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ANAS Pee AIS Sa maannnet toe" a VAY Sean = 7 a “ ‘ We = “A “WAAL | i AN a hd WAAR, ype APAAMY, gl NANN: soni he ~ ih TN ae) | (ANA, PU AEE \\ STalatalammiadl aa THT yf pata AFPAMARRARAS pe peantiene Bs SAAN nite pitta dag -- 0... of ON... ane nie ‘ av ee Re a nee if ~ Ran me me .annsha TU PA Mag Morr Ada WiA.e,. ewer WiPiasagane._- z re ; - Peery | rar a 14 | Soleil BPA | TT a TA te ee PA PEE EG PE eet oot TS ee einem ewes) ii} iP . yt tt Vunnece cS CCRCO UUM.” steal v~ Td] ao Neher ees HCeerer” anti ene, : ' : < yw a Pe bs adie Py NPN. fd \ Bw" : vuw F br wives are rytte” chee, genre SEN ears Mn Ten? 5 We Ee tees be” Oa = Eereuer” . weg ttyl” prernnie® Wew . | 1 Ty | SPN hod ‘A. \A a \ S <7 = y qe fi vy Ae rernghOe Ss SByew se yn eo wine * . wor a ad \w’> = Sot tee we cue Sey tte tn | ed Ti | \w Shy : We weve” AHG ~ weve wey ‘7 “tet it Yypses Ny ntti ry mm v Pag bdeerterten text ~~. Ago pr, everre ~ Pe VO Le “Nad. bipee ibe \ = & Len Vi . De coabh et as ta’ te! eI ree ~*~ = ‘-—— ; agree | A | i NAL ‘ wwe ’ ws AS au vO ; NM hen yc 4 a y’ ; an ey oh vee’ . aha a | Wiyr' : fa ee Lill , anvvett Naan 5 vs wn wes ‘ pds seve. “we, Mi tate ain’ “9 ey ryt d de | ute. mn a Wy vuvyTvyd ieG ue ” qd ‘wag v Vue yy? e°*y ey Sted At yer” le Wave meet se wr May) t Pd ww. —' gm ee ee nfhin ne | ail sneer. wy”: " Al Ad re N wn en KR 7 +o. het : $ ig ey iy oe Sarees oer: | : | 7 on ; es “vowel eres | | | etd by ; hath) lobdatel Ts Has é gee vet tl ba Ri ehh THO TTS LU ed | | LLU as yyy Sie ay. »EES Ay? ., ea™ ma av we . ivey a HT leah etal tll gyre one ¥ : aT TL ee rT hd ge ¥ e? r@ Seed Lad el my “y Me Vy oF ge giv grsyy544 \ / atig n 2) Vi yeu IN COOPERATION WITH THE BOTANICAL SOCIETY OF AMERICA——~ BY THE BROOKLYN BOTANIC GARDEN Ar 41 Nortu Queen Street, Lancaster, Pa. ysollal NStizy, ~ PUBLISHED F = Ei, is ” : . i | i ¢ : ne , 3 . F a ; i Py ' . et j id ‘ tei PRESS OF ; THE NEW ERA PRINTING COMPANY i : ens LANCASTER, FA. hy ~ = 7 a A | 4 * . . Oe "€ he ‘ ‘ : : ae ae ie +i ee Sa u “ he" ” Piagee i) ee sn ah 4 ' ‘ hot 1 Ps 4 : P 4 TABLE OF CONTENTS, VOLUME VIII, 1921 No. 1, JANUARY PAGE The fixation of free nitrogen by green plants (with one text figure and lls. JD) SOR PEI Sa Uo eo Oa FRANK B. WANN I Variations in the osmotic concentrations of the guard cells during the opening and closing of stomata (with seven text figures) R. G. WIGGANS 30 : The Linnaean concept of pearl millet.....-.......5.. AGNES CHASE 4I ( The effect of ck idiness on the oxygen content of water and its es 5 nificance in cranberry culture (with three text figures) / H. F. BERGMAN 50 No. 2, FEBRUARY Influence of temperature on the relations between nutrient salt pro- portions and the early growth of wheat......... W. F. GERICKE 59 The vascular anatomy of dimerous and trimerous seedlings of Phaseolus vulgaris (with twenty-three text figures) J. ARTHUR HARRIS, EDMUND W. SINNOTT, JOHN Y. PENNYPACKER, and G. B. DURHAM 63 Aspergillus flavus, A. oryzae, and associated species (with one text IMOUIGE ess. 3 CHARLES THOM and MARGARET B. CHURCH 103 No. 3, MARCH A study of Rhus diversiloba with special reference to its toxicity (with two text figures and Plate I])...::0........ James B. MCNAIR 127 The effect of salt proportions and concentration on the growth of Aspergillus niger (with six text figures)....... C. M. HAENSELER 147 Suggestions with respect to the measurement of osmotic pressure (with one text figure)...... eae L. KNUDSON and S. GINSBURG 164 Thick-walled root hairs of Gleditsia and related genera (with three eNeAMITUIGS et ks ee W. B. McDOUGALL 171 A simple method for growing plants (with one text figure) J. M. BRANNON 176 No. 4, APRIL The morphology and anatomy of Rhus diversiloba (with Plates III HG LW) os ee SRS cc AS Al AO STE nA James B. McNair 179 iV TABLE OF CONTENTS Distribution of the Malvaceae in southern and western Texas (with one stext fouse)< ha ec eee nee ane eae HERBERT C. HANSON Note on the histology of grain roots (with four text figures) GRACE A. DUNN North American Pipers of the section Ottonia (with Plates V-VIII) © WILLIAM TRELEASE Monocarpy and pseudomonocarpy in the cycadeoids (with one text figure and Plates: X— XI) 447 eee G. R. WIELAND No. 5, May Isoachlya, a new genus of the Saprolegniaceae (with Plates XIII An XA V i 4 os ee eee C. H. KAUFFMAN The transmission of Rhus poison from plant to person JAmMEs B, McNAIR The type concept in systematic botany............ A. S. HircHcock The relation of certain nutritive elements to the composition of the oat plant (with two text figures)........ JAMES GEERE DICKSON No. 6, JUNE Specialization and fundamentals in botany. . JoSsEPH CHARLES ARTHUR Certain aspects of the problem of physiological correlation C. M. CHILD Water deficit and the action of vitamines, amine-compounds, and salts;on- hydration’... .ci.9)5. 909. ee eee D. T. MacDouGAL The eusporangiate ferns and the stelar theory (with seven text figures) D. H. CAMPBELL The relation of plant pathology to human welfare..... F. L. STEVENS NOs 7 wry The relation of crop-plant botany to human welfare CARLETON R. BALL Correlations between anatomical characters in the seedling of Phaseolus vulgaris (with eight text figures) J. ARTHUR Harris, EDMUND W. SINNOTT, JOHN Y. PENNYPACKER, and G. B. DuRHAM A quantitative study of the effect of anions on the permeability of plant cells II (with one text figure) .......... OrAN L. RABER The mechanism of root pressure and its relation to sap flow JAMES BERTRAM OVERTON No. 8, OCTOBER - The vascular anatomy of hemitrimerous seedlings of Phaseolus vulgaris J. ARTHUR HARRIS, EDMUND W. SINNOTT, JOHN Y. PENNYPACKER, and G. B. DurHAM 323 Shel. 366 369 375 TABLE OF CONTENTS Vv The effect upon permeability of polyvalent cations in combination with polyvalent anions (with one text figure)........ ORAN L. RABER 382 The floral anatomy of the Urticales (with Plates XV—XXIIT). ALBERT REIFF BECHTEL 386 Genetic evidence of aberrant chromosome behavior in maize endo- epemmecwiun one text figuré)....2......0...... R. A. EMERSON 411 No. 9, NOVEMBER The interrelationship of the number of the two types of vascular bundles in the transition zone of the axis of Phaseolus vulgaris (with two text figures) J. ARTHUR Harris, EDMUND W. SINNOTT, JOHN Y. PENNYPACKER, and G. B. DURHAM 425 Area of vein-islets in leaves of certain plants as an age determinant eee CM MOLI el rs ee do M. R. ENSIGN 433 Unusual rusts on Nyssa and Urticastrum (with six text figures) E. B. MAINS 442 Miscellaneous studies on the crown rust of oats (with Plate XXIV) G. R. HOERNER 452 Comparative studies on respiration XVIII. Respiration and antagonism Mmpelodea (with two. text figures)... 2... ..00..0 00. es C.J. Lyon 458 The effect upon permeability of (I) the same substance as cation and anion, and (II) changing the valency of the same ion (with two NESS LITRES) Le cary ol ele a oe ORAN L. RABER 464 No. 10, DECEMBER ollenvand pollen enzymes......°............ Jutta BAYLES PATON 471 The embryogeny of Cyrtanthus parviflorus Baker (with Plates X XV MGM ON) Ia a: Baebes haces kta oo chai kbs Wm. RANDOLPH TAYLOR 502 Studies on plant cancers III. The nature of the soil as a determining factor in the health of the beet, Beta vulgaris, and its relation to the size and weight of the crown gall produced by inoculation with Bacterium tumefaciens (with nine text figures) MICHAEL LEVINE 507 ae POM ONODING NY Ul eek 6 ie oe ee he icteric eccapes EV ne 526 (Dates of publication: No. 1, Mar.9; No. 2, Mar. 19; No. 3, Apr. 3; INo. 4, Apr. 30; No. 5,'May 24: No. 6, June 30;. No. 7, Aug. 31; No. 8, Now. 14; No. 6, Dec. 19; No: 10, Feb. 15, (1922.) vi ERRATA, VOLUME VIII ERRATA); VOLUME. Vill Page 31, table I: Eryngium campestra should be Eryngium cambpestre. Page 145, 7th line should read: 26. Hooker, W. J. Flora boreali-americana 1: 126, 127. 8th and oth lines should read: 27. Hooker, W. J.,and Arnott,G.A.W. The botany of Captain Beechey’s voyage, part 3, p: 1327. London, ré4a; 28th line should read: Lyon, W.S. The flora of our southwestern archipelago II. Bot. Gaz. 11 330-336. 1886. Page 146, 9th line should read: London, 1838. inilime T4: New Yor should be New York. Page 195, line 26: Malva parvifolia should be Malva parviflora. Page 231, Ist line of text After Kauffman, add and Coker Page 274, 3d line should read: Wolff, E. 1871. Aschen Analysen 1: 1-149; 2: I-170. Page 451, 18th line: Urticastrt should be Dicentrae London, 1833. ‘ , } w . tes. and ‘loehg of stomata La ah x jon Ns ee UE aa me Linnean concept of jena millet a a ay : ge nm we i “AoNES Case the iH . “BERGMAN: d-class ‘matter F February 21,. 19t4, at the ‘post ies at ‘Lancaster, Pennsylvania, Is tes ie the, act) of March 3, ie Lf pe a Aira’ ti yy { “ re f , " a > | i ' : Savy y . i Y rt ya? ; : ( \ & ; } A n 5 + “K hy bos, ‘ Ee NA , : atin ¥ T hy : sit » \ Wi i { } ) it “ i i f n ee r : ‘ ‘ 4 a i \ +h a ! Y > re { x , * / Re odog ; Ay meat ; 4 yd fs Ra ve ‘ re , i ay ‘ bie sy ry | Sy “i | \ ~ ‘ E : is i yk \ i ¥ y; 3 x wiry i ‘ager ri j . { Ne B : | Rotapuisuen 1 19T4 ai A CL ea | “EDITED: BY A COMMITTEE OF THE Nae LS AR Na oc “BOTANICAL, SOCIETY. ‘OF AMERICA Pe LNG Feu Seni ie 4 "EDITORIAL COMMITTEE pat We Vy; ( Le, ds! ve om Ef aes oe | ee E ALLEN; Editor-in-Chi¢f, gh epee “i ty eainee sich a 2 oy hy University es Wisconsin Ase Oe a Dasa Cranes Ma oi Wieuaw "Caden nk! fe kB ie Nas ER: Jones, - CS teas alg hia waaay tee 1 ie is University of | Chicago iy Wea ealie, ee i ie te | Uninersity of Wisconsin CAS Ae aa Nie ‘c ‘STUART GAGER, ‘Business Manager BEY, ORLAND E: WHITE, Eo ye WEN 0 ees i oe Brooklyn Botanic Garden Nae ae Rg gs Brooklyn Botany: Garden iene R. A SLAREERG KE oo bh bale oe gat json 8. SCHRAMM, 0) tel 0 came | ( biel aye Mae ie wide e » if ne ; ‘Cornell Oniversity + ie i i a pie °C. 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The. publishers will supply missing number: Ue sins ts fey have Geet “>peen lost/in the mails, pane ek as i‘ y ih ‘Correspondence concerning ‘editorial caters should: ‘be addressed 'to Prof are ics Allen, University of Wisconsin, Madison, Wisconsin, e rg Business correspondence, including notice of change ‘of aides and direction ae ale i hae 4 “concerning reprints, should be: addressed to’ American’ ‘Journal ‘of. Botany, , Brook A re lyn Berane, ein ieee ig Ne Ne Maigret Nowth Soe ‘Street, Lat caster, AMERICAN JOURNAL OF BOTANY Vor Vill JANUARY, 1921 No. I ie EEXATION OF FREE NITROGEN BY, GREEN PLANTS FRANK B. WANN (Received for publication July 3, 1920) The ability of chlorophyll-bearing plants to utilize the uncombined nitrogen of the atmosphere has been repeatedly investigated during the last three or four decades, and the results of numerous observations and experiments, covering a wide range of species, have been quite conflicting. In some of the earlier experiments with higher plants in pot cultures the beneficial effect of a surface layer of algae was often observed, and the ability to increase the nitrogen content of the soil by free nitrogen fixation was ascribed to members of both the blue-green (Cyanophyceae) and grass- | green (Chlorophyceae) algae. Similar increases in soil-nitrogen content were observed when higher plants were excluded from the cultures, and, though bacteria were known to be present, the fixation was generally ascribed to the chlorophyll-containing forms. More recently, pure cultures of members of the Chlorophyceae have been used but the results in these cases have been almost uniformly negative. This fact, together with the discovery of widely distributed soil bacteria of the Azotobacter and Clostridium types, the ability of which to fix free nitrogen can not be ques- tioned, has led to the belief that in impure cultures fixation is due not to the activities of the green plants but to the bacteria present in the soil. Thus it has come to be very generally accepted that members of the Chloro- phyceae, as well as the higher plants, are not able to use free nitrogen. However, the number of species which have been investigated in pure culture is small, and the culture methods employed have not always been those which are most favorable for the best growth of these organisms. Accordingly the experiments reported here were undertaken for the purpose of extending the observations over a larger number of species, grown on a variety of mineral nutrient solutions under culture conditions which would insure a rapid and vigorous growth. LITERATURE A complete, and in some cases detailed, review of the literature bearing on the relation of the grass-green algae (Chlorophyceae) to free nitrogen is available in a paper by Schramm (1914 @), so that a repetition of the account is unnecessary here. Nothing of importance relating to this subject has [The Journal for December (7: 409-468) was issued January 12, 1921.] I 2; AMERICAN JOURNAL OF BOTANY [Vol. 8 appeared, so far as the author is aware, since the above-cited paper. As has already been indicated, the results of experiments with pure cultures have been pretty generally negative as regards the ability of these forms to increase the nitrogen content of the culture. In the light of results pre- sented here, some of the previous experimental work will be considered in the general discussion to follow. METHODS Seven species of Chlorophyceae were isolated and used in pure culture, In making the isolations the plate culture method, as described by Schramm (1914 0), wasemployed. With the exception of one species, Protococcus sp., all isolations were made from growths occurring on soil; the material for the Protococcus culture was secured from the bark of an elm tree. The absolute identity of all the species has not as yet been determined. Cultures have been submitted to several authorities on the group, but for some of the forms the determinations received have been somewhat at variance. For that reason no attempt has been made to apply specific names to all the organisms. Moreover, as will be apparent later, the abso- lute identity of the forms, though highly desirable, does not become of paramount importance because of the very similar way in which all the species seem to react. Unless otherwise indicated, therefore, the different species will be referred to by number and genus, or by number only, and as soon as more satisfactory determinations can be made a list will be pub- lished, if possible, in this journal. The forms used in the experiments include the following: Species number 1. Chlorella vulgaris Beyr. There seems to be no doubt about the identity of this species. Species number 2. Stichococcus sp. Species number 3. Protosiphon botryoides (Kg.) Klebs. Species number 5. Chlorella sp. A small form with cup-chaped chro- matophore. Species number 6. Scenedesmus sp. Species number 7. Protococcus sp. Species number 11. Chlorella sp. A large form with clateeme chro- matophore. All these species have been carried along on mineral nutrient agar for two or three years; they have been repeatedly transferred to media con- taining glucose or sucrose, and have frequently been examined micro- scopically. They are known to be free from bacteria and are pure cultures in the strict sense. Culture Media.—In the experiments Kjeldahl flasks of Pyrex glass and of 500 cc. capacity were used as culture flasks, because of the obvious advantage of analyzing checks and cultures without transferring the material to a digestion flask. Approximately 150 grams of mineral nutrient agar were supplied as a medium for each culture. In spite of the difficulties Jan., 1921] WANN — FIXATION OF FREE NITROGEN 3 involved in its analysis, a solid medium was chosen because of the very long-continued, vigorous growth produced on it. So far as has been observed, solution cultures, at least when unaerated, do not give a very extended or abundant development of these organisms. Since many pre- vious experiments have also shown that these forms do not grow in pure culture in the complete absence of combined nitrogen, no attempt was made to include such cultures in the experiments. Two experiments were performed, the first in the winter of 1917-18 and the second in the summer of 1919. In both cases the following mineral nutrient solution was employed as a standard for the preparation of the media: INTE LINC oh ke Gm case Be OO EY 17 fe. 0.5 gram PALE. oo 5 6's BUS See Dea et ee ease ee a A a Anca 0.2 gram ee totale (0) en nti Tere ee ks aledata dd Cale anu OM echidna 0.2 gram Cie: pie a rr 5 ouch eR ae eles SE amy Snir Rae Cre fer kt, 0.I gram PES ine 6 oo wa a onus oe ee Se ere nee rere an ere a ee ee trace Ey Steril SRNPAITSIE Sek Se a es 1000 cc. With the nitrogen content of this solution as a basis, the NH:iNO3 was replaced in the various series of 1917-18 by glycocoll, asparagine, (NH.4)eSOu, and Ca(NOs3)s, and by urea, (NH,4)2SO.u, and Ca(NOs)¢ in 1919, the nitrogen content as such being approximately the same in all media. Each of these sources of nitrogen was used in duplicate series, to one of which glucose was added. (In 1919 NHiNO3, (NH4)2SO;, and Ca(NOs3)z were also used in series to which mannite was added.) Nochange was made in the other constituents of the full nutrient solution, so that in all series these salts were present in the proportions indicated above. The 1917-18 experiment included the following series, arranged according to nitrogen sources and presence or absence of glucose: Series 1. Glycocoll (1.07 gr. per liter)—no glucose. Series 14. Same solution, with 1 percent glucose. Series 2. Asparagine (0.942 gr. per liter)—no glucose. Series 2A. Same solution, with 1 percent glucose. Series 3. Ammonium sulphate (0.942 gr. per liter)—no glucose. Series 3A. Same solution, with 1 percent glucose. Series 4. Ammonium nitrate (0.5 gr. per liter)—no glucose. Series 44. Same solution, with 1 percent glucose. Series 5. Calcium nitrate [Ca(NO3)2.4H2O, 1.475 gr. per liter]|—no glucose. Series 54. Same solution, with I percent glucose. In making up the media for the above series, sufficient nutrient solution with any one nitrogen source was prepared to supply both the series without glucose and the series with glucose. For these solutions the required amounts of the several constituents, with the exception of ferrous sulphate, were weighed out individually and dissolved in the proper volume of distilled water. (A stock solution of ferrous sulphate was prepared by dissolving 4 AMERICAN JOURNAL OF BOTANY [Vol. 8 0.1 gr. in 2,000 cc. of distilled water, 50 cc. of which were used in the prepara- tion of each liter of nutrient solution.) The solution thus prepared was divided into two equal quantities in large flasks, 1.5 percent agar being added to each, and I percent glucose to one portion. ‘The total nitrogen content of the medium with any one nitrogen source should therefore be the same per unit weight in the two series, with and without glucose, except for traces of nitrogen in the glucose or for slight discrepancies in the actual amount or composition of agar added. The nitrogen-containing compounds were not dried to constant weight nor was the agar purified in any way, all substances being added to the solution directly from the stock bottles. The chemicals used were Baker’s ‘‘analyzed’’ and Merck’s “‘highest purity’’; the agar was of the kind known as “ Difco bacto.”’ The total nitrogen content of each culture medium was determined by actual analysis of weighed portions of that medium. It is obvious that by this method any nitrogen introduced with the agar or as impurities with the glucose would be completely accounted for.! The 1919 experiment was a partial duplication and an extension of that of the previous year and included the following series: Series 6. Urea (0.375 gr. per liter)—without glucose. Series 64. Same solution, with 1 percent glucose. Series 7A. Ammonium sulphate (0.621 gr. per liter), with 1 percent glucose. Series 7B. Ammonium sulphate (as above), with I percent mannite. Series 8. Ammonium nitrate (0.5 gr. per liter)—no glucose or mannite. Series 8A. Same solution, with I percent glucose. Series 8B. Same solution as series 8, with I percent mannite. Series 9. Calcium nitrate [Ca(NO3)2.4H2O, 1.475 gr. per liter|—no glucose or mannite. Series 9A. Same solution, with 1 percent glucose. Series 9B. Same solution as series 9, with I percent mannite. Each complete solution was placed in the autoclav under 15 pounds’ pressure until the agar was dissolved. The solution was then filtered through absorbent cotton; the filtrate was free from sediment. Introduction of the Agar.—The Kjeldahl flasks were cleaned in the usual way and dried in the hot air oven. On removal from the oven, cotton plugs were inserted in the mouths of the flasks to prevent the entrance of dust. Each flask was numbered by means of a carborundum point and weighed to within 0.05 gram on a Mackenzie one-pan balance, the cotton plug being removed only during the weighing. The flasks were then stored in clean, dry boxes until required. As soon as the medium for a series was prepared the required number of flasks (1I in 1917-18, 24 in I9IQ) were arranged in one of the special wooden racks (see fig. 1, Plate I) and 150 cc. of the hot agar solution was added to 1 Numerous analyses of the agar showed the nitrogen content to be about 1 mg. for the amount present in each culture flask. It will be noticed that the analyses for the total nitrogen of the media may not have yielded exactly the calculated amounts, because of the moisture present in the nitrogen-containing compounds. Jan., 1921] WANN — FIXATION OF FREE NITROGEN 5 each flask. The flasks were then immediately weighed in the order in which the agar was introduced. The mouths of the flasks remained plugged with cotton except during the actual processes of introducing the agar and of weighing. Although water vapor condensed on the walls and necks of flasks as the agar cooled, the use, with a number of flasks, of rubber stoppers instead of cotton plugs, demonstrated that there was no detectable loss of water vapor through the cotton plug during the interval between the intro- duction of the agar solution and the weighing. Thus the actual weight of medium in each flask was known, and, as will be seen from the tables, differences resulted of one or two grams in the weight of approximately equal volumes between the first and last flask of a series to receive the medium, due to the cooling of the agar during the processes involved. After the second weighing each flask was provided with a two-hole _ rubber stopper carrying intake and outlet glass tubes, the outer arms of these tubes being adjusted so as to be readily connected in series by means of rubber tubing. Long cotton plugs were loosely adjusted in the bore of each outerarm. The flasks were sterilized at 15 pounds’ pressure for 20 minutes, the stoppers resting lightly in the mouths of the flasks but being tightly adjusted upon removal from the autoclav, the hands being moistened with alcohol for this operation. The flasks were allowed to cool in a dust-proof case. Inoculation.—The inoculations were made in the laboratory under a glass dust shield open on one side only. The inoculum consisted of a suspension of the algal cells in a test tube of sterilized nutrient solution minus combined nitrogen. Special cultures on hard (2 percent) agar were pre- pared, so that in making the suspension no agar was introduced with the cells. The tube was thoroughly shaken to secure a uniform suspension of the inoculum, of which ten drops were added to each flask in the 1917-18 experiment and one cubic centimeter was similarly added in 1919. In the former experiment four species were used and two flasks in each series were inoculated with the same species, three flasks of each medium remaining uninoculated as checks. In 1919 seven species were used, three flasks of each series being inoculated with each species with the exception of species no. 3 and no. 7, in which cases only two flasks of any one medium were inoculated; three flasks of each series remained uninoculated as checks. After the rubber stoppers were tightly fitted in the flasks, melted paraffin was run in around the flared neck, and the stopper and a portion of the neck of each flask were covered with sterilized cotton. During the two experiments eighteen contaminations occurred out of a total of 340 flasks. Aeration.—The intake and delivery tubes of the flasks of each series were connected with rubber tubing for the purpose of aeration. In making connections the free ends of the glass tubes were painted with 95 percent alcohol, which was used also in washing out the bore of the rubber tubing. 6 AMERICAN JOURNAL OF BOTANY [Vol. 8 In setting up the first experiment it was thought that in the process of aeration a loss of nitrogen from the medium in the form of ammonia might occur, especially in view of the fact that the medium was slightly alkaline, so that a tube of acid was inserted in the series just beyond each culture flask. For this purpose large test tubes, 200x 25 mm., and containing 25 cc. of standardized N/1o sulphuric acid were used. As a precaution against the backflow of this acid into the cultures, small Erlenmeyer flasks of 180 cc. capacity were placed between each culture flask and its corre- sponding acid tube. The arrangement of the apparatus can readily be understood by consulting text figure 1. Two gas-washing bottles, A and B, TEXT Fic. 1. Detail of a portion of one of the series of 1917-18. Explanation in the text. were placed at the head of each series; A contained 30 percent sulphuric acid and a quantity of pumice stone; 8 contained sterilized distilled water. Air entered the series through a calcium chloride tube filled with cotton, was washed free of ammonia by the acid in A and was moistened by the water in B. Oxides of nitrogen would also be removed by the water. Before entering the culture flask C’ the air passed through asecond calcium chloride tube containing sterilized cotton. The intake tube of each culture extended to within an inch or so of the surface of the agar medium, whereas the delivery tube merely penetrated the rubber stopper, so that in the process of aeration the air above the agar surface was completely changed. After leaving the culture flask, the air passed through the safety flask D’ and bubbled through the acid in the adjoining test tube E’. The intake tube of the latter was drawn out to a fine point which extended to the very bottom of the test tube, so that only very small bubbles were formed, insuring a thorough washing of the air before it passed into the next culture flask of the series. The delivery tube of the last acid tube in the series was connected to a filter pump, by means of which the air was drawn through the whole series at once. When the ten series of the first experiment had been completely as- Jan., 1921] WANN — FIXATION OF FREE NITROGEN 7 sembled, the delivery tubes at the end of each rack were connected in a single series by means of T-tubes so that aeration of all ten series could be accomplished by one operation. Each of these delivery tubes was provided with a screw clamp which was kept tightly closed except during the process of aeration, when it served to control the volume of air passing through the series. With a few exceptions aeration was continued for an hour every morning, it being considered that this would entirely replace the air in the apparatus. The 1917-18 experiment as completely assembled is shown in figure I, Plate I. At the end of the first experiment, titrations of the contents of the acid tubes showed that no appreciable change had taken place in the concentra- tion in any instance. It was assumed, therefore, that with these species and with the conditions realized in the experiment there was no loss of ammonia from the culture flasks. For this reason the tubes of acid were omitted from the second experiment, and in the process of aeration the air passed directly from one culture flask to the next in the series. Because of the expansion of the air in the culture flasks during the hot summer days, the liquids in the gas-washing bottles were frequently forced out through the intake tubes of these bottles;—making it necessary to place safety flasks outside the acid bottle, and between the acid and water bottles, to re- ceive those liquids. During the process of eration the acid and water were drawn back into the proper bottles so that no air ever entered the series without first passing through the liquids. Aeration was continued for an hour every other morning during the growing period. Cultural Conditions——Soon after the inoculation of the culture flasks the ten racks were transferred to the greenhouse where more uniform condi- tions of light and temperature prevailed than in the laboratory. Since preliminary tests showed that agar cultures of the organisms used were soon killed by direct sunlight, the bench occupied by the apparatus was covered with a canopy of black cloth, which reduced the actinic light intensity to about one eighth that of the normal greenhouse illumination. On cloudy days, however, this canopy was rolled up on both sides, thus permitting better illumination; on clear days the west side was open during the morning only, and during the afternoon the east side only was exposed. The arrangement of the apparatus as finally assembled in the greenhouse is shown in figure 2, Plate I, which is a photograph of the 1919 experiment. GROWTH OF THE CULTURES Length of Growing Period.—The approximate length, in days, of the growing period of each series is indicated in the headings of the tables which follow. Inoculations in the first experiment were completed on August 31, 1917, and the analyses were begun in April, 1918. The final inoculations of the second experiment were made in May, I919, and analyses were started in November of the same year. 8 AMERICAN JOURNAL OF BOTANY [Vol. 8 Method of Recording Growth.—Records of the growth of the cultures were made at intervals of three or four weeks. These consisted of written notes comparing the development of the different species on the same medium, and the difference in amount of growth of the same species on the ten different media. Charts were also prepared at about monthly intervals showing the growth in each culture flask by means of colored crayons. These were found very helpful in making growth comparisons, as they present to the eye at once the relative development in every flask. Since it was the plan of the experiments to analyze the entire contents of the culture flasks at the end of the growing period, it was not found advisable to attempt any actual weight determinations of the ‘‘crop”’ produced on the various media by the different species. Experiments with solution cultures are in progress now from which it is hoped some accurate data may be secured, showing the actual amounts of growth produced on different media and what relation the dry weight of algal material produced bears to free nitrogen assimilation. From a comparison of the written notes and colored charts, however, the following general statements may be made. 1917-18 Experiment.—In this experiment there was a remarkable sim- ilarity in the amount of development of all four species on any one of the media used. Only in a few cases did there appear to be any marked specific differences in the reactions of the organisms to the medium. In general, the presence of glucose resulted in a vigorous and rapid development of all species, irrespective of the nitrogen source. Series 1-3. The relative growth of all the cultures is indicated in the tables which follow, by means of plus signs. Since no fixation occurred in the series in which the nitrogen was supplied as ammonium sulphate or in the organic forms used, the detailed observations of these series are omitted. The results on the nitrate media, however, were so striking that a more detailed account of the growth on these media is here presented. Series 4 (ammonium nitrate, without glucose). Growth was slow but steady in all cases. Species nos. 1, 5, and 6 continued healthy to the last, giving ‘‘very fair’? growths. The growth of species no. 2 was “fair,” but the cultures were dead at the end of the experiment. Series 44 (ammonium nitrate, with 1 percent glucose). All species started with very vigorous growths, the effect of the presence of glucose being very evident. Species no. I produced a “luxuriant” growth at first, but after three months began to deteriorate, turning brown over most of the surface. Before growth had completely ceased, however, both cultures of this species began to revive, and by the end of the fourth month were again bright green. The cultures then slowly waned a second time, only small portions remaining green at the end of the experiment. Species nos. 2, 5, and 6 grew steadily from the start and remained healthy, no. 2 producing a ‘‘luxuriant’’ growth and nos. 5 and 6 ‘‘very good” growths. Jan., 1921] WANN — FIXATION OF FREE NITROGEN 9 Series 5 (calcium nitrate, without glucose). The growth was very slow with all species, but all remained healthy. Total growth of species no. 2 was ‘‘fair,’’ while for nos. I, 5, and 6 it was ‘‘very fair.” Series 5A (calcium nitrate, with 1 percent glucose). All four species gave vigorous growths on this medium, continuing healthy to the end of the experiment. The effect of the presence of glucose was apparent from the start. The growth of species no. 2 was “luxuriant” and appeared slightly better than the others, all of which were ‘‘very good.” 1919 Experiment.—So far as this experiment duplicated the previous one, the same general type of growth resulted. The presence of glucose in the medium markedly stimulated the development of all species, irre- spective of the source of combined nitrogen. It is also true, however, that death of the cultures always occurred first on the media containing glucose. The presence of mannite apparently had no effect by way of increasing the rapidity or amount of growth of any of the species on any of the three media to which this compound was added. The amount of growth produced on these media appeared practically the same as produced by the same organism with the same source of nitrogen but without either glucose or mannite. Series 6 and 7. As in the previous experiment, no fixation occurred where combined nitrogen was supplied in an organic form or as ammonium sulphate; growth observations for these media are therefore omitted. Series 8 (ammonium nitrate, without glucose or mannite). A slow, steady growth resulted, as in the previous experiment. At analysis all cultures were healthy. Species nos. 1, 6, and 11 produced ‘‘very fair”’ growths; nos. 2 and 5, ‘‘fair.’’ Species nos. 3 and 7 were not grown on this medium. | Series 8A (ammonium nitrate, with I percent glucose). All species grew very vigorously at first, but deterioration soon set in and by the end of one month all cultures of nos. 1 and 2 were dead, after a ‘‘fair’”’ growth, and species nos. 3 and 6 were rapidly waning. The cultures of no. 3 died after a “‘fair’’ growth. One culture of no. 6 also died, but the two others revived and remained healthy to the end of the experiment, giving ‘‘very good”’ growths. Species no. II gave a ‘“‘luxuriant” growth, but at the end of the experiment was turning brown. Nos. 5 and 7 remained healthy throughout the growing period, both producing ‘‘very good”’ growths. Series SB (ammonium nitrate, with I percent mannite). Growth on this medium was slow, and in general very much as in series 8. All cultures remained healthy at the end of the experiment. Species nos. 1, 3, 6, and II gave “very fair’’ growths, the development being somewhat better than with species nos. 2, 5, and 7. Series 9 (calcium nitrate, without glucose or mannite). Growth was very slow, and strikingly like that in series 8. All cultures remained healthy, species no. 6 giving a ‘‘good”’ growth, nos. I and 11 ‘‘very fair”’ IO | AMERICAN JOURNAL OF BOTANY [Vol. 8 growths, and nos. 2 and 5 “‘fair.’”’ Species nos. 3 and 7 were not included in this series. Series 9A (calcium nitrate, with I percent glucose). Development was very vigorous from the start, all cultures soon producing a complete coat over the agar surface. At the end of a month, species nos. 3, 5, 6, and II began to turn yellow. The deterioration, however, did not progress far, and after another month or two all cultures were again green, remaining healthy until analysis. Species nos. I, 2, and 7 remained healthy throughout the entire growing period. At the end of the experiment a “luxuriant”’ growth had resulted with species nos. 2 and 11, ‘very good” growth with nos. 1,5, and 6, ““zood — with no. 3, and? slight” with now 7 Series 9B (calcium nitrate, with I percent mannite). The development was very similar to that in series 9, being slow but steady. All cultures remained healthy to the end. Species nos. I and 11 produced ‘‘very fair’”’ growths, nos: 2,5, 6, and.2 fair, “and no. 7 omy slicht.” It should be noted that in the 1917-18 experiment the development of the four species on the two nitrate media with glucose (series 4A and 5A) was very similar, the growth being either “luxuriant” or “‘very good” in all cases. Of the two, however, the cultures of series 5A appeared some- what the better. In striking contrast with this condition was the growth of the various species on similar media of the 1919 experiment (series 8A and 9A). The species which gave such good growths on ammonium nitrate with glucose (series 4A) in 1917-18 showed scarcely any develop- ment on this medium in 1919 (series 8A), with the possible exception of species no. 5. However, on calcium nitrate with glucose (series 9A) the growth of these species was practically the same as was secured on the similar medium of 1917-18 (series 54). Even though a few of the flasks of series 8A were reinoculated, the growth continued poor; likewise, “reserve” flasks of this medium which were not introduced in the series at the beginning of the experiment gave very similar growths of species nos. I and 2 when inoculated a few months before the end of the growing period. This difference in the amount of growth produced in the two experi- ments may possibly be related to the difference in the seasons during which the experiments were conducted. ANALYSES At the end of a growing period of from six to eight months the cultures and checks were analyzed for total nitrogen content. After making a final record of the growth and condition of the cultures, a small loop of the algal material from each flask was transferred to a drop of sterilized nutrient solution and examined microscopically. In 1918 transfers were also made from each culture to nutrient agar containing I percent glucose, and to the same medium made acid by the addition of I percent hydrochloric acid. Two tubes of each of these media wete inoculated from each culture flask, but in no case did contaminations appear that were not apparent from the Jan., 1921] WANN — FIXATION OF FREE NITROGEN II microscopical examination. This precaution was therefore omitted in 1919, the microscopical examination being relied upon to detect contaminations not apparent to the naked eye. In all cases, however, such contaminations as occurred were perfectly evident from macroscopic examinations, the majority of them appearing soon after inoculation. After.considerable preliminary work with total nitrogen determinations, — the Gunning-Kjeldahl method was adopted for media free from nitrates, _and in the presence of nitrates the Forster modification of this method was used. In the former method the digestion mixture consists of a solution of 20 grams phosphorus pentoxide (P2O;) in 500 cc. sulphuric acid of sp. gr. 1.84, 20 cc. of this solution being added to each culture flask. After the addition of 10 grams potassium sulphate to each, the flasks were heated slowly over a low flame. The presence of the agar made the digestions particularly trying, as it was necessary to watch the flasks constantly at the beginning of the process in order to prevent the contents from foaming up into the necks. It was found advisable to start with a very low flame and to shake the flasks occasionally until sufficient agar had gone into solution to allow the ready escape of bubbles from below. At this point the full flame was used, and the water boiled off vigorously until foaming began. The flame was then turned very low again for about 30 minutes or until the appearance of dense, white fumes, at which time foaming gradually ceased. The fire was then slowly increased to full capacity and the digestion con- tinued for 15 or 20 minutes after a clear liquid resulted. About 13 hours were required for digestion after foaming ceased. In all cases the flasks at the end of the digestion were perfectly clean and the liquid was entirely transparent. 3 The distillation was carried out in the usual way. About 150-200 cc. of distilled water was added to each flask when cool, the neck of the flask being thoroughly washed down in the process. The solution was made alkaline with 50 cc. concentrated sodium hydroxide, and, after the addition of a gram of granulated zinc, the flask was immediately connected to the still, The ammonia was distilled over through block tin tubes into a 500-cc. Erlenmeyer flask containing 30 cc. standardized tenth-normal sulphuric acid, diluted with enough distilled water to cover completely the end of the delivery tube. Standard traps were used between the Kjeldahl flask and the condenser. Distillation was continued until the contents of the flask began to ‘‘bump.”’ About 125 cc. of water was distilled over in this process, and it was usually completed in about 40 minutes. During the latter part of the distillation the receiving flask was drawn away from the still sufficiently to uncover the end of the delivery tube, the inside walls of which were washed down by the remaining distillate. At the end of the distillation the delivery tube, as well as the inside wall of the receiving flask, was washed down with a small amount of distilled water. The excess acid was titrated against tenth-normal sodium hydroxide, using cochineal as an indicator. 12 AMERICAN JOURNAL OF BOTANY [Vol. 8 The Forster modification, employed in analyzing the media containing nitrates, consists essentially in the addition of sodium thiosulphate to the digestion mixture. The procedure was as follows: A considerable part of the water of the medium was boiled off in the presence of 10 cc. of con- centrated sulphuric acid, the process being continued until active foaming began. When cool, 20 cc. of the phenol-sulphuric acid digestion mixture (100 gr. phenol in 924 cc. concentrated sulphuric acid) were added and allowed to stand several hours or over night, the mouths of the flasks being covered during this time. Two grams of sodium thiosulphate were then added, and, after the reaction was completed, I gram of mercuric oxide, I gram of zinc dust, 10 cc. of concentrated sulphuric acid, and finally Io grams of potassium sulphate were added in the order named. The flasks were heated over a low flame until foaming ceased, when the flame was gradually increased to full intensity. Digestion was continued for about 20 minutes after the liquid became clear, the process requiring about 24 or 3 hours. The distillation was carried out as with the Gunning method, except that before making the solution alkaline the mercury was precipitated out by the addition of 25 cc. potassium sulphide solution (20 gr. KeS in 500 cc. H20).. Numerous “‘blank”’ determinations of the nitrogen content of the re- agents used in the two methods were made, the average of these being deducted from the determinations of the cultures and checks. Care was taken to use uniform reagents for the analyses of each series. In the preliminary determinations no difficulty was experienced with the Gunning method in securing complete recovery of the nitrogen from solutions of urea or ammonium sulphate. In the presence of agar or of agar and glucose the usual trouble with foaming was encountered, but the determinations checked readily within the limits of experimental error. The recovery of total nitrogen, including nitrates in the presence of agar, was more difficult, but consistent results were obtained with the Forster method as outlined above. It was found necessary to allow the digestion mixture to stand a considerable length of time (usually over night) in contact with the dissolved and partially concentrated agar medium before the addition of the other reagents. Following the experience of Duggar and Davis (1916), it was also found advisable to permit the flasks to stand about an hour after the addition of the sodium thiosulphate. The low results obtained when the digestions are completed more quickly may possibly be due to the rather extensive dilution of the reagents by the water of the culture medium and the consequent slower reduction of the nitrates. Tables A and B show some of the results obtained in the preliminary determinations. amy, 1927| WANN — FIXATION OF FREE NITROGEN 13 TABLE A. Recovery of total nitrogen from ammonium nitrate by Forster method Solution = 1.4285 grams dry NH4NO; in 250 cc. distilled water to Cc. Solution Made Mg. N as NH3 by : Alkaline and Distilled Mg. N as NH; Recovered Calculation Mg. Difference I 9.980 10.000 — 0.020 2 9.896 10.000 — 0.104 2 9.980 10.000 —0.020 to Cc. Solution Digested Mg. Total Nitrogen Mg. Total Nitrogen by : by Foster Method Recovered Calculation Mg. Difference I 19.812 20.000 — 0.198 2 19.540 20.000 — 0.460 @ 19.931 20.000 — 0.069 TABLE B. Recovery of total nitrogen from ammonium nitrate in the presence of agar agar by Forster method Solution of NH4NOs3. Total calculated N in 10 cc. = 26.252 mg. Total Mg. N Found in |Total Mg. N Found in zo Cc.| Mg. N Found in 2 Grams to Cc, Solution Solution Plus 2 Grams Agar Difco Agar Mg. N Recovered 26.268 27.760 1.826 25.993 26.219 27 Raifi2 Lat 25.615 26.024 26.994 b.703 25:237 It is apparent from table B that the agar interferes somewhat with the complete recovery of nitrogen. However, as the amount of agar in all series was the same in both checks and cultures, the results, though perhaps low, are still strictly comparable. RESULTS The results of the analyses of the cultures are presented in tables I-oB. These are numbered to correspond with the series, each series representing a single medium. As already explained, the only constituent of the mineral nutrient solution which was varied in the different media was the nitrogen source; the latter is indicated in the heading of each table. The presence or absence of glucose or mannite is indicated in the sub-heading. In the third column the relative growth in each culture flask is shown by means of plus marks; in nearly all cases the growth in the two or three flasks of a series inoculated with the same organism was practically the same, but a few exceptions may be noted. In the fourth column is given the total nitrogen (in milligrams) found in each flask. This figure represents the total nitrogen calculated from the titration, minus the average amount of nitrogen in the blanks. The other columns are self-explanatory. 14 AMERICAN JOURNAL OF BOTANY [Vol. 8 TABLES I AND IA. Results of analyses of mineral nutrient agar cultures of green algae. Experiment of 1917-1918. Growth period, 230 days. 1. Medium without glucose Nitrogen source, GLYCOCOLL Culture Total Mc. Wt.of |Mg.N per Mg. Loss No. Species aoe a Found wore oe ae ceue — | Check Cons 32.655 | 145.90 | 22):382)) ” — | Check Ster 32.389 147.10 | 22,018 722.204 =) Check ster. 32.796 147245. |. 22,2405) 1 | Chlorellavulgaris Beyr., +++ 31.823 | 144.90 | 21.962\ ,, 55 | _ I : +++ 32.036 145.15 | 22.0710\n ee 7 eae 2 | Stichococcus sp. ++++* | 31.859 | 145.30 | 22.926 ae 2 ® ~p+te+ 32.230 145.75. |-22:d0e pitch sho cies 5 | Chlorella (?) sp. SP Rae 31.965)| \146.15, | 21.871 )|) ee eee 186 5 +-+-+* 32.478 146.40 | 22.184 . Z 6 | Scenedesmus (?) sp. +++ 32.354 | 146.70 | 22.055 \ 4; 929 sae 6 Saaip ate 22.053 | 147.00 | 27.805) “93 ae Average all cultures 21) 5) Mee | ce ees ee eee ee 21.999 | —0.215 1A. Same medium, with 1 percent glucose — | Check Ster. 31.646 | 146.70 | 216572 | — | Check Ster. 31.841 148.00 | 21.514 + 21.502 — /|Check Ster. B15 148.15 | 21.421 1 | Chlorellavulgaris Beyr., +++-++ | 31.010 146.55 || 21.160 21.466 | —0.056 I oe te aleaian eS oso 146.60 | 21.732 a : 2 | Stichococcus sp. +++ | 31.438 | 140155.) 21-452 450| —0.052 2 tH || 31-452) | 146.05," 21 -4aees 5 | Chlorella (?) sp. +t+t++-+ | 30.656 | 147.20 | 20.826 ,, 055| —0.447 5 Spamacar am | See 147,20 | 21.283 ; : 6 | Scenedesmus (?) sp. + || (30.5400) 147-50.) | 21. 0gaee 378 | —0.124 6 ote test ate eo eoue 147.90 || 21.374 : ; Averagerailicultures: \| >) 0'+ a ee) ee ee einer 21.332 | —0.170 TABLES 2 AND 2A. Results of analyses of mineral nutrient agar cultures of green algae. Experiment of 1917-1918. Growth period, 234 days. 2. Medium without glucose Culture Z ie) Daunndwnvnv ee] | Average all cultures Species Check Check Check 7 Chlorella vulgaris Beyr. Stichococcus sp. Chlorella (?) sp. Scenedesmus (?) sp. Growth Ster. Ster. Ster. SP Se ++4++++4+4+ ++4+4+4+4+4+ +4+4++ Total Mg. N Found | 27.808 28.162 27.967 27.861 27.614 28.410 27-755 27.667 oe, e) 1) «6 Nitrogen source, ASPARAGINE Wt. of Medium, Grams 146.50 147.25 147.30 146.20 146.30 146.30 146.35 Mg. N per too Gr. Average Medium 18.982 19.125 + J9.031 18.987 19.057 18.875 18.966 19.419 18.965 | 19.192 18.885 eee \ 18.806 18.798 18.767 \ 18.783 aes, 18.937 — 0.065 +0.161 —0.225 —0.248 —0.094 * Checks or cultures which were contaminated are designated with an asterisk. { Relative growth of the cultures is indicated by plus signs, as follows: + ++ = “fair,” +++ = “very fair,” ++++ = “good,” +++++ = “very good,” +4+++4++4+ ‘“Vuxuriant.’”’ “slight,” Jan., 1921] Check Check Check DAdouuUnNN He |] | | Chlorella vulgaris Beyr. Stichococcus sp. Chlorella (?) sp. Scenedesmus (?) sp. Average all cultures WANN — FIXATION OF FREE NITROGEN 15 2A. Same medium, with 1 percent glucose Ster. 27.260 | 147.05 | 18.538 ) Ster. Lost 147.7 Ou eee j 18.655 ttt tt| shoes | goss [18402 25 tEETH | Seael | Hoge iaigs (ses) oon tti1411 | 26,888 146.90 sa secon Soe tee +4+44+4+) 27.154 | 147.80 18.372} 18:289 ene SP oes PM ear alae Ml eaeacm ee 18.299 | —0.356° TABLES 3 AND 3A. Results of analyses of mineral nutrient agar cultures of green algae. Experiment of 1917-18. Growth period, 226 days. 3. Medium without glucose Nitrogen source, AMMONIUM SULPHATE Culture , Wt. of | Mg. N per Mg. Los Total Mg. g.Np g. Loss = ee Nina) Maan ree ee eke — | Check Ster. 20.842. | 146.75. | 20.435, | ini - Check Ster. 30.338 147.65 | 20.547 st I Chlorella vulgaris +--+ 29.719 145.50 | 20.425 oF I Beyr. +++ 29.701 145.95 | 20.350 Sh sele es 2 Stichococcus sp. oe 30.055 145.80 | 20.614 2 Sel 30.090 146.30 | 20.567 20.591 | 10.150 5 | Chlorella (?) sp. “leche 30.055 | 147.00 | 20.445 5 +-+-+* 30.691 147.05 | 20.871 ACIS) | a eel 6 | Scenedesmus (?) sp. +++ 30.090 | 147.20 | 20.441 6 SP SP SR 30.284 147.40 | 20.546 20494) 21053 Average all cul- | aU ete a eM a CS ee astern solace neoeas 20.533 | +0.092 3A. Same medium, with 1 percent glucose = Check Ster. 29.400 147.10 | 19.986 — | Check Ster. 29.233 148.05 | 19.739 ; 19.957 — | Check Ster. 29.825 148.05 | 20.145 I Chlorella vulgaris) (++++)t 28.834 |- 146.75 | 19.648 \ 0 ¢ ‘: I Beyr. (CORES AE) 28.869 | 146.80 | 19.666 f 19°57} —9-300 2 Stichococcus sp. (+++-+) 29.365 146.85 | 19.996 836 ie 2 (++++) | 28.923 | 147.00 | 19.675 {79°°S cen 5 Chlorella (?) sp. +++++-+ | 29.188 147.35 | 19.809 ; el peso 5 ++4+4+4+4+ | 29.082 | 147.30 | 19.743 [19°77 6 | Scenedesmus (?) sp. (+++) 28.746 147-75 | 19.456 | 19 561| —o.606 6 (+++) 28.180 147.80 | 19.066 9 | 109 Average all cul- | ULC SMCS ange hoe a bey bw a aie, eat Scart i eerie) Go eam Le, 19.634 | —0.323 + Plus signs enclosed in parentheses indicate that the culture was dead at the time of analysis. 16 AMERICAN JOURNAL OF BOTANY [Vol. 8 TABLES 4 AND 4A. Results of analyses of mineral nutrient agar cultures of green algae. Nitrogen source, AMMONIUM NITRATE 4. Medium without glucose Experiment of 1917-18. Growth period, 270 days. Culture pine alictal Bue of |Mg. a Bea Mg. Loss| Gain rowt i edium, | Ioo Gr. vera Gain, No Species i fount eae Medium ey es GE Peseen — | Check Ster 23.569 146.90 16.044 | | — | Check Cons 23.885 | 147.95 | 16.144 > 15.788 — | Check Con ‘ 22.445 | 148.25 | 15.175 I Chlorella vulgaris SARE 22.410 | 146.20 | 15.328 XS if Beyr. Sean ' 23.674 | 146.25 | 16.188 15-758 | — 0.030 2 | Stichococcus sp. (+ + 24.921 | 146.45 | 17.017 i 2 (+--+) 23.569 | 146.70 08 16.542 |5-0. faa tee 5 | Chlorella (?) sp. +++-+ | 25.729 | 146.90 | 17.515 5 : : . Se 24.379 147.40 roast ae qi 230) gee cenedesmus (?) sp.| 25.940 | 147.60 | 17.574 6 +--+ Lost 117: er cata adie j 47-574 1a ts | Average all cul- tures _ oes ME ee een eee ae he 16.725 |--0;,0271) 50 4A. Same medium, with 1 percent glucose tei Check Ster. 24.323 | 147.75) 15-704 — | Check Ster. 23.446 | 148.60 | 15.778 } 16.032 ete Oneck Stel. 24.095 | 145.90 | 16.525 I Chlorella vulgaris | (++-++ 32.747 | 146.96 | 21.978 I Ps iA eae act 32.817 | 147.20 22.294 ( 22-130 + 6.904 ogee 2 tichococcus | 32.269 | 147.1 (21.987 2 5 orella (2) sp. |. 33:2901 | 147.090’ || 22,509 5 ee @) cea wen Sone De | 23278 16.362 | 39.6 6 cenedesmus (?) sp.| 33.624 | 148.25 | 22.681 Gia “He ab 9g2. Tsu ta s4od 21.676 { 277:179 TONAT aes | Average all cul- CUlCURES Hee See) ee eee ee eee ee 22.155 | +6.123 | 38. TABLES 5 AND 5A. Results of analyses of mineral nutrient agar cultures of green algae. Experiment of 1917-18. Growth period, 297 days. 5. Medium without glucose Nitrogen source, CALCIUM NITRATE NANAMUNNHH HS |] | Culture Species Check ‘Check (Loss) Beyr. Stichococcus sp. Chlorella (?) sp. Scenedesmus (?) sp. Average all cul- tures Chlorella vulgaris | Growth Ster. Ster.* ++ 4 a+ ++4+4+++4++ +++4++4++4++ att Total Mg. N Found 2VG2 23-797 23.586 23.990 23.902 27,210 28.079 24.850 | 147.35 148.25 Wt. of Medium, Grams 148.25 145.65 145.80 146.05 146.55 147.30 147-55 147.80 148.00 Mg. N per too Gr. Average Medium z 16.903 oes 116.903 16.338 16.177 16.258 16.425 16.310 f 16.368 18.479 19.030 18.755 18.322 16.269 } 16.565 15.105 | ena fepe se: Mg. Loss or Gain, too Gr. —0.645 | On530 +1.852 | —0.338 +0.084 Gain Percent Il. Jan., 1921] DADaUUNNHNNHH | | Check (Loss) Check (Loss) Chlorella vulgaris Beyr. Stichococcus sp. Chlorella (?) sp. Scenedesmus (?) sp. Average all cul- tures WANN — FIXATION OF FREE NITROGEN 17 5A. Same medium, with 1 percent der ; 22.603 | 148.50 |/ 15.221 ; 18.360 | 147.40 |\12.456 )tr6. 903 aaetinals tee Gore74 | 147-05 323.988 | 24 420) -7.527 145-5 =P aR ap Sear 36-572 | 147,05 |) 24.871 +++-+++)| 36.029 | 147.25 | 24.468 | 23.604|+6.701 | 39.6 Simaliate ay ae in) GS2D 19) | 147240 |..22.740 agalactiae cin ais oGo:32 7. |. 147-000) 24.402 | 247402) --7- A901) 44-3 Bling: clamine Ost 2 9147.00) (7. ee Smee alana ty e3oe502e |) l4S.25 | 23.047 Searls aiate 1) o4-007 |. 148.80 Sy me 23:956\ (27.053) 0140-7. a ee es 36.590 | 148.35 665 meee Hea ete eta eames oe 24.098) +7.195 | 42.5 TABLES 6 AND 6A. Results of analyses of mineral nutrient agar cultures of green algae. Experiment of 1919. Growth period, 165 days. 6. Medium without glucose Nitrogen source, UREA Culture No. Species — | Check — | Check — | Check I Chlorella vulgaris I Beyr. ul 2 | Stichococcus sp. 2 2 5 | Chlorella (?) sp. NESS) 3 6 | Scenedesmus (?) sp. 6 6 rT Chlorella (?) sp. II II Average all cul- tures ++4+4+4++ fra ttt | N Found 25.212 26.315 26.161 24.807 24.779 24.583 24.667 24.988 24.580 25.212 24.779 24.388 24.480 24.960 25.407 25.687 25-533 25.282 @ Te, eye 8, Total Mg. Wt. of Medium, Grams 148.05 148.30 148.45 148.15 148.15 147.95 148.05 148.05 148.05 148.05 148.15 148.20 148.15 148.25 148.30 148.20 148.30 148.30 eo © © © Mg. N per too Gr, Medium EA oe 029 17.744 a ee ec be 16.616 16.661 16.878 16.603 17.029 16.726 16.456 16.524 16. 336 17.132 17.333 1p au 17.048 AneeaeeN ce cune Ls lg 17.465 16.696 | —0.769 16.714 | —0.715 16.737 | —0.728 16.831 | —0.634 17.199| —0.266 16.835 | —0.630 + Because of the use of some old Jena flasks in completing Series 5 and 5A, a number of determinations of checks and cultures were lost. determination is taken for the nitrogen content of the media of these two series. Accordingly, the highest complete check 18 AMERICAN JOURNAL OF BOTANY 6A. Same medium, with 1 percent glucose Dnannuvnesaae| | | — = > — = QoNN | Check Average all cul- | Check Check Chlorella vulgaris Beyr. Stichococcus sp. Chlorella (?) sp. Scenedesmus (?) sp. Chlorella (?) sp. Protococcus sp. Protostphon botryoides tures Ster. Ster. Ster. +4+++++444+4+4+4+4+4+4+ +4++4++4+44+4+4+44444+ +4+++4+4+44+4+4+4+4+4+44+ +4++++4+4+4++4+4+4+44+ poppet aa ao + ++ ++ +++ 27.348 26.203 26.943 26.301 26.231 26.622 25.882 25.645 26.064 25.7560 26.762 25.784 26.580 27.390 27.209 26.860 27.363 27.306 26.846 27.893 27.069 27.265 148.40 148.50 148.90 148.35 148.40 148.35 148.45 148.45 148.35 148.35 148.40 148.45 148.50 148.45 148.45 148.45 148.45 148.75 148.70 148.70 148.70 148.90 | 18.429 | 17.645 18.095 17.676 17.945 17.435 17.275 17.569 17.366 18.034 17.369 17.899 18.451 18.329 18.094. 18.433 18.357 18.054 18.758 18.204 18.311 t 17.729 ) 18.056 17.783 17.426 17.590 18.226 [Vol. 8 —0.273 — 0.630 —0.466 -+-0.170 +0.239 +0.350 +0.202 —0.058 TABLES 7A AND 7B. Results of analyses of mineral nutrient agar cultures of green algae. Experiment of 1919. Growth period, 175 days. 7A. Medium with 1 percent glucose ENN COO Ore ere, | | II Culture Species Check Check Check | Chlorella vulgaris Beyr. Stichococcus sp. Chlorella (?) sp. Scenedesmus (?) sp. Chlorella (?) sp. Protococcus sp. Protosiphon botryoides Average all cul- tures Growth Ster. Ster. Ster. (Gr + +4++444 ++4+4+4+4++ ++++4+ +4+$>0~ +++ sl A es dodbapabo ee dedi dbdtedi feel wy peta tea Total Mg. N Found 21.385 21.986 21.190 20.729 20.799 21.148 20.561 21.651 20.799 20.380 20.771 20.911 20.310 20.799 20.366 20.212 21.162 20.450 21.036 ee we ew ew Nitrogen source, AMMONIUM SULPHATE Wt. of Medium, Grams 148.30 148.80 149.35 148.40 148.15 148.45 148.25 147.95 148.30 148.60 148.65 148.45 148.70 148.60 148.70 148.90 148.95 149.05 148.95 Mg. N per too Gr. Medium 14.421 : 14.776 14.188 13.968 14.039 14.246 13.869 14.634 14.025 13.715 13.973 14.086 13.658 13.997 13.696 13.574 14.208 13.720 14.123 \ ae 14.836 13.959 J Average| 14.462 14.084 14.176 13.925 13.784 13.834 14.032 14.398 Mg. Loss or Gain, too Gr. jane, 1927] WANN — FIXATION OF FREE NITROGEN 19 7B. Same nitrogen source. Medium with 1 percent mannite — | Check Ster. 20.520 | 148.55 | 13.814 | — | Check Ster. 21.162 148.80 | 14.222 } 13.993 — | Check Ster. 13.872 99.50 | 13.942 1 | Chlorella vulgaris +-+-+* 20.966 148.35 | 14.133 I Beyr. +-+-+- 22.070 148.40 | 14.872 } 14.321 | +0.328 I “ele te 20.729 | 148.50 | 13.959 2 | Stichococcus sp. s=45 20.938 148.65 | 14.085 2 AP = 19.570 148.70 | 13.161 7 13.658| —0.335 2 =P ar 20.408 148.65 | 13.729 5 | Chlorella (?) sp. +++ 21.064 | 148.50 | 14.185 5 +++ 20.603 148.70) |, 135855 13-700 | 0-213 5 +++ 19.765 148.60 | 13.301 6 | Scenedesmus (?) sp. +-+ 21.637 | 148.80 | 14.541 6 aR ar 20.087 148.70 | 13.508 ; 14.061 | +0.068 6 AP Ar 21.008 148.65 | 14.133 11 | Chlorella (?) sp. ++ 19.905 | 148.80 | 13.377 II + + 20.701 148.90 | 13.903 } 13.584 | —0.409 II - SESE 20.073 149.00 | 13.472 ae rotococcus sp. + 20.226 149.00 | 13.575 ~ 7, : + 20.380 149.00 | 13.678 Lier 308 3 | Protosiphon +++ 20.757 149.15 | 13.917 2 botryoides +++ 21.134 | 149.20 | 14.162 HES I) ea O07 Average all cul- es MIVA Tt Fleihan weir aa 2 got Pe cane illo. ages 13.867 |. —0.126 TABLES 8, 8A, AND 8B. Results of analyses of mineral nutrient agar cultures of green algae. Experiment of 1919. Growth period, 255 days. Nutrogen source, AMMONIUM NITRATE 8. Medium without glucose or mannite Culture Wt. of Me. N per Mg. Los Total Mg. Beecher ae Saree No. Species nea Nevoune Raaee Pepe so eae pce — | Check Ster. |= 26.704" 9) 147245. | Team) Tia — | Check Ster. 26.789 147.95 | 18.107 + 17.736 — | Check Ster. | 25.189 148.25 16.991 1 | Chlorella vulgaris| ++++-+* | 27.015 147.65) :18-202 I Beyr. ima 27.029 | 147.25 | 18.356 + 18.308 | +0.672 I +++ 26.958 147.50 | 18.277 2 | Stichococcus sp. +-+* 26.576 | 147.35 | 18.036 2 + 26.166 147.50 | 17.740 j 17.989 | +0.253 2 aie 26.888 147.80 | 18.192 5 | Chlorella (?) sp. clash 25.543- | - 147.65 | 17.300 | 5 APSF 25.413 147.90 | 17.183 } 17.198 | —0.538 5 ae oar 25:27 4 EA77O" | 17-012 6 | Scenedesmus (?)sp.. +++ 26,610) | 147.750) 18i016 6 +++ 26.194 | 147.95 | 17.705 j 17.866 | +0.130 6 +++ 26.421 147.80 | 17.876 11 | Chlorella (?) sp. +++ 20.248 | 147.90 | 13.690 II slp aliments 20.615 147.95 | 13.934 + 14.018 | —3.718 II ++++ 21.380 148.15 ASE ily oe 431 Average all cul- | tures (omitting SHCEICSENO MDI nk) ge Me wages, co dee Ne 17.840 | -+0.104 20 AMERICAN JOURNAL OF BOTANY [Vol. 8 8A. Same nitrogen source, medium with r percent glucose Culture 0 Wt.of |Mg. N per 7 Growth MeN Medium. aoe co Average ue an ney No. Species Found Grams | Medium roo Gr. — | Check ster. 25.095 | 147.70 {6001 tee 991 — | Check Ster. 25.499 | 148.00 | 17.229 } 17.178 — | Check Con. 25.529 | 147.45 | 17.314 1 | Chlorella vulgaris (+) 26.774 | 147.55 | 18.145 I Beyr. (+) 27.369 | 147.50 | 18.555 + 18.272|+1.094| 6. I (+)* 26-774 | 147.8018 115 2 | Stichococcus sp. (++) 26.180) 147-30) 7-776 25 Grr. 23.009 | 147.15 | 15-636 ; 17.417 |4-0.239)| 1.4 2 (+-+)* 27.822 | 147.65 | 18.843 5 | Chlorella (?) sp. ++++-+ | 27.161 | 147.20 | 18.452 5 +++++. | 28.658 | 147.75 | 19.396 ¢ 18.973 |-+1.795 | 10. 5 ++++-+*| 28.195 | 147.85 | 19.070 6 | Scenedesmus (?) sp.| ++—+-++ | 27.496 | 147.80 | 18.604 6 +++++ | 24.452 | 147.85 | 16.538 } 17.911 |+0.733| 4.2 6 +++++ | 27.496 | 147.90 | 18.591 11 |-Chlorella (?) sp. +++++ | 27.358 | 148.10 | 18.473 II ++++4-+ | 27.037 | 148.15 | 18. cae .293/-+1.115| 6.5 II ++++4 | 26.807 | 148215 | 182555 7 | Protococcus sp. ++++-+ | 28.377 | 148.10 | 19.161 7 ++++7 | 28.992 | 148.25 (19.556 719:202)7-2.le samee a i - ca aa 28.433 vee 19.192 3 | Protosiphon OSE © NELAS 35. Wears 2 botryoides aoe ar 26.619 | 147.30 | 18.071 18.071 | 02931 one Average all cul- te tures) 0" Se reer re (eee 18.320 !-- 1.142) |/36:6 8B. Same nitrogen’ source, medium with 1 percent mannite Culture Wt. of Mg. N per Mg. Loss Growth Total Mg. | Medium, too Gr. Average] or Gain, No: Species N Found Grams Medium too Gr, — | Check Ster 25-741 146.95 | 17.517 — | Check Ster 25.571 148.10 | 17.266 + 17.091 — | Check Con.* 24.184 146.65 | 16.491 I Chlorella vulgaris +++ 25.458 146.65 | 17.360 I Beyr. +++ 26.350 146.65 | 17.968 >} 17.587| +0.496 I Sap ae 25-033 | 143.60 | 17.433 2 | Stichococcus sp. ++ 24.467 146.80 | 16.667 2 Sear 24.000 146.90 | 16.338 ; 16.466 | —0.625 2 Sea ZAIGIS 147.10 | 16.392 5 | Chlorella (?) sp. aerate 26.746 | 147.10) | {08.182 5 +++ 24.368 147.15 | 16.560 - 17.243 | +0.152 5 +++ 25.047 | 147.45 | 16.987 } 6 | Scenedesmus (?) sp. SPAR AR 26.307 | 147.50 | 17.835 6 +++ 24.269 147.55 | 16.448 - 17.403! +0.312 6 arial | 26.449 | 147.55 | 17.926 11 | Chlorella (?) sp. +--+ )e21kO33 147.75 | 14.845 II lalate | 22.258 147.80 | 15.060 } 14.891 | —2.200 II ++ 21.848 147.95 | 14.767 7 | Protococcus sp. + 24.807 147.85 | 16.779 7 ae 25.840 | 148.10 | 17.448 { 17.114] -+0.023 3, | Protostphon ++ 25.812 148.15 | 17.423 3 botryoudes ++ 24.099 | 148.20 | 16.200 { 16.812} —0.279 Average all cul- tures (omitting SPECIES MO. TD) eee eG Cee ele eee 17.103 | +0.012 Jan., 1921] WANN — FIXATION OF FREE NITROGEN 21 TABLES 9, 9A, AND QB. Results of analyses of mineral nutrient agar cultures of green algae. Experiment of 1919. Growth period, 235 days. Nitrogen source, CALCIUM NITRATE 9. Medium without glucose Culture’ Total | Wet. of |Mg. N per Mg. eee Gan Growth Mg. N | Medium, | 1tooGr. Average | or Gain, | Percent No. Species Found Grams Medium too Gr. — | Check Ster. 24.202 | 147.90 | 16.364 | — | Check Ster. 24.146 | 147.95 | 16.320 >} 16.755 — | Check Ster. 26.045 | 148.15 | 17.580 1 | Chlorella vulgaris SR aR SR 25.822.| 147.25 |'17.536 | I Beyr. +++ 26.939 | 147.15 | 18. 307 | 17.874 +1.119 6 I Sp aRSe 26.199 | 147.35 | 17.780 2 | Stichococcus sp. =F 22.735 | 147.30 | 15.435 2 ar or 26.464 | 147.45 | 17.948 j 17.058 |+0.303 2 =e 26.241 | 147.50 a i 5 | Chlorella (?) sp. ++ 27222" 147.00 I | 5 SRaIr 26.520 | 147.90 oe 931 + 18.689 |-+1.934 | II.5 5 Maes 28.978 | 147.65 | 19.626 6 | Scenedesmus (?) sp. +--+ 22.875 | 147.85 | 15.472 6 +-+ 24.341 | 147.90 | 16.458 +; 16.522 | —0.233 6 sear 26.073. | 147.85. | 17.625 1r | Chlorella (?) sp. +++ 17.121 | 148.25 | 11.549 II +++ 17.749 | 148.15 | 11.980 ros |? 11.439 |—5.316 II SP Sear 16.046 | 148.75 | 10.787 Average all cul- tures (omitting S[OCTES Or, gL acme © en eo (nearer 17.311 |+0.556 9A. Same nitrogen source, medium with 1 percent ee — | Check Ster. 23.055 tee | 15.504 ) — | Check ster. 22.553 | 148.85 | 15.152 } 15.640 ——~ | Check Sler. 24.298 | 149.40 | 16.264 1 | Chlorella vulgaris | ++-+-++ | 34.535 | 148.70 | 23.225 | I Beyr. +++++* | 33.948 | 148.50 | 22.861 } 22.963 |+7.323 | 47. I ; +++++ | 33.864 | 148.50 | 22.804 | 2 | Stichococcus sp. +++++-+) 35.024 | 148.50 | 23.585 | 2 4 tot +} 30.020") 148.50 20.754" > 22.440 |--6.800 | 43.5 2 ae te toch te tc o46OO | E4875.) 22.982 | 5 | Chlorella (?) sp. ++++-+ | 34.849 | 148.70 | 23.436 5 Tors | 35-799: 148.900.) 24.042; ) 23.677 |--8.037 | 51.4 5 Tak tet de |S 8071, |. 248-90: 237553 | 6 | Scenedesmus (?)sp.. +++++ | 34.388 | 148.75 | 23.118 | 6 Trt | 34-975 | 149.15 | 23.450 + 23.434 |+7.794 | 49.7 6 Teich, 1359330. 149-00) (23.733 | II | Chlorella (?) sp. ++++++) 31.714 | 148.90 | 21.299 | i Sect ote at oh | S208 5. | P4QUOO 22.0342) 21. 319)|-+5.679 | 36.3 II a care 30.750 | 149.10 2 ee | a YrOLvococcus sp. 24.243 | 149.05 | 160.2 | 7 + 24.578 | 149.18 | 16.479 | 20:372| +0732 4.7 3 | Protostphon +++-+ 25.360 | 149.15 | 17.003 { | 3 | botryoides sae SUL 26.477 | 149.25 | 17.740 f 0°!" 17-372, | 11.732 | II. Average all cul- | e EUIISS Sy oo oa PAR ea ei Rc) ay eee Mey crt all age 21.082 +5.442 | 34.8 22, AMERICAN JOURNAL OF BOTANY [Vol. 8 9B. Same nitrogen source, medium with 1 percent mannite Culture - Weight of | Mg. N per Mg. Loss Total Mg. | Medium, too Gr. e i Ne oe roe | N Found | “Grams” | Medien ee yey — | Check Ster. 27.865 148.75 | 18.733 — | Check Ster. 25.360 148.55 | 17.071; ige72o — | Check Con.8 25.826 148.60 | 17.380 1. | Chlorella vulgaris sf 22.818 148.70 | 15.345 I Beyr. +++ 23.558 148.90 | 15.821 + 15.280; —2.448 I +++ 21.799 148.55 | 14.675 2 | Stichococcus sp. a Seat 21.904 | 148.70 | 14.730 2 ++ 2A 301 148.70 | 16.349 >} 16.529| —1.199 2 aE 27.511 148.65 | 18.507 5 | Chlorella (?) sp. a es De 26.973 148.60 | 18.151 5 ++ 27.029 148.60 | 18.189 } 17.764 | +0.036 5 ++ 25.189 148.60 | 16.951 6 | Scenedesmus (?) sp. ++ 19.634 | 148.55 | 13.217 6 | ++ 24.326 148.65 | 16.365 ; 15.603 | —2.125 6 | ++ 25.600" | 148,65) al 7.228 11 | Chlorella (?) sp. +++ 13.481 148.70 9.066 es +++ Lost TAS. 80m) are + 9.430] —8.298 II a uaa 14.613 149.20 9.794 7h rotococcus sp. 22.251 149.20 | 14.91 ml + Lost | 149.15 | 1... / 24914] 2.814 3 rotostphon +++ 24.481 149.1 16.41 3 botryoides +++ eae Te ie ! 17,0839) a/0n5 Average all cul- tures (omitting | Species MOTT) (i. Pe pe ea ee eee 16.192 | —1.536 DISCUSSION From a study of the foregoing tables it is apparent at once that sub- stantial increases in the total combined nitrogen content of the culture flasks occurred with the media containing nitrate nitrogen and glucose. Fixation on these media was not confined to any one species, most of the forms used showing the ability to utilize the uncombined nitrogen of the air. The cases of Protococcus (species no. 7) and Protosiphon (species no. 3) may be questioned, but it will be noticed that of all the species grown on ammonium nitrate with glucose in the I919 experiment (table 8A) Protococcus gave the highest gain in nitrogen, and that in the same experiment Protosiphon gave an increase of nearly 2 mg. on calcium nitrate with glucose (table 9A). Although an increase of only 2 mg. is a small amount to base definite conclusions upon, the evidence is certainly in favor of the assumption that these two species also possess the ability to fix nitrogen. As regards species nos. I, 2, 5, and 6 there can be no question about their ability to fix nitrogen, since the increases with these species range from a fraction of a milligram to over 8 mg. per 100 grams medium, representing additions to the total nitrogen content of the flasks of from 1 to 51 percent. The highest increase noted was in the case of one of the cultures of species no. 5 on calcium nitrate with glucose, in the 1919 experiment (table 9A); this flask showed a total gain of 12.53 mg. over the average of the checks, an Jan., 1921] WANN — FIXATION OF FREE NITROGEN 23 increase in nitrogen content of 54 percent. Fixation was secured by these four species in both experiments when grown on similar media and under similar conditions, so that there seems to be no escape from the definite conclusion that these forms do actually use free nitrogen. The amounts of fixation secured by these four species on ammonium nitrate with glucose in 1919 were considerably lower than found in the 1917-18 experiment on a similar medium. As already pointed out, however (page 9), the growth produced on this medium in 1919 was very poor, a factor which, it is believed, accounts for the smaller fixation. This leaves to be considered the case of species no. 11 (Chlorella sp.) which is of considerable interest. In series 8A and 9A (the nitrate media with glucose) substantial increases in the nitrogen content of the cultures occurred, especially on calcium nitrate, where the average of the cultures is more than 5.5 mg. above the checks. This same species, however, on both nitrate media without a carbon source, and on the nitrate media with mannite, showed distinct losses in nitrogen. These losses ranged from 2 to 8 mg. and occurred regularly on the media mentioned with this species only. This species was used only in the 1919 experiment, in which acid ‘tubes were not inserted between adjacent culture flasks, so that there is no means of telling whether the loss occurred as ammonia or as free nitrogen. However, that some sort of process resulting in a loss of introgen has taken place is apparent, and the results suggest that perhaps both the processes of free nitrogen fixation and of denitrification may be going on simul- taneously, the former overbalancing the Jatter when glucose is present. As regards the use of free nitrogen by the forms investigated on nitrate media to which no carbohydrate energy source was added, the evidence is not conclusive. In 1917-18 gains of from 0.7 to 1.8 mg. per 100 grams medium were recorded for three of the species grown on ammonium nitrate (table 4), and one of these species gave an increase of 1.8 mg. on calcium nitrate without a carbon source (table 5). In 1919 three out of five species showed very slight increases on ammonium nitrate in the absence of a carbon source, while on calcium nitrate without glucose or mannite gains of from 0.3 to 1.9 mg. per 100 grams medium occurred with three species (table 9). As has already been pointed out, however, the growth of all species in the absence of glucose is very slow, and, although the cultures remain in a healthy condition for long periods of time, the actual amount of ‘“crop’’ produced is slight in comparison with the glucose cultures. That some fixation does take place in the absence of glucose seems probable, in view of the increases cited above, and the assumption that the amount of fixation on one and the same medium is dependent, more or less, on the total amount of growth produced does not seem unwarranted. This phase of the problem is of considerable importance and is one which should be most carefully investigated, as it bears directly on the conditions as they exist in nature. It is entirely possible that the conditions realized in the 24 AMERICAN JOURNAL OF BOTANY [Vol. 8 experiments, under which marked fixation occurred, have merely amplified those already existing in the field, for soluble carbohydrates and a certain amount of nitrates would most certainly be available for the development of these organisms, especially those occurring naturally on soil. If this is actually the case, it is highly probable that fixation occurs also under natural conditions. The evidence that no fixation occurred on media lacking nitrates is quite conclusive. When nitrogen was supplied as glycocoll, asparagine, urea, Or ammonium sulphate, the increase or decrease in the nitrogen content of the culture flasks above or below that of the checks was in all cases less than a milligram, even though the growth, especially on urea with glucose, was as luxuriant as that produced on nitrates in the presence of glucose. The results were uniform for all species grown on media con- taining these nitrogen sources and were not altered by the presence either of glucose or of mannite, the latter being used only with ammonium sulphate. Although some of the cultures of these series showed higher nitrogen con- tents than the checks, there were no consistent gains, and in most cases the figures were lower than those of the checks. However, the differences in either case are so small as to be without significance, since most of them are within the usual experimental error. A comparison of the results presented here with figures secured by a large number of investigators working with the legume nodule bacteria and Azotobacter forms shows that the amount of fixation produced by the algae per unit volume of medium, under the conditions of the experiments, equals, and in some cases surpasses, the average fixation by the colorless forms. The legume bacteria especially give only slight increases when grown in pure culture outside the host plant. Fred (1912) gives a table. of the results of 25 investigators, from which it is apparent that the average fixation with this organism is about 0.9 to 2.2 mg. per 100 cc. culture medium. Fred himself secured fixations ranging from 0.4 to 1.58 mg. per 100 cc. culture solution. With the Azotobacter forms the amounts of fixation are considerably larger. Azotobacter chroococcum, which has been extensively investigated in pure culture, shows increases ranging from a few milligrams to 20 or 30 mg. per 100 cc. culture medium, the average fixation, however, being usually about 6 or 10 mg. Thus Gerlach and Vogel (1902) report 4 mg. to be the average, while Freudenreich (1903) secured only 1.6 to 2.4 mg. per 100 cc. culture solution, but on gypsum plates the increases were higher, amounting to 16 mg. per 100 cc. solution. Krzemieniewski (1908) secured increases ranging from I to 13.5 mg. per 100 cc. solution, an average of 3 to 6 mg. being fixed for each gram of glucose respired. In the presence of humus the ratio was 5 to 10 mg. nitrogen fixed per gram of glucose respired. Hoffmann and Hammer (1910) report fixations of from 4.55 to 14.4 mg. per gram of carbohydrate consumed, and in the same year Krzemieniewska (1910) published results Jan., 1921] WANN — FIXATION OF FREE NITROGEN 25 showing increases of from II to 18 mg. per 100 cc. culture solution. Remy and Rosing (1911) found from Io to 20 mg. to be the average fixation per 100 cc. culture, or 2 to 15 mg. per gram of mannite consumed. Perhaps the most extensive experiments with Azotobacter forms are those of Lipman (1903 and 1905), who secured the following gains in nitrogen, each in 100 cc. of culture solution: A. chroococcum, 4.42 mg.; A. beyerincku, 2.37 to 6.78 mg.; A. vinlandit, 1.67 to 7.90 mg. in 1903, and 8.36 to 20.99 mg. in 1905. The numerous results with mixed cultures and impure soil cultures by many investigators show even wider ranges of fixation, and the condi- tions of the experiments have been so various that it is difficult to arrive at an average, but in many cases it appears to be between 6 and 10 mg. per 100 grams of culture medium. Because of the nature of the experiments reported in this paper it is impossible to state in what form the nitrogen is added to the culture. Whether or not the process is connected with that of photosynthesis, the ratio of nitrogen fixed to sugar respired or to the amounts of nitrate absorbed, and the amount fixed per unit dry weight of crop produced are phases of the problem which can be merely mentioned at this time. The solution of these questions will be simplified by the proper development of liquid culture methods, some of which are now in progress. Since the results obtained are wholly contrary to the generally accepted idea of the relation of green plants to elementary nitrogen, it may be well to point out the conditions under which some of the earlier negative results with the green algae were obtained. The first nitrogen determinations of pure cultures of algae were made by Kossowitsch (1894), who grew a single species of Cystococcus (?) on sand moistened with 20 cc. of a mineral nutrient solution containing calcium nitrate as a source of nitrogen. The cultures, however, grew for only three weeks, because, the author concludes, of the lack of nitrates, only 2.5 mg. nitrogen as nitrate having been supplied. The addition of more calcium nitrate to the cultures caused them to revive, and it was upon this fact that his conclusion was based. However, it is not altogether clear that the lack of growth may not have been due to some other cause, such as deficiency in calcium, only a “trace”’ of which was present in addition to that added as calcium nitrate. The analysé¢s of the cultures and uninoculated checks clearly showed that there had been no increase in the nitrogen content of the flasks, either in the presence or in the absence of glucose. It is apparént, however, that conditions for a vigorous, long-continued growth were not realized in these experiments, hence it is not to be expected that appreciable increases in nitrogen should occur. As a result of a large number of analyses of pure cultures, Kruger and Schneidewind (1900) concluded that the green algae investigated were unable to use free nitrogen. They grew a large number of forms on a variety of media, using both sand and solution cultures. Two of the media contained nutrient solutions free from combined nitrogen, five contained 26 AMERICAN JOURNAL OF BOTANY [Vol. 8 organic nitrogen supplied in beef extract and peptone, beerwort, or humus, and in the two remaining solutions both ammonium sulphate and sodium nitrate were present; in none of the media was the nitrogen supplied as nitrate only. The cultures were grown in large flasks plugged with cotton, no other provision being made for aeration. No growth resulted on the media free from combined nitrogen; on those to which organic nitrogen sources were added, a vigorous development of the algae resulted in most cases but no increases in nitrogen content were obtained. On the media containing inorganic nitrogen a good growth was obtained only in one experiment with cultures of several species of Stichococcus; in the remaining experiments with these media there was no growth because of the unfavor- able reaction of the solution. No increases in nitrogen content were ob- served, however, in any case. The results of these authors compare very favorably with those presented above in the tables, no fixation having been secured when ammonium sulphate or organic compounds served as nitrogen sources. In 1903, Charpentier substantiated Kossowitsch’s results with Cysto- coccus. He grew the single species on a bean-glucose-gelatin medium; at the end of 20 days no change in the nitrogen content of the cultures was observed. However, these experiments were neither extensive enough, nor were they continued for sufficient length of time, to be conclusive. The failure to secure fixation may be attributed in this case also to the absence of proper nitrogen sources in the medium. As already pointed out in the first part of this discussion, the presence of nitrate nitrogen seems to be essential for the process of fixation. In this connection it is interesting to note that the presence of nitrates has been found beneficial for the growth of, and fixation of nitrogen by, Azotobacter forms and the legume bacteria. It has been repeatedly observed that the presence of very slight amounts of nitrates stimulates the development of the nodule bacteria. Thus, Fred and Graul (1916) report that in the pres- ence of small amounts of ammonium nitrate nodules were produced in great numbers on alfalfa and vetch by pure cultures of Bacillus radicicola; numerous nodules were formed also in the presence of small amounts of calcium nitrate, but with added amounts the degree of infection declined. In a recent paper, Hills (1918) reports that the presence of small amounts of nitrates exerted an enormous influence on the growth and reproduction of Azotobacter in sterile soil, the amount of fixation in such cultures being also increased in the presence of nitrates. In the case of Bacillus radicicola the nitrates stimulated growth and reproduction in sterilized soil, and on agar films the amount of fixation was greater when nitrates were added. The author concludes that this increased fixation may be the result of the increased growth of the organisms where nitrates were supplied. Though such observations as these cannot be claimed to have any direct application to the case of green plants, they are at least suggestive in view of the fact Jan., 1921] WANN — FIXATION OF FREE NITROGEN Za, that nitrates have long been regarded as in general the best combined nitrogen source for chlorophyll-bearing plants. The proof that green plants are capable of fixing free nitrogen is at once of real significance, both from a purely scientific and from‘a practical standpoint, even though the plants concerned are members of a low order. The question which perhaps at first suggests itself is: Do all green plants possess this ability to use the uncombined nitrogen of the air? With the exception of the legumes, the evidence is almost entirely negative as regards the flowering plants. Mbolliard (1916) has recently demonstrated that radish plants in pure culture are unable to increase the nitrogen content of the culture. The plants were grown in large tubes on nutrient solutions containing several different concentrations of ammonium chloride as a source of nitrogen, some cultures being supplied with glucose, and others being aerated with air charged with carbon dioxide in order to increase the photosynthetic activity. In no case did the total nitrogen of the crop and residual culture solution exceed that of the seed and original solution, even after six weeks’ growth. The plants produced, however, were small, bearing three or four leaves in addition to the cotyledons, the green weight varying from 0.3 to 1.3 gr. Moreover, nitrate nitrogen was not supplied as a source of nitrogen in any case, a fact which may be of considerable importance in view of the results presented in this paper. It is possible that in the case of the higher plants also the proper culture conditions have not yet been realized, since in the case of four radish plants grown with the roots under aseptic conditions and the tops in free air, gains of 1.32 mg. nitrogen per plant were reported by Molliard, but he assumes that this nitrogen has come from the ammonia of the air. The significance of the legume bacteria has recently been questioned by Beijerinck (1918) who argues that, with the exception of lupine and seradella, the number and weight of the tubercles is insignificant in comparison with the total weight of the plant. The isolated organisms fix nitrogen in pure culture only very slightly or not at all, and no fixation could be demonstrated with nodules placed in glass jars in which the changes of gases were meas- ured. In the case of Robinia the number of tubercles is extremely small and fixation in them must be very intense, yet when tested they were found to be inactive or nearly so. He concludes that ‘“‘the present accepted explanation of the behavior of legumes cannot be correct.’’ The situation in the higher plants needs to be more carefully and thoroughly investigated. ° CONCLUSIONS I. Seven species of grass-green algae (Chlorophyceae) exhibited the ability to fix nitrogen when grown in pure cultures on mineral nutrient agar media containing either ammonium nitrate or calcium nitrate as a souce of nitrogen, and glucose. The nitrogen fixed was derived from the free (un- combined) nitrogen of the atmosphere. The amounts of fixation ranged 28 AMERICAN JOURNAL OF BOTANY _ [Vol. 8 from I to 12.5 mg., representing increases in the total nitrogen content of the culture flasks of from 4 to 54 percent. _2. Five of the above mentioned species were grown on ammonium nitrate and calcium nitrate in the absence of glucose. Four of these showed slight increases in nitrogen over the original content of the medium; growth in the absence of glucose, however, was slight. 3. There was no fixation when urea, glycocoll, asparagine, or ammonium sulphate was supplied as a nitrogen source, either in the presence or in the absence of glucose or of mannite. 4. One species (no. 11) exhibited what is apparently a denitrification on media containing either ammonium nitrate or calcium nitrate as nitrogen sources, both in the absence of an organic carbon source and in the presence of mannite. The loss in nitrogen amounted to from 2.2 to 8.3 mg. per 100 grams culture medium. ACKNOWLEDGMENTS The writer wishes to express his sincere appreciation to Dr. J. R. Schramm, whose many helpful suggestions and constant interest have been invaluable aids in the completion of the experiments, to Dr. L. W. Sharp for the photographic work, and to other members of the department for kindly assistance in many ways. DEPARTMENT OF BOTANY, NEW YorRK STATE COLLEGE OF AGRICULTURE, ItTHAacA, NEW YORK LITERATURE CITED , Beijerinck, M. W. ‘The significance of the tubercle bacteria of the Papilionaceae for the host plant. Kon. Acad. Wetensch. Amsterdam (English ed.) 21: 183-192. 1918. Charpentier, P. G. Alimentation azotée d’une algue. Le Cystococcus humicola. Ann. Inst. Past! 17: 321-334. 1903. Duggar, B. M., and Davis, A. R. Studies in the physiology of the fungi. I. Nitrogen fixation. Ann. Mo. Bot. Gard. 3: 413-437. 1916. . Fred, E.B. \ Die cut a ONS / : eas = 3 \s \ aa S AIS \ Pe \ fips 4 > is | | / vies Y 2 \ \ sain f- le | 1G) PSS i x fey 4) Mea \ / 1s) \ \ oe SS / / es) aA + = ~ + — 1 ae i eet DUTCH WATER IN SHADED TUB} —-—"F-—-— 0 Fic. 3. Oxygen content of water in ditch of State Experiment Bog, East Wareham, Massachusetts, and of water from the ditch held in tubs under experimental conditions, 1919. 56 AMERICAN JOURNAL OF BOTANY [Vol. 8 Very little difference was noted as to degree of injury between shaded plants in ditch water and shaded plants in pond water. This indicates that nothing in the quality of the water aside from lack of oxygen caused the injury. The plants that were submerged but not shaded suffered little or no injury. Analyses of the water show that in these tubs the oxygen content decreased only during the night and did not remain long at a low level. The shading during the day prevented the accumulation of oxygen which would have carried the plants through a considerable part of the night. Cloudiness would have the same effect, the effect being more pronounced with greater reductions of light. | In this connection, attention may be called to the rate of respiration of. the flowers and of the young and old shoots of cranberry plants as measured by the rate of production of ‘carbon dioxide per unit of weight material. The experiments were made with Early Blacks. A weighed amount of flowers, growing tips, and old shoots was placed in closed receptacles of known volume. Determinations of carbon dioxide were made at definite intervals after the experiment was started. Analyses were made with an Allen-Moyer Orsatt apparatus. The readings were corrected to show the volume of dry gas at 0° C. and at a pressure of 760 mm. The results are given in table 2. TABLE 2. A comparison of the carbon dioxide production of flowers, growing tips, and old shoots of cranberries Caen Dioxide Date Material Ree bes | of EE oeaent aon % Ce sper ioe per | Observed Hour aaa July 7 | Flowers 11.0 250 Tle 2 hvel Sat. We22e 0.4 4.2 38.6 ‘““) Growing tips 19.0 25 oF 23h 20ers ame Ou; veel 27a “ |) Old shoots 36.5 54 Bala sles ere ys 0.5 5.2 iALG “8 | Flowers 11.0 Zine Solel Seles TO ow eae 2.0 25 22.0 4° | Growing tips 19.0 Bee ne “ 3.0 Bai) 19.3 4. Old: shoots 36.5 IY. ail die ‘< 2.6 Be 6.1 ‘14 | Flowers Deai7, 26 | 1 hour 25.5| 0.45 10.5 76.5 |-Growing tips 25/9 205) | ie a 0.5 11.6 46.2 i =- | Oldsshoots 40.5 | a i 0.5 Ties 28.3 ‘14 | Flowers 13.7 26 | 2 hours 27-5| 4.0.5 ts 42.0 — | Growing cipsa! 2510 30) s 2725s eleO 22.9 45.8 ~- = 2) Old shoots 40.5 57) o i 0.6 13.7 16.9 “14 | Flowers E2e7, 26 SF 28.0] 0.5 Pes 41.8 ~ » Growing tips |) 25:0 39 a a 0.9 20.6 41.0 os an @ldushoots 40.5 By | : z 0.5 LPs5 14.0 From the results of these experiments it is evident that under the same temperature conditions the flowers and growing tips show a much higher rate of production of carbon dioxide than do the old shoots. Nicolas (4, p. 109), in some twenty plants studied, found respiration more rapid and - Jan., 1921] BERGMAN — OXYGEN CONTENT OF WATER 57 the respiratory quotient higher in young leaves than in those fully matured. Most of the experiments indicate (see table 2) that the flowers produce carbon dioxide at a slightly higher rate than the growing tips, although in the second trial on July 14 this is not true. In these experiments the flowers and growing tips produced carbon dioxide two to three times as fast as did the old shoots. Maige (3, p. 1) has shown in experiments with a large number of plants that the intensity of respiration of floral organs is greater than that of leaves. A more rapid rate of respiration, however, means a greater oxygen requirement. This accounts for the injury to the flowers and growing tips resulting from prolonged submergence in water deficient in oxygen. From the data submitted it is evident that injury is most apt to occur to a bog by flooding during a period of cloudy weather. Naturally the injury is apt to be greater the longer the period of time during which the water is held on the vines, and especially if cloudiness prevails throughout the period. The degree of injury is probably not directly proportional to the reduction in light interfsity, for, as Brown and Heise (2, p. 85) point out, the published works on carbon dioxide assimilation “indicate a progressively smaller augmentation of the rate of assimilation for each increase in light intensity.”’ A certain, as yet unknown, reduction in light intensity is necessary to bring about a balance between the photosynthetic and the respiratory activity. After this point has been reached, accepting the conclusions of Brown and Heise (2, p. 94), each decrement in light intensity should have a progressively greater injurious efiect. A number of other factors which may affect the result are operative. Much depends upon the character of the water as it is placed on the bog. Clear pond or river water has a higher initial oxygen content, so that the oxygen is not depleted as rapidly as from a water supply initially deficient in oxygen. Where water from a cedar swamp, or other reservoir having a great deal of organic matter on the bottom, is used, the initial oxygen content may be very low. This, as indicated elsewhere, is due to the fact that organic matter absorbs oxygen and gives off carbon dioxide. In cloudy weather the initial oxygen content of water from a swamp reservoir would be considerably lower than in clear weather. All other factors remaining the same, greater injury would result during a period of warm weather than of cool, due to the increase in the rate of respiration with a rise of temperature. On the other hand, the ability of the water to absorb oxygen diminishes with an increase of temperature. In conclusion it may be pointed out that the matter of oxygen content as affected by light intensity and other factors is of great importance in combating insect pests by flooding. A combination of factors producing a low oxygen content in the flooding water at a time when the insect larvae are active is most desirable. Such conditions would, however, be highly detrimental to the cranberries. It would be necessary, therefore, to con- 58 AMERICAN JOURNAL OF BOTANY [Vol. 8 sider the state of activity of the cranberries and of the insect larvae, and, if possible, to adjust the length of the flooding period so that the larvae would be killed without injury, or at least without extensive injury, to the vines. A careful study of these problems is highly desirable. SUMMARY In a study of flooding water of cranberry bogs of Massachusetts and Wisconsin a variation in oxygen and carbon dioxide content of the water was observed. The effect of cloudiness on the oxygen and carbon dioxide content of water is indirect, resulting from the action of light on submerged vegetation. The variation in oxygen and carbon dioxide content of flooding water in the cranberry region of Cape Cod, Massachusetts, as affected by light intensity, organic matter, and abundance of vegetation, is shown. An experiment is described showing the effect of shading submerged cranberry vines. The resulting injury is due to reduction of oxygen content of the water. No essential difference was observed between the amount of injury to shaded vines in pond water and that to shaded vines in bog ditch water. The flowers and growing tips of shoots, which were the pants most seriously affected, have a higher respiratory rate than old shoots, as shown by experimental results. This accounts for their greater injury by sub- mergence in water deficient in oxygen. Injury is most apt to occur to a bog by flooding during cloudy weather. BUREAU OF PLANT INDUSTRY, WASHINGTON, D. C. LITERATURE CITED 1. Birge, E. A., and Juday, C. The inland lakes of Wisconsin. The dissolved gases of the water and their biological significance. Wis. Geol. and Nat. Hist. Survey Bulli22.. srout 2. Brown, W. H., and Heise, G. W. The relation between light intensity and carbon dioxide assimilation. Philip. Jour. Sci. 12: 85-95. 1917. 3. Maige, G. On the respiration of different floral organs. Ann. Sci. Nat. Bot. IX. 14: 1 (not seen). Abstr. in Exp. Sta. Rec.25: 729. 1911. 4. Nicolas, G. The variation in the respiration of plants in proportion to age. Bull. Soc. Nat. Hist. Afrique Nord 1910: 109 (not seen). Abstr. in Exp. Sta. Rec. 26: 628.) 1002. 5. Pettersson, O., and Sonden, K. Uber das Absorptionsvermégen des Wassers fiir die atmospharischen Gase. Ber. Deutsch. Chem. Ges. 22: 1439. 1889. 6. Roscoe and Lunt. Chem. Soc. Journ. 55: 552. 1889. . Winkler, L. W. Die Bestimmung des im Wasser gelosten Sauerstoffes. Ber. Deutsch. Chem. Ges. 21: 2843. 1888. 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University “Repeenine 4 Aierican Poses Sotiety ates : Uae is pobiaped. iouthly, except during Nt : “Subscription price, $6.00,a year. Single copies 75 cents. Back mun |. each 3'$6.00;a volume, postage extra,” Postage will he charged to al | tries, “except Mexico, ‘Enba, Porto’ Rico, Panama: ah “4 Panama, | Hawaii, Philippine ‘Islands, ‘Gaam, ‘Samoan’ I ‘Postage to, Canada, 20 cents a volume on: annual subseri tries” in) the: Bostal, Union, 40. cents. A volume on ‘annial sub ba “ onesie bated fot? Baan. iis : cases: be, subni mitted to the. Editor-in: ‘Chief, nats Oe : ‘Papers are . limited to 20 pages in fength; ‘exclu ive Jonas Babes am, De arranged) for at cost: rate to attthio ied Be eet mai ‘On. ic Garden, — should Ae “su fed tees torial rai 9 Vi consin font aa M Wyle, halide bi AMERICAN JOURNAL OF BOTANY VoL. VIII FEBRUARY, I92I No. 2 PPEURNCE OF TEMPERATURE ON THE RELATIONS BE- mevebnN NUTRIENT SALT PROPORTIONS AND THE PARE (GROW EE OF -WHEAT W. F. GERICKE (Received for publication August 5, 1920) Russell! has stated that ‘‘Potash-starved plants are the first to suffer in a bad season or to succumb to disease. The Broadbalk wheat plots receiving potassium salts give conspicuously better results than others whenever the year is unfavorable to plant growth. . . . The badness of the season may be connected with high rainfall and correspondingly low temperatures.’’ This statement emphasizes the fact that climatic condi- tions may exert no inconsiderable influence upon what may be regarded as the best set of proportions of the nutrient salts in the medium in which plants are rooted. What is a good set of salt proportions with one set of climatic conditions may not be a good one with another climatic complex, etc. Results obtained from an experimental study carried out in 1918-19, in the Laboratory of Plant Physiology of the Johns Hopkins University,’ seem to bear on this general and important proposition, and some of these results are here reported in a preliminary way. The investigation was planned to bring out the relations between main- tained temperature, on the one hand, and the physiological properties of various nutrient solutions, on the other. The germination and early seedling phases in the development of ‘‘Marquis’’ wheat were studied, the grains being supported on paraffined mosquito netting held just beneath the surface of the solution, which was renewed every 24 hours. The containers were glass tumblers having a capacity of 300 cc. Twenty-five seeds were used for each test, and all tests have been repeated at least once. Seven ‘ Russell, E. J. Soil conditions and plant growth. 2ded. 1915 (pp. 42,43). 3d ed. 1917 (pp. 43, 44). 2 This study was carried on as part of a cooperation between the committee on salt requirements of representative agricultural plants, of the Division of Biology and Agri- culture of the National Research Council, and the laboratory named above. It was partially financed from the war-emergency funds of the Council. The writer wishes to express his appreciation of much kindly interest and advice received from Prof. B. E. Livingston. [The Journal for January (8: 1-58) was issued March 9, 1921.] 59 60 _ AMERICAN JOURNAL OF BOTANY [Vol. 8 different maintained temperatures were employed, and light was excluded from the experiment chambers. The nutrient solutions used were the 126 3-salt solutions described by the committee on salt requirements,? and each solution was tested for every one of the seven different temperatures. These 3-salt solutions are of 6 types;* according to the salts employed, and 21 different solutions were tested for each type, each of these having its own peculiar set of salt proportions. The familiar triangular diagram was used to represent the difference in salt proportions. The salts employed were the nine possible combinations of the following six chemical units; K, Ca, Mg, NOs, H2PO., and SO. No iron was added to any of these solutions. As to total concentration, the solutions were all about alike and very weak, being only one tenth as concentrated as the corresponding I-atmosphere ~ solutions described in the plan above referred to. The present paper will be confined to certain points brought out for the two temperatures 28° C. and 17° C. (one about optimum and the other dis- tinctly below the optimum temperature for the early growth phases of wheat). Only those solutions of each of the six types tested will be con- sidered that gave the best growth values, determined by the criteria of total shoot elongation per culture and average elongation per seedling of the cultures, for a period of 110 hours, beginning with the placing of the seeds on the net. The best values obtained for the set of 21 solutions for each of the six types for each of the different temperatures tested were considered the ‘‘good”’ solutions, if their growth values obtained lay within the upper one fourth of the total range of values for the same temperature and the same solution type. For example, if the 21 solutions of the type con- taining the salts KHePO., Ca(NOs)e, and MgSOu., gave a growth value ranging from 1.00 to 1.80 for the average of both the criteria used, tested at a temperature of 28° C., then those solutions whose value lay between 1.60 and 1.80 would be classed as the “‘good’’ solutions for the type at that given temperature. The total-shoot-elongation value simply represented the total growth obtained from a solution. The average elongation value per seedling per culture was obtained by dividing the total shoot elongation in centimeters by the number of seedlings the culture contained. The reason these two criteria were employed was to offset any appreciable error that may accrue in the growth value from a failure of some of the seeds to germinate. The following table shows what solutions belong to the “‘good”’ class for each type and for each of the two temperatures here dealt with. In the solution designations, which refer to the triangular diagrams, the 3 Committee on salt requirements of representative agricultural plants, Division of Biology and Agriculture, National Research Council. A plan for cooperative research on the salt requirements of representative agricultural plants. -Edited by Burton E. Livingston. 2ded. Baltimore, 1919. 4For an outline of the chemical scheme of these six types, on which the committee’s plan was based, see: Livingston, B. E., and Tottingham, W. E. A new three-salt nutrient -- solution for plant cultures. Amer. Jour. Bot.5: 337-346. 1918. Feb., 1921] GERICKE — INFLUENCE OF TEMPERATURE 6I number following the letter “‘R’’ indicates the number of eighths (of the total molecular concentration) that are due to the potassium salt, the number following the letter ‘““S’’ indicates the number of eighths due to’ the calcium salt, and the difference between 8 and the sum of these two numbers is the number of eighths due to the magnesium salt. TABLEI. Good nutrient solutions of the six different salt types tested at two different maintained temperatures Type l Type Il Type III Type lV Type V Type VI Tempera- | KH2PO. K2S0s4, KNOs, K2SOu, KNOs, KH.2POsa, ture Ca(NOs) Ca(NOs)o, Ca( HePOu.)2 Ca(H2POs.)o, CaSQOu, CaSOa, MgSO4 | Mg(HePOs.)2 MgSO, Mg(NQOs3)o Meg(H2POs4)e2 Mg(NOs)2 25°. R1S3 R183 R1S3 R1S4 R1S3 RIS2 R1S$4 R1S4 R1S4 RiS5 R1S5 R1S4 R354 R251 R1S6 R2S1 R3S4 R4S1 R2SE R4S2 R4S2 R2S2 R483 R5S1 R5S2 \ oye €. R252 R3S3 R285 R4S1 R5S1 R4S3. R353 R5SI1 R3S4 R5SI1 R5S2 R5S1 R4S1 R5S2 R4S1 R5S2 R552 R483 R5S1 R5SI ‘ It is seen at once that the “good”’ group of each type of solution com- prises from two to seven different solutions. It appears that there is. generally a marked difference between the sets of salt proportions that proved good with the higher temperature, on the one hand, and those that proved good with the lower ones, on the other. For types II, III, IV, and V, low partial concentrations (1 or 2 eighths) of the potassium salt characterize the group for 28°, while high partial concentrations (2-5 eighths) of this. salt characterize the group for 17°. There is a suggestion of this same generalization for types I and VI also. For type I the potassium salt has partial concentrations of from 1 to 4 units for the higher temperature, while the corresponding values for the lower temperature lie between 3 and 5. Similarly, for type VI, the potassium-salt values lie between 1 and 5 for the higher temperature and between 4 and 5 for the lower. It is thus indicated that the proportion of the potassium salt should be high for the low temperature, and low for the high temperature, if the solution is to give good growth values. Since three different potassium salts are involved, it appears that this suggested generalization really applies to the partial concentration of potassium itself rather than to that of the salt that supplies. this element. 7 It is to be noted that the two types (I and VI) for which this statement concerning potassium is least definitely applicable, are both characterized by the fact that the potassium salt is the phosphate. But, for three other 62 AMERICAN JOURNAL OF BOTANY [Vol. 8 types (II, IV, and V), high partial concentrations of H2PO, characterize the good solutions for the higher temperature, while lower concentrations of H.PO. mark the good ones for the lower temperature. It is thus sug- gested that the H.PO,.relation (to growth and to temperature) may be the reverse of the K-relation. Potassium phosphate being employed in types I and VI as the only source of K as well as of H2POu., a sort of antagon- istic effect might be expected in these cases, and this expectation seems to have been realized. A study of the two groups of good solutions for each of these two types suggests an inversion of the H,.PO,-relation (low partial concentrations for the higher temperature, etc.) and a masking of the K-relation, as has been mentioned. Type III furnishes no evidence in this regard. It appears from these results that temperature is of prime moment in determining the mineral requirements for good germination and initial growth in this wheat, at least within the general limits of these experimental tests, and it seems safe to suppose that other climatic conditions may not be without influence. It is suggested that some of the unexplained dis- crepancies that are commonly encountered in comparative studies on plant salt requirements and on the application of fertilizers to agricultural soils, may be related to climatic influences. It seems clear that all influential conditions should be quantitatively considered in such studies. fae VASCULAR ANATOMY OF DIMEROUS AND TRIMEROUS SER DEINGS OF PHASEOLUS VULGARIS J. ARTHUR Harris, EpmMUND W. SINNOTT, JOHN Y. PENNYPACKER, AND G. B. DURHAM (Received for publication August 21, 1920) INTRODUCTORY The great majority of investigations dealing with the anatomy of plants have been purely descriptive in character. Asa result of observation, the typical or average condition of plant structures has been recorded in terms which are general and often indefinite. Comparatively few morpho- logical papers deal with the problem of the variation of the structures under consideration, treat of their correlations with one another, or even present the detailed measurements which might serve for the solution of such funda- mental morphological problems. The older comparative morphology is indispensable. It provides a general knowledge of plant structures and serves as a basis for the classi- fication of the vegetable kingdom. The recognition that description must be supplemented by the results of experimentation has, however, led to the establishment of the newer special science of experimental morphology. The time has come to extend still further our study of plant form by calling to the service of vegetable morphology the methods of measurement and mathematical analysis. These methods are particularly useful in an attack upon the fundamental problems of morphogenesis. It is by measuring exactly the various plant structures during their successive stages of develop- ment, in terms of size or number; by determining their relative variability in different organs or regions of the plant, or under varying external con- ditions, and by discovering such correlations as exist, both among the structures themselves and between them and their progenitors and their environment, that we shall be able to build up a body of fact on which morphogenetic theory may rest. The present paper gives a portion of the results of a biometric analysis of a comparatively simple morphological problem, that of the gross vascular anatomy of certain normal and abnormal bean seedlings. Our purpose has been: ; , I. A study of the vascular anatomy of normal and of abnormal seedlings from the point of view of descriptive morphology—a preliminary which we believe to be essential to a sound interpretation of any statistical results. 2. A statistical study of the number and variation of the vascular elements in different regions of the seedling. 63 64 AMERICAN JOURNAL OF BOTANY [Vol. 8 3. An investigation of the correlations between these internal characters (such as those which exist between bundle number in different regions of the seedling) and between the internal characters and external features of the plant. The results of the first and second phases of the investigation are set forth in the present paper; the third is reserved for a later publication. MATERIALS AND METHODS A priori considerations seemed to indicate that a promising line of attack upon the general field of quantitative plant morphology lay in the investigation of vascular bundle number. Such an investigation should be on a scale sufficiently large to make possible the determination of trust- worthy biometric constants, and should have as its subject a plant organ of relatively simple but variable structure. Because of the ease with which they can be grown in quantity, their sharply marked external characteristics. their convenient size for histological work, and their relatively simple internal structure, seedlings of Phaseolus vulgaris furnish highly satisfactory material for a study of variation and correlation in vascular structures. Among the many types of variant seedlings of the garden beans which may be secured by extensive plantings, two were selected for investigation: (a) normal (dimerous) seedlings, with two cotyledons and two primordial leaves, and (b) trimerous seedlings, with three cotyledons and three pri- mordial leaves. For brevity in table headings the dimerous plants will sometimes be represented by ‘‘2—2’’ and the trimerous by “3-3,” where the first figure gives the number of cotyledons and the second the number of primordial leaves. Since one of the purposes of this work is to carry out a comparison of bundle number in normal and teratological seedlings, the selection of a satisfactory control series of normal plants is a matter of primary impor- tance. It is essential that the seedlings of the types to be compared be selected in a manner to reduce to a minimum any external influences tending to bring about differences between them. It is clear that if the abnormal and the normal seedlings were taken from different series of parent plants, either genetic differences or environmental influences acting upon the parent plant might be effective in. bringing about a differentiation in the characters of the seedling examined. A normal seedling from the same parent was, therefore, taken for comparison with each abnormal seedling! in each series in which the seed was derived from individual parent plants. Closer control of the influence of innate differences in the parents and of the possible influence of parental environment hardly seems practicable since the 1 In the vast majority of the cases one abnormal seedling only was sectioned from a parent plant. When more than one abnormal seedling was available a control was taken for each. Naturally it is immaterial whether control a or 6 be compared with abnormal seedling A or B, since all are siblings. » Feb., 1921] HARRIS AND OTHERS — SEEDLINGS OF PHASEOLUS 65 pairs of abnormal and normal seedlings were, in three of the lines investi- gated, derived from the same parent plant. Furthermore, care was taken that seedlings compared were grown under essentially identical conditions, in order to reduce to a minimum the environmental influences which might possibly tend to bring about dif- ferences between them. Seeds from individual plants were germinated in flats and harvested as soon as possible after they broke through the sand. Thus all seeds not only developed under the same parental environment but were germinated under sensibly identical conditions, were collected simul- taneously, and were in consequence sectioned at essentially the same stage of maturity. 3 Because of the rapidity with which seedlings change and the great influence of temperature upon growth, it is difficult to standardize, or exactly to describe, the stage of development at which the seedlings were taken. Most of them were placed in alcohol before or very soon after the primordial leaves had unfolded. Thus a fairly uniform and early stage of development was secured.? Free-hand sections were cut and mounted temporarily. When neces- sary, phloroglucin and hydrochloric acid were used to bring out the vascular bundles. The general vascular topography of the seedlings was studied, but the data for the statistical analysis of the seedling anatomy were derived from a careful count of the number of vascular bundles at various levels in the seedling. Because of a certain amount of variation in the number of bundles with position in the organ, counts were made in definite regions only—the basal region of the hypocotyl (just at the point of tran- sition from ‘root structure’’ to ‘‘stem structure’’); the median region of the hypocotyl; and the median region of the epicotyl. In three series counts were also made of the protoxylem poles in the upper portion of the primary root. The number of data available for the several regions differs because of a change in the plan of the work. Sectioning and counting were begun by two of us at Cold Spring Harbor in the summer of 1917 and continued with the assistance of Miss Eunice Kinnear in the summer of 1918. This work was confined to the mid-regions of the hypocotyl and epicotyl. From a statistical study of these data it seemed desirable to have a further series of countings made independently by a specialist in vascular anatomy. The work was, therefore, continued at Storrs during 1918, 1919, and 1920. We are greatly indebted to Miss Flora Miller for assistance in this phase of the work. At Storrs, sections were made at the base of the hypocotyl] as well as in the mid-region of hypocotyl and epicotyl. In three series, sections were made of the root as well. The bundles vary considerably in size, the largest being well developed 2 Some of the seedlings of line 143 were allowed to become a little older, but there is no evidence of change in bundle number with age. 66 AMERICAN JOURNAL OF BOTANY [Vol. 8 and the smallest containing only one or two lignified xylem cells and a small patch of phloem. Some are even more reduced, consisting of a phloem patch alone. Any strand in which at least one well lignified xylem element could be made out was counted as a bundle. Some of the bundles are partially double in character, this condition being due either to partial fusion or to incipient division. Whenever such a strand was surrounded by one bundle sheath it was counted as one bundle; when the separation was so great that the bundle sheath itself showed signs of division, the strand was counted as two. The seedlings were harvested at a stage when the vascular tissues of the first epicotyledonary internode were not completely lignified, and the number of bundles counted was therefore possibly less than the number which would finally be developed there. None of these possible sources of error is believed to be great enough to affect the conclusions appreciably. THE STRUCTURE OF THE SEEDLING In order to provide a sound basis for the understanding and inter- pretation of our later work, it is necessary to present a brief descriptive account of the structure of the seedlings. ; The Normal (Dimerous) Seedling The morphology of the seedling of Phaseolus has received the attention of several investigators, notably Dodel? and Compton.‘ Like most of the large seedlings of the Leguminosae it is normally tetrarch in fundamental plan; that is, there are four groups of protoxylem elements in the root. At a very early stage there is associated with each of these a group of metaxylem cells. It is these groups of metaxylem elements, throughout the whole seedling, which in the present paper are counted as “‘bundles,’’ even though (as is sometimes the case) they are not associated with protoxylem clusters. At the stage when these seedlings were harvested, cambial activity had hardly begun to show itself, so that these primary bundles remained distinct and easy to identify. The condition in the upper part of the root of a normal seedling is shown in figure i. The four bundles, two in the cotyledonary plane and two in the intercotyledonary plane, are more or less V-shaped (with the protoxylem group in an exarch position at the apex of the V) and tend to extend laterally. They surround a large pith. In passing up into the base of the hypocotyl, each of these bundles divides into two (fig. 2), and typical stem structure, 3 Dodel. A. Der Ubergang des Dicotyledonen-stengels in die Pfahl-wurzel. Pringsh. Jahrb. 8149-193) ) 13872: 4Compton, R. H. An investigation of the seedling structure in the Leguminosae. Jour, Linn: S0c741n 7122-5 Tol: Feb., 1921] HARRIS AND OTHERS — SEEDLINGS OF PHASEOLUS 67 with the protoxylem in an endarch position, begins to be assumed. Each pair is subsequently referred to as a “‘primary double bundle.”’ Thus the level of transition from root structure to stem structure is low, being prac- <7 S Ss ; ‘ y) \ v “h ) "a eee oly eS a a Fic. 1. Dimerous seedling. Transverse section through the root, showing its tetrarch condition (four protoxylem poles). Fic. 2. Dimerous seedling. Transverse section through the base of the hypocotyl showing the four primary double bundles, each of which has been derived from one of the four root strands. Fic. 3. Dimerous seedling. Transverse section through the mid-region of the hypocotyl showing the normal eight- bundled condition. No intercalary bundles are figured. Fic. 4. Dimerous seedling. Transverse section just below the cotyledonary node. The four bundles or bundle groups have originated by a more or less complete fusion of the adjacent members of each of the original pairs. Each bundle, as shown by the two constrictions in it, is about to break up into the three strands shown in figure 5. Fic. 5. Dimerous seedling. Transverse section through the cotyledonary node. Each group of three strands which have arisen by a breaking up of the large bundles in figure 4 is here enclosed by a dotted line. These three strands are a cotyledonary trace (solid black), an epicotyledonary bundle, and a small bundle which will fuse with its adjacent neighbor to form another epicotyledonary bundle. Fic. 6. Dimerous seedling. Transverse section through the mid-region of the epicotyl showing the twelve bundles which have arisen by the splitting of the six original epicoty- ledonary bundles. The six strands which are to go off as traces to the two primordial leaves are solid black. tically at the base of the hypocotyl. The members of each of these four pairs soon separate until the eight bundles are approximately equidistant (fig. 3), a condition which persists throughout the hypocotyl] until the coty- ledonary node is approached. In addition to these bundles, there are in a considerable percentage of the normal seedlings studied a variable number of accessory or intercalary bundles, the ‘‘Zwischenstrange’’ of Dodel. These may make their appear- 68 AMERICAN JOURNAL OF BOTANY [Vol. 8 ance in the upper part of the root or in the lower region of the hypocotyl, some ending blindly below and others arising by division of the primary bundles. These intercalary bundles, which are not a very common feature of seedling anatomy in general, perhaps serve to increase the conductive capacity of the hypocotyl and may be associated with the large size of the seedling. They usually lack protoxylem elements. At the cotyledonary node there is a rather complex anastomosis of the bundle system. The details of this vary somewhat, but its fundamental features are as follows: The two members of each of the two original pairs of bundles in the cotyledonary plane (that is, opposite the two points where the cotyledons will later arise) become widely separated, and each member fuses with the adjacent member of the intercotyledonary pair (fig. 4). Four large bundles or bundle aggregates are thus produced. Each breaks up immediately, usually into three parts. The lateral member of each group of three which is in the cotyledonary plane approaches the corresponding bundle of the next group of three, and these two strands become the coty- ledonary traces and enter the base of the cotyledon. The lateral member of each group of three which is in the intercotyledonary plane approaches the corresponding bundle of the next group and fuses with it. The changes which are made and the resultant condition at this stage are shown in figure 5. Two strands (solid black) are here departing to each cotyledon, and six bundles are left as the basis for the vascular system of the epicotyl. The details of this nodal complex vary somewhat owing to the different | levels at which fusion and separation of bundles take place, and to the presence of intercalary bundles. These intercalary bundles, as they ap- proach the cotyledonary node, fuse with the others and are completely lost, exactly six epicotyledonary strands almost invariably emerging from the complex, quite regardless of the number of intercalary bundles which may have entered it from the hypocotyl. This fact we shall find to be of im- portance when we consider the statistical relationships of bundle number in hypocotyl and epicotyl. Above the cotyledons, the six remaining bundles approach one another, closing the cotyledonary gaps and forming a ring, the members of which almost immediately divide. The twelve bundles thus produced (fig. 6) persist throughout the first internode of the epicotyl. At the first node of the epicotyl are inserted the two primordial leaves. Phaseolus, like other Leguminosae which have been investigated, possesses a trilacunar node, the leaf being supplied by three traces, each of which causes a separate gap in the vascular ring.» The two primary leaves there- fore remove six of the twelve bundles of the epicotyl (solid black in fig. 6). The six new bundles which appear just above the cotyledonary node are, therefore, evidently downwardly extending leaf traces. These facts make 5 Sinnott, E. W. The anatomy of the node as an aid in the classification of Angio- sperms. Amer. Jour. Bot. 1: 303-322. 1914. Feb., 1921] HARRIS AND OTHERS — SEEDLINGS OF PHASEOLUS 69 understandable the almost invariably twelve-bundled condition of the first epicotyledonary internode. The structure of the normal seedling thus corresponds to the type found by one of the writers® to be characteristic of a large number of Angiosperm families, in which the vascular supply to each cotyledon, consisting of two strands, leaves but one gap in the vascular ring; and in which the foliage leaf is trilacunar. The Trimerous Seedling The seedling with three cotyledons and three primordial leaves is built on a different plan from the normal one in that it is prevailingly hexarch, six 10 Fic. 7. Trimerous seedling. Transverse section through the root, showing its hexarch condition. Fic. 8. Trimerous seedling. Transverse section through the base of the hypocotyl, showing the six primary double bundles. Fic. 9. Trimerous seed- ling. Transverse section through the mid-region of the hypocotyl, showing the nor- mal twelve-bundled condition. Fic. 10. Trimerous seedling. Transverse section just below the cotyledonary node. The,six bundles or bundle groups correspond in origin and character to the four bundles of the dimerous seedling at this level. Fic. 11. Trimerous seedling. Transverse section through the cotyledonary node. Each group of three strands bounded by a dotted line corresponds in origin and character to a similar group at this level in the dinterous seedling. Fic. 12. Trimerous seedling. Transverse section through the mid-region of the epicotyl, showing the eighteen bundles which have arisen by the splitting of the nine original epicotyledonary bundles. The nine strands which are to go off as traces to the three primordial leaves are solid black. 6 Sinnott, E.W. Conservatism and variability in the seedling of dicotyledons. Amer. Jour. Bot. 5: 120-130. 1918. 70 AMERICAN JOURNAL OF BOTANY Vol. 8 bundles occurring in the upper part of the root (fig. 7). This number is soon reduced to five and eventually to four, in passing down the root. Passing upward into the hypocotyl, the six main strands (the primary double bundles) divide to produce twelve (figs. 8 and 9). Intercalary bundles are much less common than in the normal seedlings, appearing in only a smali percentage of cases, and then being rarely more than one or two in number. At the node the same general procedure is followed as in the normal seedling, except, of course, that there are more bundles concerned. Bundles of adjacent pairs approach and fuse (fig. 10). Each of these bundles or bundle aggregates then divides, generally into three. Three cotyledons are each supplied with two bundles (solid black), and three sets of three bundles each—each formed by the fusion of two lateral bundles in the intercotyledonary plane—remain behind. The bundle changes and the final condition at the departure of the cotyledonary traces are shown in figure 11. The epicotyledonary ring which forms from the bundles which remain thus consists of nine strands instead of the normal six. Many of these divide at once, although the number is not usually doubled, as in normal seedlings, but varies from 12 to 18 or even more in the mid-region of the epicotyl (fig. 12). The bundles are much more crowded than in the normal seedlings, which may perhaps account for the failure of some of them to divide at once. A study of the first epicotyledonary node shows that three strands are given off to each primary leaf, leaving from 6 to 9 in the stem. It is therefore evident that within classes of seedlings which are uniform externally there are considerable anatomical variations and that the two classes investigated are profoundly differentiated in their anatomical organi- zation. Our next task is to subject the mass of data upon which these general conclusions are based to a statistical analysis with the object of bringing out otherwise undeterminable relationships. BUNDLE NUMBER AND ITS VARIATION AT DIFFERENT LEVELS IN THE SEEDLINGS From the statistical side we have two problems to consider. The first is that of the relative numbers of bundles at different levels, 7.e., in the root, at the base of the hypocotyl, in the central region of the hypo- cotyl, and in the epicotyl of the same plant in both normal and abnormal plants, together with the variability in bundle number in different regions. The second is that of the differences in bundle number, and in variation of bundle number, between normal and abnormal plants. Since it is impossible to consider type and variation of bundle number at different levels without noting differences in the trimerous and dimerous forms upon which the observations were based, we shall devote this section primarily to a parallel discussion of both problems. | Feb., 1921] HARRIS AND OTHERS — SEEDLINGS OF PHASEOLUS 71 We shall consider in order the levels at which sections were made, begin- ning at the root. 1. Root. Roots were sectioned in the cases of lines 93, 139, and 143. The numbers of bundles’ in the roots of normal and trimerous seedlings of these lines are shown in table 1. TABLE I Primary | Line 93 Line 139 | Line 143 Double —___- — |— a pa a ah eee Bundles ‘Trimerous Dimerous Trimerous Dimerous Trimerous Dimerous 3:-: Tae errs 2 = 4 ees A (aan 31 132 15 149 27 219 eee 87 20 53 I E13 | 2 0.5 ene 34 | = 36 == 66 | — Wipe ee — — — == I = The entries in this table show that most of the normal plants are tetrarch, although a small percentage are pentarch. In the trimerous seedlings the highest percentage are pentarch, but the remainder are distributed between tetrarch and hexarch with a few in more extreme classes. Sections made at progressively lower levels in the root show that the hexarch and pentarch conditions, in the trimerous seedlings, soon give way to tetrarch. This fact doubtless explains the relatively large number of non-hexarch cases TABLE 2. Vascular formula for base of hypocotyl of trimerous seedlings and their normal controls Line 75 Line 93 Line 98 | Line 139 Line 143 Base of , 7 | er | iy Hypocoty]| Trimer Dimer Trimer- | Dimer Trimer- | Dimer | Trimer- | Dimer- | Trimer-| Dimer- ous ous ous ous ous ous ous ous ous ous (4) = 69 = 34 at Odie | 138 2-150 ie i |, 30 ier 37 = ASB Mates | eo 3 55 ia 2) \— 10 arg 13 a 23 a a = 4 a 3 a 4 = 5 Erase ye a Regt epoa al Wages (4) +4 2 a Man 5.| — 2 = a ie = = aia |) erp Meira @+6) 1} — | —~) ~ | =~) =) =>, =] =) = (5) I 13 5 22 4 6 4 I 15 5 (5) +1 8 4 10 18 6 8 4 2 31 5 2), 2 I 3 a ae I a Tee ee ce Sl i ca I = 1 a eat | ee (6) 107 5 120 10 160 I 92 — 13H — (6) +1 12 I II 3 10 — 5 | = 25 I (6) + 2 2 — I 2 — — — — — — (7) 7 ee 4 i 1 ae = = 5 I ae a aa ai ae ae se ae AS (+2) 1} = | — | —~ | ~ J =~} = | =~ | =] = (8) I a co oa o, ae mer) ae ae (8) +1; — — — — -—— I —- | — I — 142 142 155 155 183 183 106 150 22 aie 22 7 Where the bundles were united in a ring, the number refers to number of protoxylem strands. 72 AMERICAN JOURNAL OF BOTANY [Vol. 8 observed, for the zone within which the hexarch condition persists is narrow and its level is variable; and there is necessarily more or less variation in the level at which the sections are cut. 2. Base of Hypocotyl. In the series of sections of the base of the hypo- cotyl made at Storrs, the number of double vascular strands (each of which is derived from a primary root bundle and corresponds to a pole of the root) and the number of intercalary strands were recorded separately. There is no difficulty in distinguishing between these two categories of bundles, since the latter are almost invariably without protoxylem elements and are irregularly placed. The original data for the five lines are condensed in table 2. The number of bundle pairs (the primary double bundles) is given in parenthesis, and the number of intercalary bundles, if such are present, follows the + sign outside the parenthesis. There are three outstanding features in this table. First, the wide range of variation in the number and in the combinations of primary double bundles and intercalary bundles in both normal and abnormal plants observed when reasonably large series of seedlings are sectioned. It is clear that an anatomist who deals with only a few seedlings may obtain an altogether inadequate picture of the conditions which actually prevail in the species under investigation. Second, notwithstanding the wide range of variation there are conspi- cuous modal classes in both normal and abnormal seedlings. In the normal plants these fall in all cases on four primary double bundles, without intercalary bundles, or with but one intercalary bundle; and in the trimerous plants, on six primary double bundles without intercalary bundles. Third, the plants which are externally dimerous and trimerous are also clearly differentiated in internal morphology. The internal characters are, however, transgressive. It is impossible in some cases to distinguish from sections of the hypocotyl base alone between plants which superficially fall into the strictly alternative classes of dimery and trimery. For purposes of more detailed analysis these formulae must be split up into their component elements. A. Primary Double Bundles. The distribution of the number of primary double bundles in the five lines considered is shown in table 3 for dimerous and trimerous seedlings. These frequencies, reduced to a percentage basis, are represented graphically in figure 13. This shows that in all five lines the modal number of primary double bundles is two higher in the trimerous than in the dimerous plants. In the dimerous plants the modal class is in all cases 4; in the trimerous seedlings the modal class is 6. There is, there- fore, a profound reorganization in the vascular anatomy of the seedling upon the assumption of a trimerous external organization. Limiting our attention to primary double bundles and judging from modal classes only, an increase of fifty percent in the number of vascular elements is Feb., 1921] HARRIS AND OTHERS — SEEDLINGS OF PHASEOLUS 73 TABLE 3. Number of primary double bundles at base of hypocotyl in trimerous and dimerous seedlings 4 5 6 7 8 Total Line 75 SMRimIeLOUS. -. .<7...... I II 121 8 I 142 GEGEN QA oa na | 0.70 TAS 85.21 5.63 0.70 IDMMMETOUS............. 117 19 6 — — 142 MRCRCEMEA =)... +... 2 82.39 13.38 4.23 Line 93 SGMMETOUS:.. 0... 3... I 18 132 4 — 155 PeOREeMt 5 | 0.65 11.61 85.16 2.58 WMETOUS:.. 90 50 15 — — 155 | PASS 01 ee 58.06 32.26 9.68 Line 98 | BWRIMETOUS 2 ee | I ak 170 I — 183 eneent. 6. os. | 0.55 6.01 92.90 0.55 WUMEKOUS 2. 165 16 I — I 183 IRCRCCMIU S22 cho 0 ec 90.16 8.74 0.55 0.55 Line 139 MPMeTOUS 2! ee. I 8 97 —— — 106 Renee. 25h. 6 eis | 0.94 7.55 Q1.51 DinmierOus .....6...2...5.. Le bAGy 3 = = — 150 ercemt. 2/66... 98.00 2.00 Line 143 aGtIMeTOUS..... 0... . 05. 5 46 159 9 2 221 EL CeMG 2 oy es ew | 2.26 20.81 71 OA 4.07 0.90 Dimerous........0.... | 209 10 I I —— 2211 HERCENE a. es. 94.57 4.52 0.45 0.45 PIG: 13: base of hypocotyl in dimerous (solid dots) and trimerous (circles) seedlings. Percentage frequency distribution for number of primary double bundles at TA AMERICAN JOURNAL OF BOTANY [Vol. 8 associated with an increase of fifty percent in the number of cotyledons and leaves. The distributions show, however, that this is only an incomplete, and to some extent an erroneous, statement of the condition. In the dimerous seedlings the modal number of primary double bundles is 4, and all departures from the modal number are higher. In the trimerous seedlings the modal number is 6, and the departures may be in either the positive or the negative direction. The frequency distribution for the dimerous plants is therefore wholly skew, forming a typical J-curve; that for the trimerous plants more or less symmetrical,’ but with departures occurring chiefly as smaller numbers of bundles. The variation of primary double bundle number in dimerous and trim- erous plants is, therefore, transgressive. The number of externally dimerous seedlings which might be considered to be anatomically trimerous, and the number of trimerous seedlings which might on anatomical grounds be considered dimerous is, however, very small. Turning to the physical constants in table 4, we note that the mean TABLE 4. Statistical constants for number of primary double bundles at base of hypocotyl of trimerous plants and their normal controls Standard Devi- Coefficient of Vari- Mean ation ation Line 75 Drimaerouse(N; = 142). os) 5.98 +.02 0.436+.017 7.28 +.29 Dimerous (Ne — 142) >... | 4.22.02 || ~ 0.50542.026 11.97+.49 Actual difference: I — I 4 Mean 34.18 21.81 1.97 +.02 8.84 -+.06 3.132.006 35-41 1.91 +.04 8.11 +.02 3.80+.04 46.85 1.90+.05 8.45 +.04 3.45.06 40.83 Standard Deviation 0.750+.030 1.351.054 — 0.601 +.062 44.49 0.627 +.024 1.483 +.057 | —0.856+.062 57-72 0.495 +.018 1.190.042 — 0.695 +.046 58.40 0.558+.026 0.440+.017 | +0.118+.031 26.82 T.I05+ O20 == Ore 7A ee 32-97 .035 .027 044 Coefficient of Variation 6.17 4.25 14.90+.61 —8.732—.06 5.22 =.20 15.04.59 =) 82762 4.14.15 13.47 +.48 9.33 2.50 4.69 +.22 5.43.21 —0.74+.30 LUNE Vie LV EOS, —0.56+.44 9.28+.30 9.84+.32 LIMES T 59 Fic. 16. Percentage frequency distribution of total bundles at base of hypocotyl in dimerous and trimerous seedlings. Primary double bundles counted as two. Feb., 1921] HARRIS AND OTHERS — SEEDLINGS OF PHASEOLUS 83 figure 16. Comparison of these figures with those in figure 15 shows essen- tially the same type of distribution for the dimerous and trimerous plants. The grades of the classes are merely about double what they were in the former method of treatment. The statistical constants are compared in table Io. 70 Ls Bee HYPOCOT YL OF LIWE 75 o—e = YMEROUS 60 wee 3 o—o = TRIMEROUS I 5 EE nrg 9 /0 // /2 /3 /4 eye /6 if Le re er ee ee eee Fic. 17. Percentage frequency distribution of total bundles in central region of hypocotyl. For all five lines the constants show a higher mean number of bundles in the trimerous than in the dimerous seedlings, the mean being approximately 12 in the former and 8 or Io in the latter. Thus the trimerous seedlings have from 21.8 to 46.9 percent more bundles than the dimerous seedlings. [Vol. 8 AMERICAN JOURNAL OF BOTANY Cvo | Sto | 9€'1 | 06'0 cov Te11-4| SS-et =| Vr'zo — — — I I ¢ z OI cz Iv QcI ShOnOSOs-Oo1 \tZye> | Te 11 hoSs6 .| VS 19)" | Ce 96'L Cro 060 I I iS 9 | Cz Ve QOfI VI II I z Ee r| | loo | er | Loo | £816 —- > — | — — — I Zz OI Lot | V6:0 2te°c |o9°S 1 Sz:62 |°SS°Z [BENG ~- ieee ae I ¢ 9 Tg 8 v — — 6z'0 | LQO LOI gz'6 CSG. | RSIS. ESiZ-cS — | Ea _ -— iS 6 Ze Le OI C1 | | wee _|'6079° |-6070Q §9re VL I 6z'0 ete oe A caer — | @g Iz L6z ZI 9 I — 9f°O | OTOenisecse sOOuw level I auleserOt wlnesOe, 102,01 --|-O1nG 7c) — | I QI 6¢ 96 Col 691 £6 ve roe axe) CitO ES ie —12O'O" eo LV ie aR: Sor (19725 brr I — I ZI Qe Z8 ZQC oe 8 — ~— HZ:O" | oZ.0 Oleca SO m eSZ9 £1-Gs | 5Z9:02 | OLN | LEE I ¢|; — — 6 ib 9z ge 98 €or Cri 96:04. Ve.0 Oe: (26:9 | 2o16' 1-61-02 || So7e Oz’! zZ'o bao — v I ¢ 6z ov z76z of ¢ ¢ I 3 os oe Li QI Sint br cS €1 ZI II OI 6 8 snosowig Pape EY © Seem rere quad1ag gtr ee Seen Riess SNOJOUIL |, v1 oury snosoWwiq nO a her pteceee --quaoiag Bee ay men wa ee SNOJOUWT |, 6€1 oury ea ane, ee Toe ee ee eee Roe g6 aur] ha ne hiel Mees Moat, Tarot en enhetee Saag eet C6 our] sno] er oe ee yua019g SAE ian peNae ee SNOJOUIL J, Sf oury SOULpIas SnOLIUIp PUD SnodawMidy fo JkJoI0G KY f{O UOIWIL [DA] UA U1 saypung {0 4aqQUnN ‘Il AAV], CO Feb., 1921] HARRIS AND OTHERS — SEEDLINGS OF PHASEOLUS 5 In variability as measured by coefficient of variation, the dimerous plants exceed the trimerous throughout, conspicuously so in lines 75, 93, and 98. In their standard deviation, the dimerous also markedly exceed the trimerous in these three lines, but in lines 139 and 143 the trimerous plants slightly exceed the dimerous. a Meso Bee aa <7 a a | YYPOCOTYL OF LINE 93 _! o—° = WNVFEROVS | o—o = TRIMEROUS a | T ‘oe ee 4 aaa eT | - ace Aemeea bee Poe a ee i Ds | 40 30 20 Fic. 18. Percentage frequency distribution of total bundles in central region of hypocotyl. D. Summary for Base of Hypocotyl. For the base of the hypocotyl, therefore, it is evident that in total bundle number the trimerous seedlings decidedly exceed the dimerous ones. The intercalary bundles alone (which form but a small part of the total) tend to be more numerous in the dimerous seedlings. 86 AMERICAN JOURNAL OF BOTANY [Vol. 8 In variability in bundle number at this region, dimerous seedlings in general exceed trimerous ones; although two of the five lines studied furnish slight exceptions to this rule. Fic. 19. Percentage frequency distribution of total bundles in central region of hypocotyl. 80 70+ 60 50 40 30 20 /0 | | | | | | ! | | | | | ! I ' | ! | ! | ! 1 | 1 | ! 1 | | | | | | | 1 | | | | | 1 | 1 1 1 1 1 1 | 1 ! 1 | | | ! | 1 | 1 ! 1 8 Eat Pe Sis a ” HYPOCOT YL, LIVE 139} | vi | 9 /0 /3 /4 15 Feb., 1921] HARRIS AND OTHERS — SEEDLINGS OF PHASEOLUS 87 3. Central Region of Hypocotyl. In the sections made in the central regions of the hypocotyl and of the epicotyl at both Cold Spring Harbor and Storrs, the total number of bundles was counted, no distinction being made between the bundles originating from the primary double bundles and those of intercalary origin. 60 HROCOT YL OF: LINE 143 o—- = DIMEROUS o—o = TRIMEROUS 50 40 30 20 10 Fic. 20. Percentage frequency distribution of total bundles in central region of: hypocotyl. The frequency distributions are shown in table 11. The relative fre- quencies for line 75 are shown in figure 17. The form of the distributions in line 98 is in essential agreement with those in line 75 and is not represented. The distributions for line 93 are represented in figure 18. The distribution for line 139 is shown in figure 19. That for line 143 appears in figure 20. The conspicuous feature of these distributions is the wide variation in bundle number and the conspicuous skewness of the frequencies for the 88 AMERICAN JOURNAL OF BOTANY [Vol. 8 normal plants of lines 75, 93, and 98. In these, bundle number ranges from 8 to 18 with a relatively large number of bundles in the lower classes. The modal number of bundles in the hypocotyl of normal seedlings of lines 75, 98, 139, and 143 is 8, while in line 93 it is Io. The normal plants of the five lines differ conspicuously in variability. The number of seedlings falling in the modal class is relatively small and the range of variation relatively wide in lines 75, 93, and 98 as compared with line 139. Line 143 occupies an intermediate position in this regard. In all the lines except 93 the distribution of number of bundles in the hypocotyl of normal seedlings is wholly skew, the frequency decreasing from the modal number (eight) towards the upper end of the range. In line 93 (figure 18) the distribution is also skew, but the frequency decreases from the modal number (ten) towards both ends of the range. In the trimerous plantlets of all five series the modal number of bundles in the mid-region of the hypocotyl is 12. The extent of concentration into the modal class and the range of variation differs greatly in the five lines. This is very narrow in lines 98 and 139 and relatively wide in line 143. The frequency distribution and figures bring out very clearly indeed the differentiation of the trimerous and dimerous seedlings in the number of vascular bundles. TABLE 12. Statistical constants for number of bundles in hypocotyl of trimerous and dimerous seedlings | Standard Devi- Coefficient of Vari- Mean | ation ation Line 75 Trimerous GN = 416)... 2. 12,19 4-102 0.982 +.023 8.06+.19 Dimerous(N*= 416). 2. 9.49 +.05 1.645 +.039 17-34 52-42 INCL Ua Giiherence......2 2.00. ered +2.70+.06 | —0.663+.045 |. —9.28+.46 Relabuverditerences 20 an aa 28.45 | 40.30 Line 93 | | Trimerous (ON »= 557) 40.2 12.2042.03 | | 0:922-E.01g 7.50+.15 Dimerous (N = 557)......... 10.6242,04 |" “1.5 2522-060 14.36+.30 Actialecittenences 2 ite +1.67+.05 | —0.603+.036 —6.86+.34 Relative difference... 2). 04: 15.73 | 39.54 Line 98 | @rimenrous (Ne— 345). ae 12.03 4.02 | 0.532+.014 4.42--.11 Dimerous (N = 345)......... 9.223=.04 | 1-107 -031 12.99 +.34 Net ualoditienenCe.. 24) 05. 6s. o ee ee | +2.81+.04 | — 0.665 +.034 —8.57+.36 Relative ditiereneer i. sss. 30.47 | 55-50 Line 139 | Dromerouss(N—= 106)) 2. Il.99+.05 0.694 -+.032 5.78 4.27 DimerouseCNe= 50) ee. 8.1I+.02 | 0.409.016 5.04.20 ANCEMAIGIMENeENCe.. oe eee +3.88+.05 | +0.285 +.036 +0.74+.34 Relativerditierence yaar 47.84 | 69.68 Line 143 | cEmimienOusn N — 7221) (5) 12.29+.06 1.283+.041 10.44.34 Dimenous: N= 221) 2 ee 8.7 taO5 004) Sy 2.085 13.63 4.45 ACtUalecibkeremcen whee caren... ones +3.58+.08 | +0.096+.056 —3.19+.57 Relativerdifierence 107... o aan. 41.10 | 8.09 Feb., 1921] HARRIS AND OTHERS — SEEDLINGS OF PHASEOLUS 89 The differences between the lines can best be seen from the figures. For a more critical comparison we must have recourse to statistical constants and their probable errors. The results for the hypocotyl of trimerous seedlings and their normal controls are set forth in table 12. Without exception the number of bundles in abnormal plants is higher than that in the control plants. The differences range from 1.7 to 3.9 bundles. These differences are many times as large as their probable errors and are unquestionably significant. The relative differences are about 16 percent in line 93, 30 percent in lines 75 and 98, AI percent in line 143, and 48 percent in line 139. Both the standard deviation and the coefficient of variation of the num- ber of bundles in the hypocotyl are lower in the abnormal than in the normal plants in lines 75, 93, and 98. In lines 139 and 143 the relationship of the standard deviations of the trimerous and dimerous plants is exactly reversed, that of the trimerous plants being somewhat larger than that of the dimerous series. The difference in line 143 is +.096 + .056, which is nearly twice as large as its probable error and possibly statistically significant. In line 139 the difference in standard deviation is +.285 + .036. This difference is about 8 times as large as its probable error and unquestionably significant. The percentage differences in the standard deviations in lines 75, 93, and 98 range from —40 to —56 percent. In line 143 the percentage difference is +8 percent, while in line 139 it is +70 percent. In line 143 the coefficient of variation is higher in dimerous plants (as it is in lines 75, 93, and 98), but in line 139 the trimerous show a slightly but perhaps not significantly higher relative variability. The results as a whole show that the difference in the variability of bundle number in the two types of seedlings in lines 139 and 143 is.not the same as that in lines 75, 93, and 98. In interpreting these results we must remember that each primary double bundle at the base of the hypocotyl almost invariably divides to form two bundles at higher levels in the hypocotyl. Occasionally one of these branches may further divide into two. It is impossible in sections made in the central region of the hypocotyl to distinguish with certainty in every case between bundles originating through a division of the original protoxylem strands and those of intercalary origin. The simplest working assumption is that the number of bundles in the central region of the hypocotyl will be given by twice the number of primary double bundles demonstrated at the base of the hypocotyl plus the number of intercalary bundles found at the base of the hypocotyl; or the number of bundles, 6, at the central region should be given by b=2p+1 where p = primary double bundles and 7 = intercalary bundles. gO AMERICAN JOURNAL OF BOTANY [Vol. 8 A comparison of the number of bundles calculated by this formula with the number actually observed in the central region of the hypocotyl may be best made in a table of double entry. Table 13 gives the results for dimerous and table 14 the results for trimerous plants of line 93. The TABLE 13. Comparison of actual and theoretical number of bundles in hypocotyl of dimerous seedling Actual Number Sie xo Sie) IT 12 13 14 | Totals Fheoretical,. 2p: =5\0...> voce tn 8 12. 13.4. 20 3,—);—|— 34 9 er pt” cei 3 I I I if 10 = De .22 6 5 Tn 35 II — | — I 0 al aS B I a2 12 — | — | — I [34 4 iC 28 13 Pa: eee = ele el I 3 4+ 14 a tan Gomes o = | 2 2 Totals..| 12 | 28 | 46 | 227) 200 aae 8 | 155 TABLE 14. Comparison of actual and theoretical number of bundles in hypocotyl of trimerous seedling Actual Number 10 TT | eee 13 14 1s 20 | Totals Eheoretical 2p 4. ve es eee IO I I Bl aa ee ees 5 II — | 6 3|— 1/—|— 10 12 alee | TO 2alaale eae a I 124 13 — | — | — 8 ele FI 14 ol rh |e a a 4/1 io 5 Totals..4) 71 Q | 108 | 20) 2a 4iiere I 155 frequencies for the cases in which the number of bundles at the mid-region of the hypocotyl calculated from the formula agrees with the number > actually observed are printed in blackface type. The other lines give roughly comparable results. It is clear that the number of hypocotyledonary bundles is not far from twice the number of primary root bundles plus the intercalary bundles. In rare cases the number of bundles in the hypocotyl is less than twice the root strands plus the number of intercalary bundles, since one of the root strands sometimes fails to divide. It may be, and not infrequently is, higher because of the appearance of extra intercalary bundles at a level higher than that sectioned at the base of the hypocotyl. In many cases the full complement of intercalary bundles has not appeared at this low level. In some cases it may be higher because of the secondary bifurcation above mentioned. It is worth while to give the percentage frequencies of cases in which the number of bundles of the central region of the hypocotyl is given by the formula, and for comparison the relative number of cases in which it is in defect and in excess. The percentages are calculated from double entry tables like 13 and 14. Feb., 1921] HARRIS AND OTHERS — SEEDLINGS OF PHASEOLUS QI Trimerous Seedlings N In Defect ap+z2 | In Excess Ge SSIS Sh 142 7.0 76.1 | 16.9 _ iL Of. 3) aE | 155 1.3 78.1 20.7 L1G Oo se 183 a8 86.3 | 10.4 | SE CS Ie 106 7.6 80.2 | 12.3 MEN kw ee ee [e224 0.9 TA f | 24.4 Dimerous Seedlings N In Defect apts In Excess 12 7S, ie (Ae 542 2.1 51.4 46.5 123 06 ere | 155 1.9 A757 50.3 Oe Css 8 Ara ee ey ineke 3.8 59.0 2782 Linc 20) a 150 O-7. 98.7 0.7 L0G 1G) er | 221 0.9 81.0 18.1 With the exception of the dimerous seedlings of line 139, the actually observed number of bundles is in excess of the number given by the formula. In lines 75, 93, and 98 the excess is far greater in dimerous than in tri- merous seedlings. Thus in the dimerous class about 40 percent of the seedlings show a number of bundles in the central region of the hypocotyl which is in excess of twice the number of primary double bundles plus the number of intercalary bundles at the base of the hypocotyl. In the case of the trimerous seedlings the excess is much smaller, being roughly 20 percent. Thus it is clear that, especially in the normal seedlings, a large number of the intercalary bundles do not extend to the base but appear in the axis, ending blindly below, or that a considerable proportion of the primary double bundles divide into more than two bundles. In line 143 the number of cases in which the observed number of bundles is greater than the calculated number is much more nearly equal in the two types of seedlings. Thus in the trimerous seedlings 24.4 percent of the seedlings have a number of bundles in the central region of the hypocotyl greater than 2p + 7, whereas in the dimerous seedlings there are 18.1 per- cent of seedlings of this class. In line 139 only 0.7 percent of the dimerous seedlings show a number of bundles in excess of 2p + 7, whereas in the trimerous seedlings 12.3 percent are in excess. Thus lines 139 and 143 give results diametrically opposed to those of the first three discussed. Summary for Central Region of Hypocotyl. It is evident from the above statements that the number of bundles in the hypocotyl of trimerous is decidedly higher than in that of dimerous seedlings; that in general the bundle number is more variable in dimerous than in trimerous seedlings; and that the intercalary bundles generally extend to a lower level in the hypocotyl of trimerous than in that of dimerous seedlings. ” Note that the extremely small excess in line 139 may be due to the extraordinarily normal character of the vascular system of the dimerous plants of this line. [Vol. 8 AMERICAN JOURNAL OF BOTANY Q2 Ice 1cc oSI gol cre cre LSS LoS gIt gIv [PIO | EO eedee ae 0 | 12:2 | Lob | €orbr z | 9 OF) f= Ee | | i laa i ee Ogee see ee Be 2 ae re T On O@OnGa.0" |;cO-c Tea I I LZ zZ'0 | Og°I | 91°6 — v OI 1S 0 96°0 | 96'0 | 6t'9 I v v Le le oz 61 gi LI gt €1 cI It OL snosoUng Fiteeeeeessseeee sss squgoqag Be eS Rn pee ee ee ee SNOJOWI J, €V1 our] SNOJOUWIL |, 6£1 our] Ded Ogoe co Denso Od Oho. On. 08 oom o S SNOIOUWITI g6 aur] snoJOWIg See ee esses esses equggrag FAT RHE nate iS a SNOJOUITIT, mes £6 OUI] pict sek tar Seen en Sera quaoiag Sigel 6. Femisl eu. o) \elren ie) uel pibente; Mellie Riattelice, we one) ernie “miele feniis, xeNelise] nelle! “elite Nie cieetten «ce SnNOJOUILT T SZ oury SEuripaas Snoamip Fun snodawmird fo 1&j0I1Ga fo Uda JDAJUAI Ut SajeUNG fo daqunyy “SI ATAV], Feb., 1921] 4. Central Region of Epicotyl. HARRIS AND OTHERS — SEEDLINGS OF PHASEOLUS 93 The frequency distributions of the number of bundles occurring in the mid-region of the epicotyl appear in table 15 for both the abnormal and the control plants. These distributions 80 70 60 50 40 30 20 /0 PIG. 21. epicotyl. PEIOOT VL, OF LIVE 13° o—e = D/MEROUS a o—o = TRIMERS Be ee aS UE apa ny ER ee a ee Pest ee eee a ees Ue a ee, ee ee a eee eee, Percentage frequency distribution of number of bundles in central region of reduced to a percentage basis are represented graphically in figure 21 for line 75, in figure 22 for line 98, and in figure 23 for line 143. The distributions 94 AMERICAN JOURNAL OF BOTANY [Vol. 8 He ERE BE fie RBA! Ve Pe : | EFICOT VOR LINES OS o——9) = LWIEROUS. o—o = TRP/IMEPOUS 90 80 70+ HEEEEHEEE Ba ae ne 60 50 40 30 20 é ae /3 /4 5 /6 LEN he /9 20 Zi) Fic. 22. Percentage frequency distribution of number of bundles in central region of epicotyl. for line 93 are essentially the same as those for line 75. The graph for line 139 is in essential agreement with that for line 98 and is not drawn. | In the dimerous plants the difference between the form of the frequency distributions for number of epicotyledonary bundles in lines 75 and 93 on Feb., 1921] HARRIS AND OTHERS — SEEDLINGS OF PHASEOLUS 95 the one hand and lines 98, 139, and 143 on the other is more apparent than real. All five lines agree in showing the frequencies for the dimerous plants largely concentrated in a single modal class with a slight but evident skew- Ce 70 salient A C0 OLIVE 779 sees o—e = L/MEROUS o—o = TRIMEROUS 60 50 40 30 20 /0 Vie: Fic. 23. Percentage frequency distribution of number of bundles in central region of epicotyl. ness toward higher numbers of bundles. In the case of lines 75 and 93 there is a little over 1 percent of plants with fewer than the modal number of bundles, whereas in lines 98, 139, and 143 these do not occur in series of the numbers sectioned. It is quite possible that the examination of a g6 AMERICAN JOURNAL OF BOTANY [Vol. & larger series of plantlets would result in the finding of such seedlings in lines 98, 139, and 143, thus bringing the five series into full agreement. In the trimerous seedlings the number of bundles shows rather wide, and fairly symmetrical, distribution about the modal class, which is 15 bundles. The lines differ, however, to a considerable extent in the amount of variation from the modal class. In lines 75, 93, and 98 the frequencies are to a far greater extent concentrated into the modal class, which contains from 39 to 51 percent of the frequencies, than in line 143, which contains only 24 percent of the cases. Line 139 is intermediate between these two extremes. For a more precise comparison we utilize the constants set forth in table 16. TABLE 16. Statistical constants for number of bundles in epicotyl of trimerous and dimerous seedlings Mean | Standard Deviation nee Line 75 | [Trimerous (N = 416). 2. .:..| 6 15:47s304.) 1) 9 1-o5eeerose 8.702-,21 Dimerouss(N —AUG)uwin. soe | 2.27.02 0.735.017 5.994.114 Actialvditierencen.. sa. sas nts +3.20+.04 | -+-0,6202-/036 2 ee Ielativerditierence: 20a. 26.08 | 84.35 Line 93 | ‘Brimeroust Nea 57 nes ee, 15.65 +.04 1.372+.028 8.77+.18 Dimerous\(N = 557): sues 12.19 +.02 0.615 -+.012 5.05.10 ActtialeclitteremGe 12.6. ee) ol ate 4 Oe OF +0.757 +.030 +3.72+.20 Relativevditreremce 27 55 0h sn 28.38 123.09 Line 98 | Trimenrouss( Ni = 345)e eee 14.89+.04 | 1.152.030 7.74.20 Dimerous-CN ='345)... a. oe 12.11.02 | 0.416.011 3.44.09 Actua Gimekence t,o aoe ee, eck 12276204: | +0.736+.032 “q A. gOme.22 elativerdiiterenCe. «05 is = hae ae 22.96 176.92 Line 139 Trimerous (N = 106). 2.22. 15.24+.08 1.285 +.060 8.44.39 Dimerous (N = 150)......... £2315 -.02 0.406 +.016 3.354.13 Actualeditterence -:im. Arcane +3.09+.08 | +0.879 +.062 +5.09+.41 Relative ditterence 2). 257. yee. 25.43 216.50 Line 143 Drimerous (Ny = 221) 05. 2 16.10+.08 1.750+.056 TOM 7 eno Dimerousa(Nu— 221) 28 oe 12.36.03 0.757 +.024 6313.20 Actual -ditlerences it eek ee ee +3.74+.09 +0.993 +.061 +4.74+.40 Relative difference... hen Ae eee 30.26 120 Lennie These results show that without exception the average number of bundles in the epicotyl is higher in trimerous than in dimerous seedlings. The difference ranges from 2.8 to 3.7 bundles. The probable errors of these differences are so small that there can be no reasonable doubt of their significance. In relative terms, the number of bundles in the abnormal plant is from 23.0 to 30.3 percent higher than that in the normal plant. Feb., 1921] HARRIS AND OTHERS — SEEDLINGS OF PHASEOLUS G7 The variability of bundle number, both absolute and relative, is far higher in the abnormal (trimerous) plants. The relative differences show that the trimerous plants are from 84 to 217 percent more variable than the dimerous in the number of bundles in the central region of the epicotyl. We now have to consider the relative number of bundles in the hypocotyl and in the epicotyl of the same plant. The constants for the normal plants are shown in table 17 and for the trimerous seedlings in table 18. TABLE 17. Comparison of statistical constants for number of bundles in hypocotyl and epicotyl of same plant. Seedlings with two cotyledons and two primordial leaves | Coefficient of Mean Standard Deviation Vanation Line 75 (N = 416) Mele OOCOLY eis oley sie wscehe Ss a 0 9.49+.05 | 1.645+.039 yey vers, Ale. {DDI OLEAN Ue ae 12.27+.02 | 0.735.017 5.99+.14 Pvetial GiMerenCce. ..... se ee +2.78+.05 | —0.910+.043 | —11.35+.44 Ielative difference........:...... 29.29 | 55.31 Line 93 (N = 557) | PN OCOUV cif cis a. Pes TOIO242°04 | 1.5255 :031 14,360.30 OMMCOEY Ns as ek ee bk Ss ae I2.19+.02 | 0.615.012 5.05+.10 PVODMALGINereNnCe. 2.2.6... ee bese +1.57+.04 | —0.910+.033 | — 9.31.32 Relative difference............... 14.78 | 59.67 Line 98 (N = 345) | My MOCOLV on ns ee ek es 9.22+.04 | 1.197.031 12:09 2-34. BICOL V Us ita ssc cs oe fs sw he 2.02: ee O41o OL 3.44.09 moral difrerence.... 6... 2.2. ee ey +2.89+.04 | —0.781+.033 | — 9.55+.35 xclativerdiiference, ...........5 4). 2134 | 65.24 Line 139 (N = 150) | PN MIOCOUY Wisk wks geek ee 8.11+.02 | ‘0.409.016 5.04.20 [SUCCES ARS ae re ee 12.15.02 | 0.406.016 3.35 a2 03 memlal difference. /.............. +4.04+.03 —0.003+.023 | — 1.69+.24 Relative difference............... 49.82 | O72 Line 143(N = 221) | FI MOCOLY ok sca sce ns ek a 8.71.05 1.187 +.038 13-63=-.45 | 6) [ONC(O) NA [a a a ea £2, 20s.030 "> 70:757-4-.024 O2=2.20 meumalodifierence..-. 0... 0.0.56. +3.65+.06 | —0:480==,0457) — 7.50-..40 Relative difference............... 41.91 | = 126,23 Normal and abnormal plants have in common a larger number of bundles in the epicotyl. The differences between the means for the two organs are clearly significant in comparison with their probable errors. The per- centage differences show that the epicotyl has from 15 to 50 percent more bundles than the hypocotyl. In the dimerous seedlings the variabilities, both absolute and relative, as measured by the standard deviation and coefficient of variation, are consistent in indicating a higher variability of bundle number in the hypo- cotyl. The difference is, however, very slight in line 139. The difference between the variability of the hypocotyl and that of the epicotyl in the normal seedling as measured in terms of the standard devia- 98 AMERICAN JOURNAL OF BOTANY [Vol. 8 tion is from 0.8 to 0.9 bundle, or from 55 to 65 percent of the larger value in lines 75, 93, and 98. percent. In line 143 the difference is only 0.4 bundle, or 36 In line 139 there is practically no difference in the standard deviation of bundle number in the mid-region of the first two internodes of the seedling. TABLE I8. Comparison of statistical constants for number of bundles in hypocotyl and epicotyl of same plant. Seedlings with three cotyledons and three primordial leaves Standard Devi- Coefficient of Mean | ation Variation Line 75 (N = 416) Hy pocotyl: je Seo ees L2102-.03 0.982 +.023 8.06+.19 EP picotyliik = see see eee 15.47 +.04 1.355.032 8.70 a=-21 Actual difference +; 727-3024) 13-28-2205 +0.373+.040 -Ovf0se.20 Pp Relative;difiercnce.) iy eeea ne ee 26.90 37.98 Line 93 (N = 557) LLY DOCOLY ls st nee oe) se tens 12.292-.03 | (0.922=E7059 7.50.15 FE DICOEY Ia cotscs oe an eee 15.652:,04.- |. 1.37222.028 8.7 =skS Actualidifierence.. -:e4.5e4o- ce +3.36+.05 | +0.450+.033 4.2 fie ee Relativesdifierence:: eer era 27.23 | 48.81 Line 98 (N = 345) PY DOCOLY lm aoe ee ar oe cae 12.03.02 0.532.014 A:AZ =e. TT El picoty ace: 8. nee ou eiteee 14.89 +.04 1.152 2=.020 7.74.20 Actual difference.........:..:....| —-—-2.86=£.04 +0.620+.033 +3.32+.22 Relative difference. «4.4.2... ... 22077 | 116.54 Line 139 (N = 106) Ely POCO bye tne nae ene I1.99+.05 0.694 +.032 Syise sey) PICO Lye Sar uu. eer eee Eee 1524.08 1.285 +.060 8.44.39 Actualidifterence. ..5072.....5..-) =3:253=:00 +0.591 +.068 +2.66+.48 Relativesdifterence as... 2) o. ss 27k 85.16 Pineat43n@N 9228) Ey OOCOCV ey te icee oe, neater s 12°20==.00 1.283 +.041 10.44.34 PICO Ly leer, sue ic) ear oh eee 16.10+.08 1.750+.056 10.87 +.35 Wctualiditienencer 5. 9, a.s) ees 1G .Ok lO +0.467 +.069 +0.43+.49 Relativesditterence. 6s... eee 31.00 36.40 Basing the comparisons on the coefficient of variation, we note that the coefficients for the hypocotyl range from 13.0 to 17.3 percent, whereas those for the epicotyl range from 3.4 to 6.0 percent in lines 75, 93, and 98. Thus there is a difference of about Io percent in the coefficient of variation of bundle number in the hypocotyl and epicotyl (of the normal seedlings) of these lines. In line 143 this difference is only —7.50 percent. In line 139 it is only —1.69 percent. The statistical relationship is in full accord with the anatomical findings recorded above (p. 68) where it was shown that the intercalary bundles of the hypocotyl as they approach the cotyledonary node fuse with the (nor- mally 8) bundles originating by the division of the (normally 4) protoxylem poles of the primary root and completely lose their individuality, exactly six bundles emerging from the complex irrespective of the number which Feb., 1921] HARRIS AND OTHERS — SEEDLINGS OF PHASEOLUS 99 have entered it from the hypocotyl.!! Immediately above the cotyledons the six remaining bundles approach, closing the cotyledonary gaps and form- ing a ring, the six members of which almost immediately divide, giving rise to the modal number, 12, which persists throughout the length of the epicotyl. It is apparently the disappearance of the intercalary bundles as a conspicuous feature of the topography which results in the lowered variabil- ity of bundle number in the epicotyl as compared with the hypocotyl. If this conclusion be true, we should find the least difference in the variability of number of bundles in the central regions of the first two inter- nodes in the lines in which intercalary bundles are least conspicuous as a feature of the vascular topography. As a matter of fact, this condition is strongly supported by the results for the five lines investigated. Turning back to table 6, showing the constants for number of intercalary bundles, we note that lines 75, 93, and 98 have on the average from 0.60 to 0.83 intercalary bundle per (normal) plant. These are the lines showing a relative difference of 55 to 65 percent in the standard deviations as com- pared with 36 percent in line 143 with an average of 0.31 intercalary bundle, and of only 0.73 percent for line 139 which has an average of only 0.07 intercalary bundle per plant. The differences in the coefficients of variation for hypocotyl and epicotyl are from —9.3 to —11.4 percent in the three lines with from 0.6 to 0.8 intercalary bundle per plant, —7.5 percent in line 143 with an average of 0.31 intercalary bundle, and only —1.7 percent in line 139 with an average of only 0.07 intercalary bundle. In the trimerous seedlings the relationship between the variation of the number of bundles in the hypocotyl and in the epicotyl is just the reverse of that found in the normal type. Variability as measured by the standard deviation is significantly higher in the epicotyl of all lines studied. The same is true if the coefficient of variation be used as a measure of variability, although the differences for lines 75 and 143 are not large. The anatomical explanation of this fact seems to be found in the pecu- liarities of behavior at the cotyledonary node. As pointed out above (p. 70), the epicotyledonary ring is typically made up of nine strands instead of the six characteristic of the normal plant. There is, therefore, in the modal case an increase of fifty percent in the number of bundles in the epicotyledonary ring of the trimerous plant as compared with the dimerous plant. Many of these bundles, but not all, divide to form the bundle system characteristic of the main course of the epicotyl. It is this variability in the extent of division of the bundles of the epicotyledonary ring which, in connection with the low variability of the hypocotyl due to the formation of but few intercalary bundles (except in lines 139 and 143, where the number is about the same in normal and abnormal seedlings), brings about the great variability in the bundle number of the mid-region " This statement is based on a more detailed anatomical study of a portion of the seedlings. 100 AMERICAN JOURNAL OF BOTANY [Vol. 8 of the epicotyl as compared with the mid-region of the hypocotyl, in the trimerous plants. This condition furnishes an excellent example of the importance of a knowledge of descriptive morphology as an aid in interpreting biometric constants. COMPARISON OF BUNDLE NUMBER IN THE FIVE LINES STUDIED From the genetic standpoint it seems a matter of considerable interest to determine whether the three nominally pure lines!? are differentiated with respect to their vascular anatomy. A comparison of the percentage frequency distributions and the figures of the foregoing discussion will convince the reader that certain of the lines may be differentiated either in mean number of bundles, or in variability of number of bundles, or in both average number and variability of bundle number. Since we hope to return to this problem later with even more extensive data, it seems unnecessary to consider the differences in the distributions and constants in detail at this time. The results of this brief and superficial comparison seem to indicate that while different lines may not differ greatly in respect to certain of their vascular characters they may be differentiated with respect to others. SUMMARY This paper presents the results of a comparative and biometric study of the gross vascular anatomy of the seedling of Phaseolus vulgaris. Two morphological types are considered: the normal, or dimerous, seedling with two cotyledons and two primordial leaves, and the trimerous seedling with three cotyledons and three primordial leaves. In normal seedlings, the vascular system of the root is typically tetrarch (with four protoxylem poles), and gives rise in the base of the hypocotyl to eight bundles which continue to the cotyledonary node. From the vascular complex at this point two strands are given off to each cotyledon and six are left, each of which divides into two to produce the typical twelve- bundled condition of the epicotyl. The trimerous seedlings typically possess six root poles instead of four, twelve bundles in the hypocotyl] instead of eight, and nine primary epicoty- ledonary bundles instead of six. The nine primary epicotyledonary bundles may not all divide, however, so that the number of bundles in the central region of the epicotyl is variable, ranging in general from fourteen to eighteen. 12 While the material employed in this study traces its origin from individual plants, the possibility of hybridization in the field is not excluded. Thus any comparison which may be made in this place must be regarded as preliminary merely. Feb., 1921] HARRIS AND OTHERS — SEEDLINGS OF PHASEOLUS IOI In both types of seedlings, but more frequently in the normal ones, additional or intercalary bundles appear in the hypocotyl, either de novo or as a result of division of the primary strands. The following constants" (table 19) for bundle number (at the different levels studied) epitomize the differences which characterize the two types of seedlings. TABLE 19 Trimerous Seedlings Dimerous Seedlings Neantay) j.c.D: GEVi Mean See Cov: AMMO CORO a ac ey es ae Dolce LUST, ae ce 5.02 .654 13.02 4.01 .O8I 2.03 Maine ke Ps So eh 5 eT Oe pl 2730 14.47 A13 338 8.18 J OC2SGTS 5 Ra Oe a | 5.09 .707 13-07 4.05 AZT 4.19 Primary double bundles | DER ECR 00 ea ra ae Sod .288 4.86 | 4.02 .140 3.48 MUTANT. es ke ee oe | 5.98 581 IO.01 | 4.52 5066") 14.74 SBC SE a ene ar lays Ole le G4OS 6.87 4.19 411 9.66 Intercalary bundles | Ce S000 ee | .09 .292 ‘| 156.62 | 107, .261 | 105.79 MUAY ce oes Se ee 29 .686 | 381.67 | .83 | 1.024 | 355.48 LEG OIEE. «RSS TG oy 9 -AQGI- | 274.92 .49 1687 || 182770 Mid-region of hypocotyl | | | LAL RUT 09 See | 11.99 532 4.42 8.11 .409 5.04 lesan | 12.29 | 1.283 10.44 | 10.62 | 1.645 17.34 JESUS oS eee Rn [ot2s20 .883 7.24 O23. 1.193 12.67 Mid-region of epicotyl | | MMMM ef ek ee ee TA. SO? |. 1.152 77h eel .406 B035 MPArIMIMIUI eee = os aS ees P LOO L750 >| OS 7 eee 757 G12 IW 2G05 5 pCR AI Ae a aa en Persea Te 2G2. 1) tO 2 aie. 2> .586 4.79 The variability of root pole number is distinctly higher in trimerous than in dimerous seedlings, because of the fact that in all seedlings a four-poled condition is characteristic of the main root system and prevails even in the trimerous forms up to within a few millimeters of the base of the hypo- cotyl. Sections in the upper root region in such seedlings therefore show a considerable number of four- and five-bundled individuals. The number of intercalary bundles is highly variable in both seedling types. The standard deviation is distinctly larger in the dimerous forms, but because of the generally lower average number of intercalary bundles in trimerous seedlings, the relative variabilities as measured by the coefficient of variation are higher in the trimerous type. In the central region of the hypocotyl the variability of bundle number, both absolute and relative, is far higher in the dimerous seedlings, due in large part to the generally higher standard deviation of the number of intercalary bundles in the dimerous type. In the central region of the epicotyl just the reverse is true, the varia- bility of bundle number being higher in the -trimerous than in the dimerous seedling. This is evidently due to the facts (a) that the intercalary bundles 18 Data for number of root poles are available for only three of the five lines. 102 AMERICAN JOURNAL OF BOTANY [Vol. 8 of the hypocotyl are quite lost in the cotyledonary nodal vascular complex, and thus do not affect the variability of the dimerous plants; and (6) that the doubling of the primary epicotyledonary bundles which almost invariably occurs in the normal seedling may not always take place, at least not at as low a level as the central region of the epicotyl, in the abnormal type. CONCLUSIONS The results of the foregoing morphological and biometric analyses justify the emphasis at this point of certain general considerations. 1. External differentiation such as that which characterizes dimerous and trimerous seedlings of Phaseolus vulgaris is accompanied by profound differences in internal structure. 2. Anatomical characters are by no means constant. On the contrary, they are very variable even in series of individuals which are genetically highly homogeneous. Morphological investigations based on _ limited series of individuals may, therefore, result in inadequate conceptions. 3. Variation in anatomical structure is not constant for the plant as a whole, but may differ from region to region or from organ to organ. Thus in the regions of the seedling here under consideration, hypocotyl and epi- cotyl differ widely in the variability of bundle number. Furthermore, differences in variability from organ to organ or from region to region are not constant, but may be conditioned by other morphological features. To illustrate from the case in hand, the variability of bundle number of normal seedlings is higher in the hypocotyl than in the epicotyl. In seed- lings with three cotyledons and three primordial leaves, just the reverse is true. These differences in biometric constants are readily understandable in the light of a knowledge of comparative morphology. 4. The results of this study emphasize the importance of the use of both biometric and comparative methods to supplement each other in any attack upon the problems of general morphology or of morphogenesis. Pole RGILILUS FLAVUS, A. ORYZAE, AND ASSOCIATED SPECIES CHARLES THOM AND MARGARET B. CHURCH (Received for publication August 30, 1920) Cultures of fermented food products of the Orient made from rice, other cereals, and soy beans show a number of characteristic types of Asper- gillus. Some of these are manifestly only contaminations. A few of them are so closely identified with these food products as to call for com- parative study to determine their significance in the fermentation processes under investigation. These organisms are recorded under the names A. flavus Link, A. oryzae Ahlb., A. Wentst Wehmer, and A. tamari Kita. Study of cultures from many correspondents and froma wide variety of foodstuffs shows clearly that these forms are not limited to the Oriental fermentation industries but are cosmopolitan. The numerous strains found align themselves into groups of closely related forms which may for convenience be considered here under three series names, Aspergillus flavus- oryzae, A. Went, and A. tamart. ASPERGILLUS FLAVUS-ORYZAE SERIES The saké industry of Japan is based upon the diastatic power of Asper- gillus oryzae (Ahlb.) Cohn.!. The organism as actually used is a well- marked form and its activities have been extensively discussed. Cultures of this species which have been distributed widely from the Centralstelle? at Amsterdam show the morphology and culture reactions clearly described by Wehmer*®. Among large numbers of mold cultures from many sources only one culture which might be confused with the saké organism has been received from a source unconnected with the Oriental fermentation indus- tries. : When, however, numerous cultures from the soy or shoyu industry of Japan and China are brought together, a whole series of forms are found which bridge the gap morphologically between A. oryzae as the saké organism and A. flavus as described and distributed also by Wehmer (loc. cit., p. 81). Cohn, F. Ueber Schimmelpilze als Gahrungserreger (A. oryzae). Jahresb. Schlesisch. Gesellsch. f. vaterland. Cultur 61 (1883): 226-229. Breslau, 1884. * Centralstelle fiir Pilzkulturen, Roemer Visscherstraat 1, Amsterdam. 3 Wehmer, C. Die Pilzgattung Aspergillus in morphologischer, physiologischer, und systematischer Beziehung unter besonderer Beriicksichtigung der mitteleuropaeischen Species. Mém. Soc. Phys. Hist. Nat. Genéve 332: 1-156. Pls. 1-5. 1901. This paper is commonly cited as Wehmer, Monograph (Monogr.). ‘103 104. AMERICAN JOURNAL OF BOTANY [Vol. 8 Material taken directly from fermenting vats in China by Dr. Yamei Kin, formerly of the Bureau of Chemistry, shows strains of this character. Inoculating material furnished by Dr. Teizo Takahashi for experimental work on the fermentation of soy sauce or shoyu proved to be a member of this series. Dr. Takahashi had selected his strain for this type of fermen- tation from among several recognized and studied by him in Tokyo. Our thanks are due to Dr. Takahashi for discussing his views upon the group of organisms used by the fermentation industries of his country. These forms showed variations toward the saké organism and others markedly of the ‘flavus’ type. All of these strains are regarded by him as varieties of Aspergillus oryzae, not A. flavus. In the fermenting samples examined, the dominant organism in every case has been nearer A. flavus in the sense of Brefeld and Wehmer than A. oryzae (Ahlb.) Cohn. The same condition is readily disclosed by cultures from certain of the koji products distributed under the patents* of Takamine in which the name A. oryzae is used, not A. flavus. Although the study of strains widely separated in the series gives easily measurable differences, comparison of large numbers of strains from many sources furnishes intermediate forms which break down the value of such contrasting characters. All of these forms show mixtures of yellow and green color when grown on Czapek’s solution agar which, when com- pared. with Ridgway’s plates, are found to be closely related. The whole group is found to possess conidiophore stalks and conidia with walls pitted. Stalk walls when examined with low magnification are often recorded as delicately rough, and conidia as delicately rough or spiny. Careful exami- nation with high powers shows these appearances to be due to pits. Upon the ripe conidia the pits, instead of being circular, are commonly elliptical, giving an appearance sometimes designated as areolate. Variations in length and diameter of stalk, thickness of wall, and number and arrangement of sterigmata are found, but the texture and markings of the walls, the formation, shape, and development of parts appear to link together related forms, hence to have value in characterization. Accuracy in these observations becomes, therefore, essential. Johnston® notes dis- crepancies in the description of the same culture by different workers. We find the same difficulty in our own notes. Cell walls examined with the lower powers, especially the dry objectives, may be recorded as rough or spinulose. The same cell walls examined with the apochromatic objective appear pitted. It has been found necessary to make many examinations of each species or strain studied, both separately and in comparison with other related forms. The data used in this paper have been obtained by using a Zeiss 3 mm., N. A. 1.30 apochromatic objective with a Zeiss 12 x compensation ocular. 4 Takamine, J., U.S. nos. 27401; 525.820; 525.823; 525.824. 5 Johnston, J. R. The entomogenous fungi of Porto Rico. Bull. Board Commrs. Agr. Porto Rico 10: 17. I9QI5. Feb., 1921] THOM AND CHURCH — ASPERGILLUS I05 The forms reported here have each been restudied several times, some of them at intervals of several years, to determine which characters were variable with conditions of culture and which were stable. | The following cultural descriptions of A. flavus, A. oryzae, A. parasiticus, and A. effusus are prepared as typical for races or groups of nearly related strains, which represent fairly widely separated portions of the whole series. Characterization of A. flavus Link.® Colonies on Czapek’s solution agar with sucrose, spreading widely, with floccosity limited to scanty growth of a few aerial hyphae in older and dryer areas among the erect crowded conidiophores; sclerotia at first white, then brown, hard, parenchymatous, in a few strains white-tipped, produced abundantly by some strains, scantily by others under undefined conditions, not or rarely by still others; perithecia not found. Conidial areas ranging in color in different races from sea-foam yellow through chartreuse yellow, citron green or lime green, to mignonette green, Kronberg’s green, or more rarely to wy green (see Ridgway XXXII. feereebik,m;’ approximately C. D. C. 270, 271, 266, 252, 253, 257),® persistent or changing in very old colonies toward Isabella color to brownish olwe (Ridgway XXX. 19” 1m), zinc orange (Ridgway XV. 13’), or even Saccardo’s umber (Ridgway X XIX. 17’ k); reverse (or under side) and agar either uncolored or more or less intensely yellowed, from pinkish buff, cinnamon buff, to clay or even Saccardo’s umber (Ridgway XXIX. 17” d, b, to k), or in some cases even darker brown in old and dry cultures. Stalks arising separately from substratum, 400 to 700 ,even to I000 u long, 5 to 15 win diameter, broadening upward, with walls colorless, so pitted as to appear rough or spiny with low magnification, occasionally granuli- ferous, varying in thickness, gradually enlarging to form a vesicle 10 to 30 or even 40 win diameter. Heads in every colony varying from small with a few chains of conidia to very large stellate or columnar masses, or both mixed in the same area (fig. I, c and d); small heads with small dome-like vesicles and a single series of a few sterigmata up to 10 to 15 uw by 3 to 5 yn; larger heads partly with simple sterigmata, partly with branched or double series, or with both in the same head; primary sterigmata 7 to 10 u by 3 to 4; secondary series 7 to 10 pw by 2.5 to 3.5 wu; conidia pyriform to almost globose, from almost colorless to yellow-green, with walls so thickened as to leave circular, elongated, or winding pits, giving a rough or echinulate effect? when viewed with low magnification, varying in size in different strains and even in the same culture, frequently 2 by 3 u, 3 by 4n, 4 by 5 y, or 5 by 6 uw in diameter, or even larger in some strains. Colonies grow best in starch- and sugar-containing media; some strains fruit at temperatures up to 50° C. Spores survived heating! to 57.2° C. for 30 minutes and dry heat at 110° C. for 30 minutes. This description was originally based upon culture no. 108 received from Amsterdam and identical with no. 3526 obtained directly from Wehmer. 6 This characterization is revised and extended from the form furnished to Dr. John R. Johnston and published by him (loc. cit.). ’ Ridgway, R. Color standards and color nomenclature. Washington, D. C., 1912. § Klincksieck, P., and Valette, T. Code de couleurs. Paris, 1908. 9“ Areolate’’ of Johnston. ” Thom, C., and Ayers, S. H. Jour. Agr. Res. 6: 153. 1916. 106 AMERICAN JOURNAL OF BOTANY [Vol. 8 This organism in Czapek’s solution agar is Krénberg’s green without color in the substratum and without sclerotia. The following supplementary cultures are cited: no. 3557.9 from corn (isolated by Clawson) differs by the production of sclerotia, and yellow color in the substratum; no. 128, after many transfers identifiable with no. 108, produced a sclerotium—former resembling no. 3557.9; no. 2773 from Demerara is mignonette green, produces abundant sclerotia and yellow color inreverse. In general, sclerotium for- mation is found correlated with the production of yellow color in the sub- merged mycelium and with reduced intensity of green in the conidial area. , Numerous strains with the same cultural characters have been obtained from many sources. The considerations leading to the retention of the name A. flavus for these forms are discussed later under “‘nomenclature.”’ A. ORYZAE SERIES Aspergillus oryzae has been generally accepted as a valid species. It is characterized as a group of varieties by Costantin and Lucet." In the typical form represented by the cultures and descriptions of Wehmer, the species is readily separated from cosmopolitan forms of A. flavus. In the Oriental industries in which it has been long used, the separateness of this form is largely lost. It becomes, therefore, a gigantic race in a group in which other members possess the same habits, the same essentials of structure, but differ slightly in color and greatly in size. Growth upon different substrata produces great differences in the appearance of colo- nies. The fruiting stalks on Czapek’s solution agar are commonly 2 to 3 mm. in length, much longer on richly organic media, and are reported by Takahashi" to attain a length of 20 to 30 mm. upon special rice media upon which the stalks of A. flavus reach a length of 5 m to 8 m. In contrast with A. flavus as already described, the following characteri- zation from cultures is proposed. A. oryzae (Ahlb.) Cohn. Colonies on Czapek’s solution agar spreading broadly, pale greenish yellow (at its greenest about lime green to mignonette green. Ridgway, loc. cit. XX XI. 25’’ YG—Y), with the green fading early to leave yellowish brown shades; sclerotia dark, produced occasionally, few and in clumps; mycelium and agar uncolored; stalks 2 to several milli- meters long, up to 20 to 25 win diameter; heads both large and small in the same culture, predominantly large, globose, and radiate rather than calyp- trate; sterigmata most commonly I-series, occasionally 2-series, primary sterigmata up to 8 to 12 by 5 yw, secondary when present 7 to 10 by 3 yn; conidia pyriform, colorless to very slightly yellow with walls so thickened as to leave circular, elongated, or winding pits giving a rough or echinulate 11 Costantin, J., and Lucet, L. Recherches sur quelques Aspergillus pathogeénes. Ann. Sci. Nat. Bot. [X, 2: 119-171. 1905. 12 Takahashi, T. Preliminary note on the varieties of Aspergillus oryzae. Jour. Coll, Agr. Tokyo 1: 137-140. I909. 13 “ Areolate’’ of Johnston, loc. cit. Feb., 1921] THOM AND CHURCH — ASPERGILLUS 107 effect when viewed with low magnification, varying in size, predominantly larger than A. flavus, 3 by 4u, 4 by 5yu, 5 by 64, 6 by 7 uw, occasionally 5 to 6 by 8 to 10 yn. This description is primarily based upon culture no. 113 from Amsterdam. The same form has been isolated at various times from fermented products (once from a Brazil nut), and received in exchange from various workers. A series of 3 varieties were first studied by Takahashi" in 1908. This work was continued with the accumulation of a series of strains under this name which have been furnished to us for study. These are lettered’ with the alphabet from A to P, then skip to Z, and all are regarded as A. oryzae. These cultures were transferred and grown under conditions as uniform as possible in Czapek’s solution agar. The resulting colonies were arranged into a series to correspond with our conception of the relationships involved. This may be tabulated as follows: Takahashi strains arranged in order of appearance of colonies: H. White, nearly sterile, floccose mycelium. O. Slight fruiting, predominantly yellow. B. Increase of fruiting, still a floccose colony. Near A. gtganto-sul- phureus. G. Further development of fruit at expense of floccosity. Z. Long stalks, large heads, floccose effect. D. Mycelium and long-stalked fruits, both evident. N. Abundant stalks and heads, no green color. Near A. pernictosus. F. Close resemblance to no. 113, A. oryzae of Wehmer. I. Short-stalked, form otherwise near no. I13. A. Still shorter. | M. Same morphology, green color more prominent. L. Slightly paler form with shorter stalks. C. Close to no. 108, A. flavus of Brefeld and Wehmer. P. Shorter stalks (crowded; more slender type), green passing to reddish brown. Near A. micro-virido-citrinus. 1B suggesting the same line of transformation from K. > Aberrant forms 4 strain C as is found in A. effusus, though dif- BE, fering from previously examined representatives. Similarly, Z, G, B, O, and H are progressive reductions from the A. oryzae type found in strain F. This table shows strain F to represent approximately the form already described as A. oryzae (no. 113). With almost entire loss of green color and progressively increasing floccosity, strains N, D, G, B, O, and H end at Hin almost complete loss of conidium production. The absence of all green color pclae, cit. * The lettering is maintained to correspond with Dr. Takahashi’s usage in his own _ papers. Takahashi, T., and Yamamoto, T. On the physiological differences of the varie- ties of Aspergillus oryzae employed in the three main industries in Japan, namely saké, shoyu, and tamari manufacture. Jour. Coll. Agr. Tokyo 5: 153-161. 1913. 108 AMERICAN JOURNAL OF BOTANY [Vol. 8 makes strain N conspicuous as a variation which might easily be regarded as a distinct species. From F the strains I, A, M, and L grade in appearance toward C, which is closely similar to A. flavus as already described (no. 108). The tendency toward floccosity and toward quick disappearance of green color appears again in L. Fic. 1. The photomicrographs composing this figure represent the wide variety of heads in a species and in a strain. The magnifications are various and are not given. a. Calyptrate head of A. tamari. 6b. Radiate and large head of A. tamari; the same strain as e. c. Head of no. 108, type of A. flavus. d. Columnar head of no. 129; a delicate, pale form of A. flavus. e. Radiate head of A. tamari, showing a less compact structure than 0b. f. Globose head of A. Wentiz, with heavy-walled stalk. P is a more slender, delicate form than C, with crowded stalks, and also loses its color quickly. A. oryzae var. basidiferens Costantin and Lucet!® differs from A. oryzae as described by Wehmer in having both primary and secondary sterigmata. Since the authors of this variety had no other cultural experience with A. oryzae, and since all cultures we have seen appear to show this character, it seems best to us to introduce this observation as an emendation to the description of A. oryzae instead of recognizing the validity of a variety. A. pseudoflavus Saito” appears to represent some one of the races intermediate in length of stalk between A. flavus and A. oryzae but having the color, usually simple sterigmata, and size of conidia found in A. oryzae. Aspergillus micro-virido-citrinus Costantin and Lucet'® differs from A. pseudoflavus only in its smaller conidia. A. gymnosardae Yukawa’? was found upon the fermented fish product, katsuobushi, in Japan. The description clearly marks it as also intermediate in structure between A. flavus and A. oryzae. We have not been able to identify either of these forms with certainty, although we have had several strains in culture which occupy such an intermediate position. Similarly, another strain appears in our collection once from America IO RIEOC ACU p: KOT, 17 Saito, K. Centralbl. Bakt. II, 18: 34. figs. 15-18. 1907. BOC. Ci Pp. 158. Yukawa, M. Jour.:Coll. Agr. Tokyo 1: 362. Tab. XVIII, tgs. 1-7:) 1onE- Feb., 1921] THOM AND CHURCH —~ASPERGILLUS 109 (no. 129), and once contributed by Hanzawa from Japan. This form in culture is citron green to lime green (Ridgway XXXI. 25’’). It has short, crowded stalks like A. fumigatus with small heads, with mostly a single series of sterigmata and conidia predominantly small, 3 to 4 wu, few reaching 5 to 7 w in diameter. A. parasiticus Speare. Speare”, working in Honolulu, found one of these forms parasitic upon the mealy bug of sugar cane (Pseudococcus calceolariae Mask), and described his strain as A. parasiticus. A culture with the same characters was isolated by one of us from mealy bugs obtained from Demerara; another culture was isolated from cane sugar in New Orleans by Kopeloff. However, other strains of the flavus group were isolated by Johnston”! from mealy bugs in Porto Rico; reinfection experiments by Johnston, while not conclusive, established a presumption of infectiousness as a strain or-race character unconnected with the specific morphology of Speare’s A. parasiticus. Speare’s organism when grown on Czapek’s solution agar differs from the commoner forms of A. flavus in its predominantly greener color (near ivy green Ridgway XX XI. 25”’ m), in short stalks usually 200 to 400 u long, in heads with usually a single series of sterigmata 7 to 10 uw long by 3 to 5 mu. No sclerotia have been seen. The mycelium is uncolored. Otherwise the characters are those of the A. flavus group. A. effusus Tiraboschi.?? Cultures of a cottony, floccose type have been obtained from widely separated sources (no. 130 from Dr. B. F. Lutman, Hurineton, Vermont; no. Sc. 171 in corn:meal from Indiana; no: 2750 isolated by Johnston from mealy bugs in Porto Rico). Superficially these cultures show little relation to A. flavus. Microscopic examination of heads and spores, however, shows close relationships. Characterization of A. effusus: Colonies on Czapek’s solution agar with sucrose, broadly spreading, effused floccose, or cottony, white becoming .Slowly dirty vellowish or in restricted areas greenish yellow; reverse and agar yellowish. Stalks either A. flavus-like arising directly from the sub- stratum, up to 500 uw long and frequently with large radiate heads, or pre- dominantly in the form of branching, trailing, thick-walled hyphae, each segment consisting of a long, thick-walled, fertile cell bearing a perpendicular branch (stalk) usually less than 100 uw long by 5 to 10 uw in diameter, with walls pitted and sometimes granuliferous, bearing usually columnar heads; vesicles in small heads up to 20 uw in diameter, occasionally larger, sterigmata in one series, 6 to 10» by 3 to 5 u, mostly on apex only of vesicle; larger heads with either simple or branched sterigmata as in A. flavus. Conidia pitted as in A. flavus, pale yellow, rather thin-walled, pvriform to globose, varying in size in the same culture from 3 by 4yu to 5 by 7u. Neither 0 Speare, A. T. Fungi parasitic upon insects injurious to sugar cane. Hawaiian Sugar Planters Exp. Sta. Path. and Phys. Ser. Bull. 12: 30. 1912. Oe. Ctt., p. 15. ; Tiraboschi, C. Atti Terzo Congresso Pellagrologico Italiano, p. 142. Milano, 1906; diagnosis in Annali di Botanica 7: 16. 1908. 1 6) AMERICAN JOURNAL OF BOTANY [Vol. 8 sclerotia nor perithecia were found. Colonies grew weil in all common media, grew better at 37° C. than at 20° C., liquefied plain gelatine (gelatine in distilled water) with yellow color in the liquid. The puzzling appearance of these cultures led to studies in morphology and to extensive experiments scattered over nearly ten years. Comparative study of all species obtainable shows that the stalk throughout the genus Aspergillus originates as a mycelial cell transformed into a spore-producing organ. The cell enlarges in diameter and its wall becomes thickened. The stalk arises as a branch approximately perpendicular to the course of the original cell which remains in the hypha as a kind of foot. Usually the stalk, beginning with the diameter of its foot cell, broadens upward and lengthens, hence becomes many times the size of its foot. In A. effusus the foot cell is frequently very long, branching and connected with other foot cells to form a trailing, fertile hypha from which the stalks arise as short branches. Selective transfers from Lutman’s strain (no. 130) showed the possibility of separating a race which appeared to be the usual form of A. flavus and another in which the heads were borne only on the trailing type of fertile hyphae among cottony white masses of sterile hyphae. The hypotheses of symbiosis and of parasitism were both tested through many transfers without result. The heads on all series of cultures maintained the essential morphology of A. flavus, although the colony characters diverged | widely. More recently, a transfer was made from a stock culture several months old which was grown upon Czapek’s solution with the addition of 5 percent sodium chloride, 10 percent sucrose, and 3 percent agar. The original strain (Lee 108) had maintained the cultural appearance of A. flavus as received from Wehmer, through successive transfers for about four years. This transfer produced a floccose type of colony with some A. flavus type of fruiting at the edges. Transfer from the whitest areas in this culture produced the typical white colony described above; transfer from an area showing few small heads produced a mixed colony; transfer from selected heads appearing to be A. flavus gave a pure A. flavus colony. All of the experimental work supports the hypothesis that the floccose types represent mutants from the typical A. flavus, which were probably induced in the last experiment in similar manner to the mutations of A. niger as described by Schiemann.?* The description given by Tiraboschi (loc. cit.) for A. effusus probably applies to these cultures. A. effusus was isolated by Tiraboschi from spoiled corn products. Nomenclature. Aspergillus flavus was first described by Link?* in 23 Schiemann, E. Mutationen bei Aspergillus niger van Tieghem. Zeitschr. Indukt. Abstam.- u. Vererbungslehre 8: I-35. I912. 24Tink, H. F. Observations in: Ordines plantarum naturales. Gesellschaft Natur- forschender Freunde zu Berlin, Magazin 3, p. 16. 1809. This is usually cited Link. Obs. p. 16. 1809. The description in full follows: ‘‘Caespitibus laxis, floccis albis erectis, capitulis junioribus albis, adultioribus flavis. Frequens in plantis siccis herbariorum.”’ Feb., 1921] THOM AND CHURCH — ASPERGILLUS Pit terms vague enough to baffle any attempt at certain identification. The habitat given was herbarium specimens. Our own search over many lots of moldy plants in herbaria, together with interpretation of the name flavus, suggested that some one of the Aspergillus herbariorum-repens- Amstelodami series in the sense of Mangin”® was the basis of Link’s descrip- tion. Specimens have been actually found in several series of exsiccatii labeled A. flavus but clearly consisting wholly of A. rebens. However, Link records his acquaintance with the green Aspergillus of the herbarium under the name A. glaucus. The universally distributed yellow perithecial forms which were later connected with the common green forms by De Bary were known to Link under the name Eurotium. Clearly, then, Link believed that he had some organism which he found commonly upon badly dried herbarium specimens and which was yellow enough in contrast to A. glaucus to justify the name A. flavus. The conidial forms of the A. glaucus group do not show a yellow color factor. Following De Bary,”® there is, moreover, the widespread use of this specific name A. flavus Link for our series of yellow-green forms which are universally distributed. Wilhelm,”’ Schroeter,”2 and Wehmer” base their use of this name upon Brefeld’s specimens distributed as no. 2135 in Rabenhorst’s*® Fungi Euro- paei. A comparison of Brefeld’s specimens with a culture obtained in Wehmer’s laboratory in 1905 and still maintained by us in culture shows them to be morphologically identical. This strain agrees with the characteri- zation of A. flavus in Wehmer’s Monograph. Costantin and Lucet*! reach the same general conclusion without record of having seen the speci- mens, and add the comment that this identification constitutes the perpet- uation of a tradition that this particular strain is A. flavus Link. This identification is promptly discarded by them and its name changed to A. Wehmeri Cost. et Lucet.*? With the study of the culture from Wehmer as a basis, the distribution of this and closely related strains has been followed for about ten years. - Numerous cultures have been isolated and compared from diverse sources. Molds with essentially this morphology have been sent to us in series of soil cultures made by Esten and Mason in Connecticut, by Miss Dale in England, by Johnston in Porto Rico, by Waksman in New Jersey, by Mc- Beth and Scales in Washington, by Werkenthin in Texas, and by Hartley from coniferous seed beds in Kansas. ‘They have been isolated by us many 2 Mangin, 1. Ann. Sci. Nat. Bot. IX, 10: 303-371. 1909. *6 DeBary, A. Beitrage zur Morphologie der Pilze. IIIt® Reihe, 2t¢ Abt., p. 20. 1865. 27 Von Wilhelm, K. A. Beitrage zur Kenntniss der Pilzgattung Aspergillus. Inaug. Diss. Strassburg. Berlin, 1877. 8 Schroeter, J. Cohn’s Kryptogamenflora von Schlesien 3?: 216. 1893. Beac..ci., p. 8. ; % Rabenhorst. Fungi Europaei Edit. Nov. ser. II. 1875. E200. Cib., p. 152. EeeOGrCIt., D- 169. 2 ' AMERICAN JOURNAL OF BOTANY [Vol. 8 times from miscellaneous foodstuffs, especially the cereals both as whole grain and as milled products, and more recently have been found abuaiany in the soy-bean fermentation products of China and Japan. Members of this series have been reported in the study of infections | in the human ear. Certain of these forms have produced lesions and death when injected into experimental animals. Costantin and Lucet*®® review the literature of such pathogenicity and offer a key to species based upon their review of injection experiments with rabbits and fowls. The structural characters cited by them represent fairly well the range of variation within the group. Sclerotium formation is used to separate A. flavus, attributed by them to Wilhelm, from the other forms. In our experience sclerotium formation is not limited to any morphological section of the group. More- over, A. Wehmert of Costantin and Lucet is a manuscript species based upon A. flavus of Wehmer’s monograph. It was not studied by them in culture. Both Wilhelm and Wehmer based their use of the name upon the usage of Brefeld as determined by examination of the same cultural material distributed by Rabenhorst.*4 We have seen this material, and it corresponds satisfactorily with the characters given by Wilhelm and Wehmer. If the name A. flavus is to be Heid in the sense of Wilhelm, A Wehmeri is clearly a synonym. A. flavescens of Wreden*® was not cultivated. The size of the spores and the coloration of the stalk reported caused us to believe that it belonged elsewhere.?® Wehmer*® cites Lichtheim as having compared A. flavescens with A. flavus Link as understood by Brefeld and having found them identi- cal. Lichtheim, however, uses the name as interpreted by Eidam, which is probably but not necessarily identical with the usage of Wreden. Certain organisms from ulcerated ears clearly belonged to this series. The mor- phology given by Costantin and Lucet for A. Szebenmanni and A. micro- virido-citrinus is not uncommon in cultures from the group except as to color. It will be shown later that the green factor in colony color is sup- pressed when fermentable carbohydrates are omitted from the substratum. The range of morphology cited by Costantin and Lucet was assumed by them to establish a presumption of pathogenicity to warm-blooded animals for the whole group. The infection experiments reported from different sources were intravenous with positive lesions. It is noteworthy that they found their A. oryzae var. basidiferens*® to be pathogenic also to the rabbit by the same kind of inoculation. This is consistent with a common morphology in A. flavus and A. oryzae as considered in this paper. Double sterigmata, used by Costantin and Lucet as varietal characters, are not 33506. C1l., DP. E51-103. 34 Loc. cit., nO. 2135. 1876. 3 Compt. Rend. Acad. Sci. Paris 65: 368. 1867. % Thom, C., and Church, M. B: Amer. Jour. Bot.5: 100. ~roLd: 37 Wehmer, C. Centralbl. Bakt. I], 2: 148. 1806. 38 06. Cit. p. 167. Feb., 1921] THOM AND CHURCH —— ASPERGILLUS Il3 the exception but the rule in the saké organism in which only occasional cultures show only simple sterigmata. In the rice and soy fermentation industries of Japan the workman’s eyes, ears, nose, throat, and skin abra- sions are constantly exposed to Aspergillus spores. Dr. Takahashi and Dr. Kita (personal communications), however, report absolutely no infec- tions. Our cultures show a wide range of varieties of the Aspergillus flavus series to be present. Intravenous injection, doubtless, has value in demon- strating the possible activity of an organism when so inoculated, or perhaps in lesions already established by other agencies, without proving active pathogenicity. Wreden and Siebenmann use the name A. flavescens for organisms found in the human ear. Wreden’s description lacks essentials for identification perhaps, but Lichtheim certainly had an organism of this group from infected ears. Siebenmann, using the same name, gives details which definitely ally his form with either A. flavus or A. tamari (see discussion of A. tamart later). Wilhelm clearly has a sclerotium-producing strain closely allied to the material studied by Brefeld. Wehmer, whose organism we have in culture, had a different but closely related strain which rarely if ever produces sclerotia. Costantin and Lucet*® appear also to have had but one strain of the same series, which they described as A. muicro-virido-citrinus. Cultural observations limited to single strains in a group varying as widely as this may easily lead workers unacquainted with other material to believe they have distinct species. When comparison of hundreds of cultures from separate sources has bridged the gap between these forms, it is doubtful if any effort to maintain such species is desirable. There appears to be no valid reason for rejecting the name A. flavus for the cosmopolitan organism studied by Brefeld and Wehmer and as tenta- tively covering many strains with minor variations from such a type. A. oryzae, A. parasiticus, and A. effusus are morphologically recognizable varieties or species which are certainly closely related to the cosmopolitan group of which the organism described by Brefeld and Wehmer and believed by them to be A. flavus Link may be called the type. In reaching this conclusion, many series of cultures were made with a large number of strains selected to represent the widest range of variation found in our collection. These cultures included an extensive variety of culture media; the bark of Castanea, Liriodendron, Platanus, and Tsuga, oatmeal agar with and without sugar, potato plugs, beef extract peptone agar, egg albumen, beef plugs, loam, rice, cooked soy beans mixed with ground and roasted wheat, Czapek’s solution with cerealose instead of sucrose. The following paragraphs describe some points observed which seem to be worthy of note. . Czapek with 50 percent saccharose: Twenty-seven strains of Aspergillus flavus grown on Czapek solution agar containing 50 percent saccharose grew for all practical purposes the same as if on unmodified Czapek solution eeOG, Cit., DP. 158. IIl4 AMERICAN JOURNAL OF BOTANY [Vol. 8 agar. In 3 strains of A. effusus, fruiting was increased and the conidial areas and the reverse were citrine in color. The two strains of A. oryzae were more brownish in color and the conidiophores were short as compared with the growth of the same strains on the standard Czapek solution agar. A. terricola var. Americana grew sparsely on this medium. Fish agar (halibut): Nine strains of A. flavus when grown on fish agar for two months developed only a few brown heads; nine other strains of the same species developed only white mycelium. A. oryzae and A. effusus also did not fruit. A. tamari and A. Wenti, however, showed fruiting at first old gold in color and scarcely spreading beyond the mark of the streak. Beef plugs: Plugs of fresh beef were cut with a cork borer and placed in tubes ordinarily used for potato plugs. The sterilization was fractional. At the end of two weeks six of the more common strains which develop conidial areas near in color to Krénberg’s green on Czapek solution agar were olive ocher (Ridgway XXX. 21’’) on the beef, four others ran through olive ocher to old gold (Ridgway XVI. 19’ i); three strains changed from olive ocher to other tints and shades of orange and yellow; one strain never developed any deeper color than deep colonial buff (Ridgway, XXX. 21” b). Ail the green color was, therefore, eliminated from these strains of A. flavus when grown on cooked beef, with the exception of one strain which became lime green after it had appeared olive ocher. The early growth of A. para- siticus was at first mignonette green and later olive lake (Ridgway XVI. 2’ i), a shade with no green; a yellow green strain (no. 129), possibly A. micro- virido-citrinus, corresponding with A. terricola of the brown series, was colonial buff (Ridgway XXX. 21’); and strains of A. effusus at the end of two weeks were chamois (Ridgway XXX. 19’ b). No green color was exhibited in the whole group. Plain agar (bacteriological): The A. flavus group when planted on plain agar produced color practically as when grown on beef plugs. The green factor was not suppressed as completely, however. It was more evident during the first few days of growth, and seemed to disappear except in the same instances noted under beef plugs. Synthetic agar (Currie’s):*° Thirteen of the A. flavus strains and A. parasiticus developed the green color more intensely with a reduction of yellow, when grown on this agar. They developed such shades and tints as Kildare green (Ridgway XXI. 29” b), Rainette’s green (Ridgway XXI. 27'" 1), cress green (Ridgway XXI, 29’ k), etc. Six similar strains grew as if on Czapek solution agar, as did also A. effusus. In these experiments the colors reported range from mixtures of yellow and orange to various combinations of yellow and green. The reversible factor appears to be green. Kita‘! reports similar observations. In 40 (NH.)H2PO., 2.0 gms.; KCl, 0.2 gm.; MgSQ,u, 0.1 gm.; cane sugar, 30 gms.; agar, 15 gms.; H.O,11. Formula used by Dr. J. N. Currie. 41 Kita, Gen-itsu. Ueber die Konidienbildungsfaehigkeit einiger Varietaten des Aspergillus Oryzae. Original Communications, 8th International Congress of Applied Chemistry 14: 95. Feb., 1921] THOM AND CHURCH — ASPERGILLUS II5 describing A. pseudoflavus, Saito*? observed that exposure to ammonia would destroy the green color entirely, leaving yellow or yellowish brown. Subsequent exposure to vapor of acetic acid restored the green color to this colony. The test used by Saito was applied to A. flavus (no. 108, the Wehmer strain), A. oryzae (the saké organism), and A. parasiticus, which represent the widest range of differences in our collection. Each of these strains gave the reactions described by Saito for A. pseudoflavus. The test was repeated with hydrochloric acid substituted for acetic acid. The correlation of the green color with the acid was clearly brought out. These strains grown in Czapek’s solution agar are, respectively, A. oryzae about lime green, A. flavus near Krénberg’s green, and A. parasiticus close to ivy green, all in column 25”, Plate XX XI of Ridgway’s tables. When exposed to the fumes of hydrochloric acid the green color was intensified, reaching colors given in column 29” of the same plate with the deepest areas reaching the same intensity, cress green. When the same cultures were exposed to ammonia, all of the green disappeared, and the colors remaining corresponded with combinations in column 21”’, Plate XXX, varying from deep colonial buff to olive in the deepest areas. In the experiments previously described the green colors are found present in marked degree only upon media containing sucrose or some other fermentable carbohydrate. In cultures upon beef, fish, egg, and soy beans, which lack carbohydrates or are very poor in fermentable carbohydrates, the greens were absent or nearly so throughout the series. In the mixtures of fermentable carbohydrates and proteins there are evidently simultaneous acid and alkaline fermentations which tend to neutralize each other as described by Ayers and Rupp.** The early development of conidia always shows some development of green in such cultures. When litmus is used in the culture medium, these early stages are always accompanied by the acid or red reaction. Many such cultures eventually lose all their green color, but this loss is always preceded by change in reaction. If a series of these strains grown upon a single medium show different shades of green, these shades of green are thus indications of the relative acidities reached by the culture. Some correlation between the typical color shown by a colony and the progressive changes in the reactions of the substratum, and possibly even of cytoplasm, is indicated. Thé substratum influences the development of such saprophytic fungi in numerous directions. The gross appearance of a pure mold culture may be entirely altered through the influence of the medium on which the fungus is growing. The dimensions of certain structures in an Aspergillus ® Saito, K. Microbiologische Studien tiber die Zubereitung des Batatenbranptweines auf der Insel Hachijo (Japan). Centralbl. Bakt. II, 18: 30-37. figs. 1-22. 1907. 48 Ayers, S. H., and Rupp, P. Simultaneous acid and alkaline fermentations rom dextrose and the salts of organic acids respectively. Jour. Infec. Dis. 23: 188-216. 1918. 116 AMERICAN JOURNAL OF BOTANY [Vol. 8 may be altered by a change in the medium, while those of other structures may remain constant as long as the medium is not totally inadequate or does not contain deleterious substances. Aborted or unrecognizable types of structure result from conditions positively inhibitive for normal growth. Conidial growth, sclerotia, or perithecia, each may be totally or in part suppressed or their production may be stimulated by the nutrient provided. The structure and markings of the stalk wall, general shape, markings, and range of size in conidia are fairly stable within the strain or species and fall within certain limits which for practical purposes do not vary. The length of the stalk, diameter of the vesicle, the dimensions of the primary sterigmata, and, within limits, the spore measurements are influenced by the substratum. Wall markings cannot be said to vary with the nutrient supplied, although their conspicuousness varies slightly from culture to culture doubtless through the pressure of several factors. The alterations in these latter structures are never permanent. They are dependent entirely on the substratum. Certain media stimulate in such fashion as to cause an increase in dimensions, others a dwarfing. The majority of culture media cause each strain to develop to a size falling within fairly well-defined limits. | ASPERGILLUS WENTII AND RELATED FORMS Aspergillus Wentit was described by Wehmer‘** and has been widely distributed in culture from the Centralstelle at Amsterdam. Extensive cultural studies show the species to differ from the characterization given by Wehmer in the quite general presence of both primary and secondary sterigmata. Identity of the Amsterdam strain with Wehmer’s material is hardly questionable. Cultures with the same morphology have been found by us upon moldy corn grains, upon moldy cotton cake from Georgia, (4230) within a temechee nut from Brazil, upon cubebs from Singapore, and received (4204.16c) from China through the kindness of Mr. Chung, from Hanzawa (4291.32) in Sapporo, (4186.34) from Panama collected by Dr. Thaxter, from Oregon soil (4078.0—5) collected by Waksman. One culture was received from Ohio Experiment Station, one from Prof. R. A. Harper. Although these forms differ in details of reaction and appearance, the morphological characters found mark them as a natural group. Characterization of A. Wentit Wehmer. Colonies on Czapek’s solution agar with cane sugar, deeply floccose, spreading, with sterile hyphae white or yellowish, and with heads white at first, changing through cream, cream buff, honey yellow, old gold, to light brownish olive, medal bronze, or in old cul- tures sometimes snuff brown (Ridgway, column 19, Plates IV, XVI, XXX, and Plate XXIX 15’”’ K; recorded as coffee-brown to chocolate brown by Wehmer), and in some strains producing large masses of aerial mycelium which in tubes may fill the lumen 3 cm. above the substratum; reverse of colony yellowish at first, becoming reddish brown when old; agar frequently 44 Wehmer, C. Eine neue technische Pilzart Javas. Centralbl. Bakt. II, 1: 150. 1895. Feb., 1921] THOM AND CHURCH — ASPERGILLUS ii], colored yellow; stalks 2 to 3 mm. or up to 5 mm. long, commonly 10 to 12 us or sometimes up to 25 w in diameter, inconspicuously I- to 2-septate, with walls colorless, up to 4m in thickness, and smooth, often studded with droplets in young cultures, enlarged at tips to vesicles widely varying up to 80 win diameter; heads large, yellow to brown, stellate (or globose fig. 1, f); sterigmata usually in two series, primary varying greatly, 6 to 8, occasionally to 15 wu by 3 to 5 n, in extreme cases up to 60 u by 8 to 10 uw; secondary 6 to 8 by 3 w (asingle series is recorded by Wehmer 15 by 4»). Conidia pyriform to globose, usually about 4 by 5 u, less commonly up to 5 to5.5ubv5to6yp (4.2 to 5.6 u, Wehmer), with walls thickened to leave pits or furrows on the surface arranged roughly lengthwise of the spore chain, frequently appearing smooth or nearly so with low magnifications, commonly more or less plas- molyzed when treated with 95 percent alcohol. Perithecia not found. Sclerotia limited to more or less undefined masses of thick-walled cells occurring occasionally, not uniformly. Cultural optimum below 37° C. in all strains tested. Gelatin liquefied in cultures, both with and without sugar. | The Java culture originally sent by Went to Wehmer was used in rice and soy fermentation on that island by Chinese workmen. The strains since found resemble the Amsterdam strain in their range of color changes, smooth, thick-walled stalks without pits, stellate heads, double sterigmata, and in the A. flavus-like marking of the conidial wall. The mass of sterile mycelium above the colony in typical test-tube cultures of the organism, as described by Wehmer, is present in the Amsterdam strain, but lacking or only partially or occasionally present in some of the strains. This overgrowth of mycelial masses with fruiting at several levels through a considerable period becomes more prominent upon potato plugs. A gradation from the type strain of Wehmer to those lacking this character in Czapek solution agar cultures, combined with the common structural characters cited, justifies the extension of our idea of A. Wenti to include these forms at least as varieties of a widespread natural group. , Inui*® described A. perniciosus as found in awamori-koji without giving details of stalk and spore markings but comparing the stalks with those of A. Wentit and A. luchuensis both of which we haveinculture. = se; 74 e. 3 : a \ = = é 2. $ = : = = é é E . =e : : : i 5 ~ c : % = a fat : Rs 5 . ~ A STE z S . 2 i f i LP ee : . es : in | > VBE Ss: ¥ ch Wey Bs (eile Na Py 4 ae) i A if . aie : EDITORIAL COMMITTEE : WC. B “ALLEN, Editor-in-Chief, : ay arene Ne a | Sec ahee ce Wisconsin A | Ware Cine peas ben i ah Nantes: 0 aah: » University. of Chicago! Be a "University oe ‘Sruarr Gacer, Business Monoser | Ontann E, Wuurs, “aut Brooklyn, Botanic Garden” id ae 7 _ Brooklyn Botanic CRA ‘Harper, eee 1 ae | Jacos R. ScuramM, ge Cibaesiig geen Ne € ornell Universi sat Le: aie Burcae oF Piaat Industry _ aires agi is ee Heeb eioe ae: Sonat nah ah a mae phe rae is) Palate ignehby: ee Maras: ” Subscription ; price, $6.00 a year. Single copies 75. cents. . each; $6.00 a: volume, postage extra. Postage will be’ charg doto. i “tries, except Mexico, ‘Cuba,. Porto: Rico, Panama Cana Ac e Panama, acne slope. dstaaals, (Guam, Se: S Mantseribt Offered’ for. abliadan. should be sypewrien, “and cases: be suena) to us Editor-in-Chief, ira pages may be arranged for at cost rate to authors page ) : eee . : ae Prapte: should Be Soehectae: Pinedicueee on : Journal of. pane eee ee Ga: a pe Agetitbantes aid he ade peenle “cents must be fee ttt to call, checks not a ‘ ers: shot i be addr ssed ft ale F bed sanouienths cee cede ma EB, “Allen; University. of Wisconsin, Mad son, Jiscons - ' Business. correspondence. including not cé 0 ge of address ‘concerning ee should be addréssed to American Journal of Bota ; Brooklyn, ie ‘or 4t North Queen eet, an \ AMERICAN JOURNAL OF BOTANY Vou. VIII No. 3 Pao LUDDY’ OF RHUS DIVERSILOBA WITH SPECIAL REEBRENCE TO irs TOXICITY James B. McNAIR (Received for publication September 15, 1920): Rhus Toxicodendron (L.), Rhus radicans (L.), and Rhus dwwersiloba T. & G. form a triad of plants equally regarded with aversion. The general recognition of their deleterious character is evinced in the applica- tion of the names poison iy, poison vine, and poison oak, given to them in various parts of the United States. Perhaps the earliest mention of these plants in North America is the following description by Captain John Smith in 1609: The poisonous weed, being in shape but little different from our English yvie; but being touched causeth reddness, itchinge, and lastly blysters, the which, howsoever, after a while they passe awaye of themselves without further harme; yet because for the time they are somewhat painefull, and in aspect dangerous, it hath gotten itselfe an ill name; although questionlesse of noe very ill nature. Long before the birth of Linnaeus, Cornutus in 1635 described the plant as a species of ivy in his work on the plants of Canada (Hedera trifolia Canadensis Corn. 96 from Carolina in the British Herbarium). About 1736 Linnaeus classified this plant as Toxtcodendron triphyllum glabrum. At the same time he described and named Rhus radicans. In an entry dated October 9, 1748, Peter Kalm gave an extensive and interesting description in his travels in North America of the Rhus radicans of Linnaeus. Since that time there have been many accounts of these plants and of their toxic nature. In 1820, Bigelow described Rhus radicans of Linnaeus as having _ Ternate leaves, that grow on long semicylindrical petioles. Leaflets ovate or rhomboidal, acute, smooth and shining on both sides, and veins sometimes a little hairy beneath. The margin is sometimes entire and sometimes variously toothed and lobed, in the same plant. The flowers are small and greenish white. They grow in panicles or compound racemes on the sides of the new shoots and are chiefly axillary. The barren [male] flowers have a calyx of five erect, acute segments, and a corolla of five oblong recurved petals. Stamens erect with oblong anthers. In the center is a rudiment of a style. The fertile [female] flowers situated on a different plant, are about half the size of the preceding. The calyx and corolla are similar but more erect. They have five small, abortive stamens and a roundish germ [ovule] surmounted with a short, erect style ending in three stigmas. The berries are roundish and of a pale green color, approaching to white. [The Journal for February (8: 59-126) was issued March 19, 1921.| 127, 128 AMERICAN JOURNAL OF BOTANY [Vol. 8 A plant has long appeared in the Pharmacopoeias under the name of Rhus Toxicoden- dron. Botanists are not agreed whether this plant is a separate species from the one under consideration, or whether they are varieties of the same. Linnaeus made them different with the distinction of the leaves being naked and entire in Rhus radicans, while they are pubescent and angular in Rhus Toxicodendron. Michaux and Pursh whose opportunities of observation have been more extensive, consider the two as mere local varieties; while Elliott and Nuttall still hold them to be distinct species. Among the plants which grow abundantly around Boston, I have frequently observed individual shoots from the same stock having the characters of both varieties. I have also observed that young plants of Rhus radicans frequently do not put out rooting fibers until they are several years old and that they seem, in this respect, to be considerably influenced by the contiguity of ROS objects. The attitude taken by Bigelow has been sustained by later botanists, among them Torrey and Gray (58) who consider R. radicans a variety of R. Toxicodendron. Rhus diwersiloba was first discovered by Douglas at Fort Vancouver on the Columbia river about 1830. Upon examination of this specimen W. J. Hooker (26), although he considered it ‘‘nearly allied, as this assuredly is, to the two preceding species [R. Toxicondendron and R. radicans}|,’’ neverthe- less “‘ventured to consider it distinct.’ He therefore gave it botanical significance as Rhus lobata. To support his conclusion he advances the following reasons: It’s general habit is very different, having erect straight stems and numerous small leafy branches. The leaflets besides being deeply lobed with acute sinuses are truly ovate, very obtuse, and greatly smaller than in any state of R. Toxicodendron, or R. radicans, which I have seen; the panicles, too, are exceedingly numerous. A free translation of Hooker’s Latin description of the plant is as follows: Bush erect, 3—4 feet, branches round with the youngest ones pubescent, branches numerous, short, spreading, leafy. Leaves long-petiolate, trifoliate, with little leaves ovate, 1-2 inches long, very obtuse, membranaceous, at the base sometimes acute, sometimes rotund or truncate, beneath especially pubescent, deeply and variously lobate, terminate one sub-long-petiolate, each side sub-equally lobate with lobes generally less than 3, with little lateral leaves at the exterior margin more deeply lobate. Flowers (male) yellow, in loose racemes, shorter than leaf, longer than petiole. Bracts at the base of the branches oblong, ciliate. Calyx deeply parted with oblong lappets. Petals 5, much longer than the lappets of the calyx, obovate into a tongue evidently with attenuated base, at the back veined. Stamens 5, erect, little shorter than petals. Filaments subulate. Anthers 5, somewhat more greatly ovate, pale yellow, with cells sub-opposite. Style small, extending from the center of a platter-shaped disc situated in the bottom of the calyx, margin of the disc elevated, curled. The next known discovery of R. lobata was that of Capt. Beechy (Hooker and Arnott, 27) at San Francisco and Monterey Bay about 1832. These specimens differed in no respect from the more northern ones discovered by Mr. Douglas. The observations of Nuttall (Torrey and Gray, 58) furthered the botanical knowledge of the plant. He noticed that MCNAIR——A STUDY OF RHUS DIVERSILOBA I29 Mar., 1921] JOJO MOTJaA AVIIG uayuNAYs ‘9s101}UT ayeur se yonur UMOPp pdAIND ON “WUE I “UU Z SF sv ud013 Ye podeys-onsuo fT “uu I ¢ ‘lulu V “1 c—s: IE ‘wo z—-S"I Si ‘duny fasniqg "uD V—£ UOAPUIPONXO I, SRYM ud0Is JYSIT Jo[oD MOTJIA AQIIG uayunsys ‘9st1o1}UT “Wu Got 5 ayeul se yonuw se UMOP pdAIND JON "uu G°T "tuuL © S ud015 YICC] podeys-ansuo {, “ULW S ‘tutu ¢ "uIU OI-—S WWD Sig ZI ‘du ‘9snqqg "md 9—£ (Sronee) a]euL UI se sues ay} qynoqe jJaquinu [e}0], ‘saava] se Aueu se & ud018 WY SIT | DgopIstLanip SRY MY dsIOIU < ud015 JY SIT uMOpP PoAIno ‘;eondy[y “UU Z ‘uu > S Ud018 YIeC podeys-onsuo WT "UU Z S "tu 6 “TUL: Sc (6/£) yea] SB BULLS "UID Z ‘9SOMO]T © ‘S]IXe SIMO] IO ysoysty ur auou ydao -xo ‘JooYys SUTIaMOT uo soAvo] se AueUl SV = uoupuaporxop SYM uddId WYSIT Iojoo MoOTJIA AVI | uayunNAYs ‘9s101}UT "uu S°z ¢ u9018 JY SIT uMOP podAINS ‘yeondiy]| “uu S°T “uu V S u9013 YL podeys-ansuo J, “THe S "Tit 2—V yea] SB ouIeS "UD Z ‘JSOMOT & S]IXB JSIMOT IO ysoysiy ur auou dao -xo ‘JooYys SUTIOMOTT uo saAvat se AUPUI SV uaaIs YS] DQOQLSAA2IP SHY YM a[eua ae | Jayque se 3UCT se Sout} Z Hur 2Z—Se | |icuie serge) Teh. uieie i tememelaciait Sele. cede Sti eh th cin Ayien > TOyJuyy WP ccaeai ys tuise or tnoutecns neteust oye cme not ysuaT yoquny :SUIUIRIS eee Coe eee. eee 10]05) :Ss[e19q fe AG 556. eo) se) bay cer vel yer ellie pe. ceo sea Ye! 16! Iw) re) eh a) (er we 30: ve Ue soquin yj :SdAPa XATeD Meda eee | © 18 Be: ee fe) eee. se) e be: cer ve ee Y43us] J29Ipedg o ‘ar col ee? Lat sen 6 ie ved jello: is ene. -ef uel (0 :SIOMO] HT ei cai ew Wes Se, svt ice) Je; (ee ver co. 6 te ve, ce, et te Axejoy[Aug wa) ee 0! see) col 16; fer ie: cot ie) cer se 4) 'e™ te: ‘or ca, jo yysuaT Japi1o ysIy JO SSIM} 9pIs Jo JoquinN a! vod 0) 4; Noisy @ 1ie.e ce\hes ne: fe, 8: (#0 wos YIM opsuy e 6 © (8 6. 6, si i'e se) fe. 0) 6: (0° 8) 8 os suo il ecicd eae, nan. Stic MAL ae tee yoquinyy I0[05 :sopoueg (YDQ Uosiog) vgopssaarp SHYY puD (Kay UOSIOg) UoLpuaporixoT SnYY fo S4amop] fo uostapdumo) [ee) [Vol. AMERICAN JOURNAL OF BOTANY 130 “eIUIOJI[eD ‘Aajaytog 7 pszDa][O9 Ngo71s4aatp *y JO SusUTIDedS ‘elueA[ASUUdg JO AjISIOAIU() JY} JO Uapseyy [VOIULJOG 9Y} UI paya[oo UopuaporixoT, “y JO suauIdedG Ait UdUI -Ipni are Zz YyoIyM jo ‘¢ podojaaap Ayn UOAPUIPOIIXOT SRY Axe USUI -Ipni aie Z YOTyM Jo °C podoyaAoap Ayn podeys-334 quosqy DQOTISAILIP SNYY ayewa gy AzevuswIpny UOAPUIPOIXO I, SRYM Azeqyuowipny 395] ysry “wut I dAIS -oype ‘sjjad pajutod -dieys YUM ysnoy OTE yysus] jo z/I-L/1 apy ea1e [eJUOZ “1404 Ul ‘wut *bs 00g/I DQOIISAI2IP SNYY elPN ——— = ponulju0j—(ynQ Uos10g) Dgo]isdanip snYY pun (Kal Uuostog) Uuodpuaporxoy snyy fo ssamojy fo uostavduo) ’ Mar., 1921] MCNAIR—A STUDY OF RHUS DIVERSILOBA 131 The sterile and fertile flowers of this species (which is very near R. Toxicodendron) present some notable differences. The sterile, which is figured by Hooker, has rather deeply lobed leaflets, sometimes in fives and larger flowers; in the fertile the leaflets are almost entire or slightly lobed and the flowers considerably smaller, so that it might readily be taken for a distinct species. The fruit is white, somewhat pubescent and gibbous. Torrey and Gray (58) summed up the previous knowledge of the plant and renamed it Rhus diversiloba, the name by which it is now more commonly known. . The difference between R. diversiloba and R. Toxicodendron is so small that their proper classification forms a bone of contention between botanists. Those botanists who believe in innumerable species are in favor of their separation, while the more conservative are opposed to it. Greene (21) considers R. .diversiloba ‘‘a peculiar type of Toxicodendron belonging ex- clusively to the Pacific Coast.’ Engler (15) believes dzverstloba a sub- species of Toxicodendron. ‘The only botanical ground for the separation of the two into different species is a slight difference in the shape of their leaflets (Gray, 17). A three years’ study of Rhus diversiloba and a recent study of R. Toxicodendron in Pennsylvania and Maryland for a year have enabled me to make a personal comparison of the two plants. The tracings of the outlines of mature leaves of both plants (figs. 1, 2) and a tabular account BRAG Ot Fic. 1. Tracings of mature leaves of Rhus Toxicodendron (Reduced 63% x). 132 AMERICAN JOURNAL OF BOTANY [Vol. 8 of the flowers of both plants will permit the reader to decide whether or not there is sufficient difference to constitute a separation into species. $8 i ESD x 5 se ae: Fic. 2. Tracings of mature leaves of Rhus diversiloba (Reduced 634 X). GEOGRAPHICAL DISTRIBUTION OF R. DIVERSILOBA The distribution of poison oak includes Lower California north of latitude 29° (Brandegee, 6), Santa Barbara and Santa Catalina Islands (Brandegee, 7), California, Oregon, Washington, Vancouver Island, and British Colum- bia. The region inhabited by the plant has been approximately defined by citations from botanical literature, sources of herbarium specimens, and places where birds were found that had poison oak seeds in their stomachs. From these data, the territory inhabited by poison oak embraces the Sonoran and lower transition life zones, and excludes the desert and central valley regions of California together with the upper transition and boreal zones. The inhabited area has an altitude varying from sea level to 6,000 feet above sea level. Hall’s (24) observations in the Yosemite Valley make it an inhabitant of the Hetch Hetchy and the low foothills with a maximum alti- tude of about 4,000 feet. In southern California he noticed it in the San Jacinto Mountains along the North Fork of the San Jacinto river at an altitude of approximately 3,000 feet. I have found it in Cold Water Canyon on the southwest side of Mt. San Antonio in the San Gabriel Range Mar., 1921] MCNAIR—A STUDY OF RHUS DIVERSILOBA 133 in southern California at an altitude of 4,500 feet. The lowest and highest regions of California are therefore free from poison oak. From rainfall data compiled by the United States Government the plant requires an annual rainfall of at least ten inches. Geographical Distribution of Rhus diversiloba (Poison Oak) According to Literature aud Correspondence Date Author Location HOO OUSIAS 4 6. bs a. ean Common on the outskirts of woods in dry-soils in northwest America. Plentiful at Fort Vancouver. 1832 Hooker and Arnott ..San Francisco and Monterey Bay. 1845 Lindley and Lyon ...Common everywhere in California. An inhabitant of Santa Catalina Island. re55 INewberry,......... Common throughout northern California; more rare in the Klamath Basin. Monome VOrmey 6s. oi... eke Plains and mountains near San Gabriel; Martinez. 1876 Brewer and Watson From southern California to British Columbia; in California most abundant in the Coast Range. ne7ovvheeler..>=........Common on the Pacific Coast. MOS OmmGLeCMe ook aa.) On the north side of Santa Cruz Island. ESSomebyOMms.-...,.......An inhabitant of Santa Catalina Island. HOSGmmbrancdecee = ........ In Lower California, very abundant about El Rosario. 1890 Brandegee..........Common on San Miguel, Santa Rosa, Santa Cruz, and Santa Catalina Island. ESOBM COVINE Soke oo: Found at several points on rocky hillsides in the foothill belt of the western slopes of the Sierra Nevada. ROOM AGEN a2 ho. ot os Copious in the Coast Range hills, preferring cool northward slopes and the banks of streams; absent from the more ele- vated portions of the Sierra. WOO Me GIAY <4 so ets oko Common throughout California, north to the borders of Washington. 1897 Jepson.............Fort Bragg to Sherwood Valley in redwood belt. HOGS, meowell, oe. os In forests and rocky hillsides, British Columbia to California. 1898 Jepson.............Mitchell Canyon, Mount Diablo. 1899 Jepson.............Crane Creek and Rosewood, Stiver’s ranch. TOOO epson... 2... ...€edar Creek. HO OMe EPSOM <4. cea ole Smith Mountains, Palomar, 6,000 feet. I90I Jepson.............Kaweah Range, north slopes and moist places. 1901 Jepson.............St. Helena, climbing redwood. TOOK MC EPSOM.) 34). ..Pine Canyon, Mt. Diablo, shrubs 6 feet high. 1902 Chestnut...........Common in valleys and on hillsides everywhere throughout Mendocino County. 1902 Jepson.........;...Givin Mine, Calaveras County, 1,100 feet altitude. Shrubs 12 and 13 feet high. Schoolhouse Creek, Ft. Bragg, Cahto. Redstone Park. Hawley School, Willits. Fort Seward, Ranch Ridge, Hum- boldt County, 3,000 feet. Abundant. Idolwild (near Camp Grant), Humboldt County. Climbs up redwood trunks 90-100 feet. Hawkins Bar (Dyer’s ranch). Usal to Cottonaby Creek. 134 FOO3 Greene syne 1903, Jepson... ... 1906 Piper 1907. Jepson 1909 Parsons TOO, Wi COSOnbee. TOVO, Jepsonwe 1. IQII 1911 Abrams POdds CAV Ui eee se I9II Jepson 1912 Hall LOL2 Jepson as... « LOG 1s bial 1916 Sanborn 1916 Crawford TOLOme aris 4. TOIO: Jepson = a... Alhambra Arroyo Valley Creek Berkeley Berryessa EPSOM: warty. Spares Decne a AMERICAN JOURNAL OF BOTANY [Vol. 8 A peculiar type of Toxzcodendron belonging exclusively to the Pacific Coast. ak ates Along fences in Vacaville. Vacaville to Twin Sister’s Peak. Vacaville Rock Peak. ..Washington to California in the coast regions. Humid transition zone. ...Cudahay Trail to Dutch Henry’s on Klamath River, 2,500- 4,000 feet, below fir zone, only along river. .. Throughout California, save in the high Sierras. Pepperwood, Humboldt County, in redwood trees. Hetch Hetchy (3,700 feet). te Syte. Belden, 2,000 (approximate) feet. Half Moon Bay. Arroyo Seco, Monterey County, altitude 100-500 feet. Napa Range near Atlas Peak. .. Frequent in chaparral belt throughout southern California. Common throughout the foothill region up to a height of at least 3,000 feet above sea level. _.Found in Coast Range and foothills of the Sierra Nevada, widely distributed and often abundant. ..Confined to lower end of Yosemite Valley, and to the Hetch Hetchy and low foothills. eee aaa Nelson, middle Tule River, altitude 4,760 feet. Saratoga, Santa Clara County, altitude 600 feet. ..Merced Canyon, not rare to 3,200 feet altitude. West of Wawona at 4,500 feet. Small. In Santa Cruz Mountains, dominant shrub Alma to summit, especially in redwood belt. Abundant in the hills about Eugene, and all through the western part of Oregon. In mountain canyons and valleys from sea level to about 7,000-7,500 feet elevation. Very common throughout Pomona Valley and all the valley regions between San Bernardino and the coast. Grows to some extent in damp soil in San Bernardino Valley, altitude 1,000 feet, and abundantly in the canyons of the southern slope of the San Bernardino Mountains up to 3,500 feet altitude at least. Does not grow in the higher mountains nor in either the Mojave or the Colorado Desert. Dunsmuir to Castle Rock Station along Sacramento River, 2,200 feet altitude. Locations where Birds which had eaten Rhus diversiloba Fruit were Collected Camp Meeker Chico, Tehama County Claremont Cull Canyon Guadalupe CALIFORNIA Pinte Mountains Rio Dell, 15 miles southwest San Antonio Canyon San Fernando San Jose Santa Clara County Santa Monica Mountains Santa Rosa Sierra Morena, 6 miles Mar., 1921] MCNAIR —A STUDY OF RHUS DIVERSILOBA 135 Haywards Simol Mt. Diablo Smith Creek Northwest of Pasadena South of Palo Alto Palo Alto Stewart’s Ranch Pasadena Voltas Payne P. O., Tehama County Watsonville Petrolia OREGON Ashland Coquille Bybee’s Bridge _Los Gatos WASHINGTON Garfield County (The above list was communicated to me by Dr. E. W. Nelson, Acting Chief, Biological Survey, U. S. Department of Agriculture.) Distribution of Rhus diversiloba According to Sources of Herbarium Specimens CALIFORNIA Alpine, San Diego County, Mearns 4o19. Alum Rock Springs, vicinity, Santa Clara County. Big Chico Creek Canyon, Butte County, altitude 250 feet, A. A. Heller. Black Mountain, Santa Clara County, Elmer 4785.° Blochman’s Ranch, Mariposa County, Alice Eastwood. Cantara, Siskiyou County, Alice Eastwood. Carmel, Monterey County. Casitas Pass, Ventura County, altitude 1000 feet. Chico, near, Palmer 2060. Clayton, Contra Costa County, Brewer 1068. Cow Creek Mts., Shasta County. Folsom. Forest Ranch, 1897, Mrs. R. M. Austin 1801. Fort Tejon, vicinity, Kern County. Foster Park, Ventura County, Alice Eastwood. Gasquet, Del Norte County, Alice Eastwood. Havilah, Grinnell 362. Healdsburg, Sonoma County. Kings Canyon, Lieber Mts., Los Angeles County. Little Chico Creek, Austin 749. Los Gatos foothills, 1904, A. A. Heller 7327. Los Tronus Creek, San Mateo County. Mendocino, near, H. E. Brown 750. Monterey, Bailey. Monterey, Botta in Mus. Herb., Paris. Mount Diablo, Alice Eastwood. Mutair Flat, Ventura County. New York Falls, Amador County, altitude 2000 feet. North Fork and vicinity, Griffiths 4531. Oroville, Table Mt., 8 miles north of, Butte County. Pacific Grove. Petrified Forest, Alice Eastwood. Palo Alto, foothills near, Santa Clara County. Pasadena, Jones 3206. Red Reed Canyon, Ventura County. 136 AMERICAN JOURNAL OF BOTANY [Vol. 8 Round Valley, Mendocino County. St. Helena, vicinity. San Clemente Island, Mearns 4048. San Diego, canyons near, J. J. Hernleer. San Francisco, Lone Mt. Cemetery. San Franciquito Creek, San Mateo County. : San Jacinto Mts., shade along north fork of San Jacinto River, altitude 3000 feet, H. M. Hall. Santa Barbara, Elmer 3940. Santa Clara County, J. J. Hernleer. Santa Cruz, Marcus E. Jones. Santa Cruz Island, Stanford Herbarium. Santa Cruz Mountains, 1903, N. L. Gardner. Sausalito Hills, Kellogg and Herford 332. Savage Hill, Amador County, altitude 2200 feet, Hansen 53. Shasta River, near mouth, Siskiyou County. Stanford University, Santa Clara County, Rutter 163. Stanford foothills, Baker’s collection no. 547. Sulphur Banks, Lake County. Sulphur Mountain Spring, Sulphur Mountains, Abrams and McGregor 46. Sulphur Mt. Spring, Ventura County. Table Mt., Butte County, altitude 600 feet. Tamalpais. Tassajara Hot Springs, Elmer 3178. Topsajoin (?) Hot Springs, Monterey Co. Vaca Valley, Solano County. OREGON Ashland, Stanford Herbarium. Azalea Creek, Mears. Cascade Mts., Moseley in Kew Herbarium. Columbia River, between 46° and 49° latitude. Columbia River, rocky places, Ethel I. Sanborn. Coos Bay, House 4746. Corvallis, 1898, Moses Craig. Dallas. Deschutes River, 1885, Thomas Howell. Jackson County, along Walker Creek, altitude 3300 feet, Applegate 2339. Lyall in Kew Herbarium. 8 Portland, 1885, L. E. Henderson. Portland, open hillsides, Ethel I. Sanborn. Portland, Walpole 44 and 8. Portland, rocky hillsides, 1903, J. Lunell. Rocky Butte, Multnomah County, Ethel I. Sanborn. Umpqua Divide, head of Elk Creek, altitude 1500 feet, Leiberg 4190. Umpqua River, east fork of North Fork, 6-10 miles east of Peel, altitude 1500 feet, Apple- gate 2700. Umpqua-Rogue River Divide, dry hillsides, Ethel I. Sanborn. Wasco County, 1896, L. F. Henderson. WASHINGTON American Lake, south of Tacoma, F. S. Hall. Orchard Point, Kitsap County, F. L. Pickett. Seattle, F. L. Pickett. Mar., 1921] MCNAIR—A STUDY OF RHUS DIVERSILOBA 137; Seattle, F. S. Hall. Tacoma, seashore and bluffs, F. L. Pickett. Union City, F. L. Pickett. VANCOUVER ISLAND Vancouver Island, Tolmic, Douglas in Kew Herbarium. Victoria, near Swan Lake and on the west side of Seanich Arm, J. R. Anderson. THE ORIGIN AND OCCURRENCE OF THE POISON The freshly exuded resinous sap of R. diversiloba has long been known to be capable of producing dermatitis when applied to the skin. With this in mind, investigations were carried out to see whether the poisonous portions . of the plant are limited to those portions that contain the resin canals. Microscopical examination of the staminate flower shows four resin ducts in the receptacle and pedicel, one in each petal, but no resin ducts more than half-way up the basal filaments of the stamens. Realizing the absence of resin canals in the anthers, it was thought perhaps the pollen might be non-toxic. (See Pl. II, D.) The pollen was collected by shaking the flowers over a glass funnel to the stem of which a test tube was attached. This pollen was found to be non-poisonous when rubbed into the skin of an individual sensitive to the poison. An alccholic extract of the pollen was non-toxic, nor did the pollen or the alcoholic solution assume a dark brown color when treated for five minutes with potassium hydroxide as does the poison. It is concluded, therefore, that the pollen is incapable of producing dermatitis. Similar non-toxic results have been obtained with the pollen of Rhus vernicifera by Inui (30), with that of R. Vernix by Warren (59), and with that of R. Toxicodendron by Rost and Gilg (50). C. Schwalbe (51) considered the poison of R. diversiloba to be excreted from glandular hairs on the surface of the plant. As the resin canals are not connected with the epidermis or with the trichomes, it was considered that these like the stamens might also be non-toxic. Two different forms of trichomes have been noticed on the plant, similar morphologically to those found by Mobius (42) on R. vernicifera and by Rost and Gilg (50) on R. Toxicodendron; namely, a unicellular or multicellular needle-shaped hair, and a multicellular club-shaped hair (Pl. Hl, F). Morphologically the club-shaped hairs seem to be glandular; first, the upper multicellular portion is sharply marked off from the basal portion, which resembles a stalk; second, the upper portion has thinner walls than the basal portion; third, they are found mostly on the young, rapidly growing organs of the plant, especially on the floral region and the leaves, less on the green stems, and hardly at all on the woody portions. When the green stem, pedicel, or main ribs of the leaf, which are covered with trichomes, are rubbed on skin sensitive to the poison, no dermatitis results. Care must be taken, however, that the epidermis of the plant is not broken severely enough to cause the resinous sap to exude. The fresh green leaves were placed in a finger bowl and soaked in room 138 | AMERICAN JOURNAL OF BOTANY [Vol. 8 temperature in 95 percent alcohol for ten minutes. The leaves had been examined first under a hand lens to make sure that through possible injury no resinous sap was on the surface. When placed in the finger bowl the sap was prevented from running down the pedicel from the cut end into the alcohol. The leaves when taken out of the alcohol had lost their gloss. The pale yellowish alcoholic solution remaining was concentrated by boiling in an open beaker. It was found to be non-toxic. It was not darkened by potassium hydroxide nor did it respond to other chemical tests for the poison. These results indicate that neither the plant trichomes nor their exudate are poisonous. The cork cells of the older stem were likewise found to be non-toxic either when the branch was rubbed on the skin or when an alcoholic solution was made of scrapings from the outermost cork cells of a branch as thick as a man’s wrist. As no resin ducts were seen on a microscopical examination of the pith ofa one-year-old stem of the poison oak nor in the woody stem, experiments were undertaken to determine their toxicity. The bark was carefully removed from the pith, a clean knife being used to shave off the outermost portions of the pith. The pith was then cut up in small portions and extracted in a Soxhlet apparatus with hot 95 percent alcohol. This alcoholic solution when concentrated gave neither a physiological test for the poison nor any. of the chemical tests. A similar experiment carried on with the woody xylem gave correspond- ing results. SUMMARY 1. The fresh sap emulsion is the only part of the plant capable of pro- ducing dermatitis. 2. Those portions of the plant that do not contain the resin canals do not normally have this kind of toxic effect. 3. The non-toxic portions are the anthers, pollen, xylem, epidermis, cork cells, and trichomes. LIABILITY TO POISONING RELATIVE TO GROWTH OF PLANT At just what stage in its life the Rhus diversiloba plant first contains its irritant poison has not yet been determined. After the plant has become several years old, however, all parts except the xylem, cork cells, epidermis, and trichomes are toxic. Although many persons know the sap of the stems and leaves to be poisonous, yet there are some who do not consider the sap of the roots toxic. Such is the case, however, as is attested by persons who have come in accidental contact with the broken roots of the plant while digging out other botanical specimens (Kunze, 35; Stirling, 55). The poisonous action of the roots might be expected from their structure, as they have numerous vertical resin canals encircling the xylem (PI. II, C). Mar., 1921] MCNAIR——A STUDY OF RHUS DIVERSILOBA 139 The resinous sap of the stems and roots retains its toxicity probably without much variation in amount or in the degree of virulency throughout . the year. This is evinced not only by citations from literature (White, 61; Beringer, 3) and by statistics (table 1), but also by experiments conducted with the sap by the writer. The virulency (the liability to cause poisoning) of the plant varies with the different seasons of the year in accordance with the stage of growth of the leaves, stems, and flowers. When the first leaves of the plant are unfolding in the spring they are very turgescent and easily injured. Analo- gously, the growing stems are less resistant than the mature stems. The mature leaves of the plant are not nearly as easily injured. Of the mature leaves, those that grow in the shade have a weaker structure than those which develop in the sun. From this fact one might expect the shade leaves to be less resistant to injury. The amount of poison in the plant varies with the capacity of its resin canals. Of this variation in amount, that of the stems and leaves is most commonly effective in the index of virulency. The leaf area undoubtedly makes its greatest increase in spring between the time when the leaves begin to unfold and the time when the flowers open. From this latter time the leaf area of the plant is nearly constant until the leaves begin to fallin autumn. Four weeks are generally required for the full development of a leaf. The flower and leaf buds begin to expand simultaneously, but the leaves soon expand more rapidly and reach maturity before the flowers open (PI. II, B). Thestaminate and pistillate plants begin to bloom at about the same time. At Berkeley, California, but few of the flowers were open April 4, 1915. The next spring the plants near the Greek Theatre, at Berkeley, bloomed mostly between March 22 and May 1. Either the amount or the virulence of the poison in the autumn leaves is less than that of the normal mature leaves. Of the autumnal leaves the red are less toxic than the yellow, and when the leaves have finally withered and fallen they are non-toxic (McNair, 40). Inui (30) has noticed that the amount of secretion of R. vernicifera is influenced by the conditions of light and atmospheric humidity. In potted plants the secretion lessened when carbon assimilation was hindered. Similarly, secretion was greater in damp than in dry air. This secretion therefore seems to bear a relation to transpiration and hence to turgor. As the degree of turgor varies indirectly with the amount of transpiration, other factors being equal, secretion would be least when transpiration is greatest. Turgor, too, is a necessary accompaniment of growth; flaccid tissues do not grow larger. If those influences which affect R. vernicifera ‘have a similar action on R. diversiloba, then secretion, and consequently the plant conditions for poisoning, would be greatest during that time of the year when the growth of the plant is most active and the tissues least resistant, namely, in the spring. Obviously enough, when the plant is in 140 AMERICAN JOURNAL OF BOTANY [Vol. 8 full leaf and when growth has diminished, its resistance to injury will be greater and the liability of poisoning by it less. The malignancy of the plant may also be considered in relation to its visibility or conspicuity. From this standpoint the virulency of the plant would be indirectly proportional to its conspicuity. The plant is least conspicuous when it is not in leaf, more conspicuous in the spring when the leaves and flowers are expanding, still more easily recognized when in full leaf, and most likely to be observed when its leaves have assumed their bright autumnal colors. The virulency of the plant may be summarized according to its toxic portions, the virulency of the resinous sap, the turgescence and ease of fracture of its parts, the conditions of light and atmospheric humidity, and its conspicuity. The liability of poisoning, then, by R. diversiloba tissues decreases as follows: immature leaves and flower parts (except anthers and . pollen), mature leaves, green stems, young roots, woody stems, and woody roots. According to the amount of poison in the plant, however, virulency would be greatest during the period of full leaf. This factor gives way before the far greater balance of factors just mentioned. This theoretical consideration of the liability to Rhus poisoning from a botanical point of view has its counterpart in clinical statistics. The latter lend analogous evidence to the conclusion that spring has the greatest number of cases, that a sudden decrease in cases occurs during the time of the autumn tints, and the least number of cases takes place during the dormancy of the plant from November until February. It should be noted, also, that in 1915 the greatest number of cases among Berkeley students (table 1) was in March, previous to the opening of flowers about April 4, and that in 1916 the maximum number of cases occurred during February previous to the maximum flowering period (March 22 to May 1) of that year. This evidence contradicts the belief prevalent among many people that the plant is most malignant during its flowering period. Some of the opinions expressed in medical literature in regard to malignancy are as follows: most cases usually in spring (Busey, 9); most noxious at the period of efflorescence (Yandell, 63); most cases in summer and autumn (Park, 46); greatest activity during the flowering season, from May to October (Philadelphia; Blackwood, 5); most virulent July 1 to September I (Hubbard, 29); worst in December when buds are coming out and in May when leaves fall (California; Baldwin, 2); poisonous at all seasons of the year (Philadelphia; Beringer, 3); most poisonous when in bloom (Davis, 1897); cases most prevalent at the season of the year when the foliage is beginning to show itself (Cantrell, 10); most poisonous when in bloom (Harriman, 1898); especially virulent just as the buds come in the spring (Thudichum, 56); more active (in New York) during the summer months, the last two months of spring, and the two first months of autumn (Hadden, 22), MCNAIR——A STUDY OF RHUS DIVERSILOBA I4!I Mar., 1921] eg 9 Oo oO | I I EL’ 9 9 O 0) | 6) O | O eA She) Or oT 601 OQ'II OZ “Ol “giz 61 On te oz QI | Sas cows 9 Z'OI Q Qe c v v Wit vo lv O61 LOT 22° CY SOU sey Ls 9° 1 veel Qc Sart Le rome) “6 v6 «60g Lc 0 SVL LE eon Si ty 4 i 2 cle 601 QI o Si Cz b's 6 Tet cio LV eae 98 gg If 91 VWI gl 7 ivi Cc V3 aot Ce v Go 61°89 UGe ail Odea oLl Co se o's 8 6°8 VI 1 2b ae da ol Vv 66 V6 Vo. 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Cece 1°66 LSI O fe) rer 61 |° VI=C161 Q'S el°9 Z99t gtgt ¢[°0019'°96 Ive LIF LS. 2 ti ¢ 6O1r 6 6c Et fo'v IZQz >°66 vzi rash 6 CVI QI |° €1-z161 Asewisyuy puaesua ye poyeory, fAapayiog 3 JUs013g | JaquinN | quadI19g Iaquin yusd19g iaquinNn DQO7IS AIA 22 v] 3 ~ —_ = = = ——— =. Anns Close: radar Ney ee Ln jenuuy [BIO], ea (quo! Jo 2¥q) AvP judy soltewmuing jenuuy MCNAIR——A STUDY OF RHUS DIVERSILOBA Mar., 1927 | (penutjuo)) “I ATAV 144 AMERICAN JOURNAL OF BOTANY [Vol. 8 The number of cases of dermatitis from R. diversiloba is influenced, not only by the condition of the plant, but also by those conditions which tend to make individuals come in contact with it or with substances coated with its poisonous sap. The clinical statistics, therefore, do not constitute a true index of the virulency of the plant, since the total number of persons exposed is not known. The number of exposed persons in all probability | varies at different times of the year, according to the weather conditions, state of other vegetation, individual freedom, etc. Many attractive wild flowers are found in the same locality with R. diverstloba shrubs, such as Clarkias, Godetias, Collinsias, Brodiaeas, and larkspurs (Parsons, 47). John Muir (43) “oftentimes found a curious twining lily (Stropholieion Californicum) climbing its branches.’ The desire to gather spring wild flowers is often greater than the fear of Rhus diversiloba. Circumstances thus combine to bring a person in contact with the plant at the time when it is capable of doing the most harm. LITERATURE CITED 1. Abrams, LeRoy. Flora of Los Angeles and vicinity. IgII. 2. Baldwin, A. E. A case of poisoning by Rhus Toxicondendron. Pacific Medical and Surgical Journal 30: 509, 643. 1887. 3. Beringer, G. M. Rhus poisoning. Amer. Jour. Pharm. 68: 18-20. 1896. 4. Bigelow, J. American medical botany 3: 20. Boston, 1820. 5. Blackwood, W. R. D. Some thoughts on Rhus poisoning. Abst. New Rem. 9: 278. 1886. Abst. New Rem. 10:8. 1881. Abst. Amer. Philos. Assoc. Proc. 29: 230. 1881. Philadelphia Med. Times 10: 618. 1880. 6. Brandegee, T. S. A collection of plants from Baja California. Proc. Calif. Academy OliSciences,"ser..2,, 2: 110-5 el eoG: : 7, ————_——. The plants of Santa Catalina Island. Zoe 1: 110, 134. 1890, 8. Brewer, W. H., Watson, S., and Gray, A. Botany (of California), ed. 2,1: 110. Cam- bridge, 1876. . g. Busey, S. C. Poisoning by the Rhus Toxicodendron. Amer. Jour. Med. Sci. 17: 436-442. 1873. 10. Cantrell, J. A. Relative value of certain drugs in the treatment of ivy poisoning. New Eng. Med. Monthly 17: 270-272. 1808. 11, ————————.._The Philadelphia Polyclinic 7: 20. 1898. 12. Chestnut, V. K. Plants used by Indians of Mendocino County, California. Contrib. WS: Nat. Herb. 72 364. ~ 1962, 13. Coville, F. V. Botany of Death Valley expedition. Contrib. U. S. Nat. Herb. 4: ST 1693. 14. Elliott, S. Cited by Bigelow, 4. : 15. Engler, A., and Prantl, K. Die natiirlichen Pflanzenfamilien III, 5: 168. 1897. 16. Gray, A. Botany. In “Geological Survey of California’’ 1: 110. 1826. 17. ——___, s»ynoptical flora 1, fase. $2: 382=383. 551005, 1697, 18. Gray, A., Watson, S., and Robinson, B. L. Synoptical flora of North America, ed. 2, 1': 383. New York, 1897. 19. ————————. Studies in the botany of California and parts adjacent. Bull. Cal. Acad! Sci.233203-518S0—-18387: 20. Greene, E. L. Manual of botany, p. 73. San Francisco, 1894. 21, ————————.._ Leaflets of botanical observation and criticism 1: 119. Washington, D. C., 1903-1906. Mar. ; 1921] MCNAIR—A STUDY OF RHUS DIVERSILOBA T45 22. 23 24. 25. 26. 27. 25: 29. 30. atc 22. 33- 34. i MOnGOn: 1772. Hadden, A. Poison ivy or Rhus Toxicodendron. Med. Review of Reviews 12: 764. 1906. Hall, H. M. A botanical survey of San Jacinto Mountains. Univ. Cal. Pub. Bot. E: 1-140. 1902. A Yosemite flora, p. I5I. I9Q12. Hooker, J. D., and Jackson, B.D. Index Kewensis. Part IV, p. 714. Oxford, 1895. Hooker, W. J. Flora boreali americana 1: 126, 127. London, 1831. ‘Hooker, W. J., and Arnott, J.A. W. The botany of Captain Beechey’s voyage, part 3, peta7. London, 1832. Howell, T. A flora of northwest America, p. 119. Portland, Ore., 1898. Ditto, vol. I, p. 119. Portland, Ore., 1903. Hubbard, S. Rhus Toxicodendron. Peoria Med. Monthly 6: 323. 1885-1886. Inui, T. Ueber den Gummiharz-Gang des Lackbaumes und seiner verwandten Arten (Abstr). Bot. Centralbl.;83:.352. 1900. Jepson, W. L. A school flora for the Pacific Coast, p. 38. New York, 1902. ——————. AA flora of western.and middle California, p. 250. Berkeley, 1901. 2d edition, p. 249. Berkeley, Ig1I. Kalm, P. ‘Travels into North America (Eng. transl.), ed. 2, 1: 53, 60-64, 139; 2: 20. Travels into North America, etc. (Eng. transl. by John Reinhold Forster). In Pinkerton, J. ‘‘A general collection of the best and most interesting b] voyages and travels’’ 13: 402-403, 434. 1812. . Kunze, R. E. Poison Rhus. Med. Tribune 5: 111-120. 1883. . Lindley, J. Rhus diversiloba. Various leaved poison oak. Edwards’ Bot. Reg. n. ser. 18: 38. 1845. . Linnaeus, C. Hortus cliffortianus, p. 110. Amsterdam, 1737. Species plantarum, pp. 265, 266. Stockholm, 1778. . Lyon, W.S. The flora of our southwestern Archipelago II. Bot. Gaz. 2: 333. 1886. . McNair, J. B. The oxidase of Rhus diversiloba. Jour. Infect. Dis. 20: 485-498. LOL 7: . Michaux, A. Cited by Bigelow, 4. . Mobius, M.A. Der Japanische Lackbaum, Rhus vernicifera D.C. Abhandl. Sencken- berg. Naturforsch. Ges. 20: 201-247. Frankfort, 1899. . Muir, J. My first summer in the Sierra, pp. 34, 35. New York, IgII. . Newberry, J.S. Botanical report. In U.S. War Dept., Reports of explorations .. . from the Mississippi river to the Pacific ocean, 1854-1855.6: 69. Washington, 1857. . Nuttall, T. Cited by Bigelow, 4. . Park, R. Dermatitis venenata; or, Rhus Toxicodendron and its action. Archives of Dermatology 5: 227-234. 1879. . Parsons, Mary E. The wild flowers of California; their names, haunts and habits, p. 8 (corrected edition). San Francisco, 1907. . Piper,C. V. Flora of the state of Washington. Contrib. U.S. Nat. Herb. 9: 384. 1906. . Pursh, F. T. Cited by Bigelow, 4. . Rost, E., and Gilg, E. Der Giftsumach, Rhus Toxicodendron L., und seine Giftwirkun- gen. Ber. Deutsch. Pharm. Ges. 22: 296-358. 1912. . Schwalbe, C. On the active principle of Rhus diversiloba (poison oak). Med. Record 63: 855. 1903. . Schwalbe, K. Die giftigen Arten der Familie Rhus: R. diversiloba, R. Toxicodendron und R. venenata. Muiinchener Medic. Wochenschr. 49: 1616. 1902. . Smith, J. The general history of the Bermudas, now called the Summer Isles; from their beginning in the year of our Lord 1593, to this present 1624, etc. In Book V of ‘‘ General History of Virginia, New England and Summer Isles,’’ by Captain John Smith. In Pinkerton, J. ‘‘A general collection of the best and most interesting voyages and travels’’ 13: 172. 1812. 146 AMERICAN JOURNAL OF BOTANY [Vol. 8 54. —————-—.. (1609) The Historye of the Bermudaes or Summer Islands, p.. 2. Edited by J. H. Lefroy. Publ. by the Hakluyt Society. London, 1882. 55. Stirling, E. C. An eruption of the skin caused by the poison ivy. Australas. Med. Gaz. 33: 355-359. 1913. 56. Thudichum, C. L. The alkaloidal clinic 10: 829. 1903. 57. Torrey, J. Pacific Railroad report. Route near 35th parallel explored by Whipple in 1853-54. No. 4, Report of the botany of the expedition. 1856. 58. Torrey, J.. and Gray, A. Flora of North America 1: 217-218, 681. New York and London, 1839 and 1843. 59. Warren, L. E. Some observations on the pollen of poison sumach. Amer. Jour. Pharm. 85: 545-549. 1913. 60. Wheeler, G. M. Report of the United States Geological Subse west of the 1ooth meridian, p. 42. Washington, 1878. 61. White, J. C. On the action of Rhus venenata and Rhus Toxicodendron. New Yor Med. Jour. 17: 225-249. ‘ 1873. 62. —————... ‘Dermatitis venenata. Boston, 1887. 63. Yandell, L. P. Poison oak eruption. Louisville Med. News 2: 32. 1876. 64. Young, A. H. Monotropa uniflora. Bot. Gaz. 3: 37, 38. 1878. DESCRIPTION OF PLATE II The material selected for the sections was fixed in a chrom-acetic fixative (I percent by weight chromic acid in water, 0.5 percent glacial acetic acid by weight). Suitable pieces were then hardened by placing in alcohol of different concentrations in series of 6 percent, I2 percent, 25 percent, 50 percent, and 75 percent, then in xylol and paraffine and finally in paraffine. Sections were then cut on the microtome in series, fastened to clean slides with egg albumen, stained with safranin and Delafield’s haemotoxylin, and finally washed in absolute alcohol, in xylol, and mounted in balsam. By this treatment lignified and suberized walls were stained red and cellulose walls violet. The photomicrographs were made with an electric arc as the source of light. The amount of magnification was calculated by the aid of a ruled slide on the microscope stage and of a rule on the ground glass focusing screen, using the same lenses as were used in the exposure. The photomicrographs have been reduced 2% times. A. Transverse section through a lateral leaf rib, showing a resin duct. The resin duct is 0.0053 mm. in diameter. The shortest distance to the bast ring which surrounds the resin duct is 0.0056 mm. XX 168.4. B. Young shoots showing simultaneous expansion of leaves and flower panicles (reduced one fifth). C. Transverse section through a woody root. X 7.5. D. Transverse section through a staminate flower near the apex showing ave calyx leaves with resin ducts, five petals with resin ducts, five anthers showing absence of resin ducts and presence of pollen, and the non-fertile ovule. X 40.9. E. Transverse section through a green stem, showing epidermis with its trichomes, collenchyma, cortical parenchyma, pericycle with sclerenchyma cells or bast fibers, and thin-walled pericycle parenchyma. X 40.9. F. Leaf epidermis with attached club-shaped trichomes. (Size of trichomes 0.071 X 0.0027 mm.) << 45307 G. Transverse sections of petiole. The largest resin duct has a diameter of 0.01 mm.; the smallest is 0.0044 mm. in diameter. The largest pith cells are larger than the smallest resin ducts. It is 0.02 mm. from the corner of the petiole section to the bast ring which surrounds the nearest resin duct. X 33.8. AMERICAN JOURNAL OF BOTANY. VoLumeE VIII, PLate Il. McNair: TOXICITY OF RHUS DIVERSILOBA. 7 ey hae EFRPECYT_OF SALT PROPORTIONS AND CONCENTRATION ON THE GROWTH OF ASPERGILLUS NIGER! C. M. HAENSELER (Received for publication September 21, 1920) INTRODUCTION A great deal of work has been done in recent years on the various prob- lems of plant nutrition, and especially on the physiological balance of nutrient solutions and on the salt requirements of plants. Although these questions have received some attention ever since the introduction of water cultures by Sachs and Knop, they had never been carried to such logical completeness as was done by Tottingham (9) in his work on the study of the effect upon plant growth of varying the total concentration and salt proportions in Knop’s solution. In 1915 Shive (6), using Tottingham’s systematic methods, made similar exhaustive studies of a nutrient solution containing only three salts and a trace of iron. This three-salt solution, when properly balanced, gave a better yield than Tottingham’s best four- salt solution and has the further advantage of being the simplest satis- factory water culture that had been used up to that time. Since the introduction of Shive’s three-salt solution, which, on account of its relative simplicity, is especially suitable for studies in plant nutrition, it has been used extensively by Shive (6), McCall (4), Hibbard (3), and others for various physiological studies with green plants. Fundamental problems of nutrition have also been studied extensively in certain fungi and especially in Aspergillus niger. The greater number of these studies have dealt with carbon or nitrogen assimilation and with the toxic or stimulative action of various substances. The role played by total salt concentration and by the salt proportions of the nutrient medium has apparently received very little attention. There has been no systematic study made upon any fungus which corresponds to the nutrition studies made with green plants by Tottingham, Shive, and others. The results of these authors have proven of such fundamental importance in nutrition studies in higher plants that it was thought advisable to test the adapta- bility of their methods in similar studies upon fungi. In order to make this test, a series of experiments were planned to study the effect upon Aspergillus niger of varying the total salt concentration and salt proportions in a simple nutrient solution. 1 Paper No. 21 of the Technical Series, New Jersey Agricultural Experiment Stations, Department of Plant Physiology. 147 148 AMERICAN JOURNAL OF BOTANY [Vol. 8 The methods used by Shive (6) in his studies on the physiological balance of nutrient solutions for higher plants were used as a basis for this work. A number of radical modifications in these methods, however, were necessary on account of the physiological differences between the green plants and fungi. The most essential modification was in the composition of the nu- trient solution itself. In Shive’s solution three mineral salts and a trace of iron constitute the nutrient material, and for green plants these salts contain all the necessary elements for growth. For fungi, however, a source of energy must be supplied in addition to the mineral salts, and in this work it was . supplied in the form of cane sugar. A second necessary modification was in the treatment of the solution. In water-culture work with higher plants it is neither essential nor practicable always to use sterile solutions. With the fungi, on the other hand, it is essential that the medium be sterilized thoroughly before inoculating with the desired organism. The sterilization process as used for these experiments, however, probably caused very little alteration of the medium and may be overlooked as a factor influencing the medium itself. This work was outlined primarily to see whether the methods which Shive and others have used with such marked success in nutrition studies with green plants, would prove equally useful for similar studies with fungi. This work was carried out under the direction of Dr. J. W. Shive in the Laboratory of Plant Physiology at the New Jersey Agricultural Experiment Station. EXPERIMENTAL METHODS The experiments herein discussed consist of two groups of cultures. Group I, comprising series 1,2, and 3, contains Ca(NOs)e as the source of nitrogen and will be referred to as the Ca(NO3). group... Group 2, comprising series 4 and 5, contains NaNO; as the source of ue and will be desig- nated the NaNO; group. Aspergillus niger was grown in 250 cc. Jena glass Erlenmeyer flasks on 50 cc. of a liquid medium containing three nutrient salts, cane sugar, and a trace of iron. Each culture throughout five series contained the same amount of iron and sugar, the iron being present in amounts equivalent to 0.01 gram of ferrous sulphate per liter, and the sugar in amounts equiva- lent to 38.97 grams per liter of nutrient solution. In series 1, the nutrient salts, KH:PO;, Ca(NOs3)2, and MgSOu., are present in quantities sufficient to give a total calculated osmotic concentra- tion value of 0.5 atmospheres. The series consists of 36 cultures, each dif- fering from all the other cultures of the series in the proportions of the three nutrient salts. The 36 cultures represent all the possible proportions or combinations obtainable by varying the partial concentration of each of the salts by increments of one tenth of the total concentration. A full account of this method of studying the effects of salt proportions is given by Shive (6) and need not be further discussed here. Mar., 1921] HAENSELER— GROWTH OF ASPERGILLUS NIGER 149 Series 2 and 3 differ from series I only in total salt concentration, the total ‘calculated osmotic concentration values in these series being 2.1 and 4.2 atmospheres respectively. Series 4 and 5 have the same total concentration values as series 2 and 3 respectively, but in the former NaNO; is used instead of Ca(NO3)s._ For purposes of comparison the standard nutrient solution proposed by Thom (8) was here used as a check in each series (except series 4), always with the same total osmotic salt concentration as that employed for the series in which it occurred. In Tables I and 2 are given the actual TABLE I. Partial volume-molecular concentration of the salts employed in the culture solutions of the series in group 1 Series 1 (0.5 Atm.) Series 2 (2.1 Atm.) Series 3 (4.2 Atm.) Culture No. | KHePOu Ca(NQs3)o MgsO, KHoPOx, | Ca(NOs)2 Mg3O. KHoPO, Ca(NO3)2' MgSO, RiC1......| .OIII | .00070 | .00941 | .00444 | .00302 | .04157 | .00888 | .00625 | .08648 RE C2 rs. | .OOIII | .OOI4I | .00824 | .00444 | .00604 | .03637 | .00888 | .01250 | .07567 R1C3......] .OOLII | .0O2I1I | .00706 | .00444 | .00906 | .03118 | .00888 | .01875 | .06486 150 OO Maa .OOITI | .00282 | .00588 | .00444 | .01208 | .02598 | .00888 | .02500 | .05405 RONG Bit Ff. .OOIII | .00352 | .00471 | .00444 | .OI1510 | .02078 | .00888 | .03125 | .04234 IRERCG Ay sr. h | .OOIII | .00423 | .00353 | .00444 | .OI18II | .01559 | .00888 | .03749 | .03243 RUC7......| -OOI1II | .00493 | .00235 | .00444 | .02113 | .01039 | .00888 | .04374 | .02162 IRC Sirs | .OOIII | .00563 | .0O118 | .00444 | .02415 | .00520 | .00888 | .04999 | .o1081 R2C1......| .00222 | .00070 | .00824 | .00888 | .00302 | .03637 | .01776 | .00625 | .07567 RIC 2 ei cat .00222 | .OOI4I | .00706 | .00888 | .00604 | .03118 | .01776 | .01250 | .06486 R262. sae... 00222 | .0021I | .00588 | .00888 | .00906 | 02598 | .01776 | .01875 | .05405 R2C4......| .00222 | .00282 | .00471 | .00888 | .01208 | .02078 | .01776 | .02500 | .04324 R2C5......| .00222 | .00352 | .00353 | .00888 | .O1510 | .01559 | .01776 | .03125 | .03243 RECO noe | .00222 | .00423 | .00235 | .00888 | .OI8II | .01039 | .01776 | .03749 | .02162 R2C7......| .00222 | .00493 | .0O118 | .00888 | .02113 | .00520 | .01776 | .04374 | .O1081 RZC1.... ..|) 00333 | 00070 | .00706 | .01332" .00302 | .03118 | .02664 | .00625 | .06486 IRGC 27S. .... .00333 | .OOI14I .00588 | .01332 | .00604 | .02598 | .02664 | .01250 | .05405 IRB © Beet. .00333 | .0O2II | .0047I | .01332 | .00906 | .02078 | .02664 | .01875 | .04324 RoC... .00333 | .00282 | .00353 | .01332 | .01208 | .01559 | .02664 | .02500 | .03243 RBC erin... | .00333 | .00352 | .00235 | .01332 | .OI510 | .01039 | .02664 | .03125 | .02162 Ra CGre ee... | .00333 | 00423 | .OOII8 | .01332 | .OI8II | .00520 | .02664 | .03749 | .O1081 RACT Ma... | .00444 | .00070 | .00588 | .01776 | .00302 | .02598 | .03552 | .00625 | .05405 IRAC2 Ta he | .00444 | .OOI4I | .00471 | .01776 | .00604 | .02078 | .03552 | .O1250 | .04324 R4C3......| .00444 | .002II | .00353 | .01776 | .00906 | .01559 | .03552 | .01875 | .03243 ill OUND genet | 00444 | .00282 | .00235 | .01776 | .01208 | .01039 | .03552 | .02500 | .02162 R4C5......| .00444 | .00352 | .00118 | .01776 | .O1510 | .00520 | .03552 | .03125 | .O1081 RECT a... o.. .00555 | 00070 | .00471 | .02220 | .00302 | .02078 | .04440 | .00625 | .04324 R5C2......! .00555 | .0O141 | .00353 | .02220 | .00604 | .01559 | .04440 | .01250 | .03243 R5C3......| .00555 | .0021I | .00235 | .02220 | .00906 | .01039 | .04440 | .01875 | .02162 R5C4......| .00555 | .00282 | .00118 | .02220 | .01208 | .00520 | .04440 | .02500 | .o1081 INGGr or cos. .00666 | .00070 .00353 | .02664 | .00302 | .01559 | .05328 | .00625 | .03243 INOG@ 2 sic. .00666 | .OOI4I | .00235 | .02664 | .00604 | .01039 | .05328 | .01250 | .02162 R6C3......| .00666 | .0021I | .0O118 | .02664 | .00906 | .00520 | .05328 | .01875 | .o1081 R7Ci. | 00777 | .00070 | .00235 | .02108 | 00302 | 01029 | .06216 | .00625 | .o2162 R7C2......| .00777 | .OOI4I | .0O118 | .03108 | .00604 | .00520 | .06216 | .01250 | .o1081 |itok Gl aeeneeals a .00888 | .00070 | .OOI18 | .03552 | .00302 | .00520 | .07104 | .00625 | .o1081 partial volume-molecular concentrations of each of the solutions of the five series used in this study. The culture numbers refer to the positions which the cultures occupy on the triangular diagram? graphically representing the series with respect to the osmotic proportions of the three salts employed. 2 For a description of this triangular diagrammatic scheme see Shive (6), McCall (4), Hibbard (3). I50 AMERICAN JOURNAL OF BOTANY [Vol. 8 TABLE 2. Partial volume-molecular concentration of the salts employed in the cultures of the series In group 2 Series 4 (2.1 Atm.) | Series 5 (4.2 Atm.) Culture No. j l x KH,PO, | NaNO, | MzSO, | KHyPO, NaNO; MgSO, RIG eee .00444 | .00414 | .04157 | .00888 .00860 | .08648 121 Osea, OA on, ae .00444 | .00829 .03637. | .00888 .O1721 .07567 Rat Oca ee ey er eee 00444 | .01243 03118 | .00888 .02581 .06486 IR Cer re ae .00444 | .01658 02598 | .00888 | .03442 .05405 RST 5. sco ae hd -00444- | 302072 702078 | —t00888 .04302) (in FOng2ut REO Oy te oe | 004.44 .02487 .01559 .00888 05162) > 2@s243 Ran 7a ee his, © or | .00444 .02901 .01039 .00888 .06023.' | :o2T62 RS (CS om sicm aes. .| 00444 | .03316 | .00502 .00888 .06883 | .OI1O8I RE Glew ee ce 26 00888 | .o04TA >| 102637, .01776 .00860 .07567 RO © Orr otis. | .00888 | .00829 .03118 .01776 .O1721 .06486 | Ray Bie) Sede ee ee | .00888 .01243 .02598 .01776 .02581 .05405 ROC ee ek | 00888 .01658 .02078 01770. | 03442 .04324 ROMER ye te fae .00888 502072 .O1559 .01776 .04302 .03243 RECOM ame Ni ens.4 .00888 702487 |= ,01039 01776 | | [05062 .02162 PROMG 7 hen Poe we .00888 .02901 00502 | 01776: || 700028 .O108I RG Cee ee oe ie S013 32eamia O04 04 03118 “| .02664 | _0086e .06486 RA Coe Bere Fe 5013325) 00829" | .025938 .02664 .O1721 .05405 RA OB is ic. ues 01332 01248 =| 02078. || [02664 .02581 .04324 2 Ore t.ho 8 lp 2201332 .01658 .O1559 .02664 .03442 .03243 RAG Segre oie et na. | 2-Or2a22 .02072 .01039 .02664 | 104302 .02162 IRROG et ce he orcs .01332 .02487 .00502 | .02664 .05162 .O1081 RANG Tere er kee as .01776 .00414 .02598 .03552 .00860 .05405 RAC 2 er Rta. | 301770 00829 | - .02078 .03552 .O1721 .043 24 RAC 20a ese only O10 01243 .01559 .03552 .02581 .03243 RAC TR chy oe. oS | 20107 7,6 01658 | .01039 .03552 .03442 .02162 RAK CIA ih Reagan 101776 .02072 | .00502 | .03552 .04302 .O1081 RS Clune ares chai ctac|) 2202220 00414 | .02078 .04440 .00860 .04324 IG GOs raslen she on [02220 .00829 | .01559 04440 | .O1721 .03243 ING Gare Ace .02220 .01243 | .01039 | .04440 02581" |||) 2.02762 RG OA re ke 3; .02220 .01658 | .00502 .04440 .03442 .O1081 1B<( 61 6,1 ome yak ee em .02664 00414 | .O1559 .05328 .00860 .03243 RAG OT a er ee .02664 .00829 | .01039 .05328 .O1721 .02162 IOC eee hice teres .02664 01243 | .00502 .05328 .02581 -O1081 RR Clee ire .03108 00414 | .01039 .06216 .00860 .02162 RY. C2 te eh ixs .03108 — .00829 .00502 .06216 .O1721 .O1081 [toh © haan ee eae .03552 00414 .00502 .07104 .00860 .O108I For each series a stock solution of each of the salts was made up with distilled water to such a concentration that 1 cc. of the stock in 50 cc. of the culture solution produced in any culture one tenth of its total required salt concentration. Thus, the culture in which the three salts KH»2POu, © Ca(NO3)o, and MgSO, produced, respectively, 1/10, 6/10, and 3/10 of the total concentration, received of these stocks I cc., 6cc., and 3 cc., in the order given. Each culture, therefore, received a total of 10 cc. of stock solutions, leaving 40 cc. to be supplied by other means. The sugar stock was made. up to 5/4 the concentration desired in the finished culture; thus, 40 cc. of this stock contained the proper amount of sugar for the 50 cc. culture. Ferrous sulphate equivalent to 0.01 gram FeSQ, per liter of finished nutrient was added directly to the sugar stock. The stock solutions were weighed and sterilized separately at 100° C. for one hour on three consecutive days. The loss by evaporation was Mar., 1921] HAENSELER— GROWTH OF ASPERGILLUS NIGER I5I1 replaced by sterile distilled water. After cooling, the solutions were forced by air pressure through glass tubes into burettes and the proper amount of each was carefully measured into the culture flask. All glassware and stop- pers were sterilized either in an autoclave or in a hot-air chamber immediately before using. In order to reduce the chances of outside contamination, the cultures were made up and inoculated in a dust-proof inoculation chamber. A number of uninoculated check cultures proved that they had been made under perfectly sterile conditions. It would have been more convenient to make up the cultures before sterilization, but this method was not considered advisable on account of the decomposition of the salts which occurs while heating a mixed salt solu- tion. Some of the solutions employed, especially those with a relatively large amount of KH2PQOu,, were quite unstable at the boiling point, and even at 30° C. a few of the more concentrated cultures contained a slight pre- cipitate. The stock solutions were, therefore, sterilized separately and the culture solutions were prepared from these under aseptic conditions. Baker’s analyzed salts were used throughout. On account of the un- certainty as to the exact amount of water of crystallization in the calcium nitrate, this salt was freed from its water of crystallization by carefully fusing and dehydrating at a final temperature of 150° C. A fine grade of commercial granulated sugar was used. The cultures were well shaken and inoculated heavily with spores from a one-week-old agar culture of Aspergillus niger. Enough spores were transferred in the inoculation to produce a very thin, but visible, uniform film on the surface of the culture. This heavy inoculation was found neces- sary to geta uniform growth. Light inoculations were apt to give “‘islands”’ of growth instead of a uniform film over the entire surface. tries, except Mexico, Cuba, Porto Rico, Panama Canal Zone, Republic of . Panama, ‘Hawaii, Philippine. Islands, Guam, Samoan Islands, and Shangha Re a to Canada, 20’cents a volume on annual subscriptions; to all other yes _ tries: in the Postal Union, 40 cents: a. volume on enya, subscriptions. 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VIII APRIL, 1921 No. 4 THE MORPHOLOGY AND ANATOMY OF RHUS DIVERSILOBA James B. McNair (Received for publication November 2, 1920) Rhus diversiloba T. & G. may be either a low deciduous shrub or a high climbing vine as it readily adapts itself to local conditions. In sunny ex- posures it most frequently occurs as a low shrub 3 to 4 feet high. One instance is known, however, of a tree-like plant, 14 feet high with a diameter at its base of 6 inches. In shady locations it more frequently assumes a vine-like form and by means of its aerial rootlets ascends the trunks of trees to a height of I5 to 20 feet. ; The new wood has pale grayish bark, is light in weight, brittle, and contains much pith. The ends of the young shoots and the petioles are usually red-brown in color. In the early spring groups of plants may be distinguished at a distance before fully leafing out by this red-brown of the young stems and leaves. The branches gradually become woody with the accumulation of successive annual rings, the bark thickens, roughens, and becomes the abode of many lichens and mosses. The bark on the old wood is brown-gray, furrowed lengthwise, has horizontal rows of wart-like lenti- cels, and on dead limbs peels off laterally, not spirally. The large branches have but few small lateral branches. In its shrubby form no difference can be observed between the height of the male and that of the female plants, nor can any noticeable difference be determined in the angles which the branches subtend to the trunk. The leaflets, too, generally have similar shapes on both plants. Flower panicles do not develop on the ends of young shoots, but form on the sides. The apical bud makes a growth of 4 to 5 centimeters per year. It requires about four weeks for the full development of a leaf. The flower and leaf buds begin to expand simultaneously, but the leaves soon expand more rapidly and consequently some of the leaves reach maturity before the flowers open. The expanding leaves are very tender and turgid, and sap flows quickly out of injured stem or leaf areas. These leaves are arranged alternately on the stem and have a phyllotaxy of 8/21. The leaf scars are triangular in outline. The swelling where the leaf is attached may have the function, by its growth, of turning the leaf to a better exposure. [The Journal for March (8: 127-178) was issued April 3, 1921.] 179 180 AMERICAN JOURNAL OF BOTANY [Vol. 8 THE LEAF The glossy, dark green leaflets are deepest in color when in the sun, pale underneath, generally 3 in number, although sometimes 5, orbicular to ovate or oblong-ovate, undulate or plane, entire or variously lobed, seg- mented or toothed, I to 4 inches long. ‘The 5-leaflet variety, according to Brandegee (3) is quite common on the Santa Barbara Islands. Leaves having 5 leaflets are also found on plants which have a majority of the 3-leaflet kind. Leaflets are singularly variable in size, outline, and seg- mentation, even on the same plant. This fact constitutes one of the most remarkable features of the plant and is the principal basis for its differen- tiation from Rhus Toxicodendron L. Leaf tracings (21) made from mature leaves collected by the writer at Berkeley, California, on September 27, 1916, were taken from plants within a radius of 100 feet, all of which were enjoying the same soil and exposure and had no apparent cause for such marked differences in leaf shape. Leaves in the sun differ from those in the shade, not only as regards color but also in several structural details. The young leaves are covered with hairs, which dry out and fall off as the leaves become fully matured. These hairs are apparently more frequent on leaves exposed to the sun than on those in the shade. Other differences will be described later. In autumn, as in spring and summer, the plant is singularly attractive, its leaves turning many shades of red, yellow, and brown. This color change may be induced in mature leaves in midsummer by certain insect injuries, by attacks of fungi, or by an interference with the flow of sap caused by twisting the stem. There is no apparent difference between the leaves of male and female plants in this respect. Some plants, however, particularly those in the shade, may have all their leaves yellow. Con- versely, red leaves seem to be peculiar to plants of sunny exposure, although there are many exceptions; far more frequently the leaves are mixtures of all three colors. The oldest leaves often assume autumnal tints first. The petiole in transverse section (21) has in form nearly a semi-circle for its dorsal.side and a small concave arc as a ventral surface. Under the epidermis lie two or three layers of collenchyma cells. The vascular bundles, of which there are more than 18, are arranged in a flattened circle parallel to the outer surface of the petiole. The pith consists of large, thin-walled cells with very small triangular intercellular spaces. The vascular bundles are separated from each other by broad medullary rays. Large resin ducts are found in the phloem. ‘The primary cortex is bordered internally by a starch sheath. The cells of the xylem have thick and lignified walls. The pith is enclosed by bast fibers and xylem and takes up the largest part of the section.. There are no resin ducts in the pith or in the primary cortex. The leaf in transverse section exhibits palisade parenchyma occupying about one third of the entire thickness of the mesophyl (PI. III, fig. 4). The spongy parenchyma occupies about five layers of cells. Cells with Apr., 1921] MCNAIR — RHUS DIVERSILOBA 181 crystal clusters, presumably of calcium oxalate, occur in the palisade parenchyma. The cells of the lower epidermis are similar to those of the upper epidermis but smaller; stomata are very frequent and apparently absent from the ridges. The leaves wilt very easily. It is hardly possible to bring a cut branch from the field to the laboratory without observing wilting. There are two kinds of trichomes on the leaves, multicellular club-shaped, and unicellular or multicellular bristle-shaped (21). The thick-walled bristle hairs occur mainly on the lower side on the ridges, large and small, of the leaf, although they are found also in fewer numbers on the upper side in corresponding places. The club-shaped trichomes, on the other hand, are found mostly between the ridges of the leaves. These two different forms of trichomes are similar to those found by Mobius (22) on Rhus vernicifera L. and by Rost and Gilg (24) on Rhus Toxicodendron L. Morphologically the club-shaped hairs seem to be glandular: first, because the upper multicellular portion is sharply marked off from the basal portion, which resembles a stalk; second, the upper portion has thinner walls than the basal portion; third, they are found mostly on the young, rapidly growing organs of the plant, especially the floral region and the leaves, less on the green stem, and hardly at all on the woody portion. Schwalbe (25, 26) considered the poison of Rhus diversiloba to be excreted from glandular hairs on the surface of the plant. That such is not the case can be shown by the two following experiments: . - (1) When the green stem, pedicel, or main ribs of the leaf, which are covered with trichomes, are rubbed on sensitive skin, no dermatitis results. Care must be taken, however, that the epidermis of the plant is not broken severely enough to cause the resinous sap to exude. (2) The fresh green leaves were placed in a finger bowl and soaked at room temperature in 95 percent alcohol for 10 minutes. The leaves had been examined first under a hand lens to make sure that through possible injury no resinous sap was on the surface. When placed in the finger bowl the sap was prevented from running down the pedicel from the cut end into the alcohol. The leaves when taken out of the alcohol had lost their gloss. The pale yellowish alcoholic solution remaining was concentrated by boiling in an open beaker. It was found tobe non-toxic. It was not darkened by potassium hydroxide, nor did it respond to other chemical tests for the poison. These results indicate that neither the plant trichomes nor their exudate is poisonous. The club-shaped hairs are so minute as to be hardly discernible by the naked eye. They have a length of 0.071 mm. and a maximum breadth of 0.0027 mm. Under the microscope they exhibit a clear, unicellular basal portion as an outgrowth of an epidermal cell, above which are the numerous cells that go to make up the main portion of the hair. The cells of the main portion when viewed transversely radiate from a longitudinal central axis. The apex terminates in a single cell, and the entire main portion of the hair 182 AMERICAN JOURNAL OF BOTANY [Vol. 8 is enclosed in a thin-walled sac. The hairs appear to be of two types, which apparently correspond to different stages in development: a densely granular and a sparsely granular form. This difference in granular density is interesting. In animal glands it has long been noticed that when a serous gland has been quiescent for several hours the secretory cells are granular throughout, and the outlines of the cells are only faintly marked as clear lines bounding the granular areas. When the gland secretes, many of the granules disappear and after prolonged secretion very few granules are left; 1.e., during secretion the granules normally contained by the cells are in some way or other used up, probably to form a part of the secretion. Al- though the diminution of zymogen granules is a normal occurrence in the secretion of the salivary, infra-orbital, lachrymal, mucous, and pancreatic glands, yet in the case of the mammary glands the opposite is true, v72., that granules begin to form with the commencement of secretion and do not occur during rest. In the mammary gland, the active growth of proto- plasm, the formation of granules from the protoplasm, and the discharge of these granules in the secretion appear to go on at one and the same time. Investigation of the club-shaped hairs of Rhus diversiloba has not as yet revealed a positively glandular nature, and consequently a relation between differences in their granulation can not be definitely connected with secretion. From a morphological standpoint, however, as above pointed out, the club-shaped hairs seem to be glandular. Club-shaped hairs from leaves gathered in the morning pelone. sunrise and from those secured in the heat of the day could not be differentiated. Hairs from rapidly growing leaves could not be distinguished from those > of old leaves or stems. Hairs from leaves grown in sunny exposures ex- hibited no differences, although they were present in greater number than on leaves continuously in the shade. THE STEM A transverse section of a green stem of Rhus diversiloba shows, beginning at the outside, the following tissues (PI. III, fig. 5): epidermis, with its trichomes and stomata; collenchyma; cortical parenchyma; pericycle, with bast fibers and thin-walled pericycle parenchyma; phloem, with resin ducts; cambium; xylem; medullary ray; pith. As the stem increases in diameter (fig. 3) the cortex develops a phellogen. The continuous activity of the phellogen results in an increasing thickness of the sheet of cork. The chloroplast-containing tissue beneath the cork layer maintains connection with the air by means of lenticels which have replaced the stomata. As may be anticipated, the dead cork cells are non-poisonous, 7.e., they do not cause dermatitis when rubbed on the skin of a susceptible individual and therefore do not constitute a means of transference for the poison. No resin ducts have been found in the pith of this plant. Engler (5), Apr., 1921] MCNAIR — RHUS DIVERSILOBA 183 studying Rhus Toxicodendron L., and Inui (10), studying R. Toxicodendron var. radicans, were unable to discover resin ducts in the pith. Jadin (11) cited 18 species of the genus which are provided with permanent pith resin ducts, and 9 species which do not have them. At the periphery of the pith the small outer cells acquire a thick wall and become sclerenchymatous. These thick-walled cells may assist the inner large-celled and the outer small-celled pith to maintain a circular outline. A semi-circular row of bast fibers lies external to the primary phloem and serves mechanically to protect the phloem with its resin ducts from external injury. In the phloem of the second year, new resin ducts appear. These lie neither in radial nor in tangential rows, but are so arranged as to be very nearly equidistant. The first appear in the secondary phloem between two primary resin ducts, and more are formed in a corresponding manner. It must not be forgotten, however, that the formation of resin ducts does not occur in a regular manner. New bast fibers do not appear to be formed in the pericycle. The epidermis has been almost wholly lost in the second year and is replaced by cork. The histology of the pith, wood, and bark of the older stems will be treated individually. The pith cells are polygonal and lie close together; they are generally wider than high, so that their vertical measurement is the smallest. In the specimens examined, the pith cells contained for the most part no particular substance; starch was found sparingly, and tannin sacs appeared as narrow, elongated cells. Tannin sacs, according to Engler (5), appear abundantly in the pith of the Anacardiaceae and in all species of Rhus which he investi- gated. Pith tannin sacs are not necessarily characteristic of toxic species of Rhus, as Mobius (22) was unable to find them in R. vernicifera L. The bulk of the wood consists of simple pitted wood fibers. In trans- verse section they are bordered at right angles, and are assembled in rows. The narrower and thicker-walled cells of the fall wood contain starch; the wider and thinner-walled ones of spring wood appear empty. The pits of the tracheal vessels are exclusively simple with circular or elliptical outlines. The walls are relatively thick. The structure of the vessel wall, where it is in contact with wood parenchyma, is characteristic. In these places simple pits of large size are found chiefly on the vessel wall, and, side by side with them, either transitional or true bordered pits, but no separate bordered pits-were noticed. The elliptical pits are transverse to the longitudinal axis of the vessel and parallel to one another, so that they remind one of scalariform perforations. The medullary rays are, as a rule, uniseriate; sometimes, however, they are biseriate. In tangential longitudinal section they are from three to eighteen cells high; radially their cells are joined together as are the stones 184 AMERICAN JOURNAL OF BOTANY [Vol. 8 in a wall of plane ashlar masonry. The walls of their cells are only moder- ately thickened, and their lumina are often filled with starch. The medul- lary rays are most noticeable in the lower part of the stem and in the roots. One small root had five primary medullary rays. The difference between fall and spring wood rests partly on the dis- similarity of the wood fiber cells and partly on that of the vessels. The first tracheals of spring are larger, thicker-walled, and stretched somewhat radially, while those toward the outer border of the annual ring are flattened to smaller, thicker-walled, and radial rings. The vessels in the spring wood are wider and more numerous, in the fall wood narrower and scarcer, as shown in Plate III, figure 3. The breadth of the annual rings varies. The inner wood is colored yellow or yellow-brown. A great deal of this coloring matter can be extracted with hot alcohol. This extract behaves similarly to the extract of the related species Rhus Cotinus L. (Cotinus cog- gygria Scop.) inthe following treatment: an orange-yellow solution in water was made bright yellow by hydrochloric acid, yellow-red by ammonia, orange N. with alum and sodium carbonate solution, and brown N. by calcium chloride solution. Such a behavior by no means proves that the solutes from the wood of Rhus diversiloba are identical with those from R. vernictfera, although such may actually be the case. The coloring matter is naturally attached to the membrane of the wood cells, which appear golden yellow under the microscope and assume a brown color with caustic potash. Be- sides the yellow crystals, the wood cavities contain a reddish amorphous resinous substance which is likewise soluble in 95 percent alcohol. The primary cortex contains sclerosed parenchyma. | The structure of the pericycle is characteristic. It contains many bast fibers, which, in transverse section, have the form of arcs whose convex sides are on the exterior and whose inner concave surfaces surround in each case a single, usually large resin duct (PI. IT], fig. 5). The resin canals in the later-formed portions of the bark have a lumen and are arranged more or less regularly in concentric circles as heretofore described. The old resin canals appear to be obliterated through a kind of tylotic growth. On one transverse section through the bark of an old stem which has already thrown off the primary covering there are many resin canals differing in form, outline, and dimensions. The innermost are open and nearly circular, but usually more strictly oval in shape, stretched tangentially, and of larger circumference than the outer ones. ‘The outer- most, particularly in old stem parts, are entirely or almost entirely obliter- erated through the luxuriant growth of intruding contiguous tissue. It is possible to observe at different heights of the same resin canal different states of development so that in one place it may still be open and in another closed. This occurrence of tyloses in the secretory ducts is similar to that described by Mobius (22) in Rhus vernicifera L., by Leblois in Brucea fer- ruginea, and by Conwentz in the intercellular canals of other plants. Apr., 1921] MCNAIR —— RHUS DIVERSILOBA 185 The secondary medullary rays, as already noted, are usually constituted of one row of cells. Where biseriate rays are found, it is sometimes noticed that they split apart tangentially while they remain intact radially. From this it would seem that adjacent cells of the two columns of the medullary ray are only loosely united, whereas those cells which constitute a radial row are more firmly attached. Besides what has already been said regarding the phloem, it should be added that the sieve tubes and their companion cells extend tangentially and build approximately alternating bands with the layers of phloem paren- chyma cells, as in the stem section of Aristolochia Sipho (27). The phloem apparently has but little starch, which is found deposited chiefly in the medullary rays. These cells also give a distinct reaction for tannin with ferric chloride. MorRPHOLOGY AND ANATOMY OF THE ROOT The root system in its ramifications resembles the crown, in that com- paratively few strong branch roots are formed which carry the fine, inter- laced roots. The spread of roots depends largely upon the nature of the soil, and upon the supply of food and water. ‘There is a strong tendency to form long lateral roots, particularly in shallow soil. Propagation by layering is very frequently made use of naturally by the plant to insure its food supply and reproduction. ‘The fine, interlacing rootlets are dark brown in color and are covered with fine root hairs of a lighter color. The apical tips of the rootlets are light yellow or colorless for several millimeters. As in other roots, after the secondary phloem is formed the cambium soon takes on a circular form in section, and behaves in the formation of xylem and phloem exactly as in the stem (PI. III, fig. 2). The wood of the root is less firm than that of the stem; there exist nu- merous large bundles, the fiber cells are less strongly thickened, the medul- lary rays are broader, being indeed commonly composed of two layers of cells. MORPHOLOGY AND ANATOMY OF THE FLOWERS ~ Rhus diversiloba is strictly dioecious, so far as my observations go. The male and female plants begin to bloom at about the same time. At Berkeley, California, but few of the flowers were open April 4, 1915. The next spring the plants near the Greek Theater at Berkeley bloomed mostly between March 22 and May 1. In 1917 at Pasadena I noticed some male plants at the foot of the Mt. Wilson trail in bloom on the fifth of January. February 28, 1917, the plants of both sexes were just starting to bloom in the Arroyo Seco, south of the Colorado Street bridge, Pasadena. In spite of their yellow-green color, the flower panicles are conspicuously displayed as a result of their size and their accumulation on the ends of the twigs. The presence of the staminate flowers is made very noticeable by their fragrant jasmine or hyacinth aroma. The pistillate flowers, on the other hand, 186 AMERICAN JOURNAL OF BOTANY [Vol. 8 have no apparent perfume. At this point it may be well to mention that an aromatic perfume so similar as to be perhaps identical is noticed when the fresh end of a freshly broken branch is smelled, and that this perfume, unlike that of the flowers, is not confined to the male plant, but is observed alsoin the female. Thesimilarity between the perfume of the sap and that of the flower becomes more marked upon purification. The ‘“‘aqueous solution’”’ as made and described in a previous paper (18) contains this more purified sap perfume. The panicles of the male and female flowers are somewhat differentiated as to location and structure. The flowering shoots of the male plant commonly bear as many flower panicles as leaves, in which case neither the highest leaves nor the lowest leaf develop any panicles in their axils. The lowest leaves of the flowering shoot soon fall off and more readily expose the flower panicles to insects, while the highest leaves remain and tend to protect the blossoms from the direct sunlight, wind, and rain. The panicles are 7 cm. long and stand somewhat stiffly upright at a sharp angle to the axil of the attached twig. The longer ones bear about a dozen side twigs of the first order, of which the three lowest ones are about 2 cm. long and in their turn are again richly branched. ‘Toward the tip the side twigs of the first order become shorter and are not further branched. They are formed like a bunch of grapes, and the end of a panicle is likewise visibly terminated by a flower. The same regularity, as nearly as could be determined, appears in the arrange- ment of the side twigs of the first order on the panicle stem as was noticed in the phyllotaxy. Minute woolly hairs appear on the panicles at the blooming time, particularly on the bases of the panicle stem and on those of the side twigs. The flowers are placed singly on stalks from 4 to 7 mm. long, and have a diameter of from 5 to 7 mm. when fully opened. The flowers have 5 calyx leaves, 5 petals, 5 stamens, and one rudimentary ovule; only by way of exception do 6 or 8 stamens occur, and in one flower with 6 stamens 6 petals occurred also. The calyx leaves are tongue-shaped and have broad bases. They are about 2 mm. long and have a dark green color. The petals are long-elliptical in shape, narrowed at the base and at the point, and somewhat pointed in the front. They are 4 mm. long and in the middle about 14% mm. wide. When in bloom the flowers are strongly bent downward. The color of the petals is light green, much lighter than that of the calyx leaves. The stamens are 244 mm. long. The white filaments, which are nearly twice as long as the anthers, shove themselves between the anther halves, which somewhat retreat from each other underneath. The anthers are introrse and are borne on upright but slightly curved filaments. The rudimentary ovary forms a keg-shaped pivot about 1 mm. high, and has 3 discernible stigmas. Between the ovary and the anthers is a disk, which during flowering time glistens with nectar. Apr., 1921] MCNAIR — RHUS DIVERSILOB: stand in front a the petals and are Ree rated by thei Way ne , each lobe is again slightly indented in the middle. The bioand ange / of the disk are somewhat arched toward the top; from this construction a ring-like depression appears in the middle. While just as many inflorescences as leaves are found on the blossom shoots of the male plants, the number of panicles on the female plant is only about one half as great as that of the leaves. The leaves, however, are more numerous on the blossom shoots of the female. The number of leaves fluctuated between 7 and 9 in several investigations of shoots, while the number of panicles ranged between 3 and 5. As on the male plant, neither the lowest nor the highest leaves bear inflorescences in their axils but only the middle ones. The panicles have a length of 3 to6cm. They are not stiffly erect as in the male, but on the contrary only limply placed. The — side twigs of the first order are up to 2.5 cm. in length, and have about as numerous branches, but shorter side twigs of the higher order than those of the male. The entire female panicle has about the same general outline as the male panicle. The anatomical structure of the panicle axis is essen- tially similar to that of the vegetative twig in the first year, and there is no noticeable difference in this respect between the male and the female panicle. Particular structures for tensile strength are not noticed in the axes of the fruit panicles. The stems of the pistillate flowers are not longer than 1 cm. and are often 5 mm.long. The flower itself is smaller than that of the male; its diameter, it is true, measures about 5 mm., but the petals are less curved. The 5 calyx leaves are somewhat similar to those in the staminate flower, but slightly shorter. The 5 petals are spread out flatter and do not have the curled side rims. They are approximately 3 mm. long and 1.5 mm. broad. Five stamens also occur in the pistillate flower; their anthers are of nearly the same length as the fertile ones of the staminate flower, but the filaments are about 1.5 mm. long and therefore much shorter than those of the male. The anthers are shrunken, of a dirty yellow color, with pollen absent, so that the entire pistillate flower and panicles appear darker. As seen from the broad side, the pistil originates in a somewhat compressed, egg-shaped ovary which is extended in a short style. Toward the top the style spreads out into three thick, brownish stigmas which are beset with papilli. The ovary is also to be considered as constituted by three carpels, of which, however, two are rudimentary so that they appear only in the stigmas. Between the stamens and the ovary is the disk, which is similar to that of the staminate flower except that it is narrower because of the greater expansion of the ovary. As far as the growth and the finer structure of the flower are concerned the male and female flowers show a great similarity. If one investigates young inflorescences on which the individual flowers are distinguishable as small buds, it is noticed that each flower stands in the axil of a com- 188 AMERICAN JOURNAL OF BOTANY [Vol. 8 paratively large carrying leaf which somewhat overhangs the flower. The outside of the bract, as well as the stigma and the axil, are covered with upward-bent trichomes. These trichomes are of two forms, one a single long bristle hair and the other a short, apparently glandular hair with a single-celled base and many-celled ovoid head. These hairs are similar to those previously described as found on the leaves and stems. Further developed flowers, which, with their panicles, are 2 mm. long, have a hairy carrying leaf longer than the panicle. The calyx leaves, the petals, and the stamens lie alongside each other like small enlargements and finally the carpels arise as wall-like growths. In this instance, in which one can clearly recognize the construction of the bud, the stamens are egg-shaped and are covered by the short petals and the longer calyx leaves. Finally the disk shows itself between the gynoecium and the androecium. The course of the vascular bundles may very clearly be recognized in the mounted material, as resin ducts contained in the phloem have their contents turned brown. In the calyx leaf, which is formed with a broad base, 5 ribs appear of which the middle one is the strongest and most branched. On the other hand, the petal, which has a small base, has only one short, weak or unbranched rib on each side of the strongly branched midrib. The disk appears in longitudinal section as a wide, somewhat sunken cushion. ‘Toward the bottom its tissue is large-celled; above, on the other hand, it consists of small, closely united, plasma-rich cells, of the sort com- mon to glandular tissues. Many small crystal clusters lie on the border of both tissues and in the upper, small-celled tissue, but are lacking in the lower, large-celled tissue. The epidermis consists of rather small polygonal cells and contains numerous stoma-like apertures whose guard cells are almost always larger than the other epidermal cells. A small space is found under the stoma-like opening. These openings apparently do not serve for gaseous interchange, but for the excretion of a glistening and strongly aromatic fragrant nectar whose existence has already been men- tioned. The development of the stamens in pistillate and staminate flowers is apparently similar to the time of the formation of pollen mother cells. In the pistillate flower no pollen grains are formed, the anthers remain empty, and have a shrunken appearance. The filaments of the pistillate flower remain as short as those of the staminate flower until the flowers open. The stamens naturally develop further in the latter. Pollen formation occurs in the anthers but shows nothing particularly noteworthy. The vascular bundles of the anthers contain no resin ducts, these having ended half-way up the filaments. The anther is also to a certain degree the only organ of the plant which has no resin-like or poisonous sap. It is not sur- prising then that the pollen has no toxic action on the human skin (17). Similar observations have been made by Inui (10) on the pollen of Rhus vernicifera, by Warren (29) on that of R. Vernix, and by Rost and Gilg (24) Apr., 1921] MCNAIR — RHUS DIVERSILOBA 189 on that of R. Toxicodendron. ‘The pollen sacs of R. diversiloba are composed of two coalesced sporangia, as is common in angiosperms. Their dehiscence occurs by a longitudinal slit, developed where the two coalesced sporangia join. According to Edgeworth (4), the pollen of the Anacardiaceae is oval with 3 slits. The fresh pollen grains of Rhus diversiloba are ellipsoidal, about 1/800 sq. mm. in horizontal area, with a width 1/3 to 1/2 the length. The exine is roughened by minute, sharply pointed projections. When the pollen grains are immersed tn N/4 KOH they assume a spherical form with no color change. In the material treated (which had been fixed in alcohol and xylol, stained, and mounted in balsam like the rest of the plant material), the spores assumed spherical shapes or in some instances became rounded tetrahedrons. As is common in entomophilous plants, the pollen has no surfaces so modified as to permit the wind to take hold of it, of the nature of the bladder-like appendages of the pine pollen, etc. Whereas anemophilous pollen has a dry outer covering to prevent large masses of pollen from adhering to the flower and hindering wind transportation, the entomophilous pollen of Rhus diversiloba is surrounded with a sticky sub- stance so as to adhere to the feet and other parts of the insect. In common with other entomophilous flowers, R. diversiloba has perfume-secreting glands heretofore described which may serve to attract insects. The pollen itself being non-toxic and not wind-blown, the aerial transmission of the poison by the agency of pollen is quite out of the question. As in the female flower the stamens develop to a certain advanced stage, so the ovary develops in the male flower to the extent that an almost fully developed ovule is produced. Such development of an ovule in a flower which is functionally purely staminate, borne on a purely male plant, is a phenomenon which has been but rarely observed. Each ovary contains regularly but one ovule. The funiculus becomes curved at its apex, so that the body of the ovule lies against it, and, although the axis of the body is straight, the micropyle is directed towards the surface of origin; thus the funiculus appears as a ridge along one side of the body of the ovule, and the ovule is anatropous and consequently of the form most common among angiosperms. The ovule, in the mature female flower, fills the ovarian cavity. The outer integument, therefore, occupies considerable space. The micropyle is somewhat widely removed from the upper arching of the nucellus. The inner integument is widely tubular and lengthened outwardly over the nucellus, in which the embryo sac is again somewhat pressed back toward the inside so that a wider path is prepared for the pollen tube. The advantage .of an anatropous ovule is apparent when it is remembered that the pollen. tube advances along the wall of the ovary, and that the micropyle is thus brought near the wall. It is not surprising, then, that this plant with its efficient apparatus for fertilization should have large fruit production. Numerous germinating pollen grains are found on the stigmas of open 190 AMERICAN JOURNAL OF BOTANY [Vol. 8 pistillate flowers. The pollen tubes grow inside between the stigma papillae and pass through 4 to 6 cells of which the upper one is longest and thickest. On the stigmas of the staminate flowers such papillae are not formed, so that here no pollen grains are found. The wall of the ovary is penetrated by numerous vascular bundles with resin ducts which continue to the upper end of the pistil where the resin ducts terminate blindly with pointed ends. The development of the fruit, which terminates the life of the plant, has been taken up in another paper (19). LITERATURE CITED 1. Abrams, L. Flora of Los Angeles and vicinity. Stanford University, 1911. 2. Brandegee, T. S. A collection of plants from Baja California: Proc. Cal. Acad. Sci., ' SEh. °2/522 (DAO. 8 Lao. 3: --—. The plants of Santa Catalina Island. Zoe 1: 110, 434° )189a, 4. Edgeworth, M. P. Pollen. London, 1879. 5. Engler, A. Uber die morphologischen Verhaltnisse und die geographische Verbreitung der Gattung Rhus, wie der mit ihr verwandten lebenden und ausgestorbenen Ana- cardiaceen. Bot. Jahrb. Syst. Pflanzengesch. u. Pflanzengeographie 1: 365-426. Tat.iAie S88 E. 6. Hall, H. M. A Yosemite flora, p. 151. San Francisco, 1912. 7. Hooker, W. J. Flora boreali-americana 1: 127. London, 1833. 8. ——, and Arnott,G. A.W. The botany of Captain Beechey’s voyage. Part 3, p. 137- London, 1841. 9. Howell, T. A flora of northwest America 1: 119. Portland, Ore., 1898. 10. Inui, T. Ueber die Gummiharz-Gang des Lackbaumes und seiner verwandten Arten (Abstr.)... Bot. Centralbl: 832/352. 1900, 11. Jadin, F. Observations sur quelques Térébinthacées. Jour. de Bot. 7: 382-390. 1893. 12. ——. Origine des sécréteurs. Thése. Montpellier, 1888. 13. Jepson, W. L. Aflora of western and middle California. 2nd ed., p. 249. Berkeley, SEODT: 14. Leblois, A. Recherches sur l’origine et le développement des canaux sécréteurs et des poches sécrétrices. Ann. Sci. Nat. Bot. VII, 6: 247-330. 1887. 15. Lindley, J. Rhus diversiloba. Various leaved poison oak. Edwards’ Bot. Reg. Ns, Sek. LOswo om ESAS. 16. Lyon, W. S. The flora of our southwestern archipelago. II. Bot. Gaz. 11: 330-336. 1886. 17. McNair, J. B. The transmission of Rhus poison from plant to person. Rhus diver- siloba T. and G. Jour. Infect. Dis. 19: 429-432. 1916. 18. ——. The poisonous principle of poison oak. Jour. Amer. Chem. Soc. 38: 1417-1421. 1916. 19. ——. Fats from Rhus laurina and Rhus diversiloba. Bot. Gaz. 64: 330-336. I917- 20. ——. The oxidase of Rhus diversiloba. Jour. Infect. Dis. 20: 485-498. I917. 21. ——. A study of Rhus diversiloba with special reference to its toxicity. Amer. Jour. Bot.)8: 127-140."; 1025. 22. Mobius, M. A. Der Japanische Lackbaum. Abhandl. Senckenberg. Naturforsch. Ges. 20: 201-247. 1899. 23. Piper, C. V. Flora of-the state of Washington. Contrib. U. S. Nat. Herb. 9: 384. 1906. 24. Rost, E., and Gilg, E. Der Giftsumach, Rhus Toxicodendron L., und seine Giftwir- kungen. Ber Deutsch. Pharm. Ges. 22: 296-358. I912. AMERICAN JOURNAL OF BOTANY. VoLUME VIII, PLATE III. McNair: MorpHOLOGY oF RHUS. . a , i %. han , ‘ t 2 * j ene f : ‘ . ’ ‘a pee my ‘ 1 / VoLuMeE VIII, PLATE IV. AMERICAN JOURNAL OF BOTANY. MORPHOLOGY OF RHUS. ° ° McNair Apr., 1921] MCNAIR — RHUS DIVERSILOBA IOI 25. Schwalbe, C. On the active principle of Rhus diversiloba. Med. Rec. 63: 855. 1903. 26. Schwalbe, K. Die giftigen Arten der Familie Rhus. Mtinch. Med. Wochenschr. 49: I616. 1902. 27. Strasburger, E., and others. A text book of botany. Eng. transl. by H. C. Porter. pewz2. Wondon, 1898: 28. Torrey, J., and Gray, A. A flora of North America 1: 218. New York, 1838. 29. Warren, L. E. Some observations on the pollen of poison sumach. Amer. Jour. Phat. S85: 545-549. 1O13. EXPLANATION OF PLATES PLATE III All figures have been reduced one half in reproduction and rlow show magnifications as follows: figure I, X10; figure 2, X10; figure 3, X23.3; figure 4, 470; figure 5, X91.65. Fic. 1. Transverse section through the same stem as in figure 3. Fic. 2. Transverse section through a woody root. Fic. 3. Transverse section through a stem older than that of figure 5, showing annual rings with their varied formations of spring and fall growth. Fic. 4. Transverse section through mature leaf showing cystolith in palisade paren- chyma. Fic. 5. Transverse section through stem showing cork cambium; tracheal tube (7); pericycle with schlerenchyma cells or bast fibers and thin-walled pericycle parenchyma; phloem with resin duct (R); cambium (C); pith (P). PLATE IV All figures have been reduced one half in reproduction and now show magnifications as follows: figure I, X23.3; figure 2, X 23.3; figure 3, 470; figure 4, 23.3. Fic. 1. Transverse section through a male flower near its base, showing 5 calyx leaves (C) with resin ducts (R), 5 petals (P) with resin ducts (R), 5 stamens (S), and the non- fertile ovule (O). Fic. 2. Transverse section through a female flower near its apex, showing 5 calyx leaves (C) with resin ducts (R), 5 petals (P) with resin ducts, 5 rudimentary anthers with neither pollen nor resin ducts, and the fertile ovule ((Q). Fic. 3. Transverse section through an unripe fruit near the seed, showing numerous crystals. Size of hexagonal crystal, 0.007 X 0.0025 mm. Fic. 4. Transverse section through an unripe fruit showing an abundance of resin ducts (RR). Diameter of largest resin duct, 0.0085 mm. DISTRIBUTION OF THE MALVACEAE IN SOUTHERN. WESTERN, TEXAS! HERBERT C. HANSON (Received for publication December 6, 1920) INTRODUCTION In connection with the eradication of the pink bollworm of cotton the writer was assigned the project of studying the distribution and abundance of the malvaceous plants in various parts of Texas. This work was done under the direction of Dr. W. D. Hunter, in charge of the pink-bollworm eradi- cation work of the Federal Horticultural Board. The reason for making this survey was to determine if the malvaceous plants other than cotton were of importance in relation to the eradication of the pink bollworm (Pectinophora gossypiella Saunders). Throughout the entire survey no indication of the insect was found on any of the malvaceous plants other than cotton. From June to December, 1918, and in June and November, 1919, the extreme southeastern section of the state, embracing Hardin, Jefferson, Liberty, Chambers, Galveston, and parts of Harris, Fort Bend, and Brazoria counties, were thoroughly scouted. In June, 1919, the vicinity of Hearne, 100 miles northwest of Houston, was examined. In June and July, 1919, the areas in the vicinity of Corpus Christi, San Antonio, and Pecos were studied. From January to June, 1919, and in August, 1919, a strip 20 to 80 miles wide on the Texas side of the Rio Grande from the Gulf of Mexico — to New Mexico was scouted. The species of Malvaceae found in the areas studied are discussed under the following life zones: 1. Semi-tropical Gulf Strip of the Lower Austral Zone; 2. Austroriparian Division of the Lower Austral Zone; 3. Lower Sonoran Division of the Lower Austral Zone; and 4. Upper Sonoran Division of the Upper Austral Zone. I. SEMI-TROPICAL GULF STRIP OF THE LOWER AUSTRAL The Semi-tropical Gulf Strip includes that part of Texas along the coast below the 100-foot contour line. This strip, involving approximately the Coast Prairie, is about 60 miles wide. It is divided into the Humid Section, the area east of the 97th meridian; and the Xerophytic Section, the area west of the 97th meridian. 1 Published with the permission of the Secretary of Agriculture. 192 Apr.; 1921] HANSON — MALVACEAE IN TEXAS 193 A. Humid Section Topography: Usually low and flat with occasional low hills. Drainage is poor, so there is much marshy land. Adjacent to the coast is a strip of salt marsh, often several miles wide. Sozl: Black clay chiefly.: Elevation: Most of the area is below 100 feet; Beaumont 29 feet, Galveston 9 feet. Rainfall: The annual mean in the eastern part is over 50 inches and in mee we we ee ee | | | | | | IZA | ! | | ‘oCrosbyton ? \ } } | | ( | Dallas | ] \ . Q by, : { ays | : g IFaso AHS [ 9 S a Sie : . Blanca { Peco ebarstow Ba, $ Y < ry Wes So ‘ me WA | I RATE ICI YP OI atharine Hill. TRELEASE : NORTH AMERICAN PIPERS. AMERICAN JOURNAL OF BOTANY. TRELEASE : NORTH AMERICAN PIPERS. VoLumeE VIII, PLATE VII. Kotharvine Hill AMERICAN JOURNAL OF BOTANY. VoLumME VIII, PLATE VIII. TRELEASE : NORTH AMERICAN PIPERS. = ey, Apr., 1921] 1G. ie: Fic. F1G. 2. TRELEASE — NORTH AMERICAN PIPERS PEATE, Vit Piper Neesianum (Liebmann 18). Piper Mas, the type collection. PLATE VIII Piper abalienatum, the type collection. Piper albicaule, the type collection. 217 MONOCARPY AND PSEUDOMONOCARPY IN THE CYCADEOIDS G. R. WIELAND (Received for publication December 24, 1920) Seed plants which fruit once only in the normal lifetime, and then die down to make way for the new set, are described as monocarpic. The habit is of much interest to students of ecology and succession. As ordinarily used, monocarpy is a rather fixed term which requires some extension, or even redefinition. In a stricter sense, the field of rye is monocarpic; though the usage is far less definite in its common application to plants much longer lived than annuals or biennials. There would, however, be a double reason for not calling a ‘“‘bracken fern’’ monocarpic; since after the fertile frond bears its spores, the root-stock grows on, propa- gation being only secondarily dependent on either the sexual or the asexual generation. And somewhat similarly, any seed plant which, after the wilt- ing down of a fertile stalk of one or more seasons, renews its growth from the root, is falsely monocarpic. It is an inadvertence on the part of botan- ists to cite the “century plant’’ as truly monocarpic, or any plant which does not fixedly persist by reseeding, or solely so persist. There appear to be some basic physiologic distinctions or phases in monocarpy. The Agave (or Yucca filamentosa, which regularly sends up the new buds, with very little germination of seed) does store the materials for the fertile shoot. But in such plants the length of the vegetative phase may be variable, or doubtless even local and individual. Besides, wherever the basal buds grow forward, there is some analogy to the fertile axis of limited growth in trees. The production of flowers or fruits usually means the growth of pedun- cular tracheidal or other structures old in the history of the plant, supplying greatly modified or reduced sporophylls; so that fruit maturity may mean, not merely exhaustion of the parent stem, but a heavy scar or injury to the stem not easily survived. Thus as a plant is modified from age to age, and the gap between fruit and vegetative structures widens, proliferation of the fertile axis may become difficult or quite impossible. And such has in fact become the condition in the vast majority of seed plants with renewed growth from the base of flower or cone. Particularly in the conifers the occasional appearance of proliferate cones may be regarded as reminiscent of a time when there were some normally proliferate types in the ancestral series. Contrariwise, the budding power of stems divested of their foliage or 218 Apr., 1921] WIELAND — MONOCARPY IN THE CYCADEOIDS 219 cut down to the base, is often striking in the extreme, especially in some of the Pandanales because little expected. Renewed growth from stem-base or root may then be a characteristic of singular or extreme types. It may even accompany gigantism. Undoubtedly there is a less known side to monocarpy and the monocarpic tendency; while even in the commoner instances, variations either individual or within the habitat, and details concerning any subsequent root branching, may fail. The classical example is of course the “umbrella palm”’ of Ceylon, Corypha umbraculifera. After a vegetative period of towards forty years the mature height of sixty feet is reached, when in a single short season the stem shoots forty feet higher as a much branched flowering stalk. Mean- while the foliage fronds wilt down and leave this immense terminal inflores- cence with a branch spread of thirty feet, bearing tens of thousands of flowers.! This unparalleled gigantism, however, scarcely exceeds in outright interest a Jamaica plant known as the “‘pride of the mountain,” Spathelia simplex, a member of the rue family. For this plant is nearly related to hardwood perennials like Citrus, and the Rutacee are not so generally monocarpic. The slender trunk, scarred by the fallen leaves, reaches a height of fifty feet, bearing an apical cluster of large, velvety, pinnate leaves three or four feet long. The pinnules are numerous (45-81), sessile, oblong- lanceolate, crenate. At maturity a large terminal panicle of showy purple flowers rises above the leaves, the plant dying down after the ripening of fruit. There is thus afforded an example of a tall and quite typical dicotyl of much the same habit and habitus as the ‘umbrella palm.” The primary form of monocarpy finds a notable variation along ecologic lines in the greatest of the grasses, the bamboos. The life cycle in the bamboos varies greatly. Many species bloom annually; and there are also cases of sporadic flowering, with, however, the final production of flowers on all the culms—ripening of seed then terminating the life of the plant (Arundinaria Simoni). In the bamboos of the south Brazilian prov- inces of Santa Catarina and Rio Grande do Sul, along the borders of the imposing Araucaria brasiliana forest, a thirteen-year monocarpic period occurs. For Bambusa tesselata in cultivation a not very authentic record of a sixty-year cycle is given. Floral periodicity is well attested in the Indian (Ganges) Bambusa arundinacea, reported in flower in the years 1804, 1836, 1868, [1900]. In the Blue mountains of the Island of Jamaica at an altitude of 4,000 to 7,000 feet over a region ten miles long, occurs the ‘‘climbing bamboo”’ (Chusquea 1 All the genus Corypha is perhaps more truly monocarpic than are other so-called instances. Root shoots are not sent up. There are two Ceylon species, and other repre- sentatives in Bengal and farther eastward. In this connection there should be added the interesting case of the Mauritius hemp, Furcraea americana, an agave-like plant. Suckers are not readily produced, but any such flower at the same time as the parent stock. Propa- gation is mainly by bulbils after flowering. 220 AMERICAN JOURNAL OF BOTANY [Vol. 8 abietifolia). Here, extending over more than a year in 1885-6, flowering and dying down were general; this occurred again in 1919, when 98 percent of the plants formed the dead entanglements, and the young plants 18 inches high covered the ground in long stretches (as recently described in this Journal).2, A rough thirty-three-year period is indicated, with only minor or suppressed seasonal factors. These are communal forms, and whoever has seen one of the ee dead pure stand thickets of bamboo in an open hilly region, as in parts of southern Mexico (Oaxaca), where there is a long, dry winter period, under- stands that average length of life and single flowering are complementary, intensified habits. In such plant communities the life of the individual is simple, set, and tense; and as soon as a considerable number of the plants fruit and die down, the light, heat, moisture, and soil conditions of the copse change rapidly. Only the plant behaving in the average way tends to leave its progeny. Even in the tropics there are forms which flower over wide areas on precisely the same day. Then, in the forests of Pegu, certain orchids are seen to blossom as the limbs on which they are seated lose their leaves. Yet there is the remarkable variation in that some trees bloom quite through the year, as the mango, silk cotton, and fig. This has been noted especially in orchids. Casually, this much may be said of the monocarpic habit. Plants ‘seemingly take full advantage of their environment in reaching their many forms. But they grow as they may, and reproduce as they must. The law is simple, complex though its expression may appear. Reproduction is sharply seasonal in the severe environment, and of more varied phase in the soft climate where growth factors find their favorable mean. In the cool temperate zones, the period of flowering varies for weeks as moisture and heat vary with the unusual season. In the tropics the utmost variations are found. Many of the woody plants are not dependent on their foliage in blossoming in or after the dry season. Such is the habit of various mag- nolias of the subtropics, and further north, where the burst of flowers is put forth earlier than the young leaves easily grow. These are tender and in our climate whipped by chill winds of early spring. Yet other trees are, as Schimper notes, evergreen in their flowerless youth, losing their foliage as they flower and fruit. Though such (Schizolobium giganteum of Java) suggest a certain advance toward monocarpy. EVIDENCE OF MONOCARPIC CONDITIONS IN CYCADEOIDS In few extinct plants could we hope to detect evidence for so recondite a feature as flower growth but once in a lifetime, or for any modified form of monocarpy—not even under most favorable circumstances of fossilization. Among all the plants of the past, the petrified cycadeoids alone present 2 Seifriz, William. The length of the life cycle of a climbing bamboo. A striking case of sexual periodicity in Chusquea abietifolia Griseb. Amer. Jour. Bot. 7: 83-94. 1920. Apr., 1921] WIELAND — MONOCARPY IN THE CYCADEOIDS 221 the evidence for monocarpic tendencies and pseudo-monocarpy in the numberless fruits, from the youngest stages of growth to the mature seed cones held securely packed between the leaf bases forming the heavy protecting outer ‘‘armor”’ of the short, robust trunks (note Plate IX). It was first observed in studying the petrified stems from the Black Hills that a tendency to monocarpy was present. In fact, this was con- sidered fairly proven in those instances in which the imbedded young axil- lary fruits were found throughout the armor. Thus far five of the species of Cycadeoidea are viewed as more or less completely monocarpic: 1. In the trunk fragment from northwest of the Grapevine Valley, Colusa County, California, the axils of the old leaf bases are regularly occu- pied by groups of robust bracts, few in number and apparently surrounding the ends of slender old peduncles. The trunk may of course have died down. (An illustration is given on Plate LXX of U.S. Geological Survey Monograph XLVIII; the type is in the U. S. National Museum. It is the Cycadeoidea Stantont. ‘Thin sections are not available.) 2. The type of Cycadeoidea nigra from Boulder, Colorado, is a trunk segment of considerable size from the mid-region of a columnar stem sup- posedly a meter high. Again each leaf-base axil is occupied by an old, rather slender peduncle bearing few and heavy bracts. Thin sections have been cut from the armor, and should be amplified. No basal parts of fruits have been observed; but as in the preceding instance the fruiting space appears occupied, and a final series is indicated. The flowers may have failed of conservation, or the stem may be old. This type is receiving further study. It belongs to the Colorado School of Mines, and full cita- tations and description may be found in Wieland, American Fossil Cycads, volumes I and II (Carnegie Institution of Washington Publication 34). 3. Cycadeoidea Masseiana, a remarkably fine columnar trunk segment greatly resembling the foregoing, from the ‘‘scaly clays’”’ of the flanks of the Apennines, and reposing in the Capellini Museum, Bologna. Thin sections regularly cut young, small fruit axes in the axils of the large, old leaf bases of the very heavy armor. Again the thin sections should be much amplified. But as even the lesser ones cut by both Capellini and myself show the fruits, it is certain these are very numerous, and quite uniformly young. A fine model of this specimen is in the Yale collection of study materials. (Further reference in American Fossil Cycads.) 4. Cycadeoidea Fisherae, which may be only some varietal form of the Cycadeoidea marylandica, from the Potomac formation of Maryland, exhibits a most remarkable series of very young axillary fruits. No sections have ever been cut from this specimen, the conservation being hardly equal to that of either the Italian or the Black Hills trunks; though it is certain that nearly all the way from base to apex there is a fruit of some form in the axil of each leaf. It is of course possible that two floral forms are present, one staminate, the other ovulate; and it may here be remarked that, with 222 AMERICAN JOURNAL OF BOTANY [Vol. 8 the accumulation of detailed studies of exceptionally silicified trunks, many interesting features and variations of cycadeoid fructification can yet be brought to light. 5. The most remarkable petrified plant of any kind ever recovered is no doubt the National Museum specimen found by Dr. Darton on the eastern Black Hills ‘rim,’ and illustrated by some ten plates in volume II of American Fossil Cycads as Cycadeoidea Dartoni. The type consists in the upper half of a columnar trunk perhaps a meter tall, with a finely conserved terminal bud. The armor is literally packed with hundreds of mature cones, with the dicotyledonous seeds conserved in great numbers. There are very few old or aborted axes of any kind, and, while near the summit there are a few smaller and perchance younger fruits, the great series is uniformly mature. Some further sections from about the apex remain to be made. But it is to be cited that the old leaf bases appear desiccated or shrunken, while ample sections show no trace of a succeeding foliar crown; though a fine crown of young fronds surmounts a scattered growth of flower buds in the very different species Cycadeoidea ingens— a great type of six hundred pounds’ weight from the Piedmont-Black Hawk locality some twenty miles due north of the point where the C. Darton was found. Yet other species could here be cited. In view, then, of comparable species with the full series of lateral axial fruits immature, as in C. Masseiana and C. Fisherae, and of further types with peduncle series, or with isolated fruits, the Cycadeoidea Dartont appears monocarpic. Vegetative growth has apparently ceased in a time of cul- minant fructification with the emergence of all the axes the stem can ever produce. But it must be admitted that if only a small percentage of the apical fruits are found younger or less full grown than the greater lateral series, a partial monocarpy or pseudomonocarpy is indicated. Of this more, with extension of thin section series. The available series of large tandem sections is shown on Plates 46-50 of American Fossil Cycads, volume II, so that the reader may somewhat judge for himself. It may be added that the tandem sections of C. Darton1, though not at any time regarded as complete without continuation to near the erown, traverse the trunk from the base of the recovered segment upward for 35 centimeters, cutting nearly fifty seed cones. And throughout this long distance a series of uniformly mature cones is present, and evidently extends near to the trunk summit. In the belt crossed by the sections there is an average superficial area of five to six square centimeters to the cone, indicating for this upper trunk segment not less than 500 cones with the ripe embryos. On the basal segment of the trunk, which was not recovered (and which I have twice sought for at the locality on Battle Creek), there were at the lowest estimate as many more, or 1,000 in all for the entire stem (Plate XI). If a yearly cone production is concealed above, it had become small. Among so many cones, imperfectly grown examples are to be expected, Apr, 1921} WIELAND — MONOCARPY IN THE CYCADEOIDS 223 and certainly a few of the topmost of the cones below the terminal bud appear greener and less mature. In one cone inserted 6 centimeters below the apex, the seed stem mass is split off from the receptacular cushion, with the lower portions of the stem bundles left behind adherent to the cushion, so that these bundles are seen to traverse the clear silica the full distance between the cushion and the seed stem mass. The seeds cf this cone are not conserved, but the bundles show a somewhat similar tension or tearing out in occasional cones farther down in the series. In a smallish cone five centimeters below the terminal bud, the basal seed stem mass is again all that is conserved, but it appears mature. These two bases of cones may be seen in the photograph of the full longitudinal section of the entire trunk segment (Plate 43 of American Fossil Cycads, volume I]). PSEUDOMONOCARPOUS TYPES Apparently monocarpy is a phenomenon at present confined to the angiosperms. At least no characteristic instances now occur in the gneta- leans, and none in the conifers. The latter are no longer capable of annual or biennial fructification, as perchance in some remote period of their history. The dwarf or pygmic species are simply those of the high moun- tains, such being stubbornly coniferous and longevous. It is therefore curious to find that there is in the cycadeoids a near approach and parallel to a kind of pseudomonocarpy found in several conifers of the California Sierras. The so-called ‘‘knob cone pine”’ (Pinus atlenuata) occurs in much restricted “‘close willowy groves”’ of the dry slopes —in the San Bernardino range at about 3,000 feet, or along lower forest limits. It is lower and more abundant in the Shasta region. This pine varies greatly in size, from 30 meters high down to only two, though mature. (The wood is of low specific gravity, only 0.35.) At the age of seven or eight years the large cones begin to appear, borne close to the stem or larger branches, and there persist from year to year until the older series is quite imbedded in the bark. So indurated and resistant are the sporophylls in fact that often the seeds are not shed until the trees die, the groves being periodically fire swept. Then, the cones split open and reseed as if from a single crop, the trees in the ‘“‘stands’’always being of the same age.?_ A life- like picture of this singular pine is given by John Muir. Also, Pinus Coulteri, as I noted in the San Bernardino range, bears while younger a certain resemblance to the “knob cone.’’ The very large cones are then borne only on the excurrent stems, and the first cones nay abort for some distance up the trunk, leaving near the summit only a few, or but 3 It is evident that the cones near the base of an old tree reached maturity while the stem diameter was yet small, and that if they remained in the position where they first grew, they must become imbedded in the solid stem wood. But what happens is that the quite sessile cone is torn from its seat and carried outward by stem growth, with the bases deep set inthe bark. The long persistence of the cones is partly due to an un- commonly hard resinous lacquer-like coating. 224 AMERICAN JOURNAL OF BOTANY [Vol. 8 a single pair, of the mature cones about oppositely borne. ‘The basal scales of the cones may be quite grown round by the bark. As the tree is destined to grow forward, there is of course nothing like an initial stage of true monocarpy in which apical growth must fail. Obviously in those species of Cycadeoidea such as C. dacotensis and C. Reichenbachiana, with a very heavy armor and scattering fruit axes, there may have been retention of the mature cones for several seasons, closely packed in between the leaf bases and there kept dry and free from decay by an abundant drooping ramentum. But the cones themselves did not tend to persist intact after maturity, because the Araucaria-like grouping of the sporophylls allowed easy separation and splitting free of seed stem and interseminal scale alike; while a simulated monocarpy does not suffi- ciently explain the condition found in the several cycadeoid species above cited. In order, however, to reach certain main conclusions it is necessary briefly to bring to view the xerophyllous character of the heavy-stemmed cycads, their geographic range, and also the climatic factor. XEROPHYLLOUS FEATURES OF THE CYCADEOIDS Two main series of the petrified cycadeoid stems occur in the western Cretaceous. The lower series is from the Morrison (Como of Marsh). At one point near the so-called ‘‘Bone Camp”’ in the Freeze Out Hills twenty miles northerly from Medicine Bow, Wyoming, the trunks have been found abundant in close association with the sauropod dinosaurs; but the cycad species again recur more scatteringly with the dinosaurs in the western Black Hills ‘‘rim.’’ The stems are so beset by scaly ramentum that resemblance to an ‘‘old man cactus’’ was early suggested, as some evidence of growth in dry situations, although this idea is neither con- firmed nor contradicted by the presence of a fairly primitive pine (?) which I lately found closely associated with the cycads of the ‘‘Bone Camp” (so named from the bones of the great dinosaurs there found). . The wood of these early pinaceans is dense, the wood rays are small, and the growth rings are very pronounced. Evidently growth was sharply seasonal, summer and winter, or dry and wet. The second and greater series of petrified trunks occurs in the succeeding Lakota of the Black Hills, with several hundred feet of sediments inter- vening. The horizon is considerably younger, and conifer stems are again conspicuously associated, though insufficiently studied. One is an Arau- carian, called by Knowlton Araucarioxylon Hoppertoniae. Again the growth rings are pronounced.’ | This wood offers a simple but strong structure contrast to that found with the cycadeoids in the Freeze Outs. That wood is less Araucaroid, although the stems are above spoken of as early Pinaceans or Abietineans, in only a broad or perhaps Jeffreyan sense. I do not find full identity with any described form, though a relation to Prepinus (Hollick and Jeffrey) may be indicated by an imbedded shoot with centripetal wood. Apr., 1921] WIELAND — MONOCARPY IN THE CYCADEOIDS 225 Whatever these conifers may mean, the cycads are in their entire or- ganization highly xerophyllous. There is, first, the profuse scaly ramentum thickly investing the frond bases as far out as preserved, and found scant in only a few instances (C. Stilwelli of the Black Hills, and an undescribed form from a much higher horizon in Alberta). Also, as Dr. Stopes has very lately found in a young lateral leaf crown long since cut at the British Museum, the under surfaces of the pinnules bear densely packed hairs. The feature is present in the American leaf series but varies greatly in its development with the species, of which at least four with well defined hairy leaves are known. The dense packing of pinnule hairs in Cycadeoidea imgens, as faintly preserved, simulates a tissue, in some parts of the frond as thick as the folded pinnules, and ending in a clear, sharp chalcedony line. These hairs are well cutinized, and the reduced transpiring surface, taken with the latitude (44°), suggests a warm-temperate desert climate. That these features and facts were long overlooked is quite inexcusable, though partly explained by the intention to come back to the critical study and illus- tration of cycadeoid frond structure. The character may be expected in some of the Mesozoic imprints, and is in physiologic accord with the free growth of angular scaly tomentum borne by the cycadeoid microsporophylls as they form the domelike apex of the young flower buds. The foliage crowns of five American species of cycadeoids are struc- turally known. ‘These are in order of discovery: Cycadeoidea ingens, Cyca- della wyomingensis, Cycadeoidea colossalis, C. marshiana, and C. dartont. But many more trunks with crowns remain to section. In general appearance the mature frond is absolutely determinate. The petiole was heavily clad in ramentum, varying considerably with the species, and becoming hairy instead of scaly about the insertion of the lowermost © pinnules. Thence upward the upper surface of the rachis continues hairy with the hairy growth running out more or less freely all over the under surface of the pinnules; while the lower rachial surace and upper face of the pinnules remain smooth. (Note Plate XII, and text fig. 1; also Plate X, showing comparable Jurassic imprints.) The picture, therefore, of the heavy-stemmed cycadeoids is a very well defined one of dry desert plants with all their parts, stem, fronds, and fruits beset by scales, hairs, or tomentum. And, moreover, a curious but deeply interesting bit of confirmatory geologic evidence is afforded by the presence in the Lakota of reefs of calcareous tufa accompanied by numerous polished pebbles, held to mark recedent lake shores of increasingly dry regions. Though it would be an error to view the cycadeoids as dependent on as There are no resin canals, and no bars of Sanio or other modern features in either the tracheids or the rays. The radial tracheid pits are in one row, very rarely two, and never crowded or compressed. The rays are one to twenty cells high, normally ten, and one cell thick,—or two, for a distance of a cell or two. It is probakle that both these types of wood, Araucaroid and Pinoid or more Abie- tinoid, will yet be found occurring alike in both of the cycadeoid horizons. 226 AMERICAN JOURNAL OF: BOTANY [Vol. 8 distinctly tropic climates as present-day cycads. In accord with a world- wide distribution, a distribution exceeding that of cycads all the way from Florida to near the pole, the former had plasticity of both flower and leaf. Whether to be viewed as forerunners of the angiosperms or not, the Q5 QO arFarys wk pa vA nr 8, S c) KYO” ~ 2 iS ae, LOOSE IZLE. TEXT Fic. 1. Cycadeoidea Dartoni (?). Young frond deeply imbedded in the petiolar ramentum. The arrow shows the basal ears as in Otozamites.. The two ranks of folded pinnules face the trunk axis. Finer hairs and histologic structure not conserved. Trans- verse thin section X 30. capacity to live in varied climates must be hypothesized. It is not, however, likely that the Dakota cycadeoids, fringing as they did more or less pure-stand forests of pines and araucarians, could have endured as severe a climate as that of the Argentine “‘stands”’ of Araucaria imbricata facing the dry treeless country to the west of the Patagonia plateau in south latitude 35°-38°. But with the young leafy crowns so thickly beset by ramentum and the fruits so enclosed by bracts and the armor, there was full protection from many degrees of frost and from a time of snow. RANGE OF LARGE-STEMMED CYCADEOIDS In North America, on the Atlantic-Gulf border, the evidence of an extended cycadeoid habitat is conclusive. Low down in the Potomac Apr., 1921] WIELAND — MONOCARPY IN THE CYCADEOIDS 22) formation of Maryland in the Arundel (?) occurs the characteristic Cycade- oidea marylandica in latitude 39°. -And twelve hundred miles to the southwest this same form recurs in Wise County, Texas, latitude 33°.° Abcut the same time the Arundel cycadeoids formed this southeastern continental fringe, a second and highly xerophyllous group of very small trunks finds a certain extension from the Freeze Out Hills of Wyoming to the Black Hills, or from latitude 42° to 44°. As the greatest of all American occurrences, the cycadeoids of the succeeding Lakota girdle the Black Hills in latitude 43° to 45°, while two isolated finds, which may be nearly associated, extend this range on from Colorado to California on the 39th parallel, about 1,000 miles. Correspondent to this greater North American extension, there is in Europe a triangle of occurrence with an apex in the Apennines in latitude 44°, and with a base of 1,000 miles from the Galician Carpathians and Cracow in latitude 50° to the Isle of Portland in latitude 51°. Asin America, the north-south limits are only seven degrees apart. But high in the upper Cretaceous these limits are slightly extended. In the Upson Shales of Maverick County, Texas, occurs the isolated Cycadeoidea Uddenz in latitude 29°, while in the Belly River formation of Alberta, of nearly the same age, an undescribed cycadeoid is found at the north limit, latitude 54°. Whether this represents actual late extension of habitat cannot be said. Finally, the petrified cycadeoids of India come about on the Tropic of Cancer. Whence, unless the exigencies of silicification be so great that most of the record of the heavy-stemmed cycadeoids must forever lie hidden behind the thick veil of the paleontologic past, restricted habitats are inferable, and those always with a large part of the year decidedly dry. That the cycadeoid alliance as a whole was of cosmopolitan distribution all through the Mesozoic should indicate the further capacity to live in the most varied climates, as already maintained. But that which it is espe- cially desired to point out is that the greater silicified series of early Creta- ceous time falls at a period of considerable continental emergence, in North America at least, and goes far to indicate long arid belts there covering the 35th-45th parallels, in Europe the 45th-s5ist. Elsewhere attention has been called to the fact that the record for Asia and Africa, likewise for South America and Australia, fails. > The Texas cycadeoid here referred to is so like the original Maryland type that it would be regarded as varietal if from the same locality. It is an isolated and thus remarkable find of Dr. Emilio Bose with whom I was associated as a paleontologist of the Instituto Geologico of Mexico during the years 1908-10. With great consideration Dr. Bose apprised me of his ‘‘find’”’ the day it was made, in July, 1915, and later sent me the specimen. It is from the basal Trinity Beds. These rest on the Pennsylvanian, and are generally regarded as not the very oldest Cretaceous in the broader sense, but about the age of the European Aptian. 228 AMERICAN JOURNAL OF BOTANY [Vol. 8 EARLY CRETACEOUS CLIMATE OF THE CYCADEOID HABITAT In no case is it necessary to assume a Saharan heat, or even a markedly warm temperate climate, for the North American cycadeoid belt. As above pointed out, Cycadeoidea had the habitus to resist dry cold, while the associated conifer forests might well have been as able to withstand zero temperatures [-15° to —20° C.] as the Chilo-Argentine Avaucaria imbricata forest with its lower limit undergrowth of monocarpic bamboos. Nor is it improbable that a closer scanning of the contemporaneous floras of the Lower Cretaceous “‘Rim’’ of the Black Hills as recorded from both the Morrison and Lakota, and yet destined to great extension, will compel much revision of our views of all-tropic Mesozoic climate. Even a brief notice of what is already known here may be illuminating. In the 19th Annual Report of the U. S. Geological Survey, Fontaine gives an account of the plants below the Dakota—that is from the Lower Cretaceous “‘Rim”’ of the Black Hills, immediately in, above, or slightly below the great series of cycadeoids from Minnekahta and the Piedmont- Black Hawk localities. Of dicotyls there are four, called Ulmiphyllum, Ficophyllum, Quercophyllum, and Sapindopsis. Two of these occur in the Potomac of Maryland, and all are small-leaf types. It is only by a kind of elaborate argument carrying these generalized types, only known within family limits, into other zones, that they can be made to appear to have lived solely in tropic climates. And the same is true of a few species each of more or less closely associated ginkgos, conifers, and araucaroids—all small- leaf species that by themselves would easily fit into dry, to even cool climates. While three small-leaf plants of presumably cycadeoid affinity find their nearest relatives in Oregon and Greenland. Once more it is only by cumula- tion of disconnected inferences based on series which require much further study that these plants can be brought into the all-tropic scheme. Of the fern assemblage—all small of leaf—little more is known than that according to previous interpretation they easily fit the tropic category because re- sembling their Jurassic antecedents. It is early to attempt a revised, well founded estimate of the full climatic significance of the Lakotan plants. This would in fact involve a further study of all Lower Cretaceous floras. Though it is evident that too much weight has been given to the superficial resemblances in extinct floras. These must always have a certain prevailing cast due to the average course of geologic change continental in magnitude. True, if a widespread tropic plant facies marks the early Mesozoic, the identical or closely related species must have a wider north-south range than later on; but always, the similar elements of given floras, which are a reasonably sure indication of synchroneity, are only the unsafe criteria of climatic equivalence. Apr., 1921] WIELAND — MONOCARPY IN THE CYCADEOIDS Z29 A GENERAL CONCLUSION On venturing beyond the simpler comparisons with nearly related fossil types, it becomes difficult to discuss such a highly specialized and xero- phyllous plant as Cycadeoidea. There is the immense pith, the persistent armor, and the cauliflorous habit, or production of fruits along the stem below the foliage crown. The great thickness of cortical parenchyma in some forms need not be mentioned, since in some of the types with a quite heavy woody cylinder, as heavy at least as in the Cordaiteans, the pith is somewhat reduced, the cortex markedly so. But, in the first place, Cycadeoidea in actuality nears certain conifers of robust form, and, where branched, finds some resemblance in Araucaria. In the tallest cycadeoid trunks, the height may be at least six times the stem diameter, exclusive of the armor of leaf bases. Now, in the Chilean Araucaria tmbricata forest, normally grown old trees may be found with a height of only ten or twelve times the diameter of from three to six feet, while in the Sequoias of the Mariposa grove a like ratio is found in the case of various of the larger trees. For the cypress, taking the gigantic Taxodium mucronatum of southern Mexico, the ratio is little if any higher than in Cycadeoidea, in contrast to fifty or sixty in the taller conifers. Secondly, the wood of Cycadeoidea is of a generalized type which would permit innumerable derivatives. The comparative study of tracheids suggests this, and it must be true if the complex wood ray structures of gymnosperms and angiosperms are mainly of Mesozoic origin, as the paleontologic record appears to indicate. Thirdly, there is again full sanction in the Mesozoic record for hypothe- sizing progressive pith reduction all through that age, not merely in cyca- deoids, but in gymnosperms generally. And with pith reduction, branching would set in, with great floral and reproductive, as well as foliar, change; though here a reverse process may also be conceived, dependent on habitat as tropic or high northern. Fourthly, the cycadeoid floral type permitted all the sex variation possible in any flowers. The angiosperms can vary no more. Fifthly, monocarpy which is subject to certain variations in existing plants is, in a modified, possibly in a true form, present in the cycadeoids. Great lateral series of young fruits, as well as old peduncles seen in four species afford the main evidence. Sixthly, the cycadeoids of the silicified series are so strikingly xerophyl- lous that they go far to indicate vast dry to even cool belts in the mid- temperate to northern temperate zone in both Europe and America. The organization and protective features are such that light snows and some degrees of frost could have been withstood. Seeing, therefore, that there can have been such vast change and varia- tion in the more generalized cycadeoids, it becomes evident that there 230 AMERICAN JOURNAL OF BOTANY [Vol. 8 could also be notable changes in flowering as related to length of life. Hence, even to establish the presence of a pseudomonocarpy is of deep in- terest. If a raceme were to lengthen out its life it would become pseudo- monocarpic. Evidently both mono- and pseudomonocarpy must be capable of variation with change in the length of life. And both, though in a different way, are either accompaniments of gigantism or the essential characteristic of communal forms. EXPLANATION OF PLATES PLATE IX Cycadeoidea superba. A southern Black Hills trunk with five large, low subglobular branches, 75 centimeters across. Various sparsely distributed flower buds are noted, and it is quite certain that the main fruit production would have occurred later had not the initial event leading to fossilization intervened. The crowns have never been examined for the young fronds, although this form of Cycadeoidea compares most closely ‘with the highly xerophyllous Encephalartos. PLATE Xt Portion of a slab from the Lias of the Barranca Consuelo, Oaxaca, Southern Mexico, bearing three species of Cycadeoids (?): Otozamites Reglez (the three complete fronds above), Otozamites Juarezit (large single pinnule), and Otozamites hespera (lower frond with narrow pinnules). Latitude 17°. These plants with the flora associated may be taken to indicate a Mexican Liassic climate much like that of today, with a probability of somewhat drier, or even of desert and cooler conditions. Slightly reduced. PLATE DCT Cycadeoidea Dartont. Portions of large sections traversing the many cones with the mature seeds. The dicotyledonous embryos quite fill the seed cavity, and the narrow slit separating the two cotyledons may be detected in many instances. Fig. 1 from a thin section. Fic. 2 froma polished surface. Enlarged. PLATE XII Fic. 1. Transverse section through the bisporangiate strobilus of Cycadeotdea dacoten- sts showing young central cone, staminate disc (with decurved frond tips), and outer bract husk. Enlarged. Fic. 2. Cycadeoidea ingens. Young non-emergent frond with folded pinnules. The most heavily haired frond type. The light dotted line of tissue is the pinnule, with a shaded band nearly as thick indicating the packed hairs. Transverse X 3. Fic. 3. Cycadeoidea dacotensis. Young folded frond of the lightly haired type, but with the bases of the cutinized hairs beautifully conserved, though not showing well under about fifty diameters. Transverse X 3. Fic. 4. Cycadeoidea ingens. Young folded frond with slight development of the haired feature of the pinnules. Transverse X 3. VOLUME VIII, PLATE IX. AMERICAN JOURNAL OF BOTANY. CYCADEOIDS. WIELAND AA AMERICAN JOURNAL OF BOTANY. VOLUME VIII, PLATE X. WIELAND : CYCADEOIDS. *, ' ’ iy i i i ‘ PY 0 Seni *¥ a ear : a % - ‘ < . matnle Seta gy teesey / . 3 + | ‘ ‘ A : As ‘ he Z + 76 ‘es 3 ste = bee ae courses. “i yr ; CRY ba Hine ‘Anvestizators’ ld be welcomed and: all 4 ‘facilities of the camp my pe ace Jat their disposal. fae | General Courses: in’ Botany and parent: try are offered in the Summer Ses~..]. sion of Syracuse University, July. ao 8 yy to August 12, 1921. 7), : Gea A For further. information, address the y Director of the Summer Session, Syra-. “Cuse’ University, . yor ‘Direetor | of: Me ' Summer. Camp, | ANE Ae State OER. a A cia Si aapeteine IN. 2 it Saad into a hat store, know- better service to you—more tie ‘ing exactly the kind and shaped , | hat you want; and. then have some “smart Alec of a salesman try to “sell you the kind HE likes? Makes. a Salle idler than a wet hen, doesn’ t at?! 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Postage will be charged to ‘all foreig cept “Mexico, Cuba, Porto Rico, Panama” Canal Zone, Repu ohio se osihe ee Guam, Samoan eos and ‘Sha ns Pabets ‘are ‘Tuite to 20 : pages. in: ious oer ; ditional pages may: be ‘afranged for at cost rate to author 3 er Proofs about be Ce ecad eimedintee on rece ti’ Jounal of Polenye: Brooklyn’ Botanic: Garden, one oe may. one eee at cost rates Son choe Lc abu excess’ of that amount “Yan masa oe a page for tabular ma Sper, satiate. Bet for Neher | : Af Clana: oe sing ae Paine be . ois ate of ‘mailing. ‘The publishers”. will supj they. eyes Me fost i in the aga? ie AMERICAN JOURNAL OF BOTANY VoL. VIII MAy, 1921 No. 5 ISOACHLYA, A NEW GENUS OF THE SAPROLEGNIACEAE! C. H. KAUFFMAN (Received for publication December 29, 1920) Isoachlya Kauffman gen. nov. Hyphae rather stout or slender. Zoo- sporangia formed from their tips, oval, pyriform, ventricose-clavate, the later ones (secondary) arising either by cymose or pseudo-cymose arrange- ment as in Achlya, or by internal proliferation as in Saprolegnia, both modes occuring earlier or later in the development of one and the same species, or frequently on the same main hypha. Zoospores diplanetic, as in Saproleg- nia, escaping and swarming separately, and after encystment swarming the second time before the formation of a germ tube. Oogonia terminal or toru- lose, occasionally intercalary. Oospores with centric contents, the spores filling the oogonium incompletely. Antheridia present or few to none. The genus is characterized and distinguished, in the main, by the presence of the cymose or Achlya mode of formation of secondary sporangia, coupled with diplanetic zoospores. The following species naturally fall within its boundaries: 1. Isoachlya toruloides Kauftman and Coker sp. nov. 2. Isoachlya paradoxa (Coker) comb. nov. Achlya paradoxa Coker. Mycologia 6: 285. 1914. 3. Isoachlya monilifera (de Bary) comb. nov. Saprolegnia monilifera de Bary. Bot. Zeit. 16:629. 1888. Isoachlya toruloides Kauffman and Coker sp. nov. Hyphae rather slender and short, 18-20 in diameter, later ones fre- quently smaller, straight and scarcely branched. Zoosporangia oval, pyriform, clavate-pyriform, more rarely elongated-pyriform, with a more or less distinct papilla; secondary sporangia, during the early and vigorous development, all cymosely arranged by successive basipetal formation, sometimes from the walls of earlier ones, later secondary sporangial initials appearing by internal proliferation as in Saprolegnia; zoospores diplanetic, capable of escaping and swarming separately, encysting after coming to 1 After this paper was in the hands of the editor, a letter from Prof. W. G. Coker, of Chapel Hill, N. C., indicated that he was describing the same new genus and species. An exchange of data confirmed this supposition and hence it was agreed to publish them under joint authorship. The descriptions and figures to be given by Professor Coker have been examined by me, and I believe they apply to the same fungus. [The Journal for April (8: 179-230) was issued April 30, 1921.] 231 a 232 AMERICAN JOURNAL OF BOTANY {[Vol. 8 rest, and after their second escape germinating by a germ tube. Oogonia globular, short-pyriform or pyriform-globular, with a more or less short hyphal base, produced at the ends of main hyphae in basipetal series, or mixed with the sporangia in a sporangial series, up to 6 in a series (as far as observed), persisting in series until or after maturity of oospores; their — walls firm, soon becoming pale brown, with more or less scattered, distinct, small pits, sometimes numerous. Oospores centric, I-3 in an oogonium, _rarely 4 or 6, at maturity with a thick wall and a granular central sphere separated from the wall by a layer of uniform thickness; the spores measure 16-22 w. Antheridia and antheridial branches none or very rare. Two collections: in shallow water over peat-like organic remains, shore of First Sister Lake, Ann Arbor, Michigan, and in a pool of sphagnum near by. Taken November 23. Cultivated on the house-fly, Musca domestica, from single zoospores. This species has for its nearest relative ‘“Saprolegnia monilifera de Bary,’ from which it differs in that its oogonia are persistent in the series in which they are formed until after the maturity of the oospores and the breaking down of the supporting hyphae. The oogonial pits, although scattered, are distinct, while in ‘Saprolegnia montlifera’’ they are said to be few or none. Inde Bary’s species the most general mode of proliferation of the secondary sporangia is by the method found in the species of Sap- rolegnia, while in the present species they are formed for the most part by the cymose method, and for a considerable time during my observations of fly cultures I did not see the other arrangement, not indeed until the cultures became comparatively old. The appearance of the sporangia-bearing hyphae, and the mode of proliferation of successive secondary sporangia, can be seen by referring to figure 4, a—-c (Plate XIII). This is the regular process on the house-fly in 30—40 cc. of water, varying only in minor details. The oospores in the oogonia are also less numerous than appears to be the case in de Bary’s species, but the character may vary somewhat in this group, even on the same substratum—although within limits—by reason of slight differences in the vigor of the mycelium as a result of a larger immediate food supply. In “Saprolegnia monilifera”’ the oogonia are said to be produced in great abundance, and sporangial formation is reported to be almost completely ended when the oogonia begin toform. In Jsoachlya toruloides, on the other hand, sporangia and oogonia are often formed at the same time, from the time oogonia begin to form until the age of the culture and the toxic condition of the water begin to produce an effect and chlamydospores appear instead. The zoosporangia develop in the usual manner, and spore formation as seen in a living culture indicated nothing unusual. When the zoospores are ready to escape, the papilla at the apex of the sporangium is dissolved and they swim out at a fairly rapid rate and slowly scatter to a short distance before rounding up. I have followed this procedure in normal cases. A typical case is as follows. The culture had been kept in a cool glass- a —* May, 1921] KAUFFMAN — ISOACHLYA 233 surrounded enclosure, to which a partially opened window gave access to outside temperature during the winter months. The temperature usually fluctuated between 6° and 10° C. The culture was four days old and had developed at these lower temperatures, and when examined on February 14 had been in a temperature slightly below 8° C. for at least twenty-four hours. Zoosporangia were very abundant, all primary and apical, many mature, several empty, and with motile zoospores present in the upper layer of water. These primary zoosporangia were pyriform to ventricose- pyriform in shape. In the course of half an hour or so, during examination in the warmer temperature of the laboratory, many mature sporangia opened, and in a relatively short time the water was alive with zoospores. There was every indication that they were already ciliated in the sporangium. As they lined up more or less within the sporangium, after the latter had opened, they assumed an oblong shape, obscurely curved on one side and with rounded ends, and not at all pyriform as frequently described for Saprolegnias; but, as was seen later during their evolutions immediately after escaping, they had an unequal diameter in their short axes, 7.e., they were slightly flattened. A zoospore was uninterruptedly followed from its emergence, the instant it had become free of the opening at the tip of the zoosporangium. It was seen to turn over and over on its long axis, at the same time slowly getting away from the neighborhood of the others, which were all turning somer- saults in the same curious way. Meanwhile the elongated, sub-oblong or slightly sub-reniform shape was retained. This continued for about twenty minutes, during which time every movement could be. followed. Neither this spore nor the others, as far as observed, ever exhibited the darting or rapidly swimming habit of zoospores of the species of Sap- rolegnia. At the end of this period other manifestations began to appear. The spore—always turning—began in a more or less spasmodic manner to hump itself somewhat on its convex side, then to straighten out again, and after several such performances to show signs of a shortening process. Gradually during the last five minutes, by a twisting and shrugging process, it became much shorter and one end became contracted, so that a short pyriform shape, or an oval form with a papilla, resulted; this was soon changed, however, to a more and more globular form until finally a perfect sphere resulted. No let-up of the turning which it had exhibited over its longer axis could be detected up to this time, and even for perhaps twenty seconds after it appeared to have become perfectly rounded it still continued to revolve. Then very quickly all motion ceased, and the spore assumed its first resting condition. Observations following this at intervals during the next few days showed that the spores, after rounding up, gradually sink to the bottom of the dish except when, sometimes in great numbers, they are caught in the mesh of mycelium surrounding the fly. After several days, the bottom of the 234 AMERICAN JOURNAL OF BOTANY [Vol. 8 dish is literally covered by a thin layer of such spores and an equal number of empty spore-capsules. In some cases the contents of the spores were seen emerging, but so slow did this process appear to be that none were ever observed to swim away in the second swarming stage. On the other hand, a great many spores whose structure indicated that they were the end product of the first swarming stage were seen germinating by germ tubes, which eventually became slightly branched by short outgrowths. This is not uncommon in other species of the family. By transferring these empty capsules along with germinated and ungerminated spores to a slide and adding chlor-zinc iodide, no immediate reaction occurred, although a mature oogonium in the mount took a deep stain. On adding to this a rather strong iodine solution (in potassium iodide), the characteristic color for fungus cellulose appeared. The walls of the empty capsules, of the ‘spores, and of the germinating threads, were definitely stained. The spores, during the period after the first swarming stage until they germinated, became more and more vacuolate. The scanty food supply demands that the germination tubes draw upon the protoplasmic content of the spore, and eventually doubtless they will perish, thread and spore. Although several weeks had elapsed since the first spores had germinated, no sign of sporan- gial formation at the end of the germination filaments was ever noted, although this occurs frequently in other species when food supply is lacking in the solution, and is perhaps to be found as a specific characteristic of certain species under other conditions. We may now turn to the reactions of this species under certain definite conditions. ‘The description so far is that of its “‘normal”’ behavior on a sterile house-fly in sterile conductivity water of high purity. If grown on fruit-flies (Drosophila), the rapidity with which the stages of its develop- ment follow one another is quite marked. The sporangia appear many hours earlier after exposure of the flies to the zoospores, and sexual repro- duction is several days ahead of the. time for cultures on the house-fly. The food is more rapidly diffused and exhausted. In a solution of haemoglobin 0.05 percent + KNO3 0.1 percent, four days old, at the temperature mentioned before, the culture is found to have developed very abundant oogonial initials, arranged in torulose series as shown in figure 10, a, 6 (Plate XIV), and in some cases the terminal oogonium has already formed oospheres. The latter tend to be more numerous in each cogonium than is the case in fly cultures. At this same time the house-fly was densely covered with short, straight hyphae, nearly all bearing sporangia, mature or maturing or with sporangial initials. After ten days in this solution the earlier series had produced oospheres and some oospores, but those developed later showed signs of abnormality and disintegration. In a solution (always with conductivity water) of haemoglobin 0.05 percent, in this case with an addition of 0.12 percent levulose, a culture May, 1921] KAUFFMAN — ISOACHLYA 235 four days old, under the same temperature conditions, showed the same general abundance of oogonial initials, but in this case the oogonial swellings occurred at the ends of stalks in pairs, in series of two, or with one on an adjacent short lateral stalk. After ten days the greater number of these had developed further, so that the terminal one possessed oospheres or oospores while the basal ones were initials and in many cases showed signs of never being able to complete their destiny. Later observations showed this surmise to have been correct, the apical ones maturing further, the contents of the basal ones breaking down. (See fig. 11, a-c.) In a 0.1 percent solution of leucin, under the same conditions, after four days only scattered and quite young oogonial initials were seen. After ten days they were abundant, and a large proportion contained oospheres. But in this case the globular oogonia were borne singly at the ends of long supporting hyphae, not more than I to 2 percent being in series of two. (eee fig. 12, a—c.) In all the cultures mentioned, no antheridia were ever observed. The pits of the oogonial wall were always rather far apart, not large, and in leucin unusually distinct. The torulose habit of the oogonia is reduced as we go down this list of cultures. The presence or absence of some substance in the synthetic solutions is doubtless responsible for the inability of the development of the later oogonia to run its usual course. Although no combinations with mineral salts were extensively tried with the haemoglobin and leucin solutions, work on other species has shown that there is no reason to suppose that the disintegrating effects of the haemoglobin and leucin could not be offset by the addition of proper amounts of certain salts, the oospheres thus being induced to complete their development to maturity. It will be noted that the period of time necessary for the appear- ance of the reproductive organs is not strictly an inherent quality within the species, but depends, within limits, on certain other influences. The older investigations laid great stress on the exact period of time which any process, in the life history of a species, took to complete itself. In view of many indications in this and other groups of plants, this attitude needs to be much changed before we can lay a sound foundation for plant morphology. In records of cultures of the Saprolegniaceae it is still frequent to see flies, wasps, white of egg, this, that, and the other substratum mentioned without further details, as if the organism were assumed to act alike on all. “ Saprolegnia monilifera de Bary’’ has, so far as I know, been observed and studied only by de Bary himself. He found it only in one lake, near the Black Forest, Germany, from which he obtained it repeatedly, but was never able to get it from any other of his collections made through many years. De Bary considered its nearest relative to be Saprolegnia torulosa of the “ferax’’ group; but he placed it in a separate section which he called the “monilifer’’ group. Its anomalous position was thus clearly recognized by de Bary, and since this was the only case known to him in which both 236 AMERICAN JOURNAL OF BOTANY [Vol. 8 types of secondary sporangia were present, de Bary’s native conservatism naturally induced him to attach it to one of the older genera. | An addition to the species which have this anomalous character of two modes in the formation of secondary sporangia during a sexual reproduction, was made recently by Coker (J.c.). He referred his species to the genus Achlya, as “A. paradoxa,” and felt disinclined to erect for it a new genus, on the ground that the anomalous character in question establishes merely a “narrow point of contact’’ and is not unlike similar intermediate species in Puccinia and Uromyces. While this is clearly the case, limitations to genera cannot go on indefinitely encircling more and more exceptions, else one of the main reasons for the building up of the scientific classification of plants becomes abortive. Furthermore, in a group of such a small number of species as the Saprolegniaceae, the case becomes very different, by lack of balance, from the case in the genera of rusts cited. It seems to me, then, that by the erection of the genus Isoachlya, a double objective is attained: the limitations of the old genera remain clearly defined, and the new genus takes a most natural place within the family of the Saprolegnia- ceae. BOTANICAL LABORATORY, UNIVERSITY OF MICHIGAN LITERATURE The student is referred to the extensive bibliography by Klebs for the older papers; and to Weston for more recent work. Klebs, G. Zur Physiologie der Fortpflanzung einiger Pilze. II. Saprolegnia mixta. Jahrb. Wiss. Bot. 33: 513-593. 1899. Weston, W. H. Observations on an Achlya lacking sexual reproduction. Amer. Jour: Bot. 4: 354-366. 1917. EXPLANATION OF PLATES The higher magnification was obtained with Bausch and Lomb 15 X ocular and 3 mm. objective, the lower with 15 X ocular and 8 mm. objective. All are equally reduced. Fig- ures I-9 are from cultures on sterilized house-flies, in 25-40 cc. conductivity water, from single zoospores; all cultures were free from other organisms. PLATE SGhIT Fic. 1. Sporangial initials of Isoachlya toruloides on head of a fly, two days after inoculation. ' Fic. 2, a. Young hypha before swelling. 06-h. Stages in the development and maturity of zoosporangia. Fic. 3, a-f. Escaping zoospores and form changes in the process of assuming the first resting stage. a. During first twenty minutes. 6-e. Gradual changes during next thirty minutes. jf. Completely rounded and at rest. No cilia are shown, since full data were not obtained, but they are doubtless two in number and lateral. Fic. 4,a-c. ‘‘Achlya”’ type of formation of secondary sporangia, after nine days. Fic. 5, a-e. Different types of oogonia, showing the normal variation in number of oospores as the fungus occurs on flies. After nine days. Fic. 6. ‘‘Saprolegnia”’ type of formation of secondary sporangia. The proliferation in the two empty sporangia would become the third in the series. After thirteen days. VotumeE VIII, PLATE XIII. AMERICAN JOURNAL OF BOTANY. ISOACHYLA. ° ° N KAUFFMA AMERICAN JOURNAL OF BOTANY. Votume VIII, PLATE XIV. KAUFFMAN: ISOACHYLA. May, 1921] KAUFFMAN — ISOACHLYA 227, feoeic.7. After the end of the first swarming stage. a. Empty spore walls. 6. Escaped but quiescent, naked cells. c. Act of escaping of cell. d. Germination by one or more germ tubes, the second “‘swarming”’ being omitted. PLATE XIV Fic. 8, a. Two examples of oogonia with mature oospores. 0b. A single mature oospore, much enlarged. After thirteen days. Fic. 9. One of few occurrences of an oogonium with antheridia. Antheridia nearly mature; oogonium young. After 8 days. | Fic. 10, a-c. Oogonial arrangement in solution of haemoglobin 0.05% + KNO;0.1%- Note the larger number of oospores per oogonium. After four days. Fic. 11, a-c. Oogonial arrangement in solution of haemoglobin 0.05% + levulose 0.12%. After twelve days. 90% of oogonia occur in pairs. Fic. 12, a-c. Oogonia arranged singly at ends of long stalks, frequently with vege- tative outgrowths below the oogonium, and oospores rather numerous. From culture in 0.1% leucin. d. In 0.02% leucin, In this weaker solution the} inhibitive effect of leucin for the torulose arrangement is reduced. Both after ten days. Fic. 13, a. In 0.02% peptone, after five days. 06. After ten days. The oogonia are unable to mature in this solution. Fic. 14. From peptone cultures in condition shown by figure 13, washed in distilled water for an hour, then transferred to distilled water. The initials become zoosporangia and the zoospores have escaped. After twenty-four hours. Fic. 15. From culture in 0.02% peptone. a. Note regular arrangement of the basal walls of sporangia. 06. Neat illustration of the doctrine of homology of different organs of reproduction. One of few oogonia which produced oospheres in this solution. c. An oogonial initial becoming a zoosporangium with scarcely any external morphological changes THE TRANSMISSION OF RHUS POISON FROM PLANT TO PERSON! James B. MCNAIR (Received for publication January 3, 1921) The progress of our knowledge of the transmission of Rhus poison from plant to person reflects, in a general way, the development of our under- standing of plants and plant products. This is shown prominently in tracing the history of experiments in regard to the volatility and chemical nature of the poison. In this connection it may be well to consider, besides the dermatitant from Rhus diversiloba T. & G., the similar irritant substances from R. Toxicodendron L. and from its other sub-species R. radicans L. The earliest explanation of Rhus poisoning attempted was that the plant gives off an invisible colorless vapor, or emanation, which, when breathed or permitted to touch the skin, causes dermatitis. The North American Indian and negro shared in this belief (Thompson, 44). Some early writers associated Rhus poisoning with the fabulous stories told of the effects of the deadly upas tree (Ipo toxicaria Pers., Antiaris toxicaris Lesch.) of Java (Bennett, 4).? The theory that the poison is non-volatile has also had its adherents. 1 The substance of this paper was presented before the Graduate Botanical Club of the University of Pennsylvania, May 6, 1918. 2 More light on the early physical and chemical nature of the principal irritant poison of this plant may be obtained through a study of the writings of Monti, Hunold, Gled- itisch, Achard, Willemet, Pornai, and Kriiger. All of these investigators considered the poison volatile. That this conclusion should be drawn at that time is not so remarkable if we consider that the gaseous exchange in plants was not understood at that time. Although Priestly (39) found in 1772 that plants give off oxygen, subsequent repetition of his ex- periment did not always give the same result. Ingenhousz (20) showed that the air was purified by plants in sunlight. He concluded, however, that the atmosphere is made in- jurious to animals by emanations from all plants in the shade and at night. Not only were all plants supposed to give off volatile poisons in the shade or at night, but Conradi, Acker- man, and Krauss believed the chief cause of various infectious diseases to be gaseous. As a result we have to this day the word malaria. ACHARD, F. K. Nouv. Mem. del’Acad. Royal de Sciences et Belles Lettres 1: 48. 1786. ACHARD, F. K. Nachricht von Versuchen die iiber den Giftbaum (Rhus Toxtcodendron Linn.) angestellt worden, um seine Bestandtheile zu kennen, und die Art und Weise, wie sein Gift auf verschiedene Thiere wirkt, zu bestimmen. Chemische Annalen von Lorenz Crell 1: 387-395, 494-503. 1787. ACKERMANN, J. F. Von der Natur des ansteckenden Typhus; dem Wesen des Ansteck- ungsstoffs, der Art sich gegen denselben zu sichern, und der Methode die Krankheit zu heilen. Heidelberg, 1814. ConrapI, —. Grundriss der Pathologie und Therapie I: 288. GLEDITISCH, J. G. Nouvelles expériences concernant les dangereux effets que les ex- 238 May, 1921] MCNAIR — TRANSMISSION OF RHUS POISON 239 In 1788 Du Fresnoy, experimenting with R. radicans, steam-distilled its flowers and leaves. The distillate was not poisonous, but the residue in the still remained toxic. Fontana (13) experimented with R. Toxicodendron. Because of his marked susceptibility to the poison he was forced to stop before he had determined whether or not the poison is volatile. Two years later, Van Mons (45) collected about fifteen cubic inches of gas given off by a plant of R. radicans. Chemical experiments were carried on with this. He then engaged his brother, who was very sensitive to the poison, to hold his hand for more than one hour under a glass bell jar con- taining gas from the plant obtained in the middle of the day. A month later, not having noticed any eczematous symptoms, he repeated the same experiment with gas collected under a cylinder covered with black card- board. He felt, even during the immersion, a burning sensation, and developed a typical case of Rhus dermatitis. Van Mons concluded that _ the poisonous principle of R. radicans is a gaseous hydrocarbon which ema- nates from the plant only at night, on cloudy days, or in the shade. In 1798, Horsfield, a medical student at the University of Pennsylvania, stated that some people were affected by the exhalations of R. Vernix and R. radicans to a distance of twenty feet from the plant. He also noticed that dermatitis was produced by the immediate application of the juice of the plant to the external surface of the skin. In analyzing R. radicans he placed two pounds of the flowers and leaves with several quarts of water ina small copper still. The distillate was not poisonous, but the residue in the still retained its toxicity. Lavini (25) considered the poison of R. Toxicodendron a gum resin, mixed with a “‘subtil’’ acid principle, qualified to combine with the hydro- carbon.gas which emanates from the plant after sunset. According to him, the effect of the sap squeezed from the leaves is analogous, but less intense. The effect of “‘water”’ distilled from this plant was still less intense. Khittel (22) attempted a more thorough chemical analysis of R. Tox1co- dendron. Because of his inability to find the poison by the processes out- lined, he considered it a volatile alkaloid. halaisons d’une plante de l’Amerique Septentrionale produisent sur le corps humain. Mem. de l’Acad. de Berlin 61-80. 1777. Ditto. Journal de Physique 21: 161-175. Hunotp, —. Piepenbrugs Archiv fiir die Pharmac. 1: 279. Kraus, J.C. Verhandeling over der aard en de werking det geneeswiddelen, welke ter bestrijding van zenuwkwalen en derzelver toevallen worden Aangewend. Amsterdam, 1819. KRUGER, —. Archiv ftir die Pharmac. I: 261. Mont1, GuIsEpPE. De plantis venenatis. Accademia della scienze dell’ Instituto di Bologna. Commentari II, 3: 160-168. 1755. Pornal, —. Giornale per s2vire alla storia ragionata della medicinia di questo secola. Venezia 1: 83. 1783. WILLEMET, Remi. Observations sur les effets du Rhus radicans. Jour. de Phys. 51: 369- 370. 1800. Ditto. Journ. de Med., Chir., Pharm., etc., Paris 1: 209-211. 1801. 240 AMERICAN JOURNAL OF BOTANY [Vol. 8 Millon (35), evidently unaware of the work of Khittel, also investigated R. Toxicodendron. He believed the poison a non-volatile gum resin re- quiring direct contact to cause dermatitis. He found its alcoholic solution to be toxic. | Discussing the experiments of Khittel, Maisch (33) held the poison to be volatile and said: It is natural to suppose that, during the process of drying, the greatest portion of the poisonous principle should be lost. This must be still greater, if the dried leaves are powdered, a hot infusion prepared from them, and this infusion evaporated down to the original weight of the dried leaves. It is obvious that Khittel could not have selected a better method for obtaining the least possible quantity of the poisonous principle, if, indeed, it could be obtained by this process at all. Later Maisch (34) disagreed with Khittel and denied the presence of a volatile alkaloid. He thought that he had found a new volatile acid, which he held to be the active principle and which he called ‘‘toxicodendric acid.” Maisch enclosed in a tin box a lot of freshly collected leaves of poison ivy, and introduced into this box a number of moistened test papers. The next morning he found that the blue litmus paper had been colored strongly red, whereas curcuma and red litmus paper were unaffected. He writes regarding this experiment: | This single experiment was at once a conclusive proof that the exhalations of these leaves contained a volatile acid, and that the poisonous properties were most likely due to it. Maisch describes further how he obtained an impure watery solution of his texicodendric acid by maceration of the leaves, expression and distil- lation of the expressed liquid. In preparing his acid, he suffered from a copious eruption and the formation of numerous vesicles on the back of his hands, fingers, wrists, and bare arms. He says further: Several persons coming into the room while I was engaged with it were more or less poisoned by the vapours diffused in the room, and I even transferred the poisonous effects to some other persons merely by shaking hands with them. The dilute acid, as obtained by me, and stronger solutions of its salts, were applied to several persons, and eruptions were produced in several instances, probably by the former, though not always, which was not likely owing to the dilute state of the acid. Maisch did not isolate his acid nor any one of its salts; he never had the substance in question chemically pure. He proved only the presence of a volatile acid. He noticed the characteristic eruption on his own skin while working with the poison ivy. Persons coming to the laboratory at this time were often poisoned. He observed also that an eruption some- times followed the application of the impure solution of this acid to the skin. From these very rudimentary experiments he drew the wholly unwarranted conclusion that his acid must be the active principle. By far the most valuable work on Rhus Toxicodendron is that of Pfaff (37). From aclinical study of Rhus poisoning, Pfaff came to the conclusion that the poison must be a non-volatile skin irritant. The more volatile May, 1921] MCNAIR — TRANSMISSION OF RHUS POISON 241 the irritant, the quicker is its action on the skin. Formic acid acts very quickly; acetic acid, less volatile than formic, acts more slowly, but still much more quickly than poison ivy, the latent period of which is usually from two to five days. Pfaff thought that the volatile acid obtained by Maisch might have contained some of the poisonous principle as an im- purity, but that it could not produce the dermatitis if prepared in a pure state. He therefore prepared a quantity of the acid by distilling the finely divided fresh plant with steam. The yield was increased by acidu- lating the mixture with sulphuric acid before the distillation. The acid distillate so obtained was freed from a non-poisonous oily substance by shaking the solution, with ether. Barium and sodium salts were made by neutralizing the acid and were purified by crystallization. Analysis showed them to be salts of acetic acid, and they gave the characteristic tests for this acid. The “toxicodendric acid’’ of Maisch was thus shown to be acetic acid, and not therefore the poisonous principle of the plant. Pfaff obtained the active principle by the process which he outlines. The lead compounds made in different preparations were analyzed and assigned the formula C2:1H390,Pb. The oil itself was not analyzed. Pfaff proposed the name toxicodendrol for the oil. He found that it is not volatile, is decomposed by heat, is soluble in alcohol, ether, chloroform, benzene, etc., but insoluble in water. Its effects upon the human skin were studied in many experiments upon himself and others. It was shown that an exceedingly minute quantity of the poison will produce the der- matitis, even 1/1000 milligram applied in olive oil being active. The oil was given internally to rabbits, its effects being most marked on the kidneys. Acree and Syme (1) found gallic acid, fisetin, rhamnose, and a “‘ poisonous tar, gum, or wax”’ in the extract prepared by maceration of the leaves and flowers of poison ivy with ether, and subsequent distillation of the solvent. The lead compound of this poisonous substance was found to be soluble in ether. The authors utilized this property to free the poisonous material from admixed non-poisonous substances. Lead compounds were first prepared by precipitating an alcoholic solution (of the ether extract of the drug) with lead acetate. The precipitate was washed with water, partially dried over sulphuric acid, placed in a Soxhlet apparatus, and extracted with ether until the solvent came over colorless. A green solution was obtained which was washed with water and decomposed with hydrogen sulphide. On evaporating the solvent, a black, poisonous “‘tar or gum” remained. Upon hydrolysis with 2 percent sulphuric acid, this poisonous substance gave fisetin, rhamnose, and gallicacid. The residue in the thimble was decomposed by hydrogen sulphide, shaken with ether, and evaporated. A hard, brittle, yellow, non-poisonous resin was obtained. The authors believe the poisonous pam of poison ivy to be a complex substance of glucosidal nature. Chyser in 1910 considered the poison of Rhus a toxalbumin formed by 242 AMERICAN JOURNAL OF BOTANY [Vol. 8 the combination of a liquid acid with albumin. He puts forth the following evidence in support of this conclusion: (1) the small amount of poison (0.000005 g.) necessary to produce itching and burning on the skin; (2) similarly to a toxalbumin, it loses its toxicity by heating to 50° C. on a water bath; likewise at 75° C. and 100° C. The toxicity was tested by rubbing with a probe on the skin of the upper arm. Inno case was irritation evident. This evidence is inconclusive of the poison’s being a toxalbumin, for: (1) other substances besides toxalbumins are poisonous when in such small amounts; (2) the poison remains toxic if heated on glass in a steam autoclave for one hour under twenty pounds’ pressure per square inch (temperature 126.2° C.); and (3) the poison contains no nitrogen. The work of Acree and Syme is probably erroneous for: (1) all three of the so-called constituents of the poison are found in two non-poisonous species of Rhus; (2) the natural glucoside yielding fisetin, rhamnose, and gallic acid is non-toxic; and (3) there is not sufficient evidence that the poisonous substance which Syme attempted to decompose was not a complex containing a poisonous body and one or more non-toxic glucosides in addition. McNair.(30), working with R. diverstloba, concluded that the poison of this plant is not a glucoside of rhamnose, fisetin, and gallic acid. A different method was used for extracting the poison, and none of these substances could be obtained on hydrolysis. The specific cause of skin poisoning from R. Toxicodendron L. and its two sub-species, R. diversiloba T. & G. and R. radicans L., has thus far been ascribed successively to: an emanation of vapor; a hydrocarbon gas; a gum resin, mixed with a “‘subtil’’ acid principle, qualified to combine with hydrocarbon gas which emanates from the plant after sunset; a volatile alkaloid; a non-volatile gum resin; a volatile organic acid (toxicodendric acid); an infection by bacteria (M. toxicatus, Burrill, 7); a non-volatile oil (toxicodendrol); a glucoside of fisetin, rhamnose, and gallic acid (toxicoden- drin); a toxalbumin; and finally to something other than a glucoside of fisetin, rhamnose, and gallic acid. THE TRANSMISSION OF RHUS DIVERSILOBA POISON My investigation of the transmission of the poison has been carried on from three standpoints; botanical, chemical, and pathological. The following chemical experiments were carried out: 1. One half pound of fresh, finely chopped poison oak leaves were dis- tilled normally at different temperatures up to the point of decomposition of the leaves. As a result, both the distillate and the residue were non- toxic. 2. Another lot of leaves similarly prepared was subjected to steam distillation. The distillate was non-toxic, but the residue in the retort remained toxic. May, 1921] MCNAIR — TRANSMISSION OF RHUS POISON 243 3. Distillation, either destructive or with ether, when done under reduced pressure, gave non-toxic distillates. From the results of these distillation experiments it can be safely argued that the poison is non-volatile and that if non-volatile it can not be carried by entrainment with a volatile substance. It has been considered by some _ as a non-volatile poison carried by a volatile oil. In the investigation of the smoke of the burning plant (Von Adelung, 46), leaves were placed in a glass combustion tube. The glass tube was then heated until the leaves began tosmoke. The smoke was blown against the skin of a susceptible individual. Dermatitis resulted. The experiment was repeated with the addition of cotton plugs in each end of the tube. Dermatitis did not result. It was thought that perhaps condensation of the irritant might have occurred on the cotton. The experiment was therefore repeated (McNair, 30), glass wool plugs being used instead of cotton. The glass wool was kept at the same temperature as the burning leaves. No dermatitis resulted. It is concluded, therefore, that the non-volatile poison is carried by particles of soot in smoke. | It is also possible to determine the non-volatility of the poison phys- iologically. A fresh leaf of poison oak was lightly glued to the concave side of a watch glass about six inches in diameter. The watch glass was then taped on the breast of a susceptible person (the concave side inward) and left there for half an hour. No dermitis resulted. The foregoing experiment was repeated, substituting for the leaf a drop of sap. No ill effects resulted. A drop of sap was now placed on the skin of a susceptible individual, and the area was covered by a watch glass. Dermatitis occurred after a few hours, but only in the area to which the sap was applied. It did not spread. If the poison were volatilized with moderate ease, at ordinary temperatures, it would have caused a general irritation at, as well as around, the area to which it had been applied. Volatile poisons rapidly penetrate into the tissues, and diffuse there with great ease. Such is the case with the various oils of turpentine, many ethereal oils from the vegetable kingdom, and numerous substances belonging to the aliphatic series, e.g., chloroform and ethyl chloride. Petroleum, benzol, and other compounds of the aromatic series cause local irritation in essentially the same way (Schmiede- berg, 41). In another experiment, sap was placed on the skin of a susceptible person. After dermatitis had occurred, the affected section of the skin was cut out and thin sections were mounted on microscopic slides. These sections showed that the poison had penetrated but slowly in the skin (McNair). If the poison were volatile, penetration would occur more rapidly and diffusion would be greater. In ordinary cases of Rhus poisoning, dermatitis is not noticed until 244 AMERICAN JOURNAL OF BOTANY [Vol. 8 about twelve hours or more after exposure. This long period of latency is much against the supposition that the poison is volatile. It would be much easier for a volatile poison to evaporate and diffuse through the atmosphere in twelve hours if it required a dozen hours to penetrate the skin. From the preceding experiments, it is clear that the poison is non-volatile. But we still have the question to answer as to how poisoning occurs without contact with the plant. This question has been studied by Von Adelung (46), Schwalbe (43), Hubbard (17), Hadden (16), and Frost (15). Von Adelung considered the pollen to be toxic and disseminated by the wind. Asa matter of fact, the pollen may be rubbed on the skin of a sus- ceptible person without ill effects. The skin may even be lacerated. The pollen grains, although small enough to be carried by the wind, have no wing-like projections or tissues which would aid their flight, but on the contrary are covered with a sticky substance which tends to hold them in masses to the flower. Pollination is effected by insects. Similar non-toxic results have been obtained with the pollen of other poisonous species of Rhus; with that of R. vernicifera by Inui (21), that of R. Vernix by Warren (48), and that of R. Toxtcodendron by Rost and Gilg (40). Schwalbe (43) attributed poison transmission to the trichomes of the plant. The trichomes are very minute and are found in abundance on the young stems and on the under surfaces of the leaves. The trichomes were considered to be poisonous and carried by the wind. In an investigation of this theory, fresh leaves were placed in an alembic, and a current of air was blown through. The outcoming air current was caused to impinge on the skin of a susceptible individual. No dermatitis resulted. The experiment was repeated, except that the outcoming air was caused to bubble for several hours through alcohol in which the poison is soluble. This alcoholic solution was concentrated and found to be non-toxic. In another experiment, the hairy side of an uninjured leaf (previously examined carefully with a hand lens for the absence of droplets of sap) was drawn across the skin with no ill effects. In another test an uninjured leaf was placed in 95 percent alcohol at room temperature for ten minutes. The alcoholic solution was concentrated and found to be non-toxic. Rost and Gilg (40) carried on experiments with R. Toxtcodendron to determine if the plant hairs drop off spontaneously, if they can be blown off from cut twigs, and if the poison, as in Primula obconica, can be obtained by contact from the under sides of the leaves. Two shells containing gly- cerine were placed under Rhus plants for two windy days in May. When this liquid was examined microscopically after the experiment, needle- shaped and club-shaped hairs were found. On October 17, roii, three wide glass dishes containing glycerine water were placed under thickly leaved branches of R. Toxicodendron. These were left for four days. A microscopical examination on October 21 showed no hairs in the dishes. owt gad May, 1921] MCNAIR — TRANSMISSION OF RHUS POISON 245 The preparations contained considerable dust. From the results of these experiments, it is evident that the hairs do not drop off to any great extent spontaneously at either the beginning or the end of the vegetative period. To determine whether or not the trichomes could be forcibly blown off, five experiments were conducted in I9II: A. At the end of July (Exp. S. 1 and 2); Ba xte the end of Auguste Dxp. 5. 3); C. At the end of September (Exp. 5. 4); D. After the middle of October (Exp. S. 5). A branch was firmly fastened within a rectangular glass case (100 X 75 X 180 cm.) and was exposed to an air current of about 0.3 atmosphere pressure from a distance of approximately 15 cm. so that the leaves moved as if ina storm. ‘The air current, after passing the leaves, struck an inclined glass plate on which were placed glycerine-covered slides. The current then left the case through a funnel closed with cotton. On the bottom of the glass case two more glycerine-covered slides were placed. During experiments the air current was often interrupted, especially at the beginning and towards the end. ‘This was done to secure the strongest possible distur- bances of the leaves. Each experiment lasted at least two hours. Freshly cut branches were used. ‘These branches were afterward pressed and stored, for microscopical examination as to the presence of trichomes. Trichomes were found to have been left on the leaves in abundance. The glycerine-moistened slides were examined under high and low magnifications. At the end of each experiment, preparations of the dried leaves were made in a chloral-hydrate solution to find if hairs still remained. The branches used were: In Series 1: In Series 2: Pst day 0.2... . fresh Tsteday yes 2 fresh Bada Y bk one day old 2aedanre ok nt ts: one day old Baeday sc: ... two days old Bdidaye.. ecetces three days old A. Experimental Series 1 (July 26-28, 1911). Herbarium specimens and two microscopical cross sections gave evidence of many hairs. I. Wednesday, July 26. The experiment lasted 11 hours. During the first hour the position of the branch was changed twice. A microscopical examination of the glycerine-moistened slides on July 27 showed the absence of club-shaped hairs, but the presence of needle-shaped hairs, much dust, and other impurities. II. Thursday, July 27. The branch used in the foregoing experiment was exposed to the blast again for two hours (from 11:15 A.M. to 1:15 P.M.). When examined microscopically on July 28, the preparations showed that the dried-up branch as well as the fresh one had not given off club-shaped hairs but only needle-shaped hairs. 246 AMERICAN JOURNAL OF BOTANY [Vol. 8 III. Friday, July 28. The almost entirely dried branch was subjected for the third time to the air blast (from 10:00 A.M. to 12:00 M.). A micro- scopical examination on the same day (July 28) showed the presence of needle hairs in all preparations, but of only one club-shaped hair. At the end of experimental series 1, a chloral-hydrate preparation was made of the entirely dried branch. The under side of the leaves, as is the case in the fresh leaf, were covered with many club-shaped and bristle-like hairs. B. Experimental Series 2 (July 28-31, 1911). Inthisseries of experiments a branch of densely haired R. Toxicodendron was used. A part was pressed and a chloral-hydrate preparation made of it. This showed a dense covering of both kinds of hairs. I. Friday, July 28. The experiment lasted from 12:30 to 2:30 P.M. On July 29, a microscopical examination disclosed a club-shaped hair in each of four preparations; the remainder showed many needle-shaped hairs. II. Saturday, July 29. The dried branch was blown on for two hours (from 11:00 A.M. to1:00 P.M.). A microscopical examination followed on Monday, July 30. This disclosed in Preparation 1: Three club-shaped hairs, very many needle-shaped hairs, many dirt particles, and pollen grains of other plants. Preparation 2: No club-shaped hairs. Preparation 3: Four club-shaped hairs, one with a piece of epidermis. Preparation 4: Two club-shaped hairs. . Preparation 5: Two club-shaped hairs, one containing yellow protoplasm. Preparation 6: One club-shaped hair. Preparation 7: Three club-shaped hairs. Preparation 8: No club-shaped hairs. III. Monday, July 31. The three-day-old branch was blown on from 11:30 A.M. to 1:30 P.M. A microscopical examination on August I showed one club-shaped hair in each of four out of eight preparations. Both the glycerine-covered slides on the bottom of the case were free from club-shaped hairs. The cotton in the funnel contained no club-shaped hairs (the cotton having been soaked in glycerine and the excess pressed out). At the end of the experiment, a chloral-hydrate preparation was made from the three-day-old, entirely dried branch. Club-shaped hairs were present in abundance on the leaves. The club-shaped hairs could never wound the cuticle. Three further experiments were made, similarly to the first two, toward the end of August, in the second half of September, and soon after the middle of October, 1911. The results were similar. In the first days none, or at most one or two, club-shaped hairs could be found in 8 to 10 prepara- tions. In the experiments with the twigs dried two or three days, only a few club-shaped hairs were blown loose. In many experiments in which May, 1921] MCNAIR — TRANSMISSION OF RHUS POISON 247 preparations of hairs were spread on the skin, not the slightest irritation appeared. _ The glycerine layer of one or more experiments was applied and dried on the uninjured skin of the under side of Rost’s forearm. The results were negative. Rost was susceptible to the resinous sap of the same shrub. It seems evident, therefore, that the trichomes are non-toxic and are not a means of conveyance of the poison from plant to person. Hubbard (17) and Hadden (16) thought insects might carry Rhus poison from the plant in ways similar to those by which flies carry bacteria from place to place. This method of transmission seems hardly practicable in many cases. It should be borne in mind that the insect could not trans- mit the poison by coming in contact with the uninjured plant. Recently, Frost (15) believed the poison to be bacterial. This has been refuted (McNair, 32). The methods already discussed constitute all that have been suggested for the transmission of Rhus poison to a distance. As none of them prove very serviceable, we still have to consider the question as to how poisoning occurs without contact with the plant. It has been found in an examination of the sap that: (1) The unelabo- rated sap of the xylem is non-toxic; (2) the elaborated sap of the phloem is non-toxic; and (3) the resinous sap of the resin canals is poisonous. A further examination of the plant tissues shows that the xylem, epider- mis, and trichomes which do not contain the resin canals are non-toxic. When the flowers are examined, it is evident that resin canals do not extend more than half-way up the fully matured stamens, and so it would be expected that the pollen would be non-toxic. The flower of the female plant, on the other hand, contains resin canals in the pistil, and an abundance of resin canals surround the ovule. The ovule remains highly toxic until the seed has fully ripened. The poison, therefore, acts as a protection to the immature seed. This plant thus exemplifies the natural law developed by Kipling (23) that the female is more deadly than the male. It has also been shown (McNair) that the maximum number of cases of Rhus dermatitis recorded in the University of California Infirmary occurs previous to the opening of the flowers. It has long been known that fresh leaves are more likely to produce poi- soning than are dry or fallen leaves. This difference in malignancy has been attributed to a poisonous gas given off by the plant. Van Mons (45) was convinced by the large number of cases among persons of his acquiantance, that the evil effects of Rhus were produced by a gaseous substance which escaped from the living plant, because the dry leaves or fallen leaves never caused trouble. Professor Asa Gray also held this same opinion in 1872, as the following letter to Dr. J. C. White discloses: My personal knowledge that Rhus dried specimens are harmless amounts merely to 248 AMERICAN JOURNAL OF BOTANY | [Vol. 8 this: I handle over and over dried specimens with impunity, but am very sensitive to the fresh plant. Then the poison is volatile, as shown by its affecting persons who do not touch it actually; that of the leaves, I should say, must escape and dry out in the drying process, or in the course of time. In a stem it would not volatilize so soon; but I should not expect to be poisoned from any o/d herbarium specimen, either from twigs or leaves. Likewise, Mackie (28), writing on the value of oak leaves for forage, says: 3 It would seem that the irritating and poisonous oil of poison oak is volatile at a com- paratively low temperature. In gathering the specimens the writer was badly poisoned even though gloves were worn; yet after drying at ordinary room temperature, and the leaves pressed into the mill with bare hands, no poisoning effects followed. Opposed to these opinions is the experience of Bogue (6) while investi- gating an herbarium specimen of R. venenata which had been deposited in the Ohio State University not less than three years. He was poisoned by the “sawdust”’ from the stems of the plant which was the result of borings -rom a beetle. It has previously been conclusively shown that the poison is non-volatile, and the decrease in malignancy of the leaves in drying can be attributed only to a loss of fluidity of the sap and to the loss of toxicity of the poison from oxidation (McNair). In concluding the botanical investigation, it seems evident that the plant is capable of poisoning only when injured in such a manner that the poisonous resinous sap exudes. Poisoning without contact with the plant may occur by means of smoke from the burning plant or by contact with substances that have the poisonous sap on them, such as gloves (Hunt, 18; Ward, 47; Frost, 14; Kunze, 24); pocket-knife handles, croquet balls, and botanists’ collecting cases (Hunt, 18); hands of another (Hunt, 18; White, 49; Planchon, 38; Cantrell, to; Maisch, 34); clothing (Balch, 2; White, 49; Bibb, 5; Lindley, 27; Cundell- Juler, 11); shoes one year after contact (Balch, 2; Ward, 47); instruments (Planchon, 38); leather hat bands (Leonard, 26); and firewood (Barnes, 3). Dermatitis caused by other plants is also sometimes attributed to Rhus; e.g., Cypripedium (Hurlbut, 19); eczema and other eruptions may also be confused with that caused by Rhus. CONCLUSIONS 1. The principal dermatitant of Rhus diversiloba is not volatile, for: (a) It is not distillable normally by steam or under reduced pressure. It can not be carried by entrainment with a volatile substance. (b) The smoke of the burning plant is not poisonous when filtered through glass wool at a high temperature. (c) Possible emanations from leaves are non-toxic when (1) the leaves are fastened on the concave side of a watch-glass and then to the skin of a susceptible person; and (2) when a current of air is blown over the leaves and caused to bubble through alcohol, the alcohol is non-toxic. May, 1921] MCNAIR — TRANSMISSION OF RHUS POISON 249 (d) Dermatitis occurs only on the area of skin to which the poisonous sap has been applied; a general irritation as by volatile irritants is not produced. (e) It does not diffuse rapidly in the skin, as is shown microscopically in sections of diseased skin. (f) The period of latency is too long. 2. Portions of the plant which do not cause dermatitis are: the pollen, the trichomes, the epidermis, the cork cells, and the xylem. 3. The poison is confined exclusively to the resinous sap. 4. Leaves decrease in malignancy in drying from the loss of fluidity of the sap and from the oxidation of the poison. 5. Poisoning without contact with the plant may occur from the smoke of the burning plant or by contact with substances that have the poisonous sap on them, such as clothing, shoes, cordwood, tools, the hair of animals, etc. 6. Dermatitis caused by other plants is sometimes attributed to Rhus. There is difficulty in distinguishing eczema from Rhus dermatitis. LITERATURE CITED 1. Acree, S. F., and Syme, W. A. On the composition of toxicodendrol. Jour. Biol. Chem. 2: 547. 1906-1907. 2. Balch, A. W. Poison ivy. Jour. Amer. Med. Assn. 46: 819. 1906. 3. Barnes, E. The poison ivy. Med. Rec. 30: 157-158. 1886. 4. Bennett, J. J. Plantae javanicae rariores descriptae iconibusque illustratae. Pt. 1, p. 60. London, 1838. 5. Bibb, L. B. Experimental Rhus poisoning. Texas Med. Jour. 30: 162-163. 1I914- IQI5. 6. Bogue, E. E. Garden and Forest. 1894. 7. Burrill, T. J. Some vegetable poisons. Amer. Monthly Micr. Jour. 3: 192-196. 1882. 8 Some vegetable poisons (Abstr.). Proc. Amer. Assn. Adv. Sci. 3: 515-518, 1882. 9. ——. Rhus poisoning. Garden and Forest 8: 368-369. 1895. 10. Cantrell, J. A. Unusual mode of transmission in a case of dermatitis venenata. Med. News 59: 484. 1891. 11. Cundell-Juler. The poison vine. Cincinnati Lancet and Clinic n.s. 11: 73-76. 1883. 12. Du Fresnoy, de V. Des caractéres, du traitement et de la cure des dartres et de la paralysie, etc., par l’usage du Rhus radicans, Vol. 1. Paris, an VII. 13. Fontana, F. Trattato de veleno della vipera de velem American 1: 148; 3: I14-117. Naples, 1787. Eng. transl. by Joseph Skinner. 2nd ed. 2: 181-184. London, 1795. 14. Frost, J. Remarks on the erysipelatous inflammation produced by the juice of Rhus Toxtcodendron. London Med. Physical Jour. 55: 116. 1826. 15. Frost, L.C. The bacterial etiology of poison oak dermatitis (Rhus poisoning). Med. Rec. 90: 112I-1123. 1916. 16. Hadden, A. Poison ivy or Rhus Toxicodendron. Med. Review of Reviews 12: 764. 1906. 17. Hubbard, S. Rhus poisoning. Med. Brief 32: 884. 1904. 18. Hunt, J. H. Rhus poisoning. Brooklyn Med. Jour. 11: 392-406. 1897. 19. Hurlbut, E. T. M. Antidote to poison oak. Calif. Homeopath 7: 235-239. 1869. 20. Ingenhousz, J. Experiments upon vegetables, discovering their great power of puri- fying common air in the sunshine, and of injuring it in the shade and at night. London, 1779. 21. Inui, T. Ueber den Gummiharz-Gang des Lackbaumes und seiner verwandten Arten (Abstr.). Bot. Centralbl. 83: 352. 1900. | 250 AMERICAN JOURNAL OF BOTANY [Vol. 8 22: 23. 24. 25. 26) P57 28. 29. 30. eum 22) 33. 34. 35: 36. side 28. 39. 40. Al. 42. 43. 44. 45. 46. 47. 48. 49. Khittel, J. Chemische Untersuchung der Blatter des Giftsumachs (Rhus Toxicoden- dron). Witt. Viertelj. Prakt. Pharm. 7: 348-359. 1858. Kipling, R. The female of the species. A study in natural history. Ladies Home Jour. 28: Li S1onre Kunze, R. E. Poison Rhus. Med. Tribune 5: 111-120. 1883. Lavini, M. Emanations délétéres qui émanent Rhus Toxicodendron. Jour. Chim. Méd. 1: 249-251. Paris, 1825. Leonard, W. W. Rhus poisoning. Med. Chron. 3: 21. 1884-1885. Lindley, J. S. Rhus poisoning. Amer. Jour. Dermat. and Genito-Urin. Dis. 12: 342-344. 1908. Mackie, W. W. Value of oak leaves for forage. Calif. Agr. Exp. Sta. Bull. 150. 1903. McMaster, J. B. Life and times of Stephen Girard. 1918. McNair, J.B. The transmission of Rhus poison from plant to person. Rhus diversiloba T.& G. Jour. Infect: Dis, 19: 429-432." 1916: ——. Pathology of dermatitis venenata from Rhus diversiloba. Jour. Infect. Dis. 19: 419-428. I916. {The poisonous principle of poison oak, non-bacterial. Med. Record 91: 1042- 1043. {1017. Maisch, J. M. Proc. Amer. Pharm. Assn. 13: 166-174. 1865. On the active principle of Rhus Toxicodendron. Amer. Jour. Pharm. 38: 4-12. 1866. Millon, de Revel. Recherches sur le gui de chéne, et nouvelles observations sur le sumac vénéneux, ou Rhus Toxtcodendron. Bull. Acad. Méd. Paris 26: 501-505. 1860-1861. Accidents-constatation d’immunité rapportée a la végétation. Jour. Méd. Chirug. et Pharm. Toulouse. Nov., 1862. Pfaff, F. On the active principle of Rhus Toxicodendron. Jour. Exp. Med. 2: 181-196. 1897. Planchon, L. Accidents causés par le contact du Rhus Toxicodendron (Terebinthacées Anacardiées). Montpel. Méd. II., 9: 61, 219. 1887. Priestly, J. Experiments and observations on different kinds of airs. p.324. London, 1774. Rost, E., and Gilg, E. Der Giftsumach, Rhus Toxicodendron L., und seine Giftwir- kungen. Ber. Deutsch. Pharm. Ges. 22: 296-358. I912. Schmiedeberg, O. Grundriss der Arzneimittellehre. 3te Aufl. pp. 213-224. 1895. Schwalbe, C. On the active principle of Rhus diversiloba (poison oak). Med. Record 63: 855. 1903. Schwalbe, K. Die giftigen Arten der Familie Rhus: R. diversiloba, R. Toxicodendron und R. venenata. Miinchener Medic. Wochenschr. 49: 1616. 1902. Thompson, W. Challenger Expedition. Jour. and Proc. Ham. Assoc. 8: 126. 1892. Van Mons, J.B. Mémoire sur le Rhus radicans. Actes Soc. Méd. Chirurg. et Pharm. 12: 130-837) eon Von Adelung, E. An experimental study of poison oak. Thesis Univ. Calif. Nov., 1912. Also Arch. Intern. Med. 11: 184. 10912. Ward, R. F. Severe ivy poisoning. N. Y. Med. Jour. 88: 1224. 1908. Warren, L.E. Some observations on the pollen of poison sumach. Amer. Jour. Pharm. 85: 545-549. 1913. White, J. C. On the action of Rhus venenata and Rhus Toxicodendron on the human skin. New York Med. Jour. 17: 225-249. 1873. Cae TYPE CONCEPT IN SYSTEMATIC. BOTANY! A. S. HitcHcocK (Received for publication January 8, 1921) The binomial system of nomenclature has been an important factor in the development of taxonomy. ‘The increase in the number of known species since the time of Linnaeus has been many fold; because of care- lessness and ignorance the number of names applied to the species of plants has been much greater than the number of species; the increase in our knowl- edge of genetic relationships and the diversity of opinions among botanists concerning generic limitations have still further increased the synonymy. The confusion arising from these causes soon emphasized the need of a code of nomenclature by which the naming of plants might be regulated. Many codes have been proposed, but only two have received the support of inter- national conferences: the Paris Code of 1867, and the Vienna Code of 1905. I have pointed out in another place (Science n. ser. 30: 597. 1909) that absolute stability in nomenclature is unattainable so long as botany is a growing science. The limits of genera will vary according to the knowledge and the opinions of individual workers, and the names of the plants as they are assigned to this or that genus will change in a corresponding degree. A universal code cannot bring about a permanent nomenclature, but it enables botanists to apply names according to definite rules, and this is all that we may expect of any code. The two codes mentioned have been a great help in stabilizing nomen- clature. Experience has shown, however, that they lack definiteness in directing the application of names, especially of generic names. In the early days of taxonomy a name was applied to a concept rather than to an entity. A generic name was based upon all the known species of the genus; a specific name was based upon all the known specimens of the species. When a genus was divided the original name was retained for one of the parts, usually the larger part, or was sometimes discarded altogether. The Vienna Code introduced many reforms, but the procedure for applying names when a genus or species was divided was still vague and uncertain in its application. ; About 30 years ago a new system began to receive serious attention among American botanists, the system of applying names by means of types. It is not my purpose here to give a history of this idea, but rather to point out some of the advantages of the system. The type concept lies 1 Read before the Systematic Section of the Botanical Society of America at Chicago, December 29, 1920. 251 252 AMERICAN JOURNAL OF BOTANY [Vol. 8 at the basis of modern botanical nomenclature. The type species of a genus or the type specimen of a species is the species or the specimen respec- tively that directs or controls the application of the generic or specific name. A generic name shall always be so applied as to include its type species; a specific name shall always be so applied as to include its type specimen. The old concept was that a genus was a group of species having a given combination of characters; a species, similarly, a group of specimens. The new or type concept is that, from the nomenclatural standpoint, a genus is a group of species allied to the type species, a species a group of individuals similar to the type specimen. If a genus or species is divided, that part which includes the type species or specimen retains the generic or specific name, be this part relatively large or small. The American Code? recognized the type concept as a fundamental principle. The Paris and Vienna codes do not refer to this prin- ciple. But the idea had made such headway by 1910 that it was recognized by the Brussels Congress in a recommendation as a guide for the future (an addition to Recommendation XVIII). This reads: [Botanists will do well, in publishing, to conform to the following recommendations: XVIII... ] XVIII dis. When one publishes the name of a new group, to indicate carefully the subdivision which is considered to be the nomenclatural type of the group; the type genus of a family, the type species of a genus, the type variety or the type specimen of a species. This precaution avoids the nomenclatural difficulties in the case where, in the future, the group in question comes to be divided. (Act. Congr. Internat. Bot. Brux. IQIO I: 105.) It is to be regretted that this recommendation was not made retroactive. I feel confident that the retroactive fixation of nomenclatural types is a fundamental necessity in stabilizing nomenclature. I feel confident also that this aspect of the type concept will appeal more and more strongly to the followers of the Vienna Code as its advantages are recognized, especially as there is nothing in the concept that is contrary to the principles of that code. One must carefully distinguish between the concept itself and the rules for its application. The American Code has recognized the principle of types and has also formulated rules for type fixation. One may accept the principle and reject these particular rules. The congress which adopted the Vienna Code appears to have been actuated by a desire to formulate rules that should, in a general way, preserve the current usage of generic names. I wish to point out to the followers of the Vienna Code that this laudable purpose can be accomplished with greater definiteness by applying the type concept than by applying the vague and uncertain rules adopted by the Vienna Congress. The Vienna Code contains the following rule: ArT. 45. When a genus is divided into two or more genera, the name must be kept and given to one of the principal divisions. If the genus contains a section or some other *Formulated in 1907 by a Nomenclature Commission of the Botanical Club of the American Association for the Advancement of Science. May, 1021] HITCHCOCK — THE TYPE CONCEPT 253 division which, judging by its name or its species, is the type or the origin of the group, the name is reserved for that part of it. If there is no such section or subdivision, but one of the parts detached contains a great many more species than the others, the name is reserved for that part of it. Let us apply this rule to the Linnaean genus Panicum. There are 20 original Linnaean species. Several of them, including P. miliaceum and its allies, belong to the genus Panicum as delimited by most modern botan- ists. Among the 20 are also P. italicwm and its allies, now generally dis- tinguished as Setaria or Chaetochloa. But Panicum ttalicum is the historic type of Panicum, that is, the species which was known as Panicum by pre- Linnaean authors and the one which I should interpret as, ‘‘judging from its name or its species, is the type or the origin of the group,” and therefore the segregated genus containing it should have retained the name Panicum. However, in the process of taxonomic and nomenclatural development of the various species involved, this procedure was not followed. If botanists wish to retain the name for the allies of Panicum miliaceum, the simplest method to insure this result is to select Panicum miliaceum as the type of Panicum. The Linnaean genus Holcus, presenting certain complications, illustrates the advantage of the type method. The name in pre-Linnaean literature was applied to the sorghums, but in the Species Plantarum Linnaeus unites with the three species of the sorghum group four other species of diverse rela- tionships, one of which is Holcus lanatus, the only one of the species belonging to Holcus as now recognized by European botanists. The Vienna Code provides (Art. 19) that It is agreed to associate genera, the names of which appear in this work [Species Plan- tarum] with the descriptions of them in the Genera Plantarum ed. 5 (1754). According to the Vienna Code (as well as to the American and Type- basis codes) the name Holcus should be applied to the sorghums and this I have done, since the author’s concept is most accurately interpreted by his own description. But when the aggregate included under Holcus by Lin- naeus in 1753 was divided, a century or more ago, the sorghumis and species of other genera were taken out and the name Holcus was left for H. lanatus, which until recently has generally borne that name. The followers of the Vienna Code have accepted current usage regardless of the rules of that code. Would it not be simpler and more definite to make an exception and to crystallize current usage by fixing Holcus lanatus as the type of Holcus? Examples could be multiplied indefinitely. Apparently the rules of the Vienna Code were left indefinite in order that botanists should not be too much restricted in the application of names and should have some freedom to.use personal judgment. It is impossible to foresee all contingencies and to provide for them by definite rules. As shown above, when, in par- ticular cases, the rules lead in the wrong direction they are likely to be ignored. The desired results can be accomplished with much greater 254 | AMERICAN JOURNAL OF BOTANY [Vol. 8 precision by using the type method. I commend to the followers of the Vienna Code the proposal that the International Rules be modified by a recommendation to the effect that the application of names be fixed by means of nomenclatural types, this to apply retroactively. The American Code provides for fixing the application of names by means of types. It goes further and provides rules for determining the type. It should be emphasized that the acceptance of the concept of types does not involve the acceptance of a particular set of rules for selecting types. The code formulated by the Committee on Nomenclature of the Botan- ical Society of America is called the Type-basis Code of Nomenclature. Like the American and the Vienna codes, the rules of the Type-basis Code are founded on the principle of priority. The rules for selecting types of genera and of species are in conformity with this principle, while, as stated previously, the Vienna Code omits altogether the rules for selecting types (though type appears incidentally in Art. 45 as indicated above). It will be seen then that the chief difference between the Vienna Code and the new Type-basis Code is that the one ignores the subject and the other formulates rules for selecting types. If the Vienna Code could be modified to include a set of acceptable rules governing the selection of types, the most impor- tant difference between the two codes would disappear. Attention should here be called to the fact that selecting the type of a group does not validate the name of that group. Types are selected for both valid names and synonyms. It only means that if a certain name is used it should be so applied as to include the type. I will review briefly the proposed rules for selecting the types of genera. I will pass over certain particular cases such as those in which there was but one species in the genus as originally published, or in which the type was designated originally, and refer to the troublesome cases where there were several species included in the genus as originally published. This is true of many Linnaean genera, and the typification of these is basic so far as stability of nomenclature is concerned. There was an attempt at one time to select arbitrarily the first species as the type. This would be definite, but would often run counter to the historic development of the group and would cause so many changes in names as to introduce serious and needless confusion. The new code provides for selection by applying the rule of reason, taking into consideration all the factors in each case. In preparing a recent bulletin I found it necessary to typify over 300 grass genera. I will select a few examples from these. If the genus was used in his earlier works, Flora Lapponica or Flora Suecica, the type should be chosen from among those in the Species Plantarum that are cited by Linnaeus as being in one of the earlier works, since these are the species with which he was 10re familiar. Under Andropogon in the Species Plantarum Linnaeus describes 12 species. The name Andropogon was first used in the Flora May, 1921] HITCHCOCK ——- THE TYPE CONCEPT 255 Leidensis where two species are described, both being included in the Species Plantarum. From these two Andropogon virginicus was chosen as the type because that species retained the name in its usual significance. The other species, A. hirtus, is now by many botanists referred to a different genus. Poa L. Linnaeus describes 17 species. He first used the genus in his Flora Lapponica. From among the species there described Poa pratensis is selected as the type because that retains the name of this economic species in its usual signification. Uniola L. Two species are described. One is referred now to Distichlis. The other is selected as the type, thus retaining the name in its current usage. Hordeum L. Six species are described. The reference in the Genera Plantarum is to figure 295 in Tournefort’s work, representing Hordeum vulgare, the common barley, which is therefore selected as the type. Aira L. Of the 14 species described four are included in the Flora Lapponica. ‘To take the first of these as the type would transfer the name Aira to what we now call Trisetum. Hence another one of the four, A. caespitosa, is selected in order to retain the name in its usual signification. Some botanists apply the name Aira to the last two of the 14 original species, including A. caryophyllea, and refer Atra caespitosa and its allies to Des- champsia. These two species are from southern Europe and were not included by Linnaeus in his first use of the term Aira in the Flora Lapponica, and hence did not represent Linnaeus’s original idea of the genus. In general, one should ascertain if possible what species or group of species an author had chiefly in mind in establishing a new genus. The application of the type concept to species is similar. If more than one specimen is cited, one should find which one the author had chiefly in mind. This may be shown by comparison with the description, by one having been selected for an illustration, by notes on the original sheet, by the specific name. Only when other methods fail should the first specimen cited be arbitrarily selected. The above illustrates what is meant by applying the rule of reason in the selection of types. Let us hope that soon all taxonomic botanists will accept the concept of types and that they may agree on the types to be selected. BUREAU OF PLANT INDUSTRY, WASHINGTON, D. C. ” THE RELATION OF CERTAIN NUTRITIVE EREMENaS. 1o THE COMPOSITION OF THE OAT EEzAne JAMES GEERE DICKSON (Received for publication January 8, 1921) The study of the relation of various environmental factors to the com- position of plants received its greatest stimulus when Emil Wolff published his studies on the analysis of plant ash to determine what constituents were to be found therein. Since that time an enormous quantity of literature has been contributed to the study, yet the work has never been satisfactorily concluded. Climate, availability of nutrients, water supply, and various other physico-chemical factors influence the composition of the straw greatly and of the grain or reproductive parts to a lesser extent. | W. Wolff (1864, 1865) and Hellriegel (1869) reported the first extensive study on the relation of mineral salts to plant composition. Not, however, until the voluminous work of E. Wolff (1871), did the study receive the attention of many of the best chemists in Germany. This pioneer study stimulated research until the investigations were taken up from several rather different yet related lines. Von Heinrich (1882), Atterberg (1886, 1887), Dikow (1891), Helmkampf (1892), Stahl-Schréder (1904), Jakouchkine (1915), and Sawine (1916) sought by analysis of the whole plant or of its several parts to determine the availability of the mineral nutrients in the soil. On the other hand, Lawes and Gilbert (1856, 1884), Pagnoul (1875), and more recently LeClerc and Leavitt (1910), Raymond and Paturel (1910), Hartwell and Wessels (1913 a, b), Tretiakov (1913), Headden (1916 a, 6b), Davidson and LeClerc (1917), and Maschhaupt (1918) have studied the relation of environmental factors, chiefly fertility and climate, to the composition of the whole plant and of its respective parts. Griffiths (1884), Takeuchi (1908), Chirikov (1914), and Waynick (1918) have extended the investigations still farther by studying the effects of the addition of specific substances, in many cases in varying amounts, to the composition of the plant. Kossowitsch (1909) has taken still another phase of the problem, investigating the composition of different plants grown under the same nutritive conditions. | The work referred to up to this time has been primarily a study of the influence of these factors upon the composition of the ash constituents. Although it is not within the scope of this paper to discuss the relation of environmental factors to the organic composition of the plant, yet the study would not be complete without reference to the important literature on this phase of the investigation. ‘Thacher (1913, 1917), Grisdale (1913), 256 May, 1921] DICKSON — COMPOSITION OF OAT PLANT 257 Tretiakov (1913), Headden (1916, a, 0), and others have studied the relation of nutrition and climate to the protein composition of plants. Wiley (1901) and Wilfarth and Wimmer (1903) have shown that fertilizers and climate cause a marked variation in the sugar content of the sugar beet. Seissl and Gross (1902) state that the starch content of the potato is changed quite markedly by the addition of fertilizers. Parrozzani (1908) and Jakouchkine (1915) find considerable variation in the organically combined phosphorus of plants when different fertilizers are employed. Garner (1914) and others have shown that the oil content of plants is modified by the addition of certain fertilizer elements to the soil. A review of this literature on the relation of plant environment to com- position brings out two very striking facts: first, that the plant as a whole responds quite markedly to environment by changes in its composition, and second, that for no two cases are these responses the same. Most of the work on the relation of fertilizers to plant composition has been done in the field where sufficient quantities of most of the elements have been present to supply the minimum needs of the plant; therefore, the changes in com-- position, especially in that of the seed, have not been very marked. The conclusion has thus been drawn that the composition of the grain, the reproductive part, is very constant, while the response or change in plant constituents takes place in the leaves and stems of the plants. It has been the purpose of the experiments herein recorded to study the effect of limiting certain essential nutrient elements upon the composition of the grain and straw of well matured plants when other environmental factors were controlled as far as possible. The culture work from May to August, 1915, and May to August, 1916, was carried on under climatic conditions different from those that governed the latter part of the work, which differences may in some cases explain the differences between the data recorded for the two respective periods. 0.1 Os el Oia | OG = Onn 222) I 220-0) 2:02=-0 or straw in the phosphorus- and nitrogen-deficient solutions. Therefore, the samples for analysis from these cultures were rather small, yet with special manipulation it was possible to secure results that checked very closely. 260 AMERICAN JOURNAL OF BOTANY [Vol. 8 In most cases, the quantity of plant material produced was too small to permit the analysis of the grain and straw for all the essential nutrient elements limited in the cultural series; therefore, it was necessary to select certain of these plant constituents which would represent elements having a very definite chemical combination with many of the complex plant substances, and certain others whose action was more or less secondary in nature. Phosphorus and calcium were finally selected, and determinations were made for total phosphorus and calcium present in the oat grain and straw, Analytical Methods The well matured grain and straw were ground to a finely powdered mass after drying at 90° C. for 12 hours, and stored in tightly stoppered bottles for analysis. After the samples were dried to constant weight at 110° C. and thoroughly mixed, samples were weighed out for calcium and phosphorus determinations. ‘Two-gram samples for grain and one-gram ‘samples for straw were taken for the calcium determinations, and one-gram samples of both grain and straw were used for phosphorus determinations. The official analytical methods as set forth in Bureau of Chemistry Bulletin 107, or standard-methods which had been carefully checked with the ‘official methods,’”’ were employed in. all cases. All analyses were run in duplicate, and, in case they did not check within 0.5 percent, were repeated. In the cases of three samples, however, of which there was insufficient material, only one sample was analyzed. The results given in the tables are averages of these duplicate, or in some cases quadruplicate, determinations. The samples for calcium determinations were ignited at low heat in a muffle until completely ashed, digested in hydrochloric acid, and finally filtered. Calcium was precipitated as calcium oxalate after removing the hydroxides of iron, aluminum, and phosphorus, and determined as CaO by titrating the oxalic acid with standard potassium permanganate. The phosphorus samples were analyzed by the sodium-peroxide method as originally described by Osborne (1902) and modified by Dubois (1905). The sample was placed in a nickel crucible and moistened until it formed a thick paste, after which five grams of anhydrous sodium carbonate were added and the charge was mixed immediately. Five grams of sodium perox- ide were then added in smaller portions at a time with thorough mixing after each addition. The mass was then heated until fusion was complete. Additional sodium peroxide was added to oxidize completely the organic matter. The mass was dissolved in concentrated hydrochloric acid, made up to 250-cc. and 100-cc. aliquot portions used for phosphorus determina- tions. These 100-cc. samples were evaporated to dryness over a water bath, taken up with hot water, strongly acidified with nitric acid, and phosphorus was determined by the “official gravimetric method.”’ May, 10921] DICKSON — COMPOSITION OF OAT PLANT 261 THE RELATION BETWEEN NUTRIENTS AND CALCIUM CONTENT OF GRAIN The general conception has been that there is very little variation in the composition of the seeds or reproductive parts of plants. Lawes and Gilbert (1884) state that the composition of the grain is not greatly varied by normal variations in soil composition. Their ideas of the limits of variation of the grain can best be stated in their own words: The composition of the grain only varies in any marked degree according to manure, when there is a very abnormal deficiency of one or more constituents, having regard to the amount of growth which is induced by the liberal supply of others. The composition of the grain is very uniform, notwithstanding there may be a very great excess of supply, and a relatively very great excess taken up by the plant, in which latter case a large excess remains in the straw. As previously stated, the earlier studies on composition have usually been made on plants which were grown under field conditions in soil of low or high fertility, or, in other words, where the supply of the available nutri- tive elements was not under strict limitations. It is evident from the data presented in table 4 that the calcium content of the grain varies greatly TABLE 4. The Average Calcium Content of Grain from Plants Grown in the Normal Solution and in Solutions with one Nutrient Element in Each Case Reduced to One Tenth Normal q ; Percent CaO in Grain Solution, Deficient Element eyes t915 Crop 1916 Crop 1917 Crop Average Nose re | O2I17, 0.405 1.360 0.694 oo On ee | 0.042 0.061 0.180 0.094 Lille? OST Te eee ae | 1.422 0.729 LS. O51 as te ana | 0.216 01227 1.760 0.734 POOR et tee | 0.275 0.284 0.540 0.367 RMON ie ke | 0.305 0.548 0.500 0.451 when the supply of available nutritive elements is limited to a definite small amount. The plants grown in culture solutions deficient in calcium—one tenth the amount in the normal culture solution—produced grain of very low calcium content, an average of ten percent lower than the calcium content of the grain produced in the normal solutions. Physiologists and biochemists consider the réle of calcium as secondary in the formation of seed. Therefore, possibly it may be replaced to a greater or less extent by certain other bases, notably potassium and mag- nesium. Some proof supporting this hypothesis is given in the fact that the average calcium content of the grain produced in magnesium-deficient and potassium-deficient solutions is relatively high. The low calcium content of the grain produced in potassium-deficient solutions during the first two years, however, would indicate that this is not true under all environmental conditions. The calcium content of the grain produced in the phosphorus- and nitrogen-deficient solutions during the first two seasons is quite high. 262 AMERICAN JOURNAL OF BOTANY [Vol. 8 These samples were rather small, however, and therefore the data are not as accurate as desired. The calcium content of the grain from the phos- phorus- and nitrogen-deficient solutions for the last year (1917 column, table 4) in which the total number of plants was greatly increased, making more material available for analysis, is considerably lower than in any of the other samples. The data for these last two series, namely, phosphorus- and nitrogen-deficient cultures, are too varied to draw any conclusions. In the case of the other samples, however, it is quite evident that a marked variation in the nutritive solution produces a very considerable change in the calcium content of the grain. It is held by some that calcium and magnesium function in the trans- location of carbohydrates and proteins and in the storage of these compounds during seed formation, rather than being directly connected with the syn- thesis of the carbohydrates and proteins that are later transferred to the seed. Calcium probably functions in this capacity much less than does magnesium, as analyses show a scarcity of calcium and an abundance of magnesium in most seeds. Magnesium probably functions, therefore, as the chief carrier of phosphoric and other acids entering into the chemical composition of the seed, while calcium acts more as a neutralizer of acids resulting from synthesis. Bernardini (1914) describes quite fully the functions of magnesium and its probable relation to translocation processes. This indirect function of calcium was first pointed out by Holzner (1867) and Schimper (1890), and more recently.has been supported by Chirikov (1914), Robert (1917) and others. Truog (1916) states that plants with a high protein content generally have a high calcium content and that when manganese phosphate is used instead of calcium phosphate as a source of phosphorus the plants grown in such a solution have an extraordinarily high manganese content. Robert (I9II, 1912) attempts to show that calcium is deposited directly within the fungus as a calcium salt of certain organic acids. She shows that’an in- crease of calcium in the culture solutions results in a very marked rise in the calcium content of the fungus. In general, a deficiency of calcium in the nutritive solution results in the production of grain with a very low calcium content, while on the other hand a deficiency of magnesium or of potassium in the culture solution causes a slight accumulation of calcium in the grain of the plants grown therein. The effect of a deficiency of either phosphorus or nitrogen generally results in the production of grain with a low calcium content. The calcium content of the grain of oat plants grown under varied nutrient conditions is consider- ably altered by the composition of the nutrient solutions in which the plants grow. May, 1921] DICKSON — COMPOSITION OF OAT PLANT 263 THE RELATION BETWEEN NUTRIENTS AND CALCIUM CONTENT OF STRAW The calcium content of the straw of the oat plant varies in a way in general similar to, although more marked than, that characteristic of the grain. Lawes and Gilbert (1884) have shown that the composition of the straw (leaves and stems) of various plants may be modified very markedly . by the addition of nutritive elements as well as by the subtraction of these elements. The results have been so consistent in field work of this nature as to lead certain investigators to suggest that the composition of the grain, the reproductive part of the plant, is very stable, while the variation, if any occurs, is in the straw and roots. The average percentages of calcium in the straw produced in the different culture solutions are given in table 5. TABLE 5. The Average Calcium Content of Straw from Plants Grown 1n the Normal Solution and in Solutions with One Nutritive Element in Each Case Reduced to One Tenth Normal Solution, Deficient Element ee irae oaks n Sewn a == CNen 1915 Crop | 1916 Crop 1917 Crop Average Monmal.............. 2.315 | 2.176 | 4.850 | 3.114 Ll Cli 0.520 0.220 0.485 0.408 LS Co nee a | 4.030 2.589 OOM he 2.362 | 1.666 | 4.260 2.763 MOM eke 3.175 | 1.072 | 0.935 L727 ROM a ek 1.288 1335 1.935 1.519 The average calcium content of the straw from the calcium-deficient solutions—containing one tenth of the calcium present in the normal solution —is only 13 percent of that of the straw produced in the normal cultures. The calcium content of the straw from the potassium-deficient solutions, al- though high comparatively speaking, is not as high as in the grain from the same set of cultures. The calcium content of the straw of the plants from the phosphorus-deficient cultures is considerably lower than that of the plants from the nitrogen-deficient solutions, and in both it is lower than the calcium content of the check plants. In general, a deficiency of calcium in the culture solution causes a very marked lowering of the calcium content of both grain and straw of plants grown therein. A deficiency of potassium or of magnesium does not greatly affect the intake and storage of calcium either in the grain or in the straw. A deficiency of either phosphorus or nitrogen causes a lowering of the cal- cium content of both grain and straw; especially is this true for plants in the phosphorus-deficient solutions. The average composition of the plants for the three years is given in table 8, and in graphic form in figure I. THE RELATION BETWEEN NUTRIENTS AND PHOSPHORUS CONTENT OF GRAIN Phosphorus, unlike calcium, enters into chemical combination with a great many of the plant compounds, especially with those of the seed. 264 ‘AMERICAN JOURNAL OF BOTANY [Vol. 8 Phospholipins, nuclein and nucleic acid, phytin, and possibly starch con- tain phosphorus in chemical combination. In addition to these organic compounds, some inorganically combined phosphates are stored in the seed, and often large quantities are deposited in the straw of the cereals. Probably, lecithin and phytin are the only organic compounds containing phosphorus which are accumulated as reserves in the seed, and which are, therefore, subject to variation due to the supply of phosphorus. Parrozzani (1908) found that the percentages of both lecithin and phytin phosphorus were increased by the addition of mineral phosphate fertilizers to the soil. The nuclein phosphorus, on the other hand, was quite constant, showing no change even with the most varied phosphorus fertilization. Jakouchkine (1915) stated that the amount of phytin in the grain is apparently dependent on the condition of the soil. Generally only small amounts of inorganically combined phosphorus are stored in the grain; therefore, if the total phos- phorus of the grain varies with varying amounts in the soil, it is probably due to the varying amounts of the organic reserve materials containing phosphorus. An organic analysis is necessary to disclose these relations and is planned in further pursuit of this problem. The composition of the grain from plants grown in culture solutions having a deficiency of certain nutrient elements, especially of phosphorus, is modified very markedly. The average percentages of total phosphorus in the grain from plants grown in the different culture solutions are given in table 6. TABLE 6. The Average Phosphorus Content of Grain from Plants Grown in the Normal Solution, and in Solutions with One Nutrient Element in Each Case Reduced to One Tenth the Normal Amount Solution, Deficient Element Fer cen Coe es 1g15 Crop 1916 Crop 1917 Crop Average Nionaial): ere oe ion 0.673 1.047 1.900 1.206 Gok Ox Sor reae oe oe 0.946 1.017 2.130 1.364 IAs oh OFS RAR so WR Ae 1.720 1.092 OSE OnT tee noniirea ts tek 0.625 0.963 1.665 , 1.084 Ep Oas byt ellen eres roe. 0.472 0.732 0.453 0.552 IN ONE Oe Ae ea eee ck. | 1.121 1.552 1.336 The phosphorus composition of the check plants which were grown in a complete nutrient solution varies over the three years’ experiments. These variations for the different years will be discussed later under another heading. The phosphorus content of the grain from the plants grown in the calcium-deficient solutions is the highest of any of the series, the average content for the three years being 0.158 percent higher than the composition of the grain from the checks. The explanation for this extra high phos- phorus content may be sought from two different sources: first, the calcium- magnesium ratio of the Knop’s solution may not be the best for maximum May, 1921] DICKSON — COMPOSITION OF OAT PLANT 265 translocation of phosphates, if, as is thought by a number of biochemists, magnesium is the chief carrier of phosphoric acid; and second, the calcium in the normal Knop’s solution may be present in sufficient quantity to react with the phosphates to form the less soluble tricalcium phosphate, thus making the phosphorus present less available for the plant. The nitrogen-deficient culture solutions, likewise, produced plants with grain having a very high total phosphorus content. In all probability the factors concerned here are very complicated, as the deficiency of proteins would be apt to upset normal metabolism and therefore to produce very abnormal results within the plant. It is quite possible that much of the phosphorus in this latter case may be stored as the mineral phosphate in combination with the large amounts of bases present. The nutrition of the plant in this case is undoubtedly very much disturbed and very abnormal. A deficiency of potassium in the culture solution, on the contrary, results in the production of grain with a low phosphorus content, the average phosphorus content for the three years being 0.122 percent lower than in the check. The greatest reduction in phosphorus content, however, is in the grain from phosphorus-deficient solutions. The phosphorus content of this grain is reduced to 46 percent of the phosphorus present in the grain from the checks. In all probability the reduction took place in the amount of phytin and lecithin stored in the grain. In general, then, there is a very marked variation in the phosphorus content of grain produced under different nutritive conditions. A deficiency of phosphorus or of calcium causes the greatest variation, the lack of the former element pro- ducing a very marked decrease, that of the latter a moderate increase in phosphorus content. THE RELATION BETWEEN NUTRIENTS AND PHOSPHORUS CONTENT OF STRAW The composition of the straw generally shows more marked variations than that of any other part of the plant, unless it be the roots, for the TABLE 7. The Average Phosphorus Content of Straw from Plants Grown in the Normal Solution and tn Solutions with One Nutrient Element in Each Case Reduced to One Tenth the Normal Amount Solution, Deficient Element BCU ea ein Stay pen 1915 Crop 1916 Crop 1917 Crop Average Cae) 0.309 0.734 0.832 0.628 1 Ot 0.415 0.354 1.230 0.667 rane 0.892 0.673 O52 0.265 0.334 0.682 0.427 OS 0.092 0.060 0.051 0.068 Ot 0.834 1.750 1.805 1.463 unassimilated excess salts are stored here in the case of the addition of large amounts of specific elements, and on the other hand the straw releases 266 AMERICAN JOURNAL OF BOTANY [Vol. 8 the deficient substances quite rapidly in order that they may be stored in the reproductive parts of the plant. A wider variation in the composition of the straw is, therefore, to be expected. The results, as set forth in table 7, show this to be true. The variations in composition due to varied nutritive conditions are similar to, though more marked than, those of the grain. The extremely high phosphorus content of the straw from the nitrogen-deficient solutions confirms the idea that the phosphorus meta- bolism is blocked by the deficiency of proteins, and that, therefore, phos- phorus in the inorganic form piles up to some extent in the grain, but more markedly in the straw. The phosphorus content of the straw produced in the phosphorus-deficient culture solutions is very low, the average for the three years being approximately Io percent of the amount present in the straw from the normal solutions. The variations in the composition, although more pronounced in the straw, yet in general are identical with those in the grain. TABLE 8. The Average Composition of Plants Grown in the Normal Solution and in Solutions with One Nutrient Element in Each Case Reduced to One Tenth the Normal Amount a Solution, Deficient Element a es Pouce! 20 Hercent EG Given Grain Straw Grain Straw Normal: 54) Gar exyeckion 0.697 euabit 1.206 0.628 C2 IS(01) Sayep aero 8 nantes 0.097 0.408 1.364 0.667 IMIGNOM «Fas 3 ade ine oe 0.729 2.589 1.092 0.673 KGS.) eee 0.734 2.763 | 1.084 0.427 a AAO ol x's ce genep me ee vee 0.366 1.727 0.552 0.068 IN OUT ah aoa eae aie 0.451 1.519 1,330 1.463 Insummation, comparing the composition of the plants from the modified solutions with the composition of those from the normal, a deficiency of phosphorus in the culture solution causes a very marked lowering of the phosphorus content of both grain and straw of the plants grown in these solutions. A deficiency of either calcium or nitrogen causes an increased intake of phosphorus especially in the straw. A deficiency of potassium causes a slight reduction in the amount of phosphorus accumulated in the grain and a more marked reduction of phosphorus in the straw. Magnesium deficiency in the culture solutions causes a slight decrease of phosphorus in the grain of plants grown in these solutions, and a small increase in the phosphorus content of the straw. The average composition of the plants for the three years is given in table 8, and in graphic form in figure 1. THE RELATION OF CLIMATE TO THE COMPOSITION OF THE OAT PLANT The experiments of Lawes and Gilbert (1884), Cserhati (1908), Grisdale (1913), and Tretiakov (1913) have shown very strikingly that the factors of climate and geographical location cause a wide variation in the composi- tion of plants. Lawes and Gilbert (1884) explain these variations by devia- — May, 1921] DICKSON — COMPOSITION OF OAT PLANT 267 tions from the normal maturation of the plant. They sum up their results very well in the following statements: The character of the crop left to ripen depends very much more upon season than upon manuring. There is scarcely any difference in the composition of the truly and normally ripened seed. The wide range in the composition of the ash of the grain represents a cor- responding deviation from the normal development. GITAIN = S > S ag . NORTT Ca 0/ 11g O/ CULTUNE SOLUTIONS Fic. 1. Curves showing the average composition of grain and straw of plants grown in the normal solution and in solutions with one nutrient element in each case reduced to one tenth the normal amount. Likewise LeClerc and Leavitt (1910) attribute the variations in com- position of wheat primarily to different climatic conditions and to the effect of these climatic factors upon plant growth and maturation. It is undoubt- ‘edly true that environmental factors which alter normal development and maturation have an extremely great influence upon the composition of 268 AMERICAN JOURNAL OF BOTANY [Vol. 8 crops grown in the field or in artificial culture. In artificial cultures, however, it is possible to alter the growth and maturation of the plant much more by the deficient nutrients than by climatic factors; therefore, it is reasonable to suppose that the deficient, nutrient may have as much influence upon the composition as the climatic variations. This is not, however, minimizing the effect of climate, and such environmental factors must be taken into consideration in all studies of this sort whether they be field or culture experiments. The analytical data previously discussed in connection with nutrition, when studied from another angle, that of the relation of climate and geo- graphic location to composition, show how closely the influences of these various factors are interrelated. The combination of temperature, humid- ity, and other climatic conditions greatly influenced the composition of the plants except when the reduction of certain essential nutritive elements became the predominant factor in controlling plant development. Under these conditions, variations due to other environing factors were outweighed by the deficient nutritive element. TABLE 9. The Average Composition of Plants grown under Different Climatic Conditions Composition of Plants in Percentage Based on Dry Weight Pullman, Washington | Madison, Wisconsin 1915 1916 1917 Average composition from all cultures CaO icontentotserain.-ie0 u3.50. 6. oe ee 0.231 0.305 0.960 eo OsrcOntent:Oimeralliy.. daha so oo ee ee 0.679 0.976 1.570 CaQOlcqntent-olistrawens2. 2 oa6. 5.2 see ee 1.932 1.294 2.416 i220) ;contentzonstrawe si%.2: | sae nas tne eee 0.383 0.646 0.915 Composition: of, check plants... | 68 Orr ee.s *. 60 42 48 55 60 61 60 7G 75 70 | 60 | 62 On hiss: 55 37 46 53 52 64. 62 67 69 75a O2. || 662 DOs ess. 48 27 50 49 49 68 60 61 66 77 | 66 | 64 ees 53 40 48 48 51 67 66 68 65 70°67. | 70 Diet. 51 44 50 54 a 71 65 74 O5e i 7s |) 70) | 04 123. aan 52 43 50 59 61 61 57 63 67 72 | 69 | 65 Tee 51 46 57 61 65 50 53 64 68 76 | 71 | 68 De aero l 's 49 51 68 62 69 52 58 71 70 77 ©4- | 72 MOMs... 58 56 68 61 73 56 63 69 66 ee 55 58 70 63 76 66 56 52 68 Sees. 4. 49 51 69 51 60 71 63 54 70 BOLL... Ae et OS ESS Avena OAT 09 i O2° (| “74 PO ee. 3s 50 45 50 60 45 64 73 66 76 el a 54. 42 42 63 42 67 82 66 78 22...... ; 30 52 40 70 54 59 79 65 74 eR 55% 52 53 44 We 61 59 72 64 76 Aime. x -,s 54 54 52 65 60 61 74 66 76 Be ss 52 59 56 59 66 68 WE 60 80 2 53 53 51 54 58 We 69 59 81 Bees... 61 55 52 58 59 66 66 61 75 2 57 50 54 62 49 60 66 61 78 Boils). 51 48 60 59 50 68 56 64 85 Oa: |, 58 55 55 60 63 55 70 65 71 86 Se eae 62 50 62 — = — 68 71 84 Average...! 53 Ke) 52 59 56 Ge WB 63 7 2e ea | 65 | 69 27.0 AMERICAN JOURNAL OF BOTANY [Vol. 8 TABLE 11. The Average Monthly Air Temperature at Pullman, Washington, and Madison, Wisconsin, for the Duration of the Cultural Experiments Temperature °F. at Pullman, Temperature °F. at Madison, Mn ashington Wisconsin on 1915 1916 1917 IMA yeh os 2 a, an ame aati Solid) De | 53 49 52 qune sah Ga as Oar ee CGN AE ee 59 5° 62 Uy ion oA ae ea. ears 3 72 AUISUSt ck ey re. eer | 74 65 68 | * The August temperatures are from the first to the fifteenth inclusive. TABLE 12. The Average Monthly Precipitation at Pullman, Washington, and Madison, Wisconsin, for the Duration of the Cultural Experiments | Rainfall in Inches at Pullman, | Rainfall in Inches at Madison, | Washington Wisconsin Month | IQI5 1916 1917 DY Fay ici carted ss a eee | 2G, 1.56 3.33 supe aa een eR Rt ee | 0.53 2.34. 6.47 Ail yee aS Rae os oc ew oe i OF7-7 | 0.45 3.10 PAMIOMIGEN «22 Ages aeaiieet sn V2 an, ae ee ee 0.00 | 1.24 ZN The important meteorological data for the periods during the growth and maturation of the plants are given in tables 10 and 11 and in graphic form in figure 2. The climatic differences for the three years, the first two at Pullman, Washington, and the last at Madison, Wisconsin, are sum- marized as follows: The 1915 growing season was very dry and cool, with high temperatures during maturation; the I916 season was moderately dry and cool; the 1917 period was very wet and hot. The light intensity during the last year was considerably lower than during the first two seasons. No attempt is made to correlate the variations in composition with any individual factor, for it is impossible to do more than speculate until con- trolled experiments have been run to determine the relation of such factors as light intensity, air and soil temperature, and humidity on the develop- ment and composition of plants. SUMMARY i. The calcium content of both grain and straw is reduced to about 10 percent of that of the plants from the controls by reducing the calcium in the culture solution to one tenth the quantity present in the complete nutrient solution. It is greatly reduced in both grain and straw by a similar deficiency in phosphorus or in nitrogen. 2. The total phosphorus content of the grain is reduced to 46 percent, and of the straw to 10 percent, of that in the plants from the controls by reducing the phosphate in the culture solution to one tenth of the quantity May, 1921] DICKSON — COMPOSITION OF OAT PLANT 271 present in the complete nutrient solution. It is slightly reduced in both grain and straw by a similar deficiency in potassium, and is increased by a similar reduction of calcium or nitrogen. 7 yy \ \ \ ‘ \ Beet | ake wm SLASON IHS we amm SEASONING SEASON INT DUMATION OF EXFERIMENTS a L = = é : Ko S ) ee — = ~ NS = NS Fic. 2. Graphic representation of the average monthly temperatures and average monthly precipitation at Pullman, Washington, and Madison, Wisconsin, for the duration of the cultural experiments. 3. Although the variations in composition are more pronounced in the straw, yet in general they are similar in both grain and straw. 272 AMERICAN JOURNAL OF BOTANY [Vol. 8 4. The phosphorus content of both grain and straw is modified by sea- sonal differences, except for the plants grown in the phosphorus-deficient solutions. The calcium content of the grain is modified by seasonal differ- ences even in the calcium-deficient solutions. The calcium content of the straw, however, shows no consistent response to climate. UNIVERSITY OF WISCONSIN LITERATURE CITED Atterberg, A. 1886. Die Beurtheilung der Bodenkraft nach der Analyse der Hafer- pflanze. Landw. Jahrb. 15: 415-419. ——. 1887. Die Beurtheilung der Bodenkraft nach der Analyse der Haferpflanze. Landw. Jahrb. 16: 757-761. Bernardini, L. 1914. Funzioni biochimiche del magnesio nella vita della pianta. Scuola Sup. Agr. Portici II, 12: 361-389. Chirikov, T. W. 1914. Zur Frage tiber die lésende Wirkung der Wurzeln. Russ. Jour. Exp. Landw. 15: 54-65. Cserhati, A. 1908. Die Faktoren, welche auf die Zusammensetzung des Weizens von Einfluss sind. Kiserlet. K6zlem. 11: 253-275. Davidson, J., and LeClerc, J. A. 1917. The effect of sodium nitrate applied at different stages of growth on the yield, composition, and quality of wheat. Jour. Amer. Soc. Agron. 9: 145-154. Dickson, J. G. 1918. The value of certain nutritive elements in the development of the oat plant. Amer. Jour. Bot. 5: 301-324. Dikow, U. 1891. Beurtheilung des Bodens nach den Wurzeln der Gerstenpflanze. Jour. Landw. 39: 134-147. Dubois, W. L. 1905. Determination of sulphur and phosphoric acid in foods, feces and urine. Jour. Amer. Chem. Soc. 27: 729-732. Garner, W. W. 1914. Oil content of seeds as affected by the nutrition of the plant. Jour. Agr. Res. 3: 227-249. Griffiths, A. B. 1884. Experimental investigations on the value of iron sulphate as a manure for certain crops. Chem. Soc. Jour. 45: 71-75. Grisdale, J. H. 1913. Field crops work at the Canadian Experiment Stations and Farms in I912. Can. Exp. Farms Repts. (1912): 47-53, 227-245. Hartwell, B. L., and Wessels, P. H. i1913¢a. The effect of sodium manuring on the composition of plants. R. 1. Agr. Exp. Sta. Bull. 153: 1-118, 19130. The effect of soil phosphorus on turnips as influenced by the amount available in soils. R. I. Agr. Exp. Sta. Bull. 154: 1-148. Headden, W. P. iI916a. A study of Colorado wheat II. Col. Agr. Exp. Sta. Bull. 2175 3-46. ——. 1916). A study of Colorado wheat III. Col. Agr. Exp. Sta. Bull. 219: 3-131. von Heinrich, R. 1882. Grundlagen zur Beurtheilung der Ackerkrune. pp. 77. Hellriegel, F. H. 1869. Ueber die Bedeutung der chemischen Untersuchung der Ernte- producte, namentlich der Aschenanalysen, fiir die Beurtheilung der Menge und des gegenseitigen Verhaltnisses der im Boden vorhandenen aufnehmbaren Pflanzen - nahrstoffe. Landw. Versuchsst. 11: 136-144. Helmkampf, A. 1892. Untersuchungen iiber die Feststellung des Diingungsbediirfnisses der Ackerbéden durch die Pflanzenanalyse. Jour. Landw. 40: 85-183. Holzner, J. 1867. Ueber die physiologische Bedeutung des oxalsduren Kalkes. Flora 50: 497-5SII. Jakouchkine, I. 1915. Phosphore facilement extractif et suffisance de la nutrition phosphatée. Communication préliminaire. Russ. Jour. Exp. Landw. 16: 118-139. May, 1921] DICKSON — COMPOSITION OF OAT PLANT 273 Kossowitsch, P. 1909. Pflanze, Phosphorit und Boden nach Versuchen des Landwirt- schaftlichen Chemischen Laboratoriums zu St. Petersburg. Russ. Jour. Exp. Landw. 10: 782-842. Lawes, J. B., and Gilbert, J. H. 1856. The composition of the wheat grain and its products. Trans. Brit. Assoc. Adv. Sci. 26: 173. 1884. On the composition of the ash of wheat grain, and wheat straw, grown at Rothamsted, in different seasons, and by different manures. Chem. Soc. Jour. 45: 305-407. ; LeClerc, J. A., and Leavitt, S. 1910. Tri-local experiments on the influence of environ- ment on the composition of wheat. U.S. Dept. Agr. Bur. Chem. Bull. 128: 1-18. Maschhaupt, J. G. 1918. De involed van grondsoort en bemesting op het gehalte onzer culture gewassen aan stikstofen aschbestauddeckn. Verslag. Landbouwk. Onder- zoek. Rijkslandbouwproefstat (Netherlands) 22: 25-116. Osborne, T. B. 1902. Sulphur in protein bodies. Jour. Amer. Chem. Soc. 24: 140-167. Pagnoul, M. 1875. Sur le réle exercé par les sels alcalins sur la végétation de la betterave et de la pomme de terre. Compt. Rend. Acad. Sci. (Paris) 80: 1010-1014. Parrozzani, A. 1908. Influenza di quantita progressive di concimi fosfatici sul contenuto in sostanze organiche fosforate ed azotate, e sul rapporto fra fosforo ed azoto dei semi di mais. Staz. Sperim. Agrar. Italy 41: 729-738. Raymond, B., and Paturel, G. 1910. Del’influence des engrais chimiques sur la composi- tion des graines de céréales. Prog. Agr. et Vit. (Edl. Est-Centre) 53: 777-780. Robert, Thérése. i911. Influence du calcium sur le développement et la composition minérale de l’ Aspergillus niger. Compt. Rend. Acad. Sci. (Paris) 153: 1175-1177. —. 1912. Mode de fixation du calcium par l’ Aspergillus niger. Compt. Rend. Acad. Sci. (Paris) 154: 1308-1310. —. 1917. Le réle physiologique du calcium chez les végétaux. Rev. Gen. Sci. 28: IOI—108. Sawine, P. 1916. A propos de la définition de la fertilité des sols au moyen de I’analyse des plantes. Russ. Jour. Landw. 17: 1-12. Schimper, A. F. W. 1890. Zur Frage der Assimilation der Mineralsalze durch die griine Pflanze. Flora 73: 207-278. Seissl, J.,and Gross, E. 1902. Ueber den Kali- und Phosphorsduregehalt der Blattaschen verschieden starkereicher Kartoffelsorten. Zeit. Landw. Versuchsw. Oéesterr. 5: 862-875. . Stahl-Schréder, M. 1904. Kann die Pflanzenanalyse uns Aufschluss tiber den Gehalt an assimilierbaren Nahrstoffen im Boden geben? Jour. Landw. 52: 31-92, 193-268. Takeuchi, T. 1908. On the absorption of varying amounts of lime and magnesia by plants. Bull. Coll. Agr. Tokyo 7: 579-581. Thacher, R. W. 1913. The chemical composition of wheat. Wash. Agr. Exp. Sta. Bull. irr. I-79. Thacher, R. W., and Arny, A.C. 1917. The effect of different rotation systems and of fertilization on the protein content of oats. Jour. Amer. Soc. Agron. 9: 344-348. Tretiakov, S.S.F. 1913. The influence of the mode of cultivation on the chemical com- position of the grains of cereals. Trudy Poltav. Selsk. Khoz. Opytn. Stantsii 12: 28-44. Abstr. in Russ. Jour. Landw. 15: 83-84. Truog, E. 1916. The utilization of phosphates by agricultural crops, including a new theory regarding the feeding power of plants. Wis. Agr. Exp. Sta. Res. Bull. 41: I-50. Waynick, D. D. 1918. The chemical composition of the plant as further proof of the close relationship between antagonism and cell permeability. Univ. Cal. Publ. Agr. Sci. 3: 135-242. Wiley, H. W. 1901. The influence of environment upon the composition of the sugar beet. U.S. Dept. Agr. Bur. Chem. Bull. 64: 1-64. ——_— —_— 27a: AMERICAN JOURNAL OF BOTANY [Vol. 8 Wilfarth, H., and Wimmer, G. 1903. Die Kennzeichen des Kalimangels’an den Blattern der Pflanzen. Zeitschr. Pflanzenkrankh. 13: 82-87. Wolff, E. 1871. Aschen Analy, -sen 1: I-149; 2: I-170. Wolff, W. 1864. Die Saussereschen Gesetze der Aufsaugung von einfachen Salzlésungen durch die Wurzeln der Pflanzen. Landw. Versuchsst. 6: 203-230. —. 1865. Chemische Untersuchungen tiber das Verhalten von Pflanzen in der Auf- nahme von Salzen aus Salzlésungen, welche zwei Salze gelést enthalten. Landw. Versuchsst. 7: 193-218. ne nee SLORABO fe _ Location: "Boulder, Ootstade:t at! hope hed i mountain front of the Rockies; en- ‘ ee .\ vironment “favorable for study, for eS \ Feld “work, . and for outdoor recrea- _ tion. ; Easy access to all of the life / pocusigen ‘from Plains: ue alpine: holes A Bot glaciers.) Rie inh Bota, ee en Pen © ; ology, Botany of Ornamental Esper , Ecology, Local’ Flora, Forest: Botany, oy Gollese “Beonom ¢ Boas zy. 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Allen; University of ‘Wisconsin, Madis ion, Wis onsin, a Business correspondence, ‘including of | 2 _ concerning reprints, should be addressed to ee pean: gay Brooklyn, N. AX. 2Or Fat AMERICAN JOURNAL OF BOTANY VoL. VIII JUNE, 1921 No. 6 SPECIALIZATION AND FUNDAMENTALS IN BOTANY! JosEPpH CHARLES ARTHUR (Received for publication January 10, 1921) It was my pleasure and good fortune to assist in the launching of the parent society of the present Botanical Society of America, an event which took place somewhat more than a quarter of a century ago. American botany was a lusty youngster among the sciences at that time, but was not generally regarded as capable of doing a man’s work in this work-a-day world. It was in good repute within limited circles, but was not consulted when large enterprises were in hand. Even fellow botanists abroad felt no compelling inclination to recognize the work done in America. Realizing that this condition ought not to continue, steps were taken to organize a society which should exemplify the best thought and endeavor of American botanical activity, and especially should promote higher attainments and a greater amount of original investigation. In order to finance the movement the members were willing to tax themselves with heavy annual dues. The results were increasingly encouraging. After a decade the society united with others into the present more democratic, less burdensome, and more diversified organization, which now stands as the peer of any association of its kind, either at home or abroad. It was a happy thought to introduce a banquet into the annual program of the society. There may be those who do not see how eating a good meal once a year in the presence of one’s associates can aid materially in increasing the amount and quality of scientific knowledge or give a keener zest to the pursuit of discovery. They overlook the subtle relation that exists between bodily good cheer and intellectual elation. Undoubtedly the employment of savory viands to promote fellowship is as old as the habit of eating, and why should not the same agency be carried a step further and made to promote the cause of research? I speak as if it were a new idea. Yet at a date ten times as long ago as the life of this and its parent society the great investigator, Harvey, discover of the circulation of the blood, took this means to increase interest in research. In 1656, a year before his death, he gave his estate of Burmarsh in Kent to the Royal College of Physicians of London. In doing so he stipulated that once a year a general feast should 1 Address of the retiring president of the Botanical Society of America, read at Chicago, December 29, 1920. [The Journal for May (8: 231-274) was issued May 24, 1921.] 275 276 AMERICAN JOURNAL OF BOTANY [Vol. 8 be held within the college, and on that day an oration should be delivered exhorting the fellows and members to search and study out the secrets of nature by way of experiment, and also, for the honor of the profession, to continue in mutual love and affection among themselves. Clearly there is illustrious and time-honored precedent for the Botanical Society of America to issue its invitations to “‘come and eat four grains of rice,’ as the hospitable dweller in Venice would phrase his most cordial request for your presence at dinner. I have chosen for my remarks this evening a title so inclusive that, to relieve apprehension regarding any intention to be encyclopedic, I feel it incumbent to state at the outset that it is only a camouflaged sophomoric trick to secure the opportunity for more or less disconnected comments, although on that account, I trust, not less timely or weighty. I propose to speak from the point of view of the investigator and advanced student, rather than from the more usual pedagogic one of the schoolmaster or pupil. But first of all permit me to revert to Harvey’s suggestion that for ‘‘the honor of the profession”’ the members cultivate ‘mutual love and affection.” In the earlier days of botanical organization in America, the kind of organi- zation that first attempted to embrace the length and breadth of the country, the inspiration for which came through the American Association for the Advancement of Science, there was a predominant homogeneity of sentiment and good will with mutual confidence. That was a period not so remote as to be beyond the memory of some of us. In the main that condition still exists. If in some particulars it has been violated, a remedy could be and should be applied. When making a tour of European universities and experiment stations some thirty or more years ago, I was particularly struck by the reluctance of many botanists in the German institutions to speak openly about their unpublished investigations. There seemed to be a feeling that, should they disclose any part of what they had accomplished, or had in mind to under- take, some colleague might rush into print and deprive them of their honors. It was not the precaution demanded in an older and more densely settled country against the irresponsible and lawless, causing the Germans to put two locks on each door, while in my western home we did not turn the key in the one lock that might happen to be there. It was rather a distrust of one’s fellow workers, a state of mind we have learned to associate with a certain type of bureaucracy; and every German professor was at that time a government official. It seemed to me most absurd and uncalled for, quite unbecoming highminded, conscientious, and trustworthy men of science. Since then, some of the same spirit of exclusiveness and distrust, possibly with a tinge of selfishness, has occasionally become manifest in American botanical circles, and it is not surprising to find that it crops out most from government centers. It would be natural to suppose that those who are paid and supported in their scientific work from funds derived June, 1921] ARTHUR — SPECIALIZATION AND FUNDAMENTALS 277 impartially from all the people would feel that their first obligation was to the public, at least to the scientific part of the public. But such is the insidious influence of centralized power, or, as G. R. Lyman says, ‘“‘the zeal of public service,” that in some quarters the wealth of opportunity and material are guarded with miserly oversight for the advancement of par- ticular workers and the prestige of the organization. I am not speaking of the individual. There are always a few lacking in a nice sense of propriety and the distinction of meum and tuum, who must be guarded against. My experience leads me to believe, however, that they are few in botanical circles, both in this country and abroad. What I have in mind is the spirit of exclusiveness, the dog-in-the-manger policy, which should be frowned upon, and so far as possible eradicated from all efficient and truly demo- cratic centers for scientific work. Otherwise, how are we to bear out Har- vey’s admonition ‘‘to continue in mutual love and affection” for ‘the honor of the profession?’’ The man of science should be able to appreciate and exemplify what Chaucer means when he says of his knight, that he loved chivalry, Truth and honor, freedom and courtesy. At the present day all botanists are specialists. The expansion of the science under many diversified heads, linking it up with other sciences, and the desire to excel in some restricted field, rather than to be content with the level of general information, doubtless explains much of this tendency. With increase in the number of botanical workers has come an increase in the number of organizations bearing distinctive titles. So speedy and intense has been this movement that it has carried some of the younger members of the profession quite off their feet, and in the rarefied atmosphere of their new environment they no longer see the solid earth from which the maintenance of their strength must come. Oh, no! they say, | am not a botanist, I am a pathologist, an ecologist, a geneticist, or what not. Let us hope that the allurements of the silver-lined clouds of science will not keep them from eventually considering the mists that bedew the earth and renew its verdure. Probably the most fundamental thing that, the specialist is likely to neglect is an intimate acquaintance with the plants with which he deals. When the distinctive instrument of the botanist’s labors was the vasculum, now rarely seen, and to be credited with a knowledge of elementary botany required one to pass in fifty named and mounted specimens, only the indolent missed a suitable basis for botanical advancement. When the microscope became the botanist’s chief instrument, the foundations began to be neglected in the construction of the multivaried details of the super- structure. With the advent of other instruments of research, the microtome, the auxanometer, the atmometer, etc., attention was directed more and more away from the individual plant as an interesting inhabitant of the 278 AMERICAN JOURNAL OF BOTANY [Vol. 8 organic world. The study of structure, physiology, or behavior has often proceeded with inadequate knowledge of the position that the particular plant under observation holds in relation to other plants, its resemblances and differences as compared with its kin, near and remote. When Gray’s Lessons in Botany was superseded by the general textbook that began with slime molds and ended with sunflowers, there did not seem to be time to make the acquaintance of particular plants, and when a little from each topic pertaining to the varied structure and action of plants from the cytology of the cell to the sensitiveness of a tendril had to be interpolated, the plant as an individual member of an evolutionary group was overshad- owed. The great English botanist, Sir Joseph Hooker, once wrote to Dr. Asa Gray: I content myself with a casual grin at young men calling themselves botanists, who know nothing of plants but the “innards” of a score or so. The pendulum will swing round, or rather back, one day. It is already on the way; let us hasten the movement. The botanist’s realm is the vegetable kingdom. As the man of the world is able to assign each person he meets to a particular race, country, or section, with more or less accuracy, to have some individual acquaintance with a few here and there, but knows only those within a limited circle sufficiently well to call them by name and to be familiar with some of the facts of their history, so the botanist should have a general knowledge of plants of all countries sufficient to enable him to place most of those coming to his attention within certain orders or families, to know a few by name, and with those he meets frequently, especially the flowering plants, to have the same familiar acquaintance of name, characteristics, and behavior, which he prizes for his speaking friends. In the realm of plants the botanist has a distinct advantage over the man of the world, for he has manuals which enable him to ascertain the name of the plant he wishes to know, and to be unfamiliar with such manuals is to write oneself down inadequately equipped for his duties. My attention has been most frequently called, perhaps, to the shortcomings of the cytologist, who essays to throw light on the relationship of parasitic fungi by a discussion of his observations without taking full precaution to make sure of the exact identity of the material he has used, or of the kinship of the forms he has selected for comparison. In this way laborious and extensive studies may fail to exert due influence, and may have their chief value confined to a record of the particular obser- vations. But no class of investigators need be credited with an unwar- ranted share of haphazard interest in the exact identity of the plants handled. I fancy the bacteriologist and the paleobotanist have the best reasons for being uncertain. There is a joke, with which you may be familiar, that, when puzzled about the affinities of a plant, “‘fossilize it and send it to a paleobotanist, and he will give you the genus and species at once.’’ The students of microfungi are not to be outdone in this particular. Among June, 1921] ARTHUR — SPECIALIZATION AND FUNDAMENTALS 279 parasitic forms the relation of the fungus to the host is very intimate, and the identity of the one often involves that of the other. Examples are numerous where the name of the host has been adopted for the fungus grow- ing upon it, only to learn later that due care had not been exercised by the original collector and that the host was not what it purported to be, the name thus becoming a misnomer. In general, probably, a considerable percentage of the inaccuracy and misinterpretation in various fields of botanical science is traceable to a lack of intimate acquaintance with plants as living objects having distinctive names and varied relationships. While systematic botany may never again have the place of honor in the curriculum that it held in the post-Linnaean days when Jean Jacques Rousseau wrote his delightful letters on the elements of botany, and coming down to the days of our own beloved Asa Gray, yet no man who essays to explore the domain of plant knowledge, whether as student, investigator, or philosopher, can afford to be without an understanding of its main tenets, based upon spirited contact with the plants of the field and upon ability to localize and identify individuals engaging his attention. As somewhat of an accompaniment to these thoughts, but quite as an independent theme, I bespeak consideration to the matter of names. So fully have the taxonomists been shoved aside in recent years that devotion to the task of disentangling, rectifying, and correctly assorting the mass of names in any group of plants appears to many botanists as a work of superer- ogation, largely futile, and almost finical. They are reputed to be meddlers, with a penchant for displacing well known names by unfamiliar ones, and possessed of an insatiable and egotistical desire to see their own names appended to as many Latin designations as unlimited juggling may seem to give warrant. Moreover, there is apparently a feeling that there are names enough in use, at least enough for all except a few rare species in out-of-the-way regions yet to be brought to light by explorers; and that if the nomenclaturists would let them alone we should not be obliged to learn a new set of names, and to puzzle over their identity with the old ones, every time a fresh work on botany comes from the press. There is plenty of justification for irritation over the nomenclature situation. All will agree that each plant should have its fixed name inde- pendent of any particular botanist’s certification. But we are far from that goal at present. Why? Is it an impossible goal, or do we needlessly muddle the situation and retard progress? Of course every active systematic botanist knows how false is the prevalent idea that most plants have been sufficiently studied to make their identification as species no longer uncertain. Let it be remembered how few years ago it has been since we became aware that the plantains and dandelions in our lawns were not each one species, as the botanical manuals stated, but that each comprised two species and well defined. In my garden I have grown for a number of years a delectable small fruit, 280 AMERICAN JOURNAL OF BOTANY [Vol. 8 that I have seen listed horticulturally as Garden Huckleberry, and that evidently belongs to the great genus Solanum or one of its segregates, but I have been unable to find it described in any botanical treatise at my com- mand. There may be, and doubtless are, other plants in our front yards and vegetable gardens whose naming is uncertain for lack of sufficient study, and how much more so must be the case of plants in forests, fields, and mountains, and in the botanically unexplored regions of our own and other countries. What is true of the larger flowering plants is even more appli- cable to the far greater numbers of the less conspicuous lower orders of plants. The introduction of any number of new names, when discriminately applied to really new species, is not a source of embarrassment, but an aid to better understanding. The trouble arises when two botanists in different parts of the world independently give different names to the same plant, or when a name is applied to a species supposed to be new but afterward found to have been named, or when some one ascertains that a name has been badly chosen, is inapplicable, or of faulty construction, or when the demands of classification seem to require the transfer of a name from one genus to another. Insuch or similar cases, which are exceedingly numerous, the choice of rival names is still largely a matter of personal preference, although from the days of De Candolle attempts have been made to formu- late guiding rules, which have been of more or less service, but never gener- ally accepted. It is the opinion of Mr. C. G. Lloyd of Cincinnati, whose trenchant pen has scored many present-day mycological nomenclaturists for their pedantic ways, that the value of a name should be derived from ‘historical truth and general use.’’ He believes that ‘“‘if mycological writers in general would rely on these principles alone in the selection of names, it would only be a short time until we should be in practical accord.” ‘The principles seem simple, and if they would serve to secure acceptable unity for mycological names, they would doubtless serve as well for all other plant names. Certainly the great desideratum for names is their general acceptance, so that the same name always applies to the same plant in the writings of all authors. More than a century ago, when the contro- versy was raging in this country over the comparative merits of Jussieu’s natural system and the artificial system of Linnaeus, Thomas Jefferson, “one of the six greatest men in the history of the public life of the United States,’’ as a recent historian has stated, a broad-minded statesman and a man of high scientific attainments, contended that in this connection no matter was “‘ so important a consideration as that of uniting all nations under one language in natural history.”” The committee on nomenclature ap- pointed by this society is endeavoring to aid in such a movement. As no rules to govern the names of plants can be made mandatory, their general acceptance must necessarily depend upon their appeal to precision and serviceableness, as well as upon the provision they make for authentication in doubtful cases. June, 1921] ARTHUR — SPECIALIZATION AND FUNDAMENTALS 281 As already indicated, the regulations for selecting and validating the correct name of a plant have been slow in taking shape. It has long been recognized, more and more strongly of late, that the name first given to a particular species of plant must be considered its proper and legal name. The difficulty has been to secure agreement upon the particular name to be considered as having precedence. The difficulty is somewhat the same as the courts of justice have in proving that the name on the docket properly belongs to the person before the bar. The latest move among nomen- claturists is to follow the methods of the law courts, wholly abandoning the attempt to prove that the name is correctly used and being content with making sure of the identity of the person in question, or, in botany, estab- lishing the identity of the particular specimen of plant which was in hand when a name was published. This is known as the type-basis method, and promises to bring definiteness and exactness where before was the uncertainty of individual interpretation. Nature has not provided us with species and genera, but only with individuals having greater or less resemblances. As botanists we find it convenient to treat individuals possessing a certain amount of resemblance as species, and these species we group into genera. The size and variability of the units we call species and genera will depend in each case upon the taxonomic views held at the time, but the name, according to the type-basis method, must always find its application in accord with the characteristics posséssed by the original specimen upon which it was founded. Having now said something about the desirability of knowing plants at first hand, and about the application of their names, permit me to say a few words about the names themselves. Since the days of Linnaeus, names of plants have been binomial, with tendencies now and then to become tri- nomial, quadrinomial, or even multinomial, but never monomial. E.vo- lution of the onomatology of plants has many parallelisms to that of persons. In the early days, that is, before the middle of the eighteenth century for plants, and before the tenth century for persons, names either of plants or of persons might consist of a single word, or on the other hand might be of indeterminate length. For persons there was a gradual evolution into a surname and given name, while for plants a far more rapid change brought about the corresponding generic and specific name. The names of persons are not established by law, but by usage. The first name applied to an individual, however obtained, is almost invariably accepted in after years, and yet there is no law in this country or in England, and certainly not elsewhere, against changing a name; nevertheless, certain states have provided a process by which a change may receive legal sanction, if such is desired. The case of plants is almost identical, except the last provision for validating a name. But the movement is well under way to provide fixed rules to serve as a guide in the bestowal of plant names, to indicate the correct names previously given, and to secure their maintenance, which 282 AMERICAN JOURNAL OF BOTANY [Vol. 8 should eliminate much of the present confusion; even the provision for validating a name by a fully authorized tribunal, when a change is desirable, is being considered. In other ways the usages regarding personal names and plant names show a similarity inevolution. Inthe earlier days personal names usually denoted some quality or distinction in the individual, fanciful or real; as Clovis, glorious warrior; Mathilda, mighty amazon; Adolf, the noble wolf; Cicero, the vetch-grower. When surnames came into use they were at first selected in much the same way, as Rich, Noble, Black, Brown, Archer, Goldsmith. But after a time the multiplicity of names, and the necessity of continuing their use when no longer applicable, led to the present usage of disregarding the qualifying significance in either surnames or given names, and to select them for euphony, or family association, or fanciful reasons. The course with botanical names has been much the same, but the evolution has not gone as far, doubtless because of the shorter period of time involved. For a while it was thought necessary to give descriptive or informative names to plants, and when they proved inappropriate to change them. But the practice has largely fallen into disuse in late years. Plantago major is a much smaller plant than the similar and more common plantain that grows | with it everywhere in this country east of the Rocky Mountains, yet the name has not been changed. Some taxonomists, however, who deal with the lower orders of plants, especially the fungi, are still in the dark ages with their nomenclatural practice. A rust called Puccimia Distichlidis has been renamed, because it was found that the grass on which it occurs is not Distichlis, but only looks like Distichlis. In another group of fungi a prominent writer stated not long ago that We have heretofore used Cyathus Poeppigit as the name for this species, but in the future we shall adopt the name originally applied to it by Poeppig. We do not do this on any ground of priority, but because Cyathus Poeppigit is a heathenish kind of name that ought to be suppressed. Within the last month or so a transatlantic colleague has written re- garding a species of Helminthosporium: May I point out the course we have adopted with regard to the spelling of the specific name of the barley stripe fungus? We now invariably use graminum, the genitive plural of the substantive, believing this to be more correct than the adjectival form gramineum. One might cite many instances to show that, although the latest rules of nomenclature do not sanction changes like these, we are yet slow in arriving at the stage at which the name of a plant, like that of a person, is generally considered as an appellation wholly for identification, and what- ever of descriptive or adjunct information it may convey must be considered entirely incidental or historical. Curiously, we speak of the name of a plant or person as if it were a simple designation, like a number. Yet it is a compound of two very June, 1921] ARTHUR — SPECIALIZATION AND FUNDAMENTALS 253 unequal parts. When we say that the correct name of a plant is the one first applied to it, we mean the specific name only, the one corresponding to a person’s baptismal name, and it is toward this part of the name that most of the rules on nomenclature are directed. The specific name may be transferred from one genus to another as many times as seems desirable, in order to express its relationship, just asa woman’s surname changes upon remarrying, or a man may take another surname to meet the requirement of a bequest; but the identity of the plant as of the person is inherent in the specific or baptismal part of the name, although standing by itself it would mean little. Thus the common field thistle, which we usually call the Canada thistle, was named by Linnaeus Serratula arvensis. At intervals of a few years it was successively transferred by different authors of the old time to the genera Cirsium, Carduus, and Cnicus, but at present is most . generally listed as Cirstum arvense, | believe. Again, Linnaeus called the common dandelion, that makes our lawns glorious with golden bloom in spring and later turns them into a ragged waste, Leontodon Taraxacum, the specific name being adopted from an old-time classical name. Later this genus was divided, and the dandelion dropped into the new genus Taraxacum, it being called Taraxacum densleonis, which had the same meaning as did the first name. But it is now contended that the earliest specific name is the rightful one, irrespective of meaning, and in consequence the dandelion should be called Taraxacum Taraxacum, which strangely enough is a combination that is strenuously objected to by a large number of botanists. Why should this and the like combinations, Sassafras Sassafras, Abutilon Abutilon, etc., be any worse names for plants than William Williams and Smith de Smith for persons? Until we bring our- selves to look upon plant names as simply names, not qualifying terms, our science will be handicapped by the impedimenta of prejudices whose rightful place is in the musty volumes of the antiquary. Now a word about those appendages of every Latin botanical name, which C. G. Lloyd calls the personal advertisements. In the present unsettled state of nomenclature they are as necessary for keeping names from going astray as the tail is necessary to guide a tadpole. When plants become better known, and names are more consistently applied, the caudal appendages will be dropped as burdensome and useless. To get us out of the tadpole stage in nomenclature, however, will assuredly take united effort and willingness to forego prejudices. Every botanist should be interested in hastening the day. Whatever one’s specialty, he must use the Latin names of plants. Exact names, uniformly applied, are a funda- mental requirement of the science, and it is in the interest of every botanist, as well as of horticulturists, agronomists, and all other users of plant names, to hold a favorable attitude toward attempts to secure this end. At this point I am reminded that the most prominent feature of the present movement in botanical thought is organization and codperation. 284 AMERICAN JOURNAL OF BOTANY “vote We are clearly entering a new era for scientific labors. Research has become the watchword of the hour and is to be encouraged more than ever before. In course of time it may even be acceptable and intelligent to officialdom for one to give his occupation as investigator and not to be obliged, as at present, to masquerade as a teacher, even when he does no teaching and possibly may not be connected with a teaching institution. It is now com- mendable not only to encourage the spirit of research, but to assist in pro- viding a general atmosphere favorable to its development. Naturally, as in other movements that become popular, there is sometimes more talk about the value and desirability of research, than actual accomplishment, or even hearty direct assistance in providing time and means for its prosecution. Nevertheless, we are likely to see the number of research centers, both great and small, much increased, and the ranks of those who essay the task of adding to available knowledge immensely augmented. In the re-awakening and re-orientation of the research spirit it should be possible to preserve and advance that fine democratic quality which recognizes, as we were reminded by one of our number two years ago at the Baltimore meeting, that ‘‘ botany is a world science and that its advance can not be accelerated through the usual operation of institutional or indi- vidual rivalries.’’ There must be the fullest and freest codperation between institutions, and quite as much between individuals, both as members of organizations and as independent workers. Harvey’s exhortation to mutual consideration should find practical fulfillment, both for the good name of the profession and for the efficiency of its labors. While I am pleading for individual freedom and encouragement against the encroachments of the machinery of organization, I do not undervalue the great service and importance of associations, both those of voluntary combinations of individuals and those centering about institutions. I subscribe most heartily to the views of Mr. Frederick W. Taylor, who had in mind especially the research conditions in the commercial world, but whose words are equally applicable to things botanical, when he said: The time is fast going by for the great personal achievement of any one man standing alone; and the time is coming when all great things will be done by that type of codperation in which each man performs the function for which he is best suited, yet preserves his own individuality and initiative and is supreme in his particular function, while controlled by and working harmoniously with many other men. These words breathe the true democratic spirit of personal freedom as against the bolshevistic absorption of the individual in the organization. In the movement for greater accomplishment by means of organization the class of problems which are uppermost for consideration are the economic ones, or those which can be justified by a direct popular or commercial demand. These are the ones for which money can be most readily obtained, and in which the largest number of persons can be interested. These are the ones chiefly supported by the general government, because they are june, 1921] ARTHUR — SPECIALIZATION AND FUNDAMENTALS 285 nearest to the interests of the taxpayer to whom the government must appeal for funds. They are most likely to receive attention from state institutions whose success depends upon heeding the popular demand. Even privately endowed educational institutions and detached research institutions can not help but be influenced by this tendency. Such prob- lems have almost monopolized the word specialize. Thus Dr. Lyman says: The agricultural institutions have specialized too strictly and have laid too little stress on the fundamentals of botany. With the natural instinct to be interested in the under dog, my closing words shall be a plea for greater attention to the fundamentals in making provisions for organized support. The solution of problems falling in this class furnishes the tools for the specialist. Some phases of taxonomy, of which I have already spoken, might be used as an example. The consistent, effective onward march of botany calls for careful balance between the attention given to specialization and that given to fundamentals. CERTAIN ASPECTS OF THE PROBLERIVOn PHYSIOLOGICAL CORRELATION CM: Camp (Received for publication January 14, 1921) The existence in the growth and development of axiate plants of a relation of dominance and subordination, of control and being controlled, has long been recognized. This relation is very evidently associated in some way with at least certain fundamental physiological activities of plant protoplasm and apparently particularly those which have to do with growth. The active vegetative tips are the chief regions of dominance, but other growing regions may exercise a similar dominance to a greater or less degree. That this relation is a real physiological relation and dependent on the dynamic activity of the dominant part has been demonstrated repeatedly by experiments in which the dominance is abolished by inhibition of the fundamental metabolism and growth of the dominant region, but reappears when the inhibiting factor is removed and the dominant region returns to or approaches its original condition. My work on so-called physiological polarity, correlation, and integration in animals has shown very clearly that in axiate animals as well as in plants a relation of dominance and subordination exists, not only as regards the functional activities of the fully developed individual, but also in growth and development. The evidence indicates that the functional relations of later stages as expressed in the nervous system and in the chemical inter- relation of parts are the consequence and outgrowth of a more general and primitive relation which exists before the nervous system appears and before the various parts differentiate. Since this relation in its more general form as it appears in the simpler animals and the earlier stages of development seems to be very similar to the relation in plants, I have very naturally been much interested in attempting by means of work along various lines with plants to discover whether, or to what extent, such simi- larity exists. It is because of some of this work on plants that I have been asked to take part in this program. My objection that the work cannot properly be regarded as biophysics any more than biochemics, and that it has not yet attained the exact quantitative character and formulation which would warrant its inclusion in either of these special fields of physiology, was overruled by those in charge of the program, so that responsibility rests upon them. 1 Invitation paper read before the Physiological Section of the Botanical Society of America, in the symp9sium on biophysics, at Chicago, December 28, 1920. 286 June, 1921] CHILD — PHYSIOLOGICAL CORRELATION 287 A very brief survey of certain lines of work is necessary by way of intro- duction to the experiments of which I wish particularly to speak. Study of several hundred species, including animals from all the chief groups and many algae and some other plants, have shown that physiological polarity and symmetry in their simplest terms consist of gradients in physiological condition and activity of the protoplasm or cells composing the organism. These gradients have been called axial, metabolic, or physiological gradients. That they have to do with the fundamental physiological condition of the protoplasm is clearly shown by the many different lines of evidence which demonstrate their existence. They appear as gradients in suscep- tibility to certain toxic ranges of concentration or intensity of external agents, e.g., cyanides, heavy metal salts, anesthetics, acids, alkalies, other neutral salts, COs, various dyes, extremes of temperature, and the negative condition, lack of oxygen. Within certain limits of concentration or inten- sity these susceptibility gradients are non-specific, 7.e., essentially identical in their larger features with all external agents tested, at least in the simpler animals and plants and in the earlier stages of development of higher forms. It has been shown that these differences in susceptibility are indicators of differences in rate of fundamental metabolism, particularly oxidation. The physiological gradients can be demonstrated as gradients in the rate of penetration of non-toxic or only slightly toxic vital dyes. Again, in dilute solutions of the oxidizing agent KMnQO, they appear as gradients in rate and amount of reduction of the salt. In certain cases the indophenol reaction has been used, and a gradient in the rate of appearance of the indophenol suggests a gradient in oxidizing enzymes. A gradient in electric potential is a characteristic feature of the physiological gradient in all forms thus far examined, though in some plants the electrical situation is apparently com- plicated by the occurrence of reactions which give rise to opposite potential differences, viz., the oxidations and photosynthesis. And finally, in animals in which it has been found possible to determine the oxygen consumption and CO, production of different regions along the axis, differences corres- ponding to those indicated by other methods have been found. It has not yet been possible to apply all these methods to each species examined. These physiological gradients also very commonly appear in differences in structure of the protoplasm along the axis, as in many plant and animal embryos, and they are definitely related to differences in rate of develop- ment and differentiation. It has also been possible to show that the localization and differentiation of organs and parts occur in a definite relation to the physiological gradient, in fact are determined by it. The most active region as finally determined, 1.e., the region of highest susceptibility, of greatest permeability, of greatest reducing capacity, of highest external electro-negativity, and of highest rate of respiration in the polar axial gradient, becomes the apical end of the axis, or in animals the head, and the other organs develop at different 288 AMERICAN JOURNAL OF BOTANY [Vol. 8 levels of the gradient. The question how the primarily quantitative differences in such a gradient can give rise to the qualitative differences characteristic of differentiation of cells presents no fundamental difficulties. Differences in the relation between available nutritive substance and the rate of oxidation at different levels of a gradient undoubtedly determine the appearance of certain substances in the cells at one level and their absence at another. Differences in concentration of certain substances at different levels may also determine the formation of different products, and various other factors in the complex protoplasmic system doubtless play a part in determining the origin of qualitative differences from the quantitative differences of the gradient. But whatever the local factors involved in each particular case, the physiological gradient constitutes the primary factor in determining localization and differentiation of parts along an axis. The important point for present purposes is that in such a gradient a relation of dominance and subordination exists, the high end, the most active region, of the gradient being the dominant region and determining to a greater or less extent conditions at other levels within a certain distance, which differs with the stage of development, the condition and differen- tiation of the protoplasm, and the degree of activity of the dominant region. Since this dominance is effective only within a certain range or distance, the possibility of physiological isolation exists, that is, either in consequence of increase in length of the organism, of decrease in activity of the dominant region, or through a blocking in some way of the passage of the controlling influence, certain parts may become isolated from the action of the dominant region, even though still in physical continuity with it. In the simpler ‘animals and plants such physiological isolation results, like physical isola- tion, in dedifferentiation and development of new axes, or parts already present but previously inhibited, such as latent buds in plants, become active and develop. The question of the nature and origin of physiological gradients is obviously of fundamental importance. The gradients, so far as can be determined, represent primarily quantitative rather than qualitative differences, and if this is true, as all the evidence indicates, the relation of dominance and subordination cannot be fundamentally a matter of chemical or transportative correlation, that is, of mass transportation and action upon one part of specific substances or hormones produced in another. In order that such correlation may exist the parts concerned must already be qualitatively different, and the evidence indicates that dominance and subordination exist in the absence of such differences. Unquestionably chemical correlation is of great importance as soon as qualitative differen- tiation begins, but it cannot be the primary factor in correlation and in determining such differentiation in the organism. The only other possibility appears to be the transmission of dynamic change of some sort, that is, of excitation. We must therefore inquire June, 1921] CHILD — PHYSIOLOGICAL CORRELATION 289 whether the physiological gradient shows any similarity to an excitation gradient. All living protoplasm is excitable and to some degree capable of transmitting excitation. Excitation in its most primitive form appears to be an acceleration in the rate of living, particularly as regards the energy- liberating aspects of life. Where specialized conducting paths are not present transmission of excitation occurs with a decrement, that is, the transmitted change becomes weaker and finally disappears at a greater or less distance from the point of origin. Such a process of excitation and transmission gives rise to an excitation-transmission gradient. We usually think of such gradients as temporary or reversible, but the physiological gradients show all the characteristics of excitation gradients which have become more or less permanent. Moreover, it has been shown experimentally that these gradients can be produced in cells or cell masses by subjecting them to a quantitative differential in the action of external factors. For example, new gradients and so new polarities can be determined experimentally in the simpler animals by a sufficient difference in oxygen supply, by an electric differen- tial, perhaps in some forms by a light differential, and probably also by various other differentials. A differential inhibition may have the same effect as a differential excitation or acceleration. Turning to the plants, the polarity of the Fucus egg, which is primarily a gradient, is determined by the differential action of light, and the polarity of the Equisetum spore has a similar origin. The relation of dorsiventrality and symmetry to light in the plants is a familiar fact. In order to establish a gradient suffi- ciently persistent to serve as a physiological axis, the differential action of the external factor must persist for a certain length of time dependent on the nature and intensity of the factor and the character of the protoplasm. The fixation of such a gradient in protoplasm must depend upon the occur- rence at the different levels of changes which are more or less irreversible under the existing conditions and which differ in degree according to level. Once established in a cell or cell mass, such a gradient may persist through division or other reproductive processes and become the basis of the axis of the new individual or individuals. In other cases the original gradient may disappear in reproduction and a new gradient arise. In many eggs, both animal and plant, the gradient is apparently determined by a differen- tial in relation to the parent body, such, for example, as the difference in conditions at the attached and the free end of the egg; but in some eggs it may perhaps persist from earlier cell generations, while in others, as in Fucus, it is determined by a factor external to the organism. We come now to the question of the nature of the dominance or control, and this involves the question of the nature of transmission. Many hypotheses have been advanced concerning the process of transmission, and most of them connect it in one way or another with the electric changes which are a characteristic feature of excitation. On the basis of extensive 290 AMERICAN JOURNAL OF BOTANY [Vol. 8 experimental investigation R. S. Lillie has developed an electro-chemical conception of excitation and transmission which seems to interpret and account for the various phenomena more satisfactorily than others pre- viously advanced. The unexcited surface layer of the cell behaves as if more permeable to positive than to negative, or to certain negative, ions, and is therefore electrically polarized. Excitation increases its permeability to the negative ions and depolarization results, with an increase in electro- negativity of the external surface. In this change a chemical reaction, an oxidation, is involved, whether as the primary or as a secondary factor is not at present known. The electric current arising at any point of excita- tion becomes the factor determining depolarization and excitation at all points within a certain distance, beyond which it is too weak to be effective, and each new region of excitation becomes the source of current which may, if strong enough, excite further points. At the same time the current tends to restore the polarization at the point of original excitation and so to reverse the excitation process at that point. By means of this current, then, according to Lillie, transmission occurs. With simple inorganic models he has been able to demonstrate the occurrence of transmission in this way, both with and without decrement and at different speeds, as well as the development of fixed gradients. The speed of transmission has no relation to the speed of electrical transmission, but depends on the velocity of the changes at each point of excitation which give rise to the current. The development of fixed gradients occurs when conditions determine the persistence of the region of high potential which in turn determines a poten- tial gradient extending over a greater or less distance and so a gradient in the conditions determined by the electric current. Whether this theory of excitation is in all respects correct or not, it enables us to see how a region of excitation in undifferentiated protoplasm may determine the origin of a physiological gradient. The facts indicate that these gradients do arise in this way, and if we admit this, it follows that the primary factor in dominance and subordination is transmission. The establishment of a region of high activity must affect adjoining regions within a certain distance as a region of excitation affects them, and it is difficult to believe that the electric potential characteristic of such a region is not a factor and probably the primary factor in such a relation. If the degree or intensity of excitation at each level is in any degree proportional to the strength of current, a gradient must result, and if the conditions determining the gradient persist for a certain length of time, changes in the protoplasm at the different levels may determine the more or less permanent fixation of the gradient. In most protoplasms this relation between stim- ulus and excitation does exist, and a gradient results from local excitation. In the nerve fibers of the higher animals, however, the excitation process is specialized so that any stimulus above the threshold gives rise to maximal excitation and there is therefore theoretically no decrement in transmission. June, 1921] CHILD — PHYSIOLOGICAL CORRELATION 291 The relation of dominance and subordination does not necessarily persist in its primitive form throughout life. In animals, for example, the development of the nervous system, with highly specialized paths capable of transmitting excitation so much greater distances than embryonic proto- plasm, makes possible a much more complete and extensive dominance, which nevertheless is built up on the basis of the primitive relation. On the other hand, the qualitative differentiation of different organs affords a basis for complex chemical or transportative correlation. In plants, buds which are inhibited for a time may sooner or later become incapable of development, even when physiologically isolated, either because they have not been able to develop channels for the passage to them of water and nutrition, or because of changes in the cells in consequence of the action of the dominant region upon them. On the other hand, even in plants the conductivity of certain tissues may increase with differentiation and domi- nance be possible over greater distances than at first. In short, the primitive transmissive relation may develop and attain greater importance in certain types of relation, or it may be supplemented or even replaced by chemical correlation, or, finally, with advance in differentiation of parts the correlative factors may be chiefly nutritive. In any case the situation in the plant remains much simpler than in the higher animals, in which both the trans- missive and the transportative relations are extremely complex. My work on plants, in which Dr. A. W. Bellamy assisted me, was under- taken with the hope of being able to throw some light on the question of the nature of dominance and subordination as it exists in the growing plant. Thus far I have merely succeeded in blocking by means of low temperature the correlative factor on its way without interfering to any marked degree with the flow of fluids in the plant. As I have pointed out elsewhere, the results favor the view that correlation is accomplished by a transmission rather than by mass transportation of special substances, but much remains to be done before positive conclusions are permissible. My experiments thus far have been chiefly with three plants, Bryophyllum calycinum, Phaseolus multifiorus, and Saxifraga sarmentosa. The method of experiment and the results obtained with Bryophyllum have already appeared in the Botanical Gazette. As regards method, it may be said here that the low temperature is applied by surrounding the zone to be cooled with a coil or loop of small block-tin piping which can readily be bent and adjusted and through which water of controlled temperature flows. The region to be cooled is first wrapped in tinfoil and direct contact with the piping is avoided, the space between the coil and the plant being loosely packed with slightly moistened absorbent cotton, and the whole region of the plant and the coil after adjustment well wrapped in order to reduce temperature change from external sources to a minimum. All cases in which visible external injury due to pressure or to too low temperature occurs are discarded. Temperatures ranging from 3° to 8° C. are used 292 AMERICAN JOURNAL OF BOTANY NOLES according to the plant and the particular object in view. In the bean seedling cell turgor is somewhat reduced in the cooled region after some days, particularly in the lower temperatures. On removal of the coil, however, normal turgor is reéstablished within a few hours, and if the temperature is raised gradually at the end of the experiment the turgor is normal when the wrapping is removed. In the experiments on Bryophyllum? the low temperature is applied to a zone of the petiole 2-3 cm. long, and the leaf is immersed in water so far as its position on the plant will permit. The opposite leaf of the same node and usually leaves of other nodes above and below are also immersed. In the experimental leaf all or nearly all the immersed buds develop, in the opposite leaf there is as much or almost as much development in most cases, and more or less development occurs in leaves of nodes above and below the experimental node. Controls with leaves immersed but without the low temperature often show development of a bud here and there, but the effect of the low temperature is clear and unmistakable in the experimen- tal leaf, in the opposite leaf, and to a less extent in leaves of neighboring nodes. The case of the scarlet runner bean is of greater interest than that of Bryophyllum, for here the low temperature is applied to the main stem of the seedling, and all water and nutrition passing to parts above must pass this zone. If the low temperature interferes appreciably with the flow of fluids, this should be evident in the retardation of growth above the zone, or in extreme cases in wilting. There is in some cases slight retardation of growth of the tip, particularly when the low temperature is applied to the upper part of an internode of the young seedling in which elongation is still going on and the vascular bundles are still developing. Except when the temperature is very low, however, this retardation is only temporary and the rate of growth of the tip increases even before the low temperature is removed, and in no case is the effect on the tip sufficient to decrease its dominance to such an extent that axillary buds:above the low temperature zone develop. Morevoer, that this retardation has nothing to do with isolation of buds below the cooled zone is shown by the fact that a tempera- ture of 5°-6° C., applied to the upper end of an internode, produces at first marked retardation of growth of the tip but no growth of buds below, while the same temperature applied near the lower end of the internode produces no appreciable retardation of the tip, but the buds in the axil below develop. In general, the farther away from the axils to be isolated the low temperature is applied, the less effective it is in producing growth of the buds and vice versa. These facts suggest that the inhibiting factor, if it passes. at all through a cooled zone, undergoes a gradual return or approach to its original effectiveness in its further course, so that when the cooled zone is 2 In the original presentation of these and other experimental data lantern slides were used. June, 1921] CHILD — PHYSIOLOGICAL CORRELATION 293 farther away from the buds to be isolated it is less effective. Moreover, with temperatures which are not too low, a long cooled zone is more effective as a block than a short one, but with sufficiently low temperatures the short zone is effective. In these respects the correlative factor apparently behaves to some extent like the nerve impulse. Growth of the buds isolated from the tip by low temperature is usually evident within one to two days. If the temperature is near the upper limit of effectiveness, the growth of the buds below the zone usually ceases after a few days in spite of the presence of the cooled zone. This is un- doubtedly due to the occurrence of some degree of acclimation in the cooled zone with consequently more effective passage of the block by the inhibiting factor. If the buds are allowed to grow for ten days or more before the removal of the low temperature, they usually continue to grow more or less rapidly afterward, and in this way plants with three or more stems can be produced. Earlier removal of the low temperature usually results in renewed inhibition. ‘In my experiments with the saxifrage, the low temperature is applied to a zone of a runner which has not attained its full length, with the result that the runner soon ceases to elongate and begins to develop a new plant at the tip, even when suspended in air. Here also water and salts reach the runner tip only by passing the cooled zone, and the rapid development of the new plant shows that this flow is not seriously affected. According to Loeb’s hypothesis, it seems that substances inhibiting the development of the new plant at the runner-tip must be transported by this current, but as a matter of fact the low temperature isolates the tip without stopping the current. Whatever the nature of the correlative factor may prove to be, these experiments, particularly those on the bean seedling, seem to me to offer difficulties to the hypothesis that this factor consists of an inhibiting sub- stance or substances transported in mass through the plant. Since the correlative factor can be:blocked by a zone of low temperature, we must assume, if it consists of a substance or substances, either that it is trans- ported through the living protoplasm and that its passage is dependent upon the physiological condition of the cells, or that it is of such a nature that it is precipitated out of, or otherwise removed from, the fluids of the plant as they pass the cooled zone. On the basis of the first assumption, we should expect a substance which inhibits the growth of vegetative tips to inhibit the growth of the cells through which it passes. Below the chief growing tip, for example, such a ‘substance must pass through the region of most active growth in the axis, but it does not inhibit this region. In fact, if such a substance passes through the living cells of the plant, most complex and remarkable relations of immunity and susceptibility must exist. Each growing tip, for example, or any other part producing such a substance, must be immune 294 AMERICAN JOURNAL OF BOTANY [Vol. 8 to the substance which it produces, but other growing tips are susceptible tO, it: The alternative assumption, that the substance is transported in the fluids of the plant and removed in some way from them in the cooled zone, does not serve any better than the first for the interpretation of certain experimental results. In fact, the hypothesis of inhibiting substances and their transportation, in whatever form we state it, does not account for the fact that within certain limits of temperature the effectiveness of a cooled zone of certain length in the stem of the bean seedling decreases with in- creasing distance from the buds to be isolated, even though the more distant zone may be more effective in inhibiting the growth of the chief tip. In other words, a cooled zone which has a marked inhibiting effect upon the movement of water and salts in the stem is not necessarily effective in isolating buds below, while a zone which has no appreciable inhibiting affect on regions above it may be effective in isolating buds below. Assumptions concerning the transportation of nutritive substances are not, I believe, any more satisfactory than the hypothesis of inhibiting sub- stances in aiding us to account for the facts of physiological correlation in the plant. As already noted, physiological isolation of buds below a cooled zone may be brought about without retarding the movement of water and salts to any marked degree. In the case of Bryophyllum, the leaf itself is able to produce starch, and in the bean seedling the reserves of the cotyledons are available for the buds in the axils of the cotyledons and those produced by the first pair of leaves are available for the buds of the second node. In the saxifrage runner the cooled zone may retard to some extent the passage of nutrition to the runner tip, but the results with the other species show clearly enough that this is not the primary factor in the physiological isolation of parts in plants. In the case of the Bryophyllum leaf with cooled zone about the petiole, the passage of water and salts to the leaf may be somewhat retarded, but the leaf contains plenty of starch. In the bean seedling the passage of water and salts to the buds to be isolated is not interfered with, since the cooled zone is above them, and here also plenty of carbohydrate is available. In both these cases, as well as in the saxifrage runner, physiological isolation and growth of buds occur. Again, if we cut off the stem of the bean seedling below the first foliage leaves and remove the cotyledons, the buds in the axils of the cotyledons, which are now the only buds on the plant, will develop, although in the absence of the more apical parts of the plants the movement of water and salts must be greatly decreased and the removal of leaves and cotyledons must have decreased the amount of carbohydrate available. In short, the result as regards development of the buds is essentially the same under experimental] conditions which must determine very different internal conditions as regards the movement of nutritive substances and the amount available in a particular region. June, 1921] CHILD — PHYSIOLOGICAL CORRELATION 295 Finally it may be noted that in the bean seedling the rate of reaction of the buds of the different axils to physiological isolation by a cooled zone differs according to the level of the plant. With a cooled zone of given length and given temperature at a given distance above the node concerned, the buds of a more apical node begin to grow earlier and grow more rapidly than those of a more basal node. This fact also seems to me to offer diff- culties to the hypotheses of dominance by means of inhibiting or by means of nutritive substances. On the other hand, it is apparently an expression of the physiological gradient, the primarily quantitative gradation in phy- siological condition along the plant axis, which, as I believe, is the basis of physiological correlation in the plant. In other words, the relation of dominance and subordination is also an expression of the physiological gradient, and the movements of substances in the plant are not the primary factors in physiological correlation, but rather the consequences of the differences which constitute the physiological axial gradient. WATER DEFICIT AND THE ACTION OF VITAMINES, AMINO- COMPOUNDS, AND SALTS ON HYDRATION! D. T. MAacDouGAL (Received for publication January 17, 1921) It is well known to any one who has seriously examined cells in a living condition that protoplasm is a viscous substance which at different times or in different parts of the same protoplast may vary in consistency from a liquid to that of a firm jelly, a behavior characteristic of an emulsoid colloid. This term in the present instance is applied to substances which in a con- dition of hydration exist in two distinct phases in the mass; in one, a few or many molecules of the solid substance are combined or held together by adsorption with a relatively small number of water molecules to form a denser phase, which in the more liquid condition of the mass floats in or is surrounded by a more fluid phase in which the solid particles sustain a much smaller proportion to the water. If we begin to form our picture of the colloid in this condition, which would be that of melted agar or gelatine, we shall be ready to follow its transformation to that of a jelly by visualizing an action by which the surface tension of the aggregates of molecules is increased by lowered temperatures or other causes, the aggregates coming together to form mesh- works or honeycombs, running through or partially enclosing the material of the more liquid phase. The possibilities implied in the reversal of these phases, important as they may be, may be disregarded during the present discussion. Living matter is anything but such a simple substance. The results of all of our examinations by physical and chemical methods are to the effect that the living matter of plants includes the following groups of substances which may assume the colloidal conditions described above: First, the nitrogenous substances, which include not only albumin and all its deriva- tives, but also synthetic compounds. Next in importance, and constituting perhaps the greater part of the mass, are the pentosans or mucilaginous sugars which may be formed in carbohydrate metabolism in any part of the mass when hexoses are converted into pentoses and these are condensed into the pentosans by dehydration. The presence of such fatty acids as stearic, palmitic, and oleic, and the readiness with which they form soaps by combination with potassium, sodium, and magnesium, make it also certain that such substances are an invariable component of the biocolloids. 1 Invitation paper read before the Physiological Section of the Botanical Society of America, in the symposium on biophysics, at Chicago, December 28, 1920. 296 June, 1921] MACDOUGAL — WATER DEFICIT . ZO7, In addition, a certain amount of lipins may be present, but as their possible hydration action in the colloidal mass must be very slight, they may be left for consideration in a discussion of other phases of protoplasmic action. When the general properties of these main components of living matter are reviewed, it is seen that the albuminous or proteinaceous substances are amphoteric, behaving as either acids or bases according to the hydrogen- ion concentration of the solution, that they generally unite with the highest proportion of water when in an acidified condition, and that their hydration is also facilitated in lesser degree by the action of bases and their salts. The pentosans or mucilages are weak acids and undergo the highest degree of hydration in neutral or slightly alkaline solutions, in extremely dilute solutions of the common salts of potassium, magnesium, calcium, and so- dium, or in the presence of certain amino-compounds described in previous papers. That the nitrates, chlorides, and sulphates of these metals may also increase the hydration capacity of the pentosans will be demonstrated in the present paper. The soaps are characterized by their Enea for forming films, and their high hydration capacity is altered to such degree by variations in the hydrogen-ion concentration of the solutions as to make them “‘sensitizers”’ as to acidity in any colloidal mass into which they may enter. It is to be noted that the range of acidity or alkalinity of interest to the physiologist in the present connection is that which lies between the measurements expressed by the symbols PH = 3 and PH = 11, and that of the salts as chiefly between 0.001 and 0.ooo1 M. Taking into consideration the complex conditions suggested above, it is reasonable to infer, since we have as yet devised no means of direct observa- tion, that, as the more complex proteins and the pentosans do not dissolve or diffuse in each other, they form separate aggregates, and that the sponge or meshwork of the solidified biocolloid or protoplasm must therefore consist of an interwoven meshwork of the two. This inference may not be carried safely beyond a certain point, however, nor to imply that no nitrogenous substances of the cell may engage, or be adsorbed by, the pentosans, since the results of the action of histidine, glycocoll, alanine, asparagine, and phenylalanine upon agar go far to suggest the possibility of such unions. The soaps which may be present in the cell colloids are not known to form combinations with the carbohydrates, and, although some uncertainty exists as to what disturbances may be caused by their contacts with the proteins, yet it may be assumed for the present that they form films en- closing the more solid phase of the double meshwork, being thus an inter- phase in the colloidal machine, highly sensitive to the action of the hydrogen- ion and adding greatly to the complexity of the possible action of the mass as a whole in response to various solutions and environmental con- ditions. In this my results are in accord with those of Dr. Clowes, who ascribes 298 AMERICAN JOURNAL OF BOTANY [Vol. 8 variations in permeability to effects of electrolytes and metabolic products on interfacial soap films.” Referring to the picture of the colloidal mechanism described above, it is obvious that, while the proteins or albumins and the pentosans must form separate strands or plates, the smaller aggregates in the more liquid portion or phase mingle with each other, and it might also be suggested that various unions might take place between molecular groups of pentosans with amino-compounds or salts, and that actual salts might be formed by the albumins when the colloid is immersed in hydroxides or salts such as those of potassium, sodium, or magnesium. Here then we have a crude statement of a theory of the colloidal condition of protoplasm upon which extensive experimentation as to the action of bases, salts of common metals, acids, amino-compounds, and vitamines has been carried on with the acquisition of results of positive value. These reactions are our real aim, and after we have crossed our bridge of hypothesis to the solid ground of facts, the fate of the bridge which may have served us well is of minor importance. The arrangement of living matter inferred has much to support it. It is one in which the separate components of the colloidal machine each present a separate and individualized capacity for hydration changes under any set of conditions or at any given temperature for example, and when immersed in water would move with differentiated speed to saturation or satisfaction. If the water in which the colloid is immersed holds substances in solution the ions of which may form combinations with the main components men- tioned above, their capacity for holding water in combination may be altered, and combinations or splittings in the metabolism of substances in protoplasmic colloids may also exert such action. Thus, a dissociation resulting in the freeing of hydrogen ions tends to increase the hydration capacity of the protein strands or aggregates of the mass might lessen that of the pentosans and soaps. Slightly acid amino-compounds as gly- cocoll would increase the hydration of the pentosans while exerting practi- cally no effect on the albumin or albumin derivatives. It is evident without further elaboration that in the albumin-pentosan-soap machine we have a mechanism capable of an almost endlessly diversified action in swelling and growth. With so much prelude we may now advantageously turn to a considera- tion of recently acquired results obtained by testing the action of salts, balanced solutions, amino-compounds, and vitamines on such colloidal mechanisms and on biocolloids and cell masses, living and dead. In an earlier paper? I had advanced the idea that the common metals 2 Clowes, G. H. A. On the action exerted by antagonistic electrolytes on the electrical resistance and permeability of emulsoid membranes. Proc. Soc. Exper. Biol. and Med. 15: 108. 1918. 3 MacDougal, D. T. Growth in organisms. Science, 49: 599-605. 1919. (Seep. II of reprint). MACDOUGAL — WATER DEFICIT 299 June, 1921] which enter into nutritive solutions, as potassium, magnesium, sodium, and calcium, might find their chief importance in restricting, limiting, or de- fining hydration. Such an action is exerted by these bases in the form of hydroxides when tested at 0.01 N. MacDougal and Spoehr* found later that the hydroxides of the strong metallic bases limit the hydration of agar according to their position in the electromotive series, the least swelling taking place under the action of the strongest base at concentrations of 0.01 N, with the apparent exception of rubidium. Beginning with the strongest, the series runs K > (Rb) > Na > Li. The various effects of barium, calcium, and strontium are not so clearly determined, and the quantitative relations of these metals are not known definitely. Hydration values of agar at 0.01 N were Sr(OH). = 815, Ca(OH)2 = 860, Ba(OH). = goo. These concentrations are far beyond the actual range of conditions in the cell, however, and when reduced concentrations were used it was seen that hydration of agar in calcium hydroxide exceeds that in water at 0.0001 N of the hydroxide, and this effect is also produced at 0.00001 N. Increase of hydration beyond that of water by dilute solutions of hydrox- ides of calcium, potassium, rubidium, potassium, sodium, and lithium, and excess values for aniline and ammonium hydroxides were obtained. It was also seen that the strongest of the bases, potassium, in the form of a hydroxide would increase the swelling of agar-albumin mixtures to a point beyond that taking place in water alone. The next logical step was to test the effects of salts of the common metals on swelling of the biocolloidal components. Here again the interesting fact was found that as chlorides, sodium and potassium at 0.001 M caused greater hydration of agar than water, the swelling being greater in the potassium. At 0.0001 M, sodium, potassium, magnesium, and calcium chlorides caused greater swelling than in water, the maximum swelling being in sodium, the next in potassium, and the least in calcium. When chlorides of sodium and potassium were tested in series as shown in tables 1 and 2, it was found that pentosan-albumin mixtures showed increased hydration in the potassium chloride only as indicated in the tables. TABLE 1. Hydration of mixtures of agar 3 parts, gelatine 2 parts at 14° C. Plates 0.18 mm, in thickness; swelling of sections given in thickness and volume o.or M o.oo1 M o.ooo1 M. di. Vol. Th. Vol. Thi, Vol. ACL 550 600 930 1,025 1,430 | 1,575 Or 1,900 2,015 2,270 2,530 2,440 2,640 2 Ca 920 960 1,220 1,345 2,030 2,268 Bremer 20.2... .. 2,200 2,330 4 MacDougal, D. T., and Spoehr, H. A. The components and colloidal behavior of plant protoplasm. Proc. Amer. Phil. Soc. 59: 154. 1920. 300 AMERICAN JOURNAL OF BOTANY [Vol. 8 TABLE 2. Hydration of mixtures of gelatine 3 parts, agar 2 parts at 14° C. Plates 0.18 to 0.19 mm. in thickness; swellings given in thickness and volume o.or M o.oor M o.ocor M Th. Vol. Th. Vol. Ths Vol. UC sae 1,200 1,320 650 690 860 920 TE Giosi a ce cn 800 e 98380 goo 1,010 1,620 1,850 WaGle.ii sacar ates 710 740 870 940 1,300 1,430 Water. 2.22 ems. | 1,275 1,420 The agar-gelatine mixtures are seen to have a higher capacity than the gelatine-agar, and also to react by high hydrations in greater concentration of potassium. Up to this point colloids including only two of the supposed main ele- ments in protoplasm have been used. An agar-gelatine mixture was now made to which was added a thousandth part of a soap which is probably nearly all sodium stearate. The results of the hydration swellings are given in table 3. ; TABLE 3. Hydration of plates of agar 3, gelatine 2, Ivory soap 0.005 g. at 15° C. Swellings given in thickness and volume o.ot M 0.0002 M o.coor M Th. Vol. ahs Vol. Th. Vol. SO a (ee SS eee ING Clete Oe eee crt ase hes 890 1,020 2,330 2,600 Balanced solution : (Nia 50g Cai) enon yee ne 810 842 2,460 2,660 CaCl Wee tater Remind best | 850 920 1,200 1,374 2,600 2,940 ACD Oe ene Sl ee es 970 1,050 2,750 2,050 | ie) ened Tee ae he Oe ee 1,280 1,460 1,130 1,270 1,200 1,250 WVBR ec tenn ct eters ase te elie 1,500 1,700 This biocolloid, representing more nearly the colloidal constitution of living matter, was seen to have higher hydration capacity in all salt solu- tions, and to have such capacity lessened in even the very dilute acid. A similar preparation in which the soap was pure potassium oleate gave results less marked as to the action of the salts, but the increase in the balanced solution was proportionately much greater, and the retarding effect in acids much greater. Ample justification exists, therefore, for a correction of the earlier statement as to the effect of salts of the common metals on biocolloids which have been found to offer many profitable analogies to living material. The correction implies that we may confidently look to these salts as accelerators of hydration and growth, or as increasing the water deficit of living matter. Lastly we come to the amino-acids and to water-soluble vitamine. I have previously pointed out in many papers that the commoner amino- acids, glycocoll, alanine, phenylalanine, asparagine, and histidine, which June, 1921] MACDOUGAL — WATER DEFICIT 301 have been proved to promote growth, also accelerate hydration in biocol- loids. As an additional step in this work, the effect of the water-soluble B yeast vitamine on biocolloids and living and dead cell-masses was measured. Solutions of this material at one tenth percent, having an acidity of PH = 5.25 as determined by the colorimeter method, were used, and measurements were taken by the auxograph. Taking the swelling in water as 100, the values obtained in the vitamine were as below: POravonnlmers, VOUNG. SECLIONS.. kak. sui vse le ode ode sw 8 dae ee 75 otabouvubens- large; SeCtIONS). 6. sa ce es Se. bd de sn ee wed oR be Pe 230 Squash fruits, young, sections of pulp... .......5.0.0..005...000. IIO Squash fruits, mature, sections of pulp...........).......6..0.2-:. 115 @rangce seedlings, root. tipsy living... ccc ea oa ee ete ee 150 @range seedlings) root tips; dried... 0. cc cei ee ce ek ee te bw ta es 120 Corn root tips, small, living........... rig wees eae Me, Sa Ret Oh 88 @ommuooe cips; dance: livine s,s. oi lea ee eee ee eu cbse sh eae 78 CGornaprooebips, langey dried. sf. 444 beens o's Us aris waa Seda ew ea ea 180 Sia wien sbOOt tips, WVAING <5 pais. ies so 0G we eles Weele uw dee Tee aes 133 Sunflower stems, sections of pith, mature, living.................. 150 Opuntia SECLIONS OF, VOUNE JOINS . 6 on wei ae ce ee oe eee eet 94 @pmnttas, sections of mature jOInts. ... ce ee ee ee ee 170 @ronmarieye aimed: SHCCS.)e.5 cc ee oe wk ee be ee le MMe eb haem 90 ESSIEN Gh 5st do eo es Oe RN nn 140 JSAM SUNG GIOVE 8g beats ace ge de an CEs Sas 132 PNG AteattGnpOLASSIUM-OlCALE ¢ 2s. bese ec ae bs le oS vee nb da em eoeta 80 OCEANIC MO Men VAANATIIIC! 4. i. wivlctudhe dures va 2 ales G4 ns Dass Fae uhaddes 95 omar celicine 2.cand -SaltS? <2-2 4 oi1h<< b when Whew sear cdg aoe wa 130 INCA ME CCLAUIING: 25 GOA Die ak. uh kN ik ase’ aters ecw a ne idlel nba eines bie Roe Set wn a vale wa ee Ae INGabeo celavine sang: SAltS 200% i2 a wie tbe Poe wave adele lve % 130 UE DISEASE Si At Sel inc uO a AG 7c Cn ea er 163 PelaminecaidusOd pers seas ca is tis hae arcane. | Wye! wid ApS aus? a a4 g2 The vitamine is seen to increase the hydration swelling = water capacity or water deficit of agar, agar-soap, agar-gelatine, agar-gelatine-salts, and gelatine, but to lessen hydration in some biocolloids containing soaps which would be sensitive to the free hydrogen ions of the vitamine solution. Parallel action in living and dead cells is implied, although it is to be noted that such cells may already include a certain amount of their characteristic vitamines and that the added vitamine could exert no additional effect except that of reducing hydration capacity.® SUMMARY 1. The theory as to the constitution of living matter by which plant protoplasm is taken to be a pentosan-albumin-soap colloidal mixture is > MacDougal, D. T. The effects of yeast vitamine water-soluble B on plant cell- masses and on biocolloids. Proc. Soc. Exper. Biol. and Med. 18 : 85. 1920. 302 AMERICAN JOURNAL OF BOTANY [Vol. 8 briefly restated. Its adequacy at the present stage of experimentation is confirmed. 2. The metals which form the bases of nutrient salts, as hydroxides and as chlorides and nitrates, are found to increase the hydration capacity, or the water deficit of the principal components of biocolloids, and of biocolloids of certain composition. 3. Biocolloids containing soaps show a high degree of sensitiveness to hydrogen ions, or acidity. Such biocolloids show marked action in balanced solutions of sodium and calcium as shown by data too detailed to be given in this paper. 4. Yeast vitamine water-soluble B, in a solution slightly acid, increases the swelling, hydration, or water deficit in some living and dead plant cell masses, and lessens it in others. Similar diverse action on biocolloids was found. 5. All of the substances tested which are known to facilitate growth in plants are found to increase hydration capacity or water deficit in some of the test objects. The list includes chlorides and nitrates of sodium, potassium, magnesium, and calcium in various concentrations between 0.001 N and o.ooo1 N, glycocoll, alanine, phenylalanine, histidine, and water-soluble B yeast vitamine. Hydroxides of sodium, potassium, lithium, rubidium, calcium, ammonium, and aniline also increase hydration values in some of the components of living matter. ated fee eEUSPORANGIATE FERNS AND THE STELAR THEORY D. H. CAMPBELL (Received for publication January 17, 1921) Some thirty years ago, as the result of an extensive series of investi- gations, Van Tieghem, the distinguished French botanist, brought forward his stelar theory which offered an interpretation of the nature of the tissue systems of the higher plants quite different from that which had been held by Sachs, de Bary, and other earlier investigators. Van Tieghem’s views, with some more or less important modifications, have been pretty generally accepted by the morphologists of the past two decades, and have been assumed to apply to all the vascular plants. Among the forms which have been the subjects of frequent and detailed study in regard to the nature of their stelar structures are various ferns. These studies have been directed largely toward the elucidation of the evo- lution of the stelar structures of the Filicineae and have included a study of many fossil ferns as well as the existing types. These investigations have thrown much light upon the relationships existing between the many Palaeo- zoic fern-like plants and their living relatives. An extensive literature on the subject has grown up in the past twenty years, especially in England, and with it a somewhat elaborate terminology based upon the assumption that the fibro-vascular skeleton of the fern stem is a strictly cauline ‘“‘stele’’ with which the corresponding foliar bundles are simply connected by the so-called “‘leaf-traces.”’ The general acceptance of Van Tieghem’s stelar hypothesis and its modification by Strasburger and other investigators, especially in England, are sufficiently familiar to students of plant anatomy. Van Tieghem con- cerned himself chiefly with the Spermatophytes, and his interpretation of the stelar structures of the ferns has been a good deal modified by the English investigators and by Jeffrey in this country. The latter’ has summarized these conclusions, and this has also been done at length by Bower? and Schoute.® Some of the most recent contributions to the subject* apparently accept these views as applying universally to the ferns, and quite ignore the evidence brought forward by the writer nearly ten years ago,® and amply 1 Jeffrey, E. C. The structure and development of the stem in Pteridophyta and Gymnosperms. Philos. Trans. Roy. Soc. B, 195: 119-146. 1902. * Bower, F.O. The origin of a land flora. London, 1908. 3 Schoute, J. C. Die Stelar-Theorie. Jena, 1903. 4 F.g., Thompson, J. M. New stelar facts, and their bearing on stelar theories for the ferns. Trans. Roy. Soc. Edinburgh 52: part 14, no. 28. 1920. °> The Eusporangiatae. Carnegie Inst. Washington Pub. 140. IgITI. 303 304 AMERICAN JOURNAL OF BOTANY 7 [Vol. 8 verified by the more recent work of West,° that the stelar theory, as usually understood, cannot be reconciled with the facts as revealed by a study of the Eusporangiatae. The writer has therefore thought it worth while to summarize this evidence, and also to add further facts derived from a recent study of Botrychium. From an extensive series of investigations on nearly all the genera of eusporangiate ferns, the writer was forced to the conclusion that a cauline stele is either completely wanting in these ferns, or that, where cauline stelar tissues are present, they constitute an insignificant part of the fibro- vascular skeleton. West’s studies on the Marattiaceae confirm these conclusions, which, however, as already indicated, seem to have been quite overlooked by some of the recent investigators. According to Van Tieghem’s view, most of the ferns are “ polystelic,’”’ the individual strands of the net-like woody cylinder of the stem being considered to be of independent origin, the reticulate structure resulting from the coalescence of these independent “‘steles.’”’ Most of the later students of the ferns consider the “‘dictyostele,”’ or reticulate woody cylinder, to be a single structure, 7.e., the stem is regarded as ‘‘monostelic,”’ the openings being designated ‘‘leaf-gaps’’ where the leaf-traces join the cylindrical cauline stele. In nearly all the recent studies on the stelar structures of the ferns, the strictly cauline nature of the axial fibro-vascular tissues 1s apparently taken for granted. Brebner’ first pointed out that in the very young sporophyte of Danaea simplictifolia the primary fibro-vascular bundle is common to the cotyledon and root, and that for a considerable time there is no evidence of any cauline stele. Little attention has been paid to these facts by most recent students of the ferns, but in a recent paper by West® the accuracy of Brebner’s conclusions has been fully recognized. The writer’s attention was first called to the real state of affairs in the Eusporangiatae as the result of a study of the embryology of Ophioglossum Moluccanum.® Many years ago, Mettenius"” described the young sporophyte of Ophio- glossum pedunculosum as consisting at first simply of a leaf and root, the definitive sporophyte arising secondarily as a bud upon the primary root. Very little attention was given by later students of the Ophioglossaceae to this really remarkable discovery, and it has been either forgotten or ignored. The writer collected in Java a considerable number of young sporophytes 6 West, C. A contribution to the study of the Marattiaceae. Annals of Bot. 31: 2601-41 A Ollige 7 Brebner, G. On the prothallus and embryo of Danaea simplicifolia, Rudge. Annals of Bot. 10: 109-122. 1896. On the anatomy of Danaea and other Marattiaceae. Annals of Bot. 16: 517-552. 1902. Soc cit: 9 Studies on the Ophioglossaceae. Ann. Jard. Bot. Buitenzorg II, 6: 138-194. 1907. 10 Mettenius, G. Filices Horti Botanici Lipsiensis. Leipzig, 1856. June, 1921] CAMPBELL — THE EUSPORANGIATE FERNS 305 of O. Moluccanum Schlecht, which is possibly identical with O. pedunculosum Desv. Specimens were secured in Ceylon of O. reticulatum L. or some closely related species. These agreed closely with the species described by Mettenius, and indicated that in these tropical species of Euophio- glossum, the young sporophyte is absolutely destitute of any cauline tissues, being composed at first of a simple primary leaf, or cotyledon, which merges insensibly into the root (fig. 1). A single central fibro-vascular bundle or ‘‘stele’’ extends without interruption from the petiole into the root, and its structure is essentially the same throughout, viz., “‘collateral’’ in the petiole, ‘‘monarch”’ in the root. Mettenius does not describe in detail the origin of the different organs of the embryo sporophyte. In O. Moluccanum the writer found that the sporophyte at a very early stage consists of but two portions, a large basal Fic. 1. Median longitudinal section of a young sporophyte of Ophioglossum Moluc- canum, showing the primary stele traversing the cotyledon, L, and the root, R. Pr, the gametophyte. Fic. 2. Three longitudinal sections of the bud developed upon the primary root, 7, of Ophioglosum Moluccanum. cot, cotyledon; I, /?, the first two leaves of the bud; 7, the first root of the bud; st, the stem apex. foot and an apical conical portion developing subsequently into the coty- ledon. At this stage, the embryo is strongly suggestive of that of Anthoc- eros. The growing point of the primary root arises endogenously, being formed near the center of the embryo where the base of the young cotyledon 306 AMERICAN JOURNAL OF BOTANY [Vol. 8 joins the foot. As the root grows, it pushes downward through the foot, which is practically eliminated and is no longer recognizable. The young sporophyte is thus bipolar in structure, and the stele, as we have seen, is continuous through the cotyledon and root. It is not until the cotyledon and primary root are fully developed that the bud which is to develop into the definitive sporophyte first becomes evident. This begins as a group of meristematic cells close to the stele of the root—exactly in the way a secondary root arises. The first two leaves of this bud are formed quite independently of the apical meristem of the young bud. The stele of the first leaf of the bud joins directly with the stele of the primary root, while that of the second leaf is joined to the base of the stele of the first root developed from the bud (fig. 2). It is thus clear that the fibro-vascular system in the sporophyte of O. Moluccanum begins as a single continuous strand extending from the petiole of the cotyledon into the primary root and practically of the same structure throughout, the xylem and phloem of the ‘collateral’ foliar portion being continuous with the corresponding tissues of the “monarch”’ root portion. This primary ‘‘stele’’ is not a ‘‘protostele,’’ 7.e., it is not “‘concentric’’ in structure, and, moreover, it is not a cauline structure. A study of the older sporophyte shows that much the same condition prevails as in the earlier stages. The leaf-traces unite with root bundles and with the older leaf-traces, and there is thus built up the open “dictyo- stele’’ found inthe adult rhizome." There is no indication of the develop- ment of any stelar tissues except those belonging either to the leaves or to the roots. BOTRYCHIUM In Botrychium Virginianum the structure of the axial stele is quite different from that in Ophioglossum. At an early stage,” the axis of the young sporophyte shows an almost unbroken cylindrical stele enclosing a central pith instead of the large-meshed ‘‘dictyostele’’ of Ophioglossum. In the very young sporophyte a single strand of procambium extends through the axis of the cotyledon into the primary root; but as these organs usually make a marked angle with each other, the primary vascular strand is strongly bent instead of being straight, as it is in Ophioglossum Moluccanum. Very soon the second leaf is developed, and a similar vascular strand is formed in it, which unites with the primary vascular bundle near the point of junction between the petiole of the cotyledon and the base of the primary root (fig. 3), and the fusion of the three bundles appears as a closed ring in cross section. The traces from the later leaves behave in much the same way, and the massive “‘siphonostele’’ found in the older stem is thus built up. 11 For details see ‘‘The Eusporangiatae,’”’ pp. 89-93. 2 Jeffrey, E.C. The gametophyte of Botrychium Virginianum. Trans. Canad. Inst. 5: I-32, 1808. Pe June, 1921] CAMPBELL — THE EUSPORANGIATE FERNS 307 As in Ophioglossum, the bundles are collateral, and, as is well known, there is developed a cambium between xylem and phloem, and also medul- lary rays, so that the structure of the woody cylinder in the older sporophyte is extraordinarily like that in the Gymnosperms and in many Dictoyledons. Fic. 3. A. Median longitudinal section of a young sporophyte of Botrychium Vir- ginianum, cut at right angles to the primary root. The two leaf-traces unite at the junction of the root. B. Transverse section of a similar sporophyte showing the two leaf-traces. C. Another section of the same sporophyte showing the junction of the two leaf-traces with the stele of the root. As in Ophioglossum, there is also in Botrychium no evidence of any procambial tissue in the axis above the youngest leaf-trace, 7.e., the stelar tissues as in Ophioglossum are composed entirely of the coalescent leaf- traces. A still closer resemblance to Ophioglossum is shown by the young sporo- phyte of B. obiquum Miihl., which the writer has recently examined. In this species the cotyledon and primary root, instead of forming an angle with each other, as in B. Virginianum, have a common axis, and the orien- tation of these organs is like that in Ophtoglossum Moluccanum. The writer. is indebted to Dr. H. L. Lyon of Honolulu for the material upon which hfs investigations were made, and for the accompanying photograph (fig. 4). Dr. Lyon" first directed attention to the peculiarities of this species, 18 Lyon, H. L. A new genus of Ophioglossaceae. Bot. Gaz. 40: 455-458. 1905. 308 AMERICAN JOURNAL OF BOTANY [Vol. 8 and through his courtesy the writer has been able to examine a large number of preparations made by Dr. Lyon, as well as to make a series of slides from gametophytes and sporophytes furnished by him. Fic. 4. Median longitudinal section of a young sporophyte of Botrychium obliquum, showing the continuity of the steles of the cotyledon and root, and the junction of the two leaf-traces at the base of the root. Photograph by Dr. H. L. Lyon. In B. obliquum (fig. 4), there is a conspicuous suspensor, comparable*to that in Danaea and Macroglossum.!> Moreover, the young sporophyte is bipolar in structure, and the relation of cotyledon and root is essentially the same as in Ophtoglossum Moluccanum. As in the latter, the primary root of Botrychium obliquum is endogenous in origin, instead of being superficial as it is in B. Virginianum. It grows downward through the foot, exactly 144 Campbell. The Eusporangiatae. . 15 Campbell, D. H. The structure and affinities of Macroglossum Alidae, Copeland. Annals of Bot. 28: 651-669. 1914. June, 1921] CAMPBELL — THE EUSPORANGIATE FERNS 309 as it does in Ophtoglossum Moluccanum and the Marattiales, and the foot thus is practically obliterated, instead of forming a large part of the embryo as it does in B. Virginianum. In the latter, as we have seen, the primary root and the cotyledon make a sharp angle with each other, while in B. obliquum their axes are in a straight line, and the common vascular bundle, or stele, closely resembles that of Ophioglossum Moluccanum. The stele of the primary root, however, is diarch as it is in B. Virginianum and in most of the Marattiales. The development of the cylindrical stele in the axis of the young sporo- phyte is essentially the same as in B. Virginianum, 1.e., it is formed by the union of the broad leaf traces of the early leaves. The writer was not able to obtain the youngest stages of the sporophyte in Helminthostachys, but from a study of somewhat older specimens the conclusion was reached that the stele is formed in much the same way as in Botrychium.'® THE MARATTIALES Brebner"’ first called attention to the absence of a cauline stele in the young sporophyte of Danaea simplicifolia, although the writer'® had shown in an earlier study on Marattia that there was at first a continuous procam- bium strand traversing the young cotyledon and primary root. Farmer’s figures of the young sporophyte of Angiopteris!® show the same condition. The most recent contribution to the subject is a paper by West”’ who finds that there is no cauline stele in the young sporophyte of the Marattiaceae. Fic. 5. A. Vertical section of an embryo of Danaea elliptica, passing through the cotyledon, and showing the endogenous origin of the root, 7. B. A nearly median section of a very young sporophyte of Marattia Douglasii, showing the young stele extending from the cotyledon into the root. The root apex does not show in this section. 16 Campbell. The Eusporangiatae. 1” Brebner, loc. cit. Campbell, D. H. Observations on the development of Marattia Douglasii, Baker. Annals of Bot. 8: 1-20. 1894. 19Farmer, J. B. The embryogeny of Angiopteris evecta, Hoffm. Annals of Bot. 6: 265-270. 1892. 20 West, loc. cit. 310 AMERICAN JOURNAL OF BOTANY [Vol. 8 The writer?! examined with great care the development of the fibro- vascular system in the young sporophytes of Danaea Jamaicensis Underw. and D. elliptica Smith, and also the younger stages of the sporophytes of species of Angiopteris, Kaulfussia, and Marattia. In all of these (fig. 5, A) it was found that the primary root is deep-seated, growing through the foot precisely as it does in Ophioglossum Moluccanum and Botrychium obliquum, and there is a single primary stele traversing the axis of the cotyledon and root (fig. 5, B). The young plant is thus bipolar, so far as the cotyledon and root are concerned, and the insignificant stem apex appears as a small lateral appendage near the junction of the two primary organs. No procambium can be made out in the very small mass of tissue which can be assigned to the stem. This is particularly well shown in Danaea (fig. 6), where the stem apex is seen in the angle formed by the junction of the bundle from the second leaf with the primary bundle, but no sign of procambial tissue can be seen in the region above this junction. Oy Ma cay IH 1 a B Fic. 6. A. Longitudinal section of young sporophyte of Danaea elliptica, showing the junction of the two leaf-traces. B. The stem apex of the same sporophyte with the trace of the second leaf, /?, passing to one side of it. 21 Campbell. The Eusporangiatae. June, 1921] CAMPBELL — THE EUSPORANGIATE FERNS 3II The vascular strands of the first two leaves are collateral in structure, and after their fusion near the junction of the cotyledon and primary root, the xylems of the two bundles are easily recognizable and are continuous with the two xylems of the diarch root (fig. 7). Fic. 7. Five cross sections of a series from a young sporophyte of Danaea Jamaicensts. A shows the two primary leaf-traces; B and C, the fusion of these to form the solid stele of the young plant; D, the transition region of the sporophyte; £, the stele of the root. It is clear that the central region of the young sporophyte is not strictly a cauline structure, since the cotyledon and primary root are in no sense appendages of the stem, which at this stage consists only of the very small area in the immediate vicinity of the apical meristem. Moreover, the foot contributes a considerable amount of the outer tissue in the central region of the young sporophyte. As the new leaves are formed, close to the stem apex, their steles unite with those of the older ones, and there is thus built up a ‘‘dictyostele,”’ much like that in Ophioglossum. After about seven leaves have been 312 AMERICAN JOURNAL OF BOTANY [Vol. 8 formed, this structure is complicated by the development of vascular strands inside the dictyostele, and these ‘“‘commissural’’ strands can be traced up to the apical meristem of the stem, and are therefore true cauline strucures. As the sporophyte increases in size, the number of leaf-traces increases and further commissural strands are also formed, but the greater part of the elaborate skeleton of the adult sporophyte is undoubtedly of foliar origin, only the relatively unimportant commissural strands being cauline. The history of the development of the fibro-vascular skeleton of the Eusporangiatae leads inevitably to the conclusion that in the Ophio- glossales the whole stelar system is derived from the leaves:and roots, and this is true to a great extent for the Marattiales, although in the latter the commissural strands are really cauline in origin. It may be said also that the cortical tissue of the caudex is to a consider- able extent of foliar origin, being made up of the coalescent leaf bases. This is, to some extent, a confirmation of Delpino’s theory that the leaves, instead of being appendages of the stem, are the primary organs, and that the so- called stem is formed by the coalescence of leaf bases.” It is also clear that the medullary tissue is in no case of stelar origin, but is always a portion of the ground tissue (to use the older term) which is more or less completely enclosed by the coalescent foliar steles. The great preponderance of the foliar structures over the stem in most Filicineae has not received the attention that might be expected, in the many discussions on the nature of the stelar tissues that have appeared. Few of the higher plants show this to the same extent, and it may be ques- tioned whether any Angiosperm can show leaves equal in complexity to such ferns as Angiopteris and some of the tree ferns. It is true that the leaves of some palms are bulkier, but structurally they are decidedly simpler. So far as mere length is concerned, probably some species of Gleichenia and Lygodium surpass even the longest palm leaf. Hooker” states that Lygo- dium articulatum A. Rich has stipes 50 to 100 feet in length arising from a slender prostrate rhizome. The very young sporophyte of Ophioglossum, which the writer believes to be the most primitive of existing ferns, has no stem at all, but consists simply of a single leaf and root, the stem arising secondarily as an adventi- tious bud. This is entirely in harmony with the theory of the derivation of the Filicineae from Anthoceros-like ancestors; and the predominance of the leaf, shown in the young sporophyte, is maintained throughout the whole history of the Filicineae. The assumption, therefore, that the stem is the predominant or primary organ of the sporophyte, and that the leaves are mere appendages of this, is hardly borne out by a study of the ontogeny, at least of the Eusporan- giatae; and this probably will be shown also to be the case in many, at least, of the Leptosporangiatae. 22 See Schoute, loc. cit., p. 97. : 23 Hooker, J. D. Handbook of the New Zealand flora, p. 385. London, 1867. a June, 1921] CAMPBELL — THE EUSPORANGIATE FERNS 313 When one compares the slender rhizome of such ferns as Lygodium, Gleichenia, and many Hymenophyllaceae with the large and highly developed leaves, it may well be questioned whether the leaves should be regarded as mere appendages of the relatively insignificant axis. This is particularly the case with such forms as Gleichenia and Lygodium whose leaves show almost unlimited power of continuous growth in length. It is very important that further studies upon the origin of the stelar tissues of the Leptosporangiatae should be undertaken. Most of the studies already made upon the ontogeny of these ferns have not dealt with the earliest stages of the stelar tissues, but have started with the fully developed stele of the young sporophyte, assuming that this axial stele is really of cauline origin and not a composite structure derived from a fusion of leaf-traces. In order to solve this question it is necessary to examine series of sections, both transverse and longitudinal, including the growing point of the stem and the adjacent regions. In longitudinal sections alone there is danger of misinterpretation, as the traces of the youngest leaves may be easily mistaken for a true cauline stele; but if corresponding transverse sections are examined, it is then possible to determine whether or not there is a stele of strictly cauline origin. It will not be surprising if such a test applied to the Gleicheniaceae and Hymenophyllaceae will show that the solid or tubular axial steles are in reality composed of coalescent leaf-traces as they are in Botrychium and in the young sporophytes of some of the Marattiales. It is very desirable that the many careful studies of the stelar tissues of the ferns be reviewed to determine whether the usual interpretations of the relation of the tissues of the leaves and axis are tenable. A satisfactory solution of the problem necessitates an examination of the origin of the tissues in the young sporophyte as soon as it emerges from the gametophyte, and a further study of the building up of the stelar structures as new leaves are developed, tracing the origin of the individual vascular strands to their beginning. Reconstructions from series of cross sections of the completed bundles of older stages will not suffice. CONCLUSION 99 66 The presence of a single cauline stele—“protostele,’”’ “‘siphonostele,”’ “dictyostele’’—is not borne out by the history of the stelar tissues in the Ophioglossales and Marattiales. In all of these the stelar system begins as a single strand common to the first leaf and root; the stem apex arises adven- titiously in Ophioglossum Moluccanum and O. pendulum, and is very in- significant in Botrychium and the Marattiales. No procambium is de- veloped in the stem region in the young sporophyte. In the Ophioglossales, the stelar structures of the axis are built up ex- clusively of leaf-traces to which the bundles of the roots are joined. This condition obtains also for the earlier stages of the Marattiales, but is com- 314 AMERICAN JOURNAL OF BOTANY [Vol. 8 plicated later by the formation of ‘‘commissural’’ strands, which are of cauline origin. The “‘dictyostele’’ of Ophioglossum and most Marattiales is in no sense a monostele. The “foliar gaps’’ are not breaks in a single tubular stele, but are merely spaces between the coalescent leaf-traces, and the pith is part of the ground tissue included within the cylindrical network formed by the united bundles derived from the leaves. In short, the condition found in the axis of the eusporangiate ferns is more in accord with the older theory of “‘common’’ bundles traversing a ground tissue, and united to form the woody cylinder of the axis, than with the assumption o a true cauline stele. The condition existing in the eusporangiate ferns by no means implies that the stelar hypothesis must be completely discarded. There seems to be no question of its application to the Lycopods, Conifers, and many Angio- sperms; but in all of these, the relative importance of stem and leaf is very different from the condition in the ferns; and it will not be surprising if, when the different types of the Leptosporangiatae are subjected to a thor- ough examination of the origin of the stelar tissues in the young sporophyte, it will be found that in these, as well as in the Eusporangiatae, the axial stelar tissues are largely, at least, of foliar origin. STANFORD UNIVERSITY Sab RELATION. OF PLANT PATHOLOGY TO HUMAN WELFARE! F. L. STEVENS (Received for publication January 17, 1921) In the present era of high cost it is especially fitting that one take account of all expenditures, and weigh carefully the returns. With the present underpaid and poorly equipped condition of many educational and research institutions, and especially in the light of certain criticisms that are made regarding research, I am impelled today to select from the many interesting themes which might be developed regarding plant pathol- ogy, and to direct thought to research in the science of plant pathology and its related fields, and briefly to indicate the returns therefrom. Plant pathology is preeminently a practical science, and its prime func- tion is to guide the way to an ever-increasing control over disease. The magnitude of the annual loss incurred in the United States alone through plant disease in diminution of yield and loss of produce is far greater than it is generally conceived to be. I shall not burden you with statistics, but I do wish to give a few examples, taken from the most reliable estimates that have been made, to indicate the loss. Thus, in the various Plant Disease Survey Reports we find that for the year 1919 losses from plant diseases are given as follows: For the five leading cereals 482,695,000 bushels; for potatoes 86,997,000 bushels; for tomatoes 307,168,000 bushels; for sweet potatoes the loss is put at 58,841,000 bushels, or more than one half the crop. You are all familiar with the diseases mentioned; but you fail to get the world bearing of plant-disease ravages unless you include in your vision such destructive diseases as the coffee rust, affecting in disastrous form a crop of large world value, which in two years destroyed 272,000 acres in Ceylon; the banana wilt, which is reported to have caused abandon- ment of nearly 20,000 acres of banana plantings in Panama alone and to have rendered useless large railroad lines; the cocoanut palm bud-rot, which kills the growing point of this valuable tree and which is rapidly encircling the world. Your imagination may fairly picture similar diseases as occurring throughout the world on the whole range of useful plants. Before harvest disease may devastate the crop in the field, and after harvest the inroads proceed in storage. Obviously the loss occasioned by destruction of the product at market is far greater per unit than similar losses in the field. 1 Invitation paper read before the joint session of Section G, A.A.A.S., the Botanical Society of America, and the American Phytopathological Society, in the symposium on “The Relation of Botany to Human Welfare,”’ at Chicago, December 29, 1920. 315 316 AMERICAN JOURNAL OF BOTANY [Vol. 8 Thus a carload of Georgia peaches spoiled by brown-rot in New York means loss of transportation and handling as well as of the original value of the fruit. An annual sum of $30,000,000 is said to be a conservative estimate of the loss in the United States between the field and the consumer, while in 1919 the total loss with fifteen principal food products is estimated at nearly a billion and a half dollars. Even land values are frequently seriously depreciated when the soil is so infested as to preclude the raising of the particularly profitable crop, as, for example, when the tobacco wilt pos- sesses land in the bright tobacco belt, leaving a farm which is comparatively worthless for any other crop, or again, as I have seen, when the wilts of cotton, cowpeas, and melons all occur upon the same field. As civilization advances, intercourse between regions more or less remote increases and the disease range and prevalence expand. Thus, much as with human and cattle diseases, though to much greater extent, the number of plant diseases known in any community is annually increased by additions from near-by regions or from far-away continents. Pre- sumably the potato late-blight fungus began its journey of conquest in the Andes, and as early as 1845 caused famine in Europe and much loss in many continents. The asparagus rust appeared in New Jersey in 1896 and spread until it reached California in 1901. Many other serious diseases have come to us from abroad, including the sorghum smut, grape anthrac- nose, cucurbit mildew, carnation and chrysanthemum rust. Numerous serious diseases have likewise invaded other countries from here, among them the grape black-rot and downy mildew. Of interstate migration interesting cases are afforded by pear blight, from the Hudson valley in 1792 to California in 1895, and by peach yellows from Philadelphia in 1806 to Maine and Illinois in 1886. Among the late continental arrivals are the pine blister rust, which is under such headway that it seems to be impossible of extermination. The value of the susceptible pines is such that the loss may readily reach a hundred million dollars. The chestnut-bark disease caused a loss of $25,000,000 from 1904 to 1911. Much more serious is the loss to be borne as it invades the great chestnut forests of the Appalachians. Citrus canker, imported from Japan about 1910-11, bids fair to ruin large industries. Potato wart entered Newfoundland in 1909 and was found in Pennsylvania in 1918. It is of interest to note in passing that, were agriculture not taught in the public schools, its presence might yet be unsuspected. Flag smut of wheat was un- discovered in America until May 5, 1919, and is as yet known in but one county in Illinois. This disease is said to cause loss ranging from 10 to 50 percent in Australia. As increased long-distance communication gives intercontinental trans- port to disease, so congestion of crop population creates a bridge by which. the causal organism may more readily pass from plant to plant or from farm to farm. In these two conditions, facility of transportation and June, 1921] STEVENS — PLANT PATHOLOGY 317 congestion of crop, we find, to a large degree, explanation of the fact that plant diseases are more prevalent now than formerly. The multiplicity and diversity of plant diseases are especially striking. While the physician has but one species of patient and the veterinarian but a few species, the phytopathologist has to advise regarding many species of plants each of which has, to a great extent, its own large list of diseases. Thus, on the apple alone there are 18 major diseases; on wheat 10; on potatoes 12; while for each crop the number of minor diseases is more than ten times as great. : I have attempted thus briefly to indicate the damage done by plant disease, as a background for a discussion of the part played by plant path- ology. The point of importance is not how great is the loss from plant dis- ease, but rather how much influence has the science of plant pathology had in lessening this loss. Like bacteriology the science is young, dating back barely to the middle of the last century. It was first taught in any American college in 1873 (Illinois), and first as a special subject in 1875 (Harvard). The science has grown until today the American Phytopathological Society enrolls nearly 500 members, the majority of whom are professional plant pathologists, and whereas but one paper appeared in America on the subject in 1861, each month now adds scores of titles and nearly a hundred papers are presented here this week. Large federal and state appropriations sustain its researches. What is the nature of the return that plant pathology has given? The achievements may be summarized briefly as falling within seven great categories demonstrating the value of: protective applications, sprays and dusts; excision; seed steeps; general sanitation leading to diminution of infective material; breeding for disease resistance; modifications of agricul- tural practice; quarantine restrictions. It is unnecessary to discuss these, but I wish to point out that while a modicum of the present benefit doubtless would have obtained from an empirical, rule-of-thumb procedure, the great body of our present knowledge of disease prevention is the direct outcome of truly scientific investigation. It is difficult as you journey from coast to coast today, and see spraying practiced everywhere, to realize that prior to 1885 no spraying was done in the United States. The vast sums spent for copper sulphate, lime-sulphur, etc., and the large factories devoted to making spraying machinery also attest the wonderful growth of this custom. Yet it was not the accident associated with the stealing of wayside grapes that was responsible for the discovery of the efficiency of fungicidal applications; it was the close obser- vation of Millardet followed by his keen analysis and exact experimentation, all of which would probably have failed were it not for the basic knowledge that Millardet had regarding fungi and parasitism. His receptivity of mind was doubtless dependent upon mycological studies of many decades. 318 AMERICAN JOURNAL OF BOTANY [Vol. 8 Heteroecism of apple and wheat rust and hibernation of many fruit-rot fungi in cankers or mummied fruits, which in the light of science are simple, easily comprehended facts, could without science have had but little more than the force of superstitions. The investigations which have given greatest value to seed steeps have been those that showed the part played by seedling and floral infection. Recommendations of general sanitation would be largely without force were it not that the underlying reasons were made obvious by scientific explanation. Of all the categories mentioned, perhaps the least dependent upon science and the most empirical is that relative to disease resistance, since some of our most valuable resistant varieties have been given to us by farmers, while many of the most sus- ceptible have been eliminated naturally. During recent years, however, knowledge of Mendelism and of biologic specialization has added a very important, truly scientific aspect to this somewhat empirical subject. Many crops are of such small acreage value that expensive methods of disease prevention permissible with more valuable crops are precluded. In such cases, modification of practice, as change of time of seeding, of crop rotation, of kind of fertilization, of degree of drainage, of age of seed, of depth of plowing, of proper relation of direction of rows to wind and light, has in many cases proved serviceable. The suggestion of such modifications depends upon most intimate knowledge of both crop and parasite, and full life-history studies of the ecology of the organisms are needed. It is obvious that for the establishment of proper quarantine restrictions the taxonomy and morphology of the causal organisms must be known. It is both impossible and unnecessary to assign any money values to the protection that has been given to American crop plants under the various categories mentioned. ipaab, 3 is bebliched Shoathiy: extent antine yee aye MABE bton: price, $6.00 a year. Single copies 75 cents. Back n CL each; $7.00 a volume. Postage will be charged-to all foreign c ntri s, e cept. Mexico, Cuba, Porto. 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Cepproxiinately” $6.40, a: pares ye ae per squate inch for. half-tones). % fet Separates should be ordered hen proo!) ie baote will. be ronal free; cover, and’ pale Woke “Claims. for missing haben shoud ine mac aa of mailing. co bie publishers will ‘ ia have been lost inthe mails. © ae “Correspondence ‘ ‘concerning editorial ‘matters ee 0s Allen, University, of W isconsin, Madison aia ~ Business. correspondence, including: notice o ee concerning reprints, should be addresséd to Ame Sas beeen Cate Suess dan N. oe or Aes AMERICAN \ | JOURNAL OF BOT VoL. VIII ce No. 7 ~, THE RELATION OF CROP-PLANT BOTANY TO HUMAN WELFARE! CARLETON) Re [BALE (Received for publication January 17, 1921) It is with combined trepidation and pleasure that I endeavor to discuss: before this gathering the subject of the relation of the botany of crop plants: to human welfare. Trepidation because of the difficulty of adequately presenting so important a subject. Pleasure because I believe that a vital. relation exists between the botany of crop plants and human welfare and rejoice at the opportunity to emphasize it to others. Let us define what we: shall discuss together. BOTANY What is botany? The dictionary tells us that it is the science of plant form, structure, function, relationship, and distribution. From _ this category we evolve several subdivisions of the science; for example: Phytomorphology, the science of plant form and structure; Phytophysiology, the science of plant function and growth; Phyto-ecology, the science of plant response to environment; and Phytotaxonomy, the science of plant relationships. Some of these major divisions are themselves subdivided. For instance, taxonomy includes phytography, or plant description; taxonomy proper, or plant classification; and nomenclature, or plant naming, the black sheep of the family. In like manner, plant ecology includes plant physiology and phytogeography, or plant distribution. In addition to these grand divisions of botany there are some important specialized phases of botanical science, such as phytopathology, or plant diseases; pharmacognosy, or pharmaceutical botany, dealing with medicinal plants; and phytopaleontology, or paleobotany, the science of fossil plants, sometimes called fossil botany. Phytopathology, in turn, includes my- cology, or the taxonomy of fungi; physiology, or the function of both host and parasite; and ecology, or environmental response. Finally, there is * Invitation paper read before the joint session of Section G, A.A.A.S., the Botanical Society of America, and the American Phytopathological Society, in the symposium on “ The Relation of Botany to Human Welfare,” at Chicago, December 29, 1920. [The Journal for June (8: 275-322) was issued June 30, 1921] | 323 324 AMERICAN JOURNAL OF BOTANY [Vol. 8, genetics, the science of the origin and expression of characters, the sum total of which makes up the organism as we know it. Because of the symposium arrangement, it is necessary to restrict the treatment of the theme so as not to trench on the subjects assigned to my colleagues, Dr. Cowles and Dr. Stevens. Omitting their topics, the re- lation of ecology and of pathology, respectively, to human welfare, there remain the morphology, physiology (aside from environment), and genetics of crop plants, as well as pharmacology or pharmaceutical botany. At the start we are confronted by the well-known dictum of one class of botanists that, whenever botany relates in any way to human welfare, by that very fact it ceases to be botany. One is reminded of the famous couplet written in similar vein: My name is Benjamin Jowett, Master of Balliol College, Whatever is known, I know it, Whatever I don’t, i8n’t knowledge. It may be argued that this dictum is but a theory, a state of mind. To those who cherish that delusion it should be necessary only to point out a few striking facts of the botanical past and present. Development of Botanical Science Until comparatively recent years practically all botanists studied wild and domesticated plants impartially. This was true of the Greek philos- opher-naturalist, Theophrastus, of the third century B.C., the father of modern descriptive botany. It was true of the Greek physician, Dios- corides, and of the Roman, Varro, in the first century B.C., and of the Roman essayist, Pliny, in the first century of the present era. It remained true of botanists in general until toward the middle of the nineteenth century, or some 75 to 100 years ago. With the revival of learning after the Dark Ages came a renewed ex- pression of interest in things botanical. The first important books of a distinctively botanical character were the so-called herbals, running from those of Ruel in 1537 and Fuchs in 1542 to those of Ray and Morison in 1688 and 1699, respectively. These ponderous folio volumes, written in quaint and not always accurate Latin or in no less quaint English, and illustrated with crude but often startlingly realistic woodcuts, served as repositories of popular and semi-scientific information on plants during the sixteenth and seventeenth centuries. They were based on the writings of the Greek and Roman authors named above, but contained many original observations. Many of the plants treated by the herbalists were in common cultivation, and in that period the crop plants received the same botanical attention as did the feral species. Thus we see that the important early contributions to botanical science were in the field of what, in recent years, unfortunately has been called applied or agricultural botany. July, 1921] BALL — RELATION OF BOTANY TO HUMAN WELFARE 325 Throughout the seventeenth century, botanical knowledge was growing rapidly along taxonomic lines. Master minds were laboring to formulate orderly classifications of both wild and domesticated plants. The later and more logical of these attempts are typified in the writings of Tournefort and the elder Jussieu and reached their climax in the eighteenth century in the classic works of Linné, in 1737 and 1753, and in those of the contemporaneous Jussieu the younger. By these and succeeding systematists of the eigh- teenth century and the first half of the nineteenth century, the leading species and subspecies of important cultivated plants were included and given specific rank as readily as if native and wild. The writer does not believe that the feral and cultivated species should have received identical treatment, for differences in morphological characters sufficient.to separate species of wild plants are sufficient only to separate agronomic or horticultural varieties of crop plants. The point to be em- phasized is that the cultivated plants were felt to be worthy the attention of the greatest botanists of all those centuries. Before the middle of the nineteenth century, a change of attitude had be- come fully evident. Discussion of crop plants was eliminated from manuals of botany. This change of attitude probably was due to several different reasons. The first, doubtless, was a realization of the difficulties of classi- fying cultivated crops on the same basis as wild plants. The second was the marvelous improvement in methods of transportation and communi- cation, through the development of railroads and postal facilities. These made possible the botanical exploration of the hinterlands and distributed the resulting collections to botanists everywhere for study, thus diverting attention from domesticated plants. The third and perhaps controlling reason was a growing feeling that useful plants were of a class apart, botani- cally unclean, and unworthy the best thought of the systematist and physi- ologist. The result was that, while a few botanists devoted themselves almost exclusively to studies of crop plants, the great majority shunned them entirely. That such a situation should have developed at all was most unfortunate, but that it should have come about just in that period of time was particularly deplorable. The present era of widespread appreciation and subsidization of the biological sciences was about to be ushered in. The act establishing the system of land-grant colleges was in process of formation. The founding of the state agricultural experiment stations was only 30 or 40 years ahead. The increased appropriations for research in nation and states were to follow soon after. The amazing endowment of great universities was on the horizon. The organization of such great scientific bodies as those in session here was about to become an accomplished fact. . One can almost imagine that the scientific sky must have been aglow with the coming dawn. In spite of this, Botany drew apart and proclaimed herself too sacred to be polluted by the useful. The pursuit of truth for 326 AMERICAN JOURNAL OF BOTANY [Vol. 8, truth’s sake is noble, and consecrated thousands have devoted themselves to lives of privation and sacrifice under the inspiration of this ideal. Truth for man’s sake is no less noble. If in social science, service to others is the highest form of altruism, how unreasonable the attitude that, in the © development of a natural science, no thought for humanity should be allowed to enter. From our present vantage point of perspective, it seems doubly un- fortunate that the botanical fraternity should have lost interest in domesti- cated plants just at a period when the development of teaching and re- search institutions would have given them the needed laboratory and field facilities for really effective study. Farm crops as a subject was not and is not taught by botanists. As the writer has pointed out in a previous paper, the original farm-crop specialists entered that field through many doors, including chemistry, the old-time agriculture, and even animal husbandry, as well as botany. On the other hand, when botany did deal with any phase of farm crops she called it ‘applied botany” or ‘economic botany,’’ and so erected the wall which gradually shut her off from part of her own domain. From this illogical and unfortunate separation, both botany and agronomy suffer to this day. HuMAN WELFARE Human welfare may be defined as a satisfactory condition or relation of human society, individually and in the mass. Such welfare must be both material and esthetic. It presumes a coérdination of good in the physical, mental, and spiritual realms. Can the botany of crop plants be shown to have any relation to these phases of human welfare? Let us ascertain. Upon the products of the vegetable kingdom the human race depends for the very essentials of its life. The vital needs of humans are two- fold, food and shelter, and these are important in the order named. The great classes of useful plants which minister to these two needs are cereals, fruits, vegetables, forages, saccharines, and medicinals among the foods, and fibers and forest supplies among the materials providing shelter. Human Food Human food is chiefly either animal or vegetable in origin. Primitive man doubtless was both herbivorous and carnivorous. Wild animals furnish as abundant and palatable a food supply as do domesticated animals, but the same is not true regarding feral and cultivated plants. It seems probable, therefore, that primitive man used meat as his staple food, and used vegetable materials to maintain health, to vary his diet, or as a filler. In temperate areas, at least, the necessary supply of roots, fruits, and seeds was obtained only by arduous search and tedious labor in gathering. It could have been abundantly obtainable, also, only during a portion of the year, which would have been less true of animal food supplies. On the July, 1921] BALL — RELATION OF BOTANY TO HUMAN WELFARE 327 other hand, vegetable food materials were more readily preserved for future use than were meats. The story of the first attempts at cultivation of food crops is lost in the mists of prehistoric time. Seeds dropped about a favorite camping site by members of a nomadic tribe, during preparation or eating, may have produced plants after the family moved on. Later, when they passed that way again, the mature plants with ripened seeds or fruits may have attracted their attention as providing a convenient food supply in a concentrated area. From some such chance observation may have developed the rudiment of the idea of growing the food plant where it was to be used. And no true welfare of human society was possible until man could produce and store food supplies against a time of scarcity and need. We can even imagine that the quantity and nature of the food supply had its influence on developing mentality. Perhaps there arose, even in those days, the superman who ruled by brain as well as by brawn. And perhaps also there was not wanting an envious Cassius to exclaim: “On what meat doth this our Caesar feed, that he is grown so great?” The Future Food Supply The chief problem of the world in the immediate future is the food supply. Across the pages of history one clear record runs. That nation is most secure which has, or can insure, adequate resources of food. Napoleon said that an army travels onitsstomach. Not only of the army but of the nation itself is this true. Two thousand years ago the grain ships from Egypt sailed the Mediterranean to imperial Rome. ‘Today the grain ships ply the seven seas to imperial Britain. They go from Australia and Argentina, from India and from Canada, and even from the United States itself. To- morrow they may be steaming toward our shores, carrying a similar cargo. Ever since American agriculture advanced from the forest clearing to the open prairies and the boundless plains, our country has been a heavy exporter of foodstuffs. Not once have we had to stop and consider from across what seas grain ships should come to us. But now the old order changeth and giveth place to new. With a population increasing rapidly through birth and immigration, and with no large areas of cheap and fertile lands remaining to be brought under cultivation, we come face to face with the problem of our future food supply. Our exports of vegetable food~ stuffs are steadily declining as more and more is required at home. Our imports of food materials are steadily mounting as the need increases. This is not written as a pessimistic prophecy but as a sane realization of an imminent problem in order that an adequate solution may be sought. The solution lies in one or more of three directions. First, immediate restriction of immigration and finally the restriction of the birth rate through economic pressure. There are some well recognized but unfortunate bio- logical facts which make this undesirable. Second, an endeavor to im- 328 AMERICAN JOURNAL OF BOTANY [Vol. 8, port what we do not produce. This puts us at the mercy of other nations in ways of which we have had recent illuminating examples. Third, an undertaking to increase our own production of food to keep pace with in- crease of population. This last is the only satisfactory decision. How may this result be accomplished? : There are two chief lines of attack on the problem of increasing our food supply. One is to increase the area under cultivation, by reclaiming desert areas through the more extensive and more productive use of irriga- tion waters, by the reclamation of swamp lands, and by the utilization of the untilled lands in the present tilled area. Some of these are problems in engineering, some in economics, some in soil science, and some in crop physiology. The other possibility is to increase the productivity of the areas already farmed. This requires progress in farm organization, soil science, animal husbandry, crop rotations, plant improvement, plant introduction, and the control of crop pests. Both methods present plant problems which challenge botany to her utmost endeavor. BoTANIC FAMILIES OF IMPORTANT CROP PLANTS We have seen that the crop plants vital to human welfare are those which furnish food, fodder, and medicine for man and his domesticated animals, clothing for him, and shelter for him and for them, and also for his industries. Plants from almost the entire range of the vegetable king- dom are requisitioned to provide material for one or another of these pur- poses. This realization recalls the promise of Scripture: And God said, ‘‘ Behold, I have given you every herb yielding seed, which is upon the face of all the earth, and every tree which is the fruit of a tree yielding seed, to you it shall be for food ’’ (Genesis 1: 29, Am. Rev.). The three important classes of food plants for man are the cereals, the vegetables, and the fruits. Cereals No other botanic family is of such overwhelming significance to the human race as the grass family, Poaceae or Gramineae, which contains the cereals, corn, wheat, rye, oat, barley, rice, sorghum, and millet, as well as the most important hay, grazing, and silage crops. For those who like statistics, it may be of interest to note that the estimated value of the cereals grown in the United States in 1917 was over $6,800,000,000; in 1918 about the same; and in 1919, nearly $7,300,000,000. Out of sympathy for any botanists so unlucky as to own grain farms, the figures for 1920 are omitted. A few other plants ordinarily are classed as cereals, though not truly such. Among these is buckwheat, belonging to so distant and unpromising a botanic family as the Polygonaceae, while the closely related family, Chenopodiaceae, contains quinua, a human food extensively used by the July, 1921] BALL — RELATION OF BOTANY TO HUMAN WELFARE 329 primitive Andean tribes. Other plants which are cereal substitutes, in that they furnish starch in concentrated form, such as the potato and similar plants, are discussed under vegetables. Vegetables It is somewhat surprising to note how few plant families contain the great majority of the common vegetables of the temperate world. From the Solanaceae come the potato, tomato, and eggplant, not to mention cayenne pepper, the ground cherry, and tobacco, the petunia and the matrimony vine. The Leguminosae furnish beans, peas, lentils, cow peas, soy beans, and peanuts. Another family furnishing a large number of edible roots and plants is the Cruciferae, or mustard family, containing the radish, turnip, and rutabaga, the cabbage and its congeners and deriva- tives, and the horseradish, cress, and mustard, not to mention such flowers as candytuft, sweet alyssum, wall-flowers, rockets, and gillyflower. From the sunflower family, Compositae in the broad sense, we get lettuce, salsify, chicory, endive, sunflower, and artichoke. The melon family, Cucurbi- taceae, contains many large and striking products, as pumpkins, squashes, cucumbers, gourds, gherkins, cantaloupes, casabas, watermelons, and citrons. In addition to the five large families listed above, are several with fewer economic species. Carrot, celery, parsley, and parsnip represent the Umbelliferae; rhubarb the Polygonaceae, and beets and spinach the Cheno- podiaceae. Asparagus and the various onions represent the Liliaceae, while the sweet potato is a morning glory, belonging to the Convolvulaceae, the taro and dasheen belong to the Araceae, and okra belongs to the Mal- vaceae, with cotton. Finally, the puffballs, mushrooms, and truffles are fungi, belonging to different families of that large group called Basidiomycetes. Fruits The family Rosaceae, used in the inclusive sense, probably furnishes more of the fruits grown in the temperate zone than all other families com- bined. Among its members are the apple, pear, quince, peach, apricot, plum, cherry, blackberry, raspberry, strawberry, juneberry, and almond, not to mention roses, spireas, and other flowers. Closely related is the family Grossulariaceae, containing the currants and gooseberries. The citrus family, Rutaceae, ranks next to the Rosaceae in the number and importance of its products, which include the orange, lemon, grape- fruit, citron, lime, tangerine, and others. The family Vitaceae probably stands third in rank, with its numerous and varied kinds of grapes, including the so-called currant of commerce. Other important fruits are the date and coconut, of the Palmaceae, the banana, of the Musaceae, the olive, of the Oleaceae, and the pineapple, be- 330 AMERICAN JOURNAL OF BOTANY [Vol. 8, longing to the Bromeliaceae, which includes also the so-called Spanish moss of our southern forests. Such fruits as the blueberries and huckle- berries (Vacciniaceae), the persimmons (Ebenaceae), the papaw (Ano- naceae), and the mulberry, fig, and breadfruit (Moraceae) should not be forgotten. Nor should the importance of nuts be overlooked. In nut production, the family Juglandaceae takes first rank, with its walnuts, butternuts, hickory nuts, and pecans. The family next in importance is the Fagaceae, containing the chestnuts, the beechnuts, and the numerous acorns, so im- portant as foods for primitive peoples as well as for animals. Of third rank, probably, is the family Palmaceae, if the widely distributed and important coconut is counted a nut rather than a fruit. Forages Among forages the grass family, Poaceae, has by far the largest number of representatives. Chief among the cultivated grasses are timothy, blue- grass, redtop, orchard grass, meadow fescue, bermuda, and sudan grass, -as also the cereals. A multitude of native species furnish grazing. Next in importance stand the legumes, Leguminosae, of which alfalfa, clovers, sweet clovers, vetches, cow peas, soy beans, velvet beans, and peanuts are well known and valuable representatives. Beyond these two great forage families stretches a long line of other families some of whose members are grazed, browsed, or ensiled, or otherwise enter into the animal diet. Saccharines The principal saccharines are sugar cane and sorgo, both grasses; the sugar beet, like the garden beet, of the Chenopodiaceae; and the sugar maple, belonging to the Aceraceae. Medicinal and Poisonous Plants Even to mention the many natural families yielding healing and toxic substances is beyond the scope of the present paper. Certain important examples will occur readily to all. Probably the three most important drug-producing families are the Papaveraceae, or poppy family, yielding opium and its derivatives, so useful in relieving pain, but so terrible in their effects when abused; the Rubiaceae, or madder family, producing the quinine so potent in the control of malarial fevers, as well as coffee; and the Solanaceae, or potato family, producing belladonna, capsicum, stramonium, and tobacco. Other prominent drugs are found in the Araceae; Compositae, Cruciferae, Labiateae, Leguminosae, Ranunculaceae, Rosaceae, and Um- belliferae. : Among the families containing important poisonous plants are the Anacardiaceae (poison ivy, sumach), Apocynaceae (dogbanes), Asclepi- adaceae (milkweeds), Compositae (asters, cockleburs, sneezeweeds), Eri- July, 1921] BALL — RELATION OF BOTANY TO HUMAN WELFARE 331 caceae (laurel), Leguminosae (lupines, loco weeds, milk vetches, vetches), Liliaceae (cannas, lilies), Loganiaceae (strychnin), Poaceae (grasses), Ranunculaceae (aconite, buttercups, larkspurs), Solanaceae (belladonna, henbane, nightshade, tobacco), Umbelliferae (hemlock), and Urticaceae (nettles). This takes no account of the many poisonous fungi, especti. lly among the fleshy fungi, or mushrooms. The interested reader is referred to the comprehensive manual of poisonous plants by Pammel. Fibers The most important fibers are cotton, of the Malvaceae or mallow family, and flax, belonging to the Linaceae. In addition are hemp, of the Cannabinaceae; jute, representing the Tiliaceae; sisal, of the Amarylli- daceae, and abaca, or Manila hemp, belonging to the Musaceae or banana family. Forest Materials The two most important families producing forest materials in the temperate zone are Pinaceae, including pine, spruce, hemlock, fir, cypress, and juniper, and the Fagaceae, containing the oaks, beeches, and chestnuts. Others of importance are Juglandaceae, walnuts and hickories; Acer- aceae, maples; Fraxinaceae, ashes; Betulaceae, birches; and Poaceae, in- cluding the bamboos. In the tropics many other families furnish materials of high value. ) BOTANY AND Crop IMPROVEMENT We have seen that the improvement of our present crop plants, or the finding of new ones, is a most promising means of increasing the food supply. The wide taxonomic distribution of the plant families so important to human welfare is an earnest of the complexity of the botanic problem involved. All phases of botany, including taxonomy, morphology, physiology, ecology, genetics, and pathology, must contribute largely if substantial progress is tobe made. Ecology and pathology are to have full discussion elsewhere on this program. Taxonomy The fundamental contribution of plant classification and description to human welfare has been the presentation of the vegetable kingdom as a fairly orderly series of evolving forms rather than a conglomeration of wonderful but unrelated organisms. The applications of this knowledge of relationships are many, varied, and valuable. Through such knowledge we are able to build up large plant industries with an assurance of success which otherwise would be impossible. The classification of crop plants, when accomplished with the same precision and thoroughness which have been used in the case of wild species, ~ will be of inestimable value to science and to humanity. The same prin- ciples will be applied, the same characters used, and the same results ob- 332 AMERICAN JOURNAL OF BOTANY [Voli 8, tained. The species of crop plants already are fairly well described and classified. The present need is classification and description of the seem- ingly innumerable agronomic and horticultural varieties of these plants. The duty of taxonomic botany is to make it possible for plant workers everywhere to recognize crop varieties. A classification of American wheat varieties now in manuscript has de- termined that the wheats passing under about 800 names actually represent about 200 distinct and recognizable varieties. While the proportion of synonyms in this instance may be no greater than the proportion in any other line of systematic botany, it must be remembered that the results in the case of wheat varieties are measured in bushels and not in bibliographies, in dollars and not in doubts. Such a classification of wheat varieties makes it possible to determine promptly that the so-called Superwheat of a Bur- bank is identical with the old and well-known Jones Winter Fife of New York and the Inland Empire, and that the only miracle about a ‘‘ Miracle”’ wheat is the number of suckers it attracts. : Similar results are coming out of the application of botanic classifica- tion to varieties of oats and other cereal crops, and to cow peas, soy beans, cottons, sorghums, lettuce, beans, apples, plums, peaches, and every kind of crop plants. Some years ago the writer saw at one of the largest agricul- tural experiment stations in the United States a long series of plats of cereal varieties of which less than 50 percent were under the right varietal names. Of what value will be the published results, if the varietal names are wrongly applied? Ten years ago, in his address as retiring president of the Botanical Society of Washington, Piper recorded his belief that fully 50 percent of the crop varieties published upon in varietal experiments were either untrue to name or unidentifiable. But how shall they become identifiable without adequate description and classification? And how shall they become ade- quately described and classified without botanists to study them? Large numbers of important varieties of crop plants have been pro- duced by the selection of pure lines, by the selection of mutations, and by the production and selection of hybrid forms. The intrinsic value of these important strains is great, but there is present the continual danger of their being lost by submersion among the many more or less similar varieties of the crop they represent. Careful botanic descriptions of the recognizable points of difference between them and the forms most closely related, ac- companied by adequate illustration, should make it possible for crop growers to recognize these varieties with some degree of certainty. Throughout the history of agriculture, unscrupulous dealers have sub- stituted inferior material for superior when opportunity has occurred. In- creased production of superior strains can be assured only when it is possible to detect substitutions, and this is possible only when all closely related forms are so well described as to be fairly identifiable by an intelligent layman. July, 1921] BALL — RELATION OF BOTANY TO HUMAN WELFARE 333 Another opportunity of systematic botany in relation to crop plants lies in the finding and introduction of new material. Large portions of the earth still are not well known botanically. The plants of vastly larger areas have not been critically studied with reference to their usefulness in other lands. Some will be of direct and immediate service as food materials. Others will provide hardy or disease-resistant stocks for grafting purposes, while still a third series will possess characters valuable when transmitted through hybridization. The Problem and the Challenge To place varietal experimentation on a firm basis of accurately de- scribed and easily recognized material; to insure the identity of new and valuable strains; to prevent the faker from profiteering at the expense of crop producers; and to provide new plant materials from the four corners of the earth, is the greatly needed contribution of systematic botany to crop-plant production and so to human welfare. The work previously done on the classification of varieties of crop plants and the introduction of new material has been the separate product of two different groups of workers, botanists and agronomists. You have heard the saying that no botanist will look at a cultivated plant, and no agronomist at a wild one. Granting the exaggeration, the saying is still too true. A generation of botanists must be trained to appreciate the funda- mental importance of full taxonomic knowledge of crop plants. They must recognize that characters sufficient to separate species of wild plants serve only to separate closely related field and garden varieties of domesticated plants. In dealing with the latter, they must be willing to forget Latin nomenclature, if need be, as a Latin terminology must be carried to the fifth, sixth, and seventh place in such crops as wheat or corn if one builds on the taxonomic foundations already laid. Likewise, a generation of agronomists must be produced which has had good foundation training in systematic botany, derived in large part from a study of crop plants. I put the challenge squarely up to the botanical departments of our universities and our land-grant colleges alike to work together to produce such a generation of botanists. Equally squarely are the departments of agronomy in the land-grant colleges challenged to codperate with the botani- cal departments in producing such agronomists. This challenge is to the institutions involved. A similar challenge clearly lies before the present and future personnels engaged in the investigation of plants from both the agronomic and the botanic points of view. The obligation is upon them to codperate, pooling their valuable resources of accumulated experience and information and expensive equipment in a common cause. Only by such coéperation can satisfactory progress be made in the attack on these problems so vital to the welfare of humanity. 334 AMERICAN JOURNAL OF BOTANY [Vol. 8, Crop Physiology At the present time, progress in crop improvement is waiting on a fuller knowledge of crop physiology. To the farmer, as to the agronomist, the value of crop varieties is measured in terms of their performance in pounds or bushels. We know by experimentation that one variety of any given crop yields better under a certain set of conditions than do other varieties of that crop, while under different conditions this same variety may be com- paratively unproductive. We know that crops vary greatly in their com- parative resistance to disease, to cold, to frost, to heat, to drought, to soil alkali, and to all the other unfavorable factors in the environment. In the same way, some varieties seem unable to stand prosperity. Given what apparently are very favorable conditions, they seem unable to make a proportionate response in production. These things we know, but what we do not yet know is why these things are so. That is the next and most immediate problem in crop improvement. In the practice of medicine, the detailed study of the functioning of the various members of the human body has been held indispensable to a proper diagnosis of diseased conditions. In the case of even a single one of our most important crop plants, however, no such detailed study has been made. We attempt to acclimatize them in various parts of the world, to make them productive under a wide range of climatic conditions, and to breed them to produce forms with very specialized adaptations, without this fundamental knowledge of their relations, derived from ade- quate research. The relative and actual importance of such external factors as light, air temperature, humidity, soil temperature, soil texture, and soil solution in their effect on the growing crop plant at different stages of growth, from germination to maturity, are very imperfectly known quantities today. Without doubt, the increasing determination of the values of these and other factors will have a profound influence on the practices of crop production and ultimately on the quantity and quality of the product. The recent discovery by Garner and Allard, of the effect on plant growth caused by varying the duration of the daily light period, not only is shaking the foundation of our theories and opens leads toward many unsolved problems, but is highly suggestive of results that will be obtained when other and equally fundamental researches are made in the realm of crop physiology. Some of the lines along which such research should be directed have been mentioned. Next to light, the fundamental factors are temperature, water, and food. Several unrelated studies of temperature relations have been made, but research to date has touched only the fringe of this problem. Tem- perature studies are vital to such problems as crop adaptations, including the extension of the areas of fall-sown crops, as against spring-sown; the comparative development of root and shoot and the speed of development July, 1921] BALL — RELATION OF BOTANY TO HUMAN WELFARE 335 of the resulting plant, and the determining of the conditions under which plants may escape or resist the attacks of soil-infecting and other fungi. The study of water relations is of very high importance. Studies in the water requirements of several important crop plants, as revealed through transpiration measurements, have been conducted in recent years by Briggs and Shantz and by Montgomery and Kiesselbach, but these are only a good beginning compared with the research that is needed. Investigation of the duty of water in irrigation is much more a problem for the plant physiologist than for the irrigation engineer. The first phase is the determination of the effect of applying water, at different times and in varying quantities, on the comparative and actual development of the roots, vegetation, and fruits of crop plants. The second phase is the possi- bility of increasing total crop production by making the present supply of irrigation water cover much more than the present number of acres. It is conceivable that reducing the quantity of water by one half might re- duce acre production by only two or three tenths and permit irrigation of twice the present acreage. Two acres of irrigated wheat yielding 35 bushels each may be more valuable to humanity and just as profitable to the grower as one acre yielding 50 bushels. It is not unthinkable that one day we shall see governments exercising the right of eminent domain to accomplish such results through reducing existing water rights. Studies in plant nutrition long have been known to be of fundamental importance. The chief difficulty in such research has been to control ex- perimental conditions and at the same time to approximate natural condi- tions. Solution cultures permit controlled conditions but give only sugges- tive results. Fertilizer plats approximate natural environment, but are conducive to confusing interpretations. The gulf between the two may be bridged by continued refinement of method and interpretation. Studies in the physiology of the development of seeds and fruits in~ our major food plants, such as the cereals, are of the utmost concern. The period of vegetative growth may be prolonged over several months, but usually the formation and maturing of seeds takes place in the brief period of two to four weeks. Obviously, this is an important and perhaps even critical period in the life of the plant, from the economic standpoint. Physi- ology can help to show what tillage, or irrigation, or fertilizer practices dur- ing or just previous to this period, will influence directly the quantity and quality of the product. Some preliminary studies in the deposit of protein and starch in de- veloping wheat kernels were made several years ago in the state of Washing- ton by Dr. Thatcher and his associates. Dr. Harlan, of the Office of Cereal Investigations, U. S. Department of Agriculture, is now publishing a series of papers dealing with some phases of the development of the barley kernel. Such studies are but the forerunners of what is required as a foundation for a better knowledge of the behavior of our crop plants at this critical period in their development. 336 AMERICAN JOURNAL OF BOTANY [Vol. 8, In a present study of soil-infesting rots of the corn plant, codperative between the Office of Cereal Investigations and the Indiana Agricultural Experiment Station, Funk Brothers Seed Company, and other agencies, some striking physiologic factors have been found to be involved in what was supposed to be purely a pathologic problem. Changes occurring with- in the plant result in a deposit of harmful metals and consequent severe injury to the plant. Research on the cause of the abnormal metabolism emphasizes how little we know of the functioning of the corn plant in health. And yet here is a crop worth several billions of dollars annually in our own country alone! During the last half century there has been no lack of attention to the subject of plant physiology. I draw here, however, a clear distinction between plant physiology and crop physiology, because plant physiology has been restricted very largely to studies of wild species. Physiological research in our great state and privately-endowed American universities has not lacked equipment and encouragement. Splendid results have been obtained in such research, but until recently a scanning of the titles of theses submitted in connection with the granting of doctorate degrees warrants the statement that rarely has a candidate undertaken research on a domesticated plant. The importance of fuller knowledge of crop physiology, in relation to our national welfare, warrants these universities more and more in devoting their magnificent resources of men and equipment to such research. There is no reason why this should not be done in codperation with plant workers in state experiment stations or in the research bureaus of the U. S. De- partment of Agriculture. I am sure that the universities would be met more than half way if such codperation were proposed. There is a large enough field for all, and human need does not warrant the self-imposed exclusion of any agency capable of giving effective assistance in the solution of the problems involved. Genetics Some twenty years ago, the rediscovery and interpretation of the re- markable work of Gregor Mendel created a new branch of plant physiology and ushered in a new epoch in plant improvement. As a result, important results are being achieved in two opposite directions. Looking backward, new light is being thrown on the origin of existing plant forms. Looking forward, our knowledge of somatic behavior is being used in the creation of new forms of high intrinsic or potential value. Genetic studies hold the greatest possibilities for improvement in crop production. The knowledge of the plant sources from which have been developed such tremendously variable and important crop plants as corn or wheat would greatly aid in our understanding of how to proceed in obtaining forms with needed characters. Just as fast as physiologic research can show the nature of such desirable characters as resistance to rust, smut, July, 1921] BALL — RELATION OF BOTANY TO HUMAN WELFARE 337 cold, drought, and the many other pests and unfavorable influences which reduce crop production, genetics will help in combining existing varieties to produce other better adapted ones with the desired characters. At the same time undesirable characters may be eliminated. PROPHETS OF THE NEW ORDER I have been interested to discover what the leaders of botanical thought were emphasizing a quarter-century ago. On consulting the addresses presented about that time by the retiring presidents of the Botanical Society of America and the retiring chairmen of Section G of the American Associa- tion, I was particularly interested to find that already they were foreshadow- ing or openly proclaiming the importance of the economic phases of botany. It was especially interesting to note that three such veterans as Doctors Coulter, Trelease, and Galloway, as well as others, should have had this viewpoint in common. I cite these three especially because the first has devoted his entire career to so-called pure botany, the second has divided his affiliation between the wild and the cultivated plants, while the third has been engaged continuously on various phases of applied botany. Turning then to very recent pronouncements, I was especially grati- fied to note the point of view of Dr. Coulter in his address as retiring presi- dent of the American Association in December, 1919. In this address, entitled ‘‘The Evolution of Botanical Research,’’ he noted three botanical tendencies, as follows: 1. To attack problems fundamental to some important practice, 2. To realize that botanic problems are synthetic, and 3. To recognize that plant structures are not static. He noted also three important features of future botany, namely: 1. Broader training to be required of botanical workers, 2. More extensive codperation in research, and 3. Better development of experimental control. The addresses of Dr. Flexner, three days ago, on ‘Twenty-five Years of Bacteriology,” and of Dr. Pammel, today, on ‘‘Some Economic Phases of Botany,” are striking records of achievement in applied botany but were given in your hearing and need no discussion here. AESTHETIC WELFARE So far all our discussion has been of the relation of crop-plant botany to material welfare. Its relation to the aesthetic and spiritual welfare of man is less obvious, though perhaps not so much less potent as some may think. At any rate, it is impossible to develop this phase adequately in the limits of the present paper. 338 AMERICAN JOURNAL OF BOTANY [Vol. 8, When the immortal author of Thanatopsis advised those sick in mind and spirit to “‘go forth under the open sky and list to nature’s teachings,” we are sure that the still small voice of useful plants was not excluded from the curative agencies. That scientific worker is indeed defrauded who does not get both mental exhilaration and spiritual uplift from con- templation, in crop plants, of the riotous beauty of floral color, the seductive fragrance of myriad blooms, the marvelous intricacies of structure, and the wonders of adaptation, or from the quietness of far-stretched fields of grain or cotton, and the majesty of towering forest forms, saying, in the latter case, with the dead soldier, Joyce Kilmer, ‘But only God can make AY (neers In CONCLUSION The fundamental botanic requirements in crop production are to know what we now have, to find what exists elsewhere, and to use both in creating something better than either. To know what we have requires botanic description, classification, and illustration, and a study of plant functioning. To find what exists elsewhere and to predict where it may be useful requires expert knowledge of plant relationships and plant ecology. To create the best requires in- timate genetic knowledge, and a visualizing of the plant that is to be in terms of the characters of plants that are. To no more worthy tasks can botanists devote their best endeavors. OFFICE OF CEREAL INVESTIGATIONS, U. S. DEPARTMENT OF AGRICULTURE CORRELATIONS BETWEEN ANATOMICAL CHARACTERS IN Wns oPEDLING OF PHASEOLUS. VULGARIS J. ArTHUR Harris, EDMUND W. SINNOTT, JOHN Y. PENNYPACKER, AND G. B. DURHAM (Received for publication January 17, 1921) INTRODUCTION In an earlier paper! we traced the course of the vascular bundles through- out the dimerous and trimerous seedlings of Phaseolus vulgaris and measured the variation occurring at different levels. The chief results of that paper were (a) the demonstration of the pro- found differentiation of dimerous and trimerous seedlings in their internal (vascular) as well as in their external characters, (0) the demonstration that the number of bundles at a given level in the seedling is a highly vari- able rather than a constant character, and (c) that the various organs or regions of the plant body (particularly, in the present case, those which are separated by the vascular anastomoses at the cotyledonary node) differ widely in the magnitude of their variability as to bundle number. In this paper we propose to consider in quantitative terms the degree of interrelationship between the vascular structures in the different regions of normal and abnormal seedlings. The results of such an investigation will evidently be of considerable morphological interest, since many of the problems of organic form are fundamentally problems of correlation. Two morphological problems at once present themselves for considera- tion: First, is there a high correlation between the vascular topography of two different levels of the same internode, 12.e., is the number of vascular bundles constant throughout the length of an internode or is there more or less extensive splitting or anastomosis within the length of such a con- ventional morphological unit? Second, is there a definite correlation between the vascular topography below a node and the vascular topography above it, or is the vascular system so fully reorganized at the nodal anastomosis of bundles that, in bundle number, successive internodes are practically independent of one another? With the present material, these questions may be answered by de- termining the coefficients of correlation for bundle number between (1) the base and the mid-region of the hypocotyl, and (2) between the various levels of the hypocotyl and the mid-region of the epicotyl. It is these 1 Harris, J. Arthur, Sinnott, E. W., Pennypacker, John Y., and Durham, G. B. The vascular anatomy of dimerous and trimerous seedlings of Phaseolus vulgaris. Amer. Jour. Bot. 8: 63-102. 1921. Dou 340 AMERICAN JOURNAL OF BOTANY [Vol. 8, problems which we propose first to consider. We shall also compare the normal and abnormal seedlings as to the correlations which they exhibit, and shall touch briefly on the problem of the correlation between bundle — number in seedlings from the same parent plant. The frequency distributions of bundle number are in many cases of very narrow range and very skew. There has, therefore, been consider- able question as to the formulae to be employed. It has seemed best, for various reasons which need not be detailed here, to employ the usual method of product-moment correlation. ' PRESENTATION AND ANALYSIS OF DATA The series of data considered here are in large part the same as those discussed in our earlier paper, but have in some cases been supplemented by the examination of additional sections. These have been included when the dimerous and trimerous seedlings were not true siblings. In lines 75, 93, and 98, the series compared were obtained from the same mothers. In so far as the data are the same as those used earlier, the variation constants for the different characters have already been presented and discussed and require no further comment here. The data from which measures of in- terrelationship may be computed are given in our fundamental tables A to L. We have, therefore, merely to deduce and discuss the correlation coefficients. Correlation between Bundle Number at Different Levels in the Same Internode We first turn to the problem of the relationship between the number of bundles—primary double bundles, intercalary bundles, and total bundles— at the base of the hypocotyl and the number in the central region of the hypocotyl. The reader who cares to do so may reconstruct the 24 correla- tion tables necessary for a consideration of these relationships from our fundamental tables A—L. TABLE I. Coefficients of correlation between number of primary double bundles, number of intercalary bundles, and total bundles at base of hypocotyl, and number of bundles in central region of hypocotyl Correlation for Prim rrelation for Correlation for Se me N i Double Bundled sid iReceeee Bundles Total Bundles "ph Tin Toh Trimerous | ine :75.-e eee 142 | +.378=.049 | 7:79 | =-3295-051 6.51 | +.649-.033| 19.8 Lanei93). cee 155 | +.233+.051 | 4.55| +-204.052 | 3.92 | +.469+.042] II.1 ine. O8; ster eee 183 | +.321-b.045 | 7.17| +.253=5.047 | 5.42 | -7-58022-033)buI7-0 Minei2o eae 106 | +.417-.054 | 7.71 | +.097+.065 1.50 | --.530 =:047\ rts Line tage ae 221 | +.556-+.031 | 17.8 | +.305+.041 7.40 | 1.753 22.020 gees Dimerous Line 75 cts ce 142 | +.362+.049 | 7.35 | +.668+.031 | 21.3 | +.797=+.021| 38.0 BineiO3 eee wae 155 | +.641-£.032 | 21.0 | +.390+.046 | 8.50] +.753=:.023| 32.2 Line 98... 183 | +.666+.028 | 24.0 | +.555+.035 | 16.1 | +.7864.019| 41.4 Line 13070970: 305 | +.344+.034 | 10.1 | +.898-+.008 |119.7 | +.925+.006 | 168.3 Linewa4e- nc 420 | +.530-4.023 | 22.5 | +.634+.020 | 32.2 | +.802+.011 | 68.5 THE SEEDLING OF PHASEOLUS VULGARIS 341 july, 1921] "[Ajo0Idd 10} sajaitd Aq pu [Ao90d Ay iOj S}Op prpos Aq pojuosoidar stiiveu jeoumdury ‘s8UT}paes Snoiotuti} Ut jAyooodAY jo aseq ye safpunq afqnop Azeunsd jo soquinu to [Ajoorda jo uorsear [erqus0 ur pue [AyooodAy jo UOISII [e41}UID UI Se[puNq jo JoquINU Jo UOISsoIsay «1: NVAOVIG STTINNE F7ILNOT KAY O//4aL (eu eet | l d 6GE:-0+cL9'-L/= 7 d 985-0+ %L8:8 =H d LE0-0 4 299-tl= 7 d 959:0 + L8/°8 =H ps0t Lied) =F Pere or Z/ 86 7M/7 8 L 9 iC v 6 SNV/7 d LU-OF 108-01 =F L595 80 CEC Ls =H // L 9 5 d O2F-OF bl LI=7 d 968-04 669-9 =H el 9/ (ey 8/ 66) JVI. €b/ FN/7 6/ Gh. FML7 PALOWAT MY 7TKLOIOAKH NM SI TINANE 0 L7SPLWIN NVSW 342 AMERICAN JOURNAL OF BOTANY [Vol. 8, L/VE 143 LIME, 9S H=-0:002 F 2:139 P H=/:041 F1:9935P F= /12:294 + 0-033 P F=/0-94/40:30/ P LINE 75 LINE 93 13 /2 Te 2 MEAN NUMBER OF BUNTLES IN HYPOCOTYL AND EPICOTYL Lhe // /0 eo7=— 0-487 4 2:/64 P © 5 = 9 8/440-57/P 9 F =/2 -928-0:/78 P H= 4/30 4/352 P H= 31754 [507 P Z=//:WHS54 0-090 P PRIVIARY DOUBLESBONMEES. DIAGRAM 2. Regression of number of bundles in central region of hypocotyl and in central region of epicotyl on number of primary double bundles at base of hypocotyl in di- merous seedlings. Empirical means represented by solid dots for hypocotyl and by circles for epicotyl. The correlation coefficients between the two classes of bundles which have been recognized at the base of the hypocotyl and the total number of basal bundles (7.e., the sum of the number of primary double bundles and the number of intercalary bundles in the base of the hypocotyl) and the number in the central region of the hypocotyl, appear in table 1. July, 1921] THE SEEDLING OF PHASEOLUS VULGARIS 343 The correlations are without exception positive in sign and of a material order of magnitude. They have been expressed in terms of regression on diagram 1 for trimerous seedlings and on diagram 2 for dimerous seedlings of the five lines.? Primary Double Bundles and Mid-region of Hypocotyl The constants showing the relationship between number of primary double bundles and number of bundles in the central region of the hypo- cotyl, r,,, are shown in the first section of table 1. They are positive and statistically significant in all cases in both dimerous and trimerous seedlings. The average value of the coefficient for the five lines investigated is +.3810 for trimerous seedlings and +.5086 for dimerous seedlings. Diagram 2 shows that in the case of the normal plants of lines 75, 93, and 143 a straight line represents very well indeed the changes in the mean number of bundles in the hypocotyl with variations in the number of pri- mary double bundles at the base of the hypocotyl. In line 98 the agree- ment is apparently not so good. This is, however, attributable to the fact that of the 183 plants only two have more than 5 primary bundles. Of these two, one plant is recorded as having 8, which is twice the normal number. In line 139 only plants with two classes of seedlings, those with 4 or 5 primary bundles, are available, and since the regression line must connect the two means it is idle to discuss linearity of regression. Turning to the trimerous plants represented in diagram I, we note that because of the small number of plants with other than 5 or 6 primary double bundles the distribution of the empirical means is very irregular indeed. There is some suggestion of non-linearity, but the number of seedlings in the more extreme classes is so small for every line that little stress is to be laid upon them. In both normal and abnormal plants the slope of the regression line is rather steep, showing a material change in the number of bundles in the central region of the hypocotyl with variations in the number of primary double bundles at the base of the hypocotyl. Intercalary Bundles and Mid-region of Hypocotyl The correlation between the number of intercalary bundles and the total number of bundles in the hypocotyl, rx, are shown in the second 2 The equations on the diagrams show the regression of the number of bundles in the central region of the hypocotyl, H, and in the central region of the epicotyl, Z, on the number of primary double bundles, P, at the base of the hypocotyl. The empirical means for the hypocotyl are represented by solid dots, while those of the epicotyl are represented by circles. In both cases the empirical mean number of bundles for the same organ are connected by solid lines when the number of sections averaged was five or more, but by broken lines when the number available was four or less. Fortunately for purposes of graphical representation, the mean number of bundles in both hypocotyl and epicotyl -can be drawn on the same diagram. Only the lower lines in each of the five panels of the two diagrams require consideration for the moment. ‘[Ajoorda 10j sapoat9 Aq pue [AJoo0dAy 30} s}op prjos Aq poqeseides suvow [eorduryq = ‘*s8ul[poes snosouit4j ur [Ayoood Ay jo aseq ye soypunq AreyeoJozUT Jo Jequinu uo [A}o9 -1d9 Jo worsal [e1jU90 UT pue [AJooodAY Jo uorIsea1 [e1]U9O UI seypuNq jo Joquinu jo UOIsseIsey «*& WYAXOVIG SITINNE KEV TVILSLW/ [Vol. 8, 1 VEO FLS8°b/ =Z7 EMEC # Er0-cl =F7 1 OSC-O+ 9% $/=7 4 ¢LV-OF St2-cl=H Pane Z / 006-0 + BEO-S/=F7 / $57-0—t0L-$/=7 Gi YACIE OT S00: Cl = fF / ¢LEOF 6¢0-2/=H / 140-0 + 622-5/ =F / 12-0 + 696-/1 =H cay | eee v/ AMERICAN JOURNAL OF BOTANY Sl 7ALOIAT INV TKLOIOALKH W/ SP TONNE SO 4FEWIN NYT €6 INIT €b/ INIT | 6El FN/7 344 THE SEEDLING OF PHASEOLUS VULGARIS 345 July, 1921] prjos Aq pojuesaidei suvour jeommdury [Aqoorda jo uorgas [e1}Ua0 UT pue [AJoOOdAY Jo UOIsa1 [e1]UDD UI SefpuNq jo JaquINU jo UOISSIIs2y =“ Y WVNYOVIC] | L02-0 + 00€-2/=F | 996-1 + 8b2-8=H 1 801-0 # €€6:cl= | 669:04 €00:0/=H Eb) 71 7. *tAjoo1da 10} saposra Aq pue [AJoI0dAy 10} sop ‘squeyd snosoup ur [Ajyoood Ay jo vseq ye soypung ArepeosaqUT Jo Jaquinu uo SITHINNIET KAVTWIHTLWV/ 1SL0:0 + SEl°c/= J 1 b/6-04 LOL°8 =H 1 €0:0— 26) gi =F Lh CECT EL GO'6 = 86 FN/7 / 0 Cl INL -C/20 880 C/ 7 | O¢cv:-/ + 00-8 =H SL SNI7 i ‘ 6E/ FN/7 TKLOW ALI LNY 7TKLOIOLKH MW. SFTENAI 40 YFILTWIN NVIW 346 AMERICAN JOURNAL OF BOTANY [Vol. 8, section of table 1. The straight-line equations showing the regression of the number of bundles in the central region of the hypocotyl are recorded and represented graphically on diagram 3 for trimerous seedlings and on diagram 4 for dimerous seedlings. These diagrams, like the two preceding, also give the regression equations and their graphic representation for the epicotyl which will be discussed in a subsequent section. The correlation coefficients are positive in all cases, and with one ex- ception may be considered statistically significant. They show, however, a considerable irregularity from line to line, presumably because of the varying range and distribution of number of intercalary bundles. The average value of the coefficient is +.2376 for trimerous seedlings and + .6290 for dimerous seedlings. Turning to the graphs, we may note that for the dimerous plants the agreements between the empirical and the theoretical means are very good indeed. The slope of the lines for the hypocotyl is very steep. The graphs for the trimerous plants show far greater irregularities be- cause of the generally small number of the strands but the occasional oc- currence of plants with a larger number. Reference to the tables will show that in line 75 there is one seedling with 6 intercalary bundles whereas the remaining 141 seedlings have only 0, I, or 2 intercalary bundles. In line 93 there is only one seedling with more than 2 intercalary bundles and it has 4. In line 98 all the frequencies with two exceptions fall on 0 or I intercalary bundle. The correlations and equations have been recalculated, leaving these extreme cases out of account. The regression straight lines based on all the material are represented by solid lines. Those in which the extreme class were omitted are represented by broken lines. The removal of these aberrant cases has increased the agreement between the observed and the theoretical means but the fit is still far from satisfactory. The only con- clusion which can be drawn from these diagrams is that there is a con- siderable degree of positive correlation between the number of the inter- calary bundles and the number of bundles in the hypocotyl. Total Basal Bundles and Mid-region of Hypocotyl The correlations between total bundles (primary double bundles + in- tercalary bundles) at the base of the hypocotyl and the number of bundles in the central region of the hypocotyl, 7,, are shown in the third section of table 1. The straight-line regression equations are given and represented graphically as the lower figures in each panel of diagram 5 for trimerous seedlings and diagram 6 for dimerous seedlings. As might be expected on a priori grounds, these coefficients agree with those for primary double bundles and for intercalary bundles in sign, and 3 For the curtailed series the regression equations are: Line 75, H = 12.194 + 0.6541; Line 93, H = 12.238 + 0.462 I; Line 98, H = 12.030 + 0.473 I. MEAN NUMBER OF BUNILES IN HYPOCO7 YL AND EP/COTYL July, 1921] THE SEEDLING OF PHASEOLUS VULGARIS 547 LINE 1/43 LIN LE ETS: Fi= 3*//4¢ 47064 B E = /2°89040-4/9 B fi —e2'000 7/5 /6-B B=/2°790 70-542 B 3 2 139 LINE 98 H= $:-743+/:078 B B= 18°90) —0-$29 B i= 4-790, 41-200 B H= 63314 0-349 B £ = 17:634—0-400 B E = 12-8074 0342 B TOTAL BUNTLES DIAGRAM 5. Regression of number of bundles in central region of hypocotyl and in central region of epicotyl on total number of bundles at base of hypocotyl in trimerous seedlings. Empirical means represented by solid dots for hypocot yl and by circles for epicotyl. =a | , *[Ajoo1da 30} saps Aq pue [Aoo0d Ay JO} sj}op pros Aq pojuesaider suvaur yeornduy ‘ssulppses snossutp ur JA}oo0dAy jo aseq je sefypunq jo Jequinu [e307 uo [Ajoo1da jo uorl8er [e1qUe0 UT pue JAJoOOdAY Jo UOIsa1 [eIJUa UI sa_puNq jo Jequinu jo UOIssaIsoy °9 NVAOVIG SITHNIE 7VLOL [Vol. 8, fa: S t 8 L 9 G t Z bOoO+F 505° //=7 Z 8ll-OF S69-1] =F © §LE/ + 460-6 =H Z 8S/:/ FLEE-% =H x ZT 220+ LO9-// =F : N bt I SO + [¢0-% =H a > : : (o) Oa by jaa = o- ; m on xy , S S CdS ee eae £6 INIT . Ss Z = g eo i ee S 5 I 80-0-H9E-2/=F N 2 IT SEES AOE =H FT 09/04 999://=7 8 z Raye <2 (Ay Ray ~ e S oO : as : » = ; S << Ff 0/ iS } ~ < : is HI < / < i Ky Dy i a c/ ¥ Kg S N 1 AE w 86 INIT / ae ' tl $LINIT E¢/ INIT July,*1921] THE SEEDLING OF PHASEOLUS VULGARIS 349 are in general somewhat higher than those for either of these two classes. The average value of the 5 coefficients for trimerous seedlings is +.5976 while that for dimerous seedlings is +.8126. Turning to the diagrams, we note that the straight lines and the empirical means are in excellent agreement, considering the small number of seedlings, in the case of the normal plants, but show greater irregularities in the case of the abnormal plants. This is due to a considerable extent to the greater concentration of the frequencies into two classes in the case of the trimerous seedlings. We may now consider the relative magnitudes of the three correlations which we have been studying. Table 2 shows the differences existing be- . TABLE 2. Comparison of correlations between the various types of bundles at the base of hypocotyl and the number of bundles in the central region of hypocotyl a) obec sallligs Yon — Tph Ton — Tin ph — Tin Trimerous mie 75.02... +.271+.059 | 4.59) +.320+.061 | 5.25 | +.049+.071 0.69 mie 03. ...-... +.236+.066 3.58 | -+.265-.067 | 3.95 +.029 +.073 0.40 feime 98. ....... +.265 +.056 4.73 | +.333-:.057 | 5.84 | -+.068-+.065 1.05 Line Oy seers -+.114+.071 1.61 | -+.4342+.080 | 5.43 +.320+.084 3.81 Wie 1AZ 2... . +.197+.037 | 5.32} +.448+.046 | 9.74 | +.251-4.051 4.92 Dimerous MSIE! 7 5.0 on sw cho +.435 +.053 8.20| +.129-.037 | 3.49 —.306-+.058 5.28 IIE 93... sw os +.112+.040 280i 4 -30Ss5051 127.11 +.251+.056 4.48 HEME OS"... sw +.120+.033 3.64| -+.231-+.040 | 5.78 +.111+.045 2 Ay WINE 130... 5... +.581+.035 | 16.6 +.027+.010 | 2.70 | —.554+.035 | 15.8 IME TAZ... ks +.272+.026 | 10.5 lO8e2 0227 |57.04. | -—,10425-030, | 3-47 . tween the various correlations, 7.e., the possible differences between the correlation for primary bundles and hypocotyledonary bundles, 7,,, for intercalary bundles and hypocotyledonary bundles, 7;,, and for total bundles at the base of the hypocotyl and hypocotyledonary bundles, 75,. For both dimerous and trimerous seedlings, the correlations between the total bundles at the base of the hypocotyl and.the number of bundles in the central region of the hypocotyl are higher throughout than those for either of the two separate types of bundles (primary bundles and inter- calary bundles) individually considered. In general, the differences are sufficiently large in comparison with their probable errors to be considered statistically significant. The comparison of the magnitudes of the correlations between numbers of primary double bundles and of vascular elements at higher levels, and between numbers of intercalary bundles and of vascular elements at higher levels, shows that in 7 of the 10 comparisons the closer correlation of hypo- cotyledonary bundles is with the primary double bundles. Lines 75, 139, and 143 present exceptions. In the normal plants of these lines the correlation between intercalary bundles and total bundles in the 350 AMERICAN JOURNAL OF BOTANY [Vol. 8, hypocotyl is apparently significantly higher than that between primary double bundles and total bundles in the hypocotyl. The fact that the number of bundles in the central region of the hypo- cotyl is about equally correlated with the number of primary double bundles and with the number of intercalary bundles at the base of the hypocotyl shows that both types of bundles are of about equal significance in de- termining the number of bundles in the central region of the hypocotyl. From the foregoing discussion it is clear that there is a rather close re- lationship between number of bundles at the base and the number in the central region of the hypocotyl. This might, we believe, have been ex- pected on a priort morphological grounds. The interesting feature of the results seems to be that the correlations are not larger. The results show that there is a very large amount of irregularity in the division of primary strands or in the formation of intercalary bundles, or in both, as one passes the short distance from the base of the hypocotyl to the central region. Correlation between Bundle Number in Different Internodes The data available for a consideration of the problem of the correlation between bundle number in adjacent internodes cover (A) the correlation between the three classes of bundles at the base of the hypocotyl [primary double bundles (p), intercalary bundles (z), and total bundles (d)| and the number of bundles in the central region of the epicotyl; and (B) the correla- tion between the number of bundles in the central region of the hypocotyl and in the central region of the epicotyl. : (aA) The coefficients showing the relationship between the numbers of primary double bundles, of intercalary bundles, and of total bundles at the base of the hypocotyl, and the number of bundles in the central region of the epicotyl, appear in table 3. The regression equations showing the actual change in number of epi- cotyledonary bundles associated with variation in the number of primary double bundles are given and are represented with the empirical means of arrays on diagram I for trimerous plants and on diagram 2 for dimerous plants. The graphs for the theoretical lines and the empirical means for the number of bundles in the epicotyl of both normal and abnormal plants show relatively little relationship between the number of bundles at the base of the hypocotyl and the number in the epicotyl. The differences in the slope of the lines for primary basal bundles and the number of bundles in central regions of hypocotyl and epicotyl show in a most striking manner the dif- 4Jn line 75 the range of primary double bundles is only 3 while that of intercalary bundles is 6. In line 139 the primary double bundles fall in two classes only, with but 3 of the 305 frequencies on 5 as compared with 302 on 4 bundles. The correlation coefficient in such a case can have but little value. In line 143 practically all of the primary double bundles fall in two classes while the intercalary bundles are limited to three classes. Irregularity of results must be expected under such conditions. July, 1921] THE SEEDLING OF PHASEOLUS VULGARIS 35L ferences between correlations for groups of bundles lying on the same side and those lying on different sides of the nodal complex. (1) The correlation coefficients between primary double bundles and number of bundles in the epicotyl, 7,,, as set forth in the first section of table 3, are in part positive and in part negative in sign. For the most part they can not be considered statistically significant. The average value of those for trimerous seedlings is —.0226 while that for dimerous seedlings is +.0768. (2) For the correlation between the number of intercalary bundles and the number of bundles in the epicotyl, 7;,, shown in the second section of table 3, the coefficients are not in general certainly significant in com- TABLE 3. Coefficients of correlation between number of primary double bundles, number of intercalary bundles and total number of bundles at base of hypocotyl, Correlation for Correlation for i ee ari, NV Peary Double Bundles Intercalary ‘Bundles RASS Tne Tie be Trimerous | | | eine 75........ 142 | --.053=£.056 | 0.93 | 1.126.056 | 2.27 | +.182+.055 | 3.33 iC, a 55) |) 1087 =-.054 | 1-61 | —=.055+.054 | 1.01 | —.148=5.053 | 2.70 ime OS. ....... T3585 ——-.00S42:050 | 0:70 | +4-.070=22.050 | 1.42 | --.09922.049 | 2.01 Pine 130..0:... 106 | —.105+.064 |-1.63 | +.016=.065 | 0.25 | —.095-+.065 | 1.47 Mine 143....... 22tl ea OLos.O045 | 0.40 | --.233 55.043 | 5.44 | --.190s2.044 | 4.34 Dimerous | AMO Fn kk ss 142) —,115=£.050 | 2.07 | —.043=:.057 | 0.75 | —.054=b.056 | 0.96 MOM OZ. .. 23... 155 | +.084+.054 | 1.55 | +.132+.053 | 2.48 | +.167=b.053 | 3.16 mime OG... . 5. 18@ || =-.239=5.047 | 5.08 | +-.10932.049 | 2.21 | +-.205=£.048 | 4.29 ime 130 .-... 0... BO 5a hap 04.0361) A637 =| =--14 52-038: | 3:64) | +-.175 42.037 | 4:68 We TAZ... 5. AZONGe O12 2=2032 | 0:38 |. + .134-6.032.| 4.05 | 47.121 =5.032 | 3.73 parison with their probable errors. Two of the ten are indeed negative in sign. The coefficients for line 143 in both trimerous and dimerous seedlings and possibly that for line 139 in the dimerous seedlings may be significant. The fact that eight of the ten coefficients are positive suggests that there is a slight relationship between the number of intercalary bundles at the base of the hypocotyl and the number of vascular elements in the central region of the epicotyl. The general average is +.0780 for the trimerous and +.0954 for the dimerous. | This suggestion is only slightly strengthened by inspection of the two sets of diagrams on which the regression equations are presented and drawn with the empirical means. Diagram 3 pictures the results for trimerous seedlings while the comparable representations for dimerous seedlings are shown on diagram 4. ‘These show that while the slope showing the change in the number of bundles in the hypocotyl associated with variations in the number of intercalary bundles at the base of the epicotyl is very steep, it is practically nothing for the epicotyl, thus indicating a very close relationship in the former case but the practical absence of interdependence in the latter. 352 AMERICAN JOURNAL OF BOTANY [Vol. 8, As explained above (p. 346), the slopes for the trimerous seedlings are very greatly influenced by certain aberrant individuals. When these are removed we obtain the equations represented by the broken lines in the figures. The results for the relationship between the number of inter-— calary bundles and the number of bundles in the epicoty] indicate a positive correlation in all 3 cases when the one extreme plant is removed. (3) The coefficients of correlation between total bundles (double bundles plus intercalary bundles) at the base of the hypocotyl and the number of bundles in the central region of the epicotyl, 7,., are shown in the third section of table 3, and are represented graphically in terms of regression in the upper figures of each panel of diagram 5 for trimerous seedlings and of diagram 6 for normal seedlings. The very gentle slope and the differ- ences in direction of the lines for the epicotyl of the trimerous plants, to- gether with the irregularity of the empirical means, serve to emphasize the slightness of the relationship between total bundles at the base of the hypocotyl and the number of bundles in the central region of the epicotyl. In the normal plantlets the means are less irregularly distributed about the theoretical lines, but the slope of the lines is very slight, and in one case the regression slope has the negative sign. Turning to the correlation constants for more direct numerical com- parison, we note that three of the ten constants are negative. The general average is +.0456 for the trimerous and +.1228 for the dimerous seedlings. Looking back over diagrams I-6,-one cannot but be impressed by the difference in the slope of the lines showing the changes in number of bundles in the hypocotyl and in the epicotyl respectively associated with variations in the number of bundles at the base of the hypocotyl. The lines for the hypocotyl, without exception, indicate an increase in the number of bundles in the central region of the hypocotyl with an increase in the number of bundles at the base of the hypocotyl. The lines for the epicotyl occasionally show a decrease. Furthermore, the slopes of the lines for the hypocotyl are in general conspicuously steeper—thus indicating closer dependence upon the number of basal bundles—than those for the epicotyl. Turning to table 4 for a numerical comparison of the correlations be- tween the systems of bundles on the same side and on different sides of the cotyledonary node, we note that without exception the coefficients of corre- lation measuring the interrelationship between the number of vascular elements at the base of the hypocotyl and in the central region of the epi- cotyl are markedly lower than those measuring the correlation between the number of vascular elements in the base of the hypocotyl and in the central region of the hypocotyl. (B) We now have to consider the problem of the correlation between the numbers of bundles in the central regions of the hypocotyl and of the 5 When the extreme cases are omitted the equations are: Line 75, E = 15.378 + 0.591 J; Line 93, L.= 15.670 + 0.096 J; Line 98, E = 14.840 + 0.394 L. July, 1921] THE SEEDLING OF PHASEOLUS VULGARIS 393 TABLE 4. Differences between correlations for three classes of bundles at base of hypocotyl and the number of bundles in the central regions of hypocotyl and epicotyl, respectively Character of Seedlings vr ee ee, and Line T ne Toph Tie Tin Tbe Tbh Trimerous L/S ee — .325+.074 4.39 | —.203=+.075 2.71 | —.467=5:064 7.29 Ieimero3). i... 5. —.320-.074 4.32) —.259-+.075 3.45| —.617+.068 9.07 WiMevOS:.9.02..') 2. — .313 +.067 4.67 | —.183-.069 2.65| —.487+.059 8.25 META) 5-55. —.522+.084 6.21 | —.081+.092 0.88 | —.626-+.080 7.83 Wine eTAS i... —.538+.055 9.78 | —.072+.059 1,22)| =.5034-.048 | 11.7 Dimerous nen Si. cc. > -.« —.477+.070 6.81 | —.71I1-.065 | 10.9 —.841+.059 | 14.3 TEMNE2 © Siege sss —.557+.062 8.98 | —.258+.070 3.69| —.586-.057 | 10.3 ine -O8%0, ..0:)... —.427+.055 7.76| —.446-+.060 7.43| —.581+.052 | I1.2 eament 30...) vi. —.180+.051 3.53 | —-753+.039 | 19.3 —.750+.037 |-20.3 WC TAB a 5 eo —.518+.040 | 12.9 —,500+.037 | 13.5 — .681 +.033 | 20.6 epicotyl of the plant. The correlation surfaces are given in tables A-L. The results are set forth in table 5. TABLE 5. Coefficient of correlation between number of bundles in central region of hypocotyl and central region of epicotyl Trimerous Dimerous Line N r (Ah i N r 7/Ey Fe Se. 416 5124-033 0.36 | 416 —.017 +.033 0.52 ee 557 +.075 +.028 2.68 557 =|7162'2=,.025 5.79 Qos Sar. 345 -+.090 +.036 2.50 345 +.225+.035 6.43 MAO I s,. 106 — .061 +.065 0.94 305 — 187 +.037 5.05 ee 143 +.256+.042 6.10 420 +, 107 ==.033 3.24 The correlations are positive with the exception of that for dimerous plants of line 75 and of that for both dimerous and trimerous plants of line 139, which are negative in sign. Only one of the negative coefficients may be considered statistically significant in comparison with its probable error. Several of the positive coefficients are large enough in comparison with their probable errors to be considered possibly significant. The average correlation for the trimerous plants is +.074 while that for the dimerous plants is +.058. The correlations for the trimerous and dimerous plants can not be considered to differ significantly. The generally positive sign of the constants suggests that seedlings which have a larger number of bundles in the hypocotyl have on the average a larger number of bundles in the epicotyl. This is the condition actually found in the series studied, but the difficulties in the interpretation of the probable error in cases in which the correlation coefficient is so small should make one cautious in generalizing the results obtained. How slight the relationship between the numbers of bundles in the two organs is, may be shown by the regression lines giving the change in the mean number of bundles in the epicotyl associated with variations in the 354 AMERICAN JOURNAL OF BOTANY [Vol. 8, number of bundles in the hypocotyl and in the mean number of bundles in the hypocotyl associated with variations in the epicotyl. The straight line equations are as follows: Dimerous Trimerous Line 75, H = 10.325 — .068E H = 12.055 + .oog# E = 12.347 — .oo8H E = 15.267 + .o16f Line 93, H = 5.736 + .401k£ H = 11.501 + .050£ & = 11.494 + .065Hf Eo = 14.273 +312 Line 98, H= 1.374 + 6482 H = 11.408 + .042E E = 11.388 + .078H A = 12.538 + .1905 Line 139, H = 4.105 + .338F H = 12.A920— 462332 E = 11.254 -+ .103H E = 16.591 — .113 Line 143, = 616077, 42s 1OrE H = 9.279 + .187£ B= W737 2072. E = 11.810 + .3490H All of these lines have been drawn, but it seems unnecessary to publish more than three sets. The comparison between the empirical and the theoretical mean number of bundles in the epicotyls of seedlings classified according to the number of bundles in the hypocotyl is made for three lines on diagram 7. Con- versely, the comparison of the actual mean number of bundles in the hypo- cotyl for plants with various numbers of bundles in the epicotyl is made on diagram 8. The slight slope of the lines and the irregularity of the empirical means show in a very convincing manner the laxness of the relationship between the numbers of bundles in the central regions of hypocotyl and epicotyl. These results are of decided morphological significance. The profound difference between the correlations for the hypocotyl and for the epicoty] emphasizes the completeness of the loss of individuality of the bundles at the cotyledonary node. Whereas the number of bundles in the central region of the hypocotyl is quite closely correlated with the number at the base of the hypocotyl, there cannot be asserted to be any significant correla- tion in bundle number between either the base or the central region of the hypocotyl and the central region of the epicotyl, when we deal with seedlings of the same gross morphological structure. In other words, the reorganiza- tion of the vascular system at the node is so complete that the portion of the system which is above the node shows practically no relation to the portion which is below the node. Comparison of Correlation in Trimerous and Dimerous Seedlings. In examining the results of the preceding tables the reader may have noted that the coefficients for the dimerous are preponderantly higher than those for the trimerous plants. This result is clearly brought out in table 6 July, 1921] THE SEEDLING OF PHASEOLUS VULGARIS 355 LIVE 98 pe /4 13 ii LINE 93 MEAN NUMBER OF BUNILES /N EP/COTYL YS EOCO TLL DIAGRAM 7. Regression of number of bundles in central region of epicotyl on number of bundles in central region of hypocotyl. Empirical means represented by solid dots for dimerous seedlings and by circles for trimerous seedlings. 356 AMERICAN JOURNAL OF BOTANY [Vol. 8, in which the differences between the coefficients for the two classes of plants are shown. The differences in this table are generally negative, thus indicating that the correlations are lower in the trimerous than in the dimerous seedlings. The exceptions are of some interest. 9 13 LINE 98 Wan 2 \ ‘ \ Ba x / 4 x s Be s oe ’ Gi aS £ \ 7 // /0 LINE 93 MEAN NUMBER OF BUNTLES /N HY¥YPOCOTYL LPAI CO Tigials DIAGRAM 8. Regression of number of bundles in central region of hypocotyl om number of bundles in central region of epicotyl. Empirical means represented by solid dots for dimerous seedlings and by circles for trimerous seedlings. There are only 4 exceptions among the 15 correlations between the numbers of vascular elements in the basal region of the hypocotyl and in the central region of the hypocotyl, as shown in the upper section of the table. These are without exception insignificant in comparison with their probable errors. There are 9 exceptions among the 20 correlations be- July, 1921] THE SEEDLING OF PHASEOLUS VULGARIS 357 TABLE 6. Comparison of correlations for trimerous and dimerous seedlings. Differences only (trimerous less dimerous) are given. See tables 1 and 3 for constants Correlation Coefficient Compared Vph Yih ‘ Toh LING! o/s +.016+.069 | 0.23 | —.339+.060 | 5.65 | —.148-+.039 | 3.79 loc +.408+.060 | 6.80 | —.186-+.069 | 2.69 | —.284+.048 | 5.92 imeOS. sk... 3455-053) 6.50 |. 1202421058 "15.21 —.200+.039 | 5.13 PIMESTSOM +.073+.064 | 1.14 | —.801+.066 [12.1 —.3904+.047 | 8.38 Rime GAZ... +.026+.039 | 0.67 | —.329-.046 | 7.15 | —.049+.022 | 2.23 l pe Tie Tbe [Sito to) 4+,168-+.075 | 2.24 | +.169-b.080 | 2.11 +.236+.079 | 2.99 IANO S Gi as. —.I7I+.076 | 2.25 —.187-+.075 | 2.49 —.315-.075 | 4.20 Mine OSes a <. —.231+.069 | 3.35 —.039+.070 | 5.57 —.106+.069 | 1.53 EIMeGNI3On es ks cs: —.269-+.074 | 3.64 | —.129+.075 | 1.72 —.270+.075 | 3.60 NOTE TAS es 6s -+.006=£.056 | 0.11 +.099+.054 | 1.83 | +.069-.055 | 1.25 The Meine 5 fc. s +.029+.047 | 0.62 — —- — —— MCT Re Oe as. s —.087+.040 | 2.18 — — —— —- IimeyO82 fo... —.135-.050 | 2.70 — -= — — PAMECTZO 06. ccs +.126+.075 | 1.68 — == oo — BAne@hT Ags. 2... 3. | +.149+.053 | 2.81 a —. — ——~ tween the numbers of vascular elements on different sides of the cotyledon- ary node as shown in the central and lower section. The exceptions occur, in short, among the relationships which in both types of seedlings are practically zero in intensity. | We have no explanation to offer of this greater intensity of correlation in the sub-cotyledonary region of the normal seedling. The result is stated as one of the matters of fact demonstrated by the investigation. Correlation between Bundle Number in Siblings The question will naturally arise as to whether the variability in number of bundles in both hypocotyl and epicotyl and the correlation between bundle number in these two internodes may be due to a differentiation of the parent plants from which the seeds were obtained, either in their genetic composition or because of environmental influences. This problem pre- sents many difficulties. Some light may be thrown upon it in the following manner. An abnormal and a normal seedling were taken from the same parent plant. Thus it is possible to determine in our series the correlation between the number of bundles in the hypocotyl of an abnormal plant and in the hypocotyl of a normal plant derived from the same parent. If a differentia- tion of the parent plants due to either genetic or physiological factors is the underlying proximate cause of the variability and correlation in bundle number in seedlings which we have studied, there should be a correlation between the number of bundles in the seedlings derived from the same plant. The correlations between the numbers of bundles in the hypocotyls 358 AMERICAN JOURNAL OF BOTANY [Vols and epicotyls of the normal and abnormal seedling, 4.e., of dimerous and trimerous seedlings, from the same parent plants are given in table 7.° ' TABLE 7. Correlations between bundle number in offspring of same parent plant ores Ai ea Line and Correlation Trimerous Dimerous Line 75 Line 93 Line 98 Hypocotyl ....| Hypocotyl Grou... +.0540+.0406 +.1703 +.0327 —.0512+.0529 SLOMESH 2 cna. +.2151+.0540 +.0553 +.0540 +.0853 4.0495 Bpicotylias, | Epicoty CoS. in — .0037 +.0407 — .0027 +.0336 +.1222+.0522 SCOMES 2 shy: +.0685 +.0563 +.0432+.0541 +.0401 +.0498 The coefficients are low throughout. 3 are negative in sign. Only 2 of the 12 can be reasonably regarded as significant. Both of these are positive. There is, therefore, a suggestion of a positive correlation between the anatomical characters of seedlings from the same parent. The values are too low, however, to justify the conclusion that there is a measurable differentiation in the genetic or physiological characteristics of the parent plants affecting bundle number in the offspring seedling. The absence of correlation here connotes an absence of (sororal or fra- ternal) inheritance in bundle number. Nine of the 12 are positive while SUMMARY In an earlier paper we have shown that the number of vascular elements at different levels in the seedling of Phaseolus vulgaris is subject to consider- able variation and that the amount of variation may itself differ from level to level. This is true both in normal seedlings with two cotyledons and two primordial leaves and in variant seedlings with three cotyledons and a whorl of three primordial leaves. These two types of seedlings are pro- foundly differentiated in vascular anatomy as well as in superficial structure. The purpose of the present paper is to consider the correlations between the number of bundles in the various regions of the seedling. The characters considered are (1) number of primary double bundles, of intercalary bundles, and of total bundles at the base of the hypocotyl, (2) number of bundles in the central region of the hypocotyl, and (3) number of bundles in the central region of the epicotyl. 1. There is a substantial correlation between each of the three classes of bundles at the base of the hypocotyl and the number of bundles in the central region of the hypocotyl. In the normal seedlings the coefficients 6 It has not seemed worth while to publish the tables upon which these very slight correlations are based. For purposes of comparison the series sectioned at Cold Spring Harbor and at Storrs are both given. Sie July, 1921] THE SEEDLING OF PHASEOLUS VULGARIS 359 average +.509 for primary double bundles and hypocotyledonary bundles, +.629 for intercalary bundles and hypocotyledonary bundles, and +.813 for total bundles and hypocotyledonary bundles. In the trimerous plants these correlations average +.381, +.238, and +.598, respectively. The correlations for normal plants are generally higher than those for abnormal plants. 2. The correlation between each of the three classes of bundles at the base of the hypocotyl and the number of bundles in the central region of the epicotyl is low. The coefficients are sometimes positive and sometimes negative in sign. On the basis of the data available it is impossible to assert that there is any correlation at all between the numbers of bundles in these two regions. 3. The correlation between the numbers of bundles in the central region of the hypocotyl and in the central region of the epicotyl is likewise very low. The coefficients are generally not significant in comparison with their probableerrors. If there be any correlation at all between the numbers of bundles in these two regions it is very slight indeed. These results for correlation fully substantiate the conclusions drawn in an earlier paper that there is a complete reorganization of the vascular system at the cotyledonary node. 4. The correlation between the number of bundles (either hypocoty- ledonary or epicotyledonary) in siblings is, if it exists at all, very low. The differentiation of the parent plants through either genetic or environmental factors cannot, therefore, be considered to be the source of the variation and correlation in bundle number demonstrated in this and in our preceding paper. CONCLUSIONS These results, and others for which the reader must turn back to the body of the paper, justify the emphasis at this point of the following general conclusions: a. The vascular structures of the seedling are not constant but are decidedly variable within the species. They show different degrees of variability within the individual organism. b. Seedlings differing in external form are profoundly differentiated in their internal anatomy. This differentiation is evident both in mean number of bundles and in the degree of variability in bundle number. In short, the external form and the internal structure of the seedling are highly but not perfectly correlated. c. The different anatomical characters of the seedling are interrelated with varying degrees of intensity. Between some there is a very strong correlation, but between others practically none at all. The quantitative measurement and interpretation of such relationships, by means of the biometric methods hitherto little applied in the field of vascular morphology, will make possible material advance in the investiga- tion of the fundamental problems of morphogenesis. 360 AMERICAN JOURNAL OF BOTANY [Vol. 8, TABLE A. Data for correlation between bundle number at the base of the hypocotyl and in the central regions of hypocotyl and epicotyl in trimerous seedlings Hypocotyl Epicotyl Base* Line 2 8 | 9 | Iu r1| 12 | 13) 14| rs| 16) 17| 18| 19| 20} 12 a 14/15/16 17| 18 IQ| 20| 21| 22 — | SS | — ee | SE | | SF SS | SS | SS | SS | SS | SS | | (4) +0 PAR ay ieee cre Sk Sia sitio al aval ovale | eoetl Rteslecteal eal ce eee Net ata 2 (4) +1. 139 -|.-).-) Tle. e eee |e ele eleeds cle oleete ete ste oly Tile o ltecl celle etal een eel een ean T43 |--| Tye.) Te. e-| Tec] etea|e elec lee] ee] eal sete ol oce ee Sikes lees ete pean patian ecten mee (4) +4. Be evans alee ee lncvens «|| 11S e\ ene beta ee tee ee |e 20 eee yee I (4) +5. 98 Posed ealh tese ea See Paral Ueda. aie I (4) +6. 75 8 Va eae any Pee WA eae ie Psi c hele I (5)-+o.. 75 Balter mek Wee le ers Pelt Ee cae I 93 Wet Sale w[ecfoce} Elo 0] Qicaet eit eee peal oped ates 98 Be Sele Pe Veale choi osporlo cl: 2) 139 (oti 2) ee vefee| LZ]. | Bp. 3) Thetis eee eae 143 23] 2 ie I PME aal fe iecicels op th (5)+1...] 75 BD Bilas secre ; me) ie eee healers’ | oc 93 SOS ek a Es ee ee ee eee hs whe ao al 1) 98 | B83 | 2h elec eee eee asfeve | > QD] Die caye colle weet ees ce een tas 139 rE Wf tapas i er | | oa Dojo [ace |e | Til Di) eae tea ome eee 143 2) Ea EN 2 Ale eed ee i ge ve\eis| 2h 2) Oh Gl 5 gional 2 ek eran a CEN Et Der tare maltese |e Ne ever ile a ine wel tell Ural a meals pW HDR Se alg 2 O Bev tedl easily, se Ti iaeale aa PROSE ome gie jes) 2 AO Gaal eel Cotsllteolccalraeaae Bi teal celles ols Pa re ee Bl Pleatice) oh 2 Ae Ne | AONET-Ov se 7 Salle lsc|e | Oh GO Si ai a1. 5|1Q\43 22100) Ghee alee oe cog O83) Heals oll. .\22| 102, TON ajaap 1|..| 6/14/47/25/15| 8] 3] z]..| I|z20 OS lelves AVDA SO) ea tees 6 13/36/67/26| 8} 3]..| I .-|160 139 Pea top alee Tah als 4\21/35)21| 8} 2] I). 92 143 --[--{T2T ) 7) 4) 1 I 3 7/1434 31/22 19) 3) I 134 (6)+1...| 75 MeN, all. «blac ee | Gl Meee ea eae 12 93 se Albgce ec tale Tal... | 6) 2p ea II 98 a TBleie ts he dts lle Olan I) I 10 139 Tea) 21 een (ee sail | eee ea 5 143 sole ahabedeyiesd. ol. ol 5] 5) Se sieeiee 25 {6)+2..:| 75 iret fee mui hen al pes (ies a wa [ 3.2 |e eee eg ee 2 93 Nee fre Elis alice PaaS ioe ed I {7)-+0...| 75 \--|- ae ine . sah a ater 7 93 Ale eee he Ph oie via 4 98 la Teepe: ieee I 143 he elas. Let She al Nee ar 5 (ect eelerae ee eel. 3| I Bes alg I I 4 CT ae ee! ASM hw ace ale ol soll, Elicia exe Meccllc. elec ll Miealpeyea an te ere een I (8)One fee 75 athe els « oe Dyce leilbecelltee ee eal ieeist escape seal ea eet I (S)e1)..! 148 lS od SS ae ec org Tlf, soe ea * Numbers in parentheses are of primary double bundles; those following are of iter- calary bundles. uly, 1921 | THE SEEDLING OF PHASEOLUS VULGARIS 361 TaBLE B. Data for correlation between bundle number at the base of the hypocotyl and in the central regions of hypocotyl and epicotyl in dimerous seedlings Hypocotyl Epicotyl Base Line 8 OSlETOsaT hl Le.) rs eT Al eEsel a7 eTSh ast Ow aii.) ne 3rd ers LTO hot. — |—-—. | — —_ | __._._ Loe Ww \o NO op) \O = yy Nol Xe) S) Ss = aS WwW MH ae Loe to ba | Ln Qe 75. |08.)...|. 2 = = = oo | ; : HON ANBRUWWO COORD NOAH eH BNW = _ \O (4)+3...| 75 NWNANANB OF HWW WD empeer ea elt ee te ah sala Has. Cuneo ele lee ke.|. a] .|..15 a] ole. GyresOnn-) 75: beast: tT) 8 Sic tine tesa ele perce — Geeta i 75 eee is)... |e le le El cele~s|sgl- = HHH ORB HNN B DTH BR OHDNHHNNHWAWMY ae: i | (ee «calles yee ee eee eee pa es ce pee ee CERO Mes lo ee NWeinle gle. te al wc lesc ime s(osule The Cre ee lion ee Mths los loc slbbeh eel ettlaethee) BM aloe cloacles. (6)--2 ...| 93 Paar lol (2 Thee FNSEO Ss ol WARY eI or (a eae be fee Spin reer learar||rar [role A gers eae oe SE DAES les ell. weal A tose les ak MPS ins Wl Tl a I, Bele stl de seal. wW = NQ Ln HR HOH HOUR HH HORN DHRHKHANWNANNUBYU DA Ne) [oe) 4 ey Ver pod Sac 362 | AMERICAN JOURNAL OF BOTANY [Vol. 8, TABLE C. Correlation between numbers of bundles in hypocotyl and epicotyl of trimerous plants of line 75 Epicotyl Hypocotyl I2 13 14 16 16 17 18 Lom 20 21 Totals So: To | aeewce a] ss «spas eift Gao ellie creo Sgieallle eee alee en I Oe I TL | c.c renter are pa i on blanks to te citsindd hk : i i _ Ever g gone into a hat iekore’ Lnow. ing exactly the kind and. shaped hat you want; and then have some. tl smart Alec of a salesman try to. Hert pe sell _ you the kind HE dikes?) 0°) i 1 eee | v sine habe: une eMaliea® he = fellow ‘madder than a4) | Ol wot hen, doesn’ t i a Sas me = ae to cae 4 ree { ‘Suppose, however: that sdme | lesman had told you something — “about some of his hats that meant | Rix CIAL PUBLICATION OF THE ae, : < we Pe < - vf CONTENTS EARN VPRO te NUTR WEE Ie ne emitrimerous seedlings of Phaseolus vulgaris. © Epmunb W. Sinnorr, Jorn VY. 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Le “8 Manuscript, offered. for publication’ should. be tyhewritten Sy es be’ submitted to the Editor-in-Chief. vee as Papers | are. limited: to 20 pages Jn ditional pages: may be: serene for at Ian a PTY Proofs. ahduld be. dorecnted mm dia Journal: ‘Botany, Brooklyn Botanic j aul ustrations, ‘other, than. zine | echt Zs, be réstricted to approximately ten’ ‘per ‘Authors may be charged at cost re per: square’inch for half-tones). ‘| Separates: ‘should be. ioderad wien Ben . cover will be’supplied free; cover and additional Hane: Remittances ‘should be made. payabl to” ie foe e must. be added to. all. leche: not dray date of ‘mailing, The publich ts will eee mi sen ‘they: have been lost i om, the. bite - os es m1 NaN AMERICAN JOURNAL OF BOTANY Vot. VIII OCTOBER, 1921 No. 8 THE VASCULAR ANATOMY OF HEMITRIMEROUS SEEDLINGS OF PHASEOLUS VULGARIS J. ARTHUR Harris, EDMUND W. SINNOTT, JOHN Y. PENNYPACKER, AND G. B. DURHAM (Received for publication January 17, 1921) INTRODUCTORY In an earlier paper! we discussed the gross vascular anatomy of dimerous and trimerous seedlings of the garden bean. By dimerous seedlings we understand those of the normal type, characterized by two cotyledons and two primordial leaves, both sensibly opposite in insertion. By trimerous we mean those which have a whorl of three cotyledons and three primordial leaves. The cotyledons may be, and frequently are, more or less irregular in insertion. The primordial leaves are, in the seedlings considéred, inserted in a regular whorl. | In addition to these two types of seedlings, those which are in a sense intermediate in superficial structure between the two types hitherto studied may occur. These are seedlings with a whorl of three cotyledons but with a normal pair of primordial leaves instead of three as in the case of trimerous seedlings. These we have called hemitrimerous. They are extremely rare in occurrence, but during the four years during which these studies have been under way a number sufficiently large to justify a brief discussion of their gross vascular anatomy has been secured. Our purpose in this paper is to compare the anatomy of these hemi- trimerous seedlings with the trimerous seedlings (in common with which they have three cotyledons) on the one hand and with dimerous seedlings (in common with which they have two primordial leaves) on the other. For convenience of reference the three types will in some cases be designated by the number of cotyledons and primordial leaves: 2-2 = dim- _€rous, 3-3 = trimerous, and 3-2 = hemitrimerous. MATERIALS The hemitrimerous plants and the trimerous and dimerous seedlings with which they are compared were largely secured in the series of germina- ! Harris, J. Arthur, Sinnott, E. W., Pennypacker, J. Y., and Durham, G. B. The vascular anatomy of dimerous and trimerous seedlings of Phaseolus vulgaris, Amer. Jour. Bot. 8: 63-102. 1921. [The Journal for July (8: 323-374) was issued August 31, 1921] 275) ~~ 376 AMERICAN JOURNAL OF BOTANY : [Vol. 8 tions which furnished the materials for our earlier discussion of dimerous and trimerous seedlings. The dimerous and hemitrimerous seedlings were derived from the same parent plants in lines 75, 93, and 98. In lines 209, 139, and 143 the germinations were made from mass seed instead of from the seed of individual parent plants. All of the seed, however, was grown in the same experimental field in 1917. : Since it has been shown in an earlier paper? that there is practically no correlation between the anatomical characters of the trimerous and dimerous seedlings from the same parent plant, we are fully justified in using random samples of hemitrimerous, trimerous, and dimerous seedlings for a com- parison of their vascular characters. A detailed account of the vascular topography of the dimerous and trimerous seedling is presented in a previous paper by the writers, but may be summarized very briefly here. Each primary polar bundle of the root bifurcates in the base of the hypocotyl to form a “primary double bundle,” which gives rise to two distinct and well separated strands in the central region of the hypocotyl. In addition to these, there are usually present in the hypocotyl a number of “‘intercalary’’ bundles, arising either de novo or by splitting of some of the primary strands. At the cotyledonary node a rather complex vascular anastomosis takes place, from which the coty- ledonary strands depart and out of which the vascular system of the epicoty] is organized. PRESENTATION AND ANALYSIS OF STATISTICAL DATA Base of Hypocotyl The frequency distribution of the various types of vascular organization at the base of the hypocotyl is shown for all the available data in table 1. In this table the number of primary double bundles appears in parentheses, while the number of intercalary bundles follows the + sign. Because of the relatively small numbers of hemitrimerous seedlings which can be obtained and because of the irregularity of the frequency distributions for bundle number, it has not seemed desirable in this paper to consider the frequency distributions of the numbers of bundles of the several types. Neither has it seemed desirable, on the basis of the rela- tively small series of hemitrimerous seedlings which can be obtained, to consider the relative variabilities of bundle number in the different regions of the three types of seedlings as we did in our discussion of variation in the dimerous and trimerous types. We have, therefore, limited ourselves to a comparison of mean bundle number, leaving the question of variability until larger series of countings can be obtained. 2 Harris, J. Arthur, Sinnott, E. W., Pennypacker, J. Y., and Durham, G. B. Correla- tions between anatomical characters in the seedling of Phaseolus vulgaris. Amer. Jour. Bot. 8: 330-365.: 1021. Oct., 1921] HARRIS AND OTHERS — PHASEOLUS VULGARIS SAGE TABLE I. Se Line 29 Line 75 _ Line 93 Line 139 Line 143 Hypocotyl emenoneii2e2 | 3-5 | 3-2 | 2-2). 3-3 | 3-2 | 2-2'113-3 | 3-2, 2—2)| 3-3 | 3-24 2-2 (4) +o..) — I | 83 | — | — |1o1 | — | — j117 | — 2 \270 2 20201 COn-erose—— | 4) rr | — 2NS BO) (== aA eGS I 5 | 26 4 6 |102 ec 2h: I 2 I | — 1a eile Tee 82) Veta On| 5 == 5 iej-a-e | Tr |} — | — 1 — | — 4|—-— |) — 2 eet ee el ee Game a oe) 2 |e eae be (4) +5...) —|—/{/—}]—]—] 2} r}—}/—}—]—}/—-}—-]—-|!—- ee et gy |e ee eee code go.) — bee Te ee SSS = (See Ole 7 | 11 I I Atay 43 Asia 63 7 4 | Tol 5 eel) 13 Pree rmoneer 29 hye eo | 6) 5 | 8b aly gr] 2 [ar | 20) 7 (5) +2..) —}|—}|— 2) — 2 Tene «2 eee | ee omc Gee ae es | | ee ae ey (Oia On 39-16 | — |107 | 22 TON TOO* |. 24 I }:92 | 20 | —= 340 52°) — (6) +1..) — 1 | — | 12 6 22 eo 2 I 5 2|— | 25 6 I (6) +2..) — | — | — 2 2;/—|]— I; —|— — | — (6) +4..) —}| — | — [1 — I|-—-— —) —}]~— Jr ~—] ~~) ~~ | HS | (7) +o...) ©} —{— F 2 Teal) 2Tew iy ee ete eee 5 I I G7) ti1..;$—}|—/—)—}— 1! —,— |] ri) —-tfr- — | eK 4)/—]}— (7) + 2..) — | — oat r}—}|—, —, —) — | — — | — (8) +o..) — | — |; — I}—]}]—i—]}]—f}—f}—- ef] — | — pemmen es =|) |S al =| tp=/S 56 | 43 | 99 lr42 57 NILOQs'TS3| 43226 |106 . 42) (305 \22mclira (420 Table 2 shows the average number of primary double bundles, inter- calary bundles, and total bundles in the three types of seedlings, and gives the differences and probable errors of differences in the means upon which we must depend for conclusions. The entries in the first section of this table show that the average number of primary double bundles is relatively lower in the hemitrimerous than in the trimerous seedlings. It is also relatively higher than the number in the dimerous seedlings. The differences, while small, may reasonably be considered significant in comparison with their probable errors. The differences between the hemitrimerous and the dimerous class are much larger than those between the hemitrimerous and the trimerous. Turning to the statistical constants for intercalary bundles set forth in the second section of table 2, we note that in four of the five cases the hemitrimerous seedlings have a larger number of intercalary bundles than the trimerous seedlings. These differences are small, but may be significant. In the one case in which the hemitrimerous seedlings have a smaller number of intercalary bundles than the trimerous plantlets the difference is only — 0.01 + 0.04. In two of the cases the hemitrimerous show a larger number of intercalary bundles than the dimerous seedlings, but in three lines the reverse is true. The differences are in general not so large in comparison with their probable errors as in the case of the comparison for number of primary double bundles. 378 AMERICAN JOURNAL OF BOTANY [Vol. 8 TABLE 2. Mean number of bundles at base of hypocotyl! f (Primary Double| Intercalary Total Bundles Bundles Bundles Line 29 Bria t anlik tae na hee oe al 56 5.68 + .05 Ga ay (07) 5.95 + .04 Bae Oho e ae ean te oe Neem Weed aes 43 a2 hae O8 153) == sk 5. Anse sO De Ot inp cre ee eh eM eee Vere meta AO) AL O4 pst 0% 16 4202 A20.4=)03 (Be?) = (6-3) eats hse ee a ee | — 0.47 + .09 + .26 42°14 — 0.21 a= .1T (B22) (2) eras Meck amcia. tay: + 1.17 += .08 Sr sy Se 512 =F 4 eh 10 Line 75 tot eee aR he ic een NON Aare | 142 5.98 + .02 25 = On! 6.23 == 402 ea Ee aR ce re Ph Mies Ni Bae EN a 57 Boye = O5n tl 44,32 5G7, 6.18 + .07 Peat. EN SAS NLD Ps Ae PA OO Shs Sahel (rT KOKS 4.24, E08 62 + .05 4.85 +& .05 (GOH (25) ey ere pate | — 0.24 + .05 + .19 + .08 — 0.05 + .08 (re (en ee ER ees Wo a | = be 5 Ou==)500 — Deis Oe) Slag) o= 1 0)e) Line 98 Be RO ir el. 72) boos Pe ae 183 S0)e) ae HO)! 13 ==KO2 6:00s4= 502 Oa ge ANOS SrA Sift cropeen| eee} 5.67 se .07 .53. =e 209 6:2 1921163 BaD Rene Fs toe gt ie eee i ae [22604 4.1I + .02 .62 + .03 4.73 + .04 (2D) (BAA ohne a ade a eeeme net ot | — 0.26 +.07 | + .40 + .09 + 0.15 + .08 (am aa( 2-2) ike nye th eee | a= 1256 =. 207 — ,09 + .09 + 1.48 =e .09 Line 139 | ee ay ee ee Roc Ne ESR Sasi yerOG. | 5.01 = .02 -00) E02 6.002 Bae ie Ae le nee ee) acme een 2 | 5.30 = .08: | 43 + .05 5-79) == .00 rer AE ON ie AOR NORS GES RUN 305 4.0L == 200 BH i es xis 0/21 4.14) se 302 (2-2) (33) avai paints, tea eee ae | (0,55, 2=).08 + .34 45105 — 0.21 + .06 (32) (G0) TaN A eed eae eee | + 1.25) 2=..08 + .60)==.05 + 1.65 + .06 Line 143 Ba G iy sick Heenan ae eae Cen eed 5.01 se. .03 329, == 362 6:10 E208 eo ak pe sean ce iols eee ke eee 5.45 a=) 04. 2002-02 5.73 a= 204 DOM Rah Bes ori of ea erat Erne eS YAO) A00)== 701 -20'2=5.02 4.35 S='.02 (Bq 2) (3?) a toe eerie et caste aes oe | —O:g0"== 7-05 — 101. a=0 — (0.37 25205 (B22) (22 Peary ata eee cay share te Sle 30) 5.04 — .OL + .04, “ao 3a =o We cannot, therefore, assert on the basis of the data now in hand whether dimerous, hemitrimerous, and trimerous seedlings differ in the number of intercalary bundles at the base of the hypocotyl. In so far as it goes the evidence suggests that the hemitrimerous seedlings have a larger number of intercalary bundles than the trimerous but a smaller number than the dimerous plantlets. The means for total number of bundles (primary double bundles plus intercalary bundles) at the base of the hypocotyl set forth in the third section of table 2 show that in four of the five cases the mean number of bundles is lower in the hemitrimerous than in the trimerous seedlings. The differences are, however, very slight indeed and cannot in general be considered significant in comparison with their probable errors. The differences between the hemitrimerous and dimerous seedlings on the other hand are rather large and in every case are unquestionably significant. Summarizing these results, we note that the hemitrimerous seedlings are conspicuously differentiated from the dimerous seedlings in the number of primary double bundles and in the total number of bundles. They are less conspicuously differentiated, if at all, in number of intercalary bundles. They are unquestionably differentiated from the trimerous seedlings by @et.; 1921] HARRIS AND OTHERS — PHASEOLUS VULGARIS 379 their lower number of primary double bundles and possibly by a higher number of intercalary bundles. They cannot be said to differ from the trimerous seedlings in the total number of bundles at the base of the hypocotyl. Central Region of Hypocotyl For the number of bundles in the central region of the hypocotyl we have the fundamental frequency distributions given in table 3. Considering the mean number of bundles in table 4, it appears that the number of bundles in the central region of the hypocotyl of hemitrimerous plants is slightly lower than that found in trimerous seedlings in four of the six lines available. The differences are, however, small and would not for the most part be considered significant in comparison with their probable errors. The bundle number of hemitrimerous plants is in every case distinctly higher than that of dimerous plants at this level, and these differences are con- spicuous and unquestionably significant. Thus in hypocotyledonary struc- ‘ture the hemitrimerous seedling is very close indeed to the trimerous but perhaps shows a slight deficiency in bundle number. This result is not surprising in view of the fact that so far as the coty- ledonary node and lower portions of the axis are concerned the external form of hemitrimerous and trimerous seedlings is essentially identical. Central Region of Epicotyl If a differentiation between the hemitrimerous and trimerous seedlings obtains anywhere, one would expect to find it in the epicotyledonary region, TABLE 3. Dzisiribution of number of bundles in central region of hypocotyl Steen VOR ET |) E24) 0S tA ets) TOM Ty hs a 20n) 24.) Total Line 29 Beans fe. == fe I Omar I 3 | es | ey I 56 Oat = 2 6 San 2 2/— I ITA Ngee) Gene oll Geto 43 Dire De sts 7a 20 9 2)—-}|—]m—}m ley ert es rd 99 Line 75 Cine Oe I 3 Ba 20-1292 || 207 |.2 5 I 4 | —-| —-| — | 416 Brent: == 2 Oat oa| 5a) VOU LA 2 2°) —— |a-— |. —= la-—— | 103 Las ae Piet. TOs.) 26, 1 31 14) nT I I 3 Leo) ale 510 Line 93 | SS Ra ear oS) Boe Sr lms2 13825) "82.1928. 1 ie I | — I a ale Se OS ye ae == 4 Gal 17 8 7 r1}/—{]—}]—]—]— 43 meh ie ees BOq| 9341170 \107 | 9O-| 40. |. 18 To) eres | ee 2 een a | Ok Line 98 | Coe eran = I OW I2i207 W227 8 | —}—}]—]— | — | — | 345 ee | 3 Tame 3 3 I De ae eee Ve) A 27 eee P25 ATZOS 63 37 TI Se pee) ee Dele a 2 88 Line 139 | | 2a aie = | 4 oh olf tere! 6 8 ee eee | OO Barret fot I 3 Ne oon a CAI 16) 3 1) — | 42 C=) ae 200). 22 v/ 4 I r}/—}—/}/—}]—}]—)}) — | — | 305 Line 143 | | Gaon o: 2 Peieli | 4h ote 25 6 3 I I | —|—]| 221 BGS aie Se I 2h eT Oa eS 8 5 A ea te ee eet Da) Ne BOC eos Alar |: cl? 4 3 I eee ae el AO 380 AMERICAN JOURNAL OF BOTANY [Vol. 8 since the sole superficial difference between the two types of seedlings is found at the primordial node. in the hemitrimerous than in the dimerous seedlings. The frequency distributions in table 5 show that the nodal number of bundles is in general lower in the hemitrimer- ous than in the trimerous seedlings. . It also indicates that they are higher The averages and their probable errors in the second section of table 4 show that in each of TABLE 4. Mean number of bundles in central regions of internodes ay ane (B22) lo) (3-2)-(3-3) (322)2 (252) Line 93 | Central Central Region of | Region of Hypocotyl | Epicotyl 12:36 == Om LAs Sita DEA Ol 2203 --alen 8:45=-,05) 12.05. .02 = 6222.23" 1-82=—.25 ae Scoala POOEE Ly 12.GOS=-02|5 5-472 -04 12232-2050) eo 580 Oi52 42-05): , 12. 20-102 oi) ali O0|— =I: 02 a0) ae) 2200 =-00|-\- 1592-410) 12:29 -2.03)| 9 15.05.04 D2:26eto3|) (I4e tee. io 10/02==.04)" 12.19=4=.02 ae A028 so gh3 ee Line | Line 98 GP 2 Oe (3=2)—(3-3) (3-2)-@-2) ( Line 139 Bee 20), ke 2 (3-2)-(3-3) | Ge2) ees) Line 143 Can ier ae ne 2 2 1 =. 18)(3-2)=(34) ote T.O4= 14) 92.05 =e iN Ba?) (22) Central Region of Hypocotyl 12:03 25702 12.07.2302 0:24:07) + .o4+.12 [= 2:62 sate II.99+.05 Ll. 26s i2 8. 1Gia=.02 16342203 +- 3.17 ate 12.29 +.06 [L.69ae, 10 8.66+.04 A0z=.12 te 323 Central Region of Epicotyl 14.89 +.04 EGn 2 st [202 sO = alyy Sess + 1.40/35.12 - 15.24: 08 UO e See lie 12.10+.01 Seika sie 10) boa et hes 16.10+.08 13.68.09 12, 30=—-02 =) 2:42 S202 i yee) TABLE 5. Daustribution of number of bundles in central region of epicotyl | JO Wty jot 25 |) =) iS Ase Carer Line 29 | . BOOS cid oe I | — ol es CLA es Beas Vs Ee A Boonie 2 ie [Wail 4 6 6 2 22 ee Sp 2 | OY | I I} — Line 75 | Bes uae =>) [= Sr) LOM OST OAT Rog Bohn a ots aa Bill pLOme 25 are yan en 9 PI PR se ede I Apa A225 8°"| 2 Ten TO 3 Line 93 Dar es ale] == fe 518: | Az 1226 2G BeBe dc == | == 5 4. Omar? 5 Dah 5 I C7149 37) 242) eo ol ano T Line 98 eights VANE al ee O24 | OOM Om eA@ BP ere hic = I Oita a es 8 2 Pree rie = = | ae 2 7 I I Line 139 BR ce == | = |) = Ou 2 ecm mee Bare = 2 CRr2 8 6 5 Plea Sa Done, 6 = | S278) | ee 4/—]}— Line 143 Simeghoan ont) aT a 5 Oe OR aaa et9 Reeds eis Se EY TO) 84 nal 6 OL Ane SS | SOLO 277 5 4 1 ||, Ton ZO 21 i | 4 1 cal I 27 4 + I To) Pea = 51 | 10 4b 2 2); =>) — a I I I + 2 DZ — ——a ——_ 31 9 6 2 —— 2 as =—, ar Oct. 1921 | HARRIS AND OTHERS — PHASEOLUS VULGARIS 381 the six lines the average number of bundles in the epicotyl is significantly lower in the hemitrimerous than in the trimerous seedlings, and (probably) significantly higher in the hemitrimerous than in the dimerous seedlings. In epicotyledonary structure the hemitrimerous seedlings occupy as a matter of fact almost exactly an intermediate position between the dimerous and the trimerous types. SUMMARY The purpose of this paper is a comparison of the gross vascular anatomy of hemitrimerous seedlings of Phaseolus vulgaris with those which are trimerous and those which are dimerous. By dimerous seedlings we under- stand those with two cotyledons and two primordial leaves, by trimerous seedlings those with three cotyledons and three primordial leaves, and by hemitrimerous seedlings those with three cotyledons and two primordial leaves. The hemitrimerous is, therefore, intermediate in external form between the dimerous and the trimerous seedling. In the internal structure of the axis at the transition zone, which here occurs at the base of the hypocotyl, the hemitrimerous seedling is clearly differentiated from the trimerous type by a slightly smaller average number of primary double bundles, and possibly by a slightly larger number of intercalary bundles. The total number of bundles in the basal region of the axis of hemitrimerous seedlings is not sensibly different in hemitrimerous and trimerous plantlets. The hemitrimerous are conspicuously differentiated from the dimerous seedlings by a larger number of primary double bundles and a larger total number of bundles. On the basis of the data available they cannot be asserted to differ significantly from the dimerous plants in the number of intercalary bundles. In the central region of the hypocotyl, the vascular anatomy of the hemitrimerous seedling conspicuously exceeds that of the dimerous in bundle number but agrees very closely indeed with that of the trimerous plantlet, although it may have a slightly lower average number of bundles. In the central region of the epicotyl the mean number of bundles in the hemitrimerous seedling is, roughly speaking, intermediate between that of the trimerous and that of the dimerous types. Recapitulating, it appears that in internal structure the hypocotyl of the hemitrimerous seedling is practically identical with that of the trimerous seedling with which it has in common a whorl of three cotyledons. The epicotyledonary internode in the hemitrimerous seedling, limited by a trimerous cotyledonary and a dimerous primordial node, is intermediate in anatomy between the trimerous type with three cotyledons and three primordial leaves and the dimerous type with two cotyledons and two primordial leaves. THE EFFECT UPON PERMEABILITY OF POLYVAEERN Gx — IONS IN COMBINATION WITH POLYVALENT ANIONS OrAN L. RABER (Received for publication January 20, 1921) In most of the studies carried on with a view of ascertaining how bi- valent ions (such as calcium and magnesium), and trivalent ones (such as aluminum) affect permeability, the anions employed have been monovalent ones (chloride, nitrate, etc.).. In studies in which Laminaria has been used, it has been found by Osterhout (1, 2) that wherever bivalent and trivalent cations were employed, the first effect of the salt has been to cause an increase in the resistance of the tissue (followed by a decrease), while if monovalent cations are used (with exception of acids and sodium taurocho- late) the first effect is a decrease in resistance. Since the anions, as shown in previous papers (3, 4) seem to play an important part in determining the permeability of the tissue, it seems of interest to investigate the effect of a salt composed of a polyvalent cation and a polyvalent anion. The number of such salts en are sufficiently soluble for the present experiments is exceedingly limited, because of the pronounced relation between valency and solubility. Magnesium citrate, magnesium sulphate, and aluminum citrate were finally selected for study since they possess more than most others the requisite characteristics for this work, in respect to acidity, solubility, osmotic pressure, etc. The solution of magnesium sulphate used was about 1.09 M and had the same conductivity as sea water. Its pH value is about 8 and it is hence only very slightly alkaline. Figure 1 (curve A) shows the average result of the six experiments performed. The probable error of the mean (as based on Peter’s formula) is under 5 percent of the mean. The rise in resistance at the start is seen to be very small and temporary, and at the end of five minutes the resistance has dropped to 84 percent of the original value in sea water. With magnesium citrate the difficulty of solubility was encountered. Since it was impossible to get a solution of the same conductivity as sea water, the practice of diluting with chloride was resorted to. When a solution composed of one sixth magnesium citrate 0.16 M and five sixths magnesium chloride 0.28 M is used, the results are as shown in figure 1, B (three experiments; probable error of the mean under 5 percent of the mean). If this experiment is repeated, using magnesium chloride of the same electrical conductivity as the previous mixture, viz., about 0.24 M, the 382 120 Jo ee Cc 60 ; | = F B rp) A. 20 sp 0 50 100 MIN. Fic. 1. Curves showing the resistance of Laminaria in solutions of various salts: A, in magnesium sulphate; B, in one sixth magnesium citrate and five sixths magnesium chloride; C, in magnesium chloride of the same conductivity as that represented by curve B; D,in one third magnesium citrate and two thirds magnesium chloride; £, in magnesium chloride of the same conductivity as that represented by curve D; F, in one third aluminum citrate and two thirds aluminum chloride; G, in aluminum chloride of the same conduc- tivity as that represented by curve F. Ordinates represent resistance (expressed as per- centage of the resistance in sea water, which is taken as 100 percent). Abscissae represent time in minutes. 384 AMERICAN JOURNAL OF BOTANY [Vol. 8 resistance changes as shown in figure 1, C (three experiments; probable error less than 3 percent). It is seen that without the citrate the rise in resistance is much higher than with the citrate. In other words, the citrate has a decided tendency to keep the resistance from rising at the start. If the proportion of citrate in the mixture is now increased from one sixth to one third, results are obtained as shown in figure 1, D (three experi- ments; probable error less than 3 percent). A 0.20 M solution of magnesium chloride has about the same conduc- tivity as the last mentioned solution, and if tissue is placed in it the results are as shown in figure 1, & (three experiments; probable error less than 5 percent). Here it is seen that the resistance rises slightly in the 0.20 M solution of magnesium chloride, but that if enough magnesium citrate is added the resistance does not increase, but decreases from the beginning. This is of interest since it shows the importance of the anion in studies on permeability. Figure 1, / shows the changes in resistance in a mixture composed of two thirds aluminum chloride 0.40 M and one third aluminum citrate 1.09 M. This mixture has a pH of’ about 3 and the conductiityeeiea solution of 63 percent sea water plus 37 percent distilled water. The curve shows the average of two experiments (in which the probable error of the mean is under 3 percent of the mean). Figure 1, G shows the resistance in a solution of pure aluminum chloride of the same conductivity as that represented by curve F. The number of experiments and the limit of probable error are also the same. The pH of this solution is about 4. Since it is known that acid produces an initial rise in resistance, it might be thought that the acidity of the solutions is the chief factor in the effects produced. If this were true, we should expect that the greater the con- centration of the citrate the greater would be the initial rise in resistance since the citrate is the more acid of the two salts, but this is just the reverse of what is found to be the case. Again it may be suggested that in mixtures of magnesium chloride and citrate and of aluminum chloride and citrate synergetic effects are present which cause especially rapid decrease in resistance. Since pronounced synergy has been found between the chloride and citrate of sodium (5), this is quite possible. The possibility of synergetic action can not be denied, but even if it is present it is evident that the anion has a powerful influence. Moreover, the experiments with magnesium sulphate (where there is no possibility of synergetic effects) show that bivalent cations may be pre- vented from causing an appreciable increase in resistance if they are used with bivalent anions. SUMMARY Bivalent and trivalent cations in combination with monovalent anions produce an increase in the electrical resistance of Laminaria, but when Oct., 1921] RABER — PERMEABILITY 385 combined with bivalent or trivalent anions the increase is less and may be entirely lacking. 3 LABORATORY OF PLANT PHYSIOLOGY, HARVARD UNIVERSITY. BIBLIOGRAPHY 1. Osterhout, W. J. V. The effect of some trivalent and tetravalent kations on per- meabilitv. Bot. Gaz. 59: 464-473. IQI5. On the decrease of permeability due to certain bivalent kations. Bot. Gaz. 59: 317-330. I915. 3. Raber, Oran L. A quantitative study of the effect of anions on the permeability of plant cells. Jour. Gen. Physiol. 2: 535-539. 1920. ——. The antagonistic action of anions. Ibid. 2: 541-544. 1920. 5. ——. The synergetic action of electrolytes. Proc. Nat. Acad. Sci. 3: 682-685. I917. te THE FLORAL ANATOMY OF THE URTICALES ALBERT REIFF BECHTEL (Received for publication February 9, 1921) In the search for natural relationships in the seed plants, the morpholo- gist and the anatomist have contributed much to the subject from work on the reproductive mechanism and on the structure of the vegetative organs. During recent years the breeder and the geneticist have presented theories based on experimental evidence which the taxonomist can not ignore. The subject of flower anatomy, however, has been limited in workers and in material studied. This subject should have contributions of value to offer for principles that are to be the guides in determining relationships within the angiosperms, as well as casting more light on the floral characters of the ancestors of the angiosperms. There is as yet no definite knowledge of the ancestry of the various divisions of the angiosperms. Here hypotheses must be made from the study of external form upon which the classification has been mainly based, and these hypotheses must be strengthened or weakened by evidence from internal structure. Different views have been held as to what the primitive flower was like. De Candolle (6) looked upon the primitive flower as hermaphroditic with all parts free. Engler (9) considered the simple, naked, unisexual flower as the primitive type. Bower proposed a bisexual flower with many sporophylls surrounded by one whorl of floral envelopes. The types of flowers known to us have been derived from ancestors, undoubtedly, like unto one or more of ‘those above described, by the processes of amplification and reduction, which processes students of natural history accept as the great factors in molding our present forms of life. Two cautions are suggested by Engler and Gilg (10) for students of phylogenetic relationships to heed: first, the consideration of a simple structure as primitive when it is reduced; second, the placing of reduced forms because of their reduction, too high in rank. There is a guide in this matter of interpreting reduced forms in Bower’s (4) definitions: Where the development of the natural organism, either in whole or in part, in external form or internal structure, falls short of that of the ancestry, that condition would be described as reduced. Examples from among the earlier workers of the contribution of floral anatomy to the knowledge of the morphology and relationships of angio- sperms are the following. In 1862, Darwin (5) declared the discovery of the nature of the orchid flower by studying transverse and longitudinal sections. His conclusions drawn at that time constitute our present interpretation 386 Wet 1021 | BECHTEL — FLORAL ANATOMY OF URTICALES 387 of the column and labellum. Gérard (11) in 1879 presented the anatomical features of numerous genera of the Orchidaceae. Upon Darwin’s discovery it was at once suggested that the Orchidaceae are probably in the same line of descent with the Amaryllidaceae. In all characters the genera agree anatomically except in the androecium, which varies in position and in the number of the stamens and staminodes. Van Tieghem (20) in 1868 published the results of his extensive anatomical work on the flower, par- ticularly on the structure of the pistil. This study treats of the various positions of the ovary with reference to other parts of the flower. A search into flower anatomy should aid in revealing the following points: whether the same organs in different plants have taken on different functions; whether different organs perform the same function, or whether different forms of the same organ perform the same function (10); what is the condition of the vascular supply to aborted organs and to suppressed organs; whether any amplification in the floral organs has occurred; and what is the relative position of the floral organs, normal and abnormal. The order Urticales, a study of which forms the subject of this paper, has been nearly universally accepted as a natural primitive order, but has been placed otherwise, bodily or in part, by the following students of taxonomy: Weddell (21), Lindley (13), Bessey (3), and Hallier (12). Although these students have not looked upon this order as among the very primitive angiosperms, they do agree that it belongs among the lower archichlamydeous forms. This study of the group is based upon the anatomy of the flowers and has as its object the possible determination of the phylogenetic position of this group so far as evidence may be offered by this field of research in conjunction with other parallel evidence. Two treatments of the Urticales are in use to-day: that of Bentham and Hooker (2), which places all species in one family, the Urticaceae; and that of Engler (9), which divides the order into three families: Ulmaceae, Moraceae, and Urticaceae. For convenience in this paper the latter treat- ment is used. The following characters possessed. by this order of plants have caused them to be looked upon as primitive: flowers usually unisexual; floral envelopes composed of one whorl, which is generally spoken of as the calyx, and which is inconspicuous, bracteal, with parts similar and distinct or gamophyllous; stamens isostemonous; Ovary superior, one-celled and one- ovuled, the ovule commonly orthotropous. In spite of these fairly primitive characters these plants have nevertheless been looked upon as somewhat reduced forms, the very features considered primitive being viewed as simple by reduction. The evidence from which such a conclusion is drawn is: apetaly and one whorl of stamens (Bessey, 3); a pistil with a unilocular ovary but with two styles, an indication of two fused carpels. Syncarpy is not considered primitive nor is:the solitary ovule (3, 9); these are, on the contrary, the result of reduction. In the present paper are shown cases in 388 AMERICAN JOURNAL OF BOTANY [Vol 8 which the presence of one style in this order has prompted earlier students of taxonomy to infer the presence of but one carpel; internal anatomical study, however, reveals the unquestioned presence of two carpels. MATERIAL The flowers in this study were killed in chrom-acetic acid, embedded in paraffin in the usual way, and sectioned serially, the sections being eight and ten microns thick. Because the flowers are very small and ephemeral the vascular supply is very delicate and not readily differentiated. Experi- mentation with various stains proved the safranin and light green combina- tion to be the most practical. ULMACEAE Ulmus. The species of this genus have flowers arranged in the inflo- rescence in a graded series from a short, slender, raceme-like cluster or a loose panicle-fascicle, with slender, jointed pedicels as in Ulmus americana and U. racemosa, to a very much reduced fascicle with pedicels practically eliminated asin U. fulva, U. scabra, and U. campestris. "Taxonomists state, usually, that the flowers are simple with bell-shaped perianth, which is g- to 4-lobed, imbricated, stamens opposite to, and of the same number as, the lobes, hypogynous, inserted at the base of the perianth; pistil of two carpels, each with a style, one loculus with a pendulous anatropous ovule. The one-whorled perianth of Ulmus can be called neither sepaloid nor petaloid; it is succulent to membranous-scarious with little or no chloro- phyll, possessing stomata equally distributed over its outer surface. Ulmus americana L. has a flower (Pl. XV, fig. 1) which possesses a perianth of 8 lobes, three lateral right and three left, one posterior, and one anterior. Each lobe is accompanied by a stamen. Figure I shows the ir- regular character of the flower. This isa feature usually ignored. Lindley (13) mentions this condition; Britton, in his manual of the northeastern states and Canada describes it as ‘‘calyx oblique.’”’ This zygomorphy is evident in all the elms studied. Flowers sectioned in transverse planes from the pedicel to the distal end show that the vascular supply to the posterior organs usually passes off from the stele before that of the anterior organs. Such series of sections followed through numerous flowers of U. americana reveal the following facts. The pedicel in all species of Ulmus has an ectophloic siphonostele in the form of a more or less continuous cylinder (Pl. XV, fig. 5). The first break in the stele is on the posterior side where a wide trace passes off leaving a gap in the cylinder. As nearly simultaneously with the break as can be appre- ciated, there separates from the inner face of this trace a portion composed of one or two vessels (fig. 6, m, 2). The outer bundle is destined to pass to the posterior perianth lobe, and the inner to the accompanying stamen. Fifty microns above this section (fig. 6), the outer strand divides radially — @ct-, 1621 | BECHTEL — FLORAL ANATOMY OF URTICALES 389 into three strands (fig. 7, m), and fifty microns above the latter section these strands are well away from the stele and a second trace passes off lateral to the first (fig. 8, m!) and behaves exactly like the posterior except for the subsequent branching. ‘Twenty microns above the last section a trace, in appearance like the others, passes out of the stele opposite the second trace (Pl. XV, fig. 9, m?); but twenty microns above this section, as represented in figure 9, the inner portion of this trace appears as a very faintly lignified vessel (figs. 10, 11, m7), similar in origin and position to those supplying stamens with other lobes. This weak strand aborts seventy microns above its origin. It is clearly the supply to a suppressed stamen. The remaining lateral traces pass off right and left (Pl. XV, figs. 11-13) in close succession. The supplies to the anterior lobe and to the two anterior-lateral lobes appear to pass off simultaneously (Pl. XV, figs. 14-15, m®, m®, m’), and all traces sep- arate into staminal and perianth traces coincident with their departure from the stele. This fact is revealed also by longitudinal sections (Pl. XV, fig. 3, d) When the strands to the perianth and to the stamens are definitely dif- ferentiated, such a distinct regional differentiation of tissue arises that the stamen supply is demarked from the adjacent tissues, forming what may be designated as a staminal cylinder (Pl. XVI, figs. 16, 17, c). This cylinder persists until the stamens become free from the perianth. At this level the branching of the perianth strands is usually complete; the posterior and anterior separate into two or three strands; the lateral strands rarely separate or branch (PI. XVI, fig. 18, m, m’). With a knowledge of the gross morphology of the U. americana flower, the above described structure is really what might be expected. However, anatomical study reveals additional abortive bundles present in the anterior half of the flower. These bundles arise immediately within and above the bundles to the stamens and alternate with them. They appear in the same sequence as the bundles to the perianth parts and to the stamens and are evident in transverse sections as soon as the bundles to the stamens are distinctly established in the ‘“‘cortical”’ region (Pl. XV, figs. 11-14, d', d? wed ; fig. 3, d°; fig. 4, d*, d*). These abortive bundles occurring above the bundles to the stamens and below the supply to the carpels show no lignification. They are characterized by cells of small size and by a tissue organization more close than that of the surrounding tissue. The origin of these strands from vascular bundles and the resemblance of the com- ponent cells to those of young or weakly developed bundles render their bundle nature undoubted. The centrally located cells of these demarked regions are very small and often exhibit the appearance of being crushed. They thus suggest the appearance of protoxylem as often seen in mature tissue. These abortive bundles extend upward approximately I50 microns. The question arises, are these the vestigial parts of suppressed stamens? There seems to be no alternative conclusion. The same phenomenon is found also with the strands leading to the carpels higher up in the floral axis (Pl. XVI, fig. 16, e; Pl. XV, fig. 4, e) as discussed below. 390 AMERICAN JOURNAL OF BOTANY [Vol. 8 Whenever the flower of U. americana has an equal number of perianth lobes and stamens, the number is usually 8 (Pl. XVI, fig. 22); occasionally 7, and rarely 9 parts occur in each whorl. In any case the suppression of an organ may occur. Figure 23 shows a flower with 7 perianth lobes and 6 stamens, and figure 24 shows one with 9 lobes and 9 stamens. The lowest number of lobes found in U. americana was 7 and the lowest number of stamens was 5. Small lobes of the perianth frequently exist which are unobserved by means of the hand lens. These occur between and within two main lobes (Pl. XV, fig. 2, a). Anatomically, the vascular supply to this small lobe is a branch from a strand to a main lobe. However, there is a possibility that the strand to a main lobe is a union of two or three strands which originally passed to alternating parts of the perianth, and the branch to the small lobe may be the result of a separation of an aggregate strand rather than a case of true branching. Transverse sections frequently reveal indefinite organization of a perianth strand at its origin which may be due to the passing out together of several strands from the floral axis leaving a single gap. An organized cylinder of vessels continues above the passing off of the traces to the perianth and to the stamens; this first suffers diminution, and then breaks into four strands. This latter change occurs before the stalk of the pistil is isolated from the tissue of the surrounding floral organs (PI. XV, figs. 14, 15; Pl. XVI, figs. 16,17). .Two of these four strands pass up the posterior and anterior edges of the pistil respectively (Pl. XV, fig. 3, 1,1!; Pl. XVI, fig. 17, 1, 1') and are the dorsal carpellary bundles. The two remaining bundles, the lateral strands (Pl. XVI, fig. 17, 0), bear towards the posterior side, approaching each other as they ascend and apparently form- ing one bundle (Pl. XVI, fig. 18, 0 + 0). Serial sections (Pl. XV, fig. 15; Pl. XVI, figs. 16, 17, 0) show that these strands in present development are apparently a continuation of the axis and not branches of the dorsal car- pellary bundles (see discussion, p. 404). Where the pendulous ovule originates this composite strand separates into four strands, one passing into the ovule, one soon vanishing toward the posterior side of the pistil (Pl. XVI, fig. 19, 2, 41; Pl. XV, fig. 3, 2, 21), and two passing upward) ile latter branch again sends bundles into the lateral edges of the two styles (Pl eVi, igs. 19-20,.0) and 07), This separation of the ovule-bearing strand indicates the probability of the former presence of more than one ovule. The branching at the apex supplying both styles (Pl. XV, fig. 3, 0’, 0”) indicates that these are the ~ supply of an axillary placenta tissue. The existing ovule is in the anterior carpel. The aborted bundle (Pl. XVI, fig. 19, z!; Pl. XV, fig. 3, 21) is the remnant of the supply to the ovule that was borne in the posterior carpel and which still is present in some species (Engler, 9; Bentham and Hooker, 2 Baillom, 1): eOct.,, 1927 | BECHTEL —- FLORAL ANATOMY OF URTICALES 391 Ulmus fulva Michx. is described as having perianth lobes and stamens ranging from 9 to 5. However, among the many flowers inspected by the writer, 8 was found to be the highest number and 5 the lowest, with one instance of the latter. The common number is 7 or 6, while in U. americana itis 8or 7. The perianth of U. fulva is more prominently lobed than that of U. americana, but zygomorphy is as conspicuous. The traces to the floral organs pass off from the stele in close succession, but the traces to the stamens pass off higher up and later than do those to the perianth (PI. XVII, fig. 1, 0). U. fulva has abortive bundles similar to those of U. americana, alternating with the perianth and stamen strands. However, in this species these abortive bundles exist as a distinct whorl and not merely in the anterior part of the flower as in U. americana. ‘These are organized tissues in the form of strands (Pl. XVII, fig. 2, d; fig. 1, d), but they possess no appreciable lignification. They persist to a level where the strands to the stamens are definitely isolated. The flower figured shows the anterior-lateral stamen suppressed, but its abortive trace (Pl. XVII, fig. 2, s') can be followed for 30 microns. The vascular supply to the carpels is the same as that of U. americana. Ulmus racemosa Thomas is described as having the perianth lobes and stamens ranging from 8 to 5. All the flowers studied, from one tree only, had perianth lobes 8 to 7 and stamens 8 to 6. The flower tends to be zygomorphic to the same degree as that of U. americana. ‘Traces into the floral organs originate and pass off in the same sequence (PI. XVII, fig. 3, 0; fig. 4). The presence of abortive bundles alternating with the bundles to the stamens and appearing later and above them was limited in this species to one instance (Pl. XVII, fig. 3, d, d'). Here only two such bundles were found. Ulmus campestris L. is described as having the perianth lobes and stamens varying from 5 to 3. In flowers from two trees, the perianth lobes were found to range from 6 to 4, and the stamens from 5 to 4; six lobes in the perianth are rare, and the common relationship is 5 to 4, or 4 to 4. The perianth cut away from the flower and studied under a microscope reveals the fact that the lobes are not the single structures that an observer takes them to be when inspecting the flower macroscopically. The hairy margin of the lobes obscures very small lobes on their sides (Pl. XVII, figs. 6, 7, a, 0). In figures 6 and 7, the lobes 2 and 4 are anterior and posterior respectively; lobes 1 and 3 are lateral. Such is the origin and appearance, however, of these lateral veins a and 0, that they should not be looked upon as branches of the midvein, but rather as veins separating from the vein leading to the main lobe. That is, veins a, 1, and 0 in figures 6 and 7 are the continuation of the traces, which pass out of the stele contiguously and remain in conjunction for a short distance, separating early. These bundles from their behavior (Pl. XVII, figs. 6, 7, a, 1, and 6) may be considered alternating parts of two perianth whorls, which through reduction have 392 AMERICAN JOURNAL OF BOTANY [Vol. 8 become consolidated except in their distal parts. Stronger evidence for the same conclusion is described for U. americana on page 390. The vascular supply to the flower duplicates that of U. fulva (Pl. XVII, figs. I, 2) and of U. scabra (Pl. XVII, figs. 8-10). Commonly one and some- times two perianth lobes have the stamen suppressed even in the vascular supply. Ulmus scabra Mill. is described as having the perianth lobes and stamens ranging from 6 to 5; a study of many flowers reveals that 6 perianth lobes and 6 stamens appear in the majority of cases. The vascular supply to the perianth and to the stamens arises separately (Pl. XVII, fig. 8, 0) as it does in U. fulva and in U. campestris. The strands to the perianth lobes (PI. XVII, fig. 9, m . .. m®) are well out in the “‘cortical’’ regions when the supply to the stamens is just passing out of the stele (fig. 2, 7... mn). U. scabra presents a feature not found in any of the other species. ° Alternating with the traces to the perianth lobes and arising with them are bundles that apparently lag behind (Pl. XVII, figs. 9, 10, x, x1, x?; fig. 8, x, x?; Pl. XVIII, figs. 1, 2, x, x!, x?). These are perianth bundles ime they always continue inside the strands to the perianth lobes. They do not have the number of lignified cells, nor the size of cells that the strands to the perianth lobes possess. They weaken rapidly and vanish on a level with the origin of the lobes of the perianth. Such bundles were not found in the posterior part of the flower (Pl. XVII, fig. 9). The anterior part of the flower of Ulmus is clearly the conservative part of the flower, since in this part the stamens opposite the perianth lobes are always present. Sup- pressed stamens occur in the posterior part of the flower, or here the stamen is present and the perianth lobe is suppressed. Also, the abortive bundles described in the above named species usually occur in the anterior part of the flower except in U. fulva where they make one complete whorl (PI. XVII, fig. 2, d), but the abortive stamen, s!, in the same figure, is in the posterior part of the flower. Thus, as described above, U. scabra presents an additional feature in the anterior part of the flower, namely, the weak bundles to the perianth lobes. These bundles, x, x!, x2, may be vestigial parts of suppressed perianth parts which alternated with the present perianth lobes. The origin, the position, and the appearance of these weak bundles offer no other disposition except that of a vascular supply to corolla parts which have been reduced and consolidated in the gamophyllous perianth. : Alternating with the strands of the perianth, except with the posterior strand, are organized tissue regions as in the other species suggesting bundles, but these show no lignification (Pl. XVII, fig. 9, d). The same condition has been fully discussed above in the other species. Again, there seems to be no other alternative here than to look upon these as abortive bundles to a suppressed outer whorl of stamens. Oct., 1921] BECHTEL — FLORAL ANATOMY OF URTICALES 393 The carpel supply duplicates that of the above named species. Figures g and 10 in Plate XVII and figures 1 and 2 in Plate XVIII are from a flower with three carpels developed. This may be looked upon as an abnormality, yet this extra carpel is a character parallel with the seven aborted bundles (Pl. XVII, fig. 9, d) alternating with the perianth traces instead of the five or three abortive bundles in flowers which have the usual two carpels. This flower has also three abortive bundles in the perianth (rio, figs. 9,-10;. Pl: XVIII, figs. 1, 2, x, x!, x2) which have been considered above as petal traces instead of two or no such traces in the bicarpellate flower. Celtis occidentalis L. reveals a symmetry of 6 or 5; any other rarely occurs. A unique flower was found with seven perianth lobes and five stamens. This was selected to figure on Plate XVIII. The pedicel as it passes into the flower presents a stele that organizes itself into anterior and posterior sections (Pl. XVIII, figs. 4-6). From both of these sections bulky strands pass out, and each of the latter separates into two strands passing to the perianth lobe and to the stamen respectively (fig. 3, 0; figs. 6, 7, m, n). This common origin of the vascular supply of the perianth and stamens and the persistence of this condition for a short distance is a new feature in the Ulmaceae. In addition to this is the unusual origin of the ovule supply. This arises distinctly from the dorsal carpellary strand of the anterior carpel and passes towards the posterior side of the pistil upward into the pendulous ovule (figs. 3, 8-11, 0). The two lateral strands (figs. 9-12, 01, 0”) pass up separately and vanish at the base of the styles. The course of these lateral strands is very different from that of the lateral strands in the Ulmus pistil where the lateral strands approach and join to form the ovule supply (PI. XVI, figs. 17, 18, 0). Also branches from the ovule-bearing strands in Ulmus continue in the inner lateral edges of the styles, but in Celtis there are no branches from the ovule-bearing strand. In this respect the ovule-bearing strand of Celtis is similar to that of the remaining species of the Urticales studied. The lateral strands of the pistil of Ulmus are, therefore, not homologous with those of the pistil of Celtis although they apparently originate in the same manner. In Ulmus they are the placental supply, but in Celtis they may be regarded as abortive, dorsal, carpellary bundles of suppressed carpels. The placental supply in Celtis arises distinctly from the anterior carpel supply. Evidently reduction in the gynoecium of Ulmus has proceeded to a much greater degree than in Celtis. In Ulmus the placental supply has apparently lost connection with the carpel supply and arises from the axis of the flower (see discussion, p. 404). The staminate flower of Celtis possesses an abortive pistil, a miniature of the pistil in the hermaphroditic flower, except that the lateral bundles in the pistil (figs. 9-12, o!, and o? above) are not present. The dorsal carpellary bundles of the abortive pistil continue into the two styles. The posterior style is smaller and less succulent than the anterior style. Here is 394 AMERICAN JOURNAL OF BOTANY [Vol. 8 a consistency in abortion, the posterior carpel being the sterile carpel in the hermaphroditic flower and the more greatly reduced carpel in the staminate flower; and the lateral strands of the pistillate flower (figs. 9-12, o! and 0”), regarded as dorsal strands of abortive carpels, are suppressed in the abortive pistil of the staminate flower. MORACEAE In brief, the plants of this family are woody with small flowers usually in dense clusters, unisexual; the perianth 5- to 4-parted, stamens equal in number with, and opposite to, the parts of the perianth; ovary one-celled with single pendulous ovule, styles 2 or 1. The ovule is “‘basal’’ (1, 9) in a few species. Morus alba L. presents in the pedicel of the pistillate flower a stele of four traces (Pl. XIX, figs. 2, 3). From these four traces pass off in a decussate manner the posterior and anterior traces, followed closely by the lateral (fig. 4, p, p'!). These traces supply the four perianth parts, and in each part the bundle separates into three strands (figs. 4, 5, p, p'). The floral axis above the point of departure of the perianth traces continues as four strands, posterior, anetrior, and two lateral. The anterior and pos- terior strands are the dorsal bundles of the two carpels (Pl. XIX, figs. 4-9, I, 1!) and pass on into the styles. The two lateral strands, as in Ulmus, approach each other as they ascend, unite, and pass upward into the ovule (Pl. XIX, figs. 1, 4-7, 0). In Morus alba these two strands to the ovule do not receive any evident vascular supply from the anterior carpellary strand as figured by Welsford and Benson (22) for M. nigra. The pedicel of the staminate flower (Pl. XIX, fig. 10) shows many strands in the stele which organize into four strands in the base of the flower. From these, four traces pass off decussately and each soon separates into strands to the perianth parts and to the stamens. ‘The dorsal carpellary supplies persist in the abortive pistil of the staminate flower (Pl. XIX,. HOeTO yes) An interesting difference in the vascular supply to the perianth parts of the pistillate and staminate flowers is that there are three traces to each part in the former and only one in the latter. In the pistillate flower, the perianth persists in the fruit as a fleshy organ and calls for a vigorous vascular supply. The staminate flower functions to the time of pollen production and then falls. As a result, the vascular supply to its perianth has degenerated to a single weak strand in each lobe. This is an illustration of what happens frequently in members of the Urticales. The organ de- generates to the extent that the apparent demand for it decreases. Maclura pomtifera (Raf.) Schneider has its pistillate inflorescence in a dense, succulent head, the individual flowers being sessile. A transverse section of the inflorescence axis below the bases of the flowers shows the many pedicellar steles surrounded by a continuous, extremely delicate, Oct., 1021] BECHTEL — FLORAL ANATOMY OF URTICALES 395 parenchymatous tissue. Each stele is composed of four strands. From these strands there pass off in the base of the flower four traces to the perianth (Pl. XIX, figs. 11, 12, p); the remaining four continue into the pistil (figs. 11,12, I, 11,0). The perianth parts become distinct at a level with the ovule (fig. 16), which is not far above the level where the flowers become distinct from each other (fig. 13). ; The perianth parts vary much in the amount of vascular supply. In addition to the one main bundle, there frequently exist in the same inflores- cence flowers having perianth parts with few to many small, weak bundles (Pl. XIX, figs. 14-17, ”). The peculiar feature of these bundles is that they cannot be followed to their origin because of the lack of any organization suggesting bundles in the lower part of the perianth. These small strands are either branches of the main bundle of the perianth part, or, as shown in Ulmus, they are a separation of the strands that are now passing off from the floral axis as a common trace. The presence and abundance of these faint bundles vary according to the crowding of the flowers in the dense capitate inflorescence. The two lateral strands to the pistil (Pl. XIX, fig. 12, 0) approach each other, becoming one strand (figs. 13-16, 0 + 0) which passes to the posterior side upward.into the pendulous ovule (fig. 18, 0). The anterior bundle passes up the anterior side of the pistil to the tip of the single filiform style. This is the bundle to the anterior carpel (figs. 12-18,1). The corresponding bundle passes up three-fourths of the height of the ovary (figs. 12-18, 11), on its posterior side. Comparing the vascular supply of the two carpels of Morus with that of Maclura, the conclusion is that the posterior carpel of Maclura is abortive. This abortion of the carpel and the non-actinomorphic condition of the flower make zygomorphy a feature of the Maclura flower. In the staminate flower four traces from the pedicel separate into strands to the perianth parts and to the stamens. The carpels are sup- pressed, and there are no signs of any vascular tissue in the central portion of the flower (Pl. XIX, fig. 19). Cannabis sativa L. The pedicel of the pistillate flower has four stelar strands (Pl. XX, fig.2). One of these strands (fig. 2, a) passes off anteriorly into the bract which completely envelops the flower (figs. 2-4, br). The three remaining strands pass up into the pistil of the flower. Two strands (Pl. XX, figs. 2, 3,1, 1') which have nearly the same size pass up the dorsal sides of the two carpels to the tips of the two styles respectively (figs. 2-8, 1,1'!). The fourth strand of the pedicel, which is opposite the strand passing into the enveloping bract, is twice the size of any of the other strands (Pl. XX, figs. 2-4, 0). This strand maintains its bulkiness as it passes up posteriorly into the pendulous ovule (figs. 5-7, 0). A transverse section of the flower just below the ovule shows six dis- tinctly lignified bundles in the perianth (Pl. XX, fig. 5, 7m). The appearance of the tissues of the perianth suggests more bundles than those having 306 AMERICAN JOURNAL OF BOTANY [Vol. 8 vessels. None of the bundles, even those with lignified cells in their upper portions, can be followed to their origin in the floral axis. The posterior bundle (fig. 4, m) can be followed down the farthest, that is, into the cortex of the pedicel or receptacle. It does appear that the perianth bundles are abortive in the lowest part of their courses. . Payer (14) and Zinger (24) describe and figure the cup-like perianth as having slightly developed anterior and posterior lobes. Anatomically, the author found no difference in the lobed regions as compared with the remainder of the perianth except the fact that the most prominent bundle to the perianth is the posterior bundle. A feature of the pedicellar stele not yet described is the presence of regions suggestive of bundles (Pl. XX, figs. 3, 4, x). Such a condition described in the preceding species was looked upon as one demonstrating abortive bundles. Such faint bundles and others not recognizable may pass into the perianth and become lignified in their upper parts only, a condition such that they can be followed. Also in the upper lateral ovary wall there are faint bundles with delicate, lignified cells (Pl. XX, fig. 5, 7) which cannot be followed to their origin. These must be either branches of the dorsal carpellary bundles or strands continuing from the pedicel. If the latter, they arise similarly to the two dorsal carpellary bundles and therefore suggest abortive carpellary bundles to suppressed carpels. The stele in the pedicel of the staminate flower is very different. It has many small strands which organize into five strands in the base of the flower. These pass out of the axis and each separates immediately into strands to the perianth and to the stamens. There are no signs of abortive strands to the suppressed carpels in the writer’s experience. Likewise in the pistillate flower, the stamens are suppressed and no vestiges of vascular supply are present. Pritchard (16) concludes from his experiments on the hemp plant that both the male and the female flowers are potentially hermaphroditic and that the unisexual condition is the result not of different zygotic constitu- tion, but of the lack of food supply. At what time in the life of the hemp plant the suggested feeding must be begun in order to establish organs that are suppressed, even in vascular supply, is an interesting problem to a plant anatomist. It may indicate that the unisexual nature of the hemp flower is not well established. Some of its congeners in the order still have bisexual flowers. Humulus Lupulus L. The pedicel of the pistillate flower duplicates in structure that of Cannabis except that it has fewer vessels in each of its four bundles. Anteriorly, a large trace passes out and branches profusely in a large bract which envelops the flower (Pl. XX, fig. 9, br; fig. 10, a). The three remaining strands in the pedicel, as in Cannabis, pass into the pistil. Strands (figs. 14-18, 1, 1!) pass up the dorsal sides of the two carpels into the styles respectively. These are the dorsal carpellary bundles. The Oct. 1921 | BECHTEL —— FLORAL ANATOMY OF URTICALES 397 remaining strand (figs. 9, 14-16, 0) passes up posteriorly into the pendulous ovule. The pedicel of Humulus, also, possesses in its upper portion defi- nitely organized tissues suggesting bundles (figs. 10, 11, x). One of these suggestive regions does possess a faintly lignified vessel (fig. 10, x) which must eliminate all doubt of its being a bundle. The perianth of Humulus is very similar to that of Cannabis sativa Transverse sections through the upper part of the perianth reveal many bundles (Pl. XX, fig. 16, »), varying in number from 10 to 14. These bundles cannot be traced to an origin in the pedicellar stele, but they can be followed passing into the cortex. Figures 12, 13, 14, and 15 show the traces to the perianth numbered in the order in which they become distin- guishable, e.g., figs. 12, 13, I', 17, 13. Because of the delicate cell walls, it cannot be definitely said whether there are three, five, or more original traces leading into the perianth. However, it is evident that several traces to the perianth originate at one point in the stele, or that the traces separate after they passed out as one trace (figs. 13-15). Since the perianth strands are difficult to trace owing to their delicacy, we conclude, as for Cannabis, that the basal portions are abortive. In Humulus, as in Cannabis, the abortive bundles in the pedicel may continue into the perianth and show lignification only in their upper por- tions. A fact supporting this conclusion is that these abortive bundles appear after the strand to the bract is oriented and before the appearance of the remaining strands that pass into the pistil. In all other species studied in the Urticales, the perianth bundles pass off first or lowest on the floral axis, and this is the position of the abortive bundles of Humulus and Cannabis. URTICACEAE This family of the Urticaceae contains perennial or annual herbs with very small, greenish flowers, monoecious, dioecious, or polygamous; perianth parts 5 to 2, distinct, cleft, or tubular; stamens of the same number and opposite to the perianth parts; ovary with one cell, one ‘“orthotropous”’ ovule; styles usually capitate and sessile. Urtica gracilis Ait. is figured in Plate XXI. The vascular supply in the minute pedicel of the pistillate flower appears as one strand (fig. 2) which gives off two decussate pairs of bundles which pass to the four perianth parts (figs. 3-12, m, n). ‘The remaining vascular tissue continues as four strands into the pistil. The posterior strand passes up into the sessile stigma. This strand is the dorsal carpellary bundle of the posterior carpel. The anterior strand passes up three fourths of the height of the ovary and cannot be followed further. The two lateral strands approach each other as they ascend and enter the funiculus of the basal ovule as one strand (figs. I, 5-II, 0 +0), as was found in Ulmus, Morus, and Maclura. In interpreting the vascular supply of this pistil similarly to that of Ulmus and others previously described, the conclusion is that two carpels are present 398 AMERICAN JOURNAL OF BOTANY [Vol. 8 but that the anterior carpel is partially abortive. However, Urtica has been looked upon as being ‘‘unicarpellary”’ (Baillon, 1; Bessey, 3). Transverse sections of the Urtica flower indicate zygomorphy: the two lateral perianth parts are alike; but the posterior is larger than the anterior and is the last to become distinct from the shallow perianth tube (figs. 1, 10-13, m); the pistil does not stand in the middle of the flower. Boehmeria cylindrica L. (Sw.) possesses a tubular flower with the vascular supply of the perianth confined to the anterior and posterior sides (Pl. XXII, figs. 1, 3-7, m). This fact indicates that the lateral perianth parts have been consolidated with the anterior and posterior parts and that their vascular supplies have completely degenerated. This flower shows the zygomorphic features of the Urticales. Only two strands pass into the pistil instead of four as in Urtica gracilis. These two strands arise from one strand (Pl. XXII, figs. 3, 4, 1! and o) in the basal portion of the flower. The posterior strand passes up the posterior side of the pistil into the short filiform style and is the dorsal carpellary bundle. The other strand ascends anteriorly for a short distance and then sharply curves towards the posterior; after passing horizontally in this direction for a short distance it abruptly ascends into the “basal’’ ovule (figs. I, 5,0). The anterior side of the pistil has no vascular supply. Here undoubtedly the anterior carpel is suppressed. Therefore, Boehmeria cylindrica has reached that stage in reduction having only two perianth parts, one carpel, and a “‘basal”’ ovule. The ovule from the path of its bundle (fig. 1, 0) indicates that its apparent orthotropous nature has become such by a sinking down to a basal position from a pendulous or lateral position. In the base of the staminate flower, the strands of the pedicel conjoin (Pl. XXII, figs. 9, 10, 11) and strands then pass off to the perianth and to the stamens (figs. 9, 12, m, s). The staminate flowers have an abortive pistil which possesses a weak vascular supply (figs. 9, 12, I, 1). Laportea canadensis (L.) Gaud. has a flower that is decidedly zygo- morphic. The floral structures vary from those described above. The anterior perianth part is large, the posterior is very small, and the lateral parts are alike. From a cylindrical stele (Pl. XXII, fig. 15) of the tiny pedicel a strand passes anteriorly into the anterior perianth part (figs. 14, 16, m). No strand corresponding to the anterior perianth strand passes off into the small posterior perianth part (figs. 14, 16, m!). Two strands pass off laterally to the lateral perianth parts. Evidently the decussate arrange- ment of the perianth supply as exhibited in Urtica gracilis is broken in Laportea canadensis through the suppression of the posterior perianth trace, although a very small perianth part is still present (figs. 14-19, m1). Above the origin of the perianth bundles only two bundles continue, and these in an anterior-posterior plane. The posterior bundle passes up the posterior side of the pistil into the single style (Pl. X XII, figs. 14, 16-21, @Mct., 1921] BECHTEL — FLORAL ANATOMY OF URTICALES 399 11). The anterior bundle (figs. 14, 16, 17,0 + 1) continues for some distance and then separates into two unequal strands, one passing into the anterior side of the pistil and the other, the larger, passing into the ovule (figs. 14, 18-20, 0,1). The former strand soon vanishes in the lower third of the ovary wall (fig. 14,1). This is evidence of an abortive anterior carpel. The ovule and its vascular supply again offer opportunity for specula- tion. After the anterior carpel strand separates from the single anterior strand, the main supply passes horizontally in an ascending-posterior direc- tion through a long stocky funiculus into the ovule (figs. 14, 18-20, f, 0). The position of the ovule suggests the reduction of an axillary placenta which bore ovules in a pendulous position. The ovule is past the midway stage between that of a pendulous ovule as in Ulmaceae and Moraceae and that of a basal ovule as in Boehmeria cylindrica. In fact, very little reduction in funicular tissue in Laportea canadensis is necessary to duplicate in position that of the ovule of Boehmeria (compare Pl. XXII, figs. 1 and 14). The same line of reasoning is suggested upon comparing the ovule supply of Boehmeria cylindrica and Urtica gracilis (Pl, X XI, fig. 1; Pl. XXII, fig. 1), namely: the bundle in its indirect route to the basal ovule of Boehmeria would require little reduction to duplicate the direct supply to the basal ovule of Urtica. DISCUSSION Ulmaceae. The gross floral morphology of the six species and the detailed anatomy underlying it have been presented above. The latter reveals features which warrant the disuse of the descriptive term ‘‘simple’”’ for the flowers: five sets of organs or vestiges of organs; variableness in number of parts in a whorl; zygomorphy, which is constant; and the fusion of like and unlike parts. These present a decidedly complex condition. The flowers of the genus Ulmus have a perianth ‘“‘cup”’ upon the edge of which the perianth parts and stamens have been considered perigynously inserted. Baillon (1) considered this to be the condition. Anatomical work reveals that this is not the case for the following reasons. First, the vascular supply to the stamens and to the perianth parts arises separately from the stele of the pedicel; the former passes off from the floral axis con- siderably above that to the latter in U. fulva, U. campestris, and U. scabra (Pl. XVII, figs. 1, 5, 8, 0), but approximately closely in U. americana and U. racemosa (Pl. XV, fig. 3; Pl. XVII, fig. 3, 5). Second, the tissues em- bodying the perianth and stamen traces through the perianth ‘“‘cup”’ are separable by the distinct difference in cellular structure and by a line of demarcation. These differences in the parenchymatous tissue are col- lateral and continue into the perianth lobe and stamen respectively. The line of demarcation indicates an adnation of the tissues of the perianth lobe and stamen. Here is good evidence that the ‘‘cup”’ is the fused bases of floral envelopes and stamens. Third, the lobes of the perianth are variable in length; their size is not constant, which is a character not un- 400 AMERICAN JOURNAL OF BOTANY [Vol. 8 common in hypogynous flowers. The last is among the characters used by Planchon (15) in distributing the 16 species of Ulmus into three divisions. Celtis has perianth parts distinct to the base or nearly so. The remain- ing genera of the Ulmaceae, eleven in number, have perianth parts similar to those of Celtis (2). In the Ulmaceae the receptacle is limited, then, to the pedicel of the flower, and in Ulmus, coalescence and adnation have taken place in the perianth parts and stamens. Also, the fact that the single whorl of normal vascular bundles to the perianth and to the stamens, respectively, is accompanied by whorls of abortive bundles which alternate apparently with these, enforces the conclusion that along with the coales- cence and adnation, there has been reduction in these two sets of organs. This reduction consists of the loss of an inner whorl in each. Reduction occurs not only in the number of whorls but also in the number of organs within a whorl. No constant number exists in the floral whorls of any of the elms. Greater variation occurs in those species having the greatest number of organs present per whorl; e.g., in U. americana, as described on page 390, there are 9 to 7 perianth lobes and 9 to 5 stamens. The cause of the lack of floral symmetry in a species is due to the development of a perianth lobe without its accompanying stamen. This is usually the stamen to one or the other of the posterior-lateral lobes. However, just as often, a stamen develops without an accompanying perianth lobe. The number ranges from 9 in Ulmus americana to 4 in U. campestris, and is more or less inconstant in all species. On the basis of inflorescence (which shows in Ulmus stages in reduction), the species with more floral parts are more primitive than those with fewer. Although the gynoecium of Ulmaceae is dimerous, from the presence of abortive strands to suppressed carpels it has suffered reduc- tion. Such organized tissue regions suggesting bundles were discovered in Ulmus americana. These bundles appear some distance above those to the stamens, on a level from which the strands to the carpels can be followed. Ulmus possesses spirally arranged parts (Pl. XV, figs. 7-14), though the other genera studied are cyclic. The spiral arrangement is most conspicuous in the species with the greatest numbers of stamens and perianth parts and becomes less conspicuous in those elms in which the floral characters grade into those of the Moraceae which are tetramerous and cylic. ' The spiral arrangement is an important phyletic character, but by reduction in the number of organs and in the floral axis, it has become nearly obscure. The alterations in the posterior part of the flower over those of the anterior part by modification in the relation of organs to each other, and by the suppression of organs, form a true zygomorphy. This character is perhaps the result of aggregation (23), and possibly an adaptation to insect visitation. ‘To be sure, very few species in the Urticales are known to be visited by insects, yet zygomorphism may be a character persisting from an earlier time when insect visitation was the common occurrence. There is a possibility that zygomorphism as a specialized character and as a character Oct., 1921] BECHTEL — FLORAL ANATOMY OF URTICALES 401 particularly adapted to insect visitation has been over-emphasized. Evi- dence has been presented that the angiospermous prototype (Robertson, 17) was entomophilous and that the anemophilous condition has been recently acquired. A character that in many instances accompanies the entomophilous flower is the multiovulate condition. The vascular supply of the placenta of Ulmus, the flowers of which are least reduced of those genera studied, indicates that whereas but one ovule is now borne, a multi- ovulate condition probably existed formerly. The characters gamophylly, zygomorphy, Poke ee Sere ovary, vestigial organs, indicate certain specialization and a high flower type. However, it is only in Ulmus that the gamophyllous character exists. The gamophylly of Ulmus is to be considered an isolated instance of this tendency in the Polypetalae. Finally, from the evidence gathered, the Ulmaceae are primitive forms but with many advanced characters. They should be con- sidered highly reduced and specialized forms among primitive groups. Moraceae. ‘The flowers of Morus and Maclura are anatomically alike, although the former has a pistil with two styles and the latter a pistil with one style. Anatomy reveals two carpels in each case. This reduction in the gynoecium of Maclura is no doubt a feature accompanying the dense inflorescence. For the same reason the common variation in the size and venation of its perianth parts, as previously described, occurs. The three- veined character of the perianth parts is constant in the Morus pistillate flower but not constant in that of Maclura. This suggests a palmate ve- nation which corresponds to the venation of the foliage leaves. The leaves of Morus, as in many related genera, have three basal veins, and when the foliage leaves are large they have three lobes. According to Sinnott and Bailey (19), palmate venation is the primitive type in the angiosperms, and where it occurs in the floral parts only, as it does inconstantly in Maclura, it is a ‘‘persistence of an ancient character which has been lost elsewhere.’ This sign of primitiveness is conspicuous also in the perianth parts of Ulmus (p. 388). The anterior and posterior perianth parts have three veins as a nearly constant character. The midvein departs first, and soon the lateral veins separate from it. Humulus and Cannabis form a type distinct from Morus and Maclura. The very delicate gamophyllous perianth in the pistillate flowers (p. 396) has been produced undoubtedly by the large persisting bract which envelops these flowers. Since the vascular supply is evident only in the upper part of the perianth, it is an indication that the perianth is in the process of dis- appearing. As the venation in the perianth parts of Ulmus and Morus was interpreted by referring to the venation of the foliage of the same, the perianth of Humulus and Cannabis can be so interpreted. The leaves of these two species have palmate venation and both are multi-digitately veined. Thus the many small veins of the perianth of Humulus and Can- 402 AMERICAN JOURNAL OF BOTANY [Vol. 8 nabis, the origin of which cannot be determined in the gamophyllous perianth cup, may be considered veins of several digitately veined perianth parts. They attain a weak development due to reduction. The floral envelopes have become reduced and delicate with the development of a large protecting bract. ‘The differences in the ovule supply in these genera will be elaborated upon in the general discussion (p. 404). Thus, the flowers of the Moraceae as compared with those of the Ulma- ceae have been more greatly reduced in floral axis, as described earlier in this paper, in perianth lobes, and in their vascular supply, as seen in Maclura, Humulus, and Cannabis, and in the gynoecium as illustrated in Maclura. Urticaceae. The study of three species of this family indicates the presence of that reduction which is found in its earlier stages in Moraceae, namely, the suppression of one of two carpels. In Urtica and in Laportea the anterior carpel is represented only by abortive bundles; in Boehmeria there is no trace of a bundle in this carpel. Also the irregularity of the perianth parts is slight in Urtica; it is greater in Laportea to the degree that the posterior perianth part has nearly disappeared, and has no vascular supply. In Boehmeria, an anatomical study of the gamophyllous perianth reveals two perianth parts only. There is no evidence of lobes indicating lateral perianth parts, nor bundle supply to such parts. The “orthotro- pous”’ ovule supply, as has been presented on page 398, gives evidence by its peculiar course of a change of position of the ovule from a pendulous or lateral to a basal position. The irregularity of the shape and size of the perianth parts, the number of parts, ranging from four to two, the “‘basal”’ ovule supplied by a bundle taking an ascending and then a descend- ing course, indicate a reduction in this family beyond that found in the Moraceae. Along with the floral reduction in the Urticaceae goes the herbaceous perennial or annual plant habit which character phyletically (7, 18) is in keeping with that of the flower. GENERAL DISCUSSION The Urticales present an anomalous combination of characters. These on one hand indicate primitiveness and on the other specialization. Many and indefinite organs, non-cyclic condition, preponderance of woody forms, and palmate venation, still evident in the perianth parts if not in foliage leaves, point to primitiveness. Aggregation of flowers, fusion of parts, zygomorphy, and reduction point to specialization. Therefore, they must be considered at least not highly advanced forms, though they possess a number of very advanced features. Almost any group of angiosperms possesses one or more of the characters indicating high rank. The presence of several such characters in the Urticales is not an indication of particular Oct~, 1921] BECHTEL ——- FLORAL ANATOMY OF URTICALES 403 advance over other groups. Nor is the presence of zygomorphism, for example, an indication of relationship with another group in which the same feature is present. That the Urticales are related to one of those plexuses of the angiosperms possessing types of zygomorphism, namely: that cul- minating in the Monocotyledons, that of the Rosales in the Polypetalae, and that of the Tubiflorae in the Sympetalae, can receive no support. It does seern. that a relationship more nearly correct may be discovered for the Urticales by considering the characters possessed by them that indicate primitiveness rather than those that indicate specialization, namely: many organs, non-cyclic conditions, and preponderance of woody forms. The one order of the angiosperms possessing these characters is the Ranales. The Ranales have not suffered reduction to any degree comparable with that of the Urticales. Floral anatomy of the members of the families of the Ranales may reveal important characters that macroscopic study cannot reach. However, the Urticales appear, when viewed from the standpoint of their primitive characters, to be parallel with the Ranales. The latter possesses a tendency to the pentamerous condition, and both orders possess a tendency to an unicarpellate condition. That the Urticales and Ranales are descendants from the same protoangiospermous plexus seems likely. But, since the flowers of the Urticales are greatly reduced in each set of organs, as the floral anatomy described above indicates, the Urticales are on a higher level than the Ranales. A feature of the Urticales that has caused them to be looked upon as very primitive plants among the angiosperms is the “‘orthotropous”’ ovule. This type of ovule has been regarded as the most primitive since it is apparently the common type appearing in those families classified as lowest in the Polypetalae. In the most highly reduced members of the Urticales, numbering about half the species of the order, the ovule is “‘basal”’ or “orthotropous.’’ Anatomical work reveals, however, that the ovule has become basal, as previously described, by a sinking or a sliding down from a pendulous position and that in this process the anatropous ovule has become erect. Thus the Urticaceae show a phyletic origin of the orthotropous ovule from an anatropous, pendulous, or lateral type, as Welsford and Benson (22) consider is the case in Juglans regia and related plants, basing their evidence also on anatomical study. The orthotropous ovule in this group, therefore, is not primitive. The floral anatomy of Bentham’s Incompletae (15 orders), in which the ovule, with few exceptions, is basal, is an inviting line of research. In determining the phyletic relationship of the Urticales, therefore, it is the pendulous or lateral anatropous ovule that must be con- sidered and not the erect basal ovule. A consideration of the vascular supply to the ovule, as described in the species studied, may indicate that the Urticales are not a natural order. Three types of vascular supply to the ovule were found, but these are all the results of the greatly reduced condition of the flowers. The common 404. AMERICAN JOURNAL OF BOTANY [Vol. 8 type, as found in Ulmus, Morus, Maclura, and Urtica, representatives of the three families of the order, is an ovule supply which is the result of the fusion of two lateral strands from the floral axis. The second type is found in Celtis and in Laportea, where the ovule supply is a branch from the anterior carpellary strand. The third type is in Humulus and in Cannabis, where the ovule supply is a continuation of a single strand from the pedicel. The first two suggest a foliar origin for the ovules. The two lateral strands passing to the ovule are two lateral basal veins arising with the midvein of the ar terior carpel. This condition strongly suggests the carpel to be a foliar organ with palmate venation. Passibly the ancestral condi- tion was that of a carpel with several ovules, two at least, one borne on each of these basal lateral veins; but through coalescence and reduction the two veins conjoined and one ovule was crowded out. It is likely that other lateral veins of the dorsal carpellary bundle above the two existing bearing ovules have disappeared through the same processes. The same thing seems to have happened in the posterior carpel of Ulmus. An abortive ovulary branch of the placental strand, in which are incorporated the abortive lateral strands (basal veins) in the anterior and posterior carpels, is present just opposite the branch passing into the ovule (Pl. XVI, fig. 19, a!; Pl. I, fig. 3, 74). The ovule belonging with this strand is occasionally present in Ulmus and in Morus (Baillon, 1; Engler, 9). The strand leading to the ovule in Humulus and in Cannabis arises deep in the pedicel. It is posterior and opposite to the strand that passes into the enveloping bract (Pl. XX, fig. 2, 0, a), and is the largest of the four strands in the pedicel. The single pedicellar strand to the ovule and the phenomenon present there are due undoubtedly to the greatly reduced state of the flowers, described previously (p. 402), which has altered the ovule supply to a single strand. In the anterior carpel the lateral carpellary veins have disappeared and the midrib is small, undoubted!v because of the development of the large bract. The same bundles in the posterior carpel have fused into one strand passing to the single ovule. The ovule and the ovule supply, therefore, indicate a natural order for the Ulmaceae, Moraceae, and Urticaceae. When the ovule of the Urticales is taken into consideration to determine the likely relationship of the order, the type of ovule as found in Ulmaceae must be used. That type is the anatropous, pendulous, or lateral ovule, which is the primitive type in the Urticales. The partial basal or basal- erect ovules are the result of reduction as the comparative anatomical studies previously described indicate. The accepted relationship of these three families on the part of taxono- mists is supported by this studyof floral anatomy. The Urticaceae are higher than the Moraceae, 7.e., they are more reduced in carpels and in perianth. The Moraceae are higher than the Ulmaceae, i.e., they are more reduced in number of stamens and in perianth parts. Also, the generic relationships are indicated by this anatomical study. In the Urticaceae, Laportea and @ct.,/ 1921 | BECHTEL — FLORAL ANATOMY OF URTICALES 405 Boehmeria are higher than Urtica, and Boehmeria is higher than Laportea. _In Moraceae, Maclura is higher than Morus; and in the Ulmaceae, Celtis is higher than Ulmus. In the genus Ulmus the result of these anatomical studies places species in the same groups in which they have been placed by taxonomists. U. americana and U. racemosa come in one group, and U. fulva, U. scabra, and U. campestris come together in another group. The natural position of the Urticales has been a debated subject. The common practice has been to place them in association with the Amentiferae. Jussieu, de Candolle, Endlicher, Bentham, Hooker, Engler, and Gray have assisted in establishing this arrangement. The Amentiferae, however, are coming to be looked upon as reduced rather than as primitive forms. Weddell (21), in 1840, associated the Urticales with Tiliaceae and Malvaceae, etc. One of the features that influenced him in making such a decision was the presence of “‘bast fibers’’; but on the same feature, a relationship can be established with Thymeliales, which possesses ‘several similar floral structures. Lindley (13), in 1845, placed the Ulmaceae singly in the Rhamnales. Bessey (3) and Hallier (12), in 1905, placed the Ulmaceae, Moraceae, and Urticaceae in the Malvales, as Weddell had done sixty years before. The last suggestion has received much favorable consideration from many taxonomists. The writer’s anatomical studies in these suggested affinitives have not progressed far enough to warrant any conclusive statement. The floral anatomy of the species of Ulmus reveals a feature that should be discussed at this time, namely: the staminal cylinder as described on page 389 (PI. XVI, figs. 16, 17,c). This may be considered homologous with the staminal tube of the Malvaceae. Yet, the cohesion of filaments is a character occurring in the Parietales, Geraniales, and in other small groups, and is a striking character in the Papilionaceae. The Malvales, as delimited by Engler (9), show the tendency to chorisis. Reduction, which is opposed to chorisis, is conspicuous in the Ulmaceae, Moraceae, and Urticaceae. However, it may be possible to accept a natural order exhibiting two such diverse processes. There is the danger of placing the Urticales higher than they should be, due to the greatly reduced flower condition; the caution from Engler (10) in this regard has already been stated. Such an error can possibly be avoided by considering the characters of the order that indicate primitive- ness, namely, many organs, non-cyclic condition, and preponderance of woody forms. On the other hand, zygomorphism and reduction are present in the order not as tendencies but as critical characters, 7.e., the characters present throughout the order. Therefore, these tendencies must be present in their nearest relatives, or were present in their immediate ancestors. It is doubtful that their ancestors were wind-pollinated. The progenitors of the Urticales are not in existence today. Considering their primitive characters, they are in a distinct line of descent from a protoangiospermous 4.06 AMERICAN JOURNAL OF BOTANY [Vol. 8 plexus from which also descended the Ranalian line. The Urticales have advanced parallel with the Ranalian stock to a high degree of specialization, namely, zygomorphy. Accompanying this specialization, or following it, the Urticales show great reduction in all parts of the flower. The result has been a group of plants combining characters belonging to primitive and to recent types, a combination which makes them a generalized rather than a specialized group from which no descendants seem to hive arisen. SUMMARY AND CONCLUSIONS 1. The anatomy of the flowers of the Urticales reveals a number of features extending throughout the order, which are not appreciable from a macroscopic investigation. | a. Ulmus, the primitive genus, shows evidence of suppression of a whorl of stamens and of one of perianth parts. The existing stamens are fused with the gamophyllous perianth. The parts of these whorls are somewhat spiral in arrangement and very inconstant in number. b. The bicarpellate condition has been derived from a polycarpellate condition as evidenced by the presence of vascular supply to suppressed carpels. Also, vestigial bundles indicate that the bicarpellate gynoe- cium is becoming unicarpellate by the suppression of one carpel. c. The perianth parts are reduced in number by abortion, suppres- sion, and fusion; in some cases the inner whorl has entirely disappeared, in others vestiges of its vascular supply remain. In some forms the inner and outer whorls are fused and occur as one whorl. d. Zygomorphy is a conspicuous character of all species studied; evidence of it is not only found externally but appears also on micro- scopic study of transverse sections of the flowers. e. Palmate venation, if no longer present in the foliage, is still present in the perianth parts in some forms. f. The ovules are foliar organs. The orthotropous ovule in the higher members of the order has come to its basal, erect position by a sinking from an apical or lateral position of the anatropous ovule in the primitive members. The ‘‘cauline’’ ovule in the Urticales is apparently such due to reduction. All ‘“‘cauline”’ ovules may possibly be simply the result of the same process. g. The vascular supply to the uniovulate ovary suggests a poly- ovulate ancestry. h. Accompanying coalescence and adnation, the flowers have been greatly reduced in all floral organs. 2. In plant organs suffering reduction the vascular system disappears in advance of the organs, or persists as abortive bundles after the organs have disappeared. 3. The combination of primitive and specialized characters makes the Urticales a generalized group. Oct., 1921] BECHTEL —- FLORAL ANATOMY OF URTICALES 407 4. The Urticales are probably not far removed from primitive ento- mophilous ancestors. | 5. Floral anatomy emphasizes the idea that the Urticales are a natural order which is made up of three natural families as classified by Engler. 6. The natural position of the Ulmaceae, Moraceae, and Urticaceae is at the culmination of a distinct line of descent from a protoangiospermous plexus from which also the Ranalian line descended. The writer wishes to express his gratitude to Dr. A. J. Eames, to whom he is indebted for this problem as well as for advice and assistance while the work was in progress. DEPARTMENT OF BoTANY, COLLEGE OF AGRICULTURE, CORNELL UNIVERSITY, ETHACA, "Ni Y. LITERATURE CITED . Baillon, H. Histoire des plantes. 7 vols. Paris, 1866-1880. . Bentham, G., and Hooker, J. D. Genera plantarum. 3 vols. London, 1862-1883. . Bessey, C. E. The phylogenetic taxonomy of flowering plants. Ann. Mo. Bot. Gard. 2: I0g-164. I9QI5. 4. Bower, F.O. The origin of a land flora. London, 1908. 5. Darwin, C. On the various contrivances by which British and foreign orchids are fertilized by insects, and on the good effects of intercrossing. London, 1862. 6. de Candolle, A. P. Théorie élementaire de botanique. Paris, 1813. 7. Eames, A. J. On the origin of the herbaceous type in the angiosperms. Annals of Bot. 25: 215-224.. I9gIl. 8. Eichler, A. W. Bliithendiagramme. Vol. 2. Leipzig, 1878. 9g. Engler, A. Die nattirlichen Pflanzenfamilien. Leipzig, 1897-1915. 10. Engler, A., and Gilg, E. Syllabus der Pflanzenfamilien. 7th ed. Berlin, 1912. 11. Gérard, R. Sur l’homologie et le diagramme des Orchidées. Ann. Sci. Nat. Bot. VI, 8: 213-247. 1879. 12. Hallier, H. Provisional scheme of the natural (phylogenetic) system of flowering plants. New Phytol. 4: 151-162. 1905. 13. Lindley, J. The vegetable kingdom. London, 1853. 14. Payer, J.B. Traité d’organogénie végétale comparée de la fleur. Paris, 1857. 15. Planchon, J. E. Ulmaceae. In de Candolle’s “ Prodromus’’ 17: 151-210. 1873. 16. Pritchard, F. J. Change of sex in hemp. Jour. Hered. 7: 325-329. 10916. 17. Robertson, C. The structure of the flowers and the mode of pollination of the primi- tive angiosperms. Bot. Gaz. 37: 294-298. 1904. 18. Sinnott, E. W., and Bailey, I. W. The origin and dispersal of herbaceous angiosperms. Annals of Bot. 28: 547-600. 1914. 19. Sinnott, E. W., and Bailey, I. W. Foliar evidence as to the ancestry and early climatic environment of the angiosperms. Amer. Jour. Bot. 2: I-22. I9g15. 20. Van Tieghem, P. Recherches sur la structure du pistil. Ann. Sci. Nat. Bot. V, 9: 153- 226. 1868. 21. Weddell, H. A. Urticaceae. In de Candolle’s ‘ Prodromus”’ 16: 32—235%. 1869. 22. Welsford, E. J., and Benson, M. The morphology of the ovule and female flower of Juglans regia and of a few allied genera. Annals of Bot. 23: 623-633. I9g09. G& NH 4&4 408 AMERICAN JOURNAL OF BOTANY [Vol. 8 23. Wernham, H. F. Floral evolution: with particular reference ta the sympetalous dicotyledons. IX. New Phytol. 11: 373-397. 1912. 24. Zinger, N. Beitrage zur Kenntniss der weiblichen Bliithen und Inflorescenzen bei Cannabineen. Flora 85: 189-253. 1898. EXPLANATION OF PLATES The figures in the plates are greatly enlarged. The {lower sections range from 0.5 mm. to 3 mmi. in diameter, or 2 mm. x 5 mm. in dimension. [eseINGPIEN >. AVE Ulmus americana Fic. 1. Habit sketch of flower with 8 perianth lobes and 8 stamens. Fic. 2. Portion of perianth showing small inner lobe (a) and vascular supply to lobes. Fic. 3. Longitudinal section of flower in median posterior-anterior plane. Origin of vascular supply (b) to perianth (p) and to stamens (s); I, bundle to anterior carpel; 1, bundle to posterior carpel; ov, to placenta; 7, to ovule in anterior carpel; 2, the abortive bundle to suppressed ovule in posterior carpel; o! and o?, placental branches passing through the inner faces of the styles; d°, abortive bundle alternating with those to the stamens. Fic. 4. Longitudinal section of flower perpendicular to the plane of that in figure 3; d?, d*, abortive bundles alternating with stamen bundles; e, abortive bundle in the carpel supply. Fic. 5. Transverse section through pedicel; st, the stele. Fics. 6-15. Transverse sections through a flower, 40, 50, 30, and (figs. 9-15) 20 microns apart respectively. Posterior trace passes off and separates into traces to perianth lobe (m) and stamen (s). Other traces pass off successively toward the anterior side, m!, 71, . . . m’, n', and each separates as does the posterior trace. Stamen trace n? aborts 70 microns above its origin; d! . . . d°, abortive bundles of vascular supply continuing above and alternating with that to the stamens; c, staminal cylinder; ¢, traces continuing into the pistil. PEATE. DOV Fics. 16-18. Transverse sections of flower; m...m’ and2... mn’, bundles to perianth and stamens respectively. The posterior (m) and anterior (m’) bundles separate each into 3 strands; I,to posterior, I!, to anterior carpel; strands 0 come together making o + 0 of the placental vascular system. Fics. 19-21. Transverse sections through upper part of pistil; 1 and 11, dorsal bundles to carpels; 72, to ovule; 7, abortive bundle corresponding to 2; o! and o?, inner lateral bundles of styles. Fic. 22. Section through a flower at level of stalk of pistil, an 8-merous flower. Fic. 23. Section through a flower with 7 perianth lobes and 6 stamens. Fic. 24. Section through a 9-merous flower. : Fic. 25. Section through a flower having a stamen (xk) with no accompanying peri- anth lobe. PLATE XVII Ulmus fulva Fic. 1. Median longitudinal posterior-anterior section; supply to stamens originates some distance (6) above that to perianth; o, the placental strand which branches like that in U. americana (Pl. XV, fig. 3); d, abortive bundles. Fic. 2. Transverse section through lower part of flower; , perianth; s, stamens; d, abortive strands; s', abortive (lignified) strand to suppressed stamen; ¢, continuation of floral axis above the stamen supply. Ulmus racemosa Fic. 3. Median longitudinal posterior-anterior section of flower; 0, origin of perianth (p) and stamen (s) strands; 0, placental supply. O€t.,, 1921] BECHTEL — FLORAL ANATOMY OF URTICALES 409 Fic. 4. Transverse section through lower part of flower; m...m’andun... n’, strands to perianth and to stamens respectively; d and d', abortive bundles. Ulmus campestris Fic. 5. Median longitudinal posterior-anterior section; bundles to stamens (s) originate above (0) those to perianth (p). Fics. 6, 7. Various lobing of perianth; I... 4 are strands to main lobes; a and 8, branches or separations from the main strands; s, stamen position. Ulmus scabra Fic. 8. Longitudinal lateral section of flower; }, space between origin of stamen and perianth bundles; «x, x?, abortive bundles of the perianth; d, abortive bundles above those to stamens; 0, the placental supply. Fics. 9, 10. Transverse section of flower; m...m anda... n*, bundles to perianth lobes and stamens respectively; x, x!, x2, abortive bundles of perianth; d, abortive bundles alternating with stamen bundles: ¢#, continuation of floral axis; 1, 1', 12, dorsal bundles of 3 carpels; o, the placental supply. Prats X VET Fics. 1, 2. Transverse sections above those of figures 9 and 10, Plate XVII; letter- ing the same. Perianth bundles separate into 2 to 4 strands. Celtis occidentalis Fic. 3. - Longitudinal section of flower; 6, trace from stele of pedicel which separates into perianth (p) and stamen (s) strands; 0, placental supply arises or separates from anterior carpel (1!) supply. Fics. 4-12. Transverse sections of flowers; in lower part of flower, stele is prominent in anterior and posterior regions; strands to anterior part of flower lead off, perianth (m) and stamen (”); 1, 11, dorsal bundles of carpels; 0, bundle of placental supply; o! and o?, lateral strands of the pistil. Fic. 13. Transverse section at base of styles, 0! and o? not present. PLATE, XTX Morus alba, pistillate I, anterior, and 1, posterior carpel bundles; /, posterior and anterior, and p', lateral sepals; 0, placental supply. Fic. 1. Median longitudinal section of flower in posterior-anterior plane. Fics. 2, 3. Transverse section through pedicel. Fics. 4-9. Transverse sections of a flower; 0 + 0, the union of two placenta! strands (as in Ulmus). Fic. 10. Longitudinal section of a staminate flower; 0, strand composed of stamen and perianth supply: I, 11, abortive carpel bundles. Maclura pomtfera Fics. 11, 12. Transverse sections through base of pistillate flower within the in- florescence axis; I, I', anterior and posterior carpel supply; p, perianth; 0, placental supply. Fics. 13-17. Flowers becoming distinct as well as the parts of each flower; , abortive bundles in the perianth parts. Fics. 18,19. Longitudinal median anterior-posterior section of pistillate and staminate flowers respectively. PLATE XOX Cannabis sativa, pistillate br, bract; s, bundle to bract; p, perianth; 1, 11, traces to carpels; s, stamen; a, bundle to placenta; x, abortive bundles. 410 ; AMERICAN JOURNAL OF BOTANY [Vol. 8 Fic. 1. Habit sketch of flower; ~, cup-like perianth. Fics. 2-8. Transverse sections of flower. Origin and freeing of bract and perianth; bundles to carpels and to ovule. Humulus Lupulus, pistillate (Lettering as for Cannabis) Fic. 9. Longitudinal median posterior-anterior section of flower. Fics. 10-12. Origin of bract, presence of abortive bundles. Fries. 13-18. Origin of perianth bundles numbered in the order of their appearance. Two carpel bundles continue into the styles. PLATE SOX Urtica gracilis, pistillate I, 11, anterior and posterior carpel supplies; 0, o + 0, placental supply; m, posterior and anterior, and 2, lateral perianth parts. Fic. 1. Longitudinal median anterior-posterior section of flower. Fic. 2. Transverse section of pedicel. Fics. 3-13. Transverse sections of flower; origin and the freeing of floral parts: supply to ovule. PLATE XXII Boenmeria cylindrica (Lettering as for Urtica) Fic. 1. Longitudinal median anterior-posterior section of pistillate flower; 0, bundle to ovule. Fic. 2. Transverse section of pedicel. Fics. 3-7. Transverse sections of pistillate flower; origin of floral organs and same becoming distinct. Fic. 8. Transverse section of stvle. Fics. 9-13. Sections of staminate flower; 1, 11, abortive bundles to abortive pistil’ Laportea canadensis (Lettering as for Urtica and Boehmeria) f 7? Fic. 14. Longitudinal median posterior-anterior section of pistillate flower; funiculus. Fic. 15. Transverse section of pedicel. Fics. 16-21. Transverse sections of pistillate flower; origin of floral parts and the same becoming distinct; ovule supply separating from anterior carpe! supply; 1, abortive anterior carpel bundle; m1, posterior sepa! has its vascular supply suppressed. VoLuME VIII, PLATE XV ores, aS > % AMERICAN JOURNAL OF BOTANY. ¥ nit o (=) 1) w(t Wll - ~ ~ -- FLORAL ANATOMY OF URTICALES BECHTEL 4 i AMERICAN JOURNAL OF BOTANY. VoLUME VIII, PLATE XVIII. BECHTEL : FLORAL ANATOMY OF URTICALES “ AMERICAN JOURNAL OF BOTANY. VoLumeE VIII, PLATE XIX. BECHTEL : FLORAL ANATOMY OF URTICALES i io 1 —~ ay a] ‘ ‘ , N ' ni a AMERICAN JOURNAL OF BOTANY. VoLuME VIII PLATE XX. BECHTEL : FLORAL ANATOMY OF URTICALES : 7 - Bets tae it } a ’ rad : : = v 7 7 8 i, = is 5 y ' ive 7 { ‘ 5 : —? ty ’ i ‘ - \ ‘ ™~ . i — { { . ‘ , : 2 i \ > ; } x ’ = re P ee ‘ pa — i A ‘i ‘ \ za ‘ : ' A - a a) { AMERICAN JOURNAL OF BOTANY. VOLUME VIII, PLATE XXI. BECHTEL : FLORAL ANATOMY OF URTICALES Oe etm VoLume VIII, PLATE XXII. AMERICAN JOURNAL OF BOTANY. BECHTEL : FLORAL ANATOMY OF URTICALES Y i. . Are i. ' 7 « ™— aed GENETIC EVIDENCE OF ABERRANT CHROMOSOME BEHAVIOR IN MAIZE ENDOSPERM! R. A. EMERSON (Received for publication February 26, 1921) The occasional appearance of a maize seed, the endosperm of which is in part colored and in part colorless or in part starchy and in part sugary, has long been known, and much speculation has been indulged in by geneticists in attempts to account for the phenomenon. Some years ago the writer (Emerson, 1915) reviewed the hypotheses that had been pre- viously offered as possible explanations of such seeds and suggested the further hypothesis of somatic mutation, a suggestion that has been repeated, apparently independently, by J. L. Collins (1919). It was noted also that irregular chromosome behavior might possibly be concerned. In a later paper (Emerson, 1918) numerous cases of anomalous endosperm develop- ment were reported and discussed in relation to the hypotheses of somatic mutation and of aberrant chromosome behavior. It was pointed out that the facts then at hand could be accounted for equally well by either of the two hypotheses, and the kind of evidence necessary for a crucial comparison of the two was noted. In the latter paper evidence was presented that tended to prove that aberrant seeds are not produced (1) when the dominant endosperm factor concerned, for example the aleurone-color factor C, is homozygous and therefore triplex, C C C, or (2) when the dominant factor is brought into the cross by the female parent and its recessive allelomorph by the male parent, C Cc, but only (3) when the dominant factor is contributed by the male alone, cc C. In the case of either C C C or C Cc, a single mutation from the dominant to its recessive allelomorph could result only in C Cc or C cc, respectively, and the aleurone would still be colored and no ap- parent anomaly would result. To produce colorless aleurone, ccc, two mutations in case of C Cc and three in case of C C C must occur simul- taneously or successively in the endosperm of the same seed—a chance so small that it might well be disregarded. It was noted that a single dominant mutation from c to C should change colorless, cc¢c, to colored, Cece, aleurone, but the relative infrequency of dominant mutations was thought to account for the lack of observed aberrant seeds in homozygous colorless types. Similarly, it was noted that if a single non-disjunction of the chromo- some carrying C or ¢ occurred, it could not result in a visible change in aleurone color in case of such genotypes as CC C, C Cc, or ¢ cc, but only 1 Paper No. 86, Department of Plant Breeding, Cornell University, Ithaca, New York. 4II 412 .AMERICAN JOURNAL OF BOTANY [Vol. 8 with cc C. In the latter case, if the chromosome carrying C failed to divide or if the two halves failed to separate after division, one of the result- ing daughter nuclei would be cc C or cc CC (colored) and the other cc (colorless). Thus both somatic mutation and chromosome non-disjunction might readily account for the observed cases of aberrant endosperm, and neither mutation nor non-disjunction could reasonably be expected to cause such an anomaly in genotypes where it has never been observed. It was pointed out in the writer’s 1918 paper that crucial evidence in support of one or other of these hypotheses might be obtained only from crosses in which linked aleurone and endosperm factors are simultaneously involved. It was known that the aleurone factor pair C c is thus linked with waxy endosperm, Wx wx (Bregger, 1918; Kempton, 1919), but no aberrant seeds positively known to involve both these factor pairs were available. The writer was not unaware of G. N. Collins’s case (1913) involving the aleurone factor pair J 7 with Wx wx, but the linkage relations of these factors were not known. It has since been shown by Hutchison (1921) that the factor pair J 7 is closely linked with a factor pair for shrunken endosperm, Sh sh, which in turn is linked with Cc and Wx wx. The linkage group as at present known, therefore, is made up of the pairs Cc, I 1, Sh sh, and Wx wx. Consequently, in accordance with the chromosome hypothesis, all these factor pairs are assumed to lie in one pair of homologous chromo- somes. Assuming, then, that C and W*»x lie in the same chromosome, it can readily be seen how a crucial] test of the somatic-mutation and the chromo- some-non-disjunction hypotheses is afforded by appropriate crosses. If the female parent of a cross be colorless and waxy, c wx, and the male parent be colored and corneous, C Wx, the resulting endosperm will be cc Cwx wx Wx, and the three homologous chromosomes carrying’ these genes in the “‘fecundated” endosperm nucleus will be as follows: C WX I c WX 2 ie Wx 3 DIAGRAM I Now if, at any division of an endosperm nucleus, the two halves (a and b) resulting from a longitudinal split of the chromosome carrying C and Wx (chromosome 3 of diagram 1) should fail to separate (non-disjunction) and should go together to one pole (A), the resulting daughter nuclei (A and B) would be as shown in diagram 2. Oct, 1921 | EMERSON —— ABERRANT CHROMOSOME BEHAVIOR 413 A Jp) C WH G WX ‘B“E=E_ ———EE——— 1b C WH 2a Z al 2b C Wx 3a C Wx 3b DIAGRAM 2 Obviously nucleus ‘B”’ and all its descendants would lack both C and Wx so that the resulting aleurone would be colorless and its underlying endo- sperm waxy, while the aleurone and endosperm cells resulting from the further division of nucleus ‘‘A’’ would be colored and corneous. The same results would follow if chromosome 3 failed to divide, going entire to one pole, or if after equational division one of the halves were left behind, failing to reach either pole. If, on the other hand, the colorless part of an aberrant seed be due to a somatic mutation of C to c, there is no reason to suppose that the same mutation would change Wx to wx. From what is known of the origin of factor (‘‘point’’) mutations, there is little if any more warrant for the assumption that a single mutation will ordinarily involve simultaneously two loci of one chromosome than that it will affect loci of non-homologous chromosomes. If, therefore, in the case under consideration, the coloxless part of an aberrant seed be due to a somatic mutation, the endosperm underlying it should be corneous, cc ¢c wx wx Wx, like that underlying the colored part of the aleurone, c c C wx wx Wx. It now remains to examine the evidence derived from crosses of colorless waxy individuals, c Wx, with pollen of colored corneous ones, C Wx, and to determine whether the colorless parts of aberrant seeds resulting from such crosses are underlaid with waxy or with corneous endosperm. ‘The data available from the writer’s cultures are presented in table 1. Of the 65 aberrant seeds there recorded, the part with colorless aleurone was underlaid by waxy endosperm in 55 cases, by corneous endosperm in 3 cases, and in the remaining 7 cases the endosperm texture could not be determined either because of the extremely small size of the colorless spots or because of the immaturity of the seeds. The aberrant parts of these seeds varied-in area from not much more than a square millimeter to about two thirds of the entire surface of the seed, 32 of the 65 seeds having one sixth or more and only 4 having more than one half of the surface colorless. Eight of the 65 seeds had numerous colorless spots of varied sizes but mostly small, and all the others had only a single spot each. The line of demarcation between the colored and color- 414, AMERICAN JOURNAL OF BOTANY [Vol. 8 less parts was invariably sharp but usually somewhat irregular. The correspondence in outline between the colorless aleurone and the under- lying waxy endosperm was strikingly exact irrespective of the number of spots per seed or of their irregularity (fig. 1; I, J, K, L). The waxy endo- sperm was found to extend to varying depths, the smaller spots often being more shallow than the larger ones (fig. 1; K, L). Moreover, the larger waxy parts often exhibited a somewhat irregular outline in cross-section (fig. 1, K). It is perhaps possible that the three seeds noted as having corneous endosperm under the colorless aleurone had in reality a very shallow layer of waxy endosperm, but this is not likely since in neither case did the colorless spot include less than about one fourth of the entire area of the seed. The writer has examined three aberrant seeds involving Cc and Wx wx from cultures other than his own and in each case the colorless aleurone was directly over waxy endosperm. ‘The seed described by G. N. Collins (1913), from F, of a cross of white waxy with pollen of colored non-waxy types, in which the colorless aleurone was underlaid by waxy and the colored part by corneous endosperm, involved, it is now almost certain, C c with Wx wx. Collins’s published F., records leave no doubt that he was dealing with a case of linkage between waxy endosperm and some aleurone factor. The cross certainly did not involve the aleurone factor pair J 7, for the colorless condition was recessive. Aleurone-color factors A a (Bregger, 1918) and Rr (Kempton, 1919) are.now known to be inherited independently of Wx wx, so that Cc is the only known factor pair that could have been involved. Since, however, Collins’s case appeared in Fe, and since there is about 25 percent of crossing-over between Cc and Wx wx, there is no certainty that both C and Wx were in one chromosome and c and wx in another. The evidence derived from crosses involving the linked genes C-Wx and c-wx points conclusively—in so far as genetic evidence can be regarded as at all conclusive with respect to cytological behavior—to some aberrant chromosome distribution, perhaps non-disjunction, as the cause of most cases of aberrant endosperm development; but the three instances noted above of corneous endosperm underlying colorless spots of aleurone suggest, though they do not prove, that very rarely somatic mutation may be responsible. Evidence from other linked factors in addition to Cc and Wx wx would be of great value as tending to confirm or contradict the conclusion here drawn. A number of such linkages are now known. In addition to the linkage of J 2 with Wx wx, inferred, as noted earlier in this paper, from Hutchison’s data, Hutchison has found both J% and Cc to be closely linked with shrunken endosperm, Sh sh, and Dr. E. G. Anderson (un- published data) has noted linkage between a factor pair for blotched aleurone, Bh bh, and the pair Y y for yellow endosperm. Oct., 1921] | EMERSON — ABERRANT CHROMOSOME BEHAVIOR AI5 The writer has not been able as yet to obtain from his own cultures aberrant seeds involving any of these linkages, but in Professor Hutchison’s material a single aberrant seed involving Cc and Sh sh has been observed. A colorless- and shrunken-seeded plant, c sh, pollinated by a plant hetero- zygous for these factors, Cc Sh sh, produced the aberrant seed. It was « Ji KK L Fic. 1. Aberrant endosperm in maize seeds. Factors involved: A and B, Rr Su su; Geant Cic Susu. kr Wx we; be Ava Wx wx; Gio Ssh; ty, Susur 1 to L, Cc Wx wx. In I and J the upper figures show untreated seeds with purple-colorless aleu- rone, and the lower figures show the same seeds after the pericarp and aleurone layer have been removed, the corneous endosperm appearing dark and the waxy endosperm light. In K and L, the upper figures represent parts of untreated seeds and the lower figures corresponding cross sections of the same seeds. 416 AMERICAN JOURNAL OF BOTANY [Vol. 8 colored and non-shrunken, C Sh, except for a single large spot that was both colorless and shrunken (fig. 1, G). The sperm in this case must have carried both C and Sh, for otherwise the seed would have been colorless and shrunken throughout. The evidence, therefore, so far as this one seed is concerned, is definitely in favor of the hypothesis of aberrant chromo- some behavior and opposed to that of somatic mutation. Both J 7 and Wx wx are without doubt concerned in the case of a single aberrant seed reported by G. N. Collins (1913). A colored waxy type pollinated by a colorless corneous one resulted in colorless corneous seeds. The dominance of the colorless condition establishes, so far as is now known, the presence of J in the colorless pollen parent. A single seed of this cross, though colorless and corneous in the main, had a small spot of colored aleurone which overlaid exactly a spot of waxy endosperm. It seems evident that in this one instance in which an aberrant seed involved the linked factors J and Wx, just as in the single case in which C and Sh were involved and in the great majority of the cases—55 out of 58—in which the linked factors C and Wx were concerned, aberrant endosperm develop- ment is ordinarily due to some unusual chromosome behavior possibly of the nature of non-disjunction. One unfamiliar with some of the results previously published will not have failed to observe by this time that either one of Webber’s (1900) well- known hypotheses might account for the results presented above quite as well as the hypothesis of non-disjunction. Webber, it will be recalled, suggested as possible explanations of aberrant endosperm development (1) that the second sperm nucleus on the one hand and the fused polar nuclei on the other may occasionally develop independently, each giving rise to a part of the endosperm, or (2) that the second sperm nucleus may sometimes unite with one polar nucleus, leaving the other polar nucleus to develop independently. If either of these things should happen, it is obvious that, in cases of aberrant endosperm where C and Wx come from the male and c and wx from the female parent of a cross, the colored parts must be corneous and the colorless ones waxy. The second sperm nucleus, carrying C and Wx, whether it divide independently or unite with one polar nucleus, would give rise to colored corneous endosperm, and the endosperm developed either from one polar nucleus alone or from a fusion of the two, both carrying c and wx, would produce colorless waxy endosperm. But it was shown by East (1913) that Webber’s first hypothesis, inde- pendent development of the sperm nucleus and of the fused polar nuclei, was untenable. Crosses were made between two types of maize, both with colorless aleurone but one having factor C and the other having factor R, both of which are essential to aleurone-color development. Among the numerous colored seeds resulting from these crosses, six were colored on one side and colorless on the other. It is obvious that these aberrant seeds could not have arisen in accordance with Webber’s first hypothesis, for, Oct .;-1921 | EMERSON — ABERRANT CHROMOSOME BEHAVIOR 417 since the second sperm nucleus carried C and not R and the polar nuclei R and not C, if the two elements divided without undergoing fusion, C and R could never have been brought together and no aleurone color could have developed in any part of the seeds. TAeLE I. Aberrant seeds cf maize from crosses of colorless waxy, c wx, by colored corneous, C Wx Ker ee Number of Aberrant Seeds. Endosperm under Pedigree No. ! Naas ae Colorless Parts of 9 Parent Normal] Seeds Waxy Corneous Undetermined NOOR Oh er 460 2 eee Seka sé 600 2 Gis OY a ee 500 I Occ Se eee 280 I [ OCA Re nee 10 I | CUNO) Es iol he oo eee ee 560 T I AS 250 I MOUOO a act: nae. ds 480 I | OIE O ave) See a 430 I BOW Oe elie. Se... 400 2 Bh ee ee ae 300 2 DRE AEM ct 2G 460 I Di i LH. A 4 390 4 I Bn canes cae 20 | I oe haa aang ain 70 I OLE cas Ca 460 2 I CA. cn, te a 450 | I Cees sles go" 2: 650 I Fin Thee | ee 620 | 2 I Ga eek ch, eee 340 | 2 IOS. a ae 430 2 AG ae esi oe 290 I LG eee eo... 540 | I IDG Sieh peal a ae 160 ee OE Mr emit sits te 560 3 MOM eA le ee 530 | I yen aie es hd 270 | I MSM ee Gres, 6S 630 Za | (Oot 470 | 2 | Wha eae Ole ae 190 | 2 | 256 So 240 | T | DU ce 350 2 | OS Se 140 I | Dl Sick ae eee 470 2 | ZO) os Ses ey Cee eae 430 | I LOIS 3S Oe Ie a 150 | | 2 Sata ee 240 I I | its 4 Se a | 120 | I | POVCATS a}. Pk 9,580 | O O | ) MovalGOpears... 2.2... 22816 | 55 3 fi In a similar way the writer (Emerson, 1915) was able to show that Webber’s second hypothesis is incorrect. Two colorless types, each having only one of the two complementary aleurone-color factors C and R, were crossed as in the experiments of East. In addition, the type used as the female parent was sugary and the one used as the male parent was starchy. 418 AMERICAN JOURNAL OF BOTANY [Vol. 8 Among the resulting seeds, which were colored and starchy as expected, occurred two with aberrant endosperm, one of which was part colored and part colorless but starchy throughout and the other one part starchy and part sugary but colored throughout. As in the case reported by East, no color could have developed if the second sperm nucleus had not fused with a polar nucleus. Furthermore, if the second sperm nucleus had united with one polar nucleus (Webber’s second hypothesis), the part of the endo- sperm so formed must have been both colored and starchy while the remain- ing part of the endosperm formed from the other polar nucleus alone must have been both colorless and sugary. The observed facts, namely, that the starchy-sugary seed was colored throughout and that the colored- colorless one was starchy throughout, indicated clearly that normal fusion of the second sperm nucleus with the polar nuclei had taken place. It remains to forestall the justifiable criticism that one or other of Webber’s hypotheses might still account for most examples of anomalous endosperm, the two seeds noted above being minor exceptions, just as either of these hypotheses might well be used to explain the 55 aberrant seeds with colorless spots waxy recorded in table 1, the 3 seeds with colorless spots corneous likewise being exceptions. In the writer’s 1918 paper (tables 8 and 10) were recorded 33 examples of anomalous seeds with part. colored and part colorless aleurone from crosses between types with wholly colorless aleurone but carrying complementary aleurone-color factors. These, added to the six cases reported by East (1913), a total of 39, are believed to suffice as a demonstration that division of the second sperm nucleus independently of the fused polar nuclei is quite untenable. More- over, two of the 33 anomalous seeds afforded definite evidence against the hypothesis that one polar nucleus might fuse with the second sperm nucleus and the other polar nucleus divide independently, making a total of four such instances. These two seeds resulted from a cross of a type with colorless sugary endosperm and colorless aleurone carrying R with pollen of a type with yellow starchy endosperm and colorless aleurone carrying C. The aberrant seeds were starchy and yellow throughout but their aleurone was about half colorless and half purple. | Since the publication of the writer’s 1918 paper, a sufficient mass of evidence has been obtained to remove, it is thought, any possibility of explaining anomalous endosperm development on the basis of a failure of normal fusion of the second sperm nucleus and the polar nuclei. This additional evidence is presented in tables 2 and 3. In table 2 is recorded all the available material in which corneous and waxy endosperm, Wx wx, are involved together with the aleurone-factor pairs A a, Rr,and Pr pr. The male parents of all the crosses here recorded had homozygous corneous endosperm and homozygous purple aleurone, A CR Pr Wx, while the female parents of all had waxy endosperm, wx. In addition, the female parents of all crosses recorded in groups I and 2 of @ct., 1921] EMERSON ——- ABERRANT CHROMOSOME BEHAVIOR 419 the table had colorless aleurone, a C R for group 1 and A Cr for group 2, and those of the crosses shown in group 3 had red aleurone, A C R pr. In all, 38 aberrant seeds are reported, 12 of which involved A a Wx wx (fest, F),6 Rr Wx wx (fig. 1, E), and 20 Pr pr Wx wx. In all these cases, TABLE 2. Aberrant seeds of maize from crosses of colorless waxy, a wx and r wx, by colored corneous, A R Wx, and of red waxy, pr wx, by purple corneous, Pr Wx Approximate Number of _ Group Genes Pedigree No. Number of Aberrant Seeds, Concerned of Q Parent Normal Seeds all Corneous Colored and colorless Meet hea x's beats xX ACW go6I-— 3 300 2 | 5 500 | I IOI83-— 2 500 2 8 350 3) II 520 I 14 280 2 I5 210 I I2 ears 4,610 fe) Wotales oo). 19 ears 7,100 12 Zi SESE Re ee ICR ee, IO1I87— 5 290 I | 6 440 I | 8 240 2 | 9 190 2 | 2 ears 560 O otal. 3 | 6 ears 1,720 ee Purple and red Beemer wx x Pr Wx .. 9062— 2 460 I 5 470 I IOL7I-— 4 450 I 6 650 I | 9 590 I 14 560 I 15 540 2 18 630 2 18 | 470 I 23 280 I 7s | 470 J IO182- I 390 I | 2 300 I | 5 480 2 9 390 I | TOUS2 = 7 280 I 10187— 6 440 I 8 240 I 6 ears 2,220 fe) Mota. kc: | 24 ears | 10,310 20 the recessive aleurone color of the female parent, namely, colorless in groups I and 2 and red in group 3, occurred in the aberrant part of the seed, and the dominant color, purple, of the male parent occurred in the normal part. But in every instance the endosperm was corneous throughout like that of A20 the male parent. AMERICAN JOURNAL OF BOTANY [Vol. 8 No case of aberrant corneous-waxy endosperm was observed, but there is no satisfactory evidence that none occurred among the wholly colored seeds, where waxy spots would be easily overlooked. Similarly, in table 3 are recorded all available cases in which starchy and sugary endosperm, Su su, are involved together with the aleurone-factor airs: Coc.2R 7 anger The male parents of all these crosses had homozygous starchy endosperm and homozygous purple aleurone, A C R TABLE 3. Aberrant seeds of maize from crosses of colorless sugary, c su and r su, by colored starchy, C R Su, and of red sugary, pr su, by purple starchy, Pr Su | Number of Aberrant Seeds Genes Pedigree ! Approximate All Starchy, All Colored, Group Concerned No. of 9 | Number of Golored and Starchy and Parent | Normal Seeds Colorless Sugary | ee eae GSI xX iC othe | 9063- I 260 I 23 240 I 4 320 2 5 179 I I 7 510 I 10178. 2 320 2 2 290 I 4 410 2 I 5 290 I 6 370 2 6 220 I IOI7Q— I 420 I 2 360 I 4 250 I 5 380 I 6 430 3 6 440 I 5 ears 1,750 fe) a) Potala... 22 ears 71430 20 5 CO nee WSU OS 8572-1 190 2 | LOLS5 at 420 2 2 2 320 I 4 400 2 I 5) 390 I 6 380 I 7 290 2 7 440 I 10 360 2 2 7 ears 23720 O O AROtalitages: 16 ears 5,910 13 6 | All Starchy, | All Purple, Purple and Starchy and Red Sugary ew teat Ne | br su X Pr Su| 10180- 1 360 i 5 260 I 7 220 I 5 ears 710 O fe) Pocaliey 8 ears 1,550 I 2 Oet., 1921] EMERSON —— ABERRANT CHROMOSOME BEHAVIOR 421 Pr Su, while the female parents of all had sugary endosperm, su. In addition, the female parents of all crosses shown in groups I and 2 of the table had colorless aleurone, A c R for group 1 and A Cr for group 2, and those of the crosses presented in group 3 had red aleurone, A C R pr. In all, 47 aberrant seeds are recorded. Of these, 34 had aberrant aleurone Color 20 involving Cc Su su (fig. 1, C), 13 Rr Su su (fig. 1, A), and 1 Pr pr Su su; and 13 had aberrant endosperm texture, 5 involving C ¢ Su su Mieetine)) ork 7 Su su (fig. 1, B), and 2: Pr pr Susu. Every one of the 34 seeds that had aberrant aleurone (colored-colorless) were starchy through- out, and all of the 13 with aberrant endosperm (starchy-sugary) had colored aleurone throughout. In short, there have been observed (tables 2 and 3) a total of 85 aberrant seeds involving Cc with Su su, A a with Wx wx, and Rr and Pr pr with both Su su and Wx wx. In every case in which aleurone color was con- cerned, 72 in all, the aberrant spot showed the recessive color of the female parent but was invariably underlaid with the dominant corneous or starchy endosperm of the male parent; and in all of the 13 cases in which endosperm composition was concerned the aberrant spot exhibited the recessive sugary condition of the female parent but was invariably overlaid by the dominant aleurone color of the male parent. It is obvious, therefore, that for none of the 85 seeds could the aberrant spots, though they displayed in every case one or other (never both) of the two recessive maternal characters whose genes were carried in the polar nuclei, have been produced by the inde- pendent division either of one polar nucleus alone or of the two after fusion. To explain these cases on the basis of independent division of one or both polar nuclei would require the unwarranted additional assumption that in some cases—aberrant sugary spots—the polar nucleus or nuclei alone give rise to a part of the underlying endosperm but to none of the aleurone layer, while in other cases—aberrant aleurone-color spots—they give rise to a part of the aleurone but to none of the underlying endosperm. Moreover, if such behavior of independently dividing polar nuclei were so common in cases involving the endosperm factors Wx wx with the aleurone factors Aa, Rr, and Pr pr (table 2), and the endosperm factors Su su with the aleurone factors Cc, Rr, and Pr pr (table 3), why should not the same behavior of the polar nuclei be found where there is involved Wx wx with Ciaag@able 1) or with./4 (Collins, 1913) or Cc with Sh sh (Hutchison’s data)? But the facts are that in the great majority of aberrant seeds involving Cc and Wx wx (55 to 3) where the aleurone layer is colorless (maternal), the underlying endosperm is waxy (also maternal), and the correspondence in outline between colorless aleurone and waxy endosperm is strikingly exact. Certainly no single hypothesis that assumes inde- pendent development of either one or both of the polar nuclei can be made to fit all the data now available. That the somatic-mutation hypothesis suggested by the writer (1915) does not agree with the great majority of the observed facts when C c and 422 AMERICAN JOURNAL OF BOTANY [Vol. 8 Wx wx are concerned, just as the hypotheses of independent division of polar nuclei suggested by Webber (1900) do not fit the available facts where other than these aleurone and endosperm factors are concerned, was shown earlier in this paper. The hypothesis of vegetative segregation (East and Hayes, 1911) is not sufficiently specific with respect to the mechanism of such supposed segregation to make it possible to apply crucial tests. More- over, several cases of somatic variations often referred to as cases of vegeta- tive segregation are quite as likely due to somatic mutation. There remains only the hypothesis of aberrant chromosome behavior (non-disjunction ?) which is in accord with practically all the reported cases of aberrant endo- sperm development. It was shown earlier how that hypothesis fits the cases involving the linked genes Cc (or J1) with Wx wx. That this hypothesis is not in disagreement with the cases where other endosperm and aleurone factors are concerned follows from the fact—determined by ordinary breeding tests—that these other factors are not genetically linked and that, therefore, they presumably have their loci in non-homologous chromosomes. Evidence of non-linkage for A a with Wx wx was presented by Bregger (1918), for Rr with Wx wx by Kempton (1919), and for Su su with Cc, Rr, and Pr pr by Eyster (1921); and there is indirect evidence for Pr pr and Wx wx in Hutchison’s data which show Wx wx to be linked with Sh sh and the latter to be independent of Pr pr. If none of these combinations of genes lies in the same chromosome, it is obvious that a non-disjunction of one chromosome could not affect more than one member of the combination, just as it is that both members of any combination lying in one chromosome must be affected by a single non-disjunction of that chromosome. While the writer feels that the genetic evidence in favor of the hypothesis of non-disjunction, or at least of some aberrant chromosome behavior giving a similar result, as the cause of most cases of the kind of aberrant endosperm here discussed is as convincing as such evidence can well be, it is realized that direct proof must come, if at all, from cytological studies. Whether it will ever be possible to detect non-disjunction cytologically in the endo- sperm of maize, granting that it occurs, cannot be said. The small size of maize chromosomes and their large number, 30 in the triploid nucleus, increase the difficulty of the undertaking. Moreover, the rarity of the phenomenon lessens the chance of a successful outcome. On this latter point, however, there is this to be said: non-disjunction is doubtless a much more common occurrence than are its visible manifestations. There is no reason to suppose, for instance, that, in material of the genotype cc Cwx wx Wx, such as that recorded in table 1, the chromosome carrying C and Wx is more often concerned in non-disjunction than are the other two homologous chromosomes each carrying c and wx. But a non-dis- junction involving either of the latter could result in no visible change in either the color of the aleurone or the texture of the endosperm. It may Oet., 1921] EMERSON —— ABERRANT CHROMOSOME BEHAVIOR 423 well be assumed, therefore, that non-disjunction within this one group of chromosomes occurs three times as frequently as it is visibly manifested in such material. Moreover, there is no satisfactory evidence that one group of homologous chromosomes is involved more frequently than any one of the other nine groups. It may be supposed, therefore, that non-disjunction occurs on the average 30 times for every aberrant seed observed in material hetero- zygous for a single factor pair. Certainly non-disjunction—if such be the cause of aberrant endosperm—1s not limited to the C-I-Sh-Wx chromosome. Aberrant endosperm has been oflserved to involve the additional aleurone and endosperm factors A, R, Pr, Y, and Su, all of which are inherited inde- pendently of the C-J-Sh-Wx group, and all of which, except possibly R and Pr, are inherited independently of one another. Since, therefore, aberrant endosperm has been observed to involve not less than five and perhaps six linkage groups, aberrant endosperm behavior is assumed to have occurred in at least the same number of groups of homologous chromosomes and there is no reason to suppose that it is not common to all ten groups. From tables 1-3 of this paper, it is seen that 150 aberrant seeds were observed with an approximate total of 57,830 normal seeds. Of these 150 seeds, 13 involved sugary endosperm, .a character that might easily be overlooked except when the aberrant spot is large. Since, moreover, the material involving sugary endosperm had to do also with an aleurone-color factor, the 13 seeds must be omitted if we are to deal with a single factor or linked-factor group, in other words, with a single chromosome, at a time. The observed ratio, when only one factor is involved, is 57830 : 137, or approximately one aberrant case in every 423 seeds. If now it be assumed that non-disjunction occurs thirty times as frequently as aberrant seeds in such material, non-disjunction should occur on the average once in about 14 seeds. In more than half of the aberrant seeds reported in this paper (77 out of 150) the aberrant part included approximately one sixth or more of the surface area of the seed and in about one twelfth of them it included more than one half of the seed. Consequently, non-disjunction must occur, if at all, fairly early in endosperm development in a considerable percentage: of cases. It would seem worth while, therefore, for cytologists to search for it at least in the early divisions of the endosperm nucleus. An observation noted earlier in this paper suggests that irregularities besides non-disjunction may occur in endosperm development. It was noted that 8 out of 65 aberrant seeds involving C c and Wx wx were mottled, exhibiting numerous small spots of colorless aleurone instead of a single spot. One of these mottled seeds was so immature that the endosperm texture could not be determined, but in the other seven the colorless spots were underlaid by waxy endosperm (fig. 1, L), this association indicating definitely aberrant chromosome behavior. It does not seem likely that non-disjunction would occur repeatedly in the development of a single 424 AMERICAN JOURNAL OF BOTANY [Vol. 8 seed, but if it does not there must have been very irregular migration of endosperm nuclei after non-disjunction occurred. In material involving Cc Susu and A a Wx wx, 6 of the 22 seeds showing aberrant aleurone color were mottled. Practically all of the normal seeds in material where Rr is involved were mottled, and the aberrant seeds showed mottling in the colored part (fig. 1, A and E), but mottling is known to occur commonly in aleurone heterozygous for R when R enters with the sperm and 7 r with the polar nuclei and, whatever its ultimate cause, it is not to be confused with what is here termed aberrant endosperm. SUMMARY It has been shown that, when aberrant seeds occur in crosses in which recessive aleurone and endosperm characters are contributed by the female parent and the corresponding dominant characters by the male parent, spots of the recessive (maternal) aleurone color are in the great majority of cases underlaid by the recessive (maternal) type of endosperm if the genes for these aleurone and endosperm characters are genetically linked, as shown by breeding tests, while similar recessive aleurone-color spots are always, so far as observed, underlaid by the dominant (paternal) type of endosperm and recessive endosperm parts are overlaid by the dominant aleurone color if the aleurone-color and endosperm-composition genes are not linked. These facts are held to support the hypothesis of occasional aberrant chromosome behavior—possibly non-disjunction—and are incompatible with the earlier hypotheses involving failure of normal fusion of the second sperm nucleus with the polar nuclei, and also make untenable, except in rare cases, the hypothesis of somatic mutation. LITERATURE CITED Bregger, T. Linkage in maize: the C aleurone factor and waxy endosperm. Amer. Nat. 525.5701. ifO1S: Collins, G. N. Mosaic coherence of characters in seeds of maize. U.S. Dept. Agr., Bur. Plant Inds, Cire. 132.1921. 9 191. Collins, J. L. Chimeras in corn hybrids. Jour. Hered. 10: 3-10. IgI9g. East, E. M. Xenia and the endosperm of angidsperms. Bot. Gaz. 56: 217-224. I913. ——, and Hayes, H. K. Inheritance in maize. Conn. Agr. Exp. Sta. Bull. 167: 1-141. IQII. . Emerson, R. A. Anomalous endosperm development and the problem of bud sports, Zeitschr. induk. Abstamm. Vererb. 14: 241-259. I9QI5. ——. A fifth pair of factors, A a, for aleurone color in maize, and its relation to the Cc and Rr pairs. Cornell Univ. Agr. Exp. Sta. Memoir 16: 225-289. 1918. Eyster, W. H. The linkage relations between the factors for tunicate ear and starchy- sugary endosperm in maize. Genetics 6: 209-240. 1921. Hutchison, C. B. Heritable characters of maize VII. Shrunken endosperm. Jour. Hered. 12705030 O21. Kempton, J. H. Inheritance of waxy endosperm in maize. U.S. Dept. Agr. Bull. 754: I-99. I919. 3 Webber, H. J. Xenia, or the immediate effect of pollen in maize. U.S. Dept. Agr., Div. Veg. Phys. Path. Bull. 22: 1-44. tIgoo. ak pea a mean so a Aa eee ee 0 be : sly me ity : Erect ae pre | reputation asa. Be hick qualified to write such < sens a8 Cornell, says? = | : 2 ate. | 3 aia cytologist whet only. ce both “pen and animal cyto lt PGA i x LN also. Ww th the beat of rine on. ae phe sgh ob ie oe ee i ae of ah cer =APELBD. a 7 UR : ‘ ih ER dda-e gone into a hak Sok Se Aaee cing: exactly the kind and shaped. © «hat you want; and then have some. smart. Alec oe a salesman try to . sel] you the kind HE likes? . Makes a Fellow: ease than a. wet hen, doesn’ t it? RRS y 2 | Suppose, however, shar same salesman’ had- told you something — about : some of his hats that meant) : “NEW. YORK re c “or Pack Ave. Sg you would % ‘an ol fri better : service to ¥o amare “faction for your money—> sort of feel. towards th: S ‘Salesingi iit two: is o | -when it: ‘comes to selling. 8 a house. - We dn sell; pephowe, ‘but w first sell Ricky ieee ee Ea RETNS yy Ao OF AMERIC i Herat Na iio ogi final wan EA ? ypes of vascular bundles 5) Joun Y, PENNYPACKER AN x) way, ve tii A 2 ‘fe G, B, D ‘ ; oe nd. antacopien: Hi ty ats ‘same substanc : ‘cation and anion, - ea dee) ae Okan L. RABER. i) Es) 7 i "BOTAN ICAL ‘SOCIETY. 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VIII NOVEMBER, 1921 No. 9 THE INTERRELATIONSHIP OF THE NUMBER OF PoE WO TYPES OF VASCULAR, BUNDLES iNet TRANSITION ZONE OF THE Poxts; OF PHASEOLUS VULGARIS J. ARTHUR HARRIS, EDMUND W. SINNOTT, JOHN Y. PENNYPACKER, AND G. B. DurHAmM (Received for publication January 17, 1921) INTRODUCTORY In papers! on the anatomy of dimerous? and trimerous and of hemi- trimerous seedlings we have shown that Phaseolus vulgaris is characterized by a structure of the vascular system at the base of the hypocotyl which is rather infrequent in seedling anatomy in general. This is the presence of a variable number of accessory bundles which usually lack protoxylem elements. These are the “Zwischenstrange’’ of Dodel. We have elsewhere called them intercalary bundles. They may make their appearance in the upper part of the root or in the lower region of the hypocotyl, some rising blindly below and others originating by division of a primary double bundle. These intercalary strands may be distinguished from the other bundles with perfect certainty because of their position and of the absence within them of any protoxylem elements. In another place* we have dealt with the correlations between the number of bundles at different levels in the seedling, that is, the relationship between the vascular system at the base of the hypocotyl and that in the central region of the hypocotyl and epicotyl, and between the bundle system in the hypocotyl and that in the epicotyl. Our present problem is to consider the interrelationships between the two types of bundles present in the hypocotyl just above the region of transition from root to stem struc- tures, and between each of these types and the total number of bundles in this zone. artis, |. Arthur,.sinnott, E. W., Pennypacker, J. Y., and Durham, G. B. The vascular anatomy of dimerous and trimerous seedlings of Phaseolus vulgaris. Amer. Jour, Bot. 8: 63-102. 1921. The vascular anatomy of hemitrimerous seedlings of Phaseolus vulgaris. Amer. Jour. Bot. 8: 375-381. 1921. 2 Dimerous seedlings have 2 cotyledons and 2 primordial leaves; trimerous seedlings have 3 cotyledons and 3 primordial leaves; and hemitrimerous seedlings have 3 cotyledons and 2 primary leaves. * Harris, J. Arthur, Sinnott, E. W., Pennypacker, J. Y., and Durham, G. B.- Correla- tions between anatomical characters in the seedling of Phaseolus vulgaris. Amer. Jour. Bot.8: 339-365: 1921. ; [The Journal for October (8: 375-424) was issued November 14, 1921]. 425 426 AMERICAN JOURNAL OF BOTANY [Vol. 8, Lack of space precludes the publication of the 30 individual correlation tables upon which the coefficients discussed in this section are based. These may, however, be easily formed from the schedules showing the formula for the basal bundles in other papers of this series.4 TABLE I. Correlation between Number of Primary Double Bundles and Number of Intercalary Bundles at Base of Hypocotyl Trimerous | Dimerous Line | Difference Diff. N r ideas Man r fe Feaitt. (ee | E, 75, \\ cA2 | —.5004+.0424 | 11.8 | 142 | —.1177.0558 | 2.11 | —.3827s=207004 ery ta 93 | 155 | —-6155-£.0337 | 18.3 | 155 | —-1449=2.0530 | 2.73 | —.4700=-:0024) Raise 98 | 183 | —.6515+.0286 | 22.8 | 183 | +.0643+.0496 | 1.30 | —:7158=+.0574 2A 139 | 106 | —.5053+.0488 | 10.4 | 305 | +.1364+.0379 | 3.60 | —.6417+.0618 6.0 143 | 221 | —.3184+.0408 | 7.8. | 420 | +.033822,0320)|. 1.03 | = .3522-—onae 5.4 ANALYSIS OF DATA 1. Relationship between Number of Primary Double Bundles and Number of Intercalary Bundles. We shall first consider the relationship between the number of primary double bundles and the number of intercalary bundles at the base of the hypocotyl in dimerous and trimerous plants. The correlation coefficients for the five lines appear in table 1. For ‘the trimerous plants of all five lines the correlations are negative in sign, 2.e., the number of intercalary bundles is greater in plants which have a smaller number of primary double bundles, and vice versa. For dimerous plants three of the five lines show a slightly negative coefficient, but two show a low positive correlation. The constants indicate that the corre- lations for the trimerous plants are much higher numerically than those for the dimerous plants. Those for the trimerous are of the order —.3 to —.6 while those for dimerous plants are sensibly zero, averaging + .005. The correlations for the trimerous plants are in all cases several times as large as their probable errors, while those for the dimerous plants could hardly be regarded as statistically significant if only one of the lines were available. The differences, taken with regard to sign, between the corre- lations for the dimerous and trimerous plants are in each case significant in comparison with their probable errors. Expressing these results in terms of regression we have the following equations: 4 The entries to be selected from the published tables can be determined from the values of N. In lines in which true siblings were available (75, 93, and 98) only siblings have been used, even though additional sections of one or the other type were available. In the two lines in which random samples of seed were used for the production of the dimerous and trimerous seedlings, the largest possible number of individuals available in the tables of the papers cited was employed for the constants here discussed. ° Nov., 1921] HARRIS AND OTHERS — PHASEOLUS VULGARIS Ag] Dimerous Trimerous Line 75: P = 4.255 — 0.0581 iP" = 6,050) O.4o / I = 1.641 — 0.239 P = 4.968 — 0.789 P Eme 937° P = 4.607 — 0.110 [ P= 5.902; — O445er I = 1.398 — 0.127 P I = 5.200 — 0.846 P Line 98: P = 4.099 + 0.035 I P= 5,964.1 O.302 1 1 O.U1Ae Oli 7 f= 6.559 — 1.064 7 Line 139: P = 4.005 + 0.034 1 P= 5.058 —' 0.550 1 f= — 2.038 + 0.541 P I = 2.795 — 0.457 P Line 143: P = 4.060 + 0.019 I P=" 5.024) 40.4050) I = 0.047 + 0.059 P L = "17 322 2A One The mean number of intercalary bundles associated with given numbers of primary double bundles and the theoretical means as given by the regression straight lines are shown on diagram I. For the normal plants of lines 75, 93, and 139 the agreement between the observed means and the regression line is very satisfactory. In line 98 a single seedling with 8 primary double bundles and 4 intercalary bundles gives a positive sign to the correlation and makes the agreement of theo- retical and empirical means very poor indeed. In lines 139 and 143 the correlation is also positive. It must be noted that we are dealing here with a very narrow range of both primary double bundles and intercalary bundles, and with very small frequencies in some of the classes. For the abnormal plants the agreement of empirical means and theo- retical lines is apparently very poor indeed. This is perhaps largely attrib- utable to two facts: (a) The frequencies of primary double bundles are, practically speaking, concentrated in two classes, 5 and 6 bundles. From 93 to 99 percent of the seedlings fall in these two classes. As a result of this condition, the ob- taining of trustworthy averages for the extreme classes of primary double bundles is, practically speaking, impossible. (b) The influence of the two principal classes (5 and 6) of primary double bundles is such as to throw the theoretical mean number of interca- - lary bundles for higher classes of primary double bundles on the negative side of o in four of the five cases. As a consequence, the actual mean number of intercalary bundles must lie above the line in all cases in which more than 6 primary double bundles are formed. Whether these irregularities represent a significant deviation from line- arity can be determined only when far larger series of data are available. While the primary double bundles must probably be regarded as more fundamental structures than the intercalary bundles, it seems of interest to determine the mean number of primary double bundles associated with each number of intercalary bundles. 428 AMERICAN JOURNAL OF BOTANY [Vol. 8 The lines for the regression of number of primary double bundles on number of intercalary bundles are represented with the empirical means on diagram 2. These figures show that, with the exception of the normal plants of lines 98, 139, and 143, the number of primary double bundles decreases slightly as the number of intercalary bundles increases. ‘The rate of decrease is somewhat greater in the abnormal than in the normal plants. TRIMEROUS SEEDLINGS DIME ROUS SEEDLINGS LINE S39 a LIVE 98 IIEAN NUNBER OF INJERCALARY BUNDLES LINES TS LINE 93 iy a aan RO! ee oun S 7 PRIMARY DOUBLE BUNDLES D1AGRAM I. Regression of number of intercalary bundles on number of primary double bundles, at base of hypocotyl. Nov., 1921] HARRIS AND OTHERS— PHASEOLUS VULGARIS 429 It is suggestive to note that the negative correlation between number of primary double bundles and number of intercalary bundles demonstrated here within seedlings of one class with regard to external structure is also evident when we pass froma type of seedling with a smaller to one witha higher number of primary double bundles. It has been shown in an earlier paper that (a) the number of trimerous seedlings having intercalary bundles is generally smaller than the number of dimerous seedlings with these accessory structures, and that (b) the average number of intercalary bundles is generally smaller in trimerous than in dimerous seedlings. — =TRINEROVS i 98 o—e = J/MEROVUS LINE 1/43 LINE 139 MEAN NUMBER OF PRIMARY DOUBLE BUNILES INTERCALARY BUWVILES DIAGRAM 2. Regression of number of primary double bundles on number of intercalary bundles, at base of hypocotyl. 2. Relationship between the Total Number of Bundles and the Number of Bundles of the Two Types. We may now inquire to what extent the varia- tion in the total number of bundles depends upon the primary double bundles and to what extent upon the number of intercalary bundles. Asa first step we determine the correlation between the total number of bundles and the number of primary double bundles, and between the total number of bundles and the number of intercalary bundles. These results are set forth in table 2. We note that the correlations between the total number of bundles and the number of intercalary bundles are in all cases high, ranging from +.42 430 AMERICAN JOURNAL OF BOTANY , [Welee8s to +.80 in the trimerous and from +.76 to +.97 in the dimerous plants. The correlations for the dimerous plants are in all five cases slightly higher than those for the trimerous plants. TABLE 2. Comparison of Correlation between Total Bundles and Primary Double Bundles, Yop, and between Total Bundles and Intercalary Bundles, ry;, at Base of Hypocotyl Trimerous Dimerous ; Diff. Difference ee N r ne N r We diff. E, Tad Line 75 — coe 142| +-.7788 1.0222 |.35.1 | 142 | +.8872=:.0120 | 73.9 | —:1084S2,0245)8 Ae42 Tinie aere 142| --.1532+.0552| 2,77| 142 | 4-.353632.0495| 7.14 | = .2004-— O74 mr 0 Toi —Top +.6256+.0591 | 10.6 +.5336+.0510 | 10.5 Line 93 eee noe 155| +.6934=.0281 | 24.7 | 155 | +-7628=+.0226 | 33.6 | 1000422 0301 iar -a2 Wipavous) eee 155) +-1409+.0531 2.65 | 155 | +.5292+.0390/ 13.6 | —.3883+.0655| 5.92 Toi —Top -.5525 2=.0600 | 9,2 + .2336+.0447 | 5.23 Line 98 Dict i ave 183} +.8001+.0179 | 44.6 | 183 | +.8833-.0109 | 81.0 | —.0832+.0200| 4.16 A ee 183| —.0664+.0496 | 1.34| 183 | +.5245+.0361 | 14.5 | —.5909+.0616/ 9.59 roi —Ttp- +.8665 4.0529 | 16.4 +.3588+.0374| 9.59 Line 139 Ae 106| +.4203+.0539 | 7.79) 305 | +.9721+4.002I |457.4 | —.5518+.0539 | 10.2 Rit sMontis 106] +.5707 £.0422 | 12.9 | 305 | +.3649+.0335 | 10.9 | +.2058+:.0555| 3.71 1bi—Vbp- —.1504+.0607 | 2.16 + .6072 +.0336 | 18.1 Line 143 be tad: 221| +.4382-£.0367 | 11.9. | 420 | +-.8715=4:0079 |110.2° | — 43333-0275 EES Tin okie ce 221] -+-.7126+.0223 | 31.9 | 420 | +.5196=:.0240 | 21.6 | 4-.193@032/0923 5.65 Toi —Top = 927 AA == OA420i\, 6:10 +.3519+.0253 | 13.9 The correlation between the total number of bundles and the number of primary double bundles is in general much lower. actually has the negative sign in the trimerous series. In line 98 the coefficient The differences between the correlation coefficients for total bundles and intercalary bundles, and for total bundles and primary double bundles, range from —.27 to +.87 in the trimerous plants and from +.23 to +.61 in the dimerous plants. It is clear that the two types of plants differ rather fundamentally in this correlation. The correlation between the total bundles and the primary double bundles is very low in the trimerous plants. It is a much more substantial value in the dimerous plants. Pursuing this point one step farther, we may determine by a special formula the relationship between the total number of bundles and the deviation of the number of intercalary bundles from the number which would be expected if the number of primary double bundles and intercalary bundles were in proportion to the total number of bundles formed. Determining the correlation between the total number of bundles, 0, Nov., 1921] HARRIS AND OTHERS — PHASEOLUS VULGARIS 431 and the deviation of the number of intercalary bundles, 7, from their prob- able value by the formula® Poa. 1o/V% VI — ree + (155 — 10/78)? li where 3s = 1 — —0, we have the values given in table 3. TABLE 3. Correlation between Total Bundles ai Base of Hypocotyl and Deviation of Number of Intercalary Bundles from Their Probable Number Trimerous Dimerous : ; Diff. Line : ; Difference Fee -s(1ite N Va ane N Va Bae — Fine ide 7043 5-.0225. | 32.5 | 142 | .8513=:.0156 54.6 | —.0870+.0283 2707, Oars pts 51) -0757=-.0292) | 22-2 155 | .66932-.0299 22.4 |, 00942-0412 | "0.22 GO Ss | .7944/0184 >| 43.2 ‘| 183 | .8433-.0144 58.6 | —.0489 4.0224 2.18 139 | 106] .4066+.0546 7.45 | 305 | .9701+.0023 | 421.8 | —.5636+0.540| 10.3 143 | 221 | .3841+.0386 9.95 | 420} .8510+.0090 94.6 | —.4669+03.96| 11.8 _ The coefficients are positive and high, and very consistent for the two types of seedlings. They show that within one morphological type of seedling® an increase in the total number of bundles is primarily due to the formation of intercalary bundles, rather than to variation in the number of primary double bundles, although both types of bundles contribute to the end result. SUMMARY An investigation of the interrelationship of the numbers of primary double bundles, intercalary bundles, and total bundles (primary double bundles plus intercalary buadles) at the base of the hypocotyl in dimerous and trimerous seedlings of Phaseolus vulgaris leads to the following results: 1. In the trimerous seedlings there is a negative correlation of about medium value (r = —.5 +) between the number of primary double bundles and the number of intercalary bundles. Thus the number of intercalary bundles is smaller in seedlings with larger numbers of primary double bundles and vice versa. In dimerous seedlings the correlation is perhaps also negative in sign, but practically zero numerically. 5 Harris, J. Arthur. The correlation between a variable and the deviation of a de- pendent variable from its probable value. Biometrika 6: 438-443. 1909; also, Further . illustrations of the applicability of a coefficient measuring the correlation between a variable and the deviation of a dependent variable from its probable value. Genetics 3: 328-352. 1918. ‘ 6 The differentiation of trimerous and dimerous seedlings has been shown to be due primarily to an increase in the number of primary double bundles. 432 AMERICAN JOURNAL OF BOTANY [Vol, 8. This result for seedlings of the same morphological type is suggestive in its relation to the results of a comparison of seedlings which are externally dimerous and trimerous, since in general trimerous seedlings show an increase in number of primary double bundles but a decrease in number of intercalary bundles as compared with dimerous seedlings. Asa result of this numerical compensation, most conspicuously evident in the trimerous seedlings, the total number of bundles shows a lower variability than it would if the num- bers of the two types of bundles were quite independent. 2. The correlation between the total number of bundles (primary double bundles plus ‘intercalary bundles) and the number of intercalary bundles is high. The coefficients for the dimerous seedlings are somewhat higher than those for the trimerous seedlings. The correlation between the total number of bundles and the number of primary double bundles is generally much lower. The correlation between the total number of bundles and the deviation of the number of intercalary bundles from that which would be expected if they occurred in the same proportionate frequency throughout the entire range of total bundle number is positive in sign and substantial in magnitude. In both types of seedlings variation in the number of intercalary bundles is therefore an important factor in determining variation in the total number of bundles at the base of the hypocotyl. AREA OF VEIN-ISLETS IN LEAVES OF CERTAIN PEANTS AS AN AGE DETERMINANT M. R. ENsIGN (Received for publication February 26, 1921) The available evidence in support of a theory of senescence in plants is very meager. The work of Minot (7), Child (2), Hertwig (5), Conklin (3) and others establishes quite definitely that complex animal forms are subject to a gradual retardation of physiological functions; also, that this retardation begins in the embryo and continues with more or less acceler- ation until death ensues. Benedict (1) has attempted to show that plants are subject to similar changes of physiological functions. These, he claims, are initiated im- mediately after fertilization and are registered in the increasing complexity of the vascular ramifications in the leaves of certain dicotyledonous plants. In other words, the relative age of such perennial plants as vines, trees, and shrubs can be detected by determining the relative area of the ‘“‘tissue islands”’ or vein-islets formed by the intersecting veins surrounding them. Old or senile plants, therefore, produce leaves whose vein-islets are smaller in area than those in leaves of young plants of the same species grown under similar environmental conditions. Beginning with these premises, the writer (4) studied the venation of leaves produced by polyembryonic Citrus seedlings (Citrus grandis). During the progress of this work certain questions arose regarding the accuracy of the methods employed by Benedict in determining the area of vein-islets in the leaves which he used. In order to shed some light upon this point the work herein reported was undertaken. The data collected are not as extensive as might be desired, but inasmuch as further investi- gation had to be postponed indefinitely, they are presented for what they may be worth. METHODS AND MATERIALS Leaves from the following plants were studied with reference to their venation: Berberis vulgaris L., Berberis Thunberguw DC., Castanea dentata Borkh., Quercus alba L., Fagus caroliniana Fernald and Rehder, Vitis vulpina L., and an undetermined species of Vitis growing in the physiological greenhouse at Cornell University, Ithaca, New York. The trees and vines grew in the immediate vicinity of Ithaca, and the barberries grew on the university campus. In collecting the leaves from these plants the trunk diameters were taken as an index of relative age. For comparison two or more plants 433 434 AMERICAN JOURNAL OF BOTANY [Vol. 8. growing in the same habitat were selected whose trunk diameters were indicative of youth and of old age respectively. Leaves which had approxi- mately equal light exposure were taken from these plants for study. Only mature leaves were used, inasmuch as it has been shown by Bene- dict (1) and by the writer that the area of vein-islets in immature leaves is less than that of vein-islets in mature leaves of the same species. From five to fifteen leaves from each plant were selected and taken to the laboratory. Portions of each leaf were cleared and stained, and deter- minations of the size of vein-islets were made according to a previously described method (4). At least four determinations were made from different places! on the same leaf. Thus, from each plant from twenty to seventy-five determinations of vein-islet areas were made in order 1 to reduce to a minimum the probable error due to variation. The method used by Benedict in determining the area of vein-islets is as follows: The collected leaves were taken immediately to the laboratory, measured as to length, breadth, and area, and weighed. The venation was then photographed in the following way: a heavy black paper was pasted to a clean glass plate, four by five inches in size. Ten openings, approximately four by ten millimeters in size, were then cut in the black paper. From the same part of each leaf pieces a little larger than the openings were cut, and these were laid over the openings, so that each of the ten leaves was represented. A clear glass plate was then laid over all, and the whole was bound together by elastic bands, placed in the negative holder of an enlarging camera, and photographed at an enlargement of three diameters. Negatives showing the veinlets clearly were obtained after some practice, and from these negatives velox prints were made. ... The counting (of the number of vein-islets in the opening) was done under a lens, and a sharp needle was used to prick each vein-islet as it was counted on the photograph. It occurred to the writer that such a method might be conducive to inaccuracy for the following reasons: Leaves growing on the same plant and even on the same twig vary greatly in shape and size. There is no good reason, therefore, for expecting to find such characters as leaf thickness and chlorophyll content constant. In fact, the most casual observation soon discloses the fallacy of such a premise. ‘This being the case, the pro- portion of vascular bundles visible in the uncleared leaves would vary directly with the leaf thickness and the chlorophyll content. Only one determination from each leaf appears to be entirely inadequate to overcome the probable error. It would also appear that the inaccuracy would be exaggerated by each step in the photographing, developing, and printing processes, especially when the magnification used was only three diameters. EXPERIMENTAL DATA In order to find what percentage of the vascular tissue is hidden by chlorophyll, leaves growing under as nearly identical environmental! con- 1 Benedict (1) and the writer (4) have shown that the sizes of vein-islets are quite constant in various places in a single leaf. Nov., 1921] | ENSIGN — VEIN-ISLETS AS AN AGE DETERMINANT 435 ditions as possible were selected. Mature leaves from an unidentified species of grape (Vztvs sp.) growing in the plant physiological greenhouse at Cornell furnished the necessary material. Only mature leaves were used, since it has been shown that the area of the vein-islets in immature leaves is much less than that in mature leaves. Portions of each of thirty-eight leaves from a single plant, having the same light exposure, were examined under the projection apparatus, as described in a previous article (4). The magnification. used was thirty- eight diameters. A similar portion of each leaf was cleared and stained and determinations of the vein-islet area were made with the same apparatus and at the same magnification. Figure 3, Plate X XIII, shows the vascular tissue of the cleared and stained portion of this grape leaf. Table 1 shows TABLE I. Relative Size of Vein-tslets of Uncleared and Cleared Portions of the Same Leaf (Vitis sp.) Uncleared Portion Cleared Portion ree Sec ie alg area ee ein-islets ‘ : Hidden in Number of Leaf . No. Vein- Area No. Vein- Area Uncleared veleeste Vein-islets HHS Vein-islets Leaves Unit Area ie Pas ) Unit Area Gamers) Guereerie) (4 sq. mm.) a US AeSG: mms) ot; ; BP (Ls ca Raa ae ta ee a 14 .2860 30 ees 54 DORN MMe ee Ae os rae dass 14 .2860 34 -EL7O 59 Si pe a ho ae 21 .1904 23 202 37 AP MMe dcr atk «eos ass 13 .3200 34 76. 62 Eee Ror ieee toh aa are of 5 18 2222 35 1143 49 (OR deca ct ene - a nee 25 .1600 30 “1333 17 Teed: Bont <5 soe ile .2353 exes e022 48 Ghd dicue te eee ae 21 .1904 35 -1EAZ 40 OGY as ee 15 .2666 30 pas 50 TO eg ol PASE race ae eae .3200 29 $1379 59 IC Ss eta ahah estihaadae dea eae 16 .2500 29 -1379 39 2g St Sa A a eee Nye 22353 31 .1290 46 PAVIETA CE 8) whe ow we es | 16.9 | .2468 22 .1259 47.5 Mean Average 38 WECANVCS tals a). 20 hs ds 16.9 +.135 2485 2-203 .1240 47.5% a summary of the data obtained from this study. It is evident from this study that many vein-islets are invisible in the uncleared leaves even with a magnification more than twelve times as great as that used by Benedict. Indeed, the average shows that nearly half the vascular tissue is hidden by the chlorophyll, and in some leaves as much as sixty-two percent is invisible. On the other hand, some leaves show the major portion of their vascular tissue in the uncleared, unstained condition. Benedict (1) presents data which show that the vein-islets in uncleared leaves grown in the shade are smaller than those in leaves exposed to direct sunlight. These data interpreted in the light of the experimental results shown in table 1 mean, no doubt, that the leaves grown in the direct 430 AMERICAN JOURNAL OF BOTANY [Vol. 8, light were thicker and contained a larger amount of chlorophyll, so that fewer veins were visible in a unit area. Schuster (8) records a similar con- dition in the leaves of Ampelopsis Veitchit. Inasmuch as Benedict found a direct correlation between age differences and vein-islet areas in leaves from various perennial plants, some of these plants were studied by clearing and staining. The leaves of Fagus caro- liniana were taken from plants growing in close proximity to each other yet having large differences in trunk diameters. A summary of the data from this study is presented in tables 2 and 3. Although Benedict did not study the leaves of the beech, he intimates that the leaves of all the woody perennial plants show senescence by the constantly increasing amount of vascular tissue in their leaves as they increase in age. The data in tables 2 and 3 do not show distinctive differences. TABLE 2. Kelative Size of Vein-islets of Leaves of Fagus caroliniana Fernald and Rehder, from Trees of Different Ages Trunk Diameter 5.4 cm. Trunk Diameter 22 cm. Number of Leaf; No. Vein-islets Area No. Vein-islets Area in Unit Area Vein-islets in Unit Area Vein-islets (4 sq. mm.) (sq. mm.) (4 sq. mm.) (sq. mm.) W scates 5s Meena | 63 .0635 69 .0580 PORN Oe es, ates eee ae 62 .0645 64 .0625 CRE sy eer. | 66 .0606 60 .0666 Bevo e eae oe | 63 .0635 61 .0655 Bik, slasmechee. ace 64 .0625 65 .0615 Orica scene ee, & | 63 .0635 62 .0645 a ee 61 | .0655 62 .0655 Ske NTN 2 63 .0635 63 .0635 Ol ead eee 60 .0666 — TOUR ans ere cavemen 64 .0625 — Se AWVeTave snore 62.9 | .0637 63.2 .0635 TABLE 3. Comparison of Size of Vein-islets in Relation to Age of Tree in Fagus caroliniana Diameter of Trunk (cm.) | 6.2 | Hos | 18 20 Area of Vein-islets (10 Leaves) (sq.mm.)........ | .0678 | .0640 | .0641 .0625 In leaves from specimens of Castanea dentata having a trunk-diameter difference of 49.5 cm., Benedict records a difference in vein-islet area of 0.3 square millimeter. In table 4 a summary of the findings in cleared and stained leaves is shown. Here there is but the very slightest difference shown in the area of the vein-islets. The variation found in individual leaves from the same tree shows as great differences. TABLE 4. Relation of Size of Vein-islets to Age in Castanea dentata Borkh. Diameter of Trunk (cm.) 5.2 | a7. | 50.4 | 38 8 Average Area of Vein-islets of 15 Leaves (sq.mm.)| .0897 .0697 | .0876 | .0677 \ Nov., 1921] ENSIGN — VEIN-ISLETS AS AN AGE DETERMINANT 437° A number of determinations made from the cleared leaves of white oak (Quercus alba) and of Platanus occidentalis revealed no correlation between the size of vein-islets and their relative ages. The barberry leaves studied were from plants of known age. The department of landscape gardening? at Cornell University had several hundred one-year-old seedlings in cultivation. In several places on the campus, barberry plants were known to have been growing from six to twelve years, and were probably several years old when first planted. Leaves from these plants of different ages were cleared, stained, and studied as to venation. A summary of these determinations is presented in tables 5 and 6. TABLE 5. Relation of Size of Vein-islets to Age in Berberis vulgaris L. Known Age of Plants I year 6 years + Average Area of Vein-islets of 35 Leaves (sq. mm.)........... | .2405 | (2278 TABLE 6. Relation of Size of Vein-tslets to Age in Berberis Thunbergit DC. | | Known Age of Plants | 2 years | I2 years + ino eS Average Area of Vein-islets of 18 Leaves (sq. mm.)........... | .2163 | .2196 The conclusions that may be drawn from these results are subject to two interpretations. (1) The age differences may not be sufficiently great to influence very materially the size of the vein-islets. Yet Benedict records instances in which individuals of Vitis vulpina with an age difference of not more than three to five years show a positive correlation. (2) alt may be that this particular perennial does not register its relative age in its more or less complex nervature. Figures I and 2, Plate XXIII, show the nature of venation of Berberis vulgaris. The work up to this point was done while at Cornell in 1916-17. The data presented below were obtained from leaves of Vitis vulpina which were gathered from various places near Ithaca, New York. They were preserved in 85 percent alcohol in test tubes until December, 1918, and were in very good condition. Because of the fact, however, that most of the chlorophyll had been extracted, a comparison between the sizes of vein-islets in cleared and uncleared material was not possible. The plants from which these leaves were taken were selected and marked in the same manner as that described by Benedict (1). The greatest care was taken to secure leaves for comparison that were growing under as nearly similar environmental conditions as possible. Data from cleared and stained leaves only are given in tables 7-12. The results show quite wide variations as to size of vein-islets in the leaves from different vines. The significant fact to be noted, however, is that there is no definite correlation 2 Courtesy of Professor Hunn. 438 AMERICAN JOURNAL .OF BOTANY Viol, #8, TABLE 7. Relation of Size of Vein-islets to Age in Leaves of Vitis vulpina L., Vines rt and 2 Vine 0.8 cm. Diameter Vine 5.2 cm. Diameter Ne (5 Annual Rings) (17 Annual Rings) of Leaves No. Vein-islets Area No. Vein-islets Area in Unit Area Vein-islets in Unit Area Vein-islets (2.25 sq. mm.) (sq. mm.) (2.25 sq. mm.) (sq. mm.) ee Ge el ee os ge Lan: AZT 16.6 .1389 TABLE 8. Relation of Size of Vein-islets to Age in Leaves of Vitis vulpina L., Vines 3 and 4 Vine 1.3 cm. Diameter Vine 10 cm. Diameter Neier (6 Annual Rings) (25 Annual Rings) f Leaf No. Vein-islets Area No. Vein-islets Area in Unit Area Vein-islets in Unit Area Vein-islets (2.25 sq. cm.) (sq. cm.) (2'25‘sq. cm.) (sq. cm.) | eee eae A ehE 1184 20 25 PEEIMN Sel Ee “ke23 2i .1071 BE enig osu Te .1250 20 .1125 Big cc owudanece ate 1184 18 .1250 OE ee Oe .1250 17 1322) OSes seieee 51322 19 1184 Fe ati A halceare ay Give -1125 16 .1531 oR oar RY 19 -1184 17 g23 ON siecle emit ne see 20 Is 16 153% ROW es argo re P| .1071 20 .1125 TOL peraiy tae cates 20 1125 17 51322 FF ae ene are 17, ~1323 18 .1250 AVerage ne. oe ae 18.7 .1205 18.2 .1270 TABLE 9. Relation of Size of Vein-islets to Age in Leaves of Vitis vulpina L., Vines 5 and 6 Vine 1.3 cm. Diameter Vine 5.2 cm. Diameter (6 Annual Rings) (17 Annual Rings) Number L o No. Vein-islets Area No. Vein-islets Area mS in Unit Area Vein-islets in Unit Area Vein-islets (2.25 sq. mm.) (sq. mm.) (2.25 sq. mm.) (sq. mm.) TOUS cthapar eles 13 .1740 16.6 .1389 TABLE 10. Relation of Size of Vein-islets to Age in Leaves of Vitis vulpina L., Vines 7 and 12 Vine 3 cm. Diameter Vine 6.4 cm. Diameter Nine (12 Annual Rings) (18 Annual Rings) f Pee pees No. Vein-islets Area No. Vein-islets Area in Unit Area Vein-islets in Unit Area Vein-islets (2.25 sq. mm.) (sq. mm.) (2.25 sq. mm.) (sq. mm.) TO tao ee 21.8 .1054 18.9 .1192 + Nov., 1921] ENSIGN — VEIN-ISLETS AS AN AGE DETERMINANT 439 TABLE It. Relation of Size of Vein-islets to Age in Leaves of Vitis vulpina L., Vines 9 and 10 Vine I cm. Diameter Vine 4 cm. Diameter Niner (5 Annual Rings) (15 Annual Rings) f ae No. Vein-islets Area No. Vein-islets Area in Unit Area Vein-islets in Unit Area Vein-islets (2.25 sq. mm.) (sq. mm.) (2.25 sq. mm.) (sq. mm.) 1 AT eae een a 21.8 .1042 25.2 .0898 TABLE 12. Relation of Size of Vein-tslets to Age in Leaves of Vitis vulpina L., Vines Ir and 12 Vine 1 cm. Diameter Vine 6.4 cm. Diameter Ne aiber (5 Annual Rings) (18 Annual Rings) of eons No. Vein-islets Area No. Vein-islets Area in Unit Area Vein-islets in Unit Area Vein-islets (2.25 sq. cm.) (sq. mm.) (2.25 sq. mm.) (sq. mm.) 2p a or eae ae 109 1342 18.9 «1 192 between vein-islet area and age differences. The greatest variation was found in the leaves from vines 9 and Io (table 10), where the average area of vein-islets showed a difference of 0.0144 sq. mm. The age difference here was ten years according to the number of annual rings. In contrast to these figures, the data in table 8 are interesting. In this case there is an age difference of nineteen years but a difference of but 0.0065 sq. mm. in the average area of vein-islets, the smaller islets being found in the younger plant. For a similar age difference Benedict records a difference in vein-islet area of 0.2553 sq. mm. It is interesting to make some other comparisons between the sizes of vein-islets as found by Benedict and those obtained by the writer from leaves of the same species, Vitis vulpina (table 13). Of course this com- TABLE 13. Comparison of Vein-islet Areas Obtained from Individuals of Varying Age but of the Same Species (Vitis vulpina L.) Benedict The Writer Number Annual Vein-islet Number Annual Vein-islet Rings Area (sq. mm.) Rings Area (sq. mm.) Se ya he eee Ce 0.4845 5 0.1421 Oe A ee ee 0.3983 6 0.1740 Og 6 Bo ene near 0.3684 6 0.1205 Ole Bocce ste s 0.3983 5 0.1042 HUN Ge ic) oo. ok ose, 0 0.3690 12 0.1054 "6, devate ee ee 0.2966 15 0.0898 pment etre cles Ss save 0.3310 17 0.1389 (Oe 3 > ete 0.2966 17 0.1389 Me ts gs es aes 0.3160 18 0.1192 Bre sie SS oie ws 0.2503 | 25 0.1270 PMVCTACOE ho sc. ee ee 0.3509 0.1260 440 AMERICAN JOURNAL OF BOTANY [Vol, 8, parison is not so instructive as one made from cleared and uncleared leaf portions taken from paired plants and studied by the same methods. However, these data compare favorably with the results obtained from the study of the cleared and uncleared leaves of the undetermined species of Vitis shown in table 1. It will be noted that in both cases the uncleared leaves show vein-islet areas from two to three times larger than those from cleared and stained leaf portions. DISCUSSION The above data show that any study of leaf venation made from un- cleared leaves is wholly unreliable. The varying thickness and chlorophyll content of leaves render many of the smaller veins entirely invisible. Fur- thermore, some unpublished results obtained by Heinicke® do not corrob- orate the preliminary venation studies of uncleared apple leaves made by Benedict. Heinicke finds no correlation between vein-islet area and the age of a large number of apple varieties. These are of known age, 1.e., it is known when they originated as seedlings. The results herein presented do not show a single instance in which the leaf venation might be taken as an index of-the relative ages of the plants in question. While working with yearling Citrus seedlings, a number of grape-fruit leaves were obtained from some of the oldest trees in the vicinity of Miami, Florida, and it was found that the venation of the leaves from the yearling plants was identical with that from the older trees. A similar condition was found in regard to the venation of some orange leaves taken from a plant growing in the Sage greenhouse at Cornell. This plant was probably ten or fifteen years of age. These similarities in venation seem to be indicative of something more than mere coincidences. As intimated at the beginning, it is highly desirable that more data be secured bearing upon this problem. There are certain phases which require more elucidation before satisfactory conclusions can be derived. It may be that the venation of the uncleared leaves of Vztis vulpina and other plants with which Benedict worked shows some correlation with age which the cleared leaves fail to reveal. Such a possibility, however, does not seem tenable. Just as this goes to press the following statement comes from August Henry, Royal Society of Dublin: I tried this [venation vs. age] in the various species and hybrids while working on my paper on “The Origin of the London Kane.”’ Inthis I dealt with the genus Platanus (Proc. Royal Irish Acad.) without any very conclusive results. Here the question lay in regard to whether trees produced of cuttings were as old as the original, or only as young as the time the cuttings were started. SUMMARY AND CONCLUSIONS I. From seventeen to sixty-two percent of the vein-islets are invisible in uncleared leaves of Vitis sp. 3 A. J. Heinicke, assistant professor of horticulture, Cornell University. % dt SIU St SOU eke pee Sree Pee pee ea Wt 5 al my cal Ay Pe AOL LE ale WS 1 Sug TRL ig AL WO Va pees TEE: JOR Re ctl eases ; ES ae \WYOF HOLA All i a . eee ae j a ¥. tks he arn) ie A or A i a ¢ De sy} \ "y a ae 7 e F 5 j ee a cna? : : a ; ; \ , { ~ > . gee * . + \ | } i - ‘ i ' : i \ aT S ‘ i \ 4p — ‘ a i F ‘ ‘ “ = . 7 : ~ ad & ‘ a * * . . . . ' ' “ ‘ 7 ‘ : : ~™ i é Kom, . - ; i we ‘i - ’ 7 ite Cr 7 c >. oe as 1) oe ee Nov., 1921] ENSIGN — VEIN-ISLETS AS AN AGE DETERMINANT 441 2. No correlation was evident between the age of the following plants as indicated by their trunk diameters and the vein-islet area of their leaves: Fagus caroliniana Fernald and Rehder; Castanea dentata Borkh.; Berberis vulgaris L.; B. Thunbergu DC., and Vitis vulpina L: BIBLIOGRAPHY 1. Benedict, H. M. Senile changes in leaves of Vitis vulpina L. and certain other plants. New York Agr. Exp. Sta. (Cornell) Memoir 7: 281-370. I915. 2. Child, C. M. The process of reproduction in organisms. Biol. Bull. 23: I-39. 1912. Senescence and rejuvenescence. pp. 481. Chicago, I915. 3. Conklin, E.G. Cell size and nuclear size. Jour. Exp. Zool. 12: I-98. I912. The size of organisms and their constituent parts in relation to longevity, senescence and rejuvenescence. Pop. Sci. Mo. 83: 178-198. 1913. 4. Ensign, M. R. Venation and senescence of polyembryonic Citrus plants. Amer. Jour. Bot. 6: 311-329. I9I19. 5. Hertwig, R. Ueber Korrelation von Zell- und Kerngrésse und ihre Bedeutung fiir die geschlechtliche Differenzirung und die Teilung der Zelle. Biol. Centralb. 23: 194- 223. 1903. 6. Jennings,H.S. Age, death and conjugation in the light of the work on lower organisms. Pop. Sci. Mo. 80: 563-577. I9Q12. Effect of conjugation in Paramecium. Jour. Exp. Zool. 14: 279-391. 1913. 7. Minot, C. S. The problem of age, growth and death. pp. 280. New York, 1908. 8. Schuster, W. Die Blattaderung des Dicotylenblattes und ihre Abhangigkeit von ausseren Einflussen. Ber. Deutsch. Bot. Ges. 26: 194-237. 1908. EXPLANATION OF PLATE XXIII ay figures are drawings made from the projection of the cleared and stained leaves. X 38. Fic. 1. Venation of a leaf from 6-year-old barberry (Berberis vulgaris), Fic. 2. Venation of a leaf from 1-year-old barberry (Berberts vulgaris). Fic. 3. Venation of a mature leaf from undetermined species of Vitis. Fic. 4. Venation of grape leaf (Vzizs vulpina) having a trunk diameter of 1.3 cm. and showing 6 annual rings. Fic. 5. Venation of a leaf from chestnut (Castanea dentata) having a trunk diameter of 38 cm. Fic. 6. Venation of a leaf of grape (Vztes vulpina) having a trunk diameter of 10 cm. and showing 25 annual rings. Fic. 7. Venation of a leaf from grape (Vitis vulpina) having a trunk diameter of 0.8 cm. and showing 5 annual rings. Fic. 8. Venation of a leaf from grape (Vitis vulpina) having a trunk diameter of 5-2 cm. and showing 17 annual rings. Fic. 9. Venation of a leaf from grape (Vitis vulpina) having a trunk diameter of 6.4 cm. and showing I8 annual rings. Fic. 10. Venation of a leaf from chestnut (Castanea dentata) having a trunk diameter Ol 2-7 Cm. UNUSUAL RUSTS ON NYSSA AND URTICASTRUM } E. B. MAINS (Received for publication February 25, 1921 ?) During the past year, two very interesting rusts of the family Melamp- soraceae have come to the writer’s attention. The first of these, upon Nyssa aquatica, has remained in the form genus Uredo since its description in 1890 by Ellis and Tracy under the name of Uredo Nyssae. While pre- paring the manuscript of this species for the North American Flora, the writer was fortunate in discovering the telia. A study of this stage shows that the species can not be placed in any established genus and in conse- quence the following genus is proposed. Aplopsora® gen. nov. Cycle of development imperfectly known, only uredinia and _ telia recognized, both subepidermal. Uredinia pulverulent; urediniospores produced singly, echinulate, the dores obscure. Telia lenticular, at first covered by the epidermis, soon becoming naked and cinereous from germination; teliospores one-celled, cylindric, in one layer, the wall thin, colorless, smooth, germinating shortly after reaching full size. Aplopsora Nyssae (Ellis & Tracy) comb. nov. Uredo Nyssae Ellis & Tracy. Jour. Myc. 6:77. 1890 O-+ 1. Pycnia and aecia unknown. II. Uredinia hypophyllous, scattered, round, minute, 0.1-0.3 mm. across, early naked, pulverulent, cinnamon-brown, ruptured epidermis inconspicuous; paraphyses peripheral, united below into a short, incon- spicuous pseudoperidium, clavate, incurved, 16-26 uw long, the wall Iu thick, on convex side above up to 3-4 »p, brownish-yellow; urediniospores obovoid or oblong, 13-17 by 16-26 uw; wall yellow or pale cinnamon-brown, I uw, rather closely and finely echinulate, the pores obscure. III. Telia hypophyllous, gregarious in small groups, round, small, 0.2—0.5 mm. in diameter, at first covered by the epidermis, soon becoming naked, very pale translucent yellow, becoming cinereous from germination; teliospores cylindric, 7-15 by 29-40 uw, rounded above and below, in one layer; wall colorless, very thin, 0.5 uw or less, uniform in thickness, smooth, soon germinating with typical, external basidia. 1 Contribution from the Botanical Department of the Purdue University Agricultural Experiment Station. Read in part before the Mycological Section of the Botanical Society of America at Chicago, December 29, 1920. 2 Culture results revised to July I, 1921. 3 From amdéos, simple, and Vawpa, scab, referring to the telium of one spore layer. 442 Nov., 1921] MAINS —— RUSTS ON NYSSA AND URTICASTRUM ~ 443 Missa aquatica ., Jackson, Miss., Oct. or Nov. 12, 1888, I] & III, S. 1. Tracy 1200 (type)*; Ocean Springs, Miss., Nov. 4, 1891, II, F. S. Earle; @cean Springs, Miss., Nov. 8, 1891, Il, 7. S. Harle; Great Cypress Swamp, Calvert City, Kentucky, II, W. W. Eggleston; obtained from a phanerogamic specimen, no. 5374, in the herbarium of the New York Botanical Garden by H. S. Jackson. The uredinia of this rust do not differ markedly from those of a number of genera belonging to the Melampsoraceae. The incurved paraphyses bordering the uredinium and united below into a short pseudoperidium are characteristics which are found in some species of Phakopsora (figs. I, 2). The urediniospores are borne in a very similar manner to that described by Butler® for Cerotelium Fici (Cast.) Arth. (Kuehneola Fict Butler), the Fic. 1 (Left). Paraphyses of A plopsora Nyssae showing short pseudoperidium at their bases. Fic. 2 (Right). Portion of uredinium of Aplopsora Nyssae showing peripheral position of paraphyses and manner in which urediniospores are borne. hymenium consisting of a mass of cubical cells upon the uppermost of which the spores are borne (fig. 2). The cells bearing the spores have little to distinguish them from the other cells of the hymenium except that they are separated somewhat from each other. Whether these cells are to be considered as pedicels or whether, as Butler suggests for C. Fici, they in turn may develop into spores, cannot be determined from the material at hand, but there is no evidence that the spores form chains. The telia on the other hand characterize this rust as generically distinct. The teliospores arranged in a one-layered crust of cylindric, one-celled teliospores would place this species in a group with Melampsora, Melamp- soridium, and Chnoopsora (fig. 3). From the first two it is distinct tno only in uredinial characters but in that the telium soon ruptures the epider- mis and the teliospores germinate at once. In these characters it is much like species of Chnoopsora but differs in the method of teliospore formation. In species of Chnoopsora the teliospores are produced over a period of time due to young sporogenous hyphae developing between the older ones 4The description of Ellis gives the host as N. capitata and the date as Nov. 1888. Small, in his Flora of the Southeastern United States, gives the range of N. capitata as South Carolina to Georgia and Florida. Since other collections of this rust are on N. aquatica, it is likely that this collection is also on that host. There is also some confusion as to the date of the collection, some packets being labeled 10/12/1888 and some 11/12/1888. ‘Butler, E. J. Notes on some rusts in India. Ann. Mycol. 12: 76-82. 1914. 444 AMERICAN JOURNAL OF BOTANY [Vols . and forming teliospores to replace those which have germinated; while in A plopsora Nyssae the teliospores are all formed and matured at practically the same time without subsequent spore production. umm Fic. 3. Section through mature telium of Aplopsora Nyssae showing arrangement of spores in one layer. Just what the complete life cycle of Aplopsora Nyssae may be is difficult to say. The early germination of the teliospores without a resting period, if this rust is autoecious, would apparently necessitate the production of pycnia and aecia or pycnia and uredinia immediately following infection or else the development of a systemic mycelium from which such stages would be produced the following spring. The herbarium material available does not show either condition and it is probable that this rust is heteroecious. The aecial stage and probable aecial hosts cannot be foretold definitely. The related genus, Melampsora, has for its aecial stage a Caeoma with either subcuticular or subepidermal pycnia. The alternate hosts belong to a number of genera, mostly, however, conifers. Chnoopsora has for its aecial stage a Caeoma with subcuticular pycnia, the species with known life cycle being autoecious. Melampsoridium Betulae (Schum.) Arth. has a peridermium with subcuticular pycnia on Larix. In consequence it would be expected that Aplopsora Nyssae would have a Caeoma or a Peridermium for its aecial stage, probably upon some conifer. Specimens of Caeoma strobilina Arth. on pine are in the Arthur herbarium from Gulfport and Agricultural College, Mississippi, which may possibly be the alternate stage af the Nyssa rust. Hedgcock and Hunt® have reported connecting this rust with a Uredo on Quercus from material collected in Florida. They, however, mention that some of the collections of Caeoma strobilina have pseudoperidia and that another rust is represented here, which is unconnected. There is also the possibility that this rust may produce a Peridermium on pine which is at present confused with one of the many species of Peridermium found in the south. The second rust occurs on Urticastrum divaricatum and was received by Dr. J. C. Arthur from Prof. H. W. Anderson who wrote that he had collected it for a Synchytrium but upon examination found what he thought were uredinia. An examination of the material showed that Professor Anderson 6 Rhoads, A. S., Hedgcock, G. G., Bethel, E., and Hartley, C. Host relationships of the North American rusts, other than Gymnosporangiums, which attack conifers. Phytopath. 8: 309-352. 1918. Nov., 1921] MAINS — RUSTS ON NYSSA AND URTICASTRUM 445 was correct in considering the fungus a rust and also disclosed that in addition to the uredinia abundant telia were present, most of which were white from the germination of the teliospores. A study of this rust showed that the telium consisted of a crust of one-celled, colorless teliospores, borne in chains of two or three. In these and other characters the rust seemed to belong in the genus Cerotelium. The characteristics and rela- tionships of the rust all pointed to a heteroecious life cycle. Since infection would have to occur in the fall, it appeared quite likely that the aecial stage either developed upon biennial parts of the host such as the needles of some conifer or was systemic. Since conifers were not to be found in the vicinity of the Urticastrum rust at Urbana, it appeared more likely that the aecial stage was systemic. Dr. Arthur immediately suggested Aecidium Dicentrae Trel. as the likely aecia! stage, since not only is this rust systemic but unlike the usual Aecidium of the region it possesses large, subcuticular pycnia, a characteristic of many of the Melampsoraceous rusts. This conclusion was greatly strengthened by discovering in the herbarium a specimen of the Aecidium collected by Professor Anderson in the same woods earlier in the season. The only apparently serious objection to this connection was the manner of growth of Bicuculla Cucullaria (L.) Millsp. (Dicentra Cucullaria Torr.). This plant develops and flowers early in the spring and then soon dies down, so that by the time teliospores of the Urticas- trum rust are germinating, there is nothing of the Bicuculla plant above ground except an occasional corm. This connection would therefore necessitate an unusual type of infection. Aecidium Dicentrae, however, resembles so closely what it was felt the aecial stage should be that sowings were made by placing leaves bearing germinating teliospores of the Urticastrum rust on soil containing corms of Bicuculla Cucullaria. The pots of corms were placed out of doors during the winter and then brought into the greenhouse early the next spring. No infection appeared. In spite of this, it was still felt that the rusts of Urticastrum and of Bicuculla were connected. Another attempt was made this spring (1921) by sowing aeciospores of the Bicuculla rust sent from Urbana, Illinois, by Professor Anderson. ‘This sowing produced typical uredinia upon Urticastrum divaricatum. The lack of results from the sowings of basidiospores upon the Bicuculla corms may have been due to the effect that the high temperature of the greenhouse had upon the development of the plants, since infected corms sent by Professor Anderson in the summer of 1920 when brought into the greenhouse this spring showed the rust in only a few cases and then only pycnia were pro- duced. | Cerotelium Dicentrae (Trel.) Mains and Anderson comb. nov. Aecidium Dicentrae Trel. Trans. Wis. Acad. Sci. 6: 136. 1884. O. Pycnia amphigenous, somewhat scattered, usually near the margin of the leaf, conspicuous, subcuticular, violet becoming dark chestnut or chocolate-brown, applanate or discoidal, 160-200 yw in diameter by 40-60 u high; ostiolar filaments wanting. 446 AMERICAN JOURNAL OF BOTANY [Vol. 8, I. Aecia hypophyllous, subepidermal, scattered over the entire leaf, cupulate, 0.1-0.5 mm. in diameter; peridium white, the margin remaining somewhat incurved, erose; peridial cells rhomboidal in side view, 15-20 by 24-35 uw, overlapping considerably, the outer wall 7—9 y» thick, faintly trans- versely striate, the inner wall 3-5 thick, closely and finely verrucose; aeciospores somewhat angularly globoid or ellipsoid, 12-17 by 13-21 yu; wall colorless, thin, I yu or less, closely and very finely verrucose. Bicuculla Cucullaria (L.) Millsp. (Dicentra cucullaria Torr.), Pine Hills, Union Co., Ill., April 24, 1882, A. B. Seymour 4252; Madison, Wis., June, 1884, L. H. Pammel; Decorah, Ia., May, 1886, E. W. D. Holway (Barth. N. Am. Ured. 203); May 18, 1887 (Sydow, Ured. 497); Iowa City, la., May 7, 1887, Thos. H. Macbride; Morning Sun, Iowa, April 16, 1895, Geo. W. Carver; Manhattan, Kan., May, 1888 (Kellerm. & Swingle, Kan. Fungi 2); Topeka, Kan., May 9, 1904, H. W. Baker (Ellis & Ev. Fungi Columb. 1903); Oakwood, S. Dakota, May 9, 1801, £. Ni Walco. Crawfordsville, Ind., June, 1893, E. W. Owe; Concordia, Mo., June 20, 1888, May, C. H. Demetrio (Rab.-Paz. Fungi Eur. 4335 ; Nebraska, 1899, A.A. Hunter; Nebraska City, Nebr., April, 1899, Thornber; Lancaster, Pa., May 5, 1900, A. A. Heller 4972; New York City) Aprazr, wens May 4, 1914, fF: D. Fromme; Van Cortlandt Park,’ New York Gin April 25, 1912, F. D. Fromme; April 20, 1915, P. Wilson 52; Williams- bridge, New York City, April 28, 1916, P. Wilson 230; vicinity of Grassy Sprain Reservoir, Westchester Co., N. Y., May 27, 1916, 2. Wilson 248 ; West Orange, N. J., May 9, 1915, P. Wilson 59; Brownfield Woods, Urbana, Ill., May 18, 1919, H. W. Anderson. Type Locavity: Madison, Wisconsin, on Dicentra Cucullaria. II. Uredinia hypophyllous, few, scattered or in small groups, I-2 mm. across, round, small, 0.I-0.2 mm., remaining partially covered by the epi- dermis, pulverulent, yellow, ruptured epidermis evident; paraphyses peripheral, hyphoid, 7-10 by 26-48 uw, thin-walled, colorless, incurved, inconspicuous, not projecting above the ruptured epidermis, arising from a more or less developed pseudoparenchymatous mass of mycelium; ure- diniospores ellipsoid or obovoid, 18-21 by 20-26 uw, without definite pedicels, attached to short, thin-walled, colorless cells; wall colorless, I-1.5 wu thick, closely echinulate, the pores obscure. III. Telia somewhat gregarious in groups I-3 mm. across, at first arising within and surrounding the uredinia, angular, 0.2-0.5 mm. across, at first covered by the epidermis, becoming naked just before germination, waxy, slightly tinted, becoming flocculose and white from germination, teliospores cylindric or ellipsoid, 10-21 by 29-42 yw, catenulate, in chains of 2 or 3 at the center of the sorus, usually only one at the margin; wall colorless, very thin, uniformly 0.5 « thick; basidiospores globoid, 10-13 p in diameter. Urticastrum divaricatum (L.) Kuntze (Urtica divaricata L., Laportea cana- densis Gaud,), Brownfield’s Woods, Urbana, Ill., Aug. 19, 1919, Hl & HI, Z. W. Anderson; Hannibal, Wis., July 27, 1920, Ill, J) ans, In the Urticastrum rust we have a telium differing from that of the Nov., 1921] MAINS — RUSTS ON NYSSA AND URTICASTRUM 447 Nyssa rust principally in the catenulate method of spore formation, the teliospores developing in chains of two or three (figs. 4, 5). The terminal Fic. 4. Section through young telium of Cerotelium Dicentrae showing catenulate character of the teliospores. t spore of these chains often germinates before the lower spores are fully developed, and it may be that more spores are produced from the sporog- enous cell than show at any one time. The catenulate character of the teliospores indicates that this species is related to a group consisting of such genera as Phakopsora (Physopella, Bubakia, Schroeteriaster), Uredo- peltis, Melampsoropsis, Chrysomyxa, Baeodromus, Alveolaria, and Cero- ‘telium. In Alveolaria the arrangement of the teliospores in definite layers which separate from each other, and in Baeodromus the colored thick- walled teliospores with delayed germination, taken with the short life Fic. 5. Section through mature telium of Cerotelium Dicentrae showing germination of the teliospores. 448 AMERICAN JOURNAL OF BOTANY [Vol. 8, cycle of both genera, afford reasons for excluding the Urticastrum rust from these genera. This rust is to be distinguished from species of Uredo- peltis and Phakopsora by the colorless walls of the teliospores and by their germination without a resting period. Melampsoropsis, Chrysomyxa, and Cerotelium are genera in which the teliospores germinate without a resting period and are arranged in closely compacted chains. There is no characteristic of the telium which would necessarily prevent this rust from belonging to any of the last mentioned genera, except perhaps that there are a smaller number of teliospores in a chain in the Urticastrum rust than are usually found in the rusts of these genera. The uredinium of the Urticastrum rust, however (fig. 6), is bordered by a few colorless, incurved paraphyses, and the walls of the echinulate urediniospores are colorless and the spores are borne in a manner similar to that in Aplopsora Nyssae. It therefore differs in these respects from spe- cies of Melampsoropsis (Chrysomyxa of some authors) which have catenu- late, verrucose urediniospores, usually with a surrounding peridium. Chry- somyxa as used by Arthur lacks uredinia and in consequence will be dis- Fic. 6. Section through uredinium of Cerotelium Dicentrae showing peripheral paraphyses and manner in which urediniospores are borne. regarded. This species might be placed in the genus Cerotelium as originally. described by Arthur’ (p. 30), except for the presence of paraphyses in the uredinium instead of a peridium. As a result of the study of additional material Arthur’ (pp. 505-507) brought together in each of the genera Phakopsora, Cerotelium, and Cronartium species with a peridium, species with paraphyses united below into a pseudoperidium, species with hyphoid or incurved paraphyses, and species with neither peridium nor paraphyses. In the first two genera, species can be found showing gradations, such as incurved colorless paraphyses, colored thick-walled paraphyses, paraphyses united to a greater or less extent to form a pseudoperidium, paraphyses accompanying a peridium, and a peridium only. In consequence the 7 Arthur, J.C. New species of Uredineae. Bull. Torrey Bot. Club 33: 27-34. 1906. 8 Arthur, J. C. Relationship of the genus Kuehneola. Bull. Torrey Bot. Club 44: 50I-5I1I. I917. Nov., 1921] MAINS — RUSTS ON NYSSA AND URTICASTRUM 449 presence of paraphyses seems no reason for excluding this rust from the: genus Cerotelium, the smaller number of teliospores in chains being hardly more than is to be expected in a simpler species of the genus. The Sydows® (pp. 524, 525) consider Cerotelium as a synonym of Dietelia and transfer the type species Cerotelium Canavaliae Arth. of the former genus to the latter genus. According, however, to their description of the genus Dietelia, the presence of a peridium around the telium is the most important characteristic of this genus. The telia of.C. Canavaliae as such do not have a peridium, but when they arise in the old uredinial sorus, as they often do, they are of course surrounded by the peridium there present. Neither does the telial peridium of Dietelia resemble in structure the uredinial peridium of Cerotelium, and for these reasons the writer is of the opinion that Dietelia and Cerotelium should be considered as distinct genera. In the discovery of the Aecidium of Cerotelium Dicentrae the first clue to an aecial stage of rusts of this type has been obtained. The combination of subcuticular pycnia with the cupulate aecium surrounded by a peridium is such as was to be expected from the relationship of the genus Cerotelium to other genera of the Melampsoraceae. The systemic nature of the mycelium is probably specific and accounts for the survival of Cerotelium Dicentrae in a temperate climate. The infection of Bicuculla Cucullania by the basidiospores of the rust must occur through the dormant buds of the corm, which are either exposed or immediately below the surface of the soil. Such a type of infection would apparently necessitate a close association of the two alternate hosts and probably accounts for the rather localized occurrence of the rust. | DISCUSSION OF RELATIONSHIPS The character of early maturity and germination of the teliospores has been given considerable prominence in establishing the position of these two rusts. This is a character which in some groups of the rusts is of little or no significance. Here, however, on account of the evident grouping and relationship of rusts with this character it appears to take on con- siderable importance. Thus if we consider the rusts of the Melampsoraceae which have teliospores germinating without a resting period, they would appear to group themselves in certain definite lines of development. Start- ing with Aplopsora we have teliospores in a one-layered crust which tardily breaks through the epidermis and germinates at once. In Cerotelium Dicentrae we have another step in which a number of the sporogenous hyphae cut off two and three spores in succession to form a compact crust which, on account of its continued spore formation, rather readily ruptures the epidermis. The next type is represented by Cerotelium Canavaliae in which the number of spores produced by the sporogenous hyphae is greater and in consequence the epidermis is quickly ruptured and the telium is 9 Sydow, P., and Sydow, H. Monographia Uredinearum 3: 1-726. I915. A450 AMERICAN JOURNAL OF BOTANY [Vol. 8, pushed up farther above the epidermis. The telium here is less compact and with less evident lateral coherence of its spore chains. At this point in the development of this group, or a little before, a separation into two distinct lines apparently takes place. In one line there is an increase in the teliospore production from the sporogenous hyphae and a stronger lateral coherence of the spore chains; and hair-like columns of teliospores are formed, giving us the genus Cronartium. In the other line, there is also a greater spore production resulting in longer chains of teliospores, but at the same time the lateral coherence of the chains lessens until finally in the genus Kuehneola there is a complete separation to the base and a falling apart of the spore chains. In consequence of such a development, as might be expected, there is no sharp line of separation between the genera Cerotelium and Kuehneola and some species are in consequence difficult to place, some authors referring them to one genus and some to the other, depending upon their interpretation of the limitations of these genera. As an example of this transition from one genus to the other, we have the following: Cerotelium Gossypit (Lagerh.) Arth. possesses a compact telium much like that of C. Canavahae. In Cerotelium Fici (Cast.) Arth. and in C. Vitis (Butl.) Arth., the teliospore columns are much more loosely arranged, as has been: shown by Butler,!° but the spore chains still hold together and show only a slight tendency to fall apart. In the case of Kuehneola aliena Syd. & Butl. and especially in K. Butlert Syd. we have two rusts which have been placed in the genus Cerotelium by Arthur (J. c. footnote 8, p.510). In these species, although the spore chains are short and in consequence do not separate as widely as in some species of Kuehneola, the separation is, however, definite; and it would appear best to consider both as species of Kuehneola. Although Dietel! (pp. 205-213) was the first to point out the catenulate manner of teliospore formation in Kuehneola as distinguishing it from the genus Phragmidium, yet he retained the genus in the Puccini- aceae, considering it as having developed from Uromyces species on Rubus. Kuehneola was, however, removed to the family Melampsoraceae by Arthur (J. c. footnote 7), largely on account of this catenulate character of the teliospore, and these transitional forms support such a disposition of the genus. The other genera of the Melampsoraceae with teliospores germinating without a resting period are Chnoopsora and Melampsoropsis. The former may be considered as arising from the same source as Aplopsora but diverg- ing upon what may possibly be another line of teliospore formation. The latter may have arisen from a similar source, most likely from a form resembling Melampsora but differing in the development of catenulate urediniospores. The method of urediniospore formation in the Aplopsora- Butler, E. J. The rusts of wild vines in India. Ann. Mycol. 10: 153-158. 1912. Especially pp. 156-158. Also, /. c. footnote 5, pp. 76-79. 4 Dietel, P. Uber die Verwandtschaftsbeziehungen der Rostpilzgattungen Kucaseoe und Phragmidium. Ann. Mycol. 10: 205-213. Ig12. Nov, 1921] MAINS — RUSTS ON NYSSA AND URTICASTRUM 45I Kuehneola line may possibly be considered as representing an original potentiality in the ancestral type which developed in Melampsoropsis but which in the Aplopsora-Kuehneola line became gradually weaker. Phakop- sora with the delayed germination of its teliospores may be considered as a similar but less fully developed line from a source similar to that giving rise to Cerotelium. | Both the rusts described above have offered considerable difficulty in the determination of their generic position. Itisin this, however, that their principal interest lies, for such species are to be expected as the result of evolutionary development and from such our knowledge of relationships must be obtained. With our present imperfect knowledge of the rusts of ' the family Melampsoraceae, it is perhaps impossible to gain more than a suggestion of the possibilities which exist. It is felt, however, that these two rusts, Aplopsora Nyssae and Cerotelium Dicentrae, in their evident relationships point to lines of development, the importance of which will have to be left for future information and studies fully to bring out. To Prof. H. W. Anderson credit is due for the discovery of Cerotelaum Urticastrt and for the trouble he has taken in solving the life history of this rust. The writer wishes to express his appreciation to Dr. J. C. Arthur for the opportunity of studying the Urticastrum material and for advice and criticism in the study of these two interesting rusts. To Prof. H. 5. Jackson acknowledgment is due for aid in obtaining additional material of Ablopsora Nyssae in the New York Botanical Garden herbaria and for helpful criticism of this work. PURDUE UNIVERSITY AGRICULTURAL EXPERIMENT STATION, LAFAYETTE, INDIANA MISCELLANEOUS: STUDIES ON. TEE CROWN RUSf (On OATS G. R. HoERNER (Received for publication March 7, 1921) During a somewhat extensive study of the infection capabilities of crown rust of oats, Puccinia coronata Cda. (1), the following data were collected. I McAlpine (2) ventures the opinion that crown rust of oats was probably introduced into Australia by means of seed. He does not state whether he thinks the rust was carried within or upon the seed in the form of my- celium or of urediniospores. As far as surface-borne urediniospores are concerned, it seems question- able whether under ordinary conditions urediniospores would remain viable upon the seed surface long enough to be transported to any great distance and still be able, after relatively long periods of time and probably under adverse environment, to infect the developing seedlings. In an attempt to throw some light upon this question the following experiment was devised: Twenty oat seeds of a variety known to be susceptible to crown rust (Victory, Minn. 514) were moistened in water and heavily smeared with fresh viable urediniospores. Five seeds were then planted about one half inch deep in each of four four-inch pots of a uniform soil mixture. These pots were placed in a ventilated cage in order to protect the developing seedlings from chance infection from air-borne spores. After the seedlings had been allowed to grow for ten days, a sufficient period to show evidence of infection, it was found that none of the twenty seedlings became infected. The temperature within the cage was previously determined to be an optimum one, since artificial inoculations of seedling leaves of the same variety in the same environment resulted in normal infection, and moisture was present in sufficient amounts to cause guttation from the seedling leaves. These results, though the experimental work was not extensive, would seem to indicate that in the case of Puccinia coronata Cda., urediniospores borne upon the surface of the seed do not commonly offer a favorable means of spreading the rust to the seedling plants developed from these seeds. 1 Investigations carried on while the author was a graduate student at the University of Minnesota, 1916-1918. 452 f ms | Xe nee i Fw} L a é Wei es Hl ee Nov., 1921] HOERNER — STUDIES ON THE CROWN RUST OF OATS A53 ‘ Ny 4\ oO oo | II Ntional Muse aac te In the field, the soil beneath cereals heavily rusted with P. graminis Pers. is often found literally covered with fallen urediniospores. The idea has been conceived that seedlings penetrating such soil might become infected and the rust be aided in its spread in this way. Greenhouse experiments have proven this possible, it is reported, with P. graminis Pers. In order to determine if such infection is possible in the case of P. coronata Cda., the following experiment was devised: After soaking in water for twenty-four hours, six oat seeds were planted ~ about one half inch deep in each of four four-inch pots of a uniform soil mix- ture. The surface soil was then heavily dusted with fresh viable uredinio- spores. These pots were then placed in a ventilated cage to avoid possible chance infection of the seedlings by air-borne spores. Watering was avoided in order to prevent germination of the spores before the seedlings should come in contact with them. After ten days’ time none of the twenty-two seedlings that developed showed any signs of infection, though guttation occurred from the seedling leaves, affording optimum conditions for spore germi- nation. These results indicate at least that seedling infection caused by emer- gence through soil densely covered with viable urediniospores does not occur readily. This condition may possibly be due to the fact that the sheath which surrounds the emerging seedling is not supplied with stomata and therefore affords no opportunity for the entrance of the germ tubes. III The possibility of urediniospore-producing mycelium overwintering in the host plant and producing a new crop of urediniospores in the spring, together with the overwintering of urediniospores in the field, was considered. Christman (3) found, under Wisconsin conditions, viable urediniospores at any time during the winter with a three-months’ period during which the temperature hovered about the freezing point. Urediniospores from oats, developed upon protected plants during the winter, germinated as late as January 26. Indications were that the mycelium would be as resistant as the host within which it grew. Old spores remained viable for some time, though new crops of spores from overwintered mycelium seemed to be the more important mode of spring infection. Reed and Holmes (4) found viable urediniospores on oats throughout the year under Virginia conditions. They conclude that the crown rust on oats has an enduring mycelium capable of producing a new crop of spores during much of the winter, and although spore production ceases during midwinter, the mycelium, upon the advent of warm weather, is capable of producing new crops of viable spores. The urediniospore-germination studies that the writer has performed would seem to indicate that under Minnesota conditions urediniospores 454 AMERICAN JOURNAL OF BOTANY [Vol. 8, cannot withstand the extremely low temperatures of winter. In the field, even before winter had set in, all urediniospores had disappeared and only the teliospores were in evidence. Two pots of heavily rusted oat plants were allowed to remain outside during the winter. The plants were winter- killed and when removed to the greenhouse in early April did not revive. The urediniospore-producing mycelium, if still alive, which one would naturally doubt, produced no new crop of spores. From this more or less limited observational evidence, then, it seems improbable that under Minnesota conditions a perennial mycelium exists which is capable of producing a new crop of urediniospores the following spring after overwintering on the infected oat host, though Bolley and Pritchard (5) consider it in general quite possible, even though no experi- mental data are offered to substantiate the opinion. It seems equally improbable that the urediniospores themselves can overwinter and cause infection the following spring. Just what possibility there is of the existence of a perennial mycelium or of the overwintering of the urediniospores among the wild grasses, has not been determined. IV Mains (6), working with Puccinia coronata Cda., has shown that low temperatures, lack of moisture in the moist chamber, and the absence of light retard the development of the leaf rust of oats. These same observations have been made in the present work, though only one definite experiment was performed and that to determine the effect of light upon the degree of infection and the rate of pustule formation. Four pots of oats of the same seed lot, grown under the same conditions, were inoculated on the same date with inoculum from the same source. Two pots were placed in a pan of water and covered with a glass bell jar; the other two pots were given similar moisture and temperature conditions though covered by a glass-topped metal moist chamber from which light was excluded. All four pots were removed from the moist chambers after forty-eight hours but retained for two days more under the light and dark covers. At the end of this period the plants in the dark had become spindly and dis- tinctly yellowed. Flecks appeared on all the seedlings in all the pots at about the same time. Pustules ruptured within ten days upon the plants kept in the light and within twelve days on the plants kept in the dark. Infection, one hundred percent in each case, appeared normal on all the seedlings, though not so heavy on the plants grown in the dark. The plants that had been kept in the dark, after several days’ exposure to the light showed nearly normal growth, though the effect of etiolation was evidenced by dead areas at the tips of the leaf blades. Twenty-eight days after inoculation pigment appeared on one of the plants that had been exposed to the light, while twenty-six days after Nov., 1921] HOERNER — STUDIES ON THE CROWN RUST OF OATS 455 inoculation a profuse production of teliospores was noted on every plant in one of the pots that had been kept in the dark. (See figure 1, Plate X XIV.) V The appearance of a purple pigment surrounding infected areas of the oat leaves inoculated with crown rust isnot uncommon. ‘The variety of the host plant, its age before inoculation, the length of time of infection, the history of the inoculum, its method of application, and all other externally visible environmental factors seem to have no direct correlation with this phenomenon of pigment formation. Wheldale (7), regarding this anthocyanin pigment formation in plants attacked by fungi, says: It is frequently found that the pathological conditions called forth by the attacks of fungi are accompanied by abnormal development of anthocyanin. In leaves of Tussilago, for instance, infected by Puccinia, a circular band of anthocyanin often appears surrounding the aecidium spots. . . . Injury to the living tissues of the conducting system of the veins, midrib or petiole of the leaf, or of corresponding tissues in the stem, leads to an accumulation of synthetic products in the leaves. . . . It seems likely also that parasitic growths may interfere with the progress of the translocation current through the small veins of the leaf, thereby causing congested areas to arise in which the sugar contents are above normal. But it is conceivable that the pathological condition resultant on fungal attacks may be the direct cause, in some way, of pigment formation. In view of this interpretation and of the relatively general occurrence of this anthocyanin pigmentation during the course of the studies recorded in this paper, the assumption seems justified that pigment formation, as a phenomenon connected with the infection of oats by P. coronata Cda., is not a sign of resistance on the part of the host to the attacks of the rust parasite. VI Parker (8), speaking of early production of telia on oat seedlings, says: It is certain that in the hundreds of seedlings described as very susceptible in the present experiments telia were not produced ona single one following a normal and abundant production of uredinia. In the investigations reported in this paper, this was not the case. Although certain oat varieties showing resistance to attacks of the crown rust did produce telia, other very susceptible varieties also produced telia freely and in great abundance. Super-susceptibility on the part of the host may bring about the formation of telia due to conditions as explained by Wheldale, although resistant hosts may react in some way so as to be unfa- vorable to continued urediniospore production on the part of the fungus, and thus hasten the completion of its life cycle and the early production of telia. Parker has used this phenomenon of teliospore production, in certain cases, as a basis for the classification of resistant varieties. In view of results recorded here, such a basis for the classification of resistance would seem unreliable. Ligowa oats were listed as susceptible, and yet, during the 456 AMERICAN JOURNAL OF BOTANY [Vol. 8, course of these experiments, Ligowa oats, although heavily infected, pro- duced pigment and telia both when growing under normal conditions and when subjected to adverse environmental circumstances. (See figure I, Plate XXIV.) Avena sterilis L. is considered by Parker as for the most part susceptible. In the experiments here reported it produced pigment, telia, and extensive hypersensitive areas. Figure 2, Plate XXIV, shows leaves of Avena sterilis L. infected by Puccinia coronata Cda. The strain? of rust from Saint Paul, Minnesota, indicated by ‘‘S,’’ caused a very light infection, the appearance of small scattered uredinia and large hypersensitive areas, and the early production of telia. The strain of rust from Tallulah, Louisiana, indicated by ‘‘T,’’ caused heavy, normal infection without any evidence of pigment or telia formation. Swedish Select oats Parker con- sidered susceptible, and yet in these experiments Swedish Select oats from Virginia produced telia in abundance. Appler oats Parker considered resistant. Figure 3, Plate X XIV, shows Appler oats from Alabama infected with P. coronata Cda. ‘‘T”’ shows normal infection with the strain of rust from Tallulah, Louisiana; ‘‘S’’ shows a heavy production of telia and extensive dead areas by the Saint Paul, Minnesota, strain. Parker con- sidered Burt oats susceptible. In the present experiments Burt oats from Alabama showed a similar condition to that described for Appler. Therefore, though the production of telia when associated with other phenomena indicating resistance may be additional evidence to justify the classification of oat varieties as resistant, certainly results obtained in the present work seem to show that this phenomenon of telia formation on oat seedlings is variable and largely dependent upon environmental factors and possibly also upon the strain of rust employed, to such an extent at least as to make telia formation a rather unreliable basis for the determination of true resistance. SUMMARY 1. Urediniospores borne on the surface of oat seeds do not offer a ready means of infecting seedlings developed from these seeds. 2. Seedlings of oats emerging through soil heavily covered with viable urediniospores are not readily infected. 3. Under Minnesota conditions, a perennial mycelium, capable of producing a new crop of urediniospores after overwintering, does not exist. What the situation is in the case of wild grasses has not been determined. 4. Urediniospores do not remain viable over winter on oats, under Minnesota conditions, nor does continued production take place. What the situation is in regard to wild grasses has not been determined. 5. Environmental factors influence the development of the rust on oats as well as the rate of pustule formation. 6. Etiolation brings about the early formation of telia on oat seedlings. 7. Anthocyanin pigment formation surrounding uredinia on infected 2 The term ‘‘strain’’ is used to indicate merely a locality collection. AMERICAN JOURNAL OF BOTANY. VoLuME VIII, PLATE XXIV. HOERNER: CROWN RUST OF Oats. Nov., 1921] HOERNER — STUDIES ON THE CROWN RUST OF OATS alg oat leaves is a common phenomenon though not correlated with resistance or susceptibility. 8. The appearance of telia on seedling oat leaves is not a reliable basis for determining the resistance of oat varieties. DEPARTMENT OF BOTANY AND PLANT PATHOLOGY, OREGON AGRICULTURAL COLLEGE, CORVALLIS, OREGON LITERATURE CITED 1. Hoerner, G. R. Biologic forms of Puccinia coronata on oats. Phytopath. 18: 309-314. 1919. 2. McAlpine, D. The rusts of Australia. pp. 349. Melbourne, 1906. 3. Christman, A. H. Observations on the wintering of grain rusts. Trans. Wis. Acad. Sci., Arts, and Lett. 15': 98-109. 1904. 4. Reed, H. S., and Holmes, F. S. A study of winter resistance of the uredospores of Puccinia coronata. Va. Agr. Exp. Sta. Ann. Rept. (1911-12): 18-21. 1913. 5. Bolley, H. L., and Pritchard, F. J. Rust problems. Facts, observations and theories. Possible means of control. N. Dak. Agr. Exp. Sta. Bull. 68: 608-672. 1906. 6. Mains, E. B. The relation of some rusts to the physiology of their hosts. Amer. Jour. Bot: 4: 179-220. 1917. 7. Wheldale, M. The anthocyanin pigments of plants. pp. 83. Cambridge, 1916. 8. Parker, J. H. Greenhouse experiments on the rust resistance of oat varieties. U. S. Dept. Agr. Bull. 629. 1918. EXPLANATION OF PLATE XXIV Fic. 1 (above). At ‘‘D’’ the teliospores formed on etiolated seedling oat leaves are shown; at “‘L”’ the normal production of urediniospores on the seedlings kept in the light. Fic. 2 (below at left). Avena sterilis L. infected with Puccinia coronata Cda. ‘‘S”’ shows the production of telia and of extensive dead areas by the Saint Paul, Minnesota, strain of rust. ‘‘T’’ shows normal production of urediniospores by the Tallulah, Louisiana, strain. , Fic. 3 (below at right). Oats, Alabama-Appler 617, infected with P. coronata Cda. At “T” normal production of urediniospores by the Tallulah, Louisiana, strain of rust is shown; at ‘‘S,’’ the heavy production of telia surrounded by hypersensitive areas produced by the Saint Paul, Minnesota, strain. COMPARATIVE STUDIES ON RESPIRATION XVIII. RESPIRATION AND ANTAGONISM IN ELODEA CC. ENvGN (Received for publication March 19, 1921) Previous studies in this series have dealt with the relation between antagonism and respiration, but have not included tissues of higher plants containing normal amounts of chlorophyll.!. The experiments here pre- sented were designed to test the effects of mixtures of solutions of sodium and calcium chlorides on such tissues. For this purpose the leafy stems of Elodea canadensis were selected. This has proven to be excellent material since it is hardy in respect to climatic conditions and laboratory manipulation while sufficiently sensitive to reagents. The leaves are exceedingly thin, and gaseous exchange is very rapid. All the plants were collected from one place in a slowly flowing stream and, as long as it remained open, taken fresh at least once a week to the laboratory. The material used during the late winter and early spring was provided by a quantity of plants collected in December and kept in the greenhouse in large glass jars in a cool room. It thrived well and when tested gave normal results. The method used for most of the experiments was that developed by Haas? in which the plants were immersed in solutions containing an indi- cator.2 The production of CO. was measured by the change in color of the indicator. The standard buffer solutions containing the same indicator were mixtures of borax and boric acid. The final experiments were carried out by the use of the apparatus described by Osterhout.4 The curves closely resemble those obtained by the other method. The accuracy of measurement was greater. Since the two methods are alike in all but mechanical details, only the first will be discussed in full. The procedure consisted in measuring the normal rate of production of CO, in tap water or distilled water and then testing the effect of a salt 1 The experiments on wheat reported in a previous paper of this series were made upon germinating seeds which contained little or no chlorophyll. Cf. Thomas, H.S. Jour. Gen. Physiol. 1::203. 1918. 4Haas, A. R. C. Science N.S. 44: 105. 1916. 3 The plants were thoroughly washed to remove any adhering organisms. Micro- scopical inspection showed that the plants used for the experiments were almost free from bacteria. 4 Osterhout, W. J. V. Jour. Gen. Physiol. 1: 17-22. 1918. 458 Nov., 1921] LYON — COMPARATIVE STUDIES ON RESPIRATION 459 solution on the same material in the same tube. For normal respiration the material was placed in 10 cc. of water to which had been added 5 drops of a 0.01 percent solution of phenolsulphonphthalein. Both tap and dis- tilled water were tried, and as no difference could be noted in the effects on the plant, distilled water was used exclusively, since the salt solutions were made upinit. This eliminated possible effects of the salts in the tap water. Both the water and the salt solutions were brought to the proper al- kalinity by the addition of a very dilute solution of sodium hydroxide, the same amount being added to each. _ The plants selected were healthy stems, uniform in appearance, which averaged from 3 to 4 inches in length. These were kept in running water before use (to remove any excess of COz2), and were then coiled and inserted in the tubes where the pressure of the coil held them in the middle of the tube. This kept them from interfering with the observation of the color of the solution (in the lower half of the tube) and with comparison with the color of a standard solution. The paraffined rubber tube at the top was then tightly clamped off after adding the water plus the indicator. A bubble of air, of uniform size in all experiments, was left below the clamp to aid in stirring the solution. The pH value of the water was brought to a little above 7.88, but it dropped to 7.88 shortly after the plants were placed in it. The exact time at which this point was reached was deter- mined by matching its color with that of a buffer solution of pH 7.88 (which had the same concentration of indicator). The contents of the tube were kept in constant motion by gentle stirring during the few minutes required for the evolution of enough CO» by the plant to change the color to match that of the second standard tube of pH 7.60.5 This range of 7.88 to 7.60 was used in all experiments. In getting the normal rate, the amount of material used was adjusted to give a period of from 3 to 5 minutes in most cases. That no acid other than carbonic was produced was shown by the fact that after the plant had changed the color of the indicator solution, it would rapidly return to the original color when a current of CQOs:-free air was bubbled through it. The normal period of respiration for each experiment was first deter- mined. It was usually found that this period was practically constant for two hours or more, and if this was not the case the material was rejected. At least three readings (covering a period of at least 20 minutes) were taken, previous to the addition of the salt solutions, in order to establish the normal rate. The temperature varied from 21 to 25° C. In the course of any one experiment, the temperature did not vary more than two degrees. The solutions of salts were made up in large quantities and kept in 5 The source of light was a ‘Daylight lamp.”’ Cf. Luckiesch. Science n. ser. 42: 764, 1915. 460 AMERICAN JOURNAL OF BOTANY [Vol. 8, bottles of resistant glass. It was necessary to have them well stoppered, and to keep the bottles well filled in order to avoid absorption of carbon dioxide and the consequent reduction of alkalinity and increase of buffer action when made alkaline again. Preliminary experiments showed that a concentration of 0.1 M sodium chloride was preferable for study. Anything above 0.2 M was found to give plasmolysis. A solution of 0.07 M of calcium chloride was taken as approximately isotonic with the 0.1 M sodium chloride, and all mixtures were made up with these concentrations. In order to avoid a possible error by the buffer action of the solutions, these were tested by bringing the distilled water and al! the solutions to an alkalinity of pH 8 and then adding a few drops of a solution of COs, in distilled water. All changed by approximately the same amount, which showed that there was no appreciable buffer action that would interfere in the measurements of production of COs. | The time curves of the production of CO, were plotted in the manner explained by Osterhout® and used in other papers in this series. Rate of respiration in percent is plotted against time in minutes, the normal rate (as determined before addition of salt) being taken as the reciprocal of the average period required to change the solution from PH 7.88 to 7.60; this rate was taken as 100 percent. The behavior of the plant was not quite the same in the fall and spring seasons. In early spring, while working with material kept in the green- house, it was found that the solutions of pure salts and the mixtures were giving different rates of production of CO, than the same solutions had in the fall and winter. The shapes of the curves were not changed, but there was an increase in all the ordinates of from 5 to 15 percent. This prevented the inclusion of many data, for there were not enough experiments for certain points to give a complete curve by themselves. Nevertheless, they provide confirmation of the results presented; especially of the maximum points in figure 2. Figure 1 shows the production of CO, in NaCl 0.1 M (curve A), in CaCl, 0.07 M (curve B), and in mixtures of these. It was found that all concentrations of NaCl (none above 0.2 M were tried) gave an increase,’ while all those of CaCl, gave a decrease. In some of the weaker concen- trations of CaCl. the rate showed a tendency to rise again after falling, but it remained below the normal. In low concentrations of NaCl the increase (of from 25 to 50 percent) lasted for at least 90 minutes, while in 0.1 M NaCl it fell off rapidly at first and then more slowly as shown in the typical curve. A remarkable behavior was observed when the molecular proportions 6 Osterhout, W. J. V. Jour. Gen. Physiol. 1: 171-179. 1918. 7B. Jacobi (Flora 86: 289. 1889) states that NaCl 0.0496 M causes an increase in the production of CO, by Elodea, followed by a decrease. Nov., 1921] LYON — COMPARATIVE STUDIES ON RESPIRATION 461 were 98.62 NaCl to 1.38 CaCl.; the rate increased and did not return to normal (as shown in curve D, figure 1). oe, 200 % 150 100 S$—f-5) 5U 0 30 60 MIN. Fic. 1. Curves showing the effects of NaCl and CaCl, on the respiration of Elodea canadensis. The horizontal line at the left of the point marked O on the abscissae repre- sents the normal rate of respiration before the addition of the salt. Curve A represents the rate of respiration in NaCl 0.1 M, curve B in CaCl: 0.07 M; the other curves represent the rate in mixtures of these having the following molecular percentages: curve C in 99.65 percent NaCl-++0.35 percent CaCl; curve D in 98.62 NaCl +1.38 CaCl.; curve E in 98.85 NaCl+1.15 CaCle; curve F in 98.28 NaCl+1.72 CaCl». The broken line represents the control in distilled water. Each curve represents a typical experiment. The determination of antagonism should preferably be made at a period of the experiment when both solutions of pure salts give either an increase or a decrease in rate. Since the former was impossible, an exposure of one hour was chosen, at which period both gave a decrease and the time curves had become nearly horizontal. Figure 2 shows the rates of respiration in various mixtures and in the solutions of pure salts. The ordinates represent 150 % 1004 (h 'S © ©) (Q >) Fe ee ey Na 100 98 96 1 0 Ca 0 2 4 99 100 Fic. 2. Antagonism curve showing the effect of NaCl 0.1 M-++CaCl2 0.07 M and of mixtures of these upon the respiration of Elodea canadensis after an exposure of one hour. The abscissae represent molecular proportions. Each point represents the average of 3 or more experiments; probable error of the mean, less than 4 percent of the mean (except in one case where it amounts to less than 6 percent). Nov., 1921] LYON — COMPARATIVE STUDIES ON RESPIRATION 463 the rate of production of CO, after the plants had been in the solutions for ' one hour. The figure shows that while the molecular proportions 99.65 NaCl to 0.35 of CaCl, give normal respiration, a decrease occurs at other proportions except that of 98.62 to 1.38 of CaCl». This form of the antagonism curve of NaCl vs. CaCl, is unique, as is evident on comparing it with others in this series as well as with those in which growth, length of life, etc., are used as criteria.2 In order to explain this peculiar effect additional experi- ments will be necessary, and further discussion is deferred until these can be carried out. SUMMARY I. Solutions of NaCl cause an increase in respiration, which is followed by a decrease, while solutions of CaCl, cause only a decrease. 2. After a sufficient length of exposure both NaCl and CaCl, depress the respiration. In a mixture containing 99.65 mols of NaCl to 0.35 of CaCl, the rate remains normal, while a mixture of 98.62 mols of NaCl to 1.38 of CaCl. causes a great increase in respiration. 3. The antagonism curve of NaCl vs. CaCl, is unique in that it has two maxima. LABORATORY OF PLANT PHYSIOLOGY, HARVARD UNIVERSITY 8 Antagonism curves with two maxima have been reported for other combinations of salts, but they seem to be somewhat different in character. Cf. Osterhout, W. J. V. Bot. Gaz. 48: 98. 1909. Brooks, M. M. Jour. Gen. Physiol. 2: 5. tI919. Lipman, C.B. Bot. Gaz48: 105. 1909; Bot. Gaz. 49: 41: 1910. Loeb, J. Jour. Biol. Chem. 2S 1 7.5.aRhO LO. THE EFFECT UPON PERMEABILITY OF @)7 tim SAME SUBSTANCE AS CATION AND ANION, AND (I) CHANGING THE VALENCY OF THE Sahis SON OrRAN L. RABER (Received for publication April 6, 1921) I. THE EFFECT OF THE SAME SUBSTANCE AS CATION AND ANION In a recent paper (1) facts were presented which indicate that polyvalent cations do not cause an increase in the resistance of Laminaria if they are combined with polyvalent anions, and it seems natural to suppose that the difference between the action of a salt with a bivalent cation and a mono- valent anion, e.g., MgCl., and one with a bivalent cation and a bivalent anion, such as MgSQ,, is due to the extra charge on the anion. Cations seem to Cause an increase in resistance and anions a decrease. In MgCl, the action of the cation is dominant; in MgSQu, the action of the anion. If this effect is due to electrical charge, we may ask what will happen if the same ion is used as cation and as anion. This problem seems at first glance to present great difficulties because it is necessary to work with solutions which are not strongly acid or alkaline. Certain elements (such as aluminum, arsenic, chromium, etc.) exist both as cations and as anions, but they are very weak acids or bases and solutions of their salts are not neutral. The difficulty may be surmounted in a measure, however, by adding enough acid to the alkaline solution to bring its pH value down to that of the acid solution. If necessary the conductivity may be increased by mixing with a neutral salt (e.g., NaCl). For this purpose, chromous chloride, sodium chromate, chromic acid, and sodium chloride were used. The chromous chloride is not soluble enough to make the conductivity equivalent to that of normal sea water, and in order that the osmotic pressure of the sea water (with which the solution is compared) should not be too low, the chromous chloride was mixed with sodium chloride. The final solution was then composed of 50 percent chromous chloride 0.61 M and 50 percent sodium chloride 0.52 M. This mixture has greater electrical resistance than sea water and has a pH of about 4.5 as determined by the hydrogen electrode. When tissue is transferred to this mixture from diluted sea water of the same conductivity, the following changes in resistance are observed (see fig. 1, A): 464 — Nov., 1921] RABER — PERMEABILITY 465 Percentage Probable Error of the Mean Ex- Time in of Original pressed as Percentage of the Minutes Resistance Mean (Three Experiments) 2 IOI 0% 5) 102 I 180) 92 I 20 75 6 40 51 8 60 47 II 80 46 II It is seen that there is a slight rise of resistance whichis quickly followed by a fall. That the rise is no greater may be due to the facts that (1) the chromium ion used carries only two charges, and (2) the dilution with the sodium chloride would also tend to cause an immediate fall in resistance. | RES ., 100—/ Vi 80 60 B A MIN 40 80 Fic. 1. A. Effect of 50 percent CrCl,0.61 M plus about 50 percent NaCl 0.52 M upon the permeability of Laminaria A gardhw Kjellm. Ordinates indicate percentage of the original resistance (considered as 100 percent) in sea water of the same conductivity as the solution tested. Abscissae represent time in minutes of the tissue in the solution. B. Effect of a solution of 50 percent NazCrO, 0.22 M and 50 percent NaCl 0.52 M with enough chromic acid added to produce the same pH as in A (pH 4.5). 466 AMERICAN JOURNAL OF BOTANY [Vol-P8; Sodium chromate of 0.22 M has about the same conductivity as normal sea water and has a pH of about 9.5. This is not alkaline enough to cause any appreciable effect, just as a pH of 4.5 was not acid enough to interfere with the success of these experiments, as shown by Osterhout (2). Never- theless, in order that conditions might be comparable with those in which chromium was used as a cation, a solution was prepared consisting of 50 percent sodium chromate 0.22 M, 50 percent sodium chloride 0.52 M, and with just enough chromic acid added to bring the acidity up to that of the chromous chloride solution (pH about 4.5). The acidity in the case of colored solutions was measured by diluting a hundred times, taking the pH of this diluted solution, and then making a correction for the increase in dissociation upon dilution. As a check upon this method the pH was also determined directly by means of the hydrogen electrode. | The results obtained with the sodium chromate mixture of pH 4.5 are shown below and in figure 1, B. The figures given are the average of three experiments. Time in Percentage of Original Probable Minutes Resistance Error 2 93 1% 5 88 I 10 85 2 20 78 2 40 68 3 60 62 3 80 35) 5 It is seen that the initial effect is a fall in resistance. This is to be expected since we have here a monovalent cation with a bivalent anion. It is also seen that the hydrogen-ion concentration is not responsible for the rise in resistance when the chromium has a positive charge. When it has a negative charge (even though the hydrogen-ion concentration is the same, wz., pH 4.5) the resistance does not increase but, on the contrary, decreases from the start. To what extent this is due to the presence of the oxygen in the anion can not be determined. II. THE EFFECT OF CHANGING THE VALENCY OF THE SAME ION As previously stated, the nature of the charge on the ion seems to be a very important factor in determining the initial response of the tissue to electrolytes. It is natural to inquire what the result would be if the same ion could be used with a varying charge. If an anion with one charge causes a decrease in resistance, the same ion with two or three charges should cause a more rapid decrease. Similarly, a cation with two positive charges should cause a less rise in resistance than one with three provided they are used with anions which permit this rise in resistance. Osterhout Nov., 1921] RABER — PERMEABILITY 467 found in general a greater increase in resistance with trivalent and tetra- valent cations (3) than with bivalent cations (4), but he did not compare the effects of the same ion with different charges. The choice of ions for this purpose is very limited. Antimony, arsenic, and tin can not be used because of their weak basicity which results in extremely acid solutions. The toxic action of copper is a sufficient reason for not using it. The insolubility of mercurous, chromic, and cobaltic salts almost prohibits their use. This leaves iron as a possibility among the common cations of variable valency. For this study FeCl; and FeCl, were used. The solution of FeCl; was 0.20 M and had a pH value of about 2.5. When the tissue was placed in such a solution having the same conductivity as normal sea water, there occurred a very sudden and temporary rise in resistance followed by a rapid fall. The following table and figure 2, A show the results: Time in Percentage of Probable Error Minutes Original Resistance (Three Experiments) 2 125 1% 3) _ 107 I 10 71 5 20 Do 9 40 47 8 60 44 7 80 42 7 The tissue becomes extremely hard and decidedly yellow in color, which seems to be due to the acid reaction of the solution. Inasmuch as the acidity of the ferric chloride lies outside the ineffective limit (2), a certain amount of the rise in resistance shown above is doubtless due to the hydrogen ions present. The FeCl, solution of the same conductivity as that of FeCl; used is about 0.28 M and has a pH of about 4, but in order to compare the effect of the valency the solution was acidified by the addition of HCl. The acidity of these solutions (since colored ions are present) is measured and compared in the same way as that of the solutions discussed in the previous section. The final solution used consisted of 45 g. FeCl. per liter with enough HCl added to make a solution of the same conductivity and the same pH as the solution of FeCl;. The results of this set of three experi- ments are given below and in figure 2, B. Time in Percentage of Probable Minutes Original Resistance Error 2 118 4% 5 Od ail: 5 10 68 s) 20 46 4 40 31 6 60 28 2 80 27, 2 RES 120 te 100 80 60- 16 A AO 9 B MIN 40 80 Fic. 2. A. Effect of FeCl; 0.20 M upon the permeability of Laminaria Agardhit Kjellm. Ordinates indicate percentage of the original resistance (considered as 100 percent) in sea water of the same conductivity as the solution tested. Abscissae represent time in minutes of the tissue in the solution. B. Effect of FeCl. 0.23 M with enough HCl added to produce the same hydrogen-ion concentration as in A (pH 2.5). C. Effect of FeCle 0.28 M of the same conductivity as A and B (pH 4). Nov., 1921] RABER — PERMEABILITY 469 It is to be noted that the two curves are similar. The ferrous curve lies a little below the ferric curve. Even though the two are of the same acidity, the ferric causes a greater rise in resistance at the start than the ferrous solution. ‘The additional positive charge on the cation seems to be the chief cause of the difference between these two solutions. In order to see how much of the action of these salts is due to the high acidity, a third set of experiments was performed using 0.28 M FeCle of the original pH (about 4). This concentration is the equivalent of a 0.0001 N HCl solution and, as mentioned above (2), this degree of acidity has been found to have no appreciable effect upon the permeability of Laminaria. The results of these three experiments are shown below and in figure 2, C. Time in Percentage of Probable Error Minutes Original Resistance of the Mean 2 112 1% 5 116 2 IO 114 A 20 109 e 40 85 4 60 67 7 80 51 10 100 40 10 The tissue here retains its original color and (as shown by the table and figure) the increase in resistance takes more than twice as long to reach a maximum. The general behavior and appearance of the tissue is very different from that in the two previous cases, and has in common with them only the initial increase followed by a decrease in resistance. The greater rise in the ferric chloride is not due to the concentration, since the ferric chloride is really less concentrated than the ferrous, the former being 0.20 M and the latter 0.23 M. The conclusion would seem to be that in the case of curves A and B the primary factors are the ferric, ferrous, and chlorine ions while the hydrogen ion plays a subordinate role. The acidity causes the maximum rise to be arrived at much earlier and causes the extremely rapid fall. The valency of the cation, however, determines largely the height to which the resistance rises. SUMMARY 1. Chromium has a different initial effect upon the permeability of Laminaria depending upon whether it occurs in the cation or in the anion ofasalt. If it isin the anion, the first effect is a decrease in resistance, and if in the cation, an increase. 2. Ferric chloride causes a greater increase in resistance than ferrous 470 AMERICAN JOURNAL OF BOTANY [Vol. 8, chloride, independent of the hydrogen-ion concentration. The difference seems to depend upon the valency. LABORATORY OF PLANT PHYSIOLOGY, HARVARD UNIVERSITY LITERATURE CITED 1. Raber, O. L. The effect upon permeability of polyvalent cations in combination with polyvalent anions. Amer. Jour. Bot. 8: 382-385. 1921. 2. Osterhout, W. J. V. The effect of alkali on permeability. Jour. Biol. Chem. 19: 335. 1914. The effect of acid on permeability. Ibid. 19: 493. 1914. 3. ——. The effect of some trivalent and tetravalent cations on permeability. Bot. Gaz. 59: 464. I9QI5. 4. ——. On the decrease of permeability due to certain bivalent cations. Bot. Gaz. 59: . 317. I9Q15. f n ( Rabe, i y, Ithaca ell. University, oe a bey » Ever gone. into a has Sire, knows. | ing exactly the kind and shaped .c -y—Vo% hat you want; ‘and then have some. es . cm ‘smart Alec of: a salesman ssi to. | sell you, the kind HE likes? Makes a Veale paddies, han al : ‘to the Postal. 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VIII DECEMBER, 1921 No. 10 ————— = POLLEN AND POLLEN ENZYMES JuL1a BAYLES PATON (Received for publication March 28, 1921) I. THe THEORETICAL AND PRACTICAL ASPECTS OF THE OCCURRENCE OF POLLEN ENZYMES Reasons for Undertaking the Investigation A review of the literature shows very few complete or satisfactory reports of experiments in regard to either the general chemistry or the enzymes of pollen. Our knowledge of the subject seems to be very frag- mentary. It is conspicuous by its omission from the textbooks of botany. Aside from the few references given later, up to the present time no mention of any important work has been found. Although it is generally assumed, and is stated in our textbooks, that the pollen tube digests its way through the tissues of the pistil and the ovule, yet there seems to be no experimental evidence as to the exact nature of thisenzyme action. Besides this, pollen enzymes must be very important in rendering the food stored in the grain available when the pollen ger- minates, in nourishing the tube during its passage through the style, and in stimulating the development of the embryo and the maturing of the ovary. Moreover, pollen anaphylaxis is now regarded as the cause of so-called hay fever and other forms of pollen poisoning. Pollen enzymes may be concerned in these reactions, and the proteolytic enzymes may affect the stability of the pollen-protein solutions used in pollen vaccination. In view, therefore, of the apparent meagerness of our knowledge of pollen enzymes and of the possible practical value of any contribution to this subject, it has seemed worth while to study the matter and to present the results. The Literature of Pollen Enzymes Few original, systematic experiments have been reported. Erlenmeyer (1874) found amylase, or diastase, in pine pollen. Van Tieghem (1869) reported invertase, or invertin, in the pollen of hyacinth, narcissus, wall- flower, and violet. Czapek (1905, p. 393) quotes Strasburger’s statement [The Journal for November (8: 425-470) was issued December 19, 1921.] | 471 472 AMERICAN JOURNAL OF BOTANY [Vol. 8, that the pollen tubes of Agrostemma Githago bore through the membrane of the stigma papillae as evidence for a cytase in pollen. Czapek also refers to the investigations of Rittinghaus (1886, pp. 105-122) as confirming the opinion of Strasburger. The observations of Rittinghaus may, however, be interpreted quite differently, and point quite as definitely to the presence of a pectinase, as of a cytase. Rittinghaus examined numerous flowers, including Ipomoea, Convolvulus, Alisma, Agrostemma, Lychnis, Phlox, and Silene. He writes (p. III): Die Verschmelzung zwischen der Cuticula der Papille und der Cellulosemembran des Schlauches ist ganz deutlich zu erkennen, und es leuchtet ein, dass die Liicke in der Cuticula thre Entstehung nur einer unmittelbaren Einwirkung der Pollenschlauchspitze verdankt. Das lésende Agens ist somit nur im Plasma des Pollenschlauches zu suchen. Uber die Natur desselben ist einstweilen leider nichts zu eruiren, zumal das einzige uns bekannte Cuticula-losende Reagens kochende Kalilauge ist. Vielleicht wird man spater die Erschein- ung durch die Gegenwart eines besonderen Enzymes aufklaren kénnen. J. R. Green (1891) noted amylase in pollen tubes. Green’s later re- searches in 1894 are by far the most careful and complete experiments on pollen enzymes which have so far been reported. They will be briefly reviewed on alater page. Strasburger (1886) mentions diastase and invertin as present in pollen grains prior to germination. Sandsten (1909) reports invertase and diastase. Later, Kammann (1904) found protease, diastase, catalase, and lipase in rye pollen but does not give details of his experiments. In the investigations of Green (1894, pp. 385-409) the pollen was pow- dered with glass and the powder suspended either in glycerine, or in a 5 percent solution of NaCl, to which 2 percent of potassium cyanide was added as an antiseptic. In other cases chloroform (a few drops) or oil of cinnamon was used as an antiseptic. The 5 percent NaCl solution proved preferable to glycerine. Diastase was found in the pollen of Gladiolus, Anemone, Antirrhinum, Tropaeolum, Pelargonium, Crocus, Brownea, Hel- leborus, Alnus, Tulipa, and Clivia; also in that of Zamia after germination begins. Experiments failed to show any sufficient evidence for diastase in the resting pollen grain of Zamia, and starch makes its appearance in these pollen grains only on germination. Diastase was absent from the pollen of Lupinus, Lathyrus, Eucharis, Richardia, and Narcissus. The diastase, according to Green, dissolves the starch without corroding the grains. The pollens tested for invertase were those of Eucharis grandiflora, Narcissus papyraceus albus, N. Pseudo-Narcissus, Helleborus, Richardia, Lilium pardalinum, and Zamia Skinneri. It was found in these, but was absent from the pollen of Alnus and of Clivia. He reports that A few experiments were made with a view to determining the existence of a cytolyst and a proteolyst, but in no case could either be found. In the case of Eucharis grandiflora, tested for invertase, Green says that Only the contents of three or four anthers were used, yet a workable quantity of invertase was extracted. Dec., 1921] PATON — POLLEN AND POLLEN ENZYMES Age In summarizing he says: The enzymes present in the resting pollen grains are, therefore, chiefly diastase and invertase, but their distribution is irregular, some containing one, some the other, and some both. At the onset of germination usually the amount of both diastase and invertase is considerably increased. . . . When the grain has lost the power of germinating the quantity of diastase is materially decreased. The conclusions, as will be noted later, are not entirely in accordance with the results of the present experiments. The Significance of Pollen to the Living Plant, and the Probable Role of the Pollen Enzymes A medium-sized Indian-corn plant produces about 50,000,000 pollen grains. Cat tails (Typha), which produce about 60,000 flowers to the average spike, shed enormous quantities of pollen. A near relative, the elephant grass (Typha elephantina) of East India and New Zealand, yields enough for the natives to use as a flour in bread- and cake-making. The dense cloud of pollen from a pine tree has been photographed, and many a camper has noticed the yellow powder staining the canvas of his tent when dampness has moistened the grains. Liefmanr (1904, p. 163) found 2,500,000 grains of grass pollen in one square meter. Yet so tiny and light are these pollen grains that a small amount represents millions of grains. Ulrich (1914) estimated 172,800,000 grains in one gram of ragweed pollen, and Kammann (1912) estimated 20,000,000 in one gram of timothy pollen. Pollen grains are nearly omnipresent during the flowering season. One would suppose from these figures that it is an easy matter to collect large quantities of pollen, but it is really not easy. The winged grains of pine pollen are blown away by the slightest breeze. Ragweed pollen cannot be collected easily after nine o’clock in the morning. The grain of pollen is surrounded by an oily envelope containing air. When this air is heated by the sun it causes the floating away of the pollen, or the so-called ‘smok- ing’’ of the ragweed. It is not easy to get enough for an experiment. The fact that during three fourths of the year we have pollen grains always with us makes it evident that if they have active enzyme action their im- portance cannot be lightly overlooked. Pollen grains present many types of configuration. The commonest forms are oval or spherical, but an extreme variation is seen in the extra- ordinary filamentous pollen grains of eel grass (Zostera) and of another water plant, Halophila. Although the grains differ greatly in shape and in surface markings or finish, in internal structure they are very uniform. They usually consist in the Angiosperms of two cells. One cell is purely vegetative and gives rise to the pollen tube; the other is the generative cell. Pollen grains vary considerably in size. A very extensive list of both measurements and descriptions of the pollen grains of many species and families is given by Hansgirg (1897, pp. 17-76). A74 AMERICAN JOURNAL OF BOTANY [Vol. 8, The pollen grains are very resistant to excessive heat, cold, or dryness, and certain kinds retain their viability for many years. The pollen of the date palm tested by Popenoe at the Mecca experiment station was kept seven years and still retained its power of germination. Goodale (1916) found that dry pollen could retain its active poisonous properties for twenty- five to thirty years. It is evident that pollen is an interesting physiological unit, and our knowledge of its composition should be more complete. Since one cell of the pollen grain is vegetative and gives rise to the pollen tube, food must be stored in the grain and at the time of germination ren- dered available. We should expect therefore to find enzymes suitable for the digestion of the materials stored in the grain, and perhaps capable of also digesting the inner pectin membrane (Mangin, 1893, p. 655) which envelops the grain. It is one aim of the experiments reported to determine whether such a correlation exists. The distance that the pollen tubes have to traveise varies greatly. Where a style is absent and the stigmatic surface is just above the ovary, as in Vitis and Actaea, the tube has only a little way to penetrate. In flowers with long tubular corollas and slender filamentous styles, such as Crocus, Oenothera, and Zea Mays, the tubes attain a relatively great length. The time required for them to reach the ovule also varies greatly. In some flowers the tube reaches its full development in a few hours, while in the pine, following pollination in the spring, the grains put forth short tubes which do not complete their growth for a year (Kerner, 1895, 2: 420). In certain oaks thirteen months elapse between pollination and fertilization. In regard to the Taxaceae, Coulter (1910, p. 268) writes: The tube may advance directly toward the archegonia or it may pursue a devious route, in some cases not reaching the archegonia until during the second season. Other instances are cited by Coulter and Chamberlain (1903, p. 147). Why this long delay? An interesting physiological and chemical problem is waiting to be solved. The 13-inch pollen tube of Colchicum autumnale needs only twelve hours to reach its goal, and the 9-inch tube of Cereus grandtflorus completes its growth in a few hours (Schleiden, 1849, p. 407). In Iris versicolor the male nuclei were observed in the embryo sac 79 hours after fertilization and the tubes were 14 mm. long (Sawyer, 1917, p. 163). Surely an intruding, growing tissue of such size and duration must during its period of development, profoundly affect the cells with which it comes in contact, or which are adjacent to it, in its passage through the style. It has long been customary to liken the pollen tubes to the haustoria of parasitic fungi, for they closely resemble the latter in many respects. In Pinus, according to Mottier (1904), the tube serves both as a conducting passage for the male gamete and as an absorber of nutriment. The haus- torial habit seems to be the more primitive condition, and we have survivals of it in certain Angiosperms, as in Iris versicolor (Sawyer, 1917), hazel, oak, elm, hickory, and certain mallows (Kerner, 1895), where the tube branches Dec., 1921] PATON — POLLEN AND POLLEN ENZYMES A75 frequently and serves apparently as both haustorium and directing channel. (See also Coulter and Chamberlain, 1903, p. 148.) The nature of the tube has been dwelt upon here at such length in order to emphasize the fact that we ought to know more fully how these tubular filaments make their way through the tissues of the style and ovary. We assume that they digest their way. One author of a recent textbook even states positively: Very soon after pollination, the tube cell begins to develop a pollen tube, which secretes an enzyme that dissolves the cell walls and contents of the nucellar tissue, thus facilitating the passage of the delicate tube. Is this true? Can we prove the existence of a cytase which digests the cell wall? Is one enzyme sufficient to account for the varied needs of the pollen tube in the course of its life history? There are several conditions which the pollen tubes may encounter before they reach the embryo sac. These are as follows: (1) An open stylar canal. In such cases the germinating tubes may force apart the cells of the stigma and soon enter the open space of the style without having to penetrate any cells,’at least not until they reach the ovule. The middle lamella is usually composed of pectin compounds (Frémy, Mangin, Allen, and others). A pectin-digesting enzyme might therefore be required to dissolve the middle lamellae of the stigmatic cells, but afterwards the tube has a clear course. Examples of this sort are seen in violet, mignonette, lily, rhododendron, Hypericum, Cistus, Atropa bella- donna, and iris. According to Kirkwood (1906), in the Cucurbitaceae The tubes pass chiefly over the surface of the conducting tissue lining the stylar canal and covering the placenta lobes, and this is rich in starch. The suggestion is made that the tube is directed in its course by nutrient substances secreted by the conducting tissue. This would imply the pres- ence of a diastase to digest the starch. Even if there is actually no tissue to be digested, it seems reasonable to suppose that the tubes may derive nourishment from the cells lining the stylar canal. Negative aérotropism, positive hydrotropism, and positive chemotropism, which have been fre- quently demonstrated in pollen tubes, direct their course so that they penetrate the stigma. These same responses tend in many cases to keep the tubes closely appressed to the cells lining the canal. Considering the length of time it often takes a tube to reach the ovule and its considerable growth, enzymes along with other factors in nutrition must play an impor- tant part. Frequently, as in Anagallis, the channel is only a narrow space almost completely filled with a mucilaginous substance, supposed to be secreted by the cells lining the canal. It may be pointed out here that the mucilages are closely related to the pectins. If this material is utilized by the tubes during their passage through it, we should expect a suitable enzyme to be present. (2) A mass of loose, conducting tissue in the style. The cells in the interior 476 AMERICAN JOURNAL OF BOTANY [Vol. 8, of the style frequently are loosely connected, elongated, and sometimes mucilaginous. The pollen tubes, according to most histological reports, penetrate the middle lamellae of these cells. This is the condition most frequently met with. The pollen tubes follow the middle lamellae of the cells throughout their course. The lamellae are, as has already been stated, composed either of pectin or of closely related mucilaginous substances. Here again the necessity for a pectin-digesting enzyme is evident. It has been sought for in the experiments reported later. Since this condition is the most common, many examples could be cited. It may be well seen in members of the grass family and in Salvia (Bower, 1919, p. 269). Histo- logical evidence seems to indicate that the cells of the style often remain intact. Shreve (1906, p. 115) says in regard to the pitcher plant (Sarracenia purpurea): The pollen tubes grow between the cells of the stigmatic surface and their entire passage is between the cells of the conducting tissue and never through them. Gow (1907, p. 136), describing the fertilization of skunk cabbage (Spathy- ema foetida), writes: The central portion of the style consists of a loose mass of thin-walled cells through which the pollen tube readily forces its way to the upper end of the ovary. Miller’s account of the growth of the pollen tube of corn through the silk or style is interesting (1919, p. 264): Each silk has two fibro-vascular bundles. These bundles are surrounded by sheath cells which are characterized by their dense contents and large flattened nuclei. It is between these cells that the pollen tube travels down the silk. Arriving at the base of the silk the pollen tube works its way between the sheath-like cells that extend from the fibro- vascular bundles of the silk to the cavity of the ovary. The tube enters the ovary and twists and coils in its passage along the ovule coat until it reaches the micropyle. The pollen tube then pushes between the cells of the ovule until it reaches the embryo sac. Again, in another part of his account, he says: The end of the pollen tube is greatly enlarged as it pushes its way between the sheath cells of the bundle. In its passage down the silk the tube causes but little disturbance in the position of the cells, so that after the tube disappears the cells quickly return to their normal form and position. |The emphasis here is my own.] The pollen tube so far as I have observed does not extend the full Jength of the silk at any time. It is difficult to locate it a short distance back of its growing region. It appears that the older portions of the tube are absorbed by the surrounding cells, while the growing part of the tube is apparently nourished by the dense sheath cells. Land (1907, p. 276), in explaining the fertilization of Ephedra trifurca, notes that the pollen tubes force their way between the neck cells of the archegonium, rarely destroying them in their passage. Only in two in- stances were the lower neck cells destroyed. (3) Cell walls penetrated by pollen tubes. According to most investigators this condition occurs only rarely. Perhaps it will be found more frequent if more observations are made. The classic illustration is corn cockle, Dec.;.1921] PATON — POLLEN AND POLLEN ENZYMES AT] Agrostemma. Strasburger’s illustration of the tubes actually penetrating and half filling the papillar cells of the stigma has been frequently copied. Mallow pollen tubes do the same. Recently Knight (1918, entry 964) has reported that in the apple there is no stylar canal. ‘‘Pollen tubes make their way through the tissue. There is a decomposition of the cells along this path with the extrusion of mucilage.’’ This is interesting to compare with the opinion of Grieg Smith that mucilages are decomposition products of cellulose, and with Wiesne1’s statement that all gums are produced by a diastatic ferment acting on cellulose. The writer regrets that it has been impossible to secure corn cockle and mallow pollen so as to determine whether their enzyme action is different from that of other pollens. Apple pollen has shown some differences. In histological studies of fertilization little attention seems to have been paid to the question of how much the pollen tube disorganizes the neighboring cells. It seems that it would be worth while to examine material again with this thought in mind. Many of the drawings of the passage of the pollen tubes appear very diagrammatic. In this connection it is interesting to note Kerner’s observation (1895, p. 392) that the pollen tubes of Lamium amplexicaule Peyforate the walls of the anther and grow in the direction of the stigma until they reach it. Pollen Grains as Carriers of Bacteria and Molds Nine varieties of pollen were tested to see if any contained a rennin-like enzyme, such as is found in the juices of a number of plants. Thymol had been added to the unheated and autoclaved pollen extracts, but the milk had not been sterilized. It was observed that both the unheated ragweed pollen and the autoclaved dock pollen control had strongly coagulated the milk over night at room temperature. Repetition of the test with highest grade milk (Fairlea Farm) showed that unheated corn, Easter lily, and dock pollens caused clotting, as did even the autoclaved dock pollen. The strong ‘“youghourt’’ or fermented milk odor, and the behavior of dock pollen made the reaction seem more like bacterial than like enzymatic action. Apparently the single period of heating in the autoclave had not destroyed all bacteria on dock pollen. Accordingly a number of tests were made employing the usual bacteriological methods. These tests showed that pollen grains harbor a varied flora of both bacteria and molds. It had been taken for granted that excess of toluol or of thymol was sufficient to inhibit bacteria and molds. Do the results of these tests with milk mean that in other instances it is the enzymes of bacteria and molds rather than those of pollen grains which cause the change? The writer believes that this is not true for the following reasons: a. The results were constant with the same pollen regardless of its source. Corn, pine, maple, and goldenrod pollen were collected both in New Haven, and, owing to the difference in seasons, a few weeks later on 478 AMERICAN JOURNAL OF BOTANY [Vol. 8, the hills of Vermont, six miles from a town. When this possible source of error was suspected, ragweed pollen was purposely obtained from Michigan, from two parts of New York state, and from Connecticut. It does not seem probable that the bacteria and molds carried by pollen can be so constant as to cause similar enzyme action in each instance. b. The reactions are too rapid to be due to bacteria. With the in- hibiting action of antiseptics the time required for bacteria to develop in sufficient numbers to produce similar changes would be much longer. All the enzyme reactions recorded have occurred within 24 hours, and several have been almost instantaneous. | c. Slices of wood in water over night are not in any degree sterile, yet bacteria which have free access do not destroy the middle lamellae, but pollen grains do. Pollen grains taken from unopened anthers and put into sterile Petri dishes are not likely to have peculiar bacteria, absent from the immediate environment. Besides, examination of the pollen contami- nation showed only a few omnipresent common forms of bacteria. d. Pollen solutions filtered through a Berkefeld filter gave the enzyme action of diastase on starch, and blood fibrin digestion. e. It is probable that the ground pollen added something to the milk which stimulated the growth of bacteria already in the milk, and that it was these which caused coagulation rather than the bacteria introduced by the pollen. The reason for this belief is that in all the plates poured from milk to which pollen had been added Bacillus fluorescens liquefaciens was the dominant type. The plates after standing a few days were a bright apple-green from the fluorescent growth. On other plates poured later from the pollen extracts only, not once did this form appear. In the latter it was often not until the third or fourth day that colonies of molds occurred. Doubtless there are resistant forms of spores on the pollen which endure the heat of the autoclave and develop under favorable conditions on the agar plates, but these can hardly account for digestions which occur during twenty-four hours. The Chemistry of Pollen While many kinds of pollen have been examined for certain special constituents such as starch, nitrogen, phosphoric acid, etc., only eight kinds of pollen, as far as I have been able to ascertain, have been analyzed with any degree of completeness. Czapek (1905) discusses topically the occur- rence and distribution of the principal constituents of plants; if a substance has been reported present in pollen he mentions the fact. These scattered references afford a valuable index to the original literature of the earlier analyses. According to Heyl (19194, p. 672) the walls of the pollen grain constitute 65 percent of the structure. Biourge (1892, p. 75) distinguishes four sub- stances in the wall or envelope of pollen grains: cutin, cellulose, pectic Dec., 1921] PATON —— POLLEN AND POLLEN ENZYMES 479 substances, and callose. Sometimes one, or more than one, of the four materials are present in the same grain. These substances are indicated by characteristic solubility tests and by color reactions. He examined the pollen of 19 species of monocotyledons and 26 species of dicotyledons. His plates give over a hundred illustrations of the pollen grain coats and their sculpturing, showing details brought out by staining methods and chemical treatment. Mangin (1888, p. 144) states that the membrane is formed of pectin. | Water makes up a large but variable part of the grain. Thus Koessler (1918, p. 420) found 10.5 percent of moisture in ragweed pollen, while Heyl (1917, p. 1470) reports 5.2 percent for the same kind of pollen. Braconnot (1829, p. 104) found 47 percent of water in cat-tail pollen. Lidforss (1899, p. 292) examined a number of species and found the average moisture content to be about Io percent. | The colors of pollen differ greatly. It is deep yellow in Easter lily, dark red in tiger lily, salmon in cypress, and white in petunia. Even in the same flower the color may vary, as is noted by Plimmer (1912, p. 51) in Lythrum salicaria, which has yellow pollen in the short stamens and bluish green pollen in the long stamens. Heyl (1919 0), p. 1285) states that the yellow pigment of ragweed is entirely glucosidic and about 0.6 percent of the pollen. He finds a quercitin glucoside which on melting yields a cherry-red oil; and a glucoside isorhamnetin which has beautiful character- istic crystals in the form of hexagonal prisms. So far, no other analysis of the pigments of pollen has been located in the literature. Starch has been found present in some kinds of pollen and absent in others. Molisch tested 110 varieties and found starch abundant in 45, only a trace in 9. varieties, and absent from 46. That is, about half the kinds tested contained starch. Lidforss (1899, pp. 294-298) examined 150 wind-pollinated flowers of 72 genera and 29 families of native or naturalized Scandinavian plants, and found the pollen of all rich in starch. On the other hand, he tested the pollens of a few wind-pollinated tropical plants and found them starch-free. He also calls attention to the fact that Nageli found the pollens of Alnus glutinosa and Plantago lanceolata, collected in Germany, starch-free, while pollens of the same species collected by himself in a more northerly region contained starch. Similarly, Nageli found the pollen of juniper on Swedish mountains to be rich in starch, while Molisch found little in that of the Austrian juniper. Further, Molisch states that. the pollen of Antirrhinum tortuosum is completely starch-free in summer, but in November he finds grains of three sorts, those which are normal but starch-free, little empty grains, and normal starch-containing grains. Tischler (1909, pp. 219-242), however, does not find this correlation between climate, or temperature, and the starch content of pollen. He examined a large number of tropical plants at Buitenzorg and reports that the plants growing under relatively unfavorable conditions of assimilation, for example 480 AMERICAN JOURNAL OF BOTANY [Vol. 8, on mountains 3,000 m. high and in the desert, showed no higher percentage of pollen with starch than the plants growing under the favorable climatic conditions of the tropical rain forest. He does, however, observe that there is frequently a difference in starch content between mature and immature grains. Lidforss (1899, p. 306) reports the analysis of sixteen varieties of pollen for nitrogen and P2,O;. Of these, 11 were from anemophilous, and 5 from entomophilous flowers. He found the average nitrogen content of the wind-carried pollen to be 4.63 percent, while that of the insect-carried pollen was 7.49 percent. The P.O; showed a similar difference; the average for the former pollen being 1.76 percent, and for the latter 3.03 percent. Whether or not this represents a real correlation must be established by further observations. The relative amounts of protein, fat, sugar, ash, etc., can best be seen by comparison of tables 1-7. It is interesting to note, from Stift’s analyses of the pollen of three varieties of Beta vulgaris, that the different constituents may vary considerably in the pollen of one species (Stift, 1896, p. 43; I9OT, pp. 105-106). TABLE I. Comparison of Pollen Analyses (figures indicate percentages) Kind of Pollen Authority | Protein | Fat Ash Carbohydrates Date palm. . Vauquelin, Ca3(PO.)e 1802 Meg3(PQO,.)e Gaeta... | Braconnot, 3.60 Starch Sugar 1829 Cypress #4) Church, 8.67 Toy) 3.70 85.76 1875 MAZEL seas Planta, 30.06 4.20 2:01 5.26 14.7 1885 Saccharose PING sae a8 Planta, 16.56 | 10.63 2530 7.06 Beau . 1885 Pine Kressling, 15.87 | 10.00 5.50 7.40 12.075 1891 Pentosans Beet Stift, 1896, 16.90 3.52 9.18 0.89 12:20 I90I 16.68 5.47 Fal 0.89 727 IRIC shen wink Kammann, 40.00 3.00 3.40 25 I9I2 Dextrin Sugars— Ragweed ... Heyl, 24.40 | 10.80 5.39 2.10 2.10 1917 Pentosans 720 Ragweed... Koessler, 8.25(?)| 10.30 10.60 6.89 1918 | Stoklasa (1896, p. 631) analyzed the pollen as well as various other organs of apple, horse chestnut, and beet, and concludes: Das lecithinreichste Organ der ganzen Pflanze aber ist entschieden das Pollenkorn. He found in apple pollen 5.86, in that of horse chestnut 5.16, and in that Heyl (1919 a, p. 672) discusses the chemical “building stones’’ from which the substance of pollen sperm nuclei may be of beet 6.04 percent of lecithin. Dec.,; 1921] PATON ——- POLLEN AND POLLEN ENZYMES _. 481 built, if there is a parallelism with the chemical composition of animal sperms. TABLE 2. Analysis of Pine Pollen, Przybytek and Famintzin, 1885 (figures indicate per- centages) “PY ELS ase Te aD aC NER RI ones 6.79 Ash GA CUMMRO NIC h oe no Sere a Ot atin a siecle elon ee ake cena 25223 SO CHUM ONG Cleat tatoo ct sen Sade we cae eeree taslolh, raat taxe auee mee et 3.02 IN EAS TIES tetera atte. mlascth, Goat Watcha seed eee aaah se aS aera 7.00 ANC HUTA a eee racy nc Ss eee Oe RS ace ea eee 0.88 ErOngainG calluinan MU InOXI Cl. cera is Bee cea chasse egw Ae le 5.30 NOspuenicacids(anhyGrOus)) ame wenie. sels. s dese cae Po 29.86 Sul pubic acid ManmyAnOUS)! 2.4 cues fue Ganeo ath anche neem 14.83 inl Orie re ea, A thes Se pee Ne NOR eet armies ots SI ale te a aan’ 0.99 Ira aMeSOy «0 eben SHOR, Mul oh at inn eters ands, Shy eee a a trace TABLE 3. Stift’s Analyses of Pollen from a Cattle-fodder Beet and from two Varieties of ‘Fodder Beet,) Sugar Beet, | Sugar Beet, 1895 1895 1900 POD oho Ga ee ee ee Pers 25 16.90 16.68 Nitrogenous substances not protein............. 2.50 2077, 5.82 MaweUNeIMextract)... 0)... /. ies se cme eee Be eee Brrs 3.52 5.47 Sire lmanGeG@eXUGIT) vou sa.:0 4.6 vv ca adie oe ble ala pans 0.80 0.89 0.89 IRERORZT Dg 4 5h See ea ee era 11.06 12.26 Te27, Other nitrogen-free extractives.......0....0%.-. 22470 26.27, 28.86 CRUG I TIDSIR «| ARE arene on rear a 25.45 28.21 27.95 JESUIT 0 i Ss a a a | 8.28 9.18 Fahd GUY eel tae ee Pe Pere igh earn Ree ea foe 9.78 TABLE 4. Heyl’s Analysis of Ragweed Pollen (1917) Alcohol-soluble (42.9 percent) contains (in percentages): MOU SECIRC rie ytlnt ytece ear nites or oc Uieeens Ota atte «oe. en 1.75 SUCROSE ett et Lisi see te SMR ane em iee Chop eat tele textes ab tic 22 0.40 lcoses he ee Fain ou RE I Pon 1.60 Rs tas fee es at ohn). ike eA ee rep mma aies eA Wan, feeds cuca ers 17.40 MEMELOCENOUS DASE s & 6) culpepaceunase test a) © oe cee teeMeene oe ect Sectcans trace From the above review and from the analyses given in tables 1-7 it is clear that our knowledge of the chemistry of the pollen of the very numerous species of flowering plants is very limited. It is a discouraging problem 482 AMERICAN JOURNAL OF BOTANY [Vol. 8, because of the difficulty of getting large quantities of material, as Heyl points out when he estimates that it takes 610 million grains of ragweed pollen to make a gram. ; TABLE 5. Kammann’s (1912) Analysts of Rye Pollen (figures indicate percentages) Inorganic substances 245. aia se race cenee 13.58 Water: aia fo satlt Wee lee, a oO RE asec ee le er arn 10.18 YNSID o pos cies a eG cc a ee alee ee ne 3.4 Organic Substances. 2% ieee y.5 erste teenie See oe 86.42 Afcohol-ether-soluble. 0g) 7 seis eae ke gee ae nee ee ae Carbohydrate. ..... He patna re oe Une Gene ea hate tae 25. Non-protein nitrogen: 42-3. .1. 50 aes oo eee tee 18. Probenncis & 6 aesd sede oe Pee ces ce tice a ee 40. TABLE 6. Koessler’s (1918) Analysis of Ragweed Pollen (figures indicate percentages) Inorganic substances........ RO rare Neate S Net eso, 211 IMOISCUTE 5:2 aniitaciren de Go Mates Sa ee 10.5 PSH so baila, GteeaaRinate ote the ah kine Oyues tole pti e ce eee ee eg 10.6 Organic:substances 24; 2428 Meets SP nae eee 78.9 Total reducing sugars after hydrolysis. 2...).1. see ee 6.89 Ether-soluble dipoids: 32. 23.3.8 e108 ae cane eee 10:3 Fatty acids aiter hydrolysis:. 2 9 pee ee 4.75 Phytosterol , 295 eke Ghd Pcs, chen ene Ver Eee 0.34 Insoluble in ether but soluble in 95 percent alcohol......... 12.5 Extractives, etc., soluble in alcohol (resins) and water...... 11.5 Insoluble residue (crude fiber, proteins, etc.).............. 27k TABLE 7. Purin Bases and Amino Acids in Pollen Kind of Pollen Authority Purin Bases Percentage iM ee a raknt Bh ees Planta, Hypoxanthine 1885 Guanine oo PAIZO coy od aac b See Planta, Hypoxanthine 1885 Guanine ONS PRI ers cna Se oe 2 Kressling, Xanthine 0.015 1891 Guanine 0.021 Hypoxanthine 0.085 Amino Acids Raoweeds. wee a8 o< Heyl, Histidine : not given FOL, | Arginine . | Lysine ~ Agmantine > Ragweed «2.2 Gwar. Koessler, Arginine asta 1918 Histidine 2.41 Cystine 0.57 Lysine 0.97 Other Physiological Aspects of Pollen in which Enzymes may Play a Part In certain flowers there are two kinds of pollen grains, some of which produce tubes and others which do not. Miiller (1883, p. 242) first distinguished these as ‘‘ Befruchtungs’’- and “ Bekéstigungs’’-pollens, the former being the fertile, and the latter the sterile pollen which Miiller Dec., 1921] PATON — POLLEN AND POLLEN ENZYMES 483 thought served as the food of the pollinating insects. Tischler (1910, pp. 219-242) has studied this subject and has made the interesting discovery that in certain pollens, at least, the sterile grains may be stimulated to produce tubes by the addition to the culture medium of a trace of saliva or of diastase. The lack of a specific enzyme in these pollens seems thus to be the cause of sterility. It is quite possible that in other pollens the lack of pectinase, cytase, invertase, or of other enzymes may be equally important in inhibiting the growth of the tube. In some cases the deficiency may be made good by an enzyme secreted by the stigma. The whole question has a great deal of significance in problems of plant breeding. Poller enzymes may be concerned in the production of the characteristic odors of pollen which are probably factors in insect attraction. The ema- nations from moist pollen indicate the presence of fermentation products. It also seems reasonable to suppose, as Erlenmeyer (1874, p. 206) has suggested, that pollen enzymes are co-workers with the enzymes from the body of the bee used in producing bee-bread. Gardeners commonly believe that contact with pollen is frequently the cause of the discoloration and decomposition of the petals which is often a sequence of pollination. * II. EXPERIMENTS IN REGARD TO POLLEN ENZYMES Plan of the Experiments An effort has been made to collect a large variety of pollens, representing different families of plants, and including some of the so-called ‘‘hay-fever pollens.” These pollens have been tested for twelve different enzymes. On account of the difficulties in collecting all the pollens at the start, the experiments have been made in two series. For the first the available pollens were those of (1) Easter lily, (2) Lilium rubrum, (3) red maple, (4) Norway maple, (5) Siberian crab-apple, (6) Austrian pine, (7) Scotch pine, (8) magnolia, and (9) dandelion. In the second series of experiments, in addition to some of the first nine pollens, those of the following plants were used: (10) corn, (11) daisy, (12) dock, (13) elm, (14) goldenrod, (15) rag- weed, (16) rye, (17) tiger lily, (18) timothy. Not every one of the eighteen pollens has been used in every test, but an effort has been made to use as many as possible. Methods of Collecting Pollen. Kinds of Pollen Used The work was begun in February. At this time Easter lily pollen was available in the largest quantity. Since it is customary to remove the anthers as the flower opens, to prevent the pollen from staining the petals, it was easy to find an obliging florist who would place these anthers in a clean paper box. In this way surprisingly large quantities of pollen were secured. Care had to be taken to prevent molding. A paper box was 484. AMERICAN JOURNAL OF BOTANY [Vol. 8, found to be better for collection than glass jars, as the anthers dried more readily. It was also necessary to keep the anthers spread out, and to place them in a sulphuric-acid desiccator as soon as possible after collection. When the anthers are dry, or partially dry, the large, sticky yellow pollen grains easily fall out. They can then be accumulated quickly by placing the anthers on one half of the bottom of a petri dish, moistening the other half with the finger tip, and then when the dish is covered and shaken in a horizontal plane the pollen adheres and heaps up on the mois- tened surface. When it was necessary to remove adhering masses of pollen from a dish a glass brush was found better than a camel’s hair brush, and for this purpose the glass brush from a Beegee ink eraser was excellent. The easiest way of collecting the tiny pollen from many small flowers is by drying the blossoms on large sheets of paper and shaking them through a fine sieve. The anthers usually sift out and the pollen can be separated from the anthers by sifting again through fine silk bolting cloth. (Mimeo- giaph typewriter diaphragm silk is convenient.) The microscope showed, in the case of red maple, that invisible hairs from the flower also sifted through, but the pollen from other plants appeared quite free from foreign particles. : | Wodehouse (1916, p. 430) has suggested an excellent way of collecting large quantities of ragweed pollen. The flower heads just coming into bloom are crushed in a mortar with several volumes of carbon tetrachlorid. When strained through muslin the pollen passes through with the CCl, and can be separated by filtering on filter paper. The pollen is lighter yellow since the CCl, probably removed lecithin. In collecting pine pollen it was found necessary to gather the staminate cones before they had opened, because later the slightest shaking of the branch scattered a cloud of pollen to the four winds. Cutting off the tassels of corn and allowing them to open indoors, over large sheets of paper, undisturbed by currents of air, gave the largest yield of corn pollen. Preliminary Experiments These experiments were in two parts: (1) Germination of the pollen grains, and (2) Comparison of the enzyme action of unground, ground, and germinated pollen. The results of these tests showed that the pollen ground with powdered glass was more effective in its enzyme action than either the unground or even the germinated poller. The experiments were made as follows: To secure vigorous growth of pollen tubes, Easter lily pollen was ger- minated (1) in tap water, (2) in 3, 5, and I6 percent sugar solution, (3) on agar, and (4) in Knop’s solution and modifications. The stock agar recom- mended by Crabill and Reed (1915, p. 2) was used. This contains no carbon-containing nutrient and therefore does not favor bacterial and mold growths, which are exceedingly troublesome. MWec., 1921] PATON — POLLEN AND POLLEN ENZYMES 485 Formula for Stock Agar Distilledewatebe cate osc Shae le eG ee ees Pee ae eee 1,000 cc. Nae nesranarstit AGC is sc Bsd as Dandelion............: Slow but marked ‘ : Tests for Lipase In the different methods used for testing for lipolytic enzymes the fol- - lowing substrates and testing reagents were used: 1. Substrates. (1) Ethyl butyrate. (2) Olive oil acidified with decinormal acetic acid and a little gum arabic added to make an emulsion. (3) Olive oil emulsion recommended by Zeller. 10 cc. of olive oil was dissolved in hot 100 percent alcohol. This was run through a hot separating funnel to which was attached a piece of glass tubing drawn out toacapillary jet. The stream of oil in alcohol was run into 100 cc. of cold distilled water which was stirred continually. The milky emulsion was then heated to drive off the alcohol and afterwards diluted with water. (4) Methyl acetate. 2. Actwator. Approximately N/60 oxalic acid was used, partly because free acid is needed to counteract the slight alkalinity of the ground glass and more especially because free acid accelerates the activity of lipase. 3. Alkali for titration. Approximately N/1o sodium hydroxid solution was used to which a trace of barium hydroxid was added. ‘To insure uni- formity in readings, a 3-liter bottle was filled, and the solution was drawn off as needed through a connected graduated burette. Both the bottle and the burette had soda-lime bulbs at the inlet to absorb COs. 4. Indicator. Phenolphthalein was used in all titrations as an indicator. 5. Antiseptic. Toluol was added as an antiseptic. Controls of auto- claved pollen extract were run in each case, and the digestions were carried on in small stoppered Erlenmeyer flasks kept in an electric incubator at 36 °— 38° C. Samples were titrated at different time intervals. Methyl acetate was more strongly hydrolyzed than either ethyl butyrate or the olive oil preparations. 490 AMERICAN JOURNAL OF BOTANY [Vol. 8, Austrian pine, dock, daisy, goldenrod, ragweed, rye, and timothy pollens were tested with the different substrates for lipase. The tests with ethyl butyrate were unsatisfactory. In the olive oil emulsion and methy] acetate media, Austrian pine, dock, ragweed, and rye pollens gave positive tests for lipase. The action on methyl acetate was especially marked with Aus- trian pine pollen, in which case the titrations showed nearly double the amount of acid with fresh pollen as compared with the boiled control. Tests for Proteolytic Enzymes Substrates for Proteolytic Enzymes 1. Blood fibrin. Fresh fibrin from pig’s blood was obtained at the slaughter house, and was washed for several hours with a stream of cold water to remove corpuscles. Fairly uniform and compact strands of the fibrin were selected, and portions as nearly equal as possible were placed in test tubes with 10 cc. of distilled water, plugged with cotton, and sterilized for 20 minutes in an autoclave. Other portions were stained with I percent Congo red and the color was fixed by immersion in boiling water. The red color is liberated when the fibrin is digested. The colored fibrin was also sterilized. TABLE 13. Fermt’s Gelatin Test 5 cc. Fermi’s gelatin, 5 cc. H2O, 100 mg. pollen, 37° C. Degrees of liquefaction or failure to solidify, after standing in ice water 10 minutes, indicated by signs. Unheated Pollen Autociaved Pollen Kind of Pollen 24 Hrs. | 48 Hrs. 24 Hrs. 48 Hrs. + Daisy of ecaitd oe Seok eee ee i + @ 16 <0: 6) e's) ee) .e: 'e! te 1a) oye" 0: ce. ie: eo: fe) Ampie, 'o.16 ily Hastetdccd.s 1 sds oer SMe LICer ns: 8 Seem heen TO, Pine. Austrian. ici; iwi eeee PL. gine: whites, uso. On eee ee 12 RAOWEE Mss ele, ohn. soe CHOWAN EH DH | ~ (2) Q nr + + + +- + t4+4+4++44+4++ +4++4+44+4+4+4+4+4+4+4++44+ é ve) Ss) 8 + ++ 44 ++4t4+4+4+4+4+4+4+4+44+4+4+4+ TS: Magnoliaes wet note ae be cel an Pie] ge at lf ey Pah eat a Pract elrcaleals satel oi Salicaloe lial alee Sates + 2. Fermi’s gelatin. The proportions used were those given by Dernby. 700 grams of gelatin were dissolved in 1,250 cc. of hot water over a water bath, strained through cheese cloth, and 2 grams of finely pulverized thymol were added. The solution was diluted to 2 liters and sterilized. Dernby diluted further before using, but this was not found necessary with the pollen extracts. When the gelatin was used it was melted over a bath and Dee., 1921] PATON —— POLLEN AND POLLEN ENZYMES 491 to 5-cc. portions were added 5 cc. of distilled water and 100 mg. of pollen. For the control the pollen and water were first autoclaved. The tubes were incubated at 37° C. for 24 hours or longer (see table 13). The tubes were taken from the incubator and placed simultaneously in ice water, and the failure to solidify, or degree of congealing, during 10 minutes was noted. Many investigators have used gelatin for detecting enzymes of the pepsin type, but the experiments of Malfitano, Mavrofannis, and Jordan, recently confirmed by Berman and Rettger, seem to indicate that lique- faction of gelatin by an organism is not proof of proteolytic activity. How- ever, since pollen extracts, like pineapple juice, possess this power of lique- faction to a marked degree it has been considered worth while to record the observations. : Tests for Trypsin TABLE I4 Congo red, blood fibrin, 2 cc. N/10 NaszCOs, to cc. pollen extract (50 mg. in 100 cc. distilled water, unheated and autoclaved), 1 mg. thymol in each tube. | Appearance of Solutions Test Pete el eS Sear Kind of Pollen | 24 Hrs. Unheated Autoclaved LGA) ae — No change No change 2. Coun .. + oreetae +4 Fibers disintegrated. Fibers unaltered Liquid very pink Liquid pale yellow BOM AISV pease. sale. eos - No change No change ae Wandelion...:...... — ns - S zy Sa Dols oil a A Fibers less firm. Liquid pinkish | No change Gree itnec er ee a es t i Liquid faint pink aren: ng Vegtroldentod): 5.250... ++ Fibers disappeared. Fibers unaltered Liquid red-brown Liquid yellow-brown Sralbilys Waster... .. 2. - — No change No change Ore Met eLIS CEs was — - : TO} Pine; Austrian... .:. + Fibers partly disinte- grated, liquid pink No change Mel ebiite,.WiNte, 6 Si. 2 > Fibers partly disinte- grated, liquid pink No change P2e RACWEER 056.56. + Fibers partly disinte- grated, liquid pink No change GIN Cate givis ek s.« eles + Fibers partly disinte- grated, liquid pink No change PAG OIIMOERY. 61s. s ok sea dos | + Fibers partly disinte- grated, liquid pink No change PS NaASnOMas vis wwe es © + + Fibers disintegrated, liquid pink No change 16. Maple, Norway..... — No change y - From tables 14-16 it may be seen that corn, goldenrod, Austrian pine, white pine, ragweed, rye, and timothy all gave positive results. Dock gave strongly positive results in the less alkaline medium, while magnolia and goldenrod were strongly positive in the more alkaline medium. Apple was negative with Na,CO3 added, but was positive without the addition of 492 AMERICAN JOURNAL OF BOTANY [Noles TABLE 15 Same as shown in table 14, except that no NazCO; was added. Solutions slightly alkaline from the powdered glass. Thymol added to each tube. Appearance of Solutions Test Kinds of Pollen 24 Hrs. Unheated Autoclaved 1. PAD plenya aes + ees slightly disintegrated. Liquid | No change pin 2 @ORNG eae ses ++ Fibers completely disintegrated. Liquid pink Pe : ioe DEV CN ae teens one ig Fibrin darker, liquid milky a ms Ae Dandelion: «cece — No change se oh Re DOCK: Neva ah ocala cite +--+ Fibers completely disintegrated. Liquid pink a Ge li 8 Sak ewe tee ? Slight turbidity i s 7. Goldentod ¢ scx + wee + Fibrin shrunken. Liquid dark brown | “‘ it Saalcily we aster ey.0 sme — oi e os re + < i Oy lily, gers.) cue. — oo ee o e ee ce TO}abine) Austhiatt ns ee ao Disintegration, turbid, pink i zs ti. Pine, whites .c..0.,. + i ie +: hi To, sRACWeCd.. 0.72 eae ee + , oe a i: ee ToS RV Cin Ver heats eee + A is s fe ee TA. Wimothy . 2 f240 2 ++ Complete disintegration. Liquid red; ‘ i 15) Viaonoliagvss ee. — No change = S 16. Maple, Norway..... i Liquid turbid pinkish “f = Tests for Pepsin TABLE: 16 Same as shown in table 14, except that 2 cc. of 0.2 percent HCI was added instead of Na2CO3. Appearance of Solutions Kind of Pollen Tee 24 Hrs. Unheated Autoclaved TPAD plews mutes cate tas — No change No change Bie Onn ee el et ta ty, eae + Disintegration. Liquid pink . a ii! DIGS a Ane Oe — No change a A “4. Dandelion.......... _ a * i ia eDOCS KIS itune tyne eu ~ - i “ si One ae eek tS eee ict ~ es 7..Goldenrod...5.0 3.0. = ne B s ‘ 8. lily.eHaster a. o _ a = “ ci Q.Jlily tigen eee. oe — ee o a se TO,, Pine) Austman. 15 7- _ " “ s a LL, oPane se winltere eas ieee = - ‘ a “ 12; .Raeweedare nae ene _ 3 ry ‘ a 12. ARVe@28 S.A ee ee “b Disintegration. Liquid pink s uh ia. ‘liniothy. (ee ae + - - oe oe bi 15) sMacnoliay see _~ No change st a 16. Maple, Norway..... _ > - Re * \ — eas ey C2) Gan, nee 2 sx — Bae Qa &) <7 Sey, aa oS Eee f=) aS ay Se AO ‘ates iy) x ee ‘ alk ; ; * \ { dy ) { i & u Natl { \ , yi y \ Laks Ri y y (Uf y SUE: ne: , ci f Hi 4 ; (ey Porto th Sidhe at MY C, 4 RAW. y i ’ Myer tl { 0 ; S "y i ¥} x : 5 £ : Ky 1 Sth MEK \ NR at L "Cornel L Dnbersiey: Ihaca, N. 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