) fide Aschers. & Graebn. Syn. Mitteleur. Fl. Panicum panis Jessen, in Meyer & Jess. Alberti Magni Veg. 523 in part (1867). P. viride var. vulgare Doell, im Mart. Fl. Bras. 22: 173 in obs. (1877). P. viride var. gigantea Pranch. & Sav. Enum. Pl. Jap. 2: 162 (1879). P. ttalscum var. viride (L.) Koérnicke, in Korn. & Wern. Handb. Getreideb. 1: 272, 277 (1885). Setaria viridis var. typica Beck, Fl. Nieder-Oesterr. 46 (1890). S. viridis var. typica forma communis Beck, 1. c. 46 (1890). S. viridis var. typica forma major (Gaud.) Beck, 1. c. 46 (1890). ? S. viridis var. secunda Beck, |. c. 46 (1890). Chamaeraphis ttalica var. viridis (L.) Ktze. Rev. Gen. Pl. 2: 767, 768 (1891). C. viridis (L.) Millsp. in Bull. W. Va. Agric. Exper. Sta. 2: 466 [Fl. W. Va. 466] (1892): Porter, in Bull. Torr. Bot. Cl. 20: 196 (1893): Beal, Grasses No. Am. 2: 157 (1896). ?.S. viridis var. insularis A. Terrac. in Ann. Ist. Bot. Roma 5:93 (1894): Dur. & Schinz, Consp. Fl. Afr. 5: 775 (1894). Ixophorus waidis (L.) Nash, in Bull. Torr: Bot. Cl. 22; 423 (1895). . Setaria 178 F. TRACY HUBBARD viridis var. typica Posp. Fl. Oesterr. Kiistenl. 1: 51 (1897) [an var. typica Beck?]. S. viridis var. major (Gaud.) Posp. |, c. 1: 51 (1897). Chaetochloa viridis (L.) Scribn. U. S. Div. Agrost. Bull. 4: 39 (1897): _ Scribn. & Merr. U.S. Div. Agrost. Bull. 21: 19. f. 8 (1900): Nash, in Britt. Man., ed. 1, 90 (1901); ed. 3,90 (1907). Pantcummuuae [subsp.] A. P. eu-viride Aschers. & Graebn. Syn. Mitteleur. Fl. 2!: 76 (1899). /P. viride [subsp.] A. P. eu-viride A. I. a. 1. majus (Gaud.) Aschers.. & Graebn. 1. c. 21: 77 (1899). JP. viride [subsp.] A. P. eu- viride A. I. a. 1. a. reclinatum (Vill.) Aschers. & Graebn. |. c. 21: 77 (1899). P. viride [subsp.] A. P. eu-viride A. I. a. 1. b. pygmaeum Aschers. & Graebn. 2!: 77 (1899). JP. viride [subsp.] A. P. eu-viride A. I. b. nodifiorum (Saccardo) Aschers. & Graebn. |. c. 2!: 77 (1899). Setaria viridis var. humtfusa (Lej. & Court.) Wildem. & Dur. Prodr. Fl. Belg. 3: 885 (1903). Panicum viride var. fallax (Bruhin) Dalla Torre & Sarnth. Fl. Tirol. 6!: 157 (1906). Setaria viridis subsp. eu- viridis (Aschers. & Graebn.) Brig. Prodr. Fl. Corse 1: 68 (1910). S. ttalica subsp. viridis (L.) Thell. in Mem. Soc. Sci. Nat. Cherb. 38: 85 (1911). S. viridis var. australis F. M. Bail. Compr. Cat. Queensl. P!. 61l 9i2)): An extremely variable species frequently approaching S. italica or S. verticillata in appearance. From the former it may be distinguished by the spikelets being articulate below the glumes and easily shelled out—entire—when nearing maturity, leaving a little cup at the end of the pedicel, whereas S. ttalica is articulate above the glumes and shells out the fruit only. Some few specimens show intermediate characters and point toward hybridity. S. viridis is considered by many to be the origin of S. ztalica which may well be the case, but to-day the two seem to be readily separated by the above-stated character. From S. verticillata it is at once distinguished by the antrorse barbs of the setae: S. verticillata has the setae retrorsely barbed. | There are many forms of the species which in most instances are not clearly differentiated and which pass into each other too thoroughly to seem worthy of nomenclatorial designation. Size of plant, size of panicle, color and length of setae are all variable, but not consistently enough so to separate along these lines. Seed color is usually the same, —when fully mature,—a dull straw-drab with darker mottlings, though immature it ranges from whitish-green through shades of green and yellowish-green to straw. SETARIA ITALICA AND ITS IMMEDIATE ALLIES 179 The plant is usually erect or sometimes slightly geniculate, simple or many branched at the base, 3 to Io dm. tall [depauperate specimens sometimes I dm. or even less], the culms fairly stout. The leaves are commonly fairly broad and long acuminate, 5 to 30 cm. long, 4 to 15 mm. broad. The panicles vary in size and compactness sometimes being somewhat lobulate and interrupted toward the base, but not verticillately so, more or less blunt or tapering at the base and apex, 1 to 15 cm. Jong? [usually relatively thick when short], 4 to 14 mm. in diameter, the rhachis commonly pilose. The spikelets range in size from 1.8 to 2.7 mm. long—most commonly 2.2 to 2.5 mm.,—o.8 to 1.6 mm. broad. The second glume is commonly as long as the sterile lemma, but occasionally is slightly shorter. The setae are variable in length, but usually considerably longer than the spikelets and are ordinarily green or greenish-yellow, but are sometimes purple-tipped or even all dark purple. The fruit is slightly rugulose, generally more so than in 5S. ttalica. RANGE.—Occurring pretty generally throughout the United States and Canada, usually near cultivated land. Found sparingly in Mexico and in Brazil—fide Doell. Common throughout Europe, no plants seen from Africa or Asia Minor, but in Asia it occurs in Russia, British India, China, Japan and Formosa. SETARIA VIRIDIS (L.) Beauv. var. AMBIGUA (Guss.) Coss. in Coss. & Dur. Expl. Sci. Alger. 2: 36 (1854): Boiss. Fl. Orient. 5: 443 (1884): Beck, Fl. Nieder-Oesterr. 46 (1890) [excl. syn. P. viride var. brevisetum Doe) Mure -& Schinz, Consp. Fl. Afr. §: 775 (1894): Halacsy, Consp. Fl. Graec. 3: 333 (1904). Panicum viride L. misapplied by Desf. F.. Atlant. 1: 58 (1798) [excl. syn.] fide Guss. FI. Sic. Syn. P. verticillatum b ambiguum Guss. FI. Sic. Prodr. 1: 80 (1827). Setaria ambigua Guss. Fl. Sic. Syn. 1: 114 (1842): Lojac. FI. Sic. 3: 266 (1909). S. verticillata var. ambigua (Guss.) Parl. Fl. Palerm. 1: 36 (1845): Pomel Malt) Tir (1848): Richter, Pl: Eur, 1:28 (1890): Brand, in Koch, Syn. Deutsch. FI., ed. 3, 3: 2689 (1905). Panicum adhaerens Subsp. 1 P. verticillatum antrorsum A. Br. in Ind. Sem. Hort. Berol. 1871, App.: 7 (1871). ? Setaria pseudo-verticillata Hort. ex A. Br. leer 1671, Appa: 7 (1870) in syn. S. glomerata Hort, ex A. Br. 1..c. 1871, App.: 7 (1871) in syn. Panicum ambiguum (Guss.) Hausskn. in Oesterr. Bot. Zeitschr. 25: 345 (1875): Dalla Torre & Sarnth. FI. 8 These small panicles are the secondary ones, the primary panicles are rarely under 5 cm. long. 180 F. TRACY HUBBARD Tirol. 6!: 156 (1906). P. viride var. subverticillatum Doell, in Mart. Fl. Bras. 2?: 173, in obs. (1877). Setaria decipiens C. Schimp. ex Nym. Consp. 787 (1842) in syn. sub S. ambigua. Chamaeraphis italica var. ambigua (Guss.) Ktze. Rev. Gen. Pl. 2: 768 (1891). Pan- icum verticillatum B. ambiguum (Guss.) Aschers. & Graebn. Syn. Mitteleur. Fl. 2!: 75 (1899). Chaetochloa ambigua (Guss.) Scribn. & Merr. U.S. Div. Agrost. Bull. 21: 18, f. 7 (1900). Setaria verticillata subsp. ambigua (Guss.) Brig. Prodr. Fl. Corse 1:67 (1910). Panicum verticillatum var. antrorsum A. Br. ex Briq. |. c. 1: 67 (1910) inssyn. Distinguished from the typical form of the species chiefly by the more open, verticillate or subverticillate panicle [especially toward the base] and by the main rhachis being scabrous or hispid on the angles instead of pilose. Setae antrorsely barbed otherwise in habit strongly suggestive of S. verticillata. The setae often not much exceeding the spikelets. Fruit commonly greenish. This form is decidedly intermediate between S. verticillata and S. viridis, commonly resembling the former in its verticillate or sub- verticillate panicle [occasionally S. verticillata is compact and only distinguishable from S. viridis by the retrorse barbs of the setae and quite frequently S. virzdis is somewhat subverticillate] and the latter in the direction of the barbs of the setae. Of the two characters, the second seems to me to be more stable and of greater specific weight than the form of the panicle which is extremely variable in S. viridis, in fact in most of the Setarias: consequently I consider this form better treated asa variety of S. viridis than asa variety of S. verticillata. Good botanists have treated it both ways and still others have con- sidered it to be a separate species as will be noted in the preceding bibliography. I do not find any consistent characters to separate it specifically. RANGE.—Reported as a ballast plant collected by Scribner at Camden, N. J.. (cf..Scribn. & Merr. U.S. Divs Agrost. Bullmaas (1900)) and specimens seen from Washington, D. C., and Mobile, Ala. The first a weed in the Grass Garden, the second a ballast plant. Frequent in Europe more especially southward and also known from Algeria in Africa. SETARA VIRIDIS (L.) Beauv. var. WEINMANNI (R. & S.) Brand, in Koch, Syn. Deutsch. FI., ed. 3, 3: 2690 (1905): Fernald & Wiegand, in Rhodora 12: 133 (1910). S. Weinmmanni R. & S. Syst. Veg. 2: 490 (1817). Panicum purpurascens Opiz, in Flora 5: 266 (1822) SETARIA ITALICA AND ITS IMMEDIATE ALLIES I8I nomen. Setaria purpurascens Opiz, Boehm. Gew. 12 (1823) fide Aschers. & Graebn. Syn. Mitteleur. FI. °? Panicum wride var. brevisetum Doell,t Rhein. Fl. 128 (1843): Doell, Fl. Grossherz. Bad. 1: 234 (1857): Aschers. Fl. Prev. Brandenb. 1: 809 (1864). Setaria arvensis subsp. viridis var. purpurascens Bruhin, in Bericht. Naturw. Ges. St. Gallen 1865-66: 216 (1866) nomen fide Dalla Torre & Sarnth. Fl. Tirol. 2? S. viridis b arenosa Schur, Enum. Pl. Transs. 723 (1866) ; ed. nov., 723 (1885). S. viridis.a laevigata Schur, |. c. 723 (1866); ed. Mmoy., 723 (1885). 3S. veridis var. fuscata Harz. Landw. Samenk. 2: 1260 (1885) fide Beck, Fl. Nieder-Oesterr. ?° Chamaeraphis ttalica var. viridis forma purpurascens Ktze. Rev. Gen. Pl. 2: 767 (1891). Panicum viride var. minor Koch, ex Lehm. in Arch. Naturk. Livl., ser. 2, 11: 137 [Fl. Poln.-Livl. 137] (1895) [Koch did not make the combina- tion]. ?P. viride [subsp.] A. P. eu-viride A. II. brevisetum (Doell) Aschers. & Graebn. Syn. Mitteleur. Fl. 2!: 77 (1899). P. wiride [subsp.] A. P. eu-viride B. Weinmannu (R. & S.) Aschers. & Graebn. l. c. 21: 77 (1899). ? P. viride [subsp.] A. P. eu-viride B. II. arenosum (Schur) Aschers. & Graebn. |. c. 2!: 77 (1899). P. viride var. Wein- mannii (R. & S.) Dalla Torre & Sarnth. FI. Tirol. 61: 157 (1906). P. Weinmanns “R. & S.”’ according to Dalla Torre & Sarnth. |. c. 6!: 157 (1906) in syn. [R. & S. placed the species under Setaria]. Setaria viridis var. breviseta (Doell) Hitche. in Rhodora 8: 210 (1906): Hitche. in Gray Man., ed. 7, 119 (1908) [as to plants, possibly not as to name]. Distinguishable from the common form of S. viridis chiefly in its spreading habit [base usually geniculate], its narrower leaves, 2 to 6 mm. wide and its smaller more slender panicles 0.7 to 5 cm. long, 3 to 6 mm. broad. The culms are frequently purplish toward the hase. The spikelets are commonly smaller than those of the species 2 to 2.3 mm. long with exceptions only 1.6 mm. and up to 2.4mm. The setae are either purple or green and vary in length from about the length of the spikelet to about 6 mm. long. The length of the setae is so variable (when longest only about 6 mm.) that it seems undesirable to separate var. breviseta (Doell) Hitche. as the plants so named agree in every other respect except length of setae. Whether Doell’s Panicum viride 6 brevisetum is the same I am not so certain as the only point brought out by Doell is the length of the setae which might equally well apply to the species proper. 47 can find no evidence to prove conclusively whether this is a short-setaed variation of the species proper or of var. Weinmanni; usage places it with the latter. 182 F. TRACY HUBBARD For a, further discussion of this variety cf. Fernald and Wiegand, in Rhodora 12 1235(tore): RANGE.—Occurring in northeastern and central Canada, in the ? United States in New England and Illinois. Frequent in Europe and also found in Russia and in Arabia. Specimens from Alabama, New Mexico, Arizona, Mexico, one from Hungary and several from India seem intermediate between the variety and the species. Other minor variations of S. viridis or perhaps better termed abnormalities are: A forked form of which I have seen specimens from Missouri, from northern India and from Japan. A viviparous form to which several names have been given. Pani- cum viride var. viviparum Bertol. FI. Ital. 1: 421 (1833) nomen: Doell, Rhein. Fl. 128 (1843): Lehm. in Arch. Naturk. Livl., ser. 2; rr: 238 [Fl. Poln.-Livl. 138] (1895). Setarta viridis var. vivipara (Bertol.) Parl. Fl. Ital. 1: 112 (1848) [as “b. vivzparum’’|: Lucas, in Corre- spondenzbl. Naturf. Ver. Riga, 12: 185 [Repr. 27] (1862). S. arvensis subsp. viridis var. vivipara Bruhin, in Bericht. Naturw. Ges. St. Gallen, 1865-66: 215, 216 (1866) nomen. Panicum viride monsttr. vivipara (Bruhin) Dalla Torre & Saruth. FJ. Tirol. 61: 157 (1906). The only specimen I have seen is one from Dover, Maine, collected by Fernald in 1896 and this has only one viviparous panicle, the others being normal. A few specimens show characters or combinations of characters intermediate between S. viridis and S. ttalica which point to hybridity: the specimens that I have seen showing this most clearly are: Roches- ter, New York; Baxter, no. 5—Garret County, Maryland; Donnell Smith in 1879 and Moscow, Idaho; Henderson, no. 2849 all of which are in the U.S. Nat. Herb. The following combinations have been commonly referred as synonyms to S. viridis, but I believe that they are better referable to other species. Panicum cynosuroides Scop. Fl. Carn., ed. 2, 1: 50 (1772) [com- monly referred to S. viridis, but I believe more correctly a synonym of S. glauca: cf. Roth, Tent. Fl. Germ. 2: 70 (1789) sub P. glaucum]. P. geniculatum Lam. Encycl. 4: 727 [err. typ. 737] (1798): Hornem. Cat. Hort. Hafn. 28 (1807 ?): Willd. Enum. Hort. Berol. 1031 (1809): Hornem. Hort. Hafn. 81 (1813):.Doell, in Mart. Fl. Bras. 2?: 158 (1877) [probably equals S. glauca of which Lamarck says in the original description it is perhaps only a variety]. SETARIA ITALICA AND ITS IMMEDIATE ALLIES 183 Setaria geniculata (Lam.) R. &.S. Syst. Veg. 2: 491 (1817): Kunth, Enum. Pl. 1: 150 (1833) [probably equals S. glauca: cf. P. geniculatum iam]. Pennisetum geniculatum (Lam.) Jacq. Eclog. Gram. 3: 37, t. 26 (1815-20) [the plate is an excellent picture of S. glauca]. — Panicum tejucense Nees, ex Trin. Gram. Panic. 162 (1826) in syn. sub P. glaucum: Nees, Agrost. Bras. 243 (1829) [equals S. imberbis en, Woell, in Mant) Fl, Bras. 22: 157: (1877)|- P. dasyurum Willd. ex Nees, Agrost. Bras. 241 (1829) [probably equals S. wmberbis|. Setaria tejucensis (Nees) Kunth, Enum. Pl. 1: 150 (1833) [equals S. imberbis: based on Panicum tejucense Nees]. SETARIA ITALICA (L.) Beauv. [sensu amplissimo] Agrost. 51, 170, BAe el2)o IR. & S, Syst. Veg. 2: 493 (1817): Spreng. Syst. Veg. 1: 305 (1825): Link, Hort. Berol. 1: 220 (1827): Schultes, Mant. 3, add. pad Clo iil: 598 (1827): Kunth, Rev. Gram. 1: 46 (1829): Reichb. Fl. Germ. Exctirs. 29 (1830): Kunth, Enum. Pl. 1: 153 (1833): Wood, Class-book Bot. 439 (1845); ed. 1860, 788 (1861): Gray, Man. 615 (ers) -ititche. in ed. 7, 119 (1908): Parl. Fl. Ital. 1: 113 (1848): Duthie & Fuller, Field & Gard. Crops N. W. Prov. & Oudh 2:5, t. 25 (1883) [yellow fruit]: Richter, Pl. Eur. 1: 28 (1890): Hook. f. FI. Brit. tids7:78 (1896) lexcl. syn. in part]: Hitchc. in-Bail. Cycl. Am. Hort. 4: 1662 (1902). PRELINNAEAN REFERENCES: Dodon. Stirp. Hist. Pempt. 4, bib. 1, p. 498, fig. (1583) Panics wndict spica: Lobel. Ic. Stirp. Pl. 42, fig. (1591) Panicum aliud Indicum. Panicula vilosa: Clusius, Rar. Pl. Hist: Lib. 6, p. CCXV (1608) Panicum vulgare: Parkins. Theatr. Bot. p. 1139 fig. & p. 1140 text (1640) Panicum Indicum pannicula villosa: C. Bauhin, Theatr. Bot. 518, fig. (1658) Panicum Germanicum sive panicula minore [mentions variation in color ot fruit): C..Bauhin, VPheatr.. Bot. 519, fig. (1658) Panicum Italicum sive panicula matore [mentions variation in color of panicle and fruit!: C. Bauhin, Pinax 27 (1671) Panicum Germanicum sive panicula minore |mentions color variation in panicle and _ fruit]: C. Bauhin, Pinax 27 (1681) Panicum Italicum sive panicula majore: Miorison, Hist:.3 Sect: 8, p. 188, -t. 3, Panicum 1 (1715) Panicum Germanicum sive panicula minore [mentions color variation in fruit]: iorison, Hist. 3, sect. 8, p. 183; t. 3, Panicum 2 (1715) Panicum Italicum sive panicula majore [mentions color variation in friut]: L. Hort. Cliff. 26 (1737) Panicum sativum: Royen, Prodr. Hort. Lugd.- 184 F. TRACY HUBBARD Bat. 54 (1740) Panicum spica composita aristis gluma brevioribus: L. Hort. Ups. 19 (1748) Panicum spica composita, aristis flosculo brevi- oribus: Rumph. Herb. Amboin. 5: 202, t. 75, f. 2 (1750) Panicum indicum [yellow fruit]. The plate shows a panicle with the tip digitate- divided]. Panicum italicum L. Sp. Pl. 1:56 (1753) [excl. syn. Gronov.]: Gouan, Hort. Monsp. 34 (1762): Mill. Gard. Dict., ed. 8, Panicum no. 2 (1768) [mentions different. colors of fruit]: L.. Mant. 2:.323 (1771): . Weig.. Obs... Bot. 22. (1772): Willd. Sp.: Pl 12 336mm Lam. Encycl. 4: 728 [err. type 738] (1798) [mentions different colorings of setae]: Koel. Descr. Gram. 16 (1802): Host, Ic. Gram. Austr. 4: 8 in part [excl. t.] (1809): Gaudin, Agrost. Helv. 1: 20 (1811): Mert. and Koch, in Rohl. Deutschl. FI., ed. 3, 1: 470 (1823): Metzger, Eur. Cereal. 63 (1824) [as a whole]: Trin. Gram. Panic. 164 (1826): Doell, in Mart. Fl. Bras. 22: 165:°(1877): Lehm. in_Arch: Naturk. livigicer 2, 11: 138 [Fl. Poln.-Livl. 138] (1895). P. germanicum Roth, Tent. FI. Germ. 1: 27:(1788); 2:71 (1789): Host, Ic: Gram. Austr. 27 1aaipare fexcl. t.] (1802). P. glomeratum Moench, Meth. 207 (1704). >: elongatum Salisb. Prodr. 18 (1796). P. maritumum Hort. Par. ex Lam. Encycl. 4: 727 [err. typ. 737] (1798) [possibly belongs under subvar. Metzgert, but | do not know the color of the fruit]. P.atalicum var. germinicum Koel. Descr. Gram. 17 (1802): D.C. in Lam. & D.C. FI. Fr,, ed. 3, 3: 14 (1805): [as 8 P. germamcum). ? Tino Spee Gram. 2: t. .T99 (1829): Doell,:in Mart. Flo Bras:.2?: 165 «ag777. Pennisetum italicum (L.) R. Br. Prodr. 1: 195 (1810): Eaton, Man. Bot.,-ed. 3,383. (1822);: ed. 8; [Eaton -&. Wright, No. Ant Beige (1840). Setaria germanica (Willd.) Beauv. Agrost. 51, 169, 178 (1812) [based on P. germanicum Willd.]: R. & S. Syst. Veg. 2: 492 (1817): Link, Hort. Berol. 1: 219 (1827): Reichb. Fl. Germ. Excurs. 29 (1830): Parl. Fl. Ital. 1: 114 (1848). Panicum intermedium Vahl, ex Hornem. Hort. Hafn. 1: 82 (1813) fide Trin. Gram. Panic 165 (1826): P. com- pactum Kit. ex Schultes, Oesterr. Fl., ed. 2, 1: 212 (1814) in syn. sub P. germanicum var. 8. Echinochloa intermedia (Vahl) R.& S. Syst. Veg. 2: 477 {1817). Setaria maritima (Lam.) R. & S.1. €& 2: 402;Geae Panicum sibiricum Hort. ex R. & S. 1. c. 2: 493 (1817) in syn. sub S. italica B. ..P. setosum Hort. ex R. & S. 1. c. 2: 492 (4817) im Ssyateeuis S. germanica 8 non Sw. P. aegyptiacum Hort. ex R. & S. |. c. 2: 493 (1817) in syn. sub S. italica. Pennisetum germanicum Baumg. Enum. Stirp. Magno Transs. 3: 277 (1816-46) fide Ind. Kew. 2: 458 (1895). Paspalum germanicum Baumg. |. c. 3: 277 (1816-46) fide Richter, Pl. SETARIA ITALICA AND ITS IMMEDIATE ALLIES 185 Eur. 1: 28 (1890). Panicum pumilum Link, Enum. Hort. Berol. 1: 76 (1821) fide Trin. Gram. Panic. 166 (1826) sub P. italicum f). P. macrochaeton Link, |. c. 1: 76 (1821) fide Trin. 1. c. 165 (1826) sub P. italicum d). P. melfrugum Hort. ex Mert. & Koch, in Rohl. Deutschl. Fl., ed. 3, 1: 471 (1823) in syn. sub P. italicum. P. asia- ticum Hort. ex Mert. & Koch, |. c. 1: 471 (1823) in syn. sub P. italicum. P. serotinum Hort. ex Mert. & Koch, |. c. 1: 471 (1823) in syn. sub P. italicum. Setaria macrochaeta (Link) Schultes, Mant. 2: 274 (1824): Link, Hort. Berol. 1:218 (1827). Panicum setaceum Hort. ex Trin. Gram. Panic. 166 (1826) insyn.sub P. italicum f). P.attenuatum Hort ex Trin. |. c. 166 (1826) in syn. sub P. italicum f). Pennise- tum germanicum (Willd.) Eaton, Man. Bot., ed. 5, 322 (1829); ed. 8, [Eaton & Wright, No. Am. Bot.] 346 (1840). Setaria flavida Horn. ex Reichb. Fl. Germ. Excurs. 29 (1830) in syn. sub S. germanica. 5S. violacea Horn. ex Reichb. |. c. 29 (1830) in syn. sub S. germanica. S. asiatica Hort. ex Reichb. |. c. 29 (1830) insyn. subS. italica. S. persica Hort. ex Reichb. |. c. 29 (1830) in syn. sub S. italica. ? S. globularis Presl, Rel. Haenk. 1: 314 (1830) fide Kunth, Enum. PI. 1: 152 (1833) and Hook. f. Fl. Brit. Ind. 7: 78 (1896). S. melinis Link, Hort. Berol. 2: 219 (1833). ? Panicum miliaceum Blanco, FI. Filip., ed. 1, 39 (1837) fide Hook. f. l. c. 7: 78 (1896). Setaria ttalica B germanicum (Roth) Schrad. in Linnaea 12: 430 (1838). Panicum vulgare Wallr. in Linnaea 14: 542 (1840) [based on P. vulgare Clus. Rar. Pl. Hist. Lib. 6, p. CCXV (1601)]. ?P. globulare (Presl) Steud. Nom., ed. 2, 2: 257 (1841): Steud. Syn. Pl. Gram. 51 (1854). Setaria italica B germanica (Beauv.) Ledeb. Fl. Ross. 4: 471 (1853). S. japonica Pynaert, in FI. des Serres 14: 7, fig. (1861). S. panis Jessen, Deutschl. Graser 248, 249 ex parte (1863). Panicum ttalicum c) maritimum (Lam.) Aschers. Fl. Prov. Brandenb. 1: 809 (1864). Setaria compacta Schur, Enum. Piy Yranss. 967 [index| (1866); ed. nov., 967 [index] (1885). 5S. germanica a. legituma Schur, |. c. 724 (1866); ed. nov., 724 (1885). Panicum panis Jessen, in Meyer & Jess. Alberti Magni Veg. 523 ex parte (1867). P. ttahicum var. compactum A. Br. Ind. Sem. Hort. Berol. 1871: 4 (1871) nomen. P. ttalicum var. japonicum Hort. Ber. ex A. Br. 1. c. 1871: 4 (1871) nomen. Setarta ttalica var. typica Beck, Fl. Nieder-Oesterr. 1: 46 (1890). S. italica var. compacta (Kit.) Beck, |. c. 1: 46 (1890). Chamaeraphis italica (L.) Ktze. Rev. Gen. Pa 707° (1891) in\part: Beal, Grasses No: Am: 2%: 154 (1896). ‘C. italica a sativa Ktze. |. c. 2: 768 (1891). C. italica B elobata Ktze. 186 F. TRACY HUBBARD 1. c. 2: 768 (1891). C. dialica 6 gsermanicum Ktze. |. c. 2: 768 (1891). Ixophorus tatalicus (L.) Nash, in Bull. Torr. Bot. Cl. 22: 423 (1895): Nash, in Britt. & Br. Il. Fl. 1: 127, f. 283 (1896). Chaetochloa italica (L.) Scribn. U.S. Div. Agrost. Bull. 4: 39 (1897): Scribn. & Mereis: Div. Agrost. Bull. 21: 20, f. 9 (1900):. Nash, in Britt. Man. oo/(100m)- ed. 3, 90#@(1907): Nash, in Small, Fl. Sotitheast. U.S: to7zetnoaae Panicum viride [subsp.] B. P. ttalicum (1...) Aschers. & Graebn. Syn. Mitteleur. FI. 21: 77 (1899). P. viride [subsp.] B. P. itahicum B. germanicum Aschers. & Graebn. |. c. 21: 78 (1899). P. viride [subsp.| B. P. twtahcum C. marittimum (Lam.) Aschers. & Graebn. 1. c. 21: 78 (1899). Setaria italica var. germanica “‘(Mill.) Richter,” according to Hitche. in Bail. Cycl. Am. Hort. 4: 1662 (1902)saccondine ste Hitche. in Gray, Man. ed. 7, 119 (1908) not var. germanica of Richter, Pl. Eur. 1: 28 (1890) which is based on Panicum germanicum Willd. and equals var. Hostu. S. viridis subsp. ttalica (L.) Brig. Prodr. FI. Corse 1: 68 (1910). Nota.—The preceding references apply to some form of the species, but cannot be placed with certainty under any given variation. S. italica is an exceedingly variable species which has been under cultivation for many centuries and consequently has developed in- numerable strains. Thesizeof the plant varies from well over a meter to only about a decimeter high and the thickness of the culm is equally variable. The leaves range in length from only about 3 cm. up to 50 cm. and in breadth from 5 to 30 mm.; commonly linear-lanceolate and long-acuminate, but occasionally relatively broad and _ short- acuminate. The panicle is more or less lobulate-compound and inter- rupted at the base [section Maximum of Alefeld, Landw. FI.| to dense, seemingly spicate [section Moharium of Alefeld, 1. c.] and ranges in length from I to 30 cm. or more and in diameter from 7 to 55 mm. The spikelets vary in size from 2 to 3.2 mm. long, more commonly 2.6 to 3 mm. and are always articulate above the glumes allowing the fruit to be easily shelled out. The second glume is usually slightly shorter than the sterile lemma, sometimes only three fourths as long but occasionally almost as long. The setae vary in color—being green, purple or brown—and in length from shorter than the spikelets to many times their length, but they are always upwardly barbed [sometimes very slightly so]. The fruit is of three main colors— yellowish, reddish or blackish,—but in varying shades. It is more or less rugulose, sometimes almost smooth when it is shiny. SETARIA ITALICA AND ITS IMMEDIATE ALLIES 187 Of the several lines of variation mentioned, seed-color seems to be the most stable; color of setae the next; form and size of panicle in the third degree, but very variable and intergrading; length of setae the fourth and extremely variable, but noticeable in the extremes; whereas size and shape of seed do not seem to offer any line of separa- tion. Along these lines the following variants of the species may be noted, remembering always that distinctions based on form and size of panicle are sure to show numerous intergrades and that length of setae is only applicable in the extreme form. KEY TO THE VARIANTS OF SETARIA ITALICA. a. Fruit yellowish to straw or light brown. 0. b. Setae green. c. c. Panicle more or less open lobulate. d. d. Setae noticeably longer than spikelets. d. Setae shorter than or barely exceeding spike- lets. c. Panicle dense or slightly lobulate at base. e. e. Setae noticeably longer than spikelets. é. Setae shorter than or barely exceeding spike- . lets. G-apetae purple. f- f. Panicle more or less open lobulate. f. Panicle dense or slightly lobulate at base. g. g. Setae noticeably longer than spikelets. g. Setae shorter than or barely exceeding spike- lets. b. Setae brown. h. h. Panicle more or less open lobulate. 7. 2. Setae noticeably longer than spikelets. 2. Setae shorter than or barely exceeding spike- lets. h. Panicle dense or slightly lobulate at base. a. Fruit reddish or orange. 7. j. Setae green. 2k. k. Panicle more or less open lobulate. 1. 1. Setae noticeably longer than spikelets. 1. Setae shorter than or barely exceeding spikelets. k. Panicle dense or slightly lobulate at base. | j. Setae purple. m. m. Panicle more or less open lobulate. m. Panicle dense or slightly lobulate at base. j. Setae brown. 12. > Attention is called to the fact that a, b and 7 each occur three times in the following key. subsp. stramineofructa. forma breviseta. subvar. germanica. forma mutts. var. Hostii. subvar. Metzgert. forma curtiseta. var. brunneoseta. forma brachychaeta. subvar. densior. subsp. rubrofructa. forma gigas. subvar. pabularts. var. purpureoseta. subvar. violacea. 188 F. TRACY HUBBARD n. Panicle more or less open lobulate. o. o. Setae noticeably longer than spikelets. var. rubra. o. Setae shorter than or barely exceeding spike- lets. forma aurantiaca. n. Panicle dense or slightly interrupted at base. subvar. condensa. a. Fruit blackish, brownish black or purplish black, with pale yellowish-straw fruits intermingled (these some- times predominating). pp. pb. Setae green, panicle more or less lobulate. subsp. nigrofructa. p. Setae purple-brown, panicle dense. var. atra. SETARIA ITALICA (L.) Beauv. subsp. stramineofructa nom. nov. Panicum tualicum var. A Metzger, Eur. Cereal. 63; t. 19, 12a mean ? P. aalicum Trin.’ Spec. Gram. 2:° t 198 (1820). — Pgiohemn var. longisetum Doell, Rhein. Fl. 128 (1843): Doell, Fl. Grossherz. Bad. 1: 233 (1857): K6rnicke, in Kérn. & Wern. Handb. Getreideb. I: 273 (1885): Wern. in Korn. & Wern. |. c. 2: 890 (asas iar: italicum var. californicum Kornicke, Syst. Ubers. Cereal. 18 (1873): K6rnicke, in Korn & Wern. I. c. 1: 273 (1885): Wern. in Korn. & Wern. I. c. 2: 891 (1885) [I have not been able to place this variety satisfactorily, I should judge it dealt with specimens intermediate between subsp. stramineofructa and subvar. germanica]|. P. ttalicum var. lobatum Kornicke, in Korn. & Wern. |. c. 1: 273 (1885): Wern. in Korn. & Wern. I. c. 2: 890 (1885). P. macrurum Hort. ex Kornicke, in Kérn. & Wern. I. c. 1: 273 (1885) in syn. sub: P. italicumieaas longisetum. P. frumentaceum Hort. ex Kornicke, in K6rn. & Wern. l. c. I: 273 (1885) in syn. sub P. italicum var. longisetum. Setaria chrysantha Hort. ex K6rnicke, in Kérn. & Wern. |. c. 1: 273 (1885) in syn. sub P.-italicum var. longisetum. P. viride [subsp.| B. P. italicum A. longisetum (Doell) Aschers. & Graebn. Syn. Mitteleur. FI. 21: 78 (1899). Plant stout, 5 to 11 dm. or more tall, leaves linear-lanceolate, long tapering acuminate, I2 to 50 cm. long, 5 to 30 mm. broad: panicle usually strongly lobed, variable in shape, ellipsoid, cylindrical, or elongate-ovoid, apex blunt or tapering, 10.5 to 30 cm. or more long, I4 to 55 mm. in diameter; lobes variable in size and length up to 3 or 4 cm., sometimes short-pedicelled, base of panicle more or less open-interrupted: setae green, always noticeably longer than the spikelets, up to 12 mm.long: fruit yellowish to light brownish, mature usually straw, more or less rugulose, commonly obscurely so. This form passes into subvar. germanica and many specimens are difficult to place. SETARIA ITALICA AND ITS IMMEDIATE ALLIES 189 RANGE.—Cultivated; specimens probably escapes, seen from On- tario, Canada; from Massachusetts, New York, Michigan and Texas in the United States and from Russia, British India and the Philip- pines. SETARIA ITALICA (L.) Beauv. subsp. STRAMINEOFRUCTA Hubb. forma breviseta (Doell) comb. nov. Panicum italicum var. brevisetum Doell, Rhein. Fl. 128 (1843): Doell, Fl. Grossherz. Bad. 1: 233 (1857) [excl. Host reference]: K6rnicke, in Ko6rn. & Wern. Handb. Getreideb. 1: 274 (1885): Wern. in Korn. & Wern. |. c. 2: 893 (1885). P. ttalicum var. inerme Doell, in Mart. Fl. Bras. 2?: 165 (1877) cf. Kornicke, in Korn.-c& Wern. l/c. 1: 274 (1885). P. brevisetum Doell, in Mart. lic. 27; 165 (1877) in.syn. sub P. italicum var. inerme. P. djalicum yar. brevisetum subvar. insigne Kornicke, in K6rn. & Wern. |. c. 1: 274 (1885): Wern. in K6rn. & Wern. |. c. 2: 894 (1885). P. ttalicum var. brevisetum subvar. maximum Kornicke, ex Wern. in Korn. & Wern. I. c. 2: 894 (1885). Setaria glomerato-spicata Hort. ex Ko6rnicke, in K6rn. & Wern. |. c. 1: 274 (1885) in syn. sub P. italicum var. brevisetum. S. ialica sibirica Hort. ex Kornicke, in K6rn. & Wern. |. c. I: 274 (1885) in syn. sub P. italicum var. brevisetum. Differs from the subspecies in having the setae shorter than the spikelets or barely exceeding them. RANGE.—Specimens seen from West Virginia and British India, also cultivated specimens from Massachusetts and Washington, District of Columbia. SETARIA ITALICA (L.) Beauv. subsp. STRAMINEOFRUCTA Hubb. subvar. germanica (Mill.) comb. nov. Panicum germanicum Mill. Gard. Dict., ed. 8, Panicum no. I (1768). Pennisetum macrochaeton Jacq. Eclog. Gram. 3: 36, t. 25 (1815-20). Panicum italicum Var. B Metzger, Eur. Cereal. 63, t. 19, f. B (1824). Setaria macrochaeta iWaeg-) Spreng. Syst. Veg. 1: 305 (1825): Kunth, Enum. Ph ixi 152 (1833) [as to name and syn.]. Panicum italicum var. praecox Alef. Landw. FI. 315 (1866): K6rnicke, in K6rn. & Wern. Handb. Getreideb. 1: 276, t. 8, f. 41 (1885). P. ttalicum var. macrochaetum (Jacq.) A. Br. Ind. Sem. Hort. Berol. 1871: 4 (1871): Doell, in Mart. Fl. Bras. 2?: 165 (1877) [as to name, perhaps not as to plant]. Chaetochloa ttalica germanica (Mill.) Scribn. U.S. Div. Agrost. Bull. 6: 32 (1897): Semipn: & Merr. U.S. Div. Agrost. Bull.-21: 21 (1900) [as to name, possibly subvar. Metzgert as to plant]. Panicum italicum stirps germanicum (Mill.) Dalla Torre & Sarnth. Fl. Tirol. 6!: 158 (1906) [as to name, excl. syn. S. germanica Beauv.]. 190 F. TRACY HUBBARD Differs from the subpsecies as follows,—plant usually more slender and often not so tall; leaves in general shorter and narrower; panicle variable in size, 1.8 to 13 cm. long, 8 to 15 mm. in diameter, dense or very slightly lobulate at the base, occasionally one or two small inter- rupted lobes, lanceolate-ellipsoid to elongate-cylindrical; setae notice- ably longer than the spikelets. The extreme small forms are some- times only 1 dm. tall and have panicles very closely resembling SS. vids. RANGE.—Commonly cultivated; specimens—escapes or adven- titious—seen from various parts of the United States, from Europe, Russia in Asia, British India, China and the Philippines and from Japan. SETARIA ITALICA (L.) Beauv. subsp. STRAMINEOFRUCTA Hubb. subvar. GERMANICA (Mill.) Hubb. forma mitis (Alef.) comb. nov. Panicum italacum Var. E Metzger, Eur. Cereal. 64, t. 17, f. B (1824)? P. ttalicum var. mitis Alef. Landw. FI. 316 (1866): K6rnicke, in K6rn. & Wern. Handb. Getreideb. 1: 277, t. 8, f. 42 (1885) [spelling e- mended var. mite]: Wern. in Korn. & Wern. 1. c. 2: 898 (1885). Differs from the subvariety only in having the setae shorter than or barely exceeding the spikelets. RANGE.—A single specimen from Quebec, Canada, also one from France and one from China. A somewhat digitately forked form of subsp. stramineofructa is described and pictured by Rumphius, Herb. Amboin. 5: 202, t. 75, f. 2 (1750) and is also spoken of by Ko6rnicke, in Korn. & Wern. Handb. Getreideb. 1: 278 (1885). This form is represented by the following specimen from Texas, Nealley in 1886 in U.S. Nat. Herb. SETARIA ITALICA (L.) Beauv. subsp. STRAMINEOFRUCTA Hubb. var. Hostii nom. nov. Panicum italicum Host, Ic. Gram. Austr. 4: 8 in part, t. 14 (1809) [the text covers much more than this form; the plate is an excellent representation of the form]. P. germanicum Willd. Sp. Pl. 1: 336 (1797) fide description of specimen in Will- denow’s Herbarium and fragment of the same. Setaria ttalica B germanica (Willd.) Ledeb. Fl. Ross. 4: 471 (1853) [as to name]: Richter, Pl. Eur. 1: 28 (1890): Beck, Fl. Nieder-Oesterr. 1: 46 (1890) [excl. syn. var. brevisetum Doell]. Panicum ttalicum var. macrochaetum Kornicke, in Korn. & Wern. Handb. Getreideb. 1: 273 (1885) [as to plant and descr., not as to syn.]. Differs from the subspecies principally in having purple setae: SETARIA ITALICA AND ITS IMMEDIATE ALLIES IQI rarely only purple-tipped. The leaves are the same as in the sub- species; the panicles are always lobulate, but sometimes more dense than in the subspecies and almost cylindrical, 7.5 to 22 cm. long, 15 to Ao mm. in diameter; the setae are noticeably longer than the spikelets, up to 18 mm. long. This form passes into subvar. Metzgert1 and many specimens are difficult to place. RANGE.—Commonly cultivated. Specimens not strictly cultural seen from eastern and central United States, from Germany, Russia in Asia and from British India. SETARIA ITALICA (L.) Beauv. subsp. STRAMINEOFRUCTA Hubb. var. Hostrt Hubb. subvar. Metzgeri (K6rnicke) comb. nov. Panicum ger- manicum Host, Ic. Gram. Austr. 2: I2 in part, t. 15 (1802) [the description covers more than this form, the plate is a perfect representa- tion of the commonest strain of the subvariety]. P. ttalicum var. Metzger1 Kornicke, in Korn. & Wern. Handb. Getreideb. 1: 276 (1885) : Wern. in Korn. & Wern. 1. c. 2: 896 (1885). Setarta ttalica var. Metzgert (K6rnicke) Hack. ex Gramina Hungarica Exsiccatae 7: no. 303° and in Exsiccatae List published in Magyar Bot. Lapok 10: 462 (I9II) nomen. Differs from var. Hostit principally in its shorter, denser panicles [rarely slightly lobulate toward the base] which are often clavate and truncate or sometimes penicillate at the tip and frequently taper at the base owing to empty clusters of setae. Plant commonly not so tall; leaves usually shorter and narrower; panicle 1 to 13 cm. long, 7 to I7 mm..in diameter; setae always noticeably longer than the spikelets, up to 14 mm. long. Certain specimens resemble purple- setaed specimens of S. viridis, but are readily distinguished by the free fruit and ordinarily by the larger spikelets. RANGE.—One of the most commonly cultivated strains and widely escaped in many parts of Canada and the United States. Specimens also seen from Italy, Hungary, Russia in Europe, India and the Philippines. SETARIA ITALICA (L.) Beauv. subsp. STRAMINEOFRUCTA Hubb. var. Hostit Hubb. subvar. METZGERI (K6rnicke) Hubb. forma curtiseta forma nov. Differt a subvarietate Metzgeri setis quam spiculis brevioribus vel vix superantibus. 6 Prof. Hackel wrote me that the combination was published on the label of this set of exsiccatae and synonymy is given on the printed label. 192 F. TRACY HUBBARD Differs from subvariety Metzgeri in having the setae shorter than barely exceeding the spikelets. RANGE.—Only specimen see, Italy—-Venetia—Patavium (Padova), FI. Ttalica Exicc., ser. 2, no. 1002 [Typein Gray herb:]) SETARIA ITALICA (L.) Beauv. subsp. STRAMINEOFRUCTA Hubb. var. brunneoseta var. nov. Differt imprimis a subspecie colore setarum. Setae flavidi- brunneae, longitudine variabiles, ad 17 mm. longae, semper quam spiculis multo longiores. Panicula lobulata, 9-27 cm. longa, 15-30 mm. in diametro. Fructus aliquando fuscostramineus. Spiculae 2-3 mm. longae. Differs from the subspecies chiefly in the color of the setae which are yellow brown, variable in length, but always noticeably longer than the spikelets, up to 17 mm. long. The panicle is lobulate, 9 to 27 cm. long, I5 to 30 mm. in diameter. Fruit sometimes dark straw. Spikelets 2 to 3 mm. long. RANGE.—Philippines, Luzon, Mangubat in 1906, Bur. Sci. Philipp. no. 1344 [Type in Gray herb.]. I have also seen a specimen from Tennessee, Robertson in 1873 and several cultural specimens. SETARIA ITALICA (L.) Beauv. subsp. STRAMINEOFRUCTA Hubb. var. BRUNNEOSETA Hubb. forma brachychaeta forma nov. Varietate similis sed setae quam spiculis breviores vel eas paullo superantes. Panicula interdum pro longitudine tenuior. Like the variety, but the setae shorter than or slightly exceeding the spikelets in length,—panicle sometimes relatively more slender. RANGE.—India—Mont. Khasia, Hooker & Thomson [type in Gray herb.| and Formosa—Bankiang, Henry, no. 571 are the only specimens of this form I have seen. SETARIA ITALICA (L.) Beauv. subsp. STRAMINEOFRUCTA Hubb. var. BRUNNEOSETA Hubb. subvar. densior subvar. nov. Differt imprimis a varietate panicula densiore compactiore elobata vel paululim ad basin lobata, 4-15 cm. longa, 8-23 mm. in diametro. Folia saepe grandia usque 40 cm. longa,37 mm. lata. Fructus inter- dum fusco-stramineus. Differs from the variety chiefly in the denser more compact panicle which is not lobed or very slightly so at the base, 4 to 15 cm. long, 8 to 23 mm. in diameter. The leaves frequently large up to 40 cm. long, 37 mm. broad. The fruit occasionally dark straw. RANGE.—Massachusetts—Weston, Wiliams in 1895 [Type in ) i ; . SETARIA ITALICA AND ITS IMMEDIATE ALLIES 193 Gray herb.]. I have also seen specimens from Maine, Louisiana, Russia in Asia and China, besides several cultural specimens. SETARIA ITALICA (L.) Beauv. subsp. rubrofructa nom. nov. Panicum, italicum var. erythrospermum Kérnicke, Syst. Ubers. Cereal. 19 (1873) [not P. erythrospermum Vahl, ex Hornem.]: Koérnicke, in Koérn. & Wern. Handb. Getreideb. 1: 274 (1885): Wern. in Korn. & Wern. heac.-2;: 802. (1885). This form according to Kérnicke, in K6rn. & Wern. |. c. 1::274 (1885) has a long slender, rather open, lobulate panicle up to 21 cm. long and 16 mm. in diameter; the setae are green and very long; the fruit intensive light red, shiny and is said to be small; the last state- ment according to Werner, in Kérn. & Wern. I. c. 2: 892 (1885). Nota.—Ko6rnicke names this form Panicum italicum var. erythrospermum, but it is inadvisable to take up this name as it is apt to lead to confusion owing to P. erythrospermum Vahl, ex Hornem. Hort. Hafn. 1: 82 (1813) which has purple setae and is referable to subvar. zzolacea. RaNGE.—I have seen no specimens; Koérnicke says from Botanic Gardens. SETARIA ITALICA (L.) Beauv. subsp. RUBROFRUCTA Hubb. forma gigas (Kornicke) comb. nov. Panicum italicum var. gigas Kornicke, in K6rn. & Wern. Handb. Getreideb. 1: 275 (1885): Wern. in Korn. & Wern. |. c. 2: 896 (1885). P. frumentaceum Hort. ex K6rnicke, in Korn. & Wern. I. c. 1: 275 (1885) in syn. sub P. italicum var. gigas. Kornicke, in Korn. & Wern. |. c. 1: 275 (1885) describes this form as having a very large and thick panicle up to 23 cm. long and 30 mm. in diameter. Setae less numerous and scarcely exserted: green [cf. Key on page 272]. Fruit dark red. RANGE.—I have seen no specimens; KG6rnicke says he received it from Hungary as Panicum frumentaceum and that this strain was derived from the Austrian East-Indian expedition. SETARIA ITALICA (L.) Beauv. subsp. RUBROFRUCTA Hubb. subvar. pabularis (Alef.) comb. nov. Panicum italicum Var. C Metzger, Eur. Cereal. 64 (1824). P. ttalicum var. pabularis Alef. Landw. FI. 315 (1866): K6rnicke, in K6rn. & Wern. Handb. Getreideb. 1: 276 (1885) [emended spelling var. pabulare]. This form is described by K6rnicke, in Kérn. & Wern. |. c. 1: 276 (1885) as belonging to the Moharium group,—that is small, dense- panicled,—having green setae and orange-yellow fruit. RANGE.—I have seen one cultivated specimen from Europe which 194 F. TRACY HUBBARD I have tentatively referred to this form; K6rnicke says that this form is unknown to him, but that it has been experimentally cultivated in Germany and France. SETARIA ITALICA (L.) Beauv. subsp. RUBROFRUCTA Hubb. var. purpureoseta var. nov. Differt a subspecie setis fusco-purpureis. Folia 30 cm. longa, 20 mm. lata. Panicula crebre lobulata ad basin interrupta, 23 cm. longa, 20 mm. in diametro; setae longae, purpureae. Fructus rubro- aurantiacus. Differs from the subspecies principally in having purple setae. Leaf 30 cm. long, 20 mm. broad. Panicle close-lobulate, base inter- rupted, 23 cm. long, 20 mm. in diameter. Setae longish, purplish. Fruit red-orange. RANGE.—India—Himalayan Herbarium Strachey and Wainter- bottom, no. 3 [Type in Gray herb.]. I have also seen cultural speci- mens in the Economic Collection of the U.S. Bur. Pl. Ind. SETARIA ITALICA (L.) Beauv. subsp. RUBROFRUCTA Hubb. var. PURPUREOSETA Hubb. subvar. violacea (Alef.) comb. nov. Panicum erythrospermum Vahl, ex Hornem. Hort. Hafn. 1: 82 (1813): Jacq. Eclog. Gram. 3: t. 24 (1815-20) [setae represented more pink than usual]. Echinochloa erythrosperma (Vahl) R. & S. Syst. Veg. 2: 477 (1817). Pennisetum erythrospermum (Vahl) Jacq. Eclog. Gram. 3: 34 (1815-20). Panicum italicum Var. D Metzger, Eur. Cereal. 64 (1824). Setaria erythrosperma (Vahl) Spreng. Syst. Veg. 1: 304 (1825) [excl. syn. laevigatum]. Panicum ttalicum var. violaceum Alef. Landw. Fl. 316 (1866): Kornicke, in Kérn. & Wern. Handb. Getreideb. 1: 276 (1885). P. ttalicum var. erythrospermum (Horn.) A. Br. Ind. Sem. Hort. Berol. 1871: 4 (1871). Panicle dense, often somewhat lobulate and interrupted at the base, but empty; 8 to 13 cm. long, 10 to 15 mm. in diameter. Leaves usually narrower and plant probably more slender and not as tall [specimens examined run from 5 to 8.5 dm. tall]. Setae purple or sometimes only purple-tipped, noticeably longer than the spikelets, but rather short, up to about 7 mm. RANGE.—The only specimens of this form which I have seen are cultural. SETARIA ITALICA (L.) Beauv. subsp. RUBROFRUCTA Hubb. var. rubra (Kérnicke) comb. nov. Panicum italicum var. rubrum Kornicke, in Korn. & Wern. Handb. Getreideb. 1: 274 (1885): Wern. in Korn. & Wern. |. c. 2: 892 (1885). : SETARIA ITALICA AND ITS IMMEDIATE ALLIES 195 Varies from the subspecies mainly in having brown setae which are short, but at least twice the length of the spikelets about 6 mm. long. Panicle rather slender, lobulate, somewhat loosely so and interrupted at the base, 13 to 17 cm. long, 13 to 15 mm. in diameter. Spikelets small. Fruit orange-brown. This form undoubtedly passes into subvar. condensa. RANGE.—I have seen specimens from Assam and the Philippines. SETARIA ITALICA (L.) Beauv. subsp. RUBROFRUCTA Hubb. var. RUBRA (K6rnicke) Hubb. forma aurantiaca (K6rnicke) comb. nov. Panicum italicum var. aurantiacum Kornicke, in Korn. & Wern. Handb. Getreideb. 1: 275 (1885): Wern. in Korn. & Wern. I. c. 2: 895 (1885). Setaria persica Hort., S. brevifolia Hort., Panicum macrurum Hort., and P. ertogonum Hort. all ex K6rnicke in K6rn. & Wern. |. c. £3275 (1885) sub P. italicum var. aurantiacum. Setae shorter than or barely exceeding the spikelets; panicles 9g to 10 cm. long, 8 to 10 mm. in diameter, otherwise like the variety.’ RANGE.—Only specimen examined, Philippines—Luzon, Ramos in 1909, Bur. Sci. Philipp. no. 7842. K6rnicke mentions the form from Algeria. SETARIA ITALIACA (L.) Beauv. subsp. RUBROFRUCTA Hubb. var. RUBRA (Ko6rnicke) Hubb. subvar. condensa subvar. nov. Differt a varietate panicula breviore non lobata. This form differs from the variety in having shorter, dense, not- lobed panicles, with some scattered empty tufts of setae below the ‘dense portion: measurements without these tufts 4.8 to 6.5 cm. long, about 10 mm. in diameter. Plant about 5 dm. tall and rather slender: leaves smaller and narrower. Fruit rather more orange-red than the variety. RANGE.—Only specimen seen, Assam, Chatterjee in 1902° [type in Gray herb.]. SETARIA ITALICA (L.) Beauv. subsp. nigrofructa nom. nov. Panicum italicum var. nigrum Kornicke, in Korn. & Wern. Handb. Getreideb. I: 274 (1885): Wern. in K6rn. & Wern. I. c. 2: 893 (1885). According to K6érnicke, in Korn. & Wern. |. c. 1: 274 (1885) this form has thick panicles somewhat shorter and less lobulate than Panicum italicum var. erythrospermum Kornicke—which is a synonym 7 Kornicke, in Korn. & Wern. |. c. 1: 275 (1885) gives somewhat larger measure- ments—panicle up to 17 cm. long, and up to 20 mm. in diameter. 8 Material of this collection in the U. S. Nat. Herb. is var. rubra. 196 F. TRACY HUBBARD of subsp. rubrofructa—; has green setae of medium length; the fruit mottled and violet-black. RANGE.—I have seen no specimens; K6rnicke reports it from Hungary. SETARIA ITALICA (L.) Beauv. subsp. NIGROFRUCTA Hubb. var. atra (K6rnicke) comb. nov. Panicum italicum var. atrum Kornicke, Syst. Ubers. Cereal. 19 (1873): Kérnicke, in Korn. & Wern. Handb. Getreideb. 1: 277 (1885): Wern. in Korn. & Wern. I. c. 2: 899 (1885). Setae brownish-purple, noticeably longer than the spikelets, up to 7 to 8 mm. long. Panicles dense, occasionally somewhat lobulate and interrupted at the base, I.5 to 10 cm. long, 7 to15 mm. in diameter, frequently with clusters of empty setae below the dense portion of the panicle. Fruit brown-black to black when mature and well developed. [There are always present in larger or smaller proportion less well developed pale-straw colored fruits.] | RANGE.—AIl the specimens which I have seen are cultural. The cultural names applied to the various forms of S. ztalica— collectively known as Foxtail Millets—often cover more than one form of the variants. Sometimes no distinction is made in setae color sometimes the density of the panicle is not a factor. Certain strains in cultivation have been given names and frequently these names seem to have been used for more than one strain. For the application of the following cultural names I am indebted to Mr. H. N. Vinall and Mr. M. A. Carleton of the U. S. Dept. of Agriculture. GERMAN MILLetT.—A large lobed form with long setae, green or purple in color, no distinction being made. Applies indiscriminately to subsp. stramineofructa and var. Hostii. Cf. Carleton, in Bail. Cycl. Am. Agric. 2: 469, f. 695 (1907). GOLDEN WONDER MILLET.—A large lobed form with setae shorter than or barely exceeding the spikelets. Applies to forma breviseta. CoMMON MILLET.—A compact form with dense ; vike-like panicles of the Moharium group. The setae are long, but no distinction of color is made. Applies indiscriminately to subvar. germanica and subvar. Metzgerit; probably also to stout-panicled, Jarge-fruited specimens of subvar. densior. Cf. Carleton, in Bail. 1. c. 2: 469, f. 694 & 470, f. 697 (1907). : AINo MILLET.—Sometimes termed Japanese Millet, a misleading name, as it covers various strains. Commonly a slender lobulate form with brown setae and small fruit, about 2 mm. long, but also used SETARIA ITALICA AND ITS IMMEDIATE ALLIES 197 for slender, small-fruited, compact-panicled specimens. Cultivated by the Ainos of Japan and also occurring in the Philippines. Applies to the slender-panicled, small-fruited specimens of var. brunneoseta and to similar specimens of subvar. denstor. Cf. Carleton, in Bail. ieee 2: 469, 1. 696 (1907). TuRKESTAN MILLET.—A robust growing form with lobulate panicles, orange or reddish fruit and purple setae. Similar to Kursk Millet, but much coarser and ripening later. Applies to var. pur- pureoseia. Kursk MILLET.—Sometimes called Russian Millet, but this term seems to have been used also for the Siberian Millet. A form, belong- ing to the Moharium group, with dense or at base slightly lobed pan- icles, red or orange fruit and long, purplish setae. Said to be the result of breeding selection from Siberian Millet. Applies to subvar. violacea. SIBERIAN MILLEtT.—A form with large, more or less open lobulate panicles, orange or red fruit and long, brown setae. Applies to var. rubra, but judging by the figure under the name Red Siberian Millet it has been also used for subvar. vzolacea, cf. Carleton, in Bail. |. c. 2: 469, f. 693 (1907). HUNGARIAN MILLET or Mouar.—Also called Hungarian Grass. A form, belonging to the Moharium group, with dense, spike-like panicles, fruit blackish or brownish-black with pale yellowish-straw fruits intermingled (these sometimes predominating) and long purplish- brown setae. The panicles average smaller than those of the other forms commonly cultivated [a small strain of subvar. Metzgeri is apt _ to beas small]. Applies correctly to var. atra, but seems to have been used also for subvar. Metzger1, cf. Carleton, in Bail. 2: 470, f. 698 (1907). The following combinations have’ commonly been refered as synonyms to Setaria italica, but I believe that they are better referable to other species. Panicum indicum Mill. Gard. Dict., ed. 8, Panicum no. 3 (1768) [cited by Hook. f. Fl. Brit. Ind. '7: 78 (1896) asa synonym of S. ttalica. The Miller description points to Pennisetum americanum (L.) Leeke and the Bauhin reference given by Miller is certainly P. americanum. Mr. A. B. Rendle writes me that there is no act of Panicum indicum in the Miller herbarium]. P. laevigatum Muhl. Cat. 9 (1813) nomen: Muhl. in Ell. Sk. 1: 112 198 F. TRACY HUBBARD (1816): Muhl. Descr. Gram. 100 (1817) [I believe this is undoubtedly a form of S. wmberbis, cf. Scribn. & Merr. U.S. Div. Agrost. Bull. 27: 2 (1900)]. Setaria rubtcunda Dum. Obs. Gram. Belg. 139 (1823) [this is referred to S. ttalica, but seems better referable to S. glauca, cf. Hook. 1 Al Brit mceeza7© (soo) |. S. multiseta Dum. |. c. 138 (1823) [referred in synonymy to S. italica by Richter, Pl. Eur. 1: 28 (1890), but I believe it is more probably S. glauca though ‘“‘setae . . . deorsum scabrae”’ does not fit S. glauca and does apply to S. verticillata). Panicum viride var. rubicundum (Dum.) Lej. Rev. FI. Spa 217 (1824): Lej. & Court. Comp. Fl. Belg. 1: 51 (1828) [cf. Setaria rubi- cunda Dum. on which it is based]. Setarta viridis var. rubicunda (Dum.) Th. Dur. in Wildem. & Dur. Prodr. Fl. Belg. 3: 80 (1900) [Durand did not publish the variety in Cat. Fl. Liége as he states, in Prodr. Fl. Belg., that he did: cf. S. rubicunda Dum.]. S. viridis var. multiseta (Dum.) Th. Dur. im Wildemee Win Prodr. Fl. Belg. 3: 80 (1900) [Durand did not publish the variety in Cat. Fl. Liége as he states, in Prodr. Fl. Belg., that-he did: cf. S. multiseta Dum. on which it is based]. Resp oe ‘anita aie Bolsbicd) Botiety of America a een i the sochtty* ~ for. Plant. Morphology. and Frcleny eee) : oF the American Mycological _ Sodety (1903). : ap Ne a ices ee Pans fra th i Bs OFFICERS FOR 1915 2, se x 5 President: Tonx. M. ‘Counter. ee, De eo : Chisego, I Hl. oe aes Vise residents R os ioe: < ee ie) Ss UE a ai New York, N, a | | Treasurer: “Aon Hobie: re New ese, N. oe aaa fe Nee eee Maa oe inte ‘Secretary ay ‘HL. Barrverr’ ere Ee aC AGES pa Se ace aaa! Washingion, D. es ae Cy Raabe Councilors: ee : : ONE pe et cs ae Moe ek Geoace Eg le aa i a ey ee - ugk ae - Ithaca, N. Yy. ey PT ig pn me er GIN EFS Davin Pike: Poe ek dae EE EL ery a, oe en «Washington, D. c es ec as WwW. rR Ganonc : ae Pe ho 5 a Ue = a _ Northampton, Mass. : The die: consists 7 ee and snembers: id any one sei in- ie sro in Ce boc is ae for reeeneohir Candidates for. member- me at ave to s the Hakegicaw: Pee OF Borany, : ‘Caudiaater for tines u _proposed and approved by. the Council, may receive the Journa at $3. 00 pee oe s “ae A gene es gt tee thei = election at the x winter meeting of the Society. n 1907, ‘we. eid. Sr Mr Rutherford Trowbuides ge,: New. ven, Conn., this. on€ compartment house, 23 feet wide by feet long, Last year,two ‘more com ah ole were added as shown, above. ‘Let us plan some su plan for you, - spas ans pees. a — becaus ey P u with hha in vow. ying a U. tee ‘) mone oS UBAR ct t OFFICIAL PUBLICATION. oF THE: BOTANICAL SOCIETY OF AMERICA. eae : | t \ ete > a : fe irs = rd ° x Me ia ws ; f 3 i Aimy *! : . mn o x r i) hae ele bj i z = Sore , "ait f y iV re le LF : 4s Le 4 (saree eT EE] oe 4 A URIs Puan cma < = 1) ss en ¥ K dana ery 7 i Bs Rn CONTENTS | The sorphology and epstematic position. of Bodomietua : ‘ Doveias Houcuton CAMPBELL 199) | 2 aii hase On the selation of root owe and development to the temperature and : Sore _ aeration of the RO Ng aceon eo We A. CANNON 211. “The anatomy of a hybrid Rquisetum............)-...,.-Rom HoLpEN Ree a Some’ features in the anatomy of the Malvales. ee ee C. ForsairH | 238 : "The absorption o of ic ions by Bases tar dead roots... PEON Na au V. JOHNEON 250° ; a Wee ee: eS : PUBLISHED. : ey : IN COOPERATION wit THE E BOTANICAL SOCIETY OF » AMERICA oe ro p Ss ‘BROOKLYN BOTANIC GARDEN | Ar 41 Noara Quaew Street, Lancasrex, Pa. “Batered as ssecond-) I r ;; ig ins a ¥ rT . i The Society. consists of. iallows Pen members), and any one ¢ actively ir ine ss ion " terested i in botanical work i is eligible for membership. Candidates for member a pe ‘ ae are recommended by three’ members, oy members: of the Council, on gs ty ‘ pe to be obtained from the ogra ie ne ae ae : a on Ae o i% SWE A SSA SS SSS i 1907, we ded for Mr. Ruthedeid Towbadee New f Haven, Caras this one compartment house, 23 feet wide by |Z 33 feet long, Last year, two more. compartments were; odds as shown above, Let us plan some ss plan for You. Ne and regrets are tooling aw ay your satisfaction. Send oR catalog ¢ or r send for us. Or both, e Se _UBAR_GREENHOU SES. ie PIERSON U-BAR CO | ONE MADISON AVE. NEWYORK. ‘CANADIAN OFFICE, 10 PHILLIPS PLACE. mona» ’ ea “a ap ; by y ‘| : f i" : ‘ \ Lf 1 Tet y Ties i j ‘ ay 0 Mus ‘ : Phy OMAR aE i is vy ! \ \ ‘ f, ig 1 I at i 4 i Ni See We co ch i ve 5 hs st ea" * i Y, f i; - “BOTANICAL, § SOCIETY OF AMERICA _ ae “NOV 5 195 te wins? | Capa paeea aN EN earn hans ne i pone of “ ety of en water. Shab es ae P. Hipparp 389. ri vg "pressed vegetable saps from the depression of the freezing point Beer Sikes ne eG ROR Gn “Je ARTHUR HARRIs 418 "Cale Ibypochlarite asa seed sterilizer, Leahey tlle James: KY Witson ‘420° i re f : , Zz 5 fs and others that pentosans as such disappear in * Published by permission of the Secretary of Agriculture. 1Swartz, Mary Davies. Nutrition Investigations on the Carbohydrates of Lichens, Algae, and related substances. Trans. Conn. Acad. Arts and Sciences 16: ZAg-352. IOI. 2Lindsey, J. B., and Holland, E. B. Concerning the Digestibility of the Pentosans. Ann. Rep. of Mass. Agr. Exp. Sta. 1894: 175-188. 8 Slowtzoff, B. Ueber das Verhalten des Xylans im ThierkGrper. Zeitschr. Physiol. Chemie 34: 181-193. 1901-2. 4 Goetze, K., and Pfeiffer, Th. Beitrage zur Frage iiber die Bildung resp. das Verhalten der Pentaglykosen im Pflanzen- und Tierkérper. Landw. Versuchs- Stationen 47: 59-93. 1896. 5 Swartz, Mary Davies. Loe. cit. [The Journal for July (2: 311-374) was issued 18 Aug I915] S/S) 376 LON A. HAWKINS passing through the alimentary canal of higher animals, but it seems doubtful from the work of Seilliére® and of Swartz that the digesting enzymes are secreted by the higher animals themselves. Seilliére attempted to show that the digestion of xylan by various animals was not carried out by the intestinal or pancreatic juices of these animals but by the bacteria present in the intestines. His work seems to be corroborated by the investigations of Swartz. Seilliére’ showed further, however, that the extracts of the digestive organs of snails hydrolyzed xylan, and later obtained similar results with other molluscs’ and also with the larvae of certain Coleoptera.?® The question of the utilization of pentoses and pentosans by plants, especially by fungi, has received some attention. Behrens!? has shown that Botrytis vulgaris and some Penicilliums can utilize both arabinose and xylose as a source of carbon. Went" obtained good results when growing Monilia sitophila (Mont.) Sacc. upon xylose. Cross, Bevan and Smith,” Schéne and Tollens,™ and Cross and Tollens“ consider that the disappearance of pentoses in fermenting mixtures of sugars is due to the utilization of pentoses in the building up of the yeast plant. Kriiger’™ has recently shown that a species of Gloeosporium parasitic upon apple, which he calls Gloeosporium fructigenum germanicum, can utilize arabinose. Other writers have 6 Seilliére, G. Sur la digestion de la xylane chez quelques mammiféres herbi- vores. Compt. Rend. Soc. Biol. 64: 941-943. 1908; Sur la digestion de la xylane chez les mammiféres. Compt. Rend. Soc. Biol. 66: 691-693. 1909. 7 Seilliere, G. Sur la présence d’une diastase hydrolysant la xylane dans le suc gastro-intestinal de L’Escargot. Compt. Rend. Soc. Biol. 58: 409-410. 1905- 8 Seilliére, G. Sur la présence de la xylanase chez différents Mollusques gastéro podes. Compt. Rend. Soc. Biol. 59: 20-22. 1905. 9 Seilliére, G. Sur une diastase hydrolysant la xylane dans le tube digestif de certaines larves de Coléoptéres. Ibid. 58: 940-941. 1905. 10 Behrens, J. Beitrage zur Kenntnis der Obstfaulnis. Centralbl. Bakter. und Parasit. Abt. II 4: 547-553. 18098. 1 Went, F. A. F. C. Monilia sitophila (Mont.) Sacc., ein technischer Pilz Javas. Centralbl. Bakter. und Parasit. Abt. II 7: 591-598. 1901. 12 Cross, C. F., Bevan, E. J., and Smith, Claude. The Carbohydrates of Barley Straw. Journ. Chem. Soc. 73: 459-463. 1808. 18 Schéne, A., und Tollens, B. Ueber die Garung der Pentosen. Journ. Landw. 49: 29-40. 1901. 14 Cross, W. E., und Tollens, B. Versuche tiber das Verhalten der Pentosen in garenden Mischungen. Journ. Landw. 59: 419-428. I9II. 16 Kriiger, Frederich. Beitrage zur Kenntniss einiger Gloeosporien I und II. Arbeit. Kaiserl. Biol. Anstalt Land und Forstwirt. 9: 233-323. I913. THE UTILIZATION OF CERTAIN PENTOSES B77. shown that the pentoses can be used by fungi, but it is generally agreed that they can not be fermented. In regard to the utilization of pentosans by fungi it seems probable from the work of Czapek" and Schérstein’’ that certain components of the cell walls of woody tissues are rendered soluble and utilized by some fungi. The last mentioned writer reaches the conclusion that xylan is digested by Merulius lachrymans. He bases his conclusion upon the observed difference in optical rotation of extracts of wood before and after it had been acted upon by the fungus. Swartz!§ found apparently that the pentosans of Rhodymenta palmata, a marine alga, were hydrolyzed to a reducing substance by ‘Taka diastase.”’ Duggar and Davis!® recently announced that they were unable to demonstrate the presence of a pentosanase in Fucus. It seems then that pentosans and pentoses are of some value as food for higher animals but that these organisms probably secrete no enzymes capable of hydrolyzing pentosans. Some of the invertebrates can utilize pentoses readily and seem to be able to hydrolyze some pentosans. Pentoses have been found to be a good source of carbon for certain fungi and there is evidence that some pentosans may be broken down through the action of enzymes secreted by fungi. The products of such enzyme action have apparently not been identified. It was to obtain more information upon the effect of parasitic fungi on pentoses and their compounds that the experiments described in this paper were planned and carried out. The study includes a series of experiments on the effect of Glomerella cingulata (Stonem.) S. & v. S. upon pentose compounds in the apple fruit, a series on the utilization of pentoses and certain of their compounds as sources of carbon for this fungus, and experiments upon the effect of the extract of the fungus mycelium under aseptic conditions on xylan. The fungus was isolated from apples kindly furnished by Dr. C. L. Shear and was maintained in stock culture in tubes of cornmeal agar throughout the study. In the experiments on the effect of the fungus upon the pentose-containing compounds in apples practically 16 Czapek, F. Zur Biologie der holzbewohenden Pilze. Bericht. Deutsch. Bot, Ges. 17: 166-170. 1899. 17 Schorstein, J. Zur Biochemie der Holzpilze. Centralbl. Bakter. und Parasit. Abt. II 9: 446-447. 1902. 18 Swartz, Mary Davies. Loc. cit. 19 Duggar, B. M., and Davis, A. R. Enzyme Action in Fucus vesiculosus L. Ann. Mo. Bot. Gard. 1: 419-426. I914. 378 LON A. HAWKINS the same methods of sampling and analysis were used as in a former study of peaches.2° The different compounds were determined in the two halves of the same fruit, one portion of which had been inoculated with the fungus while the other was retained sterile as a control. In the determination of the furfurol-yielding material it was found that the percentage of this substance in the rotten half was considerably less than in the sound portion. The fungus appar- ently used the furfurol-yielding constituents of the apple fruit. Sev- eral apples were then prepared and inoculated. After two weeks the sound and rotten halves were sliced up and extracted with alcohol. The furfurol-yielding material was then determined in the extract and solid portions separately. The results are given in Table I. All data were calculated as percentage of the original wet weight of the portion of the apple used. TABLE | Percentage of Alcohol-soluble, Alcohol-insoluble, and Total Furfurol-yielding Material in Sound and Rotten Halves of Apple, Each Substance Determined in Sound and Rotten Halves of Same Fruit — — Alcohol-soluble Alcohol-insoluble | Total ex | ae Z sarees (eee Bee : Hes Sound half | Rotten half Sound half | Rotten half | Sound half Rotten half 0.12 0.18 | 0.62 0.49 0.74 | 0.67 0.12 0.16 | 0.71 0.50 0.83 | 0.66 0.14 0.27 | rT 0.93 1.25 1.20 From the data in Table I it is apparent that the total percentage of furfurol-yielding material and the percentage of alcohol-insoluble furfurol-yielding material were higher in the sound portion than in the corresponding rotten half of the apple, but that the percentage of alcohol-soluble furfurol-yielding material was higher in the rotten. half. Pentose sugars are readily soluble in 80 percent alcohol while most of the furfurol-yielding material in the sound apple is not. The increase in alcohol-soluble furfurol-yielding material during the early stages of the rot therefore indicates that some compounds con- taining pentoses were broken down. That this was due to the action of the fungus and not to the autolysis of the dead apple tissue was evident from the fact that when portions of apple in which the cells had been killed with chloroform were allowed to stand under aseptic 20 Hawkins, Lon A. Some Effects of the Brown Rot Fungus upon the Compo- sition of the Peach. Amer. Journ. Bot. 2: 71-81. I915. TS Bd Pe en easy, THE UTILIZATION OF CERTAIN PENTOSES 379 conditions for three weeks they contained practically the same per- centage of both alcohol-soluble and alcohol-insoluble pentosans as control portions of the same apples in which the cells had not been killed. When the fungus was allowed to act upon the apple four weeks or more the percentages of furfurol-yielding material in the alcohol ex- tract and in the solid residue were both higher in the sound half. Itis evident then that the fungus is able to break down the pentose- containing compounds, whether they be pentosans or compounds of the pentoses with other sugars, and to use the pentoses. There is no evidence at hand, however, to show which pentoses can be used or what compounds of the pentoses are most readily broken down by the fungus. This was made the subject of further experimentation. An attempt was made to determine the value of xylose, arabinose, xylan and arabin as compared with glucose as sources of carbon for this fungus. The fungus was grown on a solution of nutrient salts to which the sugars and compounds of the pentoses were added. The medium was prepared according to a formula used by Hasselbring” in his work on Penicillium. It was composed of NH.zNO31 g., KHePO, 0.5 g.. MgSO, 0.25 g., and 100 cc. H.O. To this was added the carbohydrate usually in sufficient quantity to make a I percent concentration. Three hundred cc. Erlenmeyer flasks were used for the cultures. In each of these 100 cc. of the culture solution was placed. These solutions were inoculated with the fungus by trans- ferring conidiospores from the stock cultures with a sterile needle. After a suitable period the felt of mycelium was removed from each flask separately, washed with a little water, placed in a tared, glass- stoppered weighing bottle and covered with alcohol. The alcohol was driven off at low temperature, about 60° C., and the weighing bottles with mycelium dried to constant weight in a vacuum drying oven at 78° C. The sugars, the arabin, and a small part of the xylan used in these experiments were purchased from a chemical supply house. The rest of the xylan was prepared from rye straw, most of it according to a method similar to that used by Schéne and Tollens* for the prepara- * 2 Hasselbring, H. The Carbon Assimilation of Pencillium. Bot. Gaz. 45: 176— 193. 1908. *3 Schone, A., und Tollens,.B. Untersuchungen tiber die Pentosane der Jute, der Luffa, und der Biertreber. Journ. Landw. 49: 21-28. tIgot. 380 LON A. HAWKINS tion of xylan from jute. The ground straw was extracted with 2 percent solution of ammonia and then digested 48 hours with a 6 percent solution of KOH, 6 liters to each original half kilogram of straw. The extract was pressed out, filtered through cloth and the xylan precipitated by adding an equal volume of 95 percent alcohol to the solution. It was then filtered and the precipitate neutralized with a solution of HCl in alcohol. The xylan was washed in a Buch- ner funnel with 60 percent alcohol until the washings gave no test for chloride with silver nitrate. It was then dried with absolute alcohol and ether, ground up in a mortar, passed through a fine sieve and was ready for use. In order to prove that the preparation was a compound of xylose sugar, a quantity of the xylan was hydrolyzed with sulphuric acid according to the method of Wheeler and Tollens.”* The mixture of xylan and 5 percent sulphuric acid was heated on the steam bath for six hours. The acid was then neutralized with magnesium-free calcium carbonate, filtered, and the filtrate evaporated nearly to dryness under reduced pressure. The residue was taken up with hot alcohol and treated with animal charcoal to decolorize. The solution was concentrated in a vacuum desiccator over sulphuric acid and in a few days the sugar crystallized out. It was recrystal- lized from alcohol several times. The crystals were white and had the characteristic form of xylose. They melted at 143°-144° C., uncorrected. The sugar had a specific rotation, [a]p, of + 18.92 as compared with + 19.22” for xylose. It reduced Fehling’s solution, formed furfurol which was precipitated as the phloroglucid when boiled with a hydrochloric acid solution of phloroglucin. The osazone was prepared in the usual way and crystallized as bright yellow needle- like crystals, insoluble in cold water, soluble in hot, and melting at 160° C,, uncorrected, as compared with 160° C. for xylosazone according to Tollens.2 The sugar was apparently xylose. In the preparation of xylan, Salkowski’s method?’ was also used. 24 Wheeler, H. J., und Tollens, B. Ueber die Xylose oder den Holzzucker, eine zweite Penta-glycose. Zeitschr. Vereins Riibenzucker-Industrie Deutsch. Reichs 39: 848-868. 1889. | 2 Parcus, E., und Tollens, B. Ueber die Mehr- oder Weniger-Drehung (Multi- Rotation oder sog. Birotation und Halbrotation) der Zuckerarten. Liebig Ann. 257: 160-178. 1890. : 26 Tollens, B., et al. Untersuchungen iiber Kohlenhydrate. Zeitschr. Vereins Riibenzucker-Industrie Deutsch. Reichs 41: 885-911. 1891. 27 Salkowski, E. Uber die Darstellung des Xylan. Zeitschr. Physiol. Chem. 34° 162-180; T9q0t—02; THE UTILIZATION OF CERTAIN PENTOSES 381 According to this method the ground straw was boiled in 5 percent KOH for 1 hour, the extract pressed out with a fruit press and filtered through cloth. Fehling’s solution was added in the proportion of one half liter to every 300 g. of straw used. The precipitate was separated by filtration, washed and treated with an alcoholic solution of hydrochloric acid. It was washed several times with 60 percent alcohol, dried and dissolved in dilute KOH and again precipitated with Fehling’s solution. It was then washed and acidified as before and finally freed from chloride and dried. Salkowski considered that xylan prepared according to this method was practically free from araban and other hemicelluloses, cellulose and starch, and that it was nearly pure. This preparation yielded 74.0 percent xylan, according to Krober’s tables,?2 when analyzed according to Tollens’ phloroglucid method for the determination of pentosans, which is one percent more than was found in the xylan prepared according to Schéne and Tollens’ method and analyzed in the same way. The commercial preparation of xylan used in these experiments gave 86.1 percent of the the- oretical amount. The xylan prepared according to Salkowski’s method dissolved in I percent KOH had a specific rotation, [a]p, of — 83°. It contained 0.5 percent ash. Swartz?? mentions some which she prepared which yielded 72.0 percent of the theoretical amount according to Krober’s tables. She found the specific rotation of one sample to be — 83°. Tollens gives the specific rotation of xylan from wheat straw as — 84.1°. Swartz’s determinations of ash in the xylan were somewhat higher than those obtained in the present study while Salkowski reports that some of his preparations contained as low as 0.7 percent ash. The xylans prepared by both Tollens’s and Salkowski’s methods were tested for galactan and methyl pentosans with negative results. No starch was present. It is apparent from the above described experiments that the compounds prepared were composed largely of xylose. The first series of experiments was made with the two pentose sugars as compared with glucose as a source of carbon. The sugars were added to the solutions of nutrient salts in quantity to make 28 Wiley, H. W., et al. Official and Provisional Methods of Analysis. Asso- ciation of Official Agricultural Chemists. U.S. Dept. Agr., Bur. Chem. Bull. 107. 1907. 29 Swartz, Mary Davies. Loe. cit. 382 LON A. HAWKINS one percent concentration. In preparing the culture media the solutions of salts were made up at a somewhat higher concentration than finally desired and sterilized in the autoclave, then diluted to the proper concentration by the addition of the sugar solutions which had been sterilized separately in a steamer. The culture solutions were inoculated and the fungus allowed to grow twelve days in an incubator at about 28° C. At the end of this time the mycelium was removed and dried to constant weight. The culture solutions were tested for reducing sugars, and the solutions which originally contained xylose or arabinose were tested for pentoses. Traces only were found. The sugar had been almost entirely removed from the solution by the fungus. The weight of the mycelium from the twelve flasks is shown in Table II. TABLE II Comparative Yields of Mycelium, Expressed in mg. Dry Weight, from Cultures of Glomerella cingulata on Nutrient Salt Solutions with Glucose, Arabinose or Xylose as Source of Carbon Glucose Arabinose Xylose 365.1 382.0 401.4 347-5 37203, 4. 383.8 217.3 337.2 380.1 365.3 349.5 386.1 The pentoses seem to be slightly better sources of carbon than glucose. Xylose is apparently the best, though the difference is not great. It is very evident that the pentoses are readily utilized by this fungus. A series of culture solutions was next made using arabin and two xylans, one prepared from rye straw and the other the commercial preparation. The yield in dry weight of fungus mycelium with the amount of furfurol-yielding material used in each case is given in Table III. The amount of arabin or xylan used was determined by TABLE III Comparative Yields of Mycelium and Amount of Pentosan Used with Cultures of Glomerella cingulata Grown upon Nutrient Salt Solutions with Xylan or Arabin as the Source of Carbon Commercial Xylan Xylan Prepared from Rye Straw Arabin | Sse ? 2 } : g Z f Arab wclaeoe Herceuiaes et Xylan Vislde mes Bee Xylan Vielduene Perce rabin AOD | cia, Ang cS eae DOP 2 wa 86.0 lin 280:9 88.0 TAs 15.0 196.0 | 85.0 Fie Na oe ere 64.6 14.0 179.9 90.0 Neorg sboe 98.2 13.0 205.4 | 87.0 eran ie owe ys or 57920 oul et OLm THE UTILIZATION OF CERTAIN PENTOSES 383 difference between the amount remaining in the culture solutions and the amount recovered from uninoculated control flasks, prepared in exactly the same way as the flasks for cultures. The flasks were kept in an incubator at 28° C. throughout the course of the experi- ment, which lasted fifteen days. | It is obvious from Table III that these compounds of pentose sugars can be utilized by the fungus as sources of carbon. It is apparent that the yield of mycelium was less after fifteen days with either arabin or one of the xylans as the source of carbon than in the case of any one of the three sugars in twelve days. A much larger amount of the compound remained in the solution than in the case of the sugars which were removed almost quantitatively. It is evi- dent then that xylan and arabin are not as readily available for the fungus as the pentose sugars themselves. A comparison of xylan and arabin shows that the yield of dried mycelium was considerably more than twice as great from the cultures containing xylan, while a much higher percentage of the material was removed from the solution. The conclusion is obvious then from the table that xylan, although not as good a source of carbon as xylose, arabinose, or glucose is readily available and can be utilized by the fungus. It also seems probable that in the utilization of the xylan it is broken down into simpler compounds and that one or several steps in its digestion and assimilation is the hydrolysis of xylan to xylose, the sugar. If this is the case there is probably an enzyme secreted by the fungus which brings about this hydrolysis. A series of experiments was planned and carried out to see if the - extract of the fungus was able to hydrolyze xylan to xylose under aseptic conditions. “The fungus was grown on the mixture of nutrient salts already described with gum arabic or glucose as a source of carbon. The cultures were allowed to grow about three weeks before the mat of mycelium was removed. It was separated from the culture solution, washed with a little water and ground up in a mortar. The resulting pulp was then placed in a flask with water and a little chloroform and allowed to stand with frequent shaking for 24 hours. It was then filtered and the extract used in the digestion experiments. The xylan was prepared by weighing small quantities, 0.2 or 0.3 g., into 100 cc. flasks to which 25 cc. of water was added. The mixture was then boiled, cooled, and 25 cc. of the extract from the fungus mycelium was added to each flask. The mixtures were neutral to 384 LON A. HAWKINS litmus. Several of the flasks containing the xylan and extract of the fungus were again boiled as controls. Chloroform was finally added to all the preparations as an antiseptic. The flasks were then placed in an incubator at a temperature of approximately 30° C. and allowed to remain with frequent shaking for the required time. To see whether the preparationss were contaminated with microorganisms frequent inoculations were made from the mixtures to various culture media. No organisms were found. The first series of experiments was carried out with six preparations for each experiment, all alike excepting that three had been boiled. After these had remained in the constant temperature chamber from five to eight days they were removed, and the contents of the flasks washed into beakers with 95 percent alcohol. Sufficient alcohol was added to bring the mixture up to about 80 percent alcohol, thus pre- cipitating the unchanged xylan. The mixture was filtered and the alcohol evaporated from the filtrate on the steam bath. One pair of preparations consisting of one unboiled and one boiled control was used for the determination of the furfurol-yielding substance. A similar pair was used for the determination of the reducing substance. These last were cleared with neutral lead acetate, filtered, the excess lead precipitated as oxalate, and the mixture filtered again. The sugar was determined in the filtrate, using Allihn’s modification of Fehling’s solution.2® The dry cuprous oxide was weighed directly. The third pair of preparations was usually used for the preparation of the phenylhydrazine derivative which will be taken up later. It has been shown earlier in this paper that xylose is soluble in 80 per- cent alcohol, while xylan is not; also xylose is a reducing sugar, forms furfurol when boiled with HCl and reacts with phenylhydrazine to form a characteristic osazone. It is apparent then that if the amount of alcohol-soluble furfurol-yielding material and reducing substance is greater in the unboiled preparation it will be evidence that a pentose sugar results from the action of the extract of fungus mycelium upon the xylan. This evidence will be strengthened if the phenylhydrazine derivative is similar to xylosazone. The results of this series of experiments are shown in Table IV. The phenylhydrazine derivative was prepared in the usual manner in the third unboiled preparation in all the experiments given in Table IV. In all cases it proved to be the same, bright yellow needle- 30 Wiley, H. W., et al. Loc. cat. THE UTILIZATION OF CERTAIN PENTOSES 385 TABLE IV Comparative Effect of Boiled and Unboiled Extract of Fungus Mycelium upon Xylan from Rye Straw as Shown by Alcohol-soluble Furfurol-ytelding Material and Substance Reducing Fehling’s Solution Cuprous Oxide enved from . . bli ? Duration of | Amount of Xylan in bess) Bea Bae. See Amount of Pentoses Experiment Each Preparation cs we Unboiled Boiled Unboiled Boiled g. mg. mg. mg. mg. 6 days 0.3 123-8 31.5 67.5 9.2 Shoe 0.3 93.6 18.1 52.1 8.3 8 z 0.2 70.5 1251 46.7 8.1 ee 0.3 133.0 31.4 67.8 9.5 10 0.2 108.2 17.6 47.4 9.5 shaped crystals soluble in hot water, insoluble in cold, and melting at 161°-162° C. uncorrected, as compared with 161° C. for xylosazones prepared from the xylose sugar obtained from rye straw and from the commercial xylose as used in the experiments already described. Some of the control preparations were treated in a like manner, but the quantity of osazone formed was so small that it was impossible to identify it with certainty. It is obvious from Table IV that there is much more alcohol- soluble substance which reduces Fehling’s solution and furfurol- yielding substances in the unboiled preparations than in the boiled controls. That this increase is not due to the autolysis of the filtered extract of the fungus mycelium itself was proved by the fact that when the extract was accorded exactly the same treatment as the unboiled preparations no appreciable amount of reducing substance was found and no measurable amount of furfurol-yielding material. The above mentioned considerations and the fact that the phenylhydrazine deriv- ative is similar to the osazone of xylose seems to show that xylose re- sults from this action of the unboiled extract of fungus mycelium upon the xylan. Whether this hydrolysis takes place immediately or the amount of alcohol-soluble reducing substance and furfurol-yielding material present at the completion of the experiment is the result of a gradual breaking down of the xylan is now shown by the results already given. Further experimentation was necessary to obtain evidence upon this point. In these experiments twelve or more similar preparations were made, as already described, several of which were boiled as 386 LON A. HAWKINS before for controls. The flasks were placed in an incubator and shaken frequently. At intervals some of the preparations, both unboiled and boiled, were removed from the incubator, the xylan precipitated and alcohol-soluble furfurol-yielding substances determined. In the experiments, the results of which are given in Table V, both TABLE V Production of Alcohol-soluble, Furfurol-yielding Material, and Substance Reducing Fehling’s Solution, by Action of Extract of Fungus Mycelium upon Xylan from Rye Straw. 0.3 g. Xylan in Each Preparation. Yield in mg. Pentoses and mg. Cuprous Oxide | Unboiled Preparations Boiled Preparations Duration of Action a —- Cuprous Oxide Pentoses Cuprous Oxide Pentoses mg mg mg mg Besinning : orcs Niet ory Ont ee 227, 9.5 4 days ye 3 eee. 7 akg Sy ae eM hn It cae 99.8 63.5 19.4 9.0 OPI iment d, oS 100.4 6625). orl Ss ee 1Ooy | 100.8 66.3 20.6 9.4 the reducing substance and the furfurol-yielding material are given. In the other experiments (Table VI) only the latter was determined. It is evident from Tables V and VI that the hydrolysis of the TABLE VI The Production of Alcohol-soluble, Furfurol-yielding Substances by Action of Extract of Fungus Mycelium upon Xylan. Yield as mg. Pentoses j - t of Xylan ; Unboiled No. iene ance veer on pene | Boiled Control in g. I ° 2 | mg mg. mg I 0.3 At beginning i Gree ieee | 9.7 20 hours | 34.1 35.0 irae Aa | 40.0 28.2 10.5 es chethoeur | 41.2 Aaa | a eee PST OG wien | AGES 44.0 | 8.8 2 0.3 At beginnings se eteres |e re ere | 9.9 | 20 hours 19.0 FO,0 70? 5| eee Pie: i 250 25:4 A eee 6Or i) 31.0 LT oe SO) ea 38.7 BAe | 8.1 2 | Oe At beginning is ieee eee | 8.8 | 12 hours 13.5 13.8 (4: Si eee We icon 15.9 LOR yh any ire eee 7O oR se 1722 galt Pome ere O4 at 17-810 tenet ean 8.8 THE UTILIZATION OF CERTAIN PENTOSES 387 xylan did not take place immediately but that the amount of alcohol- soluble furfurol-yielding substance (xylose) increased the longer the extract was allowed to act. The rate of hydrolysis was much more rapid at first and gradually decreased almost, if not quite, to zero. In contrast to the unboiled preparation there seemed to be no increase in the alcohol-soluble furfurol-yielding material in the boiled controls; that is, heating to 100° C. apparently rendered the extract incapable of affecting the xylan. While it seemed probable that xylose was liberated by the action of the extract of the fungus upon xylan it was deemed advisable to attempt to crystallize the sugar from the alcohol-soluble portion of the preparation. In order to obtain a sufficient quantity, a number of flasks of the xylan and extract of the fungus mycelium were pre- pared in the usual manner excepting that larger quantities were used. These preparations were kept in the incubator for about a week, the unchanged xylan precipitated as before, the alcohol extract con- centrated under reduced pressure, decolorized with animal charcoal and filtered. The filtrate was allowed to evaporate slowly over sulphuric acid in a desiccator and crystals were formed. These crystals were separated from the mother liquor and recrystallized. The substance did not melt sharply but apparently fused between 141° and 144° C. This was probably due to impurities present. The crystals reduced Fehling’s solution and formed the characteristic phloroglucid when boiled with a hydrochloric acid solution of phloro- glucin. The phenylhydrazine derivative was similar to the osazone of xylose as prepared in this study in color, crystal form, solubility and melting point. The compound when dissolved in water and treated with cadmium carbonate and bromine formed the charac- teristic crystals of the double salt of cadmium xylonate and cadmium bromide described by Widstoe and Tollens*! and observed in the present study with xylose. These properties all agree closely with the properties of xylose. It is evident then that xylan is hydrolyzed under aseptic conditions by the extract of the fungus and that xylose is formed. No attempt was made to determine whether intermediate products of hydrolysis were present as might well be the case. In these experiments on the effect of the fungus Glomerella cingulata 31 Widstoe, J. A., und Tollens, B. Ueber Arabinose, Xylose und Fucose aus Traganth. Bericht. Deutsch. Chem. Ges. 33: 132-143. 1900. 388 LON A. HAWKINS upon pentoses and compounds of pentose sugars it has been shown that the amount of furfurol-yielding material in the apple is decreased when the apple is rotted by this fungus. This decrease is brought about by the action of the fungus on the compounds in the apple which contain pentoses. These compounds are broken down and the furfurol-yielding material at least is used by the fungus. In this process the alcohol-soluble furfurol-yielding material is increased, which would seem to indicate that the pentose sugars are split off from the compounds in which they exist in the fruit. The fungus is able to utilize either glucose, xylose, arabinose, arabin or xylan as a sole source of carbon. The three sugars are most efficiently utilized, xylose perhaps the best. The fungus grows better on xylan than on arabin. The filtered extract of the fungus mycelium is able to act on xylan under aseptic conditions with the formation of alcohol-soluble sub- stance which reduces Fehling’s solution, forms furfurol when boiled with HCl and possesses other properties of xylose. This ability of the extract is lost on boiling. The breaking down of the xylan takes place gradually and the alcohol-soluble furfurol-yielding material is found to increase the longer the extract acts upon the xylan, the rate being much more rapid, however, during the early part of the action. A crystalline compound was obtained from the alcohol-soluble portion of the preparation of xylan which had been acted on by the extract of the fungus mycelium. This compound was apparently xylose. It is evident then that there is a xylanase present in the extract of fungus mycelium which hydrolyzes xylan to xylose. OFFICE OF PLANT PHYSIOLOGICAL AND FERMENTATION INVESTIGATIONS, BUREAU OF PLANT INDUSTRY, U. S. DEPARTMENT OF AGRICULTURE wie OUBRSTION OF THE TOXICITY OF DISTILLED WATER * R. P. HIBBARD INTRODUCTION Attention has recently been drawn to the. necessity of a physio- logically balanced salt solution for water cultures. This necessity arises from the fact that in order to know the effect on organisms of certain single salts or mixtures of salts in solution, one must use a control or check solution, called by True,! a normal physiological solution. This solution or medium should cause no disturbance of the regular functions of the seedling. Such a culture would afford an ideal standard for comparison and one much to be desired. The usual method, however, has been to use distilled water. That such cultures for biological studies are unsuitable was shown some time ago. In the first place, ‘distilled water’ is an indefinite term. As soon as the seedling roots are introduced the conductivity of the water rises rapidly, showing that salts are leached from the roots and the water remains no longer pure. Secondly, the extraction of salts, it is affirmed, suggests the means by which pure samples of distilled water exert their harmful effects. Some authors affirm a starvation theory; others, like Loeb,? the loss of certain necessary ion-proteid compounds; and still others, as True,’ that certain necessary con- stituents, partly inorganic, are dissociated from their proper attach- ments in the complicated chemical and physical mechanism of the living cell. Thirdly, the harmfulness of distilled water is attributed by others to the presence in it of toxic substance derived from the apparatus or because of the method of preparation. * Received for publication April Io. 1True, R. H. Distilled Water in the Laboratory. Science n. ser. 39: 296. Igt4. ; Loeb, J. On Ion-proteid Compounds and Their Réle in the Mechanics of Life Phenomena. I. The Poisonous Character of a Pure NaCl Solution. Amer. Journ. Physiol. 3: 327. 1899-1900. 8’ True, R. H. The Harmful Action of Distilled Water. Amer. Journ. Bot. 1: 270. I9QI4. 389 390 R. P. HIBBARD The two views, namely, that the distilled water is toxic per se, or that it is toxic because of the presence of poisonous substances in it, have been the principal ones held to the almost complete ex- clusion of any other. In this paper the writer wishes to draw attention to two other aspects of the distilled water problem. It seems that the problem is more a dynamic one than otherwise and in all proba- bility more than one factor enters into the explanation of why seed- lings cannot survive in distilled water. Right at this point it must be said that certain seedlings because of various physiological charac- teristics are less susceptible to distilled water injury than others. It is very probable that different varieties of seedlings and even different individuals of the same variety react differently to the same external conditions. From the work done in this laboratory but not reported the conclusion can be drawn that, providing one selects disease-free seed uniform in size, color; etc., one can detect as much individual variation in the behavior towards toxic substances in solu- tion of seedlings of pure line seeds from one plant as in that of seed- lings of a mixed progeny from unknown source. It follows from this that a medium which affords optimum conditions for one organism will not be the best for another. A normal physiological solution as men- tioned above will perhaps afford optimum conditions for one kind of seedlings but not for another. Certain other salts or other proportions are better. In all probability there is no universal physiologically balanced solution. Loeb‘ states that salt water is a physiologically balanced salt solution for such organisms as thrive in 1t and the blood of normal, healthy subjects can also be considered a physiologically balanced solution. From our knowledge of balanced solutions it may be assumed that the réle of the various salts is one of antagonistic action, a neutralization of the toxic effect of a single salt or of the harmful proportion of the various salts in the medium. That distilled water cultures of seedlings are not physiologically balanced solutions, can be assumed without much danger of contradiction. Wherein, then, lies the inappropriateness of distilled water for control cultures; and wherein lies the explanation of the injurious effect of such waters, and still further injury as the seedlings continue to grow in it? This preliminary paper will endeavor to show, first, that the adjustment of 4Loeb, J. The Relative Toxicity of Distilled Water, Sugar Solutions and Solutions of Various Constituents of the Sea Water for Marine Animals. Univ. Calif. Publ. r: 62. and 69.) 1903. THE QUESTION OF THE TOXICITY OF DISTILLED WATER 391 the seedlings to a distilled water medium is an important factor. It seems probable that injury is due to the greatness of the change. It will attempt to show secondly, that the possible injury which might arise from toxic substances excreted by the roots while the seedlings are growing in the water is another important factor to be considered. In lieu of any historical resumé, mention is here made of the literature and references cited by Livingston,’ Hoyt® and True.’ These papers give a very complete summary of the history of the work done up to date by both animal and plant physiologists. It is interesting to note that the work is about evenly divided between these two divisions of biology. MATERIALS AND METHODS The distilled water used in these experiments was of low con- ductivity, 2.5 —1.4 X 10-8. A battery of stills was set up and kept in operation all day and on the following days until the required amount of water was obtained. Then the experiment was started. The waters were stored in clean Jena bottles for no longer than the period of experimentation. Each individual still consisted of one 3-liter Jena glass flask, one condenser with spiral Jena glass inner tube, Jena glass tubing for connections where necessary and cotton plugs. The water was collected in either 5-liter, ground-glass- stoppered Jena bottles, or 3-liter ground-glass-stoppered Jena flasks. As indicators, the roots of Lupinus albus were selected. The seeds were kindly given me by Dr. True. These were soaked over night in either tap water or distilled water and suspended in damp chambers so as to form straight roots. No trouble was experienced in obtaining straight roots. When of the proper length the roots were marked at a point 10 or 15 mm. behind the tip and inserted in the loop of paraffined copper hooks. These were never allowed to touch the water, and only the radicles were immersed. These hooks were then arranged around the rim of Jena beakers of 250 cc. capacity. All glassware was previously thoroughly washed, rinsed, soaked in sulphuric acid-potassium dichromate solution for a few hours, then 5 Livingston, B. E. Further Studies on the Properties of Unproductive Soils. Wes. Dept. Agr., Bur. Soils Bull. 36: 57, 71. 1907. 6 Hoyt, W. D. Some Toxic and Antitoxic Effects in Cultures of Spirogyra. Bull. Torrey Club 40: 333-360. 1913. 7True, R. H. The Harmful Action of Distilled Water. Amer. Journ. Bot. 1: 253-273. 1914. 392 R. P. HIBBARD washed, thoroughly steamed and finally rinsed with the test water before being used in the experiment. The growth rate was chosen as the criterion. The control solution was tap water. This was allowed to run freely from the faucet for at least 15 minutes so as not to use the water that had rested in the supply pipes. It was then collected in clean Jena glass flasks and put aside near a radiator to come quickly to the room temperature, the temperature at which the distilled water was maintained. The cultures were set aside on a table away from the windows and covered with paper. PREPARATION OF DISTILLED WATER Only water which was prepared in contact with laboratory air of average purity is to be here considered. Absolute water prepared and used in vacuo®; water obtained by the combination of oxygen and hydrogen in the presence of an electric spark® or water distilled from a quartz distilling apparatus in contact only with air from out- of-doors dried and rid of CO, by the usual methods,’ is not here used. A comparative study of all the available methods was made. Various combinations were used for the purpose of eliminating this or that source of trouble. In all twenty-six methods were tried. Livingston" showed that good water could be obtained by shaking distilled water with finely divided carbon black. By a process of adsorption both volatile and non-volatile substances toxic in nature are taken up. E. P. Lyon” obtained water of excellent quality by adding a little H.SO, to the tap water in an automatic still consisting of a copper vessel and glass condenser, providing none of the condensed water touched the metal. It is suggested that the volatile toxin, ammonia, is eliminated by the H.SO, treatment. Hoyt made an improvement on Livingston’s method by distillation with animal charcoal in the retort. A combination of the last two with an additional treatment 8 Kohlrausch & Heydweiller. Wied. Ann. 53: 209. 1894. ® Any of the good text-books on general chemistry. 10 Method used in the research laboratory of physical chemistry, University of Illinois. Not yet reported. 11 Livingston, B. E., Jensen, C. A., Breazeale, J. F., Pember, F. R., Skinner, J. J. Further Studies on the Properties of Unproductive Soils. U.S. Dept. Agr. Bur. Soils Bull. 36. 1907. : 2 Lyon, E. P. Biological Examination of Distilled Water. Biol. Bull. Marine Biol. Lab. 6: 198. 1904. 18 Hoyt, W. D. Some Toxic and Antitoxic Effects in Cultures of Spirogyra. Bull. Torrey Club 40: 338. 1913. THE QUESTION OF THE TOXICITY OF DISTILLED WATER 393 mentioned below gave the best water in contact with air that the writer could get and one that had a conductivity of 1.4 X Io~*. About 5 liters of tap water were first shaken up with animal charcoal and allowed to stand, with frequent shakings, for several hours, and then transferred to the retort for distillation. This length of time is hardly necessary since the adsorption equilibrium is reached in a comparatively short time. The first 500 cc. distilled over was discarded and the last 800 cc. was not distilled. The part left, the middle portion, was poured into a second retort to which a little concentrated H2SO,4 was added and distillation started. The first 300 cc. (about) was discarded and the last 800 cc. not distilled. The middle portion proved to be a medium in which lupine seedlings grew very well and it was thought, since previous work had shown that dilute solutions of H.SO, stimulated growth, that some sulphates might have passed over. Three tests were made for sulphates but none were found. It was thought that here was a suitable water, but to allay all suspicions it was again distilled in a third retort to which was added a little Ba(OH)2. The middle portion from this distillation was caught in a 5-liter Jena bottle, thoroughly cleaned and steamed. The water was then aerated. The air was drawn from out-of-doors through tubes of calcium chloride, sulphuric acid and potassium hydroxide solution. This bubbling process was continued for about twenty-four hours. In this water, lupine seedlings made good growth and showed not the least sign of injury for three weeks providing the distilled water was changed at least twice daily. In the following experiment the records were kept for a week. The experiment was continued, however, and at the end of three weeks the seedlings were in good condition but not as far advanced as the checks in tap water. There were 10 seedlings in each beaker. At the end of the first twenty-four hours the total average growth in distilled water was 33.7 mm. On the following day the average increase was 4.5mm. The increase for each following day is 3.6 mm., Festi. 4.3 Itty, 7.0 mm. and 7.3 mm. The data for tap water follow: At the end of 24 hours the total average for 10 seedlings was 34.6 mm. Increase during successive 24-hour periods: II.7 mm., 15.2 mm., 13.5 mm., 9.6 mm., 11.6 mm., 9.1 mm. It may be noted here that the growth is a little better in tap than in distilled water, but the rate of increase is of the same order. The growth in tap water is quite luxuriant. Analysis of the tap water kindly given me by the station chemist, Prof. A. J. Patten, is here appended. 394. R. P. HIBBARD Total Solidse i... 2% CaO MgO SO; Cl Parts per 100,000. . .33.9 11.06 00 .025 THE PROBLEM OF ADJUSTMENT It seems that marked and sudden changes from waters of low resistance to waters of high resistance would affect the equilibrium between the physical and chemical mechanisms of the living cell. When the changes are not so great the deleterious effects will not be so great and the organism can survive in a medium, in which, if thrust suddenly, it would die. This fact also suggests another gen- erally admitted but so often overlooked in biological studies. If comparative results are desired, the organisms used must be living under uniform conditions previous to the time of experimentation. Their future behavior depends as much on their internal nature, which is the result of their previous surroundings and past history, as on the condition of the surroundings into which they will be placed. One will not be offering a too daring interpretation if he regard this feature of adjustment as a very important one in the consideration of the injurious effect of distilled water. My experiments agree in general with the results obtained by Daniels't who concluded that death of Paramecia in distilled water was caused by the sudden change in media. Peters states, ‘that if a change from culture liquid to pure water be made as rapidly as in these experiments (with Stentor and Para- mecia) a fatal effect ensues. On account of the conception I have formed of the mode of action of the pure water, I would expect the same effect to occur even if the changes occupied a longer period of time.” The inference is that the factor of adjustment is not im- portant. To test this theory of adjustment for lupine seedlings and distilled water, various mixtures of tap and distilled water were made. The seedlings were first placed in a beaker of tap water and allowed to grow in that for a definite time. Then all but 5 were removed and placed in a beaker containing a mixture of tap water and distilled water in the ratio of 1:4. After a definite time all but 5 of these were removed and placed in a mixture in the proportion of 1: 8. This operation was repeated until 5 were placed in distilled water from 144 Daniels, J. F. The Adjustment of Paramecia to Distilled Water and Its Bearing on the Problem of the Necessary Inorganic Salt Content. Amer. Journ. Physiol. 23: 48-63. 1908-9. THE QUESTION OF THE TOXICITY OF DISTILLED WATER 395 the last dilution of 1:64. The experiment was then allowed to run 24 hours longer. Now the total growth was recorded in each beaker. At the same time that this series was set up a duplicate one was arranged and placed on the table beside the first. At the beginning of the experiment 5 seedlings were placed in each beaker. The total growth of the roots of these seedlings was recorded at the same time as the other set. In the last beaker of the first set were five seedlings that had been placed in the distilled water after they had been grow- ing for a stated interval in 5 different dilutions of distilled and tap water. In the last beaker of the second series were five seedlings that had been immediately placed in distilled water. In this way one set of seedlings was exposed to the influence of distilled water after gradual changes and the other immediately. In another experi- ment the method was varied, in one way, by using mixtures containing different ratios of tap and distilled water, and in another way by arranging the experiment so that 10 seedlings could be put into the last beaker of the series. The change was first made daily, in another experiment twice a day, and in the final experiment four times a day. In one experiment the records of root growth were made at every change in every beaker. The results of these experiments show that better root growth was obtained in distilled water when changes from tap to distilled water were made gradually instead of suddenly. In the following table T and D stand respectively for tap and distilled water. Ue OBES are SCE LT 48D suyp aes BD | Be T+ D aT + 2D | Dist. alee Tap ——_. ae Paced = | Total growth in mm.,... | 34.3 36.5 ue B7ee Ble 2125 | 27.5 Where Chane are Cla Total growth in mm..... | 38.3 | 43-4 | 38 | 36.4 26,2 | 34.3 Greater differences might have been obtained had a more sensitive indicator been selected. As far as our experience goes lupines are not very sensitive to distilled water injury. When exposed to sudden changes they react in a moderate way. When in distilled water fre- quently changed to rid the medium of the toxic substances or un- balanced condition of the organic and inorganic material therein, they live until the nutritive substances in the cotyledons have been exhausted. Frank’s!® statement that lupine seedlings are poisoned 396 R. P. HIBBARD by distilled water has been questioned by Schulze!* who states that in all probability the effect produced is the result of impurities in the water. From our experience with lupines we would prefer to agree with Schulze. It may also be observed from a study of the experimental data above that the change from the last dilution to distilled water affects the growth rate more than changes in the other dilutions. Were the cause of injury due to osmotic effects, this would show up in the first or greater change but here we always have a better growth rate than in any of the other dilutions. That osmotic effects can be ruled out has been shown by Loeb, True and others. Can the same be said of low salt content? Is the low salt content of the medium the cause of injury? J. F. Daniels'* showed that the destructiveness of distilled water cannot be due to its low content of inorganic salts. Two drops were diluted with equal amounts of distilled water. Into one having the high original salt content of the culture solution many paramecia were placed, into the other with a very low original salt content few organisms were placed. The paramecia in the first drop died quickly, while in the second drop they lived for days. Obviously the salt content was not the important factor. Here no mention is made of the possible deleterious effects of toxic excreta from so many organisms. If lupine seedlings are grown in distilled water with frequent daily changes the salt content will be kept quite low and in addition the toxic excretions will be more or less eliminated. Under these condi- tions lupine seedlings grow well until they show signs of injury from lack of nutrient salts. The experiments mentioned above, besides others not reported here, lead us to the belief that the problem of adjustment is so important that it must be taken into account in culture experiments. THE PROBLEM OF Toxic EXCRETIONS For a little more than ten years the Bureau of Soils of the United States Department of Agriculture has sought to explain soil infertility, by the presence of deleterious substances of organic nature in the soils. These substances may be given off by the roots, or arise from 16 Frank, B. Untersuchungen tiber die Ernahrung der Pflanzen mit Stickstoff und iiber den Kreislauf desselben in der Landwirthschaft. Landwirthsch. Jahrb. 17: 421 -553am LOCGam 16 Schulze, E. Uber das Verhalten der Lupinenkeimlinge gegen destillirtes Wasser. Landwirthsch. Jahrb. 20: 236. 1891. THE QUESTION OF THE TOXICITY OF DISTILLED WATER 397 the decay of organic matter. It must be admitted that these investi- gators have collected a vast amount of facts to substantiate this theory. In Bulletin 23!” it is shown that the bad characteristics of a soil are transmitted to its aqueous extract. The injury to wheat seedlings grown in this as compared with their growth in an aqueous extract of a good soil, could not be attributed to a difference in nutritive salts for they analyzed practically the same. The conclusion was drawn that the solution contained injurious substances which checked the growth of plants. In Bulletin 28!8 it was shown that wheat seed- lings give off substances which are toxic to themselves. This is true when seedlings are grown in nutrient solutions which have been previously used for growing plants. Carbon black or ferric hydrate corrects the used solution so that it produces nearly as good growth as the fresh one. The same is true when distilled water is used instead of the nutrient solution. ‘These results with distilled water,”’ the author states, ‘“‘seem to make it very certain that the roots of seedling wheat plants do give off substances which are poisonous to themselves, and these substances can be corrected by carbon black or ferric hydrate.”’ This recalls to mind the experiments by Macaire!® in 1832, who noted that peas grew poorly in water containing excre- tions from the roots of the same plants, while wheat on the other hand did well in water charged with the excretions from pea roots. Later, 1845, Boussingault?® studied the question of root excretion and came to the conclusion that plants do not normally excrete materials from their roots, but may do so in water culture. For a more extended account of the earlier literature and especially that of the work of the Duke of Bedford and S. U. Pickering on the deleterious effect of grass on fruit trees, the writer wishes to refer the reader to Bulletin 36 by B. E. Livingston, and the works of Schreiner and his assistants, found in bulletins of the Bureau of Soils, U. S. Department of Agriculture, Nos. 47, 53, 70, 74, 75, and 80. What we are most concerned with at the present time is the possi- 17 Whitney, M., and Cameron, F. K. Investigations in Soil Fertility. U.S. Dept. Agr. Bur. Soils Bull. 23. 1904. 1 Livingston, B. E., Britton, J. C., and Reid, F. R. Studies on the Properties of an Unproductive Soil. U.S. Dept. Agr. Bur. Soils Bull. 28. 1905. 19 Macaire-Prinsep, Memoire pour servir 4 l’histoire des assolemens; Mém. Soc. Phys. Hist. Nat. Genéve, 5: 282-302. 1832. 20 Boussingault, J.B. J.D. Rural Economy. Trans. by George Law (London), 345. 1845. 398 R. P. HIBBARD bility of deleterious root excretions from lupine seedlings. We must admit in the beginning that we have not isolated any toxic substance from the water in which these seedlings have been growing. The quantity of poison is obviously so small that it will necessitate the evaporation of 10 or 12 liters of solution to get enough residue for a chemical test. The difficulty of this problem is still further increased since organic chemists can give us no universal method of treatment applicable to all unknown solutions. Previous literature gives us assurance that the excretions are probably organic in nature. The probability is that the substances excreted are compounds of multiple function, and if they do contain an acid radicle, this radicle must be internally compensated with some basic group, since the solution gives no reaction to the ordinary indicators. If an aldehyde is sought for we meet with a difficulty in that we have no differential test to indicate what aldehyde it may be. On the other hand, we have one or two indirect methods to attack this subject of toxic excretion. After a certain length of time in distilled water lupine roots display certain characteristic features not noticeable in tap water cultures. Roots in tap water are long, slender, grayish white, usually straight and firm. A large conspicuous root cap covers the tip. The water does not become turbid. In distilled water the roots present quite a different appearance. They are covered with a felt-like coating which at first gives the appearance of a mass of root hairs or a weft of fungus mycelium. It is believed that this is nothing more than root cap tissue hanging on the root as the tip pushes forward. On closer examination under the microscope this material does not present the characteristic structure of root hairs or fungus hyphae and resembles in every particular the cells of the outer layer of the root cap. No reason is hazarded for its abundance in distilled water. It has not been noticed in tap water, and appears less abundant in distilled water that has been changed two or three times daily. This weft of tissue falls off and gives the water a turbid appearance. In distilled water the roots are shorter, thicker and usually more or less bent near the tip, showing the tendency for fishhook formation. The lateral roots appear earlier and are more abundant in distilled water cultures than in tap water cultures. They show all the appearances of roots suffering from slight doses of poisons. In all probability the roots excrete a toxin which retards growth. If this toxin is removed by frequent changes of the water THE QUESTION OF THE TOXICITY OF DISTILLED WATER 399 the growth rate is increased. This can be shown in gradation. If the water is changed once a day, the growth rate changes but little in the earlier stages of the life of the seedling in the water. If changed twice a day, the growth rate is increased. When changed four times a day the best results were obtained. Again a large number of seed- lings growing in the water suffer less apparently than a few in the same amount of water. It seems that the greater amount of salts leached out in one case acts in an antitoxic manner and so corrects the water, or the roots might actually reabsorb some of the salts. Some such explanation might be given for the condition reported by True to the effect ‘that the leaching process seems to have been somewhat less active in the cultures containing the larger number of roots than in that containing the smaller number of roots.’’ Some such conclusion is more reasonable than to assume that roots under the same external condition do not lose salts at approximately the same rate. In soils where oxidation is hindered we are told that those sub- stances inimical to growth are more apt to be found. The very nature of water cultures produces this condition of poor oxidation and organic matter collects more rapidly than it can be oxidized. A renewal of the medium in which the plants are growing eliminates to a greater or less extent the toxic substance present therein, and at the same time the roots are living in a solution continually aerated. When an experiment is set up with these facts in mind lupine seedlings will survive in distilled water until injury from lack of nutritive sub- Distilled Water Changed Distilled Water Not Changed Seedling No, Growth in Mm. Seedling No. Growth in Mm. I 44 I 25 2 34 2 at 3 42 3 31 4 38 4 34 5 32 5 35) 6 43 6 oF 7 41 ff 29 8 37 8 a9 9 39 9 36 Be 10 46 o 10 37 A Av. Growth in mm. 39.4 Av.Growth in mm. 32.7 stances sets in. The rate of growth is also greater. Ten seedlings were suspended, as described above, in distilled water. The water 400 R. P. HIBBARD was changed four times daily. As a control for this experiment I0 seedlings were suspended in another beaker of distilled water. The water in this beaker was not changed. At the end of 30 hours, even, these seedlings surpassed the growth of those left in distilled water without change. This can be seen from the obove table. The table below gives the data on another experiment extending over a period of four days. Distilled Water Changed Distilled Water Not Changed TSti24y)| 2d\j24 3d 24 | 4th 24 | rst 24 2d 24 | 3d 24 | 4th 24 Seedling No. Hrs. Hrs. Hrs. Hrs, Hrs. Hrs. Hrs. Hrs. Growth | Growth | Growth Growth | Growth | Growth | Growth } Growth inMm./in Mm.| n Mm. inMm./in Mm.|in Mm.| in Mm, |in Mm. I 27, 34 43 46 33 39 43 48 2 34 37 41 45 25 33 40 46 3 37 42 48 55 31 35 36 4I 4 33 37 47 55 37 40 43 49 5 32 40 51 58 35 38 43 51 6 35 38 A7 55 34 37 42 48 7 31 34 40 45 35 40 43 48 8 37 45 50 60 36 Al 45 50 9 38 44 51 61 35 39 Al 45 10 38 45 53 60 36 40 42 45 Av. growth in mm... ...../.34:2 | 39:6 | 47.1,| 54.0 (32-7 | 2820 eae ean CONCLUSION In regard to the question as to the toxicity of distilled water, the writer wishes to draw attention to the importance of considering the problem from the dynamic standpoint. To describe how the distilled water produces its effect, it is necessary to know what physical and chemical conditions prevail-in the organism and what relation exists between the organism and the medium in which it lives. Our present knowledge of the physical and chemical nature of protoplasm, the basis of all organic life, is too fragmentary upon which to lay the foun- dation of any theory. However, it seems that one will not stray far away from the truth if he concludes the harmfulness of distilled water due not to any one predominant factor but rather to a resultant of many. These factors may be conceived of as forces bringing about a disturbance of the normal equilibrium of the various chemical and physical interactions within the organism and between it and its environment. In this paper only two aspects of the subject have been considered. THE QUESTION OF THE TOXICITY OF DISTILLED WATER 40I First, there is the problem of adjustment. Some organisms because of their inherent physiological characteristics possess a low order of resistance to harmful agents and are injured, sometimes fatally, by slight deviations from optimum conditions. However, in the case of lupine seedlings a slow process of adjustment or acclimatization to distilled water fits them better for life within it than if they were thrust into the water immediately. That the change from tap water or culture solution, whatever it may be, to distilled water is great, can be demonstrated by the determination of the electrical conduc- tivity of the waters. It appears that such marked differences disturb the equilibrium between the physical and chemical functions of the organism. Several experiments were made and one was reported to test the theory of adjustment. The conclusion was reached that better root growth was obtained in distilled water when changes from tap to distilled water were made gradually rather than suddenly. This problem of adjustment is so important that it must be taken into account in all cultural work. The second aspect of the distilled water question dealt with in this paper is that of toxic root excretions. The idea that roots of plants excrete injurious substances, harmful to their further growth, has persisted from the time of De Candolle. The investigations of the Bureau of Soils of the U. S. Department of Agriculture appear to set this theory on a more secure foundation. In this paper are reported the results of experiments to show that, in all probability, seedling roots of Lupinus albus excrete a substance that inhibits growth. The toxin was not isolated from the turbid solution but it was shown that when the water was changed four times daily the growth rate of the roots increased. Roots that had grown continually in distilled water presented the appearance of roots injured by dilute solutions of toxic salts. The roots were thick, short, more or less blunt and crooked near the tip, when grown continually in unchanged distilled water. In tap water the roots were long, slender, firm and usually straight. The lateral roots appeared later in tap water than in distilled water. MICHIGAN AGRICULTURAL COLLEGE, EXPERIMENT STATION, East LANSING, MICHIGAN AN ANATOMICAL STUDY OF GYMNOSPORANGIUM GALES ALBAN STEWART GYMNOSPORANGIUM JUNIPERI-VIRGINIANAE ON JUNIPERUS VIRGINIANA The common “cedar apple’’ on Juniperus virginiana L., caused by Gymnosporangium juniperi-virginianae Schw., is one of our most common galls on coniferous plants. While the organism that causes this gall has been widely studied, but little work has been done on the changes brought about in the tissues of the host through the activities of this parasite, and the results obtained are contradictory. Sanford (8) was the first one to investigate the anatomy of the cedar apple caused by G. juniperi-virginianae. He considers the gall to be formed by an abnormal growth of leaf tissue, which carries the apex of the leaf up as the gall develops, and pushes the branch bearing it to one side until the gall appears to be terminal. He further states that the vascular system enters the knot as one single bundle, given off from the vascular bundle of the branch of the tree, and further, that as the knot increases in size the vascular bundle develops rapidly until it appears like the vascular system of a branch. Wornle (11) who has done more work than any other investigator on the anatomy of Gymnosporangium galls, considers that the gall arises from a swelling of the stem induced by the fungus, that leaves may also enter into its composition, and, if dormant buds are en- countered, they also may be stimulated to development. Kern (6), who probably bases his statement on the work of Sanford, gives the cedar apple as an example of a gall originating in the leaves. Engler and Prantl (4), on the other hand, describe both G. junipert- virginianae and G. globosum as causing woody galls on Juniperus virginiana. Heald (5, pl. 10) has recently figured very young galls of this kind as arising near the axils of the leaves, and Coons (1, figs. I and 5) has figured galls some of which appear to be axillary in origin, and others arise from the upper surface of the leaf. These rather conflicting statements in regard to the morphology 402 AN ANATOMICAL STUDY OF GYMNOSPORANGIUM GALLS 403 of this gall seemed to offer an opportunity for further investigation. As the juniper trees in the vicinity of Madison, Wisconsin, furnished abundant material of this gall in various stages of development I concluded to determine, at least for my own satisfaction, which of these statements was correct. I soon discovered, from external exami- nation, that some of the older-galls had remains of more than one leaf attached to them, a fact which in itself would indicate that stem tissue was involved to a greater or less extent. I found still further, that normal branches are attached to the gall, apparently growing out from it at times (fig. 7), a condition that would be rather unusual if the gall were composed entirely of modified leaf tissue. Rather thick hand-sections showed that when a gall was located on the end of a stem, the stem continued into it for some distance before it broke up. This hasty examination seemed to justify a more careful study, the results of which are given in the following pages. Before entering into the description of the anatomy of the gall it seems desirable to describe briefly the anatomy of both the leaf and young stem, as both of these are involved in gall formation. The normal leaf is triangular in cross section except towards the base where it is rather four-sided. The epidermis is heavily cutinized and forms a smooth layer on the outer (morphologically lower) side of the leaf. Towards the edges and inner side, however, the indi- vidual cells are somewhat raised towards the outside causing them to appear more or less papilliform when seen under the microscope. The stomata occur mostly in the inner (morphologically upper) epidermis, but they may appear occasionally in the outer epidermis near the edges of the leaf. In addition to the epidermis, the outer side of the leaf is protected by a hypodermis which may extend entirely or only part way across this side. The hypodermal cells are usually arranged in one or two layers, although three layers may be present at times over a space. Sanford (8) describes a double epidermal layer for this side of the leaf but he evidently mistook the hypoderm for an inner epidermal layer. The interior of the leaf is filled with rather thin-walled paren- chyma cells (text fig. 1) which are loosely arranged towards the inner side near where the stomata are borne, and resemble very much ordinary spongy leaf tissue. Towards the outer side of the leaf the parenchyma cells are elongated perpendicular to the surface and form a palisade tissue. The base of the leaf is united with the stem for some 404 ALBAN STEWART distance. A large resin cavity occurs in this region of the leaf. A single bundle enters the upper part of the leaf, xylem above and phloem below. On each side of this bundle are peculiar tracheids with bordered pits and bar-like thickenings (text fig. I, xxx). For further consideration of these tracheids see De Bary (2). It is sometimes difficult to determine in the young stem which of the cells belong to the leaf and which to the outer bark of the stem as the leaf and stem tissue blend together more or less at the point of union of leaf and stem. Between the attachment of the opposite leaves, however, the stem is exposed and is covered with epidermal cells similar to those which occur on the inner side of the leaf. There is a single layer of rather thick-walled parenchyma cells inside the epidermis, followed farther in by one or more layers of large, thin- walled, oval-shaped parenchyma cells. The inner bark is composed of concentric rings of bast fibers between each two of which there are three layers of cells. According to De Bary (2), two of these layers are of sieve tubes which are separated from each other by a layer of parenchyma cells. | The xylem of the young stem is composed of small triangular groups of tracheids, separated from similar adjacent groups by uni- seriate rays. The rays extend outward into the inner bark, thus dividing the phloem and bast region into segments. The rays are broader opposite where the leaf trace bundles leave the xylem portion of the central cylinder. This broadening comes about -usually by an enlargement of the ray cells, but sometimes there is an inter- polation of additional ray cells. As two leaf traces leave the stem about opposite each other, the gaps caused by the broader rays divide the woody portion of the central cylinder into two nearly symmetrical halves. A small V-shaped depression occurs in the xylem where the leaf trace leaves it. A small group of pith cells occupies the center of the stem. A diagrammatic drawing of a very young cedar apple gall and the stem that bears it is shown in figure 1. This gall has arisen from the axil of a leaf, a portion of which is shown to the right of the figure. I have examined a large number of young galls and have been unable to find any which were similar to the two figured by Coons (1, right side of fig. £), which according to the figure have arisen evidently from the surface of the leaf. I have also been unable to find any young galls which were terminally located, and which might have AN ANATOMICAL STUDY OF GYMNOSPORANGIUM GALLS 405 arisen from the infection of a terminal bud such as has been suggested by Wornle. They were always distinctly axillary in position although on the same tree with them there were older galls which were appar- ently terminal. According to Sanford (8), the branch is pushed to one side during the development of the gall. If this be true the terminal position of many of the older galls is probably more apparent than real. The leaf, from the axil of which the gall arises, is pushed outward to some extent so that a small shelf is formed on which the gall rests. Apparently the fungus has not entered the stem at this stage as no abnormalities appear beyond a slight production of cork just above the gall (fig. 1). In the stage figured, the gall consists entirely of eset ete ee Deas Le Oe OM ee ev ce 2s” ie 7. 2 ae . eas XO aA yh ‘\ WM cat iN | NWN SO ally) es earqell| 3: ef dss ( ) e Ba I= LSA CAAT KN 5208s )s Z “10 Cy we @ AO ~e YX 20 D> eS: Cp 6 ACEH eat exe ¢ See C/ > 6 meseeenaees : 2ete.e-0191@ O) sivesines: SREB eo () 68 17 X) {7 Ex F6. a) RY @ % a Or 5! Ys Of ,

ISO) son OMe 21 DQUP>m™B AA v9" Ti | \ STEWART: GYMNOSPORANGIUM GALLS. AN EXTENSION TO 5.99° OF TABLES TO DETERMINE THE OSMOTIC PRESSURE OF EXPRESSED VEGE- TABLE SAPS FROM THE DEPRESSION OF THE FREEZING POINT J. ARTHUR HARRIS Some months ago Dr. Gortner and I published in this journal (Amer. Journ. Bot. 1: 73-75, Feb. 1914) tables of the value of P.— 12,0004") —),02me for freezing point lowerings of .o1° to 2.99°. At that time this range of values seemed to us, as well as to others, Hundredths of Degrees, Centigrade A ° I 2 3 | 4 5 6 7 8 9 3.0 | 35.99;| 36.11 | 36.23 | 36.35 |. 36.47 | 36.59 | 36.71). | 36.83, | 36:05:5)(37,.00 3.1 | 37.18 | 37.30 | 37-42 | 37.54. | 37.66 | 37.78. 37.90 ||) 38.02 ge Tawa es26 3-2 | 38.38 | 38.50 | 38.62 | 38.73 | 38.85 | 38.97 | 39.09 | 39.21 | 39.33 | 39.45 3.3 | 39-57 | 39-69 | 39.81 | 39.93 | 40.05, | 40:17 | 40:28 |. 40:40 | 40:5255|) 40°64! 3.4| 40.76') 40.88 | 41.00 | 41.12'| 41.24 | 4h.36 |: 41-48: | 4160.0) 41 7a es 3.5 | 41.95 | 42.07 | 42.19 | 42.31 | 42.43 | 42.55. | 42.67 | 42.79") 42:01 Aste? 3-6 | 43.14 | 43.26 | 43.38 | 43.50) 43-62 | 43.74 | 43.86 | 43.98 | 44.10 | 44.22 3:7 | 44-33 | 44-45 | 44-57 | 44-69 | 44.81 | 44.93 | 45.05 | 45-17 | 45.29 | 45.41 3.8 | 45.52 | 45.64 | 45.76 | 45.88 | 46.00 | 46.12 | 46.24 | 46.36 | 46.48 | 46.60 3-9 | 46.71 | 46.83 | 46.95 | 47.07 | 47-19 |. 47.31 | 47-43 | 47-55 | 47-67 | 47-79 4.0] 47.90 | 48.02 | 48.14 | 48.26 | 48.38 | 48.50 | 48.62 | 48.74 | 48.86 | 48.97 4.1 | 49.09 | 49.21 | 49.33 | 49-45 | 49-57 | 49.69 | 49.81 | 49.93 | 50.04 | 50.16 4.2 | 50.28 | 50.40 | 50.52 | 50.64 | 50.76 | 50.88 | 50.99 | 51.11 | 51.23 | 51.35 4.3 | 51.47 | 51.59 | 51.71 | 51.83) 51.94 | 52.06 | 52.18 | 52.30 | 52.42 | 52.54 4.4 | 52.66 | 52.78 | 52.89 | 53.01 | 53-13 | 53-25 | 53:37 | 53-49 | 53-61 | 53-73 4-5 | 53-84 | 53-96 | 54.08 | 54.20) 54.32 | 54.44 | 54.56 | 54.68 | 54.79 | 54.91 4.6 | 55-03 | 55-15 | 55-27 | 55-39 | 55-51 | 55-62 | 55.74 | 55.86 | 55.98 | 56.10 4.7 | 56.22 | 56.34 | 56.46 | 56.57 | 56.69 | 56.81 | 56.93 | 57-05 | 57-17 | 57.29 4.8 | 57-40 | 57-52 | 57-04 57.76| 57.88 | 58.00 | 58:12 | 58:23.) 56:35. I) 56:47 4.9 | 58.59 | 58.71 | 58.83 | 58.95 | 59.06 | 59.18 | 59.30 | 59.42 | 59.54 | 59.66 5-0 | 59.78 | 59.89 | 60.01 | 60.13 | 60.25 | 60.37 | 60.49 | 60.60 | 60.72 | 60.84 5.1 | 60.96 | 61.08 | 61.20:| 61.32 | 61.43 | 61.55 | 61.67 |. 61.79 | 61.g1 | 62703 5.2 | 62.14 | 62.26 | 62.38 | 62.50 | 62.62 | 62.74 | 62.85 | 62.97 | 63:09) 63am 5-3 | 63.33 | 63.45 | 63.56 | 63.68 | 63.80 | 63.92 | 64.04 | 64.16 | 64.27 | 64.39 5-4 | 64.51 | 64.63 | 64.75 | 64.87] 64.98 | 65.10 | 65.22 | 65.34 | 65.46 | 65.58 5-5 | 65.69 | 65.81 | 65.93 | 66.05 | 66.17 | 66.29 | 66.40 | 66.52 | 66.64 | 66.76 5.6 | 66.88 | 67.00 | 67.11 | 67.23 | 67.35 | 67.47.| 67.59 | 67.71. | 167.82) O7-e" 5-7 | 68.06 | 68.18 | 68.30 | 68.41 | 68.53 | 68.65 | 68.77 | 68.89 | 69.01 | 69.12 5.8 | 69.24 | 69.36 | 69.48 | 69.60 | 69.71 | 69.83 | 69.95 | 70.07 | 70.19 | 70.30 5-9 | 70.42 | 70.54 | 70.66 ' 70.78 ' 70.90 710 rpm lies 71.25 TT. 71.49 418 TABLES TO DETERMINE OSMOTIC PRESSURE 419 quite sufficient for biological work. Since then, however, Mr. John V. Lawrence and I in determinations of freezing point depressions on the juices of the plants of the southwestern deserts about Tucson and of the Jamaican coastal deserts have repeatedly found values far beyond the range of the table there published.! It has seemed worth while therefore to extend the tables to 6°. In using such a table one must of course remember the difficulties which surround the problem of the osmotic pressures of concentrated solutions—even those of pure solutes in pure solvents under the controlled conditions of the physico-chemical laboratory. But the physiologist cannot neglect the many problems presented by the concentration of vegetable saps until physical chemists are in full agreement concerning all details. Quite to the contrary he must use the best methods he can under the peculiarly complex and difficult conditions which surround his work and express’ his results in consistent terms, remembering that methods and formulae which are the best that are practicable at present may soon have to be replaced by others. STATION FOR EXPERIMENTAL EVOLUTION, CoLD SPRING HARBOR, LONG ISLAND, N. Y. 1 These results confirm those obtained in cryoscopic studies by Cavara and by plasmolytic methods by Fitting, in that they show that very high concentrations may occur in plants growing in their natural habitats. As yet we have not found concentrations quite so high as some they report. CALCIUM HYPOCHLORITE AS A SEED STERILIZER: James K. WILSON For certain physiological experiments seeds and plantlets free from active bacteria and fungi are necessary. Most attempts to secure such seeds or plantlets in any considerable number have re- sulted in failure, usually because of the harmful effects of the germi- cide, its low efficiency, or the complicated methods required for treatment of the seed. While it is recognized that no germicide will give perfect satisfaction under every condition, some are more effective in this respect than others. The treatment of seed to remove bacteria and fungi, especially the latter, has been practiced for a considerable period of time; and while one investigator has secured fair results with a particular method, another one has considered it a failure, or nearly so, when tested on another kind of seed. As a result many methods have been pro- posed for seed sterilization with the final condition that most of them can not be relied upon to yield a very large percentage of sterile plantlets. In the following compilation the methods of treatment employed by various investigators are summarized together with the seeds treated. AUTHOR SEED DISINFECTANT Brown and Escombe. Hordeum vulgaris. CuSO., 1%, one to two hours. Combes. Radish. HgCle. Czapek. Corn. HgCle, 1%, two minutes. Godlewski and Pisum sativum, Vicia Faba, HgCle, 1/100%. Polzeniusz. Triticum vulgare, Zea mays, Ricinus communis, Brassica napus. Griiss. Grain. HOH 48 hours. Then in HgCle 45 minutes. Hansteen. Zea mays and others. Washed in absolute alcohol then in HgCl, I-1,000. Kehler. Peas, beans, blue lupine, yel- HgCle, CH20, various low mustard, red clover, sugar beet, wheat, alfalfa, and others. strengths of each. * Contribution, Laboratory of Plant Physiology, Cornell University. 420 CALCIUM HYPOCHLORITE AS A SEED STERILIZER Laurent. Lefevre, J. Lewis and Nicholson. icutz, L. Maze and Perrier. Molliard, M. Nabokich, A. Nobbe, F. AUTHOR Pinoy and Magrou. Polowczow, W. Prazmowski, A. Puriewitsch, K. Robinson, T. R. Schroeder, H. Schroeder, H. Schulow, Iw. Staklasa, J. Stoward, F. v. Ubisch, G. de Zeeuw, R. Corn, peas, lentils and other seeds. Lepidium sativum, Ocimum minimum, Tropaeolum na- num. Leguminous seed. Cucurbita maxima, Zea mays, Cucumis prophetarum, Heli- anthus annus and others. Zea mays. Raphanus sativus, Allium cepa, Ipomaea purpurea, Nastur- tuum officinale. Zea mays and others. Wheat, timothy and others. SEED Sinapis alba, Lupinus albus, Zea mays, Orobus tuberosus. Lepidium sativum, Lupinus luteus and others. Garden peas. Endosperm of Zea mays, Triticum sativum, Secale cereale, Hordeum distichum, Oryza sativa and others. Alfalfa, crimson clover, gar- den pea, soy bean, wheat, oats, radish. Wheat. Wheat. Corn, peas, barley. Hordeum distichon. Hordeum, Zea and Ricinus. Spore capsules of moss. Lupinus albus, Pisum sait- vum, Triticum vulgare, Hor- A421 HgCl, 1-500 alone and in combination with other substances. HgClo, 2 to 3 pts. per 1,000, I5 min. and longer. Phenol 5%, 50 min. HgCl, 1-2,000, 5 min. HgCl, 1-1,000, 15 min. Soak seed in sterile water un- til wrinkled then in abs. alc. for a short period, then I min. in 1% HgCle. Bromine water I-1I,000 20 to 30 min. CuSO, various strengths for 24 hours. DISINFECTANT Br. water and H2QOx. Br. water. HgCl, 2%, then absolute alcohol and burned off alcohol. CH,O, 214% parts—I,000, 3 min. CH.O, HgCly, H.0, in vari- ous strengths. AgNO; 5%, 14 hrs. HNOs, AgNOs, HgCle, and other substances in combi- nation. Br. water 20 min. HgCl. On G: CuSOu,, HgCle, CH.O, Sat, sol. chloroform. Sat. sol. toluene. Alcoholic HgCle. H2Os, HgCle, cleaning fluid. Potassium dichromate, am- 422 JAMES K. WILSON deum vulgare, Zea mays, and monium persulfat, and bro- Sinapis alba. mine water. It will be seen from the above compilation, to which others might be added, that about two dozen methods have been used. Of these mercuric chloride, alcohol, formalin, hydrogen peroxide, or combina- tions of these have served in the main as the germicide. The writer tried various of the above methods and finding them unsatisfactory resorted to the use of other substances among which was bleaching powder (calcium hypochlorite). The favorable results which have been secured with bleaching powder and the many re- quests for the method have prompted the author to present this brief description. The method is simple: Ten grams of commercial chloride of lime (titrating 28 percent chlorine) is mixed with 140 cc. of water. The mixture is then allowed to settle for five or ten minutes and the supernatant liquid decanted off or filtered. The solution or filtrate which contains about 2 percent chlorine is used as the disinfectant. Dilutions from this known strength may be used as well as the full strength. The volume of solution employed should be about five times or more the volume of the seed. In order to find the exposure at which the ability for germination is affected seeds were placed in sterile test tubes and covered with a one percent chlorine solution, obtained as above, for different lengths of time. Germination tests were then made with the seed: which were removed at these different times and compared with untreated seed germinated in the same way. Seeds were also removed from the disinfecting solution at various intervals and tests made with respect to freedom from bacteria or fungus organisms. The most trustworthy tests were made by planting the seed on the surface of peptone agar and into bouillon. Tubes thus prepared were kept at room temperature, at 30° C., and 37° C. Observations were made on these tubes at intervals up to four weeks. In addition to these tests of sterility many plants which were germi- nated on agar were used in experimental work where sterility was required and was apparently maintained for several months. In making transfers the seed was removed from the disinfecting solution by means of a small hand-wrought spoon, momentarily drained and sown in the culture vessel. No attempt was made to remove completely the disinfectant from the seed since it does not seem to interfere with the germination unless the period of treatment CALCIUM HYPOCHLORITE AS A SEED STERILIZER 423 is exceedingly long or a noticeable amount is carried over with the seed. In making the transfers, however, an effort was made to leave behind as much of the germicide as possible. That the quantity of disinfecting material carried over with the seed into the bouillon was not sufficient to act as a germicide was proven by placing along with some of the treated seed in the bouillon an untreated seed. In every case within three days there was ample bacterial growth. With large seeds such as corn, beans, peas, and wheat, only those seeds were used which appeared normal and seemed capable of pro- ducing vigorous plants. With other seed, such as timothy, alfalfa, clover, etc., no such selection was made. The number of seeds placed in each series of six to ten tubes varied from one to three with corn and from four to one hundred with other seeds. The number, how- ever, varied with the size of the seed and the amount of available agar surface in the test tube. In several cases as many as 4,000 timothy seeds were treated at a time and transferred to a single container. The following table presents some of the data. Time Required for Time Required to Cause Sterilizing Seed. Injury to Seed. Seed Hours Hours Kaos LEO )S Vay. OS ee ere 8 18 MER TG OONSOUIUT Ne ee ie le eee 6 18 LEU IBN DINED ANN ee rr 15 JE OUETR SE DANES Os 8 EGRESS: ISN WR ES ae 4 IPOS ACO) SOUT, De en a ea 9 a eh 2 I 5 Petroselinum satigum Wothms. 0. .........5 mals Sec VEL TE ADT AP LGR Cn oe Ter (COS OS 02 POON SER i a JO LEX PORSSHGOR OWA, CHG TOE Se rr 2 eae i IO AQUATIC 24 eT DEAIUNE USULGLUSSUI HY Vee ee ge ee 10 1a Fagopyrum esculentum Moench.’.............. No success at all 40 ADA TORSTEN 558 8 cos ony 5 Se en 7G a 4 OLA IUNY TD CrOSm ales ee oe ob Ns ee oe No success at all 2 Seeds hulled before treatment. 3 Tested only on peptone agar and at room temperature. 4 Cuttings of tuber. The data show that the time required in most cases for sterilizing seed and the exposure necessary to produce injury are many hours apart. This allows considerable latitude with respect to the time during which the disinfectant may be allowed to act. It is evident 424 JAMES K. WILSON also that the different seeds are not sterilized in the same length of time, alfalfa requiring about s:x hours while wheat requires more than twice as long. Other tests of the efficiency of the method have been made. In one experiment thirty or more carefully selected seeds of each of the different kinds used were placed in test tubes and covered with a calcium hypochlorite solution containing approximately two percent chlorine. After treatment the seeds were transferred into test tubes on the surface of a medium which contained tap water and one per- cent each of agar and saccharose. After germination of the seed each tube was inoculated with a pure culture of Bacillus radicicola and placed in the greenhouse where it remained 45 days before examination. The seeds used and the time of treatment of each are as follows: Seed Time of Treatment Medicavo sattd As ea ote oe ee ae ee 7 hours hispida Gaertn’, i. Gane ee ee 7 “ INCU Bey a ea ae Pees rey a Aete es 25 © 7 oe LCF (dey Denn rs Semen O RD SRA RO Ba te. 7 . lupuling eve ae ee eee 7 Melalotus alba lADests ince ee ee ee 7 5 gndicata (es) Ae we ne ee sor oficinalis(L..)) Wwains ees. case Ore ee 7 Vaeta. villosa Rotisie ie ee ee ee 614% Se SOMO orev an wants eke se cat ant en 6% OREM? 101/102) DLE tee ees eR aesy Pee Seria’: ONG At 2 TEQUNY TUS: SVLUCSETESICS sai oe Me es ed en ee 5 i SOLLUUS HD invoke tense. he. ieee erence neat: Pea ts 4 The above treated seeds were planted in 184 test tubes which were reopened when inoculated. On examination only three tubes showed signs of contamination. Two of these were contaminated with molds while the other was with a bacterial organism. In addition to the foregoing tests the following data on the efficiency of the method were contributed by Dr. Knudson from experiments made on the organic nutrition of plants. These data are valuable because they indicate the thorough sterilization of the seed as is shown by the freedom of cultures from contaminations even after 30 days or more of culture. The duration of immersion of the seed in the disinfectant and the culture media employed are indicated in the following table. CALCIUM HYPOCHLORITE AS A SEED STERILIZER A25 Immersion Media. Pfeffer’s Solution % Strength to which was Seed ' Hours Added in Separate Lots PaeUm Provensé Iu... 2. . 2s. osu 74% Saccharose, glucose, levulose, maltose and lactose each 2%. PLEO JG (OA es eR ae IO Glucose, levulose, maltose and saccharose 2%. One series in the laboratory and one in the greenhouse. Vicia villosa Roth. (100 seeds)... 6 Lactose, maltose, saccharose, 1% each. Sterilization not always successful. mapnanus-satious Lo... 3. 6 Lactose, maltose, dextrose, and_ sac- charose, each 1%. pRassica oleracegi\......3...... II One series in greenhouse and one in laboratory on _ saccharose, maltose, levulose, lactose, each in four concen- trations from one tenth percent to 2%. Linum usitatissimum L........ Ti Maltose and lactose each 1%. Pesumi sativum L..........--.- 4 Saccharose, lactose, maltose, glucose each 1% and checks. Satisfactory sterilization was secured in every case with the exception of the vetch seed. In certain experiments where only a few vetch seeds were employed no contaminations occurred in the cultures except occasionally where it was clearly evident that con- tamination was due to causes other than the failure of the hypochlorite. In certain experiments on the influence of sugars on respiration, 100 seeds were required for each culture chamber. Failure to sterilize occurred in 20 out of 40 trials and in each case it was due to intro- duction of one or more dead seed along with others. The considerable number of experiments made in which this method of seed sterilization has been employed and in which sterile plantlets have been secured and maintained over a period of thirty days or more conclusively demonstrate the efficiency of the method as an aid for securing sterile plantlets. The ease of operation and the fact that the solution does not injure the seed except after long exposure to the hypochlorite solution make the method particularly desirable. The effect of the solution is probably due to the hypochlorous acid, as suggested by Hooker, which acts as the toxic agent. In conclusion it should be added that the method not only offers a way for securing sterile plantlets for physiological experiment but also for eradicating such plant diseases as may be controlled by treating the seed. 426 JAMES K. WILSON BIBLIOGRAPHY Brown, H., and Escombe, F. 1898. On the Development of the Endosperm of Hordeum vulgare during Germination. Proc. Royal Soc. (London) 63: 3-25. Combs, Ruola. 1912. Sur une methode de culture des plantes superieures en milieux steriles. Compt. Rend. Acad. Sci. (Paris) 154: 891-893. -Czapek, Fredrich ; 1896. Zur Lehre von den Wurzelausscheidungen. Jahrb. wiss. Bot. 29: 321- 390. Godlewski, M. E., et Polzeniusz 1901. Sur la respiration des graines placés dans de |’eau et sur la production de l’alcool pendant la respiration. Bull. Acad. Sci. Cracovie. 1901: 227-276. Griiss, J. 1897. Ueber die Secretion des Schildchens. Jahrb. wiss. Bot. 30: 645-664. Hansteen, Berthold 1894. Ueber die Ursachen der Entleerung der Reservestoffe aus Samen. Flora 79: 419-429. Hooker, A. H. 1913. Chloride of Lime in Sanitation. John Wiley & Sons, N. Y. Kehler, Walter 1904. Ueber Methoden zur Sterilisation von Erdboden und Pflanzensamen und iiber zwei neure thermoresistente Bakterien. K6nigsberg Dissertation. 1904: I-54. Lutz, M. L. 1898. Recherches sur la nutrition des vegetaux. Ann. Sci. Nat. VIII. Bot. 7: I-103. Laurent, J. 1904. Nutrition carbonée des plantes vertes a l’aide de matieres organiques. Rev. Gen. Bot. 16: 14-48. Lefevre, Jules 1906. Sur le development des plantes a chlorophylle, a l’abri du gaz carbonique de l’atmosphére dans un sol amidé, a dose non toxique. Rev. Gen. Bot. 18: 205-219. Lewis, L. L. and Nicholson, J. F. 1905. Soil Inoculation: Tubercle-forming Bacteria of Legumes. Okla. Sta. Bull. 68: 1-30. Maze, P., et Perrier, A. Recherches sur l’assimilation de quelques substances ternaires par les vegetaux a chlorophylle. Ann. Inst. Pasteur. 18: 721-747. Molliard, Marin 1907. Action morphogenique de quelques substances organique sur les vegetaux supérieurs. Rev. Gen. Bot. 19: 241-291. Nabokich, A. 1901. Wie die Fahigkeit der héhern Pflanzen zum anaeroben Wachsthum zu beweisen und zu demonstriren ist. Bericht. Deutsch. Bot. Ges. 19: 222-2206; CALCIUM HYPOCHLORITE AS A SEED STERILIZER 427 Nobbe, Friedrich 1872. Ueber die Wirkungen des Maschinendrusches auf die Keimfahigkeit des Getreides. Landw. Versuchstat. 15: 252-275. Pinoy, et Magrou 1913. Sur le sterilisation des grains. Bull. Soc. Bot. France 59: 609-612. Polowczow, W. 1903. Untersuchungen tiber die Pflanzenathmung. Bot. Centralbl. 93: 462- 464. Prazmowski, Adam 1891. Die Wurzelkndllchen der Erbse. Landw. Versuchstat. 38: 5-62. Puriewitsch, K. 1898. Physiologische Untersuchungen tiber die Entleerung der Reservestoff- behalter. Jahrb. wiss. Bot. 31: 1-76. Robinson, T. R. 1910. Seed Sterilization and its Effect upon Seed Inoculation. U.S. Dept. PNGi oUt: bela nce Circ.O71n—Tilis Schroeder, H. 1910. Die Widerstandsfahigkeit des Weizen- und Gerstenkorns gegen Gifte und ihre Bedeutung fiir die Sterilisation. Centralbl. Bakter. und Parasit. 2. Abt. 28: 492-505. Schulow, Iw. 1911. Zur Methodik steriler Kulturen hoherer Pflanzen. Bericht. Deutsch. Bot. Ges. 29: 504-510. Schulow, Iw. 1913. Versuche mit sterilen Kulturen hoherer Pflanzen. Bericht. Deutsch. BOv.Ges, 31. 97-127. Staklasa, Julius 1898. Uber die Assimilation des elementare Stickstoffes durch den Bacillus Megatherium bei Gegenwart der Phanerogamenvegetation. Cen- tralbl. Bakter. und Parasit. 2 Abt. 4: 511-513. Stoward, Frederick 1908. On Endospermic Respiration in Certain Seeds. Annals of Botany 22: 415-448. v. Ubisch, G. 1913. Sterile Mooskulturen. Bericht. Deutsch. Bot. Ges. 31: 543-552. De Zeeuw, Richard 1912. The Comparative Viability of Seeds, Fungi, and Bacteria when Sub- jected to Various Chemical Agents. Centralbl. Bakter. und Parasit. 2UAbt. 3h: 4-23. ‘AS it Se ae to you that eae be ad stin i ? sig eas ‘6 a pikes bok thought that prac-. | ad gins ane) ess in’ (aid a ‘I } ee | ou _ cyte dee, it t there Bari is ‘the oe af vant an a WAY “Te. the other, builders are. con structing their : i 4 =~ ba 1A 4A Ww ¢ of transpiration to the Lancasrer, Pa. as BAI Yee ere 1c W. AMERICAN JOURNAL OF BOTANY ———a tas WoL. lI NovEMBER, 1915 No. 9 Se asONAL DURATION OF ASCOSPORE EXPULSION OF ENDOTHIA PARASITICA* F. D. HEALD AND R. A. STUDHALTER The effect of moisture upon the expulsion of ascospores of the chestnut blight fungus, Endothia parasitica (Murr.) And., was first studied by Rankin (1). Later the same writer (2) made the following report: ‘‘The speaker found the ascospores being shot from mature pustules during every rainy period last summer’ (1911). A more detailed study of ascospore expulsion was made during the following summer by Anderson and Babcock (3). The main conclusion of these writers on the moisture relation can best be presented in their own words: ‘‘We may say then in general that, as long after a rain as the surface of the bark remains wet, the spores will continue to shoot.” That certain temperature conditions must be fulfilled to insure spore expulsion was first brought out by laboratory tests reported by the senior author (4). Later in a discussion of ‘‘The relative prevalence of pycnospores and ascospores of the chestnut blight fungus during the winter,’’ Heald and Gardner (5) report the results of field tests made during the winter of 1912-13, showing that asco- spores were not expelled during the warm winter rains. A brief statement of the relation of temperature to the expulsion of ascospores of Endothia parasitica has been published by Walton (6) and a more detailed consideration by Heald and Walton (7) of the temperature relation based on laboratory tests in constant temperature rooms has substantiated the earlier results. The only previous work bearing on the seasonal duration of ascospore expulsion is that of Rankin to * During the first part of the period covered by this paper, Mr. M. W. Gardner was associated with the writers, and he deserves special mention for careful and painstaking work in connection with the records and observations made at West Chester. [The Journal for October (2: 375-428) was issued 4 Nov 1915.] 429 430 F. D. HEALD AND R. A. STUDHALTER which reference has been made, and observations made by Anderson. and Babcock (3) during every rain period for the month of August, IQI2. It seemed desirable to have continuous records of the behavior of definite groups of perithecial pustules through one or more seasons under natural field conditions. For this purpose the work presented in the following pages was undertaken. METHOD AND PLAN In carrying out this work the first requisite was a means of holding a glass slide over a definite area of perithecia-bearing bark. For this purpose a wooden bracket was devised. ‘This is a small rectangu- lar block, the width of an object slide, carrying a slot at one end for FS Mar. eae Apr. Paes Feb. hee BiG.*2. FIG. 2. Fic. 1. Diagram of ascospore traps, showing the two positions employed; }, bracket; ba, bark bearing perithecial pustules; s, object slide for catching the ex- pelled spores. Fic. 2. Diagram showing the relative intensity of ascospore expulsion at West Chester, Pa., during the season of 1913. the insertion of the slide, and attached to the tree by a brass screw passing through the center (Fig. 1). On some trees the bracket was directed downwards, in which case the object slide was held in position by a screw turned into the bark (Fig. 1, 4). In both cases the object slide was always wedged firmly into the slot. Ascospore ASCOSPORE EXPULSION OF ENDOTHIA PARASITICA 431 expulsion was recorded equally well by slides in either position. Form A was designed to prevent excessive washing of the slide by the rain, while form B allowed a more ready penetration of the water. This device has been designated as an “ascospore trap’’ to distinguish it from other kinds of spore-traps (5, 8). The original plan called for the placing of sets of ascospore traps on typical lesions under three different field conditions: first, in young chestnut coppice; second, in a chestnut orchard; third, in a forest. Owing to lack of assistance it was necessary to omit the traps from forest trees. A suitable chestnut coppice was found near West Chester, Pa., and the orchard of the Paragon Fruit and Nut Co., Martic Forge, Pa., was selected for the second set of traps. A set of standard weather instruments, including a rain gauge, Friez thermo-. graph, and Friez hygrograph was installed at each station. Following: each rain or rain period the slides were collected and brought to the: laboratory and a record made of the amount of ascospore expulsion: shown for each trap. ite TRAPS AT-\WWESTD ©HESPER A total of 76 ascospore traps were set on trees 4 to 8 inches in diameter, in a badly diseased coppice growth of native chestnut. Trees were selected so as to give varying conditions of the lesions, with perithecia in different stages of development. Some of the pustules showed perithecia which were just reaching maturity when the traps were set while others judging from the conditions of the pustules had probably expelled spores during the previous summer. The first traps (I-16) were set on November 26, 1912; a second lot (17-32) were placed December 3, 1912; another addition (33-36) was made December 18, 1912; and the number was further increased (37-49) on December 31, 1912. Nos. I-49 inclusive were under con- tinuous observation during all or part of the winter of 1912-13. During the spring several more traps (50-52) were set. A fence made of chestnut rails gave an opportunity to test ascospore expulsion under different conditions, and on May 1, 1913, traps (53-56) were placed over areas of perithecial pustules on several rails. The last traps (57-76) were set August 15, 1913. Nos. 50-76 included no perithecia that had reached maturity and expelled spores during the previous season (I9QI2). 432 F. D. HEALD AND R. A. STUDHALTER THE RESULTS FROM THE TRAPS AT WEST CHESTER From November 26, 1912 to March 19, 1913, there were 19 rain periods with precipitation varying from 0.01 to 1.8 inches, and maxi- mum temperatures from 37° to 64° F. during the days of the rain, with no expulsion of ascospores (Table I). The first expulsion of TABLE I Ascospore Trap Tests, Records for West Chester, Pa., Winter and Spring, 1912-13 Rainfall Temperature Percentage of Traps Showing Date Amount, Inches Max, Min. Expulsion Nov. 28 0.01 38 30 ce) Dec. 2 0.36 56 38 0) pedo 0.5 68 36 traces bed HTL 0.03 43 a7 o) oS oA NiO seen eee 0.2 46 30 O : BQ) a hte honey eee? 0.89 53 33 O 1, RRO Ra Ne os. 9.5 (snow) 34 25 oO ES ee ae he 1.02 27 32 0) SD BOs ae en eee 1.8 46 36 ce) Naty 3a Sore oe 1b 10:08 58 a7, fe) ane. Cera ENN 0.1 (snow) 39 27) O ys Jhansi 0.78 60 36 O PL ALONE ra els 3 cueaena ee 0.13 40 22 0) ol DES eee | 0.24 59 32 O 5) STG tO thin ensoee | 0.81 46 26 O mee Uae Sol rea a nee O:3 1 57 23 0) et Ol stle sui Uk cert 0.24 54 22 fe) boy 27 Ot aan pe ieee | 0.88 57 28 O AGM e\ 0 aan & ara sate eer pth a] 0.15 35 15 0) A WD ON wie cuted Nantes erent 0.07 58 45 O ig TED VTBD ohm hh ahem eal 0.78 60 38 0) VI cite ail Op geen ee ere: 0.53 49 38 O 4 TOG nh. beeen: 1.375 64 29 fe) E20 ean oe 1.64 63 49 47 ascospores for the season of 1913 occurred on March 20 following a rainfall of 1.64 inches and expulsion continued through the following months, but was checked by unfavorable temperatures early in November (Table II). From November 16, 1913 to May 4, I914, there were 18 rain periods with precipitation varying from 0.10 to 1.95 inches and maximum temperatures from 34°-68° F. during the days of the rain, and no expulsion of spores. The first expulsion of ascospores for the season of 1914 occurred on May 5, following a rain of 0.71 inches (Table III). It may be noted that the first ex- pulsion for 1914 was about six weeks later than in the spring of 1913. The records were continued to the end of July, 1914 (Table IV), when there was a forced discontinuance of the work. ASCOSPORE EXPULSION OF ENDOTHIA PARASITICA 433 TABLE II Ascospore Trap Tests, Records for West Chester, Pa., 1913 Rainfall . Temperature Pereentaseoiirars Date Amount, Inches Max, | Min. SH wing te pulsion METER ZO eth coe 6 lasek cbaoeze xs | 1.64 63 49 47 Be 2027) a ee 1.18 69 38 47 PNPM Mack Ese ck aa, 0.56 76 44 21.5 Ma BOT 36 eas. 2.49 58 27, 36.5 (OST IN | C eat aa 157, 67 4I 4 Be 72 Oe wu es a 2.43 74 48 65.4 (CES) es O.1I 56 54. 8 WMlaty TG=17 60. i ee en 0.65 80 48 46.4 10 GSS eH ee era 3.20) oa | 46 70.3 Oo NGA EPA ae ee ae 0.69 72 53 83.3 | A oer 0.12 83 64 re) Be OS re hiee ied ao 0.08 88 62 50 9.” GO ca ean ana 0.02 82 65 11.1 CO ae a 0.19 78 65 52 OO Gag aoe he ee ae ae 0.22 86 66 66 ty 85-0 me 0.16 92 69 28.8 WANG ess 8 2s ed Sas 0.30 75 56 Aven He SIO ise ence ea 0.75 86 56 88.5 Gn cae 0.20 87 57 27.5 eR Te AS oe ge 0.05 82 61 4 RRR AR Pi. 2e eh 0.04 88 66 4 i Ge) eee eae 0.06 gI 70 O EASON ees ln 0.74 96 68 60.8 ENCE 0S, et 0.05 83 72 11.5 ° Ts OG ot aa 1.98 83 62 74 MLO he che Bethe ees 0:37 93 ve 75.5 RMU Pes Shah", oe 0.03 93 70 0) ELON as 0.40 80 62 67.6 BE 2 Oe oece Cae 0.25 76 50 63.4 WZ: | doleseese col aaeierer nec 0.175 85 63 50.7 Meme ORO eas ot at = hen 1.10 81 62 74.6 SDE ie i asa ieewe dh 0.37 86.5 66.5 62.8 ND fo ean ae er re 0.095 70 58 18.5 Me eNO Fos 0.26 74.5 52 577, BETO NOG) cache es ee. 0.68 74.5 58 62 LOS 2Oial. Ges os 0.09 65.5 57 34.2 2O PO Oe ak 1.26 79 48 77k Ocoee eee 1.30 71 52 54.3 Meer Ten hoo mete ce ts Ki, 0.73 69.5 62 67.1 PRO 16 ote een nara ar ee 0.86 69 50 64.3 eNOS ON ney cess 0.08 69 54. 2.8 BOA 268 8. ans Wee 1.44 64 49 743 NOW 26 6015 -Fusc8 Shida 1.00 60 37 54.3 The percentage of traps showing expulsion for any rain period during the season of perithecial activity varied from 0 to 88.5 (Tables I-IV). The failure to get expulsion of spores from all traps depended on the following conditions: 434 F. D. HEALD AND R. A. STUDHALTER . Temperature below the optimum for expulsion. . Insufficient rainfall. . Location of the trap with reference to the direction of the rain. . Condition of the perithecia (not in proper stage to expel spores). BW NbN & TABLE III Ascospore Trap Tests, Records for West Chester, Pa., Winter and Spring, 1913-14 Rainfall Temperature Percentage of i Traps Showing Date Amount, Inches | Max. Min. Expulsion INOW. “8=0 a ae ie ok 1.00 60 37 54.3 ~ oF SOg.. err oe 0.96 58 28 ce) eee re | cate 0.62 44.5 33.5 O DCC iets eee | 0.53 52 AI e) Os ails sear tcnte be Sa 0.05 47 | okeey re) Fe ee Sa 1.95 50 32 0) Jan Lincs ee eer ae 39 25 ce) ie EAN Is Soca Ree 1.35 50 4O re) ea BRAT st Reon ee | 0.88 Key 2 O RGD 6-7 = tina ne yee | 0.62 £5 vile 27 O ie Ee eee | 4.5 (snow) 32 re) e) Us © MOR. naan eet eee 1.5 a 3 25 D fe) iia BLOQU can caterer ee aae 0.68 34 29 fe) Mare 31-25 aise | 4.50 (snow) 38 I5 e) . BOM ht teenie 6.00 “ B30! Vii 26 O aie! Ricaer arene Ae e traces ‘“ 29 20 fe) Dey hcl aR Lee 0.10 AC a ane O SO BOO 2 is esi ree | 3.5 (snow) ZA oa |b Oar O ee) ee. Cee eae 0.35 58 Ba O AO Te SDs igs ee ea 0.25 48 aT O Pane oh ome wc ri 0.60 67 4I 0) oe AML Bh eae Sk ac ae ae 0.49 45 41 (0) SD Oe Waive sie Mone eae 0.44 68 45 ) Pe, SO Sona ch tase teen 1.57 50 44 O os 72 Olle fat Cees an 0.10 50 45 0) Mayet 5 2 ee ae | 0.71 65 ae 50 Ths) There was a marked example of the influence of the temperature following a rain of 1.57 inches on April 13-16, 1913, when only 4 per- cent of the traps showed expulsion. The maximum rainfall following which there was no expulsion of ascospores when temperature con- ditions were favorable was 0.12 inches on June 7, 1913. A small percentage of traps sometimes showed expulsion when the rainfall was less than 0.12 inches. (See Tables I-IV.) The number of rains for the entire period covered by our investigations following which there was expulsion of ascospores is shown in Table V. Perithecial activity in 1913 began in the spring as soon as tempera- ture conditions became favorable, but since the temperatures at this ASCOSPORE EXPULSION OF ENDOTHIA PARASITICA 435 TABLE IV Ascospore Trap Tests, Records for West Chester, Pa., Spring and Summer, 1914 Rainfall, Temperature Percentage of Traps Showing Date Amount, Inches Max. Min. Expulsion AS i 0.71 65 59 11.8 OO SAS ae 0.21 58 52 traces “O | TG a eae 0.56 72 45 a 10 Dk ES a ee 0.19 92 65 2 aie oe nee eae | 0.30 70 61.5 9.2 3 Ss rr 0.08 75-5 58 0) oO 77 1.08 80 48 26.1 ME Re nce c ss | 0.68 86 60 29.2 Miya cro WAS Sor s| 1.15 82 64 24.6 . Ce dbs a ie 0.35 Gig} 61 24.3 of Se ee 0.70 70 59 31.1 GES ioe es 3.14 85 66 36.6 ORS ae eee a 0.25 83 69 40 MME coe sons. ae So 2 0.31 16.6 MS 3.58 | 23.8 TABLE V Summary of Number of Rain Periods and Number of Times Spores Were Expelled No. of Rains Period No. of Amount of During Which Rain Periods Rain in Inches Expulsion Occurred Nov. 26, 1912—Mar. 19, I913.’...... 19 12.125 e) WMar-20, 19013—Nov. 15, 1913....... 42 30.01 39 INGy. 16;,1913—May (4, 1914....... 18 11.64 oO Mayes elOLd aI uwly 31; 1OR4:,:.... 15 13.04 14 TABLE VI Records for Ascospore Traps, Martic Forge, Pa., Winter of 1912-13 Rainfall Temperature Percentage of ; ‘Traps Showing Date | Amount, Inches Max, Min. Expulsion atte 2 Oe ok ae. ak. | .08 38 31 fe) Bem G Erac ES ae. ea | .06 62 42 O 1G! Oy er Le ee | 70 My) 28 fe) ee EOe Marr acetals: | 03 22 18 fe) ho a A die eee aie oe 04 35 29 fe) re by | .09 56 49 e) ee ys | .36 58 38 oO Mar. 5 | .02 45 ae 0) MPL OP eeu. ours sis. 3 | 55 52 4I O MRT Ara cts cod. es Sous 10 55 53 fe) Ot a oe ae 1.29 67 53 2 436 F. D. HEALD AND R. A. STUDHALTER time were generally below the optimum for spore expulsion, the highest activity was not reached until the month of May. There was a decline in spore expulsion during the months of June and July, but there was abundant expulsion in August, September and October. In November, with the return of unfavorable temperatures, the perithecia ceased to expel spores. represented graphically in Fig. 2. TABLE VII This seasonal relation is Records for Ascospore Traps, Martic Forge, Pa., Spring and Summer, 1913 Rainfall Temperature Date | Amount, Inches Max. Min Maier cen ae ee a 1.29 67 53 ss coll yee pence eae ee As eee ted ras ae .Q2 61 56 ee 2Or .62 66 49 ci iD iT ee NAN oer aac Rc e te 102 68 52 : 1 6 et 17 NRE TERE a MS PE, BAG, 65 30 BT a. eaten eee stern ee .04 69 49 Apr. ROT RUS Nia a ea eee eee | 10 fe 39 Se atresia wh Ms Pe, ee cae 23 59 40 M 1 i eRe eae aera are ye aan 1.78 57 51 HOP fies tishaveeootton han ee eee ay 1.15 59 45 7 7) saat er mae iets Aine Mie base f 8.09 2°32 58 45 DOR Marries Om as eee ee .07 49 35 Wi ayceS Reiiectas, osttearwicnt ets rae eee Sti 74 42 STR) ENO Terie aeaens Rae ee eae Baie 84 49 PES OT rh gee ance ee aaah ee a2, 64 50 Sy 20-20 ei ne ene .69 75 44 JMNESTOH OTE ek eens eek eres 34 92 52 Se POA ean era rence Cteiiot 2 oa ae 26 75 61 Ae, ee ne Teas eM aT, | 7. IOI 74, July.’ toganicl 5. eeerne, goede 1.97 97 66 HS © RO epee penn, tal ee 59 69 63 Se) 7 TAG DORE ee ee Roce ees 12 89 57 Fe (ams Cece remy oy RM ro), cL ied 2.68 87 68 % Par aaa sgmarniar areas Oke eek 1.37, 82 64. ME CORT en ee etek renee coat 1.13 97 70 Pe DOU ofa at enact Gros noe 17 73 58 PS SOOO Woh pe. eae ae mee .30 79 68 Sept.) Fiano tage ee ae .67 92 69 abn (Meri hve erp Ar Na SCR 2 .09 67 56 he “CEPT SG att oe ocr ger ere .19 75 54 Che 7D O- OIE ane een Reet ae eena ee Paes 80 57 Bae 10 Pie ah ee ene er neg | .02 68 55 Sept: 30-Octy2enw ee ere 1.63 Pez, 50 OCts TEA Gein eure Ses | .67 68 56 PES SGT Qin Mehsana tae oa, PE eRe 1.05 57 46 WME 710 BRR hen ona SS OS eh er ae 15 68 42 2S =O Otis, erent mers eaten 1.04 61 50 NOV: SO=O 8 al nee ee eee OI 58 Ba SSS TST oo Nee eng eee .83 40 25 Percentage of Traps Showing Expulsion Tiable are! ae: R= BE OREEE srageceasieats TTT entire : Sun taal sie tt tT Ht BRBRBEH Sku : PCERASEEREE 4 Ht nanan Jan |Feb ct a Ea ct. |Nov.|Dec. eptlO. ic) ez Ltn Eiko = T NI | 8 Dis G Heed Ean be UL EESEROC CES CR PARE ASEEE BD eae om Sem eB 8 Ae 0G SIN] La SS a aT STA Ee ee Lea a Sea a eB a PNR PRP EES SiS SSS SSS SS SSS SEAS SES foe SIS SISIS a SSSA a aA S SSRIS SSS Chart showing the seasonal duration of ascospore expulsion at West PIG-3. Chester, Pa. The vertical parallel lines indicate the approximate time when the D following two parallel lines indicates the discontinuance of a trap. The figures below each month refer to the number of rain periods for the traps were set. month, while the figures opposite each trap number represent the number of rain periods for the month following which there was an expulsion of ascospores. 438 F. D. HEALD AND R. A. STUDHALTER At the beginning of our work it was an open question as to whether perithecial pustules would exhaust their power to expel spores during the course of a single season. Our records show that bark bearing stromata with perithecia that probably were active during the season of 1912, gave an abundant expulsion during the season of 1913 (Traps I-49). For these same traps there was either no expulsion or it was irregular and spasmodic during the season of 1914. Somewhat more certain evidence of the continuation of perithecial activity during two seasons is shown by the records for traps 57-75 from which there was an abundant spore expulsion during the season of 1913 and the part of the season of 1914 covered by our observations. This duration of ascospore expulsion from the different traps and also their seasonal activity is represented in an accompanying chart (Fig. 3). Records were obtained on the majority of the 76 traps for the entire period covered in this report. Sixteen traps were discon- tinued before the expiration of the work for various reasons. In 9 cases the bark was shed from the tree due to the action of blight and accessory agencies, in 4 the bark was overgrown by sporophores of sap rot fungi, while one was overrun by a foreign mycelium. THE TRAPS AT MARTIC FORGE A total of 75 ascospore traps were set in the chestnut orchard at Martic Forge on Paragon trees ranging from 3 to 12 inches in diam- eter. The pustules over which the traps were placed were in varying stages of development, but were on the whole much younger than those at West Chester. The first traps (1-41) were set on January 21-24, 1913; four (42-45) were added on January 31; three (46-48) on February 10; and two more (49-50) on February 12. An addition of twenty-five traps (51-75) was made early in October 1913, and the first record on these was taken for the rain of October 11. These last 25 traps were placed over pustules which had just reached sufficient maturity to expel spores during the season of 1913. THE RESULTS FROM THE TRAPS AT MARTIC FORGE There were 10 rain periods between January 29 and March 13, 1913, with a precipitation ranging from 0.02 to 0.70 inches (Table VI). No expulsion of ascospores occurred during this period. Tempera- ASCOSPORE EXPULSION OF ENDOTHIA PARASITICA 439 tures favorable for perithecial activity were first offered on March 14, 1913, when 2 percent of the traps shot spores following a rain of 1.29 inches. From March 14 to November 9 there were 38 rain periods with precipitation varying from 0.02 to 3.47 inches (Table VII). During most of these rain periods there was abundant expulsion of ascospores, and following seven of them all of the traps expelled spores. After November 9 the temperatures were again unfavorable TABLE VIII Records for Ascospore Traps, Martic Forge, Pa., Fall and Winter, 1913-14 — Rainfall ‘Temperature Percentage of 5 Traps Showing Date Amount, Inches Max. Min, Expulsion INGO OG Bi ds ve a ee ees | OI 58 22 S223 MINS NOM res fas no Mat oe 83 40 25 O ZORA ah Saco en's, oe 54 Ke) 32 fe) ILS Re ae oa ae ca ac .06 44 20) fe) Rl cs UO ie cs aa 51 52 37 0) MONA ce i es Se. Big 49 26 fe) ME BaD eS ais sys wucsig sd atte ¢ Qn2i 49 28 fe) tee eFC Dalal 41 25 O DEO Ae hoe od ota etme che gs 1.21 47 39 fe) Eh GUO oe et ie eae a .83 61 34 fe) [1S 082 ONC rota ie ena ee a naan ae .48 46 24 fe) MME eM Be A Pa ciatee coc hee s3 lent 2 30 Bie) O “SSO Ee Se a eo ae 102 31 2 O eb a2 8— Nate Te ry .h ay as file 0%. 38 40 18 re) DMeMicmert Ss OFS ce oie Mikawc d stcae ss | 72 26 17 fe) te TALS ie Ae Re eae ce rena | .03 30 18 O eMC O22 Lc niah, Ae ROL huh hac | 34 ay 17 O PED Z Gre Srna rate Ow oe Wee kes | .36 59 36 e) INGO? 7 1a a tea ticet aR a a echoing 28 45 35 O i (see ari: LMR as ek hang ae ee ler 59 65 35 0) SSO cre or eaten aa | 07 59 aa O TaN A Sin Pose uate Secs | 70 45 39 fe) Peed OR nar sey Mery ev homie aad. 5's 59 67 53 10.8 for perithecial activity, and no expulsion occurred for any of the 21 rains between November 10, 1913 and April 15, 1914 (Table VIII). Beginning with April 20, with the return of higher temperatures, there was again an abundance of expulsion following every rain period until July 31, 1914, when the work was discontinued (Table IX). It will be noted (Tables I and VI) that in the spring of 1913 expulsion began at Martic Forge a few days earlier than at West Chester. In the fall of 1913 perithecial activity ceased with the same rain period at both places, but in the following spring the Martic Forge traps were again the first to expel spores. There was very 440 F. D. HEALD AND R. A. STUDHALTER little difference between the temperatures at the two stations. The earlier start at Martic Forge is accounted for by the fact that the pustules over which the traps were set were on the whole much younger and in better condition to expel spores than those at West Chester. During the seasons of perithecial activity for both 1913 and 1914, a larger percentage of the traps at Martic Forge expelled spores than at West Chester. Furthermore a much more copious expulsion was obtained from individual traps at Martic Forge. TABLE IX Records for Ascospore Traps, Martic Forge, Pa., Spring and Summer, 1or4 Rainfall Temperature Percentage of Traps Showing Date Amount, Inches Max. | Min. Expulsion Apr i200 eat. cake a .59 67 se 10.8 256 er ae eee 1.57, 68° | az 27 May eh5 uth oars co gine 66° 4| = 60 27.0 Pee or mre CEES sig “12 62 50 22-9 SOS TD cies ta ae ee ee er .40 81 a7. 16.2 i DOE ay setae (ees eee tae ee Ae 27 23 e60 5.6 Me! Fe. ect vie oe eee Ae 72 |". 164 46.6 Oaks ean od ee ae (22 97 |= 65 20.8 ro TG ancl 20 23-0", eee ee .97 gI 50 43.1 = 23-25. andi28.. ee ee 2:10 94 62 68.6 Naly: S233 he ie ec ee ee 1.63 80° = 54 55.6 if eee ee ce A ee hrs 28 73 57 78.1 sTOsand S22. 2. St eae ae .95 90 66 69.2 ‘ E3214 it. oe ee oe 84 65 69.9 TAS V5) a see 53 86 66 75-3 STATO) 8. 1 ie een ee eee eens Deg 86°27 |) 166 53-4 See) C7 amie em Ree As es fee ou gI 67 45.8 SPA ZOU CUS co ng Dt ile Eee 2.61 81 55 56.2 TABLE X Summary of the Number of Rain Periods and Number of Times Spores were Expelled at Martic Forge, Pa. Period No. of Rain Amount of Rain NO ee eeaeenie Periods in Inches Occunced jian-20,,19013—Miar. a, Toler a 0) 2.03 0) Mar: 04, 1913—-Nov. 0) 1013... aes 38 31.94 33 Nov. 10, 1O13—-Apr.. 19, 1014... 23 14.16 O Apis 20,1014 |uly 39, tol eee 18 17.64 18 Under laboratory conditions the minimum temperature for asco- spore expulsion was found to be between 50 and 55° F. While it was impossible to get the maximum and minimum temperatures for ASCOSPORE EXPULSION OF ENDOTHIA PARASITICA 441 the exact period during which ascospores were expelled after each rain, still the maximum and minimum temperatures during each rain period (Tables VI-—X) show that the minimum temperature for expulsion in the field corresponded quite closely with that in the laboratory. The rain of April 14, 1913, offers a good example of a heavy rain with no expulsion, due to low temperatures (Table VII). Although copious expulsion occurred both before and after this date, and the precipitation had been heavy (1.15 in.), the temperatures were below the minimum for perithecial activity. . @ : eee Sager eur ge” Eien oes eS 0 oe Os ey ae eee ore ee ee Fic. 4. Diagram showing the relative intensity of ascospore expulsion at Martic Forge, Pa., during the season of 1913. The number of rains for the entire period covered by our investi- gations and the number of rains following which ascospore expulsion took place, are shown in Table X. The seasonal relation of expulsion at Martic Forge for the year 1913 was somewhat different from that at West Chester. At the latter place the monthly percentage of traps showing expulsion in- creased suddenly from March to a maximum in May; during June and July there was a decided decrease, but after July there was abundant expulsion until November (Fig. 2). At Martic Forge there 4A2 BE. D. HEALD? ANDUR. A. STUDHALTER Fis. 5. Photographs of slides from trap 52 (upper) and trap 62 (lower). Slide from 52 was taken following a rain of 1.63 inches on July 2-3, 1914; slide 62 following a rain of 0.95 inches on July 10-12, I914. 443 ASCOSPORE EXPULSION OF ENDOTHIA PARASITICA cau eM TLL Trea ck HE | is eens Pecastencreaosnesecrerorcac agenenenctoeaenartraroras0vaTsevirc7070 E SSN Sty SISSON ISN Sofeiniolotiofeiniotojabataay}otoiei|atotoka =) an Lo f,oly = mala RID Rens Qhahohs MIS. SUN = x Pe ere eased UU EGE suvacgeusnd lDacuestt ssuececeaicaeseeecuaeeias : Aad A021 cHANUSHAAAGRUIGNAOFONGTOMNGECECNSCHTSeL=z2crsforT*= eT EE : See adccaieentmaiian CECE OES ULE EEEETTL LATER HEHE EERO CREE EOS iGR CER BSE EEREEEREe an HCBE REE ARR ERS UNECE cED EES bEoE GAB see TeEBEBRePBeNEees Pe BEDE EERE ANOS EERE EER HiE SEHR BREE EAIE EERE RR EERSRaaReR REDE ACNE OREaReneUneRe Se en eitin ateeeeets ot Ei HEBER REMEG ea za 4aTzZ |4 14 414 Ez, 473 rae 413 35 wa AZ Rae, 4 A [4t4 rat Z| 3 4 (a Z| Aa ra ra Zs 4 Sue OOP U RR RERAEEAR AMARA Pirlo ibe beret TEEPE CEE CE okpeptoprppptpppoa}pohebopohopohstshopaporstapopopspopobrplspopopohoporohoporspotomors TT LLL TT TTT TTT TTT Pfehopshobohobbaistohtetohopotoftobotopapstobopopstopbstomptoitcboaboborapbtorrsttorl LT TT TTT TTT TTT Eres Mepst-siporosenisrnborppppolmebtcberpersttby tert | TTT TTT TTT TTT TT —l—!) See Rennenined UE eens sei a Sei aeeEsS Pe EES te sel en | LI oO] Bae ee MOST THY St |LohLo taahshalshok IBIS RS OSS) NS) REIN Fic. 6. Chart showing the seasonal duration of ascospore expulsion at Martic (See legend of Fig. 3 for more detailed explanation.) Forge, Pa. 444 F. D. HEALD AND R. A. STUDHALTER was a gradual increase from the beginning of expulsion in March to a maximum in August, followed by a more sudden, though steady, decrease until November (Fig. 4). The maximum at Martie Forge (98.8 percent) was also much higher than that at West Chester (67.3 TABLE: Xf Record of Expulsion from Typical Young and Mature Pustules at Martic Forge for the Active Season, 1913 Rainfall Trap Numbers and No. of Active Ostioles eroune Young Pustules Mature Pustules Date Inches 21 23 37 40 II 17 28 43 Mar. I5.. 1O2 0) 0) 0) fe) re) re) 6 0) So 2OR ean ke .62 O O fe) 10 fe) O O O up2O-27 hat BAG, e) N 14 re) 15 8 N 28 ADT Stee te [10 oO e) oO O e) 0) 0) 0) ry Rat ates 23 fe) O O 27, fe) I fe) O De WAR a renee 1.78 fe) N led ie 31 N I 27 Ca Pe BOn hoe oe 1.15 O O O O fe) O O O leo nn tn Ae ae 2123 2 N N 19 N N_ | 100 N Nai et Sein ftyes iL) fe) O I re) re) O 0) e) A (6 be ees 7A 25 N N N N N N N ges kb 27 fe) I O I 14 16 21 N Sen 2O=30h fe .69 N N 64 N N N N II June To=2i. 2). 34 F: |e: 0) 95 N N I 7 UE FZ ar ere ee ee 26 N N 6 N N N N N Wale (27 Paes ihe 37 N 13 N N N N N N July 2and5 107. 100 58 | 100 N N N N 73 spun oer eS 59 N N_ | 100 N 70 50 N 50 Se izaand.20 s1i3 18 4 I 5 2 41 6 2 aN Sagal Loy Ber ee 2.68 N N 47 N 48 P P 54 Ste Ol eartr pac 1.37 N 32 38 36 22 18 8 65 Sera O Mere nor 1s) 35 N N 40 26 24 18 27 Oe ele noe LZ 22 66 4 23 34 22 II 21 C20. eee 30 N 43 4 49 BY, 2 15 20 Septet (7 see ae: .67 N N 29 27. 10 5 24 N Be NLD ees ai oe .09 N 18 5 19 4 P 1? 5 my OE TT Ouaye oot .19 N N 3 27 22 P 7. P b NEQOR2 Ver fe 2.27 N N N N 17 P 5 33 =) "30-Oct:2 1.63 N N N N 14 6 Pp 34 Oct ahi era. .67 N N N N 17 5 II 26 Db reclOnan ees 1.05 N N N 26 10) 27 3 5 Pe UE2 OG 15 O fe) re) o) fe) 0) 0) fe) METZ 5260, 1.04 N N N N 15 2 9 8 Novy. 8-9)4.50 OI N 65 N N 19 0) 10 8 Ben Coo (oye 153 0) 0) O re) 0) fe) re) O percent). N = numerous. It is difficult to explain this seasonal difference except to suppose that it might have been due to the difference already men- tioned in the condition of the pustules. ASCOSPORE EXPULSION OF ENDOTHIA PARASITICA 445 The fact that perithecial pustules would not exhaust their power to expel spores in the course of a single season is even more clearly shown by the traps at Martic Forge than by those at West Chester. Pustules tested by traps I-50 must have expelled spores during 1912, before the traps were set. All of these traps showed active ostioles during 1913, and all but five of them expelled some spores during 1914. Pustules tested by traps 51-75 probably expelled their first spores during the spring and summer of 1913. All of these showed perithecial activity during 1914, most of them expelling spores in large numbers. Fig. 5 represents slides taken from traps 52 and 62 during July, 1914, and shows how copious the expulsion may be during the second season. No heavier shooting was obtained from any of the traps even during their first season of activity. The accompanying chart (Fig. 6) represents the duration of expulsion and. the seasonal activity for the traps at Martic Forge. In some instances the tree was entirely dead and the bark peeling off at the time the trap was set, early in 1913. Nearly all of these cases showed continued perithecial activity during 1913, and most of them had not entirely lost their power to eject spores in I9QI14. This is best shown by traps 10, 43 and 50 (Fig. 6). There is a difference between the continued activity of young and mature pustules. The former continue to shoot spores unabated throughout the entire season, whereas the maturer pustules eject a larger number of spores during the early part of the season and show a gradual decline toward the winter months. In Table XI is pre- sented the record of expulsion of ascospores for each rain period during the active season of 1913 for typical young and mature pustules. EXPULSION OF ASCOSPORES FROM BARK SHED FROM OLD INFECTIONS Trap 41 at West Chester was discontinued in April, 1914, the bark to which the trap was attached being shed from the tree. In order to determine whether perithecia in bark shed from trees would continue to expel spores, five representative pieces from bark col- lected on the ground beneath trap 41 were brought to the laboratory and tested for ascospore expulsion. Each of the pieces (1 X 3 inches) showed active ostioles, the maximum number for any day during the progress of the test being forty. This activity was less than from similar bark taken directly from cankers. Bark shed from the rail 446 F. D. HEALD AND R. A. STUDHALTER fence adjacent to traps 53 and 54 was also tested for spore expulsion with positive results. These results give added emphasis to the statement already made (7) that the chestnut blight fungus has a most remarkable power of ascospore production. DISCUSSION A comparison of the results obtained at the two stations shows a very general agreement in the more important features. All of the traps at West Chester were set on native coppice while those at Martic Forge were on Paragon trees grafted on native coppice. Some of the traps at the latter station were set below the graft unions. The greater activity of the perithecia during the second season at Martic Forge as compared with the results at West Chester is probably due to the host relation, permitting a somewhat more rapid maturing of the fungus at the latter place. Since the prolonged perithecial activity established by our obser- vations for Endothia parasitica, is not in accord with what is known in regard to many other pyrenomycetous fungi, an explanation for this peculiarity should be sought. There seem to be two important features in the development of the fungus that contribute to prolonged perithecial activity. First, the period of successive maturing of asci is very extended. This fact is substantiated by the work of Heald and Walton (7) on the expulsion of ascospores under laboratory condi- tions, and is in agreement with the study by Anderson (9) of the morphology of the perithecia. The second feature not so clearly revealed by the morphological studies (9) appears to be the successive maturity of the perithecia in a given stroma. All perithecia in a given stroma do not reach maturity at the same time, some being still buried deep in the stromata when the necks of others adjacent have reached the surface and are in a condition to expel spores. Add to the above features the fact that there is no seasonal relation to the maturing of perithecia and one must admit that Endothia parasitica is remarkably well equipped for the continuous production of asco- spores. During the progress of our work detailed examinations were made of the perithecial pustules included under each trap, their condition being recorded at the beginning of the tests and at intervals there- after. Asa result it can be definitely stated that the development of new stromata was the exception, the bulk of all expulsion recorded coming from stromata that were present at the beginning of the tests. ASCOSPORE EXPULSION OF ENDOTHIA PARASITICA 447 SUMMARY 1. Expulsion of spores from the perithecia of Endothia parasitica begins in the spring with the first warm rains, and increases to a maximum of activity as conditions become more favorable, to be followed by a decline in the fall when lower temperatures prevail, and ceases entirely during the cooler portions of the year, although there may be an abundant rainfall. During 1913 the period of expulsion extended from about the middle of March to the middle of November. For 1912-13 there were I9 rain periods during the fall, winter and spring with no expulsion of ascospores, and in 1913-14 there were 18 rains which induced no expulsion of ascospores. During one third to one half of the year there is then no expulsion of ascospores, but during the remainder there is an abundant expulsion with each rain of any consequence (0.10-+) except in a few instances when the temperature drops below the minimum. 2. Perithecial pustules of the chestnut blight fungus show a re- markable power of spore production. This power is not exhausted during the course of a single season, and in many cases is as marked during the second season as during the first. Our results point also to the conclusion that pustules first producing mature perithecia in the fall may continue to be a source of spores during the two follow- ing seasons of perithecial activity, the maximum of production occur- ring during the season following their maturity, with a gradually dimin- ishing production of spores during the next season. This remarkable power is due to successive maturity of asci, to successive maturing of perithecia, and the successive maturing of stromata throughout the season. LITERATURE CITED 1. Rankin, W. H. The Chestnut Canker Disease. Abstract. Phytopathology 2: 99: I912. 2. Rankin, W. H. How Further Research May Increase the Efficiency of the Control of the Chestnut Bark Disease. Proc. Pa. Chestnut Bl. Conf. 46-48. 1912. 3. Anderson, P. J. and Babcock, D. C. Field Experiments on the Growth and Dissemination of the Chestnut Blight Fungus. Bull. Pa. Chestnut BI. Comm. 3: 1-45, pls. I-15. 1913. 4. Heald, F. D. Pathological Investigations. Rep. Pa. Chestnut Bl. Comm., July 1 to Dec. 31, 1912: 40-42, figs. 22-38. 1913. 5. Heald, F. D. and Gardner, M. W. The Relative Prevalence of Pycnospores and Ascospores of the Chestnut Blight Fungus During the Winter. Phyto- pathology 3: 296-305, pls. 26-28. 1913. 448 F. D. HEALD AND R. A. STUDHALTER 6. Walton, R. C. The Relation of Temperature to the Expulsion of Ascospores of Endothia parasitica. Abstract. Phytopathology 4: 52. 1914. 7. Heald, F. D. and Walton, R.C. The Expulsion of Ascospores from the Perithecia of the Chestnut Blight Fungus, Endothia parasitica (Murr.) And. Amer. Journ. Bot. 1: 499-521, figs. I-2. I914. 8. Heald, F. D., Gardner, M. W. and Studhalter, R.A. Air and Wind Dissemination of the Chestnut Blight Fungus. Journ. Agr. Research, 3: 493-526, figs. I-3, pls. 63-65. 1915: g. Anderson, P. J. The Morphology and Life History of the Chestnut Blight Fungus. Bull. Pa. Chestnut Bl. Comm. 7: 44, pls. I-17. 1913. DIMORPHISM IN CONIOTHYRIUM PIRINUM SHELDON! GC. H.*CRABILE Several years ago the writer began a study of the Phyllostictas and Coniothyriums associated with the frog-eye leaf spot of apple. In a previous paper”? the morphology, cultural features, and host relationships of Coniothyrium pirinum were briefly dealt with. The following report is the outcome of pure culture studies of C. pirinum isolated from apple leaf spots. The first cultures of this fungus, Strain I, were secured in I9I1I from Black Ben Davis leaves at Blacksburg, Va. In 1913 another strain, Strain II, was obtained from the same source. Although the mycelium, pycnidia and spores of the two are in no wise to be dis- tinguished from each other under the microscope, the two strains are readily distinguished when grown in Petri dish culture on artificial media. Starch agar? has been used almost exclusively in these studies. Plates of this agar were poured, cooled, and inoculated at the center by the use of a platinum wire. Tubes of this medium were used in making dilution and stock cultures. Strain I, four years after isolation, exhibits the same characters as when first secured. It may be described as follows: PETRI DisH CULTURE: STARCH AGAR Mycelium pure white or pinkish until about the 1oth day, acquiring then in spots, which spread gradually throughout, an olivaceous color. Pycnidia very few, large, multilocular, prolific, irregularly distributed about the point of inoculation, appearing 8 to 30 days after inoculation (Fig. 1). Strain II when first isolated was described as follows: 1Paper No. 40 from the laboratories of Plant Pathology and Bacteriology, Va. Agr. Exp. Sta. 2 Crabill, C. H. Studies on Phyllosticta and Coniothyrium occurring on apple foliage. Rep. Va. Agr. Exp. Sta. 95-115. IQII-I2. 3 Made according to the following fermula: H2O, 900 cc.; MgSOu, .5 g.; Ke H POs, 1.0; NaNO, 2.0; KCI, .5; FeSO.u, .1; Agar agar, 20.0. Cook, filter, and while still hot add the following paste: H2O (cold), 100 cc.; cornstarch, 10 g.; and sterilize. 4 Unless otherwise stated, cultures were incubated in moist chamber. 449 450 C. H. CRABILL PETRI DiIsH CULTURE: STARCH AGAR Mycelium pure white or pinkish until about the 5th day, when an olivaceous color begins to develop just inside the margin of the colony. Pycnidia multitudinous, small, prolific, uniformly distrib- uted over the colony, appearing 3 to 6 days after inoculation (Fig. 2). As the pycnidia develop, the olivaceous color is absorbed, so that the greenish zone near the margin of the colony is continually advancing. Fic. 1. Strain I in about the 43d generation, 24 days old. The tardy pro- duction of relatively few large multilocular pycnidia is characteristic of this strain. A few young pycnidia have just appeared in this culture. Note: The photographed plates here figured may be located on the chart shown in Fig. 6 by sub-numbers which correspond to the numbers of the figures. For a year after isolation, Strain II was grown in test-tube cultures and transferred to fresh media every two months. At the end of that time plates of starch agar were center inoculated with it. Mycelium, as well as spores, was transferred. All of the colonies which developed showed fruiting and non-fruiting sectors. The fruiting sectors were in all respects typical of Strain I]. The non- DIMORPHISM IN CONIOTHYRIUM PIRINUM SHELDON 45I fruiting sectors produced typical mycelium which however never developed either color or fruiting bodies, although they were kept for two months. Other plates were then inoculated with spores and with mycelium from the fruiting sectors and with mycelium alone from the non- fruiting sectors. These plates were labeled II + and II — respec- tively. The use of plus and minus to designate different strains of a fungus species is not new. Blakeslee’ in 1904 applied these terms to Fic. 2. Plus and minus cultures of Strain II, 7 days old. This plate was inoculated directly from the fruiting and non-fruiting sectors of the plate culture in which the first variation was noticed. sexual strains of the Mucorineae which when bred together produced zygospores. Edgerton® in 1914 followed Blakeslee’s example and used plus and minus to differentiate strains of Glomerella which when grown together produced the ascogenous stage in abundance. The present paper deals with a fungus reproducing asexually. Plus and minus must therefore not be interpreted as representing 5 Blakeslee, A. F. Proc. Amer. Acad. 40: 203-321. 1904. 6 Edgerton, C. W. Plus and minus strains in the genus Glomerella. Amer. journ. Bot. 1: 244. 1914. 452 C. H. CRABILL male and female strains. Plus is used in the present instance to denote a strain fruiting abundantly and minus to denote a relatively poor fruiting strain. In the II + colonies on the plates referred to above, the growth was typical of Strain II as it appeared when first isolated. The II — colonies grew rapidly but remained white for 12 days. They then began to take on the olive-green color characteristic of the species Fic. 3. Same as Fig. 2,17 days old. Note that II — has produced numerous pycnidia from which the black spore masses are oozing. It was necessary to open the plates when photographing. The contaminations around the edge of the plate got in when the plate was photographed the first time. and in 20 days from the time of inoculation produced a few large pycnidia about the center of the culture (Figs. 2 and 3). Microscopic examination showed the mycelium, pycnidia and conidia all typical of C. pirinum. The — colonies were exactly like Strain I in morpho- logical and cultural features. It appeared that from Strain II there had arisen a poor fruiting strain identical with Strain I. Poor fruiting is here used in a relative sense. The minus strain produces great multitudes of spores from the few large pycnidia; but, when compared with the enormous numbers DIMORPHISM IN CONIOTHYRIUM PIRINUM SHELDON 453 of pycnidia and spores produced by the plus strain, the prolificacy of the minus strain falls into insignificance. To test out the evidence that a minus strain had split off from a plus parent, dilution plates were poured, using spores from both the plus and minus strains. All the progeny of the plus strain were typically plus. All the progeny of the minus strain were typically minus (Figs. 4 and 5). The former produced abundant pycnidia and Fic. 4. Second dilution plates poured from the colonies shown in Fig. 3. These plates are 5 days old and of the second generation after the mutation. They therefore have the labels II + 2 and II — 2. Note: Wherever two or more cultures appear in the same figure they were inoculated at the same time, incubated the same time under the same belljar, and photographed on the same plate. fruited in 4 days. The latter fruited sparingly only after 14 days. Spores from these dilution cultures were used to pour other plates. Again all progeny of plus parents were typically plus and all progeny of minus parents typically minus. For 12 generations the plus strain remained entirely plus. Then suddenly it gave rise to a minus sector in a petri dish culture. The progeny of thissector were typic- ally minus, whether poured or transferred by wire. The progeny of 454 C. H. CRABILL the plus sectors of the same colony were typically plus and remained so for 5 generations when a third minus strain developed. Fig. 6 will show better than words the history of the development of these strains. On November 5, 1914, a culture of C. pirinum was received from Mr. J. W. Roberts, of the Bureau of Plant Industry at Washington. It was labeled III and immediately subjected to test to see if it would also develop plus and minus strains. Fic. 5. II +2 and II — 2 of the same parentage as those in Fig. 4. These cultures are 8 days old. A single large pycnidium has developed at the point of inoculation in the II — 2 colony. When spores alone are transferred the formation of pycnidia begins much later. When mycelium is transferred as in the present case a few pycnidia often develop at an early age due to the fact that young pycnidia are sometimes transferred on the mycelium. These naturally hasten to maturity as soon as food is supplied. The first set of subcultures was made on Petri dishes of starch agar. Every colony showed fruiting and non-fruiting sectors (Fig. 7). Progeny of the minus sectors have remained constant, for 9 genera- tions or until the present time. Progeny of the plus strain remained constant only 4 generations, when a small minus sector appeared in DIMORPHISM IN CONIOTHYRIUM PIRINUM SHELDON 455 one of the plus colonies (Fig. 11). Progeny of this minus sector have bred true until the present time. These results indicate that the C. pirinum from the Bureau of Plant Industry was dimorphic in stock culture when received. The separation of the plus and minus forms was accomplished as in Strain LT 0-0-0-dat 25 |-0-0-0-0-0-0-0-0-0-0-0-0-0-0-0-0-0-0-0 8-®-8-8-8-0-6 9-0-0-0-@ Q-@-O-©-©-O-O-O-O-8-O CHSRORS IT @-©-@-|dboit 7 U |-@ O=O-O0-0-O0-O0-O0-O Q-9-0-0-0-0-0-0-0-0-9-0-0-0-0-0 f-9-8-8-8-8-8-9-® B- @-Q-O- un 0-0-0-0 9-0-9-9-0-0-0-0-0 Q-©-@-0-0-O-9-9--@-9-O ©-0-6-9-0:0:0 Y U-9-0-0-0-0-0-0-0-0-9 Fic. 6. Chart showing the history of 5 cultures of C. pirinum. O Petri dish culture of minus strain. ran) 66 66 66 b6 plus 6 Sy ee ‘“ “mixed strains. It is in such plates that the plus and minus sectors have appeared. U Test tube culture. = Succeeding plates poured from spores of preceding culture. The plus strains suddenly give rise to minus strains which are subsequently constant generation after generation. It will be noticed that fewer generations of minus strains than plus strains have been grown in nearly every case. This is due to the fact that the plus strains fruit more quickly than the minus strains and more generations can be produced ina given time. I and II were isolated by the writer, III (IV) and V by workers of the Bureau of Plant Industry. Subnumbered plates have been photographed. The numbers correspond with the numbers of figures appended. II. The minus formremained minus. The plus form threw off another minus form in the following 4th generation. On January 2, 1915, two other cultures of C. pirinum, B. P. I. 227 and B. P. I. 345, were received from Dr. J. S. Cooley and desig- 456 C. H. CRABILL nated IV and V respectively. The latter, B. P. I. 345, was isolated by Dr. Cooley from a soft rot of apple on December 3, 1914. The behavior of these cultures is shown in the diagram. Very likely IV is of the same parentage as III, received from Mr. Roberts of the same laboratory. The first subcultures showed it to be dimorphic, 1. €., producing plus and minus sectors (Fig. 12). The first subcultures of V (B. P. I. 345) showed it to be a minus strain (Fig. 12), and it has so remained for Io generations as shown in the diagram. It is identical with Strain I and with all the other minus strains developed in culture. Fic. 7. C. pirinum III. First subculture. 19 days old. Plus and minus sectors were more numerous in these cultures than in any previously or subsequently examined. These plates were opened for examination five days previous to photo- graphing, hence the contaminations around the edges of the cultures. The plus strains are all identical in appearance and behavior. The fact that the minus strain of C. pirinum, viz., I and V, and the plus strain, viz., II and III (IV), have been twice isolated by investi- gators working separately indicates that plus and minus strains exist in nature. The cultural studies show that minus strains may arise from plus strains by a sudden sporting or mutation. An objection might DIMORPHISM IN CONIOTHYRIUM PIRINUM SHELDON A57 be raised that these cultures were impure, 7. e., mixtures of two strains. In anticipation of such an idea it seems desirable to state that frequent pourings of dilution cultures were used to preclude such a possibility. Progeny were then selected only from well-isolated plants, micro- scopic examination of which showed that each was derived from a single spore. By consulting the chart, the frequency of these pourings may be considered. In each instance the poured plate is preceded by the symbol =. It will be evident from this that the possibility of either of the strains being constantly a mixture is eliminated. Both strains have repeatedly arisen from the progeny of a single Fic. 8. III + and III —, 8 days old. These plates were inoculated from the plus and minus sectors respectively of the colonies shown in Fig. 7. plus spore. When once purified the minus strains remain constant from generation to generation. The variation apparently is occurring in only one direction. Attempts to determine the factor which disturbs the stability of the plus strains and causes the liberation of new minus strains have proved fruitless up to the present time. Attempts have been made to develop a minus strain from a plus strain by artificial selection alone. The following methods have been employed: 458 C341. ‘CRABIBI 1. Mycelium was continuously selected from the extreme edge of plus colonies where no pycnidia were yet forming. 2. Mycelium was continuously selected from colonies which were subjected to temperatures so low and so high that fruiting was poor. 3. Mycelium was continuously selected from colonies grown in such dry atmosphere that fruiting was poor. Several generations of such selections gave no promise of the development of a minus strain. Attempts have also been made to develop a plus strain from a | minus strain. - Fic. 9. Same as Fig. 8, 16 days old. In all the minus strains studied there appear colonies which with age show greater prolificacy in some sectors than in others (Fig. 9). By the transplantation of spores only from the pycnidia of the pro- lific sectors generation after generation it was thought possible to build up a plus strain. Selection of this sort however has in no way altered the minus strain. Pedigreed cultures after such selection are in no respect different from pedigreed cultures obtained by con- tinuous selection of mycelium from the poorest fruiting sectors of the minus colonies. These experiments show that selection is not a factor in the origin DIMORPHISM IN CONIOTHYRIUM PIRINUM SHELDON 459 of the two strains. Since C. pirinum reproduces asexually segre- gation from heterozygous parents cannot explain the origin of the two strains. The only explanation which remains is that the minus strain is a sport or mutant arising from the plus strain at irregular and unprognosticable intervals. What makes it arise and what are the controlling factors of such mutation are worthy of speculation and experiment. The writer’ has reported a somewhat similar mutation in a fungus belonging apparently t the genuso Phyllosticta. re sie Fic. 10. Third dilution plates of III + 3 and III — 3, 6 days old. DEVELOPMENT OF PLUS AND MINUS SECTORS A single isolated plant of Contothyrium pirinum like most fungi grows radially. A germinating spore may produce one or two hyphae which, by a continuous dendritic branching, may give rise to large numbers of branches all of which however are nearly equal in diameter for their entire lengths and many of which end at the growing margin. Each hypha with its many branches then covers an area shaped like the sector of a circle. Therefore if something should happen to a hypha which would change its growth characters, its branches would 7Crabill, C. H. A mutation in Phyllosticta. Phytopathology 4: 396. I914. 460 CeH? CRABIEL no doubt also exhibit this change and a sector unlike the rest of the plant would be the result. Similarly with a colony. Different spores might give rise to hyphae unlike in their growth characters and sectoring of the colony Fic. 11. 24 days old cultures of minus and plus strains of II and III for com- parison. II — is in the 11th generation. II + is in the 3d generation after the splitting off of the third minus strain. III — is in the 4th generation. III + isin the 5th generation and shows the small minus sector which gave rise to the 2d minus strain from III. The cultural characters of the two minus and the two plus cultures are identical. In the minus cultures it will be noticed that some sectors produce more pycnidia and more color than others. This fact was taken advantage of in trying to develop a plus strain from a minus strain by selection. would result. In the present studies sectoring in a single plant has not been observed. The sectoring has so far always occurred in colonies. Although the data are insufficient for positive proot the DIMORPHISM IN CONIOTHYRIUM PIRINUM SHELDON 461 evidence points to the conclusion that the variation which gives rise to minus strains occurs in the spore rather than in the mycelium which develops from it. The study of plants of C. pirinum on Petri dishes shows beyond a doubt that the fungus is chemotactic, 7. e., growing only in the direc- tion of its food supply or that it produces some toxic substance detri- mental to its own growth. Whichever the case may be, two plants in close proximity never grow entirely together (Fig. 13). The mycelial threads of the two do not interlace. They are to that extent antagonistic. Fic. 12. IV and V, respectively. First sub-cultures, 24 days old. Some simple tests indicate that this apparent antagonism is not due to excreted toxins. The fungus was grown for two months on a liquid medium containing starch. This medium was then filtered through a sterilized Chamberland filter. The extract, which was found sterile on plating, was used as follows: 1. A large drop of the plain extract was placed at the margin of a thrifty colony on each of several Petri dishes. 2. Boiled extract was used in a like manner. 3. Plates were poured with plain extract and cooled agar and subsequently inoculated. 462 @; H..CRABILE 4. Boiled extract was used likewise. In no case was the growth of the fungus hindered by the presence of the extract. It appears then that it is a lack of food rather than a toxic secretion which keeps colonies of C. pirinum from growing together. A microscopic examination of thin Petri dish cultures shows that the mycelial strands exhibit a similar chemotaxis. They tend to E> 1" PIO ee ont Ye See ee ge ag 3 Say Fic. 13. Two colonies of a plus strain somewhat enlarged. The dark zone is olive green in color and just inside the white advancing margin. Inside of this zone the mycelium is hyaline. The colored oil has been withdrawn from it and deposited in the spores. The two colonies are antagonistic and do not grow together on adjacent sides. diverge continually. If somewhat crowded they grow parallel but seldom converge or cross. It is doubtful therefore if plus mycelium ever crosses over into minus sectors and vice versa. In this way the integrity of the plus and minus sectors is preserved. Some experiments were conducted to throw light on this question. DIMORPHISM IN CONIOTHYRIUM PIRINUM SHELDON 463 I. Small pieces of agar containing plus and minus mycelium respectively were placed side by side on starch agar. In the resulting Fic. 14. Culture of a minus strain with the same enlargement as Fig. 13. The mycelium is dark throughout the greater part of the culture. The pycnidia are few, large, producing shining black masses of spores. The greenish color has been removed from the mycelium immediately surrounding the pycnidia. Some white fluffy aerial mycelium is present on the surface. growth the two sectors were entirely distinct and typical. The minus portion was in all cases much larger than the plus portion. 2. Spores of plus and minus strains were mixed and then used to 464 C. H. CRABILL inoculate plates. In the resulting colonies sectoring was prominent. The plus sectors were about equal to the minus in number, somewhat less in width and were decidedly distinct as usual. In some of the photographs appended it is evident that the minus sectors of certain colonies are not quite sector-shaped in outline but narrow somewhat toward the margin (Figs. 7 and 11). As stated Fic. 15. Enlarged photograph of the IV colony shown in Fig. 12. The mycelium in the minus sector is almost entirely hyaline and fruitless. In the plus sector the color zone which was much broader a few days previous is very narrow and surrounds the outermost ends of the mycelium. Some of the color has diffused out into the agar. above, when pieces of agar containing mycelium of the two were used to inoculate simultaneously the minus sectors were larger than the plus sectors. These observations together with measurements of the rate of growth in colonies of like age indicate that unless the plus strain has a relatively large amount of inoculating material to start from, the minus strain will predominate in growth volume. This is DIMORPHISM IN CONIOTHYRIUM PIRINUM SHELDON 465 accounted for by the fact that the prolific spore production in the plus strain requires food material and energy which would otherwise be used for vegetative growth. CoLOR PHENOMENA AND SPORE PRODUCTION IN RELATION TO TEMPERATURE The production of an olive green color is characteristic of the mycelium of C. pirinum. On some media color production is more pronounced thanon others. The present observations have been made on the two strains growing on starch agar in Petri dishes. An interesting correlation between color production, sporulation and incubation temperature has been studied. It was found by experience that vegetative growth was most rapid at 25° C., and that sporulation was most active at 18-20° C. It was therefore customary to grow the cultures in moist chambers for 3-6 days at 25° C. and then at 18°-20° C. The moist chambers, each consisting of a plate of water and a tall belljar, maintained an atmosphere of absolute humidity and prevented the drying of the culture medium. The dishes were incubated upside down to prevent moisture from running over the surfaces of the cultures. Under the above conditions the following observations have been recorded. Plus Strain.—Plus colonies grow rapidly but remain white 2-5 days. In three days pycnidia are usually present in abundance. Most of them are pink at this time but a few may be black due .to the dark color of mature spores within. About the 5th day the mycelium just back of the growing ends begins to take on an olivaceous color. Later as the pycnidia mature in abundance this color is all absorbed, leaving the mycelium hyaline (Fig. 13). At the same time more color is formed farther out toward the margin of the radi- ating colony. The result is that an olivaceous zone migrating con- tinuously outward always exists just behind the growing ends of the mycelium. Microscopic study shows that the olivaceous mycelium is replete with refringent droplets of oil. In the inner zone of spore production the mycelium is hyaline and contains no oil drops. It is evident that the oil supplies the color. It is manufactured and stored temporarily in the mycelium. Later it is withdrawn and passes into the spores as they develop the dark color characteristic of the species. In. cultures only 1-3 days old, spore production precedes color formation 466 CP H. (CRABIEE No oil is at that time stored in the mycelium but passed directly into the spores. In some old cultures the greenish color diffuses out into the agar (Fig. 15). This is especially noticeable in colonies which have reached the edge of the Petri dish in which they are growing. In many cases it has been subsequently absorbed on sporulation. Cultures 20 days old kept at 13°-15° C. for 10 more days become quite black in the outer zones. This temperature is too low for active production of spores but does not materially hinder the growth and manufacture of oil by the mycelium. The oil is therefore not used as rapidly as produced and necessarily accumulates in the mycelium and imparts to it a dark color. | Cultures incubated constantly at 25° C. produce much less color, as well as fewer pycnidia, than those incubated at lower temperatures. Minus Strain.—Minus colonies remain white 10 to 20 days. The olive green color then appears in minute spots usually around very young immature pycnidia. The color spreads gradually some- times appearing more pronounced in some sectors than in others. The pycnidia on maturing absorb the color from the mycelium im- mediately surrounding them but owing to the fact that these are so few in number, the cultures retain most of the olive color which darkens with age until the culture is quite black (Fig. 14). Minus cultures incubated constantly at 25° C. produce very little color. The optimum temperature for spore production is 18°-20° C. Some cultures will scarcely fruit at all at 25° C. but fruit typically at 18°- 20° C: Color production and sporulation are both maximum at the latter temperatures. Mixed Cultures.—In mixed cultures in which plus and minus strains grow in respective sectors, the plus sectors develop and later absorb color as described for pure plus strains. The minus sectors on the other hand may never develop this color or if they do it is in small amounts and very tardy in appearing (Fig. 15). In many cases there is no spore production whatever. Normally a few pycnidia appear with characteristic tardiness. INOCULATIONS The plus and minus strains of Coniothyrium pirinum have been used to inoculate apple leaves. Holes were seared in the leaves with a hot needle, wetted and smeared with spores. The leaves were then bagged. DIMORPHISM IN CONIOTHYRIUM PIRINUM SHELDON 467 In two weeks the spots inoculated with each strain turned whitish and bore several pycnidia from which it was easy to reisolate the strain. The cultures show that both the strains retained their diag- nostic characteristics. One month after inoculation no enlargement of the seared spots has taken place. In fact some of the dead tissue bearing pycnidia of the fungus has fallen out producing a ‘“‘shot hole”’ ' effect. This is further evidence that Coniothyrium pirinum is a saprophyte. The above experiment was conducted under controlled conditions in the greenhouse. CONCLUSIONS 1. Coniothyrium pirinum is sometimes dimorphic in culture and probably also in nature. 2. Two distinct strains have been isolated, viz., a plus strain, which fruits abundantly, and a minus strain, which fruits poorly. 3. The minus strain arises in artificial culture by sudden sporting from the plus strain. This phenomenon has been observed in four separate instances. 4. Minus strains never give rise to plus strains but remain constant, generation after generation. 5. Attempts to develop the strains from each other by continuous selection of extremes have been unsuccessful. 6. Attempts to determine the cause of the sporting have been fruitless. VIRGINIA AGRICULTURAL EXPERIMENT STATION, BLACKSBURG, VA. THE GENUS ESPELETIA * PauL C, STANDLEY The genus Espeletia is a member of the Asteraceae, placed by Hoffman in the tribe Heliantheae, subtribe Melampodinae, in his treatment of the family in Engler and Prantl’s Natiirlichen Pflanzen- familien.! Although it has often been referred to other groups, systematists are now agreed as to its proper disposal. The plants are among the most conspicuous of the composites, because of the long wool which closely invests the leaves and inflorescence of most species, and because of the peculiar habit of growth of some members of the genus. The woolly covering is not peculiar to the Espeletias, however, some species of the genus Culcitium, for example, a member of the Senecioneae, closely simulating in their general appearance certain species of the present genus. » Two species of Espeletia are tall, much branched shrubs. Others have a large tuft of radical leaves borne upon the surface of the ground from which one or more flowering stems rise. A few, like Espeletia grandiflora,” develop tall, thick, erect caudices a meter high or more, which are naked below but bear at the summit a large cluster of leaves from which several flowering stems rise. In their habit of growth this group suggests some of the Senecios and Lobelias which abound in similar situations in the high mountains of East Africa.’ The species are not numerous and are confined, so far as known, to the paramos of the high cordilleras of Colombia and Venezuela, occurring usually at elevations of 3,000 metersor more. The southern- most species, E. corymbosa, was collected not far from the southern border of Colombia, and it is not improbable that it or some related species may occur in Ecuador. The genus Espeletia was founded by Humboldt and Bonpland in 1809,* three species being described and illustrated. The name was * Published by permission of the Secretary of the Smithsonian Institution. 1 45: 216.) 1800: 2 See Eng}. & Prantl, Pllanzenfamy: a°> f. 209: 3 See the National Geographic Magazine 27: 194, 196, 197, 200. I9QI5. 4 Pl. Aequinm:. 2%. 0: * 468 THE GENUS ESPELETIA 469 given in honor of Don José de Espeletia, at one time viceroy of the Kingdom of New Grenada, who encouraged botanical explorations about Bogota during his term of office. Bonpland expressly states that the generic name should be credited to Mutis, who discovered FE. grandiflora in the vicinity of Bogota. It is thus apparent that Espeletia grandiflora is the type of the genus,—a matter of no very great importance, since only one species has ever been referred to any other genus. Libanothamnus was based by Ernst upon the plant now known as Esbeletia neritfolia, which differs in habit from other members of the group and had previously been referred to two other genera. Ernst did not recognize the relationship of Humboldt’s Trixis nerufolia with Espeletia, nor was he aware that Weddell had already transferred it to its proper position, otherwise he probably would not have made it the type of a new genus. No additions were made to Espeletia after 1809 until Weddell published his elaborate treatment of the Andine flora in 1855.° That author lists eleven species, seven of which are described as new, while one is transferred from another genus. Since 1855 the only published addition to Espeletia is a subspecies described in 1905. That so little concerning the genus is found in later literature results from the fact that few botanists have visited the Andine regions of Venezuela and Colombia in recent years. Lately the U.S. National Herbarium has received twenty-two sheets of Espeletia, representing nearly as many collections. This is probably more material than is found in any other herbarium and has led the writer to undertake a review of the genus as a whole, in the course of which no less than six species apparently new have been discovered. Nearly all these sheets were included in a large set of Venezuelan plants collected by Dr. Alfredo Jahn, mainly in the high cordilleras, in regions difficult of access and not visited by any other recent collector. The col- lection includes many highly interesting plants, but no genus is so completely represented as the present one. Of the species of Espeletia previously described from this region Doctor Jahn has recollected all but two, and one of these is a plant of doubtful standing. Of the shrubby species of Espeletia one is known as “‘incienso,”’ since the branches produce a large quantity of resin which is burned as incense in the churches. The non-shrubby species are commonly 5 Chloris Andina, Essai d’une flore de la région alpine des cordilléres de L’ Amér- ique du Sud, in Castelnau, Expédition dans les parties centrales de |’Amérique du Sud, part: 6. 470 PAUL C. STANDLEY ’ known as “‘frailején,’’ their dense covering of white or gray wool recalling to the Venezuelans the cloaks of the brothers of some of the religious orders. Humboldt and Bonpland state that the resin of E. grandiflora was used at Bogota in the manufacture of printers’ ink, a purpose for which it was highly prized. The resin, they state, was known at Bogota as ‘“‘trementina (turpentine), although it did not possess either the odor or the consistency of the turpentine of commerce. KEY TO THE SPECIES Shrubs or trees with much branched, woody stems. Leaf blades sinuately and subspinosely denticulate. 1. E. banksiaefolia. Leaf blades entire. 2. E. neritfolia. Herbs, often with much elongated, 2rect caudices, but these simple, never with much branched, woody stems. Heads usually solitary on each scape, rarely more than one. Leaf blades linear or narrowly ligulate, attenuate, 25 to 30 cm. long; heads 4 to 4.5 cm. in diameter; pubes- cence rufous or fulvous. 3. E. mortiziana. Leaf blades linear-oblong, 4 to 6 cm. long; heads about 2 cm. in diameter; pubescence nearly white. 4. E. weddellit. Heads several or numerous, the stems not scapose. Cauline leaves opposite; bracts of the inflorescence often opposite or verticillate. Pubescence of stems and leaves white, -silvery; heads radiate or discoid; basal leaves 4 cm. wide or less. Bracts of the inflorescence verticillate; leaves 3 to 4 cm. wide. 7, E. argentea. Bracts alternate; leaves 1.8 cm. wide or less. 8. FE. grisea. Pubescence usually fulvous or rufescent; heads radiate; basal leaves 4 to 10 cm. wide. Involucral bracts broadly ovate; nerves of the leaves mostly obscured by the wool. 5. EL. grandtflora. Involucral bracts lanceolate or narrowly ovate; nerves of the leaves prominent. 6. E. schultz. Cauline leaves and bracts of the inflorescence all alternate, rarely subopposite. Heads in an elongated simple raceme. 9. HE. spicata. Heads in a more or less compound panicle or corymb. Leaf blades of nearly uniform width through- out, not noticeably narrowed toward the base. : Leaf margins strongly revolute, the blades 3 to 6 mm. wide, soon glabrate on the upper surface; inflorescence congested. 10. HE. jahnii. THE GENUS ESPELETIA 471 Leaf margins flat or slightly revolute, the blades 7 to 12 mm. wide; inflorescence openly corymbose or paniculate. Heads 12 mm. broad, their pubescence brown, short. 11. EL. pannosa. Heads 20 to 25 mm. broad, their pubes- cence white, very long. 12. E. floccosa. Leaf blades evidently broadest above the middle, conspicuously narrowed at the base. Leaves sericeous beneath, with short ap- pressed hairs; heads about 8 mm. broad. 13. E. paltonioides. Leaves more or less lanuginous beneath, with loose hairs; heads more than 8 mm. broad. Leaves in age glabrous on the upper surface or sparsely pubescent. Heads 25 mm. broad; leaf blades nar- rowly oblong, attenuate at the apex, slightly narrowed below; in- volucral bracts glanduliferous but nearly without pubescence. 14. E. lindenit. Heads about 12 mm. broad; leaf blades elliptic, attenuate below to a winged petiole; involucral bracts copiously pilose. 15. E. bracteosa. Leaves copiously lanuginous on the upper surface. Involucral bracts ovate or elliptic- ovate, sparsely pilose outside. 16. E. corymbosa. Involucral bracts lanceolate, densely pilose outside. Vf Jap UCR. 1. ESPELETIA BANKSIAEFOLIA Schultz Bip. & Ettingh.; Wedd. in Cast. Expéd. Amér. Sud Bot. 1: 67. 1855 TYPE LOCALITY: Sierra Nevada de Mérida, Venezuela, at an alti- tude of 3,500 meters. Type collected by Funck and Schlim (no. 1550). This differs from all other species in having denticulate leaves. Weddell stated that he had seen only a fragment of the plant. It is more than possible that it has been referred to the wrong genus. 472 PAUL C. STANDLEY 2. ESPELETIA NERIIFOLIA (H.B. K.) Schultz Bip.; Wedd. in Cast. Exped. Amer. Sud Bot. it (67. 1635 Trixis nervifolia Humb. Voy. Rég. Equin. Rel. 1: 605. 1814, Bailleria ? nerufolia H. B. K. Nov. Gen. & Sp. 4: 289. 1820. Chibadium ? nerufolium DC. Prodr. 5: 507. 1836. Libanothamnus nerufolia Ernst, Vargasia 186. 1870. TYPE LOCALITY: Silla de Caracas, Venezuela. Type collected by Bonpland. Weddell reports several collections from the Silla de Caracas and the Sierra Nevada de Mérida, Venezuela. A specimen in the U. S. National Herbarium was collected by Otto Kuntze in 1877 at the type locality. In May, 1913, Mr. Pittier collected the plant in the upper belt of the Pico de Naiguata, State of Miranda, at an alti- tude of 2,400 to 2,765 meters. The common name is “‘incienso.”’ The species differs from most others in being a shrub 3 to 4 meters high with numerous leafy branches. The heads are very numerous, in broad corymbs, and comparatively small, I cm. broad or less. They are nearly destitute of the long wool characteristic of most of the other species. Upon this plant Ernst founded his genus Libano- thamnus (“‘incense tree’’). He erred in referring it to the Senecioneae and was apparently unaware that Weddell had previously referred it to Espeletia. The shrub differs so conspicuously in habit from other members of the genus, with some of which Ernst must have been acquainted, that it is not surprising that he did not associate it with Espeletia. 3. ESPELETIA MORITZIANA Schultz Bip.; Wedd. in Cast. Expéd. Amér. Sud Bot. 1: 65. 1855 TYPE LOCALITY: Sierra Nevada de Mérida, Venezuela, at an altitude of 4,200 to 4,500 meters. Type collected by Moritz (no. 1416). Two collections of this have been secured by Doctor Jahn, one in the Paramo de Timotes, State of Tachira, at an altitude of 3,000 to 3,500 meters (no. 150); and the other in the Paramo de la Culata, Cordillera de Mérida, at an altitude of 3,500 meters (no. 235). The common name is given as “‘frailején dorado.” This species, like E. weddellii, is characterized by monocephalous stems. The heads are much larger than in that species, however, being 4 to 5.5 cm. in diameter. The bracts are covered with very THE GENUS ESPELETIA A73 long and dense pubescence, the unopened heads appearing to be globular masses of wool. Weddell says the heads are ‘‘perpaucis, interdum solitariis,’’ and in the present specimens they are always solitary. The following notes may be added from the recent collections: Pubescence ferrugi- nous, especially on the younger leaves; leaves 25 to 30 cm. long, 8 to I4 mm. wide, coriaceous; flowering stems stout, 40 to 60 cm. high; Fic, 1. Espeletia moritziana. 'Paramo de Timotes, Venezuela. Photo by Dr. A. Jahn. paleze of the disk linear or oblong-linear, acuminate, minutely serru- late near the apex; achenes 2 to 2.5 mm. long, nearly black, smooth, sharply angled. The inner involucral bracts are longer than in any other species, their apices being long-attenuate. 4. ESPELETIA WEDDELLII Schultz Bip.; Wedd. in Cast. Expéd. Amér. Sud Bot. 1: 66. pl. 75, B. 1855 TYPE LOCALITY: ‘Venezuela: Paramo de Niquitao!, dans les Andes de Truxillo, 4 une élévation de 4,000-4,500 métres.”’ Type collected by Linden (no. 1443). 474 PAUL C. STANDLEY Two specimens of this are in Doctor Jahn’s collections, one from the Paramo de Timotes, State of Tachira, at an altitude of 3,000 to 3,500 meters (no. 152); and the other from the type locality, the Teta de Niquitao, the highest peak of the Sierra Nevada de Mérida, at an altitude of 3,000 to 4,000 meters (no. 151). The common name is given as “frailej6n de batata.’’ The word batata (tuber or bulb) refers to the large tuber-like roots which, however, are not present upon the specimens. The species is distinguished by its very numerous short leaves which form a dense basal cluster, and by its monocephalous stems. Weddell says that it is the lowest plant of the genus and this is prob- ably true. From the specimens it can be seen that the caudex is not elongated and that the leaves rest upon the ground. Weddell states that there are one or two heads on each stem. In these recent specimens there is never more than one. The leaves are described as “acutatis’’ but the tips are very blunt, and Weddell so figures them. In Doctor Jahn’s specimens the leaves are 4 to 6 cm. long and 5 to 10 mm. wide; the flowering stems are 6 to 30 cm. long, evidently elongating in age; the palez of the disk are oblong-linear, densely villous at the apex, and bearded at the base with long, white, erect hairs; the achenes, which seem not to be full developed, are about 2.5 mm. long. 5. ESPELETIA GRANDIFLORA Humb. & Bonpl. Pl. Aequin. 2: 9. pl. 7o. 1809 Espeletia hartwegiana Schultz Bip.; Wedd. in Cast. Expéd. Amér. Sud Bot. 1: 62. 1855, as synonym. Espeletia oppositifolia Schultz Bip.; Wedd. loc. cit., as synonym. TYPE LocALiTty: Near Bogota, Colombia. Type collected by Bonpland. ILLUSTRATIONS: Wedd. in Cast. Expéd. Amér. Sud Bot. 1: pl. 15, A. Collected by Mr. H. Pittier in the Paramo de Buena Vista, Huila Group, Central Cordillera, State of Cauca, Colombia, at an altitude of 3,000 to 3,600 meters, in January, 1906 (no. 1116). Weddell! reports several collections from Colombia and a single one from the Sierra Nevada de Mérida, Venezuela (Linden 398, in part). Schultz’s manuscript name hartwegiana was based, doubtless, upon a specimen collected by Hartweg (no. 1137) in the Paramo de 1In Cast. Expéd. Amér. Sud Bot. 1: 63. 1855. THE GENUS ESPELETIA 475 Guanacas, in the Andes of Popayan, Colombia. The name oppositi- folia was based upon Linden’s no. 398 from the Sierra Nevada de Mérida, Venezuela. Weddell states that this collection is a mixture, the inflorescence (from which the specific name was taken) being of E. grandiflora and the leaves those of FE. moritziana. Fic. 2. Espeletia grandiflora. Paramo de Culata, Venezuela. Photo by Dr. A. Jahn. 6. ESPELETIA SCHULTzII Wedd. in Cast. Expéd. Amér. Sud Bot. 1: 63. 1855 ( TYPE LOCALITY: Paramos of the Cordilleras of Mérida, Venezuela, at an elevation of 3,200 to 3,500 meters. Type collected by Linden (no. 370). Weddell also cites Moritz’s no. 1419 from the same region. Three of Doctor Jahn’s collections are referred here: no. 245, from La Culata, Cordillera de Mérida, altitude 3,500 meters; no. 236, from Pan de Aztcar, Cordillera de Mérida, altitude 4,000 meters, 470 PAUL C. STANDLEY and no. 153, from the Paramo de Timotes, State of Tachira, altitude 3,000 to 3,500 meters. The common name is given as ‘“‘frailején.”’ The leaves are said to be used for wrapping cheese and butter, to secure the flavor and aroma which they impart. The species appears to be very closely related to E. grandzflora, and Weddell’s diagnoses do not afford a satisfactory means of sepa- rating the two. £. schultzw has paler wool, however, more con- spicuously veined leaves, narrower involucral bracts, and presumably Fic. 3. Espeletia schulizit, Paramo de la Cristalina, Venezuela. Photo by Dr. A. Jahn. : a broader inflorescence. Whether this last distinction will hold when a large series of specimens is brought together is doubtful. The leaves are almost ligulate, not ‘‘oblongo-lanceolatis’’’.as..Weddell described them in his diagnosis, a characterization which is somewhat amended in his discussion of the species. In the present speciimens they are 18 to 31 cm. long and 2.5 to 6 cm. wide, being scarcely at all narrowed at the base, slightly broadest above the middle, and from obtuse to acute. The heads vary considerably in size with age, ranging from 2 to 3.5 cm. in diameter. The palez of the disk are nearly linear, attenuate, furnished near the apex with numerous short, stiff hairs. THE GENUS ESPELETIA A77 FO ESPELETIA ARGENTEA Humb. & Bonpl. Pl. Aequin. 2: 12. pl. 71. 1809 TyPE LocaLity: Near Zipaquira, north of Bogota, central Colom- bia, at an altitude of 1,300 meters. Type collected by Humboldt and Bonpland. Weddell reported two forms of this species: The first ‘‘capitulis radiatis,’ and the second ‘‘capitulis discoideis,’’ the typical form, known only from the type collection. To the first form, with radiate heads, he referred three specimens from the mountains of Mérida, Venezuela, at elevations of 3,200 to 3,900 meters, collected by Linden (no. 401), Funck and Schlim (no. 1072), and Moritz (no. 1418). He cited as a synonym Fspfeletia nivea Moritz, mss., this name doubtless referring to Moritz’s specimen. It is probable that none of the Venezuelan specimens are really E. argentea, but belong rather to Espeletia floccosa, E. grisea, or E. pannosa, which are described as new in the present paper. To which one of these three the name nivea was applied can not be determined without examination of Moritz’s specimens. Bonpland illustrated and described the heads as discoid. From his diagnosis and plate we also find that the leaves are opposite and the bracts of the inflorescence opposite or verticillate. Weddell describes the cauline leaves as “‘interdum oppositis,’”’ this modification of the original description probably necessitated by the inclusion of the Venezuelan specimens. Certain other modifications of Bonpland’s diagnosis introduced by Weddell into his notes seem to indicate that he had before him perhaps the plant described here as Espeletia pan- nosda. 8. Espeletia grisea sp. nov. Caudex stout, 20 cm. long, 2 to 2.5 cm. in diameter, from a stout elongated tap-root, covered with closely imbricated leaf bases; radical leaves narrowly linear-oblanceolate, 15 to 21 cm. long, 13 to 18 mm. wide, acuminate at the apex, gradually and slightly narrowed toward the base, dilated at the point of insertion, subcoriaceous, flat, or the margins slightly revolute, abundantly lanate on both surfaces with grayish hairs but more densely so beneath, the midrib not prominent, the lateral veins scarcely perceptible; flowering stem about 120 cm. high, stout, densely lanate below, the pubescence less abundant above and somewhat deciduous, corymbosely much branched, the branches long, ascending or erect, slender, the stems furnished below with several pairs of opposite leaves, these similar to the basal ones 478 PAUL C. STANDLEY but smaller; branches of the inflorescence all alternate, the bracts resembling the cauline leaves but smaller; heads very numerous, on slender peduncles 2 to 4 cm. long, these viscid and rather sparsely tomentose with long loose whitish hairs and shorter brown ones; heads 15 mm. broad in age, subglobose, the outer palez of the re- ceptacle and the involucral bracts reflexed; involucral bracts narrowly lanceolate to narrowly obovate, acute, pilose outside with brown hairs, glabrous within; palez of the disk cuneate-obovate, acutish, densely viscid-pilose outside near the apex; corolla tube sparsely villous out- side; achenes not seen. Type in the U. S. National Herbarium, no. 602351, collected on the Sierra Nevada de Mérida, State of Mérida, Venezuela, at an altitude of 3,000 to 4,000 meters, in January, 1911, by Dr. Alfredo Jahn (no. 157). Additional material of the same collection, consisting of a caudex and basal tuft of leaves, is mounted on sheet no. 602352. Fic. 4. Espeletia spicata. Sierra Nevada de Mérida, Venezuela. Photo by Dr. A. Jahn. Espeletia grisea is evidently related to E. argentea but is dis- tinguished by the shorter, narrower leaves (which are less narrowed toward the base), the villous corolla, and the alternate branches and bracts of the inflorescence. The heads are too far developed to determine whether they are radiate or discoid. THE GENUS ESPELETIA 479 9. ESPELETIA SPICATA Schultz Bip.; Wedd. in Cast. Expéd. Amér. Sud Bot. I: 65. 1855 TYPE LOCALITY: Sierra Nevada de Mérida, Venezuela, at an altitude of 4,500 meters. Type collected by Linden (no. 400). Collected by Doctor Jahn on the Sierra Nevada de Mérida, at an altitude of 3,000 to 4,000 meters (no. 158); on Pan de Aztcar, Cor- dillera de Mérida, at an altitude of 4,000 meters (no. 237); and in the Paramo de Timotes, State of Tachira, at an altitude of 3,000 to 3,500 meters (no. 149). In the first specimen the inflorescence is lacking, but the leaves show that it is of this species. The second specimen bears only part of an inflorescence, with a few heads about I5 mm. in diameter; the leaves are 27 to 40 cm. long and 18 to 27 mm. wide; and the whole plant is densely woolly with fulvous hairs. The third specimen shows a complete inflorescence about 95 cm. high; the stem is very stout and densely lanuginous; the peduncles average about 5 cm. in length, and the heads 3 to 3.5 cm. in diameter. 10. Espeletia jahnii sp. nov. Leaves all basal, 18 to 35 cm. long, 3 to 6 mm. wide (excluding the strongly revolute margins), linear or nearly so, acute or acuminate at the apex, widened at the sheathing base, coriaceous, the margins strongly revolute, densely clothed on the upper surface with long, white, nearly straight wool when young but becoming glabrate in age, densely tomentose on the lower surface, the bases of the leaves more densely furnished with wool; flowering stems stout, 20 to 25 cm. high, naked up to the inflorescence, covered by a dense, matted, slightly fulvous wool; bracts of the inflorescence 2 to 5.5 cm. long, similar to the leaves but broader; inflorescence corymbose, of 12 to 15 crowded, short-pedunculate heads, these about I cm. in diameter; involucral bracts few, lanceolate, obtuse or acutish, densely covered with long wool; palez of the disk 3.5 mm. long, linear, slightly broadened up- ward, densely glandular near the apex; rays wanting; styles much exserted. Type in the U. S. National Herbarium, no. 602482, collected in the Paramo del Batallén, State of Tachira, Venezuela, at an altitude of 3,000 meters, in March, 1911, by Dr. Alfredo Jahn (no. 155). This is well distinguished from all the other species by the very narrow leaves which are soon glabrate on the upper surface and strongly revolute. The flowering stems, too, are lower than in most species. It is not possible to determine from the specimens whether the plant has an elongate, erect caudex. 480 PAUL C. STANDLEY In the Paramo de las Rosas, State of Trujillo, at an altitude of 3,200 meters, Doctor Jahn collected another plant which somewhat resembles this, the leaves being slightly broader and glabrous on the upper surface. Unfortunately this specimen is without flowers. It is doubtless an undescribed species. Fic. 5. Espeletia jahnit. Paramo Malpaso, State of Tachira, Venezuela. Photo by Drs Aw J ahi 11. Espeletia pannosa sp. nov. Caudex short, about 9 cm. long, 8 cm. wide at the crown, from a very thick ligneous root, bearing very numerous erect leaves; radical leaves broadly linear, of uniform or nearly uniform width throughout, except for the slightly dilated point of attachment, 16 to 30 cm. long, 7 to 8 mm. wide, coriaceous and rigid, the margins slightly or strongly revolute, obtuse to acute at the apex, densely covered on the upper surface with long, straight but somewhat matted, silvery white hairs, densely tomentose beneath with long, matted, white or slightly yellowish hairs, the midrib very broad and prominent beneath, the lateral nerves not apparent; flowering stem 45 cm. high or more, THE GENUS ESPELETIA A8I stout, bearing a few alternate leaves similar to the basal ones but much smaller, corymbosely branched above, the branches few, erect or nearly so, densely tomentose with dark brown hairs, the pubescence of the lower part of the stem lanate, white; heads rather numerous, I2 to 15 mm. broad, on stout erect peduncles 2 to 5 cm. long, these densely tomentose with brown hairs; bracts of the inflorescence I to 2 cm. long, oblong-lanceolate, alternate; involucral bracts numerous, linear-lanceolate to narrowly ovate, acute or acuminate, glabrous within, densely villous outside with long matted brown hairs; rays numerous, 5 to 6 mm. long, shallowly bilobate at the apex; palea of the disk densely villous outside; corolla lobes densely pilose; achenes 1.5 mm. long, obtusely angled, dark brown. Type in the U. S. National Herbarium, no. 602484, collected in the Paramo del Jabén, State of Trujillo, Venezuela, at an altitude of 3,000 to 3,200 meters, in October, 1910, by Dr. Alfredo Jahn (no. 165). This is related to Espeletia argentea but is easily distinguished by the radiate heads, narrow radical leaves, and alternate cauline leaves. It is a handsome plant by reason of its silvery leaves which contrast with the brown pubescence of the inflorescence. Closely related to this is a specimen collected by Doctor Jahn (no. 244) at La Culata, Cordillera de Mérida, at an altitude of 3,500 meters. - The form of the leaves is almost exactly the same as in E. pannosa, except that they are 40 cm. long and acuminate. The inflorescence is represented only by a few lateral branches, each sub- tended by a large, silvery bract, and terminated by several sessile heads. This specimen probably represents an undescribed species, but it is not complete enough to determine all the essential characters. 12. Espeletia floccosa sp. nov. Radical leaves broadly linear, of nearly uniform width throughout, slightly dilated at the point of attachment, 30 cm. long, 9 to II mm. wide, acute or acuminate, coriaceous, rigid, erect, flat, densely sericeous on the upper surface with long, matted, silvery white hairs, densely tomentose on the lower surface with matted, white or yellowish hairs, the midrib large and prominent beneath, the lateral nerves not visible; flowering stem nearly a meter high, very stout, bearing a few long alternate leaves below, corymbosely branched above, the branches numerous, stout, erect, alternate, densely covered throughout with very long white wool, this loose and floccose; heads numerous, 18 to 25 mm. in diameter, on stout peduncles I to 4 cm. long; involucral bracts numerous, linear, attenuate, I0 to 15 mm. long, glabrous within, densely covered outside with long loose white wool; palez of the disk narrowly linear, rigid, erect, 5 to 6 mm. long, acute, pubescent 482 PAUL C.. STANDLEBY outside; corolla villous, white; achenes 2.5 mm. long, compressed, very broad, obtusely angled. Type in the U. S. National Herbarium, no. 602483, collected in. the Paramo del Jaboén, State of Trujillo, Venezuela, at an altitude of 3,000 to 3,200 meters, in October, 1910, by Dr. Alfredo Jahn (no. 154). The collector gives the common name as “‘frailején plateado,”’ and states that the flowers are white. The heads have been badly eaten by insects, and if any rays were present they have disappeared. The collector states that the same plant was observed in the Paramo de Timotes and the Sierra Nevada de Mérida. It may be, however, that this was not distinguished from EF. grisea and E. pannosa. Fic. 6. Espeletia floccosa./ Sierra Nevada de Mérida, Venezuela. Photo by | Dr. A. Jahn. Espeletia floccosa is a most distinct species, especially because of the abundant, long, white wool. The palez of the disk are narrower than in any other species. The plant is related, perhaps, to £. argentea, but it has much larger heads, longer, narrower bracts, and narrower leaves. 13. Espeletia paltonioides sp. nov. Caudex elongated, stout, 3.5 cm. in diameter, densely covered with leaves, their bases imbricated; leaf blades narrowly linear- THE GENUS ESPELETIA 483 oblanceolate, 20 to 28 cm. long, 13 to 18 mm. wide, attenuate to the apex, long-attenuate to the broadly margined petiole, this abruptly dilated at the point of attachment, the margins strongly revolute, the upper surface grayish green, closely covered with very short, stiff, yellowish, almost scabrous hairs, densely sericeous on the lower surface with very dense, short, white or fulvous, appressed hairs, the blades coriaceous but not much thickened, the midvein very prominent beneath, the lateral veins inconspicuous, irregular, somewhat reticu- late; flowering stem tall, erect, densely sericeous, furnished with numerous alternate bracts or leaves; inflorescence of many heads in a rather compact corymb 16 cm. high and 12 cm. broad, the branches 3.5 to 11 cm. long, the heads corymbose at the end of each branch, on peduncles 2 to 12 mm. long; heads 8 to 10 mm. broad; bracts few, oblong-linear to narrowly oblong, acute, glandular-puberulent at the apex, densely pubescent with mostly appressed hairs, these abraded by weathering; palez of the disk oblong or oblong-spatulate, rounded or subtruncate at the apex, puberulent, glandular above; corolla 3.to 3.5 mm. long, glabrous; achenes 2.5 mm. long, sharply trigonous. Type in the U. S. National Herbarium, no. 602354, collected in the Paramo de las Rosas, State of. Trujillo, Venezuela, at an altitude of 3,200 meters, in October, 1912, by Dr. Alfredo Jahn (no. 159). The material consists of a stout caudex bearing a thick cluster of leaves and a weathered inflorescence. Unfortunately, because of the age of the latter, it is impossible to describe the pubescence of the involucral bracts or the form of the rays, if these be present. All the leaves and bracts of the inflorescence have disappeared, too. Never- theless, this is easily distinguished from all other species by the short, close pubescence of the leaves. The heads are very numerous and small. The species is most closely related, probably, to E. corymbosa. The form of the leaves suggests the fronds of a fern, Paltonium lanceo- latum (L.) Presl; hence the specific name. 14. ESPELETIA LINDENII Schultz Bip.; Wedd. in Cast. Expéd. Amér. Sud Bot. 1: 66. 1855 TYPE LOCALITY: Paramos of the State of Mérida, Venezuela, at an altitude of 3,250 to 3,900 meters. Type collected by Linden (no. 1414). This is the only species described from Venezuela which is not represented in Doctor Jahn’s collections. Judging from the descrip- tion it must be similar to the plant here described as Espeletia bracteosa. It is said to have heads twice as large, as well as leaves of somewhat different form. The heads are radiate, the rays being yellow. 484 PAUE C.” STANDEEY 15. Espeletia bracteosa sp. nov. Basal leaves numerous, 20 to 25 cm. long, 3 to .4 cm. wide in the broadest part, the blades elliptic, acute or acutish at the apex, long- attenuate at the base to a stout margined petiole, this gradually dilated at the base, at first abundantly lanuginous on the upper surface, glabrous in age, very densely matted-tomentose beneath with fulvous hairs, with additional long loose white lanuginous hairs, the midvein prominent beneath, white-woolly, the lateral veins numerous, parallel, conspicuous beneath, visible also on the upper surface; bases of the petioles densely covered with long silky fulvous hairs; flowering sems 40 to 50 cm. high, bearing a few small, alternate, narrowly lanceolate or oblanceolate leaves below the inflorescence; inflorescence corymbosely branched, the branches erect or ascending, bearing numerous leaflike bracts 2 to 6 cm. long, these lanceolate, attenuate, pubescent like the leaves; branches of the inflorescence slender, densely lanuginous with brown hairs; peduncles 2 to 4 cm. long; heads rather few, about 12 mm. broad; involucral bracts oblong to linear, acute to attenuate, densely pilose with brown hairs; rays not seen; paleze of the disk attenuate, densely covered above with short, stiff, appressed, viscid, reddish brown hairs; corolla tube densely viscid- pilose; achenes dark brown, smooth, sharply angled. Type in the U. S. National Herbarium, no. 602350, collected in the Paramo de la Cristalina, State of Trujillo, Venezuela, at an altitude of 2,900 meters, December 20, 1910, by Dr. Alfredo Jahn (no. 156). This appears to be most closely related to Espeletia corymbosa, but the leaves are shorter, relatively broader, and less acute, while the inflorescence is more open, more branched, and bears broader, less pubescent bracts. The corollas are densely pubescent, while for E. corymbosa they are figured as glabrous. 16. ESPELETIA CORYMBOSA Humb. & Bonpl. Pl. Aequin. 2: 13. pl. 72. 1809 Espeletia rigida Humb. & Bonpl. op. cit. pl. 72, nomen nudum. Espeletia platylepis Schultz Bip.; Wedd. in Cast. Expéd. Amér. Sud Bot. 1: 64. 1855, assynonym. TYPE LOCALITY: Vicinity of Almaguer, near the southern boundary of the State of Tolima, Colombia, at an altitude of 2,268 meters. Type collected by Humboldt and Bonpland. This species is apparently the southernmost representative of the genus. Weddell also refers here specimens collected by Goudot and by Linden (no. 1291) in the mountains near Bogota. Judging from Bonpland’s original illustration and description the plant is not far THE GENUS ESPELETIA 485 removed from some of the species here described as new from Vene- zuela. In the text of the original description the species is named Espeletia corymbosa, but the accompanying plate bears the name Esbeletia rigida. 17. ESPELETIA FUNCKII Schultz Bip.; Wedd. in Cast. Expéd. Amér. Sud: Botat 2645 71355 TYPE LOCALITY: Andes of Pamplona, Colombia, at an elevation of 3,400 meters. Type collected by Funck and Schlim (no. 1290). The type locality is near the Venezuelan boundary and the species may be expected to,occur in that country. Weddell compares this with E. corymbosa, saying that it is a larger plant with a less regular, more open inflorescence, and with more elongate achenes having sharper angles. AMERICAN JOURNAL OF BOTANY. VOLUME II, PLATE XVII. Espeletia grandiflora IN THE PARAMO DE BUENA VISTA, STATE OF CAUCA, COLOMBIA. PHOTOS BY Mr: HH. Pitter, Peo Y OF THE RELATION OF TRANSPIRATION TO THE SIZE AND NUMBER OF STOMATA WALTER L. C. MuENSCHER Considerable work has been done in determining the quantity of water lost from various plants under various conditions. Several extensive investigations have been conducted with a view of deter- mining stomatal values for various species. Although several im- portant researches have been conducted concerning the stomatal regulation of transpiration, very little has been done on the amount of transpiration in relation to the size and number of stomata per unit of transpiring surface. It was my aim to determine whether any relation exists between the amount of transpiration and the number of linear units of stomatal aperture per unit of leaf surface. i OHISTORICAT Probably the earliest attempt to determine quantitatively the amount of water lost in plants was by Hales (7) as early as 1727. Hales not only determined the amount of transpiration per square foot of leaf area for the sunflower, cabbage, grapevine, lemon tree, and apple tree, but he also found that the amount of transpiration varied during the day and night hours and also with changes in physical factors. Clapp (4) determined the quantity of water in grams transpired (GM?H) per square meter of leaf surface per hour for day and night for thirty common greenhouse plants both under greenhouse and under “standard’’ conditions, but she used only one individual of each species in her experiment. In computing the transpiring surface she considered only the upper surface of the leaf. She also found that there are two extremes of transpiration, the greatest amount in the early afternoon when the light is strongest and the minimum amount during the night when the stomata are closed. The work on transpiration up to 1904 is summarized in Burger- stein’s excellent work on transpiration in plants (3). He discusses the various phases of transpiration, methods for its determination, 487 488 WALTER L. C. MUENSCHER the effect of leaf structure and of external factors on transpiration, and whether transpiration is a process of vital significance to the plant or whether it is a necessary evil to the plant. The work on the size and number of stomata for various species is rather limited but several rather extensive investigations have been conducted along this line of work. Several early investigators— Humboldt, 1786; Hedwig, 1793; Kieser, 1815; Lindley, 1832; Krocker, 1833; Meyer, 1837; Unger, 1855; and Morren, 1864—determined stomatal values for a number of different species, the results of which are given in a summary table by Weiss (1865). Weiss in his very extensive work (13) gives the length, breadth, and area of stomata as well as the number of stomata per unit of area both on the upper and lower surface for 167 of the more common European plants. The results of these early investigators do not always agree and often their figures are very different. This may be explained as being due to the insufficient instruments for measuring, but more probably because different men used plants which were grown under different conditions or even used different varieties. Weiss observed that stomata may be present on underground stems and aerial stems as well as on leaves. He also states as a result of his observations that the presence of stomata is not limited by the surrounding medium; in other words, stomata may be present on parts of plants which are in air, water, or earth. Eckerson (5) determined the stomata-quantities for about 38 common greenhouse plants. She gives a valuable table in which are recorded the length of the guard cells and of the pore of the stomata and the number of stomata per sq. mm. of upper and lower leaf surface for each species. _ The most important work on transpiration in relation to stomatal movement is by Lloyd (10). In his experiments on cuttings of Verbena ciliata and Fouquieria splendens he discovered that the rate of transpiration may undergo sudden and wide changes without any corresponding changes in the size of the stomatal aperture. From this he concludes that stomatal regulation does not occur, though, of course, conservation of contained water follows upon complete closure of the stomata; but it has not yet been proven that this ever occurs. The plan of my work was to determine the quantity of transpiration simultaneously for a number of species with various stomatal values RELATION OF TRANSPIRATION TO STOMATA 489 and then determine what relation, if any, exists between the amount of water lost and the amount of stomatal aperture in linear units, per unit of leaf area. Il. MrtHop Various methods have been devised and employed for the quanti- tative determination of transpiration both directly and indirectly. It seems that the results, in order to be of much value, should be obtained from plants which at least approach the natural conditions of the plant. Some investigators have confined their investigations I-ZEA 2-PRIMVULA 3-PELARGONIU ¥-TRITIGUM 5-RICINUS b-PHASEOLVS THELIRNTHUS 8-IMPATIENS 2.00 --- = CVTTINGS — = ROOTED PLANTS Fic. 1. Showing the amount of transpiration in mg. per hour per sq. de. leaf surface for cuttings and rooted plants of the several species. entirely to observations upon removed parts of plants, small cuttings of branches, or even single leaves. Lloyd (10) worked only with cuttings in his experiments on stomatal movements. Freeman (6) questions the value of determinations of water loss made from detached parts of plants except where only comparative results are desired, which was the case in Lloyd’s work (10). However, it is doubtful whether the limited number of experiments performed by Freeman (6) 490 WALTER L. C. MUENSCHER would warrant us in drawing the conclusion which he makes in regard to the relation of the amount of transpiration from rooted plants and detached cuttings of plants. In order to satisfy myself as to the possibility of a relationship between the amount of water lost from plants with their roots and plant-parts without roots, an experiment was conducted with five cultures of cuttings in water and ten cultures of rooted plants in jars for each of eight species. The two sets of jars were run simultaneously for 24 hours from 6 o'clock P. M. January 4, to 6 o’clock P. M. January 5, 1915. -The results of the experiment are given in the form of curves in figure I. The two curves show that the water loss in cuttings is about 20 or 30 percent less than the amount lost by the same plant under similar conditions when rooted ina jar. In every case the amount of transpir- ation was less per unit of leaf surface from cuttings than from rooted plants. The cuttings consisted of short stems with from two to five leaves and were removed from the plants under water. The quantity of transpiration is recorded in milligrams per hour per square deci- meter of leaf surface. In brief, the method employed in the following experiment con- sisted in sealing jars so as to prevent all loss of water except that which is lost through the plant; determining this loss by weighing; and then computing the amount of loss per unit of leaf area and stomatal aperture. 1. Selection and Preparation of Plants In selecting the species used in this experiment several important considerations had to be kept in mind. Only plants with simple stomata were taken; that is, plants with stomatal elaborations such as pits, plugs, many hairs or extremely sunken stomata could not be used in this work so as to bring us face to face with the one factor, namely, the relation of the size and number of stomata to the amount of water lost. The species had to be chosen so as to present the widest possible range in size of stomata. Only those plants,could be selected which could be readily grown under greenhouse conditions such as those under which the experiment was performed: lilacs and cottonwoods were started, but shed their leaves and failed to send out a new set of normal leaves; plants of Datura stramonium were started — v7 RELATION OF TRANSPIRATION TO STOMATA AGI . from seed but the plants grew very slowly and were so much stunted they they could not be used in this work. Lastly only those plants could be used which would attain a fair size and maintain a reasonably uniform rate of growth while the experiment was performed. (See Table I.) 2. Preparation of the Jars Most of the plants with the exception of Pelargonium zonale and Primula sinensis were started in flats from seeds. While the plants were still young they were carefully transplanted into the jars where they remained unsealed until they were large enough to use in the determination of transpiration. The plants of Primula and Pelar- gonium were healthy greenhouse plants which were transplanted directly into the jars and then were left on the greenhouse bench for several weeks before sealing for the experiment. Large stone crocks of two sizes were used for containers; gallon crocks were used for the large plants, while one-half-gallon crocks were used for the smaller species. On the bottom of each jar was placed an inverted three-inch flower pot filled with gravel. Around the flower pot about one centimeter of sand was spread over the bottom of the jar. A glass tube with a 1 cm. bore was inserted into the hole in the bottom of the inverted flower pot and the tube extended about 10 cm. above the surface of the paraffin when the jar was sealed. This tube was used for watering the plant. The flower pot with gravel in it rapidly absorbed the water and the sand spread the water evenly over the bottom of the jar. Another tube was put in the jar so as to act as an aerating system for the soil when the jar was sealed. - Then the soil was placed in the jars and the plants were planted in them. The number of individuals of each species that were placed in one jar varied from one to several, depending upon the size of the plants. (See Table I.) The cotyledons which still happened to be on the plants at this time were removed when they were transplanted. After the plants had stood in the jars from one to several weeks, and had become well established they were ready to be sealed. The kind of seal used is that which was used by Briggs and Shantz (1) except that a little harder grade of paraffin was used. A mixture of eight parts of about 60° melting paraffin and two parts petrolatum was taken; however, the exact proportions were not determined in every case. The mixture was heated to melting and was then poured over the surface of the soil so that upon cooling it made a perfect seal. 492 WALTER L. C. MUENSCHER In order to prevent the warm paraffin from doing any possible injury + to the stem of the plants partially cooled strips of paraffin were first wrapped around each stem at the surface of the soil where the hot paraffin would otherwise come in contact with the stem. Each jar was marked by writing a number on the paraffin surface. After the jars had been continued for several weeks the paraffin began to draw away from the edges of the jar slightly. In order to determine any possible error due to evaporation from the edge of the jar a number of jars were reweighed several times after the plants had been removed to see whether any water was lost by evaporation, but this was found to be very small or sometimes so small that it could not be detected by weighing from day to day. After removing the paraffin from the jars I found that it had run down between the edge of the soil and the stone crock several centimeters and still formed a very tight seal near the surface of the soil so that very little water could escape. 3. The Determination of Water Loss A week after the jars had been sealed I took the first weighing for determining the amount of water lost. The amount of water lost was computed from the decrease in the weight of the jar between the intervals of weighing. At first the jars were weighed twice daily, between six and seven o’clock A. M. and between six and seven o’clock P. M. Later the weighings were taken only once each day in the evening, while some of the final weighings were taken at intervals of two or three days; but the jars were always weighed between six and seven o'clock P. M. For weighing I used a “micrometer scale”’ which has a capacity of 18,000 grams and weighs very accurately down to two or three grams. The loss of water from the jar was replenished at intervals of two or three days through the glass tube which was inserted for that purpose. The amount of water added at one time was approximately the amount that was lost by the plants during the interval since the last addition of water, and was added in the evening between seven and nine o'clock. 4. The Determination of Leaf Areas Numerous methods have been employed from time to time for the determination of leaf areas, but it was found impracticable to use RELATION OF TRANSPIRATION TO STOMATA 493 any of these methods without modification. It was impossible to use the solio paper method which was employed by Sampson and Allen (12), for it necessitated the making of many thousands of leaf prints. I made a number of preliminary determinations of the leaf areas of a number of plants by comparative weights, that is by weighing a given area of leaves which was determined by a Ganong’s leaf-area cutter and then weighing the entire leaf area of a plant, from this proportion I determined the total leaf area of the plant. Then I also determined the leaf area of the same plants by making leaf traces and then com- puting the area by a planimeter. I found that the planimeter method was not only the most accurate but also the most practicable method for this problem. I found that the weight of one square centimeter of leaf varied from I to 100 percent from the average weight, depending upon the part of the leaf from which it was taken. The loss of water from the cut leaf sections is also very great especially if the leaves are not weighed immediately. By the weighing method the leaf area had to be found at the same time in all the plants of one species because the amount of photosynthate in the leaf at any given time varies with the time of day as well as from day to day. This would necessitate the determination of the weight per unit area each time it was desired to determine the leaf area for a number of plants. By the use of the planimeter one can find the leaf area of a plant regardless of the amount of water or photosynthate in the leaf and one can use the same method for almost any number or kind of plant at any time. But, because of the enormous number of leaves of which I had to find the area, it was found necessary to modify the method somewhat. I found that each species had a large number of leaves in each plant which were almost of the same size, so I sorted the leaves of each plant into five piles and measured the area of one leaf of each pile and then multiplied the area of one leaf by the total number of leaves in that particular pile and then I computed the total leaf surface for each plant in square centimeters, considering both the upper and lower surface of each leaf. Petiolar and stem areas were not considered as transpiring surfaces. It was found that these organs were practically free from stomata and the water loss from the same was very small in the plants used in this experiment. The leaf area was determined for each plant at the end of the experiment. Since most of the plants were small and had a small 494 WALTER L. C. MUENSCHER amount of leaf surface to begin with, compared with the leaf surface at the end of the experiment, the increase being due to growth, I had to make a correction for the increased leaf area. I considered the increase in transpiration as being proportional to the increase in leaf surface and divided the total leaf area by two to find the average leaf area during the entire period when the plants were used. This would be taking the leaf surface as zero to begin with but this discrepancy is balanced by the leaves which died and were removed from the plants during the progress of the experiment. This method was employed for ali of the species which were started from seed. In the case of Pelargonium and Primula, two slow growers, where mature plants were used, I used the entire leaf surface as was determined at the close of the experiment. What little growth took place was offset by the loss of several dead leaves which were removed from each plant. 5. The Determination of the Number of Stomata For determining the number of stomata I used a Spencer micro- scope with a micrometer scale. The value of the scale and of the field of the microscope were determined with a stage micrometer. The field was divided into quadrants by drawing very fine threads of balsam over the eyepiece micrometer. In those plants which had an epidermis which could be easily removed I mounted pieces of epi- dermis in absolute alcohol and then stained them with a weak solution of iodine. In the plants from which the epidermis could not be removed readily I counted the stomata “in situ’’ by the method suggested by Lloyd (9). The stomatal counts were determined from an average of 30 to 50 sq. mm. each for the upper and lower surface from five or more leaves taken from as many different plants. Only fairly mature leaves were used and from these the fields were taken at random on the different parts of the surface. 6. The Determination of the Size and Area of Stomata After determining the number of stomata for the several species the next process was to determine the size of the stomata for the same. In this process I employed the method so successfully used by Lloyd (10), Eckerson (5), Renner (11), and Livingston (8), in fixing the stomata so that they will not shrink and lose their shape and actual RELATION OF TRANSPIRATION TO STOMATA 495 dimensions. This method is based upon the rapidity with which alcohol dehydrates the guard cells when bits of epidermis are dropped in absolute alcohol immediately after they are removed from the leaf. When once dehydrated these cells become hard and remain permanent in size and shape even if they are afterwards mounted in water. Since Lloyd (10) and Eckerson (5) found that the stomata are open widest at about 10 o’clock A. M., I removed bits of epidermis at this time of the day on bright clear days, placed them in vials of abso- lute alcohol and then determined the length and breadth of both guard cells and pores for about 25 to 50 stomata for each leaf surface for the several species used. From these data I also computed the area of one stoma and the amount of stomatal aperture and the number of linear units of stomatal pore per unit of leaf area for upper and lower surface. Tit. The following tables with short explanations and discussions represent briefly the scope and results of this investigation. EXPERIMENTAL TABLE [| Showing the Species and the Number of Individuals of Each Used and When Each Were Studied No. of Plants | Total Series Species Date No. of in No. of Jars | Each | Plants Jar Hiechanthus annuus L.... 0... 11 days—Nov. 4-15, 1914 10 2 20 Hehanthus annuus W......... 5 days—Dec. 20-24, 1914 10 4 40 Impatiens sultant Hook...... | 30 days—Nov. 4—Dec. 4, I2 | 2-3 30 I9I4 . Impatiens sultant Hook...... 25 days—Dec. I0, 1914- 6 3 18 Jan. 3, 1915 . Pelargonium zonale Willd..... | 20 days—Nov. 4-24, 1914 10 I 10 . Pelargonium zonale Willd..... 40 days—Dec. 10, 1914- 10 I 10 | Jan. 18, 1915 » Phaseolus vulgaris L......... | 25 days—Nov. 4-29, I914 IO , 2-4 22 mehascolus vulgaris Ls... 40 days—Dec. I0, I1914- Io | 4-6 48 | Jan. 18, 1915 . Primula sinensis Sabine .....' 40 days—Nov. 30, 1914- 10 I 10 an. 8, I9I5 DINICTIUS COMMUNES. Ti... 5... s.- | 60 days—Nov. 4, 1914- 10 | 2-3 2a Jan. 4, 1915 . Triticum sativum Lam. ...... 20 days—Jan. 3-23, I9I5 8 10 | I0O MAC ENUE SON orion. feasted ae dis how +s 30 days—Nov. 4—Dec. 4, 10 4 40 I9QI4 »_ AGT Ee eee heer 30 days—Dec. 19, I1914- 10 | 4-5 45 Jan. 18, 1915 “INGOs Ai See Nera ease Seen, | ae cn aye eA ea ene 128 424 496 WALTER L. C. MUENSCHER Table I briefly shows the extent of the work performed. The first column of the table gives a list of the species used. The number before the name indicates the number of the series for that species. With several exceptions two series were performed for each species. The second column indicates for how many consecutive days the tran- spiration was determined for each series and also the date when the work was carried on. The third column gives the number of indi- viduals in each jar and the last column contains the total number of plants used in this investigation. TABLE II Showing Average Amount of Transpiration from the Various Species Used in Series I Average Trans- | Total Time in Total Water | Total Leaf Area eras na | Days Loss in Gm. in Sq. Cm. Sq. De. Leaf Surface Species . Helianthus (unuus II 8027 20,000 I5I TE DAUICTES: SULUL IL rae ete | 30 10,862 6,200 243 Pelargonium zonale......... | 20 8,930 38,000 50 Phaseolus vulgaris.......... 25 12,440 13,400 155 URACINUSICONMUMUTIS Sr ee eae 60 25,897 8600) 209 LEUNG Siren Roy) hee 30 23,047 41,000 78 TABLE III Showing Average Amount of Transpiration from the Various Species Used in Series II Average Trans- Total Time in Total Water | Total Leaf Area es ra Days Loss in Gm, in Sq. Cm. Sq. De. Leaf Surface Species FICHONiNUSONNUUS ©. eee a | 5 1,440 7,200 166 Unt Paiiens SULLG711 eae ey ae tn 25 5,805 3,800 255 Pelargonium zonale. 2.22.3. 40 11,610 18,600 65 PRGSEOlUS VULZATIS set (oc: 40 15,200 10,100 156 VATU SINCNS See | 40 6,580 9,200 75 UAULVCUTIE, SCIUUILIN 20 1,900 5,100 79 ZL CGRIN GY Sneath ee ee oe 30 4,700 8,024. 82 Upon examining Tables II and III it will be found that the abso- lute amount of water lost for the various species varies very much because of the variation in the total amount of leaf surface in the several species. It will also be noted that the average quantity of water lost per square decimeter of leaf surface, average of upper and lower surface, varies from a minimum of 50 mg. in Pelargonium to a RELATION OF TRANSPIRATION TO STOMATA 497 maximum of 235 mg. in Impatiens per hour, average of day and night transpiration for the average of ten jars for about 40 days. The third column of Table II shows the total amount of water lost in grams by all the jars of each species used in series I. Column four shows the total, upper and lower, leaf surface in square centi- meters for each species used in series I. Column five shows the average amount of water transpired for each square decimeter of leaf surface per hour for each species. Table III records the same data for the second series (Series II). A comparison of the results of the two tables (Tables (II and III shows that the average amount of water transpired by each species as determined in the two series of plants varies but slightly. The largest difference is found in Pelargonium zonale and this is well accounted for by the fact that I accidentally used a different variety of Pelargonium for the second series. The results of Helianthus annuus may have been modified by an early attack of Erystphe cichora- cearum, which prevented me from continuing the cultures for a longer period than five days in the second series. The first eleven days of weighings which were obtained from the first series were taken before there was any evidence of Erysiphe and, I think, represent normal transpiration. TABLE IV Showing the Minimum, Mean, Average, and Maximum Number of Stomata in One Square Millimeter Leaf Surface Lower Surface Upper Surface Name of Species | Min. Mean Avy. Max. Min. Mean Av. Max. PRGSCOIUS VINEETES... UST | e269 250° 327 28 40 40 50 Ricinus cummunis........ 79 | 156 | I2I 172 35 68 52 76 LAD GUUS en ek ae ee gI 102, | TOL 110 51 61 60 83 PVIMULG SINCNSIS.... 0... 49 87 84 | 112 ) Pelargonium zonalel...... B2 52 52 72 9 18 19 24 Pelargonium zonale 2...... L904} .290 .| 215% ,2232 4 9 8 II Impatiens sultanit......... HOS, W140) Laan GO 12 277 29 70 Lrvicum SAUVUM.......... 12 21 21 27 35 46 46 55 Helianthus annuus........ 125 T7Om lye 198 27 70 71 90 Table IV shows the number of stomata per square millimeter leaf surface for each of the several species. It will be noticed that all but one species, Triticum sativum, have fewer stomata on the upper surface than on the lower surface. I found that the number of stomata varied considerably in different parts of the same leaf. On 498 WALTER L. C. MUENSCHER the other hand, I noticed that for similar areas of the various leaves examined the number of stomata was more or less constant for the species. I tried to take the upper and lower counts for each leaf from similar or corresponding areas. The number of stomata per square millimeter varies from an aver- age of a maximum of 250 in Phaseolus vulgaris to a minimum of 21 in Triticum sativum for the lower surface and from a maximum of 71 in Helianthus annuus to a minimum of zero in Primula sinensis on the upper leaf surface. (See Table IV.) In Table IV I recorded the minimum, mean, average, and maxi- mum number of stomata determined per square millimeter for each species. The average number in each case represents the average of thirty or more single counts. The minimum and maximum are rather widely separated but the mean and average are nearly always equal or nearly so. These figures do not compare exactly with any figures found for the same species by Weiss (13), or Eckerson (5), but the differences are not so large but what they may be accounted for by differences in conditions under which the plants were grown, or different varieties or strains might have been employed by the different investigators. In Impatiens sultant I found 29 stomata per square millimeter -on the upper leaf surface while Eckerson reported that no stomata were found by her on the upper leaf surface of the same species. Prob- ably I used a different variety of Impatiens sultant. The table also shows two sets of figures for Pelargonium zonale, number I was used in the first series and number II was used in the second series. These are two different varieties, the former has fewer and much larger stomata than the latter variety. Table V records the size of the stomata for the upper and lower surface for the various species. The length and breadth of the guard cells and of the pore is recorded in microns. Each number represents the average of thirty or more measurements. I found that all the stomata as well as their pores were more or less elliptical. The length of the pore is about one half of the total length of the guard cell apparatus. (See Table V.) The width of the pore is usually less than one half its length. The largest stomata were found on the upper epidermis of Triticum sativum, with a pore 39 microns in length; the smallest stomata were found on the upper side of Impatiens sultam with a pore six microns in length. The stomata which were measured RELATION OF TRANSPIRATION TO STOMATA 499 were taken from several pieces of epidermis from several leaves from at least three plants. It was noted that individual stomata of the same plant or even leaf vary somewhat in size depending upon the part of the leaf in which they are located. TABLE V Showing the Length and Breadth of Stomata in Microns Lower Surface Upper Surface Name of Species Pere aero eras Guard Cell Pore Guard Cell Pore Phaseolus vulgaris.......... | 20.655 8 X 3 DOG Seer belt Xe 3 RICUMUS COMMUNIS. . 0... sss 26 X 18 10 X 3 22° X15 10 X 3 LEDS TOSS 26) G27, 19 X 3 Bei oN HOM nie 1 kOn 3 PUNT SINCNSIS. ..0..0. 04: [2 eADr Sesar 20 X 4 — — Pelargonium zonale 1........ ln, 138i px 27, 20 X 4 ais 6 Py Lj 3 Pelargonium zonale 2........| 28 X 18 Pees BOn 2 len a 12S UQUPANCHSISHUGNE. . oc. asks le 20 KeG oes 7G EA 4 6a. 3 TIMCUNESQUUUM: .. 2... 622720 BAX C5295 a5 3058 Helianthus annuus.......... 27 X20 I9 X 4 30 X 24 1A exX3 Upon comparing the results of Tables ITV and V, I noticed several general facts: 1. The size of stomata on the same plant may vary considerably with the upper and lower leaf surface. 2. In general plants with few stomata have large ones and plants with many stomata have small ones. From the data in Tables IV and V, I computed the average number of units of stomatal pore length in microns per square millimeters of leaf area for the upper leaf surface and for the lower leaf surface by multiplying the number of stomata per unit of area by the average length of one pore. Then I computed the average number of microns of stomatal pore length for the upper and lower surfaces by adding the two values and dividing by two. The linear units of stomatal pore and not the area of the stomatal pore is important since Brown and Escombe (2) have shown that the length of the pore and not its area is important in transpiration. | I also computed the area of the stomatal pore of one stoma and then the area of the total amount of stomatal aperture per square millimeter, average of upper and lower leaf surface, by considering the stomatal pore an ellipse. (See Table VI.) The data show that it is not necessarily the plant with the largest stomata or the plant with the greatest number of stomata that has 500 WALTER L. C. MUENSCHER the largest area of stomatal aperture per unit of leaf surface. The largest number of linear units of stomatal pore, average of upper and lower surface, was found in Helianthus annuus, 2056 microns, and in Zea mays, 1530 microns, while the smallest number was found in Pelargonium zonale, 682 microns, and in Impatiens sultant, 731 microns. (See Table VI.) TABLE VI Showing the Number and Size of Stomata, and the Linear Units of Stomatal Pore in Microns per Square Millimeter Leaf Surface Average No. of Stomata | Average Length of Pore | Average No. of Linear | per Sq. Mm. Surface in Microns Units of Stomatal Pore Species Average of Upper and Lower Upper Lower Upper Lower Surface in Microns per Sq. Mm. aseolus a ea eo en | 8 ai 1,220 RACUNUS © ee a Pern = Tote i egs20 se TOm ea 865 LOWE Wp eng iene es |: OE 60 19 | 19 1,530 PYUMIULO ease ye nae: | 84 e) 200) Gi) See 840 PCLGN CONTA Loe 52 19 20 | U7. | 682 Pelarzonvm 2.5... | 205 8 It | I2 | 1,230 LUN POWEHSE te se es [a TAs ee 20 ‘ony @ 6 731 TPALUCU IO ene co ons a a6Rr lee oy 25 1,254 Hehanthuse | 8s Were yy erty all, Sk) 1A 4 2,056 I now arranged the several species in Table VII in the order of their greatest number of linear units of stomatal pore and also placed: opposite each species in the third column the average amount of TABLE VII Showing Relation Between the Amount of Transpiration and Stomatal A perture | : , | Amount of Area of Stomatal | Linear Units of | Transpiration per Aperture in Sq. | Stomatal Pore in | Sq, Dm. per Hour |Microns per Sq. Mm. ptertetss per Sq. Mim. in Mg. of Leaf Surface Helianthus GnWUuus a oe. ee 2,056 | 156" 6,433 ZECCA Se ACCS ese dae 1,530 80 3,864 I DTCC SO GITOS SAS oe OG RR OAT | 1,254 79 2,998 PelargOnium BOUGIe 2. ween. te | 1,230 65 2,868 EWGSCOLUS VUICOTUS yt er te eae | 1,220 | 156 | 2,890 RAICUNUS, CONIMUNIS:. = ee | 865 | 209 1,989 Primula sinensis.........+..+. 840 | 75 2,614 Tn patiens SULLONIR he eo 7a | 249 1,705 Pelargonium ZOngle Tae ee 682 | 50 1,992 water transpired per square decimeter per hour in milligrams. This average amount of transpiration was obtained from the data in Tables II and III. RELATION OF TRANSPIRATION TO STOMATA 501 From this table (Table VII) I could determine whether any rela- tion exists between the amount of water lost and the amount of linear units of stomatal pore. These results do not show any constant relation between the amount of transpiration and the number of linear units of stomatal pore. The two species, Impatiens sultant and Ricinus communis, both of which have a small amount of stomatal pore per unit of leaf surface, have the two highest amounts of transpir- ation per unit of leaf surface. Phaseolus vulgaris also has a high transpiration in proportion to the amount of stomatal pore per unit of leaf surface. - =STOMATAL PORE = TRANSPIRATION IH HELIRNTHYS +PELRRGONMIUMa 7-PRIMVLA -ZER 5-PHASEOLVS 5-IMPATIENS s-TRITICUM = -&-RICINS 9-PELR RGON VM. Fic. 2. Showing the amount of transpiration in mg. per hour per sq. dc. leaf surface and the number of linear units of stomatal pore in microns per sq. mm. leaf surface. The data of Table VII are also shown in graphic form by two curves in figure 2. The twocurves are drawn to separate scales. In the transpiration curve the side of each square represents fifty milligrams. In the curve showing stomatal pore the side of each square equals 500 microns. In other words the latter curve represents values ten times as large as the former, or each unit equals ten microns in the curve which represents stomatal pore. In Table VIII I have arranged the species in the order of the 502 WALTER L. C. MUENSCHER TABLE VIII Showing Length, Area, and Amount of Transpiration for One Stoma Ave, T i Ave. No. of Ave. Length Ave. Area of baa aa eee | Stomata per o' Stomatal i Stoma in Sq. per Hour in 5 in Microns Microns Sq. Mm. Pore in Grane Phaseolus vulgaris.......... | 145 9.5 22 .OOOIO Helianthus ORNS oe a. | 122 16.5 47 | .00013 Pelargonium zonale 2........ 112 Tas, 35 | .00006 Ricinus communts.......... 87 IO 23 |. 0@022 Lin Patten S| SUILONL..) tents es | 86 Fels! 18 .00029 ZED INOS eRe vay | 81 19 48 .00009 PYUMULG SINCNSIS= 2.10. 2). aos | 42 20 62 .00018 Pelargonium zonalel........ 36 18.5 51 .OOO14 EP UCUNT SUMUNM. en ne oe 34 36.5 85 .00023 largest number of stomata, the average of the upper and lower surface. I also recorded the average length of the pore of one stoma in microns, the area of one stoma in square microns, and the amount of transpira- tion from one stoma in hundredths of a milligram. I-PHRSEOLUS b-ZER aHELIRN THUS 7-PRIMULA 3-PELRRGONIUM(@ 8-PELRRGONIV H-RICINUS q-TRITICUM s-AMPRTIENS ~--= LENGTH OF 1 PORE — -=TRANSPIRATION Fic. 3. Showing the length of one pore in microns, and the amount of transpiration in hundredths of mg. per hour for one stoma in the several species. The data, namely the length of the stomatal pore and the amount of transpiration per stoma for the various species, are represented in graphic form by two curves in figure 3. These figures do not show RELATION OF TRANSPIRATION TO STOMATA 503 any relation between the length or area of the pore of a stoma and the amount of water lost for the several species. SUMMARY 1. There was found no constant relation between the amount of water lost and the numbers of linear units of stomatal pore, 7. e¢., the number of stomata per unit of leaf surface multiplied by the length of the average pore, in the various species studied. 2. There is no relation between the amount of transpiration and the length of the pore of one stoma. The number of stomata per unit of leaf surface however varies at the same time that the length of the pore varies for the several species; so in this case we have two variables. 3. There-is no relation between the amount of transpiration and the number of stomata per unit of leaf surface in the different species investigated. ) 4. From the above results it would seem that the amount of transpiration is not governed entirely by stomatal regulation, and that the variations in the amount of water loss in different species cannot be accounted for by the size and number of stomata but must be explained perhaps by a complex of several factors. This investigation was suggested by and conducted under the direction of Professor Raymond J. Pool, of whose kindly advice and suggestions as well as the encouragement and suggestions of Dr. C. E. Bessey, I wish to express my sincere appreciation. UNIVERSITY OF NEBRASKA. LITERATURE CITED 1. Briggs, L. J. and Shantz, H. L. The Wilting Coefficient for Different Plants and its Indirect Determination. U.S. Dept. Agr. Bur. Pl. Ind. Bull. 230. I9I2. 2. Brown, H. T. and Escombe, F. Nature 62: 212. 1900. 3. Burgerstein, A. Die Transpiration der Pflanzen, Jena, 1904. 4. Clapp, Grace Lucretia. A Quantitative Study of Transpiration. Bot. Gaz. 45: 254-267. 1908. 5. Eckerson, Sophia H. The Number and Size of Stomata. Bot. Gaz. 46: 221. 1909. 6. Freeman, Geo. P. A Method for the Determination of Transpiration in Plants. Bot. Gaz. 46: 118. 1909. 7. Hales, Stephen. Statical Essays, Vol. I, London, 1727. 504 WALTER L. C. MUENSCHER 12. 13: . Livingston, B. E. and Estabrook, A. H. Observations on the Degree of Stematal Movements in Certain Plants. Bull. Torrey Club 39: 17-22. Ig12. . Lloyd, F. E. Leaf Water and Stomatal Movement in Gossypium and a Method of Direct Visual Observation of Stomata in Situ. Bull. Torrey Club 4o: 1-20, fOba: . Lloyd, F. E. The Physiology of Stomata. Carneg. Inst. Washington Publ. 82. 1908. . Renner, O. Beitrage zur Physik der Transpiration. Flora 100: 451-547. IQIO. Sampson, A. W. and Allen, L. M. Influence of Physical Factors on Transpira- tion. Minn. Bot. Stud. 4: 33-64. 1909. Weiss, A. Untersuchungen iiber die Zahl und Gréssenverhdaltnisse der Spalt- offnungen. Jahrb. wiss. Bot. 4: 126-196. 1865-1866. Perr < me is age shee = Hse it ever occurred to you. thar ‘ “greenhouse constructions differ 2: ‘greatly, and that a large share of the. "success of your flowers depends « on : construction # 2) Bo “Haven't you Pee that prac-. | “tically the only difference in green- | houses, aside from design, was the : A difference i in price? ; With ‘everything elses isn " tihere. PO 4 always some one kind. that is gener- ~ .ally conceded to be better than the — rest, and taken. as a standard for. com- . ; s ower ee. fe ‘parison, . eas Se Se) 2 pisos experts oie |- these. advantages . are >worth sence in cost is a question Then i isn’t it aly fogicati it should es Before’ you Sue any Be sO with greenhouses? - ie oe premslpiee it might = When other ‘greenhouse ‘baiiders: ne ane the matter a. er : claim. their Dore: are ‘‘as light” as ; : =the U- Bar’ s’’ it’s ‘significant that a ; U-Bar i is the lightest of them all. Bhat | PS most featitate if the other builders. are. con: oe a eae ge ee : ; i structing their houses with curved . ONE MADISON’ AVE. NEWYORK. Lae >CANADIAN OFFICE, JO'PHILLIPS PLACE. MONTREA ©) io Hae fe "PIERSON. UBAR CO SS oo Prite ell phenomena. ATES 4 MY SEED Ben f SY a 1 lad? ye ae ADRIAN J. PIETERS 529 POM ae EET, LANCASTER, . & 8 sine SC onl Pane e i Bussey Plus 3 AEN AMERICAN JOURNAL OF BOTANY. VOLUME II, PLATE XVIII. AMERICAN JOURNAL OF BOTANY —— Vor.Ll DECEMBER, 1915 No. 10 Pb RiEE Sith TCH OF THE. LIFE AND WORK OF CHARLES EDWIN. BESSEY (WITH PORTRAIT) RAYMOND J. POOL - Charles Edwin Bessey, professor of botany and head of the depart- ment of botany in the University of Nebraska since 1884 and a con- spicuous figure in American science and education, passed away at his home in Lincoln on February 25, 1915, after a critical illness of four weeks. The Bessey family is of French extraction, the original form of the name being Bessé. The tradition is that the early members of the family, who were Huguenots, were compelled on account of religious persecution to flee to England from the old home near Strassburg in Alsace. This exodus occurred in the latter part of the seventeenth century after which the “y’’ was added to the name. The Besseys remained in England for several generations. Among the first of the family to come to America was Jacob Bessey who, - about the middle of the eighteenth century, emigrated from England. Jacob Bessey married and settled near Doylestown, Pennsylvania. One of the sons of this union was Michael, who was a weaver by trade. This son married Mary Wismar, a descendant of a family from the Palatinate on the Rhine. Michael-and Mary Bessey were the parents of Adnah Bessey who was born in eastern Pennsylvania in 1812. In 1832 Adnah Bessey’s family with many others from the vicinity of his home migrated westward and settled in Wayne County, Ohio. Adnah Bessey married Margaret Ellenberger. Emmanuel Ellen- berger, the father, died when Margaret was but a young girl and the widowed mother married again. Margaret’s stepfather was Jacob Kimmel, a widower with a large family. Margaret was accordingly [The Journal for November (2: 429-504) was issued Dec. 16, 1915.| 595 506 RAYMOND J. POOL ‘put out to live” with James McKinly and wife, an estimable couple who had also migrated from Pennsylvania to Ohio. This young lady was sent by her foster parents to a school which happened to _be taught by Adnah Bessey, and some time later (1841) teacher and pupil were married. Adnah Bessey and Margaret Ellenberger Bessey were the parents of Charles Edwin Bessey, who was born in a log house on a farm in Milton Township, Wayne County, Ohio, May 21, 1845. Much of the early education of Charles, the boy, was received under the direct attention and supervision of Adnah, the father. In his seventeenth year he received a certificate to teach school. He did not teach at that time, however, as he had made all arrangements to enter the academy at Seville (Ohio) in order to fit himself for college. His educational training was interrupted soon after this by his father’s ill health, but even then he attended the district school and studied algebra by himself. His father died in 1863 and that fall Charles attended the academy for five weeks. He then began teaching at Wadsworth, Ohio, where he remained for a term of four months. Following this (March, 1864) he again entered the academy where he studied for two months, when the breaking up of the school precipitated another halt in the young man’s educational career. After several years of broken but persistent preparation in the country school and academy young Bessey entered the freshman class of Michigan Agricultural College in July, 1866. He completed his college course at the age of twenty-four and graduated from the college with the degree of bachelor of science, November 10, 1869. When Bessey entered the college he firmly intended to return home to follow the profession of civil engineering or surveying. But he came to love the plants of the fields and forests. Finally his marked attention to such things became noticeable to others; for, after two years or so in college, the President (Abbott) and some of the professors (especially Prentiss) advised him to specialize in botany. At first the advice was rejected. But after weeks of reflection the young man found that his inclination had changed from engineering to botany. Immediately after graduation Bessey was awarded an assistant- ship in horticulture and was placed in charge of the greenhouse at the Michigan Agricultural College. This position was held for a very short time because in December of that year (1869) the offer of an instructorship in botany and horticulture came from the Iowa State LIFE AND WORK OF CHARLES EDWIN BESSEY 507 College of Agriculture at Ames. The call was accepted and he began his work at Ames, in February, 1870. An interesting point of his- torical note is seen in the fact that he attended and took part in the first Farmers’ Institute held in Iowa; this was during the winter of 1870-1871. These were among the very first meetings of the kind ever held in any part of the United States. It also appears that he was the guiding spirit in the founding of the old (the first) Iowa Academy of Sciences. He was president of this organization in 1875 and was re-elected regularly for several years. Bessey was elected to membership in the American Association for the Advancement of Science at the Dubuque, Iowa, meeting in August, 1872. This was the first occasion of his meeting with Dr. Asa Gray, who was then the retiring president of the Association. Here He also met Dr. How, Dr. Winchell, M.S. Bebb, and others who were to be some of his closest scientific friends and co-laborers for many years. The first fruits of this acquaintance with Gray were gathered during that winter of 1872-1873 when he spent the long vacation of three months at Harvard University studying under Professor Gray. Under the watchful eye and kindly direction of the great botanist he laid the foundations of his knowledge of systematic botany especially in its philosophical aspects. The genial physician led the young man beyond the threshold of a “life among species and phyla”’ which was probably Dr. Bessey’s most constant scientific enjoyment. Bessey returned for another period of study at Harvard in the winter of 1875-1876. In 1872 he received the degree of master of science from his alma mater and was promoted to a full professorship at Ames. In Novem- ber, 1873, he was offered the chair of botany, zoology, horticulture and pomology but this was refused. Later he was made professor of botany and zoology. Early in the year 1874, at the suggestion of Dr. Gray, President Gilman, of the University of California, wrote to Professor Bessey asking him to give his views in regard to certain problems relating to agricultural education then demanding solution. His reply, along with those from Louis Agassiz, Andrew D. White, Samuel W. Johnson and others, was printed in a bulletin issued in the spring of 1874 by the University of California under the title: ‘Recent Information respecting Agricultural Education Elsewhere.’’ This correspondence led to an invitation from President Gilman to Professor Bessey to go 508 RAYMOND J. POOL to California to give a series of botanical lectures in the university the following winter. The invitation was accepted and resulted in the appointment of Professor Bessey as a temporary “Lecturer on Botany.” In 1879 the University of Iowa conferred the degree of doctor of philosophy upon Professor Bessey in recognition of his publications in botany and as a partial reward for what he had already accomplished for the state of Iowa. After having served the state of Iowa and its Agricultural College faithfully and efficiently for a period of fifteen years there came in June, 1884, notification that he had been elected professor of botany in the University of Nebraska. He went to Lincoln but found nothing in botany at the institution that had called him, and_he was reluctant to leave the accumulation of his labors at Ames to go to a new state to build up another department from the very beginning. So he declined. A second offer was made in August of the same year. After another trip to Lincoln and a consultation with the regents this posi- tion was accepted and his inaugural address was delivered in Septem- ber, 1884. In Nebraska he began at once to collect data with reference to Nebraska grasses and the other plants of the state, and to make ad- dresses. With the late Governor Furnas he organized the first series of Farmers’ Institutes, which thereafter were periodically enlivened by his presence. Bessey was a power among his fellow scientists, who were to be found in all parts of the world. From the time he first met Gray at Dubuque he seldom missed one of the annual gatherings, and he always took a keen interest in the administration of the various societies and in the scientific programs. The esteem of his associates was often reflected by the offices to which he was elected. In 1872 he became a member of the American Association and in 1880 was elected a fellow in the same. In 1889 he was president of the Society for the Promotion of Agricultural Science, and also of the Western Society of Naturalists. He was vice-president of the American Association and chairman of Section G (botany) in 1893, 1894, 1902, and 1907. He was also a charter member of the Botanical Society of America, of which he was president in 1895. That same year he was also president of the department of science of the National Educational Association. He was chosen to be botanical editor of LIFE AND WORK OF CHARLES EDWIN BESSEY 509 the American Naturalist in 1880, a position which he held until 1897 when on invitation he accepted a similar position on the staff of Science. The crowning scientific honor came at the Minneapolis meeting of the American Association for the Advancement of Science in I910- 1911, when he was elected president. The following winter (1911I- 1912) at Washington, D. C., he presided over the deliberations of this organization. When he returned to Lincoln after the meetings he was given an ovation by the students, faculty and the regents in the university chapel. The next winter (1912-1913) at the Cleve- land meeting of the Association Dr. Bessey gave his address as retiring president. The paper which he read at that time was entitled: “Some of the Next Steps in Botanical Science.” Bessey was one of the pioneers who did much to lay the founda- tions of the present superstructure of American botany. The training with Gray naturally increased his love for taxonomy which is reflected in many of his publications. But he was thinking of other phases of botany also and some of his early papers show that he took advanced ground as to what should constitute the content of botany. In his paper on ‘‘The Diseases of Plants’”’ (1882) we have abundant evidence that he was doing more than recording species and making herbaria. At this time the heterecious nature of rusts was not accepted by all botanists as conclusively demonstrated but Bessey felt that De Bary’s conclusions were correct. The comments upon bacteria as disease- producing organisms are also interesting. When in the early eighties the United States Department of Agri- culture was considering the proposition to establish federal aid for state agricultural experiment stations he was consulted in regard to the wording of a bill for that purpose. He plunged into this work with the usual readiness and vigor and he finally wrote the paragraph defining the duties of such experiment stations verbatim as it was adopted later and became a part of the law known as the Hatch Act. He also wrote the first and second annual reports of the Agricultural Experiment Station of Nebraska in 1888 and 1889, and from that time he did not cease to concern himself actively with the progress of these institutions which were to become important seats of learning and research throughout the country. Much of the time that Dr. Bessey gave to study and writing was devoted to a painstaking survey of the structure and evolution of all 510 RAYMOND J. POOL of the main groups of the plant world. This monumental work was begun many years ago, the first paper in the series dealing in particular with the higher plants being entitled: ‘“The Phylogeny and Taxonomy of the Angiosperms”’ which was read as the address of the retiring president of the Botanical Society of America at its third annual meeting at Toronto, Canada, August 17, 1897. This paper contains a further statement of the thought and principles of a still earlier production—*‘Evolution and Classification’’—which was given as the vice-presidential address of the chairman of the botanical section of the American Association at the Madison, Wisconsin, meeting in 1893. For a quarter of a century Professor Bessey worked upon the “‘phyletic idea” in taxonomy, the above papers being among the earlier ones upon that subject. His last paper entitled: ‘‘The Phylo- genetic Taxonomy of Flowering Plants’? was one of a memorable series of papers read by invitation at the Twenty-fifth Anniversary Celebration of the Missouri Botanical Garden, at St. Louis, October 15,1914. The gathering at St. Louis at that time was his last meeting with the botanists of America. | But after all has been said about all of the other features of this great man’s life we still must conclude that the most powerful and far-reaching effects of his captivating magnetism were recorded in the classroom, in the laboratory, in the college and in the university as a teacher and guide for the young. Professor Bessey utilized this potential to the limit, for he was one of the greatest teachers that the world has known. His powerful presentation of subject matter in the classroom was magnified by a personality which, because of its quaint paternal cordiality, won the admiration of thousands of students. His lectures, delivered with a heavy clear voice and in non-technical phraseology, were always illustrated by means of quickly executed blackboard sketches which served at once to portray morphological features graphically and to drive home to the students’ understanding exactly what he was talking about. He was so skilled with this method that he seldom used charts or diagrams prepared before class time. His small alarm clock and the black cloth-covered record book with pencil attached were inseparable adjuncts to every lecture. Among the many services Professor Bessey rendered to the teaching of botanical science that might be mentioned was the introduction in 1873 at Iowa Agricultural College of the laboratory method of instruc- LIFE AND WORK OF CHARLES EDWIN BESSEY Bit di tion with the use of compound microscopes. It is said that the laboratory method for advanced students was introduced at Harvard the year before, but this was wholly unknown to Bessey. During the month of July, 1881, he gave the first course in botany in which laboratory work was offered at the University of Minnesota. He used to tell with much pleasure of reminiscence how he “carried the first compound microscopes to Minnesota.’ These instruments were borrowed from the college at Ames for that particular session, since the University of Minnesota possessed no such microscopes at that time. There were none at Ames when he went there. Neither were there any at Nebraska when he went there. Bessey’s students were numbered by the thousand and one of his greatest pleasures was to look over the lists of former students of his department and to picture them, oftentimes in distant lands, contributing of their thought and life for the betterment of mankind. Dozens of instructors in American schools and many investigators in . the offices and laboratories of many institutions of learning and re- search owe their very life-ambition to the initial boost administered by Dr. Bessey at some critical moment. This was true not only of the botanically inclined but also of others whose primary inclination had drawn them into other fields. After a careful estimate made a few months before his death Professor Bessey came to the conclusion that in his forty-five years as a college professor he had had over 4,000 students in his classes. This was the work closest to his heart and in this he felt that he had fulfilled a divine commission. The love for young people and for the work of the teacher kept him young in . spirit and vigorous in action to the end. A large factor in moulding Bessey’s standing as a botanist and in bringing him success as an educator was his series of text-books. In 1878 he began the preparation of a work on “Botany for High Schools and Colleges.’ The publishers had asked Dr. Gray to write this book, but he declined and recommended Professor Bessey. The manuscript was finished in January, 1880, and the book appeared August 1 of that year. This text went through two later editions, one in 1881 and another in 1883. Then followed ‘The Essentials of Botany,’’ the briefer course, of 1884. This book, which went through seven editions, the last being in 1896, enjoyed the enviable reputation as one of the most popular and most widely used texts in America. His last book, ‘‘The Essen- 9 512 RAYMOND J. POOL tials of College Botany”’ which he wrote (1914) with the assistance of his son, Dr. Ernst A. Bessey, was really another (the eighth) edition of the first elementary book. At Nebraska, Bessey’s ability as a college administrator was recognized at various times by the regents. At one time he was also acting president at Ames. He went to Nebraska as professor of botany and dean of the Industrial College. This deanship he held from 1884 to 1888 and again from 1895 to 1909, when he was made head dean. He was dean of the college of literature, science, and the arts from 1888 to 1891. He was made acting chancellor of the university in the summer of 1888, being called home from Europe, where he had gone with Mrs. Bessey to spend the summer in study. This position he held until 1891 when he succeeded in obtaining the appointment of Professor Canfield as chancellor. He was made chancellor ad interim again in 1899, and still once more, in 1907. On December 25, 1873, Charles E. Bessey was married to Miss Lucy Athearn of West Tisbury, Martha’s Vineyard, Massachusetts. To them three sons were born: Edward, Ernst, and Carl. All of the sons graduated from the University of Nebraska. Edward and Carl specialized in electrical engineering. The former was assistant professor of electrical engineering in the Colorado Agricultural College at Fort Collins at the time of his death on July 12, 1910. Carl is at present assistant chief engineer of the firm of Byllesby and Co., Chicago. Ernst specialized in botany and is at the present time professor of botany at the Michigan Agricultural College, his illustrious father’s alma mater. Mrs. Bessey retains her home at 1507 R Street, Lincoln. THE UNIVERSITY OF NEBRASKA, LINCOLN. A PartTiAL List OF BOTANICAL ARTICLES AND Books PUBLISHED BY CHARLES E. BEssEy! 1871. Natural science in common schools. Jowa Instructor and School Journal 12 268-2402, Loy’. Contributions to the flora of Iowa. Report to Board of Trustees Iowa Agricultural College for 1871. 1872. Lemna polyrrhiza. Amer. Nat. 6: 636. 1872. 1873. Directions for collecting mosses and lichens. Ames, Iowa. 1873. Sensitive stamens in Portulaca. Amer. Nat. 7: 464-465. 1873. 1] wish to thank Dr. Ernst A. Bessey for help in connection with the prepara- tion of this sketch, also Professor T. J. Fitzpatrick for a number of titles that have been included in the bibliography.—R. J. P. 1876. 1877. 1880. 1881. 1882. 1883. 1884. 1885. 1886. 1887. LIFE AND WORK OF CHARLES EDWIN BESSEY 513 On a scientific course of study. Read before Iowa State Teachers’ Associa- tion Dec. 28, 1876. Reprinted from The Aurora. The Erysiphei. Seventh Biennial Rep. Ia. Agr. Coll. 1-16. 1877. Observations on Silphium laciniatum, the so-called compass-plant. Amer. Nat. 11: 486-489. 1877. The supposed dimorphism of Lithospermum longiflorum. Amer. Nat. 14: 417-421. 1880. A new species of insect destroying fungus. Amer. Nat. 17: 1280-1281. 1881. Simblum rubescens Gerard, in Iowa. Bull. Torrey Club 8: 126. 1881. A sketch of the progress of botany in the United States in 1880. Amer. Nat.215:°947.. 188r. The diseases of plants. Ja. State Hort. Soc. 1882. Some observations on the action of frost upon leaf-cells. Proc. Amer. Assoc. Adv. Sci. 31: 464-465. 1882. The coffee-leaf fungus, one of the Mucedineae. Amer. Nat. 16: 584-586. 1882. The asparagus stem for laboratory study. Amer. Nat. 16: 43. 1882. The periodical cicada in southeastern Massachusetts. Amer. Nat. 17: 1O7Ory 1883: A suggestion in regard to the publication of new species. Amer. Nat. 18: Wl fer LOo4: A real Yankee puff-ball. Amer. Nat. 18: 530. 1884. Preliminary lists of Cryptogams of the Ames flora. Bull. Ia. Agr. Coll. Bot. 132-150... 1884. The wheat smuts. Bull. Ja. Agr. Coll. Bot. 118-126. 1884. The smut of Indian corn. Bull. Ia. Agr. Coll. Bot. 127-129. 1884. Adventitious inflorescence of Cuscuta glomerata. Amer. Nat. 18: 1145- 1147. 1884. The ergot. Bull. la. Agr. Coll. Bot. 130-132. 1884. The injuriousness of porcupine grass. Bull. Ia. Agr. Coll. Bot. 116-118. 1884. Sexuality in the Zygnemaceae. Amer. Nat. 18: 421-422. 1884. The rattle-box. Bull. Ia. Agr. Coll. Bot. 111-115. 1884. Science and Practice. Ann. Rep. Nebr. State Board Agr. 1884. The abundance of Ash-rust. Amer. Nat. 19: 886. 1885. Injurious fungi in their relation to the diseases of plants. Amer. Pomolog. Soc. 1885: 35. The grasses and forage plants of Nebraska. Ann. Rep. Nebr. State Board Agr. 1886. The rust of the ash tree (Aecidium Fraxini). Amer. Nat. 20: 806. 1886. The roughness of certain uredospores. Amer. Nat. 20: 1053. 1886. Eastward extension of Pinus ponderosa in Nebraska. Amer. Nat. 21: 928. 1887. A meeting-place of two floras. Bull. Torrey Club 14: 189-191. 1887. Westward extension of Juglans nigra in Nebraska. Amer. Nat. 21: 229. 1887. 514 1888. 1889. 1890. 1891. 1892. 1893. RAYMOND J. POOL The use of English names for fungi. Amer. Nat. 21: 264. 1887. The growth of Tulostoma mammosum. Amer. Nat. 21: 665. 1887. The ash-rust again. Amer. Nat. 21: 666. 1887. The study of lichens. Amer. Nat. 21: 666. 1887. The grass flora of the Nebraska plains. Amer. Nat. 22: 171. 1888. First Annual Report of the Nebraska Agricultural Experiment Station. 1888. ; Report of the Botanist on the grasses and forage plants, and the catalogue of plants. (With H. J. Webber.) Ann. Rep. Nebr. State Board Agr. 1889. Second Annual Report of the Nebraska Agricultural Experiment Station. 1889. The flora of the upper Niobrara. Amer. Nat. 23: 537-538. 1889. The diseases of farm and garden crops. Nebr. Farmer 14: 89. 1890. Black knot. [Plowrightia morbosa (Schw.) Sacc.] Nebr. Farmer 14: 129. 1890. Stinking smut [Jzlletia foetans (B. & C.) Trel.]. Nebr. Farmer 14: 130. 1890. Grain smut [Ustilago segetum (Bull.) Dit.]. Nebr. Farmer 14: 151. 1890. Corn smut [U. maydis (D. C.) Cord.]. Nebr. Farmer 14: 165. 1890. Sorghum smut [U. sorghi (Link) Pers.]. Nebr. Farmer 14: 189. 1890. The strawberry leaf spot, Ramularia Tulasnet Sacc. Nebr. Farmer 14: 209. 1890. Grain rust (Puccinia graminis Pers.‘and other species). Nebr. Farmer 14: 250. 1890. The rust of Indian corn (Puccinia sorght Schw.). Nebr. Farmer 14: 293. 1890. ee The raspberry stem fungus. Nebr. Farmer 14: 333. 1890. A preliminary list of the grasses of Nebraska. Ann. Rep. Nebr. State Board Agr. 1891. The bearberry in central Nebraska. Amer. Nat. 25: 1130. 18091. On the fertilization, crossing and hybridization of plants. Ann. Rep. Nebr. State Hort. Soc. 100-114. 1891. The hybridization of plants. Gard. & For. 4: 466-467. 1891. A second report upon the native trees and shrubs of Nebraska. Ann. Rep. Nebr. State Hort.-Soc. 154-185. 1892. A preliminary description of the native and introduced grasses of Nebraska. Ann Rep. Nebr. State Board. Agr. 1892. The rules of botanical nomenclature. (Rochester Rules.) Amer. Nat. 26: 860-861. 1892. A second edition of Webber’s ‘‘ Appendix to the catalogue of the flora of Nebraska’’ with a supplementary list of recently reported species. Contr. Bot. Dept. Univ. Nebr. n. ser. 3. 1892. Transpiration, or the loss of water from plants. (With A. F. Woods.) Contr. Bot. Dept. Univ. Nebr. n. ser. 4. 1892. Evolution and classification. Proc. Amer. Assoc. Adv. Sci. 43. 1893. Fungous diseases of sugar beets. Rep. Nebr. Agr. Exp. Sta. 6. 1893. 1894. 1895. 1896. - 1897. 1898. 1899. 1900. LIFE AND WORK OF CHARLES EDWIN BESSEY 515 The Russian thistle in Nebraska. Nebr. Agr. Exp. Sta. Bull. 31: 67-77. 1893. The reforesting of the sandhills. Ann. Rep. Nebr. State Board Agr. 1894. Homologies of the Uredineae. Amer. Nat. 28: 989-996. 1894. The botany of the apple tree. Ann. Rep. Nebr. State Hort. Soc. 7-39. 1894. Third report on the native trees and shrubs of Nebraska. Ann. Rep. Nebr. State Board Agr. 1894. Some facts in vegetable physiology related to problems in irrigation. Ann. Rep. Nebr. State Board Agr. 1894. The structure of the wheat grain. Nebr. Agr. Exp. Sta. Bull. 32: 100-110. 1894. The structure and composition of bran. Nebr. Agr. Exp. Sta. Bull. 32: POEL. 1394. A protest against the ‘‘Rochester Rules.’’ Amer. Nat. 29: 666-668. 1895. Notes on the distribution of the yellow pine in Nebraska. Gard. & For. 8: 102-103. 1895. The botany of the grape. Rep. Nebr. State Hort. Soc. 7-26. 1895. The botany of the plums and cherries. Rep. Nebr. State Hort. Soc. 163- i7o. — LSos. : A preliminary list of the honey-producing plants of Nebraska. Nebr. Agr. Exp. Sta. Bull. 40: 141-152. 1895. Were the sandhills of Nebraska formerly covered with forests? Publ. Nebr. Nats Sel, 5:7. . 1896. | \(Abstract.) The origin of the flora of Nebraska. Publ. Nebr. Acad. Sci. 5: 33. 1896. (Abstract. ) The box-elder on the plains. Gard. & For. 9: 33. 1896. The condition of forests and forestry in Nebraska. Proc. Amer. For. Assoc. Ir: 83-89. 1896. Notes on the botany of the strawberry. Ann. Rep. Nebr. State Hort. Soc. 1896. Are the trees receding from the Nebraska plains? Gard. & For. 10: 456- 457. 1897. The forage problem in Nebraska. Ann. Rep. Nebr. State Board Agr. 1897. The systematic arrangement of the Protophyta. Amer. Nat. 31: 63. 1897. Erysiphe communis. Bull. Torrey Club 24: 421. 1897. The phylogeny and taxonomy of the Angiosperms. Bot. Gaz.°24: 145-178. 1897. The nomenclature of the Nebraska forest trees. Proc. Collect. Nebr. State Hist. Soc. II. 2. 1898. Some facts in plant physiology bearing upon horticultural practices. Ann. Rep. Nebr. State Hort. Soc. 1898. The forests and forest trees of Nebraska. Ann. Rep. Nebr. State Board Agr. 1899. The physiology of the apple tree. Ann. Rep. Nebr. State Hort. Soc. 1899. Some agricultural possibilities of western Nebraska. Ann. Rep. Nebr. State Board Agr. 1900. 516 IQOI. 1902. 1903. 1904. 1905. RAYMOND J. POOL Vegetation of the sandhills. Ann. Rep. Nebr. State Board Agr. 1Igoo. The natural spreading of timber areas. Forester 6: 240-243. 1900. The modern conception of the structure and classification of Diatoms, with a revision of the tribes and a rearrangement of the North American genera. Trans. Amer. Micr. Soc. 21: 61-86. 1900. One thousand miles for a fern. Asa Gray Bull. 8: 2-6. 1900. A preliminary account of the plants of Nebraska which are reputed to be poisonous, or are suspected to be so. Ann. Rep. Nebr. State Board Agr. I9OI. Old world contributions to western orchards. Proc. Soc. Prom. Agr. Sci. 22. {1901 Notes on the apple scab. Nebr. State Hort. Soc. Bull. 1. 1got. More about fungus spores as bee-bread. Pl. World 4: 96. Igor. The botanist’s journey to the Denver meeting of the A. A. A.S. Science, n. ser. 24: 185-187. I90OI. Early winter colors of plant formations on the great plains. Science, n. ser. 24: 721-724. I90I. The modern conception of the structure and classification of Desmids, with a revision of the tribes, and a rearrangement of the North American Genera. Trans. Amer. Micr. Soc. 22: 91-96. 1901. Baptisia tinctoria as a tumble-weed. Rhodora 3: 34-35. 1901. Home made wall charts. Journ. Appl. Micr. 4: 1195. Igot. The structure and classification of the Conjugatae, with a revision of the families and a rearrangement of the North American genera. ‘Trans. Amer. Micr. Soc. 23: 145-150. 1902. A word as to indexes. Science, n. ser. 16: 476-477. 1902. The morphology of the pine cone. Bot. Gaz. 33: 157-159. 1902. Tree growing in Nebraska. For. & Irr. 8: 453-456. 1902. List of Nebraska trees. Nebr. Park & For. Man. 1903. Evolution in microscopic plants. Trans. Amer. Micr. Soc. 24: 5-12. 1903. The structure and classification of the Phycomycetes, with a revision of the families and a rearrangement of the North American genera. ‘Trans. Amer. Micr. Soc. 24: 27-54. 1903. Preliminary paper on diseases of grapes in Nebraska. Ann. Rep. Nebr. State Hort. Soc. 1903. Distribution of forest trees on the Nebraska plains. Atl. Slope Nat. 1: 21. 1908. The classification of Protophyta. Trans. Amer. Micr. Soc. 25: 89-104. 1904. Notes on the agriculture of the Caucasus Mountains. Proc. Soc. Prom. Agr: Sci. 25.5 1904; The chimney-shaped stomata of Holacantha emoryi. Bull. Torrey Club 31: 523-527. 1904. The grasses of Nebraska. Ann. Rep. Nebr. State Board Agr. 1904. Some foreign botanical gardens and parks. Ann. Rep. Nebr. State Hort. Soc. 1904. Life in a seaside summer school. Pop. Sci. Monthly 80-89. 1905. s 1906. 1907. 1908. 1909. IQIO. IQII. 1913. IQI4. IQI5. LIFE AND WORK OF CHARLES EDWIN BESSEY ley How much plant pathology ought a teacher of botany to know? PI. World 8: 187-189. 1905. Plant migration studies. Univ. Nebr. Stud. 5: 11-37. 1905. The structure and classification of the lower green algae. Trans. Amer. Micr. Soc. 26: 121-136. 1905. The forest trees of eastern Menraska. Proc. Ia. Acad. Sci. 13: 75-87. 1906. The Carolina poplar. Ann. Rep. Nebr. State Board Agr. 1906-1907. The growing importance of plant physiology in agricultural education. Loc. 50c. Prom. Agr. o61..274) 1000, Twinned pistils in partridge pea. Amer. Bot. 17: 103. IQII. Crop improvement by hybridizing wild species. Ann. Rep. Nebr. State Hort. Soc. 117-123. 1906. City Trees. Ann. Rep. Nebr. State Hort. Soc. 153-157. 1906. Field work in botany in grammar and high schools. Nature study Review. BeeOqlon (L907. A synopsis of plant phyla. Univ. Nebr. Stud. 7: 275-373. 1907. The taxonomic aspect of the species question. Amer. Nat. 42: 218-224. 1908. (Also in Bot. Soc. Amer. Pub. 34: 218-224. 1908.) Physiology of pruning. Nebr. State Hort. Soc. Bull. 18. 1908. The phyletic idea in taxonomy. Science, n. ser. 29: 81-100. 1909. Outlines of plant phyla. Univ. Nebr. Dept. Bot. 1909, with two later editions with corrections and additions. Laying the foundations. Annals of Iowa 9: 26-44. 1909. The phyla, classes and orders of plants. Trans. Amer. Micr. Soc. 29: 85- 96: 1910. Some European forest notes. For. Quart. 8: 201-209. I9I10. Selected rules of botanical nomenclature. (Especially for the use of foresters.) Univ. Nebr. For. Club Ann. 2. I9I0. On the preparation of botanical teachers. Science, n. ser. 33: 633-639. IQII. Literature of North American Systematic botany. Univ. Nebr. For. Club Ann: 5. 1913. A preliminary paper on drought endurance. Ann. Rep. Nebr. State Hort. Soc. [or3. Tea growing in Transcaucasia. Pomona Coll. Journ. Econ. Bot. 3: 441-445. LOL. Some of the next steps in Botanical Science. Science, n. ser. 37: I-13. LOR" Synopsis of the conjugate Algae-Zygophyceae. Trans. Amer. Micr. Soc. 33: II-50. I914. Revisions of some plant phyla. Univ. Nebr. Stud. 14: 37-109. I914. The phylogenetic taxonomy of Flowering Plants. Ann. Mo. Bot. Gard. 2: 109-164. I9I15. Textbooks Botany for high schools and colleges. Henry Holt and Co. First edition 1880, second edition 1881; third edition 1883. 518 RAYMOND J. POOL The essentials of botany, briefer course. Henry Holt and Co. First edition 1884; second edition 1885; third edition 1886; fourth edition 1889; fifth edition 1892; . sixth edition Feb. 7, 1896; seventh edition July 23, 1896. Elementary botanical exercises for public schools and private study. Lincoln 1892. Elementary botanical exercises and an elementary manual of Nebraska plants (second edition of above). Lincoln 1894. New elementary agriculture. (With Bruner and Swezey.) Univ. Publ. Co. 1903. Elementary botany, including a manual of common genera of Nebraska plants. Univ. Publ; Go; Lincoln,*1904: Essentials of college botany. (With E. A. Bessey.) Henry Holt and Co. 1914. Ben EDITY AND MUTATION AS CELL PHENOMENA* R. RUGGLES GATES Heredity is frequently defined as the tendency of like to beget like; and the degree of resemblance between parent and offspring is often considered to be a measure of inheritance. The modern Men- delian work with dominant and recessive characters has, however, rendered such definitions incomplete and therefore untenable, since a plant or an animal may inherit in a predictable manner characters which its immediate ancestors did not exhibit at all. Two white races of sweet pea may on crossing give rise to a purple race, a re- version to an ancestral type which in the absence of definite knowledge might have been looked upon as an unexpected variation. But breeding experiments with the two white races will show that their germinal constitution is different. The interpretation then follows that the purple character is not a variation but is a result of inheritance. What has been inherited, however, is not a similarity but a difference. In similar fashion the French zoologist Cuénot found that in certain cases where a wild gray mouse is crossed with an albino the second generation of offspring contain not only the two original types but black as well, the frequency being 9 gray: 3 black: 4 albino. Breeding experiments with the albino parent disclosed the fact that some of the germ cells of the albino carried the potentiality of pro- ducing black under certain conditions, 7. e., when meeting a germ cell containing the capacity or factor for developing color. Again we are dealing with phenomena of inheritance not of similarities but of differ- ences. In the same way, in all sexually reproduced organisms it is not the similarities but the differences between the ancestors, or between the offspring, that we remark upon as being inherited. We can only speak of a boy inheriting his father’s shape of nose or his mother’s color of eyes when his parents differ in these attributes. It has therefore become necessary to reverse in a sense the usual point of view with regard to heredity. Since it is inaccurate to say that heredity is measured by the degree of resemblance between * Presented by invitation at the Genetics Conference, San Francisco, August 4, IQI5. RI9g 520 R. RUGGLES GATES parent and offspring, we may state that heredity consists in the perpetuation of the differences between related organisms. Inheritance is then the process by which these differences are perpetuated from generation to generation. This manner of statement is particularly useful when we contrast heredity and variation from the evolutionary point of view. For we may then define variation as the process by which new differences arise, and inheritance as the process by which they are perpetuated. These definitions have much more biological usefulness than may at first appear. They enable us to compare the phenomena of heredity and variation from a different point of view. Though the above definition of variation applies to all kinds of variations, yet it has in view particularly mutations, which are com- pletely inherited. We may classify variations as regards their herit- ability, into three classes: (1) those which are completely inherited, (2) those which are non-inherited, (3) those which are partially inherited. These three classes of variations must then have very different evolutionary significance. It is obvious that completely inherited variations, or mutations, are immediately effective for evolution or at least for species-formation, though it does not neces- sarily follow that they are of greatest evolutionary significance. It is not necessary to enter into this question here, nor discuss the relative importance of classes (1) and (3),—matters which are still to a large extent in dispute. We may, however, point out that bathmic varia- tions, such as the rectigradations of Osborn, may come in a different category still as regards their relation to evolutionary processes. The relative evolutionary significance of mutations and continuous or partially-inherited variations can be determined in part by com- parison of specific differences in particular groups with the mutations which occur in those groups. Although a mere beginning has as yet been made in this direction, yet it would appear that mutations have probably played adarger part in specific differentiation in some groups than in others. But it will be a long time before these questions can be definitely decided. We may, however, affirm without doubt that in many groups mutations have played an important and even a pre- ponderant part in species formation. Therefore whatever place may ultimately be assigned to mutation in the hierarchy of evolutionary factors in relation to the paleontological history of organisms, it has undoubtedly played an important réle in the process of speciation. HEREDITY AND MUTATION AS CELL PHENOMENA 521 Considering now certain features of mutations as we know them, one of the most interesting is their variety. Many attempts have been made to explain all mutations in terms of one idea. These universal explanations have involved (1) redistribution of Mendelian char- acters, (2) loss of factors, (3) reduplication of gametes, and many other hypotheses. They have nearly all, however, involved the idea of the mere loss of qualities or the recombination of those already existing. Such views have been brilliantly advocated, particularly by Mendelian writers. But they consider the phenomena of muta- tion largely from the outside. The cytological and anatomical combined with the experimental investigation of particular mutations already reveals that mutations belong in various categories and are the result of different types of change.! Germinal changes are not due merely to plus or minus variations, in the loss or addition of Mendelian factors; but on the other hand each change results from a morphological or physiological alteration affecting one element of the germ plasm. This being the case, there is plenty of material at hand for progressive and divergent evolution, and it is unnecessary to imagine that the endless variety of organic structure has resulted from successive germinal simplifications by means of loss. In a brief analysis of the various known types of germinal change, they may be classified into (1) those which are fundamentally morpho- logical and (2) those which are primarily chemical. It may be ex- pected in general that these two classes of changes will frequently be inherited in different ways. This brings us to the question of the relation between heredity and particular variations in another aspect. We have already observed that variation has to do with the origin of differences between organisms, and heredity with the perpetuation of those differences. It may here be pointed out that in the inheritance of any character-difference such as we are considering, there are two features to be taken into account; (1) the nature of the character it- self, and (2) the mechanism of its inheritance. It is true that in Mendelian inheritance the mechanism by which the germinal deter- miners of the various character-differences are distributed in the germ cells during meiosis is the chief feature to be considered. But since it is now clear that in certain cases these characters are diluted and 1 For a discussion of this subject see Gates, The Mutation Factor in Evolution, Chap. IX. MacMillans: London. 1915. 522 R. RUGGLES GATES otherwise modified by crossing, the nature of the character and the possibility of its modification through hybridization can never be wholly neglected. In certain cases this feature of change in the character becomes of great importance, overshadowing the mechanism by which the fundament of the character is transmitted. I am, of course, speaking here of permanent modifications in a character, and not merely of the temporary effects which may be produced in a heterozygous organism through the presence of other factors. When we study the nature of mutations which are fundamentally morphological it first becomes evident that each mutation is essentially a cell change, transmitted as such to every cell of the organism through mitosis. Before considering this aspect of mutation let us classify some of the known types of morphological mutations. For this purpose we will consider chiefly the genus Oenothera where these types of change have been most fully studied. It seems clear that all or nearly all the changes to which I refer have originally taken place in the nucleus of the cell. Referring first to chromosome changes, in the genus Oenothera the original number of chromosomesis 14. This is true of Oe. Lamarckiana and many other species. Duplication of one of these chromosomes through an irregular meiotic division has led to 15 in Oe. lata, a charac- teristic mutation which has occurred both in Oe. Lamarckiana and in certain races of Oe. biennis. The same chromosome number occurs in semilata and in a very different form from Sweden which I have called znmcurvata.2. DeVries’ has recently described still another form having 15 chromosomes. It was derived from Oe. biennis semigigas pollinated in part from Oe. biennis, and has flat leaves, whitish foliage, white veins, longer spikes, slender buds, small, erect flowers, thin cylindrical fruits and few seeds. Hence we may say that whenever a germ cell having 8 chromosomes fertilizes a normal germ cell a new form is produced, though what its characters will be depends upon various circumstances which need not be considered here. One of the most important of these factors 1s probably the particular combination of chromosomes received. It is perhaps not inappropriate to speak of all these mutants as belonging to the /ata series, or the series with an extra chromosome. Oe. mut. gigas is the prototype of another series of still more 4Op. Cit.,; p: 147. 8’ DeVries, Hugo. 1915. The coefficient of mutation in Oenothera biennis L. Bot. Gaz. 59: 169-196. HEREDITY AND MUTATION AS CELL PHENOMENA 523 closely parallel mutations in which the chromosome series is doubled— 28—the plant being a cell giant and not merely gigantic in its external dimensions. This mutation has not only occurred independently in two different strains of Oe. Lamarckiana, but a somewhat different giant having the same chromosome number occurred in Heribert- Nilsson’s cultures of the Swedish Lamarckiana. Recently Bartlett* has found a tetraploid giant in his experiments with Oe. stenomeres which is an exact parallel to the previous cases in every particular. A third series of morphological mutants is the semugigas series, having 21 chromosomes. This type of mutation has now been found in several species, including Oe. Lamarckiana, Oe. biennts, and hybrids of Oe. Lamarckiana or its derivatives with such species as Oe. cruciata, Oe. muricata and Oe. Millerst. Triploidy is thus much more frequent in its occurrence than tetraploidy, though the latter condition was discovered first. Derivative from the above three types of chromosome change are various others. Thus a mutant from Sweden which I have called latescens® probably has 16 chromosomes, and by crossing and other- wise several additional numbers have been obtained. The truth is obvious, not only that parallel mutations occur inde- pendently in different species, but that the type of change which gives rise to the extra-chromosome series is entirely different from that which produces the tetraploid series. I wish to point out that the nature of these changes is probably limited, and in this sense determined, by the structure of the germ plasm. In one group of organisms the mutations are of one kind, in another group they are wholly different in nature. This must be because the germ plasm in each group has its own particular lines of cleavage. Tetraploidy is a phenomenon which has occurred in a great variety of organisms, because the nuclear structure of almost any organism allows of its occurrence. Chromosome duplication, as in the /afa series, is appar- ently much less common, but it may be expected to occur wherever the pairing of chromosomes in meiosis is weak and therefore liable to irregularities. Mutations then, in a sense, indicate where lines of weakness exist in the germ plasm, and it is these lines of weakness which define the particular directions which the mutations will take 4Bartlett, H. H. 1915. The mutations of Oenothera stenomeres. Amer. Journ. Bot. 2: 100-109, figs. 4. AO OClie pall 7. 524 R. RUGGLES GATES in any genus. Some of these “‘cleavage lines”’ are structural, some chemical in nature. Another important feature of mutations which has not hitherto been emphasized is the fact that each is the result of a cell change which is represented in every part of the organism. This change originally occurred in the nucleus of a single cell, and the mitotic mechanism is responsible for handing it down to every part of the organism. The cells of Oe. lata constantly have 15 chromosomes, in whatever part of the plant they have been examined. Similarly in Oe. gigas even the most specialized tissues retain the double number of chromosomes transmitted to them,* though in the tapetal cells of all the forms secondary changes may take place, through fusion of nuclei and similar causes. The conclusion follows that, with non-significant exceptions, every cell receives the number of chromosomes transmitted to it from the original fertilized egg. The blunt-pointed, deeply crinkled leaves, short stature, irregular branching, nearly sterile pollen, rounded buds and other features of Oe. lata are then an external expression of the fact that an extra chromosome is present in every cell. The real mutation was a cell change and is transmitted by mitosis as a cell change. Although we have at present practically no knowledge of the relation between cell structure and external form in organisms, yet we can at least affirm that the original change from a 14- toa particular 15-chromosome complex has resulted in the various external differ- ences which we observe between Jata and Lamarckiana. The organism is different because its every cell is different, and if in any part the extra chromosome should be dropped out into the cytoplasm through a slip in mitosis we should expect in that part a reversion to the foliage and other characters of Lamarckiana. Viewed in this way, it is clear that we must consider the peculiar- ities of /ata a result and not merely an accompaniment of the presence of the extra chromosome. We must, moreover, visualize the change as a cell change and the special features of /ata as its external expression. The same point of view applies probably to all other mutants. This makes comprehensible the fact that in many cases the mutants differ from the parent as strikingly in the early seedling stages as in the mature plant. The ontogeny of the new form does not witness a 6 These and similar facts point to the conclusion that the chromosome divisions during ontogeny are not differential in nature, as Weismann supposed, but equational. HEREDITY AND MUTATION AS CELL PHENOMENA 525 gradual drawing apart from its parent, except in so far as different organs may be unfolded in the later development. Already in the fertilized egg the difference is present, and makes itself felt as much in the first leaves asin the last. It is therefore important to remember that a mutant is such because not only its germ cells but every one of its somatic cells contains a certain peculiarity. This is probably as true of animals as it is of plants, though an important difference is introduced here by the fact that in animals the germ cells are very early set apart from the somatic cells, while in higher plants this only happens with actual flower production. If we compare the dimorphic condition of the cells in the males and females of certain insects with the difference between the cells of Oe. lata and Lamarckiana, it is clear that these differences correspond in certain features. The females of Anasa tristis have two members of a certain pair of chromosomes where the males have only one. Though the germ cells are set apart very early in the ontogeny in insects, yet it is probable that these chromosome differences occur not only in the young embryos, where they have been actually ob- served, but throughout the somatic tissues. A female animal, like a mutant, is somatically distinguished by having a different chromosome content in all its tissues, and not merely by the possession of female sex glands or secondary sexual characters. So far as I am aware, the fundamental significance of these facts in their bearing on heredity, embryology and variation, to say nothing of the structure of the cell, has not hitherto received attention, except in the case of cell giants. The fact that mutants differ from the parent in their every cell carries with it many important implications. Giant mutations are now known in various organisms in the absence of tetraploidy. In such cases, although the cells are gigantic the chromosome number is unchanged. There is at present no clear indi- cation of the fundamental nature of this type of cell gigantism. One of the best known instances is that of the giant variety White Queen Star,.of Primula sinensis, described by Keeble.’ This appeared in the normal variety and breeds true, but crossing experiments indicate that it may result from the presence of three independent factors. Various tetraploid giants of Primula have also been obtained. No doubt there are many other types of chromosome change 7 Keeble, F. 1912. Gigantism in Primula sinensis. Journ. Genetics 2: 163- 188, pl. IT. ; 526 R. RUGGLES GATES besides those I have mentioned. These for the most part await dis- covery or investigation, although the cytological literature abounds with cases which are probably of this nature. One such instance, recently described, may be referred to here. In one of the grass- hoppers, Tettigidea parvipennis, Robertson*® found that certain indi- viduals possess an abnormally long chromosome mated with a short one, while in other individuals the corresponding pair are of equal length. The explanation appears to be that a portion from the end of one chromosome became attached endwise to its mate. A study of the variability of these grasshoppers should be made, to discover whether any corresponding somatic change can be detected in those that have the long chromosome. It is suggested that negative muta- tions might be due to the loss of a portion from the end of a chromo- some in this way. In the remarkable phenomena of mutation in Drosophila studied by Morgan and his pupils, the new forms appear to result from changes in the nature of certain portions of particular chromosomes. The new characters are grouped in four series according to their hereditary behavior, corresponding to the four pairs of chromosomes, and in giving rise to each mutation a particular part of one chromosome may be assumed to have undergone a change.® This change is probably chemical in nature, at least in the series giving rise to the color varieties. The ‘crossing-over’? phenomena, which have been studied in such detail by Morgan and his collaborators, are accounted for on the theory of the chiasma type of chromosome reduction, in which the chromosome pairs become looped around each other and so exchange segments of their substance. This process has not, I believe, been observed actually to take place in Drosophila.’ In preparations of Drosophila chromosomes exhibited by Mr. Bridges, the somatic chromosomes appear to be remarkably closely paired and twisted about each other during the prophases of mitosis. Waai.® Robertson, W. R. B. 1915. Chromosome studies III. Inequalities and ateciercinst in homologous chromosomes: their bearing upon synapsis and the loss of unit characters. Journ. Morph. 26: 109-141, pls. 3. ° The recent observations of Chambers (Some physical properties of the cell nucleus, Science, n. ser., 40: 824-827. 1914) on the living spermatocytes of a grass- hopper lend direct support to the view that the chromosomes are composed of discrete and more or less independent particles. He found that stimulation of the cell in- duces chromosome formation in the resting nucleus, and that the chromosomes are formed by the aggregation of definite granules in bunches about a hyaline core. HEREDITY AND MUTATION AS CELL PHENOMENA S27, The suggestion therefore occurs to one that it may be in the premeiotic rather than the meiotic divisions of the germ cells that the ‘‘crossing- over’’ of material from one chromosome to its mate occurs. Person- ally I have never seen such an intimate relation between the members of chromosome pairs as is to be observed in the somatic cells of Dro- sophila. The absence of crossing-over in the male is, however, a difficulty with any hypothesis yet proposed. Returning now to the subject of Oenothera, there is at least one mutation which is fundamentally chemical in nature. In the origin of Oe. rubricalyx we see exhibited the type of change which must occur whenever a new monohybrid Mendelian character appears. In all such cases it is only necessary to assume that one chromosome, or a portion of one, underwent a change in its chemical nature. The meiotic mechanism performs the function of distributing this chromo- some and its descendants so that a Mendelian 3 : 1 ratio in the off- spring will result.!° There have been many suggestions as to why the new character is dominant in one case and recessive in another. Cases like that of Oe. rubricalyx, in which the new character is dominant, are rare. It is possible that dominance may occur whenever the change results in the increased production of a substance, and recessiveness whenever there is loss of or reduction in the capacity for producing such a substance in the cell. This would apply to both dominant and re- cessive whites. Thus in the case of dominant White Leghorn fowls a substance is present in quantity which inhibits color production, while in recessive white flowers it is clear that the capacity for pro- ducing color has in one way or another, been almost completely lost or suppressed. One may suppose that this color-inhibiting sub- stance is one of the substances produced by the cells in all Leghorn fowls, but that in the White Leghorn it has been very largely increased through a chemical modification on the part of a chromosome. In the present state of our knowledge it is impossible to determine the precise chemical nature of the change which produced Oe. rubri- calyx from Oe. rubrinervis. The difference consists in an enormously increased capacity for anthocyanin formation in every cell of the 10 For an explanation of later 15:1 ratios, see Gates, 1915, On successive dupli- cate mutations. Biol. Bull. 29: 204-220. 11 Of course this suppression may occur in various ways. Thus it has been suggested (Robertson, J. B. 1914. Bloodstock Breeders’ Rev. Nos. 1, pp. 16-31; 2, pp. 91-107; Reviewed in Exp. Sta. Record 32: 361. 1915) that in the gray horse, 528 R. RUGGLES GATES organism, but especially in the buds. When the chemistry of the nucleo-proteins is better known, it may be possible to determine what chemical change one of them would have to undergo in order to in- crease the amount of anthocyanin produced by interaction with the cytoplasm of the cell, but these matters are as yet too complex for analysis, though much is being learned concerning the chemistry of anthocyanin and the physiology of its production in the cell. Like the morphological mutations to which reference has already been made, there can be little doubt that Oe. rubricalyx is also a cell mutation, the nuclei in all parts of the organism containing a de- scendent of the original changed chromosome. Parallels to this mutation are found in such plants as the copper beech and the red sunflower, which belong to widely separated groups. | In conclusion, our complete lack of knowledge of the relation between internal cell structure and external form in organisms may be pointed out. Except in the relatively simple case of gigantism through tetraploidy it is quite unknown how a change in the nucleus of the cell-unit results in the external modification of characters. Why are the buds of Oe. rubricalyx more conspicuously red than any other part, and why are the leaves of Oe. lata blunt, the buds rounded, the pollen sterile, etc.?2 Before an answer to such questions can be attempted, something must be learned of the way in which the meta- bolism of the cell—-a complex series of chemical activities—-expresses itself in the form of structure involving relationship between differ- entiated cells and tissues. which is a recessive, the grayness results from a structural modification in the canals which connect the pigment-producing cells with the hair follicles, rendering them too narrow for the passage of the pigment granules, Sie RELATION BEAWEHEN VEGETATIVE VIGOR AND REPRODUCTION: IN’ SOME SAPROLEGNIACEAE! ADRIAN J. PIETERS HISTORICAL INTRODUCTION Advances in science have been made by workers arriving at new points of view, as witness the work of Mendel, Darwin, deVries and others. So also the writings of Klebs during the last twenty-five years have served to open up a field, the cultivation of which is already exercising a profound influence on biological thought. Klebs was not the first to suggest that the forms, to the development of which a plant might at any one time devote its energies, were conditioned by ex- ternal factors acting upon the plant; but it will be neither necessary not desirable to review the older literature. This has been adequately done by Klebs in his various papers. (See especially ’0o0, ’03, 04.) The idea underlying all of Klebs’s work is that every species has a sum total of potentialities constituting the specific nature of that species, but that the chemical and physical conditions prevailing at any one time within the organism determine which of the particular forms that the species is capable of producing shall appear, and that these inner conditions may be controlled, to an extent at least, by regulating the external conditions. In other words the changes in form which we witness in the course of the development of an individual are the results of chemical and physical interactions. Since 1890, when Klebs (’90) showed that the formation and discharge of zoospores in Hydrodictyon could be encouraged by providing certain conditions, he has established the truth of this principle in the case of other algae (96), of fungi (’908, ’99, ’00), and also for flowering plants (’03, ’04). Other workers have built upon 1 Contribution 150 from the Botanical Department of the University of Michi- gan. This work was begun at Heidelberg, Germany, under the direction of Prof. Dr. Georg Klebs, and was continued at the University of Michigan under the direc- tion of Dr. C. H. Kauffman. To both of these inspiring instructors the writer wishes to return his thanks and to them he desires to express his appreciation of the mental stimulus he owes to their discussions and suggestions. 529 530 ADRIAN J. PIETERS the foundations laid by Klebs and the present writer believes that the proposition that the succession of forms in the development of any plant is conditioned by environment, needs no further support than that furnished by the work of Klebs, Véchting (00), Kauffman (’08), Freund (’08), Obel (’10), and Raciborski (96). It has seemed worth while, however, to answer a question raised in the writer’s mind by a study of Klebs’s paper on Saprolegnia mixta. In this paper Klebs describes the production of sporangia and oogonia on a mycelium transferred from one solution to another, and states that such a mycelium, to react properly, must have been ‘well nour- ished.’’ He does not define more exactly what he means by this term. Obel (10) also emphasizes the need of a vigorous mycelium and uses a solution of peptone, sucrose and salts instead of the pea extract favored by Klebs. Obel agrees with Freund (08), who states that, in algae, the conditions favoring the production of reproductive bodies depend upon the preceding conditions of growth and Obel states that this conclusion is confirmed by an examination of the Saprolegniaceae (10, p. 427). Freund does not, however, give the results of any experiments to show the importance of the vigor of growth in the algae, and apparently makes the statement as a general one, meaning that the alga must be in good health and not in poor condition. Neither Obel, Klebs nor Kauffman gives any record of experiments on this point. In his paper on S. mixta (99) Klebs repeatedly refers to the necessity for a well nourished mycelium and states (p. 585) that during vegetative growth a part of the nutritive substance is stored, to be used later when reproduction shall have commenced. Further on the same page the statement is made that the production of oogonia requires a nutritive plasma (Nahrplasma) of a somewhat different composition from that needed for the production of sporangia. Klebs fully recognized that the chemical nature of the solution to which a mycelium is transferred, is important. He found that haemoglobin and leucin were the substances most favorable to the production of oogonia and that the addition of certain phosphate salts increased both the number of oogonia and of antheridia. Kauffman (08), in his work with S. hypogyna and with two cultures of S. mixta, did not find any marked increase in the number of oogonia due to the addition of salts, and the number of antheridia in the case of S. mixta was as marked in the solutions containing magnesium sulphate, as in those containing phosphates (1. c., p. 368). However, neither RELATION BETWEEN VEGETATIVE VIGOR AND REPRODUCTION 531 studied the effect of various foods consumed by the mycelium before transfer. THE PROBLEM The question was raised therefore, ‘‘What is a ‘well nourished’ mycelium?” and naturally the question of what constitutes a good nutritive solution followed. This work was commenced at Heidel- berg, under the direction of Prof. Dr. Georg Klebs, who agreed with the writer that a culture medium, in which the fungus can attain the greatest dry weight in a given time under favorable conditions, is generally to be considered the best nutritive medium. The question which the writer set himself to answer therefore, is the following: Will a nutritive medium in which a species of water mold produces the greatest amount of dry matter in a given time also give to that myce- lium when transferred to a suitable medium, the power to form more reproductive organs, than are produced by a mycelium grown in a poorer medium, measured by dry weight of mycelium produced? Or, in other words, is there a direct and necessary relation between vegetative growth and reproductive capacity? Generally in cultures of other fungi the reproductive bodies, when formed under these conditions, are produced while the mycelium remains in or on the culture medium in which its vegetative growth has taken place; not so with the Saprolegnias. If satisfactory results in the production of sporangia or of oogonia are to be obtained, these plants must be transferred from the solution in which they have been grown, to another and weaker solution. When the plant is trans- ferred to the second solution, it finds itself in a new environment. This may differ from the old merely in concentration and in the absence of the poisonous products of metabolism, which have accumu- lated in the old solution, or it may differ from the latter in chemical composition. As Klebs has pointed out (’99), whatever the chemical nature of the second solution may be, it must, if oogonia are to be formed, be of such character and concentration, that a slight growth can take place. If the solution is too poor in food, sporangia will appear and perhaps exhaust the mycelium, while if the food is too good vegetative growth will prevent reproduction. If the protoplasm produced by the consumption of all kinds of foods is alike we may expect that the nature of the changes observed after transfer will depend wholly on the new environment; but if the food used during vegetative growth can affect the character of the 532 ADRIAN J. PIETERS protoplasm, as well as the weight of the product, such effects should become evident when mycelia, grown in different culture media, are transferred to solutions of identical composition and concentration. The new and uniform environment acting upon the protoplasmic product of varying environments may be expected to bring out differences if any exist. Data will be presented in this paper to show that, in some cases at least, such an effect, not to be measured in terms of dry weight produced, has resulted from the use of special culture media and that there is no direct and necessary relation between weight of mycelium and the production of oogonia. It was realized at the outset that there were two aspects of the problem—the quantitative and the qualitative. Either a nutritive substance of one kind might affect the organism because of its greater abundance, or substances of various kinds might affect especially the reproductive capacity or the vegetative growth. Solutions were therefore prepared with these two aspects in view, different concentrations of peptone being used on the one hand and. various carbohydrates and salts on the other. The number of oogonia produced on the mycelia after transfer, together with the weight of mycelium in the duplicate lots left in the nutrient solutions, make up the evidence on which the conclusions reached are based; this evidence can best be presented in a series of tables and as tables are rather uninteresting reading, the writer has pointed out briefly after each table the conclusions that seem warranted by the evidence, leaving a general discussion of these conclusions to be given at the end of the paper. The chief contributions which it is believed are made by the present work to our knowledge of the physiology of these forms are the recognition of a minimum concentration of food necessary for a full development of the plant, and the fact that above this mini- mum an increased concentration during vegetative growth does not necessarily increase the reproductive capacity of the fungus. It developed further, as will be shown in the following pages, that certain carbohydrates are readily used while others can not be utilized, and that of those that are used some are of more value to the plant for the formation of reproductive organs than others. MATERIAL The material used for this study consisted of four species of Sapro- legniaceae, selected from among a larger number isolated and studied. RELATION BETWEEN VEGETATIVE VIGOR AND REPRODUCTION 533 Two of these belonged to the genus Saprolegnia. S. ferax (Gruith) Thuret was collected at Heidelberg in the fall of 1911; S. monoica Pringsh. was secured from alga material out of pools near Ann Arbor. Achlya racemosa Hildebr. was collected at Heidelberg and was unfortunately lost in a fire which destroyed a large part of the botanical laboratories at Ann Arbor in the spring of 1913, at which time also a large number of notes and other records were lost. The records of this species are therefore incomplete. Achlya prolifera De Bary was obtained from alga material out of pools near Ann Arbor. Physiological studies on several other species, together with descriptions of some new species and remarks in regard to certain points of relationships, will be discussed in other papers. METHODS For isolating the forms Kaufiman’s (’08) method of single spore cultures was strictly followed. While the writer is not disposed to say that men like Maruzio (’94) and Obel (1. c.) did not work with pure cultures it seems more than likely that the results claimed to have been gotten by Lechmere (’I1) may be due, in part at least, to mixed cultures. Moreover, a writer with so little appreciation of the worthlessness of sporangia and gemmae as specific characters,? can hardly be taken seriously when he states that the sporangia charac- teristic of six different genera were found in one supposedly pure culture. While Lechmere says that he used Kauffman’s method he has apparently quite overlooked the fact that the essential part of this method is the isolation of one spore; it does not appear that he attempted to do this in his work. All the cultures of each species made in connection with the present problem were descended from an original single spore and if there was the slightest reason to doubt the purity of a culture, a single spore culture was again made and carried through on a fly, so that the specific characters might be compared with the original. All the species that were not being used for experimental work were kept alive on flies or in pea extract. In either case cultures can be set aside for two or three months without any danger of loss. 2See |. c., p. 176, where Lechmere concluded that two species of Saprolegnia were identical because of the similarity of habit, gemmae, and shape and peculi- arities of the sporangia, although the one produced oogonia readily and the other did not produce them at all. 534 ADRIAN J. PIETERS For the experiments cultures were made in Erlenmeyer flasks of Jena or of resistance glass except in the few cases that will be noted, the flasks containing 100 or 200 cc. of culture fluid. This medium was inoculated by placing in it small bits of pea agar in which the fungus was actively growing. Pea agar was used instead of the beef gelatin used by Kauffman as it was found more convenient, both to make and to handle. It is easily made by putting about 100 peas in a liter of water together with sufficient agar to make the strength desired. This is heated in an autoclave for an hour at about I5 to 20 pounds pressure and, after filtering, is ready for use. It has been found a good culture medium, and has a further advantage over gelatin in not becoming soft in warm weather and in not being lique- fied by the action of the fungus. When a set of cultures was to be inoculated the bits of agar were cut as nearly of the same size as possible, about I square millimeter in size. After having grown for the necessary length of time the entire mass of mycelium was placed in a dish of sterile distilled water and allowed to stand for half or three quarters of an hour. It was then cut into pieces of as nearly the same size as possible and usually of about one square centimeter in area. These pieces were transferred to fresh sterile distilled water and were sometimes washed a third time before being placed in the solu- tions in which sporangia or oogonia were to be made. For the latter solutions glass capsules containing 25 cc. of solution were used. All of these cultures were in duplicate, and sometimes in quadruplicate. Condition of the myceltum.—-For studying the effect of food previ- ously consumed, on a mycelium after transfer to haemoglobin, or to some other suitable medium for the production of oogonia, or to water for the production of sporangia, it is important that the mycelium be actively growing, and that, in all the experiments, it shall be as nearly as possible in the same physiological condition. If a flask con- taining 100 cc. of pea extract be inoculated, the resulting growth will reach the surface after four or more days, depending upon the species. When the surface of the liquid is reached, a mat is formed, which, after a few days, may become quite thick and firm. At this stage the mycelium is in a very different condition from that in which it is just before reaching the surface. In the latter case the hyphae are all actively growing, the tips being, of course, the most active; when a mat has formed, and especially if the mat has been allowed to become thick, the lower parts of the mycelium are dead or dying while the RELATION BETWEEN VEGETATIVE VIGOR AND REPRODUCTION 535 parts in the mat have taken on something of a resting condition. If such a mat is cut up and the pieces placed in another solution, new growth must take place in that solution before sporangia or oogonia can be formed, but if the mycelium is used earlier the hyphae not only grow but respond at once to the stimulus of the new environment by the production of appropriate organs. It was found that the most advantageous stage at which to use a mycelium was just as the mat started to form, for at that stage masses of actively growing hyphae, all in uniform condition, can be readily secured. Temperature.—Temperature is a factor of the utmost importance. Klebs states that a temperature of about 18 degrees Centigrade is the optimum for S. mixta, but this is certainly not true for S. ferax. It has not been possible to determine the optimum for each of the various species studied, but enough was learned to warrant the state- ment that the temperature requirements of the different species differ. In Achlya racemosa a high temperature favors the production of oogonia and inhibits the formation of sporangia, while the tempera- ture at which oogonia are produced with certainty by S. ferax is in the neighborhood of 15 degrees. S. monoica seems to do about as well at 18 degrees as at 15; but at 20 degrees or over there is a distinct decrease in the number of oogonia. Achlya prolifera seems to prefer a temperature of 18 to 20 degrees; at lower temperatures as well as at higher, 22 to 24 degrees, the production of oogonia is uncertain. Unfortunately it was not possible to determine the optimum tempera- tures from lack of apparatus by means of which constant low tempera- tures could be maintained. For the purposes of the present work, however, it was sufficient to maintain the temperatures near the optimum for each species. It is interesting in this connection to note that Peterson (’10) in his study of the Danish freshwater Phyco- mycetes states that A. racemosa produces oogonia with difficulty, and only at low temperatures. In the writer’s experience A. racemosa produces oogonia very readily and particularly so at relatively high temperatures. Method of expressing the number of oogonia.—Klebs, in comparing the value of different substances for the production of oogonia ex- pressed the number of oogonia by the Roman figures I, II, and III, the relative number of oogonia present being in that order; Kauffman and Obel merely stated that few or many oogonia were present, while Horn (04) followed the precedent set by Klebs. Such a method 536 ADRIAN J. PIETERS does very well when one wants only to say that a food is poor or very good, but obviously if the number of oogonia formed is to be used as a measure of the value of a food, a more exact method of determining that number is necessary. In the present work the method of Klebs was used to some extent, especially in the earlier part of the work, but it soon became evident that this was not sufficiently accurate. When the value of various foods for vegetative growth is to be deter- mined, the mycelium is dried and weighed at the conclusion of the experiment, but it is not practicable to weigh the oogonia produced; these were therefore counted. For this purpose the mycelium was removed from the dish in which it was growing and laid on the inside of a Petri dish cover. Here it was carefully spread out by means of a jet of water from a pipette till it lay as a circular flat mass, the older, and thicker, portion at one end or near the middle and the younger portions forming a fringe around this. ee ee) a ee ee Fic. 1. Diagram showing the arrangement of the spaces in which the oogonia were counted. The bottom of the Petri dish was then inverted and laid on the mycelium, so that the whole lay flat and firm between the two halves of the Petri dish. A square ruled micrometer had been fitted to the RELATION BETWEEN VEGETATIVE VIGOR AND REPRODUCTION 537 eyepiece of a Leitz compound erecting body and the number of oogonia in a square, having an area of 25 square millimeters, was determined under a Leitz number two objective. The oogonia are, of course, not unifornly distributed over the mycelium, and in order to find the average number, the oogonia in ten squares were counted, beginning at one edge and following a line across the mass. As a rule the oogonia are more numerous a little back from the edge than in the middle, but:this is not always the case. Slo biod g £5 tao Lists | | Clo lol LU d dG Pa Cf oh am 26m Fic. 2. Diagram showing the arrangement of the oogonia in one of the spaces. Magnified about 100 times. The diagrams, text figures I and 2, show the method of counting and Table I gives some counts showing the distribution of the oogonia on the mycelium. This matter will be referred to again. ‘TABLE I Illustrating Method of Counting Oogonia. Each Number Represents the Oogonia in One Square of 25 Square Millimeters. Oogonia out of Haemoglobin 0.075 Percent or Haemoglobin 0.05 Percent From Set III Peptone and levulose........... AO; 85; 63, 52, 81, 87,1144, 115, 82, eve. 78.6 IBROSSIGONELE « cans cud See ee 14, 41, 26, 145, 92, 76, 76, 86, 66, 49— 67.1 Miuplicate........ Ly ee 44, 80, 88, 72, 79, 85, 70, 76, 76,85— “ 75.8 BOMIBDCA cc 7.c50 Ss. Cas Mea dara cee SelOw Wael 25a76.0 70; 100, OF; 72, 37-— Aaa PISS COMME tlc eo tee Ae 8,0NS,) Is, 22,70) 122, 07,37; 27) a OENDUIC AUC 6 lise Fas wea oe eeeans 0; G52 60, 780)1575 755 00, 23, 13, 10 49.3 538 ADRIAN J. PIETERS From Set VIII Peptone and levulose......... 42, 84, 97, 87, 120, 72, 135, 150, 209, 191—Ave. 118.7 The advantage of this method lies in the fact that the mycelium studied is practically all in one plane of focus, only a slight turn of the focusing screw being required to enable the observer to count every oogonium. That such counting of oogonia was extremely tedious and time consuming is quite obvious; consequently the quicker method of Klebs was also used. In the following tables wherever the Roman numerals are used they should be interpreted as follows: I. A fair number, about a dozen in the field of a Leitz number two objective. Ii. Many, at least four or five times as many as I. III. Very many, at least twice as many as II. To express intermediate gradations, the following have been used: o-I, a few in each field; I-II and II-III to indicate numbers not properly expressible by either figure alone. Transferring the mycelium.—In each set of experiments the my- celium was removed from some flasks five to six days after inoculation, washed, cut up and transferred to haemoglobin for the production of oogonia, or to some other solution as will be indicated in the proper place. The mycelium in the duplicate flasks was allowed to grow for some time longer and was then washed, dried at 100 degrees and weighed. The length of time the fungus was allowed to grow before being weighed was, of course, precisely the same for every lot of a series; with the exception of series I this time was thirty days. The capsules containing the haemoglobin or other solution were always in duplicate, and sometimes in quadruplicate, and were kept at a temper- ature as nearly uniform as possible. This varied roughly between 13 and 20 degrees, the extremes being only occasionally reached; usually the temperature was between 15 and 18 degrees. The first experiments were made at Heidelberg and the mycelium was not weighed, nor were the oogonia counted. The later experiments were made at Ann Arbor. Klebs showed that the most important condition for the production of oogonia by S. mixta was a gradual decrease in the food supply and that for the formation of sporangia a sudden, or at least a rather rapid decrease in the concentration of the food, was necessary. ‘To secure oogonia, then, the mycelium must be placed in such an environment RELATION BETWEEN VEGETATIVE VIGOR AND REPRODUCTION 539 that, while no rapid growth takes place, the formation of sporangia shall be almc.<+ or entirely prevented. It is evident that a mycelium, exhausted by the formation of sporangia, cannot produce the number of oogonia that it could have produced under other conditions. Hae- moglobin, in concentrations to suit, offers such a medium, but un- fortunately the commercial article is not of uniform purity, always leaving more or less of an insoluble precipitate. Klebs (’99, p. 520) states that in 0.05 percent haemoglobin only vegetative growth took place and that even in 0.01 percent sporangia were but sparingly produced. The forms of S. mixta collected by the writer formed sporangia readily in 0.05 percent haemoglobin and some even in 0.075 percent, and this was also true of S. ferax and of S. monoica. While Klebs’s form may have developed sporangia less readily than those which the writer used, the difference in the results is doubtless due largely to the relative purity of the haemoglobin. As a conse- quence concentrations thought to be the same are not always so, and, owing to the delicate balance between growth and sporangia forma- tion existing in a weak solution, a slight difference may result, either in the formation of many sporangia, and in the consequent weakening of the mycelium, or in an undue amount of growth with the attendant formation of the poisonous products of metabolism. In either case the formation of oogonia will be interfered with. | Purified water.—The earliest tests made in Heidelberg brought out the fact that distilled water from the chemical laboratory had a harmful effect on the mycelium. Horn (’04) had already observed this and had attributed it to the presence of copper, or of some other metal in the water. He found that when common distilled water was re-distilled out of hard Jena glass, it lost its poisonous properties... This was not found to be so in the present case. Distilled water was twice re-distilled out of hard Jena glass, but, although the latter was better than the distilled water as it was received from the chemical laboratory, it was by no means as good as freshly collected rain water. The action of the distilled water was not uniform, sometimes wholly preventing the formation of sporangia, while at other times some sporangia were formed but did not discharge normally. This was also found to be true sometimes of the rain water, which, though often satisfactory, was not infrequently quite as toxic as the distilled water. Much of the work first done had to be discarded because of this un- reliable action of the water. The use of carbon black as described by 540 ADRIAN J. PIETERS Livingston (’05) was then tried and it was found that this gave uni- form and satisfactory results. Out of the many records available on this point only one table will be given to show the effect of the water on the formation of sporangia. Tap water was also found to be quite as unsatisfactory as distilled water. TABLE II Achlya racemosa, Grown Three Days in Culture Solution N*; Transferred to Water for the Formation of Sporangia 2 3 4 Sporangia in r2 Hrs. In 24 Hrs. 48 Hrs. Distilled water from | chemical laboratory..... 0, Mycelium dying. Dead. Distilled water shaken up with carbon black...... _I + discharged. III Exhausted. Distilled water re-distilled and filtered through car- bom black tance os ees I + discharged. III Exhausted. Double distilled rain water.| Few formed, none | Few discharged. | Some gemmae. discharged. Double distilled rain water filtered through carbon blacks. etme gers te rea 350% II + discharged. III Exhausted. Similar results were secured with other species. All species are not equally sensitive to the water, however, but all gave more uniform results when water purified with carbon black was used. In all the following experiments, therefore, whenever the ability of a mycelium to produce sporangia was to be tested purified water was used. To determine whether the use of this carbon black would intro- duce an unknown food element into the water some carbon black was carefully washed by shaking it up with a small quantity of water, which was then filtered off. Mycelium was transferred to this water but in no case did any growth result, though sporangia were at once formed showing that the water contained no food. Culture media.—With the exception of pea extract, only synthetic media were used. Many of the various natural media, such as ants’ eggs, larvae, and so forth, might have been used and might have given interesting results. It was desired, however, in the first place to use, so far as possible, only media of known composition; and in ’ This solution contained 0.1 percent peptone, I percent sucrose and 0.1 per- cent salts. RELATION BETWEEN VEGETATIVE VIGOR AND REPRODUCTION 54I the second place to weigh the mycelia. Any insoluble substances would introduce an element of error by adhering to the fungus hyphae. When pea extract was used this was prepared by sterilizing about six peas with a hundred cubic centimeters of distilled water. If carefully sterilized, the peas did not break up. Any flasks in which the peas had broken up were discarded for these experiments, as the loose starch grains would surely adhere to the fungus and increase the weight. Peptone was used as a source of both nitrogen and carbon and in a few cases haemoglobin was used. Asparagin and leucin were tried but the growth was so poor that these substances were not considered satisfactory; Klebs (’99, p. 520-523) also found that growth took place in lower concentrations of peptone than of leucin. The latter is very expensive to use for so large a number of experi- ments as had been planned, and is moreover of a rather uncertain quality. In the course of the work there was occasion to try the leucin from three prominent German makers and no two were found to give like results. Synthetic leucin was once used, but proved to be so toxic that little growth took place init. Carbohydrates were added as an additional source of carbon. At first sucrose alone was used, but later maltose, levulose and dextrose were added. For salts, potassium nitrate, magnesium sulphate, and potassium phosphate, either mono- or di-basic were added. Wherever in the tables the term ‘‘salts’’ is used, the above salts, in concentration to total 1/100 or 1/200 mole- cular, are meant. In the earliest experiments these salts were used in a strength of 0.1 percent or 0.05 percent but later molecular weights only were used. In some cases each salt was used separately. EXPERIMENTAL Experiments with Achlya racemosa This species offers advantageous material because it will produce in water oogonia with oospores and antheridia. It is not necessary to transfer to another food solution; it is only necessary to prevent the too rapid formation of sporangia. This can be done by placing the mycelium in a temperature of 24 degrees, at which temperature few sporangia are formed, while the formation of oogonia is not interfered with. The mycelia for the following experiments were grown for seven days in 50 cc. of the following solutions. (1) Peptone 0.1 percent, sucrose I percent, salts 0.1 percent; (2) peptone 2 percent; (3) peptone I percent; (4) peptone 0.5 percent; 542 ADRIAN J. PIETERS (5) peptone 0.1 percent; (6) peptone 0.05 percent; (7) peptone 0.01 percent; (8) peptone 0.1 percent, salts 0.1 percent; (9) peptone 0.1 percent, salts 0.05 percent; (10) peptone 0.1 percent, sucrose I per- cent; (II) peptone 0.1 percent, sucrose 0.5 percent. At the end of seven days a mat had been formed on the surface of the solutions, numbered 1, 8 and 9 while the quantity of mycelium was in the following order, numbers 1, 8, 9, 2, 3, 4, 10, II, 5, 6, and 7. As before stated, no weighings were made for this species, but careful notes were taken of the apparent mass and condition. From these it appears that the complete solution, such as was suggested by Obel, solution number 1, produced the best growth; that a mat was formed only in those solutions containing salts and that those solu- tions were much better for growth than those containing the same amount of peptone and sugar, but no salts. There was very little difference between the mass of mycelium produced by solutions of 0.5 percent or I percent peptone or by peptone and sucrose, but the mass of mycelium out of 2 percent peptone was many times greater TABLE III Achlya racemosa. Mycelia Grown Seven Days in Media Given in Column 2. Trans- ferred to Water at 18° 3 4 5 6 7 I 2 : Sporan-| Oogo- Z “Boge | Bie Ps | on | Om 1 | Peptone 0.1%, sucrose 1%, salts:o:1975 2. 6. II* | oI I ile DB APP EpPLONe 20,8 antoe ke ee II II o-I o-I o-I 2 (Peptone, Lore cemere nee s II-III I o-! I II Ay Peptones0:5 pew ee II III I I II 5) | Reptone.O;t an. aa oe o-I e) I-II II IIT 6 |pPeptone O:050p2 541... .-e = 0) fe) II II-III III 73\ ne ptOHeO,Ol Votes. re aa Very few; oO II III III oogonia mostly empty 8 | Peptone 0.1% + saltso.1%| II-III | o-I I Tit III 9 | Peptoneo.1% + saltso.05%| II+ I fo) I I 10 | Peptone 0.1% + sucrose 1% ) e) I II II] 11 | Peptone 0.1% + sucrose 0.5%......... 0) fe) II II III oogonia | | | | mostly empty 4 The number of sporangia given for the second day is in addition to those first recorded. >The number of oogonia recorded for each day represents the number then present and not a number in addition to those previously recorded. RELATION BETWEEN VEGETATIVE VIGOR AND REPRODUCTION 543 than that out of 0.01 percent peptone, as was to be expected. After being washed the mycelia from these solutions were transferred to water at the laboratory temperature, about 18° C. Sporangia were readily produced and Table III shows the relative number of sporangia and oogonia from mycelia grown in various culture media. With the exception of the mycelium out of 2 percent peptone, there is roughly an agreement between the mass of mycelium and the number of sporangia formed, and it is clear that a poorly nourished mycelium will not make many sporangia. The addition of salts to the peptone not only increased the vegetative growth but also the number of sporangia, a result not secured with sugar. So far as could be observed the mycelium from the 0.5 percent peptone developed as many sporangia as did that from I percent or from 2 percent, while that from 0.1 percent showed a falling off. This would seem therefore to be the critical concentration, below which a still more decided falling off in the number of sporangia is to be noted. The same mycelium developed both sporangia and oogonia in water, and the TABLE IV Achlya racemosa. Mycelia Grown Seven Days in Media Under Column 2. Trans- ferred to Water at 24° 2 3 4 5 6 7 8 I : Spo O : Oo Oo Media. Sporangia, rangia, ogonia, | gonia, | gonia, Remarks. t Day. j|2Days.| 1 Day. |. Days. | 5 Days. I | Peptone 0.1%, sucrose 1%, salts O.1%.-..- Few e) II 1 III | Many without oospores. eapPeptone 2%....- I I I I II | Oogonia larger than in No. I and well filled. 2) \Peptone1%..... o-I o-I II II III | Oogonia large, mostly with one large oospore. 4 | Peptone 0.5%....| I-II O Tee lie et 5 | Peptone 0.1%....| Very few| o II-III Ill III | As in number 3. 8 | Peptone 0.1%, Salts O.-7G). «ee O Rare | II-III | III | III | Some empty oogonia. 9 | Peptone 0.1%, salts 0.05%.... 0) O II-III _ | III | III |About 20% empty. 10 | Peptone 0.1%, sucrose 17%... .| \ o—I O III III | III |About 30% empty. II | Peptone 0.1%, sucrose 0.05%. . O O Ill III | III |About 50% empty. 544 ADRIAN J. PIETERS table shows that there was no agreement between mass of mycelium and number of oogonia, which fact may, however, have been due to the exhaustion of the mycelium by reason of the sporangia produced. By keeping the mycelia after transfer to water at a temperature of 24°-25° C. the production of sporangia could be almost wholly prevented. This was done and the result is given in Table IV. The cultures used in this case were duplicates of those used for the previous experiment. It will be noted that at this temperature the production of sporangia was almost inhibited. The number of oogonia present at the end of five days was in every case great; but it was also noted that the oogonia on mycelium from I percent and from 0.1 percent peptone were large, though containing but one oospore, while in all other cases, except the mycelium from 2 percent peptone there were many empty oogonia, sometimes as many as 50 percent. In this series the largest number of oogonia, containing oospores, was produced by the mycelium having the smallest mass. From the same culture series another set, now seventeen days old, was taken and placed in water. After five days the number of oogonia found was as shown in Table V. TABLE V Achlya racemosa. Mycelia Grown Seventeen Days in Media Under Column 2. Trans- ferred to Water at 18° I 2 3 Media Oogonia in 5!Days 2: sPeptone 1 Oitgce ten cere ioe eee III, oospores 2-3 in an oogonium. A*Peptoneso15 [5 es area eee eee ere III, large, 2-4 oospores. 5; Peptone Om Volek ee eee II-III large oospores. 6° "Peptone' 0105 Fos, dive tae eles Orie oe II, smaller than in 0.1 and few oospores. 7 aPeptone:O:Ol pia teeek. ctor ate tre I-II, few with oospores. &.) Peptoneio:r?,, saltsior jn. - ee eee II-III, large and good. There have also been many sporangia. g Peptone 0.1%, salts 0.05%........+. o-I, there have been many sporangia. 10. Peptone:o J, suctose £7,— 0 II, small, but with oospores. Ir Peptone 0.1%, sucrose 0.5%.......¢+ II, mostly with oospores. The table shows that, while above a certain concentration there is no relation between mass of mycelium and the number of oogonia produced, there is a concentration below which the lack of food is manifested in the smaller number of oogonia formed. In the case of RELATION BETWEEN VEGETATIVE VIGOR AND REPRODUCTION 545 Achlya racemosa, this limit lies between 0.1 percent and 0.05 percent peptone. Above this limit there was an increase in the growth, whether the concentration of the peptone only was increased, or whether salts or sugar were added, but there was no corresponding increase in the number of oogonia produced. Results similar to those given in the above tables were secured from other tests and we may conclude that for Achlya racemosa there is a concentration of food which, while it will enable the fungus to make some growth, will so reduce its vigor as to interfere markedly with the production of spor- angia and of oogonia. Above this critical concentration the effect of the food will be marked by an increase in weight without a corre- sponding increase in the number of sporangia or of oogonia formed when the mycelium is transferred to water. For the production of the maximum number of sporangia this concentration lies above 0.1 per- cent peptone while for the production of oogonia it lies between this and 0.05 percent peptone. Experiments with Saprolegnia ferax (Gruith) Thuret This species was collected at Heidelberg in the fall of 1911. One spore was isolated, and all the cultures made during the past three years have been with material derived from this original culture. This species was first clearly separated from other members of the ‘“‘ferax group” as S. Thurets by DeBary in 1881 (’81) when he called attention to the fact that antheridia were scarcely ever found. Later (88) he distinguished S. mixta as having antheridia on about one half of the oogonia and as having a more slender mycelium than that of S. ferax. A careful study of the form collected as number 25 failed to disclose antheridia and a comparison of this form with another collected as number 17 led to the conclusion that number 25 should be identified as S. ferax while number 17 had the characters of S. mixta. Humphrey (’92) has discussed the nomenclature of these species and since his point seems well taken the name S. ferax (Gruith) Thuret is used here instead of S. Thureti as this species was called by DeBary. The fact that antheridia were found later has been recorded in a paper read before the Michigan Academy of Science at the meeting of April 1915, together with a discussion of the “ferax group” of the genus Saprolegnia (see Pieters, 15). S. ferax furnishes favorable material, since the oogonia are quite large and are readily produced. Klebs had already shown that 546 ADRIAN J. PIETERS S. mixta produced oogonia readily in 0.05 percent haemoglobin and Kauffman that S. hypogyna produced oogonia readily under similar conditions. Preliminary tests showed that S. ferax behaves like the above species and in all subsequent work haemoglobin, either alone or with salts and sugars, was used as the transfer solution when it was desired to test for oogonia. Unfortunately commercial haemoglobin is not of uniform purity and it sometimes happened that a solution made up to 0.05 percent was stronger or weaker than expected and thus either encouraged unusual growth or the formation of sporangia. Either event worked disadvantageously for the production of oogonia. The different tests could not of course be carried on at the same time and hence were subject to variations in temperature. Klebs showed that a temperature of 18° C. was favorable for the production of oogonia in S. mixta but under the conditions prevailing in the laboratories of the University of Michigan such temperatures are almost impossible to maintain. The room temperature is generally 20° C. and above, temperatures at which oogonia are not readily produced. Some tests were therefore carried on in the basement of a private house while others were conducted in a small closet built in from a window in the botanical laboratory and opening to the out- side by a swinging window. By doing the work in the winter and by using an electrically heated incubator, fairly satisfactory temperatures could be maintained but not exact enough to reach any conclusions as to the optimum temperatures for reproduction. The following record will, however, show the difference between the number of oogonia produced at lower and at higher temperatures. Mycelia TABLE VI Saprolegnia ferax. Mycelium Out of Media Under Column 1. Transferred to Haemo- globin and to Haemoglobin and Salts at Different Temperatures. Average Number of Oogonta in 25 Square Millimeters I 2 3 4 5 209-239 C. | 149-189 C, Y 209-23°C, | 149-18°C. Haemoglo- | Haemoglo- Medium Haemoglo- Haemoglo- | pin 0.05%, bin 0.05%, bin 0.05% bin 0.05% | Saltso.r% | Salts 0.1% Peptone 2%. ssc ee ee er eee ne eee 12 70 — 198 Peptonert eee ee anaes ieee em 19 67 15 142 Peptone 0.1% ++ Knop’s Sol. 0.05%....... 15 176 5 157 Peptone 0:2 %' => suctose one), hear 5 51 4 163 Peptone 0.1% + sucrose 0.1%.......... at 59 15 155 RELATION BETWEEN VEGETATIVE VIGOR AND REPRODUCTION 547 from various solutions were transferred February 14, 1913 to 0.05 percent haemoglobin and to haemoglobin and salts, one set being kept in the laboratory where the temperature ranged from 20° to 23° C. and often higher while another set was kept in the basement of the writer’s home at a temperature varying between 14° and 18° C. Production of sporangia.—The optimum temperature conditions for the production of sporangia are quite different, a high temperature causing the sporangia to develop more rapidly than a low one. Klebs found this to be the case in S. mixta also. Ata temperature of 28° C. S. ferax formed young sporangia in five and one-half hours and these were discharged in seven hours from the time the mycelium was put into water. A duplicate lot from the same culture, kept at laboratory temperature (about 20° C. at that time), formed young sporangia in seven hours and these were discharged in nine hours. The maximum temperature at which sporangia were formed in this species was 30°-+ C. at which some swarming took place in six and three quarter hours but the spores left the sporangium slowly and were evidently feeble. In another test duplicate lots of mycelium were placed in water at 28° C., at laboratory temperature 18°-20° C., and in an ice box where the temperature was between 6° and 8° C. Table VII shows at the same time the effect of the higher temperature on the production of sporangia and also the relative number of sporangia formed in mycelia out of different foods. TABLE VII S. ferax. Mycelia Out of Media Under Column 1. Transferred to Water at Different Temperatures for the Production of Sporangia . 2 3 4 ie 5 ep ee tee , 6°-8°, 16 Hrs. 6°-8°, 22 Hrs. Peptone 0.1%, sucrose 1%, Sas OT: -ce east mss Ill III | II, none discharged | III, just discharging. Peptone 2% ii. ee es III III | I, none discharged I, none discharged. IPeptone 0:5.% o's... <: II+ | II+ | I, none discharged I, a few discharged. Peptone 0.1%... ....- II+ | II+ | I, none discharged I-II, a few dis- charged. Peptone 0.05%....... I I fe) o-I, a few discharged. Reptone O:01%...:.... o-I On kO 0, a few discharged. Peptone 0.1% + sucrose 1%...... III II | o-I, none discharged | I-II, discharging. Peptone 0.1% SE ISalts Opes os. a III III | o-I, none discharged | I, discharging. 548 ADRIAN J. PIETERS The fact that no examination was made before sixteen hours ex- plains why there is no apparent difference between the number of sporangia produced at 20° and at 28° C. It may also be noted how the higher temperature helps to conceal the difference in strength between the well- and the poorly-nourished mycelia. When many sporangia are formed at high temperatures it is really impossible for the observer to make a close comparison between them. At the low temperature, however, the difference in vigor is more clearly brought out. Here again, as noted before for Achlya racemosa, the difference between mycelia grown in 2 percent, 0.5 percent, and 0.I percent peptone is not very marked though the first had food at a concentra- tion twenty times greater than the last. But as soon as the con- centration of the food falls below 0.1 percent the effect is quite marked; the critical concentration lies between 0.1 and 0.05 percent. Production of oogonia.—It will be necessary before presenting the results of the experiments in which oogonia were counted to discuss more fully than was done earlier the reliability of the method of counting the oogonia. When a mycelium is spread out between two glass plates the oogonia are sometimes scattered quite uniformly over the surface of the plate, but more often the largest number of oogonia lie near but not just at the margin and again near the thickest part of the mass, which is the original piece put into the dish of haemo- globin. When the oogonia are uniformly scattered the count can be made along any line, but sometimes the observer must select a line that will lie across an average field or must count along a second line at right angles to the first. It may be asked whether such a count will accurately represent the number of oogonia on a given mycelium. The answer to this is that with few exceptions two counts of oogonia on lines at right angles to each other agree closely. It may also be asked whether such a count means anything, whether it is not rather wholly fortuitous and whether another lot under the same conditions might not show a very different number. It must be ad- mitted that there is an element of uncertainty here which the writer would have been glad to eliminate. Living forms do not always behave with apparent uniformity; cases occurred in which the my- celium in one dish of a pair showed many oogonia while on the dupli- cate not one was to be found. Such instances however were excep- tional. Asa rule the number of oogonia was nearly the same in each dish of a pair as will be seen from the tables and it is believed that a atl te i Me a le Ve 6 = ie ee RELATION BETWEEN VEGETATIVE VIGOR AND REPRODUCTION 549 where duplicate pieces of mycelium placed under identical conditions give similar results, such results may be assumed to correctly repre- sent the response of the plant to these conditions. In Tables VIII and IX a number of figures are given to show that counts at right angles to each other agree substantially and that the counts from dupli- cate dishes also agree pretty closely. While the duplicate dishes were not always counted, because of the great amount of labor involved in this work, they were always examined and when the number of oogonia was strikingly different from that in the dish counted this fact was noted. In all other cases the oogonia were either counted or the fact noted that the two dishes were in substantial agreement. No oogonia were counted that did not contain oospores. Table VIII has been compiled from the record of Series III transferred November 19, 1914. In no case is there a great difference between the counts at right angles to each other, and only in numbers 6, 7 and 12 is there any considerable difference between the number of oogonia in the two dishes of a set. Such a difference was often due to contamination of one dish by bacteria, but whatever the cause, whenever there was as great a variation as is shown by numbers 6, 7 and 12, the highest average was used in the final tables. TABLE VIII S. ferax. Mycelia Out of Media Given Under Column 2. Transferred to 0.075 Per- cent Haemoglobin. All Sugars in the Culture Media 1/50 Molecular. Temperature 16°-18° C. All Figures Given to the Nearest Whole Number First Dish Second Dish | 3 + 5 6 7 8 ; Ae (en =v ; : Aver From Medium Eos [Gately Gaia etc HM gee coe Shen A ogi t Tate oe + ouefo, 6 SM aeons a2 AZ a a2. AG, | =— |) 4081 46 PREC HEONE ONO. . veloc. wee alees boas dee LO. ty ALO) | 2 2ae | —— Poa oT SMe BEOMC O27 po cls ns dS ns Win haus weed ate 49 | 44 | 47 | 45 |— | 45 | 46 PAE CEOME O15. pir sctuel.. of oia viens wich s oe Sonne BAPE Ago 2a 9T. | 20U ae BMMIZCEGOTC Los 5 wsdl s a 4lho .aveceie eas vee Bearer Atal ZOmneAOy WAG |= Agee Al Gr beprone 0.1% -- SUCTOSE 025 bb ace ene Zo aArie 2 Op || ele WT So an oy eo 7 | Peptone 0.1%. + maltose.............. 65 || 6565. 25° | 28 || 27 |. 46 Babeeptone OLL7p -- dextrose: 2... oes 29 | 28 | 29 | 28 | — | 28 | 28 9 | Peptone 0.1% + levulose.............. aCe oe OF Vie | One eA Mn PECpLOne:0;2 7) --/SUuCTOSe. 62... 6). 2.05. HOM 526) /022.) 36" | 329134. 28 PielePentone 0.2% -- maltose. 2.0.6). 2... +: 46 | 46 | 46 | 4o | — |-40 | 43 12) \"Peptone 0.2% +- dextrose........2...1. eRe) eas (eae an ee cle le ey ¥3°\ Peptone 0.2% ++ levulose 2.0.2... 0.5.8. 33 \|-20 31 | 55 | —- | 55. |) 43 2i0)(| lelevesonteyed Ke) oy bale) (A) a erin Pal Aare Onn oye 220 e222 3.) 21 k=. || Stevie o2 550 ADRIAN J. PIETERS In Table IX another record is given to show that there is sub- stantial agreement between the number of oogonia found on mycelia from like origin and that mycelia grown in different media may vary in their capacity to produce oogonia. It is not urged that the figures obtained have the value of those commonly secured in a seed test, for example, for there are too many unknown and uncontrollable factors; but they are offered as a more exact statement of the relative number of oogonia produced on various mycelia of a given series than could be made in any other way. TABLE IX S. ferax. Part of Record from Set I Showing Agreement Between Count. and Cross Count. Mycelia in 0.05 Percent Haemoglobin. Temperature 16°-18° C. Figures to Nearest Whole Number 3 4 5 ist Count |Cross Count] Average Tal Peal o 2 aes oct te c oan een peck hae oe een ainee ea Dene 40 44 42 2 >|. Peptone sk Yo sine uK ue cpodau<. tenes tne cnet, sean eceonaee 37 31 34 ‘ TU duplicate «ae ner sie eae 43 45 44 2. SPENLONE O25 Vo. ety as eee tei ee ee 36 22 34 Anil MReptonelO2 7p... stk aie Peay oe ee edd i 27 34 31 O29 duplicatesier 2) he er sy ie 26 23 25 5 i, Peptone- ON Gp ike cs ce eee eee 16 EG 16 6 re =f="SUCEOSE fi4)c4 ce trees oe Re coe ae ee 19 17 18 7 rs “MMA bOSEL, aise stabs Soa te Sew an eh dh One 43 38 AI 8 = SuCKOSe = iSaltshs a. oo ee eee 27 28 28 9 | Peptone: -- maltose 4- salts..0.. 3... Jn. 3 2 ee 44 40 42 LO (|: Peptone; 0.2.97) a1SUCKOSe arte teas 55 52 54 ib a 0.20521 maltose sani e cd eae eee eee 45 50 48 12 i 0.2% + s -ErSdit Soh fan Ae 64 A7 56 103) ‘ O07) salto Van ee eros sae eee ee 22 33 23 14 es 0:29) Se tae Bee ore 32 38 36 Record of Series I, October, 1914.—For this experiment the food solutions were contained in tubes each holding 50 cc. of liquid. These were inoculated and allowed to grow for seven days and transferred to 0.05 percent haemoglobin October 1, 1914. At the time the mycelium from one tube was transferred to haemoglobin, that from a duplicate was washed, dried and weighed, and a third tube was left for forty-eight days and the contents then washed, dried and weighed. Table X gives the complete record of this test. Unfortunately a rather large number of sporangia were produced in the haemoglobin, especially by the mycelium from pea and from peptone I percent and this interferred materially with the production of oogonia. In the Ao un RELATION BETWEEN VEGETATIVE VIGOR AND REPRODUCTION 55I following discussion the record of these two will be disregarded as being probably too greatly affected by the production of sporangia. TABLE X Series I. Saprolegnia ferax. Mvycelia Out of Media Under Column 2, Transferred to 0.05 Percent Haemoglobin; Sugars and Salts 1/100 Molecular Except as Noted. Weight in Milligrams of Mycelia Out of 50 cc. Liquid. Record of Sporangia and Oogonia Produced. Figures for Number of Oogonia to Nearest Whole Number 13 4 5 6 7 | 8 Spo- : ; . s Weight, | Weight, |rangiain| Sporangia) _Sporangia, Oo- 7 Days. | 48 Days. | Water, |i8 Water | Haemoglobin, | gonia, I | PGE hens sas epee Ure ae 0.018 | 0.056 NG ie Stil; cogonia)|))" 42 started 2 Peptone 0.0%. .0s26-: 0.002 | 0.005 II II-III I+ 16 2 e (OY ase EO: 0.003 | 0.OII I-IT II [+ 30 A ‘ ORO ta oe COO Lara e II+ | II-III I-II 34 5 yi Lee eae aE 0.0036 | 0.037 | J-II | II-III II-III 36 6 ~ 0.1%, sucrose | 0.0035 | 0.007 | o-I I+ 0, oogonia | 18 started 6m 7. 0.1%, maltose 0.0044 | 0.033 II | II-III I Al vi i 10.1.7, sucrose amdesaltss: \ a. | 0.0054 | 0.010 | o-I I+ Hes 28 7m se 0.1%, maltose | andsalts ae. 0.004. | 0.033 I 1 ess i o-I 42 8 ‘* 0.2%, sucrose | 0.0054 | 0.013 | o-I II o-I 53 8m a 0.2%, maltose | 0.005 | 0.036 |, o-I II II 43 9 ia 0.2%, sucrose | and salts... .. | 0.006 | 0.020 | Few | J-II II 51 om i 0.2%, maltose | and salts..... 0.005 | 0.044 | Few | II-III Atl 50 10 “0.1%, salts. ..| 0.002 | 0.0066 | _0 I-II I 36 II Me 0.2%, “ ...| 0.0023 | 0.007 | Few | [II I 28 12 i O20F | 1/333 mole- | GUAR 6 ti..5 eras HOLOO2r tsete sede. ) tur O 43 If these records are arranged in two groups, the first containing those in which 16-36 oogonia were found in 25 square millimeters, and the other those with 41-53 oogonia per square it will be noted that the first group contains those culture media without sugar, except for numbers 6 and 7, in which sugar was used. The second group includes all those with sugar except 6 and 7 and also one (12) to which no sugar was added. See Table XI. The weights for the seven-day-old mycelia are, as a rule, higher in the second than in the first group and this is even more striking 552 ADRIAN J. PIETERS TABLE XI Series I. Saprolegnia ferax. Record of Table X Arranged in Two Groups According to the Number of Oogoma Per Square of 25 Square Mullimeters. Weights in Milligrams I 2 3 4 5 No. Number of Oogonia| Weight in 7 Days | Weight in 48 Days | Increase in Weight Group I Picked Pua iA Ae 16 .002 .005 X2.5 Onpetsr ee 18 .0035 .007 X2 Freee Ie et Ni 28 .0054 .O10 X1.85 1B) Re eae ae 28 .0023 .007 X3 Fee neces eed: 30 .003 .OII X3°3 ae ee eines 34 .004 — ROA Een Tits sere 36 .002 .0066 x33 Group II: OM a ey agains 4I .0044 .033 X75 TU My code 42 .004 [022 x8 Ae ree ee 43 .005 .036 X7 DD toe ee AAR. 43 .003 — = ON eas Wien 50 .005 .044 X9 OR. rede s® 51 .006 .020 X3:3 Se Se ee 53 .0054 013 X2.5 when the weights of the mycelia forty-eight days old are compared. Number 12 has the one disturbing record in this series. With a small weight, it combines a rather large number of oogonia. From the weight of mycelium as well as from the composition of the food, it was to be expected that this lot would appear in a group with numbers 2, 3, 10, and 11. The explanation of the peculiar behavior of this lot doubtless lies in the fact that no sporangia were produced in haemoglobin. As has already been shown, the suppression of spo- rangia, in a solution of this sort, reacts very favorably on the formation of oogonia, but why no sporangia were formed in this case it is im- possible to say. A study of the record in Table XI would seem to show an agreement between weight of mycelium and the number of oogonia produced. But this is true only as showing a trend toward a minimum food requirement. In group I, for example, 0.1 percent peptone is clearly too poor a food to produce vigorous mycelium and the addition of sucrose alone does not add to the vigor of growth. When, however, salts are added, or higher concentrations of peptone used, the number of oogonia at once approaches that of the lower numbers in the second group. The separation of the groups along the line between 36 and 41 oogonia is, of course, arbitrary and only RELATION BETWEEN VEGETATIVE VIGOR AND REPRODUCTION 553 for the purpose of illustration. Differences as great as between 28 and 41 might well be found between two counts of the same dish, but no two duplicate dishes would, without contamination or other accident, show as great a difference as that between 28 and 53. While the upward trend in the number of oogonia, with the in- crease in the food furnished, is evident, there is no corresponding increase in the weight of mycelium. It will be noted also that the amount of increase in the weight of mycelium between seven and forty-eight days is by no means uniform and a study of this feature brings out a rather interesting point. In Table XII these data are _ arranged in the order of the increase in weight of each mycelium due to the growth from the seventh to the forty-eighth day. TABLE XII Series I, S.ferax. Record of Table X Arranged in the Order of the Increase in Weight, Due to Growth Between Seven and Forty-eight Days I 2 3 4 5 Ree cee | pba | 48 Days Number of Oogonia Wn eas X1.85 0054 O10 28 Cea). x2 0035 .007 18 | Group I. Average number of oogonia 20 Ee eae X25 002 005 16 29. Average excluding No. 8—21. (Sh Ab ae ae X2.5 0054. O13 53 a ane x3 0023 .007 28 MO eae x28 002 0066 | 36(Group II. Average number of Bu ea XK 3:8 .003 OI! 30 oogonia 36. (oe Maar BiG .006 020 51 cit ee 7, .005 .036 A3 | GH, ..'.. X7.5 .0044 033 41 | Group III. Average number of Tallin cs x8 .004 033 42 oogonia 47. OT oars e's x9 .005 .044 50 Of the first group, those showing an increase in weight of not more than 2.5 times, number 8 is the only one credited with a large number of oogonia; of the second group number 9 is credited with a much larger number of oogonia than the others. The third group differs strikingly in the much greater increase in weight shown by all the mycelia. Reference to Table X will show that the culture medium for all those in group III contained maltose, and that this group in- cludes only those in which maltose was added to the culture medium. The record of numbers 2 and 6 may be compared since in both cases 0.I percent peptone was available, but sucrose had been added 554 ADRIAN J. PIETERS to solution 6. It is apparent that the fungus was not able to utilize sucrose in the presence of this strength of peptone while it made free use of maltose as shown by the record of number 6m which also contained 0.1 percent peptone but to which maltose had been added instead of sucrose. Numbers 8 and 9 contained the same amount of sucrose as 6 had and in addition 0.2 percent peptone, and 0.2 per- cent peptone and salts respectively, and on these mycelia were de- veloped the largest number of oogonia of any lot in the series. This large number of oogonia cannot be accounted for by the higher per- centage of peptone alone nor by the presence of salts alone, as then numbers 3 and 11 should have given comparable results. It is evident that in numbers 8 and 9 the sucrose was used but the weight record indicates the use of only a small part of it. During the first seven days’ growth the fungus was very well nourished as shown by the fact that the weights at the end of that time are among the highest. This rate of growth, however, was not maintained as is shown by the small increase in weight. The explanation is doubtless that in numbers 8 and 9 part of the sucrose was changed to invert sugar owing either to too prolonged heating at high pressure while sterilizing, or to the presence of salts in the solution together with a high temperature. If this is true the fungus would find invert sugar present as soon as new growth began; development would then be rapid in the next few days or as long as the invert sugar lasted. Upon the exhaustion of the invert sugar growth would slow down and the final weight would be less than that from solutions containing more available carbo- hydrates than sucrose. Further, if the fungus were removed before the invert sugar present was consumed it would be in the same physi- ological condition as it would have been if all the carbohydrate con- sisted of invert sugar. These theoretical considerations are supported by the facts of the experiment. After seven days numbers 8 and 9 had made as large a growth as any lot, but at the end of forty-eight days the total increase was 2.5 times the weight at the end of seven days, as compared with 7—9 times the weight in the case of 8m and 9m. Growth was at first rapid, then slowed down; the invert sugar was used up. That the mycelium at the end of seven days was in good condition is shown by the large number of oogonia produced. At the end of seven days and while the fungus in solutions 8 and 9 was in the full enjoyment of an abundant supply of invert sugar the RELATION BETWEEN VEGETATIVE VIGOR AND REPRODUCTION 555 mycelium was transferred to haemoglobin; that this mycelium was then in good condition is shown by the large number of oogonia pro- duced. We may then conclude that invert sugar is an especially valuable food for the subsequent production of oogonia; that sucrose cannot be utilized by this fungus, but that maltose is used and is a good food both for vegetative growth and for the production of oogonia. (In series II the oogonia rarely developed oospores. This was due probably to contaminated haemoglobin. The entire set was dis- carded.) Series III.—This series was prepared to repeat the work in series I and also to determine if possible which of the two sugars present in sucrose, dextrose or levulose, was most used by the fungus. Solu- tions containing salts as well as sugars were also prepared and cultures grown in them, but since the large number of cultures thus set up would make it impracticable to handle all at one time those with salts were transferred in a separate lot. For some reason not deter- mined, all of the mycelia from the solutions containing salts failed to make oogonia or at most produced but a few oogonia each. Since this result was obviously due to some abnormal condition the entire lot was discarded and the results with the sugars alone are presented. Table XIII gives all the data for this series. TABLE XIII Series III. Saprolegnia ferax. Mycelium Out of Culture Media in Column Two. Grown Seven Days Before Transfer to 0.075 Percent Haemoglobin and Thirty Days in 200 cc. Liquid Before Weighing; Weights in Milligrams. Oogonta Count Recorded to Nearest Whole Number. Tem- perature 15°-18° C. Sugars All 1/50 Molecular Record After 2 Days Oogonia After No. Food Weight Spee — rangia Oogonia 3 Days |17 Days Tl LEO 3 Sc eee ae ara aoe a Pa 0.041 lise I] IIT 46 PEPE MEONe: OFLU e . sac. cea Cee kee 0.0172 | o-l O Few on 2 Se i Oso ee ios beg aj chy oo 0.0376 I o—I I 46 4 4 ONS a ee een Tee 0.079 II fe) o—I 38 5 : EY eee eee nnn PAOD seer 0.124 I] re) I 4I 6 O85) -i- SUCLOSG45. 3. 0.024 Tit 0) o-I 29 7 5 Only or aic maltOse 0.068 II O I 65 8 ‘ 0.1% + dextrose..... 0.023 II ) o—I 28 9 % Onl 7G ae leVulose wae. |. 0.061 I I II-III 72 10 ue 0.2% + sucrose...... 0.0404 II fe) i 28 II a 0.2% + maltose..... 0.099 II I II 43 12 0.2% + dextrose..... 0.0474. TI O Few 24 13 0.2% + levulose..... 0.065 II_ | Starting o-I | 55 200 tlaemoslobin O.195.-..0...... 0.0199 | I-II fe) Few 22 556 ADRIAN J. PIETERS It will be noted that not so many sporangia were formed in the 0.075 percent haemoglobin as in the 0.05 percent used in series I, and that the numbers of sporangia were nearly the same in all the dishes, so that any influence on the production of oogonia can be disregarded. This table shows, as did Table X, that there is no constant relation between the amount of dry weight a culture medium will produce and the number of oogonia formed by the mycelium after transfer. In only one case do these figures agree; the mycelium out of 0.1 percent peptone is at the bottom of the list in both instances. The greatest weight was made by mycelium out of 1 percent peptone which was seventh in the production of oogonia. Mycelium out of solution number 9 had the largest number of oogonia and was sixth in weight. The second heaviest mass of mycelium was out of solution number Ir and this was sixth in the production of oogonia. The results secured in the first experiment are therefore confirmed so far as concerns this point. The value of maltose is also confirmed, and the great difference between the value of dextrose and levulose in the conditions of this experiment is brought out. Dextrose added to peptone increased the weight of mycelium slightly, but the number of oogonia not at all, while the addition of levulose produced a my- celium with a tendency toward the production of oogonia quite out of proportion to its value for vegetative growth. In the course of this work many tests were made to determine the effect on the production of oogonia when salts or sugars were added to the haemoglobin. If it is true that levulose is an especially favor- able form of carbohydrate for the production of oogonia, this fact should also be brought out if levulose is added to haemoglobin. This was done in a number of cases and the results confirm the conclusion that for S. ferax levulose has an especial value for the production of oogonia. In Table XIV the results of one test are brought together. TABLE XIV Saprolegnia ferax. Mycelium Out of Pea Extract in Haemoglobin and in 0.025 Per- cent Haemoglobin and sugars. In Every Case Concentration of Sugar Equals 1/200 Molecular. Figures Represent the Number of Oogonia Formed in 25 Sq. Mm. of Mycelium After Nine Days Haemoglobin Haemoglobin Haemoglobin Haemoglobin Haemoglobin Haemoglobin 0.025% 0.05% + Dextrose + Levulose + Maltose + Sucrose 21 34 49 129 14 92 ) | RELATION BETWEEN VEGETATIVE VIGOR AND REPRODUCTION 557 It will be noted that here sucrose has also had a marked effect, due doubtless, as before suggested, to the inversion of a part of the sucrose. In dextrose the growth was short and dense but did not fill the dish, while in maltose the growth was very dense and rapidly filled the dish with a mass of delicate hyphae. Possibly a lower concentration of maltose might have given better results. The growth in the haemoglobin-levulose solution was open but vigorous; oogonia appeared earlier than in the other dishes and the number formed was so much greater than the number produced by mycelium of the same origin in haemoglobin alone or in haemoglobin and dextrose that there can be no mistaking the conclusions that are indicated. The fact that these results confirm those secured when levulose was offered as a food during vegetative growth only make3) them especially significant. ill The results with S. ferax support the general conclusions arrived at in the experiments on A. racemosa. The weight of mycelium secured from any culture is no measure of the number of oogonia which that mycelium may be expected to produce when transferred to a suitable environment. There is however a minimum concentra- tion of food at and below which both the weight of the dry matter and the number of oogonia decrease. This minimum is about at 0.1 percent peptone for the production of both sporangia and oogonia. Levulose is especially valuable as a food for the formation of oogonia; dextrose is not used as freely and sucrose not at all unless previously inverted by other agencies, while maltose is readily used. Experiments with S. monoica S. monoica was used because it normally has an antheridium on each oogonium and oogonia are freely produced, even under some- what adverse conditions. This species is closely related to S. ferax, and experiments with it might be expected to confirm the conclusions drawn from the results of previous experiments. It was also thought that some light might be thrown upon the kind of food that is best for the production of antheridia. Cultures were made in flasks containing 200 cc. of liquid in the same manner as for S. ferax series III; after growing five days these cultures were transferred to 0.05 percent haemoglobin. Table XV gives the complete record for this set. 558 ADRIAN J. PIETERS TABLE XV Serres VIII. S. monoica. Mycelium From Media Under Column 2, Transferred to 0.05 Percent Haemoglobin. Weight of Mycelium in Milligrams after Thirty Days’ Growth in 200 cc. Liquid. In Numbers 6-17 the Amount of Peptone is Always 0.1 Percent, of Sugar 1/50 Molecular, and of Salts 1/200 Molecular Weight of My- Weight, Sporangia Oogonia | Antheridia, | celium Out of No. Food Milligram | 2 Days to Days Percent | Haemoglobin, Milligram TTP ed eee eee .0398 Mit 80 21 .0157 2. | Peptone.O:5 Vo. -e .O146 I 170 6 Lost 2 a O29 ee .0256 | I 146 9 .O109 4 i ONS eae sr .0561 I-I] 79 9.5 {0132 5 - TE Sree .0863 II 108 7 .0138 6 4 pemlicrose...| .O15 II 62 6 .O120 v4 x + maltose...| .0598 II 58 I2 .0144 8 + dextrose...| .0281 II 96 10 .0128 9 o + levulose...| .0581 rt LIOn we a2O .O152 14 7 -++ sucrose Galt acti: .3645 II 79 6 .OIQI 15 . + maltose a SaltSirscten 4 2r51 o-l 2. ey Tih eis .0219 16 i + dextrose | +isalts..%...5 .181I0 II 105 13 .0188 17 . + levulose... ap Salts) owen. .2100 II 103 64 .O186 A striking feature of this record is the very large number of oogonia found on the mycelium from 0.1 percent and from 0.2 percent peptone. The preliminary tests showed that in this species mycelium from 0.I percent peptone produced as many oogonia as that from I percent but such a large number as recorded in Table XV was quite unexpected. In this series the tests in haemoglobin were conducted in quadruplicate. Before making the final records all dishes were carefully examined and the number of oogonia in each was found to be practically the same. Probably the large number of oogonia is connected with the smaller number of sporangia in these lots, for, as has been said, a mycelium producing few sporangia is in better condition to produce oogonia than one that has produced many sporangia. This fact introduces an element of uncertainty into this work that is always present and must be taken into consideration in interpreting the results. Leaving the record of numbers 2 and 3 out of consideration it is apparent that levulose is a food favorable for the formation of oogonia out of pro- portion to its value for vegetative growth, and that sucrose with 0.I percent peptone alone is probably not available to the plant. RELATION BETWEEN VEGETATIVE VIGOR AND REPRODUCTION 559 The weight of mycelium out of solution number 6 is practically the same as that out of number 2. When salts are added to peptone and sucrose the number of oogonia produced is materially increased and the weight is much greater than from solutions without salts. As before suggested, this is probably due to the formation of invert sugar from the sucrose by the action of high temperature and salts. There is evidently no relation between weight of mycelium and number of oogonia even when the record of numbers 2 and 3 is dis- carded. The weight of mycelium was about equal out of solutions 4, 7 and 9 but the number of oogonia varied from 58 on mycelium out of solution 7 to 119 out of solution 9. Again, mycelia out of solutions 15 and 17 show practically the same weight but vary in the number of oogonia recorded from 52 to 103. For vegetative growth S. monoica does not use dextrose as freely as it does maltose and levulose, agreeing in this with S. ferax. The difference in weight is striking and appears consistently in all the records, but it is doubtful whether it is safe to draw more than the most general conclusions from the record of the number of oogonia, since this species produces oogonia more readily and abundantly than any other species the writer has isolated. In general, however, the conclusions previously drawn seem warranted by the results of this experiment also. The minimum food required for an abundant supply of oogonia is less than that demanded by S. ferax, the critical concentration evidently being below 0.1 percent peptone, but there is no uniform relation between weight of mycelium and number of oogonia. The value of levulose is also confirmed. Klebs found that when S. mixta was placed in 0.05 percent haemo- globin oogonia were readily formed but they were not accompanied by antheridia. With the same treatment S. monoica gave similar results although some oogonia with antheridia were always present. The number of oogonia accompanied by antheridia was determined by counting I00 or 200 or sometimes a larger number of oogonia under a 16 millimeter objective and recording the number to which antheridia were attached. While the figures cannot be considered final there is a consistent trend toward an increase in the number of antheridia when the culture medium has contained a form of invert sugar, especially levulose. Number 14 is the only exception to this. In this solution much of the sucrose was probably converted into invert sugar and if so the number of antheridia should approach that in the case of the solutions with invert sugar. The larger number of antheridia present 560 ADRIAN J. PIETERS on mycelium grown in the presence of levulose is especially interesting, since when this sugar is added to haemoglobin the resulting growth produces oogonia nearly all of which are accompanied by antheridia, while when dextrose is substituted for levulose the oogonia are larger but only a few of them have antheridia. In this series mycelium from each culture solution was trans- ferred to four dishes of haemoglobin and especial care was taken to have these transferred masses of as nearly the same size as possible, as it was intended to weigh the mycelium from the haemoglobin after the final oogonium record. This was done to see whether there was any relation between the number of oogonia and mass of my- celium produced in the haemoglobin. The column at the right in Table XV shows the total weight of the mycelium in the four dishes. It is true that slight differences in the masses originally placed in the haemoglobin probably did exist but when it is remembered that the figures represent the total weight from four dishes after several days growth, it will be seen that the small piece of mycelium originally put into the haemoglobin could in no case have exceeded one milligram in weight. The difference in weight between any two such masses there- fore would be represented at most by one unit in the fourth decimal place and could have no influence on the general result. Here again the evidence confirms the conclusion previously reached that there is no relation between the weight of. mycelium and the number of oogonia produced by it. On March first another series was set up with S. monoica to confirm the results obtained by series VIII. Pep- tone and sugars only were used and the resulting data are presented in Table XVI. The cultures in this series were made in Joseph Kav- alier Bohemian glass flasks. TABLE XVI Series IX. S. monoica. Mycelia Out of Solutions Under Column 2. Transferred to 0.05 Percent Haemoglobin After Five Days. Weighed After Thirty Days tn 200 cc. Solution. Weight in Milligrams. Number of Oogonta in 25 Square Millimeters. Sugars All 1/50 Molecular I 2 | 3 4 | 5 Media we ee s : tien ‘: ss I. | Peptone:nt Opis wise rr ener ened .0980 re) 89 2 fs OT ort a a Nine a ee 0244 A few 48 3 EEO OER GHOSEs syne iy tener .0270 A few 52 4. ms OTF oe levulose saps aa .0196 A few 85 5 _ 0.1%: timaleose nc. ee ee 10773 A few 57 6) OT Tp as SUGLOSe ee eee 02675, | Plow to RELATION BETWEEN VEGETATIVE VIGOR AND REPRODUCTION 561 .In this series the small weight of the mycelium out of peptone- levulose is difficult to account for. Unfortunately the duplicate flask failed of inoculation, and the weight represents but one culture in- stead of an average of two as in the other cases. The number of oogonia on the mycelium out of levulose-peptone was however again greater than that on mycelia out of other solutions containing sugars and was practically the same as that out of I percent peptone. Mal- tose was freely used for vegetative increase but the mycelium out of maltose-peptone did not develop a large number of oogonia. At the same time that the cultures recorded in Table XVI were set up, another series was planned to determine what effect salts added to the peptone alone, or to peptone and sugars, would have on the vegetative growth or on the number of oogonia. The time available was insufficient for repeating this experiment and the results are offered as suggestive rather than as proof of the conclusions that seem to be warranted. Table XVII gives the results of this experiment. The mycelia out of the solutions marked with an asterisk were overheated during the experiment, and the number of oogonia formed cannot be regarded as fairly representing the capacity of each mycelium to produce oogonia. The figures have, therefore, been omitted. It would appear from the record that the addition of phosphate salts, and, to a much less degree, potassium nitrate, stimulates growth while calcium nitrate, potassium sulphate, and magnesium sulphate have no marked effect. The only conclusion that seems fully justified by the somewhat uncertain results of one trial, however, is that phosphates exert a marked effect both on the amount of dry matter and on the number of oogonia produced. Comparing numbers 5 to 10 with 16 and 17 it will be seen that while in the latter the use of phosphates with peptone alone has increased the weight materially over that when peptone only was used (21), it still falls short of the weight of the mycelia out of the solutions containing sugar, peptone and phosphates. The number of oogonia produced on the various mycelia is, however, of the same order of magnitude in all cases, in which a phosphate was used, and this number is much larger than that obtained when other salts were used (compare I9 and 20). The conclusions seem warranted, therefore, that in the case of S. monoica also there is no necessary relation between weight of mycelium and the number of oogonia that may be produced; that 562 ADRIAN J. PIETERS levulose is especially adapted to the production of oogonia and that phosphate salts influence the number of oogonia produced to a greater degree than they do the increase in weight of mycelium. TABLE XVII Series IX. S.monoica. Mycelia Out of Culture Media in Column Two, Transferred to 0.05 Percent Haemoglobin After Seven Days’ Growth, and Weighed After Growing Thirty Days in 200 cc. Solution. Wetghts in Malligrams. Sugars All 1/50 and Salis All 1/200 Molecular; Peptone 0.1 Percent I : 2 3 4 5 No. Media Weight Sporangia Oogonia 1 | Peptone + dextrose + KNQ3..... .0423 0) | 4I, oogonia small 2 a + dextrose + CaNO3....) .0281 O 80 3 zs + levulose + KNO3..... | .03675 o-I 93 4 + levulose + CaNO3....) .02095 o-I 40 5 ‘++ dextrose + KH2PO,..| .2011 o-I 94 6 is + levulose + KH2PQ....| .2084 8) 79, large and well filled of ee + dextrose + NaHePO.:| .13835 fe) 90, many small - ones 8 i + levulose + NaH2PO...| .17145 o-I 89, mostly small ones 9 % + dextrose + Ca(HePO.)e| .15954 o-I 110 TOns| . + levulose + Ca(H2POx)2) .1093 I 93, large well filled II | 4 + dextrose + KSO...... .0291 o-I = 12 es + levulose + KSQO,..... .O1855 Few ‘i 193 + dextrose + MgSQz....| .0404 ef] : 14 . + levulose + MgSQO,....| .02225 o-I 54 15 in SAN On. ase ee eee 0183 Few ‘ 16 : att oP Ogee 5 se eee .0426 III 7 . Spe NCE > POme ewe eee .O401 101, small 18 2 + Ca(H2PQ,z)2 wl Gomaue rer ee .0416 - 19 os sO Oe. os eee ee .0213 68 20 i se MIGS@a ss) a cee .0276 57, small 21 a Ost Gail cha eae Sey eee .0244 48 Experiments with Achlya prolifera Several sets of experiments were carried on with this species along the same lines as those before described. The results however were not satisfactory but are presented because so far as they may be accepted at all they seem to confirm most of the conclusions arrived at by a study of the previously considered species. The oogonia could not be counted as the mycelium was so thickly matted that many oogonia could be but dimly seen and the distribution of the RELATION BETWEEN VEGETATIVE VIGOR AND REPRODUCTION 563 oogonia was often so irregular that it was not possible to secure a fair average. The number of the oogonia was therefore estimated. The most important cause of the lack of good results with this species is that no entirely satisfactory solution was found to which the my- celium could be transferred for the formation of oogonia. Early experiments showed that haemoglobin was worthless, oogonia were formed but no oospores were ever developed. Many experiments were then made with peptone and sugars as suggested by Obel (’Io) and also by placing masses of mycelium into pure water or water to which salts had been added. A. prolifera will form oogonia in water as is the case with A. racemosa, but unfortunately the formation of oogonia is prevented by a temperature lower than that at which sporangia continue to be freely formed. If however a mass of mycelium is left in the water in which it was placed on being taken from the culture flask, it may find suf- ficient food present to prevent any free formation of sporangia and thus permit the production of oogonia. This method was therefore used with the others, but the results have not been uniform. Many solutions containing peptone and sugars were tried and at tempera- tures from 10° to 24° C. A few of the results of these tests are given in Table XVIII; it is thought unnecessary to extend the record as in most of the other cases only vegetative growth took place with an occasional oogonium, sometimes with and sometimes without oospores. Oogonia were formed under most of the conditions presented in the table but they were seldom well filled, often only a small number of the oogonia containing oospores. The failure to produce oospores appeared generally to be connected with excessive vegetative growth although the specific value of the carbohydrate is clearly seen when the record of numbers 3, 4, 6 and 7, each of which contained 0.01 percent peptone, are compared, or when the record of number 8 is compared with that of number 11. Both of these solutions contained 0.02 percent peptone but when this only was present the fungus made a dense growth which rapidly filled the dish and no oogonia were produced. The addition of sucrose restricted the growth and caused the development of many well filled oogonia In nearly all peptone-sucrose solutions oogonia were freely produced; maltose and dextrose were also generally satisfactory; levulose always encouraged a great growth of delicate hyphae while few oogonia were formed and those were usually empty. 564 ADRIAN J. PIETERS TABLE XVIII Achlya prolifera. Mycelia Out of Pea and Peptone-Sucrose-Salis Solution into Solu- tions Under Column 2. Estimated Number of Oogonia and Oospores. The Figures for Oospores at Temperatures Given are Estimated Percentages Pea Extract, 200-229 C. Peptone-Sucrose-Salts, 200-229 C. 100-159 C. Pea Extract, 169-189 C. 5 6 7 8 9 Media. Oosp. Oosp. Oog. Haemoglobin 0.1%. a 0.05% Peptone.o;01%. .-: Peptone 0.01% + dextrose M/500 -Peptone 0.01% + levulose M/500 Peptone 0.01% + maltose M/500 Peptone 0.01% + sucrose M/200 Peptone 0.02%.... Few Few III 50 III 90 Great growth only 9g | Peptone 0.02% + dextrose M/1000 Peptone 0.02% + levulose M/250) I Peptone 0.02% + sucrose M/300 III Peptone 0.02% + sucrose M/500 Few IT 25 Great growth only 95 Great growth only Peptone 0.03% + dextrose M/300 I Peptone 0.03% + maltose M/300 Peptone 0.03% + sucrose M/300 50 Few 60 95 IT 50 The experiment with 0.02 percent peptone and sucrose, number II, was among the earlier ones and it seemed reasonable to conclude that this was a favorable medium for the production of oogonia and that experiments at room temperature would give reliable results. Series V was therefore planned and carried through, the mycelia being grown in the culture media for seven days and then transferred, after being twice washed, to a solution containing 0.02 percent peptone and 1/300 molecular sucrose. through at room temperature. xX: The entire experiment was carried The results are presented in Table In all cases more or less growth took place in the peptone- RELATION BETWEEN VEGETATIVE VIGOR AND REPRODUCTION 565 sucrose solution and in some the formation of oogonia and oospores was all that could be desired. The results, as a whole, however, are not regular enough to warrant the statement that the failure of some mycelia to produce oogonia was due to the preceding conditions of growth. This however does not affect the general value of the results as pointing to the conclusions previously reached. It is true that the mycelia with the largest number of oogonia and oospores are also among those having the greatest weight, but not all of the latter class have developed large numbers of oogonia (see numbers 12, 15, 17), while the records of several numbers show a good develop- ment of oogonia together with weights one half to one third as great as those from other lots. When the solutions containing sucrose are not overheated there is clearly little invert sugar; the fungus makes practically no more growth in such solutions than in peptone alone and the number of oogonia is about the same on mycelia from all these solutions; compare the record of numbers 2 and 6, 3 and Io. The weight of mycelium out of levulose-peptone solution was nearly the same as that out of peptone alone and the mycelium out of the former solution responded to the peptone-sucrose environment by an unusually vigorous growth of delicate hyphae which soon filled the dish. This phenomenon was observed in two out of three series of experiments while in a third oogonia were formed on mycelium out of levulose-peptone but these were not nearly so numerous as on other mycelia. The influence of levulose when used as a culture medium for Achlya prolifera was certainly against the formation of oogonia. The addition of salts produced a marked increase in growth with all the sugars, but most with the sucrose, doubtless because of the ex- tensive inversion of the sucrose under the action of the high tempera- ture and salts. Two other series of experiments were made with A. prolifera, and in both the results were similar to those recorded for series V. In one series a transfer solution weaker in peptone (0.01 percent) and stronger in sucrose (1/200 molecular) was used in the hope that this would lead to a better development of the oospores. The results were, however, no more uniform than before as only a small pro- portion of oogonia in any lot contained oospores. In another series various salts were added to the peptone-sugar solutions and to the peptone alone. The salts used were various phosphates, nitrates and sulphates, the same as were used in the experiments with S. monoica, 566 ADRIAN J. PIETERS TABLE XIX Series V. Achlya prolifera. Mycelia Grown Seven Days in Media Shown in Column 2, Transferred to Peptone 0.02 Percent + Sucrose M/300. Weight of Mycelia Grown Fifteen Days in 200 cc. of Media Shown in Column 3. Numbers of Oogonia and of Oogonia with Oospores Estimated, the Latter in Percentages 3 4 5 I 2 ; : Oogonia Weight in : . Niiieraee Oogonia Gee Tal Pea ei ee seas ac Boe cee Re panera ne .O9I II 25% 2:| Peptonet0.1 92a. 2. see ee eee .025 IT LO:+ 3 i O12 G6 a via ok A A ee ee ee 0332 I] HOje 4 s Os5 Tos wscka nibs he BRS a ORC Ce ee .039 II-III 20 5 iH TOY cc Sok aes oR re es | 0405 II-III aes 6 ey O.0 7 - SUCLOSE anh. ha eet a ee .0270 Il 10 7 a Onl Gott anal t seven eck 3 tec ae heen ae .0476 II Be 8 7. ON Joe eX trOSe:.05 00 eae re adele ete .036 O= | Few 9 O19, - levulose cys seen ee .0286 O 1K0) ey O12 7G " SUCT OSE |cc key ie, emcee .0324 I Few II OF Grate mal tOSe! eo alse ae ei oe) sae .058 II 10% Heh as 0:20) = GeEXtrose, 2 aly, Ae Baer aoe .O412 I Few 13) O12 07; --weviloses = acne Pena .0346 o-I ig 14 a O31 %:- sucrose’ salts a. eee .I140 EA | 75% 15 e 0.1% -= maltose: saltse=) 2e ee eee .0950 I | Few 16 oi 0:1-%. + dextrose: = saltsietes- nc ee 71234 II-III | 90% 17 ik o:1% = levulose -— salts.) 2 .085 I AO, 18 | O1,95 Salts hoe ee ee ee .0466 II Few 20: Whiaemoglobinio.*..: io: oc. 5 ee ae ee eee .0242 ae | 10% see Table XVII. In every case the mycelia grown in solutions con- taining potassium, sodium or calcium phosphate, either with or with- out sugar, when transferred to peptone-sucrose solutions grew rapidly and filled the dish with a mass of fine hyphae on which few or no oogonia were formed. Mycelia out of solutions containing nitrates or sulphates, on the other hand, made little growth when transferred to the peptone-sucrose solution and the number of oogonia was recorded as I, II, or III on various mycelia. As in the case of S. monoica, the addition of phosphates to the solution increased the vegetative growth markedly, and the great growth without oogonia may be because the peptone-sucrose solution used was not the best for the production of oogonia by this kind of mycelium. DISCUSSION In the preceding pages attention has been called to such con- clusions as seemed warranted by the record of each set of experiments. RELATION BETWEEN VEGETATIVE VIGOR AND REPRODUCTION 507 These conclusions will be briefly discussed under the following heads. 1. The relation between the weight of mycelium and the number of oogonia produced. 2. The expression in one environment of tendencies acquired in a former environment. 3. The value of levulose. 4. The value of sucrose. 5. The value of phosphates. The Relation between Weight of Mycelium and the Number of Oogonia Produced The question which was stated at the beginning of the paper, whether any constant and necessary relation exists between the vegetative growth and the reproductive activity in several species of the Saprolegniaceae must be answered in the negative. The record of experiments with Achlya racemosa, A. prolifera, Saprolegnia ferax and S. monoica all agree in showing that a mycelium out of a relatively poor solution, measured by vegetative growth, may produce more or larger oogonia than are produced on a mycelium from a solution that is much better for vegetative growth. This has, of course, nothing to do with the well-known fact that after a mycelium has been transferred to a new solution, vegetative growth may prevent the formation of oogonia. Whena mycelium of S. ferax is taken from pea extract and placed in a haemoglobin solution that is stronger than O.I percent, few if any oogonia are produced; vegetative growth is excessive and prevents reproduction. Strong vegetative growth and reproduction cannot go on at the same time in the same solution but the problem under discussion is the relation between vegetative growth in one solution and reproduction by such a mycelium when transferred to another solution. It might be expected, a priori, that if a vigorous mycelium will produce a given number of oogonia when transferred to a suitable medium a more vigorous one, that is, one that had made a greater weight of dry matter in a given time in a fixed quantity of solution, would produce a greater number of oogonia. To produce oogonia after being transferred the fungus must draw upon its reserve materials and it might be thought that the dry weight of the mycelium would serve as a measure of the reserve materials available for the production of oogonia; but this was found not to be the case. 568 ADRIAN J. PIETERS When only one food, peptone, was offered in varying concentrations there was a steady increase in the dry weight with the increase in the concentration of peptone but the mycelium out of 0.2 percent, 0.5 percent and I percent produced practically the same number of oogonia. See Series II, Table XIII and Series VIII, Table XV. When sugars or sugars and salts were added to the solution the absence of any fixed relation between weight of mycelium and the number of oogonia was even more marked. This does not mean, however, that a mycelium need not be vigorous in the sense in which this term seems to have been used by Klebs, Kauffman and Obel. They evi- dently meant that a mycelium must have been well enough nourished, and that a poorly nourished mycelium was to be guarded against in experimental work. It is very clear that a fungus can grow in a solution that will nourish too little to enable it to reproduce well; such a solution is to be considered as being below the minimum con- centration necessary for satisfactory growth. For S. ferax a peptone solution containing 0.I percent peptone, with or without sucrose provided none of the sucrose has been inverted, represents such a sub-minimum concentration. The minimum concentration for the production of a “ well-nourished’’ mycelium may vary with the species. For S. monoica 0.1 percent peptone is enough to produce a well nour- ished mycelium if the number of oogonia produced be accepted as determining whether or not a mycelium has been well nourished, while S. ferax needs a stronger solution. Solutions containing nutrient substances at higher concentrations than the minimum will enable the fungus to produce an increased yield of mycelium but not a propor- tionate increase in the number of oogonia. Above the minimum concentration an increase in the number of oogonia will only be secured by changing the quality of the nutrient solution. The Effect of a Given Environment May Not Become Evident Until the Plant Has Been Transferred to Another Environment Nutritive substances do not, however, all have the same importance for the development of the different parts of the plant. Pfeffer (’97) states this in general terms when he says (Oxford Ed., 1900, p. 387) ‘‘the importance of a substance to a plant is not to be measured solely by the amount of growth which it induces.”’ While there is as yet no evidence that new species can be produced by changing the conditions, we can safely say that a plant is the RELATION BETWEEN VEGETATIVE VIGOR AND REPRODUCTION 569 creature of its environment in the sense that the potentialities of the species may be called forth in greater or less degree, or the expression of them may be retarded by the conditions under which it grows. The effect produced may be due to the environment as a whole or to certain factors. Klebs and others have already proved that conditions determine what phase of the plant’s growth shall appear and it has been shown in the preceding pages that these phases may be influenced by the conditions prevailing during vegetative growth. The plant therefore not only responds to the immediate environment but is molded by it in ways that can find expression only after that environ- ment has been changed for another. While growing vegetatively a mycelium may acquire a tendency toward the development of sporangia and oogonia and such a tendency may be carried over to the new environment into which the mycelium is transferred. This fact was recognized by Klebs who said (’99, p. 585) that in all investigations on the production of sporangia the nutrition conditions of the mycelium have a specific importance as upon them depends the ability of the mycelium to respond to the change in nutrition by the formation of reproductive organs. Klebs further states that these two forms of reproduction make different demands on the protoplasm (Nahrplasma), both quantitative and qualitative. So far as the present studies show, the minimum con- centration of peptone needed to grow a mycelium that will develop sporangia or oogonia is about the same, but the addition of sugars favored the production of oogonia and that of salts the production of sporangia. In this connection reference may be made to some observations not given in the data above. Mycelium of Achlya prolifera grown in solutions of peptone and potassium nitrate, when transferred to water developed sporangia more freely than that from any other solution and the sporangia were strikingly large, with dark walls. A similar observation was made on Dictyuchus monosporus, mycelium of which from solutions with potassium nitrate developed large dark sporangia, while that out of solutions with mono-potassium phosphate formed small light colored sporangia. This phenomenon was observed a number of times and proved quite constant and char- acteristic. The view that the fungus may, during vegetative growth, acquire a tendency toward the development of oogonia irrespective of the vigor of growth is strongly supported by the results obtained with 570 ADRIAN J. PIETERS levulose as a food as well as by those with phosphates. The fact that the effect of both levulose and phosphates was different on Achlya prolifera, from what it was on Saprolegnia serves to support this view and to show that the specific characters of different plants will react differently to similar environmental conditions. The close relation- ship existing between S. ferax and S. monoica made it reasonable to expect that similar responses would be made by these species to like conditions. But Achlya is, in habit and in choice of habitat, very different. As Petersen (’10) has pointed out, the Achlyas, though growing on dead animal matter, are frequently found on decaying plant parts, habitats not affected by the Saprolegnias. It seems probable that the specific response of the plants grown in levulose and in phosphates may be due to a storage of reserve materials which are drawn upon as soon as the fungus finds itself in a poorer nutrient medium. Klebs suggests this but seems also to hint at a more intimate change in the character of the plasma. It is sufficient for the present purpose to point out the fact that the specific effect of one solution may be to awaken an appropriate reaction when the mycelium is transferred to another solution. The Value of Levulose The results of the experiments on both Saprolegnia ferax and S. monoica support the statement that levulose is used more readily by these species than other sugars except maltose and that it has a much greater effect in developing a tendency toward the production of oogonia than maltose has. This effect on the vegetating mycelium is confirmed in the case of both maltose and levulose by the effect of dilute solutions into which mycelia are transferred for the production of oogonia. In this case also maltose promotes vegetative growth to the almost entire exclusion of oogonia while levulose encourages the production of both oogonia and antheridia. The remarkable effect of levulose on some species was shown in the case of a still unde- scribed form which was collected as number 74. The details of the experiments with this form will be given in another paper but here it may be said that while, during almost two years of cultivation on natural and synthetic media, no oogonia were produced, these ap- peared when a mixture of leucin and levulose was used. The result was undoubtedly due to the levulose, as leucin alone or with any other carbohydrate or with salts failed to produce a like effect. RELATION BETWEEN VEGETATIVE VIGOR AND REPRODUCTION 571 An apparent exception to the statement that levulose is as good a food for vegetative growth as maltose is found in the results shown in Table XVI. At the completion of this series (IX) it seemed difficult to account for the small amount of dry weight out of the peptone- levulose solution compared with that out of peptone-dextrose and peptone-maltose. On comparison of the conditions under which the various series of experiments had been made, it developed that the levulose used in the earlier experiments had been from a certain lot from Kahlbaum. The supply being exhausted and no more from the same source being available, the solutions for series IX were made up with levulose from Schuckardt. It seemed probable therefore that the smaller weight could be accounted for by the difference in the quality of the levulose. To test this the small amount remaining in the original Kahlbaum bottle was carefully collected and two lots of two times 100 cc. each of a solution containing 0.1 percent peptone and 1/50 molecular levulose were made up, one set with the Kahlbaum and one with Schuckardt levulose. After being inoculated with S. monoica the solutions were allowed to stand 20 days and the mycelia were then dried and weighed. The average weight of one lot out of the Kahlbaum levulose was .0212 milligrams, and out of the Schuckardt lot .0082 milligrams; practically. the same degree of difference as that existing between the weights of mycelia out of peptone-levulose solu- tions recorded in Tables XV and XVI. It was not possible to under- take an analysis of the sugars from these two sources but the organism clearly recognized a difference between them. This experience per- haps suggests one of the reasons for the sometimes contradictory results secured by different workers. The reactions of living things to their surroundings are extremely delicate. Not only must the inherent characters of different species be considered, but in each species there are most probably a number of strains each of which makes its own peculiar response to a given set of conditions. Add to this the effects produced on a certain organism by supposedly chemically pure sub- stances from different manufacturers and the conditions are present for varying results from which conclusions may be drawn leading to endless controversy. Possibly the result which Klebs reported, that S. mixta made a better growth in sucrose, dextrose and maltose than in levulose may have been due to the use of a poorer levulose than that which the writer used in the earlier series. 5/2 ADRIAN J. PIETERS The Value of Sucrose The records of the tests with solutions containing sucrose have been difficult of interpretation throughout the work. Under the discussion of Table X it was pointed out that the close agreement in the weight between the mycelium out of 0.1 percent peptone and that out of peptone and sucrose, as compared with the results when maltose was used, showed that little if any sucrose was utilized by the plant. The mycelium out of solution 8, however, showed not only a considerable increase in weight, but also in the number of oogonia formed over that out of No. 11, the difference between the two solutions being the addition of sucrose in number 8. The results recorded in Table XIII are similar, the fungus growing in a solution containing sucrose having made little more weight than that grown in peptone alone, and the number of oogonia formed on the mycelium out of sucrose and peptone is nearly the same as that from the mycelium out of peptone alone. At the time the experiments were catried on the writer was not aware of the work of Noel Deerr (10) on the effect of high temperatures on the inversion of sucrose. Deerr found that when sugar solutions in pure water were heated in an autoclave to a temperature of II10° no invert sugar was formed but that above that temperature a rapid increase in inversion with equal increments of temperature took place. Salts, as the nitrates of potassium, sodium and others, and the sulphates, as that of mag- nesium, increased the amount of inversion. The above facts offer an explanation of the results obtained with sucrose. The solutions used were always sterilized in an autoclave at a temperature of about I12°-120° C., but occasionally the temperature rose to 125° C. Ac- cording to Deerr’s results a small amount of invert sugar should have been made in practically every solution in which sucrose was used and it will be noted that the weights secured from mycelia out of peptone and sucrose are consistently a trifle greater than those from mycelia out of peptone alone. When salts were added, and occasion- ally when peptone and sucrose alone were present, a more marked increase in weight resulted. In order to determine whether invert sugar was formed under the conditions prevailing in the above described experiments a series of solutions was made up containing in each case 0.1 percent peptone and 1/50 molecular sucrose. One set was sterilized at 110° C., one at 115° C. and the other at 125° C. Another series was also prepared RELATION BETWEEN VEGETATIVE VIGOR AND REPRODUCTION 573 in which the following salts were added to the peptone and sugar, magnesium sulphate, potassium nitrate, and di-hydrogen’ potassium phosphate, each salt being present in 1/600 molecular. A third series was like the second except that mono-hydrogen potassium phosphate was substituted for the di-hydrogen. The two series containing salts were sterilized at 110°, 115°, and 125° C. These solutions and tem- peratures were selected because they represent the conditions of the foregoing experiments and not for the purpose of making a detailed study of the reactions involved; quantitative determinations of the amount of invert sugar present in each lot after being sterilized for 30 minutes were made by Mr. R. B. Harvey, PhC. Of the solutions sterilized at 110° C. those with peptone and sugar alone and with the mono-hydrogen phosphate showed no inversion, while in that with the di-hydrogen phosphate 1.2 percent of the sucrose had been in- verted. Sterilized at 115° C. the peptone-sugar solution and the solution containing mono-hydrogen potassium phosphate showed a trace of inversion, not enough to express in percentage, while in that containing the di-hydrogen 1.9 percent of the sucrose had been in- verted. When the temperature at which the solution was sterilized went up to 125° C. 1.2 percent of the sucrose had been inverted in the solution containing peptone and sucrose only, 0.6 percent in that containing mono-hydrogen potassium phosphate, and 4.7 percent in that with di-hydrogen potassium phosphate. The fact that more inversion took place when the di-hydrogen potassium phosphate was used is interesting because this was used in practically all of the solutions made up for the experiments with Saprolegnias. On the whole the results correspond to those obtained by Deerr: at 110° C. there was no inversion, while at the higher temperatures an increasing amount of sucrose was inverted and when salts, including the di- hydrogen potassium phosphate were used a considerable amount of invert sugar was present before the solution was inoculated. Invert sugar was therefore available but varied in amount in different solu- tions and under different conditions. We may conclude that S. ferax does not contain invertase and cannot make use of sucrose. In sterilized culture media, however, there is sure to be more or less invert sugar present owing to the inversion caused by heating and by the presence of salts. What has been shown to be true of S. ferax is equally true of the other Sapro- legniaceae so far as they have been studied by the writer. 574 ADRIAN J. PIETERS The Value of Phosphates No attempt was made to determine the need of the fungus for inorganic nutriment. This would have been difficult, if not impossible, with peptone as the source of nitrogen, as the peptone itself contains a large percentage of total salts. An analysis of the Witte peptone used showed 2.78 percent total salts and a qualitative test revealed the presence of phosphates, sulphates, chlorides, and of potassium, sodium, magnesium, calcium, aluminum, and of ferric iron (K3(Fe- Cu)s), so that it was manifestly impossible to exclude the presence of all of these salts except the one wanted for a particular experiment. However, the tests in which certain salts were added to the culture medium showed that with an abundant supply of phosphates Sapro- legnia developed a greater capacity for the production of oogonia than when other salts were added, and this irrespective of whether potassium, sodium, or calcium phosphate were used. This result is in general agreement with the conclusions of Klebs, that the phosphates favored the production of oogonia, though Kauffman secured as good results from some of the other salts as from the phosphates. In Achlya prolifera the increase in growth due to the phosphates was marked, but in this case the mycelium may have carried over - sufficient reserve material to produce a vigorous growth in the peptone- sucrose solution, and thus prevented the formation of oogonia. SUMMARY 1. There is no constant and necessary relation between vegetative growth and sexual reproduction when the food offered exceeds the minimum concentration necessary for the given species. : 2. The minimum concentration of food necessary varies with the species but lies, in general, in the neighborhood of 0.1 percent peptone for the production of both sporangia and oogonia. 3. While growing vegetatively a mycelium may develop tendencies that may affect the number and character of the reproductive organs produced under subsequent and different conditions. 4. Of the carbohydrates used maltose and levulose are especially useful for vegetative growth and the latter has an especial value for the production of oogonia. 5. Sucrose is probably not used by species of Saprolegnia or of Achlya unless it is first inverted by some other agency. RELATION BETWEEN VEGETATIVE VIGOR AND REPRODUCTION 575 6. Phosphates in the culture solution tend to increase the repro- ductive capacity of the fungus. 7. In preparing a culture solution for the Saprolegniaceae due regard must be paid to the fact that the conditions present during vegetative growth may affect the mycelium qualitatively as well as quantitatively. Not only is a vigorous mycelium needed but the composition of the medium must be accurately given for each experi- ment if workers are to arrive at comparable results. LITERATURE CITED] Bary, A. de (’81). Untersuchungen tiber die Peronosporeen und Saprolegniaceen. Beitrage zur Morphologie und Physiologie der Pilze. IV Reihe, 1881. (’88). Species der Saprolegniaceen. Bot. Zeit. 46: 1888. Deerr, Noel (’10). The Effect of High Temperatures on Cane Sugar in Solution. Hawaian Sugar Planters’ Exp. Sta. Bull. 35. (19102). Review in American Chemical Society, Chemical Abstracts 5: 2574. I9gII. Freund, H. (’08). Neue Versuche tiber die Wirkungen der Aussenwelt auf die unge- schlechtliche Fortpflanzung der Algen. Flora 98: 41-100. 1908. Horn, L. (’04). Experimentelle Entwickelungsanderungen bei Achlya polyandra de Bary. Annales Mycologici 2: 207-241. 1904. Humphrey, J. E. (’92). The Saprolegniaceae of the United States with Notes on Other Species. Proc. Amer. Phil. Soc. 17: 63. 1892. Kauffman, C. H. (’08). A Contribution to the Physiology of the Saprolegniaceae, with Special Reference to the Variations of the Sexual Organs. Annals of Botany 23: 362. 1908. Klebs, G. (’90). Uber Vermehrung bei Hydrodictyon. Flora 73: 351-410. 1890. Klebs, G. (’96). Die Bedingungen der Fortpflanzung bei einigen Algen und Pilzen. Jena, 1896. Klebs, G. (’98). Zur Physiologie der Fortpflanzung einiger Pilze I. Jahrb. wiss. DOts32+/1:.~ 1898, Klebs, G. (’99). Same title, II. Jahrb. wiss. Bot. 33: 513-593. 1899. Klebs, G. (’00). Same title, III. Jahrb. wiss. Bot. 35: 80-203. 1900. Klebs, G. (’03). Willkiirliche Entwickelungsanderungen bei Pflanzen. Jena, 1903. Klebs, G. (’04). Ueber Probleme der Entwickelung. Biol. Centralbl. 24: 1904. Klebs, G. (’13). Ueber das Verhaltnis der Aussenwelt zur Entwickelung der Pflan- zen. Sitzungsb. Heidelb. Akad. Wiss. 1913. Lechmere, A. E. (11). Further Investigations of methods of Reproduction in the Saprolegniaceae. New Phytologist 10: 167-203. IQII. Livingston, B. L. (’05), Britton, J. C., and Reid, F. R. Studies on the properties of an unproductive soil. U.S. Dept. Agr. Bur. Soils Bull. 28. 1905. Maruzio, Adam (’94). Zur Entwickelungsgeschichte und Systematik der Sapro- legniaceen. Flora 79: 109. 1894. 576 ADRIAN J. PIETERS Nageli, Carl ('79). Ernahrung der niederen Pilze durch Kohlenstoff und Stick- stoffverbindungen. Botanische Mittheilungen, p. 395. Miinchen, 1881. Obel, P. (’10). Researches on the Conditions of Forming Oogonia in Achlya. Annales Mycologici 8: 421-443. 1910. : Peterson, Henning E. (’10). An Account of the Danish Freshwater Phycomycetes with Biological and Systematical Remarks. Annales Mycologici 8: 494-560. Pfeffer, W. (’97). The Physiology of Plants, 1897. Oxford edition. 1900. Pieters, A. J. (15). The Ferax Group of the Genus Saprolegnia. Mycologia 7: 307-314. Tucker, J. H. (’12). A Manual of Sugar Analysis. Ig12. Voéchting, H. (’00). Zur Physiologie der Knollengewachse. Jahrb. wiss. Bot. 34: 1. 1900. INDEX -TO VOEUME II. (New names and final members of new combinations are in heavy-face type.) Abnormal and normal permeability, 93 Absorption of ions by living and dead roots, 250 Achlya prolifica, 562; racemosa, 541 Agropyron sericeum, 309 Agrostis exarata ampla, 303; exarata microphylla, 303; exigua, 303; Schiede- ana, 304 Algae, fixative and stain for, 89 Anatomy of a hybrid Equisetum, 225 Anatomy of the Malvales, 238 Anatomical study of Gymnosporangium galls, 402 Ancestry of Angiosperms, I Andropogon stolonifer, 299 Angiosperms, phylogeny, ancestry, and early climatic environment of, I Apple, host of Coniothryium, 449 Arabin, as source of carbon, 382 Arabinose, as source of carbon, 382 Aristida adscensionis, 301 Ascospore expulsion of Endothia para- silica, 429; traps, 430 - Asteraceae, embracing Espeletia, 468 Auxanometer, a simplified precision, 95 BaILey, IrRviING W. (See Sinnott, Edmund W., 1) BARTLETT, HARLEY Harris. (See True, R. H. 255; 311); The mutations of Oenothera stenomeres, 100; The experi- mental study of genetic relationships, 132 BESSEY, CHARLES Epwin, life and work of, 505 Bibliography of writings of Charles Edwin Bessey, 512 Biological spectrum of flora of New York, 27 Se Birds, dissemination of spores of chestnut blight fungus by, 167 Botryodiplodia, probable non-validity of, 324 Bovie, W. T. A simplified precision auxanometer, 95 Brown, WILLIAM H. The development of Pyronema confluens var. inigneum, 289 Brown-rot fungus, effects of, upon the composition of the peach, 71 Calamagrostis scabra, 304 Calcium hypochlorite as a seed sterilizer, 420 Calcium nitrate, experiments with, 261, 312, 316; sulphate, experiments with, 264 CAMPBELL, DouGLAS HouGHToN. The morphology and systematic position of Podomitrium, 199 Campulosus floridanus, 306 Cannon, W. A. On the relation of root growth and development to the tem- perature and aeration of the soil, 211 Castela galapageia, notes on the forms of, 279; f. albemarlensis, 280; f. bind- loewsts,. 283: f. carolensis, 2843 f. duncanensis, 286; f. jacobensis, 287 Chaetochloa lutescens, 299 Chaetodiplodia, probable non-validity of, 324 Chamaeophytes, 24 Chestnut blight fungus, 162; 429 CLAUSEN, R. E. (See Goodspeed, T. H., 332) Climatic distribution of foliar types, 15 Climatic environment, early, of Angio- sperms, I 578 CoLLEY, REGINALD H. (See Curtis, Otis F., 89) Color phenomena in Coniothryium, 465 Coniothyrium pirinum, dimorphism in, 449 | CRABILL, C. H. Dimorphism in Conz- othyrium pirinum Sheldon, 449 Culture of Pyronema, 289 Curtis, OT1s F. and COLLEY, REGINALD H. Picro-nigrosin, a combination fixative and stain for algae, 89 Danthonia Cusickii, 305; Macounii, 305 Development of Pyronema_ confluens var. inigeum, 289 Development, on the relation of, to temperature and aeration of the soil, 211 Dimorphism in Coniothyrium pirinum, 000 Diplodia, characters of genus, 329 Diplodia gossypii, 326; natalensis, 326; tubericola, 328 Diplodiella, probable non-validity of, 324 Distilled water, the toxicity of, 389 Durio zibethinus, transverse section, 246 EHLERS, JoHN H. The temperature of leaves of Pinus in winter, 32 Endothia parasitica, 162; 429 Equisetum, hybrid, on the anatomy of, 225 Equisetum variegatum, var. Jesupi, 225 Eragrostis Barreliert, 308; cilianensis, 309; floridana, 308 Espeletia argentea, 477; banksiaefolza, 471; bracteosa, 484; corymbosa, 484; floccosa, 481; funckit, 485; grandzflora, 474; grisea, 477; jahnii, 479; lindenzz, 483; moritziana, 472; nerifolia, 472; paltonioides, 482; pannosa, 480; schultzit, 475; spicata, 479; weddellit, ‘473 Excretions, toxic, by roots, 396 Expressed vegetable saps, osmotic pres- sure of, 418 . INDEX TO VOLUME II Factors influencing flower size in Nicoti- ana with special reference to questions of inheritance, 332 Fagus grandtfolia, transverse section, 246 Fixative and stain for algae, 89 Flora of New York and vicinity, 23 Floral parts in Angiosperms, 8 Foliar evidence as to ancestry of Angio- sperms, I Foliar evolution, 13 ForsaitH, C. C. Some features in the anatomy of the Malvales, 238 Fouquierta splendens, daily growth rate of roots, 220; hourly growth rate of roots, 221 Fremontia californica, transverse section, 246 FRoMME, F. D. Negative heliotropism of urediniospore germ tubes, 82 -Galls of Gymnosporangium, anatomical study of, 402 GATES, FRANK C. A woody stem in Merremia gemella induced by high - warm water, 86 GaTEs, R. RuGGLes. Heredity and mutation as cell phenomena, 519 Genetic relationships, experimental study Of, 832 Genetic relationship of parasites, 116 Geophytes, 24 Germ-tubes of urediniospores, 82 Glomerella cingulata, utilization of pen- toses by, 375 Glucose, as source of carbon, 382 Gnetum, morphology of, 161 GOODSPEED, T. H. and CLAUSEN, R. E. Factors influencing flower size in Nicotiana with special reference to questions of inheritance, 332 Grasses, new or noteworthy, 299 GREENMAN, J. M. Morphology as a factor in determining relationships, Se Growth-forms of the flora of New York and vicinity, 23 Gymnopogon Chapmanianus, 306 INDEX TO VOLUME II Gymnosporangium galls, anatomical study of, 402 Gymnosporangium globosum, galls of, 411; juniperi-virginianae, galls of, 402 Harris, J. ARTHUR. An extension to 5.99° of tables to determine the osmotic pressure of expressed vege- table saps from the depression of the freezing point, 418 Hawkins, Lon A. Some effects of the brown-rot fungus upon the composi- tion of the peach, 71; The utilization of certain pentoses and compounds of pentoses by Glomerella cingulata, 375 HEALD, F. D. and STUDHALTER, R. A. Seasonal duration of ascospore expul- sion of Endothia parasitica, 429 Heliotropism, negative, of urediniospore germ tubes, 82 Helophytes, 25 Hemicryptophytes, 24 Heredity and mutation as cell phe- nomena, 519 HIBBARD, R. P. The question of the toxicity of distilled water, 389 Hibiscus tiliaceus, transverse section, 246 Hitcucock, A. S. New or noteworthy grasses, 299 Holcus halepensis, 299; sorghum, 299 HoLpDEN, RuTH. The anatomy of a hybrid Equisetum, 225 HvuBBARD, F. Tracy. A _ taxonomic study of Setaria ttalica and its im- mediate allies, 169 Hybrid Equisetum, on the anatomy of, 225 Hypochlorite of calcium as a sterilizer, 420 seed Inheritance in Nicotiana, 332 Ions, absorption of, by living and dead roots, 250 Ions, exchange of, between roots of Lupinus albus and culture solutions, containing one nutrient salt, 255; containing two nutrient salts, 311 979 Jounson, H. V. The absorption of ions by living and dead roots, 250 KERN, FRANK Dunn. The genetic relationship of parasites, 116 Lasiodiplodia, probable non-validity of, 324 Lasiodiplodia tubericola, 326; theobromae, 326 Leaves of Pinus in winter, temperature of, 32 ; Lupinus albus, use of in distilled water experiments, 391 Lupinus albus, exchange of ions between the roots of, and culture solutions, 255; 311 Magnesium nitrace, experiments with, 266, 316; sulphate, experiments with, 268 Malvales, anatomy of, 238 Manisuris, 299; fasciculata, 299 Merremia gemella, a woody stem of, induced by high warm water, 86 Millets, 196; 197 Morphology and systematic position of Podomitrium, 199 Morphology as a factor in determining relationships, III Morphology of Gnetum, I61 . MUENSCHER, WALTER L. C. A study of che relation of transpiration to the size and number of stomata, 486 Mutation and heredity as cell phenom- ena, 519 Mutations of Oenothera stenomeres, 100 New or noteworthy grasses, 299 New York and vicinity, flora of, 23 Nicotiana, factors influencing flower size of, 332 Nicotiana Tabacum vars. X N. syl- vestris, 334; var. macrophyllum, 334; flower size of and factors influencing, 332 Node in Angiosperms, 5 Normal and abnormal permeability, 93 580 Notes on the form, of Castela Galapageza, 279 Notholsus mollis, 304 Nutrient solution for plants, 157; agar, 449 Oenothera stenomeres, mutations of, 100 Oenothera stenomeres mut. gigas, 104; 108; mut. lasiopetala, 101; 102; f. typica, 108 Opuntia versicolor, daily growth rate of roots, 220 Orcuttia californica, 308 Osmotic pressure, tables to determine, 418 OsTERHOUT, W. J. V. Normal and ab- normal permeability, 93 Palaeobotanical evidence, 4 Panicularia erecta, 309 Pappophorum bicolor, 308 Parasites, genetic relationship of, 116 Peach, some effects of the brown-rot fungus upon the composition of, 71 Pennisetum glaucum, 300 Pentoses, utilization of, by Glomerella cingulata, 375 Permeability, normal and abnormal, 93 Pfitzer, use of picro-nigrosin, 89 Phanerophytes, 24 Photosynthesis at low temperatures, 37; in winter, 64 Phylogenetic evidence, 10; conclusions, 18 Phylogeny of Angiosperms, I Pinus, leaves in winter, temperature of, 32 Picro-nigrosin, 89 PIETERS, ADRIAN J. The relation be- tween vegetative vigor and_ repro- duction in some Saprolegniaceae, 529 Plus and minus strains, 449 Poa Merrillana, 309; Wrightii, 309 Podomiurium Malaccense, 199; 200 Podomitrium, morphology and _ syste- matic position of, 199 PooL, RaymMonp J. A brief sketch of INDEX TO VOLUME II the life and work of Charles Edwin Bessey, 505 Potassium chloride, experiments with, 275; dihydrogen phosphate, 273; ni- trate, 270, 312, 320; sulphate, 272 Precision auxanometer, 95 Preliminary note on the morphology of Gnetum, 161 Prosopis velutina, daily growth rates of roots, 220; hourly growth rate of roots, 221 Puccinia Rhamni, 82, 83, 84 Pycnospores of chestnut blight fungus, 162 Pyronema confluens var. inigeum, de- velopment of, 289 urediniospores of, Raunkiaer, system of temperature fac- tors, 23 Relationships, study of, 132 Relationships, morphology as a factor in determining, III Relationship of parasites, 116 Reproduction, in some Saprolegniaceae, 529 Reversions of Angiosperms, 9 Root-growth, on the relation of, to the temperature and aeration of the soil, 2m Roots, absorption of ions by, 250 Rytilix granularis, 299 genetic, experimental Saprolegnia ferax, 545; monoica, 557 Saprolegniaceae, the relation between vegetative vigor and reproduction in some, 529 Sclerotinia cinerea, cause of brown-rot of peach, 71 Seed sterilization, by calcium hypo- chlorite, 420; historical summary of methods, 420 Seedling of Angiosperms, 8 Setaria ttalica and its immediate allies, a taxonomic study of, 169 Setaria italica subsp. stramineofructa, INDEX TO VOLUME II 188; f. breviseta, 189; subvar. ger- manica, 189; f. mitis, 190; var. Hostii, 190; subvar. Metzgeri, 191; f. curtiseta, 191; var. brunneoseta, 192; f. brachychaeta, 192; subvar. densior, 192; subsp. rubrofructa, 193; f. gigas, 193; subvar. pabularis, 193; var. purpureoseta, 194; subvar. vio- lacea, 194; var. rubra, 194; f. auranti- aca, 195; subvar, condensa, 195; subsp. nigrofructa, 195; var. atra, 196 SHIVE, JOHN W. A three-salt nutrient solution for plants, 157 SINNOTT, EDMUND W. and _ BaILeEy, IRvING W. _ Investigations on the phylogeny of the Angiosperms. 5. Foliar evidence as to the ancestry and early climatic environment of the Angiosperms, I Sodium chloride, experiments with, 275 Sphenopholis pennsylvanica, 304 Sporobolus contractus, 303; macrus, 303 Stain and fixative for algae, 89 STANDLEY, PAuL C. The genus Espe- letia, 468 Starch agar, 449 STEWART, ALBAN. An anatomical study of Gymnosporangium galls, 402; Notes on the forms of Castela Galapageia, 279 Stipa lepida, 302; lepida Andersoni, 303; pulchra, 301 Stomata, relation of transpiration to size and number of, 486 Strains, + and —, in Coniothryium, 449 STUDHALTER, R. A. (See Heald, F. D., 429) STUDHALTER, R. A. and HEALD, F. D. The persistence of viable pycnospores of the chestnut blight fungus on normal bark below lesions, 162 581 Tables to determine osmotic pressure, 418 TAUBENHAUS, J. J. The probable non- validity of the genera Botryodiplodia, Diplodiella, Chaetodiplodia, and Lasi- odiplodia, 324 Taxonomic study of Setaria italica and its immediate allies, 169 TAYLOR, NORMAN. The growth-forms of the flora of New York and vicinity, 23 Temperature of leaves of Pinus in winter, 22 Therophytes, 25 THompson, W. P. Preliminary note on _ the morphology of Gnetum, 161 Three-salt nutrient solution, 157 Tilia americana, transverse 246 Torresia, 300; alpina, 300; macrophylla, 300; mexicana, 301; odorata, 301; pauciflora, 301 Toxicity of distilled water, 389 Transpiration, relation of, to size and number of stomata, 486 TRUE, RopDNEY H. and _ BARTLETT, HarteEy Harris. The exchange of ions between the roots of Lupinus albus and culture solutions containing one nutrient salt, 255; The exchange of ions between the roots of Lupinus albus and culture solutions containing two nutrient salts, 311 section, Urediniospore germ tubes, negative heliotropism of, 82 Willkommia texana, 307 WiLson, JAMES K. Calcium hypo- chlorite as a seed sterilizer, 420 Clarke Memo Paper A Permanent Record Paper with Half Tone and Color Printing Qualities CLARKE & COMPANY 741% Brunswick Building 225 Fifth Avenue NEW YORK ~ MANUALS OF POLYPORES AND BOLETES By WILLIAM A. 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According to engineering authorities, the more severe the weather conditions, the harder and stronger these shingles become, because such conditions tend to hasten the setting or crystallizing of the cement or binding material. Supplied in sizes and shapes to meet all requirements, in colors of gray, Indian red and mottled brown. Our latest J-M Transite Asbestos Shingle booklet will be forwarded upon re- quest. Write our nearest branch. ko One Big Heating Plant Costs Less to Operate than a Dozen Little Ones Institutions, colleges, etc., that have a small heating plant for each building can often save thousands of dollars by installing one central heating system and convey- ing steam pipes through J-M Sectional Conduit to all buildings. J-M Sectional Conduit carries steam thousands of feet with practically no radiation or condensation. It lasts indefinitely because made of special tile, salt glazed inside and out. Not affected by acids, oils, fire or the action of the earth. Readily removed and re- placed. Write nearest Branch for Catalog No. 112. Bey) H. W. JOHNS-MANVILLE CO. Manufacturers of Roofings; Stucco; Pipe Coverings: Cold Storage Insulation; Waterproofing; Sanitary Specialties; Acoustica! Correction; Cork Tiling, Etc. Akron Columbus Milwaukee St. Louis Albany Dallas Minneapolis St. Paul Atlanta Dayton Newark Salt Lake City Baltimore Denver New Orleans’ San Francisco Birmingham Detroit New York Seattle Boston Duluth i Omaha Syracuse Buffalo Galveston Philadelphia Toledo Chicago Houghton Kansas City Pittsburgh Washington Cincinnati Houston Los Angeles Portiand Wilkesbarre Cleveland indianapolls Louisville Rochester Youngstown Memphis THE CANADIAN H. W. JOHNS-MANVILLE CO., Limited Toronto Montreal Winnipeg Vancouver RECENT PUBLICATIONS OF THE NEW YORK BOTANICAL GARDEN JOURNAL, Vol. XVI, No. 181. An illustrated monthly containing notes and non-technical articles of general interest. $1.00 a year. MYCOLOGIA, Vol. VII, No. 1. An illustrated bimonthly publication devoted to fungi and lichens Contains /zdex to American Mycological Literature. $3.00 a year. BULLETIN, Vol. VIII, No. 31. $3.00 per volume. A separate, in advance, on Philippine Mosses by R. S. Williams, containing a list of 240 species, 27 of which are described as new. MEMOIRS, Vol. 5. $2.00. Flora of the vicinity of New York: A contribution to Plant Geography, by Norman Taylor, nearly 700 pages, containing a discussion of the factors affecting the distribu- tion of the local flora and a catalogue of the flowering plants and ferns occurring within 100 miles of New York City. NORTH AMERICAN FLORA. Descriptions of the wild plants of North America. To be com- plete in about thirty volumes of four or more parts each. Subscription price for entire work, $1.50 per part; separate parts $2.00. Twenty three parts have been issued, eight on fungi, twelve on flowering plants, one on ferns, and two on mosses, Parts recently issued are: Vol. 10, part I, issued July 28, 1914, Agaricaceae (pars). Vol. 29, part 1, issued August 31, I914. Clethraceae, Lennoaceae, Pyrolaceae, Monotropaceae, Ericaceae. Vol. 34, part I, issued December 31, 1914. Carduaceae. CONTRIBUTIONS. A series of technical papers by students or members of the staff. $5.00 per volume; 25 cents each. BRONX PARK, NEW YORK CITY Clarke Memo Paper A Permanent Record Paper with Half Tone and Color Printing Qualities CLARKE & COMPANY 731 Brunswick Building 225 Fifth Avenue NEW YORK Annals of the Missour1 Botanical Garden A quarterly journal, published by the Board of Trustees of the Missouri Botanical Garden, containing scientific contributions from the Missouri Botanical Garden and the Graduate Laboratory of the Henry Shaw School of Botany of Washington University in affiliation with the Missouri Botanical Garden. Editorial Committee J. RK. Schramm The Annals of the Missouri Botanical Garden appears four times during the calendar year, February, April, September, and November Four numbers constitute a volume. G. T. Moore B. M. Duggar Subscription price, $3.00 per volume The publication of the Annals of the Missouri Botanical Garden was begun January, 1914, and the completed volume (1914) may. be had, unbound, at the regular subscription price. CONTENTS OF VOLUME I, I914 (432 pages) No. 1 The effect of surface films and dusts on the rate of transpiration. B. M. Duggar and J. S. Cooley. Some pure culture methods in the algae. ‘ J. R. Schramm. The identification of the most characteristic sali- vary organism and its relation to the pollution of air. A. G. Nolte. The Polyporaceae of Ohio. LE ©: Overholts: No. 2 . A contribution to our knowledge of the relation of certain species of grass-green algae to ele- mentary nitrogen. J. R. Schramm. The Thelephoraceae of North America. I. E. A. Burt. Indications regarding the source of combined nitrogen for Ulva Lactuca. G. L. Foster. The effect of certain conditions upon the acidity of tomato fruits. B. M. Duggar and M. C. Merrill. A method for the differential staining of fungous and host cells. R. E. Vaughan. Two trunk diseases of the mesquite. H. von Schrenk. A trunk disease of the lilac. H, von Schrenk. No. 3 Descriptions of North American Senecioneae. J. M. Greenman. A study of the physiological relations of Sclero- tinia cinerea. J. S. Cooley. The Thelephoraceae of North America. IT. Craterellus. EAS, Burt: The effects of surface films upon the rate of trans- piration: experiments with potted potatoes. B. M. Duggar and J. S. Cooley. No. 4 The Thelephoraceae of North America, III. Craterellus borealis and Cyphella. FE. A. Burt: Some Oenotheras from Cheshire and Lancashire. R. R. Gates. A Texan species of Megapterium. R. R. Gates. Diagnoses of flowering plants, chiefly from the southwestern United States and Mexico. © J. M. Greenman and C. H. Thompson. Enzyme action in Fucus vesiculosus L. B. M. Duggar and A. R. Davis. Address all communications to the Missouri Botanical Garden, St. Louis, Mo. BOTANICAL SOCIETY OF AMERICA Reorganized 1906, uniting the Botanical Society of America (1893), the Society for Plant Morphology and Physiology (1896), and the American Mycological Society (1903). OFFICERS FOR 1915 President: Joun M. CouttTer Chicago, Ill. Vice-President: R. A. Harper New York, N. Y. Treasurer: ArtHuR Ho.uick New Br ghton, N.Y. Secretary: H. H. Bartietr Washington, D. C. Councilors: GeorceE F. ATKINSON Ithaca, New Davip FarirRcHILD Washington, D.C. W. F. Ganonc Northampton, Mass. The Society consists of fellows and members, and any one actively in- terested in botanical work is eligible for membership. Candidates for member- ship are recommended by three members, not members of the Council, on blanks to be obtained from the Secretary. The annual dues are $5.00 $1.00 remitted in 1915), including subscrip- tion to the AMERICAN JOURNAL oF Botany. Candidates for membership, proposed and approved by the Council, may receive the JOURNAL at $3.00 per year, pending their election at the winter meeting of the Society. Make the Roof | FIREPROOF | as well as ARTISTIC J-M Transite Asbestos Shingles are madeof Asbestos (rock) fibres and Baraga High School, Baraga, Mich.. covered with Portland cement, both minerals som) Trans|tevAshestos Shingles They are by nature immune to the destructive action of fire, water, acids, gases, chemical fumes, and climatic conditions. ‘The longer they are exposed to the weather the harder and tougher they become. J-M Transite Asbestos Shingles are made of Asbestos and Portland Cement moulded into a homogeneous mass under hydraulic pressure. They provide a roof of indestructible rock—a roof that actually becomes more durable with age—a roof that is artistically beautiful. Supplied in sizes and shapes to meet any condition; one-eighth inch thick with smooth edges, and one-quarter inch thick with rough edges; in gray, mottled brown and Indian red. Write our nearest Branch for ‘‘J-M Transite Asbestos Shingle ’’ Booklet Buildings of Metropolitan Sanitarium heated from Central Power Plant through J-M Sectional Conduit This model institution has done away with the dirt and labor of individ- ual heating systems and is saving large sums of money by the above plan. Many of the buildings are heated from the central power plant and al- though they are at a considerable distance, they get almost 100% efficiency out of the steam where the pipes are laid in J-M Sectional Conduit. This is the most perfect underground pipe insulation on the market. Steam Pipes Laid inJ-M Absolutely watertight. Acids, gases and the chemical Sectional Conduit action of the earth do not affect it. Can be taken up i and relaid without injury. Write nearest Branch for particulars and Catalog No. 112 H. W. JOHNS-MANVILLE CO. Manufacturers of Asbestos Roofings; Stucco; Pipe Coverings: Waterpreofing; Sanitary Specialties; Acousticai Correction; Cork Tiling, Etc. Atlanta Columbus i Milwaukee Pittsburgh Baltimore Denver Minneapolis Portland Boston Detroit Newark St. Louis Buffalo Galveston New Orleans _— Salt Lake City Chicago indianapolls New York San Francisco Cincinnati Kansas City covers 2 Omaha Seattle Cleveland Los Angeles ENE, Philadelphia Toledo Louisville Memphis THE CANADIAN H. W. JOHNS-MANVILLE CO , Limited Toronto Montreal Winnipeg Vancouver 2239 BOTANICAL SOCIETY OF AMERICA Reorganized 1906, uniting the Botanical Society of America (1893), the Society for Plant Morphology and Physiology (1896) and the American Mycological Society (1903). OFFICERS FOR 1914 President: Joun M. Courter Chicago, Ill. Vice-President: R. A. Harper New York, N. Y. Treasurer: ArTHUR HoLuick New Brighton, N.Y. Secretary: H. H. BartLetr Washington, D. C. Councilors: GerEoRGE F. ATKINSON Ithaca, NAY: Davip FAIRCHILD Washington, D. C. W. F. Ganone Northampton, Mass. The Society consists of fellows and members, and any one actively in- terested in botanical work is eligible for membership. Candidates for member- ship are recommended by three members, not members of the Council, on blanks to be obtained from the Secretary. The annual dues are $5.00 ($1.00 remitted in 1915), including subscrip- tion to the AMERICAN JOURNAL OF Botany. Candidates for membership, proposed and approved by the Council, may receive the JoURNAL at $3.00 per year, pending their election at the winter meeting of the Society. Notice to Contributors It is well known that the paper commonly used for scientific periodicals, and manufactured from wood pulp by modern rapid methods, is not durable. Such paper, in publications not now more than 15-20 years old, already shows signs of disintegration, and the common magazine paper now being made is of even less permanent quality than that made 15-20 years ago. We are assured by paper experts that the scientific publications to which we are now devoting sc much care, time, and expense, will not be available to our successors of one hundred years from the date of publication. This is especially true of the illustrations issued as plates on coated paper. The Editcrial Committee of the American Journal of Botany have had this matter under careful consideration, and are now able tc secure a memoir paper, specially made from pure linen fiber, and guaranteed by the makers as permanent, so far as concerns deterioration resulting from composition and method of manu- facture. This paper will have to be made to order for the JOURNAL, and will, of course, be more expensive than the cheaper stock paper hitherto employed. In order to secure an equal degree of permanency for illustrations and text the same paper must be used for both, but the new paper will give results quite as satis- factory for all line-cut and half-tone reproduction as the coated paper which we have been using. In the future the half-tone cuts will be prepared specially for use with the new paper. The JourNAL will, therefore, until further notice, print all line-cut and half-tone illustrations, whether full page or otherwise, as text figures, on the ‘‘permanent”’ paper, backing them up with text or other illustrations, as the makeup of the issue may require. Plates on coated paper will no longer be used. The Committee believe that the advantage of permanency thus secured will ccmmend itself to every contributor who is desirous, not alone that the results of his investigations shall be artistic when published, but that they shall remain avail- able to as many future generations of readers as possible. We believe that the new plan will not involve any sacrifice of clearness and accuracy of illustration, worth considering, in view of the assured permanency of the publication. THE EDITORIAL COMMITTEE. THE NEW ERA PRINTING COMPANY LANCASTER, PA. is prepared to execute in first-class and satisfactory manner all kinds of Printing, Electrotyping and Binding. Particular attention given to the work of Schools, Colleges, Universities and Public Institutions Books, Periodicals Technical and Scientific Publications Monographs, Theses, Catalogues Announcements, Reports, etc. All Kinds of Commercial Work Publishers will find our product ranking with the best in workmanship and material, at satisfactory prices. Correspondence solicited. Estimates furnished. THE New ERA PRINTING COMPANY HUN Ma ag BY AAS cf a Oe We AGS SEAS be x [T "rummaging around in Grand- mother’s garret did you ever come’ - across an old, old umbrella having heavy reed bows or frame, instead of the” light steel ones now used. 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