Title: Contributions from the Botanical Laboratory and the Morris Arboretum of the University of Pennsylvania, vol. 14 Place of Publication: Philadelphia Copyright Date: 1939 Master Negative Storage Number: MNS# PSt SNPaAg244.2 CONTRIBUTIONS FROM THE Botanical Laboratory and The Morris Arboretum OF THE UNIVERSITY OF PENNSYLVANU VOLUME XIV 1937-1938 PHILADELPHIA 1939 » f < 4. » ♦- •r t *• 4~ T •• .T. f A- "i' I 4^ . t CONTENTS ADAMS, J. W. : New stations for Lophotocarpus spongiosus in south- ern New Jersey. Bartonia, No. 19:41-42, (1938). Populus heterophylla in southern New Jersey. Bartonia, No. 19:45-46, (1938). BEALL, RUTH : Exchange of electrolytes between roots and acid solutions. Plant Physiology, 72:455-470, (1937). BOESHORE, IRWIN, and JUMP, JOHN AUSTIN: A new fossil oak wood from Idaho. American Journal of Botany, 25:307-311, (1938). CHILDS, THOMAS W.: Variability of Polyporus schweinitzii in culture. Phytopathology, 27:29-50, (1937). COHEN, ISADORE: Structure of the interkinetic nucleus in the scale epidermis of Allium cepa. Protoplasma, 27:484-495, 2 pi., (1937). CONSTANCE, LINCOLN : See Edgar T. Wherry. FENDER, FLORA S.: The flora of Seven Mile Beach, New Jer- sey. Bartonia, No. 19:23-41, (1938). FOGG, JOHN M., JR.: A station for Hymenophysa pubescens in the eastern United States. Rhodora, J9:191-192, (1937). FOSBERG, R. F. : A central repository for type-specimens. Journal of Botany, 76:327-330, (1938). The Lower Sonoran in Utah. Science, 57:39-40, (1938). Notes on plants of the Pacific islands. L Bulletin of the Torrey Botanical Club, 65:607-614, (1938). Two Queensland Ixoras. Journal of Bot- any, 76:233-237, (1938). Additional note on Queensland Ixoras. Jour- nal of Botany, 76:276-277, (1938). GRAY, WILLIAM D.: The effect of lighting on the fruiting of myxomycetes. American Journal of Botany, 25:511-522, (1938). — , A note on Stemonitis fusca Roth. Proceed- ings of the Indiana Academy of Science, ^7:86-87, (1938). Observations on the methods of stipe-forma- tion in Stemonitis and Comatricha, Proceedings of the Indiana Academy of Science, '/6:81-85, (1937). JUMP, JOHN AUSTIN: A new disturbance of red pine. Science, 57:138-139, (1938). ^ . A new fungus on Ficus nitida Thunb. Jour- nal of Agriculture of the University of Puerto Rico, 27:573-576, (1937). A study of forking in red pine. Phytopa- thology, 25:798-81 1, (1938). See Irwin Boeshore. LEPOW, SAMUEL S.: Some reactions of slime mould protoplasm to certain alkaloids and snake venoms. Protoplasma, i7; 161 -179, (1938). RUSSELL, MARY A.: Effects of X-rays on Zea mays. Plant Physi- ology, 72:117-133, (1937). SEIFRIZ, WILLIAM: Methods of research on the physical proper- ties of protoplasm. Plant Physiology, 12:99-116, (1937). That word "emulsoid". Science, 57:212- 214, (1938). . A theory of protoplasmic streaming. Science, 86 '397-39S, (1937). TRUE, RODNEY H. : Francois Andre Michaux, the botanist and explorer. Proceedings of the American Philosophical Society, 75:313-327, (1937). . Lichens from the New England coast. Bry- ologist, 40:71-73, (1937). TRUE, R. P.: Gall development on Pinus sylvestris attacked by the Woodgate Peridermium, and the morphology of the parasite. Phytopathology, 28 :24A9, (1938). WEAN, ROBERT E.: The parasitism of Polyporus schweinitzii on seedling Pinus strobus. Phytopathology, 27:1124-1142, (1937). WHERRY, EDGAR T.: A hybrid-fern name and some new com- binations. American Fern Journal, 27:56-59, (1937). . — Midland fern notes. American Fern Jour- nal, 28:28-30, (1938). and CONSTANCE, LINCOLN: A new Phlox from the Snake River Canyon, Idaho-Oregon. The Ameri- can Midland Naturalist, 79:433-435, (1938). Notable Pennsylvania ferns. Proceedings of the Pennsylvania Academy of Science, 77:52-54, (1937). — Notable Pennsylvania orchids. Proceedings of the Pennsylvania Academy of Science, 72:42-45, (1938). .., The Phloxes of Oregon. Proceedings of the Academy of Natural Sciences of Philadelphia, P(?:133-140, (1938). Southern occurrences of Dryopteris clinton- tana. American Fern Journal, 27:1-5, (1937). — Two plant-geographic notes. Journal of the Southern Appalachian Botanical Club, 2:1-2, (1937). YORK, HARLAN H.: Inoculations with forest tree rusts. Phyto- pathology, 25:210-212, (1938). ZIRKLE, CONWAY: Aceto-carmin mounting media. Science, 55:528, (1937). The plant vacuole. Botanical Review, 3:U 30, (1937). i -4* f ■1- ( ■ i, I 4 I » ♦ » ^ -* - -• » T • * <.|. i « • CONTRIBUTIONS FROM THE Botanical Laboratory and The Morris Arboretum OF THE UNIVERSITY OF PENNSYLVANIA VOLUME XIV 1937-1938 PHILADELPHIA 1939 f » »« CONTENTS » -< >•• # h • I • ^ • •- * \ • ADAMS, J. W.: New stations for Lophotocarpus spongiosus in south- ern New Jersey. Bartonia, No. 19:41-42, (1938). — ■ Populus heterophylla in southern New Jersey. Bartonia, No. 19:45-46, (1938). BEALL, RUTH: Exchange of electrolytes between roots and acid solutions. Plant Physiology, 72:455-470, (1937). BOESHORE, IRWIN, and JUMP, JOHN AUSTIN: A new fossil oak wood from Idaho. American Journal of Botany, 25:307-311, (1938). CHILDS, THOMAS W.: Variability of Polyporus schweimtzii in culture. Phytopathology, 27:29-50, (1937). COHEN, ISADORE: Structure of the interkinetic nucleus in the scale epidermis of Allium cepa. Protoplasma, 27:484-495, 2 pi., (1937). CONSTANCE, LINCOLN: See Edgar T. Wherry. FENDER, FLORA S.: The flora of Seven Mile Beach, New Jer- sey. Bartonia, No. 19:23-41, (1938). FOGG, JOHN M., JR.: A station for Hymenophysa pubescens in the eastern United States. Rhodora, 59:191-192, (1937). FOSBERG, R. F. : A central repository for type-specimens. Journal of Botany, 7^:327-330, (1938). The Lower Sonoran in Utah. Science, 57:39-40, (1938). Notes on plants of the Pacific islands. I. Bulletin of the Torrey Botanical Club, ^5:607-614, (1938). Two Queensland Ixoras. Journal of Bot- any, 7^:233-237, (1938). Additional note on Queensland Ixoras. Jour- nal of Botany, 7^^:276-277, (1938). GRAY, WILLIAM D.: The effect of lighting on the fruiting of myxomycetes. American Journal of Botany, 25:511-522, (1938). A note on Stemonitis fusca Roth. Proceed- ings of the Indiana Academy of Science, 47 :S6-S7y (1938). Observations on the methods of stipe-forma- tion in Stemonitis and Comatricha. Proceedings of the Indiana Academy of Science, 46-M-^S, (1937). JUMP, JOHN AUSTIN: A new disturbance of red pine. Science, 57:138-139, (1938). -^^^— — — — — A new fungus on Ficus nttida Thunb. Jour- nal of Agriculture of the University of Puerto Rico, 21 :573-576, (1937). A study of forking in red pine. Phytopa- thology, 25:798-811, (1938). I I See Irwin Boeshore. LEPOW, SAMUEL S.: Some reactions of slime mould protoplasm to certain alkaloids and snake venoms. Protoplasma, i/: 161 -179, (1938). RUSSELL, MARY A.: Effects of X-rays on Zea mays. Plant Physi- ology, 72:117-133, (1937). SEIFRIZ, WILLIAM : Methods of research on the physical proper- ties of protoplasm. Plant Physiology, 72:99-116, (1937). ■ That word "emulsoid". Science, 87:212- 214, (1938). A theory of protoplasmic streaming. Science, Sd:397-398, (1937). TRUE, RODNEY H. : Francois Andre Michaux, the botanist and explorer. Proceedings of the American Philosophical Society, 75:313-327, (1937). Lichens from the New England coast. Bry- ologist, 40:71-73, (1937). TRUE, R. P.: Gall development on Pinus sylvestris attacked by the Woodgate Peridermium, and the morphology of the parasite. Phytopathology, 25:24-49, (1938). WEAN, ROBERT E. : The parasitism of Polyporus schweinitzii on seedling Pinus strobus. Phytopathology, 27:1124-1142, (1937). WHERRY, EDGAR T. : A hybrid-fern name and some new com- binations. American Fern Journal, 27:56-59, (1937). Midland fern notes. American Fern Jour- nal, 25:28-30, (1938). and CONSTANCE, LINCOLN: A new Phlox from the Snake River Canyon, Idaho-Oregon. The Ameri- can Midland Naturalist, 79:433-435, (1938). Notable Pennsylvania ferns. Proceedings of the Pennsylvania Academy of Science, 77:52-54, (1937). Notable Pennsylvania orchids. Proceedings of the Pennsylvania Academy of Science, 72:42-45, (1938), The Phloxes of Oregon. Proceedings of the Academy of Natural Sciences of Philadelphia, 9(7:133-140, (1938). Southern occurrences of Dryopteris clinton- tana. American Fern Journal, 27:1-5, (1937). Two plant-geographic notes. Journal of the Southern Appalachian Botanical Club, 2:1-2, (1937). YORK, HARLAN H.: Inoculations with forest tree rusts. Phyto- pathology, 25:210-212, (1938). ZIRKLE, CONWAY: Aceto-carmin mounting media. Science, 55:528, (1937). The plant vacuole. Botanical Review, J:l- 30, (1937). ' t n M ♦ ) •> *• I k -^f «.< "^ Reprinted from Bartonia, No. 19, issued March 8, 1938 New Stations for Lophotocarpus spongiosus in Southern New Jersey J. W. Adams The recent explorations by Dr. John M. Fogg, Jr., and Mr. Bayard Long along the Delaware River have shed light on the distribution of a number of native species once considered rare in southern New Jersey and in one case unknown. Two outstanding examples are Lilaeopsis chinensis (L.) Kuntze and Lophotocarpus spongiosus (Engelm.) J. G. Sm. Since its first discovery along the Delaware River, Lilaeopsis chinensis ^ has been found at so many stations and in such abundance that now it is considered common along the brackish tidal marshes. In 1936 Dr. Fogg records the first substantiated addition of Lophotocarpus spongiosus ^ to the flora of southern New Jersey. This present note places on record two more of what may eventually become a series of stations paralleling those of Lilaeopsis chinensis in frequency. On June 17, 1937 the Torrey Botanical Club made a field trip to study the tidal vegetation along the Delaware in Salem County. The writer while riding in the company of Dr. Fogg, who acted as guide, and Miss Flora S. Fender noticed along the exposed tidal mud, 2.5 miles southwest of Salem, plants suggesting Lophotocarpus. Since it was decided best not to stop at the time, the return trip was made by the same route. Examination proved the plants to be a colony of Lophotocarpus spongiosus in all its leaf-shapes and plant sizes. While most of the plants were in full bloom, a great number still had quite young flower-buds tightly nestling among the leaves; other plants were young and immature: these indicated that the colony had from days to weeks more of anthesis. There was a conspicuous absence of other more fully developed plants, there being only a few clumps of Peltandra virginica and young plants of Ranunculus sceleratus growing nearby. On the mud at the bases of some of the Lophoto- carpus were tangled mats of Spirodela polyrhiza abandoned by the receded tide. Between the time the above mentioned station was observed and our return to it Dr. Fogg and the party found another locality which had as fine a display of the Lophotocarpus as could be expected anywhere. On an extensive tidal mud flat, behind the shore of the Delaware River at Elsinboro Point, 4 miles southwest of Salem, were countless plants in bloom. It is safe to say there were acres of them. The plants in general appeared to be more fully developed than those of the previous locality, both in size of the plants and in their stage of bloom. This 1 Bartonia 16: 51. 1934. 2 Bartonia 17: 21. 1935. % t 42 BARTONIA species dominated that particular mud flat at the time. As before, the two associated plants were Peltandra virginica and Pontedena cordata with Baccharis halimifolia along the margin. One seeing such an impressive abundance of a plant, once unknown to south- em New Jersey, cannot help but wonder, as suggested in the first paragraph, how much more of it is yet undiscovered. It also brings to mind the fact that while concentrated effort was being made to explore the Pine Barrens by local (and distant) botanists, Salem and Cumberland counties, and especially along the Delaware, seem to have been neglected. Anyone at all within the area of this plant, from the middle of June until frost, could not possibly have over- looked such an abundantly conspicuous display of it. Specimens of Lophotocarpus spongiosus from both stations were collected and distributed to the following herbaria: J. W. Adams No. 3309, University of Pennsylvania, Philadelphia Botanical Club, Gray Herbarium, University of Mon- treal, Dr. Walo Koch in Zurich, Switzerland; J. W.Adams No. 3296, Gray Her- barium, University of Montreal, Dr. Walo Koch; John M. Fogg, Jr. No. 11,890, University of Pennsylvania, Philadelphia Botanical Club. Morris Arboretum of the University of Pennsylvania • <♦ -• i# ^ k J y^ ^ WITHOUT P " " T T " "~ - I234 56789DIII2I3 DAYS Fig. 1. Conductance (millionths recip. ohm) of HjO with and without seedlings. Circles mark end of rapid initial increase in pH ; arrows mark onset of visible root injury. ductance values for HoO with and without seedlings at 22° C., and for HoO with seedlings at 28° C. Since no considerable conductance increase oc- curred without plants, it may be inferred that the increases shown by water with plants were due to outward movement of material from the roots. There was continuous exosmosis of electrolytes, in excess of any absorption that may have occurred. Net exosmosis, which was at first relatively slow, became markedly accelerated about the fifth day at the higher temperature and about the eleventh day at the lower temperature. It was at all times more rapid at the higher temperature than at the lower. At the lower tem- perature the pH value of HoO with seedlings increased from 5.4 to 5.7 in »/ -- •♦ — ■^ .1 V « T » BEALL: EXCHANGE OY ELECTROLY'TES 457 the first day, and then to 6.1 in the following three days, after which it re- mained practically unchanged. At the higher temperature the changes were very similar, but more rapid, from 5.0 to 5.5 in one day, and to 6.1 in three days. This may indicate either that there was some absorption of hydrogen ion, the effect of which on conductance was overbalanced by rela- tively greater exosmosis of electrolytes, or that exosmosis acted to reduce the hydrogen-ion concentration of the surrounding liquid. Mineral acid solutions. — The manner in which conductance of the min- eral acid solutions in contact with seedling roots first decreased and subse- quently increased is shown by the graphs of figures 2 to 4. Each graph 60 50 40 O ^20 < 1- ^ 10 D Q Z 0 o o -10 -20 -30 :i J / 1 H< \ 1 / / - 1 1 i 1 i 1 1 / 0 J 7 ^o 9- / / f — \ r > y^ J — 2i^ i^ / k — 0-- 7 t~ 1 - \\.(e)no2»jg. l?t-^ o > V "^^ ^ ^ — Mil 2 3 4 5 6 7 8 9 10 II 12 13 DAYS Fig. 2. Conductance change (millionths recip. ohm) in HCl solutions with seedlings. Circles mark end of rapid initial increase in pH j arrows mark onset of visible root injury. starts at zero on the scale of ordinates (which, in each case, represents the initial conductance of the solution in question) and conductance decreases are plotted as negative while increases are plotted as positive. The three average initial conductances, expressed as reciprocal ohms x 10 **, for each of the three concentrations of each acid were as follows : HCl, 8.9, 26.8, 52.8 ; HoSO,, 8.8, 26.9, 56.2; HXO^. 8.5, 31.3, 64.5. « 1 • 458 PLANT PHYSIOLOGY Some special measurements made at the end of the first liour showed, in many instances, a slight initial increase in conductance, relatively greater as the original concentration of the solution was higher, which suggests that there may have been an immediate exosmosis of electrolytes from the roots. This detail is not shown on the graphs ; it was not seriously studied and war- rants no more than mention here. Each of the graphs shows an initial decrease in conductance, which was very slight and of very short duration for the solutions of lowest concentra- tion, but progressively more pronounced and of longer duration as concen- tration was higher. Considering the two graphs for each concentration togelher, it may be said that the period of conductance decrease was shortest Fig. 3. Conductance change (millionths recip. ohm) in H..SO4 solutions with seed- lings. Circles mark end of rapid initial increase in pH; arrows mark onset of visible root injury. (about 1 day or less) for the lowest concentration, longer (about 2 to 3 days) for the intermediate concentration, and longest (about 3 to 4 days) for the highest concentration. Similarly, the average amounts of conduc- tance decrease were approximately as follows, the unit being 1 x 10-« recip- rocal ohm : for the lowest concentration, 2 units (all three acids) ; for the in- termediate concentration, 11 units (HCl), 13 units (HoSOJ, and 17 units (HNO3) ; for the highest concentration, 26 units (HCl), 28 units (H2SO4), and 41 units (HNO3). It is notable that HNO,, showed greater conductance decrease at the two higher concentrations than was shown by either of the other mineral acids at these concentrations. - 4 f t r 4 V -^- V •- • BEALL: EXCHANGE OF ELECTROLYTES 459 «» ^ ♦ y > 1- i * ^ '\ V i As to the temperature relations of the amount of conductance decrease, the available data permit no satisfactory generalization for all three mineral acids. From the graphs of figure 2 it is clear that the amount of conduc- tance decrease for solutions of HCl was regularly somewhat greater at the lower than at the higher temperature. For the lowest and highest concen- trations of HNO3 (fi^- 4) the same temperature relation apparently holds, but for the intermediate concentration of HNO3 and for all tested concen- * *|»> . il. , Fig. 4. Conductance change (millionths recip. ohm) in HNOj solutions with seed- lings. Circles mark end of rapid initial increase in pH; arrows mark onset of visible root injury. trations of H2SO4 (fig. 3) conductance decrease was more or less markedly greater at the higher than at the lower temperature. When conductance decrease was relatively large in amount its rate is seen to have been rapid at first and then progressively more gradual. For the intermediate and highest concentrations the initial period of decrease was followed by a more or less prolonged period during which conductance was nearly maintained. This period was longer for the lower temperature , » w 460 PLANT PHYSIOLOGY than for the higher one. After conductance began to increase, its rate of change sometimes remained low to the end of the experimental period, but in many cases (notably all the graphs of figure 2) its rate was soon rapidly accelerated, so that the final portions of the graphs ascend very steeply. As to changes in hydrogen-ion concentration, the pH values of all tested solutions — and that of H2O also — increased from the beginning of the experimental period onward. This increase was most rapid during the first day and became progressively slower till a value of about 6.0 was reached, when hydrogen-ion equilibrium with the roots was apparently established. Eventually the solutions became slightly more basic. The initial pH values of the solutions, corresponding to the initial conductances given above, were as follows : HCl, 4.6, 3.9, 3.6 ; H0.SO4, 4.5, 3.9, 3.6 ; and HNO3, 4.5, 3.8, 3.5. After one day the following values were reached: HCl, 4.8, 4.6, 5.3 ; H,SO„ 5.1, 4.6, 5.5 ; and HNO3, 4.9, 5.1, 5.6. The time required, at the lower temperature, for each medium to attain a pH value of about 6.0 is shown by the position of the circle on the corresponding graph of figures 1 to 4. That time was in general somewhat shorter for the solutions of highest concentration than for the other solutions and for H..0. The two lower concentrations of the three acids were essentially alike, reaching this value in four days; for the highest concentration the change was somewhat more rapid in HNO3 (2 days) than in IICl (3 days) or H2,S04 (4 days). Organic acid solutions. — Conductance changes shown by the three organic acids in contact with seedlings are set forth by the graphs of figure 5, which are plotted like those of figures 2 to 4. For each of the three con- centrations of each acid, the initial conductance (each plotted as zero on the ordinate scale) was as follows: HCOOH, 6.2, 16.2, 29.3; CH,COOH, 5.5, 10.6, 16.3 ; C0H5COOH, 4.2, 9.6, 14.1. For each solution the two tem- peratures tested showed no essential differences in conductance change; the graphs of figure 5 represent the average values for the two temperatures. As in the case of the mineral acids, conductance measurements made within the first day of the experimental period indicated in some instances a very slight immediate increase in conductance, of very short duration, which is not shown on the graphs. These organic acid solutions showed an initial decrease in conductance, similar to that shown by the mineral acid solutions, but less pronounced. For the lowest concentration of each acid this decrease was of one day's duration or less. It was scarcely noticeable in the case of the most dilute solution of propionic acid, only very slight in the weakest acetic acid solu- tion, and somewhat more pronounced in the lowest concentration of formic acid. In all these cases conductance subsequently increased gradually for about three days, after which it remained approximately unchanged. For i \ t -^ » *- ♦ r / yj ♦-•'■«• s' r y I < > 7*^ N BEALL : EXCHANGE OF ELECTROLYTES 461 solutions of higher concentration the initial conductance decrease lasted somewhat longer, from one to three days, after which there was, in most cases, some increase, but this was never sufficient to raise the conductance of the solution above its original value. In general, conductance remained unchanged after five days. As was shown for the inorganic acid solutions, initial decrease was greater in amount as solution concentration was greater, the amounts of decrease (in conductance units) being as follows: HCOOH, 2, 11, 20; CH3COOH, 1, 4, 8; C.H.COOH, 0.1, 3, 6. \i Fig, 5. Conductance change (millionths recip. ohm) in organic acid solutions. Circles mark end of rapid initial increase in pH; winged arrows mark onset of visible root injury at 23° C, naked arrows at 28° C. Only the graphs for the lowest concentration of acetic acid and of pro- pionic acid and the ones for the lowest and intermediate concentrations of formic acid show any considerable conductance increase within the 8-day period, but of course the conductance of the other organic acid solutions might have increased eventually had the period been longer. To make possible satisfactory comparisons among all six acids, with reference to con- 1 ♦ ♦ 462 PLANT PHYSIOLOGY BEALL : EXCHANGE OF ELECTROLY^TES 463 ductance increase, it would apparently have been necessary to employ experimental periods longer than 8 days. Turning to the changes in hydrogen-ion concentration of the organic acid solutions, these were very nearly like the corresponding changes that occurred in the mineral acid solutions, except that the original solutions were generally less acid and the amount of change was correspondingly less. The initial pH values of the three concentrations of each of the three organic acids were as follows : HCOOH, 4.5, 3.9, 3.6 ; CH..COOH, 4.8, 4.5, 4.1 ; and C^H.COOH, 4.9, 4.5, 4.2. In general there was rapid' decrease m acidity during the first day, the following pH values being reached- HCOOH, 5.6, 5.7, 5.8; CH3COOH, 5.5, 5.3, 5.3; and C,H,COOH, 5.3, 4.5, 4.3. Subsequently the decrease in acidity continued, but was more grad- ual, the solutions attaining a value of about 6.0 by the end of 3 days (inter- mediate and highest concentration of HCOOH and all three concentrations of CH3COOH) or at the end of 4 days (lowest concentration of HCOOH and all concentrations of C^H.COOH), after which pH was approximately maintained. These times are indicated by the positions of the circles on the graphs of figure 5. Physiological effects Controls.— On the roots of the control plants, in nutrient solution, lateral branches always appeared within the first day of the experiment and these subsequently increased in number and enlarged as the primary root continued to elongate. On the second day, when the hypocotyl was well extended, the cotyledons diverged, and later the plumule opened and elongation of the epicotyl began. No chemical injury was apparent and these plants appeared to be quite healthy throughout the entire experi- mental period of 14 days, excepting that, being in darkness or very weak light, they were markedly etiolated. Plants with roots in water.— Seedlings with roots in H^O developed more slowly than those with roots in nutrient solution. Development of lateral roots was similar to that in nutrient solution except that these did not appear until after 2 days and they did not elongate so much. After 2 days also the cotyledons began to diverge and gradually development of the epicotyl proceeded. Injury at the tips of the roots was evident after about 9 days at the lower temperature and after about 4 days at the higher temperature, as is indicated by the arrows of figure 1. Injury to hypo- cotyls followed in many cases. Plants with roots in mineral acid solutions.- In solutions of min- eral acids development was still more retarded than in water and injury was apparent earlier. Injury was first noticeable as a slight constriction in the primary root, 1 to 2 mm. from the tip. The tip region soon became •» 1 » f > ' y X flaccid and translucent, then very soft and brown. These changes gradu- ally spread upward until, in the most severe cases, only the upper centi- meter or less of the root was apparently free from necrosis. Injury to the hypocotyl usually followed soon after the onset of root injury. It was first evident as a translucent region 1 to 2 cm. long, about the middle of the hypocotyl, and necrosis spread both upward and downward. Subsequently the injured region of the hypocotyl turned brown and collapsed, sometimes with exudation, whereupon the cotyledons and plumules drooped. The plumule developed only slightly or not at all. Development in the mineral acid solutions was roughly in inverse rela- tion to the original concentration of the solution. Neither primary root nor hypocotyl elongated to any considerable extent in the solutions of high- est concentration and both showed less elongation in the solutions of inter- mediate concentration than in the most dilute solutions. Lateral roots appeared after 2 days in all cases ; in solutions of lowest concentration they developed throughout practically the entire length of the primary root, in solutions of intermediate concentration they were confined to the upper two-thirds of the main root, and in solutions of highest concentration they were confined to the upper one-third or less. When the region of their development was restricted they were unusually crowded. At the end of the experimental period they were longer in all mineral acid concentrations than in water, and their length was greater as the region supporting them was shorter. At either temperature, injury to the roots became evident earlier and progressed more rapidly as solution concentration was higher, and for any tested concentration it appeared earlier and progressed more rapidly at the higher than at the lower temperature. At the end of the experimental period injury in the least concentrated solutions was confined to the terminal region of the primary root, in solutions of intermediate con- centration it occupied from one-third to one-half of the length of the main root, and in solutions of highest concentration the primary root was visibly injured throughout practically its entire length. Injury to the hypocotyls also appeared earlier and progressed more rapidly as the solution was more concentrated and as the temperature was higher. If the times required for the onset of root injury in these solutions (as shown by the positions of the arrows in figures 2 to 4) are considered roughlj^ as reciprocally pro- portional to the toxicity of the solutions, then it appears that, for the two lower concentrations at the lower temperature, the decreasing order of tox- icity for these three mineral acids was HNO.^, H2SO4, HCl. For the high- est concentration at this temperature HNO^ and HgSO^ were about alike and produced injury sooner than HCl. For the lowest and for the inter- mediate concentration at the higher temperature HNOo and HCl were about alike and produced injury more promptly than HoSO^. Finally, all three acids were approximately alike, on the basis of this time criterion, for the t »' 464 PLANT PHYSIOLOGY BEALL: EXCHANGE OF ELECTROLYTES 465 highest concentration at the higher temperature since all had produced injury within the period before the first examination, but in these cases injury was noticeably more severe in HNO., solutions than in the other acids. It thus appears that both temperature and concentration took part —along with the chemical characteristics of the acids— in determining the promptness of the onset of injury. Plants in organic acid solutions.— Development was more rapid and more extensive in the solutions of organic acids than in the corresponding solutions of mineral acids. With formic and acetic acids development was somewhat greater in the lowest concentrations than in the others • with propionic, however, development in the solution of intermediate concen- tration equalled or exceeded that in the most dilute solution, and develop- ment m the most concentrated solution was less rapid than in either of the other solutions. By the second day lateral roots had emerged in all cases They appeared earlier, however (after only 1 day), in the most concen- trated solutions of formic acid and acetic acid at the higher temperature and m all solutions of propionic acid at this temperature. Like the min- eral acids tested, the organic acids permitted the development of laterals throughout most of the length of the primary root in solutions of lowest concentration. As the solution was more concentrated laterals were in- creasingly restricted to the older region of the primary root, but the re-ion bearing laterals was not so limited as under the corresponding influence of the mineral acids. As with the inorganic acid solutions, but to a lesser extent, lateral roots were finally more closely crowded and longer the more restricted was the region from which they arose. Injury by the organic acids was similar, in inception and nature to that produced by the mineral acids, but for any tested concentration and tem- perature It generally appeared somewhat later with the most rapidly in- jurious organic acid than with the lea.st rapidly injurious mineral acid as IS shown by comparing the positions of the arrows on the graphs of fi-iires 2 to 5. (It should be noted that in figure 5 winged arrows refer to the lower temperature while naked arrows refer to the higher temperature ) Acetic acid generally appears to have been less promptly injurious than either of the other organic acids, which were apparently about alike in this respect. In general, injury by any solution was less prompt at the lower than at the higher temperature and it appeared sooner as the concentration was higher. The six acids and H.O compared as to toxicity Although the available data are inadequate for a satisfactorv compara- tive study of the degrees of toxicity of all six acids, it may be^of interest to summarize the lengths of time required for the onset of visible root injury, as follows : f m.* >»-* r I ^ V I * 4 1 ^ \ ^ V (J 1. At 22° or 23° C. the descending order of indicated toxicity for each concentration is: a. For lowest concentration: HNO3 (3.5 days), HGOOH and CH3COOH (5 days), H^SO^ (6 days), HCI (8 days). b. For intermediate concentration: HNO^ (1 day), HCOOH (3 days), H^S04 (4 days), CH.COOH (5 days), HCI (6.5 days). c. For highest concentration: HNO3 and H-^O^ (1 day), HCOOH (2 days), HCI (2.5 days), CH3COOH (3 days). 2. At 28° C. the descending order of indicated toxicity for each con- centration is: d. For lowest concentration: HCI (2 days), HNO3 (2.5 days), HCOOH and C^HsCOOH (3 days), H^SO^ (4 days), CH3COOH (5 days). e. For intermediate concentration: HNO3 and HCI (1 day), H2SO4 and CACOOH (2.5 days), HCOOH (3 days), CH3COOH (4 days). f. For highest concentration: HNO3, H0SO4, HCI and HCOOH (1 day), CoH,COOH (1.5 days), CH3COOH (2 days). For each combination of temperature and concentration the reciprocal of the injury-time for each acid may perhaps be taken as an index of its relative toxicity. If for each combination all six reciprocals are expressed in terms of the reciprocal for HNO3 and if the resulting relative toxicity values for each acid are averaged, the final outcome is a single series of tentative toxicity indices that may represent all the tested combinations of temperature and concentration; that new series is as follows: HNO3, 1.00; HCI, 0.71; H,SO„ 0.66; C,H,COOH, 0.63; HCOOH, 0.61; CH3COOH, 0.41. The corresponding value for H,,0 is 0.22. According to these aver- ages, it appears on the whole: (a) that HNO3 was the most toxic, while (b) CH3COOH was least toxic of the six acids considered, (c) the former being about 2.5 times as toxic as the latter; (d) that HCI and H.SO, were nearly alike in toxicity (about 69 per cent, as toxic as HXO3) ; (e) that CoH.COOH and HCOOH were nearly alike in toxicity (about 62 per cent, as toxic as HNO3), (f) only slightly less toxic than HCI and H21SO4, and (g) about 50 per cent, more toxic than CH3COOH; and, finally, (h) tliat H.O ap- peared to be about half as toxic as CH3COOH and about one-fifth as toxic as HNO3. While these statements seem to agree in general with some con- clusions that are suggested by superficial scrutiny of the graphs and their appended arrows, it must be remembered that they are intended only to represent a sort of summary of the toxicity phase of the present study, for it is almost certain that the degrees of toxicity of these acids in any test depend on temperature and concentration as well as on chemical nature. Conclusion The initial reduction in conductance of these acid solutions was evi- dently due to absorption of ions by the roots. This absorption was prob- ably accompanied by some exosmosis in all cases, as was shown for HoO. — y^ MM 466 PLANT PHYSIOLOGY BEALL: EXCHANGE OF ELECTROLYTES 467 While it is conceivable that the conductance of a solution might be dimin- ished by exosmosis alone, if the materials extruded from the roots were of such nature as to remove from solution the ions present, consideration of the nature of the solutions examined in this study and of the kinds of mate- rial that might possibly escape from plant roots leads to the conclusion that the observed conductance decrease must have been due to direct removal of ions by the plants. This was no doubt accomplished principally by absorption of material into the cells, but adsorption of ions on to root sur- faces may have occurred also. Since solutions of all the acids tested were reduced in conductance, and hence in ion concentration, it seems safe to conclude that ions were absorbed from both inorganic and organic acid solutions. The amount of net absorption from the several solutions seems to have varied directly with ion supply. For any acid, the higher the ionic con- centration of the original solution, the greater and more rapid was the removal of electrolytes. And among the six acids tested, the greater the degree of ionization, in general, the more rapid was net absorption. A general index of absorption from these acid solutions under all the condi- tions represented by these experiments may be derived by expressing the average amount of net absorption from each concentration of each acid in terms of the average net absorption from the corresponding concentration of HNO3, and averaging the three values for each acid. This index is as follows: HNO3, 1.0; H,,SO„ 0.83; HCl, 0.77; HCOOH, 0.73; CH3COOH, 0.30 ; and C0H5COOH, 0.12. That is, considering the average of the results of the several experiments, there was greatest absorption from HXO3 solu- tions, and least from those of CoJIr.COOH. Net absorption from the organic acid solutions was generally less than from the corresponding con- centrations of mineral acids, but HCOOH was only slightly below HCl and H0SO4 in this respect. This is in general agreement with the order of dis- sociation of these acids. Absorption was, however, apparently influenced by other factors to some extent. Examining the results for the two temperatures separately it may be seen that, for the several mineral acids at the lower temperature, net absorption from HNO3 solutions was greatest and that from H0SO4 solutions was least, net absorption from HCl being intermediate. This order of penetration (HNO3 > HCl > H.3O4) is in agreement with Davidson and Wherry's (9) results with wheat seedlings. On the other hand, in the present study the order of penetration at the higher tempera- ture is: HNO3 > H^SO, > HCl; this is the order reported by Hind (15) from her experiments with potato tuber. Net absorption was apparently increased by rise in temperature in the case of all solutions of H0SO4 ^^d the intermediate concentration of HNO3, t»ut in the case of the other solu- tions (namely, all concentrations of HCl and the lowest and highest con- 4 1^ centrations of HNO3) net absorption was apparently decreased. Delf (10) and Skeen (20) found the permeability of plant tissues to increase with rise in temperature, and Stiles and J0rgensen (24) report greatly augmented absorption of hydrogen ion under similar conditions. Eckerson (13), on the other hand, reports that the effect of rise in temperature on permeability of root cells varied according to the species considered. Studies on this subject reported in the literature have been carried out with a variety of materials and by means of a variety of experimental pro- cedures. It is safe to presume that rates of penetration of materials into plant roots, and rates of absorption, depend on the kind and condition of the roots as well as on temperature and the nature of the solutions from which the absorbed substances are derived. The pronounced decrease in hydrogen-ion concentration which accom- panied the initial decrease in conductance of tliese solutions may be taken as evidence that there was rapid absorption of hydrogen ion in the early part of each experimental period. As was the case with absorption in general, this was always greater the higher the concentration of the original solu- tion, and greater the first day than in any succeeding day. Absorption of hydrogen ion by plant cells has been reported by many observers (5, 14, 18,19,23). The toxicity of the hydrogen ion has long been recognized. About forty years ago Kahlenberg and True (17) pointed out that the toxic influence ex- erted upon plants by dilute solutions of strong acids was probably largely due to this ion. Since that time Breazeale and LeClerc (3), Hoagland (16), and Dunn (12), among others, have reported on acid toxicity for plant tissues. As to the effect of such toxicity within plant cells, Addoms (1) observed that the protoplasm of root hairs injured by an acid medium appeared to have been coagulated. It seems probable that the toxic effects produced by the acid solutions of the present study were due chiefly to the hydrogen ion. Similar injuries appeared in all experimental plants and this was the only ion, other than the hydroxyl ion, which was common to all solutions. Also the hydrogen ion is more active, and probably more toxic, than any of the anions present. In general, degree of toxicity followed hydrogen-ion concentration. For any acid the higher the concentration and the greater the ionic absorption therefrom, the more promptly did injury occur, and in the acids as a group it was generally true that the more completely dissociated the acid the more toxic it was. Propionic acid was, however, more toxic than would be ex- pected from its ionic concentration or from the amount of ionic absorption that occurred from its solutions. Apparently the propionic molecule also was toxic. This is in agreement with True's (25) idea that the harmful action of formic and acetic acids is due principally to hydrogen ion, whereas that of propionic acid is due chiefly to the undissociated molecule. Other r * r 468 PLANT PHYSIOLOGY BEALL : EXCHANGE OF ELECTROLYTES 469 workers also have attributed the toxicity of organic acid solutions to undisso- ciated molecules (6, 8, 12). Even in the case of the acids whose toxicity seems to have been fundamentally due to excess of hydrogen ion, other ions or molecules may not have been without influence on toxicity. The toxicity of these acids was generally greater at the higher tempera- ture than at the lower. Injury was apparent earlier and the plants became more severely affected at the higher temperature. Skeen (21) also has found toxicity to be increased by increase in temperature in some cases. Although, as has been intimated, it appears likely that the initial de- crease in conductance of the acid solutions was fundamentally due to hydrogen-ion absorption by the roots, there is indirect evidence that other ions and molecules also were absorbed. Furthermore there seems to be no reason to doubt that there was in all cases some exosmosis of ions or mole- cules, or both. That the initial lowering of solution conductance ceased — generally after 1 to 3 days for the mineral acids, and after a somewhat shorter time for the organic acids — must have been due to decreased absorp- tion, to increased exosmosis, or to both together. Either of these altera- tions in the rate of movement of material across the root periphery may well have been occasioned by increasing toxic action. Indeed this increased exosmosis has been used as a criterion of injury by some workers (8, 22). AVith the mineral acids excessive exosmosis from the poisoned plants even- tually became greatly pronounced, as was shown by increase in solution conductance, but no rapid outburst of electrolytes had occurred at the time the experiments with the organic acids were discontinued. Nor did rapid exosmosis occur within 8 days in solutions of aromatic acids tested in a similar way by Mother Chrysostom (8). In those cases where conductance increased rapidly toward the end of the experimental period, the sudden increase in exosmosis that is indicated may have been due to a corresponding sudden increase in outward perme- ability of the root cells to electrolytes, such as might result from some sorts of poisoning, or perhaps poisoning may first have caused general proto- plasmic coagulation, followed at length by a sudden disintegration. It is possible that the anions or undissociated molecules of the organic acids acted in some way to retard the poisoning process or hinder protoplasmic disintegration. Summary 1. Roots of Liipinus alhus were found to absorb electrolytes from very dilute solutions of certain inorganic acids— HCl, H^^O^, HNO3— and cer- tain organic acids— HCOOII, CH,COOII, C,H,COOH. Absorption was greater in amount and more rapid the higher the original concentration of the solution. For any tested concentration it was greater as dissocia- tion was more nearly complete. I - w AVy 2. There was apparently a rapid absorption of hydrogen ion, probably accompanied by some absorption of other ions, of molecules, or of both. 3. Injury to the plants resulted in all cases. This is probably to be attributed to absorbed hydrogen ion in each instance except that of pro- pionic acid, whose undissociated molecules seem to have been largely responsible for the injury. 4. In many cases rapid exosmosis of materials from the roots eventually occurred, indicating increased permeability of cell membranes or rapid dis- integration of cell contents. 5. Influence of temperature, solution concentration, and hydrogen-ion concentration on absorption and exosmosis and on growth and development of the seedlings are among the topics discussed. This problem was suggested by Professor Rodney H. True, Director of the Morris Arboretum, who maintained a continuous interest through- out the study. In the analysis of the results and in the preparation of this paper the writer received many helpful suggestions from Dr. Burton E. Livingston, of the Johns Hopkins University, and from Dr. Charles A. Shull, of the University of Chicago. University of Pennsylvania Philadelphia, Pennsylvania LITERATURE CITED 1. Addoms, Ruth M. The effect of the hydrogen ion on the protoplasm of the root hairs of wheat. Amer. Jour. Dot. 10 : 211-220. 1923. 2. Beall, Ruth. A new cover for culture jars. Science n. s. 84 : 70. 1936. 3. Breazeale, J. F., and LeClerc, J. A. The growth of wheat seedlings as affected by acid or alkaline conditions. U. S. Dept. Agr. Bur. Chem. Bull. 149. 1912. 4. Britton, Hubert. Hydrogen ions. Chapman and Hall Ltd. Lon- don. 1929. 5. Brooks, :Matilda M. Studies on the permeability of living and dead cells. I. New quantitative observations on the penetration of acids into living and dead cells. Pub. Health Rpts. 38: 1449- 1470. 1923. 6. Clark, J. F. On the toxic effect of deleterious agents on the germina- tion and development of certain filamentous fungi. Bot. Gaz. 28 : 289-327, 378-404. 1899. 7. Clark, W. Mansfield. The determination of hydrogen ions. Ed. 3. The Williams & Wilkins Co. Baltimore. 1928. 8. Chrysostom, Mother Mary. The influence of several benzene deriva- tives on the roots of Lupinus alhus. Amer. Jour. Bot. 23: 461- 471. 1936. 1 ' ^ 470 PLANT PHYSIOLOGY 9. Davidson, Jehiel, and Wherry, Edgar T. Changes in hydrogen-ion concentration produced by growing seedlings in acid solutions. Jour. Agr. Res. 27 : 207-217. 1924. 10. Delf, E. Marion. Studies of protoplasmic permeability by measure- ment of rate of shrinkage of turgid tissues. I. The influence of temperature on the permeability of protoplasm to water. Ann. Bot. 30 : 283-310. 1916. 11. DoAK, K. D. Effect of mineral nutrition on reaction of wheat varie- ties to leaf rust. Phytopath. (Abs.) 21: 108-109. 1931. 12. Dunn, Marin S. Effects of certain acids and their sodium salts upon the growth of Sclerotmia cinerea. Amer. Jour. Bot. 13: 40-58. 1926. 13. EcKERSON, Sophia. Thermotropism of roots. Bot. Gaz. 58: 254r-263. 1914. 14. Heald, F. D. On the toxic effect of dilute solutions of acids and salts upon plants. Bot. Gaz. 22 : 125-153. 1896. 15. Hind, ]\Iildred. Studies in permeability. III. The absorption of acids by plant tissue. Ann. Bot. 30 : 223-238. 1916. 16. Hoagland, D. R. The effect of hydrogen and hydroxyl ion concentra- tion on the growth of barley seedlings. Soil Sci. 3: 547-560. 1917. 17. Kahlenberg, Louis, and True, Rodney H. On the toxic action of dis- solved salts and their electrolytic dissociation. Bot. Gaz. 22 : 81- 124. 1896. 18. Pfeffer, W. Osmotische Untersuchungen. \V. Engelmann. Leip- sig. 1877. 19. RuiiLAND, \V. Beitrijge zur Kenntnis der Permeabilitiit der Plasma- haut. Jahrb. wiss. Bot. 46: 1-54. 1908. 20. Skeen, John R. The tolerance limit of seedlings for aluminum and iron and the antagonism of calcium. Soil Sci. 27: 69-80. 1929. 21. ' . Experiments with Trianea on antagonism and absorp- tion. Plant Physiol. 5 : 105-118. 1930. 22. Stiles, Walter. The exosmosis of dissolved substances from storage tissue into water. Protoplasma 2 : 577-601. 1927. 23. , and J0rgensen, I. Studies in permeability. I. The exosmosis of electrolytes as a criterion of antagonistic ion-action. Ann. Bot. 29 : 349-367. 1915. ^ and . Studies in permeability. II. The 24. effect of temperature on the permeability of plant cells to the hydrogen ion. Ann. Bot. 29: 611-626. 1915. 25. True, Rodney H. The toxicity of molecules and ions. Proc. Amer. Phil. Soc. 69 : 231-245. 1930. r t •; "1 I- * • r A « » x > * 4 • ' i' Reprinted from the American Journal of Botany, Vol. 25, No. 5. 307-311, May, 1938 Printed in U. S. A. A NEW FOSSIL OAK WOOD FROM IDAHO ^ Irwin Boeshore and J. Austin Jump Quercus, although it would appear that less confusion Although several hundred species of fossil oaks from the United States have been described from their leaf impressions, comparatively little work has ])een done on the fossil wood of the genus. This neglect may be attributed in part to the uncertainty of specific differentiation on the V)a.sis of characters of the wood anatomy, since po.sitive identification of oak species often cannot be made from wood struc- ture alone. However, the sul)-genera of the genus Quercus may be determined by the microscopic ex- amination of their wood anatomy, and many of their included sjiecies also may be identified by this means. Thus it seems reasonable that well iireserved speci- mens of fossil wood might be expected to show simi- lar distinctions. Most of the work on fossil oak wood has been done l)y German investigators who described a number of western American si^ecies as well as European ones. This work is so little known that Trelease (1924), in monographing the oaks of America, stated that the only fo.«*sil oak wood to receive a specific name was Quercus Marcyana Pen., although at that time more than a dozen species had been described. However, the descriptions of these oaks might have been over- looked very easily since they were not published in widely distributed journals. The first descrii)tion of fossil oak wood was pub- lished in 1839 by Gopi)ert, who described fossil wood from Silesia and northern Germany and named it Klodenia qucrcoides. At that time he mentioned the nearly complete agreement of A', qucrcoides and Quercus pedunculata and six years later changed the generic name to Quercites, the genus in which fossil Feaves of oak are now jilaced. However, linger in 1S42 had proposed the genus Quercinium for fossil oak wood and had pointed out that probably Klo- denia (iopp. .should l)e changed to Quercinium. Very little notice was taken of the genus until Felix (1SS3, 1884) ])ubli I ^ 0 > I 7-f « < <^ ^ »> o e<9 CO o i SQ "3 O O O o tn fclj en CO a ^ & CO C -^ d =1- ■z o ;- CO _S o< X OS .d W -'. f; Si N ^-r-^ I 73 d ^^ -2 % ^-:3 •^ --^ P - d u. d e 0 « .-^ CO-Q HH bii apart, 400/* ■fa »3 JO d t^ • ■— 1 T— t a -2 s =* OJ 0^ d _o d a. o_ »-H m^^ »f5 ;i. i o Of o d o 73 »" ^ X r-l _4 p' lO CO ^ r: Ph 5"^ o o T3 03 c S d - d ? ^^ ^^ tiN 3. ' ' 8d ^ = -^ I d ^_ o V. rr d o X .2 J^ - ^ i d ►5.2 d (^1 *- C3 Fie. 1-0. — Fiff. 1. Tr.insxcrso .^rclion of ()ii( rri) sifion from s|>rin<: to suiniiicr wood.— Fij;. 2. Tr Ih iintn (iihintt s lio\viii-. I)ro;i(l r.iys. nnd .'ihnipf fr;i Fip. o. HM(linl scclioti of (J. (ilhmn -howine pits in liMchcid uififMiliid sfM'tioii of (J. alhiun showiii^r pits in n- \v;ill oi a \rs.sf<|. — w wood of (J. filhiitn. — Fitr. 5. Wood dis. — Fie. i. 'r_\ l% « / ^ f T # > '• ^ V > * * • f ♦ f <► May, 19381 ^ C ^ %i g r/3 72 fct BOKSnoUF. AM) JIM I' — i'USSlL OAK UOOD ;s e C TT" % c ^ C -* c ■-: sS tt ■«- ;- J: ~ tt GQ «! ac »o o tt Z.S * ^ 5 ^-s •« ^ 72 K' « i-. (M 3 1^ cc tt & r- 2 m i:^ :^ «? — w a 1^ S • •i - tt '^ i =*• xP !>. lO . ,;.: -~ O ::; — OC I- <. ■r ^O r o :=; c >. T. .= — • — w C T. - T' -T^ Ci O ^ 00 »5^1 I* " - - - o — : ,. *- ►-■ • • hJ i - ?: = w S it .- . O 4 S H, -r z', ■^ c ^9 o o ;- a ~ M 1^ M IP C:,_^" j;£ ^' O -- - :t o ^■' CI ;i; ► '^ o ^ JL i. c< H £ JK ■A 0-3 -: - fee S5X* 14 « Cj K c c t ^ i tt ~ i ^. ^ TTs: ■'• a iC =i- i - — = O a =*• »*► r: tt r' .- « ro « V) % 4 >> ^ * o^ o - a. o -^ c, If^ ''. •r (M ^* T £3 -r ^w. 5 Jl. ^- ,— * a. 7 - r i o > a - i a c s^ i i o o ep i fiQ o ^ X -*f*4 & GO »- ^ o* o* o INTENTIONAL SECOND EXPOSURE 310 AMERICAN JOURNAL OF BOTANY [Vol. 25, May, 19381 CRIBBS — FOSSIL PLANT 311 00 e aa l\i ^ bC Q o.s =="5 "•*-' ^ "- "^ • ^ > ^ S3 ."^ •- S <•«. t» 5- "^ c 2 ^^ the •^ ^H c w ^ "^ r/J « C 50 ^ 5 s r:^ ;= b " - S ""' "' , S ''- '^■ v/i 1— 1 ^ Mo3 t« o C CO o S 02 •T3 C O is o o u O - ^ = C3 e CO — o c - - o • - - «- J-. 0) = is iw c CO "O a. 5-3 T , — »o iO -3 7-r -J 0, 10 radi ntia a. CI bt c 0 ^ >-H ^ ^f-^ i; =L "^^ r. S •fi I'^^L • 1.11 >. J3 o »9 jn if P is I— I >o CO o :l :l 1 ^ v. to d 4-) 5S «>2 5 CO O o . I 5, Vi s s >, § w .w «M ;^ o ^ 5 t^ 5! ry: ^ cc X "C c 2 * fl ^ §"^''^ . "^ bt - t: £ s ^ 7 « - t' O »■' .^ '- T G-^ = >, !>5 2 fl 5 6 CO ►— -S -" bC • ^ I I *- -2 o L. '~' ^ S c ^ v" C ^ bC b o o c C ^ »2 S3 'f. c — o — -3 CO O a o «* m ^ ^.^ q; c -c 7. c >■ ^ =1. c 0 I I S a. •/. ff 0 »* , ( — 5 "^ f^ 5 C O i, "^* ^^ ^ a.. 2 ^ 0-3 CO '/: = a. ^ t:^ ^ x= '■..=. a. a. 0 0 1 bt ^ S 0 c ^ ^ f^ "*~ 1^ >. en ^ -Prf "~ ^ • «t j= ^, s: 7. f. b». ^ ^ ^ 0 f,. '^^ C ^i* "^ ^' ^ bt >* I -^ V ,1. rs" ■1. r: 4' >■ • i> CoNWENTZ, H. 1876. t)ber die versteinten Holzer aus dem norddcutschon Diluvium, p. 30, Diss. Breslau. Felix, J. 1883. Untersuchungen iibcr fossile Holzer. Zoitschr. Deutsch. Geol. Ges. Band 38. . 1884-87. Die holzopale Ungarns in paleophyten Hinsicht. Mitt, aus dem Jahrb. d. kgl. ung. Geol. Anstalt. Budapest, Bd. 7. GoPPEKT. H. R. 1839. In Bronn und Leonhardt, Neus. Jahrl). etc. p. 518. Knowlton, F. H. 1899. Fossil flora of the Yellowstone National Park. U. S. Geol. Sur. Mont. 32, pt. 2, 771. Mekcklin, C. E. vox. Paleodendrologicon rossicum. St. Petersburg. Penh.\llovv, D. P. 1891. Trees from the post-glacial of Illinois. Proc. and Trans. Roy. Soc. Canada Sec. 4: 31. Platen, P. 1907-08. Sitzungsberichte der Naturforsch- ende Gesell. zu Leipzig, 34. ScHLEiDEN, M. J. t)ber die organische Structur der Kieselholzer. p. 37. Trele.\se, W. 1924. American oaks. Nat. Acad. Sci. Mem. vol. XX. UxciER, F. J. A. X. 1842. In P:ndlichor, Gen. Plant, suppl. sec. (Append.) Webber. I. E. 1933. Woods from the Ricardo Pliocene of Last CMianco (Julch. C^arnegie Inst. Pub. Wash- ington 412: 96. O' O O O^ O o» O^ V [Reprinted from Phytopathology, January, 1937, Vol. XXVII, No. 1, pp. 29-50.] ■^ > ■r m * 19- ♦ 4 ^V VARIABILITY OF POLYPORUS SCHWEINITZII IN CULTURE* Thomas W. Childs2 (Accepted for publication May 1, 1936) INTRODUCTION Slight cultural differences within various species of wood-attacking fungi have been briefly mentioned by Long and Harsch (8) and by Fritz (3), but these workers, primarily interested in the diagnosis of decay, attached little importance to the variability they encountered. Schmitz (14) has described definite morphological and physiological differences between 4 isolations of Fomes pinicola (Sw.) Cke. Mounce (11), also working with F. pinicola, has discussed differences between cultures from a number of sources and ascribes these differences to individual variation rather than host influence. Aside from these studies, intraspecific variability in the Polyporaceae seems to have received little or no detailed attention, although the possibility of its occurrence often has been suggested. When Polyporus schweinitzii Fr. was found causing a destructive root rot in 20- to 25-year-old plantings of northern white pine, Pinus strohus L., on unfavorable sites near Springwater, New York,^ the possible implication of a heretofore unreported parasitic strain of either native or foreign origin could not be ignored. Cultures of this fungus, therefore, were requested from several American and foreign sources, and a number of isolations were made from diseased white pines at Springwater. A few isolations also were made from white pine and larch in New England. As this material accumu- lated it became increasingly apparent that there was no morphological basis for subdividing the species into local, parasitic, or host-specialized strains. Some of the cultures resembled each other rather closely, and all of them had a few characters in common, but with the exception of those taken from within a few feet of each other no two were identical, nor did those from any given host or locality manifest any special similarity. In the past, Polyporus schweinitzii has been known principally as a cause of heart rot in butts of over-mature trees ; with the disappearance of 1 Presented to the Faculty of the Graduate School of the University of Pennsylvania in partial fulfillment of the requirements for the degree of Doctor of Philosophy. 2 This study was begun during the tenure of a Morris Arboretum Fellowship in the Department of Botany of the University of Pennsylvania, and was completed at the Morris Arboretum with the assistance of a fellowship granted by the Charles Lathrop Pack Forest Education Board. The writer also wishes to acknowledge his indebtedness to Dr. H. H. York and Dr. Conway Zirkle, to Edith Adams Cliilds for laboratory assis- tance, and to the many workers who furnished cultures. 3 York H. H. A study of resinosis and root rot in forest plantings of Pinus strohus and P. resinosa. Unpublished manuscript. 29 IRREGULAR PAGINATION •» ^ 30 Phytopathology [Vol. 27 virgin stands it was expected to become relatively unimportant. Its de- structiveness at Springwater and in a Douglas fir plantation near Biltmore, North Carolina, (6) suggests, however, that it may constitute a serious obstacle to the use of coniferous species in artificial reforestation, at least on certain sites. An investigation of its variability has, therefore, been un- dertaken to determine to what extent specialized strains may be responsible for damage to young trees. Certain preliminary observations are pre- sented here. Pinal conclusions will be possible only when the results of field studies, already inaugurated, become available. SOURCES AND DESCRIPTIONS OF CULTURES It was the writer's practice to make several isolations from each sporo- phore or infected tree from which a culture of the fungus was desired. More than 100 isolations, representing 25 sporophores and 9 trees, were thus secured and cultured on nutrient agar under closely comparable conditions. With 2 exceptions, isolations from any given tree or sporophore were indis- tinguishable from one another, but differed more or less distinctly from those from any other source. It, therefore, was assumed that similar isolations from the same source were derived from the same individual mycelium, i.e., were members of the same clone, and that dissimilar isolations were derived from genetically different mycelia. These mycelia, together with those obtained from other workers, will be referred to by the following numbers : 1 to 30, inclusive. From Pinus strohus ; Springwater, N. Y. 31. From Pinus strohus (lesion on 0.5 cm. root of an 8-year-old planted tree) ; Honeoye Lake, N. Y. Weston, Ontario. Lake Timagami, Ontario. Harvard Forest, Petersham, Mass. Brattleboro, Vt. silvestris; Stralsund, Germany. ** ; Skjaerningsfjell, Ringebii, Norway. ** ; Great Britain. rigida ; Medf ord, N. J. mughus; Central Experimental Farm, Ottawa, Ontario. Pieea canadensis ; Lake Timagami, Ontario. sitchenis ; Moresby Island, British Columbia. ** ; Gahrenberg, Germany. 44 and 45. From Pseudotsuga taxifolia ; Berlin, Germany. 46. From Larix eiiropea ; Island of Visingso, Lake Vetter, Sweden. 47. ** ** ^ancma ; Franconia, N. H. 48. ** Thuja plicafa ; Vancouver, British Columbia. 32. ' 33. * 34. * 35. * 36. * 37. * 38. ' 39. * 40. * 41. * * Pic 42. ' ( << 43. * it n a {( 1937] Childs: Variability of Polyporus schweinitzii 31 M ■ ^^ il i 4 4> <^4 V ♦ Fio. 1. Cultures showing differences between individual mycelia and between cultures of the same mycelium exposed to different environments, and similarities between parallel cultures of each mycelium. Numerals refer to the numbers given the various mycelia on pages 30 and 32. '^ » i ^ • 30 Phytopathology [Vol. 27 A >■• k. 1937] CniLDS: VaRLVBILITY of PoLYPORUS SCnWEINITZII 31 virgin stands it was expected to become relatively unimportant. Its de- structiveness at Springwater and in a Douglas fir plantation near Biltmore, North Carolina, (6) suggests, however, that it may constitute a serious obstacle to the use of coniferous species in artificial reforestation, at least on certain sites. An investigation of its variability has, therefore, been un- dertaken to determine to what extent specialized strains may be responsible for damage to young trees. Certain preliminary observations are pre- sented here. Final conclusions will be possible only when the results of field studies, already inaugurated, become available. SOURCES AND DESCRIPTIONS OF CULTURES It was the writer's practice to make several isolations from each sporo- phore or infected tree from which a culture of the fungus was desired. More than 100 isolations, representing 25 sporophores and 9 trees, were thus secured and cultured on nutrient agar under closely comparable conditions. With 2 exceptions, isolations from any given tree or sporophore were indis- tinguishable from one another, but differed more or less distinctly from those from any other source. It, therefore, was assumed that similar isolations from the same source were derived from the same individual mycelium, i.e., were members of the same clone, and that dissimilar isolations were derived from genetically different mycelia. These mycelia, together with those obtained from other workers, Avill be referred to by the following numbers : 1 to 30, inclusive. From Pinus stroMis ; Springwater, N. Y. 31. From Finns strohus (lesion on 0.5 cm. root of an 8-year-old planted tree) ; Honeoye Lake, N. Y. ; Weston, Ontario. ; Lake Timagami, Ontario. ; Harvard Forest, Petersham, Mass. ; Brattleboro, Vt. .^ilvestris; Stralsnnd, Germany. ; Skjaerningsf jell, Ringebii, Norway. ; Great Britain. rigida ; Medf ord, N. J. mufjhus; Central Expei'imental Farm, Ottawa, Ontario. Picea canadensis; Lake Timagami, Ontario. sitchcnis ; Moresby Island, British Columbia. ** ; Gahreiiberg, Germany. 44 and 4;"). From Pseudotsuga taxlfolin ; Berlin, Germany. 46. From Larix europea ; Island of Visinjrso, Lake Vetter, Sweden. 47. ** ** Zflr/cfna ; Franconia, N. II. 48. ** Thuja plirafn; Vancouver, British Columbia. 32. 33. 34. 35. 36. 37. 38. 39. 40. 4L 42. 43. a (< ( ( t < << i < < ( < ( < ( n It ti it < < < i ( < < < < ( i i I i i i it ti it ' 4 * SERIES I. A » fi :i. 4 « p SERIES II FiO. 1. Cultures showing differences between individiial mycelia and between cultures of the same mycelium exposed to different environments, and similarities between parallel cultures of each mycelitmi. Numerals refer to the numbers given the various mycelia on pages ."iO and .32. INTENTIONAL SECOND EXPOSURE 32 Phytopathology [Vol. 27 49. Host not specified ; Timagami Forest Reserve, Ontario. 50. Host not specified ; Kyota, Japan. One isolation of each mycelinm was maintained as a stock culture from which all subsequent cultures of that mycelium, except when otherwise noted, were derived. Cultures of these mycelia were under the writer's observation for periods of from 6 to 20 months, and in no instance was any alteration of cultural characters detected. At the same stage of development, cultures of any given mycelium differed in appearance only when they had been subjected to different environments. This is shown in figure 1, where growth char- acters of each of 3 representative mycelia are seen to be consistent in dupli- cate cultures raised under the same conditions (i.e., in the same series) but are not consistent in cultures of tlie same mycelium in different series. The appearance of a given mycelium cultured in a given environment was deter- mined entirely by the inherent nature of that particular individual my- celium and by the factors of that particular environment; i.e., the cultural history of the material from which transfers were made had no appreciable effect upon the characters of the derived cultures. Schmitz (14) and Mounce (11) have described and illustrated the line of demarcation that usually develops when different mycelia of Fomes pini- cola approach each other on the same artificial substratum. An apparently identical reaction occurs between mycelia of Polyporus schweinitzii. When isolations from different sources were subcultured on the same slant or plate of nutrient agar, a well-defined dark line about 1 mm. wide usually ap- peared between them soon after the advancing margins had come in contact. This line did not form between subcultures taken from a single isolation or from similar isolations from any single source, nor did it form when mono- sporous mycelia were paired with other monosporous mycelia of either the same or different ancestrv. Where not otherwise specified, cultures were made on nutrient agar con- sisting of 20 g. of malt extract (either Fleischmann's Diamalt or Trommer's Diastasic) and 20 g. of agar per litre of distilled water. Sterilization was effected by autoclaving for 15 minutes at a pressure of 15 pounds. Growth was somewhat more luxuriant and fruiting was more frequent on media made with Diamalt. Trommer's extract was variable, some bottles being caramelized to a much greater extent than others, but the line of demarca- tion formed between mycelia from different sources was generally darker and more distinct when this brand of nutrient was used. In other respects these two brands of malt extract did not differ perceptibly in their effect on growth characters. As Fritz (3) has remarked, Polyporus schweinitzii is quite sensitive to if ¥ 1937] CniLDS: Variability of Polyporus schweinitzii 33 1 ♦ * SERIES II. I ^ > • •^ »- 1 r > J. • » L v/' Fig. 2. Cultures showing differences between individual mycelia. Note sporophores in Nos. 16 and 41. 32 Phytopathology [Vol. 27 49. Host not specified ; Timagami Forest Reserve, Ontario. 50. Host not specified; Kyota, Japan. One isolation of eacli mycelinm was maintained as a stock enlture from which all .subsequent cultures of that mycelium, except when otherwise noted, were derived. Cultures of these mycelia were under the writer's observation for periods of from 6 to 20 months, and in no instance was any alteration of cultural characters detected. At the same stage of development, cultures of any given mycelium differed in appearance only when they had been subjected to different environments. This is shown in figure 1, where growth char- acters of each of :J representative mycelia are seen to be consistent in dupli- cate cultures raised under the same conditions {i.e., in the same series) but are not consistent in cultures of the same mycelium in different series. The appearance of a given mycelium cultured in a given environment was deter- mined entirely by the inherent nature of that particular individual my- celium and by the factors of that particular environment; i.e., the cultural history of the material from which transfers were made had no appreciable effect upon the characters of the derived cultures. Schmitz (14) and Mounce (11) have described and illustrated the line of demarcation that usually develops when different mycelia of Fames pini- cola approach each other on the same artificial substratum. An apparently identical reaction occurs between mycelia of Pohjporus schwrinitzii. AVhen isolations from different sources were subcultured on the same slant or plate of nutrient agar, a well-defined dark line about 1 mm. wide usually ap- peared between them soon after the advancing margins had come in contact This line did not form between subcultures taken from a single isolation or from similar isolations from any single source, nor did it form when mono- sporous mycelia were paired with otiier monosporous mycelia of either the same or different aiicestrv. Where not otherwise specified, cultures were made on nutrient a-ar con- sisting of 20 g. of malt extract (either Fleischmann's Diamalt or Trommer's Diastasic) and 20 g. of agar per litre of distilled water. Sterilization was effected by autoclaving for 15 minutes at a pressure of 15 pounds. Growth was somewhat more luxuriant and fruiting was more frequent on media made with Diamalt. Trommer's extract was variable, some bottles being caramelized to a much greater extent than others, but the line of demarca- tion formed between mycelia from different sources was generally darker and more distinct when this brand of nutrient was used. In other respects these two brands of malt extract did not differ perceptibly in their effect on growth characters. As Fritz (3) has remarked, Polyporm schwetnitzii is quite sensitive to I - i" I i " r • ^ * » < ■♦ < ^^J»■ - < '^ « > 1937] ClIILDS: VakIAHILITY of PoLYi'OKUS SCUWEINITZII 33 SERIES I SERIES II Fig. 2. Cultures showing diflforencM'S Itotwocn individual mycelia. Note sporophores in Nos. 16 and 41. INTENTIONAL SECOND EXPOSURE 34 No. 1. 5. 7. 12. 18. Phytopathology [Vol. 27 TABLE 1.— Descriptions of some cultures of representative mycelia Series I. II. I. II. I. II. I. II. I. II. II. II. II. Color and character of growth Near inoculum Intermediate region Sienna to olive brown; short pile (occasionally with 1 or 2 indistinct concentric zones of longer and darker pile). Farthest from inoculum Dark greenish- brown; uniform and moderately long pile. Brown; uniform and moderately long pile. Pale and sparse. Light red brown to lemon yellow ; uniform and moderately long pile. Concentric zonation Indistinct. (Fig.l). Quite distinct. (Fig.l). l*ale and sparse. Yellow and light brown; fairly even mat. Mostly greenish or reddish brown, with some lemon yel- low to creamy yellow; regular and moderately long pile (occasionally slightly massed, tufted, or nuffy). Red brown; numerous very small tufts (some areas with short and fairly regular pile). Lemon yellow. None. Fairly distinct. Light orange brown; fairly regular pile be- coming very slightly and uni- formly tufted. Pale to lemon yellow; sparse to moderately long and irregular pile becoming slightly fluffy near edge of culture. Pale to lemon yellow. Yellow brown (sometimes with one or two concentric zones of lighter yellow) ; short, matted pile. Olive to greenish or reddish brown; moderately long and uniform pile. Pale lemon yellow to reddish brown; an uneven mat. Yellow to orange ; moderately long and irregular pile (some concentric zones of tufted, compacted, or fluffy growth. ^^_^__ Indistinct. Fairly distinct. Fairly distinct. Fairly distinct. (Fig. 2). Mostly rather pale but with 2 or 3 fairly distinct con- centric zones of lemon yellow to light brown; sparse except in regions of darker color, where regular pile is fairly well developed. Vague. Generally indis- tinct. (Fig. 2). Lemon yellow; short and irregu- lar pile with some semicom- pact or slightly fluffy growth. Pale and sparse. Zones of reddish brown, short, and regular pile alternate concentrically with zones of dark yellow to orange, longer (sometimes slightly fluffy) pile. Very pale; very sparse. Lemon yellow; moderately long and fairly regu- lar pile (some- times slightly fluffed). Fairly distinct. None. (Fig. 2). Quite distinct. " ^1^' - K > ♦ t. ) > V- V ^/i^ ^ - ,' ^. 1 ^ s y ♦ V -t 1937] Childs: Variability of Polyporus sciiweinitzh TABLE 1. — {Continued) 35 No. 31. Series ' Color and character of growth Near inoculum Intermediate region II. Dark, mottled, yellow brown; ir- regular, matted pile with frequent fluffy growth. Farthest from inoculum Concentric zonation 42. Greenish to red- dish brown; rather long and irregular pile. Dark creamy yel- low; small to medium large rounded tufts. Pale and sparse. Pale and sparse II. Pale and sparse. Pale and sparse. 44. I. II. Pale and sparse. Pale to yellow green (with some reddish brown) ; sparse, irregular, or fluffy. Generally pale and sparse but with numerous long, cobwebby, lemon yellow to brown wefts or fluffy masses. Generally pale and sparse but with zones of loose, creamy wefts and some creamy to yellow green fluffs at edge of culture. None. (Fig.l). Fairly distinct. (Fig.l). 47. I. 49. Pale to yellow to short, sparse, pile edge of culture). Dark cream to orange; irregu- lar (sometimes fluffy) pile. reddish brown; very thin mat with (some lemon yellow, fluffy growth at Light to dark yellow green ; pile ir- regular (fluffy in some central areas and near edge of culture). II. Yellow brown to reddish brown; short, irregular, pile and occa- sional small yel- low fluffs. Generally pale and sparse but usually with some pale lemon yellow growth near the edge of the culture. Greenish yellow, orange, and red- dish brown ; fairly long and regular pile (with a few small tufts). A narrow zone of creamy, mod- erately fluffy growth. Creamy to yel- low green; sparse pile with some fairly well developed fluffy growth. None. Rather vague. None. (Fig. 2). Indistinct. (Fig. 2). Vague. Rather indistinct. 36 Phytopathology [Vol. 27 light, growing more slowly and displaying darker and more varied colora- tion when exposed, even for very short periods, to indirect daylight than when kept in darkness. High humidity stimulates the development of fluffy superficial growth. Temperature, concentration and kind of nutrient, etc., also affect the appearance of cultures. The continuous nature of these environmental variables and the apparently great number of hereditary fac- tors involved make it impracticable to describe in detail all the mycelia studied. Partial descriptions of a few representative mycelia cultured in Petri dishes are presented in table 1, however, to indicate the range and kind of cultural variations encountered during the study. In this table, and in figures 1 and 2, series I consists of cultures stored at a constant tem- perature of 22°* and exposed to electric light, but never to daylight, for a few seconds every 12 hours, while series II consists of cultures stored at room temperature (10° to 25°) and exposed to diffuse daylight for a few seconds every 4 or 5 days. Descriptions of series I were taken on the 19th day and photographs on the 40th day of growth. Descriptions and photo- graphs of series II were taken on the 29th day of growth. From 2 to 20 cultures were made of each mycelium in each series. Cultural characters of each mycelium in each series were consistent in all cases where not other- wise indicated. From the descriptions in table 1, and from the photographs in figures 1 and 2, it is apparent that differences between mycelia may sometimes be large enough to make difficult the diagnosis of wood decay by cultural methods. Identification may be facilitated by culturing on agar slants in ordinary test tubes and by storing cultures where they will be exposed to daylight of low intensity, since these conditions promote the formation of the more or less leathery mat and hasten the appearance of the ''range of yellow tints" (3) typical of this species, but, even under these conditions, occasional mycelia are not readily identifiable on the basis of their cultural characters. The writer has not investigated the microscopic features of the various mycelia to a sufficient extent to determine their reliability as criteria of specific identity. Hasty and superficial observations indicate, however, that differences in microscopic characters are neither so great nor so fre- quent as are differences in gross cultural characters. A number of mycelia were noticeably destructive to the agar, in some spots lowering the level of the surface 3 mm. or more below that of the rest of the culture (Fig. 3, 40K). Such lowering usually w'as confined to the region near the inoculum, but sometimes occurred in other portions of the culture. This destructive effect upon the agar, and the particular region in which destruction was visible, were quite as characteristic of certain mycelia as were the color and character of growth in culture. In cultures * All temperatures recorded in this paper are in °C. ^ - 1937] Childs: Variability of Polyporus schweinitzii 37 I >. "♦ «i — ^ < - / « « * V > •» Fig. 3. Cultures showing individual diflferences between nionosporous mycelia. Note aporophoies in No. 40B and destruction of agar near inoculum in No. 40K. 36 PlIYTOPATIlOUMJY [Vol. 27 light, growing more slowly and displaying darker and more varied colora- tion when exposed, even for very short periods, to indirect daylight than when kept in darkness. High humidity stimulates the development of fluff. V superficial growth. Temperature, concentration and kind of nutrient, etc., also affect the appearance of cultures. The continuous nature of tliese environmental variables and the apparently great number of hereditary fac- tors involved make it impracticable to describe in detail all the mycelia studied. Partial descriptions of a few representative mycelia cultured in Petri dishes are presented in table 1, however, to indicate the range and kind of cultural variations encountered during the study. In this table, and in figures 1 and 2, series I consists of cultures stored at a constant tem- perature of 22°* and exposed to electric light, but never to daylight, for a few seconds every 12 hours, while series II consists of cultures stored at room temperature (10° to 25°) and exposed to diffuse daylight for a few seconds every 4 or 5 days. Descriptions of series I were taken on the 19th day and photographs on the 40th day of growth. Descriptions and ])hoto- graphs of series II were taken on the 29th day of growth. From 2 to 20 cultures were made of each mycelium in each series. Cultural characters of each mycelium in each series w-ere consistent in all cases where not other- wise indicated. From the descriptions in table 1, and from the photographs in figures 1 and 2, it is apparent that differences between mycelia may sometimes be large enough to make difficult the diagnosis of wood decay by cultural methods. Identification may be facilitated by culturing on agar slants in ordinary test tubes and by storing cultures where they will be exposed to daylight of low intensity, since these conditions promote the formation of the more or less leathery mat and hasten the appearance of the ''range of yellow^ tints" (3) typical of this species, but, even under these conditions, occasional mycelia are not readily identifiable on the basis of their cultural characters. The writer has not investigated the microscopic features of the various mvcelia to a sufficient extent to determine their reliabilitv as criteria of specific identity. Hasty and superficial observations indicate, however, that differences in microscopic characters are neither so great nor so fre- quent as are differences in gross cultural characters. A number of mvcelia were noticeablv destructive to the agar, in some spots lowering the level of the surface 3 mm. or more below that of the rest of the culture (Fig. 3, 40K). Such lowering usually was confined to the region near the inoculum, but sometimes occurred in other portions of the culture. This destructive effect upon the agar, and the particular region in which destruction was visible, w^ere quite as characteristic of certain mycelia as were the color and character of growth in culture. In cultures * All temperatures recorded in this paper are in °C. ■ > H - T "• 1937] Guilds: Variability ok Polyporus sctiweinitzii 37 i. \ ♦ * -* > s Fig. 3. Cultures showing individual diflferem-es between nionos[)orous mycelia. Note aporophores in No. 40B and destruction of agar near inoculum in No. 40K. INTENTIONAL SECOND EXPOSURE 38 Phytopathology [Vol. 27 of mycelia that did not alter the level of the surface of the substratum the agar retained its original gelatinous consistency until dry, but mycelia that caused any perceptible lowering of the agar surface also rendered the rest of the agar definitely crumbly, even in regions not adjacent to spots of visi- ble destruction. Schmitz (14) has shown that some mycelia of Fomes pini- cola secrete larger quantities of certain enzymes than are produced by other mycelia of the same species. The phenomenon just described suggests that mycelia of Polyporus schweinitzii also differ in enzyme production. In only one instance during the entire study did repeated isolations from the same source fail to yield identical cultures. Two isolations each of Nos. 23 and 24 were made from a single sporophore. These mycelia differed so markedly in appearance as to be distinguishable from each other during very early stages of development. No. 23 was in no way noteworthy, dis- playing the usual cultural characters of the species and differing from most of the other mycelia only in relatively minor details. No. 24, however, be- came established very rapidly at ordinary temperatures and quickly filled the space above the agar slants with a permanently fluffy mass of hyphae. Portions of this mass ranged from a faint lemon yellow to a very light red brown, but most of it remained white throughout the life of the culture. In many respects this individual resembled some of the single-spore cul- tures described in a subsequent section. No. 47, the only mycelium found in more than one individual host, was isolated several times from each of 3 wind-thrown larches separated from each other by distances of 20 to 30 feet. Since previous experience had shown that even neighboring hosts of the same species usually were in- fected by different mycelia, isolations from each of these trees were kept under close observation for about 6 months. At no time during this period were any greater differences detected between them than were to be found between parallel cultures derived from any single mycelium, nor was a line of demarcation formed when they grew together on a common substratum. Fruiting bodies have not been produced by these isolations and the possi- bility that they represent different mycelia belonging to a definite and per- haps specialized strain, therefore, has not been tested. Their homogeneity suggests, however, that a single mycelium has spread vegetatively to trees near the one originally infected. RATE OF GROWTH ON NUTRIENT AGAR During the growth of the cultures in series I, described in the preceding section, the radius of each culture was determined every 12 hours by measur- ing along the diameter of the Petri dish from the edge of the inoculum to the margin of the colony. Table 2 contains growth data derived from these measurements for 15 representative mycelia. (Nos. 4a and 35a were started ■ -f k. > \ - > % ¥ ■• > * < ' > ■» r: f * <*► ► t f< J ^ ,.-> 4 v - ^ * 1937] Childs: Variability of Polyporus schweinitzh TABLE 2. — Growth of representative mycelia in series I 39 Mycelium No. Days since establishment of cultures 8 Difif . be- tween max. and min. on 8th day 1. 2. 3. 4. 4a. 5. 31. 35. 35a. 38. 40. . 41. . 42. . 43. 46. . 48. . 50. . Average radius of mycelium — mm » 11 17 23 28 35 43 51 10 16 21 27 33 39 47 10 16 22 29 37 45 53 10 16 21 28 34 41 48 11 17 23 29 34 40 46 10 16 23 30 37 44 52 10 16 28 36 44 52 10 15 21 27 33 40 47 10 15 21 27 34 40 48 9 18 28 37 46 55 64 8 11 14 18 22 25 30 11 18 25 33 42 51 60 12 21 30 38 46 55 64 12 19 27 33 40 50 59 13 22 30 38 46 54 62 10 15 21 28 34 42 49 10 16 21 27 34 41 49 i 58 55 61 53 54 60 60 55 56 74 34 69 74 68 70 56 56 mm. 7 2 8 13 9 3 3 3 3 3 13 4 6 2 2 4 6 one week later than the rest of the series, but were otherwise identical in origin and treatment with Nos. 4 and 35, respectively). Averages for each mycelium are based on 4 cultures of that mycelium. The principal diffi- culty encountered in studies of growth rates in this species was the differ- ence in time required by the various mycelia for establishment on new sub- strata. In most instances, this difference was correlated with the character of the growth in cultures from which transfers were made. Other factors being equal, establishment was rapid when the transferred material included fluffs of loose aerial hyphae ; less so when the aerial growth was compacted or matted ; and slowest when the projecting hyphae were sparse and short. Mycelia that were to be transferred for comparative purposes were invari- ably cultured under the same conditions, but, because of inherent morpho- logical differences, it was impossible to secure the same type of material for transfer from different mycelia. For this reason, the age of each mycelium in table 2 is computed from the time when the 4 cultures of that mycelium attained an average radius of 5 mm. (arbitrarily taken to indicate establish- ment) instead of from the time the transfers were made. Averages have been calculated, where necessary, by straight-line interpolation between the original semi-daily averages. The close correspondence between Nos. 4 and 4a, and between Nos. 35 and 35a, shows that, within rather wide limits, the >»•.> 40 Phytopathology [Vol. 27 1937] Guilds: Variability of Polyporus schweinitzii 41 age of the agar is not an important factor, and that the arrangement of the data in this table may therefore be considered legitimate. Differences between mycelia are clearly apparent in table 2. It is evi- dent that Nos. 38, 41, 42, 43, and 46 have attained a greater radius and are growing more rapidly than the other mycelia, and that the growth rate of No. 40 is relatively low. Relative growth rates of mycelia cultured at higher or lower temperatures than series I were, however, frequently not consistent with the classification suggested by this table. For example, at 27° No. 43 grew more rapidly than No. 42, while at room temperature (Series II) No. 42 grew more rapidly than did No. 43. The cultures of No. 48 included in series II increased their average radius from 8 to 84 mm. in 14 days, while the cultures of No. 50 in the same series increased from 7 to only 65 mm. during the same period: at 27° the growth of No. 50 was ap- preciably more rapid than was that of No. 48. In every case where 2 mycelia differed significantly in growth rate at the extremes of the tempera- ture range (app. 10° to 27°) to which they were exposed, but not at one or more of the intermediate temperatures, the mycelium that grew relatively rapidly at one extreme grew relatively slowly at the other. These observa- tions indicate a difference between mycelia either in the form of the tem- perature-growth curve, or in the location of the maximum ordinate of this curve {i.e., the optimum), or both. Inclusion of the 3 mycelia from spruce in the group of rapidly growing individuals in table 2 is probably the result of coincidence rather than of any host-correlated relationship, since these 3 mycelia differed rather widely from one another in growth rate at other temperatures. Mycelia from Springwater, with one or two exceptions, maintained their relative rates of growth more consistently at the different temperatures to which they were subjected, and resembled each other more closely in absolute growth rates than did mycelia from other sources. This tendency toward similarity, even if not fortuitous, does not necessarily imply the existence of a special- ized strain at Springwater. It may indicate only the closer degree of rela- tionship that might logically be expected between isolations from a limited area as compared with isolations from widely separated points. EFFECT OF ACIDITY ON GROWTH Nutrient solutions adjusted to 9 different acidities were prepared by mixing 3 parts of 3 per cent Trommer's malt solution with 2 parts of KH phthalate buffers compounded according to the formulas of Clark and Lubs (2). The buffered solutions were placed in 38 x 200-mm. test tubes (app. 65 cc. of solution per tube) and autoclaved 15 min. at 15 lbs. pressure. The acidity of a sample from each lot was then determined potentiometrically, and the remaining tubes were inoculated with various representative my- 1^ <* I <♦ f * y *.< celia. One culture at each pH was made of each mycelium, except No. 1, which was cultured in duplicate. Twenty-three days after inoculation the depth of the mycelium and the acidity of the medium in each tube were determined (Table 3). Since in no case did the change in pH during the TABLE 3. — Effect of acidity on growth of mycelium of Polyporus schweinitzii Initial pHof Mycelium No. Final Duffered nutrient 1 1 8 31 40 42 47 49 50 pH Depth of mycelium — mm. 2.5 9 10 5 ; 5 8 8 6 6 15 2.6 3.0 18 18 13 i 12 15 15 10 10 28 3.0 to 3.2 3.5 26 27 28 30 22 34 16 25 45 3.6 to 3.7 4.0 40 43 41 44 45 46 37 39 52 4.2 4.3 42 44 50 45 46 52 50 42 52 4.5 4.7 44 46 55 39 40 50 43 42 55 4.8 to 4.9 5.1 43 43 53 36 7 48 39 46 55 5.2 5.4 22 21 28 17 9 20 20 34 33 5.4 to 5.6 5.7 10 10 10 1 7 4 11 7 12 15 5.6 to 5.9 growth of the fungus exceed 0.2, and since the 2 cultures of No. 1 at each acidity were reasonably consistent, the data in this table are considered approximately indicative of the effect of acidity on the growth of these mycelia. It will be seen that in every instance the optimum pH for growth was greater than 4.0 and less than 5.4, and in most cases probably lay be- tween 4.2 and 5.0. Within these limits, however, the acidity permitting maximum growth was somewhat different for different mycelia. For ex- ample, Nos. 31 and 40 obviously made their best growth in more acid media than did Nos. 49 and 50. RATE OF DECAY OF WOOD BLOCKS IN VITRO Thirty-two white pine blocks (all from the same tree) approximately i" X J" X 4" were air-dried in the laboratory for several weeks, given indi- vidual numbers, weighed to the nearest half -gram, placed in 38 x 200-mm. test tubes half full of water, and autoclaved 30 min. at a pressure of 15 lbs. They were then transferred to similar tubes, each of which contained a pure culture of the fungus growing actively in about 25 cc. of 2 per cent malt- extract solution, and stored in darkness at 22°. Two months later an addi- tional 30 cc. of sterile distilled water was added to each tube. After a total incubation period of nearly 9 months the blocks were removed from the tubes, air-dried for 3 weeks, and reweighed. Average losses in weight, expressed as percentages of the original air-dry weights, were as follows : 42 Phytopathology Heart wood 9 blocks inoculated with No. 1 7 " ** '' '' 39 Difference Sapwood 9 blocks inoculated with No. 1 7 ** '' '* ** 39 Difference [Vol. 27 1937] Childs: Variability of Polyporus schweinitzii 43 4.9 ± 0.7% 3.7 dtz 0.7% 1.2 ± 1.0% 17.7 zh 1.0% 24.9 ±: 2.7%, 7.2 ± 2.4% The difference between the 2 sets of heartwood samples cannot be considered significant, since it is only slightly greater than its probable error.^ The difference between the 2 sets of sapwood samples, however, is 3 times its probable error, and in spite of the small basis it may, therefore, be concluded that there probably is a real difference in the ability of these 2 mycelia to attack sapwood under the conditions of this experiment. Schmitz (14) presents evidence of similar differences between mycelia of Fomes pinicola. In his studies, as in the present instance, the relative order of destructiveness in heartwood is not the same as that in sapwood. SPOROPHORES IN ARTIFICIAL CULTURE Eleven mycelia, including isolations from both sporophore tissue and decayed wood, fruited in vitro during the course of these studies. Nine of the 11 fruited under conditions to which most or all of the 39 sterile mycelia also were exposed, while the remaining 2 produced sporophores only under conditions to which less than half of the other mycelia were subjected. A few of the fertile mycelia fruited commonly : in others, fruiting was rare or sporadic. In the series of wood-block cultures described in the preceding section. No. 39 formed sporophores consistently on sapwood but not on heartwood! It also fruited several times on white-pine sawdust and at the surface of cultures growing in a 2 per cent solution of malt extract. The only other mycelium cultured on these media was No. 1, which failed to fruit on any of them. On Trommer's malt agar, Nos. 39 and 50 produced one sporophore each. Nos. 23, 30, 34, and 41 occasionally formed a few sporophores, and No. 40 fruited rather commonly. On this medium, sporophores usually appeared only in rather old cultures. Fruiting, therefore, might have been less sporadic had it been possible to retain the cultures longer. Diamalt agar was apparently a somewhat better medium for sporophore 6 Standard deviation = /2(fd2) \ n-1 Probable error of average = 0.6745 S.D. Probable error of difference = VP.E.,2 + p.E.b2 4 production. No. 1 fruited rarely ; Nos. 9, 13, 23, and 41 fruited occasionally ; and No. 16 fruited fairly regularly on this medium. No. 34 never produced sporophores in test-tube cultures but did so invariably in Petri dishes except when the layer of agar was quite thin. The most consistent and prolific fruiting encountered during the entire study was afforded by No. 40. This mycelium commonly produced sporophores in test-tube cultures: in Petri dishes, sporophore rudiments appeared soon after the surface of the agar was overgrown and functioning sporophores were often present in cultures only 3 weeks old. The largest sporophores were those of Nos. 39 and 50, which averaged 1 cm. in diameter and ranged from 5 to 15 mm. in thickness : the smallest were those of No. 40, which averaged 3 mm. in diameter and 2 mm. in thick- ness. Color varied from yellow (Nos. 34 and 40) to reddish brown (No. 50) or dark brown (No. 39). Neither stipes nor recognizable pilei were present in any instance. Sporophores that developed above the culture medium consisted principally of short spines (produced by the majority of the fertile mycelia), small and disarticulated lamellae (Nos. 34 and 40), or longer hydnoid processes, frequently once- or twice-dichotomized (formed only by No. 39). Pores occurred infrequently, and then only in sporophores borne laterally, as on wood blocks and vertical agar slants, or vertically, as in in- verted Petri dishes. Average pore dimater varied from less than 0.5 mm. in No. 40 to slightly more than 1 mm. in No. 39. Small pores were approximately circular but the larger ones were quite angular. Young fruiting bodies that had recently become spiny or lamellate could sometimes be induced to form pores by reversing their orientation with respect to gravity. Neither sporo- phore production nor pore formation appeared to depend on exposure to daylight. The microscopic structure of hymenia in artificial culture did not differ essentially from that of hymenia developed under natural condi- tions. Sporophores formed by different mycelia displayed some microscopic dissimilarities — for example, cystidia of No. 34 were relatively large and sometimes terminally swollen, while those of No. 40 were smaller and of more nearly uniform diameter throughout. These variations, however, were no greater than those occurring normally in **wild" sporophores. It seems probable that the well-known variability of the sporophores of many species of Poljrporaceae under natural conditions (9, 15) is simply another example of differences between individuals. MONOSPOROUS AND POLYSPOROUS MYCELIA Basidiospores of Polyporus schweinitzii are so small and fragile that it is extremely difficult to secure uninjured single spores by Hanna's dry needle method (4), even with micromanipulation apparatus. Progressive dilution of material from spore prints is much less laborious, but the serviceabilitv 44 Phytopathology [Vol. 27 of this method is impaired by the persistence with which many of the spores remain aggregated in small groups throughout the process of dilution. The procedure devised by Mounce (11),^ however, proved quite satisfactory and in a slightly modified form was followed in obtaining all single-spore cultures except those from No. 39, which were secured by dilution. Polysporous cultures were obtained by scraping an uncontaminated spore print with a sterile spear-pointed needle and planting the material thus obtained on a slant of nutrient agar. Both monosporous and polysporous cultures were derived in all cases from spores produced by pure cultures of the fungus. Fresh spores germinated readily, and numerous monosporous cultures were obtained from Nos. 34, 39, 40, and 50. These monosporous cultures resembled the stock mycelia in many respects. The mat developed on the surface of the agar seemed, however, generally thinner and more delicate, and superficial growth was often much fluffier. The following, cultured under the same conditions as series II, are fairly representative (Figure 3) : No. 34A.^ Orange brown and rather irregular pile with numerous short, fluffy, pale lemon yellow tufts (Fig. 3). No. 34B. Growth sparse and pale near inoculum ; becoming pale yellow and loosely fluffed at other side of culture (Fig. 3). No. 34D. Orange to dark olive brown pile near inoculum; pale lemon and loosely tangled fluff at other side of culture. No. 39A. Reddish brown near inoculum ; reddish brown pile with numer- ous short tufts and fluffy cream-color masses at other side of culture (Fig. 3). No. 39B. Reddish brown near inoculum ; a mixture of light orange and dark cream at far side of culture. No. 39C. Zones of pale, sparse growth alternate with zones of orange pile near inoculum and with zones of cream-color fluffy growth at far side of culture (Fig. 3). No. 40 A. Growth near inoculum mostly pale and scantily fluffy ; rest of culture consists of light to dark yellow fluff (Fig. 3). No. 40B. Sparse and pale near inoculum, with some destruction of agar ; rest of culture dark lemon yellow to olive or red brown (Fig. 3). 6 ** A sporophore was removed from a culture with sterile forceps and quickly fastened to the cover of a sterile Petri dish by means of a drop of melted agar. Then the cover was removed from a Petri dish containing lactose gelatine, the cover bearing the sporophore was substituted, and slowly rotated. Then the cover was replaced and as many Petri dishes as desired were inoculated in this way. The plates were kept at room temperature for from four to five days. By that time, the germinating spores appeared as tiny de- pressions in the surface of the clear gelatine. A circle was drawn around each mycelium, it was examined under the microscope, and if it proved to be monosporous, and if there were no other spores within the circle, it, surrounded by a small square of gelatine, was removed with a sterile spear-shaped needle, placed on a potato-dextrose or malt agar slant, and grown at room temperature." 7 Numeral refers to parent mycelium — e.g., No. 34A is the first monosporous mycelium secured from No. 34. i\ 1937] Childs: Variability of Poly^porus schweinitzii 45 No. 40C. Mostly sparse and pale near inoculum; rest of culture rag- gedly fluffy and pale yellow to dark creamy yellow or light orange. No. 40F. Zones of sparse and pale growth alternate with zones of short, lemon yellow tufts (Fig. 3). No. 40K. Most of agar destroyed in immediate vicinity of inoculum; rest of culture consists of light to dark yellow fluff (Fig. 3). From these descriptions, and from table 4 and figure 3, it will be seen TABLE 4. — Growth of representative mycelia of both monosporous and natural origin (Series II) Mycelium No.a Age of cultures — days 11 16 21 Diff . between max. and min. on 16th day 1. . 3. . 4. . 34. . 34A. 34C. 34D. 34F. 39. . 39A. 39C. 39D. 40A. 40B. 40E. 40F. 40K. 41. 43. 46. 48. . Average radius — mm. 15 2 18 6 22 18 22 16 8 9 12 10 12 13 12 13 11 13 6 7 8 27 13 33 17 39 36 40 28 25 27 26 23 32 31 32 30 29 32 19 23 24 49 41 64 41 65 67 68 48 56 65 54 38 63 60 69 56 62 63 39 56 51 77 69 82 63 b b b 63 79 74 54 83 81 b 76 b 82 62 90 84 WTO. 2 4 4 8 0 8 S 4 0 % S 8 8 7 S 7 1 7 9 2 6 a Data for each mycelium are based on 2 cultures. b Entire surface of culture overgrown (radius greater than 90 mm.). that differences between monosporous mycelia are of the same general type and of about the same order of magnitude as those between isolations secured from different sources in the field. Cultural differences are ill-adapted to quantitative expression, and an exact comparison of the extent of variability in the different groups of monosporous mycelia of common descent is, there- fore, impracticable ; in general, however, it may be said that mycelia having the same immediate ancestry appeared to differ less than did those of more diverse descent. Individual differences were apparent, however, even be- 46 Phytopathology [Vol. 27 tween mycelia descended from the same sporophore. When cultured under the same conditions, hyphal-tip transfers from opposite sides of a given monosporous colony were culturally identical. Twenty-four of the 41 monosporous mycelia secured from No. 40 pro- duced sporophores. From one of these fertile monosporous mycelia (No. 40L) 33 single-spore cultures were made, of which 30 produced sporophores. These third-generation cultures resembled each other very closely, much more so than did those of the preceding generation, but could not be consid- ered identical in appearance. The only other sporophores that appeared in either monosporous or polysporous cultures were developed by a few descen- dants of Nos. 39 and 50. Some of these were presumptively of single-spore origin, but a few of the supposedly monosporous cultures (including the fertile ones) derived from No. 39 showed striking similarity to polysporous cultures from the same mycelium: In view of the defects of the dilution method (used with spores from this mycelium only) the monosporous origin of these cultures is doubtful. Polysporous cultures from Nos. 34 and 40 were similar in general appear- ance to isolations secured in the field, but polysporous cultures from Nos. 39 and 50 displayed none of the gross characteristics of Polyporus schwein- itzii. The hyphae in the latter two were hyaline, giving cultures on malt agar a dirty grey appearance, and were almost entirely confined to the medium within 1 mm. of the surface of the agar slants. Polysporous cultures from No. 39 produced a few short aerial hyphae that looked like very fine cotton lint sparsely sprinkled on the surface of the agar. Those from No. 50 produced no aerial growth. In other respects these cultures were indis- tinguishable from one another. After repeated transfers, several of the polysporous cultures from No. 39 developed thin, irregular, dark reddish brown mats on the surface of the agar slants : such cultures invariably pro- duced sporophores. DISCUSSION Morphological or cultural characters and more purely physiological quali- ties are not necessarily correlated (1). Nevertheless, the wide range of variability apparent between the cultures from Springwater, a range almost as great as that found between cultures from widely separated points and unlike hosts throughout the Northern Hemisphere, indicates that damage in this locality cannot now be attributed definitely to infection by a race or strain of special virulence. The planting stock used in the infected areas at Springwater came from nurseries in which seedlings from European nur- series had been transplanted, and other plantings in the immediate neighbor- hood have been made with stock of European origin, but the large number of different mycelia isolated from the infected trees precludes the possibility of infection having originated entirely by vegetative spread of the organism 1937] Childs: Variability of Polyporus Schweinitzh 47 T>' ■m. • » * •*•»•> ^ y ^ S y i ■ -,-. T from one or a few foci in nursery beds. Fruiting bodies of Polyporus schweinitzii have been rare in these plantings until quite recently.® It seems unlikely, therefore, that a few original infections by a virulent strain have been spore-propagated, with consequent morphological diversification of the organism, to a sufficient extent to account for the large and varied population of this species now present in these areas. Wean® has shown that infection may occur in very young trees, but his studies also indicate rather definitely that parasitic activity of this fungus is favored by certain conditions, such as those at Springwater, which are suboptimum for white pine. Pending the completion of inoculation studies in the field it must accordingly be con- cluded that the Polyporus schweinitzii epiphytotic in this locality probably is attributable to edaphic factors that have permitted extensive infection by native representatives of the species. The possibilities of future specialization created by the wide variability exhibited by this fungus must not be overlooked. It may be assumed that the majority of the individuals constituting this species are now adapted to a quasi-parasitic life in the basal heartwood of older trees, but extensive infection of younger trees growing under more or less unnatural conditions might conceivably result in the gradual selection of lines best adapted to such existence and a consequent increase in the average virulence of the species to planted trees even on good sites. The likelihood of such a change and the time necessary for it to occur must remain problematical, but the immediate values involved are great enough to justify the development and adoption of preventive and control measures. Variability of the type described in this paper is by no means peculiar to the Polyporaceae. Recent pathological literature contains numerous ref- erences to similar instances of intraspecific variability in widely separated species of fungi. The work of Magie (10) on Coccomyces hiemalis, Johnson and Valleau (7) on Thielaviopsis hasicola, Palmiter (12) on Venturia in- aequalisy and Hansen and Smith (5) on Botrytis cinerea may be cited as random examples. All of these workers observed differences in cultural appearance between isolations, and in some cases also found differences in such characters as growth rate, spore production, size and shape of conidia, pathogenicity, reaction to toxic substances, and reaction to acidity of the medium. None of these differences were correlated with the host or locality from which the isolations were made. Palmiter 's (12) statement seems gen- erally applicable to all of the above-mentioned species: "No two of the 36 isolations studied appeared exactly alike when grown under standardized conditions, yet duplicate cultures could easily be recognized V. inaequalis 8 York, H. H. A study of resinosis and root rot in forest plantings of Pinus strohus and P. resinosa. Unpublished manuscript. « Wean, R. E. The parasitism of Polyporus schweinitzii on seedling Pimis strohus. Unpublished manuscript. 48 Phytopathology [Vol. 27 is not a homogeneous species or one composed of a few well-defined forms with definite cultural and pathogenic reactions but is one made up of many strains that differ in various degrees in their morphologic and pliysiologic characters. ' ' Kesearch on such highly heterozygous species can be of maxi- mum usefulness only when the sampling errors inseparable from studies of varied populations have been reduced by the use of a large and representative basis. Small bases may be sufficient in species characterized by slight vari- ability, but in most instances a high degree of variability must be assumed until investigations of numerous isolates have shown the species to be essen- tially uniform for the character in question. It seems probable that dis- crepancies between quantitative data secured by competent workers often may be charged directly to the basing of studies on only one or a few indi- viduals from species in which wide variation may exist. For example. Lager- berg and Melin,^" Robak (13), and Weis and Nielson (16) disagree rather widely as to the optimum pH for growth of Fames annosiis (Fr.)Cke. in arti- ficial culture. This disagreement may be the result of differences in experi- mental conditions, but the observations of Palmiter (12) and the present writer suggest that it is caused by differences between the individual mycelia used in the various studies. The term ' * strain ' ' often is employed by pathologists to designate a single original isolation and its subcultures (with the exception of saltants, etc.). Its use in this particular connection is unfortunate, since it frequently leads the reader to an erroneous conception of the relationships involved, even when it does not indicate a misunderstanding on the part of the user. ** Strain" suggests a racial grouping^^ of genotypically distinct individuals that are homozygous for one or more physiologic or cultural characters serv- ing to distinguish them from other members of the species. In many in- stances the term is, of course, correctly used, but it is not applicable to any one of the different mycelia of Polyporus schweinitzii herein described. Genetic irregularities and the frequent occurrence of asexual reproduction obscure the individualization of fungi, but in this group, as in any other group of organisms, individuals are the concrete units into which such ab- stract conceptions as strains and species are ultimately resolvable. The situation is made more complex but is not fundamentally altered by such anomalous phenomena as mixochimaeras (heterocaryotic cells), ** discon- tinuous variations", and physical discontinuity of individual genotypes. For practical purposes, separated fragments of an organism or new colonies 1" Not seen by the present writer; fide Weis and Nielson (16). 11 << When the isolates are compared on various culture media and under different environmental conditions they fall into a number of groups each of which contains isolatee which are alike in their cultural characters and cultural behaviour. Each of these groups constitutes a Strain or Race, and equates with Lotsy's 'Jordanon'. . . . Cultural races may be equated with biological races in the more strict sense." Brierley (1). 1937] Childs: Variability of Polyporus schweinitzii 49 - » I i •f ->■* '■■f v) y <" • Reprinted from PROTOPLASMA Vol. XXVII, 1937 Verlag von Gebriider Borntraeger, Berlin W35 STRUCTURE OF THE INTERKINETIC NUCLEUS IN THE SCALE EPIDERMIS OF ALLIUM CEPA by ISADORE Cohen (Harrison Fellow, Botanical Laboratory, University of Pennsylvania, Philadelphia, U.S.A.) With Plates VI and VII Received for publication July 2, 1936 Although the interkinetic nucleus has been intensively investigated for more than half a century, there is at present little agreement among cytologists as to the properties of the nuclear membrane or the structure of the chromatic elements. The fact that the genes can be assigned definite loci in particular linkage groups has raised the question as to the possible persistance of such gene- strings throughout the interkinetic phases, and has focussed attention on such structural details in the resting nucleus which might serve as the physical basis of such linkage groups. It is interesting that some of the earlier investigators described individual coiled threads in such nuclei, although they were unaware of the bearing of their findings upon any possible theory of the mechanism of heredity. Recent advances in cytological technique, micro-manipulation, etc. have made it probable that a re-investigation of the resting nucleus would reveal new facts which might have some bearing on the genet ical problems involved. MATERIAL AND METHODS In the experiments designed for the study of the nuclear membrane, rectangular strips of epidermis from the onion-scale were mounted on a slide and flooded with the various reagents. Then, with a sharp razor the strips were cut across the longitudinal axis of the cells. Thus the nuclei were either extruded into the mounting medium or the various reagents had immediate access to the nuclei in the cut cells. The nuclei in similar pieces of tissue mounted in a hanging drops against the surface of cover slips were dissected with a Chambers' micro-manipulator, while the uncut specimens were placed directly into the various fixatives tested. In order to investigate the osmotic properties of the nuclear membrane, extruded nuclei were mounted in solutions of M/4 glycerine and M/4 sucrose, isotonic with the cell sap, and in distilled water. The effects of the presence and absence of calcium ions on the per- sistence of the membranes and the internal structure of the nuclei were observed in isotonic solutions of glycerine, sucrose and once distilled water, all containing traces of either CaClj, C2H04Na or C2Hg04. These solutions were used over a wide pH range, the hydrogen-ion concentration being measured colorimetrically. The nuclei were also extruded into M/8 CaClj and M/8 KCl in order to observe their responses to calcium and potassium in high concentrations. The well known cytological fixatives were used, such as Navaschin, weak Flemmino, BouiN, potassium dichromate, nickel/copper bichromate-propionate, chromic-formic-acet- 1 \ i I . k > structure of the interkinetic nucleus in the scale epidermis of Allium Cepa 485 aldehyde, Erlikt's mixture modified with ethylene diamine and some new mixtures, such as aluminum/uranyl nitrate-acetaldehyde-alcohol, calcium chloride-hydrochloric acid and tribasic sodium phosphate-formaldehyde-sucrose. The fixatives were selected to note the effects of those which are extremely acid and of those which are decidedly basic. Fixation in all cases was for a period of forty-eight hours, but frequent examinations of the specimens were made during the process. The specimens were then washed and stained in haematoxylin, Feulgen nucleal stain, aceto-carmine, or with Cloth Red B dissolved in HCl. Cloth Red B differs but slightly in chemical composition and in staining reaction from Orseillin B. B. reported by Cohen and Doak (5). A few drops of M/8 HCl half-saturated with Cloth Red B added to 10 c.c. of M/10 CaClg stains the nucleus sharply and the finer details of material fixed in this mixture can readily be photographed. RESULTS Many investigators have indicated that the nuclear membrane is not a simple structure. Schwarz (22), Robyns (16), STRtJOGER (24, 25) have pictured the membrane as double, while Scarth (18) and Klingstedt (11) reported that the outer membrane is cytoplasmic in origin. In addition, Scarth noted differ- ences in the osmotic properties of the two membranes. When nuclei of onion epidermis are. freed into distilled water, exposed to air to reach a pH of 6.5, they may react in one of two ways. They either swell in such a manner that no details of their structure become visible, or, more frequently, they swell so that two distinct membranes separate. At first, only small areas of the outer membrane form what appears to be small blisters but, later, as more of the outer membrane becomes involved, these blisters coalesce into a common vesicle, encompassing the nucleus with its inner membrane. The nucleus then shrinks, while the outer membrane, now clearly visible and presumably of cytoplasmic origin, expands, the two membranes becoming well separated (Figs. 1, 2, 3, 4). These changes occur so rapidly that it is a matter of a few minutes before the nucleus shrivels, while the outer membrane becomes progressively thinner, finally bursting when its contents have attained a volume approximately twenty-four times greater than that of the original nucleus. When nuclei are placed in M/4 glycerine, an isotonic solution, their reactions are similar to those placed in distilled water but much slower, the swelling oc- curing apparently as the glycerine penetrates. As the glycerine slowly penetrates the nuclei, it raises their osmotic value while their surrounding medium becomes hypotonic. In M/4 sucrose no swelling occurs and the nuclear membranes do not separate, the sucrose apparently not penetrating at all (Fig. 5). In these experi- ments the hydrogen-ion concentration did not vary significantly. The solutions, although unbuffered except for slight impurities in the glycerine, remained at pH 6.4 — 6.5. In order to note the effect of free calcium on the persistence of the nuclear membranes and their reactions, the experiments were repeated with a trace of C2H04Na added to the reagents to remove traces of calcium. When nuclei are placed in distilled water containing a trace of C2H04Na, at pH 9.0, the membranes separate, but in M/4 glycerine with a similar addition, the pH now being 6.4, no such separation occurs. In M/4 sucrose with a trace of C2H04Na, at pH 8.0 — 9.0, the nuclei swell and burst at one side, out of which the liquified IRREGULAR PAGINATION 486 Cohen nuclear contents then flow. In order to vary the pH a sufficient quantity of C2H2O4 was added to bring to pH 6.4 the mixture of M/4 sucrose and C2H04Na. Thus the effects of the oxalate ion in sucrose could be compared at the same pH with its effects in glycerine. In such a mixture the nuclei do not swell, but lose all indications of internal structure, and moreover the membranes do not separate. If the M/4 sucrose solution containing the mixture of C2H04Na and C2H2O4 at pH 6.4 is replaced by one containing only C2H04Na at pH 9.0, the nuclei swell and burst as they did in the previously cited case. To differentiate further the responses shown by the nuclei to changes in the hydrogen-ion concentration, C2H2O4 was used in place of C2H04Na. When a trace of C2H2O4 is added to distilled water, M/4 glycerine and M/4 sucrose, bringing all to pH 3.0 — 4.0, the nuclear membranes do not separate, while in distilled water with C2H2O4 at pH 4.6, the isoelectric point of many proteins in the cell, the membranes separate but slightly on one side of the nucleus. In M/4 glycerine with C2H2O4 at pH 3.0 — 4.0, the nuclei behave in a peculiar fashion. Intranuclear alveoles arise and break through the nuclear membranes, the nuclei thereby becoming irregular and ragged. The membranes do not separate in the nuclei liberated into distilled water, M/4 glycerine or M/4 sucrose to which HCl had been added to bring their pH to 3.0. In the presence of CaCl2 there is a significant difference in the behavior of nuclei placed respectively in distilled water, in M/4 glycerine and in M/4 sucrose, all at pH 6.4 — 6.6, as contrasted with their behavior when immersed in the same solutions without traces of calcium. The membranes of those nuclei immersed in the M/4 sucrose with an added trace of CaCla do not separate. More- over, the nuclei here do not noticeably differ from those placed in M/4 sucrose with added traces of HCl, bringing the pH to 3.0 (Fig. 6). At first, the nuclei in distilled water and in M/4 glycerine, pH 6.4, with calcium appear to be no different than those freed into the calcium-containing sucrose, but in a short time the membranes separate and the nuclei shrink, although they maintain their internal structure (Fig. 4). The outer membranes of the nuclei so treated are much thicker, possibly due to the adhering cytoplasm coagulated by the calcium, than those nuclei in distilled water with a sufficient quantity of the oxalate ion to insure the absence of calcium, provided the acidity is less than pH 4.6. In order to observe the effects of incidental changes in the hydrogen-ion concentration in the presence of free calcium, nuclei were placed respectively in distilled water, in M/4 glycerine and in M/4 sucrose to which a trace of a mixture of CaCl2 and HCl had been added to pH 3.0. The behavior of the nuclei here did not differ essentially from those freed into the similar liquids containing only HCl. On the other hand, nuclei extruded into a saturated aqueous solution of Ca(0H)2, having a pH of 8.4, may react in one of two ways. They either swell in such a way that no details of their structure become visible or, less frequently, they swell so that two distinct membranes become separated from each other. The outer membranes around the latter nuclei are much thinner than those separating from the nuclei in distilled water to which traces of CaClg (pH 6.4 — 6.6) had been added. This suggests that the coagulative action of calcium on the Structure of the interkiiietie nucleus in the scale epidermis of Allium Cepa 487 cytoplasm is definitely influenced by the decrease in the hydrogen-ion concen- tration. In order that some insight might be gained as to the action of the hy- droxyl ion in high concentrations, nuclei were freed in very dilute NH4OH (one drop of concentrated ammonia water to fifty c. c. of distilled water) having a pH 9.8. Here the nuclei were constant in their response, merely swelling without showing any separation of the membranes. Since the presence or absence of calcium in certain of the described mixtures of distilled water, glycerine and sucrose produced such marked reactions on the part of the extruded nuclei, it was considered to be of sufficient importance to follow their behavior in solutions containing large amounts of calcium. When nuclei were placed in M/8 CaClg, pH 6.8, they reacted in one of two ways. More frequently they became reticulated from the outset and then solated, thus appa- rently losing all internal structure. The Brownian movement was very vigorous during the solation of the reticulum. The nuclei were rigid and appreciable amounts of coagulated cytoplasm adhered to their unseparated membranes (Figs. 12, 13). Less frequently their internal structure was preserved generally in one of two ways, the chromonemata being either accentuated or changed into a reticulum. The membranes slowly separated and the nuclei gradually shrank to a small size within the outer membrane, which attained a volume several times greater than the original nucleus (Fig. 14). By extruding nuclei into a M/8 KCl solution an opportunity was afforded to follow the reactions of nuclei in high concentrations of potassium and where the concentration of calcium, was too dilute to produce any characteristic response on the part of the nuclei. The nuclei were found to respond in one of three ways. First, they may become reticulated with the two membranes separating. The nuclei, still delimited by their inner membrane, shrink within the vesicle bounded by the outer membrane (Figs. 8, 11, 15). The separation of the membranes is rarely completed and the entire structure collapses at the end of five hours. Secondly, the outer membrane may lift from the nuclear surface, but soon col- lapses, the nuclei remaining reticulated without any further change (Figs. 9, 10). Finally, the nuclei may become reticulated without any separation of the mem- branes occuring. When uncut strips of onion epidermis are mounted in M/8 KCl, cytoplasmic streaming contiiuuMl without any apparent change in the nuclei within the intact cells. Evidently at approximate isotonic concentrations KCl does not materially affect the cells. In M/5 KCl, however, the cells are plasmolyzed within two miiuites and the nuclei become reticulated, indicating a disruption of the colloidal phases in the iniclei. When such strips of epidermis are fixed and stained in aceto-carmine, it is possible to arrange a series of nuclei with reticula of nuuiy interstices to reticula with but few interstices (Figs. 24, 25, 20, 27). A careful examination of the luielei in healthy onion epidernus does not lend support to the view that the interkinetic chromatin is distributed in a reti- culum, showing it to be present rather as entangled chromonemata. From a meie cursory examination, one could readily gain the impression that the chro- matin is disposed as a reticulum in the resting nucleus. This apparent reticulum 488 Cohen is however clue in no small measure to the fact that the chromonemata lie in the lower limits of microscopic visibility and the points where they pass over one another can accordingly be scarcely distinguished from points of fusion. In addition to the fact that the gyres of a chromonema are only partly visible at one focal level, the chromonemata may dip sharply into the nucleus and be partly superimposed upon each other, thus increasing the difficulty of tracing their entire length. As early as 1885, Rabl (14) was unable to decide whether he saw in the interkinetic nucleus a real network or independent threads which merely passed over one another. In most onion nuclei it is possible to single out a chromonema which lies at a high focal level for a greater part of its length and by gradual adjustments in the focus to follow the coiling, and even changes in the direction of coiling in the chromonema (Figs. 6, 20). The chromonematic structure is accentuated in nuclei freed in M/4 sucrose to which a trace of HCl, yielding pH 3.0, or of CaCla, pH 6.4—6.6, had been added (Fig. 6); and in nuclei placed in M/8 CaClg with HCl, the chromonemata are shown most clearly. The chromonematic structure of the same nuclei can be made to disappear, the chromatin apparently disssolving, by replacing the solutions with M/4 suLrose containing the above described amount of C2H04Na at 8.0—9.0. When HCl is then added, structure reappears, but this resultant structure is clearly a reti- culum. That the disruption of the true chromonematic structure in the above cited cases was not due to the incidental changes in the hydrogen-ion concen- tration is shown by the fact that nuclei which had been placed previously in a saturated aqueous solution of Ca(0H)2 and which appeared to be optically empty would revert to their original chromonematic structure when HCl was added. The effect of calcium removal from nuclei placed in solutions of high acidity is most clearly demonstrated by the nuclei placed in M/4 sucrose to which has been added C2H2O4 at pH 3.0—4.0. The chromonematic structure is visibly altered in a series of changes suggestive of Belar's (3) description of an " Entmischung ". The formation of the reticulum in the nuclei so treated follows directly upon the collapse of the chromonemata and the accompanying changes. The formation of the reticulum in nuclei first treated in M/4 glycerine containing C2H04Na, pH 6.4, so that the chromatin is apparently dissolved, is due to a precipitation when the glycerine mixture is replaced with one containing HCl. The reticulum thus formed bears no resemblance to the original structure present before the solation of the chromatin in the presence of the oxalate ion. When the chromatin dis- appears in this case, it must be assumed to have dissolved, for the structure which reappears with the change in the hydrogen-ion concentration differs markedly from that originally present. However, the chromatin in what are apparently empty nuclei is not necessarily dissolved, for in the case of nuclei placed in Ca(0H)2, the chromatin can be made to reappear in its original from. Thus the description of nuclei as optically empty is of no particular significance unless it be accom- panied by evidence showing that the nuclei under discussion really have no internal structure. Although the nucleoli remain visible in the nuclei immersed in distilled water containing a trace of CgHO^Na, they disappear in those nuclei placed in M/4 glycerine to which had been added a comparable amount of CgHO^Na or Structure of the interkinetic nucleus in the scale epidermis of Allium Cepa 489 C2H2O4. In the nuclei extruded into very dilute NH4OH, the nucleoli disappear as they in Ca(0H)2; when treated with the latter reagent, however, they can be made to reappear with acidification, thus indicating a marked difference in the action of the two hydroxides. Usually two nucleoli are visible at different focal levels in the living interkinetic nucleus. In nuclei treated with M/4 sucrose containing a mixture of C2H04Na and C2H2O4, at pH 6.4, these nucleoli fuse, and the resultant fusion nucleolus is of irregular outline, bearing no resemblance to the individual participating nucleoli. In fixed and stained material the chromonematic structure of the nucleus can be best observed in strips of onion epidermis fixed overnight in a mixture of M/IO CaClg (10 c.c.) and a few drops of M/8 HCl half -saturated with Cloth Red B. The imclei are delicately stained and a permanent mount can be made easily. Acid salts of metals such as aluminum, as well as uranyl nitrate, when combined with acetaldehyde and alcohol, give fairly reliable fixation images. With such fixa- tions, however, the chromonemata show a tendency to collapse in part, producing incidentally artificial chromatin nucleoli (Figs. 17, 18). Since they may collapse through only a small portion of their length, such artifacts could readily be mistaken for plasmosomes were it not for the fact they stain with the Feulgen technique, and are thus unlike true plasmosomes. Moreover, in the living nucleus such chromatin nucleoli are not visible, whereas the real plasmosomes, having an index of refraction different from the chromatin, can readily be seen. While no evidence is presented here that contradicts the reality of prochromosomes, it is necessary to demonstrate that the chromonemata have not collapsed into such artificial chromo-centers (Heitz 8, 9). As a rule most of the acid fixatives tested preserved the nuclei with reticula (Figs. 19, 20) but, in a few instances, the true chromonematic structure was retained in some nuclei while in others the chromatin was precipitated in many intermediate forms. Such was the case in three of the most widely used cytological fixatives, Bouin, Navaschin and weak Flemmino. These fixatives, containing large amounts of acetic acid, usually preserve the nuclei in material of several cell layers in thickness as hollow structures with a halo between the nucleoli and the reticulum. That such reticula and haloes are not due entirely to fixation, but in part to the methods employed in paraffin infiltration, is indicated by a comparison of onion epidermis and of root tips fixed in these fluids. While the collapse of the chromonemata is one of the most important factors contributing to the formation of the reticulum, the fact should not be overlooked that acetic and chromic acids have already been reported to produce artificial reticula (Bal- BiANi 1, Nebel 12). The lymph of the nuclei in onion epidermis fixed in Bouin, Navaschin and weak Flemmino solutions was better preserved than in those nuclei examined in the root tips. In addition, there was no appreciable halo between the reticulum and the nucleoli. Zirkle (30) has already stressed the fact that the fixation image is greatly influenced by the relative rates of ixjne- tration of the various components of a fixative. In smears and in material of a few cell layers in thickness, such as onion epidermis, there is of course no such difference in the fixation image caused by this factor. Protoplasma. XXVII 490 Coh en The fixatives which are most commonly used to preserve mitochondria if made more alkaline, preserve the nuclei as if devoid of chromatin. In some cases there is an actual dissolving out of the chromatin, as when nuclei are treated with a mixture of M/8 tribasic sodium phosphate.4 M/3 formaldehyde-M/4 sucrose (Fig 23). Such nuclei stain diffusely with the Feulgen technique, acidulated Cloth Red B or carmine, while with haematoxylin they stain with great difficulty, if at all. If strips of onion epidermis have had a prior treatment with dilute NH4OH and then are fixed and stained in aceto-carmine, it is possible to arrange a series of nuclei indicative of the various stages in the dissolution of the chromatin and other nuclear structures (Figs. 28, 29, 30, 31). On the other hand, nuclei fixed in chromic sulphate-formaldehyde, a reported basic fixative, show a fine preservation of the chromonemata and the maintenance of the structural rela- tionships of the parts of the nucleus which can not be successfully stained with haematoxylm, but stain very easily with acidulated Cloth Red B Nuclei fixed m Erliki's mixture modified with ethylene diamine and in potassium dichromate alone reacted similarly in being hard to stain with haematoxylin, and showed several stages in the disorganization of the chromatin structure. In several instances cytoplasmic alveoles may indent nuclei fixed in potassium dichromate, so that after dehydration the nuclei are distorted (Fig. 22). The primary purpose of micro-dissection was to attempt to pull the chromo- nemata out of the nucleus and count them. The nuclei from onion epidermis were freed in the usual manner against the surface of the cover slip in a hanging drop of M/4 sucrose with a trace of CaCl^, the cover slip serving as the roof to the moist chamber of the micro-manipulator. Such nuclei can be pushed about with the point of the micro-needle since they are quite rigid, but if they are punctured, the nuclear lymph, though rather viscous, then flows out through the puncture and the structure collapses. The collapsed nuclei prove to be sur- prisingly elastic when seized by both needles and stretched. When the nuclei are being stretched, the chromonemata are deformed and, before breaking into small segments, become alligned parallel to the direction of pull. If the chromo nemata are pulled out of the nucleus, which thereuiK)n collapses, they become immediately rounded up into shapeless blobs at the end of the micro-needle or on the surface of the cover slip. The presence of cytoplasmic streaming in uncut cells at the end of the experiment was used as a check of the original healthiness of tissue. In the literature there are several reports on the effect of wounding being transmitted to nuclei far removed from the involved area. In onion epidermis, cells next to those which had been cut open were not affected and cytoplasmic streaming contmued m them without any alteration in the appearance of the nuclei. DISCUSSION Although the existence of a nuclear membrane was suggested by Robert Brown as early 1833, it was noted by few other workers prior to 1884 In fact many believed that there was no such membrane and the entire question became I Structure of the interkinetic nucleus in the scale epidermis of Allium Cepa 491 highly controversial. Tischler (26) and Heidenhain (7) have both reviewed adequately this earlier literature. It is of interest that Schwarz (22) reported the nuclear membrane to be osmotic. He subjected onion nuclei to the action of distilled water and observed that blisters were produced on their surfaces. These he termed " Randvakuolen " and noted that the blisters coalesced to form a single, large peripheral vacuole which enclosed the entire nucleus, thus indicating that the nucleus had two distinct membranes. Furthermore, Scarth (18) and Klingstedt (U) confirmed the existence of these two membranes. Scarth explains the observed differences in the nuclear membranes of his material (cockroach oocytes) as due to their osmotic differences. In general, the reaction of nuclei placed in distilled water or in M/4 glycerine indicates that they are surrounded by two membranes, the outer of which, as observed by Schwarz, is truly osmotic. Although the inner membrane does not seem to be semi-permeable, it is quite possible that it is semi-permeable in the living cell and that this property is lost as soon as it is taken out of the cell. The outer membrane is very thin and apparently there is no cytoplasm adhering to it. In addition, it does not persist for as long a time in distilled water alone as it does when the nucleus is extruded into distilled water containing a trace of calcium (Fig. 4). When the outer membranes separate in those nuclei placed in distilled water or in M/4 glycerine with traces of CaClg, they are much thicker, probably due to the coagulated cytoplasm adhering to them, than those membranes which separate in solutions containing an excess of oxalate ions or in M/8 KCl where the absence of calcium is assured. It is well known that calcium plays an important role in membrane formation, and its absence from solutions in which nuclei are freed might permit the outer membranes to be dissolved or be inhibited from separating. The outer membranes of nuclei placed in distilled water with a trace of C2H04Na separate before there any action of the oxalate ion is noted. In cases where the separation of the membranes is normally slow, the outer membranes may be dissolved before they separate. This is apparently what occurs when nuclei are subjected to the action oft he oxalate ions in M/4 glycerine and M/4 sucrose. The coagulative action of calcium is probably influenced by the changes in the hydrogen -ion concentration. The membranes which separate from nuclei placed in Ca(0H)2 at pH 8.4 are much thinner and obviously lack the coagulated cytoplasm seen with those thicker membranes of nuclei placed in distilled water or in M/4 glycerine with added traces of CaCl2 at pH 6.4 — 6.6. Optical analysis of protoplasm is inadequate to determine whether or not structure or a definite pattern exists. Although the investigations with polarized light may reveal a fundamental arrangement of submicroscopic particles in proto- plasm (Schmidt 19, 20), the usual optical systems employed in microscopy are not able to show these structures. For example, nuclei freed in Ca(0H)2 appear to be optically empty but are really not so as is proven by their behavior when treated with dilute HCl. When thus acidified, the nuclei reveal a chromonematic structure which is essentially their form in the living cell. Changes from optically homogeneous structure in nuclei to those which show visible detail are well 32* 492 Cohen known in tlic living cell. Weber (28) found that the nuclei in the guard cells of the stomata underwent reversible changes depending upon stomatal activity. The view that the chromatin does not differ in its arrangement in the inter- kinetic nucleus from that of the chromosomes is rapidly gaining ground, and is in harmony with the results of several modern re-investigations of the mitotic cycle. If the chromonemata persist through the interkinetic phases in the nuclei of higher plants, we can readily see how the linear order of the genes is preserved in the linkage groups. In well fixed nuclei of onion root tips, the chromosomes are visibly coiled in the earliest prophase stages. The chromonematic structure of the interkinetic nucleus can be demonstrated best in thin strips of onion epi- dermis and in well fixed smear preparations (Nebel 13, Sax and Sax 17). Sax and Sax (17), by prior treatment of the resting nuclei in Tradescantia microspores with ammonia-alcohol, were able to preserve the chromonemata even with acetic acid fixation. The formation of the reticulum in the interkinetic nucleus can readily be interpreted as to the collapse of the chromonemata which are appreciably coiled at this stage. Nuclei behave in this manner when treated in M/4 sucrose containing traces of C2H2O4. A comparative study of nuclei in the same strip of epidermis of from different strips fixed in the same fluid reveals a series of fixation images from well preserved nuclei with chromonemata to badly preserved •nuclei with reticula. The cause for such variations in the specimens may be due to local differences in the vacuolar contents of the different cells. Linin was not observed in either living or fixed nuclei. Although no attachments were seen between the chromonemata and the nucleoli in either living or fixed nuclei or those fixed in the copper or nickel salts of chromic and propionic acids, there is no evidence here that disproves the possibility for such an attachment. The observations and results reported here may not be generally applicable to animal nuclei or even to the nuclei of the lower plants. The formation of vesicular nuclei in which the chromosomes retain their pellicle into interkinesis has been demonstrated by Wenrich (29) and Richards (15). In addition, the processes extending from the telophasic chromosomes in certain animal nuclei have been shown to be real structures, and these structures coalesce to form what is apparently a true reticulum. SUMMARY 1. Two membranes invest the extruded, interkinetic nucleus of Alliutn Capa. In (listilled water or in M/4 glycerine, an outer membrane, probably cytoplasmic in origin, separates from an inner one which remains intimately associated with the nuclear contents. The rate at which the membranes separate IS related to the rate in change of the osmotic values within the nuclei. The be- havior of the outer membrane indicates that it possesses semi-permeable proper- ties which the inner one lacks under the same experimental conditions. 2. The outer membrane is not preserved in the presence of oxalate ions except when nuclei are placed in distilled water containing C2H04Na at pH 9.0. Here the outer membrane is preserved temporarily since the change in the I • : structure of the interkinetic nucleus in the scale epidermis of Allium Cepa 493 osmotic value of the nucleus is faster than the dissolving action of the oxalate ion. 3. The outer membrane is preserved by calcium ions over a wide pH range notwithstanding the decrease in the coagulative properties of calcium ions in alkaline media. 4. Independent, coiled chromonemata were observed in living, in moribund and in fixed nuclei. Through micro-dissection the chromonemata can be pulled out of the nucleus, which thereupon collapses. 5. When nuclei are placed in distilled water, in M/4 glycerine and in M/4 sucrose, all of which contained C2H04Na, their chromonematic structure dis- appears, the chromatin apparently dissolving. If the same nuclei are then treated with dilute HCl, the chromatic structure reappears as a reticulum. 6. When nuclei are placed in Ca(0H)2 at pH 8.4, their membranes are preserved but their chromonematic structure is rendered optically homogeneous. Treatment with dilute HCl causes the same nuclei to return to their original chromonematic structure. 7. The results obtained by the use of various fixatives indicate that the chromonemata and the nuclear membranes are preserved when the fixation image is conditioned by either hydrogen or -calcium. 8. The reticulum, reported in the interkinetic nucleus, does not occur in the living nuclei of the epidermal cells of Allium Cepa, but originates through the collapse and fusion of the chromonemata caused by either experimental injury or by certain types of fixation. 9. Artificial chromatin nucleoli were produced in certain nuclei by fixation when the coiled chromonemata partially collapsed. The author is indebted to Professor Conway Zirkle who assisted him in this investigation, and also to Doctor Kenneth Davis Doak and Mr. Vernon DoNEY who assisted him in preparing the photographic illustrations. LITERATURE LIST 1. Balbiani, E. G., Zool. Anz. 4, 637, 662, 1881. 2. Belar, K., Zeitschr. f. ind. Abst.- u. Vererbgsl. Sup.-Bd. 1, 402, 1928. 3. — , Protoplasma 9, 209, 1930. 4. Brown, R., Trans. Linn. Soc. 10, 685, 1833. 5. Cohen, I. and Doak, K. D., Stain Techn. 10, 25, 1935. 6. Dehorne, A., Arch. f. Zellforsch. 6, 613, 1911. 7. Heidenhain, M., Plasma und Zelle, Jena, 1907. 8. Heitz, E., Ber. d. Deutsch. Bot. Ges. 47, 274, 1929. 9. — , Planta 18, 1933. 10. Kato, K., Mem. Coll. 8ci. Kyoto Imp. Univ. B. 10, 4, 251, 1935. 11. Klinostedt, H., Mem. Soc;. Fauna et Flora Fetmica 4, 32, 1928. 12. Nebel, B., Zeitschr. f. Zellforsch. 10, 251, 1932. 13. — , Cytologia o, 1, 1933. 14. Rabl, C, Morph. Jahrb. 10, 214, 1885. 15. Richards, A., Biol. Bull. 32, 249, 1917. r 494 Cohen 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. RoBYNs, W., La a'llule 34, 367, 1924. Sax, H. V. and Sax, K. J., Arnold Arboretum 26, 423, 1935. ScARTH, G. W., Protoplasma 2, 189, 1927. Schmidt, W. J., Zool. Jahrb. 4o, 177, 1928. — , Arch, Protistenk. 77, 463, 1932. ScmwAGO, P., Biol. Zentralbl. 46, 679, 1926. ScHWARZ, F., CoHNs Bcitr. z. d. Biol. d. Pflanzcn 5, I, 1892. Strohmeyer, G., Planta 24, 470, 1935. Strugger, S., Planta 18, 717, 1929. — , Protoplasma 10, 363, 1930. I'lscHLER, G., Allegemeine Pflanzenkaryologie, Berlin, 1921. Wada, B., Cytologia 1, 404, 1930. Weber, F., Protoplasma 2, 305, 1927. Wenrich, D. H., Bull. Mus. Com. Zool. Harvard Coll. 00, 155, 1916. ZiRKLE, C, Protoplasma 19, 565, 1929. DESCRIPTION OF THE PLATES VI AND VII Figures 1-23 magnified 1150 diameters, figures 24-31 magnified 1400 diameters. (Plates reduced to three— fourths of original size) I Fig. 1. Nucleus extruded into distilled water containing traces of C^HO.Na, pH 9.0, showing ^^^ separation of the two nuclear membranes and the absence of nuclear structure. *ig. 2. .Nucleus extruded into M/4 glycerine, pH 6.4, showing the separation of the two membranes and the absence of nuclear structure except for the nucleoli *ig. 3. Nucleus m the same solution as above. Later stage in another nucleus. *ig. 4. Nucleus freed into distilled water containing traces of CaCl,, at pH 6.4-6.6, showing the coagulated cytoplasm around the outer membrane and the maintenance of the internal structure in the shrunken nucleus. Fig. 5. NucleuB libcraUd into M/4 sucrose, pH 6.4, showing no api«.rent structu.., except for the vacuolate nucleoli. Fig. 6. Nucleus extruded into M/4 sucrose containing trax^cs of CaCl,, pH 6.4-6.6. showing an accentuated chromonematic structure. Fig. 7. Nucleus fixed in CaCl,-HCl and stained with Cloth Red B. The chromoncmata are well preserved. Fig. 8. Nucleus extruded into M/8 KCl, approximately at neutrality, illustrating the solation of the reticulum. Kig. 9. Nucleus freed into M/8 KCl, showing the separation of the two nuclear n.eM.hranes and the solation of the reticulum. Fig. 10. Same nucleus in Fig. 9, but fifteen minutes latc-r. showing the collapse of the outer membrane. Fig. 11. Later stage of nucleus extruded into M/8 KCl, illustrating the course of solation. VUr ,9 ^.*^^7"^"!- '"«'»b^^»^" '« very thin in comparison to the unseparated membranes. P t \i' I r ^^■'^''l '" ^/' ^^^^^' ^* ^PP^<^^""^^' neutrality, illustrating a reticulum. Fig. 13. Nucleus extruded into M/8 CaCl,, showing an advanced stage in the solation of the reticulum. Note the thickness of the unseparated membranes as compared with tboee that have separated in Figs. 1, 2, 3 Fig. 14. Nucleus freed into M/8 CaCl, showing'the separation of the two nuclear membranes and the maintenance of the nuclear structure (slightly out of focus) J?ig. 16. A late stage in the solation of a nucleus extruded into M/8 KCl. I Structure of the interkinetic nucleus in the scale epidermis of Allium Cepa 496 II Fig. 16. Nucleus fixed in CaCla-HCl and stained with Cloth Red B, showing the chromo- ncmata. Fig. 17. Nucleus fixed in uranyl nitrate-acetaldehyde- alcohol and stained with haematoxylin, showing the artificial chromatin nucleoli at the periphery produced by fixation. Fig. 18. Nucleus treated the same as in Fig. 17, showing the abundance of the artificially induced "chromatin nucleoli". Fig. 19. Nucleus fixed in chromic-formic-acetaldehyde and stained with haematoxylin, showing the reticulum in which traces of the chromonematic structure is preserved. Fig. 20. Nucleus fixed in CaCl^-HCl and stained with Cloth Red B, illustrating well preserved chromonemata. Fig. 21. Nucleus preserved in chromic-formic-acetaldehyde and stained with haematoxylin, showing a reticulum. Fig. 22. Nucleus fixed in potassium dichromate and stained with acidulated Cloth Red B. The greater part of the fixed nucleus is preserved nuclear lymjih., the chromatin being dissolved. Fig. 23. Nucleus fixed in tribasic sodium phosphate-formaldehyde-sucrose and stained with acidulated Cloth Red B, illustrating a swollen nucleus of the basic tyjie in which the chromatin is not preserved. Figs. 24 — 27. Nuclei treated in M/5 KCl, fixed and stained with aceto-carmine, showing the various degrees of reticulation. Figs. 28 — 31. Nuclei treated with dilute ammonium hydroxide, then fixed and stained with aceto-carmine, showing various stages of disorganization. PROTOPLASMA vol. XXVII Plate VI -44- V 41 — » ■» * W \ > 9 » 4 'm -' 1 ISADORE COHEN Verlag von Gebruder Borntraeger in Leipzig PROTOPLASMA vol. XXVII Plate VI \ r r f - iiA' ^ m. i « - ISADOHK ("OHK.X Verhuj von (lehriiihr lionitraiHjer in Lt'tpzuj INTENTIONAL SECOND EXPOSURE PROTOPLASMA vol. XXVII Plate VII r ' I * 't I-* >- ■- ♦• X ►1» It «~.i -^ ISADORE COHEN Verlag von Oebriider Borntraeger in Leipzig ♦ PROTOPLASMA vol. XXVII Plate VII / - ^ 4' * * r • -■••.* c ' ■ • i . tf i. n 18 19 % f ^•'•* ISADOHK COHKX Verlay vott (kbrildtr liorntraeyer in Leipzig INTENTIONAL SECOND EXPOSURE rrt Reprinted from Bartonia, No. 19, issued March 8, 1938 •^ 4 M. >. -'l < I "♦ f ".*■,>» .^v^ ' ♦> I — .^ The Flora of Seven Mile Beach, New Jersey Flora S. Fender Part I: General Discussion INTRODUCTION Seven Mile Beach is one of the coastal islands off the shore of Cape May County, New Jersey. It lies parallel to the coast in a NE-SW direction, and has accumulated the greatest land-mass on the northern end. No detailed study of the plants of the island has previously been made, either as such or in any work dealing with the flora of the entire state. The present survey was therefore undertaken with a twofold aim: (1) to compile a comprehensive list of the plants of the island, and (2) to compare the flora of Seven Mile Beach with that of southern New Jersey as a whole, laying particular stress on the coastal islands of Cape May County. EARLY BOTANICAL WORK In the earliest botanical surveys of New Jersey, Willis's " Catalogue of the Plants Growing without Cultivation in New Jersey " (2nd ed., 1877), and Brit- ton's catalogue of the same area (1881), no coast island is mentioned except Atlantic City. Stone's "Plants of Southern New Jersey" (1910) includes 149 species of native plants from the island, based on specimens which are at present, almost in their entirety, in the herbarium of the Philadelphia Botanical Club. RECENT BOTANICAL WORK Since Stone's publication (7), occasional collecting on the island by W. M. Benner, R. R. Dreisbach, J. M. Fogg, Jr., C. Knopf, B. Long, F. W. Pennell, H. E. Stone, and a few others brought the number of species and varieties of plants, both native and introduced, to 255. Further collecting from April, 1935 to September, 1937, has increased this figure to 367. Those reported by Otway Brown, who has botanized on the island since 1899, bring the total number to 415. According to Mr. Brown's statement, the best stations have been destroyed, but he has unfortunately not kept specimens from them. In connection with the present study, the herbaria of the Philadelphia Botan- ical Club and the University of Pennsylvania were inspected for plants from Seven Mile Beach, and, for comparison, from the other coastal islands of New Jersey. In the former herbarium, 253 species were represented from Seven Mile Beach. All but two or three of the additional collection of 111 species have also been deposited there. The University herbarium contained only 23 specimens from the island, which number has grown to 505, representing 277 species. These 23 IRREGULAR PAGINATION 24 BABTONIA -^.» THE FLORA OF SEVEN MILE BEACH, NEW JERSEY 26 were obtained on collecting trips taken on an average of every two weeks during the growing season from early April, 1935, to June, 1936. Scattered tnps have been taken since then. An attempt was made to cover all habitats at all seasons. TOPOGRAPHY There are three towns situated on Seven Mile Beach, Avalon and Peermont contiguous on the north end of the island, and Stone Harbor about a mile from its southern tip. Since the towns are not very large, much of the land even within their limits seems undisturbed. Between the two settlements are houses scatterwl along the connecting highway, but to the east is the very interesting region of the tall dunes. This region has apparently been little disturbed m the past four or five years, but was much cut when the now-abandoned railroad ran through. Directly below Stone Harbor is another patch of woods, but a great deal of the land behind the dunes to the southwest of the town is covered with brackish marsh. HABITATS Beach: Gently sloping beach edges the island on the side toward the sea and up the bay shore at each end. In common with other sand beaches scattered plants grow above the reach of the storm waves. These are chiefly Cakde eden- tula and Xanthium echinatum, Arenaria peploides var. robusta, an occasional Euphorbia polygordfolia, and Salsola Kali and Atriplex arenana wherever the l)p&ch is low Salt Marsh: The land side of the island is edged with extensive salt marshes, characterized by Spartina patens and DMchlis spicata, with frequent patches of Salkomia ambigm, S. herbacea, and S. mucronaia. The edge of the marsh is fringed with thickete of Bacchant halimifolia and Iva frutescem var. oraria, and near them grow less frequent but just as typical marsh plants, notably Limonium carolinianum, Pluchea camphorata, and Aster tenwifohus. Spergularm leiosperma and Suaeda linearis grow in more sandy places. ^ . , ^ _, Salt Marsh Islands: The wide marshes have many scattered islands. Iwo of these, Crab Island and an unnamed one to the north of it, have been explored. They are much like the well developed thickets of the mainland, consistmg mamly of Quercus stellata and Juniperus virgini^na. The latter is in such abundance that the islands are locally called " cedar ridges ". ^ . ^ u a Sand Dunes: The sand dunes are of two types, grass-bound and tree-bound. The former are those nearest the sea, and none has been observed more than 25 feet high, although the average is about 15 feet. The sand-binder is marram or beach grass {AmmophUa brevUigulata) , but other characteristic dune species are: Tnplasis purpurea, Spartina patens, Panicum amarum, Euphorbia polygonifolm, Oenothera biennis, and Solidago semperviren^. The dunes farther from the sea are most frequently formed around a clump of Prunus serotina. After the dune has been started by the cherries, there is sufficient protection for Sassafras vam- folium Rhus Toxicodendron (the bush form), Vitis aestivalis, V. labrusca, and \N ♦. -^^ * V "«. V w. ^ L - - < ^ > ^ ■♦. •» -l^fc V • ^ Psedera quinquefolia to become established. These, in turn, prevent the west wind, which is the prevailing one on this coast, from blowing back the sand. As a result, tree-bound dunes become as high as 45 feet. They are usually bound by AmmophUa on the side facing the sea, except when they occur within the woods, where they are rarely very tall. Sand Dune Hollows: These are of three types, open sand, grass-filled, and thicket-filled. In the first grow scattered plants of Panicum Lindheimeri var. jasciculatum, Cenchrus paucifiorus, C. tribuloides, Juncus bufoniu^, Strophostyles helvola, S. umbellata, Euphorbia polygonifolia, and Polygonella articulata. The second are thickly covered with AmmophUa breviligulata, with scattered Myrica carolinensis and Vitis aestivalis. The bayberry forms the thickets of the third type. In them grow AchUlea Millefolium, Erechtites hieracifolia, Solanum nigrum and Phytolacca americana. The central bushes of these thickets often die off from some undetermined cause, and in the protected spot thus formed grow Achillea Millefolium, Erechtites hieracifolia, Aster laterijiorus, and Oenothera fruticosa. They are the forerunners of the woods formation into which such a thicket ultimately grows. Of course, every intermediate form exists. Woods: Woods have developed behind the tall dunes. In fact, woods and dunes are interdependent, each affording the necessary protection for the other. Harshberger (4) observes that the dune finally destroys the woods, and it is true that one can observe a great deal of buried forest. However, the advance cannot be as swift as he implies, since then there would be a succession of very short- lived woods, and the present one, by tree-ring count, must be over 200 years old. The dominant trees are Juniperus virginiana var. crebra, Quercus stellata and Q. falcata. Sassafras variifolium, Amelanchier oblongifolia, Prunus serotina, and Rhv^ copallina. Rhus Toxicodendron is the most frequent underbush, and Smilax rotundifolia the most constant vine, sometimes making the woods abso- lutely impassable. The soil beneath these trees is often dry sand, when the woods are somewhat open, but just as often it is a rich, dark leaf-mold, made light in texture by the unusual percentage of sand. Low places in the woods are most likely to support Myrica carolinensis, Nyssa sylvatica, Vaccinium atrococcum, V. corymbosum, and Diospyros virginiana. One large and well established swamp is dominated by Acer rubrum var. tridens and contains a clump of Liquidambar Styraciflua. Fresh Marshes: These occur at intervals along the edge of the woods and in various other dune-protected spots. They usually contain Typha latifolia, T. angiLstifolia, Hibiscus Moscheutos, Sium suave, and, occasionally, Scirpus Eriophorum or 7m versicolor. The more extensive ones are often merely wet, and are covered with a mixture of Spartina patens and Panicum virgatum. 26 BARTONIA Elements in the Flora — New Jersey Undifferentiated Coastal Plain The most prominent element in the flora of Seven Mile Beach is that of the region of southern New Jersey which is called here, for want of a better name, the Undifferentiated Coastal Plain. This name is used to distinguish it from specialized pine barren and maritime districts and to link it with the similar widespread formation of the Atlantic Coast of North America from New Jersey to Florida. This region includes the Middle District, Cape May District and Coast Strip of Dr. Stone (7). The combination of these undeniably similar dis- tricts has been made because the basis of Stone's division was the Piedmont element rather than the characteristically coastal plain plants, whereas the other two districts (Pine Barren and Maritime) are on such a " native " basis. More- over, the comparative absence of Piedmont species from the Coast Strip and abundance of them in the Middle District seems rather a question of area avail- able than of ability to support them. More than two-fifths of the plants on Seven Mile Beach belong to the region of the Undifferentiated Coastal Plain, a proportion which would be expected from the situation of the island. Of this element may be noted: Thelypteris palustris, Juniperus virginixina var. crebra, Typha latifolia, T. angitstifoliaf Elymics virginicus, Panicum clandestinuniy P. virgatum, Cyperus Grayi, Carex Swanii, C. comosa, Juncus effusus var. solutuSy Iris versicolor, Salix nigra var. falcata, Quercus phellos, Rumex verticillatus, Ranunculus sceleratus, Liquidambar Styraxnfiua, Rubu^ argutu^, Rosa virginiana, Prunus serotina, Strophostyles helvola, S. umbellata, Oxalis stricta, Rhus copallina, R. Toxicodendron, Euonymus americanus, Hibiscus Moscheutos, Oenothera fruti- cosa, Diospyros virginiana, Convolvulus sepium, Salvia lyrata, Lycopus ameri- canus, Tecoma radicans, Galium pilosum, G. Claytoni, Mitchella repens, Sambucus canadensis, Mikania scandens. Aster laterifiorus, Helianthus giganteus, and Bidens connata. Because of their great interest, the Piedmont plants have been separated out as a subdivision of the Undifferentiated Coastal Plain. Thirteen of these have been discovered on the island: Poa cuspidata, Smilacina stellata, Moras rubra, Silene stellata, Aquilegia canadensis, Phaseolus polystachyus, Celas- trus scandens. Geranium Robertianum, Verbena urticaefolia, Teucrium canadense var. virginicum, Scrophularia lanceolata, Galium circaezans, and G. trijlonxm. This is a small list as compared with the 265 species in West Jersey, but they are included in an area 1.5 miles long and 500 feet wide. Furthermore, it is interest- ing that some of these are very well established. Particularly is this true of Poa cuspidata, Aquilegia canadensis, and Celastrus scandens, which range almost the length of the woods. General About one third of the plants of the island are of widespread occurrence in southern New Jersey. Among these have been included species which are not strictly universal, i. e., those which commonly enter the western and southern THE FLORA OF SEVEN MILE BEACH, NEW JERSEY 27 r-*,? J ^ ■r f s%i * - » rr"' ^ * * •*«^ f » '41 > * A. -■» » < A> part of the Pine Barrens, not its eastern, more thoroughly " barren " areas. Some of the most common general species on the island are as follows: Triplasis purpurea, Cyperus cylindricus, Carex Longii, Scirpus americanus, S. Eriophorum, Juncus dichotomus, J. canadensis, Smilax rotundifolia, Myrica carolinensis, Quercus stellata, Q. falcata, Polygonella articulata. Magnolia virginiana, Sassa- fras variifolium, Lepidium virginicum, Pyrus arbutifolia, Amelanchier oblongi- folia, Rubus cuneifolius, Prunus maritima, Apios tuber osa, Linum medium, Acer rubrum var. tridens, Helianthemum canadense, Lechea Leggettii, Ludvigia altemi- folia, Nyssa sylvatica, Vaccinium corymbosum, V. atrococcum, Linaria cana- densis, Eupatorium album, E. hyssopifolium, E. rotundifolium var. ovatum, Soli- dago suaveolens, S. nemoralis, S. tenuifolia. Aster novi-belgii, Erigeron ramosus, Gnaphalium obtusifolium, and Krigia virginica. Maritime Nearly one-fifth of the native plants in number of species, and by far the greatest number of individuals, are maritime. A characteristic list includes: Ammophila brevUigulata, Andropogon scoparius var. polycladus, Cenchrus tribu- loides, Distichlis spicata, Elymus virginicus var. halophilus, Festuca rubra, Panicum amarum, Spartina altemiflora var. pilosa, S. patens, Cyperus filicinus, Juncus Gerardi, Atriplex arenaria, Salicomia ambigua, S. herbacea, S. mucronata, Salsola Kali, Suo^da linearis, Arenaria peploides var. robusta, Spergularia leio- sperma, Cakile edentula. Euphorbia poly goni folia, Limonium carolinianum, Saba- tia stellaris, Plantago oliganthos, Baccharis halimifolia, Iva frutescens var. oraria, Pluchea camphorata, Solidago graminifolia, S. sempervirens. Aster tenuifolius, and Xanthium echinatum. Elements in the Flora — Introduced Fifty introduced species have been collected from the island. The most promi- nent of these are the yarrow {Achillea Millefolium) in the thickets and the Japanese honeysuckle {Lonicera japonica) in the woods. Most of the others are in local patches near house-sites or on fiUed-in ground. Elements in the Flora — North America By far the greater part of the plants on the island represent species which are widespread in eastern North America, that is, they range from Labrador, Nova Scotia, or Maine to Virginia or Florida. About one quarter (26%) belong to the southern or coastal plain element, which reaches its northern limits in New Jersey, Long Island, and southeastern Massachusetts. Only seven per cent of the species are northern, that is, reaching their southern limits in New Jersey or the adjacent states. One per cent belongs to a restricted element which grows only in New Jersey and adjoining states. Except for a slightly greater emphasis on the widespread plants, these percentages correspond in general to those ob- tained from Stone's (7) figures for all of southern New Jersey: .,f- . L. 28 BARTONIA THE FLORA OF SEVEN MILE BEACH, NEW JERSEY 29 Southern New Jersey 54% 35% 9% Seven Mile Beach Widespread 64% Southern 26% Northern 7% Restricted 3% It is to be noted that the widespread and southern elements include certain Piedmont species which have traveled down the mountains to Georgia, and which therefore have a northern affinity despite their range, and a corresponding signifi- cance when growing on the coastal plain in southern New Jersey. These include: Poa cus-pidata, Moras rubra, Silene stellata, Aquilegia canadensis, Phaseolus j>olystachyus, Cela^trus scandens. Verbena utricaefolia, Teucrium canadense var. virginicum, Galium circaezans, and G. trijlorum. Summary The results of the study are summarized in the following table: Total number of species and varieties known from Seven Mile Beach 416 Introduced 63 Native 353 Species added by this study to Seven Mile Beach Introduced 37 Native 74 Species added by this study to the coast island flora of N. J.. . Introduced 17 Native 17 New Jersey elements in the native flora General Undifferentiated Coastal Plain Pine Barren Maritime ACKNOWLEDGMENTS The writer's deepest gratitude is due to Dr. John M. Fogg, Jr. for the sym- pathetic guidance which has made this paper possible. She also wishes to express appreciation for the invaluable suggestions of Mr. Bayard Long. Part II: Annotated List of the Vascular Flora EXPLANATION OF THE TABLES Range: The range in North America is self-explanatory. North and south, rather than westward extension has been considered. The terms employed in the New Jersey ranges have been discussed under Elements in the Flora. No 111 34 128 36.2%, 162 45.8% 1 0.2%, 61 17.8%) *4 attempt has been made to give the New Jersey range of introduced species be- cause the collections are very inadequate. Habitat: The habitats are those in which specimens were collected, except when the plant was not personally observed or there was no habitat on the label of the collector. In that case, the plant's usual habitat was combined with those available on the island to make the probable one. Localities and Collectors on Seven Mile Beach: Directions have been omitted in these in order to save space. A combination of " woods " and " Peer- mont " usually means south of Peermont. The sand dunes and marshes are only rarely within the towns. On the other hand, the swamp and woods in Avalon are surrounded by houses. The initials of the collector follow in parentheses. Nomenclature and Synonymy: The International Rules of Botanical Nomen- clature have been followed. Synonyms are included in parentheses when the name used differs from that given in the last edition of Gray's Manual (1908). Symbols Employed in the Tables Range — North America (= NA) N = northern (southern limit in N.J., Del., Md., or Va.) S = southern (northern limit in N.J., L.I., Conn, or Mass.) W = widespread along the Atlantic seaboard of N.A. R = restricted to N.J. and adjoining States Range — Southern New Jersey (= NJ) PB = Pine Barren CP = Undifferentiated Coastal Plain Ma = Maritime G= General in S. N.J. In = Introduced Habitat Beach = beach Dr Op Sd = dry open sand Ft Mar = fresh marsh Sd Dune = sand dune Sd Du Ho = sand dune hollow Sal Mar = salt marsh Localities on Seven Mile Beach Ava = Avalon Peer = Peermont St. Har = Stone Harbor Collectors and Herbaria WB = Walter Benner (University of Pennsylvania, Philadelphia Botanical Club) JBB = J. B. Brinton (U of P) SB = Stewardson Brown (PBC) RRD = R. R. Dreisbach (PBC) FF = Flora Fender (U of P, PBC, Morris Arboretum) Sal Mar Bord = salt marsh border Swamp = swamp Thick = thicket Wood = woods Wet Sd = Wet sand Open Wood = dry, sandy, opening in woods O. I. = Crab Island nCr. I. = island north of Crab Is. 30 BARTONIA THE FLORA OF SE^^N MILE BEACH, NEW JERSEY 31 JMF = John M. Fogg, Jr. (U of P, PBC) AJ = Albrecht Jahn (PBC) IK = Ida Keller (PBC) CK = Charles Knopf (U of P) BL = Bayard Long (PBC, U of P) Species POLYPODIACEAE Asplenium platyneuron (L.)Oakes Thelypteris palustris Schott (Aspidium Thelypteris) Pteridium aquilinum (L.)Kuhn (Pteris aquUina) Onoclea sensibilis L. OSMUNDACEAE Osmunda regalis L. O. cinnamomea L. OPHIOGLOSSACEAE Botrychium dissectum Spreng. f. obliquum (Muhl.) Femald (B. obliquum var. dissectum) EQUISETACEAE Equisetum arvense L. LYCOPODIACEAE Lycopodium inundatum L. var. Bigelovii Tuckerm. PINACEAE Pinus rigida Mill. Junipenis virginiana L. var. crebra Femald TYPHACEAE Typha latifolia L. T. angustifolia L. NAIADACAE Ruppia maritima L. Zostera marina L. GRAMINEAE Poa annua L. P. pratensis L. P. cuspidata Nutt. (P. brachyphyUa) P. compressa L. Eragrostis caroliniana (Spreng.) Scribn. (E. PurshU) E. pectinacea (Michx.) Steud. var. spectabilis Gray Distichlis spicata (L.) Greene Uniola laxa (L.) BSP. Phragmites communis Trin. Triodia flava (L.) Smyth (Tridens flavus) Bromus tectorum L. FWP=:F. W. Pennell (PBC, U of P) HS = Hugh Stone (PBC) WS = Witmer Stone (PBC) SSVP=:S. S. Van Pelt (PBC) CSW = C. S. Williamson (PBC) ,t Range NA NJ Habitat Localities and Co! W G W CP Woods Fr Mar Peer(WS,CSW) Peer(FF,FWP,WS) W G Thick Peer (FF) W CP Swamp StHar(FF) W G W G Swamp Swamp St Har {¥h') Ava (FF) S G Woods Peer(FF,CK) W CP DrOpSd R G SdDuHo W G DrOpSd N CP Woods Thick W CP FrMar W CP FrMar W Ma W Ma W In W In Sal Mar Pools Sal Mar DrOpSd Open Woods S CP Woods W In W G W Ma W Ma 8 CP W CP S CP W In Woods SdDune SdDune Sal Mar WetSd WetSd Woods Sd Dune, DrOpSd Ava (FF) Peer (FWP) Peer (FF) Peer (FF, WS), St. Har (FF), CrI(FF,JMF) Ava (FF,JMF,SB), Peer (FWP), St Har (WB) Ava (SSVP), Peer (FF, JMF, SSVP) Peer(WS), St Har (SSVP. CSW) reported by 0. Brown Ava (BL) Peer (FF) Peer (FF) Peer (FF) Ava (FF,JMF,HS) Ava(JMF), Peer (FWP), St Har (FF) Ava (FF, HS), Peer (SSVP), StHar(FF,RRD) Peer (FWP) St Har (FF) Ava (FF) Ava (Clarke), Peer (FF) Vi 1 Range Species NA NJ Festuca octoflora Walt. W G F. myuros L. W In F. rubra L. W Ma F. elatior L. W In Glyceria septentrionalis Hitchc. W CP G. obtusa (Muhl.) Trin. N G G. pallida (Terr.) Trin. W CP Triplasis purpurea (Walt.) Chapm. W G Agropyron repens (L.) Beauv. W In Elymus australis S & B var. W Ma glabriflorus Wiegand {E. canadensis, in part) E. virginicus L. W CP E. V. var. halophilus (Bickn.) N Ma Wiegand Sphenopholis obtusata (Michx.) S CP Scribn. Aira praecox L. R In Holcus lanatus L. W In Danthonia spicata (L.) Beauv. W G Ammophila breviligulata Femald W Ma {A. arenaria) Agrostis stolonifera L. {A. alba var. vulgaris) A. perennans (Walt.) Tuckerm. W CP Cinna amndinacea L. W CP Aristida purpurascens Poir. S G Cynodon Dactylon (L.) Pers. S In Spartina cynosuroides (L.) Roth S Ma S. alterniflora Loisel var. pilosa W Ma Men*. (S. glabra var. pilosa) S. patens (Ait.) Muhl. W Ma S. p. var. juncea (Michx.) Hitchc. W Ma Anthoxanthum odoratum L. W In Leersia oryzoides (L.) Sw. W CP Digitaria sanguinalis (L.) Scop. W In Paspalum setaceum Michx. W G P. pubescens Muhl. S G Panicum verrucosum Muhl. S G P. microcarpon Muhl. S CP P. Lindheimeri Nash. var. fasci- W CP culatum (Torr.) Femald (P. huachucae, P. tenneaseense) P. meridionale Ashe S P. lanuginosum Ell. S P. villosissimum Nash S P. Addisonii Nash S P. sphaerocarpon Ell. W P. scoparium Lam. S P. clandestinum L. W P. dichotomiflorum Michx. W P. amarum Ell. S Habitat DrOpSd DrOpSd Dr Op Sd, Thick Dr Op Sd FrMar Swamp Swamp Sd Dune, DrOpSd DrOpSd Sal Mar Bord Dr Op Sd, Thick DrOpSd, Thick DrOpSd DrOpSd DrOpSd Open Woods SdDune N In DrOpSd Woods Woods Open Woods DrOpSd Sal Mar Sal Mar Sal Mar, SdDune Sal Mar, SdDune Dr Op Sd Swamp DrOpSd DrOpSd Dr Op Sd WetSd Woods Woods, Sd Dune Woods Sal Mar Bord, Woods Woods DrOpSd DrOpSd Woods, Thick Thick WetSd SdDune G CP G G G G CP CP Ma Localities and Collectors Peer (FF) Peer (FF) Ava (BL), Peer(FF), Crl(FF), St Har (FF, SSVP) reported by O. Brown Peer(WS), St Har (SSVP) Peer (HS) Peer (FF) Ava (FF, JMF, HS), Peer (SSVP), St Har (FF) StHar(WB) Ava (JMF, HS), St Har (WB) Ava(FF), Peer (FWP), CrI(FF,JMF) Peer (FF,FWP), CrI (FF) Peer (FF, HS), St Har (SSVP) Ava (BL) Ava (SSVP), Peer (FF, HS) Peer(FF,WS) Ava(JMF,Kaji), Peer(FF), St Har (FF, HS) St Har (WB) Ava (FF) Peer (WS) Peer (SSVP) Ava (BL) Ava (HS) Ava (JMF), Peer(FF), StHar(RRD,FF) Ava(FF,HS), Peer(FF), St Har (FF, SB, Jahn) Ava(JBB,JMF,Witte), St Har (FF) Ava (FF), Peer (CK) Peer (WS) Ava (FF, JMF), StHar(WS) Peer (FWP) StHar(WB) reported by O. Brown Ava (SSVP), Peer(WS) Ava (SSVP), Peer (FF,SSVP) Peer (HS) Ava(HS), Peer (SSVP, WS) Ava (SSVP) Peer (WS) Ava (SSVP), Peer (SSVP) Ava (FF, HS), Peer (SB, FF), St Har (WB, Jahn) Peer (FF, SSVP) Peer (FWP) Ava(FF,JMF,HS), St Har (FF) 32 Species Panicum amarulum H. & C. P. virgatum L. P. V. var. spissum Linder P. longifolium Torr. P. condensum Nash Echinochloa Walter! (Pursh) Nash Setaria geniculata (Lam.) Beauv. (S. imberhis var. perennis) S. viridis (L.) Beauv. Cenchrus pauciflorus Benth. (C carolinianus) C. tribuloides L. Andropogon scoparius Michx. var. polycladus S.&B. {A. 8. var. littoralis) A. glomeratus (Walt.) BSP. A. virginicus L. CYPERACEAE Cyperus filicinus Vahl (C. NuttaUii) C. ferax Rich. C. esculentus L. C. strigosus L. C. cylindricus (Ell.) Britton C. ovularis (Michx.) Torr. C. Grayi Torr. C. filiculmis Vahl. var. macilentus Fernald Eleocharis parvula (R.&S.) Link {Scirpus naniLs) E. Smallii Britton (E. palustris, in part) E. halophila Fernald and Bracken iE. palustris, in part) E. calva Torr. (E. palustris var. calva) Stenophyllus capillaris (L.) Britton Fimbristylis castanea (Michx.) Vahl. F. mucronulata (Michx.) Blake (F. autumnaUs) Scirpus americanus Pers. S. validus Vahl S. robustus Pursh S. Eriophorum Michx. Rynchospora capitellata (Michx.) Vahl (R. glomerata, in part) Mariscus mariscoides (Muhl.) Kuntze (Cladium mariscoidei) BABTONIA Range NA NJ S Ma W CP N G W G S CP S Ma S Ma W In W G S S Ma Ma Habitat DrOpSd Dry or WetSd DrOpSd WetSd FrMar Sal Mar Bord Sal Mar Bord DrOpSd Sd Dune, DrOpSd Sd Dune DrOpSd S G DrOpSd S G Dr Op Sd W Ma S Ma W CP W CP S G S CP R CP W G W Ma N CP N Ma (V CP W CP S Ma S G W G W CP S Ma S G Sal Mar. FrMar Sd Dune, DrOpSd Sd Du Ho FrMar WetSd DrOpSd DrOpSd Sd Dune, DrOpSd Dr Op Sd Sal Mar FrMar FrMar Fr Mar, DrOpSd WetSd FrMar Fr Mar, WetSd FrMar Sal Mar FrMar W G WetSd W G FrMar Localities and Collectors St Har (FF) Ava(JMF), Peer(FF), StHar(WS) Ava (FF) reported by O. Brown Peer (WS) Ava(FF,CK) Peer (FWP) Ava (FF) Ava(FF), Peer(FF) Ava (FF, JMF, BL), St Har (RRD) Ava (FF), Peer (FWP), St Har (FF) reported by O. Brown reported by O. Brown Ava (FF, JMF,HS), Peer (FWP.SSVP) Ava (FF, JMF), Peer (FWP, SSVP), St Har (FF, SSVP) Ava(FF,JMF,HS), Peer SSVP) Peer (FWP), St Har (FF) Peer (FF, SSVP), n Cr I (FF) reported by O. Brown Peer (FF, SSVP), St Har (SB, FF,SSVP) Peer(FF), StHar(WB) Ava (FWP) Peer (reported in (7), but no specimen seen) Peer(FF), StHar(FF) Peer (SSVP) reported by O. Brown Ava (HS), Peer (FWP, SSVP). St Har (SB, SSVP) reported by O. Brown Ava (RRD, JMF), Peer (FF, WS),StHar(WB,HS, (SSVP) Peer (SSVP) St Har (SSVP) Peer (FF.FWP), Ava (FF), St Har (FF) Peer (FWP) reported by 0. Brown ^ THE FLORA OF SEVEN MILE BEACH, NEW JERSEY 33 Species Carex scoparia Schkuhr C. Longii Mackenzie (C alholutescens) C. hormathodes Fernald C. silicea Olney C. alata Torr. C. vulpinoidea Michx. C. crinita Lam. C. Swanii (Fernald) Mackenzie (C. virescens var. Swanii) C. nigro-marginata Schwein. C. pennsylvanica Lam. C. lanuginosa Michx. C. comosa Boott C. lurida Wahlenb. JUNCACEAE Juncus bufonius L. J. Gerardi Loisel. J. tenuis Willd. J. dichotomus Ell. J. effusus L. var. solutus Fernald & Wiegand J. canadensis J. Gay J. scirpoides Lam. J. acuminatus Michx. J. aristulatus Michx. Luzula campestris (L.) DC. var. bulbosa A. Wood LILIACEAE Hemerocallis fulva L. Lilium superbum L. Asparagus officinalis L. Smilacina stellata (L.) Desf. Maianthemum canadense Desf. Polygonatura biflorum (Walt.) Ell. (P. commutatum) Aletris farinosa L. Smilax rotundifolia L. S. glauca L. IRIDACEAE Iris versicolor L. Sisyrinchium gramineum Curtis S. atlanticum Bicknell ORCHIDACEAE Spiranthes vemalis Engelm A Gray S. cemua (L.) Richards. SALICACEAE Salix nigra Marsh. (Pursh.) Torr. Populus alba L. Range NA NJ Habitat W G WetSd W G Thick, DrOpSd N Ma Sal Mar N Ma Sd Du Ho W CP Sal Mar Bord W CP FrMar W W S w S w w w s s w CP Fr Mar CP Fr Mar CP G CP CP G W CP W Ma CP G W G W G Woods Woods Sal Mar Bord Swamp Swamp Sd Du Ho, DrOpSd Sal Mar, SdDuHo Open Woods FrMar FrMar Fr Mar, WetSd S CP Fr Mar W G Fr Mar S G Fr Mar S CP Wet Sd W In Dr Op Sd W G FrMar W In Woods N CP Open Wood W CP Woods W CP Woods W G Woods W G Woods S G Thick W CP FrMar W CP FrMar W G WetSd CP G var. falcata N CP W In WetSd WetSd Swamp, SdDuHo DrOpSd Localities and Collectors St Har (CSW) Ava (JBB), Peer (FF,WS), St Har (JBB) Ava (FF, SSVP), Peer (SSVP) Peer(WS),StHar(Jahn, SSVP) Ava (SSVP), Peer (FF, WS) Ava (SSVP), Peer(WS), St Har (FF) reported by O. Brown Ava (JBB, SSVP), Peer (FF, WS) Peer (FF) reported by O. Brown Peer (FF, SSVP) Ava (JBB), Peer (FF, SSVP) Peer (SSVP) Ava (RRD,BL), Peer (FF, WS), St Har (CSW, SSVP) Peer (FF, SSVP), St Har (WB) Peer (FF) Ava (RRD), Peer (FF,FWP), St Har (JBB) Peer (FF) Peer (FF, FWP) Ava (RRD), Peer (FF, WS) StHar(WB,Jahn) Ava (SSVP), Peer (FWP, WS) Peer (FWP) Peer (FF) St Har (FF) Peer (FF, FWP) Peer (FF) Peer (FF, SB) Peer (FF) Ava (FF,FWP), Peer(FF,WS> St Har (SB) Peer (FF) Peer (FF) Peer (FF,WS,SSVP), Ava(FF> Ava (FF), Peer (FF, SSVP) Ava (SSVP), Peer (FF) Ava (IK, FWP), Peer (FF) Peer (FWP) Peer (FF,WS), Ava (FF) StHar(FF) I 34 Species MYRICACEAE Myrica carolinensis Mill. M. asplenifolia L. FAGACEAE Quercus stellata Wang. Q. falcata Michx. Q. phellos L. URTICACEAE Celtis occidentalis L. Morus rubra L. Boehmeria cylindrica (L.) Sw. POLYGONACEAE Rumex verticillatus L. R. crispus L. R. Acetosella L. Polygonum prolificum (Small) Robinson P. aviculare L. P. pennsylvanicum L. P. punctatum Ell. (P. acre) P. Persicaria L. P. sagittatum L. Polygonella articulata (L.) Meian. CHENOPODIACEAE Cycloloma atriplicifolium (Spreng) Coult. Chenopodium ambrosioides L. C. album L. C. Boscianum Moq. C. leptophyllum Nutt. Atriplex patula L, var. hastata (L.) Gray A. arenaria Nutt. Bassia hirsuta (L.) Aschere. Salicornia mucronata Bigel. S. herbacea L. (S. europaea) S. ambigua Michx. Suaeda linearis (Ell.) Moq. Salsola Kali L. PHYTOLACCACEAE Phytolacca americana L. CORRIGIOLACEAE Scleranthus annuus L. BARTONIA THE FLORA OF SEVEN MILE BEACH, NEW JERSEY 35 Range NA NJ W G W G Habitat Sd Du Ho, Woods DrOpSd S G Woods S G Woods S CP Woods W CP Woods W CP Woods W CP Woods W w w CP In In W Ma W G Swamp DrOpSd Woods, DrOpSd Sal Mar Bord Sal Mar, FrMar S G WetSd S CP Fr Mar W In Sal Mar Bord W CP FrMar W G SdDune, WetSd W In Beach W In Beach W In SdDuHo W CP Woods N CP Open Wood W CP Sal Mar Bord S Ma Beach R Ma Sal Mar W Ma Sal Mar W Ma Sal Mar W Ma Sal Mar W Ma Sal Mar W Ma Beach W CP Thick W In DrOpSd Localities and Collectors Peer (FWP), StHar (FF), Crl(JMF) Ava(FF) Ava(BL,HS), StHar(FF) Ava(SSVP), Peer (SB, FF), nCrl(JMF) Peer (FF) Peer (FF) Peer (FF, SSVP), Ava (FF) Peer (FF, WS) Peer (FWP) Ava (FWP), Peer (FF,SSVP) Peer (FF) Ava(FF,Kaji), Peer (FF), StHar(FF) Peer (FF, WS), St Har (SSVP) Ava(FF,HS), StHar(FF) Ava (Kaji) Ava (FF, JMF), Peer (FF, WS) StHar(FF) Ava(RRD,JMF) Peer (FWP) Peer (FF), StHar (FF) Ava (FF, JMF), St Har (SB, FF) Peer (FWP), CrI (FF, JMF) Ava (FF, BL) reported by O. Brown Ava (FF, BL) Ava(FF), Peer(FF), StHar (FF), nCrI(FF,JMF) Ava (BL), Peer (FWP, WS), StHar (FF, SSVP), CrI (FF,JMF) Ava (FWP) Ava(FF), Peer (FF, FWP), St Har (SSVP) Ava (FF, JMF, Kaji), Peer (FWP), nCrI(FF,JMF) Peer (SSVP), St Har (FF), nCrI(FF,JMF) Peer (FWP), StHar(FF), nCrI(FF,JMF) Ava(FF,JMF,RRD), Peer (FWP), StHar (FF) Peer(FF), StHar(WB) Ava (RRD) ^ %* 1 rf # 'f ' Species AIZOACEAE Sesuvium maritimum (Walt.) BSP. CARYOPHYLLACEAE Spergularia leiosperma (Kindberg) F. Schmidt (S. marina, in part) Sagina decumbens (E11.)T.&G. Arenaria peploides L. var. robusta Fernald Stellaria media (L.) Cyrill Cerastium vulgatum L. C. semidecandrum L. Silene pennsylvanica Michx. S. stellata (L.) Ait. f . Dianthus armeria L. RANUNCULACEAE Ranunculus sceleratus L. Aquilegia canadensis L. MAGNOLIACEAE Magnolia virginiana L. LAURACEAE Sassafras variifolium (Salisb.) Ktze. CRUCIFERAE Draba vema L. Lepidium vii^inicum L. Capsella Bursa-pastoris (L.) Medic. Cakile edentula (Bigel.) Hook. Raphanus Raphanistrum L. Sisymbrium Thalianum (L.) J. Gay Cardamine parviflora L. Arabis lyrata L. CRASSULACEAE Sedum acre L. HAMAMELIDACEAE Liquidambar Styraciflua L. ROSACEAE Pyrus arbutifolia (L.) L. f . P. a. var atropurpurea (Britton) Robinson m j. r« \ Amelanchier oblongifolia (T. & U.) Roem. Fragaria virginiana Duchesne Potentilla recta L. P. pumila Poir. P. canadensis L. Geum canadense Jacq. Rubus argutus Link Range NA NJ Habitat S Ma Sal Mar N Ma Sal Mar N CP Sd Du Ho N Ma Beach W In DrOpSd W In DrOpSd R In Dr Op Sd S CP Woods S CP Woods R In Dr Op Sd W CP Fr Mar W CP Woods S G W G S In W G Swamp Woods, Sd Dune DrOpSd Dr Op Sd, Thick W In Open Woods W Ma Beach N In DrOpSd S In Dr Op Sd W CP Woods W CP Woods W In DrOpSd S CP Swamp S G Woods W CP Swamp W G Woods W N W W W CP In CP CP CP W CP Woods DrOpSd DrOpSd DrOpSd Woods, DrOpSd Woods Localities and Collectors Ava (FWP), StHar (FF) Peer(FF), StHar(WB) Ava(CSW),StHar(CSW) Ava (FWP), Peer (FF, WS), StHar(SSVP&CSW) St Har (FF) Peer(FF), StHar(FF) Peer(CSW) Peer (FF, CK, CSW) , St Har (McCadden) Peer (FF) St Har (FF, WB) Peer (WS), Ava (FF) Peer (FF, WS), StHar (CSW) Peer (FF, WS & SSVP) Peer (FF,WS),StHar (FF) StHar(FF) ^^^^ Ava (FF, JMF), Peer(FF), StHar(FF), nCrKFF, JMF) Peer (FF) ^ ^ Ava (RRD, JMF, Witte), Peer (FF),StHar(WB,SB) Ava (FF) Peer (CK) Peer (CSW) Ava (SB), Peer (WS) Ava (Kaji, BL) Peer (FF) Peer (FF) Ava (FF) Peer (FF,WS), Ava (FF) Ava (FF), Peer (FF,WS) Peer (FF) reported by O. Brown reported by O. Brown Ava (JBB,BL, FWP), Peer (FF, WS) Peer(SB,FF,WS,CSW), St Har (CSW) I( 36 Speciea Rubus cuneifolius Pursh R. hispidus L. R. Baileyanus Britton (R. vUlosus var. humifuans) R. flagellaris Willd. (R. villosus) Rosa palustris Marsh. {R. Carolina) R. virginiana Mill R. Carolina L. {R. humilia) R. rugosa Thunb. Prunus serotina Ehrh. P. maritima Wang. LEGUMINOSAE Cassia Chamaecrista L. C. nictitans L. Baptisia tinctoria (L.) R. Br. Trifolium arvense L. T. pratense L. T. repens L. T. hybridum L. T. procumbens L. T. dubium Sibth. Melilotus oflficinalis (L.) Lam. M. alba Desr. Robinia pseudo-acacia L. Desmodium Dillenii Darl. D. paniculatum (L.) DC. D. obtusum (Muhl.) DC. Lespedeza procumbens Michz. L. frutescens (L.) Britton L. capitata Michx. L. striata (Thunb.) H.&A. Vicia villosa Roth Apios tuberosa Moench Phaseolus polystachyus (L.) BSP. Strophostyles helvola (L.) Britton S. umbellata (Muhl.) Britton Amphicarpa Pitched T.&G. LINACEAE Linum medium (Planch.) Britton OXALIDACEAE Oxalis stricta L. GERANIACEAE Geranium Robertianum L. POLYGALACEAE Polygala cruciata L. P. verticillata L. BARTONIA THE FLORA OF SEVEN MILE BEACH, NEW JERSEY 37 Range NA NJ S G W G S CP Habitat Woods FrMar Woods W G SdDuHo W CP Swamp N W N W CP G In CP W G SdDune DrOpSd Sd Dune SdDune, Woods SdDune S CP DrOpSd W CP W G W In W In W In W In W In R In W In W In R In W G W CP S G W CP W G W G S In W In W G S CP S CP S CP S CP Woods edge DrOpSd DrOpSd DrOpSd DrOpSd DrOpSd DrOpSd DrOpSd DrOpSd DrOpSd DrOpSd WetSd Swamp edge SdDuHo DrOpSd DrOpSd DrOpSd DrOpSd DrOpSd FrMar Woods DrOpSd, Sd Du Ho DrOpSd, SdDuHo Woods W G DrOpSd W CP Woods N CP Woods W G FrMar W CP DrOpSd Localities and Collectors Ava(SB), Peer(FF), StHar. (Jahn) reported by 0. Brown Peer (FF) Peer (SB, FF), St Har (CSW) Ava (JBB), Peer (FF, FWP) Peer(FWP),StHar(FF) Ava (SSVP), Peer (FF, WS) StHar(FF) Ava (FF, HS), Peer (FF, WS). St Har (FF, CSW) Ava (HS), Peer (FF) Peer (FF, CK, FWP), St Har (FF) Peer (FF) Peer(FF), StHar(WB) St Har (FF, WB) Peer (FF) reported by 0. Brown reported by 0. Brown Peer (FF) Ava (SSVP), Peer (CSW) St Har (FF) reported by 0. Brown Peer (FF) reported by O. Brown Ava (FF), Peer (FWP) Peer (FWP) Peer (FWP) Peer (FWP) Peer (FF, WS) Peer (FWP), St Har (SSVP) Peer (FF) Ava (HS), Peer (FF, FWP) Ava(HS), Peer(FF,WS) Ava (FF, JMF), Peer (FWP, FF) Peer (FF) reported by O. Brown Ava (RRD), Peer (FF, WS), StHar(WB,SB,FF,WS) Ava (SB,FF), Peer (FF), St Har (CSW) Peer (SB, CK, FF, CSW) St Har (Jahn) reported by O. Brown Ava (FWP), Peer (WS), St Har (SSVP) a I Species EUPHORBIACEAE Euphorbia polygonifolia L. E. nutans Lag. {E. Preslii) E. maculata L. ANACARDIACEAE Rhus copallina L. R. vemix L. R. Toxicodendron L. AQUIFOLIACEAE Ilex opaca Ait. I. verticillata (L.) Gray CELASTRACEAE Euonymus americanus L. Celastrus scandens L. ACERACEAE Acer rubrum L. var. tridens Wood BALSAMINACEAE Impatiens biflora Walt. VITACEAE Psedera quinquefolia (L.) Greene Vitis labrusca L. V. aestivalis Michx. MALVACEAE Kosteletzkya virginica (L.) Preal Hibiscus Moscheutos L. HYPERICACEAE Hypericum mutilum L. H. canadense L. H. gentianoides (L.) BSP. H. virginicum L. CISTACEAE Helianthemum canadense (L?) Michx. Hudsonia tomentosa Nutt. Lechea minor L. L. maritima Leggett L. Leggettii Britton & Hollick CACTACEAE Opuntia vulgaris Mill. LYTHRACEAE Decodon verticillatus (L.) Ell. MELASTOMACEAE Rhexia virginica L. R. mariana L. Range NA NJ Habitat W Ma SdDune, Beach S CP Dr Op Sd W CP DrOpSd W CP Woods W G Swamp S CP Woods, Sd Dune S G Woods, Thick W G Swamp S CP Woods W CP Woods S G Swamp W CP Swamp W CP Woods, SdDune W CP Woods, SdDune W G Woods, SdDune S Ma Sal Mar S CP Sal Mar, Fr Mar W CP Swamp W G Wet Sd W G DrOpSd W G Fr Mar Localities and Collectors Ava (RRD, FF, JMF), Peer (FF,FWP), StHar(WS) Ava (FF) reported by O. Brown Peer(FF,WS) reported by O. Brown Ava (FF,HS). Peer (FF.FWP), St Har (FF) Peer(FF,WS), StHar(FF, WS) reported by O. Brown Peer (FF) Ava (FF), Peer (FF.WS). StHar(FF) Peer (FF, WS), St Har (FF) Ava (FF), Peer (FF), StHar (FF) Peer (FF, FWP, WS) , St Har (FF) Peer (FF, FWP), StHar(FF) Peer (SSVP, FWP), St Har (FF) Ava (CK, Witte) , Peer (SB, IK), StHar (FF) Ava (FF, JMF,IK), Peer (FF. WS). StHar (FF, RRD) Peer (CK, FWP), St Har (FF) Peer (FWP) Peer (FWP) Peer (FF) W G Open Wood Peer (FF, SSVP) N Ma SdDune W G DrOpSd W Ma DrOpSd S G Dr Op Sd reported by O. Brown reported by 0. Brown StHar(FF) Peer (FF, FWP) S CP Open Wood Peer (FF) , St Har (FF) W G Swamp W G S G FrMar FrMar reported by O. Brown reported by O. Brown Peer (FWP) 38 Species ONAGRACEAE Ludvigia alternifolia L. L. palustris (L.) Ell. Oenothera biennis L. O. humifusa Nutt. O. laciniata Hill. 0. fruticosa L. O. linearis Michx. HALORAGIDACEAE Proserpinaca palustris L. UMBELLIFERAE Eryngium aquaticum L. Hydrocotyle umbellata L. Ptilimnium capillaceum (Michx.) Raf. Cicuta maculata L. Sium suave Walt. (S. cicutifolium) Daucus carota L. CORNACEAE Nyssa sylvatica Marsh. ERICACEAE Gaylussacia baccata (Wane.) C. Koch Vaccinium corymbosum L. V. atrococcum (Gray) Heller PLUMBAGINACEAE Limonium carolinianum (Walt.) Britton PRIMULACEAE Samolus parviflorus Raf. (S. floribundiis) Lysimachia quadrifolia L. L. terrestris (L.) BSP. Trientalis americana (Pers.) Pursh EBENACEAE Diospyros virginiana L. GENTIANACEAE Sabatia stellaris Pursh APOCYNACEAE Vinca minor L. Apocynum cannabinum L. ASCLEPIADACEAE Asclepias tuberosa L. A. incarnata L. var. pulchra (Ehrh.) Pers. A. syriaca L. CONVOLVULACEAE Convolvulus i^pium L. C. 8. var. pubescens (Gray) Fernald Cuscuta Gronovii Willd. BARTONIA THE FLORA OF SEVEN MILE BEACH, NEW JERSEY 39 Range NA NJ Habitat S G Fr Mar W CP FrMar W CP SdDune, DrOpSd S Ma Sd Dune S G Dr Op Sd W CP DrOpSd, Thick S G Dr Op Sd W CP FrMar S CP Fr Mar 8 CP Fr Mar S CP Sal Mar, FrMar N CP Swamp W CP FrMar, Swamp W In DrOpSd W G W G W G W G Swamp Woods Woods Woods W Ma Sal Mar W Ma SdDuHo W CP FrMar W G Swamp N G Woods S CP Swamp. WetSd S Ma Sal Mar W In Woods W CP Thick W G DrOpSd W CP Sal Mar Localities and Collectors Peer (FF) Ava (JBB) Ava (HS), Peer (FF, FWP, WS) reported by O. Brown reported by O. Brown Ava (RRD, JMF), Peer (FF, WS), StHar(WB,SB) Peer (FF) Ava(SSVP), Peer(WS) Peer (FWP) Peer (WS) Ava (FF, JMF, WS, Witte), Peer (FF, CK) reported by O. Brown Peer (FF, CK, WS) Ava (BL) Peer (FF, WS) Peer (FF, WS) Peer (FF) Peer (FF) Ava (FF, JMF, IK), Peer (FF, FWP), StHar(RRD), nCrl(JMF) Peer (SSVP) reported by O. Brown reported by O. Brown Peer(FF,CSW), StHar (McCadden) Ava(FF), Peer(FF,WS) Ava (FF, JMF, Witte), Peer (FF, FWP), StHar (SB, FF) Peer (FF) St Har (FF) Peer (FF) Ava (FF, JMF), Peer (WS) W G Dr Op Sd reported by O. Brown W CP DrOpSd W Ma DrOpSd, FrMar W CP Thick Peer(WS), StHar(FF) Peer (FF), StHar (SB) Peer (FWP) - A* t <9B» V r ^. 'r •'i^*p» •I o > #«•♦ ^ ^ • tr*( Species BORAGINACEAE Myosotis virginica (L.) BSP. VERBENACEAE Verbena urticaefolia L. V. hastata L. Lippia lanceolata Michx. LABIATAE Teucrium canadense L. (T. c. var. littorale) T. c. var. vii^inicum (L.) Eaton (T. canadense) Trichostoma dichotomum L. Salvia lyrata L. Monarda punctata L. Lycopus uniflorus Michx. L. americanus Muhl. SOLANACEAE Solanum dulcamara L. S. nigrum L. Datura Stramonium L. D. Tatula L. SCROPHULARIACEAE Verbascum Thapsus L. V. Blattaria L. Linaria canadensis (L.) Dumont Scrophularia lanceolata Pursh Agalinis purpurea (L.) Pennell {Gerardia purpurea) A. maritima Raf. {Gerardia maritima) BIGNONIACEAE Tecoma radicans (L.) Juss. PLANTAGINACEAE Plantago major L. P. arenaria L. P. oliganthos R. & S. P. lanceolata L. P. aristata Michx. RUBIACEAE Galium Aparine L. G. pilosum Ait. G. circaezans Michx. G. Claytoni Michx. G. tinctorium L. G. triflorum Michx. Diodia teres Walt. Mitchella repens L. CAPRIFOLIACEAE Lonicera japonica Thunb. L. sempervirens L. Range NA NJ Habitat W CP Woods Localities and Collectors Peer (FF, CK) W CP Woods Peer(FF) W CP SalMarBord Ava (FF, JMF) S CP FrMar Ava (JBB) W Ma Sal Mar, SdDuHo W CP W G s s w CP G CP W CP W In W G W In S In W In W In W G W CP S CP Woods Dr Op Sd Woods Open Wood FrMar SalMarBord Woods Thick DrOpSd DrOpSd Dr Op Sd DrOpSd DrOpSd Woods SalMarBord Ava (FF, JMF), Peer(FF,WS), StHar(FF,WB),nCrI (JMF) Peer (FF) Ava (IK), Peer(HS), StHar (FF) Ava (FF.SSVP), Peer (WS) Peer (FF, FWP) Peer (FWP) Ava(FF,JMF,HS), Peer (FWP, WS),StHar(FF) Peer (SSVP) St Har (FF) reported by O. Brown reported by O. Brown StHar(WB) Peer (FF) Ava (Clarke), Peer (FF.FWP), StHar(FF,WB) Peer (SB) Ava(FF,Kaji,FWP), Peer (FF.FWP), StHar(WB, RRD, FF) W Ma SalMarBord Peer (SB, SSVP) S CP Woods N N W W Ma In Ma In In W CP W CP W CP W CP W CP W CP S G Sal Mar DrOpSd Sal Mar Bord DrOpSd DrOpSd Woods Open Woods Woods FrMar Woods Woods DrOpSd W CP Woods S In Woods W CP Woods Peer (FF, SSVP) reported by 0. Brown Ava (BL) StHar(FF) Peer(FF,CK) Ava(FF). Peer(FF), StHar (WB,SB) Peer(FF), StHar(FF) Ava(BL), Peer(FF), StHar (Jahn) Peer (FF) Ava (FF, JMF, FWP), Peer (FF,WS),StHar(FF) Peer (FF) Peer (FF) Ava (FF, JMF), Peer (FWP), StHar(FF) Peer (FF.WS, Walter) Peer(FF),StHar(FF) Peer(SB,FF,BL) I i.. 40 Species Viburnum scabrellum (T. & G.) Chapm. V. dentatum L. Sambucus canadensis L. COMPOSITAE Eupatorium maculatum L. E. album L. E. hyssopifolium L. E. verbenaefolium Michx. E. rotundifolium L, var. ovatum Torr. (E. pubescens) E. perfoliatum L. Mikania scandens (L.) Willd. Liatris graminifolia (Walt.) Willd. var. dubia Gray Solidago bicolor L. S. sempervirens L. S. suaveolens Schoepf (5. odora) S. fistulosa Mill. S. rugosa var. aspera (Ait.) Feraald (5. aspera) S. nemoralis Ait. S. altissdma L. S. graminifolia (L.) Salisb. S. g. var. Nuttallii (Greene) Fernald S. tenuifolia Pursh. Aster patens Ait. A. undulatus L. A. ericoides L. A. dumosus L. ' A. vimineus Lam. A. lateriflorus (L.) Britton A. novi-belgii L. A. umbellatus Mill. A. linariifolius L. A. tenuifolius L. A. subulatus Michx. Erigeron ramosus (Walt.) BSP. E. annuus (L.) Pers. E. pusillus Nutt. E. canadensis L. Baccharis halimifolia L. Pluchea camphorata (L.) DC. Gnaphalium obtusifolium L. (O. polycephalum) G. purpureum L. Iva frutescens L. var. oraria (Bartlett) Fern & Grisc. (/. oraria) BARTONIA THE FLORA OF SEVEN MILE BEACH, NEW JERSEY 41 Range NA NJ S CP Habitat Woods W CP Woods W CP Woods W CP Fr Mar S G Thick S G SalMarBord. Thick S G SdDuHo N G Sal Mar Bord W W S w w w S s CP Fr Mar CP Sal Mar, Swamp G DrOpSd Localities and Collectors Ava (FF), Peer (FF), St Har (FF) Peer (FF) Peer (FF, WS) reported by 0. Brown Peer (FWP) Ava (JMF), Peer (WS), St Har (FF) Peer (FF) Ava (FF, JMF), Peer (FWP. FF) reported by O. Brown Ava (FF, IK), Peer (SB,SSVP), St Har (FF) Peer (SB, FF) G Ma G G G W G W W W CP Ma CP Woods Peer (FF, FWP) Fr Mar, Sd Ava (FF, HS) , St Har (FF) Dime, Sal Mar Woods Peer (FWP) Fr Mar Peer (FF, WS) Woods Peer (FWP) Thick, Ava (FF), Peer (FWP), St Har Woods (FF) Dr Op Sd reported by O. Brown Sal Mar Bord Ava (FF) , Peer (FWP) Sal Mar Bord St Har (FF) r • r S G W G W G W CP W G W CP W CP W G W CP W G S Ma W Ma W G W CP S CP W CP S Ma S Ma W G W G Sal Mar Bord Woods Woods Woods Dr Op Sd Thick Woods, Thick Swamp edge FrMar Woods Sal Mar Sal Mar FrMar DrOpSd DrOpSd DrOpSd Sal Mar, SdDuHo Sal Mar DrOpSd, Thick DrOpSd R Ma Sal Mar Ava (FF, IK), Peer (FWP) Peer (FWP) Peer (FF) Peer (FWP) reported by O. Brown Ava (FF) Ava(FF), Peer (FF, FWP). StHar(FF) Ava(FF,SB),Peer(WS) reported by O. Brown Peer (FWP) Ava(FF,WS), Peer (FWP), StHar(FF) St Har (FF) Peer (FF, Walter) reported by O. Brown Ava(FF,IK), Peer (FF) reported by O. Brown Ava (JMF, IK), Peer (FF. FWP), St Har (FF) Ava(FF,IK,Witte), Peer (SSVP) Ava (IK), Peer (FF), St Har (FF), CrI(FF,JMF) Ava (RRD), Peer (WS), St Har (FF, CSW) Ava (FF, JMF, HS), Peer (SB), St Har (RRD), Crl(FF) •'i( >-/ ^^ ♦ l»^% Range Species NA NJ Ambrosia artemisiifolia L. W G Xanthium italicum Moore W In X. echinatum Murr. W Ma Helianthus giganteus L. W CP Coreopsis lanceolata L. W In Bidensdiscoidea (T.&G.) Britton S CP B. frondosa L. W CP B. connata Muhl. N CP B. bipinnata L. S CP B. coronata (L.) Britton S G Achillea Millefolium L. W In Chrysanthemum leucanthemum L. W var. pinnatifidum Lecoq & Lamotte Artemisia Stelleriana Bess. Erechtites hieracifolia (L.) Raf. Cirsium spinosissimum (Walt.) Scop. C. discolor (Muhl.) Spreng. Krigia virginica (L.) Willd. Hypochaeris radicata L. Taraxacum officinale Weber Lactuca canadensis L.^ L. c. var. integrifolia (Bigel.) Gray L. hirsuta Muhl. Prenanthes serpentaria Pursh P. trifoliolata (Cass.) Fernald Hieracium venosum L. H. scabrum Michx. H. Gronovii L.^ N W N W W R W W s w s N w w s In Ma G CP CP G In In CP CP CP PB CP G CP G Habitat Dr Op Sd Dr Op Sd Beach, SdDune Sal Mar, Fr Mar, DrOpSd Dr Op Sd Swamp Swamp FrMar Open Wood FrMar Dr Op Sd, Thick DrOpSd SdDune Thick Woods, DrOpSd Dr Op Sd DrOpSd Dr OpSd DrOpSd DrOpSd DrOpSd DrOpSd Woods Woods Woods Woods DrOpSd Localities and Collectors Ava (FF, JMF), StHar(FF) reported by O. Brown Peer (FF, FWP), St Har (FF, SSVP) Ava (FF, JMF), Peer (FF, SSVP), CrI(FF,JMF) Ava(BL), Peer(FF) reported by O. Brown StHar(FF) Ava (FF), Peer (FWP) Ava (FF) Peer(WS), StHar(WS) Peer(FF) StHar(FF,WB) StHar(FF) Ava (FF, JMF) Peer (FF, FWP), St Har (FF) Ava (Clarke), Peer (FF, WS) Peer (FF) Ava (Clarke, SSVP), Peer (FF) Peer (FF) Peer (CK, FF) Ava (FWP), Peer (FF, FWP) Peer(WS), StHar(FF) Ava (JBB), St Har (Jahn) Peer (FF, FWP, WS) reported by O. Brown Peer (WS) reported by O. Brown Ava (JBB), Peer(FF,WS) BIBLIOGRAPHY 1. Britton, N. L., A Preliminary Catalogue of the Flora of New Jersey. Geol. Surv. New Jersey, 1881. 2. Fogg, J. M. Jr., The Flora of the Elizabeth Islands, Massachusetts. Rhodora, 1930. 3. Gray, Asa., New Manual of Botany. 7th edition 1908 (Revised by B. L. Robinson and M. L. Fernald). 4. Harshberger, J. W., Additional observations on the Strand Flora of New Jersey. Proc. Acad. Nat. Sci. Philadelphia, 1902. 6. Hitchcock, A. S., Manual of the Grasses of the United States. U. S. Dept. Agric. Misc. Publ., 1935. 6. Smaix. J. K., Manual of the Southeastern Flora, 1933. 7. Sit)NE, Witmer, The Plants of Southern New Jersey. New Jersey State Museum, 1910. 8. Willis, O. R., Catalogue of the Plants Growing without Cultivation in New Jersey. A. 8. Barnes & Co. 2nd. edition, 1878. University of Pennsylvania 1 Recorded in Stone (4) as L. aagittifolia Ell. 2 Recorded in Stone (4) aa H. marianum Willd. Reprinted from Rhodora, Vol. 39. May. 1937. > ■• --* « A -kW • » ^ -^W • A STATION FOR HYMENOPHYSA PUBESCENS IN THE EASTERN UNITED STATES John M. Fogg, Jr. Late in April, 1936, I first noticed, from the window of a passing train, a colony of cruciferous plants growing on a high embankment along the tracks of the Pennsylvania Railroad a few blocks northwest of the 30th Street Station in Philadelphia. The broad leaves and flat- topped inflorescences strongly suggested Lepidiwn Draba, and as this is not a common introduction in the Philadelphia area the locality was visited a few weeks later for the purpose of collecting material. Al- though upon this date, May 18, the plant was only in bud, its corym- bose inflorescences and oblong, clasping leaves still contributed to its superficial resemblance to Lepidium Draba. On May 27 the plant was in full bloom, its corymbs of whitish flowers forming an attractive sight as seen from the car-window. Specimens collected on this date revealed a few immature silicles, and as these were ovoid rather than flattened it was apparent that the species must belong to some genus other than Lepidium: By June 8 its fruits were well developed and the still corymbose heads were crowded with upright, purplish, ovoid to globose, mucronate pods. It was now evident that the plant could be referred to no species included in our current manuals for eastern North America. Comparison with herbarium material, aided by reference to standard Old World treatments, established its identity as Hymvnophym pv- bescois C. A. Mey., a species said to be native to Siberia. A casual survey of recent literature disclosed the fact that the plant had already twice been recorded from North America. In 1925 Dr. P. C. Standley published a note on its occurrence in Idaho,' and the following year Dr. B. A. Walpole called attention to the fact that he had previously (1919) collected and distributed specimens of Ilymnw- physa from Ypsilanti, Michigan.^ In an effort to ascertain whether the species had be- • « 192 Rhodora [Extracted from The Journal of Botany, November, 1938.] [May the Gray Herbarium, the Missouri Botanical Garden, the New York Botanical Garden and the U. S. National Museum. To the authori- ties of these institutions I am greatly indebted for their kindness in having examined the material in their respective collections and sup- plying me with all the available records. From the evidence thus accunmlated it appears that Hymcnophysa puhesccns has become well established in several of the far western states, e. g., California, Ore- gon, Washington, Idaho, Wyoming and Colorado, but that it has not before been collected east of Ypsilanti (Washtenaw County), Michigan. The Philadelphia locality, then, is apparently the first to be noted for any of the eastern states. The plant occupies the crest of a grassy embankment, at 31st and Baring Streets, overlooking the tracks. Although twice cut back and once burnt over by railroad workers, it continues to put up new shoots from its perennial bases. It seems reasonable to suppose that the plant will persist and that in the future it will be reported from addi- tional stations in this area as well as elsewhere on the Atlantic sea- board. Specimens of five separate collections (Fogg Nos. 10347, 10440, 10527, 1104G, 11145) have been deposited in the herbarium of the University of Pennsylvania and duplicates of one or more of these numbers are being distributed to the Philadelphia Botanical Club, the Gray Herbarium, the Missouri Botanical Garden and the U. S. Na- tional Museum. University of Pennsylvania. •f I \ -4 ^* <9 \ 4 I A CENTRAL REPOSITORY FOR TYPE-SPECIMENS. By F. R. Fosberg. Type-specimens are the basis of stability in botanical nomenclature and one of the main factors in taxonomic inter- pretation. This fact has come to be recognized in the International Rules and is realized, I think, by all taxonomists of any experience whatever. It must also have occurred to many what a catas- trophe to the science of botany it would be if one of the major herbaria, or, for that matter, some of the smaller ones, were to be destroyed or seriously damaged in any way. Modern mili- tarism has thoroughly demonstrated its irresponsibility and utter disregard for the welfare of humanity, and its willingness to destroy anything in its path to gain a military objective. The current war in Spain should be given careful consideration by scientists as well as war-experts as a rehearsal for future wars. It has shown conclusively that educational institutions and botanical gardens, in spite of their international character and peaceful inclinations, will receive no special consideration whatever. The state of war is an emergency and all sense of human values is lost. With world politics in its present unsettled condition and armaments piling up in all of the major nations, it seems time for systematic botanists and others interested in the science to give some thought to the safeguarding of the precious type-specimens on which our nomenclature rests. A plan calculated to accomplish this end is here presented for the consideration of the botanical pubUc. It is not claimed that this is the best possible plan. It is expected that this or any similar plan will be bitterly opposed by all who place the prestige of their own institutions above the welfare of the scienc^e of botany. The suggestion is merely made to stimulate thought and discussion, and with the idea that if it is considered of sufficient importance it may be brought up for discussion at the 1940 international congress at Stockholm. Perhaps by this means something practical and acceptable to all botanists may be evolved and put into operation. 4 IRREGULAR PAGINATION 328 THB JOITBNAI. OF BOTAJfY ^^\^fly' «iy /"ggestion IS this. That a modem central herbarium be estabhshed for the housing of all types in a locality sufficiently remote from densely populated centres, industrial areas or points of stragetic importance, to be reasonably safe from destruction m case of any war whatever. This herbarium should be admmistered by a director and board of regents appointed by the international botanical conf^ress If there were a reasonable degree of'' unanimity anion- botanists as to the desirability of such an institution, a fund fo? establishing and endowing it could doubtless be built ud bv requests to the various research foundations, national research councils, and even to the governments of the important nations Institutions with a large number of types to be cared for would likely be willing to contribute certain sums for annual running expenses. Gifts from private individuals with an interest iS botany would probably not be entirely lacking. An international association of systematic botanists might be organized to promote the welfare of the central herbarium, with small dues to go to maintenance and service. ^ The administration of this herbarium should, I think be placed m the hands of a salaried director, appointed by 'the mternational congress for a term of five years, subject to renewal at the discretion of the congress. He should be assisted and advised by a council or board of regents of six members, also appointed by the congress, who would have the power by a two-^thirds majority vote, to veto any action of the director that they considered detrimental to the welfare of the institution or the collections housed there. Facilities should be provided for working and for inexpensive living for visiting botanists who wished to work on the collections housed in the herbarium Loans of types to accredited institu- tions would naturally be permitted. Photographs of types would be made at the request of institutions or individuals and distributed at as small a cost as possible. A working collection of specimens other than types and historically important specimens should be maintained, the specimens in so far as possible compared with the types, both for use of resident and visiting botanists and for loan to those interested in special groups. This w^uld prevent shipping damage to more important specimens m all cases where a carefully compared siJecimen would serve m place of a type. This collection might t built up by the distribution, by exchange, of specimens checked against types, to institutions m all parts of the world. In thi.s uav a great service to botany would result at the same time that the central collection was being built up. The library, necessary in connection with any herbarium would m time, naturally become a repository for rare and important works, as well as a very complete collection of all A CENTRAL REPOSITORY FOR TYPE-SPECIMENS 329 t iif 1 documents of interest to systematic botanists. A self-supporting photostating or microfilm copying service could be established in connection with the library. Also, a natural outgrowth of this would be a universal bibliographic and abstracting service for systematic botany. A probable result of the maintenance of such a library would be that many of the extensive private libraries (and also herbaria) might be kept together by bequest of the owners, rather than dispersed at their deaths. Choice of a location for such an institution should only be made after careful deliberation and study of all factors involved, and perhaps only after consultation with military men of several different nations. The place must be remote from any possible military route or battle-ground. Yet it must be reasonably accessible to botanists, without entailing too much travel expense. The climate must not be so extreme in either cold or heat as to discourage botanists from visiting the institution and working there. The region should, if possible, be interesting botanically. Needless to say, deliberation on this subject should be carried on in an atmosphere free as possible from all national or regiona feeling. Switzerland, with its record of hundreds of years of peace, would be a logical choice as a location for such a herbarium if it were not situated in the midst of potentially antagonistic powers. Certain localities m Scandinavia, or perhaps Iceland, would seem likely to remain peaceable, though perhaps the winter climate might be a deterring factor. Sparsely inhabited areas in the southern Rocky Mountain region of the United States might give reasonable safety combined with a moderate climate. Perhaps the most important question of all, after that of finance, would be that of the status of the types and other historical specimens housed in this herbarium. Of course, some institutions might be willing to give their specimens outright^ in the interests of botany, but the majority would probably want to see how the plan worked, before losing control of their specimens. This very reasonable desire might be satisfied in either of two ways. The specimens might be deposited on per- manent loan. Or an institution might endow or provide a table and herbarium cases sufficient to house the number of specimens of historical importance in its possession, and make this unit a department of its own herbarium. Its collection would not, however, be maintained in these cases as a unit, as this would be a source of confusion and extra labour for everyone. This would make possible the participation of institutions where the loaning of specimens or of types is forbidden. Needless to say, the construction and maintenance of such a herbarium could profit by the hundreds of years' experience and the mistakes and successes of all other herbaria combined. Working out of details could be left to the first director and his 4RREGULAR PAGINATION 330 THE JOURNAL OF BOTANY advisory council, who would, naturally, welcome suggestions Irom experienced persons the world over. This plan is not presented under any illusion that it is perfect It seems workable. The benefits that would result to the science ot botany would seem to outweigh, by far, any conceivable detriments. If worked out properly it should insure reasonable satety for tyije-material as long as civilization itself will last, or at least as long as types are necessary to the stability of botanical nomenclature. University of Pennsylvania. Since the above was ^^Titten definite news has been received of the deliberate and total destruction of at least two of the largest universities in China, and of some damage by bombs to a third. What happened to their botanical collections, or for that matter, to the botanists, is not known. Certainly ' the promising start that the Chinese have made in systematic botany IS seriously retarded. " '^ 111 Europe the most serious crisis since the world war has ]ust been passed. The days during which war seemed practically inevitable must have made many botanists wonder why some such scheme as that presented above was not put into effect long ago. The settlement reached, though peaceable, seems little more than a truce that will enable the powers to build up their mihtary strength still further. No thinking person should be deceived into thinking that a permanent peace has been achieved 1 he history of the past year has demonstrated beyond a doubt the desirability of taking some action similar to that outlined above, also the danger entailed by delay and procrastination. By good fortune the herbaria of Great Britain, Prague, Vienna Pans, and the various German institutions, rich in type-material' escaped exposure to war for the present, but who can predict what may happen next time international politics reaches a boilmg point ? J Reprinted from Science, January 14, 1938, Vol 87 No. 2246, pages 39-40. ' THE LOWER SONORAN IN UTAH Readers of Professor Cottam'si interesting article on the effect of the heavy frost in January, 1937, may be pleased to know that, after all, Utah is in no danger of losing her claim to the Lower Sonoran life zone. Stimulated by the above-mentioned article and hav- ing an opportunity to drive through southwestern Utah in August, 1937, the writer took note of the condition of the Lower Sonoran vegetation, both in the region around St. George and in a considerable area on the alluvial fans west of the Beaver Dam Mountains. Particular attention was paid to four typical Lower Sonoran plants — creosote bush, Larrea divaricata; mesquite, Prosopis chilensis; burro weed, Franseria dumosa; and desert willow, Chilopsis linearis. All these species showed the effects of the frost, but not a single plant was noticed which was completely dead. Numbers of plants of Larrea and Prosopis had been frozen to the gi'ound, but all these observed were sending forth a vigorous growth of shoots from the base which were already 10 to 50 centimeters tall. The wholesale destruction of the vegetation implied by Professor Cottam's observations of "brown water- soaked cambium layers even at the crown of most shrubs" was not evident along any of the forty or fifty miles of road traveled by the writer in this life zone. Also the bronzing of the junipers in the Upper Sonoran zone noted by Professor Cottam had disap- peared, at least in the Beaver Dam Mountains and the regions of Zion and Bryce Canyons. Interesting was the evident variation in resistance to the frost of plants even of the same species. Some 1 W. P. Cottam, Science, 85: 563-564, June 11, 1937. > f of the difference in effect was doubtless due to differ- ence in altitude and exposure. The areas at the ex- treme upper edge of the Lower Sonoran, 900 to 1,000 m in the St. George area and somewhat higher west of the Beaver Dam Mountains, in general suffered more, except where sheltered. But of specimens of Larrea growing side by side, with no apparent differ- ence in conditions or size of plant, one would be frozen to the ground, while another might have only the tips of the branches nipped. Every degree of injury excepting actual death was present in a small area of uniform altitude and exposure. However, all plants of Larrea had evidently produced far less fruit this year than is usual for this species. Contrary to Professor Cottam's conclusion that this occurrence "emphasizes the inadequacy of Merriam's theory of zonation in its failure to take into considera- tion temperature data of the dormant period," these later observations would indicate that, in this instance, at least, the ignoring of the dormant period was quite justified. And a week of sub-zero weather would seem to be quite a severe test. The facts suggest that the life zones are states of equilibrium reached by the vegetation as a result of the action of definite climates over a long period of time, and that they are not likely to be profoundly disturbed by brief intervals of "unusual weather." F. R. FOSBEBG University op Pennsylvania I f IRREGULAR PAGINATION i -1 J I I f « Separately printed, without change of paging from Bulletin of the Torrey Botanical Club 65: 607-614. December, 1938. Notes on plants of the Pacific Islands. I. F. R. FOSBERG To record miscellaneous and fragmentary observations accumulated while studying and determining plants from various islands of the Pacific it seems worth while to start a series of papers, to appear at irregular intervals as sufficient material accumulates. The first of these is presented here, treating species of the genera Gouania (Rhamnaceae), Diospyros (Ebenaceae), and Randia (Rubiaceae). Thanks are due to the authorities and staff of the New York Botanical Garden for the privilege of working in the herbarium and for various courtesies extended to me while working there, and also to the authorities of the Bernice Pauahi Bishop Museum, of Honolulu, for the loan of some of the specimens treated in this paper. GOUANIA Gouania mangarevica Fosberg, n. sp. Frutex scandens hirsutus vel tomen- tosus, foHa dimorpha piloso-hirsuta supra bullata serrata membranacea vel chartacea, flores in spicas glomerata, spicae maxima 12 cm. longae, bracteoli minuti caduci, flores maxime 3 mm. latae pentamerae, styli 3, discus sub stylis barbatus in lobos triangulares productus. Liane 20 m. or more long, older parts woody, younger parts hirsute-pilose with tawny spreading hairs, older parts becoming rusty-tomentose; leaves alternate, dimorphic, sparsely hirsute-pilose on both sides, especially beneath on the veins, bullate above, those on young sterile parts of the plants oblong or oblong-ovate, up to 9 cm. long and 4 cm. wide, truncate at base, acuminate at apex, coarsely serrate, especially above, membranous, those on older parts broadly ovate- or elliptic-cordate, obtuse to acuminate, usually shorter than those on sterile branches, as much as 6 cm. wide, chartaceous; tendril-bearing branchlets in the axils of the leaves, these sometimes bearing one or two small leaves, the tendrils slender on young branches but becoming much thickened and woody on the older parts, 2-3 tendrils borne on one branchlet, on older parts these branchlets more robust, sometimes occurring 2 at one node, bearing the inflorescences as well as tendrils. Inflorescence densely yellowish- tomentose, spicate; spikes as much as 12 cm. long, often borne as many as 6 on a branchlet, forming a panicle; glomerules small, subtended by minute caducous bractlets, often scarcely manifest; flowers usually sessile, rarely short-pedicelled, pentamerous, about 3 mm. across when spread out; calyx tomentose externally, its lobes ovate, 1 mm. long, obtuse, glabrous within; petals alternate with the calyx lobes, clawed at the base, about 1 mm. long, the limb suborbicular, saccate, surrounding the upper part of the stamen; stamens opposite and almost equalling the petals; filaments flat, dilated to- ward the base, inserted beneath the disk between its lobes; anthers almost 607 IRREGULAR PAGINATION 608 BULLETIN OF THE TORREY CLUB [VOL. 65 circular 0 2-0.3 mm. long, the anther sacs terete; disk thin, produced into triangular lobes with strap-shaped apices, opposite the calyx lobes and two- thirds their length, glabrous except for a conspicuous tuft of hair immediately below the styles; styles 3, distinct to the base, divergent, glabrous, fleshy, about 0.7 mm. long; ovary inferior but indistinct in flower; fruit unknown. Gambier Islands, Mangareva: South side Mt. Mokoto, upper forest climbing over bushes and trees, alt. 320 m., June 2, 1934, Si. John 14844 (type) (B) ; northwest slope Mt. Duff, in woods on low bushes, alt 110 m May 24, 1934, St. John 14488. (B). ' Mangarevan name "tara koa" (ace. Theophile, a native informant). Ihis species seems to resemble most closely the widespread Indo- Malaysian G. tiUaefoUa and G. javanica and also certain similar American species but differs from all in the dimorphic leaves and in the tuft of hair surrounding the styles. This whole group of species of Gouania badly needs revision. Perhaps all might be united into one large species with many varieties, as they closely resemble each other. Howt^ver, certain details in the formation and hairiness of the disk, and especially in the length and degree of union of the styles seem significant. All specimens of C. liliaejolia and G. javamcu examined except one had very short styles. Most of them had more prominent bracts and less paniculate inflorescences than G mangarcnca. More distantly related are G. vilifolia of Hawaii, with shorter spikes and quite different leaves, and G. Richii of Fiji, with almost glabrous leaves and with the styles united part way up Hooker and Arnott (Bot. Beechey Voy. 61, 1832) report Gouania domtngucnstsL., an American species, from the Society Islands, citing a Lay and Collie collection, and many subsequent authors have credited the species to these islands, presumably on the basis of this report To my knowledge no other collection has been cited. The authorities at Kew are unable to locate this Lay and Collie specimen, so its identity may not be determined. However, remembering that Hooker and Arnott regarded most of southeastern Polynesia as included in the Society Islands, and smce no other Gouania has been found in southeastern Polynesia, I think It quite probable that the Lay and Collie specimen belonged to G. man- garmral he expedition spent some time in Mangareva and is known to have collected several other plants there. It is unlikely that it was an American species, unless there was a confusion in labeling, as with several other plants collected by this expedition. Fruiting specimens are much to be desired. DIOSPYROS While working up the Hawaiian species of Diospyros I found it neces- 1938] FOSBERG: PLANTS OF THE PACIFIC ISLANDS 609 i I 1 sary to determine the status of the Maba sandwicensis reported by See- mann and subsequent authors from Fiji. This did not prove as easy to settle as was expected, and led to a considerable study of various Pacific Ebenaceae, and comparison with available Malaysian and Asiatic material. No attempt was made at a revision of the Pacific species of this group, but some observations were made which seem worth recording. The specimens studied are mainly in the herbaria of the New York Botanical Garden and the Bishop Museum, designated here respectively as (N) and (B). Dates of collections are not cited when collectors' numbers are available. The recent study of the Malaysian Ebenaceae by R. C. Bakhuizen van den Brink (Bull. Jard. Bot. Buit. ser. 3, XV^ 1936; XV^, 1937) treats some of the Pacific species and makes the combinations for several of them under Diospyros, but touches upon the extra-Malaysian ones only incidentally. Bakhuizen's reasoning (Gard. Bull. Str. Sett. 7, pt. 2, 1933) in reducing Maha to Diospyros seems, in the light of these further studies of Pacific species, to be thoroughly sound, and necessitates the transfer of several Pacific island species from Maha to Diospyros, Other transfers are doubtless to be made, but material has not been available to determine their exact status. Diospyros subgen. Maha sect. Ferreola Diospyros L. subgen. Maba (Forst.) Bakh sect. Ferreola (Roxb.) Fosberg, n. comb. Ferreola Roxb. PI. Corom. 1 : 35, t. 45, 1795. Maha sect. Ferreola Hiern, Trans. Cambr. Philos. Soc. 12: 108, 1873. Diospyros subgen. Maha sect. Forsteria Bakh. Bull. Jard. Bot. Buit. ser. 3, XV^: 50, 1937. Hiern's name Ferreola, based on Roxburgh's genus Ferreola (credited to Koenig by Index Kewensis), seems to be the earliest to have been used in the sectional category for this group of species, and so by the rule of priority must replace Forsteria. It may be argued that Hiern's section of Maha was more inclusive than Bakhuizen's section of Diospyros, but the type species of Roxburgh's genus was Ferreola buxifolia {Diospyros ferrea) which was also included in Hiern's section Ferreola and is the principal species of Bakhuizen's section Forsteria, though no type species was desig- nated. This seems to make all three nomenclatorially equivalent, and therefore the earliest name must take precedence. The present studies show that there are at least five distinct species belonging to this section, and a number of additional varieties in the two original species. 610 BULLETIN OF THE TORREY CLUB [VOL. 65 Diospyros ferrea (WiUd.) Bakh., var. nandarivatensis (Gillespie) Fosberg, n. comb. Maba nandarivatensis Gillespie, Bish. Mus. Bull. 74: 13 1930 Bakhuizen reduces this species outright to D. ferrea var. sandwicensis, but It seems to me to present differences enough to be maintained as a variety. Ihe leaf-shape is not that of var. sandwicensis and the fruit is more nearly that of var. liltorea. The branched inflorescence (in the stami- nate plant as shown by Smith 544 as well as in the pistillate as described by Gillespie) suggests that it may have some relationship with D. ellipiici- foha and, indeed, except for the small size of the fruit, might equally well be placed in the latter. It certainly connects the two. As recognized here, the size of the leaves and fruits varies considerably but there seems no place to draw a line between them. Those with large leaves tend to have large fruits, but this is not constant. Specimens examined: ^^T^^Y.' ^^'' ^'" ^^''"' '^^°^° ^°'^^ ^'°^-' Nandarivatu, Gillespie 3764 )nll ;!^^P''"' Thakaundrove, Natewa Peninsula, Uluingala, Smith mz (N); Vanua Levu, Thakaundrove-Malhuata boundary crest of Korotmi Range, bet. Navitho Pass and Mt. Ndelaikoro, Smith 544 (N)- Vanua Levu, Mbua, southern portion of Seatovo Range, Smith 1564 CN)' Kandavu, hills above Namalata and Ngaloa bays, Sm.ith 126 (N). Caroline Is.: Yap, Kanehira 11840 (N). Palau Is. : small islands near Korror, Herre 29 and 36 (N) The last four collections cited have much larger leaves than the first four. Diospyros ferrea (WiUd.) Bakh., var. GiUespiei Fosberg, n. var Ramuh glabrati; folia coriacea elliptica vel oblonga subsessilia canaliculata nervis supra impressis; fructus subsessilis 1.5-2 cm. longus; calix in fructu' gl3,Der. Branches and branchlets gray, beset with prominent lenticels, strigose when very young, but early glabrate; leaves stiff-coriaceous, elliptical to oblong, subsessile, canaliculate, the veins prominently impressed above the blade up to 8 cm. long and 3.5 cm. wide, glossy, minutely appressed puberulent when very young, but soon becoming glabrate; flowers not seen; fruits sub- sessile, cylmdric to ellipsoidal, sparsely appressed hirtellous, 1.5-2 cm. lone tipped with the persistent style; fruiting calyx cupulate, 7-8 mm. across, its lobes rounded to obtuse, low, glabrous inside, glabrous or almost so outside. Fiji Is.: Viti Levu, Naitasiri Province, woods near road beyond Tamavia village, 7^ mi. from Suva, alt. 150 m., Gillespie 2450 (B, N) ; same loc, Gtllespte 2146 (B), (type). Referred by Gillespie to Maba sandwicensis, by A. C. Smith to Maba buxtjolta, and by Bakhuizen to Diospyros Jerrea var. Itttorea. f — ^ - ■* ■^'^ -^ t.-,.^^ r " ► r t • '^ - » • - ► • • r-— '""^ 4'* f ^ f "■**> 1938] FOSBERG: PLANTS OF THE PACIFIC ISLANDS 611 Diospyros ferrea (Willd.) Bakh., var. subimpressa Fosberg, n. var. Ramuli glabrati; folia coriacea elliptica glabrata, nervis supra sub- impressis; fructus ovoideus vel ovalis strigosus, calix in fructu planus vel reflexus strigosus. Branches brownish, beset with prominent lenticels, sparsely strigose when young, glabrate; leaves stiffly coriaceous, elliptical, acute at base, rounded-acute at apex, sparsely strigose beneath when young, glabrate, the veins somewhat impressed above, the petiole about 2.5 mm. long; flowers not seen; fruits solitary on peduncles 2 mm. or less long, ovoid to broadly oval, up to 15 mm. long and 13 mm. wide, tipped with the persistent style, rather persistently strigose; fruiting calyx flat or with lobes reflexed, about 7 mm. wide, the lobes low, obtuse, strigose inside and outside. Fiji Is.: Viti Levu, Rewa Province, woods near summit of Korom- bamba mountain, Gillespie 2324 (B), (type). Very close to var. GiUespiei, differing chiefly in the shape of the fruit and in the less conspicuously impressed veins. Referred to Maha sand- wicensis by Gillespie. Diospyros ferrea (Willd.) Bakh., var. savaiiensis (Christ.) Fosberg, n. comb. Maba savaiiensis Christophersen, Bish. Mus. Bull. 128: 173, 1935. Characterized by coriaceous leaves, reticulate on both sides, abruptly contracted, then shortly attenuate into a petiole, blade ovate-acute, fruiting calyx reflexed, appressed puberulent inside and out, and fruit appressed puberulent. Plant otherwise glabrous or very early glabrate. Samoa: Savaii, Falealupo-Fagalele, Christophersen 3328 (N). Diospyros ferrea (Willd.) Bakh., var. angustifolia (Miq.) Bakh. Schlechter 19205 from '^Walder bci Alexishafen, Kaiser- Wilhelmsland" (N) is, so far as I know, the first record of this variety from New Guinea. It agrees well with material of this variety {Thwaites; DeSilva 43 from Ceylon (N), cited by Bakhuizen. The other two specimens cited by him, Stocks from Malabar Concan (N) and Kajewski 932 from the New Hebrides (N) do not agree so well, and probably do not belong to this variety. Diospyros ellipticifolia (Stokes) Bakh., var. elliptica (Forst.) Fosberg, n. comb. Maha elliptica Forst. Char. Gen. PI. 122, /. 61, 1776. Ferreola ellipticifolia Stokes, Mat. Med. 535, 1812. This, the typical form with elliptic obtuse leaves, dull and venulose above, may be reported, I think for the first time, from Niuafou, {Jaggar, Oct. 1930, (B) ). This variety is widespread from Samoa to Malaysia and southeastern 612 BULLETIN OF THE TORREY CLUB [VOL. 65 Asia. Specimens were examined from Samoa, New Caledonia, and Am- boina (all N). Forster's original material came from Tonga. Diospyros ellipticifolia (Stokes) Bakh., var. iridea Fosberg, n. var. Maba aff. elliptica Christophersen, Bish. Mus. Bull. 128: 173, 1935. Arbor, ramuli aureo-sericei glabrati; folia lanceolata; flores paniculatae; fructus 23-25 mm. longus leviter umbonatus; calix in fructu cupulatus glabratus. Tree 6 m. tall, branches terete, grayish brown, rather smooth; leaves not congested; young parts at first golden-sericeous, very early glabrate; leaves lanceolate, slightly acuminate, blunt, up to 11 cm. long and 3 cm. wide, chartaceous, glossy, drying an almost iridescent gray-brown, the petiole 3-5 mm. long; staminate flowers in loose 4-5 flowered panicles about 10-13 mm. long, panicle and calyx densely sericeous; calyx tube 2 mm. long, lobes 1 mm., acuminate; corolla tube cylindrical, 4 mm. long, 1 mm. wide, sericeous just beneath the lobes, otherwise glabrous, lobes ovate, 1 mm. long, sericeous; pistillate flowers not known; fruit on a peduncle 1 cm. long, red when ripe, yellow when immature, 23-25 mm. long, 15 mm. wide, slightly umbonate at apex; fruiting calyx glabrate, cupula te, 7-8 mm. across, lobes obtuse. Samoa: Savaii, near Tufutafoe, alt. 10 m., Christophersen 2272 (B, N), 3310 (B, N) (type); (type sheet B). No. 2272 is in flower and no. 3310 in fruit. DifTers from var. elliptica in the shiny thin lanceolate leaves, light colored smooth twigs, acuminate calyx lobes, pedicellate flowers, much smaller calyx and corolla, and red fruit. Diospyros lateriflora (Hiern) Bakh. This species may be reported from Uvea on the basis of Burrows W. 23 ''inland, edge of forest, el. 15 m." (B). Burrows gives the aboriginal name as ma pa. This specimen was labeled Maba sandwicensis in the herbarium. Parks, 16291 from Liku Terrace, Eua, Tonga Is. (N) and a collection from Tonga, without island or locality, McKern 99 (B), also belong to this species. Some doubt was expressed by Bakhuizen in assigning this species to his section Forsteria. The material at hand shows that the ovary is definitely 3-celled, the cells 2-ovuled, the ovary hairy, the style trifid, and staminodia absent; seeds were not available, so the endosperm could not be examined. There seems little doubt that it belongs in this section, here called Ferreola. Diospyros globosa (A. C. Smith) Fosberg, n. comb. MabaglobosaA. C. Smith, Bish. Mus. Bull. 141: 121, 1936. This is close to D. lateriflora, as Smith says. It may eventually prove only a variety, if further collecting should reveal intergrades, as the dif- ferences are no greater than the extremes in D.Jerrea and D. ellipticifolia. I 'a »*• . I ^~ i' \-' 1 > -V - A 4 •- ^ ■•, 1938] fosberg: plants of the pacific islands 613 Material now available does not justify any change in its status, other than the generic transfer. The fruits on Smith 1242 are not mature and would probably approach in size those of D. lateriflora at maturity. Fiji Is.: Kambara, Smith 1241 (isotype), 1242 (both N). Diospyros Christophersenii Fosberg, n. name Maba samoensis Hiern, Jour. Bot. 15: 99, pi. 186, 1877. Not Diospyros samoensis Gray. Amply characterized by Hiern. Known only in fruit. Probably to be placed in sect. Ferreola, though in absence of flowering specimens this is uncertain. Samoa: Savaii, Siuvao-Auala, Christophersen 3379 (N). Diospyros subg. Maba sect. Cuptdifera Diospyros L. subgen. Maba (Forst.) Bakh. sect. Cupulifera Fosberg, n. sect. Calix in fructu insigniter accrescens, fructum usque ad medium cingens. Fruiting calyx extremely accrescent, woody, surrounding the lower half of the fruit; ovary trilocular; fruit and calyx copiously rusty pubescent. Diospyros rufa, characterized by its peculiar acorn-like fruit, was placed in Maba sect. Ferreola by Hiern, and would go, according to Bakhuizen's key, into sect. Forsteria, but seems to have little in common with the rest of the members of the section. Those who follow a narrow genus concept would doubtless refuse to include them in the same genus. So far as I know, the type species, D. rufa, in the sense accepted below, is the only species in the section described above. Diospyros rufa (Labill.) Fosberg, n. comb. Maba rufa Labill. Sert. Austr. Caled. 33, t. 36, 1824. Maba sericocarpa F. Muell. Fragm. 5: 164, 1866. Maba cupulosa F. Muell. Fragm. 5: 164, 1866. Diospyros sericocarpa F. Muell. Austr. Veg. in Intcrcol. Exh. Ess. 1866- 67:35, 1867. Diospyros cupulosa F. Muell. Austr. Veg. in Intercol. Exh. Ess. 1866- 67:35, 1867. Maba yaouhensis Schlecht., Engl. Bot. Jahrb. 39: 226, 1906. }Maba parviflora Schlecht., Engl. Bot. Jahrb. 39: 226, 1906. Material of this species from New Caledonia has generally broader and more obtuse leaves than that from Australia. Schlechter's species cannot, on the basis of material available, be maintained as specifically distinct. The small flowers of the material upon which M. parviflora was based suggest that if more material were available it might prove to be a good variety. 614 BULLETIN OF THE TORREY CLUB [VOL. 65 RANDIA Randia cochinchinensis (Lour.) Merr. Tr. Am. Phil. Soc. 24 (2) : 365, 1935. Aidia cochinchinensis "Lour. Fl. Cochinch. 143: 1790. Stylocoryne cofeoides Gray, Proc. Am. Acad. 4: 309, 1860 (excl. syn.). Randia cofeoides (Gray) B. & H. Gen. PI. 2: 88, 1873. Randia Graefei Reineke, Engl. Bot. Jahrb. 25: 683, 1898. Full synonymy in Merrill, Tr. Am. Phil. Soc. 24(2) : 365, 1935. The widespread Pacific Randia cofeoides does not seem to differ sig- nificantly from the above tropical Asiatic and Malaysian species. Plants from Polynesia and Micronesia tend to have the buds slightly bent and sharper at the apex than most Asiatic specimens, but some, even from Ceylon, cannot be distinguished from those from the Pacific. Neither can I distinguish R. Graefei on the basis of the description and a number of collections from Samoa. I have not seen the type. It is considered by Kane- hira (Bot. Mag. Tokyo 45: 349, 1931) to be a synonym of R. racemosa (Cav.) F.-Vill., itself a synonym of R. cochinchinensis. Stylocoryne cofeoides Gray was so inadequately characterized that it is doubtful whether it could be considered validly published. Neither speci- mens nor localities were cited, and both synonyms given by Gray have been shown by Seemann (Fl. Vit. 123) to belong to other genera altogether. The U. S. Exploring Expedition specimens and a collection by Harvey labeled by Gray are the only means of positive identification of the species. Duplicates of these that I have seen in the herbarium of the New York Botanical Garden are certainly R, cochinchinensis. Seemann (I.e.) con- sidered these identical with Stylocoryne racemosa Cav. which Gray defi- nitely excluded from his species. Merrill (I.e.) considers S. racemosa Cav., of the Philippines, to be a synonym of R. cochinchinensis. This seems to add the weight of Seemann's opinion to my reduction of R. cofeoides to R. cochinchinensis . The range of the species in the Pacific extends from the Society Islands westward through most of the major island groups to Malaysia, and be- yond through tropical Asia. It has been collected from most of the high island groups including the Societies, Samoa, Wallis, Fiji, Tonga, New Hebrides, Solomons, Marianas, Carolines, Palau, etc. It does not occur in the Hawaiian Islands, and has not been collected in the Cook Islands or the Marquesas, and, strangely enough, apparently not in New Caledonia. It may here be reported from the island of Niuafou on the basis of a specimen in the Bishop Museum collected by T. Jaggar, Oct. 1930. Botany Dept., University of Pennsylvania Philadelphia, Pa. 1 4- r»* r r ... ji A, I I i a. X i. [Extracted from The Jodbnal of Botany, August, 1938.] TWO QUEENSLAND IXORAS. By F. Raymond Fosbbbo. During work on the Rubiaceae collected by the Maugarovan Paixarno 1q' ?r,W"^''""l<'^ Fo.s berg, Bishop Mus. Occ. l-ap. xui. no. 19, 1937) the question arose of which of the two speces warned Ixom triflora had priority. Both were said to have been pubhshed m 1866. The fascicle of Seemann's ' Flora Vitiensis ,n which Ixom tnflora (Forst.) Seem, was published «„n^w'^P7' ^' '-'^^ I''q»'--y,^t Kew Suited in the'^^nJorma tion that volume lu. of Bentham's ' Flora Australiensis,' in which 234 THE JOURNAL OF BOTANY Ixora triflora R. Br. was published, though dated 1866, really did not appear until January 5, 1867, givmg Seemann's species clear priority. Since Ixora triflora R. Br. is closely related to other species of Ixora sect. Phyleilema I thought it best, before renaming it, to examine any available material to make certain that it is distinct. Several sheets were borrowed from the herbaria of the Botanic Garden, Brisbane, the Arnold Arboretum, and the Bishop Museum, Honolulu. Thanks are here expressed to Dr. C. T. White, Dr. E. D. Merrill, and Mr. E. H. Bryan, in charge of these collections respectively, for the privilege of examining this material. In all, three collections were examined, with several sheets of two of them, which had been credited to this species. These specimens were found to represent two entirely distinct species, the /. triflora of R. Brown, here renamed /. qiteemlaTulica, and another species so different that it is a matter of some doubt whether it is correctly placed m the genus Ixora. Original descriptions of both are presented below to faciHtate comparison. Ixora queenslandica Fosberg, nom. no v. Ixora triflora R. Br. in Benth. Fl. Austr. iii. 416, Jan. 5, 1867 (in part). Non Ixora triflora (Forst.) Seem. Fl. Vit. 133, April 2, 1866. Small tree, branchlets glabrous, slender, intemodes up to 2-5 cm. long ; leaves obovate to elliptic, blunt-obtuse at apex, cuneate at base, up to 7 cm. long and 3-5 cm. wide, thin-coriaceous, glabrous, petiole 4-6 mm. long ; stipules only shghtly connate, ovate, firm but with a noticeable thin wing at each side, strongly carinate toward the apex, not so near the base, beak very strong and slightly incurved, sharp or blunt, the whole stipule up to 4 mm. long, caducous ; cyme of 3 sessile or subsessile flowers, the central one with a pedicel about 1 mm. long, peduncles up to 22 mm. long ; bracts almost orbicular, cordate, apex rounded, petiole less than 1 mm. long, but lower 5 mm. of mid-rib thickened (as in /. Setchellii), bracts up to 2 cm. long and wide ; hjrpanthium glabrous, 1-5 mm. long ; calyx about 1 mm. long, rather sharply but remotely denticulate, sUghtly ciUate ; corolla-tube 10-13 mm. long, or less in stunted cymes, 0-8 mm. thick, the four lobes up to 8 mm. long, 2 mm. wide, oblong-lanceolate, acute, somewhat contorted in bud ; anthers up to 4 mm. long, linear-lanceolate, acuminate, sagittate at base, attached in the sinuses of the corolla, filament less than 1-5 mm. long ; style filiform, up to 18 mm! long, exserted 4-5 mm., upper 1-5-2 mm. thickened and bifid. Moore (Joum. Bot. Ixiv. 216, 1926) gives the following information not available on the specimens before me : " corolla white ; fruit subglobular, 6x5 mm." Specimens seen : Queensland, Percy Is., Pine Island, scrub, March 1906, Tryon (Brisbane Herb. ; Am. Arb.) ; another TWO QUEENSLAND IXOBAS 235 specimen, fragmentary and without data, determined in F M Bailey 8 writing (sec. C. T. White) (Brisbane Herb.). Although I was unable to borrow the type material of Brown's species I thmk there is little doubt that the Tryon specTrTns are Identical with it, as they were compared by Moore (se^Joum Bot. Ixiv. 216) and considered identical. As originally described /. triftcyra R. Br. represented a con- Z^Mt Thi« f °'^*'"^* ^'^^ specimens of Diplospora Tc Sfran^th. '^"^ r' P^^"*"^ ^"^ ^y Spencer Moore m cit.), and the species as here renamed includes only /. triflora Ben^ham ^ ' ''''^ '"^ *^^ """^^^^ '^^'^ ^' pubhshed by It is difficult with the present lack of material of most of the species oUxora sect. Phyleihirm to make any suggestion as to the relationship of /. queer^luruUca with any of th! other species Certam similarities with I SeU^hellii Fosberg are probably merely the result of parallel development. When more complete collections are available from Melanesia and the Papuan area relationships in this section oi Ixora may become more obvious Among collections distributed by the Arnold Arboretum is a plant coHected by L. J. Brass in Queensland, labelled ^^ Ixora rlT ^' ^^i-^- ^'; ^' ^: "^^^ ^"^^^^^^ '^^' '^^ plant J^t represent a different variety. Open flowers and fruits are not present on the specimens, but careful dissection and examination ol the flower parts shows features possessed by no other Ixora known to me. There is some doubt that it even belongs in this genus, but m the absence of fruit it would be unwise to set un a new genus for it, and the aspect and most other characters are those of an Ixora. Ixora biflora Fosberg, sp. nov. Frutex, folia elliptica vel oblonga vel obovata acuminata cymatermmahs bracteatareducta ad flores aolitarias vel geminates' hypanthium glabrum tarde hirteUum, coroUa glabra 4-loba' antheres lata oblonga 1-2 mm. longa in alabastris dehiscentes * "Shrub about 4 ft. tall," branchlets cylindrical, glabrous woody almost to the tips, intemodes up to 3-5 cm. long fre' quently under 1 cm. on branchlets ; leaves oblong to elliptical or slightly obovate, acuminate, - thin and soft, paler on under surface, up to 9 cm. long and 4 cm. wide, glabrous, base con- tracted, petiole 6 mm. long ; stipules broadly ovate, not carinate except at apex which is prolonged into a sharp beak, the whole 4 mm. long : ultimate branchlets much condensed with imbricate bracts and their stipules, producing an appearance of termmal ieaty buds, from which the corollas project singly or in pairs between a pair of leaf-like bracts, ovate, acuminate at Lex 1-1-5 cm. long, up to 8 mm. wide, obtuse at ba.se, with a slight winged petjole Jess than 1 mm. long, the imbricate bracts sur- IRREGULAR PAGINATION 236 THE JOTTRNAIi OF BOTANY rounding the flowers thin, pale green, elUptical, sharply acuminate, about 1 cm. long, 5 mm. wide ; flowers terminal, single or paired, sessile ; hypanthium about 1-5 mm long, glabrous at first, but after shedding of corolla becoming wooUy-hirtellous ; calyx about 1-5 mm. long, membranous, cup-shaped, irregularly lobed, minutely fimbriate-ciliate, otherwise glabrous ; corolla white, glabrous, not known except from buds almost ready to open, typically Ixora-\ike but not or only slightly twisted, evidently nodding in bud, tube up to 14 mm. long, enlarged slightly at base and slightly funnel-form at throat, 0-8 mm. thick, lobes 6 mm. long, imbricate in bud, but not or only slightly contorted ; anthers broadly oblong, 1-2 mm. long, attached in the sinuses of the corolla, dehiscing, evidently introrsely, while the bud is still closed, showering the inside with pollen ; style completely undeveloped in the buds at this stage, represented only by a small conical prominence on the disk, but evidently deciduous, as it is represented only by a circular scar on an ovary which had just shed its corolla, the disk on this a raised somewhat irregular ring ; ovary of buds too young to make out anything with certainty, even with thin sections, the older ones attacked and destroyed internally by insects ; fruits unknown. Specimens seen : North Queensland, slopes of Mt. Demi, rain-forests, alt. 2000 ft., Feb. 6, 1932, L. J. Brass 80 (type in Brisbane Herb., duplicates in Arnold Arb. and Bishop Mus.). The unusual feature of the ovary becoming woolly after the shedding of the corolla is evident from the presence of glabrous young ovaries and woolly older ones on the Bishop Museum specimen. The only plant that I can find mentioned which could be related at all closely to this is a little-known species from New Guinea, Ixora coffeoides Valeton, of which only the fruit and very young buds are known. It is excluded from Ixora by Bremekamp (Bull. Jard. Bot. Buitenz. ser. iii. xiv. 348, 1937), who suggests that it may come nearer to Diplospora. The latter genus, however. so far as I know, never has a sclerified endocarp, which Ixora coffeoides has, in common with other species of Ixora. The lack of a concavity on the inner side of the pyrene is the most obvious difference which would exclude it from Ixora. Features of Ixora biflora different from other Ixora species are the short and broad anthers and the extreme retardation of development of the style. A delayed opening of the style-lobes to expose the stigmatic surfaces is a feature of the whole tribe Ixoreae. Also there is a vague suggestion in the sections of the very young ovaries available of more than one ovule in a cell, but the material is too young for the point to be determined with certainty. It would seem best to retain these two species in Ixora until flowers of /. coffeoides and fruits of /. biflora are available. Then their relationship and generic afiinities may be determined more TWO QinCENSLAND IXQRAS 237 satisfactorily. At present neither can be placed in any of Bremekamp's subgenera. The sheet of/, biflora in the Brisbane herbarium is designated the type, as it has the most flowers, though the more mature ovaries are on that in the Bishop Museum. The sheets in the Bishop Museum and the Arnold Arboretum may be considered isotypes. Botany Department, University of Pennsylvania Philadelphia, Pa., U.S.A. m * -v [Extracted fr(m The Journal of Botany, September, 1938.] t ADDITIONAL NOTE ON QUEENSLAND IXORAS 277 IxoRA BiFLORA Fosberg var. Fleckeri Fosberg, var. nov. Folia oblongo-lanceolata acuminata, hypanthium persistens glabra, calix valde 4-dentatus. Differing from var. typica in having oblong-lanceolate acu- minate leaves up to 9 cm. long and 25 cm. wide ; hypanthium not becoming hirtellous, but remaining glabrous, calyx margin not fimbriate-ciliate, but glabrous and prominently 4-dentate ; fruit maturing later, those on specimen young and green, about the size of small peas. Specimen seen : Queensland, Mossman Gorge, jungle at intake, June 20, 1937, Flecker 3521 (type, Brisbane Herb.). ADDITIONAL NOTE ON QUEENSLAND IXORAS. By F. R. Fosberg. After the manuscript of the article on " Two Queensland Ixoras " (Journ. Bot. Ixxvi. 233-237, 1938) was submitted for publication, Mr. C. T. White was kind enough to send for my examination two additional recent collections of Ixora biflora with the suggestion that one of them might represent a new variety. This turned out to be so, and the variety Fleckeri is described below and also a varietal name is given to the typical form. One of the collections, Mossman Gorge, Queensland, June 20, 1937, Flecker 3509 seems identical with the type of the species, and, fortunately, bears a mature fruit, from which the following may be added to the description of the species : fruit " bright scarlet," somewhat flattened, slightly grooved on the flat sides, bearing a large calyx scar at apex, subsessile, 11 mm. high, 13 mm. wide, 6-7 mm. thick when dry, surface when dry ruguiose-papillose, subtended by two or more persistent bracts, these not, or only slightly, accrescent. IxoRA BIFLORA Fosbcrg var. typica Fosberg, nom. nov. This is the typical form of the species as described on p. 235. *M^ IRREGULAKPAGl SSfimSI .i- ,^ w« -* t m.'*n T J -• » — '% Reprinted from the American Journal of Botany, Vol. 25, No. 7, 511-522, July. 1938 Printed in U. S. A. '-• ♦ - s r V U '^ THE EFFECT OF LIGHT ON THE FRUITING OF ]\IYXOMYCETES ^ W illiam D. Gray ALTHOUfJH THE myxomycetcs are unique in that they unite in themselves a nunil)er of characteristics of both animals and i)lants, they have l)een investi- gated hy relatively few hiolo2;ists. As a result of this neglect there are many gaps in our knowledge of their simpler physiological processes, and a search through the literature reveals the fact that little is known of them excei)t i)erhaps the taxonomy within the group. This situation cannot he attributed to a lack of in- terest or inclination on the part of biologists, al- though it might l)e explained to some extent by the rather micertain i)osition of the myxomycetes. One reason for so little being known is probably the fact that only a few s])ecies have been cultured. Our present knowledge of the })iology of this group has been obtained from the few forms which can !)e easily cultured in the lal)oratory and from observa- tions made on species collected in their vegetative phase. The limitations of this latter procedure are obvious: No matter how assiduous a worker may l)e, there are man\' species which lie may never see in the vegetative state; in fact, the plasmodia of only about one-third of the described species have ever been seen and recorded. Tlie problem that imme- diately confronts u-^ then is that of culturing species of myxomycetes which have never been grown in the laboratory. 1 "Received for ptiblicntion AprM 25. 19''S. The wri+er is indobtod to Dr. Harlan H. York and Dr. Conway Zirkle for helpful criticisms in the preparation of the manuscript. Certain species have been cultivated successfully, but these form a very small percentage of the total. P/njsanun poli/ccphalum 8chw. can be most satisfac- torily cultured at i)resent. Howard (Wm) first suc- cessfully cultured this species in the laboratory, and further imi)rovements by Camp (lO.'iO) made the cultivation of P. vohjcephalum nuich simpler than that of most fungi. Skupienski (1920, 1928) has cul- tured both Di(Ujmhim difjorme (Pers.) Dul)y and D. wqripes (Link.) Fr. on jwtato and carrot decoction ngar, and Lister (1S88) has descriljcd the cultivation of Badhnmla iitricularis (Bull.) Berk, on the sporo- phores of various agarics. The limitation of Lister's method is olniously the fact that such si)oroi)hores are not available at all times of the year. Klebs (19()0) cultured D. difjonne and D. ci]usum (synonym of D. squamidosmn (Alb. & Schw.) Fr.), and Cay- ley (1929) cultured 1). nigripcs and I), xanthop'us (Ditm.) Fr., in addition to the two species of Didij- mlum used by Klebs, on Knop agar. A few other forms have also been cultured, l)ut, considering the fact that there are al)out four hundred sj^ecies of myxomycetes, it i> clear that comparatively few have been grown in the laboratory. .Many attempts of the writer to cultivate various species (largely of the order Physa rales) from living j)lasmodia, coilecred in their natural habitats, have generally failed because of the rapid formation of fruiting bodies by the plasmodia when brought into the laboratory, despite efforts to simulate conditions 512 AMERICAN JOURNAL OF BOTANY [Vol. 25, July, 19381 GRAY — FRUITING OF MYXOMYCETES under which they were growing in nature. Such re.sults lead to the .susse.stion, already advanced hv Cayley (1929) and Seifriz and Russell (imV), that there is a definite rhythmical fruiting exhibited l)y myxoniycetes. Cayley found that Didnmium difforme pos.«jesses a nuich shorter ^•esetative jiha.se than either D. jiigripes or D. squamulosum hut stated that the factors causing plasmodia to form fruiting l)odies are "still a complete mystery" (p. 2.U). If such rhyth- mical fruiting does exist, it is highly possible that the species ol)served by the writer may i)osse.ss rhythms of relatively short duration in so far as the vegeta- tive phase is concerned. I'nless this rhythm is so innate as to be unalterable, it should be possible, by controlling various environmental factors, to vary the rhythm one way or the other so that plasmodia would vegetate for a longer or shorter i)eriod than normal. Thus it is entirely possible that a larger number of species might be brought into and maintained in cultivation if methods of prolonging the vegetative phase might be found. Of course, the prol)lem of nutrition must also be considered, but evTn if the nutritional requirements of a sjiecies be known, it would be an extremely arduous task to maintain it in culture if it remained in the vegetative state for only a short period. Undoul)tedly, a Plasmodium about to form fruiting l)odies differs fundamentally from one which is beginning its vegetative existence, even though the exact nature of the difference is not known. If the change from vegetative to fruiting ])nase is due to chemical factors, physical factors, or a combination of ])oth, it should be possible to hasten or delay it. As mentioned above, the factor of nutri- tional requirements is an important one, and no attempt is made to minimize it; however, this can undoubtedly be ascertained by experimentation with various species. The pre.sent series of exj^eriments are concerned with the influence of light upon the length of time required to develop fruiting bodies in various si)ecies of myxoniycetes. There is a po.-^sibility that these findings may have a bearing upon the culture of other si)ecies as well as contribute to our knowledge of the physiology of the groiiji. It is quite probable that temperature, moisture, texture of .substratum, nutri- ent materials, and degree of acidity or alkalinity of the medium are also important, but in the i)resent work the influence of light alone is described. Almost no investigations have been made of the effects of light upon myxomycete plasmodia. The mosi; extensive work in this field was that Baranetzki (1876), who, however, limited himself almost entirely to studies of the heliotroiiic responi^es of plasmodia of Fuliqo septicn (L.) Weber and the interrelation- ships of heliotropic and geotropic responses. The remaining references in the literature are merely scattered observations made by various workers in the course of other stuflies; these observations will be referred to in the discussions of the ex])eriments conducted by the writer. 513 Materials and methods.— With the exception of ])lasmodia of Phymrum polijcrphalum Schw., which were deriveil from sclerotia, i)lasmo(lia of all species cultured in the laboratory were obtained from si)ore cultures. Since there is not enough surface moisture on agar slants to insure good spore germination, cul- tures were made by adding 1 cc. of sterile distilled water to each slant; sjiores were then sown directly m the water. Such cultures were i)lace(l in the dark, as various workers have shown that light destroys swarm cells. When i)lasmo(lia developed, they were maintained in culture l)y means of plasmodial trans- I)lants to fresh nutrient media. Three culture meth- ods were used: (1) the moist chamber method develoi^ed by Camj) (fig. 7), (2) Petri i)late culture (fig. U), and (.S) beaker culture (fig. <)). With P. poh/rcp/taltnn, all three methods were employed, whereas only Petri dish cultures were made for /^ tenerum Rex, P. comprcssum Alb. & Schw., Didy- mnnn xanthopus (I)itm.), Fr., Hemitrichia vcsparimn (Patsch) Macbr., Fidigo scptica (L.) Weber, and Ij'ornrpus Jraqilis (Dicks.) Rost. In moist chamber cultures of P. polyccphahnn, the Plasmodia were fed every twenty-four hours by sprinkling ground-up rolled oats directlv on them; in l)oth beaker and I'etri dish cultures of this .same species, three i)er cent rolled oat agar was used. P. teiwnnn, P.couipressum, F. scptica, and H.vcsparimn were grown on two and five-tenths per cent corn de- coction agar, adjusted to pll 5.4-5.0; D. xmithopus was grown on five per cent carrot decoction agar; L. frmidis was grown on both corn decoction agar and three per cent hay infusion, one i)er cent dex- trose agar. In exjieriments involving light intensity, the cul- tures were placed under light bulbs of various watt- age, the bulbs being suspended three feet above the open top boxes in which the cultures were placed. All experiments involving such artificial light were conducted in a room in which the temperature varied from 23' to 2r,T^ Experiments involving direct sunlight were conducted in a greenhouse where the temperature remained at 21X\ in the daytime, drop- I)mg to as low as 15C. at night. Species herein considered are divided into three groups, based on plasmodial color: yellow pigmented non-pigmented. and variable plasmodial tyjie"-, which ranged from white to cream-colored and various light shades of yellow. YKLLOW-PIfiMENTKD PLASMODIAL TYPES.— This groUp includes the species whose plasmodia are normally yellow at all times, except for the period immediately previoiH to the formation of fruiting bodies. In this frroup are includcl Phymrvm voh/crnhnlum Schw P. tcncrum Rex.. Fjdinn scptica (L.) Weber, and Leo- carn,is jraqilis fDicks.) Rost. Phtfsanmi pnli/ccphalum Schw.— It was nossib'e to »>> r\*^r> "i I >l I J 1 ' til J 1 ^ * ^oi I jiii iji ' uiiii iiiis species tbnn with any of the others, because of the ease with which it can be cultured, the rapidity of its growth, and the .subsequent formation of relatively large f 4 f I A fi 4 ■•'*» amounts of protoplasm. As has been previously mentioned, all three tyi)es of culture methods were utilized, but the ma.iority of experiments was con- ducted with moist chamber cultures which were fed equal amounts (0.5 gm.) of inilverized rolled oats everj' twenty-four hours. It was soon discovered that the jjlasmodia could not be kept in direct sun- light, although whether the destruction was due to the intensity or (luality of the light is not yet known; possi})ly both factors were concerned. Preliminary exi)eriments revealed that, excei^t in a few cases, i)lasmo(lial cultures of the beaker or Petri dish tyi)e would not form fruiting bodies in the com- l)lete absence of light if they were inoculated from stock cultures that had been maintained in the dark. This was also found to be true for moist chamber cultures that were fed every twenty-four hours. Several hundred cultures of this type were set up in the dark at various times and only three or four fruited, while check cultures grown in artificial or dilTuse natural light nearly always formed s])orangia. Tho.-^e few cultures that did fruit in the dark ])assed a considerable time (15 days or over) in the vege- tative stage. In cultures inoculated from stock pla.s- modia maintained in strong natural light, a delayed effect was frequenth- shown, for cultures of this tyi)e often fruited in the comjilete al)sence of light. Sixty cultures were inoculated from stock plasmodia which had l)een ke])t in the light; of these, seventeen (2S ]:er cent) i)roduced sporangia in the dark. In order to overcome any delayed effect, all sulxsequent experiments were conducted with cultures inoculated from stock ])lasmodia maintained in the dark. For the sake of uniformity, five-day old stock cultures were arbitrarily chosen as sources of inocula. Plasmodia grown in light were always a paler yellow than those kejit in the dark. Hofmeister (1807, \\ 21) stated that plasmodia of Fidiqo scptica (L.) Weber which were kei)t in the dark were paler than those grown in the light; however, Baranetzki (1870), working with the same sjiecies, stated that the reverse was true. The observations of the writer on yellow-])igmented ])lasmodia tend to confirm the findings of Baranetzki rather than those of Hof- meister. It was also noted in P. pohjccphnhnn that jilasmodia grown in the dark showed a tendency to sjiread, whereas the ]irotoi)lasm of jilasmodia grown in light showed a decided tendency to form clumps and large, swollen jilasmodial " veins." These two ty]ies of plasmodial configurations are shown in figures 4 and 5. The finding that light was nearly always essential for the fru t'ug of this spec'es, under the conditions outlinefl, naturally suggested that the intensity of light misrht condition the time of fructification, and it WIS sulisequentlv found that a definite correlation exists between light intensity and diration of vesre- tative phase. Over a period of two years moist chamber cultures were grown in open top boxes unripr 00. 100. 150. 200. and 400 (Mvo ?00's) watt bulbs, suspended three feet above the cultures, and it was fountl that under each type of light (with the excei)tion of the most intense) a rather definite fruit- ing rhythm was exhibited. The following list sum- marizes these data: Cultures under 60 watt light fniKod in 12 days 100 " " " "11 150 " " " '' 10 200 " " " •' 9 « u ii 400 " &-9 There were some variations from the above figures, but, in the great majority of cases, the fruiting occurred with .such regularity that the day of spor- angium formation of a given plasmodial culture could be correctly i)re(licred on the day it was started. The analyses of lights from the various types of bulbs used in the experiments show that variations in the length of time spent in the vegetative phase were due to the factor of intensity rather than to differ- ences in (luality of the various lights. Analyses of the types of bulbs used are shown in table 1.- Tabi.k 1. Analyses of electric Uyht bulbs used in light inlcn.sily studtes. Lamp size (watts) A|)i)roxi- nKif(> lunien.s Porccntage of total light Violet Orange and blue Green Yellow and red 60 100 150 200 760 1530 2535 3400 2.25 2.3 1.9 2.5 40.25 40.7 41.0 41.5 27.5 27.4 27.0 27.2 30.0 29.6 30.1 28.8 In discussing various features of the plasmodial behavior of Didymium difforme (Pers.) Duby, Sku- pienski (PI2S, p. ;^2!)) made the statement: ''. . . . if one submits a well-exi)anded pla.>or mnt in the arpf^n and yellow, increasing in the orange and red. The light transmission of the yellow plate was roughly similar to that of the red plate; however, it trans- mitted a greater percentage of light in the green, yellow, and orange bands. Six cultures were used in the preliminary experi- ment, the results of which are given in table 3. This experiment showed that the shorter wave lengths of the visible spectrum stinuilate fruiting, while the longer wave lengths either retard or prevent fruiting. In later experiments in which Wratten Filters were u.^ed, only one, whose transmission analysis is shown m figure 1, induced fruiting. This would tend to supi)()rt the view that the shorter wave lengths are necessary for fruiting. On the other hancC in the longer wave lengths of the visible spectrum or in mfra-red rays, the i)lasmo(lia never fruited under conditions of continuous exposure. .1^ 3 100^ 200 300 400 V.'avo Length 500 milimlcrons 600 700 Fig. 1. Light fransini.ssion analvsi.s of golatin filter under which fod cultuio« of P. polyeephalum formed iruiting bodio.s. A number of investigators have maintained that the fruiting of myxomycetes is due solely to the de- l)lotion of nutrition. Camp (1937) has shown that Plasmodia of P. polyeephalum may be induced to fruit by starvation. The jjresent studv indicates that cultures which are not fed will spend less time in the vegetative phase than tho.^e which are fed but this will scarcely exi)lain why i)lasmodia will Table 4. The effect of li.ht intensity on fruiting in ynfrd cultures of Physarum polyeephalum. La in I ) (watl.s) Nunibor of culturo.s 60 \V 8 100 W 5 150 W 21 400 W 21 Avorajrolonjithof fiine roquirod for fruiting 87 hours 81 " 78 " 68 " often fruit when they are supplied with abundant food materia s In order to determine whether or not light would have any effect on plasmodia to which no nutrient was .'supplied, a number of cultures were «et up. As in previous t^AiR-jiinonts, nve-dav old stock cultures, which had been fed twentv-four hours pre- viously, were used as .sources of inocula. With few exceptions the light reactions were the same as thosQ I I « i I 'V July. 1938] GRAY — FRUITING OF MYXOMYCETES 515 Placed in light: Remains of stock culture actively vegetating Aug* 8 (\) Fruited July 26 (4 days) (5) Fruited July 27 (5 days) — (3) Fruited July 28 (6 days) (4) Actively vegetating Aug, 8 (?) Placed In light Aug. 5 (J) Fruited Aug. 5 (2 days) ©Divided and placed In light July 28 6^ Fruited July 30 (2 days) ffi) Fruited Aug. 1 (4 days) PHYSARUM TENERUM REX. Fig. 2. Diagrammatic ropro.sontation of tho effect of diffu.so natural lijiht upon the fruiting of P. tenerum. .shown by fed cultures. A higher jiercentage of unfed cultures (inoculated from .stock i)la.smodia maintained in the dark) formed sjiorangia in the complete ab- sence of light than did fed cultures of the .same type, Kesults of light intensity studies conducted with cultures of this type are summarized in table 4. Exi)eriments concerned with exposing unfed cul- tures to various wave lengths of light did not produce results which were in agreement with the results obtained when fed cultures were u.sed. Sporadic fruiting occurred in such cultures, regardless of the light used, so the inference is that the influence of .starvation is much stronger than any possible inhibi- tory effect of light in the longer wave l)ands. Phi/sarutn tenerum Hex. — This species has the same type of bright yellow ])lasm()dium as P. polyeephalum, but as yet has not been induced to form large ma.sses of i)rot()i)lasm. Like P. polycepltalum, i)lasmodia of P. tenerum grown in the light were a paler shade of yellow than those grown in the dark. A clumping of i)Iasmo(lia occurred in the light but to a lesser degree than in !\ polycepltalum. Numerous jilasmodial cultures were expo.sed to direct .sunlight in the greenhouse, and after forty- eight hours all had lost their color. When the.se cultures were i>laced in the dark, they failed to revive, thus showing that jila.smodia of P. tenerum, like tho.se of P. polyeephalum, are killed by direct .sunlight. In no case did i)lasmodial cultures fruit in the com- l)lete ab.sence of light; over two hundred cultures were kept in the dark, and all remained in the active vegetative state as long as the nutrient agar re- mained moist. Many i)lasmodia remained active for as long as sixty days, and one persisted for seventy- six. Due to the fact that it needs trans])lanting so infrequently, this s])ecies is excellently adajjted for lal)oratory cultivation, althouuh, as mentioned above, »i V.v/V., iiwv 2i4ijiiiii V .11 u 1 ,1 _ Vjkiil 41 li Live \Jk l^iUlAJ'" plasm as /-*. polyccphaluw. Preliminary experiments indicate that it can probably be adai)ted to moist chamber culture, using i)ulverizcd rolled oats as nutrient. In diffuse natural light in the laboratory, different lengths of time were required for the formation of fruiting bodies by various i)la. hours later. X 3. — F'ig. 13. Petri dish culture of D. xantlio/ms on carrot agar. Natural size. 516 grown as large as 1 cm. in diameter (fig. 0). Bara- netzki (1870) mentioned the clumping of i)lasmo(lia of F. septica exposed to light but failed to mention the i)ro(luction of large plasmodial swellings such as tho.-^e just described. Plasmodial cultures of F. sep- tica on corn decoction agar were killed within twenty- four hours when exi)o.^ed to direct sunlight. l^LA>i,ll.> fj itfi jiiiyiLio \ l_yiL*k.';. ; Ai,uct. xik ^u kui us u source of small plasmodia is concerned, this species is an excellent yellow-pigmented type for laboratory cultivation. Spores germinate readily, and plasmodia can generally be obtained in ten days or less from group herein discusi^ed change color l)efore the fruit- ing bodies mature, but the change occurs after spor- angial delimitation has begun. Physarum compressum Alb. & Schw. — The pearly- white Plasmodium of this sjiecies was obtained easily from spore sowings on corn decoction agar. Plas- modia formed typical sporangia in the absence of iiglit aijuiii ^lA *4ii_>a iiiici iiifv »cic iijigu t'liOugiJ lU be visible without the aid of a lens. Attempts to maintain the species in culture by means of plas- modial transplants were, on the whole, unsuccessful. Disturbing the plasmodia markedly inhibits growth July, 1938] GRAY — FlU ITINC OF MVXOMYCKTKS oi: V\l'<"anj..a ol /. />o..../... x 2.-Fig. 9. lioukcr cultur. of />. ;>./ ral .% 1 4 1 4 IMasiiiodia of /''. scptica show the plienoincnon of (•hinij)iny; in a i('inarkal)Ie dciircc when oxposed to hjilit. For this study, ])':i'vv> of wood, on which active l)las!M()dia were spread, were placed in moist cliamher culture, and in a few hours most of the jjlasniodia had moved to the moist pajx'r on which the j)ieces of wood were ])!aced. Wlien such i)lasmodia were exposed to liome of tliem had sj)()re sowintis on e'.ther corn decoction airar or hay inlusion airar. 'iliese plasmodia need tran>i)lantin«!; infre(|uently (e\.ry 2 or 'A week^i and hence are easily maintained. No fruitinu" has been obtained a.s >-et, and the su^ucstion is made that neither of the two media used i^ adapted for it- cultivation. Like the yellow-])iiiinciiied t>pes previously discussed, the l)lasinodia of L. jr/ufllls clumi) and are bleached when exjjosed to liuht .md ;ire killed when exi)o>ed to direct sunliuht. No.N-lMOMKX III) I'LASMODIAL TVI'llS. — In this <»;r()up are ])laced tho-c -pecies in which the plasmodia are t\i)ically water\- or i)ear!y-white. All species in the ¥iiz. 10 13.— Fiir. 10. IMasinodiuni of S. jusm. x 3. — Fi<:. 11. Same :i> fit:, id - O'-j linnr> lairr. ■ ;',._ Fl-r. 12. Siinic a.s fiji. 10 — Vj, Ijoins later. X 3. — Fif;. 13. Petri d.-h culture of I), xdiithiiinis on c.iirot a k 11 ,^l> 111 I il."> it source of small ])la-modia is concerned, this speeie.s is an excellent yellow-piiimented tyj)e for laboratory cultivation. Spore- germinate readily, and ])lasmo(ha can generally Ije ol)tained in ten days or less from gHMip lierein di-cu-sed chantre color before the fruil- iny; bodies mattnc, biit the change occurs after spor- antrial delimitation lias betrun. Phiisdnnn rotnprcssinn Alb. i\: Schw. — The jiearly- white Plasmodium of thi> species was obtained easily from sj)ore sowings on corn decoction airar. I'la.s- modia formed t\]»ical sporangia in the ab.-ence of i;,,l,* ,i.,.,,t .;,. I , . . «'*r,». ♦I,,,,. ,,.„^,, I .„„„ „,,,,,,„u ♦_ ii„ijl ilinHU ."-i-l 11. (>.^ .11 111 i ill _\ U I 1 I J.MUl I l|t>tl^ll I i) be visible without the aid of a len>. Attem|)ts to maintain the species in culture by means of plas- modial transplant- were, on the whole, unsuccessful. Disturbing the plasmodia markedly inhibits growth INTENTIONAL SECOND EXPOSURE 518 AMEKICAN JOCK.NAL OF BOTANY and fnrt.Hg, nnci cultures nia.lc by means of nlas- mod.m uan-planls nearly ai«a)s U.ed wuhin twentv- lour liou.s, a.tnougii m a lew cases the lile c\-cle w^* con.),lo,ca. Lal.er the plasum.hun. of l\ conLe.sut IS cxcept.cnallv- dehcate or eise corn deeoct.on a-'ur IS not uie most su.tawe meduini for its cultivation Jlo«ever, atteu.pts to grow tins species on various otlier types ot ined.a j-.cKled no better results nim-ion ,„ita„. J'ers.-Only one observation was mauc on ,l,e hght relationshi],s of this spec.es \ small, white, net-like i.lasiiiocl.um, found under the bark ol a uecay.ng log of I'runus sentum, was trans- ported to the m„oratory, kept in complete darkness, and, alter three days, lormed t>pical sporangia o ..«/"«.. ihe plasmod.uai received no light except 01 a lew nioiiionts when it was first discovered, so IS sale to mier that light is not j.rerequisite for tl e c„iii,,let,on ol its life cycle. Since the age of the l)la>iiioc ,111,1 was unknown, no definite length of vec^e- tativc jihase inav be set ° hall'or',l,n"l"'''r'!'" "'^''"""''^' "f npproximately Imc , ' 7"'«^l -^l-ecies in the genus Stemo,uts h. not jet been obser^■e.l, but in the majoritv of kno«n cases they are white. This is true or both ;*•■,'' ' • ^- ■ ""•' I'lasniodial behavior of which ].».,<. IMO- All conclusions concerning these species io ,e rr' 1 "" "'^*";',''""-^ ">^"le on Plasmodia col- 1- ■; ,: '"■"•. "^'["■•^'1 ''''l-ita.s, as there are no records to r;^' ''■:,?■'"" '"'''" "'i«""ll>- cultured, and lev I >:• '"","'' "" "'^'^ "'"ivation have been d0M>e, . J-rom ob.vervations made bv the writer it ■s evident that light is not neces..arv for fruttiU s.nce Plasmodia which arose on decayed wood in moi.::;; c lanibcrs were able to complete their life c^•cles in fhc absen,.e of light. Se,eral observations have also been ma.lo o„ plasmodia which were collected under the bark ol decaying logs and transported in ,la,k- noj., to . he laboratory; fruiting of .such plasmodia I >o,,uonlly occurreil i„ the complete absence of nh bi, '!l '" ? '"*■'■'*'■■'• '" "•'"<•'» "'« Plasmodia Mn,\ to establish a definite lengih of time for the eget. n-e ph.i.se: ho^-ever, data at han.l in.licate that i.lasmodia of N. f,,,r„ remain in the veget.-,ti\e stage somewhat longer than do pl.ismodia of S. ax:fer„. naLTIr i'!'^''''''^ *'*""•» H"'t— In n recent paper (.ray, 1!W, ) the writer describerl the develop- "Z "^.""^;'"'''i'-' f™'" "■'"•<- Plasmodium to mature ^Por.uigi.i. The time re(|uired for completion of fruit- ing m the specimen described was twenlv-three hours. Anolhor such observation has been .nnde, and the mimrcd time was fifteen hours. This variation in t.me was probabl>- ,Iue to difference in ages of plns- modi.a, a„,l .s,„ee i.la.sniodia of this .species are similar "ill be difficult to determine the normal length of r' V'"," '■'!'•' "'"■"" '" '"<" ^'fSPtative stage. . t,iphou!es aiiparently does not need light in order to frmf, since plasnio.lia have frequently ap- IVoI. 25, reared and eventually formed sporangia on moist I'.eces 01 uecayeu wood kept in the darlc. Uimproderma arc,jr,o„ema Host. -Spores of this si.eces may be germinated easily in e.ther tap- or ..stilled water, but as yet no plasmod.al development h.» been obta.ned on any of the artificial media used, i ..i.smoil.a ha^■e been obtained, however, by sowing spores 01, small pieces of n,oist, decayed wood kept / he dark ^porang,a have been obtained in this re .n",i!!n """'''"' ''^'^'"'^ °'' ''S'"- ''"t »o data are .nai able concerning the e.xact length of the ve"e- ,, 1 ltd" , i"'""'!"^:'"''' ^- "'^y'"'"^"^'^ possesses Jil.ismo, ha which inhabit the interior of lecayin" »;ood; hence the age of plasmodia cannot be a cu ! ately determined. Since m several instances s nial C^: ;';■' '"'?"•''" "'■ •■- ■^•-■- -- on ;;o t //c ,W.;° " "'■ "" ^"''''='*'^"« ^f'-'S" '^ concerned. Hemdiichia vespanum (Batsch) Macbr.-The „las- mo.hu,,, 01 this species is li.sted in most taxo,, , e ork, a, being deep red or purj.le re,l, but the writer 1 as never observed anything but white plasmo.ha unng the active vegetative phase. It is true that the color becoiues red before the fruiting bodies am n.atu,;e, but this color change occurs after spo an.^al < ehniitation has begun an.l not while plasn o.ha Ire l.:r;''t^.„.iL*,! -«-'-''.'- PlasniLha! July, 1938] GRAY — FRUITING OF MYXOMYCETES 519 H;r::.irof 'v^t:'"' S'"'^^ ^^i^'^^^z Pha.se ,s due simply to the engulfiug of differeU colored particles of the .substratum. Xatur.al ? Plasmoihum should engulf red particles it wo^i for a "ne, appear nvl. This type of change in pl.l " o'lial color ,s exemplified bv the photograph of -. 1 .;s„,od,iii„ of /■■. .,,,tiea (fig. 6);'at L „p he ,,' 1 'l .'"'"";'^''--""»- ''"« to the engulfiu, of Lmv, .-■n,.,ll, b,„wn fnigments of decaye.l woo.l on which it ".as growing before it was allowed to .spread to the Jiaper in the moist chamber Jf ve.p„ram nee.ls no light in or.Ier to form spor- angi.,. Numerous instances have occurred in which Plasmodia have come to the surfaces of pieces of le ca.ve,! woo,l, wluch had iK-en place.l in the .lark .and h re ormed sporangia. Sporangia of H. .c^V, ,4 a^e also been fo.ind on the walls and ceili„.r, of :w ste;;!:"^ '"^ °"'^ '-'^ »- - -:. Si>ore sowings in tubes of corn .lecoction agar have .^.e,led peary-wlnte plasntodia which grow rapid v are easily maintained through pla.si^odial an l e nic ~n 1 ■"' "' "'^'""- f""'i"= «•■■>■' very en, tic, -o no .lata concerning rhvthm or effect of mdi";,: ';':','L'i ™, ';:;i,^^;r^ - /■•'■- p'--'o.iium one sporangium was produced by a .small bit of r,ut mo.l>um and then was engulfed and .lest roved Imtt "^■cihately by the rcnaining pla.smodiali i s ( •im-^ ^Mf /W ^ yet the factors inducing fruiting in H. vespanum in this t3'i)e of culture are unknown. Hemitrichia spp. — Except for the species just dis- cussed, no other si)ecies of the genus Hemitrichia were grown in the hd)oratory. However, several naturally occurring plasmodia of H. clavata (Pers.) Rost. (fig. S) and H. stipitata (Massee) Alacbr. were ob.served to form sjiorangia in the complete ah.^ence of light. Both si)ecies have been found in nui.>;hroom houses where they had fruited in dark- ness. The writer has observed only white i)lasmodia of either species, although Lister (1925, p. 221) states that the i)lasmodium of //. clavata may be either watery-white or rose-red. Si)orangia of both species have been observed to pass through a rose-red stage, but this change of color occurs after sporangial de- limitation has started. made on twcnt\-i\vo plasmodia of this species which fruited in the dark. Variable plasmodial types. — One species, Didy- inium xanthopufi ( Ditm.) Fr., has been i)laced in this sei)arate group because of the varial)ility of its i)las- modial color. In the discu.^sions of various yellow- l)igmc'nte(l i)lasm()(lial types it was noted that the bright yellow color fades to a certain extent when l)lasmodia are ])l,i('ed in the light. Plasmodia of D. .vnutJiopus did not exhibit this tyi)e of variability, as any given i)la.>nu)(lium might vary from white, through various shades ol" creamy- or greeni.-^h-yellow to bright yellow, indei)en(lcntly of light conditions. For in- stance, two i)larry lojj; 1 lender bark of docay- inj? wild cherry log 3 Appeared on moist wood in dark chamber 7 Under bark of decay- ing wild cherry Ion 3 T^nd«T bark of decay- ing wild cherry log 4 Under bark of decay- ing wild cherry loji; Hours Time of collection Fniilinji conii)l(^le(l re piiied 2:00P.M. Sept. 8 8:00 A.M. Sept. 9 3:00P.M. Sept. 13 8:00 A.M. Sept. 14 3:00 P.M. Sept. 13 8:00 A.M. Sept. 15 10:00 A.M. Sept. 14 8:00 A.M. Sept. 15 1 :30 P.M. Sept. 15 11 :00 P.M. Sept. 15 1:30P.M. Sept. 15 8:00A.M. Sept. 16 2:00P.M. Sept. 17 8:00 A.M. Sept. 18 18 17 31 22 9.5 18.5 18 Trich>a pcrsimilis Karst. — The i)lasmodium of this species is typically watery-white and does not change color until the .separate si)orangia have taken form (fig. 8). After the sjmrangia have assumed the .' t'\X>' > . -^ - . as shown by previous workers, but coincided exactly with the periods of maxinmm intensity of illumination. This latter worker hypothecated that Dictijota, in adajiting itself to differing conditions at various localities, has acquired its habit of i)erio(licity in re- si)onse to different factors. Tahara (1000) described an identical i)erio(licity in the fortnightly liberation of oogonia of Sargai>sum enerve ; in a later work (1013), Tahara showed that the initial liberation in S. inicrvc, S. Horneri, anil ('ystopIiiiUuni sisynibrioides bore a relationshij) to the highest spring tide, but that the intervals between successive liberations varied, thus suggesting the i)ossible influence of other environmental factors (illumination?). Keeble (1010, J). 33) found that light was a very important factor in the egg-laying of Convoluta wscojjvnui.s. He found that alternation of six hours of exi)osure to light with eighteen hours of darkness was the optimum for egg-laying and noted that this was the same exposure received by C roscofjcnsis during spring tidal periods at which its eggs are laid habitually. In relation to the rhythmical fruiting of myxo- mycetes, Williams' view that i)eriodicity might be hereditary is sui)i)orte(l in i)art by the fact that cer- tain species exhibit longer vegetative i)hases than others. For exami)le, Phymrum tenerum, under a 100-watt light, required eighteen days for fruiting, whereas, P. polycephaluni, under identical conditions, re(|uired only eleven days. While rhythmical fruit- ing may i)ossil)ly be hereditary to a certain extent, it is certainly not so iimate as to be unalterable with respect to environmental factors. The writer has shown that in fed cultures of P. polycephaluni fruit- ing rh>thm may be changed by varying the intensity of illumination. Camp (1037) showed that by star- vation of Plasmodia the vegetative phase of this species could be markedly redticed. Exi)eriments of the writer, involving both starvation and intensity of illumination, .showed that both factors inter-reacted in influencing the time of fruiting. Differences in rhythms of species, then, tenil to support the view that rhythmical fruiting is hereditary; yet experi- ments on yellow-j)igmented plasmodia, involving lights of different intensities, show that this periodicity of fruiting may be modified. SUMMARY Our lack of knowledge of the biology of the myxo- mycetes is due to the fact that so few species have l)een cultured. The problem of inuncdiate interest is the devel{)i)iuent of methods for culturing many s])ecies under laboratory conditions. The effects of light upon the fruiting rhythms of four yellow-plasmodial types, ten non-i)igmented i)las- modial tyi)es, and one variable plasmotlial type (Didy- m'luni xanthopus) were investigated. It was found that, with the species stuilied, yellow-i)igniented tyi)es reciuire light in order to complete their life cycles, while non-jiigmented types and D. xanthopus fruit equally well in liglu or darkness. Under controlled conditions of temi)erature, light, and nutrient, fruiting periods of ])igmented types assumed great regularity. Under conditions of con- stant tenqieratiire and continuous illumination the lengths of the vegf^tative i)hase of Phymrum poly- cephaluni and P. tenerum are conditioned by the total amount of light received. Under conditions of inter- mittent illumination the vegetative i)hase of P. poly- cephaluni may be lengthened, although the total amount of light necessary for a cycle to be completed may be greatly reduced. Fed cultures of P. poly- cephalum, when i "laced in lights of various wave lengths, formed sporangia only when expo.«ed to the shorter wave lengths of the visible spectrum. The ])Iasmo(lia of all ye.Mow-pigmented tvpes clumped and faded when they were exposed to light. The laboratory cultivation of Physarum comprcs- sum. P. tenerum, ind Hemitrichia vesparium is re- ported for the first time. Dkpartmknt of Botany. UnIVKRSITY of i*KNNSYLVANIA, Philadelphia Pa. litp:raturk cited Baranktzki. .T. 1876. Influonce do la Imnicro sur los Plasmodia dcs Myxoiuvcct(>s. Mom. Soc, Nat, Sc. Xatiir. (niorboiirg 19: 321-360. BuANDZA. M. 1926. Sur la polychronro dps nivxo- mvct^tos vivant en ploin sclcil. Compt. Rend, Acad. Sci. Paris 182: 987-989. Camp. W. (J. 1936. A mothod of cultivating myxo- mvccto Plasmodia. Bull. Torroy. Bot. Club 63: 205- 210. . 1937. The fniitinj; of Phi/sunnn j)oJ}/rrj)haJum. ill i(>lation to nutrition. Amor. Jour. Bot. 24: 303- 303. Caylicy. Dorothy M. 1929. Some ob^orvations on in>'coto5!oa of tho frcniis Dith/minm. Trans. British Mvm!. Sor. 14: 227-'>4X. D.AVKNPOHT, C. B. 1897. Kxprrimental morpholo;rv. Vol. I. New York. Enolemann. T. W. 1879. Cbor Rrizuntr contrakti'on Protonlasmus duich plotzliche Beleuchtung, Arch. Gcs. Physiol. 19: 1-7. FiESKR, L. F, 1036. The chomisfiy of natural i)rodiicts roliitod to ph(>n,inthrene. New York, Gray. W. D. 1936. Notes on plasmodial behavior of Sfrnwnitk jusca Roth. Proc. Indiana Acad. Sci. 45: 74-76. . 1937. Observations on the methods of stipe- formation in Strmfnilli>< and Comntrichn. Proc. Indiana Acad. Sci. 46: 81-85. HoFMEii^TER. W. F. B. 1867. Lehre von dw Pflanzen- zello, Leipzig. Howard, F. L. 1931. Laboratory cultivation of myxo- mycete plasmodia. Amer. Jour. Bot. 18: 624-628. Hoyt, W. D. 1907. Periodicity in the jtioduction of the .sexual cells of Dirtyola dirhotoma. Bot. CJaz. 43: .•V«_,?92 Keehle. F. 1910. Plant animals. A study in symbiosis. University Pre.ss. Cambridjre. Klems. G. 1900. Zur Physiolonie der Forti)flanzen einijrer Pilze. III. Allg. Betrachtungen Pringsh. Jahrb. 35: 80-203. 522 AMERICAN JOURNAL OF BOTANY [Vol. 25, Lkwis. I. F. 1910. Periodicity in Dlclyola at Naples liot. Caz. 50: 59-64. LisTKu. A. 188S. Xotos on tho iilasmodium of Bad- Ikuu'ki and lire fi hi id. Annals Jiof. 2: 1-24. . 1925. Monojiiaph of tho niycolozoa. 3rd. odilion, rovis(-d by G. Li.sfor. Oxford University Press. LoKH. J. 1904. The control of heliotropic reactions in fresh water crustaceans by cheniicals. (^specially COa. Univ. (\ilifornia Puhl. Physiol, 2(1): 1-3. M.\sT. S. (). 1911. Li<;ht and the behavior of orjianisms. \e\v York. Skifhiz. W.. .AM) M. RrssKLL. 1936. The fruiting of niy.xoniycetes. New Phytol. 35: 472-478. , .AND M. Zktz.manx. 1935. A slinie-niould pig- n)enl as indicator of acidity. I^rotopla.snia 23: 175- 179. SKri'iKNSKi. F. X. 1920. Recherthes sur le cycle cvo- lutif de certains niyxoniycctes. Paris. 1928. liadania Jiio-cytolicze nad Didymimn diljonnc. Czesc pierwsza. (fitude bio-cytologique du Didymiam difjonnc. Premiere partie.) Acta. Soc. Bot. Poloniae 5: 255-336. . 1934. Sur I'existence de races physiologiques chez les Myxoniycctes. Annales de Protistologie 4. 121-132. SoLACOLU, T. 1932. Sur les matieres colorantes dc qucl- quos niyxoniycct(>s. Le liotaniste 24: 107-140. Stahl, K. 1884. Zur liiologie der Mvxonivccten. Zeitschr. Bot. 42: 146-155, 162-175, 187-191. Stobhk, H. 1908. Phototroi)ieerscheinungen bei Fulgi- den und anderen StofTen. Licbig's Annalen der Cheniie, Bd. 359: 1-48. Tahaka, M. 1909. On tho periodical liberation of tho oospheres in Sargassmn. Bot. Mag. Tokyo 23: 151- 153. . 1913. Oogonium lil^eration and the ombryogony of some fucaceous algae. Jour. Coll. Sci. Univ. Tokyo 32 (9) : 1-13. Williams, J. L. 1898. Reproduction in Dictyota dicho- loma. Annals Bot. 12: 559-560. . 1905. .Studi(>s in the Dictyotaceao. III. The ])eriodicity of the .^exual cells of Dictyota dichotuma. Annals Bot. 19: 531-560. ,- < ■^ • >• f- *' ' T A Note on Stemonitis fusca Roth William D. Gray, University of Pennsylvania The phenomenon of fairy-ring formation by certain fungi has long been known to mycologists, and numerous workers have reported and figured this type of sporophore arrangement among various agarics. Among the Myxomycetes, however, there have been few reports of fairy- ring formation. Macbride (1899, p. 35) reports such formations as being common to Physa/rum cinereum (Batsch) Pers., stating that the rings are small, measuring but a few inches in diameter. More recently, Poteat (1937) reports the finding of quite large fairy-rings of P. cinereum. This author states that he has found rings as large as fifteen feet in diameter. As far as can be deteratiined from available literature, these are the only records of the production of fairy-ring formations by the fruiting bodies of Myxomycetes. Of the several species of Stemonitis which occur in Indiana, perhaps the two most frequently encountered are Stemonitis fusca Roth and Stemonitis axifera (Bull.) Macbr. During the spring and summer months, except in exceptionally dry weather, sporangia of one or both of these species may be found in almost any shaded woodlot where moist, decaying twigs and logs occur. In the course of collecting trips through- out the southern part of the state, both species have been observed many times. Fairy-rings, formed by sporangia of S. fusca, have been noted \ V .« v_ -», }■* ■-* ""^ Mair^fication^r2/3.^'* °^ Stemonttw fusca Roth showing two incomplete fairy-rings. numerous times, but in no instance were sporangia of S. axifera nor any other species of Stemonitis found to be arranged in ring fashion. Such rings are quite small, never approaching the large size reported by Poteat for P. cinereum; the largest ring did not exceed three inches, (86) Reprinted without change of pages from the proceedings of the Indiana Academy of Science, Vol. 47: pages 86-87, 1938. " > I I .VJl.' a:mi:i{I('a\ jotrxal of botany rVol. 25, Lkwis. T. F. 1010. l*(ii()(licity in Dirlijohi at Naples. I'.ot. Ciz. .')(): oO 04. l.!>Ti;i{. A. ISSS. Xolcs oil 111,, iilasinodiuin of ]iac<'lo/.oa. 3iiol 2(1): 1-3. M\sT. S. <). liUl. Ja^lit and tlic b('lia\ ior of oiyiaiiisiiis. .\(\v \"ork. Si::fi{:/. W.. and M. I^sskm.. IfelJO. Thr fruit inc(«tcs. New IMiyloI. 3'): 472-47S. ■ . AN!) M. Zktzmwn. !<);)'). A slinic-iMould jtiy;- mciit a> indicator of acidilw ^Motoiilasnia 23: 175- 17U. Skii'Iknski. F. .\. 1020. i^'(•ll( rdics -c>l()!()y:i(jii(' du Uitlijniiinn (lijlonin. JMcniiiTe parlic.) Ada. Soc. liol. Poloniac 5: 250-336. . 1031. Siir rcxistcncc dc laccs ijliysiojojiiqucs chcz Ics Myxoniycctcs. Annalcs dc I'lutistcjlooie 4. 121-132. Soi.uoi.r. T. 1932. Sur Ics niati(:-rcs coloranfcs dc> fiucl- <|U( s ni.\'\()in.\('ctcs. Fc Hotaiiistc 21: 107-140. Staiii.. !•;. 1SS4. Zur Hiolojric dcr Myxonivcctcii. Zcitsclir. Hot. 42: 140-155. 102-175. 187-101. Stomhi;. H. lOOS. IMiototropiccrsclicinunjifn hci Ful einhryojrcny of sonic fucaceous aljiac. .J(jui'. Coll. Sci. Vuiv. 'I'okyo 32 (0) : 1 13. Wii.iiAMs. .1. 1.. ISOS. i^cproduclion in Dlrhjohi dirlio- hniKi. Annals Hot. 12: 559-500. . 1005. Sludi.'s in the I)ictyotacca(\ IIF Tho periodicity of the sexual cells of Dlclyula i/irliolonia. Annals Hot. 19: 531-5G0. -" • '■• rr A Note on Stemonitis fusca Roth William D. Gray, University of Pennsylvania The phenomenon of fairy-ring formation by certain fungi has long been known to mycologists, and numerous workers have reported and figured this type of sporophore arrangement among various agarics. Among the Myxomycetes, however, there have been few reports of fairy- ring formation. Macbride (1899, p. 35) reports such formations as being common to Physanim cinereum (Batsch) Pers., stating that the rings are small, measuring but a few inches in diameter. More recently, Poteat (1937) reports the finding of quite large fairy-rings of P. cinereum. This author states that he has found rings as large as fifteen feet in diameter. As far as can be determined from available literature, these are the only records of the production of fairy-ring formations by the fruiting bodies of Myxomycetes. Of the several species of Stemonitis which occur in Indiana, perhaps the two most frequently encountered are Stenioniti^ fiitica Roth and Stemonitis axifera (P>ull.) Macbr. During the spring and summer months, except in exceptionally dry weather, sporangia of one or both of these species may be found in almost any shaded woodlot where moist, decaying twigs and logs occur. In the course of collecting trips through- out the southern i)art of tho state, both species have been observed many times. Fairy-rings, formed by sporangia of S. funca, have been noted . I r * -^, ^ > M .ik'Jification^x'TvS.*^''' "^ ^^'''''"'''*''* ^"■'"'" ^''^'^ showing two incomplete fairy-rings. rN numerous times, but in no instance were sporangia of S. axifera nor any other species of Stemonitis found to be arranged in ring fashion. Such rings are quite small, never approaching the large size reported by Poteat for P. cinereum; the largest ring did not exceed three inches, (86) ■D I- r P*^P'*'"tfrl without change of iiaKcs from the rroceedinpTs of the Indiana Academy of Science. Vol. 47: paf?i's 86-87. 1938. - % '4 INTENTIONAL SECOND EXPOSURE IRREGULAR PAriTMATTOM Botany 87 and the majority were about two inches in diameter. The explanation of such a ring formation is simply that the Plasmodium arises at a certain point on the substratum, grows evenly, and creeps at an even rate from the point of origin. When the vegetative phase of the Plasmodium is terminated and it enters into the fruiting phase (Seifriz and Russell, 1936), the Plasmodium is in the form of a circle; naturally when sporangia are formed, they are arranged thus. In the case of S. fusca, fairy-rings were found only on uniformly decayed wood which possessed a fairly even surface. This is prerequisite for ring formation because any obstacle in the path of the advancing Plasmodium, such as an uneven place in the surface of the substratum or an area where the texture of the substratum is different, leads to a change in growth rate of the Plasmodium at that particular point and hence disrupts the circu- lar arrangement; such rings are shown in Fig. 1. The fact that S. fusca forms rings, whereas no other species of this genus has been observed to do so, leads to the conclusion that such a characteristic is probably a specific one. Literature Cited 1. Macbride. Thomas H., 1899. The North American species of slime-moulds. Mac- millan. New York. 2. Poteat. William L., 1937. A lawn marvel. Science 86:155-156. 3. Seifriz, William and Russell, Mary, 1936. The fruiting of myxomycetes. New Phyt. 35:472-478. f7-t Observations on the Methods of Stipe-formation in Stemonitis and Comatricha William D. Gray, University of Pennsylvania During the course of the writer's observations on myxomycete Plas- modia, it has become apparent that many generic as well as specific be- havior differences are to be expected. By behavior is meant the various conformations and color changes that are passed through in the develop- ment of sporangia from Plasmodia; the time* required to complete fructification is also taken into account, although it is less likely to be a fixed character. Myxomycete fructifications* are of three types: aethalioid, plasmodiocarpous, and sporangiate; the last may be either sessile or stiped. Naturally the behavior of the Plasmodium of a plas- modiocarpous species would differ from that of either a sporangiate or an aethalioid type. In addition to such differences, however, there occur differences between forms which possess the same general type of fructification; this is shown particularly well by members of the genera Stemonitis and Comatricha. Stemonitis and Comatricha are closely-allied genera belonging to the family Stemonitaceae, and mature forms of members of the two genera, when examined m&croscopically, show many resemblances — so many, in fact, that at one time the representatives of both genera were included in the same genus. Plasmodia of Stemonitis and Comatricha character- istically inhabit the interior of rotten wood, emerging only for fruiting; in the majority of known cases, the typical plasmodial color is white. A general feature in sporangia of both genera is the presence of a long central columella, which is simply an elongation of the stipe. Observa- tions on species of both genera show that the process by which the stipe and columella are developed in Stemonitis differs somewhat from that process in Comatricha. It is hardly to be expected that stipe- and columella-formation of all the species of Comatricha would be uniform; this is also to be said for species of Stemonitis, since there are forms which may be considered more or less border-line species between the two genera. A review of the literature yields but few references to the subject of stipe- and columella-formation. DeBary (1884) presented some obser- vations concerning this process in Stemonitis axifera (Bull.) Macbr. (S. ferruginea of Ehrenberg) , but his method of viewing the fruiting bodies was to harden them with alcohol and then render the protoplasm trans- parent with glycerine. By this method, according to deBary's figures (Figs. 2a and 2b), it may be shown that formation of the stipe and columella begins at an early stage in the development of the fruiting body. Bisby (1914), working on the development of the capillitium 'Macbride and Martin (19.34) define these as follows: aethaliutn : a fructification in which all or a considerable part of a jariven Plasmodium is involved and in which differentiation has not proceeded to the delimination of separate sporangia ; pfaawiodiocorp." a fructification which has the interior structure of a sporanRium, but which retains the netted form and outline of the Plasmodium ; this type is formed by the aerKr^Kation of protoplasm into a few of the larRer veins of the Plasmodium as it rests upon the surface of the substratum; siwranuium : typically an erect fructification of definite form and structure for a Riven species, each sporangium representing only a small part of the protoplasm of a given Plasmodium. (81) lieprinted from the ProreedingH of the Indiana Academy of Science, Vol. 46, i937 IRREGULAR PAGINATION 82 Proceedings op Indiana Academy op Science tails of the development of sZcaTo^'J''"^ '^'^"'^ cytological. De- described by the writer (Gray 1936) Tnv""^""" '° ^P-^angia, were the developing sporangia wer7;re„ed Ja^n m,?.""" "» "^-"-^^ "' accompanied by excellent photographs „f 1 ^ ^'^ ^^''^ "" «<^<'°""t, ment of Comatricha mera(pZT)fl,uT ''"«*" '" *e develop. (1899) on this same specie!" ili^ont ^i^^^^^^^^^^ Were it possible to grow members of ttil f -1 "*'«»«<« Preuss. of observing the development oTall ,„1 't "' *'"' "^e matter present, however, one must, e'vunoifiT^' T"''' •>* ^^Plified; at haunts, removing them to he"^ raborator'^a'H'r"'" '" '"^'^ "«'-« during the fruiting process. It has b'l I*- "Ix^rving them ordinarily to find emerging nlas„„di»r*,, ^='P«"«n<=e of the writer noon or evening; this is by „fn ea„s ho"we '' '""" ^'"^^^ '" *e after- summaries and the accompanying dkgramrwiH "' ■""'" "'"* ^o""*'"^ point out the main features of the sttrfr,'' '" " """''"'^- *» mental processes observed in the two .en„ T""^ ^"^ "^^"'^ "'^^'op- exception of 2a and 2b) are diag ri^o? 'n' -^'^t '' '' """ ' <«"•• the Figure 4 is redrawn from photo™, by jlhr '"•"'"^''' "' '"^ *"'- = tivelytr."'^ '""" ''""'• •'^"^- ^' «-> P'-"-d-™ Pearly-white, re.a- r Pis' 1 s* • • *-* * JL*^ ^** 7 1 3lvi^'r?.^iv"'?lT"'^^^ -^^^^^^^^ ^erh^-"- '•" the various sta.es of longitudinal sectfons of vn.'"* °^'^'''-« Reprinted from Science, February 11, 1938, Vol. 87, No. 2250, pages 138-139. .V I ll^ i A NEW DISTURBANCE OF RED PINE Red or Norway pine {Pinus resinosa Ait.) has been regarded as a particularly suitable species for refores- tation in the northeastern and lake states. The suscep- tibility of the more valuable white pine {Pinus strobus L.) to tip weevil and blister rust and the relative free- dom of red pine from pests have caused the latter species to be given preference in many cases. The investigations now being carried on by the writer upon an unreported disturbance of red pine indicate that its freedom from disease is more apparent than real in many sections of the northeastern United States. The external symptoms of the disease were first noticed by James A. Brock, assistant superintendent of the Rochester Municipal Watershed, in a plantation of young red pine in Ontario County, N. Y., during the summer of 1933. Since then the writer has found it or had reports of it throughout most of New York State, Connecticut and two counties in southeastern Pennsylvania. In gross aspect the symptoms resemble some types of insect injury, as the most conspicuous external characters are the extra-seasonal growth of one or more lateral buds in the terminal bud-cluster and the subsequent "forking" of the tree. The extra- seasonal growth of the lateral buds begins in June or July of the year that they are set, and may continue through August in the region of Hemlock Lake, N. Y. One or more buds are formed at the tip of these pre- cocious shoots before growth ceases. The terminal bud of the parent shoot seldom takes part in this extra- seasonal growth and usually elongates at the normal time the following year. Since the abnormal or pre- c^ocious laterals assume a more vertical position than is normal, the subsequent growth of the terminal bud causes a forked appearance of the tree. In some cases the original terminal may be forced to take the posi- tion of a lateral. This phenomenon, although undesir- able, per se might be of no great consequence were it not for the fact that organic union of the wood usually fails to take place between the forked members during the later growth of the tree. Dissection of a tree, in which forking had occurred, ordinarily showed that the forked branch had failed to unite on its upper surface with the bole of the tree. A resinous pocket or fis- sure, surrounded by discolored wood, usually occurs at these areas of non-union. Such defects afford an ideal environment for many species of fungi, some of which are known to be parasitic. Representative plots from approximately 800 acres of red pine, ranging in age from 5 to 25 years, have shown forking in 68 to 94 per cent, of the trees. The affected trees seldom, if ever, remain permanently forked, as the more vigorous member of the fork as- sumes a completely vertical position after a few years' growth and the other member tends to take the posi- tion characteristic of a normal branch. Hence the presence of the disturbance in a stand of red pine may not be evident to a casual observer until the in- vasion of parasitic fungi takes place. Forking has been found on soils varying in pH from 4.5 to 7.5 and ranging in texture from sandy to clay loam. It has been observed in young natural repro- duction as well as in plantations and in both mixed and pure stands. Investigations are being carried on in an effort to determine the primary cause and mecha- nism of forking. The evidence at hand strongly sug- gests a fungous origin. John Austin Jump Department op Botany, University or Pennsylvania — ^ ^^ ♦ .N '■^ ♦ *-^ * '4 A NEW FUNGUS ON FICVS NITIDA THUNB John A. Jump, Graduate Student, University of Pennsylvania. While visiting at the Agricultural Experiment Station in Rio I*iedras during the summer of 1936 tiie writer became interested i;i a fungus appearing upon Fieus nitUla Thunb. Avliich had been sent to the hiboratory for identification. Subsequent study of tho organism at the botanical laboratory of the University of Penn- sylvan a indicated that it was an llnrel^(.rtod spc-ios of Hupoxylon. The name Ilypojylon horlnqucnsis is proposed by the writer and tho species is herewith described : Tlypoxylon hoyinqucnsis sp. nov. Stroma latissime elTsa, carbo- naceo, nitida, subsupcrficiale in trunci ramonunque cortice, aliquid seabra propter ostiolis papillis, extui intusque nigrum. Peritheciii proiique, ovoides-oblongis, .2— .4 mm. h:ta. Ascis cylindraccis, den- sissimc stipat::;, l)i'evissimc stipatatis, apai'a[)hys;itil)us, loO-lGO )< 7-8}.i, octosporis. Sporidlis monostichis, subacutis, fusiformibui y. ovatis, saepe inequalatcrall)us, subflavi'^ lG-18 X 3.5-5^i. Ilab. in ramis truncibusque Fici nitidi in Puerto RicO. Stroma broadly eiTused, carbonaceoi'S, shining, subsuperflcial in bork of the trnuk and bi'aui-ho^^ ; somewhat rough due lo tlie papillatQ )stiolos; bhul: v,ilhi]i find without. Perithecia uumutous, asoid- ol)lonu-, .2 .' r Vsci cylindrical, very densely crowded, veryshoil ,", . t-- ■ ) .; 7-8u, oi<,rlit spored. As^o.si'ores monos- tiehous, s I;; . li-.i'iiin or ovate, often inequilateral, yellowish, 16-18 X 3.;') -5|_i. Habitat in branches and trunks of Picus nitida in Puerto Rico. The strr.ma in gross form bears some reseml)lance to both Eutypa onitnjKus Ma.^s'^-e and XviniHulxria fii'clor lierk. and. since these fungi are also found in many <>f the islands of the Wset Indies, a careful examination should be made of diseased Ficus in order that the orgaid'^m involved may be correctly determined. Eutypa ennn- pcns has l)een reported as occurring on Ficus nitida and Cacao in Trinidad. Is is quite diffcult in many cases to identify the genus of a fungris in the group of tho Sphcriales which includes Eutypa, Diatryps. Uypoxylon and Nummiilana, as they are similar in many respects 5T3 .074 Liiii JOUK.NAL i.n- AUKICULTURE OF THE UNIVERSITT CF P. R. A Ni:w ruNcrs ox fictis vilida tiiuni! To a. id have transitional icvm% of wliioh the generic status is debatable !J >jl}OX'j'on lorinqiicntis corresponds in certain r(s;>ec'.s to Xic.nnU' laria thictor nnd ncrliaps should be rcgaiilcd as - . /' ,/.,;; V/r:na. Asci oih.ttcHl. Loriiiquen^is n unace o: ■1 aagraiiUh ■ -As.' n.s (if 7/ -Ascospores. • -* I*-* i i *W A ^ m 4t» IRREGULAR PAGINATION PLA.TE XIX 'A A STUDY OF FORKING IN RED PINE «<<*► ^ A DISSERTATION IN BOTANY PRESENTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF PENNSYLVANIA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY »- - f -^ > r »■ ^**- fm JOHN AUSTIN JUMP Reprint from PhrtefalMoey, Vol. 28. No. 11 PHILADELPHIA 1938 ^T-^" PLATE XIX 1, ^ 'A *# -f~' -> ^ -* ««r^ ¥ *» — -» A STUDY OF FORKING IN RED PINE A DISSF.RTA'i ION IN I {OTA NY PKF.SF.XTKD TO TIIK FA( ILTY OF TllK (iKADIATK SCHOOL OF THE UNIVERSITY OF PKXXSYTAANIA IN PARTIAL FILFILLMKNT OF TIIK KKQllKF.MKNTS FOR TMK DF.GKKK OF DOCTOR OF PHILOSOPHY r. 1 \ I 0 '^^ m r r -f- T JOHN AUSTIN JUMP Rt-print from Thytofathrih^y, Vol. 2«, No. 1 1 PHILADF.LPHIA 1938 INTENTIONAL SECOND EXPOSURE ► .'-< - f T [Reprinted from Phytopathology, November, 1938, Vol. XXVIII, No. 11, pp. 798-811.] -r --r^ >» f T I I \ '- f I ->^^» A STUDY OF FORKING IN RED PINE^- ^ John Austin Jumps (Accepted for publication July 12, 1938) INTRODUCTION A high percentage of false bifurcation of the current season's growth of red pine, Pimis resinosa Ait., was noted in a forest planting on the eastern side of Canadice Lake, N. Y., by J. A. Brock in the spring of 1933 (9). The writer found the same disturbance in 3 counties in western New York State, in the northern Adirondacks, and in 2 counties in southeastern Pennsylvania in the summers of 1936 and 1937, although no systematic attempt was made to scout for it. H. H. York has reported it as occurring in plantings in Connecticut and several counties in central and eastern New York. Unless specifically designed otherwise, all data included in this paper were obtained from plantings on the Rochester Municipal Watershed in Livingston and Ontario Counties, N. Y. Trees in these plantings varied from 5 to 25 years of age. Kienholz (10) described a type of fasciation that might be considered as a tendency of the species to susceptibility to morphogenic disturbances, but there seem to be no references in the literature to the phenomenon of forking. Witches '-brooms do not appear to have been reported as occurring on red pine, although the writer observed one near the top of a tree in a 22-year-old planting. SYMPTOMS The first apparent symptom of forking is the extra-seasonal growth of lateral buds in the winter-bud cluster of the terminal shoot of the tree (Fig. 1, B). Extra-seasonal growth becomes noticeable in late June or 1 A dissertation presented to the faculty of the Graduate School of the University of Pennsylvania in partial fulfillment of the requirements for the degree of Doctor of Philosophy. 2 The writer wishes to express appreciation to Prof. H. H. York for advice and criticism during the course of this investigation, and to Prof. J. R. Schramm and Prof. Walter Steckbcck for criticism of the manuscript. Grateful acknowledgement is made to Mr. J. A. Brock for cooperation in the field investigations. 3 The greater part of this investigation was carried on during the tenure of a George Leib Harrison Feliowsiiip at tiie TJiiiveisil^ of rt;iiiiajivuuiu. 3 - 4 f-- • •* buds t!,kci Jul • I"^^^. r SpHnLwaS'' N Y V"^''- ''.• E^trasoasonal growth of lateral tree with dcpreiionmmed^ateW above D V?^Z"""'\ '"'J'"'^- ^'"r'' "" » 22 vear-old short needles and two buds at anex P Pe^nT f T' f'""^ "^'"^ ''"'"" '""' "'"»'" ^ith Cross section near a ?ork p™ Si, se^rd' rv br"-. neh LT h"^/""""""'? •"•"""hing- F. tion of trunk showing effeet^f ice breSge i ;L^rf": '^ brlr" n'-EiSet'*";''"' "7; of ice upon two forked branches near top of 22-veir oM tree T W, r "i !■' °',^'='«'>t characteristic appearance of a fork, with' reddle.,- Wo^n'sUin e.\eSg'iru^ toT'^ "• ^ ' A ■ 7 - ♦ July, when 1 or 2, and in some cases as many as 4 lateral buds commence to grow. The resulting precocious shoots may attain a length of 3 to 4 inches, with 1 or more winter buds at their apices, before becoming dormant (Fig. 1, D). Needles develop on such shoots, but seldom attain more than half of their normal length and fail to elongate further at the initiation of growth the following spring. The precocious shoots may continue to elon- gate until quite late in the current season, although, according to Gustafson (5), elongation in pine shoots has been completed by early July in Michigan. Measurements of 10 precocious shoots in a planting of 8-year-old red pine were taken on August 15, 1937, and again on October 25. All but one of them showed an increase in length from i to If in. The formation of a bud cluter at the tip of the precocious shoot (Fig. 1, D) interfered with normal subsequent growth, as it caused secondary branches to be formed within a few inches of the bole (Fig. 1, E). The terminal bud of the cluster in which the extra-seasonal growth oc- curs seldom takes part in the abnormal development, and usually elongates at the normal time the following season. The laterals, however, during the course of their extra-seasonal growth, react as if the terminal bud had been injured or removed and grow in a more or less vertical direction. Thus the shoot, originating from the terminal bud the following season, grows in competition with one or more laterals that had commenced growth the preceding summer. This results in a bi- or trifurcated appearance of the tree, which will be referred to as ''forking" (Fig. 1, A). This condition does not remain conspicuous for more than 2 or 3 seasons in most cases. Either the true leader or one of the precocious laterals will assume domi- nance and the other member, or members, of the fork will tend to the more or less horizontal position normally characteristic of red pine branches. Forking may occur repeatedly in a tree. One 22-year-old specimen, when dissected, showed this phenomenon in at least 12 nodes (Fig. 2). An examination of nodes where forking had occurred at some previous time frequently showed a slight depression in the trunk immediately above the insertion of the branch (Fig. 1, C). There seems to be no relation between the age of a tree and its suscepti- bility to forking, in stands up to 25 years of age. No data have been obtained upon older trees because of the difficulty of making certain, without felling the tree, that a fork might have been induced by insect or mechanical injury. DAMAGE RESULTING FROM FORKING The structural injury that results from forking usually has its incep- tion during the season following the formation of the fork, and is caused by the failure of the forked members to completely unite as they increase in diameter. Because of the acute angle at which they stand in relation to each other, they are unable to slough off, or push aside, the bark that lies on the inside of the fork between their cambiums, in the manner that the growth of the trunk displaces the bark of a normal lateral branch. This w . troo with doprossi,,,, inn.UM a d> : /oVV if Vn^^^ "'', ^'Z^'-^- '"''"'"'' "" •'' -- .vc'nr old »J« -■^ -^ •< r - 4 '♦ « - • July, wlien 1 or 2, and in some eases as many as 4 lateral buds commence to o-row. The resultino- precocious shoots may attain a lenj^th of 3 to 4 inches, witli 1 or more winter buds at their apices, before becominj? dormant (Fig. 1, D). Needles develop on such shoots, but seldom attain more than half of their normal leno-th and fail to elonofate further at the initiation of g-rowth the followinj*' sprint-. The precocions shoots may continue to elon- gate until quite late in the current season, although, according to Gustafson (5), elongation in pine shoots has been ccmipleted by early Jnly in Michigan. Measurements of 10 precocious shoots in a planting of 8-year-old red pine were taken on August 15, 1937, and again on October 25. All but one of them showed an increase in length from ] to VI in. The formation of a bud cluter at the tip of the precocious shoot (Fig. 1, D) interfei-ed with normal subsequent growth, as it caused secondary branches to be formed within a few inches of the bole (Fig. 1, E). The termiiud bud of the cluster in which the extra-seasonal growth oc- curs seldom takes part in the abnormal development, and usually elongates at the normal time the following season. The laterals, however, during the course of their extra-seasonal growth, react as if the terminal bud had been injured or removed and grow in a more or less vertical direction. Thus the shoot, originating from the terminal bud the following season, grows in competition with one or more laterals that had commenced growth the preceding summer. This results in a bi- or trifurcated appearance of the tree, which will be referred to as ^'forking" (Fig. 1, A). This condition does not remain conspicuous for more than 2 or 3 seasons in most cases. Either the true leader or one of the precocious laterals will assume domi- nance and the other member, or members, of the foi-k will tend to the more or less horizontal position normally characteristic of red pine branches. Forking may occur repeatedly in a tree. One 22-year-old specimen, when dissected, showed this phen(mienon in at least 12 nodes (Fig. 2). An examination of nodes where forking had occurred at some previous time frequently showed a slight depression in the trunk immediately above the insertion of the branch (Fig. 1, C). There seems to be no relation between the age of a tree and its suscepti- bility to forking, in stands up to 25 years of age. No data have been obtained upon older trees because of the difficulty of making certain, without felling the tree, that a fork might have been induced by insect or mechanical injury. DAMAGE RESULTIXO FROM FORKIXG The structural injury that results from forking usually has its incep- tion during the season following the formation of the fork, and is caused by the failure of the forked members to completely unite as they increase in diameter. Because of the acute angle at which they stand in relation to each other, they are unable to slough off, or push aside, the bark that lies on the inside of the fork between their cambiums, in the manner that the growth of the trunk displaces the bark of a normal lateral branch. This 6 area of non-union of cambium remains in tlie main axis of tlie tree as a fissure or poeket tl,at becomes filled with a heterogeneous material composed largely of resm and fragments of the bark (Pigs. 1, p and 1 I) These fissures are m a more or less vertical plane for several years until one of the torked members assumes dominance, as described above. While in this ver- tical pos.t.on they are easily penetrated by water, and a favorable environ- ment ,s thus established for the development of many species of parasitic and saprophytic bacteria and fungi. The presence of this flora may be an additional factor in causing the fissure to remain open. Although it is not within the province of this investigation to evaluate the damage caused by forking from the point of view of the defects in the timber of forked trees, mention should be made of the increase of winter damage in forked stands. Since part of a forked branch may not be united organically with the trunk, the mechanical resistance to stress caused by weight of snow or ice is reduced. A branch that breaks in such a manner (*ig. 1, Gr) produces a more serious injury to the tree than does the break- !r *^'r"'l ''i*^""''• "' '* '*^' "P^" " '^"^"y *•'«* »«y <'^te"d almost to the pith (Pig. 1, H) . Normal branches are able to stand much greater stress and. If breaking occurs, usually snap off at a point beyond the trunk. FORKING IN RELATION TO ENVIRONMENT There appears to be no marked correlation between forking and site con- ditions, except a possible relationship with the mean seasonal temperature 4 1 ^ |J^ i of the locality. Determinations of soil pH in plots of forkin- red pine showed a range from 4.5 to 7.5 without appreciable difference hi the per- centage of forked trees. The disturbance occurred on soils varyin- from sandy types m parts of the Adirondacks to the clayey loam upon which the pine had been planted in Montgomery County, Pennsylvania. The most prevalent soil types on the Rochester Watershed were Volusia loam and Volusia shale loam. Degree and aspect of slope likewise seemed to be of no significance. There was no relation between forking and extremes of pre- cipitation, or years of abnormal temperatures. The lowest percentages of forking encountered were in natural stands and plantations of red pine near Mt. Discovery, Essex County, New York. This locality was the most north- ern and the highest in altitude of those studied, which suggested that tem- perature and latitude might be possible limiting factors of the disturbance. Numerous observations of comparable sites, however, are necessary before any positive statement can be made on this point. Obviously, a shorter growing season would tend to minimize the effects of extra-seasonal growth, even though it might have no effect upon the primary cause. FUNGI FOUND ASSOCIATED WITH FORKING Isolations Cultures were made from red pine wood immediately after sawing. The surface of the wood was sterilized by swabbing with alcohol and flaming an area of several square inches in the vicinity of the place from which'' the sample was to be taken. A small piece of wood in the desired locality was then excised with a sterile woodcarving gouge, flamed briefly, and thrust into a tube of 2 per cent Fleischman 's Diamalt Agar. Cultures from buds were made by dipping the bud in 70 per cent alcohol and flaming it until the alcohol was burned off and the bud scales were partly charred. The bud was then split longitudinally with a sterile scalpel and the freshly cut surfaces were placed in contact with an agar slant. Blue-green molds commonly occurred in cultures from discolored wood from the interior of a bole in the vicinity of a fork. The majority of these are believed to be inhabitants of the wood, and not the result of poor labora- tory technic, for they usually did not appear for ten days or more following inoculation of the tube of medium with the wood sample. Bacteria were occasionally present, but no attempt was made to examine them in detail, although it was ascertained that no given form was consistently isolated! While only a limited number of trees above 20 years of age could be removed from the plantings for detailed examination, some indication of the internal condition of about 30 forked trees was obtained by means of an increment borer. Frequently, it was impossible to remove a complete core with the borer because of wood rot in the vicinity of old forks about waist high on the tree. Forked areas were almost invariably accompanied by reddish brown streaks within the wood (Figs. 1, I and 2) from which fungi could be isolated. These streaks often were found surrounding the pith in forked trees. area of non-union of cambinm remains in the main axis of the tree as a fissure or pocket that becomes fille,l with a l,etorogeneons material composed la.gely oi resn, and fragments of the bark (Pigs. 1, F and 1, I) These fissures are m a more or less vertical plane for several years until one of the torke. pullulans. Resistance of the hyphal wall to Cartwright's stain (1), which characterizes the chromatic hyphae of D. pullulans, was also typical of the pith-inhabiting organism. Several members of the Dematiaceae have been recognized for some time as wood-inhabiting organisms. Siggers (13) refers to Torula ligniperda as a cause of structural weakness and brown staining in tulip poplar and white ash. Reddish-brown staining was usually present in the vicinity of old forks in red pine wood and was of frequent occurrence surrounding the pith in trees affected by the disturbance (Fig. 2). Dematium pullulans is readily grown upon a w^ide variety of culture media, both liquid and solid. Cultures in flasks on sterile red pine sawdust, moistened with distilled water, produced a mycelium and conidia, but the yeasty conidial masses, characteristic of its growth on other media, w^ere not evident. A series of cultures was made by placing a block of agar 5 mm. square, containing the fungus, upon the surface of steam-sterilized soil in large culture tubes. Growth took place readily and the hyphae penetrated the soil to the bottom of the tube : a depth of about 6 cm. Single-spore cul- tures were made by means of the Kienholz technic (11), and the following observations were made on sub-cultures of these on prune agar and on liquid 2 per cent malt. MORPHOLOGICAL OBSERVATIONS UPON DEMATIUM PULLULANS Dematium pullulans de Bary: The mature conidia are typically ovoid to ellipsoidal, averaging 7.4 x 4.0 p, 2-guttulate and nearly hyaline. Varia- tions from this type occasionally occur (Fig. 4, A), especially in liquid cul- ture. In hanging-drop cultures the spores may reproduce by budding or germinate directly, forming hyphae upon which conidia may be borne lat- erally (Fig. 4, E). Freshly transferred cultures on prune agar produce conidia so abundantly that a yeasty mass, varying from white to light violet, is formed upon the surface of the culture. The vegetative mycelium is usually somewhat submerged and black in macroscopic appearance, forming a compact stroma-like layer at the surface. The mycelium varies from a dark Torula-\\^s.e form, frequently found in liquid cultures, to a hyaline, distantly-septate type. It may contain granular material or drops of oil in some instances (Fig. 4, E). Round, black bodies, usually less than 1 mm. in diameter, often appear on the surface of prune-agar cultures 2 weeks or more old. These structures are composed of a black, highly-compacted outer layer with a pseudo-parenchymatous, readily stainable central core. Such bodies will be referred to as microsclerotia. Dark-ringed tapering hyphae often appear in tufts on the surface of microsclerotia, although they may also arise directly from the mycelial surface. Bulbilous structures, appar- ently of similar nature to the rings, frequently occur at the base of a hyphal branch. A type of exospore, resembling spores of the genus Coniothecium, 10 was found among these hyphae and was abundant also in liquid cultures These spores were ovate to orbicular and usually very dark. They averaged 24 M in length, and often had one or more hyphal cells attached to their distal end. A graded pH series was set up with Elliott's medium (3), which contains asparagin as a nitrogen source ; and a second series was run with 2 per cent '"1*ttJ,''* J^^^^ °'""*^'^ *''°'" *''"'^ '"^''■^ «"d the pH was adjusted with HCl and NaOH. The malt series of 12 pH values from 1.8 to 8.5 showed growth from 1.8 to 7.2, although growth was slow at the extremes of the range, particularly at the acid end. Optimum growth occurred between 5 5 and 6.5. On Elliott's medium at pH values from 2.2 to 8.4, growth was obtained from 2.2 to 7.2, with an optimum range betweeen 5.0 and 6 5 The malt series was inoculated with mycelium, while conidia were used with Ji»lliott s medium. Growth of different cultures of the fungus collected from various locali- ties showed no tendency toward morphological variation when grown upon 2 per cent malt agar in Petri dishes. Numerous varieties of Dematium pullulans have been described, but the variety with which we are here con- cerned cannot be placed satisfactorily among these. In the character of its nodose hyphae it resembles var. fimicola Marchal, but differs markedly from It m spore characters. EXTENT OF FORKING IN STANDS A partial summary of data obtained from some of the plots studied on the Roches er Watershed is given in table 1. It is evident from data upon plots L and D that the more vigorous trees are the most susceptible to fork- Zn.T/' 'Tr^ '" ^^^^^ *' '"''' ^^ ^^^ ^^ '^' ^^' ^^^^P« ^t^^died, although quantitative data are not available for trees of the older classes. wateli^^ 1-^-o.n. of forlcing with related data on sample plots^ on the Rochester Plot Trees ex- amined Age Trees forked Trees with precocious buds in terminal 1937 cluster Trees with secondary lateral near main stem Aver- age height of stand A B C D E F G H No. Tears 417 1 10-11 100 1 7-8 404 6-8 100 1 5-7 60 5-7 88 6-10 100 23-25 100 1 7-9 Per cent 92 63 69 78 88 92 94 87 Average height of forked trees Per cent 30 10 30 41 Per cent 40 38 51 22 57 In. 121 27.1 33.1 37.1 In. 30.0 34.5 37.1 324 : sams SSKxrs l-.-- '•»"■'•".".«».». .». 11 A map was made of two plots (C and G, Table 1) to determine whether foci of infection occurred in forked stands. The map indicated that fork- ing was evenly distributed throughout the stand, as no evidence of ''islands'' was found. Two stands of natural red pine within 30 miles of the watershed showed percentages of forking that fell in the range of those of the plantings. In most cases the plantings were of pure red pine, although in some of them there was a light admixture of Norway spruce and eastern white pine, which apparently had no effect upon the prevalence of forking. The disturbance seems to reach a maximum after a period of years and then gradually subside, although there are indications that there may be a second cycle in the same planting. Annual occurrence of forking when plotted on coordinates (Fig. 3) may be considered additional evidence that an organism was the causal agent of the disturbance. 1933 1934 1935 1936 Year 1982 25 24 25 Zf, 27 28 29 30 * ^ ^r' ^' o^^^^^^ occurrence of new forks in plantings of red pine. A. Distribution of forks on 852 trees, 6-7 years old, from five plantations. B. Distribution of forks on 100 trees from a plantation in which the trees averaged 23 years old. HISTOLOGICAL STUDIES Material for histological examination was fixed in a chromic acid— forma- lin—salcylic acid solution (2), dehydrated with the N-butyl alcohol technic, and cut on a sliding microtome in sections varying from 5 to 15 |j in thick- ness. It was found necessary in working with the older tissues to extend the period of dehydration and infiltration to 10 days or 2 weeks in order to insure complete penetration by the alcohol and paraffin. Buds, precocious shoots, wood from the older portions of the trunk, and roots were examined for abnormal anatomical features and for the presence of fungus hyphae. The most successful stain employed was Cartwright's picro-analine blue combi- nation (1), although the chromatic hyphae that appeared in the old wood 12 13 I •5^ t^l '^MifL^^ s A> Fig. 4. Dematium pullvlans dp Rnrv a t',,^ ^ left is the typical form. B. Section fhrou^hl; Ifn^. f comdia; the spore in the upper ConiotheciumAike spores. C CoSLci^nL.r. ''/''*'"."' ^^**^ "«^««« hyphae and in liquid culture. D. Nodose hyXrETv^^^^^^ found among nodose hyphae and of D, .unmans in pith cells of "^reJ^Ji^^^^^P^ caLr^fe)^;^^-^^ *« ^« ^r were satisfactorily demonstrated in unstained sections. No hyphae were observed in the root sections, but chromatic Torula-W^Q hyphae were abun- dant m the pith of the wood taken near a twelve-year-old node (Fig 4 P) and occasionally penetrated the wood rays. Hyphae occurred both inter- and intra-cellularly in the pith, and were observed entering pith cells through the simple pits in the walls. Minute hyphae also occurred in the procambmm and primary xylem of precocious buds, but were not found in the undifferentiated tissue of the promeristem toward the apex of the bud. Such hyphae appeared to be entirely intercellular. There was no evidence of destruction of cells or of the occlusion of vessels in any of the infected material. INOCULATIONS A suspension of conidia of Dematium pullulans in distilled water was used to inoculate a series of potted red pines 5 years of age. These trees were brought into the greenhouse from coldframes in early January and inoculated about a week later. Seasonal growth was initiated in the trees, in most cases, by the second week of February. The conidial suspension was introduced into the bud, and into the phloem and pith of the stem by means of a hypodermic needle. Distilled water alone was injected into 3 trees of the series to serve as a control. The site of the needle puncture was ringed with a colored wax pencil, and 5 weeks after inoculation the buds and sec- tions of the stem were surface-sterilized, split longitudinally, and placed in tubes of nutrient agar. Sections of the stem were taken from the site of the inoculation and at points 1 and 2 cm. above and below it. Each tree bore 3 inoculations: in the terminal bud; 1 to 2 in. below the bud; and 8 to 14 in. below the terminal. D. pullulans was recovered from all of these sites and, in some cases, from sections 1 and 2 cm. above and below the inoculation. Another series of potted pines was inoculated by the following method. A small area of the stem was surface-sterilized with 70 per cent alcohol and a slight wound was made in the bark with a sterile needle. This wound pene- trated the cells of the cortical parenchyma but did not reach the cambium. A small piece of agar upon which Dematium pullulans was growing was placed in contact with the wound ; absorbent cotton, wet with distilled water, was placed over it ; and a band of cellophane was wrapped around the cotton and tied at both ends to insure a moist atmosphere around the inoculum. The cotton and cellophane were removed after 2 weeks, and five weeks from the date of inoculation an attempt was made to recover D. pullulans from the stem in the manner previously described. The failure of D. pullulans to develop in any of these cultures leads to the conclusion that it is unable to enter the tree through wounds in the bark or cortical parenchyma. Since resin canals are abundant in the latter tissue, it is possible that the copious resinous exudation may have a fungicidal action. DEMONSTRATION OF A GROWTH SUBSTANCE PRODUCED BY DEMATIUM PULLULANS Dematium pullulans when grown in liquid culture produces abundant 12 'if f> >e7 C Fig. 4. T)rmalium pullnlans {\i^ \\-tr\- at,. ^ left i, the ty,,i,-.nl f„rm. ' IS. See io , t 'nLl, ^ ,!?"' f "'!'"»»;."'« spore in tl.e upper in li.|„i,l ,.„ltn,e. J). X,„lose Inpl,,,.. E Tvne', , f 1 • *""»'' ""'""K >>o ';,"r; ean;e"r id;)'r'"'^""' *" "" Ai M it I 13 were satisfactorily demonstrated in unstained sections. No hypliae were observed in the root sections, but clironiatie Tonda-Wki^ liyiiliae were abun- dant in the pith of the wood taken near a twelve-year-old node (Pio- 4 F) and occasionally penetrated the wood rays. Hyphae occurred both iiiter- and nitra-cellularly in the pith, and were observed enterino- pith cells through the simple pits in the walls. Minute hyphae also occurred in the procambuim and primary xylem of precocious buds, but were not found in the uiulifferentiated tissue of the promeristem toward the apex of the bud. Such liyphae appeared to be entirely intercellular. There was no evidence of destruction of cells or of the occlusion of vessels in any of the infected material. INOCULATIONS A suspension of conidia of Dcmatium pullulans in distilled water Avas used to inoculate a series of potted red pines 5 yeai-s of age. These trees were brought into the greenhouse from coldframes in earFy January and inoculated about a week later. Seasoiud growth was initiated in the trees, in most cases, by the second week of February. The conidial suspension Avas introduced into the bud, and into the phloem and pith of the stem by means of a liypodermic needle. Distilled water alone was injected into 3 trees of the series to serve as a control. The site of the needle puncture was ringed with a colored wax pencil, and 5 weeks after inoculation the buds and sec- tions of the stem were surface-sterilized, split longitudiually, and placed in tubes of nutrient agar. Sections of the stem were taken from the site of the inoculation and at points 1 aiul 2 cm. above and below it. Each tree bore 3 inoculations: in the terminal bud; 1 to 2 in. below the bud; and 8 to 14 in. below the terminal. D. puUulanH was recovered from all of these sites and, in some cases, from sections 1 and 2 cm. above and below the inoculation. Anotlier series of potted pines was inoculated by the following method. A small area of the stem was surface-sterilized with 70 per cent alcohol and a slight wound was made in the bark with a sterile nee. puJJuJans from the stem in the manner previously described. The failure of I). puJhdnns to develop in any of these cultures leads to the conclusion that it is uuable to enter the tree through wounds in the bark or cortical parenchyma. Since resin canals are abundant in the latter tissue, it is possible that the copious resinous exudation may have a fungicidal action. DEMONSTRATION OF A HROWTII SUBSTANCE PRODUCED BY DEMATIUM PULLULANS Dcmatium pullulans when crrown in liquid cnltuTv produces abundant INTENTIONAL SECOND EXPOSURE 14 growth substance according to the lupine test developed by Granick and Dunham (4) Th.s technic briefly consists of measuring increase of growth in length of the upper centimeter of a 7-8 em. etiolated lupine hypocotyl decapitated at the junction of the hypocotyl and the cotyledons The sub stance to be tested is applied to the cut surface in lanolin paste or agar and a mark ,s made with India ink 1 cm. below the cut. The increase in length of the original centimeter is measured after 4 days exposure to light. For this study Z). pnllnlans, Tympanis sp., Fnsarium sp. and Polyporus sehwmmUn were grown on liquid 2 per cent malt in 500 cc. Erlenmeyer flasks until a thick hyphal mat covered the surface of the medium The mat was then removed, washed in water to free it from as much of the malt as possible, and dried thoroughly at a temperature not exceeding 80° C Fleisch man s yeast was grown on Pasteur's solution instead of malt, and freed of the medium by washing in a funnel lined with filter paper. The dried mats were ground in a mortar, and the resulting powder was mixed in a propor- tion of 2 parts by weight of lanolin to 1 part of the fungus powder The increase of growth produced by the powder of Dematium pullulans was com- pared with that of other fungi and with control plants upon which lanolin alone was applied. Several series of tests were conducted at intervals of 2 weeks and it was found that the average increase of growth produced by a given organism varied slightly in the different series, but the relative amounts of growth produced by the fungi employed remained constant. This variation might well have been caused by increasing greenhouse tem- perature and change of sea.son during the experimental period. Fifteen to 30 test plants were used for each species of fungus tested. Dematium pullu- lans produced a greater increase of hypocotyl elongation than any of the ItT nr^T f "' ;''"'"' growth-promoting activity of the powdered mats of the fungi may be expressed as follows: Dematium pullulans > DISCUSSION The causal agent of forking cannot definitely be determined from the experimental data available at present, but the theory of a fungus Zilin phys ological disturbances of a nutritional nature do not appear to provide a satisfactory explanation for the phenomenon ^ Complete serial sections of buds were examined, and no traces of larvae or the puncture of insect probosci could be found. There was no evidence of insects on the buds in the cour.se of daily field observations made during late June and the month of July. Even if this evidence were disregarded an hypothesis of insect injury would have to imply a selective feedTn: of the' growth, biich a circumstance seems highly improbable A physiological disturbance from the standpoint of a nutritional unbal- ance or excess cannot be correlated satisfactorily with the wide range of soil ^i> 15 types of varying hydrogen-ion concentrations upon which forking occurs Gus afson (6) found that red pine, brought into the greenhouse in January would commence growth at once and form 2 bud clusters by July. Appar' ently the terminal bud develops normally in this case, and there would seem to be no connection with the type of growth that results in forking It cannot be proved conclusively that a genetic factor might not be causally connected with forking, but several circumstances are contrary to such a possibility. The wide geographic range of the occurrence of forking together with the fact that forking in neighboring stands may gradually attain a maximum in different years and at different ages of the plantings l-big. J), indicates genetic influence to be unlikely. The presence of fungi in the buds and internodal pith, as indicated by the results of cultural and histological studies, forms the basis of the writer's suggestion of a theory of the fungus origin of forking. Research on phyto- hormones has proved that bud inhibition and activation are regulated, at least in some instances, by auxin. It also is well established that certain fun-i produce auxin in considerable quantities. Thus, tlie extra-seasonal growth of laterals might be attributed to the production of auxin within the bud by fungus hyphae. Le Fanu (12) showed that in two shoots of Pisum, one of which was being strongly inhibited by the other, the dominant shoot indi- cated a high concentration of auxin with the Avena test, while the inhibited shoot showed only slight traces. If a similar condition occurs in the bud clusters of pine, it would follow that the presence of a phytohormone pro- duced by a fungus in the lateral buds might conceivably act as a stimulus to extra-seasonal growth. Production of the hormone in the terminal bud would not necessarily lead to extra-seasonal stimulation if the hormone were normally present in considerable quantities. The abundant production of growth substance by Dematium pullulans and the proof of its ability to in- habit red pine might be regarded as indicative of the possibility that the fungus could be causally connected with the disturbance. It is impossible to compare the microflora of normal trees with those of this investi elation Since forking may be latent in a tree, it could not be determined with cer- tamty that a given specimen was completely normal. SUMMARY An abnormal bifurcation of red pine trees is prevalent in New York State and other sections of the northeastern United States. This condition is caused by extra-seasonal shoot elongation in which the lateral buds take part while the terminal remains in the dormant condition. Failure of the inner surfaces of the resulting fork to unite completely during subsequent growth of the tree causes an open fissure in which wood-destroying fungi may enter. Tympanis sp. also frequently gains entrance in this manner. No correlation was found between rate of forking and site conditions. Trees at high altitudes in the northern part of the area studied showed the lowest percentage of forking. Dematium pullulans de Bary w^as isolated from Ill 16 abnormal buds in the majority of cases, and hyphae of the characteristic form of the organism were found in the wood of abnormal trees. Young red pines were inoculated hypodermically with conidial suspensions of D. pullu- lans and, after 5 weeks, the fungus was reisolated from sections of the stem 1 and 2 cm. from the site of inoculation. D. pulMans was found to contain a growth promoting substance by means of the lupine technic in a quantity exceeding that of other fungi tested. The theory is advanced that abnormal growth of a pine bud may be initiated through the action of a phytohormone produced by the fungus living within the tissues. Botanical Laboratory, University of Pennsylvania, Philadelphia, Pa. 1. 4. 6. 7. 8. 9. 10. 11. 12. 13. LITERATURE CITED Cartwright, K. St. G. A satisfactory method of staining fungal mycelium in wood sections. Ann. Bot. 43: 412-413. 1929. Cohen, I. and K. D. Doak. The fixing and staining of Liriodendron tulipifera root tips and their mycorrhizal fungus. Stain Techn. 10: 25-32. 1935. Elliott, J. A. Taxonomic characters of the genera Alternaria and Macrosporium. Amer. Jour. Bot. 4: 439-476. 1917. Granick, S. and Dunham, H. W. Growth substance determinations. Science (n.s) 87: 47. 1938. GusTAFSON, F. G. When does a pine tree complete its seasonal growth? Papers Mich. Acad. Sci. 22: (1936) 83-84. 1937. . The influence of length of photoperiod upon the initiation of growth in Pinua resinosa seedlings. (Abstract) Amer. Jour. Bot. 24: 738. 1937. Hansbrouqh, J. R. A new canker disease of red pine caused by Tympanis pinastri. Science (n.s.) 81: 408. 1935. . The tympanis canker of red pine. Yale University School of For- estry Bull. 43. 58 pp. 1936. Jump, J. A. A new disturbance of red pine. Science (n.s) 87: 138-139. 1938. KiENHOLZ, R. Fasciation in red pine. Bot. Gaz. 94: 404-410. 1932. . Isolating single spores without special equipment. Phytopath. 27: 950-951. 1937. ^ ^ Le Fanu, B. Auxin and correlative inhibition. New Phytol. 35: 205-220. 1936. SiGGERS, P. V. Torula ligniperda (Willk.) Sacc, a hyphomycete occurring in wood tissue. Phytopath. 12: 369-374. 1922. '*i SOME REACTIONS OF SLIME MOULD PROTOPLASM TO CERTAIN ALKALOIDS AND SNAKE VENOMS by SAMUEL S. LEPOW With 3 Text-figures Received for publication, March 23, 1938 INTRODUCTION The study of the effects or pharmacodynamics of alkaloids and snake venoms has received much attention resulting in a great body of information insofar as organisms and tissues are concerned. The field of protoplasmic pharma- codynamics has received some attention, but there are few data on the effects of snake venoms and alkaloids on protoplasm. Barber (1911) determined the influence of alkaloids, strychnine, and quinine upon the viscosity of the protoplasm of Nitella. Hopkins (1922) investi- gated the effects of morphine on the swelling and coagulation of the protoplasm of protozoa. These workers did not consider changes in the elasticity, extensi- bility, tenacity, or colloidal state of protoplasm, qualities to which workers in the field of protoplasm generally attach considerable significance. Balbach (1936) observed the effects of urea solutions on the protoplasm of a slime mould (Chondrioderma difforme)- He noted, among oth^r responses, granule aggregation, changes in protoplasmic streaming, and swelling or shrinkage of the strands. The Plasmodium of the myxomycete, Physarum polycephalum, afforded favorable material for this investigation. A fine pipette controlled by a micro- manipulator was used to apply limited quantities of solutions to small areas of protoplasmic strands. The reaction of the treated protoplasm was compared to that of untreated adjacent to it in the same strand (Figs. 3, 9). Attention was focussed on: 1. The effects of alkaloids and snake venoms on the physical properties of protoplasm. 2. The structure of protoplasm as revealed by these effects. 3. The modus operandi of the substances applied. MATERIALS AND METHODS The Plasmodium of Physarum polycephalum was cultured according to the methods developed by Howard (1931) and by Camp (1936). Camp's technique appeared preferable because of the greater facility in culturing, the richer yield of the material and its greater vigor. The organism is grown on powdered oatmeal in a moist chamber. The optimum growth occurred in a water saturated atmosphere at a temperature of 25 ^ C. The original Protoplasma. XXXI || IRREGULAR PAGINATION 162 Lepow material was obtained from Dr. Howard, and has been successfully cultured for four years. The Plasmodium, when growing under optimum conditions, easily overcomes and feeds upon molds and bacteria which may appear as contaminants. Ingested ciliates have been observed in vesicles resembling the food vacuoles of protozoa. Cultures showing contaminants were discarded. Cultures about to change from the active vegetative to the spore or "fruiting" stage were not suitable for study. It was easy to anticipate the fruiting of cultures by noting the tendency of the protoplasm to coalesce into small clumps and the change in color of the agar substratum to brilliant green just prior to fruiting due to acidity, as recorded by Seifriz and Zetzmann (1935). A small portion of the plasmodium was removed from the glass wall of the culture chamber and placed on a moist coverslip. After two to four hours, the protoplasm spread into a sheet or a network of strands. Protoplasmic masses which did not spread were dis- carded. A suitable coverslip-culture was inverted over the micromanipulating chamber, the interior of which was kept moist during the observations. Possible injury by heat from the source of illumination was minimized by the use of a water filter. The micropipette was introduced into the moist chamber, and varying quantities of the solutions were gently applied to one side of a strand. It was necessary to keep the micropipette from touching the strand and to prevent forceful flow of the solution, since the protoplasm reacts to a purely mechanical stimulus by exhibiting a sudden cessation of motion and a temporary or reversible coagulation. The micropipettes were discarded after use with each individual concentration of the reagent. One to two drops of each sample sufficed usually to produce a result. Strands possessing the least adherent water film were selected in order to prevent dilution of the applied solution. When fully immersed in water the protoplasm contracted and showed sjoieresis, therefore the immersion method was not utilized. Injection of the solutions into the Plas- modium brought on localized coagulation about the region where the micropipette penetrated and this coagulated material interfered with the flow of the solution into the protoplasm. To overcome this, greater force had to be exerted on the hypodermic syringe. Accurate control of the quantity being introduced could not therefore be attained. The mere insertion of an empty pipette into the protoplasm caused coagulation. B£la& (1930) noted similar mechanical effects due to micromanipulation. For these reasons microinjection was not continued. Different concentrations were used in order to determine the toxic range of the sub- stance being studied. The treated protoplasm was observed for at least one hour after the application of the solution so as to observe a possible reversal of pathological changes, slow responses, or other subsequent reactions. Seifriz (1935) reported that high concentrations of acids and alkalies have a pro- nounced effect on the plasmodium of Physarum polycephalum, and Crane (1921) found that alkaline solutions of alkaloids are more toxic than acid solutions for Paramecium. A series of studies were, therefore, made with HCl solutions of different pH values. It was found that H+ within a range of 4.0 to 7.0 exerted no effects comparable to those obtained with the toxic solutions with pH values from 4.1 to 7.1, epinephrine being the only exception with a pH of 3.0. Buffers were not used because of toxic effects. Observations were made on strands of varying thickness, size, and rate of streaming in order to eliminate misinterpretation. Strands shoAving signs of degeneration were not used. NORMAL SLIME MOULD PROTOPIJVSM A Plasmodium is a mass of relatively undifferentiated protoplasm. The plasmodium of Physarum polycepfialum is bright lemon-yellow in color. Seifriz and Zetzmann (1935) •I* J J. ) i i ^> 1— 4% t ' - t I i Ta t Some reactions of slime mould protoplasm to certain alkaloids and snake venoms 163 showed that the pigment responsible for this color is an indicator of pH. The pigment is present in granules and not dissolved as in colloidal suspension in the matrix, for all non- granular areas are colorless. The protoplasm contains pigment granules, oil droplets, and other globules with well defined contours including some few colorless vesicles resembling oil globules and probably of the nature of vacuoles, all of which may collectively be termed macrosomes. With these occur minute particles or microsomes. The protoplasmic stream is in one direction only at a time and reverses itself repeatedly. The viscosity of the quiescent protoplasm is that of an elastic gel whereas the flowing protoplasm is liquid. The elasticity (that property of protoplasm by virtue of which it tends to recover its original form after subjection to a distorting force) is low, while extensibility (stretch as distinct from elasticity) is great or low depending on the size of the strand and condition of the protoplasm. The tenacity or cohesion of the protoplasm as determined by the ease with which it can be torn is moderate, but varies somewhat. The criteria used to indicate toxicity were: (1) Effects on streaming (6) Syneresis (2) Swelling and shrinkage (7) Outflow of matter (3) Changes in color (8) Changes in physical properties (4) Coagulation (9) Reversibility of changes (5) Brownian movement (a) Alkaloids Cocaine hydrochloride Quinine hydrochloride Quinine sulphate Atropine sulphate Morphine sulphate Ephedrine hydrochloride Strychnine sulphate COMPOUNDS USEDi (b) Venoms Bothrops atrox Crotalus atrox (c) Other siibstances Chloral hydrate Epinephrine hydrochloride EXPERIMENTAL RESULTS The following results are general for all solutions used except where a specific reagent is named. Effects on streaming The initial effect on streaming was a temporary stimulation resulting in an acceleration of the rate. The subsequent reaction varied. Quinine hydro- chloride caused complete cessation of motion a few seconds after the initial stimulation. Morphine eliminated all motion in two to three minutes. When streaming was not abolished in five minutes, no reaction to the added solution could be observed. Frequently, movement would continue at a reduced rate on the untreated side of the strand until the reagent had penetrated this region * Dr. Schmidt, of the Department of Pharmacology, School of Medicine, University of Pennsylvania very kindly furnished the alkaloids used in this investigation. The venoms were obtained through the kindness of Dr. Githens, of the Sharp and Dohme Biological Station. 11* IRREGULAR PAGINATION 164 Lepow (Fig. a). At times the protoplasm would draw away from the coagulated site and then produce two streams which flowed in opposite directions but did not show reversal (Fig. b). Often the protoplasm would move in jerky spurts as if encountering a barrier, rebounding and then pushing forward. An unusual response was noted in one strand (Fig. c) in that the actively flowing protoplasm became less opaque, due apparently to precipitation of some of the protein; as shown by coagula which appeared within the streaming protoplasm. Another type of flow occurred especially after treatment with cocaine which is of quite a different kind from the normal flow. The hyaline substratum moved very sluggishly in an irregular path through the coagulated, granular mass. The movement does not undergo reversal and appears to be due to mechanical pressure, possibly shrinkage of the protoplasm on coagulation. stream treated side coagulated matter stream stream coagulated matter treated side stream stream coagulated matter treated side SWELLING AND SHRINKAGE Swelling is to be distinguished from normal enlargement which occurs with growth and movement ; the latter is reversible, while swelling is permanent. With increase in volume due to a pathological change, there is no recovery, no return to the original volume after swelling (Figs. 8, 18). Abnormal swelling is characterized by the presence of hyaline, granule-free areas at the treated site. Shrinkage was caused by the removal of protoplasm from the treated region to adjacent untreated regions, and by contraction of the coagulated protoplasm accompanied by the production of syncretic vesicles (Fig. 16). \ V I I ■I' I \ >> I f Some reactions of slime mould protoplasm to certain alkaloids and snake venoms 165 CHANGES IN COLOR The yellow pigment of Physarum changes to yellow-green with alkalinity and to orange-red with acidity. Epinephrine with a pH of 3.0 caused the color to change to orange, i. e. to the acid side. Cocaine and several of the other reagents altered the color to a greenish yellow indicating an increase in acidity. Other changes in color occur which are not due to the pigment, but to aggregation of granules, swelling, and shrinkage which alter the depth of color or opacity. Coagulation The coagulation of protoplasm concerns primarily the proteins dispersed in the aqueous substratum, but agglutination or aggregation of particles may occur with the coagulation of the proteins. Any reagent which has an ill effect on protoplasm is likely to cause coagu- lation. Coagulation is most readily recognized by the aggregation of the visible protoplasmic particles. Cocaine causes aggregation of the particles into one irregular mass (Fig. 8). A coarsely granular coagulum due to aggregation of particles into large and scattered groups was produced by quinine sulphate (Fig. 17), atropine sulphate (Fig. 21), and ephedrine hydrochloride (Figs. 19, 22). A moderately coarse coagulum of aggregates in uniform distribution was caused by epinephrine hydrochloride (commercial solution). A finely granular coagulum was produced by strychnine sulphate (Fig. G), chloral hydrate (Figs. 14 and 20) and the venom of BofJirops atrox. A very finely granular almost homogeneous appearance due to aggregation of particles into very small groups was produced by quinine hydrochlorkle (Fig. 4) and U. S. P. epinephrine (Fig. 1). Crotalus afrox venom dkl not appear to modify granule dispersion or to profoundly influence the protoplasm in any way (Fig. lo). BROWNIAN MOVEMENT Brownian movement of microsomes was not visible in streaming portions of the protoplasm but could be seen when streaming ceased if phase separation occurred. The degree of fluidity was roughly indicated by the amplitude of vibration of the microsomes. The duration of the movement varied with the substance used (cf. table I). Brownian movement was not obersved when coagu- lation took place without phase separation. The granules rlid not undergo Brow- nian movement. Although phase separation was a common reaction, the vibration of the microsomes failed to occur in every instance. SYNERESIS The squeezing out of a fluid with the contraction of a gel is known as syneresis. It occurs in protoplasm on coagulation (Table I). Syneresis assumed several forms: 1. Vesicles which contained a hyaline fluid only (Fig. IGrt, 22a). 2. Vesicles containing hyaline fluid and other protoplasmic constituents (Figs. 2rt, 7, 10, 15a, 16, 22). 166 Lepow 3. Irregular extensions of hyaline fluid and protoplasmic inclusions (Fig. 6). 4. Peripheral extensions of hyaline fluid. Syneresis is an entirely different response from that which is here termed outflow (cf. Fig. 22a and 226). OUTFLOW OF MATTER Normal protoplasm may rupture the surface and produce an outflow as a result of pressure created by a stream block. This matter undergoes reversible coagulation, recovers and produces a new surface. There is no separation of the protoplasm into several phases. Quinine sulphate caused a different type of outflow v\hich appeared to be a toxic effect. The surface possessed a ragged opening which was probably caused by death changes and loss of elasticity. This outflow consisted of hyaline fhiid and microsomes. There was, therefore, a protoplasmic separation in this type of outflow. The microsomes were under- going Brownian movement. Later, the microsomes aggregated and the hyaline fluid was found to be miscible with the aqueous solution on the coverslip. Ephe- drine and atropine also produced an outflow. CHANGES IN PHYSICAL PROPERTIES a) I'iscos \'iscosity changes were caused by all the reagents. The outer treated regions were affected more than the inner ones. The changes differed with each reagent (cf. quinine hydrochloride and atropine, Table I). b) Elasticity The elasticity generally increased on application of the toxic substances, but it was to be observed only after the protoplasm underwent structural changes. c) ExtensihUity An increase in extensibility due to treatment was noted in protoplasm which had not coagulated. Quinine hydrochloride produced a rather uniform coagulum which possessed little extensibility. d) Tenftcious qualities There was an apparent relationship between extensibility and tenacity. A coagulum was rather difficult to tear and scarcely stretched. Protoplasm which had not coagulated throughout was less difficult to tear, the torn surface was not smooth and the extensibility was increased. SPECIFIC EFFECTS OF REAGENTS Cocaine hydrochloride SoLLMAN (HK3f)) described cocaine as a typical if rather weak, protoplasmic poison, paralyzing all sorts of cells, without producing any gross chemical changes. Cocaine in high concentrations, from 1—10 caused, first, liquefaction, and later i i i I ♦ S I .1* 1 i k 4. Some reactions of slime mould protoplasm to certain alkaloids and snake venoms 107 gelation. This resembles the death change in protoplasm reported by Chambers (11)17). The co-existence of apparently normal protoplasm side by side with dead matter was seen after cocaine had acted (Fig. 8). A mechanical barrier appeared to separate these regions. A 1—1000 solution pnxluced a reversible coagulation, which suggests the hy})othesis of Beknakd (1875) namely, that narcosis is a reversible coagulation of protoplasm. Fig. 1. Effects of Bothrop-s ofrnx, 1—20. a. TreaU'd strand showing ^'ratiule a<;s the death change in proto|)lasm reported by Chamhkks (11)17). The co-existence of a|)parently normal |>roto|)lasm side by side with dead matter was seen after cocaine had acted (Fig. S). A mechanical barrier appeared to separate these regions. A I — 1000 solution produced a reversible coagulation, which suggests the hypothesis of Bkknakd (1875) namely, that narcosis is a reversible coaguhition of protoplasm. Fi«i. I. Kffccts of liollno/is f,lro.r, 1-20. II. Treated strand showing' <:rannle ahate, I — 20. (f. Treated strand mto small groups and dispersion medium altered in appearance. IMg. 13. 1 ecuhar effects of Crotalus atrox venom, 1—200. a. dead matter alternating with knots of living matter (/>). c. venom particles. Chloral hydrate This compound was the only suh.stance of synthetic origin nse.! in this investigation: it is a nerve depressant an.l hypnotic. Chloral hydrate was used m concentrations ranging from 1-10 to 1-300. An acceleration of streaming «ith no other apparent toxic effects was pro.luced by a 1-3(M» solution. Although cocaine and chloral hy.lrate are both nerve depre.s.sants, their effects on slime mould protoi)lasm are not identical. Quinine 1)IX(,N and ,>E Pkkmankuu (1927) working with Paramecium, conehulod mat all quuiine derivatives are protoplasmic poisons. a) Quinine hydrochloride Quinine hydrochloride was distinctive in producing a more rigid coagulum li Table T ""^'"* *""'■ ^""' ^'"'™' '"'"*' "^ *'"'' ''"^«^"* ^^^ «'^«" r T ^ ^^^ \ b) Quinine sulphate This substance was used to c'onij)are the toxicity of different salts of tlie same alkaloid. The results indicate tliat tiie sulphate, in the same concentrations as the hydrochloride, is less toxic (Table 1). Several explanations are: 1. The hydrochloride contains from 7 to 8 per cent more of the quinine radicle, by weight. 2. The different anions may have niodified the effect: see Kainio (1921), Keznikoff and Chambers (n)27). 3. The dissociation constants of the saHs differ. Strychnine sulphate This alkaloid has been described as a weak protoplasmic poison on amoebae and yeasts. A 1 — KM) solution was found to be scarcely toxic. Strychinne, com- pared with the other alkaloids, is destructive to slime mould proto[)lasm only at relatively high concentrations (1—40). Syneresis is particularly characteristic of this compound (Fig. 0). Morphine sulphate This alkaloid has been said to be one of the weakest in its action. A con- centration of 1 — 100 produced no apparent effects. So high a concentration as 1 — 20 was not lethal. Data suggesting a fibrous structure for protoplasm were found in the following observations: The protoplasm, adjcU-ent to the treated area, moved away and hyaline fibril-like structures with the uranules arran«'ed in a linear fashion were produced (Fig. 18). This behavi(jr was also noted with untreated pr()t()f)lasm and can scarcely be credited to toxic effect. The response is apparently an evidence of irritability. Atrophine sulphate Atropine in a concentration of 1—500 did not give rise to any apparent effects. More concentrated solutions, to 1 — 100, [)roduce(l a hyaline band along the periphery of the protoplasm which appeared to result from syneresis. Atropine-treated protoplasm failed to coagulate" completely; the outer regions j)ossesse(l high viscosity whereas the internal matter had but moderate consistency. Outflow of the protoplasm occurs as the result of a break at the surface. Ephedrine hydrochloride Atropine and ephedrine are grouped together by pharmacologists. Kphedrine in a concentration of 1— oOO had no pronounced influence beyond a slight tem- porary reversible effect on streaming. A 1 — 1(H)0 solution caused no apparent effects. Ephedrine was similar to atropine in its influence on protoj)Iasm (Table I) Figs. 19, 21, 22). in com[)arable solutions (1 — 2(M) ephedrine influenced the inner protoplasm in such a manner that the microsomes failed to exhibit Brownian movement — indicating high viscosity — but atropine-treated protoplasm showed this motion — indicating low viscosity. 170 Lepow Table Substance used Cocaine hydro- chloride Chloralhydrate Quinine hydro- chloride Quinine sulphate Strychnine sul- phate Morphine sulphate Atropine sulphate Ephedrine hydio- chloride Bothrops atrox venom Crotalns atrox venom Minimum lethal concen- tration l^:ffcct on streamitifj; Svvellinj^ (sw.) and shrinking (sh.) 1— ICOO 1—200 1—500 1—250 1— 100 1—75 1—250 1—300 1—300 I-IO temporary stimulation, then cessation except for trace temporary stinmla- tion, then cessation ceased after several seconds ceased after one minute ceased after one minute cessation except for slug(ln,r „ ,.„||„i,|M| sus|K-iisi„n wlicn pla.v.l in distillcl water holhroi,. „ln,.r ,ans(.,l a transit.,ry sthnnlati.,n in streaming rate vvlion at a .•o,„-,.nfrati.,n of U-IOdd. Tl„. tiral,.,! n.-ion «as al,an,lon,..l l.v th. stroanun.- pn.toplasn, att,.,- twenty nnnutcs. A I- 200 suspension prodncr.l a r<.a.-fion sMn.lar to that reportci in tal.l,. I witi, the ,.x,-,.ption that traces of reeovorv «.Te note, „, isolate,! spots «h<.r.. th.. injnr.v »as apparently of sli-ht ..ons,'- <|n,.n,-e. .M„r,. .-oiKcntrat,.,! suspensions pr,„|nee,l irr.'versil.le ehan.'es \ f.' 1 : r i; J. . f '< . Sonic reactions of sliuu' iiu)ul(l protoplasm to certain alkaloids and snake venoms 17:^ h) Crotalus atrox Essex and Mahkowitz (1030) stated that tliis venom is a specific proto- plasmic poison which affects pr()to])lasm wherever it contacts it. A 1 20 sus- pension slowly influenced the i)eriphery, the protoplasm becoming less active although streaming did not cease (Fig. 15). Tlie treated regions were eventually abandoned by the active protoplasm. A j)eculiar reaction occurred in several experiments. There were alternating areas of apparently normal and dead protoplasm in the same strand (Fig. 13). This may be a different type of adjust- ment to the presence of injured protoplasm. It was not possible to note any pronounced changes in physical pro])erties where streaming had not ceased, but in the abandoned areas the i)r()toplasm was of butter-like consistency, scarcely extensible, showed little tenacity, and was moderately elastic. Description of Figs. 14 to 22 (p. 172) Fig. 14. Effects of chloral hydrate, 1—10. a. mottled coagulum resulting from granule clumping with hyaline areas. 6. an "artery" of streaining protoplasm : note granule aggregation m\ side bordering dead matter. Fig. 15. Fffeets of Crotalus atrox venom, 1 — 20. {icking of granules. Fig. 17. Fffects of ((uinine sulphate, 1—150. II. normal protoplasm. b. large granular aggregates, syneresis, and phase separation. Fig. IH. Strand treated with rcsorcinol, 1 — 10. (I. living matter showing fibrillar stnu-ture. 6. swelling and phase separation. Fig. 19. Effects of ephedrine hydrochloride, 1 — 10. a. ])hase separation and large granular groups. 6. living matter. Fig. 20. Effects of chloral hydrate, 1—10. n. slight swelling, hyaline were area and mottled apfK'arance. Fig. 2!. Effects of atropine sulphate, 1 — 10. a. large graiuilar aggregates and phase separation. b. living matter. c. sei)aration l)etween dead and living regions. Fig. 22. Effects of ephedrine hydrochloride, 1—10. (I. hyaline syncretic vesicles. 6. hyaline residue outflow with microsomes. c. phase separation. INTENTIONAL SECOND EXPOSURE 174 Lepow DISCUSSION a) Comparison of toxicity The greatest toxic effect on any reagent is evident by the production of a coagulum which fails to show phase separation. The latter response results when less concentrated solutions are used. Quinine hydrochloride appears to be the most toxic alkaloid since it produced the greatest toxic effect at a com- paratively low concentration. There is no relationship between toxicity and molecular weight of the poison. Members of the same group did not cause parallel degrees of destruction (e. g. the quinoline group, quinine and strychnine). Very similar effects were produced by atropine and ephedrine which belong to different chemical groups. The degree and type of dissociation is an important factor in toxicity of alkaloids according to Botazzt (1928). He noted that toxicity of alkaloids depends on particle size and the presence of undissociated molecules. While quinine is more destructive in its effects on protoplasm, and therefore in this sense more toxic, cocaine produces harmful effects at much lower concen- trations (e. g. 1 — 10,000 as compared with I — 500 for quinine). The morphine effects compared favorably with the data of other workers in being the least toxic of the alkaloids investigated. Slime mold protoplasm is more sensitive to the venom of Bothrops atrox than to that of Crotalus atrox. Philpott (1930) showed the same to be true for Paramecium. Githens (personal communication) noted similar results for the pigeon. Slime mold protoplasm is more sensitive to the alkaloids than to the venoms. Several possible reasons are: 1. The mechanism of action differs. 2. The venoms were used in the crude state and the concentrations were therefore not comparable. 3. If slime mold protoplasm is animal-like, then the suggestion of Macht (1930), that poisons of plant origin are more toxic for animals than poisons produced by plants, may be the explanation. 4. Slime mold protoplasm contains cholesterol (Seifriz, 1936), Phisalix (1897) observed that cholesterol exerts a detoxifying action on snake venoms. The last two suggestions re-emphasize a principle established by True (1930) for plants; namely, that the protoplasms of different species have different toxic equivalents because they are chemically dissimilar. h) Effects on protoplasmic structure The most significant structural change in this investigation is the separation of the protoplasm into the coagulated phase and the hyaline, fluid phase. Addoms (1927) reported Brownian movement in clear spacer between flocculent masses. This response closely resembles phase separation. Lepeschkin (1927) noted that poisons act on the dispersed protein phase when coagulation occurred. ScHACKELL (1923) investigating protoplasmic poisoning by phenols, assumed that a factor in the maintenance of dispersion of tissue proteins was the ad- F>7 ^ I ^ Some reactions of slime mould protoplasm to certain alkaloids and snake venoms 175 . sorption of lipins at the water-protein interface, the inference being that ( substances which interfere with the adsorption of these lipins would permit change m the dispersion of tissue proteins and therefore would be toxic Since coagulability is a characteristic of proteins and as these constitute the major portion of the dispersed phase, the occurrence of coagulation in proto- p asm indicates a change in the dispersion of proteins, an alteration in proto- plasmic structure, changed physical properties, and resulting toxic effects. The coagula may, as has been noted, be of several types. Changes in pH were detected by differences in color of the pigment The change was in all cases an increase in acidity which might result from the acidity of the solution, from injury, or from the reaction of the protoplasm to the solution Changes in pH are indicative of physical and chemical effects; therefore it appears that the view of Sollman regarding cocaine is untenable because he stated that this alkaloid produces no gross chemical effects. Seifriz (1918) pointed out that normal protoplasm is not miscible with water but that degenerated living matter is miscible. Chambers (1928) observed the dissipation of the inner matter of Echinoderm eggs in the surrounding medium The evidence obtained from abnormal outflow in slime molds indicates that the dispersion medium of protoplasm is an aqueous solution. There was further no evidence to indicate phase reversal such as occurs with emulsions. 4 f I « - « i I 10. The pigment in this organism is associated with the granules. 11. The results indicate that the micromanipulation of slime mold proto- plasm treated with toxic agents should be of value in pharmacodynamic studies. ACKNOWLEDGMENTS I wish to express my deepest appreciation to Professor William Seifriz for his critical supervision, impersonal criticism, and unfailing interest. I am also indebted to Professor Rodney H. True for extending to me the facilities of the micromanipulation section of the Botanical Laboratories. Botanical Laboratories University of Pennsylvania Philadelphia, Pennsylvania. LITERATURE CITED Addoms, R. M., The effect of the hydrogen ion on the protoplasm of the root hairs of wheat. Amer. Journ. Bot. 10, 211—220, 1923. — , Toxicity and protoplasmic structure. Amer. Journ. Bot. 14, 147 — 165, 1927. Balbach, H., Die Wirkung von Harnstoff-Gymnoplastcn. Protoplasma 26, 192 — 204, 1936. Barber, M. A., The effects on the protoplasm of NiteUa of various chemical substances and microorganisms introduced into the cavity of the living cell. Journ. Infect. Dis. », 117—129, 1911. Bayliss, W. M., Reversible gelation in living protoplasm. Proc. Roy. Soc. 91 B, 197 — 201, 1920. — , Principles of General Physiology. Longmans, Green and Co., 1924. B£la&, K., Dber die reversible Entmischung des lebenden Protoplasmas. Protoplasma 9. 209—244, 1930. Bernard, C, Le9ons sur les anaesthetiques et sur I'asphixie. Paris, 1875. BoTAZZi, F., The surface tension of colloids with special reference to protein colloids. Colloid Chemistry. Chemical Cayalogue Co., 1928. Camp, W. G., A method of cultivating myxomycete plasmodia. Bull. Torrey Bot. Club 63, 205—210, 1936. Chambers, R., The visible structure of cell protoplasm and death changes. Amer. Joum. Physiol. 42, 596—597, 1917. — , The physical structure of protoplasm as determined by microdissection and injection. Cowdry, Cytology, Univ. of Chicago Press, 1924. — , Action of electrolytes en physical state of protoplasm. Amer. Nat. 60, 121 — 123, 1926. — , The nature of the living cell as revealed by micromanipulation. Colloid Chemistry, Chemical Catalogue Co., 1928. Clark, A. J., Mode of action of drugs on cells. Williams and Wilkins Co., 1933. Ceane, M. M., The effect* of the hydrogen ion concentration on the toxicity of alkaloids for Paramecivm. Journ. Pharm. and Exp. Therap. 18, 319 — 339, 1921. Dixon, W. E., and Premankur De., The action of certain quinine derivatives with special reference to local anaesthesia and pulmonary oedema. Joum. Pharm. and Exp. Therap. 81, 407-^52, 1927. Essex, H. E., and Markowitz, J., The physiologic action of rattlesnake venom (Crotalin). IV. The effect on the lower forms of life. Amer. Journ. Physiol. 92, 342, 1930. Heilbrunn, L. v., The colloid chemistry of prottiplasm. Colloid Sym. Mon. 3, 136 — 151, 1925. • # * .V Some reactions of slime mould protoplasm to certain alkaloids and snake venoms 179 HoBER, R Physikalische Chemie der Zelle und der Gewebe. Engelmann, Leipzig, 1926. HOPKINS, H. S. Protoplasmic effects of papaverine, histamine and other drugs in relation to the theory of smooth muscle contraction. Amer. Journ. Physiol. 61, 551—561, Howard, F L., Laboratory cultivation of myxomycete plasmodia. Amer. Journ. Bot. 18, 624—629, 1931. Kahho H Zur Kenntnis der neutralen Salzwirkungen auf das Pflanzenplasma. II Biochem. Z. 120, 125—142, 1921. Lepeschkin, W. W., Some aspects of the cause of narcosis. Physiol. Zool. 6 (3), 479^90, Lecomte du NotJY, p., Considerations generates sur I'adsorption dans les ph^nom^nes biologiques. Extrait du Bull, de la Soc. de Chimie biologique 18 (6), 790—796, 1936 Macht, D. I., Contributions to phytopharmacology or the appUcations of plant phvsiology to medical problems. Science 71, 302—308, 1930. Phisalix, C, La cholesterine et les sels biliares vaccins chimiques du venin de vipere Compt rend. 126, 1053—1055, 1897. ' Philpott, J., Effects of toxins and venoms on Protozoa. Journ. Exp. Zool. 56, 167 1930 Reznikoff, p., and Chambers, R., Micrurgical studies in cell physiology. III. The action of CO2 and some salts of Na, Ca, and K on the protoplasm of Amoeba dubia. Journ Gen. Physiol. 10, 731—738, 1930. SCARTH, G. W., and Lloyd, F. E., Elementary Course in General Physiology. John Wilev and Sons, 1930. ^ e. j Seifriz, W., Observations on the structure of protoplasm by microdissection. Biol Bull 34, 307—324, 1918. — , Viscosity values of protoplasm as determined by microdissection. Bot. Gaz 70 l&) 360—386, 1920. • v ;, — , The effects of acids and alkalies on the viscous and structural properties of protoplasm Physics 6, 159—161, 1935. — , The structure of protoplasm. Bull, de la Soc. de Biologic de Lettonie 6, 87—99, 1936a. — , Protoplasm. McGraw Hill Co., 1936. — , Elasticity as an indicator of protoplasmic structure. Amer. Nat. 60, 124—136, 1926. — , and Zetzmann, M., A slime mould pigment as indicator of acidity. Protoplasma 28 176—179, 1935. Shackell, L. F., Studies in protoplasmic poisoning. I. Phenols. Joum. Gen. Phvsiol 6, 783—806, 1923. True, R. H., The toxicity of molecules and ions. Proc. Amer. Philos. Soc. 69 (4) 231—245 1930. 12* Reprinted from Plant Physiology, 12: 117-133, 1937. •■I! if • f» ^/^ IRREGULAR PAGINATION EFFECTS OF X-RAYS ON ZEA MAYS Mary A . Russell (with eight figures) Introduction Since the discovery of x-rays, their effects on both plants and animals have been studied as important biological problems. Many reports of early work are conflicting because quantitative results have been possible only in recent years, after methods of accurately measuring the dosage were per- In the study of many general problems, plants are ideal material because ot the large numbers which can be used and the ease with which environ- mental conditions can be modified or kept constant as occasion demands, bamples can be taken for cytological studies as the experiment progresses without detracting from the final results. Numerous reports on plant ex- periments have come from hospital laboratories where the work has been done with the conviction that many of the results will be applicable to prob- lems of animal x-ray technique. The work reported here was started for the purpose of finding out how Zea mays would react to x-ray treatment in various aspects of its growth and appearance, in order to use it eventually as a test material for studying effects of irradiation as they might be influenced by changing environmental condi- tions. Zea mays has been used occasionally for special radiation problems Stadler (14) found it valuable for studies of mutations produced by x-rays because its genes had been more thoroughly mapped than those of many other plants. Bersa (2) reported Zea mays to be relatively more resistant to irradiation than many plants which he tested. He used it extensively for cytological examination of irradiated root tips. Materials and methods The source of radiation was a standard air-cooled Coolidge tube. A con- stant potential of 180 kv. was used and the filament current was maintained at 3.7 ma. The distance from the center of the target to the material to be irradiated was 30 cm. The only filters were the heavy pyrex glass of the tube and the paper over the seedlings. The actual strength of the x-rays used was measured during radiation by the usual ionization-galvanometer ar- rangement, the ionization chamber having been calibrated previously with a standard chamber as described by Taylor and Singer (17). The calibra- tion of the apparatus gave a factor of 1.7 r units per centimeter of deflection of the galvanometer. Thus at a deflection of 15 cm. for 40 minutes, the dose 117 s - 118 PLANT PHYSIOLOGY would be 1.7 X 15 X 40 or 1020 r. The Roentgen unit or "r" was defined at the Second International Congress of Radiology in 1928 as folZ ' ' the and the wall effect of the chamber is avoided, produces in one cubic cm of a mosphenc aar at 0° C. and 76 cm. mercury pressure such a degree of con rent '- ae) "' "'"'*'*" ""* "' "'''''' " "^^^"^^'^ '' ^^*"-" ^ -" Improved Golden Dent field corn was used in all experiments The drv g aans were planted between layers of moist paper toweling inrfass chambers placed in a dark incubator at 24° C After 72 hn„r« Z.f * tf «*»a"»bers roots were slightly more than 1 cm. lonrThl^'rrwT ^XeTi^ ItoiS" ^^ '^'""'''" '" *^^ ^^^"'"" ^* ^"""t *"« «ame houi in order o avoid differences in sensitivity due to possible natural mitotic rhythm These A Tnf 'fT T" f ''*"^ "'^''^ ^^^ ^^^^y ^t^^'g'^t roots 10 to 15 mm long tjZ /^ ""' "'''"' '^"''y ^" '"'«• fr^™ tl'e tip of ea^h root and all future measurements were made from this point. In most experi ments 20 seedlings were used in each group. The plants to be treated were individual y placed on damp filter paper over the bottoms of glas diles The side of the grain where the shoot was about to emerge was aSys P aeed up in order that all of the embryos might be equally exposed to T™ The dishes were closely covered with three layers of moist Scott Tissue towe' mg to prevent evaporation from the seedlings during radiat on The con trols were kept under the same conditions outside of the x-ray apparatus Immediately after treatment the seedlings were placed eitheMn noK „f damp earth for observation in the greenhouse or in7ar^ o oo" ^ Pa ked sphagnum to be put into the incubators for root study. The irradiated p ants grown in soil had the normal greenhouse conditions of light and tem perature A large number of pots were prepared at one time with the slme lot of soil to insure uniformity of nutrition. Results Effect op x-bats on aerial parts of Zba mats The following observations were made on a typical series raised in th» ofZl'A / ? ^^ ^^^""^ "^"^ "" •> I 40 36 43 39 47 45 63 68 67 96 97 42 66 96.5 mm. 112 106 82 73 ]29 115 95 91 104 108 104 96 90 99 119 122 100 mm. (mean) 112 94 104 98 103 122 with increasing temperatures the time required for the appearance of lateral roots decreases, while the length of the primary root at this time is very constant. For instance, in a group raised at 22° C, after the usual germina- tion period at 24° C, the lateral roots appeared 97 hours after the first measurement. In another group raised at 85° C. the rootlets appeared after 48 hours, the average length of the primary roots being the same in each case. Figure 2 shows the very regular rate of decrease in time necessary for the appearance of rootlets as the temperature increases. t .1 \ >i 1 ^ If 1^ i t i RUSSELL: EFFECTS OF X-RAYS OX ZEA MAYS 123 100 90 SO 70 60 50 If) i 40 O z Z 30 UJ 2 20 I- (0 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 TEMPERATURE IN DECREES C Fig. 2. Effect of increase in temperature on time required for appearance of secon- dary roots on seedlings of Zca mai/s. Careful examination of the control seedlings showed that the rootlets first appeared on the primary root immediately below the grain in all cases, no matter how long their appearance was delayed by cold and the slow growth •^- y uiiUjj 's'!,>i'|r I I'll '* •>-. . Fig. 3. 1500 r. Section showing abnormal growth of secondary root after an x-ray dose of INTENTIONAL SECOND EXPOSURE 124 PLANT PHYSIOLOGY accompanying it. It can be seen from column VI of table II that, when doses of 300 r and more were used, the average distance of the first lateral root from the grain increased from 6 mm. to 31 mm. after a dose of 1500 r. The roots grown in soil showed this same type of behavior after irradiation. With the higher doses the primary root had only a tassel of rootlets very close to its tip. Histological sections made from roots which had received a dose of 1500 r showed primordia of lateral roots in the region just above the level where rootlets were growing out in a normal manner. In some cases these primordial rootlets were subnormal in size and in a few examples they had turned either up or down, growing parallel to the longitudinal axis of the primary root, and they were entirely inclosed within its tissue. Figure 3 illustrates this condition. Effect of temperature on growth after x-ray treatment Experiments performed to test the effect produced by keeping the corn seedlings at 6° C. during irradiation with doses of 200 to 1000 r and then allowing them to grow at 24° C. showed no differences between such treated seedlings and those similarly irradiated at room temperature. Following the failure to produce differences in sensitivity to x-radiation by means of low temperature during treatment, a number of tests were made to find out whether varying the temperature after x-ray treatment would affect the amount of injury due to irradiation. Since corn was found to thrive at temperatures of 17° to 36° C, it was possible to observe the behavior of roots whose growth rates varied widely according to the temperature selected. In the first experiment three groups of seedlings were irradiated at the same time with 2000 r, and then the separate lots were placed, with their controls, in three incubators at varying temperatures. Care was taken to measure the growth at very frequent intervals. Figure 4 shows the curves made by the 2000 R 30 40 SO 60 70 TIME IN HOURS 60 90 100 Fig. 4. Curves of growth at three temperatures after equal amounts of radiation. Length of life increases as temperature decreases. RUSSELL: EFFECTS OF X-RAYS ON ZEA MAYS 125 ' l f" growth of the primary roots of these seedlings exposed to 36°, 24°, and 17-21° C. after all were irradiated under exactly the same conditions. The shape of the curves is very significant, showing that the time of death was 90 60 *) 70 tt Li u 60 2 -I d 50 2 Z 40 Z 30 O u 20 K) 24* 1500 _R -5 ^-H. _241.20pO_R 10 20 30 40 50 60 70 •l«i 10 20 30 40 50 60 70 60 90 TIME IN HOURS Fig. 5. Growth curves showing effects of different temperatures after doses of 1500 and 2000 r. Increasing doses decrease the latent period and the effect is exaggerated by increase in temperature. \Wh 20 30 40 50 60 70 80 90 100 TIME IN HOURS Fig. 6. Growth curves of primary roots of Zca mays showing effect of exposure to 24° and 36° C. after a dose of 800 r. At 20 hours the irradiated group at 24° C. shows no injury while at 36° C. roots are 4 mm. shorter than their controls. 126 PLANT PHYSIOLOGY O 20 30 40 50 60 70 Fig. 7. Curves showing comparative effects of exposure to 24° and 36° C. after exposure to various doses of x-rays. delayed as the temperature decreased. Similar experiments were performed by repeating the dose of 2000 r and also using 1500 and 800 r, in each case exposing part of the irradiated group to 24° and part to 36° C. with their respective controls at each temperature. Figure 5 shows that after both the 1500 and 2000 r treatments, the roots grown at 24° lived longer than those in the group at 36° C. Growth after 1500 r extends over a longer period of time at the lower temperature, and, also, the roots reach a considerably greater length before death when grown at 24° than at 36° C. Figure 6 shows that when the dose is lowered to 800 r, the irradiated groups grown at 120 100 90 u ao UJ J 2 60 Z 50 X O 40 z u -" 30 20 10 / / 4 / 24" 2000 R i / T 10 ao 30 40 50 TIME 60 70 10 IN HOURS 20 30 40 50 Fia. 8. Growth curves showing effect of temperature on length of latent period after an x-ray dose of 2000 r. «Va RUSSELL: EFFECTS OF X-RAYS ON ZEA MAYS 127 the two temperatures grow roughly parallel to each other, the 36° group growing the faster just as the 36° controls grow faster than the 24° controls. In figure 7 the curves of the growth following the three doses have been rear- ranged to show more graphically the effect of the two temperatures on each. The curves in figures 4, 5, 6, and 8 show that with all the doses used, the latent period was longer at the lower temperature. As the dose increases, the difference between the temperature effects is increased. Discussion The coleoptile was the only part of the corn seedling which failed to show any effects of irradiation. Cattell (3) reported it to be the least sensitive part of wheat, as after a dose of 1200 r the coleoptiles showed a reduction of 8 per cent, in height while the primary roots were reduced to 45 per cent, of the length of the controls. It would appear that the coleoptile of corn is less sensitive to irradiation than the coleoptile of wheat. The apparent lack of sensitivity to x-rays of this organ may be partly explained by the results of cytological studies of Zea mays by Tetley and Priestley (18) . They found that cell division stops in the coleoptile earlier than in any other part of the embryo. The fact that cell division has probably ceased long before exposure to x-rays may account for this organ showing no injury after doses which seriously interfere with the growth of parts of the seedling in which there has been rapid cell division at the time of irradiation. Skoog (13) , using moving pictures to study the behavior of the coleoptile in Avena, found that its growth stopped when it was ruptured by the shoot pushing through it. If it is natural for the growth to continue only as long as the sheath is intact, and if the rupture is delayed when the shoot is stunted by irradiation, then this tendency towards excess growth in height may mask the effect of x-ray treatment on the corn coleoptile. The fact that the coleop- tiles of treated corn seedlings were a trifle longer than those of the controls may indicate that the stunting effect of x-rays was a bit less influential than the opposite tendency towards increased growth. The chlorotic disturbance noted in the corn leaves is typical of the results reported for many species. Noguchi (10) examined sections of leaves from irradiated seedlings of Helianthus annuns and found that the light color was due both to a reduction in the number of chloroplasts and a deficiency in their chlorophyll content. The fact that irradiation in any amount between 4000 to 10,000 r failed to produce more stunting of the primary root than did 4000 r, shows that corn responds to x-rays in this respect in very much the same manner as does wheat. Francis (5) reported that doses increasing from 3390 to 13,000 r fail to produce any increase in damage to the seedlings either in affecting the growth or in reducing the rate of respiration. Many workers have tried in vain to find a dose of x-rays strong enough to kill the plant immediately. 128 PLANT PHYSIOLOGY _ In an early experiment, where it was necessary to compare the effects of ondi'tSnTthe?'' T"' "' "^'""^ "'^''^'^ '^^'^ "^^ ^-^^^ under diSre. Ttt star tint li Hh 'T^""°" "'' '^''°''" "'^'^ ''°*'^ ^"-""P^ '^^d lateral oth r oitS • °"' ''* ^"■' *'"'**'^"' ''«"«'°P«'^ than those of the other. Quantitative measurements were made at first by carefully euttin-- off the rootlets, obtaining the dry weights of those of the two groups and hen computing the average weight of the lateral roots of each gr^7 Tht laborious method was discarded when further study showed that small' diffe" by noting the time at which lateral roots appeared and also by recordin-^ the d, tance below the grain at which the first rootlets appeared. The u^e of the time factor is the more exact of the two methods It is necessary to decide on a given proportion, such as one-third, of the plants to be requLd to show rootlets before the time of appearance is recorded. This helps to prevent mistakes being made when the group contains one or more freak plants which, for some unknown reason, are unusually resistZt Sue J individuals have been omitted in computing averages when they were few n number and extremely different from most of the group When preliminary experiments have been carried out at the same tem- perature and near the dose to be used, the time interval during whTh Zy frequent observations are necessary to establish the time of appearance of rootlets can be fairly accurately predicted. In order to compare the sensTtiv ity of two groups of corn seedlings, a dose of 300 r would have th '"2 teJe' of yielding data both on the time of appearance and the spacing ofTter;! roots below the grain, and it would furnish this information i^ a shorte time than vv-ou.d be required for the same observations after higher dose" WiGODER and Pattek (19) ob.served that lateral roots of Vicia fabaTre delay d in the time of their appearance and were formed more and more distany from the seed as the dose of x-rays was increased. It I therefore possible that this type of reaction could be expected in many kinds of p am and could be used as a general method for comparing sensiliv ity to rrldia tion between groups of the same species. The fact that the appearance of the first lateral roots is delayed bv irradia tion might indicate that time is required for the repair of cLtaL Im "e before these roots can be formed in a normal way. The few se tL wh^fh were prepared from seedlings which had been irradiated wUh 1500 r showS no indications of lateral roots being formed in the region just below the seed t'^onL7'Z K "■*'""■ '""'^ *""•' ^'^^^ a''ortive\ttempts al lit S forniation followed by a region producing apparently normal rootlets Since irradiation is generally considered to have an aging effect on tTssues thP failure of the lateral roots to reach the exterior alonf the upper part of It root might be due to the same type of conditions which prL'eiZeeolry RUSSELL: EFFECTS OF X-RAYS ON ZEA MAYS 129 '4 » 4 root formation m old, tough roots. Further study may determine whether the rootlets ni the x-rayed material changed the direction of their growth because of the mechanical difficulty of penetrating the outer layers of the primary root. In normal seedlings there was a quite definite relation between the length of the primary roots and the time of appearance of lateral roots, but this relationship was upset in the irradiated groups. Here the rootlets tended to appear while the primary root was relatively shorter, but if the plant's main root never reached a length of 60-70 mm. it wa^ unlikely that any rootlets would be formed. It may be possible that as the plant begins to suffer from the slow growth of the x-rayed primary root, the lateral roots tend to appear earlier in proportion to the length of the main root as an attempt at com- pensation for the lack of absorbing power of the main root. Johnson (7) reported that irradiated seedlings of Helianthus annum lack ability to absorb as much water as their controls. This condition is all the more serious for the plant, because the aerial parts, not being stunted by irradiation to the same extent as the roots, are in need of a supply of water approaching that of a normal plant. There are reports of several experiments on the effect of irradiating living material at low temperatures. Crabtree and Cramer (4) treated" mouse tumor with radium at 0° C. and found that its sensitivity to irradiation was greater than when it was treated at room temperature. Mottram (9) obtained similar results, using Yida faha (a very small number of plants) treated with both radium and x-rays. On the other hand, Henshaw and Francis (6) failed to find any difference in the sensitivity of wheat seedlings when irradiated at different temperatures. The same type of experiment was tried in this laboratory with corn, with the idea that this plant, being more sensitive to cold than wheat, might give somewhat different results. The findings were negative. It was found that growing corn seedlings at different temperatures fol- lowing irradiation produced differences in the length of the latent period, the ones grown at the higher temperature showing the damaging effects sooner than those which were allowed to grow more slowly at the lower temperature. This held true for groups of roots which had received a dose of 800 r and grew in a manner very nearly like the normal. The effect on the latent period became more and more noticeable as the dose was increased to 2000 r. It would be of interest to test higher doses to determine whether a stage could be reached where the damage done by irradiation would be so great that a comparatively low temperature could have no effect towards lessening the amount of injury. With all biological material the time required for the effects of irradiation to appear varies according to the dosage, the type of organism, and probably 130 PLANT PHYSIOLOGY many other facto.;s. It is generally considered that biological changes are he result of chemical changes set in motion by ionization during the actull nne of arrad.ation Conditions which would speed up or modify' the ehemt re uirof r Z :T''^ *° ''""''''' '''' '^*^«* P^™'^ -1"-ed for the results of the biological changes to appear. Heat increases the speed of most mer«rrr '"'' ''''-' " *°"^" ''' '^*-* ^-^°^ ^' *^« -p-i- Although irradiated seedlings grown at 36° C. begin to show the effects 0 irradiation at an early age, they grow fairly rapidly until they suddenly die. When groups of irradiated plants grown at successively lower tem- peratures are compared, it appears that the slower the plants grow, the longer they are likely to live. However, the dose of 2000 r has set a lim t perlial within a range of 6 mm., to the amount of growth possible following ha amount of irradiation. The seedlings grown at each temperature group dL when their primary roots have reached this limit no matter how long a time IS required lor each group to grow roots of that length. This limit may be determmed by any one of several factors or a combination of them. A given amount of food material may be available for the building of new root tissue and irradiation may prevent the formation or transportation of more o tha substance after the original supply is exhausted. If the root is grow W rapidly, it will use up its supply and die sooner than a .slow-growingroot which uses up Its food more slowly. This theory might help explain why the primary root can grow to the same length, when either 4000 or 10 000 r have been apphed. If there is the same amount of food material ava:aable 10 oTr dot' """T "' "?' ^"^ '"^ ' '"^ '^ ^-t - ««-«- a th 10^00 r dose in preventing any further growth after the original supply is exhausted. As the dose was reduced below 4000 r this food formation was apparently less and less disturbed. Since growth of the plant as a wh^e proceeds quite normally after a dose of 800 r, it may be assumed th^t thire early recovery if this amount of irradiation interferes with the food supply Another cause of death may be the accumulation of mildly toxfe substances until they interfere with natural physiologic processes or at products as their decomposition proceeds. Either of these conditions would be aggravated and cause earlier death with increase in temperature anT^e ttT 7?/ ■"''t^bolism accompanying it. The difference between the ecet n*; 00 r't' b^f'/* ''"^T' ^^P^*"- - the case of pllit recening 800 r might be due to the difference in the rate at which toxic materials were formed. It is possible that some of these poisonous substance may diffuse out; this would help explain part of the recovery whlh takes place when the doses of radiation are not too heavy The comparative lack of irradiation injury shown in the early stages by plants grown at 24° C. and below might be due to the time elemeL of dilay RUSSELL: EFFECTS OF X-RAYS OX ZEA MAYS 131 made possible by the use of lower temperatures. Several authors have tested the effect of allowing time to elapse between irradiation and the beginning of growth processes following it. Strangeways and Fell (15) reported that when chick embryos were kept in the refrigerator for 24 hours after irradia- tion and then allowed to grow in incubators at their normal temperature, the injury due to irradiation was less than the injury suffered by embryos similarly irradiated but allowed to continue their normal growth rate imme- diately following exposure to x-rays. There may be a certain amount of recovery going on during the period before growth is resumed. After living material has been irradiated there is a certain amount of residual effect which continues at a steadily decreasing rate. According to Kinqery (8), the rate of decrease of this effect with time follows a loga- rithmic curve. If growth processes are delayed for a time, the amount of residual effect will be growing less and less in the meantime. Therefore, when the rate of metabolism is allowed to return to normal there will be less damage done to the cells which are again beginning to divide than would occur if they continued dividing immediately after the irradiation period. In like manner, the long, slow growth made possible by low temperature could be assumed to reduce radiation injury by allowing more time for the reduction of the amount of residual radiation. Packard (1^) performed a set of experiments on Drosophila eggs which very nearly parallel the tests reported here on growing corn seedlings at different temperatures following irradiation. After the same dose of x-rays, groups of eggs which were incubated at 18° C. hatched in 80 per cent, of the cases, while in the group kept at 28° C. only 53 per cent, hatched. Low temperatures after irradiation also reduced the amount of injury shown by chick embryos in experiments reported by Ancel and Vintemberger (1). The fact that corn seedlings, Drosophila eggs, and chick embryos all react to temperature effects after irradiation in the same general way would seem to indicate that temperature during this period controls one or more funda- mental conditions involved in determining the ai)iount of irradiation injury shown by the living organism. Summary Seedlings of field corn were x-rayed with doses of 60 to 10,000 r and their subsequent growth observed under varying conditions. The results are as follows : 1. The height of the coleoptile was not affected by doses up to 5000 r which was the highest dose used in these particular experiments. 2. Shoots of plants which received over 800 r showed the effects of irradia- tion by delay in bursting through the coleoptile and by failure to reach the height attained by their controls during the time of the experiment. » -0 r 132 PLANT PHYSIOLOGY RUSSELL: EFFECTS OF X-RAYS ON ZEA MAYS 133 3. All of the greenhouse plants irradiated with 200 r and more showed a ehlorotic disturbance in the early leaves. 4. Leaves of all plants which received 800 r, and the small number of sur- vivors from the 1200 r group, showed a strong tendency to curl their edges toward the under surface. 5. The rootlets growing laterally from the primary root were the most sensitive part of the seedlings. Doses of 75 r and higher delayed the appear- ance of these secondary roots increasingly as the dose was raised, while doses of 300 r and higher increased the distance between the grain and the first of these roots. Comparison of the time of appearance and spacing of these secondary roots is suggested as a method for comparing the degree of sensi- tivity to x-rays of various groups of seedlings. 6. Doses up to 10,000 r failed to prevent at least 20 mm. of root growth after radiation was applied. 7. Plants irradiated with doses between 800 and 2000 r showed a much shorter latent period before x-ray eifects were measurable when grown at 36° C. after treatment than when grown at 24° C. and lower temperatures. 8. After a dose of 800 r corn seedlings showed the same amount of injury whether they were grown at 24° or 36° C. After 1500 r the 24° group grew faster than the 36° group after 50 hours, and the roots attained considerably greater length during their longer life. After doses of 2000 r the 24° groups surpassed the 36° groups after 35 hours and lived longer, but after this dose the final length was never more than 9 mm. greater than that of the 36° group. On the whole, lower temperatures after irradiation tended to reduce the amount of radiation injury. The writer wishes to thank Professor William Seifriz for his interest and helpful criticism. Dr. Ellice McDonald for the use of the x-ray equipment and other laboratory faciilties of the Biochemical Research Foundation of the Franklin Institute, and Professor Rodney H. True for the use of the greenhouses of the Department of Botany of the University of Pennsylvania. University of Pennsylvania Philadelphia, Pennsylvania 1. 2. LITERATURE CITED Ancel, p., and Vintemberger, P. Influence de 1 'activite cellulaire sur la manifestation des lesions produits dans le blastoderme de I'oeuf de poule par les rayons X. Compt. Rend. Soc. Biol. 91 : 1425-1428 1924. Bersa, E. Strahlenbiologische Untersuchung. II. Ueber die Wirkung der Roentgenstrahlen auf die Kernteilung der Wurtzelspitzen von Zea mays. Wien Akad. wiss. Sitzungsber. Math.-Naturw. Kl. Abt 1136:383-401. 1927. #1 y f>|? 4»i KWm / ^^^ 6. 7. 3. Cattell, W. The effects of x-rays on the growth of wheat seedlings. Science n. s. 73 : 531-533. 1931. 4. Crabtree, H. G., and Cramer, W. The action of radium on cancer cells. II. Some factors determining the susceptibility of cancer cells to radium. Proc. Roy. Soc. London B 113 : 238-250. 1933. 5. Francis, Dorothy S. The effects of x-rays on growth and respiration of wheat seedlings. Bull. Torrey Bot. Club 61 : 119-153. 1934. Henshaw, P. S., and Francis, D. S. Growth rate and radiosensitivity in Triticum vulgare. Jour. Cell. & Comp. Physiol. 4: 111-122. 1933. Johnson, E. L. Effects of x-rays upon growth, development, and oxi- dizing enzymes of Helianthus amiims. Bot. Gaz. 82: 373-402 1926. 8. KiNGERY, L. B. Saturation in roentgen therapy; its estimation and maintenance. Arch. Dermat. & Syphilol. 1 : 423-^33. 1920. 9. Mottram, J. C. On the alteration in the sensitivity of cells towards radiation produced by cold and by anaerobiosis. Brit. Jour. Radiol. 8 : 32-39. 1935. 10. NoGUCHi, Y. Modifications of leaf structure by x-rays. Plant Physiol. 10 : 753-762. 1935. 11. Packard, C. The relation between division rate and the radio-sensitiv- ity of cells. Jour. Cancer Res. 14 : 359-369. 1930. 12. Schwartz, G. tJber die Latenz-Zeit. Acta Radiologica 7: 452-460. 1926. 13. Skoog, F. The effect of x-irradiation on auxin and plant growth. Jour. Cell. & Comp. Physiol. 7 : 227-270. 1935. 14. Stabler, L. J. The frequency of mutation of specific genes in maize. Anat. Rec. 47 : 381. 1930. 15. Strangeways, T. S. P., and Fell, H. B. A study of the direct and indirect action of x-rays upon the tissues of the embryonic fowl. Proc. Roy. Soc. London B 102 : 9-29. 1927. 16. Taylor, L. S. The precise measurement of x-ray dosage. Bureau Standards Jour. Res. 2 : 771-785. 1929. 17. Taylor, L. S., and Singer, G. An improved form of standard ioniza- tion chamber. Radiology 15: 637-646. 1930. 18. Tetley, Ursula, and Priestley, J. H. The histology of the coleoptile in relation to its phototropic response. New Phytol. 26 : 171-186. 1927. 19. WiGODER, S. B., and Patten, R. E. P. Variations in the growth of irradiated bean roots. Brit. Jour. Radiology 2 : 588-596. 1929. Reprinted from Plant Physiology, 12: 99-llG, 1937. 1, < > ^ \ > ». I 4 METHODS OF KESEARCH ON THE PHYSICAL PROPERTIES OF PROTOPLASM^ William Seifriz (with five figures) Much excellent research has been done with a test tube and a Bunsen burner, but certain problems cannot be successfully attacked without the aid of intricate apparatus. It is the latter type of research, in so far as it applies to studies on the physical properties of protoplasm, with which this report deals. Obviously, the first instrument with which the student of protoplasm must become familiar is the microscope. It had its beginning in the primi- tive use of lenses by the ancients. In the first book devoted exclusively to microscopical studies, *'Micrographia,'' which was written by Hooke in 1660, his compound microscope is illustrated (fig. 1). It is not within the scope of this report to present a detailed description of the compound microscope. A knowledge of this instrument is extremely useful and it is suggested that the diagrams and descriptions of their opti- cal systems appearing in the catalogues of leading microscope manufac- turers be studied. The microscope has a limit fixed, not by the magnifying power of lenses, but by the wave length of light and other factors which determine its resolv- ing power. The highest powers of the microscope reveal objects which are about 0.1 |j (0.0001 millimeter) in size.^ Greater magnification could be obtained, but it would be of no use, because it would then be impossible to distinguish between two particles or lines which are closer together than the length of the wave of light used. It is not greater magnification, but 1 One of the purposes of this committee is to make available those methods of physical measurements which have proven useful and have passed their preliminary experimental stages. However, all methods should be regarded as steps in the progress of research which undoubtedly will be modified and improved from time to time as new facts are discovered. Obviously these reports cannot be exhaustive treatments. They are intended to point out fundamental principles and include a bibliography which should serve as a starting point for those interested in a more comprehensive study. In the present report of the physical methods committee, organized for the committee by Dr. SEirEiz, the immediate objective has been to discuss briefly the approved methods and apparatus used in determining the physical properties of protoplasm and cell structure. — Earl S. Johns- ton, Chairman. 2 \i = micron = 0.001 millimeter m^i = millimicron = 0.001 micron nil = micromicron = 0.001 millimicron A = Angstrom unit = 0.1 millimicron \Hi is often erroneously used for mji. 99 IRREGULAR PAGINATION 100 PLANT PHYSIOLOGY Fig. 1. The microscope of Robert Hooke (1660) AJS. greater resolving power that is needed. Resolving power is the capacity of a lens to separate one line or point from another lying very close to it. Let us take a specific case: A diffraction grating, as used for the formation of spectra, may contain 1000 lines to a millimeter. Microscope lenses of the highest power easily distinguish these as individual lines clearly separated from each other. But ,f there are two lines where there was but one before, then each line will be separated from its neighbor by 0.5 p. It would, in this case, be barely possible to distinguish individual lines if the microscope lenses and the eye of the observer were of the best. We can go no further with direct vision. As a result, efforts to increase the powers of the micro- scope have of late been directed toward methods of illumination. Illumination wTT^i/""^ °^f ^ V r ^^' P'""""" °^ microscopic illumination have been followed ; either light of other wave lengths is used, or white light is applied in differen ways. The light, other than composite white light so far most successfully used is ultraviolet. The ultraviolet microscope «nZt' "'1^''"^ """"^ °' ^ '""' '^ proportional to its numerical aperture and inversely proportional to the wave length of the light used. In other '/ ^l^- SEIFRIZ : PHYSICAL METHODS OF RESEARCH ON PROTOPLASM 101 Tr^L*^! ^'■^^*^'* *^^ numerical aperture and the shorter the wave length o± light, the smaller the structure which can be imaged by the lens. Written m the form of an equation : Smallest structure .^Vhl^-^^^^ length of light numerical aperture The average length of a wave of white light is 0.55 p (5500 A) ; accordingly, a lens with a numerical aperture of 0.65 will reveal an object— p or 0.85 p in size. This means that if the lens is used to examine para^llel lines 0.5 p apart, the image produced would not contain separate lines, the struc- ture would not be resolved, and higher magnification would be of no avail. The resolving power of a lens increases with the angle at which light enters it, and with the refractive index of the medium intervening between the lens and the object viewed. The increase due to the angle of the enter- ing light IS proportional to the sine of i of this angle. If the maximum angle at which light can enter is 80°, the sine of * of 80°, or 40°, is 0 64 If the medium between lens and object is water with a refractive index of 1.33, the resolving power will be 33 per cent, greater than if it is air, the refractive index of which is 1.00 ; and if the medium is cedar-wood oil with a refractive index of 1.51, the resolving power will be 51 per cent, greater than m the case of air. Abbe, German optician, introduced the term ''nu- merical aperture" (abbreviated N.A.) to include both of these factors. The numerical aperture is the product of the sine of i the maximum angle at which light enters the objective, and the index of refraction of the medium between the front lens of the object and the cover glass. To in- crease the resolving power of a lens, we can (as the above formula shows) decrease the wave length of light, or increase the numerical aperture. The latter has been done by lens manufacturers as far as practicable, with 1 6 as the upper limit. As the numerical aperture is increased, chromatic and spherical corrections are more difficult, and the working distance of the lens becomes less. We then turn to the possibility of using light of shorter wave length. Blue light is shorter than the average (yellow) light of the sun. It should, therefore, increase the resolving power of lenses. Ultra- violet light, having shorter wave lengths than the blue, should serve the purpose still better. For this reason ultraviolet has been used, and the method has given rise to the ultraviolet microacope. ^ By using ultraviolet light with a wave length of 275 mp, as compared with 550 mp, the average wave length of white light, it is possible practi- cally to double the resolving power of a lens. The theoretical limit of the resolving power of an objective of numerical aperture 1.40, is 0 16 p for a wave length of 450 mp, and 0.13 p for a wave length of 365 mp. This theo- 102 PLANT PHYSIOLOGY retioal value of 0.13 p is only slightly above the lower limit of microscopic visibility. An objective of numerical aperture 1.40, with the 365 mu ultra- violet spectral line (of mercury) will give 19 per cent, more resolving power than the best microscopic system with white light. But there are difficulties Glass IS practically opaque to light waves of less than 300 mp, i.e., to ultra- violet light; furthermore, such light is invisible to the human eye The first difBculty is overcome by using quartz lenses, and the second by sub- stituting the photographic plate for the human eye. The ultraviolet microscope was presented to the biological world some thirty years ago by Kohler of the Zeiss scientific staff at Jena. The instru- ment has a potential resolving power twice that of the best optical systems using visible light, which means that it gives twice as much detail as do the best lenses with ordinary light. It also gives greater optical differentiation ^.e greater contrast, for example, between the translucent, glass-like parts ot a cell. It thus obviates the need of staining protoplasm, which is diffi- cult of accomplishment in living matter. The method was originaUy in- tended for metallographic studies, but in spite of a promising future, little ontstanding work was done with the ultraviolet microscope. Lucas (14) of the Bell Telephone Laboratories in New York, has revived its use. Ultraviolet light of a known wave length is obtained by passing light from a suitable source, such as a spark between cadmium or magnesium electrodes, through a quartz prism which resolves or breaks it up into its component wave lengths. Ultraviolet rays of about 275 mji wave lengths are selected. As these are invisible, the optical image cannot be seen in the ordinary way. It must either be photographed or visualized on a fluores- cent screen. The fluorescent screen commonly used is made of uranium glass, and is mounted interchangeably with a camera just above the micro- scope. Magnifications of 5000 diameters result in sharp brilliant images with a degree of resolution surpassing by far that achieved with any other known optical system. With such an equipment, Lucas has taken some excellent photographs of living cells. Any wave length shorter than the average of white light will increase .^.-rfr^ •'';'?' f I ''°'- Consequently, light just leyond the visible CIV." Til 'I *''' 'P'"*™"" (*'"' ^'^'"^ ^P*^*™"' «toP« at about 390 mp) should be better than white light, and it retains the advantage of white light in passing through glass. Ultraviolet of 365 mp is, therefore used with glass lenses. ' The use of ultraviolet light for photographing living tissues is open to criticism because of the harmful effects which it may have on the cells Certain microorganisms are destroyed almost instantly by ultraviolet light- others are mildly excited, and still others remain normal ' i. * .1 > » 11 *■ V • SEIFRIZ: PHYSICAL METHODS OF RESEARCH ON PROTOPLASM 103 Vertical illumination Direct light, i.e., rays coming from below the microscope stage, is the time-honored method of illuminating transparent material through the microscope. Obviously, opaque material cannot be so viewed, consequently it must be illuminated from above. The types of apparatus designed for illumination from above are numerous; the following are representative. The illuminator of W. A. Beck (fig. 2) is a simple and inexpensive optical REFLECTED LIGHT MICROSCOPE OBJECTIVE Fig. 2. The Beck illuminator. Light enters from below, is totally reflected by the prism C, and directed down upon the opaque material F, system in which light comes from below, enters a circular glass prism from the upper silvered surface of which it is totally reflected to the opaque material lying in the center on the microscope stage. Similar, and as simple, is the ''epi-mirror" of Carl Zeiss; it is part of the "epi-" equipment of Zeiss for dark-field illumination by incident light. One of the earliest of vertical illuminators is that invented by Silverman (manufactured by Spencer Lens Co.) ; instead of a reflector or prism, the light source is directly above the material surrounding the objective. Of a like design is the surface illumi- nator of Bausch and Lomb. It consists of a ring of electric lights surround- ing the objective. Other more expensive devices for vertical illumination are the ''Ultropak" of Leitz, and the *'epi-condenser" of Zeiss. These latter instruments use light from a small and concentrated electric source placed close to the objective and entering it from above. The vertical illuminator is a development of a metallurgical microscope which is used to advantage mostly in connection with the observation of highly-reflecting surfaces, such as those of polished and etched metals. Introduced by the metallurgist, the method has been adopted by the biolo- gist (32) who finds it of advantage in studying not only opaque but also transparent material by viewing it in another light. P^ 4l 104 PLANT PHYSIOLOGY Dark-field illumination h. T^'l^^^'T'' f"""'^ *^' ^°^*^ ^"'"''^y « suspension of colloidal gold r«v >, r. ^'\.*' '""•' ^^''''''' "' ^°^*J ''^ «"^P««-°n scattered figh rays which strike then., so that a brilliant cone of light is produced. dL- field Muvumtu>n and the ultra-microscope are based upon this phenome- non. Col oidal matter is illuminated laterally against a dark background St w7r.l'' "'"'"' P'""""''^^ *"^ ""''^^ "-^ible" because of the 2L . tJ^^^ ^^^tte'- The direct beam of light from the source of illu- mination does not enter the eye of the observer ; only the scattered rays from the particles are seen. The colloidal particles thus illuminated appear as brilliant spots against a black background. Two main types of lens sys knInTs Z H '"" ''''''' illumination-the cumbersome equipmTnt SZil^:f"'"~''^^' ""*^ '""^ ^-P''' -'•^-'^ -»''— or dark. TOPrt'r,? 7 '""""'"* °* !!?' Ultra-microscope is due to the Germans, Sieden- ZeZ. f ^T""" J'' '°'*''""^"* •^^^•^"^'^ ''y t'^^- i« a large and expensive apparatus which others have attempted to simplify. This type STsmaTr: " ""'"•"" "'^""'^ *° ^^ "- ^»* «"~P«' the sH ened room il'^rT T^^P""'^'"^ *" the hole in the shutter of a dark- the air The ,T "" "' "^'^^ *"'^^^ ^"^^ '""'"'"^t^^ *« ""otcs in tne air. The ultra-microscope consists further of a series of lenses and ZTr'/r^" '™'" '"' '"'"°^<"'P« P™P-. -h-'' di-ct a Hgh ray into ft\:l-!lZT '"' ''"^ '"""•^"''*^ " •^*''-"^- '^'>« TvNOA.'eo„ scalrW of , ..r' "' '''" ^""^ *'"' ""'^^'^ 'y'' '^ tl^« total effect of the In the, ft ^ ^ "'"^ P"""'''*^' "° ^"dividual particles being visible burst of Sir' tT"'- *'%'"''''•'"''' P""'''^^ «- "--" - -"ters of bursts of light. Thus viewed, a colloidal solution resembles the Milkv Wav at^«.ght, but with every "star" dancing about in active BrownianCv^ shoS'in'Z!? r'^A^^^f'"'" " ''"'^ ^''^ ^'^'^ '"'^'■•««t illumination is shoHn in figure 3. Another simple method is the central stop diaphrasm to be used with an Abbe condenser. It excludes all rays within the field bj remtL^rthe t''"'"T / '"' """^ "''' ''^ ^« P-''"-'^ -^-te easily dLvT^r f P^""* "* *'" "^''^^ condenser and substituting for it a the . t / r'"*:. ^^' ^'''"' °P*'''*' ^y^tem for indirect illumfnatLn is he dark-field condenser. The same fundamental principles underl Hh s uTZZVaZ"' 1%'''' '''' "' «'tra-microscope,\amely, a black background, and indirect illumination. The light, however, enters the con- denser from below as in an ordinarj- microscope. bol2'Z7irT *''^'' '""^ '"O'Jiflcations, such as the cardioicl, the para- rli aiid the change-over condenser. The first two names indicate the nature of the curve of the reflecting surface. The light rays strike and r r/ **' ft' •» ) SEIFRIZ: PHYSICAL METHODS OF RESEARCH ON PROTOPLASM 105 Fig. 3. A simple arrangement for dark-field illumination. (From Bausch and Lomb.) reflected by two successive mirrored surfaces which direct and concentrate them at a point in the colloidal solution (fig. 4). Thus does the illumi- nating beam of light not enter the microscope, only the scattered rays from the colloidal particles being visible. The change-over condenser permits Fig. 4. The optical system of a cardioid dark-field condenser. rv » ^' 106 PLANT PHYSIOLOGY II going from direct illumination, as in the ordinary microscope, to dark field without changing condensers. A special dark-field condenser is manufactured by Bausch and Lomb which m Itself takes the form of a moist chamber; it has been designed for work in microdissection. The Spierer lens The Spierer lens (26, 28), an ingenious development of dark-field illumi- nation (o, fig. 5), is based on the principle that a colloidal particle scatters XA\.V\.V.V\-T ^ y^M^^MW/A V////^^/^/W///. ^ riG. 5. The optical system of a Spierer lens : 0 = objective m = Spierer mirror 1 = lens r.r. = reflected rays s.r. = scattered rays e = cover slip f = material g = slide c = cardioid condenser d = diaphragm a = aperture for direct rays d.r. = direct rays for Spierer lens b = slot for cardioid condenser rays c.r. = cardioid condenser rays light unevenly, i.e., there are more rays given off in one direction than in any other, namely, from that side which is away from the source of illumi- -rtS- v >*' ^ 1 >, « * T i> p J • -it« vie^ving a liv- our is St t " T T'''''''^ ^* ^'^"^ 'P'""- The microscope, of u t for he tr'T' tl *^' ""'•'""' P^'^^^^ '''"''' ''' ^ «^*t flashes on made un Jf '•.*^'* ""' °''*"°' ''^ apparently continuous picture ^ranh The""^"! T '""'''''^' ^^^''''"'^^ P'^^'^^'^ ^' '" ^^e cinemato- graph. The gradual precipitation of granules due to centrifugal force can thus be observed while it is going on. The polariscope hJJ''' F^^t'''\ ^"P""^'^ properties of crystals differ in different direc- 1T'/m^ ^'^"u '''".*'"'°"^'' '"■'"''^ ''y'^^^' ^'-S-' Iceland spar) appears to be double This is known as double refraction, and is believed to be due ou from r rV'"''*- """''""^ "^''* "'""'"^ ■" -» Pl-«« -tenZg out from the point of propagation, but the vibrations of polarized light are res ncted to one plane. If a crystal polarizes light, it will allow ligh already polarized to pass through in one plane only, and two crystals will permit polarized light to pass through only if their planes of polarization t SEIFRIZ: PHYSICAL METHODS OF RESEARCH ON PROTOPLASM 109 are parallel. If two such crystals are crossed, more or less of the polarized light will get through, depending on the angle of rotation. When the crossed crystals are at right angles to each other, no light can pass through, buch crossed crystals when built for the study of polarized light are known a^Nicol prisms; mounted in an instrument they constitute a polariscope. The presence of double refraction and the polarization of light by crystal- line matter may be detected and studied with the polariscope. The two prisms, the polarizer and the analyzer, are inserted, the one below the micro- scope objective (usually in conjunction with the condenser) and the other above the ocular, the crystalline substance being placed between them on the microscope stage. Rotating one of the prisms, which is mounted on a scale, gives in degrees the angle of the plane of vibration. If the material is not crystalline there will be no decrease in light intensity as the Nicol prism is rotated. By means of the polariscope, chlorophyll within a living cell has been found to be crystalline, and as it is fluid, it must be of the nature of a liquid crystal. Frey (10) has ascertained the crystalline structure of cellu- lose m plant tissues by means of the polariscope, and Bailey and Kerr (2) have added to our knowledge of the lamellar structure of cell walls, with a series of excellent photographs taken through Nicol prisms. It is of fundamental importance to consider the possible crystalline char- acter of protoplasm. The polariscope has not shown protoplasm as such to be anisotropic, but it has shown striped muscle and contractile tissue in general to be so {i.e., not having the same properties in all directions, as is true of crystals). Microdissection Microdissection and microinjection, with all forms of micromanipula- tion, are now included in the term micrurgy. The method, like all scientific discoveries, developed from primitive manipulative efforts on the part of pioneer workers. The first satisfactory instruments were constructed by Barber (3) and by Schouten (23). Barber built his pipette holder for the purpose of isolating single bacteria from cultures. The instrument is now replaced by several other types, each of which has its own peculiar ad- vantages. The best known instruments which are commercially obtainable are the Zeiss-P^terfi (21) and the Leitz- Chambers (6) instruments. The Taylor (31) micromanipulator, privately manufactured, was an early de- sign built for stability. A recent commercial model is that of Fitz (8), manufactured by Bausch and Lomb. A new but as yet little known model is that of DE Fonbrune (37). It promises greatly to facilitate microdissec- tion technique. It consists of a universal joint which controls three pistons operating against air. By means of three tubes, the pressure exerted by y 110 PLANT PHYSIOLOGY the pistons is conveyed to a separate machine where three metal dia- phragms, similar to those of aneroid barometers, are forced out or drawn in, and thus control, by means of levers, the rod to which the microneedle or pipette is clamped. The manipulator operates with remarkable precision, being wholly free from lost motion. The independence of operating and receiving mechanisms eliminates vibration. The hand of the operator and the needle-point move in the same direction and the operator's hand need never leave the one lever with which all movements are performed, de Fon- BRUNE has also constructed an instrument for automatically drawing needles and pipettes under the microscope lens. In addition he has devised a simple method for protecting preparations of living material from dehydration without the use of a moist chamber. He simply places the material under an oil drop (nujol) either on the upper side of a slide or as a hanging drop. The oil is harmless, and lack of air has no ill effect for some time. Unfor- tunately the DE FoNBRUNE micromanipulator is not yet commercially obtainable. It is impossible to say which instrument is the best. The last mentioned gives promise of being so, but it has not yet been used by anyone except its inventor. Among the others, it is primarily a question of the instrument upon which one has learned. The Leitz-Chambers instrument is stable; the Zeiss-P^terfi has the greatest freedom of movement. Accessories to micromanipulators are many. The fewer bought the better (certain ones sold are worthless). Exceptions are the needle-holders of Zeiss and the micropipette equipment of Leitz. Needles are best made by the individual investigator, because the type of needle should fit the work to be done, and the various types can be made only by hand. However, automatic devices such as drawing needles and pipettes are to be had (manu- factured by E. Leitz). Microscalpel, microcauter, micromagnet, micro- electrode, and microthermocouple are some of the appurtenances to micro- manipulation. The construction of each requires a special technique. The problems of micrurgy are many, and some have been quite success- fully attacked. Certain of them are of interest not only in themselves, but have important bearings on related problems, thus the elastic qualitV of protoplasm, ascertained by micromanipulative methods, is of value as an indicator (the best we have) of the structure and fundamental nature of protoplasm. Other problems which remain untouched, or as yet unsolved by this method, are the structure of the chloroplast and of other plastids (is the chloroplast a droplet or a sac?), the removal of a chloroplast from one cell and its injection into another cell, the reality and nature of spindle fibers, the physical properties of the huge, motile, male gametes of cycads and the eternal problem of the outer protoplasmic membrane and the tono- plast which is not yet fully elucidated to the satisfaction of all. ! I J. I * f • -J- 4 *- -^ seifriz: physical methods of research on protoplasm 111 Electrical properties The determination of electrical forces in organisms and cells is a fasci- nating study which is as yet in its infancy. These forces reach as high as a thousand volts, as, for example, in the ray-fish or electric eel, while that existing between the parts of plants, or between one cell and another, is of the order of 50 millivolts (0.050 volt). It is toward the measurement of these small electrical forces that our attention is here directed. This in- volves the use of microelectrodes (15) and a potentiometer circuit with amplifiers for greatest accuracy. Circuits for such measurements have been used and described by Lund (15) and his coworkers in the determination of potentials in trees. Electrical investigations on cells and protoplasm require microelectrodes controlled by micromanipulative methods. Metal electrodes, even when of platinum, are likely to cause disturbances within the protoplasm; agar electrodes (agar with salt solution in a micropipette) are to be preferred. While in this review of methods we are concerned only with apparatus, it should be said that extensive work on other forms of potentials, namely, oxidation-reduction potentials, has been done with the aid of indicator dyes such as are used for pH determinations. ^ Cataphoresis studies have yielded some fundamental results on the elec- trical properties of living cells. The method involves subjecting suspen- sions of cells (or colloidal particles) to a difference in external potential; that is to say, the cells are placed in an electric field, and as all suspended particles, whether of metallic gold, oil, or living cells, possess an electric charge (with few exceptions), and, if free to move, they will migrate. The direction of migration is toward the pole of opposite sign to that of the charge on the particle. The rate is proportional to a number of factors including the potential of the external field and the potential (the inter- facial potential) of the particles. These factors are all taken care of in the Helmholtz formula for cataphoretic migration (37). With as yet no con- vincing evidence to the contrary, it can be definitely stated that all living cells are negatively charged when suspended in a solution similar (in re"^ gard to salt concentration and pH) to that in which the cells normally live. The interfacial potential of colloidal particles and cells in suspension is of the order of 30 to 50 millivolts; settling out (for example, of bacteria) occurs at about 11 millivolts. The apparatus so far most extensively used in America for cataphoretic work is the Northrop-Kunitz (20) model. It consists of a very shallow glass chamber open at the two ends to receive the electrolytic solution which connects the chamber with two poles of zinc immersed in zinc sul- phate. A more recent model is that of Abramson (1) which is less expen- sive and has the advantage of rigidity as it is constructed of one piece. The 112 PLANT PHYSIOLOGY NoRTHROP-KuNiTZ model is commercially obtainable. The Abramson model is as yet only privately made. To make simple and relatively crude cataphoretic observations is an easy task, but to make accurate measurements is quite another matter. The beginner is cautioned against the use of simple types of cataphoretic cham- bers which have been offered for sale. They are well-nigh useless and the results obtained with them are wholly unreliable. Practically all work pur- porting to show that cells are positively charged is due to the use of such, or home-made apparatus. Failure to measure pH is sometimes responsible for the trouble, for the sign of the charge on many suspended particles can be reversed by a change in the pH of the solution. Simple comparative and reasonably accurate experiments in catapho- resis can be performed without calibrating the instrument, but for exact work this must be done. The best method for doing so is that of Abram- son (1). Work in cataphoresis has yielded some of the most interesting and pos- sibly far-reaching results of recent experimentation on the physical proper- ties of cells. Only brief reference will be made to this work here, merely to call the attention of the student to articles wherein technique as well as results are described. Some of the earliest research on the cataphoretic migration of colloidal particles and bacteria was done by Freundlich (9) ; then followed work by Northrop and Kunitz (20) establishing the critical potential of bacteria. Falk (7) correlated the pathogenicity of bacteria with their potential. (This work may prove in part erroneous, but it was highly suggestive, pointed the way, and may yet be right to a degree.) More recent is the important work of Mudd (17) on sperms and bacteria, of Abramson (1) on protein-covered quartz particles, and of Moyer (16) on latex. The last-mentioned investigation has made a fundamental contribu- tion by showing that kinship or species relationship of Euphorbias is re- lated to the shape of the mobility curves, and, primarily, to the iso-electric points of the latex particles. The centrifuge The centrifuge has played a part in studies on protoplasm chiefly in regard to determinations of viscosity. The first work of this kind was done by Nemec (18). He found that changes in the viscosity of protoplasm occur during cell division. In the young dividing cells of the onion, bean, etc., resting nuclei are, by centrifuging, thrown against the apocentric cell wall, while spiremes and asters remain situated in the center of the cell. Metakinetic and telophasic figures are only slightly displaced. The most easily disturbed stages of cell division are those coincident with the forma- tion of the cell wall. In other words, the protoplasm of the resting cell f T .-,1. ■< » f * I ( t ■i- •»- SEIFRIZ: PHYSICAL METHODS OF RESEARCH ON PROTOPLASM 113 preparatory to division is of low consistency ; the protoplasm of the cell in mid-mitosis and immediately following is of high consistency, and the proto- plasm of the cell in the last stages of division is of minimum consistency. Recent developments have resulted in the construction of a small air- driven centrifuge which attains the highest speed yet known for centri- fuges (5). Centrifuging is but one of several ways of determining protoplasmic consistency. Other methods are microdissection (24), magnetic properties (37), the fall of statoliths (37), plasmolysis (34), and Brownian movement (4) . Misunderstandings which have arisen, and a convincing clarification of them, have recently been set forth by Fry (11) ; he employed the centri- fuge method. All determinations of protoplasmic consistency by whatever method in- volve a consideration of the important question of the applicability of the single constant laws of Newton, Poiseuille, et al, developed for true solu- tions and pure liquids, to systems such as protoplasm, the behavior of which is anomalous. The problem is an important one and involves recent rheo- logical theories on non-Newtonian fluids (37). Investigations on the vis- cosity of protoplasm are many and the bibliography extensive; these have been reviewed by Weber (33) and others (25). Photography Photographs supply an authentic record of an experimental result. Photomicrography is now so highly developed a science and art that a dis- cussion of it here would be wholly inadequate. A few remarks of interest and perhaps value may, however, be made. The small, 4^ x 6 or 9 x 12 cm. camera which rests directly upon the microscope tube is very convenient, and excellent results can be obtained ; but many workers still prefer to use the old-fashioned camera-box swung upon an upright support. The latter equipment is certainly the more flexible of the two, but it lacks one very convenient attachment of the former— the beam-splitter. It is a decided advantage to be able to focus through an ocular rather than upon a ground glass. Certain new but expensive photographic instruments combine the advantages of the old type camera-box and the new microscope camera with beam-splitter. The production of good photomicrographs is to a consider- able degree dependent upon a correct choice of light filters. These can be had in considerable variety. The infrared filter is a recent addition and is to be used with special infrared sensitive plates. The photographer must try out the various combinations of lights, filters, and negatives suitable for his particular material. The introduction of cinematography into microscopic work has yielded some fascinating results which are of real value to the research worker as 114 PLANT PHYSIOLOGY well as to the teacher or student. With the aid of moving pictures, details in mitosis heretofore unsuspected have been discovered. (A word of warn- ing should be given in regard to the misleading impression created by speeding up films ; pictures taken at the rate of one a second or one a minute then shown at the rate of 32 a second often give a highly entertaining but scientifically inexact impressions.) Motion pictures of tissue-cultures show the migration and division of fibroblasts and the movement of chromosomes in culture; others illustrate the development of the growing mammalian embryo from the one-celled stage to the sixteen-celled stage. Very striking are moving pictures taken with dark field. For botanists, moving pictures have made their chief contribution in such studies as those on the mechanism of conjugation in Spirogyra, and on the operation of the animal-trap of Utricularia. These studies have been carried out by Lloyd (13). Through them is the method of movement of the male gamete in Spirogyra for the first time fully understood (motion is by contraction of vacuoles) . A recent application of the cinematograph to protoplasmic studies has involved photographing protoplasmic streaming. Streammg protoplasm is always fascinating to watch, and becomes even more so when seen on the screen. But the method has a practical applica- tion ; events which take place but once and too hurriedly to permit careful observation can be seen again and again if they have been photographed as moving pictures and are then shown at slow speed. The equipment neces- sary for taking moving pictures can be expensive but need not be. An essential feature is the beam-splitter so that material may be seen while it is being photographed. Other than this, any type of amateur camera for cinematography is all that is needed. The 8-mm. film is fairly satisfactory, but better results are to be had with the 16-mm. or professional 32-mm. film. The saving in cost of the 16-mm. over that of the 32-mm. film is to be considered in regard to the film, camera, and projector. Perhaps in no other way can an idea of the perpetual activity of protoplasm be so dra- matically conveyed as through a moving picture of the streaming proto- plasm of myxomycetes. LITERATURE CITED 1. Abramson, H. a. Electrokinetic phenomena and their application to biology and medicine. New York. 1934. 2. Bailey, I. W., and Kerr, T. The visible structure of the secondary wall and its significance in physical and chemical investigations of tracheary cells and fibers. Jour. Arnold Arboretum 16 : 273-300 1935. 3. Barber, M. A. The pipette method in the isolation of single micro- organisms and in the inoculation of substances into living cells. Philippine Jour. Sci. B, 9 : 307-360. 1914. f j^. i -N SEIFRIZ : PHYSICAL METHODS OF RESEARCH ON PROTOPLASM 115 4. Baas-Becking, L. G. M., Bakhuijzen, Henriette van de Sande, and Hotelling, Harold. The physical state of protoplasm. Verh. k. Akad. Wet. Amsterdam, Afd. Natuurk. (2de sec ) 25- 1-53 1928. 5. Beams, J. W., and Weed, A. J. A simple ultra-centrifuge. Science n. s. 74 : 44-46. 1931. 6. Chambers, Robert. Microdissection studies. T. The visible structure of cell protoplasm and death changes. Amer. Jour. Phvsiol 43 • 1-12. 1917. ■ ' 7. Falk, I. S., Gussin, H. A., and Jacobson, M. A. Studies on respira- tory diseases. XXI. Electrophoretic potential and virulence of pneumococci. (Types 1, 2, 3 and 4.) Jour. Infect. Diseases 37: 481-494. 1925. 8. FiTZ, G. W. A new micro-manipulator. Science n. s. 73- 72-75 1931. 9. Freundlich, H. Kapillarchemie. Leipzig, 1932. [Eng. transl., ''Capillary chemistry.'' London, 1926.] 10. Frey-Wyssling, a. Mikroskopische Technik der Micellaruntersuchung von Zellmembranen. Zeitschr. wiss. Mikrosk. 47 : 1-46. 1930. 11. Fry, H. J., and Parks, M. E. Mitotic changes and viscosity changes in the eggs of Arhacia, Cumingia, and Nereis. Protoplasma 21 : 473- 499. 1934. 12. Har\t:y, E. N., and Loomis, A. L. A microscope-centrifuge. Science n. s. 72 : 42-44. 1930. 13. Lloyd, F. E. Maturation and conjugation in Spirogyra longata. Trans. Roy. Canadian Inst. Toronto 15 : 151-193. 1926. 14. Lucas, Francis F., and Stark, Mary B. A study of living sperm cells of certain grasshoppers by means of the ultraviolet micro- scope. Jour. Morph. 52 : 91-113. 1931. 15. Lund, E. J. Electric correlation between living cells in cortex and wood in the Douglas fir. Plant Physiol. 6 : 631-652. 1931. 16. Moyer, Laurence S. Species relationships in Euphorbia as shown by the electrophoresis of latex. Amer. Jour. Bot. 21 : 293-313. 1934. 17. MuDD, Stuart. Agglutination. Cold Spring Harbor Symp. Quant Biol. 1 : 65-76. 1933. 18. NfiMEC, B. The mechanism of mitotic division. Proc. Fourth Inter- nat. Congr. Plant Sci., pp. 243-249. Ithaca. 1926. 19. Northrop, John H. The flocculation and stability of colloidal suspensions. Colloidal Behavior (ed. R. H. Bogue) 1: 70-97. McGraw-Hill, New York. 1924. 20. , and Kunitz, M. The swelling and osmotic pressure of gelatin in salt solutions. Jour. Gen. Physiol. 8: 317-337. 1926. 116 PLANT PHYSIOLOGY 21. P...^.x, T. ^^^^r^,i.^eMemii,. Handb. biol. Arbeitsmeth., Abt. 22. SCARTH G. W. The structural organization of plant protoplasm in the light ot micrurgy. Protoplasma 2 : 189-205 1927 23. SCHOUTEN S. L. En methode voor het maken van reinculturen. Handelmgen V. h. 7e Nederl. Nat. en Gen. Congr. 1899 24. Seifbiz, W. Viscosity values of protoplasm as determined by micro- dissection. Bot. Gaz. 70 : 360-386. 1920. ^"' '•'^^ viscosity of protoplasm .- Molecular physics in rela- 26. 27. tion to biology. Bull. Nat. Res. Counc. 69 : 229-261. 1929 — -. Spierer lens and colloidal structure. Ind. & En^ Chem. 28 : 136-140. 1936. ^* , and Plowe, Janet. Effects of salts on the extensibility ^^ protoplasm. Jour. Rheology 2 : 263-270. 1931 28. SpiEREB,a Un nouvel ultra-microscope a eclairage bilateral. Arch. Sci. Phys. Nat. (Geneve), 5 S. 8: 121-131 1926 Taylor, C. V. Cataphoresis of ultramicroscopic particles in proto- plasm. Proc. Soc. Exp. Biol. & Med. 22 : 533-536. 1925 mT^M ^ ^'''?fr!l?.'^'' ^''"^ micromagnets. Proc. Soc. Exp. 15101. & Med. 23 : 147-150. 1925. Improved micromanipulation apparatus. Univ. Calif. 29 30. 31. Publ. in Zool. 26 : 443-454. 1925 32. VoNwiLLER, p. Ober indirekte Beleuchtung in der Mikrosknie im senkrecht auiTallenden Licht. Protoplasma 1 : 177-188 1926 ^*' Z' ~- ,^''^^,^f Beurteilung der Plasmavikositiit nach der General References 35. Abdebhalden^E_ Ilandbuch der biologischen Arbeitsmethoden. Ber- 36. '"^^;^^^<^ r^^rn, P. Handbuch der Mikrobiologie. 37. Seifriz,W. Protoplasm. McGraw-Hill. New York. 1936. \ .!. I » t I M •* € I * <*J^ ■ V ' I ^ Reprinted from Science, March 4, 1938, Vol. 87, No. 2253, pages 212-214. THAT WORD "EMULSOID" Shakespeare wrote, "What's in a namet"; but then, he had never heard of "emulsoids." No word has caused more confusion in the colloid-chemical thinking of physiologists than "emulsoid." Biologists continue to take it at its etymological value, while most chemists have long ceased to regard it in this, its original sense. The term was coined by Wolfgang OstwaJd in the early years of the present century, when colloid chemistry was still in its infancy, as sciences go. Originally "emulsoids" included coagula, jellies and emulsions, the term being based on the assumption that jellies are fine emulsions. Ostwald's only evidence for assuming that jellies are "emulsion-like" was that both they and emulsions increase in viscosity with increase in concen- tration of the dispersed phase, while "suspensoids," or solid colloidal suspensions, do not do so. The evidence on which the term was based is correct, but it tells a very small part of the whole story and is entirely mis- leading. To this day, the concept that jellies are fine emulsions clings, in spite of repeated and substantial proof to the contrary. My own interest in this matter rests on the mis- chievous influence of the word "emulsoid" on interpre- tations of protoplasmic structure. All agree that pro- toplasm is an "emulsoid," but those biologists who know of the word only from the reading of old and standard texts conclude that protoplasm must be a liquid-liquid system, for that is what the word "emul- soid" means, i.e., "emulsion-like," and emulsions are liquid-liquid systems. Other workers, knowing the historical backgpround of colloid chemistry, realize that the word "emulsoid" has long since lost its original meaning, for no chemist well grounded in his knowl- edge of the colloidal state believes jellies to be like emulsions, except in that one property which Ostwald selected for the basis of his classification. In every other respect, dispersions of gel-forming substances are quite distinct from emulsions. Whatever their structure, jellies are not liquid-liquid systems. Though there are some interesting experiments which indicate that protoplasm behaves like an emul- sion, many physiologists regard the similarity as purely incidental and superficial. The emulsion hypothesis of protoplasmic structure satisfies but few conditions, and is wholly contrary to many of the most significant properties of living matter. Among speculations on the emulsion nature of protoplasm, there IS one that has recently come to my attention, namely, that the human brain is an emulsion; not only this, but It IS said to reverse from oil-in-water to water- in-oil. While I know little about the histology of the brain, I am convinced that the hypothesis is based on the keen sense of humor of its author, for a brain that IS an emulsion of water dispersed in a continuous phase of oil is a brain through which little can pene- trate. Perhaps the author of the theory has some colleague in mind!-The brain consists of cells, or so-called cell-bodies, with their fibrous axons and dendrites, interwoven by a supporting framework of neuroglia, the whole bathed in fluid: this is not an emulsion. ^^ It is not, however, my purpose here to discuss the emulsoid" hypothesis of protoplasmic structure, but rather to plead with physiologists to give up ideas on gel structure which most chemists have long since dis- carded. The controversy is an old one, but I had rather assumed that it was amicably settled, yet on reviewing of late a number of text-books on physiology and chemistry, the persistence of the theory was brought to my attention in a striking way. A note, therefore, seems worthwhile. None of us treads with security when wandering far from our chosen field of research, as every physi- ologist must if he covers his subject in a broad way, but an author may save himself and his readers from' old and discarded ideas by simply turning to an authoritative source. In the present instance, the col- loid chemist is the man to whom physiologists must turn. An outstanding authority such as Freundlich* says that the word "emulsoid" must either be dropped or Its original and etymological meaning ignored. Selections from five new books, four in physiology and one in chemistry, illustrate how poorly biologists and some others have heeded this advice of Freundlich In one text, emulsoids are defined as liquid-liquid systems, and milk cited as an example. On the next page, the author states that in addition to "these vari- ous types of colloidal suspensions, mention should be made of emulsions," which he again defines as liquid- 1 "Capillary Chemistry," ]926. ^V \\ X Hquid systenis with nulk as an example. What is the student to make out of this, for not only is an emulsoid given the twenty-five-year-old and discarded definition of Ostwald, but the author tells the student that another type of system will now be considered, namely, emulsions, and then gives the same definition and the same example already stated for emulsoids. In the second text, "colloidal theories" are intro- duced by a reference to "emulsoids," then, without any break in thought, the emulsion hypothesis of cell per- meability is presented. Two other text-books publish the diagrams first used by Bayliss and intended to illustrate a hydrosol and a hydrogel. The first figure is of black spots on a white background and labelled "hydrosol"; the second is of white spots on a black background and labelled "hydrogel." These are typical diagrams of an oil-in- water and a water-in-oil emulsion. Bayliss refers to the scattered black spots of the "hydrosol" and the black background of the "hydrogel" as solid matter and thus regards the hydrogel of gela- tine as similar to that of silica. This is in keeping with the micellar theory of gel structure which Bachmann thought he had shown to be true for gelatine. The error of Bayliss lay not so much in supporting the micellar hypothesis of gel structure but in assuming that water is firmly held in a jelly because it is "im- prisoned" between "solid walls." Water is firmly retained in jellies not because it is trapped in pockets or in isolated drops, but because it is bound by adsorptive or other forces. Those who have copied the black and white diagram of Bayliss misinterpret the situation still more by re- ferring to jellies as fine emulsions and to the setting of gelatine as involving phase-reversal. The figure is labelled "a diagram to illustrate the reversal of phases during sol-gel transformation." With this statement the assumption is again made that "enormous pres- sure is required to squeeze water out of a set hydro- gel," because "the previously continuous phase — becomes the dispersed phase." The author of a recent book on the colloidal state of matter asserts that liquid-in-liquid systems include the "emulsoids," and these in turn "contain the emul- sions." The author realizes that emulsions are not good "emulsoids" and suggests that they be put in a separate group, but he gives as reasons that many emulsions are outside of the colloidal range because of ( the size of the dispersed drops, and many are not simple two-phase systems; all of which is true, but these are not the reasons why emulsions are not "emulsoids," as the term is now generally used. The structure of jeUies is not known with certainty, though much progress on this subject has been made in' the past quarter of a century. At the first CoUoid Symposium (in 1923) a preliminary (and as yet un- published) report on the structure of gelatine was given. The speaker referred to the gelatine molecule as a fiber of great but definite length, with a cross- section of not more than a few Angstrom units. It is only necessary to postulate an interweaving or other specific grouping of these molecular fibers to obtain the type of structure now very generally appUed to jellies and to protoplasm. Whether the fibrillar (chain molecule) or micellar theory of gel structure is the correct one is unimpor- tant so far as the point under discussion is concerned. Here we are concerned only with the generaUy held pomt of view that jeUies are not fine emulsions and gelatmization does not consist in a reversal of phases. EUis^ and Donnan stated in 1913 that neutral oil emulsions are model suspension colloids; that is to say they are "suspensoids," and not "emulsoids." Four years later, Hatschek,» on the basis of a neat mathe- matical-physical analysis, asserted that "the theory that gels consist of two liquid phases must be pro- nounced untenable." McBain* has since found that there is no change in the electrical conductivity of a soap sol when it sets to a gel, which means that the continuous and conducting phase remains the same; there is, therefore, no reversal of phases. The work of the x-ray investigators, Sponsler,^ Astbury," et al, should be sufficient to convince one that gels and jeUiw are not emulsions. Much confusion and erroneous instruction wiU be avoided if the word "emulsoid" is discarded. It was based on a misconception and is no longer used by well-informed chemists in its original sense. So with Lady Macbeth, I cry, "Out, damned emuU soidal spot I out, I say I" William Seipriz University op Pennsylvania 2 Transactions of the Faraday Society, 9: 14 1913 3 Transactions of the Faraday Society 12- 17 1Q17 * Joiir. Phys. Chem., 28: 706, 1924. ' 5 Jour. Gen. Physiol., 9 : 221, 1925. 6 '* Fundamentals of Fibre Structure," Oxford 1933 Reprinted from Science, October 29, 1937, Vol. 86, No. 2235, pages 397-398. 1^ A THEORY OF PROTOPLASMIC STREAMING Protoplasmic movement is now recognized as a general and fundamental property of metabolically active cells. It has risen to this position from the con- troversial one held not many years ago when the streaming of protoplasm was looked upon by some physiologists as a pathological condition in opposition to the view that it is evidence of a normal and healthy state. The force responsible for protoplasmic flow has been the subject of much lively and stimulating specu- lation. My earliest memory goes back to the days when surface tension, the sine qiia nan of so many cellular activities, was regarded as the energy source of protoplasmic streaming. There was also the sug- gestion that the one-way "shuttle" type of flow in the filaments of coenocytes, where movement is first in one direction and then in the other, may be due to hydration at one end and dehydration at the other end; but the protoplasm flows equally well in both directions, even when fully submerged. Obviously, dehydration can play no part. Physiology next entered the colloidal epoch when it became apparent that many cellular activities had their counterpart in colloidal systems. There thus arose the suggestion that protoplasmic streaming is a cataphoretic migration of particles or the electro- endosmotic flow of an aqueous medium; if either, it is the latter, for in streaming protoplasm the entire mass of material moves and not just the suspended particles. In spite of some attempts to prove that streaming protoplasm is associated with electric poten- tials, there has been no convincing evidence that the potentials measured are real, in the sense of innate to the protoplasm, and if real, are the cause rather than the result of the streaming. Most electrical determina- tions made on cells and tissues are subject to the criticism that the potentials measured may not reside in the living material alone, but are produced by the expenmental equipment as a whole. This statement is in no way intended to deny or question the existence ot electric potentials in organisms; on the contrary, all necessary conditions for the production of potentials are present, but do the potentials measured exist in the living material? Should the source of energy responsible for proto- plasmic streaming be found, there would still remain the question, why the direction of flow reverses in the shuttle type of streaming. The protoplasm of slime- molds flows but one way at a time. Were a potential responsible there would have to be a reversal of it every forty or fifty seconds, and so the problem takes on a two-fold aspect—why the streaming, and why the reversal? The slime-molds, or myxomyoetes, sometimes re- ferred to as mycetozoa, possess a body or Plasmodium which IS a non-cellular protoplasmic mass with ever- shifting channels of flow. A brief study of slime-mold Plasmodia is sufficient to suggest the presence of a nearly perfect rhythm in the reversal of direction of flow. The average of many time records gives forty to fifty seconds for each period of flow. The minimum time IS twenty seconds, and the maximum sixty be- tween reversals. (The average maximum rate of flow IS 0.07 mm per second.) The foregoing limits in time of flow suggest a lack of rhythm, but most often the penods of flow vary little from the mean. Pro- nounced divergences from the average of forty-five seconds are to be attributed in part to physiological disturbances in an artificially mounted specimen of Plasmodium. Hi 3 My own motion pictures of slime molds had all been taken at moderately high magnification and at normal speed. I therefore failed to see the essential feature of the rhythm. This first became obvious to me on viewing the excellent cinematographs of slime-molds taken by Drs. J. Comandon and P. de Fonbrune, of the Pasteur Institute at Garches, France. My visits to these laboratories have always been profitable. The first films of slime-mold plasmodia by Dr. J. Coman- don and Professor P. E. Pinoy were made as long ago as 1912. When the moving-picture of a small Plasmodium is taken at one hundredth of the normal speed and pro- jected at the customary rate, the entire Plasmodium is seen to go through rhythmical contractions and expan- sion similar to the pulsation of a heart. At each contraction, the direction of flow reverses. The mech- anism of protoplasmic movement in slime-molds is, then, one of rhythmical contraction and relaxation of the Plasmodium as a whole, with a period of 40 or 45 seconds for each pulsation. If the area covered by a Plasmodium, or a branch of it, is noted with the aid of a micrometer, it will be seen that the Plasmodium contracts with outgoing protoplasm and expands with incoming protoplasm. I had interpreted this swelling and shrinking as the result instead of the cause of streaming. Contraction is not due to the exit of fluid, and expansion to its entrance, but on the contrary, as in the case of the heart, the exit and entrance of fluid are due to the contraction and expansion of the living substance. The beat of the heart is controlled by a sympathetic nervous system. There is no nervous system in the sense of nerve-centers and nerve-fibers in slime-molds, but these are not necessary in order to have response and control. Some modern workers are inclined to strip proto- plasm of all the attributes of higher organisms, for- 43 IRREGULAR PAGINATION getting that many of these attributes exist because they are properties of protoplasm. The tendency to regard the protoplasm of more primitive forms of life as less intricate, less responsive, if not "less living*' than that in more highly developed forms is evident in such statements as, "there is considerable objection to the use of the word 'injure* in reference to plants." I recall the delightfully courteous remark of the English chemist, H. R. Proctor, who, finding my mechanistic interpretations of protoplasmic behavior rather harsh, asked if he might not still be allowed to regard life as, so to speak, a new departure. In my turn, I ask the reader merely to admit that protoplasm is alive, for in so doing he tacitly grants that it exhibits irritability, in other words, nervous response. It is interesting to contemplate the possible rela- tionship between the rhythmical pulsations responsible for protoplasmic streaming in myxomycetes and the rhythmical contraction of sympathetically controlled muscle-tissue. William Seifriz University or Pennsylvania / >— 1 r ■ "• FRANCOIS ANDRE MICHAUX, THE BOTANIST AND EXPLORER RODNEY H. TRUE Director of the Morris Arboretum, Philadelphia (Read April e4, 1937) Abstract anH f^^f'TfT" '^^*?v.^' in outline the chief events in the life of Andre Michaux and that of h,s son, the subject of the sketch. The two are considered because their work was so very thoroughly intertwined. because «.^ ^^^ !u-^^ botanical explorations of Francois Andr6 Michaux are outlined sonstaJ^d '"^ '*^"^'^^ accomplishment of both the father and th^ The relation of the son to the American Philosophical Society is pointed blghfup'tf SIL'' ''' ^"''"' ^^""^^'^^ ^^^^^ ^" ^^•^'"-"^ Park ha's be:' The person who finds himself called upon to write on Michaux, the botanist and explorer, may well find himself perplexed as to the course to be pursued. There were two Michauxs, both eminent in our early history for their botanical accomplishment and for their wide-ranging explorations. Father and son, they have left records of achievement that will make the name ever illustrious in the annals of American botany. Andr^ Michaux, the father, is the man who comes first to the mind of the botanist. His name was rendered memo- rable by his discovery and description of a host of plants that had not been brought to the attention of science before his time. By virtue of close observation and by his clear descrip- tions of the new plants found, he is now credited with the discovery of over three hundred species of plants. I have looked through the 7th edition of Asa Gray's Manual of Botany for plants credited to Michaux, senior, in an area bounded on the north by the 48th parallel from the Gulf of St. Lawrence to Lake Superior and the international PROCEEDINGS OF THE AMERICAN PHILOSOPHICAL SOCIETY VOL. 78, NO. 2, DECEMBER, 1937 ' gjg ^^P^*"i Printed in U. S. A. IRREGULAR PAGINATION 314 RODNEY H. TRUE thTw 7k r'' *° ^^' northwest corner of Minnesota; on the west by the western boundary of Minnesota and north- western Iowa then southward along the 96th meridian; and on the south by the southern boundaries of Kansas, Mis ouri Kentucky and Virginia. This area was but p^rt of the country traversed by him. He traveled from Hudson Bay to Florida along the eastern side of the continent, and penetrated westward to the Mississippi River. «^'«»iea Accredited to Andre Michaux in that portion of the coun- try seen by h.in and included in the narrower range of the Manual, I find 24 new genera of flowering plants He discovered and first described 293 species of flowering plants of which 91 have since been referred to genera othe^ thin those to which Michaux assigned them, leaving 202 species unclianged in the present botanical record, in spite of over a Z Z t-^t"" ""'^ "'*''^' '*"^y- '^•^e persistence of the name of Michaux in textbooks now in daily use is due almost entirely to the explorations, critical study and accurate His son, Francois Andr6 Michaux, however, is the main subject of my sketch, although their lives and work were closely interwoven until the death of the father. AniT6 Michaux, like his son, Francois AndrcS Michaux was born on a royal estate at Satory, near Versailles, France! Ihe Michauxs for generations had been in charge of this estate. Andre, pere, with whom our interest begins, was born here in 1746, and grew up on the estate under his father's nstruction. His marriage in 1769 to Cecil Claye, the daugh- ter of a wealthy farmer, was followed after eleven months by her death leaving Andr6 with the infant son. It is said that as a relief to despondency he turned to an intensive study of botany under the eminent French botanist, Bernard de Jussieu. This brought him nearer to the Royal Gardens of p "f" J ! ^°°" ^^^" ^''^'''^'y t° '^o'lect plants, visiting England Auvergne and the Pyrenees. A diplomatic appoint- ment took him to Persia and Mesopotamia, where he collected T irf*f FRANCOIS ANDRE MICHAUX 315 seeds and plants for introduction into France. After three years in the Near East (1782 to 1785), Andre returned to France, and was immediately sent by the Government to North America, then little known to science, to study the forest trees, with the object in view of introducing into France such species as might prove useful in naval and other con- struction work. On October 1, 1785, Andre iVIichaux, his fifteen-year-old son, Frangois Andre, and a practical gardener, Paul Saulnier, landed in New York, and proceeded to establish a nursery near Hackensack, New Jersey, in which American trees were to be grown for shipment to France. The interest in applied science at that time centered in Rambouillet, and to the Rambouillet nurseries Andre Michaux's trees, to the num- ber of 60,000, were sent. After a year and a half spent in botanical explorations and in establishing the northern nursery near New York, father and son went to Charleston, South Carolina, leaving Saulnier in charge of the first nursery. The year 1787 saw the purchase of 220 acres of land about ten miles from Charleston, on which a second nursery was to be developed, and on which trees of more southern origin were to be grown for shipment from the port of (Charleston. In the year 1787, exploring expeditions were made into Florida and to the Bahamas, also a strenuous excursion up the Savannah River to the head waters, over the Appalachians mto the Tennessee Valley and then the return to Charleston. A visit was paid to the northern nursery in that busy year. The son was retained at Charleston, probably much of the summer, either because of ill health or to manage the new nursery in his father's absence. His troubles were increased m 1789 when he was shot by a companion hunter, the pro- jectile injuring his left eye. This accident seems to have been much above the horizon during the year and longer. Troubles at home in France began to interfere with plans in America. Remittances ceased, the faraway scientists were out of sight, and much trouble was near at hand. Michaux, 316 RODNEY H. TRUE Ph-e, advanced what money he could to continue the work and pledged h.s property in France to secure loans ' r3u1ion H^e'^"'''' 'JT^" *° ^''''''- ""^ ^ ^ '^ate of revo ution He espoused the popular cause, although the family had for generations been closely attached to the Royal mterests and his own childhood had been spent on a part o the Royal domaan. Perhaps his course of action was some blmet Tf 'Lr" ^^^ - *^^ — - at Ram. bouiUet. He found there but a few of the trees that his father had striven so earnestly to secure for France. Half the tota number had been given by the Queen to her imperial fathe itmsT'th''"^ ''' '"" ^'^■^" '" '"^-'^- °f '>.e CourUo embellish their private estates, and those that remained in the Royal Nurseries had been shamefully neglected Francois seems to have spent several succeeding years in the study of medicine, with the plan in mind of returnTng to trTmlT ^'■"*"" ™^ P'^'^ "'^^ «'^- "P' however, wLn m 1796 his resources were exhausted. After years of strenn ous exploration following his son's return to'^^rlnce HZ too sought the homeland. He sailed from Charleston L August with the botanical materials and records tL" ha" made his name imperishable in the annals of American botanical science, but he was financially exhausted. of ^"J A ^° «' *''°"''''''' ^'' ^°^^ ^^ ^'•^'^'^ed off the coast V. ur u . u^ "^^^ '^''"^^ ^^t^"" floating on a board to Si . ?'■'? t'' '"' '" ^'""^ """^h °f h- water-soaked botanical material also was rescued and dried out. Unfortu- nately, packages of priceless notes and records were lost ^avmg gaps in the story of his strenuous life in the New World He arrived in Paris after a time, where he was mosT favorably received, but the government ;as not inteTeledt his plans for further explorations. With the help of Frangois, the year 1796 and several fdlowmg years were devoted to the cultivation of the material rom America, and m the preparation for publication of his luxuriously illustrated volume on the North American Oaks FRANCOIS ANDRE MICHAUX 317 V 1 * ■^ ^ il ^ '. and his volumes of the ''Flora Borea'i- Americana:' These publications set up a landmark in American botanical history. Andr^, still strenuous for the bright reward of additional scientific knowledge, through ministers and through the Central Society of Agriculture, tried to persuade Napoleon, then First Consul of the French Republic, to authorize further explorations in remote lands. Only after great effort was Andrd appointed naturalist in a scientific exploration to Australian seas, under the command of Captain Baudin, it being agreed that Michaux should be permitted to remain at the Isle de France if he desired. After the freedom the un- controlled explorer had enjoyed in his American years, he was disgusted with the haughty and discourteous manners of Captain Baudin toward the scientists associated with the expedition, and abandoned his commission at Mauritius. After six months, he decided that Madagascar offered a better chance to accomplish something for France and for Science, and set about estabhshing a nursery at Tametave for trees and other plants. He died there of fever in December, 1803. Frangois, now arrived at vigorous young manhood, sharing his father's objectives and feeling strongly as Andr6 had done that there was still much unfinished business in America, and relying on his experiences and acquaintanceships there, solicited a commission to resume work in the United States! De Chaptal, the Minister of the Interior, dissatisfied with the course of things in the nurseries still hngering near New York and near Charleston, gave Frangois charge of them, with orders to ship to France such trees as still remained in them and to sell the land. He set out on October 10, 1801, from Bordeaux for Charles- ton in the same boat, in charge of the same captain that had brought him home to France years before. Thus, a rather remarkable thing was happening, the father who had rendered his name and that of P>ance illustrious in the history of botany, was denied the opportunity to re- turn to continue this work, being rather sent to his death in Madagascan wilds, while his son, with whom he had begun 318 RODNEY H. TRUE i-ran^ois arrived at Charleston on October Q Tsni a spent much of the winter shipping stock from the' Oh«' T nursery. He also visited Pauf S a'ulnier eft'n chai^flhe Hackensack nursery near New York Sfl„ln; ^ ^ fact, spent the rest of his life at thf, ,''^"'"'"'"' ^s a matter of that he was still serving Vifmlte-^rLI^-'H^^ 't^ have introduced the Lombardv Donlar infl a '' '''"^ *° remains a living reminder of tL^.^T •"'""*• ^^ ««' '^' remain faithfufovTa farthings. "'' '^'"' ^^"'^"^ '"^ Francois had accompanied his father on ,«„ i wearisome and exciting journeys. Now he TasTo nl T' own course and to travel much Hp h^ ! 7 ^ '^^ ^'^ Jersey and along the Hudrn Rive" Si'^'T" '" ''*'" and hickories and sent the seeds to Prafc^ H T^ "'^ °'*'^' .nd. ,„ „„. case., b.ri,. „,efui for u„„i g™ IS 2 1 • J \ trance, ut the forest trees of Franrp nnUr qa kinds reached a height of "^0 (^m nf .u ^^^^^^^ ^"^y 36 up .Ke bull „, „e ,f^'J. ^'^^ « ^^ ^ ^^kl'/uS othet'"lHn'B''sV'f''\?''^''^'P'>'^' --*-« --ong Bartram h' ^ ""i:*""' ^^^^ghan, Peale and William Wirm%aS;r;^.roXnl^?r^ — '« O^^^- and Kenhtr^ '^ J«l "months at his disposal, he decided to go to long ,o„r„y. Cing b, »., „, La„c„ ™ci2, Y„* •'/ ► • ^ ( FRANCOIS ANDRE MICHAUX 319 Carlisle and Shippensburg, he crossed the Alleghenies, arriving in Pittsburgh in ten days, having traveled by stage, horseback and on foot. On July 14th, he set out for Wheeling, where he bought a canoe in which he descended the Ohio River in company with an American officer named Craff. On July 17th he was at Marietta, and on July 24th he landed at Limestone (probably now Maysville) in Mason County, Kentucky. From here he walked alone to Lexington, arriving there on August 10th. He continued on to Nashville, where he spent four weeks collecting in the vicinity or along the banks of the Cumberland River. His return trip was to take him to Charleston. Starting on September 5, 1802, he entered upon a more interesting route than that followed in going westward. Fort Blunt, West Point and Knoxville, Falls of Roaring River and Jonesboro, in Tennessee, brought him to the mountains on the North Carolina boundary line. Soon he reached the farm of old Davenport, who had guided his father in those mountains in former years. He continued on by way of Morgantown, Winnsboro and Columbus, finally reaching Charleston, South Carolina, after a journey of 1800 miles, made in three and a half months. This trip furnished material for a book Voyage a r Quest des Monies Allegheny, published in 1804, and trans- lated into English soon after. In this work one has an ac- count not only of the vegetation, but comments on the conditions and methods of agriculture, on the nature of the soils, particular plant products, and the commercial relations between these backwoods regions and the seaboard cities. On March 1, 1803, he sailed for France, landing at Bor- deaux on March 26th. On going to Paris he found the publi- cation of his father's volume on the oaks delayed. Although the text had been printed in 1801, the plates were still lacking. He also attended to the pubUcation of his father's great ''Flora Boreali- Americana.'' He associated the botanist, Claude Richard, with the work in a literary capacity. Mi- chau::'s efforts at this period also resulted in the publication in 1804 of his own ''Voyage.'' I 320 RODNEY H. TRUE timber trees fo'und fnTslttty 2^^^^^ Tf"'^''' and contrasted the willow o«V .1-7 f ^ ^ °^ America, with the short, scrubb'^t^h fl';^^^^ France. Sandy areas o^ Amela with tu! *^^ ^?* '^"^^ «f valued in naval construction were Tn^rl.t 7'-?f '" '^'^'^'^ tive sands of France m. contrasted with unproduc- desirability of iTuTali J ^ ^^reXefof ^N 'Tr ''' was warmly sunnorfpH ir. 7 ^^ ^^^^^ America A.Hculture^b;r^^^^^^^^^ the head of the Department of R f" ' *"'' temporarily at following the deatHf BeLlin S%'\ ''^ '^'^'''''''y proposed that a naturalist be s2 ff^Kr?"' .^^' P'*" collect seeds and young tL of r-f^' ^*^*'' *° the national nurser^ef of France S^ ""'''l *° "^ ''''' *» Duke de Gaete, favored the nrolcf.T"*"'" ''^ ''"^"*'^' was entrusted with thereZnlSl'Tf '"'°'' '^'"''^'^"'^ American trees to France ^ ^'■^•"« ''»*'" *° ''""8 1806':;^ltr^Tee'ryr:tsra?t^r °" ^^"^-^ ^' the British man-of!war "ider' IntT "''"^^^ "P ''^ on board. After forTvthrtw « '^ ^"'''^"'^ ^^ taken Bermuda Island wt^te '^^^^^^^ ''^ '^"^^^ ^ *he detour in his route ^Ive hU k "^ *° ^"^ ^'^'"■«- This and new scenes Thes he dl 1"!.' '° ''"'^ "^'^ P'^"*« - ,es Isles BermZi' He trtall" V'T "^"'^'^^ mitted to resume his journev Hp « ^ "^"^ ^"^ P^*- States near the end of Ma™ ^^^"^ '" *^' ^"'*^^ Easfe^ uS^Tat: "^S^^^-^r ^™« '"-'^ «^ *•>« he traversed the states of thf AH V^' ^'''"'' °^ Maine, Georgia, covering ttl^lfc:^, ^^^^ ^th- furC tm FRANCOIS ANDRE MICHAUX 321 *•» northeast to southwest. Along the coast he visited shipyards to see the timbers and methods used in American ship con- struction. He talked less with botanists and other scientists and more with artisans, and sought to meet the most skillful workmen in wood. He made five explorations into the interior. He penetrated Maine along the Kennebec and the Sandy Rivers, perhaps to the Rangeley Lakes. He crossed New Hampshire and Vermont, going from Boston to Lake Cham- plain. Farther south he traversed the present state of New York from the city of that name to Lakes Erie and Ontario. Proceeding from Philadelphia, he reached the Monongahela, Allegheny and Ohio Rivers, and again following a route that must have been better known to him from his earlier travel, he went from Charleston to the sources of the Savannah and Oconee Rivers. In these journeys into the interior, he studied the trees making up the bulk of the forests, with special reference to the nature and uses of their woods, their com- mercial values in the exchanges between the states and in their relation to demands for exportation. Barks used in tanning, qualities and prices of woods, values for fuel, cabinet work and otherwise, were objects of his inquiry. A complete collection of polished specimens of different woods was made. He remained in America about three years on this quest, making many new acquaintances among the prominent men of the day, Muhlenberg, William Hamilton, Barton, Hosack and Alexander Wilson being among the number. This may have aided in bringing about American recognition. On April 14, 1809, Michaux was made an honorary member of the Philadelphia Society for Promoting Agriculture, a Society organized in 1785 to advance agricultural interests. The membership of this Society overlapped that of the Ameri- can Philosophical Society in many instances, and for years the two organizations met in the same rooms. In the minutes of the meeting of the Philadelphia Society for Promoting Agriculture, held on January 10, 1809, Dr. James Mease, of Philadelphia, a member of this Society, proposed the name of 322 RODNEY H. TRUE M. Andre Michaux, of Paris, as an honorary member, his election following at the meeting held on February 14th of the same year. Although he later lived near Paris, his interest in the affairs of the Society continued. Repeated references to communications received from him, pamphlets, books, and other publications, now on the shelves of the Society's library, offer substantial evidence of this interest. On April 21st, of the same year, Michaux was elected to membership in the American Philosophical Society, an honor highly esteemed during the rest of his life. The Michaux Fund gives eloquent testimony on this point. He now returned to France, presented himself to the Central Society for Agriculture that had sponsored his enter- prise, and reported what he had done. The results of his efforts in America had been reaching France in shipments from time to time in the form of seeds and young trees. In this effort he had better success than his father. From the seeds sent over by the son, about 250,000 trees were obtained. A committee was appointed with Corra de Serra as chairman to report to the Central Society on his effort. The report was most flattering. Michaux was complimented on the faithful execution of his trust, and a vote of thanks was passed in recognition of the important service rendered. With his great task of travel and physical hardship in a new and untamed world successfully accomplished and appreciated by his sponsors, Michaux, now about forty years old, still had long years before him in which to aid through efforts in France to bring to the homeland the solid result toward which the efforts of both his father and himself had been directed. For two years following his return to France, he was busy in securing the publication of his great work ''Histoire des Arbres Forestiers de VAmerique du Nord.'' Volume I appeared in 1810, volume II in 1812, and the third volume in 1813. The text was illustrated with 144 copper plates, designed by the eminent French artists, the Redout^s. In 1817, an English translation in four volumes by Augustus L. r- ^ I ♦ t \ 4|> 1 I I- f I: FRANgOIS ANDRE MICHAUX 323 Hillhouse was published in Paris under the title of ''North American SylvaJ' WilHam Maclure bought the plates in Paris and brought them to the United States. American editions followed, the first brought out in New Harmony, Indiana, in 1842; a second appeared in 1852 in Philadelphia with notes by J. J. Smith. After the appearance of his great work, Michaux devoted his attention to the cultivation and propagation of trees for public use in France. The Central Society of Agriculture still supported him by entrusting the use of a large estate to his care for experimental purposes. On his own country place near Pontoise and on the extensive lands of M. Dela- marre, he carried on work of this character until his death. After a strenuous young manhood, the quieter life of the experimentalist must have had its rewards. He had the time and opportunity to carry to a successful conclusion the projects that had sent him to the wilds. He continued to take a special interest in the use of the so-called ''heaths," of which France had at that time two million unproductive acres. As a result of forty years of experimentation by Michaux and the Central Agricultural Society, it was shown that these lands could be improved and used productively by the growing of ''certain resinous trees," undoubtedly pines. This seems to be indicated by him in a letter written in 1852 to Isaac Lea, then President of the American Philo- sophical Society, in which he mentioned the Russian pine {Pinus sylvestris), known to us as the Scotch pine. He recommended this tree for special consideration in the northern and middle states of this country. It may seem somewhat strange that one who had studied the trees of this country and the conditions prevaihng here, as Michaux had done, should have found any considerable use for this inferior species in the regions named. In his letter to Isaac Lea, just referred to, written at the ripe old age of 82 years, he glances over the past. Speaking of his Sylva, he says: "The science of botany was the principal object of my father's explorations in North America and the 324 RODNEY H. TRUE Flcra Boreali-Americana' vias the result of these explora- tions As for me, I took another view of the vegetable kingdom whilst in our country— a view more limited and less scientific, It IS true, but perhaps more generally profitable to the farmer and the landholder, as well as to that class of society, so numerous in the Northern States of the Union who employ wood in so many different ways. I do not consider my Sylva Americana as complete as it might be- thus, for instance, I have omitted several species which grow in lower Louisiana and in the two Floridas. In the second place, I have described and figured some trees that are deficient m the flowers and in the fruits. Had circumstances permitted, I would have returned to the United States, and m a new edition, have corrected the errors and filled up the omissions. I would thus have been able to present to the American nation a work worthy of her great name, but now that I have arrived at a very advanced age— nearly 83 years —I can do nothing in this respect other than to express my regrets and the hope that some native arboriculturist may com- plete my researches on the plan which I have adopted The publication of such a work would be attended with much benefit to the country, and afford particular honor to him who would undertake it." Although he would be hardly called an arboriculturist, an Enghsh-born botanist, who also attained scientific fame in America, may have come in answer to Michaux's prayer. Thomas Nuttall, a botanical disciple of Benjamin S. Barton ot the Botamcal Department of the University of Pennsyl- vania, later himself professor of botany at Harvard Uni- versity, brought out a book in three volumes dealing with about 120 kinds of trees, mainly from the far west, a region not vis.ted by Michaux. The two works supplemented each other in the way that Michaux had hoped, and the set of vol- umes makes a very important contribution to our knowl- edge of the forests of the United States a hundred and twenty-fave years ago. Michaux's interest in America dealt principally with trees, TRANgOIS ANDRE MICHAUX 325 ■--J - ^■. h J^ .Vi ^ I ! but he concerned himself with other matters also. He happened to be planning a trip along the Hudson River in 1807, and hearing that a steamboat was to make its trial trip soon, found his interest aroused. He was pleased that he and another Frenchman were passengers when Fulton made the first trial trip of the '^ Clermont." That they were the only passengers may also have added to his satisfaction. Not long before his death (1852), he added his contribution to the bulky literature that grew out of the terrible yellow fever epidemic that visited Philadelphia (in 1821) under the title of '' Causes of Yellow Fever in the United States.'' Michaux's life as a bachelor ended in a rather sudden decision at an advanced age to marry his housekeeper, a remote relative who had long managed his household. Elias Durand, a Frenchman residing for a time in America, who had known Michaux in his later years, and who had aided him by collecting and forwarding American tree seeds to France, writes in his account of Michaux, published in the Transactions of the American Philosophical Society in 1890, that he was carried off with frightful suddenness by a stroke of apoplexy on the 23rd of October, 1855, ''after a busy day among his American trees." He was in comfortable financial circumstances, and deposited a will with Isaac Lea four years before his demise, providing for the now well-known Michaux Fund, to be used by the American Philosophical Society after his wife's future had been provided for. After the death of this lady, the fund was used in part for the creation of a monument to the generous donor, and this monument still exists as the Michaux Memorial Grove of oaks in Fairmount Park. This grove was planted near Horticultural Hall at the opening of the Centennial Exhibition in 1876, when Fairmount Park was in the earliest stage of its formation. The Chairman of the Committee on Trees and Nurseries in the Fairmount Park Commission, Mr. Eli Kirk Price, who was also chairman of the Committee on the Michaux Fund in this Society, reported to the Society on 326 RODNEY II. TRUE June 16, 1870, that "he had visited today the Fairmount Parle nursery and found the ground well taken care orand he large s ock of trees in a flourishing condition. Of the acorns planted last winter, nearly all have grown except those of the 'Bartram Oak,' and of the fifty or sixty of those planted Jl^ThylfJ >^"- '^ ^^^ ''' '-^' ^« ^" ^— o^ ^hat oak It seemed to me a matter of interest at this time to see what has happened to this Grove in the sixty or more years smce It was established. ^ Accordingly, the area indicated to be that occupied by ExhStTon of TstTv,"' ''' ''r' P"P"^^^ ^°'- '"^^ C'-tennial J^xhib tion of 1876, shown us by Mr. S. N. Baxter, the present Arbonculturist of the Commission, was visited and the Z of a number of trees now present determined by the use of an jncrement borer^ Mr. R. D. Forbes and his asstrt, Mr O M. Wood, both of the Allegheny Forest Experiment Station, made the test on trees selected by Mr. Baxter as representative specimens. Out of 7 tested, 6 showed an aee estiniated to be 00 years. These trees undoubted^ belong ear thaTt^M ^'•"^r.^"'" '""'^ e..min.Uon ft seems clear that the Memorial Grove is still in large part made up of the oaks reported on by Mr. Price in 1876 and, judging from the uniformity of the Grove, relatively few of he original trees have had to be replaced. Michaux Avenue now passes through the Grove and presents one of the most beautiful spots in the Park. Thus prime, and, if spared by weather, disease and other hostile mflue„c.s, should delight generations still to come before S age in the end brings them down. In closing this sketch, I will take the liberty of making some suggestions regarding ways in which the Society may continue to carry out the will of the donor, and at the s^me time advance the significance of the organization. Michaux was an ardent advocate of a public policv of support for the scientific study of wild plant iffe. He stuLd FRANrOIS ANDRE MICHAUX 327 ^^ f ^ '^ '/" - * i ♦ ^ I > r r X it with equal interest with reference to human utility. Prob- ably influenced to a considerable extent by the needs of France at that time, his special efforts were centered on trees in nature and under cultivation. Something perhaps re- mains in the forests of France of the trees introduced by the Michauxs. How much they actually did for their country in this respect I do not know. For us and for the rest of the world, their greatest accomplishment is seen in the writings they have left. With the world changing as rapidly as it is, authentic records of the past should be sought out and held with great care by repositories like this Society. Hence, accounts of explorations and records of earlier days in the fields and forests are basic material. As the scene changes, succeeding accounts will continue the story. For example, the question of soil conservation, a very old one and now much in the public mind, is presenting itself as a series of problems in agriculture, forestry, wild life preservation and human welfare in all of their many aspects. Old records tell of nature and of early human life. We should have those early records. The story should be con- tinued by the important writings of later days. Thus, the early contributions of the Michauxs, father and son, remaining in our hbrary, become a priceless possession as the record continues to unfold. Memorial trees in time die and their places are forgotten. The practical accomplishment of the past must be sought in books and other lasting sources of information. Exploration, forestry, botany, plant introduction and plant utilization, the problem of the soil that supports all life, are subjects that may fittingly claim the earnest attention of the Society in building up and preserving its collection of records. Utility may be immediate. Certainly there will be a priceless value for the future if civilization persists and present ideas of worth continue to be held. * • m ^ ^i.-^ - A Reprinted from The Bryologist. Vol, XL, July-August, 1937 l„ A .^^ . if LICHENS FROM THE NEW ENGLAND COAST Rodney H. True Some years ago, the writer spent parts of two summers on the islands of Casco Bay and collected a few specimens of lichens that were sent to Dr. C. C. Plitt, of Baltimore, for identification. Fortunately, the list of names given to them was received in Dr. Plitt's lifetime. The specimens named have been ' 0 ^ - I. • > —72— tZtfJl]'' '"'""■"" °' ^'" ^^P^^^"^^"' ^f ^«^-y of the University of The list that follows is prepared from Dr. Plitt's manuscript. The several locations at which collections were made with dates are indicated also the nT^ bers of the several collections given by the collector. """ Orr's Island, Cumberland Co., Maine izTy:::f::LTp'^^^^^^^ -^s is^s) Aug. .,, .,... /'. '9-^' --.head of Longrov"er lTt^Z^\%''r ''""" ^^^^^ J"'^' C/at/oma cnspata (Ach.) Fit. f. tnfundibultjem (£haer ) Wainio -On H^ • stump, dry ndge. (P55) July 30, 192^. ^"^""^^'-^ Wam,o.-On decaymg '''"1r^)7ulT;^^ ,?2f -^-^^ '"^^'"^ ^-^ -^ h-- -" - dry clearing. Cladonia fimbriata (L. f.) var. cornuto-radiata Coem fnrohahlv'^ — Hn • u earth near low shrubbery. {950) July 30 IQ2V CProbably).-On rich Cladoma gracilis (L.) Willd. var. elongatd Qacq ) Flk -Amonc mn««.c schist rocks near Great Island BHdge.^(^6o) Aug 21 iSf v fSS^'^nn'^H ^' shaded rock Aug. 20 1923 » v ^f ^"g. -fi, 1923, {^9^4) on dry C/ado^.^^3«^ML.m. var. «,;..,a (Flk.) Mass.-Rich soil, in edge of woods Cladonia^ran^ferina (L.) Webb.^n dry ground under spruces (867). Aug. .4. "'""'Aur.t^"'" "'"""'" Wainio.-On dry rocks, ridge east of village (83s). Ctadania squamosa (Scop.) Hoffm. var. denlkolHs (Hoff m ) Floerke-On ri.h earth under shrubbery (947), ((,^8) July 30. 192? i-'oerke.— On rich "'"rnd%:?iSrnder- path to CWo«MMr/«(//oM^?^;,^ i 1 DMistes scruposus (L.) Nor™.^"n^ d^ mTclSsTi^.l^/^J;)',''!"','^?^. "ntrge^(f^;):'(^ffi„,^",','; ^J/.-"- '"---'- -^= -^ ^-t ,s.and ^7.;ra,''(^ioMrA%.T-,^^^^^^ --^^ -- °' ^'"- (*^*). (*->. Lm^a granulosa (Hoffm.) Ach.— On dry mica-schist (S2^) Amc t^ 'nr.nu'gV?;t/ <'- '•' <^---<>" 'S--^^'rilt"r'^fvi,,age. east of viflage W), (^j), Aug^^T^ ."gT^ '•^" ""'=^-^'"'' ™^''^- """^ Par«W. M^.*. a.)^Ach.-Co™.on on branches of spruce (pdo), July 30, V t- '•» . ft *^ ^ ■>, * * » • —73— Peltigera apthosa (L.) Hoffm.— On damp shaded soil among mosses, path to David Orr's cove. (J885), Sept. 7, 1922. Peltigera polydactyla (Tuck.) Hoffm.— Dry soil under blueberries, (o^d). Tulv 30, 1923. Physcta ohscura (Ehrh.) Th. Fr. var. endococcina (Kbr.) Th. Fr.— On mica-schist rocks. (8 1 pa), August 19, 1922. Pertusaria amara (Ach.) Nyl.— On mica-schist rocks. {816), Aug. 19, 1922. Rhisocarpon petraeum (Nyl.) Zahlb.— On dry open mica-schist ridge. (817) (824), (827), {828), Aug. 19. 1922. ^ ^^ Stereocaulon paschale (L.) Fr. — On mica-schist rocks. (829), Aug. 19, 1922. Umhilicaria puslulata (L.) Hoffm.— On mica-schist rocks near Great Island bridge. {861), Aug. 21, 1922. Scattering collections were made at other points in Casco Bay during the same years. Determinations were also made by Dr. C. C. Plitt: Cladonia gracilis (L.) Willd. var. elongata (Jacq.) Flk.— Dry hillside, north of entrance to New Meadows River Basin, Sagadahoc Co., Me., July 18, 1923, {936). Cladonia pyxidata (L.) Fr. var. chlorophaea Flk. — Moist shade. Indiantown Island, Sagadahoc Co. (1006), Sept. i, 1923. Cladonia rangiferina (L.) Web. — Dry hillside, north of entrance to New Meadows River Basin, Sagadahoc Co., Me. (926), July 18, 1923. Cladonia reticulata (Russell) Wainio. — On the ground, Small Point Harbor, Cumberland Co. (qqj), Aug. 23, 1923. Cladonia squarrosa (Scop.) Hoffm. var. denticollis (Hoffm.) Flk. — Under spruce trees, Ragged Island, Cumberland Co. {768), July 15, 1922. Cladonia uncialis (L.) Web. — Open dry ground. Small Point, Sagadahoc Co. (PP7), Aug. 23, 1923. Gyrophora Dillenii (Tuck.) Mull. Arg. — On mica-schist outcrop. Small Point Harbor, Sagadahoc Co. (1002), Aug. 23, 1923. Umbilicaria pustulata (L.) Hoffm. — On shaded rock face, entrance to New Mead- ows River Basin, Sagadahoc Co. (Q2j), July 18, 1923. Usnea dasypoga (Ach.) Nyl. — On dead spruce, entrance to New Meadows River Basin, Sagadahoc Co. (Q37), July 18, 1923. On branches of conifers, Ragged Island, Cumberland Co. (772), July 17, 1922. Nantucket Island During a brief visit to Nantucket Island in 1923, a small collection of lichens was made. The names by Dr. Plitt and numbers follow: Cetraria islandica (L.) Ach. — Dry soil. {(;67e), Aug. 8, 1923. Cladonia alpestris (L.) Rabenhorst. — Common on dry sandy uplands. {p6i), Aug. 5, 1923. Cladonia cristatella Tuckerman. — On dry sandy soil of Commons. {962), Aug. 5. 1923- Cladonia furcata (Huds.) Schard. — Dry soil of Commons. {964), Aug. 5, 1923. Cladonia rangiferina (L.) Web.— Common on soil of dry uplands. {963), Aug. 5, 1923; {967c), Aug. 8, 1923; (,967d), Aug. 8, 1923. Cladonia reticulata (Russell) Wainio. — Dry soil of Commons. {967), Aug. 5, 1923; {967.0, {967 g), Aug. 8. 1923. Cladonia verticillata var. evolutu Th. Vr. — Dry soil of Commons. (967a), Aug. 8, 1923. Botanical Department, University of Pennsylvania c^Tm i\ ^' IRREGULAR PAGINATION -* * I - ^ [Reprinted from Phytopathology, January, 1938, Vol. XXVIII, No. 1, pp. 24-49.] GALL DEVELOPMENT ON PINUS SYLVESTRIS ATTACKED BY THE WOODGATE PERIDERMIUM, AND MORPHOLOGY OF THE PARASITE^ R . P . T R U E 2 (Accepted for publication Aug. 26, 1937) INTRODUCTION A Peridermium attacks Scotch pine, Pinus sylvestris L., plantations bor- dering Round Lake near Woodgate, New York, causing susceptible trees to form globoid galls. Since its discovery there by York (13) in 1925, it has been called the Woodgate rust or the Woodgate Peridermium. York's observations led him to believe that the rust has been present since 1895 in these plantations, some of which, planted in 1870, are among the oldest in America. Some trees of P. sylvestris do not form galls after natural infection or inoculation with the fungus. Hutchinson (6) has recently in- vestigated this problem of resistance. The present study deals with the reactions of susceptible trees and the morphology of the rust fungus. INOCULATIONS AND FIELD NOTES One hundred forty field inoculations were made upon 27 trees of Pinus sylvestris, ranging in age from 9 to 30 years and located in the infection area, supplemented the naturally infected material available for study. The inoculated trees varied widely in susceptibility, as indicated by the varying number of galls they bore from natural infection. The inoculations were made between the 7th and 30th of June from 1930 through 1933. Suscep- tibihty was found to be highest during the first half of June and to decline toward the end of the month. The aecial inoculum was obtained from fruiting galls collected in the vicinity. Spores for immediate use were carefully removed from the galls as soon as the latter were collected. Galls whose spores were to be used in later inoculations were placed indoors in a dry shaded place and the viabil- ity of the spores was tested periodically by placing samples of the spores on fresh well water and noting their percentage germination. Inside storage was resorted to because the spores of galls left late in the field often are blown away or washed out, or they germinate in the aecium. The method of inoculation was that employed by York,^ using the cellu- d — r« <> 1938] True: Gall Development on Pinus Sylvestris 25 f T -* < «i 4s- 4 «- ♦ » < I > » ri^> r-iir » ) loid iceless refrigerator. Early inoculations were made upon the stems of the current season 's shoots at the time when their elongation separated the developing needles sufficiently to permit a direct inoculation upon the sur- face of the stem. The needles borne on the young shoots had elongated to more than half their mature size before the last inoculations were made. Field observations and collections of artificially and naturally infected materials were made each year. During the summer of 1933, observations were recorded and collections made daily for periods of from 3 to 7 days after inoculation and thereafter at weekly intervals. In other years, after the month of June, the intervals between collections varied from 1 to 2 months until dormancy. Notes and collections also were made in late No- vember and in May to reveal the developmental stages present shortly after entry of the host into the winter condition and its breaking dormancy. anatomical studies Methods Newly inoculated shoots, developmental stages, and mature galls were fixed in the field. Bouin's solution, formal-acetic-alcohol, and formalin- alcohol proved the most satisfactory of the 13 fixatives tried. The first and the last gave the best fixations of the fungus. During fixation, air was removed from the tissues by placing the vials containing the specimens in a closed chamber and evacuating it with a suction pump. After 12 to 48, and, in some cases, 72 hours of fixation, most of the material was dehydrated and transferred into pure hard paraffin by Zirkle's (14) butyl-alcohol method. Tissues not imbedded at once were stored in the dehydrating mixture made up of 30 parts of water and 50 to 20 parts of ethyl and butyl alcohol, respectively, where they remained reasonably soft until dehydration could be completed. Early infection and developmental stages for histological study as well as three-year old galls were cut from 8 to 15 p thick. Material with sori in the bark was cut as thin as 3 p. Land's method, using a saturated solution of gum arable followed by a 1 per cent potassium dichromate solution to float out the ribbons, was found most satisfactory in fixing the sections to the slide. The stains that most clearly differentiated the mycelium in its relation to the host tissue were Orseillin BB in a 3 per cent aqueous solution of acetic acid, counterstained with a similarly acidulated aqueous solution of anilin blue, or with a saturated solution of crystal violet dissolved in clove oil. By following a procedure recommended by Doak,* crystal violet in clove oil was used alone to show excellent detail in hyphae and haustoria, but it often necessitated destaining the host tissues too far to show them clearly. The combination of Heidenhain's haematoxylin and iron alum was used to show nuclear phenomena and, in combination with safranin, to show the histo- logical details of the gall. * Personal communication. IRREGULAR PAGINATION 26 Phytopathology [Vol. 28 For field study, the following rapid method was used to obtain permanent slides : Fresh material was cut in water on a portable sliding microtome and placed at once in Bouin's fixative, where it remained for 10 minutes. It was then washed in water until no further color of the fixative came from it and set to stain for five minutes in Orseillin BB. Excess stain was washed out in the early steps of dehydration and, after a brief clearing, in clove oil fol- lowed by xylol, the material was mounted in balsam. The whole schedule may be completed in from 30 to 45 minutes. External Morphology of Gall Development In order to make it easier to follow the sequence of internal events de- scribed under histological studies, it seems desirable to give a brief summary of the external phenomena that often follow infection and accompany gall development, as observed in inoculated material and supplemented by obser- vations of galls resulting from natural infection. While the phenomena here considered often occur as described, gall formation is very variable and the times and sequence of events show such variation that this brief outline must be considered only as an aid to the understanding of the histological phe- nomena involved. Infection occurs primarily through the epidermis of the current season's shoots. Points of infection often are marked by spots that usually range from orange to dark brown. These sometimes appear in the first week of July. Their margins are often sharply defined, but frequently are less definite, and may appear water-soaked. Infection spots do not necessarily indicate successful invasions. Infections whose spots show indefinite or water-soaked margins seem more apt to produce galls than those with more sharply delimited spots. As observed, slight swellings may result in the season of infection below many infection spots and spot-free points of infec- tion. In the second season, dead shrunken cortical areas may appear in the central portion of the surfaces of the swellings, which by the end of that season may become distinct spherical or hemispherical galls. In May of the third season, two years after infection, slight colorless exudates are at times found near the margins of the sunken areas, which, in some cases may be associated with internal pycnia. Toward the end of the third season the central sunken area occupies most of the gall surface, and is bounded by a slightly raised rim. During the fourth season the gall usually acquires those external charac tenstics which typify it during its further enlargement (Fig. 1, A) Aecia are produced in the central sunken areas of the roughly spherical or hemi- spherical galls (Fig. 1, D). As the aecia develop, they push up the dead tissues, which usually scale off, eventually exposing the often confluent fruit- mg surface (Fig. 1, C). Since the layer of aecia-bearing tissue is in turn usually exfoliated before winter (Fig. 1, E), the central portion becomes more and more sunken in comparison with the peripheral rim, which then appears as a collar delimiting the gall (Fig. 1, A, C, and E). On an occa- r J r 1938] True: Gall Development on Pinus Sylvestris 27 t ♦- ■4 I A X« -^ n > { sional tree, the periderms of the galls are not sloughed off but, as swelling continues, are cleft by deep cracks in which the aecia may be borne (Fi» 1, F.) Fig. 1. A. Typical spherical gall with sporulation well advanced. B. Young fusi- form swellings due to multiple infections. C. Gall with sporulation completed. Note complete exfoliation of periderm external to aecial layer. D. Hemispherical gall. E. Gall with aecial layer being sloughed away by a new periderm formed benath. F. Bibbed gall with adhering periderm. Galls on an occasional tree become necrotic and frequently develop abor- tively, or are misshapen, often through necrosis originating in their central areas, which then appear much more deep-sunken than in normal galls. Such necroses may well at times be caused by invading secondary fungi. Mass infections resulting from inoculations of twigs of highly susceptible trees may cause an extensive killing of the outer layers, which, with subse- quent swelling, crack deeply into the woody tissues forming longitudinally 20 PllYTOI'ATlIOLOCJY [Vol. 28 For field study, t]io followino- rapid method was used to obtain permanent slides: Fresh material was ent in water on a poi-table slidin<> microtome and placed at once in Bonin's fixative, where it remained for 10 minntes. It was then washed in watei- until no further color of the fixative came from it and set to stain for five minntes in Orseillin BP>. Excess stain was waslied out in the early steps of dehydration and, after a bi'ief clearino-, in clove oil fol- lowed by xylol, the material was mounted in balsam. The whole schedule may be completed in from 30 to 45 minutes. External Morpliolooy of Gall Development In order to make it easier to follow the sequence of internal events de- scribed under histolooi^al studies, it seems desi.-able to o-ive a brief summary of the external pheuomena that often follow infection and accompany ^all develojnnent, as observed in inoculated material and supplemented by obser- vations of o-alls resultino' from natural infection. While the phenomeua here considei-ed often occur as described, jrall formation is verv variable and the times and sequence of events show such variation that this'brief outline must be considered only as an aid to the nnderstandin- of tlie histological phe- nomena involved. Infection occurs primarily throu-h the epidermis of the current season's shoots. Points of infection often are niarked l)y spots that usuallv ranf?e from oranjre to dai-k brown. These sometimes appear in the first week of July. Their mar-ins are often sharply defined, but frequently are less definite, and may appear water-soaked. Tufection spots do not necessarily indicate successful invasions. Infections whose spots show indefinite or water-soaked maro-ins seem more apt to produce -alls than those with more sharply delimited spots. As observed, sli-ht swellin-s mav result in the season of infection below many infection spots and spot-free points of infec- tion. In the second season, dead shrunken cortical areas mav appear in the central portion of the surfaces of the swelliuo-s, which by the end of that season may become distinct spherical or hemisj)herical -alls. In ^Fay of the third season, two years after infection, sli-ht colorless exudates are at times found near the margins of the sunken areas, which, in some cases may be associated with internal pycnia. Toward the end of the third season the central sunken area occupies most of the -all surface, and is bounded by a slig-htly raised rim. Durin- the fourth season the jrall usually acquires those external charac- teristics which typify it durinpr its further enlargement (Fi- 1 A) Aecia are produced in the central sunken areas of the rouo-hlv spherical or hemi- spherical ^n]h (Fi- 1, D). As the aecia develop, they push up the dead tissues, which usually scale off, eventually exposin- the often confluent fruit- mn: surface (Fi- ], C). Since the layer of aecia-bearin- tissue is in turn usually exfoliated before winter (Fi- 1, E), the central portion becomes more and more sunken in comparison with the peripheral rim, which then appears as a collar delimitino- the -all (Fi- 1, A, C, and E). On an occa- 1.. - i - '.^ t t: • • A A 1", ) < > i 4 • '40 r 4 , J. . ^ t ' 1938] Tkue: Gall Dlvelopmkxt ox Pints Svlvkstkms 27 sional tree, the periderms of the -alls are not slou-hed off but, as swelling continues, are cleft by deep cracks in which the aecia may be borne (Fi- 1,F.) Fig. 1. A. Typical splKTical jjnil with sporulntioii well ndvaiicciL H. Young fusi- form swellings duo to multiple infections. ('. (Jail with sporulation completed. Note complete exfoliation of periderm external to ai'cial layt-r. I). Hemispherical gall. E, Gall with aecial layer being sloughed away I»y a new periderm fornu-d henath. F. Ribbed gall with adhering periderm. Galls on an occasional tree become necrotic and frerpiently develop abor- tively, or are misshapen, often throu-h necrosis ori-inatin- in their central areas, which then ai)pear much more deep-sunken than in normal -alls. Such necroses may well at times be caused by invadin- secondary fun-i. Mass infections resultin- from inoculations of twi-s of hi-hly susceptible trees may cause an extensive killin- of the outer layers, which, with subse- quent swellin-, crack deeply into the woody tissues formin- lon-itudinallv INTENTIONAL SECOND EXPOSURE 28 Phytopathology [Vol. 28 sparsely, if at all. " ^ ^- ^' ^^' ^^'^'ch fruit very Histology of Invasion and Host Reaction Epidermis and Primary Cortex. Most suceessfnl ,-nf.„f ing periderm formation. During thistenod t^! L ""'"' ^'""^- epidermis whose outer walls are, i^ general heavH f'";iP™*««t''d by an with a thick cuticle (PI I i) aTTC ^ '^"'^^'^ ''"'* «■•« «o^ered walls of the epidermacells'areunffL!!^ \T, "'""' ^*''^''^'«^' however, fied throughout, and ustlly a^e S^il^hTr"^^^ wallsofeventhesubeDiderm«llin I.' ^*'"'°'^ ^^'^^' *e outer appear. The eellfoTtheTr^t s" S^^^^ "'"''^' ^""^ ^""'^^ «*"-*« elongate (PI. I n other .. ™''?"'«™*' J^y"'' are large and radially typical o/parenchU in^^' 1^0;';"''"^^ T^^^ '" '' m the nner cortex are vprt;„oii ,*''''*'• ^^"» of the resm ducts found stances, as do aTw „gnS Lt"'>l' '"' f ^" •^<'"*«'" *--"-»ke sub- through the cortica7plreSra Ih.^'lf'' """'' "''''='» ^"-^ -ottered arises from the subepfdermal eX „. f * t" "'""''' °* J»"« « "^^tem 2 or 3 rows of thin-waU tal„ ,L T *'*' '''" "^•^^'^ "^n^^th, forming to Which the cells die a^ tClt^Ltr (Vll^/) ^''^""="- -*--' portions. Appressoria'hrnttTernr;^^^^^^^^ ^ ^^^ "^^"'^ penetrating the cuticle and outer wall, of ti , ^' ' ^- ^^^ ^«™ tube narrowed, but, once inside k broad tf ''"'^'™*" '^"^ '^ considerably before leaving the cell tr;lr the "1 T'"^^"''''^''' ^"'^ '"^^ branch 3, A). In the cases of cells Jen.Vfu''™^' mtercellular space (Pig outer walls was atlil^tc'Trp^^Sn'^.r"' *^ *''■•=''«'""=" "^ *e boring epidermal cells. Thisseems n„t r "^ '" ""P'"^*™*^^ ""^h- fungus, since no unpenetrated lTl« f "^ '"""''■ ''^ ^'''««tion by the the same or .similar m'aS1fT:lT„uTf"' thinness were found in actual dissolution of the wall or of prevenW / "^"', '"^*"°"' ^^''^''- "f duced quickly, since it is already obvlus in " "Tf ^J''""'"^' '* '' P"-"" lat.on. Once the infecting hypha paTes IZT "".'' " ''"^^ ''"" '"««»■ tercellular space below it brancL r!! r. ] ' «P>dermal cell into the in- ing epidermal and sub ^ dermatc , L S " /'u'' '^"""^ '"*" ""^''bor- haustoria, but, unlike the typS hlu tori fo' ""fr ""'•"" *° '""'^"°" «« are often septate and ^ay^ven^rrnlTpS fB) ^^^^^ ^'d "^*"- ^''^^ intracellular development is shnrf i,-, i 7 , ^' ^'^ tendency toward ..r "r;:s.r..ttn- sir;-, t™ »- ^ — «*. . most susceptible trees Histolo.,v! ? r . ^ formation, even on the .. .- ..« .... ....r,'s' r r„,s.'„tr.,;' r:rn: 1938] True: Gall Development on Pinus Sylvestris 29 •^ # > Fig. 2. A. An invasion resisted partially by cell hypertrophy as at A. B. An inva- sion spreading vertically along the resin canal (a) has stimulated the adjacent tissues to cell division and radial cell orientation accompanied by marked reduction of intercellular space. C. An invasion being effectively walled out by a cicatrizing zone. D. Radial longitudinal section of infected phloem and xylem. Note broad rays with abundant intercellular hyphae (a) ; short, blunt irregularly pitted tracheids (b) ; abundance of phloem parenchyma (c) ; and resin-containing cavity (d) associated with radial resin canal not shown in this section. E. Transverse section showing wedge-shape area of gall wood with adnormalities intensified at the edge. Note also the internal and exter- nal areas of killing in the cortex. F. Longitudinal section showing a late stage in the development of the '* collar" with living tissues considerably isolated radially by the death of cells interior to them. resistance, others are constantly opposed by partial barriers, and yet others may at once be surrounded by barrier zones and walled out completely. Tn the course of the present study, a shoot no more than 2 cm. long, sectioned serially a month after inoculation, was found to have 24 infections. Five of these showed no trace of reco^izable mycelium among the very abnormal 28 Phytopathology ^v [Vol. 28 confluent furrowed swelling's. These somefimp. f -w develop into rou.h.y f„.ifo4 eon-pt LrS'piJ"' 3, ^,7 7. "'"^ °- sparsely, if at all. ^ ^' ' ^'' ^^J^'*'' fruit very Ilistolos- of Invasion and Host Reaction Epidernm and Primary Cortex. Most suecessfnl inf».r ."f. peridern, fonnation. Durin. tl.is pe Id th! tT " "''"'"' ^"''''^- epidennis whose outer walls arP ,C ' ,T ^" " Protected by an with a thick cutic. H 1 ' YfTi' "■''■ """■■''"^ «"'' -« — d walls of the epider„,a cells; eunffllri """" ''""'"' '"""^"'■' fled throughout, and usual,; a ^ i if ihTT"" *,''r''''™'^' "'^''"- walls of even the subepidermal 111 l[' ""'"''"' '""^''"' *>'« outer appear. The cenfof eX J ^''"' " ^""'"^ ""' '""'''" •'"'™«*^ elongate (PI. I Ottr no '"''?"""™«' "'J'- «re large and radially typieal 0/ pare; J, v.r i ir r « ' ^S; of^'tl '''''^ T'^' "" ^ in tlie inner cortex are vertic«IIv »i * , °^ ^^^ '"*"" •^"'^t« found stances, as do a f^uS^i:^::!i'"'''r'''''''""^'''''^^"'- through the cortical pareTchyma Ih .'^f, '■?'"' '''^"'^ '''' '*««"<^'-«d arises fron, the snbepfdermal eelk n. f " "'"'''"' "' '^"'"'^ '^ '"^''-tem 2 or .3 rows of thiu-w J ta,''en IK T "'" ''" '"•^''''•^ '^^"'"'t''' f""""'? to w^.icb the cells die a^d^^ ^ ^t^^^^^^^^^^^ "■■«"-"• ^ ' dent::;trr:;r!tTo'r;t:S !^-" '"""^ "-="'' ^''e epi- Po.-tions. Appres.soria' W n tTe i^^^^ the less Hgnified penetrating the cuticle and outer Sis of ! " ' ^ ' ^'"' ^"'•"' *"''« nan-owed, but, once inside i broad nsf ''''''•''''""' '''^' '' considerably before leaving the cell to ;rterth"V;'''''"f •"''''•''''• '"" '"«>■ •'•anch 3. A). In the cases of cells pel latedbv'™! ■"*<"•-""'- «P-e (Fig. outer walls was at times m„c!^, S Jlon, T\ '•' *''" """''•"""-" "* «'« boring epidermal cells. T seems not t? "" '" """""''*'•«*«' neigh- fungus, since no unpenetra ed tv^ls 7 V^**'^'' "' ^•"'"«*'"" V the the .same or similar maten^ If i:.;::/':"''''^ "'■'•""" ^"" ^■•^""" '" actual di.ssolution of the wall or of nrel ? / ^'^"' '"^^'^'on, whether of duced quickly, since it is allrobv ' - T •","""' *'"''^^'^''"'''- " - P-'O" lation. Once the infectin ! hvoL t T "'?'"'' °"'^ ^"^ '"^-^''^ ««er inocu- tercellular space beW V 2 ~i '''''" I'" "';''"""" '^'"' ■"*" *"<■ "- in. epidermal and sub piden^a , s T ""' "',"" ''"'""'^ "■*" '""^""""- haustoria, but, unlike the tvD ca l1 \ T *''"'^'' '"^P"" *" ''""<'tion a.s are often septate and n^.^'^^ btrrr.vT^BrT^- '\ ''^ ^''^"^' '"^^ i=t^:i;;z5-;;;\-:-;--r^^ most susceptible trees. Histological st„,li . " ^•"""'t'on, even on the «. - .^. „„. .„„„ „,.,, s «" isr4f:.;.' r.r~ V ^ ^ < ► 4 ■ i- ♦ 1938] True: Gall Development ox Pixrs Sylvesthis 29 Fig. 2. A. An invasion resisted partially by cell hypertrophy as at a. B. An inva- sion spreading vertically along the resin canal (a) has stimulated the adjacent tissues to cell division and radial cell orientation accompanied by marked reduction of intercellular space. C. An invasion being effectively walled out by a cicatrizing zone. D. Radial longitudinal section of infected phloem and xylem. Note broad rays with abundant intercellular hyphae (a) ; short, blunt irregularly pitted tracheids (b) ; abundance of phloem parenchjTna (c) ; and resin-containing cavity (d) associated with radial resin canal not shown in this section. E. Transverse section showing wedge-shape area of gall wood with adnormalities intensified at the edge. Note also the internal and exter- nal areas of killing in the cortex. F. Longitudinal section showing a late stage in the development of the '^collar" with living tissues considerably isolated radially by the death of cells interior to them. resistance, others are constantly opposed by partial barriers, and yet others may at once be surrounded by barrier zones and walled out completely. Tn the course of the present study, a shoot no more than 2 cm. long, sectioned serially a month after inoculation, was found to have 24 infections. Five of these showed no trace of recogrnizable mycelium among: the very abnormal INTENTIONAL SECOND EXPOSURE 30 Phytopathology [Vol. 28 cell a^n'-sletuent^TnU^StU^^^^^^ penetration showing vesicle in epidermal a subepidermal cell. C. Mycelium and haustorium S fhf TF '"P^^*^ haustorium within with short germ tube entering host shoSL ab^nc/nf ? ''^"^ Parenchyma. D. Spore lular type of penetration. E? Diafframmatif ro^Zl\ i^'^^l^T''"'^ ^"^ ^^e intercel- Uum of a typical unresisted infectfonh^on^h T. *^*'^'' ""^ *^^ «P^^^^ «f the myce- spread is shown and the appearance of transv^^^^^^ T'*'^- ^^^^^^ longitudinal cated: a, point of penetration; b extent If «nh!-r' ^^^^"^ ^* ^^"«"« ^^^^Is is indi- growthjD, extent of Wread adjacent tfrLinLct^^^^^^^ T"^^' ^' P^*^ «^ radial r, the phloem. j no resm auct in inner cortex; e, cortical resin duct; >r > *t ->. ^^ r 4 ^ ► '■*■ ^> ri- •^ w*** <* f ^ A 1 ^ n> 1938] True: Gall Development on Pinus Sylvestris 31 and often dying cortical cells. Most of them showed isolated groups of living hyphae penetrating the more normal portions of the cortex and the inter- cellular remains of others where tissue abnormalities were more severe. In the case of one infection, discovered in sectioning, where no external symp- tom was present, the abundant and continuous spread of the large inter- cellular mycelium had produced almost no abnormality in the areas it pene- trated. This infection had penetrated as deeply into the cortex as most of the others, some of which were still confined to the outer cortex. Infections to which little or no resistance apparently is offered are those that cause but slight early abnormality in the host. The abundant large hyphae pass between or encircle nearly every cell in the infected area (PL I, 2 and 3), and many cells are penetrated by the ellipsoid to cylindric haus- toria, which are uninucleate and greatly attenuated where they pass through the cell wall (Fig. 3, C and Fig. 6, E). The haustoria often appear to seek out the nucleus of the host cell and those touching it appear at times to be flattened at the point of contact. The host cells in the infected zone are more spherical and regular in outline than those in the normal cortex, and there is a tendency toward hypertrophy, which is most pronounced in the cells invaded by haustoria. The hyphae of a mycelium whose invasion apparently is unresisted advance in characteristically different directions (Fig. 3, E). Some run parallel to the axis of the twig in the spaces between the first one or two cortical cell layers or just beneath the epidermis. The major portion of the mycelium, however, spreads radially inward through the outer cortex until the inner cortex is reached, and there a second vertical spread occurs, which is most pronounced in the intercellular spaces adjacent to and near the epithelial cells of the resin ducts. These cells often show a tendency to hypertrophy, increasing in size until they may partially or completely fill the ducts. (PL I, 2). The mycelium often spreads a considerable distance about the margin of the phloem before entering it. (PL I, 3). Fresh shoots of the current season's growth, collected September 20, 1933, and tested microchemically, indicated a possible physiological explanation for the diversified paths followed by the mycelium in unresisted infections. The layer of cells just below the phellogen, and at times the cells of the phellogen itself, well established at that date, were found containing starch, particularly near the bases of the needle fascicles, where every cell was filled with it. Elsewhere in these peripheral tissues starch was less abundant, appearing only in scattered cells. Internal to this layer the cells of the outer cortex seldom showed starch except below needle fascicles where starch-con- taining cells are few and scattered. The inner primary cortex, however, had numerous scattered starch-containing cells in the vicinity of the phloem and near the resin ducts. Droplets of fatty substances were present in scattered cortical cells, but were especially abundant in the cells surrounding the resin ducts. Compounds giving a positive reaction to the ferric chloride test for tannins were found in scattered cells in the inner cortex, but especially in the 32 Phytopathology n , " [Vol. 28 cells boraerin«- thp vaoir, a x With rj:^]:'^^^:^^::^' "r -''^^ ■-- ^^ --^-d rapid s„,,ests that the loe.u7JlZ oodS °l*^\'«^-«»- - «ost ence the paths of invasion in the cases jf *'^' ^f "> ^^e host may influ. Partially resisted invasions te„dtofn, r*'*^ '"'*<'*'°"«- progress is unopposed, but they "„e t St^ VT ^'^''^ *^ *^°^« ^''««e s srr ' ^^^^^ -- -- - --rr --^r tt :s"oi in. »t^.r Ti'STrS— ^^^^^^^ °r^-- - *^e invad- and often a less pronounced e,^rar.e!H "^f ?''^ °* *''^ <=«»« '"vaded This diminishes the space betwe Hh e. I ''"*!^"°"' "'''' ^^'^- ^' ^)- i'feration of the mycelium. C TsIpreTsS 1^"" ''' -tercellular pro- abnormality have been observed wedS TetwL ^^^^ '", ^''■^'"" "*«"«« "* m areas of severe cell hypertroply t 1 °^ ""'^'^^'^ ««"« t.ps may grow beyond the enlarging cell before tT' !"' '''"""•''"^ ''^P''-' trophy are attained and so escan» ft *^ extreme stages of hyper- difference in degree of h^^ertronltt T"" °* '"-hanical resistance. The measure of the eompatibCoST "« J^17'J«^.h-t cells seems to be one A second structural resjons usual vlffe""""''"''- and like excessive cell enlargelr seems ^ T'/''''*'"'^^ *° '"-««'"« patibility between the host and^tb!' I '"'^'"^'^ considerable incom- eells in and near the inf^ ion arfa dSfin'Tf """• ^'''"- -«-' occupied with a larger number o7ZaZl^7f '^' '''^'' '^'y f^^erly mg from the infected area (fL 2 B) ''""V^^^" °"«»t«d '« rows radiat- cells might appear to favor a swift inva ion bf /'* arrangement of the by the almost con.plete elimination of >/ , , "'"^"^ '^ accompanied appears to be the morphologTcalba "rier trr.t"'"'' '''"''' "''''='' «="-» trophic and hyperplastic reac^ioi app "r to '!?";"^^^"°"- 2"*^ hyper- ing fungus. ^PP®*'^ '° be direct responses to the invad- of pitiio'^: SL^Xdirrir ? r ^"^^^^°" ^" ^ ^''^-^ -- ^ row duces a distinct zone of ciclt^tt e "^V'^ '''""''' "'^'^'^^ P- have been killed. This layer cuts off thrdearcll, "."' V''"''' '^"^^ ''''' the very abnormal cells adiaeent tn T f ' *°»«ther with some of This cicatrix often serves as a :ll'Sr,t^^ the more normaf tissues does so only when the host cells attlkidr *"''"^' ^"'^^^ evasion but -e walls out the invading Z::^l:'::,r,::ZTV'' "'^'^^^ 2, C) In ease of an infection by a mvceliZ f! T ^ *° 'P''**'' (^'^S- mvaded areas remain nearly normTt -^ *°™P**''"« ^ith the host which the invading mycelLms^rS/lir/r.^ ''*" '"^^^'^ ^--^ M>crochem.cal tests made upon material shlw^^,,, ,,,,,, ,_ 1938] True: Gall Development on Pinus Sylvestris 33 4i^ h >« 4 ■► f 4, -^ T 1 ^4 y ^ 1 «. r« vj ^-\ > ^ l> ^ 4| > 4 sions revealed that abnormalities in the visible cell contents appear in tissues before they show the structural deviations described above. Starch is un- usually abundant in the early stages of physiological abnormality. It is present in large quantities in the morphologically normal cells at the margin of the infection, both before and after the tissue has been invaded and its cells penetrated by haustoria. The presence of these large deposits of starch within and adjacent to the infected areas is accompanied by a scarcity of it elsewhere in tissues in which it normally occurs. Fatty globular substances appear abnormally abundant in the pathic living cells and seem abnormally scarce in adjacent uninvaded tissues. Tannin or tannin-like substances are universally found in cells that have passed the early stages of abnormality ; and their appearance often is synchronized with the complete disappearance of starch and fatty substances from the pathic cells. They appear in the wall or lumen of nearly every dead cell. In the case of barrier formation, the thin- wall cells of the barrier are not cutinized nor lignified, nor do they contain starch, fatty substances, tannin, or tannin-like compounds. They give a negative reaction to tests with Scar- let R; but Hutchinson (6), using ammoniacal gentian violet as a reagent, found them to be suberized in the material with which he worked. A globu- lar fatty metabolite, staining pink with Scarlet R, is often present in the meristematic cells below the barrier, and scattered cells of the adjacent cortex, and starch grains appear similarly distributed. Whether the invasion is resisted or not, invaded cortical tissues eventually die. Their death occurs generally in the same chronological order as their invasion, so that, in cases of at least moderate compatibility, an external area of killing appears close beneath the epidermis to correspond with the early vertical spread of the mycelium there, and a similar but more extensive inter- nal area is located in the inner cortex, where the internal vertical prolifera- tion took place. These are united opposite the point of epidermal penetra- tion by a smaller area of killing in the outer cortex, where the centripetal spread occurred. (Fig. 3, E). External areas of killing usually involve the epidermal cells, as well as those directly interior to them, and are clearly visible to the naked eye as infection spots. When these spots are minute or have sharply defined mar- gins, it may be taken as an indication that the invasion is being at least partially opposed by the host ; particularly, if they appear within a month or six weeks after inoculation. Larger spots having indefinite margins often appearing water-soaked are likely to mark successful infections, and espe- cially so if their appearance is considerably delayed. In cases of extreme compatibility, swellings may appear before their infection spots. Most infec- tion spots on susceptible trees at Woodgate show intermediate characters. Peripheral and longitudinal spread of the fungus is limited, at first, almost entirely to the primary cortex, and, later, to the phloem. The myce- lium spreads more swiftly in the inner than in the outer cortex, and the death of the invaded cells of the former cuts off the partially invaded and 34 Phytopathology [Vol. 28 inner cortex, formin-. a Z2.7.T iJ f "°"''P'' ^S*'"^* t^e dead the swelling xTe p^Lher J^ . ..' ™''^"'' "' '''' ^^^^t'^' P°rtio„ of not at all s^o isola ed^n^ala^^^^^^^^^^^^ T' '""''''' °"'^ P^'''-'^^' - becomes .ore PronouneedX [hVLdTo X Tnl o1":,°^ """• '^''^^ when the infected cambium usually has cut offn/^ . '''°"'' ''*'°° which serves thereafter in th.ZT /I considerable phloem tissue, tieal and peripWa, spr^:^^^^^^^ t »; e ^7^ ^^ "^^'^ ^^""'^" ''^ ^- ing, the dead tissues of the central sSln. !""^ ''^'''"' °" ^""'t- theeolIarevenmorcpromiLnt pt 2 pT T.T T'"^'" '^'^' '"''^"^ gradually dies back as its ed"es a e" further .^V^.".!"'"^'"-" ^"" ^'^'^ '=''"^'- of the tissues internal to it h. * . °'**^'' ""^ *''« continual dying Of most .naturetl.:ev:n^t^';riTeL^^^^^^^^^^ ^^'""'"^"* ^" ^''^^^ ryrr:^-irrthr±,^''^^ delaying gall fonnation The mS^m J ^ '"' ""t "/.'"-^ ™-. ">- penetrating the cambium on T^ly 0 of he ~^^^^ August 1, the hyphae of several of that seasonW. t ''"''' ^^ had been resisted, had penetrated beyond th cTmbi'n n^^^^ """ **' "'^''^^ in which a resisted infection was just enteriLtre IT' T' ""^^ "°*''* season following that of its epidermi, penet "ado^ ' "" "" '""^ '^ ''^ *'"' spac!SSS:^cd'?^ "'"""'" ~ *''-"^'' *''« »*-""'ar direction. Th ZurTl res„„ ? 7."''?,'' '''""''•"" '"'•^^'^ '" ^ radial ence of the fun^usTs Z t.7 ., *''' ''"' ''^ P'"'^*'" «««»« *« the prcs- someof thelXd^JLT^gri:::^^^^ *•- a divisio^n of formation of a barrier have hZ,.\T T invasion, and even the mostly along the J lo'em rav he ""1-'" '""''"''"'' '^''- Penetrating angles and Ltinu T rZilZ^ltZr'V'''' '^^'"'^'"•" '' "'"''^ but a short distance into the xyle'ii where ft Sl7to "''"""'' ^^^'^ *"' structural abnormalities. *° ""a"*^ ^^^ appreciable logic troLTblTiTflhf' ^''"" """" ""^ ""''^^^^ « -r'^ed physio- Starch grains and farllprr^^^^^^^^^^ ''! P"-"^™'*^ °^ '"e fi Jgus. chymatous cells of botii nhlo m ;ndTvf '""'' f'^''' « ^^e normal paren- many large amounts „ tki,!! , ^ ' **'' °**'" P''^^*"* t^ere in abnor- mality in the matured ;mI,^ """'^^ ''"* -'"sht structural abnor- stimuLes th clrbtum to the "'Tf' """ '" *''« ^^'«"'' ^*« P-ence •<' * ^fl-^ ■^1 1 >>» v^ > ■■■■ r ' < ► 4- A ^4- if t . ."*• 4 .A 1938] True: Gall Development on Pinus Sylvestris 35 of their cambial initials, it seems likely that these abnormalities reflect abnor- malities in the cambial initials induced by the presence of the fungus in the cambium. The cambium usually is first penetrated at a point or over a limited area adjacent to the point of infection, and subsequent cambium invasion comes always from the phloem, never from vertical or peripheral spread of the mycelium within the cambium itself (PL II, 4 and 5, and PI. Ill, 6). Since the cambium is stimulated rather than killed by the invasion, normal and invaded portions are found functioning side by side and producing normal and abnormal phloem and xylem adjacent to each other (PI. II, 1). Newly invaded cambium, i.e., that at the edges of the invaded areas, produces phloem and xylem cells showing greater abnormality than those at the center of the areas that have been invaded for some time. This fact, together with the relatively slow rate of invasion of the adjacent normal cambium areas, often results in the formation of a more or less wedge-shape body of gall wood whose edges show greater abnormalities than its center (Fig. 2, E). Among the abnormalities found in tissues derived from invaded cambium are the following: 1. Increase in radial and tangential diameter of both phloem and xylem elements. In the case of the xylem this often is accompanied by a pro- nounced shortening of the tracheids, which are often blunt (Fig. 2, D, PI. II, 4 and PL 111,6). 2. An 80 to 100 per cent increase may be shown in the number of xylem cells formed. No comparable increase is shown in the number of phloem elements. 3. Increase in the proportion and number of parenchymatous cells in both phloem and xylem. In the phloem these almost completely replace the sieve tubes (PL II, 1), while, in the xylem, the number of parenchymatous cells associated with the resin ducts is so increased that often continuous areas of parenchymatous cells extend from one duct to another, particularly in the areas at the edges of the abnormal wood. 4. Increase in the number of vertical resin ducts in the xylem. This increase, however, is roughly in proportion to the increase in the volume of gall wood, and, though their diameters are abnormally large, many of them are partlj^ or entirely closed through the swelling of their epithelial cells (PL II, 5). 5. Frequent production of radial resin passages in the fusiform rays of the phloem, which terminate next to the cortex in resin-containing cavities, somewhat spherical as seen in transverse and in radial longitudinal sections. These have been observed very seldom in normal wood (Fig. 2, D, and PL II, 3). 6. Increase in the number, height, width, and cell volume of linear rays in both phloem and xylem. (Fig. 2, D; PL III, 4 and 5). The number of xylem rays may be increased by 50 to 60 per cent, as seen in cross section. While the height of normal xylem rays is from 1 to 5 cells and the rays are 36 Phytopathology [Vol. 28 comparatively widely separated vertically, abnormal rays vary from 2 to 6 cells hxgh and often appear almost vertically confluent one wilh an'the a een m tangentaal longitudinal sections (PI. Ill, 4 and 5). Width of nor„.a hnear rays of the .yle™ is 1 and very exceptionally 2 cells, while tho seTnl ga I wood vary from 1 to 3 and even 4 cells. The much greater volume o L« ir™''' -""^ '''-'''- ''' ^^-«-« «^ '^^ ^^-paTci:; noted i~r ra'r "^' """^'" "' "'^ ^"^^^°™ ™^^ -^^-^^'^ *« that 8. The radial rows of xylem and phloem lose .something of their orderlv appearance as seen in cross sections (PI. II, 1), a phenomenon notedt a lesser degree in normal wood making fast growth wall's (pfm luo wv\"'; " '°""f °" "" *'"-"^""«' ^^ "«" - «- -*3-l th,t"f .?" ^alls of abnormal tracheids vary in width but often are twiee hat of the normal. Walls of the elements produced at the end of the l7Z mg sea.son do not differ appreciably in thickness from those of the snU wood and the diameter of the cells is much the same throughout the a^""! nng, so that these rings are often difficult to distinguish in laU lood. Discussion of Anatomical Studies Meinecke (8) and Klebahn (7) have shown thaf rAr.oo+- reinfect their pine hosts througl L y:£::;^JZT^ZZZ::i shoots. In the present study, as in the work of Hutchinson ^G) fhTlT , penetration of the epidermis by aecial germ tubes was Zer v ' ''' "'"^' While no detailed histological studies have previouslv been madp nf „»!, formed by the Woodgate Peridermium the Z\JTr. ?• ^ (Berk ) Mivpho fr „. i, «;™""'"' ">e galls of Cronarhum quermium iserko Myabe C. cerehrum) have received considerable attention Weir (12) considered the annual growth of the galls to be stimulated by the pre and Adams (4) found the mycelium of C. quercuum (C. cerebrum) in galls on Pmusno^da Mill, to be intercellular and uninucleate, abundint in the cortex and apparently following the rays in the phloem a id xylem Haus the piw;cotx'" ^"^ " *'^ '''"'^'"' ^"-^ -"P-atively scarce in Stewart (11) found that many of the tracheids in the -rail wood of r quercuum (C cerebrum) had blunt ends and were diff ent.a Jfrom t£ wood parenchyma cells only in their pitting, which, at times was irregul I Araucarian,' with the bars of Sanio seeminglv lackin- tTp niL J ZZIX Thre! •„ !;, r"^ ''"'"'""'' "•'» '=°'"P°-d of unusuaUy large cells. Three times the usual number of resin canals appeared in cross y1 ' •^:'% 4.- J> » 1938] True: Gall Development on Pinus Sylvestris 37 section often so close that only the ray cells separated them. Above and below the gall, the normal number was present. The present study has indicated the similarity of the galls produced by the Woodgate Peridermium to those of C. quercuum (C. cerebrum) as de- scribed by Stewart. He, however, interpreted the discolored nonconducting core as resnious in nature, while, in the case of the Woodgate Peridermium, the discoloration is probably due to tannin derivatives. Resin was found limited to the canals and did not impregnate the wood in any general fashion. It is well to bear in mind, however, that the term 'Hannins" as used in this paper includes the broad range of substances that give the ''tannin'^ reaction with ferric chloride. Unlike the galls studied by Stewart, those of the Peri- dermium at Woodgate can, and often do, influence the structure of the host beyond them where no mycelium is present. Beyond the gall, the stem is often of larger diameter than below it, probably because of the fact that the abundance of phloem parenchyma in the gall has cut off much of the normal downward passage of elaborated material, which expresses itself in this diameter increase. With this diameter increase goes an increase in the num- ber of resm canals. At times a tendency toward brooming may arise beyond or on the distal portion of the gall. EFFECT OF GALL DEVELOPMENT UPON CONDUCTION THROUGH THE XYLEM On August 18, 1933, conduction experiments were made with eosin to determine in some degree the effect of the gall upon the conduction of water Pig. 4. A. Transverse section through an unstained gall showing dark color of the core (A) and wide area of conducting tissue (b) between it and the cambium at (c). B and C. Longitudinal sections through galls through which eosin has been conducted staining the conducting tissue as at A and b. Note the contrast between the con- ducting eosin-stained tissue and the non-conducting naturally discolored core (c) and the nearly complete absence of conducting tissue between the core and the cambium of gall C The branches beyond A and B appeared normally vigorous while that distal to C was dwarfed and dying. D Young gall in which whole^f gall wood still functioned fn conduction and showed the eosin stain. E. Young gall in Ihich the core faUed ?o show the stam as strongly as the outer portion and whose core is inferred to have becLr^ least partially non-conducting, though as vet no nntnrnJ .U«..oi.rof;.„ 1,0^ !fJli^!!??! ;. 36 PlIYTOI-ATHOLOGY [Vol. 28 cornparatively ^^•ii'~'"-"^ m^ season do not differ appreciably in thickness from tJio^P nf fi,. • wood and the dian.eter of the cells is much the san t W on tie Zu!^ nng, so that these rings are often difflcult to distinguish in 5 1,!! ''''' Discussion of Anatomical Studies (-.<.) Mjyahe (c. ^...n,.;'hr;:;i^:.f :!!:i::r: ;::^ ana Adams (4) found the mycelium of C. qucrcum (C. cerchrum) in .-Su on Pu,„s rujula Mill, to be intercellular and uninucleate alnmhnt n t e coKex and apparently following the rays in the phloem ai'idx ., ' tona were constricted where they penetrated the cell wall were fo md to he ts:;;.;".::r ' — - ^"^ '''-' -^ ' ^i^^zixz •: Stewart (11) found that many of the tracheids in the .^all wood of C s w^s rrtula Iv Araucarian,' Mith the bars of Sanio seen.inglv lackin.^ T . e n f ! 'T r;::;'t:::ro'';::,r"-"T'"v -- '- *"^ -^^^-^'^^^^ la.^e . dis. Tlnee times the usual number of resin canals appeared in cros; 1938] \-f % <3> > • A I 4 Thue: Gall Dlvlloi'mlxt on Pinus Sylvesthis 37 section often so close that onlj^ the ray cells separated them. Above and below the gall, the normal number was present. The present study has indicated the similarity of the o,ilis produced by the Wood-ate Pendermium to those of C. qncrciium {C. cnrhnim) as de- scribed by Stewart. lie, however, interpreted the discolored nonconducting core as resinous in nature, while, in the case of the Woodoate Peridcrmium, the discoloration is probably due to tannin derivatives. ]{esin was found hmited to the canals and did not impreo-nate the wood in anv oeneral fashion It is well to bear in mind, however, that the term ''tannins" as used in this paper includes the broad range of substances that give the ''tannin" reaction with ferric chloride. Unlike the galls studied by Stewart, those of the Pni- dermuim at AVoodgate can, and often do, influence the structure of the host beyond them where no mycelium is present. Beyond the gall, the stem is often of larger diameter than below it, probably because of tlie fact that the abundance of phloem parenchyma in the gall has cut off much of the normal downward passage of elaborated material, which expresses itself in this diameter increase. AVitli this diameter increase goes an increase in the num- ber of resin canals. At times a tendency toward brooming may arise beyond or on the distal portion of the gall. EFFECT OF GALL DEVELOPMENT UPON CONDUCTION TIIKOUOII THE XYLEM On August 18, 19;}:i, conduction experiments were m;ide with eosin to determine in some degree the effect of the gall upon the conduction of water Fig. 4. A. Trnnsvcrsc section tliioii^rh ;,n nnst.-rmcd ^^■^l core (a) aiul wide nrou of coiKhictinjr ti.ssue (h) bctuccii i B and ('. Lt.iig^itiidinal Hoctioiis tliroii^ri, ^r,.,||s tliioiijrh wliici stnining tlio conducting: tissno ns at A and n. Note tlic ductin^r eosin-staini'd tissue and tlio non-c(.ndnctin^ natnrally nearly complete al)sonce of conchictin^r tissue between the core The brandies beyond A and B ajtpeared n<.rniallv vi^rnrons dwarfed and dyin^r. D. Vounjr ^r.-ill in wliidi whole of ^r;i conduction and sin. wed the eosin stain. K. Vonn^r jr;,ii j,, ^vl the stain as stronj^jy .,s the „nter portion and whos^ core is least partially non condnctiny^. thongJi •)« v,.f n„ .io>,,|.j,i (],v, I showing' dark color of the t and the canibinni at (c). I eosin has been con. ■ ^1 ^^' ^ » V- !► 4 - 4 7 i»*. Tb>» r * J > . ^ * 4. * ^ ^ ♦ » While all the gall wood of the 1-year-old swellings was active in con- duction (Fig. 4, D), an undyed, or lightly stained, central core often ap- peared in 2-year-old galls (Fig. 4, E). In some of the 2-year-old and all of the 3-year-old galls, the cells of this nonconducting core were filled with brownish substances (Fig. 4, A). These cells and nondiscolored cells close to them contained tannin-like compounds instead of the starches and oils so abundant in the parenchymatous cells of the major part of the nondiscolored gall wood (Fig. 5, A, B, and C). In three-year-old normal stems, however, the whole woody cylinder was stained, and all the tracheids seemed to have been active in conduction. The nonconducting discolored core of mature galls may be relatively small (Fig. 4, A and B) or may extend outward from the center of the gall and include all the cells of the gall wood except those most recently derived from the cambium (Fig. 4, C). If the first condition exists, the galls may cause little or no abnormality beyond the areas of local swelling. In the second condition, however, the branch beyond the gall may often be dwarfed or dying. ASH ANALYSES^ In the hope that analyses of the ash of galls and rust-free portions of susceptible trees would perhaps indicate some differentiation between the physiological phenomena of gall formation and normal branch development, analyses were made on December 1, 1933, of fresh material from susceptible trees that was divided into two lots. The first contained galls from 2 to 4 years old, and the second consisted of the rust-free portions of the same branches from which the galls were taken. Bark and wood were taken together in both samples. These lots were ashed and analyzed by the method of H. S. Washington.* TABLE 1. — Results of ash analyses of 2 and 4-year old galls and rust-free portions of stems of Pinus sylvestris^ Constituent Rust-free portions Galls 2Yr8. 4Yrs. 2Yrs. 4Yrs. CaO MgO KoO 17.76% 5.38 35.03 4.46 0.51 5.33 13.09 15.54 2.92 0.53 0.41 0.2 1.31 26.39% 7.22 28.39 2.64 0.31 4.10 10.83 11.28 3.37 6.10 0.03 trace 0.65 18.57% 7.25 30.62 1.58 0.70 5.15 19.68 7.99 2.68 5.75 0.18 0.10 1.38 22.88% 6.50 25.90 NajO 4.29 N SO3 P„Ob 0.95 7.46 18.59 SiOa 6.09 FcOa 2.72 ALO, 5.34 MnO CI % Ash 0.12 trace 0.88 • Besults reported on the basis of a carbon and COj free ash. 5 The author wishes gratefully to acknowledge the research grant from the Univer- sity of Pennsylvania Chapter of Sigma Xi, which enabled him to have the an.alyses made. 6 Analyses were made by H. J. Hallowell, consulting chemist, Philadelphia, Penn- sylvania. 38 Phytopathology [Vol. 28 1938] Tkue: Gall Development ox Pixus Sylvestkis 39 and dissolved substances to tlie branch beyond. One-, 2-, and 3-year-old portions of stems, with and without ^alls, were cut ott' below the surface of an eosin solution. The galls aud uoiiiul'ected areas of the <^alled t\vi- •* » « if r s \ » • • * ♦ » AVhile all the gall wood of the 1-year-old swellings was active in con- duction (Fig. 4, D), an undyed, or lightly stained, central core often ap- peared in 2-year-old galls (Fig. 4, E). In some of the 2-year-old and all of the 3-year-old galls, the cells of this nonconducting core were filled with brownish substances (Fig. 4, A). These cells and nondiscolored cells close to them contained tannin-like compounds instead of the starches and oils so abundant in the parenchymatous cells of the major part of the nondiscolored gall wood (Fig. 5, A, B, and C). In three-year-old normal stems, however, the whole woody cylinder was stained, and all the tracheids seemed to have been active in conduction. The nonconducting discolored core of mature galls may be relatively small (Fig. 4, A and B) or may extend outward from the center of the gall and include all the cells of the gall wood except those most recently derived from the cambium (Fig. 4, C). If the first condition exists, the galls may cause little or no abnormality beyond the areas of local swelling. In the second condition, however, the branch beyond the gall may often be dwarfed or dying. ASH analyses'' In the hope that analyses of the ash of galls and rust-free portions of susceptible trees would perhaps indicate some differentiation between the physiological phenomena of gall formation and normal branch development, analyses were made on December 1, 1933, of fresh material from susceptible trees that was divided into two lots. The first contained galls from 2 to 4 years old, and the second consisted of the rust-free portions of the same branches from which the galls were taken. Bark and wood were taken together in both samples. These lots were ashed and analyzed by the method of II. S. Washington.^ TABLE 1. — Results of a.sh analyses of 2 and 4year-old (jails and rust-free portions of stems of Pinus sylvcstris'^ llust-free portions Galls Constituent 2 Yrs. 4 Yrs. 2 Yrs. 4 Yrs. CaO 17.70% 20.39% 18.57% 22.88% MgO 5.38 7.22 7.25 6.50 XVnV-^ ...,«,.........*..*.«*****«•»• 35.03 28.39 30.02 25.90 xN «jl2^<^ •■•*»•*«* •-*»»•■ ••■■*••>»» 4.40 2.04 1.58 4.29 ^^ .,,,.. «IHI*(iH**«4ii*»»«**»»*^*«4*> 0.51 0.31 0.70 0.95 i^V./f ,,.,,,,trr*irttf t^rt^f— ****** 5.33 4.10 5.15 7.46 P,0, 13.09 10.83 19.08 18.59 0 1 Vy 2 »»«»**»is»*4****t*P*M*t*4i». 15.54 11.28 7.99 6.09 JT "'iV/g .....#!«»*#••**•»«*«*■*■ 2.92 3.37 2.08 2.72 J\- loV^3 «»..*.t»»^j»«4ti>«M«»»iff*84»* 0.53 0.10 5.75 5.34 UnO _.» . 0.41 0.03 0.18 0.12 01 .>M..mHMM.HH.. 0.2 trace 0.10 trace % Ash 1.31 0.05 1.38 0.88 » Results reported on the basis of a carbon and CO2 free ash. 5 The author wishes gratefully to acknowledge the research grant from the Univer- sity of Pennsylvania Chapter of Sigma Xi, which enabled him to have the analyses made. 6 Analyses were made by II. J. JIallowell, consulting chemist, Philadelpliia, Penn- sylvania. INTENTIONAL SECOND EXPOSURE 40 Phytopathology [Vol. 28 Table 1 shows some divergences between the ash of the galls and the rust- free wood. The greater amounts of nitrogen, sulphur and phosphorus in the galls can perhaps be correlated with the large quantities of food sub- stances stored in them. The lesser amounts of silicon present in the galls appear to add a chemical reason to the structural basis of the mechanical weakness shown by gall wood. Most of the results, however, show differences whose explanation is at present difficult. Perhaps when the complexities of the physiology of the host-parasite relationship are better understood these figures may appear more significant but here the methods of ash analysis appear to shed little light on the physiological aspects of the problem. R]gsUME OF THE LIFE CYCLE AND MORPHOLOGY OF THE WOODGATE PERIDERMIUM The Woodgate Peridermium is autoecious and has but 2 known spore forms, pycnia and aeciospores. The pycnia were first found during the course of this study. They were discovered in material collected on May 17, which had been selected to show developmental stages of the gall and in choosing and fixing the material, the pycnia had been passed over unnoted. Later, other inconspicuous internal pycnia were found in sections of material from several young galls collected in the same season some of which had shown external traces of exudate at the time of collection. The aeciospores reinfect the host directly. They are roundly and irregularly rhomboid with verrucose walls. No germ pores have been observed in the walls of the aecio- spores. (Fig. 3, D). They may germinate on water or upon the host in from 12 to 24 hours. The germ tube protrudes often but not always from an apex of the spore and broadens to form a septate branching hypha, which within the inoculation chamber, may apparently penetrate the host at once or grow luxuriantly over the epidermis for a considerable distance before entering. In most cases the mycelium vegetates within the host for 2 seasons and in May of Its third season may produce pycnia from small hyphal wefts beneath the periderm of the central sunken portion of the gall often near its edge (PI III, 3) . In late May and early June of the fourth season, approximately 36 months after infection and in successive seasons thereafter, aecia are pro- duced. They arise from larger and denser wefts arising deeper in the tissue than the pycnial wefts and presumably radially interior to them, though the exfoliation of the pycnial weft precedes aecia formation. As the aecial weft develops, the host cells within and adjacent to it are separated widely from each other, but often do not appear to die for sometime, in consequence. The peridial cells of the young aecium push up the periderm and the upper portion of the hyphal weft, which may then disappear entirely or remain partially adherent to the edges of the mature aecium. The peridium varies from 1 to 4 cells in thickness (Fig. 6, A and PI. Ill, 1) and stalactiform structures of the nature of the peridium are sometimes found uniting the dome of the peridium with the base of the aecium (PI. Ill, 2). *J"^ ■^ : ^ » >4 I « «\ 4 ^ ^ > i 1938] True: Gall Development on Pinus Sylvestris 41 Fia. 6, A. A portion of the dome of the peridium with cells drawn in outline. B. Portion of the bases of aecial spore chains showing binucleate and trinueleate condition. C. Young aeciospores, some binucleate and some trinueleate. D. An apparent binucleate condition in a cell of a vegetative hypha. E. Hyphae and haustorium in the cortex. Note the effects of shrinkage upon the nucleus at its point of contact with the haustorium. The usual number of nuclei in the vegetative cells and of those making up sterile portions of the wefts is 1, and, while 2 have been exceptionally found in vegetative cells (Fig. 6, D), it seems likely that they do not indicate the initiation of a distinct stage in the life cycle. A 2-, 3- and, exceptionally, 4-nucleate condition arises in the basal cells of the enlarged hyphae that cut off the young aecial spore chains (Fig. 6, B), and the young aeciospores are in turn usually binucleate but often possess 3 (Fig. 6, C) and at times 4 nuclei. The nuclei in maturing spores appar- ently become grouped, but no actual fusion has been observed. The young germ tubes, however, often appear to have 2 or more nuclei per cell, but only 1 has been observed in each cell at the time of host penetration. Until further cytological studies can be made upon this organism, the interpreta- tion of these nuclear phenomena would appear hypothetical. Dodge and Adams (4) found that of a collection of Cronartium querciium (C. cerebrum) galls on Pinus rigida from New Jersey, some produced only 42 Phytopathology [Vol. 28 galls on P. virginiana Mill., however boft 1. T"'- '" '''' ''''' «* different parts of the same eallTn 7^.' ^""^ P^''"'^ developed on western rusts m.y produraTel 11^0^5. rT'": ?."^ °^ ''''^'^^' are exceedingly rare. ^' ^^^ ^^P^^*^ that their pycnia GENERAL DISCUSSION WoJdtrStrfrteTt^L'r^"-*'' "^ '^^«"^*^'- -*«~ at feet the host without the product o„ of slwd" '"""'T', ^'™ *"'^^ *''«* ^«»- we have here to do with the mierote a of f ?"f "'"' *^ ^'"""^'''^y that Pmrf.mm«, then, in the eharleter of its UfT'f '"«=>-''«ye«c rust. This to the two western repeating forms stud !VhM' T"^ °"'^ comparable by him as CronartnL ka^ ^^Zt'/^ZT^i ^'^ -^"^ "'"'^'^ *° Memecke, and now „.rouped together byArthur ^'^^,7*"'" 'f^*'"'''''^^ *P«m.d., (D. and H.) Arth. and to / pili(wfmTiTr "'/• ""'"■ repeatnig pine rust that does not iovm.I\ ^ }V '*"'' ^ European Woodgate Peridermium seems hov^rr f f'"^ ''^ '^'«''«''" ^^^^ The quency than either of the western flrl ^"l T ^^""'^ ^'* ^••««t«'- ^e- thickne.ss, contrasts marked^ S haTofTh. '\"''"'"' ' *" ^ <^«"^ ^ c«^mp«nWd«. iC.harknessU) whose peridumisTT"^, ''"'' '"^ "^ ^• Intracellular penetration was founTl , " ^ ''"' '" *'''«'^"«^« (D- between epidermal cells. TheTrly intra^r, "r^""^ *''^" penetration Hum in the epidermis and its Sion of T "' , r'"'''"^"* "' *»>« ^y^^' toria in the subepidermal el ssT^r///*?'^'' '"'^""hing, septate haus- shown the early tLdencv of infcE tow« H ^"' °n ''"'^ ('^' ^''<' ^'^ be even more pronounced in tLe 2rTe ' 1?/ '"*'•*'=«»"'«'• development to The coincidence of the paths of e^f ?' '"'* °* ^"'"'*- the regions more richly supSw^ltr^'f'. ''°^"''^' ■"^-'^ -*•> there may be a relationsh^ betJ^t T. """^ "'"^ '""'"'' *« ^'^^^ that rust and the supply of delnS L .U XTJoi s'^t' -^^^"^^'^ *« ^''^ note that the tannin-like compounds prlntTn , " "'*«'-««ting to vacuoles of many cells in these reWonsT! "/°™al quantities in the of the mycelium along these paths ' * '"'^'^'"' ''''^ ^^e migration to tStT iZ^l 1^^^^ "' -T-^- - 'oods available and found that trees whose Th contained litrT •""' """ *"« ^ost ceptible than those with larger amolts of H w Potassium were more sus- that fail to produce galls inTuscTp b ree, didT '" -f *^* '"^-«''- petition and lack of nutriment. In th sTaner '7"'"«"'y through com- Woodgate, susceptibility varies not only with thl\ A'' *'*'^'" *''^*' «' parasite, and that there is a question of eomnthr. ^"* "^'° ^'th the host and each mycelium whose^el tub pTeC^^^^^ rm^^*^ 'Tj'^'^ *he in mind, too, that the entrance of seeon^prvT • """^ "^^^ he borne -.H..0.. T. w„„a«a.p:^z:r,;;— ^ --^^ ^'^ -'•^^'^ have 1938] True: Gall Development on Pinus Sylvestris 43 • 4h^ * "t >' «. «* ^ II > » 4 ^t - 0 < quite frequently been noted in histological preparations of young galls, might influence the reaction of otherwise compatible hosts. It is true, as Hutchinson has pointed out, that different parts of the same tree may produce galls more readily than others following infection. The host tissue of a single shoot is a variable factor, since portions of the young stem, such as areas at the needle bases, may be better supplied with starches, oils, etc., than others. Yet it seems unlikely that a commandeering of the food supply by the first mycelium to penetrate could explain the wide varia- tion of host reaction to the different invading mycelia in inoculated twigs where the infections may be considered to have taken place at about the same time. In these cases, too, as in others studied, there seems to have been no relation between the compatibility of the mycelium with its host and the region of the shoot attacked, though definite regions of abundance and scarcity of demonstrable stored foods do exist in the shoot as indicated. Also, areas invaded by incompatible mycelia and those adjacent to them, rather than being free of demonstrable foods, are abnormally full of starch and oil in the early stages of invasion. Compatibility in susceptible trees, then, seems to vary not only with the host but with some property of the infecting mycelium. That this variable property is a physiological one seems clear. It appears likewise unlikely that a variation in vigor of the invading mycelium is the sole cause of the varia- tion in the host reaction. Rather, it seems likely that some mycelia are genetically better adapted than others to achieve and maintain the metabolic balance with the host so necessary to obligate parasites. Perhaps the varying number of nuclei in the aeciospores may indicate such an instability in the genetic constitution of the Peridermium at Woodgate. The question is here raised whether we do not, then, have in this case of a forest tree rust some- thing comparable to the physiologic races recognized among cereal rusts. Even in the cases of most complete compatibility, the cells of all the invaded areas except the cambium eventually die and, in contrast to phe- nomena sometimes exhibited in the cereal rusts, die sooner than cells of comparable but uninvaded tissues. This is true of the invaded cortical tissues as well as of the phloem and xylem. The length of time intervening between invasion and death of the cortical tissues usually measures the com- patibility of the reacting organisms and consequent susceptibility of the host, since an infection that reaches the cambium seldom fails to cause gall forma- tion. The more compatible mycelia at first cause but little abnormality in the structure or contents of invaded parenchymatous host tissues, but grad- ually these become filled with starches and oils and, just previous to death of the cells, unusually large amounts of tannins appear in them in the place of the oils and starches. Where compatibility is less, this series of events fol- lows in swifter succession. Coincident with, or shortly after the appearance of an abnormal abundance of tannins in cortical cells, less compatible mycelia meet the resistance of hypertrophy or of hyperplasia or both and the degree of structural abnormality of the tissues is proportional to the physiological 44 Phytopathology 4 , ~ f Vol 28 grow beyond the zone of cicatrization ' '"^*='''""' ^"^ "" «me to ^he P"ele:rt?'tr^t:rs:;r S^'""^*^ ^^^^ ^^^^ae are influenced by than norma, and these are .ooTmTS T '"""' ^'■*"'^'- Proportions starches, oils, and, in the case of fhe phTol "'""'•7"^ 'arge amounts of the parasite appears to cause its host ,fn. 7 Parenchyma, tannins. Thus but also with extra food Jn It, olu V^ '"f ^ '' "'*" «*- fo^d pronounced in ti.s.sues derived from n! • '^ abnormalities are most -«esuggestionof areturn tota^The":^^ T^'^ ''*'"''"'"' «"d therTL m cambium areas that have bee^TnJaded f or " *"'""' ""^'««" ^ ^o- ^nitia s of t,me which vary in different mature IS f """T "'" """''■ ^^^^ periods years after their formation invaded t-J '' *''*" ^ *" as much as 3 '--^" at Woodgate. °"« Physiologic race of the Pcridermmtn Mycelia causing little earl^ .i, f eredascompatibl With thSos^Th! 'y '" *^ *™ '"vaded are con- along characteristic diverging pa L flun^ r"'*'"*' '''' '"'''^ ""ore swiftly ^" xn t :r s-mS: ~^ iLxr "^-- "-- fatty s„,,,,„,^,_ and' ta^y TaniTiir^"''^ "'* ^''^ '>-*«' starches rated in eells near the hypL FoUt „;2°T'' '" '^P''"^ -ncen large quantities of the last, these celt dT a J thr""°" "' ^•'»°™ally The more compatible mycelia r,Jl . *'"' ^""S"s dies with them succeed in forming galls, but llhyTertrS °' "»^ '^'"'« « « < <( • • « 4r i * h < \ 'i ♦ . ♦ ». If ♦ f 1938] True: Gall Development on Pinus Sylvestris 45 the first season, while others may be delayed. Phloem and xylem are invaded mostly along medullary rays. Stelar tissues, matured before invasion, show comparatively slight structural change, but their parenchymatous cells become abnormally full of starches, fatty substances, and tannins. The presence of the fungus in the cambium causes the production of abnormal secondary tissues containing a high proportion of parenchymatous eells that become in turn filled with starches, fatty substances, and, finally, tannins. Increase in cell size characterizes both phloem and xylem, while only the latter shows a marked increase in cell number. Irregularities in size, shape, and pitting of the tracheids also are produced. Portions of the cambium penetrated for the first time produce the most abnormal secondary tissues, so that areas of gall wood are outlined by tissues showing the greatest abnormalities. Abnormal parenchyma tissues in the xylem at first contain large amounts of starch and fatty substances, which are replaced by tannin-like compounds usually during the third season in the oldest gall wood, which then ceases to conduct. In cases where the nonconducting core occupies nearly all of the xylem, the branch beyond the gall often dies. Meanwhile, the death of the inner cortex and of the abnormal phloem cells leaves portions of the cortex external to them isolated from radial conduction. These portions die, collapse, and later are exfoliated except at the edges, where, by contrast, they appear as a raised collar. Early in May 2-year-old galls often bear pycnia hidden directly beneath the periderm below the sunken central area. This is the first report of pycnia for the Woodgate Peridermiiim. Three-year-old galls often produce confluent aecia from extensive dense mycelial wefts in the sunken area arising slightly below those of the then exfoliated pycnia. Each year the tissues containing the aecial weft are exfoliated by a periderm arising internal to them and the next year aecia are formed below the new periderm. Ash analyses of 2- and 4-year-old galls and rust-free portions of sus- ceptible trees showed the galls to be significantly lower in silicon and higher in nitrogen, phosphorus, and sulphur. Other discrepancies were slight or difficult to interpret. Preliminary nuclear studies revealed the perennial vegetative mycelium to be predominantly uninucleate. Two, 3 and occasionally 4 nuclei appear in the cells at the base of the aecial chain, the young aeciospores, and many portions of the germ tubes. The latter, however, are markedly uninucleate when they penetrate the host. Department of Botany. University of Pennsylvania Philadelphia, Pennsylvania LITERATURE CITED 1. Arthur, J. C. The plant rusts. ... 446 pp. John Wiley & Sons, Inc., New York; Chapman & Hall, Ltd., London. 1929. 46 Phytopathology [Vol. 28 '■ °"'pTyToSh'^2st8l!Sr"7935. '''""' ^^'^^^'"'^ '" " gall-forming Periaern^iurn. 7. Klebahn, H. Peridermium pini CWilld ^ K^^^h i,t,^ o«,-« -^vu ^ 1929. i"'Perments w.th repeating pine rusts. Phytopatl.. 19: 327-342 ,'• ^'""^'uficl'^- ptIoTX ^:"^J^ ''^Zr''"' """'""^'^ '^ P™^'-«» of the ■ 7''Gt„™"na"co.%„ftor"lt7'" "■' ^"'^ ^-'-» gymnosperms. ... 374 pp. 11. S™.,_A^ Note^, „„ j,„ ,„,i„„^ „, ^^„.^^^^,.^^ ^^^_^ ^ ^_^^^ ^^^^ ^^^ 12. WEia, X R._^ Observations on the pathology of the .jack pine. U. S. Dcpt. Agr. Bull "■ l°"Aioo%1ttTe "'-' '" *'''' N-"'-t"n United States. Science ■ '""^e^be'ddin^g"." i%ti':tf,rr^oUs,'xr'''' ^""^ «-- ^^ p-am™ > 4 • ?^ V « :: - •<♦ J »* -)■ > k PLATE I 1. Transverse section of a normal twig of P. sylvcstris as it appears in early June. Note especially the thickness of the walls of epidermal and subepidermal cells at a, the base of the needle fascicle supplied by the bundle trace at b as contrasted with the thinner walls elsewhere as at c. 2-3. Transverse sections showing unresisted invasions. 2. shows a very limited necrosis at point of penetration (A), scarcity of hyphae in the outer cortical region as compared with the inner, especially in the vicinity of the resin canal and the closing of the canal by the hypertrophy of the epithelial cells. The mycelium is shown entering the stelar tissues through a bundle trace at b. 3 shows a resin canal still open in spite of the abundance of hyphae near-by. The mycelium enters the phloem of the main stele at a. 4. Early stage in the formation of the "collar". Necrosis has spread from near the center of infection (a) largely in the irmnr cortex to b and this, together with pronoujiCCu €iuiiOi'ni3,iitiG5 3.risiiig m i/iiG imicr n .iiu plijooui i.ruiiA B lu c, ijus pHi'liciiiy isolated the outer cortex external to it. Following its isolation, it has become severely necrotic from a to d and appears to be dying also from d to e where a barrier from p to o is forming to separate the dying portion from the more normal cortex at the left in the photograph. 46 Phytopathology [Vol. 28 zu 1 ^'^-S^-r^^'^^^^^^ - - - 403-400. 1932. '^^ *" "* Peridcrmitim cercbroidcs. Jfyeologia 24: "■ """""^^^^i^ ,^^:^'^^''''"'^ ^^'-^'"■^ to a gall.f„™i„g P„,V7.„,u-„™. 7. Klebaiin, ir. Ptr.e I..-...,„.„«y of ,„o .ia,.k Pino. V. S. Pept. Ag,- B„n "■ !""%.'') '^i:^„o!ro,*";'^^' -"• *" "- ^^".•H.oa.s.o,•„ Unite., State. Seience • -"^^k^! SeiereT.'.'"!r7l"'.'T03i,'04"^';i3';-'""« "-"^- 'i-- ^or paraffin \ 4 > ^ f '• - > > ^ . .u K PLATE I 1. Trnnsversc section of a nornial twi^ of P. .si/Jrt siris ;is it ;i|i|it'ar.s in early June. Note especially tlie tliickncss of the walls of epidermal and siihepiclcrnial cells at A, the base of the needle fascicle snpjdicd by the bundh" trace at H as ccnitrasted with the thinner walls elsewhere as at C. 2-.'{. Ti'ansverse sections showing unresisted invasions. 2. shows a very limited necrosis at ]ioint of penetration (A), scarcity of liyjdiae in the outei- c(n'tical region as compared with the inn<'r, especially in the vicinity of the resin canal and tlie closing of the canal l)y the hypertrophy of the (-pithelial ceils. The myceliimi is shown entering the stelar tissues through a >)undle trace at B. o shows a resin canal still open in s])ite of the nl)undance of liypliae near-l»y. The mycelium enters the phloem of the main stele at a. 4. Early stage in the formation of the "collar", Necrosis has sju-(\ad from near the center of infection (a) largely in tlie inner cortex to n and this, together with pronoiHK'co ahMornialities arising in the inner c(»iic.\ and piiincm troni H to c. lias partially isolafi'fl the outer cortex external to it. Following its isolation, it lias liecome severely necrotic from A to i) and ajtjxN'irs to he dying also from I) to K where a harrier from D to r is forming to separate the dying ]tortion from the more normal cort(>x at the left in the photograph. k *»- •f* - 4 ! ♦ * I * * ' ^J- t > * PLATE II ..oJa 'r^f."8v^'*se section showing the edge of an infection with invaded and uninvaded Z^raJlwZ!- •'^' ^^f ^f • /^x^\^ *^^* ^" *^^ P^**^^*^ P'^^««°^ the parenchyma cells with their deeply staining contents (a) have almost completely displaced the sieve tubes (b) which comprise most of the phloem in uninvaded tissuesf 2. A cicatrizing zone formed from A to B to c to d has proved ineffectual in resisting the invasion as has the simTlar zone from a to e to f which formed later. Note that the mycelium is already present in the phloem o and xylem h. Eadial resin duct in uninvaded tissue uniting the res n cavity A in the phloem with the vertical xylem resin duct. 4. Radial longitudinal section showing edge of infection where the mycelium is advancing vertically and radially iito uninvaded phloem (a) and cross ng the cambium to enter the very abnormal xylem ^ Note that abnormalities in the secondary tissues are produced at some distance in pdv.n.p '? it'-t^**^r''""li *"• //f/'^Y^'^se section showing mycelium crossing the cambium and dis- tributed mostly radially m pathic phloem and xylem though not always confild to the * k » ,♦ • ^ PLATE III 1. Section through a small aecium showing external periderm, the peridium of the aecium, the darkly stained basal cells of the spore chains, and the widely separated host cells below the aecium. 2. Section through the filaments occasionally found showing their double character and suggesting that they are separations between otherwise confluent aecia. 3. Section through a pycnium showing the very limited pycnial weft and its posi- tion below the periderm. 4. An approximately tangential section through gall wood and uninvaded wood adjacent showing the irregularities and large size of the cells of the nfOll ^^OAO O «rt +H#»f ^ <^»fl»T\t»/>r\f>*^i AT^'>40^''* To T-iTPrS O *^-» *-*<-»*^4- ^^ Y>nv<<^Vt rtl^T**^^ ^4-^*^,^ 4-*nr-wri ^ A higher magnification of a portion of the gall wood shown in 4, showing particularly the tangential pitting of one of the walls of one of the tracheids at a. 6. Radial longitudinal section showing the mycelium oriented radially in and near the cambium. 7. Epidermis and periderm of normal P. sylvestris as they appear in early August. PLATE II 1. Tninsvor.sc section showing the ("dge of an infection with invafk-a and uninvadod 8cconresent in the phloem G and xylem ii. J?adial resin d,ut in uninvaded tissue uniting the resin cavity a ,11 the phh.em with the vertical xylem r,.sii, duct. 4. Kadial longitudinal section showing edge of intc-ction where the mycelium is advancing vertically and radially into uninvaded ].hloem (a) and crossing the cambium to enter the very abnormal xvlem ' Note that abnormalities in the secondary tissues are produced at som'o divtnn^o J,," ..,i..;„ce of liie niyceiiuin o. ^1 ransverse section showing mycelium crossing the cambium and dis- tributed mostly radially in patliic phloem and xylem though not always confined to the rays. • ' ^^ ^ f * / 1' * 1 f . / , • ^ M* "i.- r r ^ ^ -4 r r » » .^ T PLATE Til 1. Section through a siiiall aecium slu»wing external pciidcnn, the peridium of the aeciuin, the darkly stained basal cells (if the sp(»re chains, and the widely se])arated host cells below the aecium. 2. S(M-tion through the (ilaiueiits (tccasionally found sliowing their double character and suggesting that tliey are scpniations between otherwise confluent aceia. I!. Section through a pyciiium showing the wxy limited pycnial weft and its posi- tion b(>l(»w the ]»eriderin. 4. An a|»proxiinately tangential secti(»n through gall wood and uninvaded wood adjacent showing the iri'egnlarities and large size of the cells of the rrnll t.-^.N,! ,, . I 4l -. i; .,,.,, ,•,.«: .ill < /' I , ,• — 4 fe"" '• ' " » It . I I ii 1 , 1 < 1 i.i|ii U|»\)J i Mdi.l I 1. i_> 11(1^1 il 111 tMl II I Oi J >(4.~l ( irtr^iu', o, I\. higher magnitication of a portion of the gall wood shown in 4, showing jiart icularly the tangential pitting of one of the walls of one of the tracheids at a. (5. Radial longitudinal section showing the mycelium oriented radially in and near the cambium. 7. Epidermis and periderm of normal P. sjfli-r.^lris as they a[»pear in early Atigiist. INTENTIONAL SECOND EXPOSURE [Repiiiited from Phytopathology, December, 1937, Vol. XXVII, No. 12, pp. 1124-1142.] THE PARASITISM OF POLYPORUS SCHWEINITZII ON SEEDLING PINUS STROBUS^ Robert E. Wean2 (Accepted for publication August 24, 1937) INTRODUCTION Polyportis schweinitzii Pries, as a cause of root rot in coniferous trees, was reported by Sargent (23), in 1897, to be more destructive in the United States than in Europe. Von Schrenk (25), in 1900, stated that this organ- ism was very common in our northern forests of spruce and fir. In 1933, York^ discovered P. schweinitzii causing root and root-crown decay in forest plantings of white pine, Pinus strohus L., established in 1912-1914. It is generally believed that this fungus attacks mature and over-mature coniferous trees by invasion of the roots through wounds. The penetration of root parasites into woody plant tissue has been extensively studied by several investigators (6, 11, 18, 25, 29). However, our knowledge pertain- ing to Polyporus schweinitzii is based entirely upon field observations. From this information it appears to have a wide range of coniferous hosts (2, 8, 12, 13, 21, 25, 34). It also has been reported on hardwood trees (15, 22). The writer is not aware of any published record of the ability of this fungus to enter its host directly or of infecting and injuring the living roots of seedling conifers. York's conclusion {op. cit.) that this organism is, apparently, a direct parasite and that there seems to be a close relation between the amount of infection and the pH of the soil suggested this investigation. The influence of temperature, hydrogen-ion concentration, and mineral nutrition on the 1 A dissertation in botany, presented to the Faculty of the Graduate School, Univer- sity of Pennsylvania, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. 2 The writer wishes to acknowledge the advice and criticism of Dr. H. H. York during the progress of the work and the facilitation of chemical analyses indicated. Apprecia- tion IS expressed to Mrs. Minnie Taylor York, Dr. Conway Zirkle, and Dr. W. G. Hutchin- son for their suggestions regarding the manuscript. 3 York, H. H. A study of a rcsinosis and root rot in forest plantings of Pinus strohus L. and P. resinosa Ait. [Manuscript.] k- ' % ■1 i -^ f* »i t , T r " V .* 0 1937] Wean: Parasitism of Polyporus schweinitzii 1125 susceptibility of various plants to attack from fungous parasites has been demonstrated by numerous workers (3, 7, 9, 16, 27). Since Polyporus schweinitzii is now known to be exceedingly destructive in the Rochester watershed plantings of northern white pine in the State of New York, young trees of that species were utilized in this study in order to determine whether such factors as soil acidity and nutrition influenced the suscepti- bility of the host. A study of the physiology of the fungus also was at- tempted with a view^ to a more complete interpretation of host reaction. materials and ]METII0DS The original isolation of Polyporus schweinitzii used in this study was made from tissue of a sporophore collected from a severely infected white- pine plantation near Hemlock Lake in the state of New York. The inoculum was prepared by filling Petri plates two-thirds full of crushed white-pine needle duff to which was added sufficient 2 per cent Trommer's plain malt solution to secure a saturated condition. Having been autoclaved, they were inoculated at the center of the dish and held at room temperature in diffused light. The medium was ready for use when the fungus showed good growth over and through the duff. The determination of the parasitism of young trees by this fungus in- volved the following procedure : 1. The use of seedlings immediately follow- ing germination of seed, and 2-year-old trees from a nursery. 2. Plants grown under 5 different nutritive conditions in the greenhouse, i.e., a com- plete nutrient solution adjusted to the pH points of 4.5, 6.0, 7.0, and 2 con- ditions of nutrient deficiencies at pH 4.5. 3. For each of the 5 conditions 12 pots composed a series; 8 of these pots were inoculated with Polyporus schweinitzii, and 4 served as controls, the latter having been added non- inoculated duff. In such an arrangement there were 60 pots of young seed- lings and 60 of 2-year-old trees, each nutrient series being thus run in dupli- cate with the exception of the difference in age of the trees. Pot cultures of quartz sand, to which the nutrient solutions were supplied by the drip method, were deemed advisable for this study. The sand, after being thor- oughly washed in running water, was placed in 6-inch clay pots for the young seedlings and 8-inch pots for the 2-year-old trees. The white-pine seed used in this study was collected in 1933 in the State of New York. Prior to being placed on sterile sand in large moist cham- bers for germination, they were scarified with sand and were then surface- sterilized with 1-1000 bichloride of mercury for 2 minutes, and washed twice in 2 washings of sterile distilled water. A half plate of the medium was placed at a depth of 1 inch below the surface of the sand before planting 12 germinated seed in each container. The 2-year-old seedlings were obtained from the Saratoga Nursery of the New York State Conservation Department. They were "lifted" from IRREGULAR PAGINATION i. . 1126 O O 00 to) ;;$ o o Q ^~ e si o ••• o < to -♦^ o o M QQ Phytopathology 4-1 -OOOC . m .•• ft 05CQO00l0^t-0'^«0 O C4 CO oo OOCOOOCCIOIOOO ?DO^lO^»~i'~<^COO ^ ■«*HO^O»OQOeOCOQOO S OiO'^^OtOCOOfMOif-H oio«oioot^»o»ooo lo id w w »c lo Tt5 -^ '<* TjJ s coeoco'^c> t^ w s i-Hi-iOiWWior^t-^'^'i** ft C5 3 O .T3 oO «mPQ [Vol. 27 ♦ ^> 1937] Wean: Parasitism of Polyporus schweinitzii 1127 the seed beds in the fall of 1934, shipped to the University of Pennsylvania, potted in soil, and held over the winter in a cool greenhouse. On March 1, 1935, just as new growth was beginning, the plants were carefully removed from the soil and all foreign material washed off of the roots. Two plants were placed in each 8-inch sand culture. In this case a Petri plate of the needle-duff medium was put directly in contact with the root systems when the plants were potted. Since few facts were available on the nutrition of white pine in sand cultures, the search for a satisfactory nutrient constituted a problem in itself, for it was necessary to provide one whose acidity might be altered over a wide range. The basic nutrient decided upon, following a personal communication with Dr. J. W. Shive* consisted of the following: Ca(N03)2 5.0 cc; KH^O, 2.5; MgSO, .7H,0 2.5; (NHJ.SO, 1.5; Fe.,(C,HA)3 1.0; H^O 1000.0. " -V 4 4 0/3 The salts were made up as molar solutions with the exception of the ferric tartrate of 1 per cent concentration. A nutrient solution composed of the above elements was used for Series A, B, and C after adjustment to pH 4.5, 6.0, and 7.0, respectively, by the addition of 0.1 N NaOH (Table 1). Two series with a reduction in the amount of an essential element completed the arrangement ; namely, Series D in which the NO3 ions were partially replaced by CI ions through the reduction of CaCNO^), to 1.25 cc. and the addition of 3.75 cc. of CaCL, ; Series E in which the PO^ ions were partially replaced by CI ions through the reduction of KH.PO^ to 0.625 cc. and the addition of 1.875 cc. of KCl. The two latter series were adjusted to pH 4.5. The methods utilized, whereby the nutrient might drip continuously into the pot-cultures, were of two types. For the young-seedling experi- ment the technique of Shive and Stahl (28), as modified by Trelease and Thomson (30), was followed. Because of the time and labor of maintenance in an experiment of this size, a different system was devised for the series containing the older plants. This system, as previously described by the author (33), consists essentially of a central reservoir for each series, an automatic flow meter, and a main feed line with a capillary tube leading to each pot. The plants were maintained in culture for 220 days. During this time, root isolations were made for the organism and data upon the plant reac- tions were obtained. At the end of the period, the plants were carefully lifted from the sand, washed, and the growth and infection data recorded. The "typical plant," as outlined in Table 1, is a result of an average for 10 plants selected at random in the control and inoculated pots of each series. In the study of the 2-year-old plants (Table 2), 8 trees from the inoculated pots and 4 from the controls, selected as above, were carefully * Shive, J. W. New Jersey Experiment Station, New Brunswick, New Jersey. 1128 Phytopathology [Vol. 27 expeHmfli? 2.-Dafa showing the average growth of the 2-year-old trees during the Series Nutrient Initial pHof nutrient Shoots Needle length Stem length New growth Roots Number of roots Total new root growth A-c B B-c C C-c D D-c E E-c Complete Low nitrogen Low phosphorus 4.50 it 6.00 < ( 7.00 ti 4.50 < < 4.50 mm. mm. mm. 60.0 151 166 265 81.0 156 200 239 68.3 109 142 200 70.5 139 215 305 74.3 106 106 374 75.1 153 171 441 77.9 125 161 163 80.0 140 183 218 65.6 148 155 161 81.9 144 189 277 mm. 2116 2264 1614 2497 2251 3746 1915 2541 2068 2692 bSerTes a" B '"p 'n ^^^'^i ^5*^ ^ '^"!'^^ P^'^"*' ^^' ^^^^ «^"^« ^^l^^t^d at random, oeries A, ii, 0, D, and E— inoculated plants; A-c, etc.,— controls, noninoculated. examined in each series. The extract for pH determinations was secured by grinding the plant tissue with damp quartz sand in a mortar. To the ex- tract was added 10 cc. of distilled water prior to making the readings elec- trometrically with a quinhydrone electrode. Root material was fixed for histological study in a saturated aqueous solution of salicylic acid plus chromic sulphate and formaldehyde (4). Representative plants for each series were reserved for photographic purposes and ash analyses. Since reddish areas have been reported occurring in roots infected by Pohjporus schweinitzii, an injection experiment was made in an attempt to determine whether a fungous extract would produce a similar change in woody tissue of young white-pine trees. The extract was made by grinding fungus mats with sand in a mortar and filtering the liquid, plus distilled water, through a Seitz filter. This extract was introduced by capillary in- jection into the stems of 3-year-old trees. In similar manner sterile-needle punctures and injections of distilled water were made in control trees. After a period of 3 months, sections from plants thus treated were made for microscopic examination. In order to determine whether during growth the fungus altered the pH of a medium, duplicate liquid cultures of surface and submerged type were arranged over a pH scale of 2.59 to 7.0. The nutrient consisted of a 2-per cent Trommer's plain malt solution adjusted to the various pH points by the addition of hydrochloric acid and sodium hydroxide. For the purpose of determining whether an acid was produced by this fungus during growth, culture solutions, buffered by potassium hydrogen phosphate and citric acid, were arranged over a pH scale of 2.0 to 8.0. All cultures of the fungus were held at room temperature and secluded from >. 9^ 1937] Wean: Parasitism of Polyporus schweinitzii 1129 direct light. Examination for changes in the medium was made after 14 days. RESULTS Vol Cultures of Young Seedlings. The plants in the control cultures showed that distinctive growth reactions occurred when young white-pine seedlings were grown under various nutritional conditions. Physiologically, the only variables for the different series were introduced by varying the pH and the mineral content of the nutrient solutions. The character of growth response for each series and the results from inoculations with the fungus are shown in table 1. The apparently normal seedling plants in the control cultures of Series A, receiving the full nutrient at pH 4.5, developed sturdy stems and bright green needles, and their light brown roots bore many root hairs (Fig. 1, A). The response of the inoculated plants in this series was not strikingly different from that of the controls. Root and shoot development was not seriously impaired, although 20 per cent of the lateral roots were infected. In a few cases (Fig. 1, A) the plants sustained severe root injury. The infection of the roots was characterized by localized brownish areas, and the portion toward the root tip ultimately died. The control plants of Series B (Fig. 1, B), with the full nutrient ad- justed to pH 6.0, were apparently less healthy than those of Series A. While the shoot growth was about equal, the needles were a paler green. The root system of the plants in Series B was more extensive and bore more laterals with well-developed root hairs. The inoculated seedlings of this series were more heavily infected than those of Series A, since infection occurred on 37 per cent of the roots. Root and shoot formation was noticeably less than that of the controls ; death of the tap root was characteristic of these plants (Fig. 1,B). The seedlings of Series C, grown at a pH of 7.0, differed decidedly from those of Series A and B (Fig. 1, C). The needles, although well developed, were a pronounced pale yellow-green. Although the stem growth was sur- prisingly vigorous, the root system was dark brown, and consisted of many fine, short laterals. Root hairs were short and sparse. The inoculated plants showed a higher degree of susceptibility, as infection occurred on 52 per cent of the roots. The roots were near the surface of the sand (Fig. 1, C), as in the controls, thus penetration of the root system was not noticeably altered. Although the number of lateral roots increased with the adjustment of the nutrient solution toward pH 7.0, the number of infected roots increased in still greater ratio. With a reduced supply of available nitrate nitrogen, the seedlings of Series D (Fig. 1, D) appeared, during the first 4 weeks of growth, to de- velop in height at a greater rate than those of the other series. This agrees 1130 Phytopathology [Vol. 27 ^ ®. -e ©42 iS a p T3 Qj 03 r* S Fi.^ -^ ^^ C.2- ^ Ph g o (D Xi o ^ § § o i 5 fl S ci- o s o fl » ^ s OQ rt o o iS -c S^ " O o 0!!_, "^ « (U o 3 CS be O "^ ^ r^ «4/ « « :S s o jii on i" I' 2 « fl « s 2-S (=^-5= s •y H O 'Ji "S X ^ -^^ -t- c C ^ ^ fl O S QJ O o W -^.2 ' n .4^ ^.. o'° rn ^ ^ >r. ■4^ ^^ 1-^ f~ 09 F^ 00 K C w ■r-l O CD O eS « u bo '^ O ^ Q ij an 3 *" .S 00 2 'O O -M .S +j 2r rt Ph c V ui s C rr » 3 oi . t; * f- » r V . h'^ ly*^ '•►A V ,ir >4 1937] Wean: Parasitism of Polyporus schweinitzii 1131 with the growth of Corsican pine as recorded by Aldrieh-Blake (1). This shoot reaction, however, was barely perceptible by the end of the experimen- tal period. The diameter of the stems was slightly less than that of those receiving the full nutrient and the needles of a slightly paler green. The amount of root injury in the inoculated cultures, 28 per cent, was greater than under conditions of higher nitrogen concentration, and may be inter- preted as showing that nitrogen deficiency may induce an increased sus- ceptibility. The reduced supply of phosphorus greatly retarded the growth of roots and shoots in the controls of Series E (Fig. 1, E). The needles, although of a deep green, were of normal length. Fifty-two per cent of the roots of inoculated plants were infected, which is identical with that occurring under conditions of a full nutrient at pH 7.0. Pot Cultures of 2-Year-Old Trees. In many respects the older plants responded in a manner similar to that of the young seedlings in the different series. The control plants of Series A showed good shoot development, and the character of the root systems indicated favorable growing conditions. This was the only instance in which the number of new roots per plant in the inoculated cultures exceeded that of the controls (Table 2). From table 3 it is plainly evident that infection by the fungus was not severe on roots of the current season or on those of greater age. The fact that the needles of the control plants at pH 6.0, Series B, were slow in assuming a normal green indicated less favorable growing conditions than those described above. Shoot growth was little affected except in color. There was an increase in number of roots and in the extension of the root system. Infection in this series was apparent on roots of both the current and previous seasons. Open lesions of decay were of common occurrence. The poor color of new shoot growth at pH 7.0, Series C, was very marked ; in fact, the color did not become a healthy green during the entire experimental period. The lateral roots were greatest in number and in total length of the 5 series, but they were very slender and a dark brown. The infection was severe (Fig. 2, C). The percentage of infected roots of the current season's growth was more than twice that of Series B and 5 times that of Series A. In the roots of previous seasons' growth, the percentage dead was nearly 3 times that of B and 6 times that of Series A (Table 3). In Series D, at pll 4.5, low nitrogen appeared to be a factor affecting the growth of trees at this age, for shoot growth was less than that of Series A. The number of roots also was less, but the root system was of greater total length. The per cent of dead and infected roots of the current season and of the older roots was similar to that of Series B, but the development of lesions was less pronounced. Under conditions of reduced phosphorus, shoot growth was again less than that of the full nutrient series at pH 4.5. The root systems were char- •r ► 1130 Phytopathology [Vol. 27 -= ^ t: o i2 ^'^ " '- 2 •— >—-♦-,_ .— (J X :• -J CL,f— ^ «-H ^ O O f^.«w - C X (V .i = o n '^ tX ^ ^ ^ -• ^ 1.^ S r* . "■' " X R O o •*-' t- .a w ■? I i "Sc x- . ~ " s ti -'••r- o — ^ ^. -- — ,-. - ^ 'T x = O = 7z U z, s ±? C >-i — s s »= ■*- ■*- C " cu • taD *- '^ TjZ- s E » 5 > ^ s tt o r = =^ o = 5S x; "w j^ ^ «'!;:: ^ X X C C • — c "" = r X ^ — ~ C J< r '^ ^ -^ C' o = - « .i ;r .2 ^ "" ^ r-^J ^ ■^ • 2 13 ^.H'Z'^ - So (3 r .:: -M fct j5 Ph = .^ ^' = c ~ TB 2 . f* v** '•v A r' 1937] AVean: Parasitism of Polyporus sciiweinitzii 1131 with the ofrowth of Corsican pine as recorded by Aldrieli-Blake (1). This shoot reaction, liowever, was barely perceptible by the end of the experimen- tal period. The diameter of the stems was slio-htly less than that of those receivino- the full nutrient and the needles of a sli«,ditly ])aler j»reen. The amount of root injury in the inoculated cultures. 28 per cent, was "-reater than under conditions of hi«iiier nitro«»en concenti-ation, and may be inter- preted as showin«»- that nitroj-en deficiency may induce an increased sus- ceptibility. The reduced sui)i)ly of phosphorus nreatly retarded the orowth of roots and shoots in the controls of Series E (Fin-. ], p]). The needles, althou-'h of a deep oreen, were of normal length. Fifty-two per cent of the roots of inoculated i)lants were infected, which is identical with that occurring; uiuler conditions of a full nutrient at pll 7.0. Pot Cultures of 2-Year-()l(l Trees. In many respects the older plants responch'd in a manner similar to that of the younjr seedlinjrs in the different series. The control plants of Series A showed jrood shoot development, and the character of the root systems indicated favorable ••rowinj; conditions. This was the only instance in which the mnnber of new roots per plant in the inoculated cultures exceeded that of the controls (Table 2). From table 3 it is plainly evident that infection by the fungus was not severe on roots of the current season or on those of prreater ajre. The fact that the needles of the control plants at pll 6.0, Series B, were slow in assumin«r a normal jrreen indicated less favorable jirowinj? conditions than those described above. Shoot jrrowth was little affected except in color. There was an increase in number of roots and in the extension of the root system. Infection in this series was apparent on roots of both the current and previous seasons. Open lesions of decay were of connnon occurrence. The poor color of new shoot growth at pll 7.0, Series C, was xovy marked; in fact, the color did not become a healthy green during the entire experimental period. The lateral roots were greatest in number and in total length of the 5 series, but they were xovy slender and a daik brown. The infection was severe (Fig. 2, V). The jiercentage of infected roots of the current season's growth was more than twice that of Series B and 5 times that of Series A. In the roots of previous seasons' growth, the percentage dead was nearly 3 times that of 15 and fJ times that of Series A (Table 3). In Series D, at pi I 4.5, low nitrogen appeared to be a factor affecting the growth of trees at this age, for shoot growth was less than that of Series A. The number of roots also was less, but the root system was of greater total length. The per cent of dead and infected roots of the current seasfm and of tke older roots was similar to that of Series B, but the deveIoi)ment of lesions was less pronounced. Under conditions of reduced ])hosphorus. shoot growth was again less than that of the full nutrient series at pll 4.5. The root systems were char- ♦ » 1132 Phytopathology [Vol. 27 c« o I V. Q o •»» e K Q ?»> Q u '? o o eo < K O O o 03 o 03 1-:) Q o >o O lO o [^ Co' Tji in rH «d T3 O o o Ol o o »q lo o o ^ o t' CO tH d T3 Ol J M Per reMt 2.4 5.5 13.4 6.0 5.2 a ft Per cent 1.4 5.5 12.6 3.0 2.0 OQ ■4J o o O lOO •«** CO 1-1 ►> o o t^ «o CO **-• (M (M CO 1-1 i-l Initial pHof nutrient o o o o o iq O o iq iq ■^* «o' t>^ '^* •^' •4J g •c •♦J Complete < < Low nitrogen Low phosphorus 2 a 1 ) 1 < ) t3 #2 ^« «« ^ ^ ^ « >. ^ V. o a - v a> OQ 00 hi (U SD c3 n3 » * > «• * # • - ¥ «r - S ^ . . c3 'S tJ w O M -s -S <" S -f? Cuo . . 2 2 ^ 0)0^73 M . o -^^ C3 o ^ ^ I"* C3 .F^ •*- GQ %l si > ^' C3 CM J- o OS CO a> aj X CJ CIS OQ OQ c3 P4 to •r? f3 1*^ tH ^j f"" c3 « X «-^ s >^ c o • fl 5 "" -5 ■r< o . ^-^^ Ol •O ,a GO ^^ i3 - ^ s^ ^ (u cj^£^ J3«2 g ^ W +- -^ cj ,^ p 2 o ^^ b S 2 *^ «o 9 W W!«w rt 2 g^ X 4) 3 O • ^ O Eh .ti g bcrt S ^^ fl ■•-> o »-< cs o .S o c4 c« 03 -M 1132 •2 Si o o ft4 W w 'A l*IlVT()I'ATll(>L()(iY Old roots .2 ^' o o O l.O o i-- Co' 't lO r-i O •*— c: & P CI CC O '>C; CO .'' t" c; — * od LO T- I— 1 C fz; o Qc o o - ^- ^- r^ -V ^1 ?- .— CO M CI c w c -t •■0 ^ -+< CI ►J t-: 1 - CO CO Ci '^1 CO C 1 O CO -f Roots of current season S o o C/ -^^ IS o c Ft r cent 2.4 5.5 13.4 6.0 5.2 •*• ^ 'IJ l.-O o o o j^ 1-i iri ci co' ci ;;, 1—1 (^ O '"J O -t* CO ^ << CI CI CO r-i rH Initial pH of nutrient o o o o o to O O »q lO t' d t- -t ^ 4^ g CO ^•* •* O ^ w ^ o •— o 5. ; 1 *« s« %« \« s« j; - - - c < c: o fi w o 00 u Q CO U CS V so r3 S o § CS pa Vol. 27 «' ■. -* »> t tl. • r / •■ • - P 1937] Wean: Parasitism of Polypohus schweinitzu 1133 • ' . ■ [i] .> '^ '^ 'w ^ ^.r-i ^-^ o o O g P «M N^ Ph .S 03 - 1; 2 O HH -K -3 := o - r:3 r— W ■/- '^ >- ^ -^^ "■■■ I ■* ,-K ■«-' j^ j^ *^ •::;cc o "^ sa tt-3 _ - ? cj "^ "? 5 ^ J E - :- ' "^ ^ S X .w ,-^ t: ^- ^ .S .^ ^ "^ -^ "-^ -2 'E ^ ^ N»4 S W -^ •- V_ . . ■ m -^^ "^ «^ • ;l^-.— s ? c: ^ •"S -*- -5 ^ -J '"' S «w « 5 r .^ rS r" =• •-: c3 tl ei; O -J '-2 Ci) S s ^ • ^ pi. — p— .-- — '; J f^ "^ 1j -IIj ■-' ^-* s = 5 '= ^ :^ U^ "^" ^ "^ •* ^ ij r 5^ .;:i '^- ~ 2 < 2: i:- > Hi; - - CO ej^Xrzj.^ ^ xn T t "=■ •=■ fc. c c: ti--^ ^ - ^ - c- *- - r" -'- '^ s '^ <«-i -*- "^ SP ^ • :; S V S C X S*- *^ ? ^ " s s o - J2 — A" " ^ -^ ^ ^ - tx— = « 1; .= '^ " ■= — '? • * ^"^ r I- ,r |i ■*^ '-< _ t- C/ c . — — ~ s ^ '*- *- "^ "• ••' "^ ; V = c :s = '/■ s X '- 'f ^* -^ ■ ^' ^ "^ - >. S *- ° o ? ^ •= ■:: a» o -fc' _• x . o = ? ^ £; 2 INTENTIONAL SECOND EXPOSURE 1134 Phytopathology [Vol. 27 1937] Wean: Parasitism of Polyporus schweinitzii 1135 acterized by short laterals of larger diameter than those of the other series. The percentage of infection on the roots of the current season was similar to that of the low nitrogen series, but that of the older roots was similar to that of Series C at pH 7.0 (Fig. 2, E). It is significant that all inoculated cultures in the various series yielded plants with less new-shoot growth and smaller root systems than were found under similar conditions in the controls. Fewer lateral roots, likewise, developed in the former cultures, with the exception of the 2-year-old plants of Series A. The results from pH determinations of juice extracted from the roots of both age classes of the plants showed a correlation with the shift in pH of the nutrient solution. Juices from the needles and stems, however, failed to show any substantial deviations. Infected roots (Fig. 2, K) showed injury of various degrees of severity. In some instances the mother roots bore lesions (Fig. 2, F) with large open areas of decay. In others it appeared that certain root tissues had become meristematic in an attempt to wall out the fungus. Direct infection had often taken place through epidermal and cortical cells (Fig. 3, A and B), although the typical mode of entrance appeared to be either through a lat- eral or at its base (Fig. 2, H and I). Portions of roots killed by the fungus showed a dark red discoloration of the central region (Fig. 2, J) extending upward in the mother root toward the root-crown, occasionally even into the stems. The fungus could not be isolated from tissues very far distant from the point of initial infection, even though the discoloration in the central portion of the root extended beyond such areas. Chemical Analyses.^ York (op. cit.) found upon comparing the ash analyses of wood from normal white pine with that of trees infected with Polyporus schweinitzii, that the diseased trees contained less calcium than the healthy ones. The results from ash analyses*' of plants in the inoculated and control cultures are shown in table 4. The outstanding points in these results are the correlation in the absorp- tion of calcium and phosphorus and the greatly reduced amount of calcium in plants grown at pH 7.0. This would appear to confirm the belief of Truog (32), namely, that the feeding power for phosphorus is related to the calcium content of the plant. The fact that York (oj). cit.) found, as a result of several hundred pH tests and analyses of water extracts of the soil, that there was on the aver- age one-third more calcium in areas of diseased trees than in disease-free plantings suggested the possibility that Polyporus schweinitzii may inhibit the entrance of calcium into the roots of trees. Chemical analyses were made 6 These data were loaned to the writer through the courtesy of H. H. York. 6 The chemical analyses were made by H. J. Hallowell, Consulting Chemist, Phila- delphia, Pa. t -t-r TABLE 4:.— Ash analyses of seedlings and sand, indicated on a percentage lasis of carbon and CO 2 free ash Seedlings Series Total ash SiOa CaO P.O5 lb 2 1 2 1 2 1 2 Aa A-C ... B B-c ... C C-c ... D D-c ... E E-c . 3.89 4.02 3.94 3.74 4.20 5.32 4.06 4.48 4.14 4.61 3.99 3.50 3.03 3.31 3.33 2.81 2.93 3.22 2.87 3.32 2.48 2.29 2.39 3.14 3.77 4.20 1.60 1.51 2.66 2.22 7.90 7.89 7.07 6.82 8.68 6.23 5.25 6.80 8.68 9.40 8.72 10.08 10.02 10.63 6.23 6.72 9.86 10.34 7.93 10.80 12.93 11.05 12.02 16.80 8.35 13.88 7.68 10.84 7.59 15.40 23.75 23.76 23.51 25.87 18.35 21.35 25.38 28.63 22.85 26.76 21.49 28.75 21.93 27.05 21.99 24.75 22.02 25.82 20.02 25.28 Series B — Inoculated B — Control 0.054% CaO 0.0025% CaO Sand •r • ► a A, B, C, D, and E— Inoculated ; A-c, etc.,— controls, uninoculated. b 1 — Seedlings, 220 days old ; 2 — Two-year-old trees. of water extracts for calcium in the quartz sand removed from the imme- diate vicinity of the roots of plants grown at pH 6.0. The results (Table 4) showed a percentage of only 0.0025 CaO in the control cultures, compared with 0.054 per cent about the roots of inoculated plants. Ordinarily, a root parasite is not considered as influencing the availability of nutrient material in the medium surrounding the roots of the host, but, under the conditions established in the sand cultures, it appears that P. schweinitzii caused the calcium to become less available to the plants. Histological Study. Healthy and diseased roots were tested for cellulose and lignin content. For cellulose, potassium iodide with iodine and sul- phuric acid were used, and for lignin, phloroglucin and hydrochloric acid. The infected roots showed a marked reduction in cellulose in the discolored areas, while the same zone evidenced heavy lignification of the cell walls. Compared with normal roots of similar age and size, the lignification of the woody elements was perceptibly advanced in the infected roots. It would thus appear that Polyporus schweinitzii produces an effect on the host that may hasten lignification of the cell walls (Fig. 3, C and D). Since the above tests gave similar results on tissue in which the hyphae were not present, but were discolored as a result of near-by infection, it seems that some product of the parasite was responsible for such host reaction. When cross sections of diseased roots were stained with pH indicators, as that of the Hellige set and bromthymol blue of the Lamotte Chemical Company, the cell walls of the dead tissue acquired the color of the dyes in ^p 1136 Phytopathology [Vol. 27 1937] Wean: Parasitism of Polyporus schweinitzii 1137 their acid range, while the walls of the living, uninjured cells acquired the color of the dyes in their basic range. The acid color reaction also was found to occur upon treating normal white-pine wood known to be heavily lignified. These results indicate that the presence of Polyporus schweinitzii within host tissue causes a substantial change in the chemical constitution of the cell walls. Fio. 3. Penetration of Polyporus schweinitzii into root tissue. Drawings made with the aid of a camera lucida. x 650. A. Hyphae in the living cells of a young root. Note the break-down in cell contents of cells * * a " in contrast with adjoining cones unaffected by the presence of the fungus. B. The direct penetration of hyphae through the heavy cortical layer of cells in a 2-year-old root. C. Heavily lignified cells from affected tissue illustrating the thick walls and small lumen of xylem cells. D. Structure of conducting tissue in the root of a control plant. \ * k. »h .4 ''»>■* \ ^ * n . ^9 An examination of stem tissue near the root-crown, into which a fungus filtrate had been injected by means of a capillary tube, showed death of the cells about the point of entrance and reddish streaks in the surrounding wood cells. This was in contrast with the normal meristematic action of tissue about wounds made by the injection of water and needle punctures. Physiology of the Fungus. A number of media proved satisfactory for the pure culture of this fungus. Growth typical of Polyporus schweinitzii occurred on oatmeal agar, Trommer's plain malt agar, whole buckwheat, white-pine-needle duff, and noncrushed white-pine seed. Little growth occurred on rice, and none on potassium silicate plus nutrients that had been found satisfactory for growth when added to agar medium. Trommer's plain-malt agar proved to be the most satisfactory for root-isolations and the storage of cultures. Liquid cultures of 2 per cent Trommer's plain malt showed growth over the wide pH range of 2.59 to 7.0. After a 14-day period, however, the solu- tions made above pH 4.0 were found to be decidedly more acid than when inoculated (Table 5). The fungus assumed a green-yellow at points lower than pH 3.5 and a brown-yellow as the conditions were more alkaline. The effect of the fungus upon the solutions in surface and submerged cultures was not comparable in the more alkaline pH range. TABLE 5.— Liquid culture data of Polyporus schweinitzii Fries, Culture acid Item Initial pH after Diameter of surface Character equivalent after 14 days compared pH 14 days growth in mm. of growth when buffered. CC. N/10 acid 2.59 2.55 50 sb.a 3.20 3.20 70 sr. 4.00 3.60 82 sr. Surface 4.65 4.00 90 sr. cultures 5.18 3.65 85 sr. 6.00 3.62 65 sr. 7.00 4.20 40 ar. 2.59 2.55 sb. 3.20 3.21 sb. 4.00 3.60 5 sr. Submerged 4.65 3.55 45 sr. cultures 5.18 3.49 7 sr. 6.00 4.32 sb. 7.00 5.00 sb. 2.0 9 45 sb. 5.25 3.0 50 sb. 6.00 Buffered 4.0 emained same 60 sb. 6.00 solutions 5.0 2 sb. 3.75 vol. 37.5 cc. 6.0 1 sb. 1.50 7.0 8.0 In a Sb. — submerged growth, Sr. — Surface. 1138 Phytopathology [Vol. 27 Liquid cultures, buffered by potassium hydrogen phosphate and citric acid, showed a sharply defined pH optimum for this fungus (Table 5). Growth occurred at pH 2.0, but none above 6.0, while 3.0 to 4.0 appeared to be the optimum. The titratable acidity of the solutions was determined be- fore inoculation, and 14 days later, using O.IN NaOH, with phenolphthalein as an indicator. The amount of acid was found to be increased in all solu- tions where growth had occurred. From chemical analyses, it appeared that the increase in acidity was due to the production of succinic acid by the fungus. DISCUSSION An attempt has been made in this study to correlate the parasitism of seedling white-pine roots by Polyporus schweinitzii with growth conditions in sand cultures. For this study the 2 drip methods of supplying the nutri- ent, irrrespective of the system, proved very satisfactory in maintaining growing conditions for the plants. The development of corky excrescences around points of emergence of laterals indicated that the sand had been kept well moistened (Fig. 1, F and G). Reactions to the modifications in the nutrient solutions were rather sharply defined, and it was quite evident that the growth response of young white pine can be determined best by a study of both shoot and root reaction. There was a definite increase in the length of the root system and in the number of lateral roots as the nutrient solution was made more alkaline. However, with this increase in length and number of roots, the average root diameter became less, and thus the total root surface would not increase by so large a ratio as might seem to be the case. The development of the roots near the surface of the sand at pH 7.0 may have been due to the greater amount of phosphorus in that area. It is probable that a large percentage was precipitated into a less available form of ferric phosphate before pene- trating far into the sand. Moreover, with the increase in alkalinity of the nutrient solution, the amount of calcium and phosphorus absorbed by the plants was decreased, and there was an increase in the alkalinity of the root extract. Since the susceptibility of white-pine roots to infection by this fungus appears in this experiment related to the chemical nature of the nutrient medium, the amount of calcium and phosphorus available must have an important bearing on host resistance. True (31) has contributed an explanation of the role played by calcium in the normal absorption of essential ions by plants, while Loew (17) has shown that phosphorus is of primary importance in the formation of new cells. At pH 7.0, the roots were typical of plants low in calcium (10), and reduced phosphorus proved to be a limiting factor to the increase in the size of seedling plants. In the case of the older plants when grown at pH 4.5, a reduction in the amount of either phosphorus or nitrogen in the nutrient solution resulted in , -^ V* :'■ ;»kU 1#« ¥ '•wr * T^n > H « - — — » 1937] Wean: Parasitism op Polyporus schweinitzii 1139 greater extension of the root system. These results are in accordance with several previous studies (14, 19, 20, 24, 26). Low nitrogen in the case of the young seedlings produced a similar reaction, but low phosphorus unfa- vorably affected root length extension. The root systems o plants m the latter 'ease were decidedly reduced in length, -"though ash an^^^^^^^^ showed phosphorus absorption to be nearly equal to that of the plants re ce vtog the full nutrient at pH 4.5. It appears that the reduction in amount of available nitrogen and the increase in alkalinity of the nutrient so ut^n affect the extension of the root systems of young white pines in similar ™* The'len^th of the shoots of the young seedlings was about equal, regard- less of the Icidity, when receiving the full nutrient The older Plants we- more sensitive, however, with the least new shoot grow h of the 5 series occurring at pH 7.0. Low nitrogen did not impair shoot development of the younger plants, although the stem diameter was less. A reduction of phos- phorus suppressed shoot growth of young seedlings, as did nitrogen and phosphorus in the case of the older plants. From the above it appears that the effect of nutritional variations influences the development of roots more than that of the shoots of 1-year-old white-pine seedlings, while older plants may be expected to show rather sharply through their shoot development the influence of the medium in which they are grown. The needles, regardless of the age of the plants, showed similar color reac- tions to the modifications in the nutrient solution. The needles of plants receiving the full nutrient at pH 4.5 were apparently normal in color. The reduction of phosphorus resulted in a deeper shade of green, hut not a purple-green, as has been reported for phosphorus-starved plants The yellow-green formed at pH 6.0 and 7.0 seemed to indicate low availability " The results of this investigation show that Polyporus schweinitzii is capa- ble of parasitizing the roots of seedling white-pine trees under the conditions of this study Parasitism occurred on both roots of the current and previous easons- growth. Injury was evidenced by death of the small roots, the pro- duction of lesions, and red discoloration in larger roots. The presence of he funlus decidedly reduced the total length and number of lateral roots on the root systems of inoculated plants. Entrance into living ho^t tissue was ind to occur directly through epidermal and cortical cells, through the bae of lateral roots, and through the corky excrescences at the base of some of the lateral roots. The intracellular passage of this fungus from one nost 1 to another occurred without evidence of mechanical pressure or constr^- tion of the hyphae. Its entrance into the living roots of seedling plants indi- cates he pSiUty that this fungus may be distributed by nursery stock to f est p antings, and, furthermore, that it may be a factor affecting the sur- V vd of naturSiy reproduced white pine and possibly other conifers. 4? 1140 Phytopathology [Vol. 27 The seedlings grown under alkaline conditions and also those with low phosphorus and nitrogen were the most susceptible to attack. Hence, it appears that where these conditions exist singly or together, Polyporus schweinitzii may be expected to be especially destructive. The effect of this fungus upon absorption of mineral elements by its host is important, since the ash of infected plants showed a lower percentage of calcium than that of the controls, and an analysis of water extract of the sand from inoculated cultures showed a higher percentage of calcium than in the controls. Whether the calcium was made less available by the presence of the fungus, or whether the plants for some reason were unable to absorb the element, is problematical; the former interpretation seems the more logical, however, since it appears that the fungus produces succinic acid in liquid cultures. Since an acid was produced by Polyporus schweinitzii during growth on buffered solutions, the red discoloration that occurred within infected roots may have been due to the acid liberated by the parasite within the host tissue. Furthermore, it seems that this excretion caused prematurity and death of the cells it penetrated. This may explain the observations on structural weakness of wood beyond the areas of visible decay (5). That this fungus produces a substance causing wood discoloration and change in cell character is further indicated by the fact that reddish streaks developed when a filtrate prepared from the mycelium was injected into woody tissue of 3-year-old white-pines. SUMMARY A physiological and pathological study of white-pine seedlings, inocu- lated with Polyporus schweinitzii, has been made in order to determine whether the fungus may become parasitic to living root tissue. Root and shoot growth of the plants was found to be correlated with the pH of the nutrient solution and reductions of inorganic nitrogen and phos- phorus. Low phosphorus proved to be a limiting factor in young seedling growth, while the reduction of nitrate nitrogen did not unfavorably affect it. The absorption of calcium and phosphorus was reduced at pH 7.0 in both control and inoculated cultures. The presence of the fungus lowered the absorption of calcium by inoculated plants, as evidenced by the occurrence of relatively large amounts of calcium compounds in the immediate vicinity of the roots and a lower calcium content in the plant ash. The hyphae of this fungus were found penetrating directly through liv- ing cortical cells of the root and corky excrescences at the base of lateral roots. Parasitism increased with the alkalinity of the nutrient solution and with the reduction of phosphorus. Infection of roots was accompanied by a reddening of host tissue. Such areas seemed to be prematurely lignified in young roots, and appeared to be X r4 V"** 4 Jf V 4 JL V A la •• ^ f •«»«^i < - -» 4 ^ "■ I 1937] Wean : Parasitism OP Polyporus SCHWEINITZII 1141 acid in character. The total length of the root system and the number of lotPrnl roots were less in the case of infected plants. ^^X ^alasL of living roots on plants of seedling ^f^^^^^^^^^ J^ dhilitv of this fun-US being spread by nursery stock to forest plantings and full inJ^^^^^^^^^ it Ts I factor affecting the natural reproduction of '^S^mZt.U, in liquid cultures, produces an -id capable of increalg the acidity of nonbuffered solutions. On buffered solutions, the optimum point for growth is pH 4.0. LITERATURE CITED 1 Ai.»a,CH.B..K., B. N. The plasticity of the root system of Corsiean pine in ea,^ , ^o.^tyk-T:t:ZtlT.Z%Zl'^ .en.ei.U.U Pr. Phytopath. U = 3 CAvi e'e.- The relation of temperature to the F«ari»m wilt of the tomato. Amer. Jour. Bot. 10: '1-88- l^l^/ „ ,„^ .tainine of Liriodendron tulipifera 4. Cohen, I., and K D. Doak. The fl-»g/"|j^ ^'¥S=1mol. 10: 25-32. 1935. 5 CO J.r/^H ^\V X"^:* iSlnt fe^y on the mechanical properties of a.rp.ane e r,7f B ^Trr^lSrortV^^^ott rJ« in relation to conifers, .our. Forestry 21 : 9-21. 1927 ^^^^^.^^ ^^ ^^,^,^t varieties to leaf T'^tl^^^"'^''^^ concentration-soil type-common »• «'"^ ui^^abl'^SoirSci.e: 219-236^ 1918. g„„„ , enlture ^«- ^-^iZion^s rachSrni«:rt?= '^^ ^'^Z^r.^ n. HAKTio r Textbook of the diseases of reeT^-n^-^^^^^^^^ 1. H.,>o1oc'k^ G. g: ^Zts on l^e dSes of trees in our national forests. IV. Phy topath. i: 181-188. 1914. Marshall. Polyporus schweinitzii Fr. on 13 - > G- F. GBAVATT, and R. P. MARSHALL. »g 568-569. 1925. 15. HCBFRT t.^^^ ^^^ ^ Ltd London. 1931^ ^.^ ,^ture to disease in ''■ ^"^"^„?.- tZI Cr B^ull. 45 1903 ^^^ ,,,, ,„a ^«- "-^cT'EinS: ?9\TBVi«^ und der -• -^^^ h^TH^ftrS p'«an^.™Sr., «n. u. BodenU. (A) 10: 329-347. 1928^ „j ,„^^, ,„„ fertility. . . • Black Rock Forest :: 3^ H-Uro. of the halsam «r in .uehec Province. (Ahstract) Phy- ,- ,.w 1142 Phytopatholog\ [Vol. 27 23 Sargent, C.S. The silva of North America. . . . v. 11. Comferae (Pmus). Hough- ton, Mifflin and Co., Boston and New York. 1897. 24 SCHREIBER, M. Beitrage zur Kenntnis des Wurzelsystems der Larche und der iiehte. Centbl.Gesam.Forstw. 52: 147-162. 1926. 95 ScHRENK, H. VON. Some diseases of New England conifers: a preliminary report. U. S. Dept. Agr. Div. Veg. Physiol, and Path. Bull. 25 1900. 26 Schwartz, F. Ueber den Einfluss des Wasser- und Nahrstoffgehaltes des Sand- bodens auf die Wurzelentwickelung von Pinus sylvestris im erstern Jahre. Ztschr. Forst.u.Jagdw. 24: 88-98. 1892. , . ^ . .u * • -u ^ 27. Sherw^ood, E. C. Hydrogen-ion concentration as related to the tusarium wilt ot tomato seedlings. Amer. Jour. Bot. 10: 537-553. 1923. 28. Shive, J. W., and A. L. Stahl. Constant rates of continuous solution renewal for plants in water cultures. Bot. Gaz. 84: 317-323. 1927. 29. Thomas, H. E. Studies on Armillaria mellae (Vahl) Quel., infection, parasitism, and' host resistance. Jour. Agr. Res. [U. S.] 48: 187-218. 1934. 30. Trelease, S. F., and J. R. Thomson. Regulating the flow of solution for plant cul- tures. Science (n. 8.) 81: 204. 1935. 31. True, R. H. The significance of calcium for higher green plants. Science (n. s.) 55: 1-6. 1922. . , ^. Truog, E. The utilization of phosphates by agricultural crops, including a new theory regarding the feeding power of plants. Wisconsin Agr. Expt. Sta. Res. Bull. 41. 1916. Wean, R. E. Automatic flow-meter for drip solutions in plant nutritional studies. Science (n. s.) 82: 336. 1935. 34. Weir, J. R. Polyporus schweinitzii Fr. on Thuja plicata. Phytopath. 11: 176. 1921. 32. 33. ^1' • f ^- <7 -y A- 1 ry /_»-*•» .... AMFRicAN Fern Journal, Vol. 27, No. 2, A Hybrid-fern Name and Some New Combinations Edgar T. Wherry' In the course of compilatiou of a popular guide to the f ^^ of the Middle Atlantic States, wh.ch it is hoped cepted to be a nyoi u Article 32 of the Inter- and Camptosorus rl»zophyllus. Article -J^ o :lnal Lies of Botanical ^^^^^^^I'.^Z.^^Ti that bigeneric hybrids are to be Wted whe _^^ seems useful or --<^^^-^y'\]Zly aclibination of new 'generic- name usually formed "y " « ^^j^h r -r u^rn^;"po:eT:o des^rrthe fer„ in tins plan, it '« ""J J J^^^ ebenoides (Scott), nom. Kt" A?:nCropriate common name, Wa.Uing Spleenwort may be used. . . , „ reUuM of the University of Pcu..»,lva.ua. Botrychium obliquum var. pemisylvanicum Graves. Does not differ essentially from the original B. obliquum Muhl. ex Willd., which was, indeed, described from Penn- sylvania; will be taken up as representing the earliest name for this in varietal status. Two other well-marked varieties are var. oneidense (Gilbert) Waters and var. tenui folium (Underw.) Gilbert. There are also many forms, one of which requires further discussion. This is a mutant with the marginal serrations more or less in- tensified, and, as shown by Tryon,^ may develop from tlie same root which another year produces a normal oh- hquum frond. It has been regarded as the plant to which Sprengel gave the name B. dissectum, and, when reduction to the status of form is made, the rules of pri- ority require the combination to read B. dissectum f. ob- liquum, as pointed out by Clute. The more southern plant to which Sprengel's name was applied is, however, much more deeply cut, with divergent teeth, and grows nidependently ; in the writer's opinion it is to be classed as a distinct species. Anyone who feels that forms must have technical names can coin a new one for the serrate obliquum. When a species has become segregated into two or more geographic varieties— or subspecies, as they are coming to be termed by an increasing number of authors— all of the segregates should be regarded as of equal rank and assigned trinominals. This has already been done for some of our eastern species, but apparently not for those named below. When there is no name in varietal status already on record, the Rules permit, for the subdivision which includes the type, either a duplication of the spe- cies name or the use of *'one of the customary epithets, typtcus, genuinus, originarius, etc.," the latter alterna- tive being preferred here. 2 This Journal 26: 2G. 1936. ^^'V \ s-^— ' < V ■<* . ^ ?< 'Ti * r ^- r-^ J> < - •** •> 3 Botrychium simplex var. cordatum (Fries), comb. nov. B. lunaria var. cordatum Fries. This is the variety rep- resented by the original B. simplex Hitchcock. Others are var. tenebrosum (A. A. Eaton) Clausen and var. laxi folium Clausen. Osmunda claytoniana var. vera, iiom. nov. The basis of 0. claytoniana L. A varietal name is needed to make this coordinate with the Asiatic var. vestita (Wall.) Milde. Adiantum pedatum var. originarium, nom. nov. The plant named A. pedatum L. It grades into var. aleuti- cum Rupr. Pteridium latiusculum var. verum, nom. nov. The original P. latiusculmn (Desv.) Hieron. ex Fries. An- other is pseudocaudatum (Clute) Maxon. Onoclea sensihilis var. genuina, nom. nov. The Amer- ican 0. sensibilis L., with an Asiatic counterpart, var. interrupt a Maxim. Athyrium angustum var. ttjpicum (Butters), stat. nov.^ A. a. forma typicum Butters. The epithet is needed in varietal status for coordination with vars. elatius, rubel- lum, etc. Lycopodium lucidulum var. verum, nom. nov. The variety on which was based L. hicidtdum Michx. Its counterpart is the more northern var. occidentale (Clute) Wilson. Lycopodium inundatum var. typicum, nom. nov. The basis of L. inundatum L. Here there are two other varie- ties, var. bigelovii Tuckerm. and var. robustum R. J. Eaton. Equisctum sylvaticum var. multiramosum (Fernald) stat. nov. E. s, var. pauciramosum forma multiramosum 3 The category term is placed in bold face here because it is the noveltv, rather than the third epithet; the latter is some- times so treated even when merely changed in status, but in my opinion this may be misleading. * - • ,1 r *. 1 «i^' IRREGULAR PAGINATION Fernald. Since I interpret the term form as applying to a peculiar plant which appears sporadically in the midst of normal ones, and variety to a plant of abundant occur- rence over a definite range, I can only place this Horse- tail in the latter category. Philadelphia, Pa. <^% -^ i*^ ^ * „ '> -^ 1/* Ronnnted from American Fern Journal, Vol. 28, No. 1, Jaiiuaiy-Maich, 1938, pages 28-30. Midland Fern Notes 29 Midland Fern Notes Edgar T. Wherry During the summer of 1937 the writer drove from Philadelphia to Salt Lake City and back, visiting en route a number of fern localities; Mr. J. E. Benedict, Jr., of Washington, D. C, took part in the first half of this trip. Notes as to species observed which seem of general interest are here placed on record, except for the state of Colorado, on which a separate article has been prepared. Spcci- ineiis obtained have been placed in the herbarium of the Academy of Natural Sciences of Philadelphia. Michigan.— We barely entered this state late in May, paying a visit to Mr. and Mrs. Boydston, who live at Thorir Acres about four miles south of Niles, in Berrien County. Here in ponds and on sandy slopes at the edge of tlie floodplain of the St. Joseph river there is an un- usual abundance of Equisetums, of which five species were recognized: arvcnsc, fluviatile, laevigatum, palustre,ana prcaltum. This appears to represent a southern limiting station for E. paliistre (var. americanum), for although it is here within a mile of the Indiana line the thorough exploration of the latter state by Mr. Deam has never turned it up there. Iowa.— A visit to Pikes Peak State Park in Clayton County yielded Selaginella riipestris, not heretofore re- ported from this county, and two varieties of Gystoptens fragilis. Examination of their rootstocks and indusia showed these to be vars. genuina and protrusa, the former growing on steep bluffs, the latter in humus-rich soil on gentler slopes. * f 4« I «* At Decorah search was made for the two rarities there reported. One, Phegopteris rohertiana, was soon found west of town on a moist, crumbly, north-facing limestone bluff ; unfortunately it is in danger of destruction, since the road close to which it grows leads to a state park, and may at any time be 'improved" by landscrapers. The other, Woodsia scopuUna, could not be located, and m view of the dominance of limestone in the region its occur- rence there seems improbable. The Woodsia which grows on the quartzite rocks iu Gitchie Manitou State Park, in Lyon county at the ex- treme northwest corner of the state, has been reported both as W. scopulina and W. oregana ; but a visit there disclosed that it is neither, as it shows instead the dense glandularity characteristic of W. cathcartiana. " South Dakota.— There being reports of both Woodsia oregana and W. mexicana in the Black Hills, some atten- tion was paid to this group there, especially in the Custer State Park. No trace of the glabrous fern bearing the former name, or of the downwardly narrowed, thick- margined, conspicuously hairy-indusiate Mexican Cliff- fern could be found. Instead the plants seen were all more or less intermediate between these two extremes, the margins being more or less thickened— never markedly so -and short-stalked glands being present on vascular parts, but sparse or lacking on laminar tissue. Here, as in western Iowa, W. cathcartiana seems unciuestionably represented, although some material appears to lie inter- mediate between that and some other species, and will require detailed study for proper classification and nam- Oklahoma.— During August a brief visit was made to Cimarron, the northwesternmost county, to look for the ^* 4 - « * IRREGULAR PAGINATION 30 American Fern Journal unusual ferns which have been reported there.^ From Kenton highway No. 64 was followed eastward for 6 miles, and a private lane northward about a mile farther, until cliffs became prominent. The native vegetation in low moist places proved to have been largely destroyed by grazing animals, making search for Dryopteris fihx-mas hopeless, but small ferns could still be seen in crevices of the sandstone rocks. North-facing cliffs and ledges yielded Cheilanthes eatoni, C. feei, and C. wootom, to- crether with Selaginella wnderwoodii, not previously re- corded for this region. On south-facing ones the yellow globules formed by the rolling up of the fronds of Notho- laena standleyi were conspicuous, and while examining these several small but unmistakable plants of Pellaea wrujhtiana were found. Texas.— In the hope that some of these Cimarron valley specialties might also turn up in the Texas panhandle, the Palo Duro State Park east of Canyon, Randall County, was next visited. Here, however, only the widespread Cheilanthes feei and Pellaea atropurpurea could be found on the rocks, and Equisetiwi prealtum along the creek. Louisiana.— Under the guidance of Miss Caroline Dor- mon, of Chestnut, three species not listed by Brown^ for this state were obtained : Cheilanthes lanosa, in crevices of sandstone north of Kisatchie (15 miles south of Proven- cal) ; Selaginella riddelUi, in a sand-barren 2 miles south- east of Goldonna, both in Natchitoches Parish ; and Lyco- podium prostratum, in a wet thicket 1^ miles north of Lucky, Bienville Parish. Philadelphia, Pa. 1 Greene, This Journal 17: 125. 1927; Bush. Am. Midi. Nat. 12: 91. 1930; Fcatherly and Still, Okla. A. & M. Coll. Expt. Sta. Circ. 80. 1934. 2 La. Cons. Rev. 5: 12. 1936. { I 4f*- • ■*■ * .( - T- I "* if* ><>* -f \ Mr f- x^ T t ' J ' "• • « ~ RpprinUtl from "The Amciican Midlantl Naturalist .' Vol. 1st, No. 2, |)i). !:!:{- i:'.rj. Maich. i'.i;is A New Phlox from the Snake River Canyon Idaho-Oregon Edsar T. Wherry and Lincoln Constance In the course of the study of the genus Phlox being carried on by the senior author, the existence of a hitherto undescribed member of the genus in the Snake River region was recognized. Specimens were found to be pre- served in several herbaria, variously identified as P. Imeartfoba, P. longtfolta, or P. whitedit, although it differs markedly from these. Its nearest relative is P. speaosa var. mt,da Suksdorf, with which it agrees in glabrous herbage and short styles, but on the average its leaves are narrower flowers fewer pedicels longer, calyx-membranes more carinate, and corolla-lobes longer. Moreover, while in all normal P. speaosa the corolla-lobes are notched, becoming entire only in rare mutants, in the present plant these lobes are consistently notchless and terminally obtusish, acutish or mucronate. In addition, ^t occupies a range remote from that of the Pacific-border var. nitida so may well be classed as an independent species. A name for it has been derived from one of the Latin terms for snake, in reference to its region of occurrence. Phlox colubrina sp. nov. Plant a slender glabrous shrub 15 to 50, averaging 30 cm tall; leaves thinnish or with somewhat thickened margins, narrowly Imear; the largest 40 to 80, average 50 mm. long, and 1 to 2 (rarely 2.5), average 1.5 mm wide; inflorescence 1 to 6 or rarely 12-flowered; pedicels 18 to 70, average 35 mm. long; sepals 8 to 12, average 10 mm. long, united 1/2 to 2/3 their length: membranes of calyx-tube rather distinctly carinate, the lobes glabrous exter- nally but pilose on their inner surface; corolla-tube 9 to 15, averaging 11 mrn^ long, sometimes constricted near its orifice, externa ly glabrous; lobes 9 to 20 by 4 to 7 mm., usually 2 to 3 times as long as wide, terminally varying from obtusish to acutish or to mucronate; stamens deep within the tube, rarely ess th'.r. 3 mm. below th: orifice; styles 1.5 to 4, averaging 2.5 mm. long, tree and stigmatic for ' /;{ to 'Vs their length. Frutex parvus, glaber; folia circa 1.5 mm. lata; corollae lobi lat.tudine 2-3- plo longiores obtusiusculi, acutiusculi vel mucronati; styli 1.5-4 mm. lon^i. Type in Herbarium Academy of Natural Sciences of Philadelphia, col- lected by Lincoln Constance, H. Heggencss, D. Hedrick, and D. Peters, No^ 1823 Mav 13 1937, 1 mile above the mouth of Sheep Creek, a tributary ot the Snake River, Idaho County, Idaho, latitude 45° 27i// N., longitude 116" 321/2' W. L L ■ u- Other collections known to date are listed herewith, the herbaria being abbreviated to groups of letters which obviously represent them: 433 IRREGULAR PAGINATION f-4M 434 THE AMERICAN MIDLAND NATURALIST Idaho. Idaho Co.: Snake River Canyon 2 m. n. of Sheep C". 45 29 , 116 331/2 , Constance. RoUins & D.llon 1595, 5/16/36 (Wash. St. and ANSP) ; mouth of Wil- low Creek. 45°29!/2'. 1 16°331/2'. Constance, Hedrick & Peters 1821. 5/13/37. Oregon. Wallowa Co.: near mouth of Battle Creek 45° 18//, Jlo!o?'4',vff?!' 18143. 3/27/'34. (Will. U. & ANSP) ; 4 m. ne. of Buckhom Spr.. 45 48. m6 47 , Peck 18286 6/27/34 (Will. U. & ANSP); headw. left fk. Cache Cr.. 45 59 , I16°58'. Sheldon 8185. 5/28/'97 (NYBG. USNH); Cache Cr Bar alt. 800 45° 59' 116° 54'//. Constance. Rollins & Dillon 1548. 5/14/36 (Wash. St. & ANSP); Cher y^O. at 2000'. 45°50'. 116°50'. Jardine 388 WW (U. S. For. Serv.); Snake Canyon near Deep Cr.. 1000'. 45°47'. 116°40'. C R & D- J 567, 5/15/*36 (Wash. St. & ANSP); Imnaha. 45°33l/2'. i>6 50'. Sherwood 73. 6/4/73 (Will. U. & Field M.); m. Imnaha R-. 45°49'. > l^^t^!' ,<^- N- J-es 6440. 4/11/-31 (U. Wash. & ANSP); n. slope Mt. Wilson. 45 49 . 117 5. Sheldon 8094, 5/20/97 (NYBG. USNH). Fig. I. Phlox coluhrina in the Snake River Canyon. Fig. 2. The type specimen of Phlox coluhrina. This is the common Phlox of the Snake River Canyon and its tributary streams from about the mouth of the Grand Ronde River to th2 north edge of Hell's Canyon, where the river cuts its channel between the Wallowa and Seven Devils ranges. Doubtless the species extends still farther south but the canyon has not been explored above this point. Within this deep trough. Phlox coluhrina ranges through a variety of habitats, appearing to be equally at home in the timbered and timberless Arid Transition and the badly over- grazed Upper Sonoran Zone. A list of its associates would comprise a con- siderable proportion of the highly endemic canyon flora. In the timber belt it is found under Pinus ponderosa and Amelanchier CusickH in company with J# A l * 1^ »-^ ^-y T- i < * f • - "i* ^<. . »> l_ «<» •i- i A NEW PHLOX FROM THE SNAKE RIVER CANYON 435 Cas title ja angustifolia, Brodiaea Douglasii and Arabis crucisetosa, and spreads down through the bunchgrass climax with these and other herbs. Perhaps its frequency is highest on the dry basaltic ledges of the Upper Sonoran whose sparse vegetation is dominated by Celtts Douglasii Amelanchier Cusicku, Rhus glabra, R. Toxicodendron, Ribes Gooddtngii and Opuntia polyacantha. In the most severely grazed sectors, the Phlox often persists solely withm the protective presence of Opuntia (see Fig. 1) and Rhus glabra, surrounded by such unpalatable herbs as Amsinckia, Leptotaenia Cogswellia fragrans, trig- eron concinnoides and Lupinus sericeus. Plants of the last habitat, growing in crannies in the volcanic rock, are usually more obviously shrubby and more numerous flowered than the lax and sprawling, few-flowered mdividuals found in the partial shade of shrubs. The soil reaction is practically neutral (pH 6.8 to 7.2). Corolla color is somewhat variable, but most of the fresh material showed the outside of the lobes and tube to be (terminology according to Ridgway) "Pale Laelia Pink," and the inside to range from "Light Rosolane Purple to "Rosolane Purple." The lobes are frequently bimaculate at the throat with deeper hues of the same color, and a few plants were found with pure white corollas. The flowers of Phlox longifolia Nutt., a species which replaces F. coluhrina in the Snake River Canyon in the vicinity of the mouth of the Clear- water River, have the lobes "Pale Rose-Purple" outside and Light Mallow Purple" within. University of Pennsylvania. Philadelphia, Pa. AND University of California. Berkeley, California. * V 434 THE AMERICAN MIDLAND NATURALIST Idaho. Idaho Co.: Snake River Canyon 2 m. n. of Sheep Cr45°29'. n6°33«/2'. Constance. Rollins & Dillon 1595. 5/T6/'36 (Wash. St. and ANSP; mouth of Wil- low Creek. 45"29l/2'. 1 16''33»/2'. Constance, Hedrick & Peters 1821. 5/13/37. Ork.goN. Wallowa Co.: near mouth of Battle Creek. 45° IS'//. ' J o°i?'4',vff^!' Ittl43. 3/27/-34, (Will. U. & ANSP); 4 m. ne. of Buckhorn Spr.. 45 48, 116 47. Peck 18286. 6/27/34 (Will. U. & ANSP); headw. left fk. Cache Cr.. 45 59 . 116 58'. Sheldon 8185. 5/28/97 (NYBG. USNH) ; Cache Cr Bar alt. 800 45^^59' 116^ 541/. Constance. Rollins & Dillon 1548, 5/14/36 (Wash. St. & ANSP); Cherry'Cr. at 2000', 45^50', 116^50', Jardine 388, WW (U. S. For. Serv.); Snake Canyon near Deep Cr., 1000', 45M7', 1 16°40 . C R & D 1 567, 5/l5;'36 (Wash. St. & ANSP); Imnaha, 45°33« /. 1 16 50^, Sherwood 73, 6/4/73 (Will. U. & Field M.); m. Imnaha R., 45^9'. 1I6°46, G. N. Jones 6440, 4/ll/'3I (U. Wash. & ANSP); n. slope Mt. Wilson. 45"49'. n7°5 , Sheldon 80^M, 5/20/-97 (NYBG. USNH). %W **^. l-i". 1. Phlox coluhrina in the Snake River Canyon. r_'r.i;i. jes.s* •^u-.. Fig. 2. The type specimen of Phlox coluhrina. This is the common Phlox of the Snake River Canyon and its tributary streams from about the mouth of the Grand Ronde River to the north edge of Hell's Canyon, where the river cuts its channel between the Wallowa and Seven Devils ranges. Doubtless the species extends still farther south but the canyon has not been explored above this point. Within this deep trough, Phlox coluhrina ranges through a variety of habitats, appearing to be equally at home in the timbered and timberless Arid Transition and the badly over- grazed Upper Sonoran Zone. A list of its associates would comprise a con- siderable proportion of the highly endemic canyon flora. In the timber belt it is found under Pinus ponderosa and Amelanchier Cusickii in company with 'V - *•- < "T' «H. - - »T m aI • ^ .^ - -y »^"%,' jif* 1» •■ '-• i *,.. ' •< **■■ .-1 m ^ ..I •^ A NEW PHLOX FROM THE SNAKE RIVER CANYON 435 Castilleja angustifolia, Brodiaea Douglasii and Arabis cruasetosa, and spreads down through the bunchgrass climax with these and other herbs. Perhaps its frequency is highest on the dry basaltic ledges of the Upper Sonoran whose sparse vegetation is dominated by Celhs DougUsii, Amelanchier Cusickn, Rhus glabra, R. Toxicodendron, Ribes Gooddingii and Opuntia polyacantha. In the most severely grazed sectors, the Phlox often persists solely within the protective presence of Opuntia (see Fig. 1) and Rhus glabra, surrounded by such unpalatable herbs as Amsinckia, Leptotaenia, Cogswellia fragrans, trig- eron concinnoides and Lupinus sericeus. Plants of the last habitat, growmg in crannies in the volcanic rock, are usually more obviously shrubby and more numerous flowered than the lax and sprawling, few-flowered mdividuals found in the partial shade of shrubs. The soil reaction is practically neutral (pH 6.8 to 7.2). Corolla color is somewhat variable, but most of the fresh material showed th- outside of the lobes and tube to be (terminology according to Ridgway) "Pale Laelia Pink," and the inside to range from "Light Rosolane Purple to "Rosolane Purple." The lobes are frequently bimaculate at the throat with deeper hues of the same color, and a few plants were found with pure white corollas. The flowers of Phlox longifolia Nutt., a species which replaces 1 . colubnna in the Snake River Canyon in the vicinity of the mouth ot the C^ear- watcr River, have the lobes 'Tale Rose-Purple" outside and ' Light Mallow Purple" within. University of Plnnsylvania, Philadelphia. Pa. AND University of California. Berkeley, Caliiornia. INTENTIONAL SECOND EXPOSURE Reprinted from Pkocekdincs of the Pennsylvania Academy op Science, Vol. XT, 11)37, pngos 52-54. NOTABLE PENNSYLVANIA FERNS By Edgar T. Wherry University of Pennsylvania Although the early botanists did not usually designate a type locality for their species, there seems reason to believe that at least ten of our native ferns may be regarded as having been first described from this State. These are : Asplenutm ehenoides R. R. Scott. Described in 1865 from the south- west bank of the Schuylkill River, in Montgomery County. A. pinmitifidum (Muhl.) Xuttall. This was discovered by Muhlen- berg, no doubt in southern Lancaster County, and named by him A. rhizophyllnm B. pinnatijidmn in 1813. It was raised to species rank by Nuttall 5 years later. A, trudelli Wherry. Founded in 1925 on specimens collected at Cully, just below Holtwood, along the Suscpiehanna River in Lancaster County. Botrychium oUiqmim Muhl. ex Willd. Muhlenberg collected this, in all probability in Lancaster County, and sent it to Willdenow, who described it in IHIO. Dryopteris intermedia (Muhl. ex Willd.) Gray. This had the same history as the next-i)receding, although it was at first mistakenly de- scribed as a Poly podium, transfer to the genus Dryopteris being made by Gray in 1848. Lygodium palmatum (Bernh.) Swartz. Another discovery by Muhl- enberg ; some reference to it is made in his journal, now preserved at the American Philosophical Society in Philadelphia, but just where he ob- tained it is not clearly stated. He sent it to Willdenow% who announced it as a Hydroglossum, but this name was first published as a synonym for Gisopteris palmata by Bernhardi in 1801. Five years later Swartz trans- ferred it to his genus Lygodittm. Lycopodium ohscurnm L. John Bartram collected this near Philadel- phia and sent it to Europe, where it was figured by Dillenius in 1741. Linnaeus based his species upon this figure. Another Clubmoss with the same early history was misunderstood by Linnaeus, and grouped under PENNSYLVANIA ACADEMY OF SCIENCE 53 tf A, ♦ 4 - ■«— L'-> • ^ - - T '\ - > **V^ A "I *■» J- .X. *; r* . - »• the northern L. complanatum, not being recognized as deserving species standing until 1919, when Blanchard named it L. flahelUforme. Pohjstichum acrostichoides (Michx.) Schott. Michaux observed this fern in ''Pennsylvania, Carolina, and Tennessee" and named it Nephro- dium acrostichoides in 1803. Schott transferred it to Polystichum 31 j^ears later. Selaginella apoda (L.) Fernald. This was probably sent to Europe by Bartram, for when he figured it in 1741, Dillenius gave the range as "Pennsylvania, Virginia, and Carolina." When Linnaeus named it Lycopodium apodiim in 1753, he listed the same States, but in reverse order. Fernald made the correct combination in 1915. Woodsia ohtusa (Spreng.) Torrey. When Sprengel named this Poly- podium ohtusum in 1804, he attributed it merely to Pennsylvania, but as he is known to have been receiving material from Muhlenberg at the time, the probabilities are that it had been collected by the latter, in Lancaster County. Transfer to the correct genus was made by Torrey in 1840. Several ferns are noteworthy in that, although in part discovered else- where, they reach a range-limit in this State. Here may be enumerated : 1. CiRCUMBOREAL PLANTS Botrychium multifidum (var. silaifolium). This enters from the north, being known in Erie and Monroe Counties, and reaching a limit in central Berks County. (One station in Monmouth County, New Jersey, being slightly farther south.) Botrychium simplex. Known in 10 counties: Berks, Bucks, Craw- ford Dauphin, Indiana, Lehigh, Monroe, Northampton, Pike, and Wayne. A station near Finland, Bucks County-lat. 40° 23' N.-is apparently the southernmost known for the species. Three varieties are represented, but their individual distributions have not been worked out. Cryptogramma stelleri. In cool ravines from western Lycoming to eastern Wayne counties. Known further south only at one place in Bergen County, N. J. Polystichum hraunii (var. jrurshii). Found in ravines tributary to that of Kitchen Creek in western Luzerne County, and to that of Fishing Creek in eastern Sullivan County; also near Lake Shehawken, Wayne County. The first named is the southern limit for the species, lat. 41° 18' N. Equisetum litorale. The taxonomic status of this plant is not clear, but at any rate it reaches its southern limit here, along the Susquehanna River in York and Lancaster Counties, and at Chester in Delaware County. 4 . k .> «, - # IRREGULAR PAGINATION 54 PENNSYLVANIA ACADEMY OF SCIENCE 2. Appalachian Mountain Plants Lygodium palmatum. The Climbing Fern has a sporadic distribu- tion, but becomes locally abundant in several northeastern mountain counties, reaching a western limit near Lopez, Sullivan County, long. 76° 20' W. Asplenium hradleyi. Several stations along the Susquehanna valley in York and Lancaster Counties and one in Carbon County. The latter is a northwestern limiting occurrence, although the species is known at two places each in New Jersey and New York. A. gravesii, the hybrid between this and the next, reaches its northeastern limit in Lancaster County. Asplenium pinnatifidiim. Scattered through the more southern coun- ties, Lancaster County being in the lead, with 15 recorded stations. An old specimen from Berks County, if authentic, represents the northeastern limit of the species, reports from New Jersey apparently referring to the next following. Asplenium trudelli. One station in Fayette, and several each in York and Lancaster counties. This presumable hybrid between A. pinnati- fidum and A. montanum reaches a northern limit in Warren County, N. J. 3. Southern Calcareous Rock Plants Asple7iium resiliens. This Mexican species follows limestone outcrops into Missouri and North Carolina, and has been especially successful in traversing the Shenandoah valley. It recently turned up on a single cliff near Mercersburg, in Franklin County, Penna., lat. 39° 47' N., apparently the absolute limit of its range. 4. Eastern Bog Plants Thelypteris simidata. The northwesternmost authentic occurrence of the Bog Fern is that in Bear Meadows, Centre County, long. 77° 45' W. Reports farther northwest have proved to be based on misidentifications. Little is known as to the limits of the range of this plant in the south, and it may have entered the continent from a former land mass lying off our coast, now buried beneath the sea. Lycopodium adpressum. Besides being found along the Delaware, this coastal lowland species extends inland to White Oak, in northern Lancaster County. •«- t-~«- * m.'^ ■ ■y Jw-'* < s . *. ^ 1 -A - ^ Reprinted from Proceedings of the Pennsylvania Academy of Science, pages 42-45. Vol. XII, 1938 NOTABLE PENNSYLVANIA ORCHIDS Edgar T. Wherry University of Pennsylvania Last year there was presented a discussion of the ferns native to Pennsylvania which are of special interest in having been dis- covered in this State. A similar study of the native orchids is now offered. Aplectrum hyemale (Willdenow) Nuttall. This species was collected by Muhlenberg, no doubt in Lancaster County, and sent to Willdenow under the manuscript name Cymbidium hyemale, which was published by the latter in 1805. Nuttall based a new secondary genus Aplectrum on it in 1818, this being raised to full genus standing by Torrey in 1826. Corallorrhiza wisteriana Conrad. Early taxonomists rarely gave definite localities for their plants, but the present species was exceptional in that its describer stated it to have been collected by Charles J. Wister in 1828 along the Schuylkill River "between the Falls and the mouth of the Wissahickon Creek," in what is now northwestern Philadelphia County. Cypripedium candidum Muhlenberg ex Willdenow. Muhlen- berg's manuscript name for this was published by Willdenow in 1805, the source being merely stated as Pennsylvania. It may well have come from a locality in Lancaster County designated on a label accompanying a specimen collected by A. P. Garber in May, 1865 as "Little Conestoga near Millersville." (Porter Herbarium, now at the Academy of Natural Sciences of Philadelphia.) There is no other record of the species in the State. Habenaria blephariglottis (Willdenow) Hooker. This orchid was early found by Clayton in Virginia, but never described on his material. In announcing it under the name Orchis blephariglot- tis, Willdenow ascribed it to Pennsylvania, without recording its exact locality or collector. Since sphagnum swamps, in which it grows, are widespread, it may have come from almost any part of the State. Assignment to the genus Habenaria was made by Hooker in 1824. Habenaria orbiculata (Pursh) Torrey. When he described this species under the name Orchis orbictdata in 1814, Pursh knew it "on the mountains of Pennsylvania and Virginia." Torrey trans- ferred it to the genus Habenaria in 1826. PENNSYLVANIA ACADEMY OF SCIENCE 48 t •i m r - . T» *-!»-• '^• I J . --r r "r-^ 1 T ♦ Habenaria viridis var. bracteata (Willdenow) Gray. Being unfamiliar with European orchids, Muhlenberg considered this plant, which he presumably found in Lancaster County, an inde- pendent species, and named it Orchis bracteata. The same view was held by Willdenow, who published this name in 1805 ; by K. Brown, who transferred it to the genus HabenaHa in 1813; and by Parlatore, who placed it in the supposedly distinct genus Coeloglos- sum in 1858. Meanwhile, in 1851, Reichenbach recognized it to be a mere variety of the circumboreal species which he termed Platanthera viridis. His view is now coming to be accepted, al- though following Gray (1867) we now use the genus name Habe- naria for it. Isotria verticillata (Willdenow) Rafinesaue. (Poponia v. of Gray's Manual). This species was listed as from Maryland by Plukenet in his Mantissa, published about 1700, and figured in his plate 348, but received no name for over 100 years. Then Muh- lenberg collected it, no doubt in Lancaster County, and named it Arethusa verticillata; this name was published by Willdenow in 1805, his diagnosis being based chiefly on the Pennsylvania ma- terial. Three years later Rafinesque made it the type of his genus Isotria, and he is now followed by most botanists, although some prefer the assignment to Pogonia, made by Nuttall in 1818. Orchis obsoleta and 0. virescens Muhlenberg ex Willdenow. These supposed species were collected by Muhlenberg in Lancas- ter or neighboring counties, and sent to Willdenow who publish- ed their names in 1805. They apparently represent variations of the comprehensive species named by Linnaeus from Virginia Or- chis flava, and now known as Habenaria flava. The first Muh- lenberg name is usually ignored, but Fernald has published the second in varietal status under H. flava, in 1921. Certain species are of interest in another way, namely in reaching significant range-limits in Pennsylvania; they may be listed under three geographic headings: 1. Northern Spekibs Goodyera tesselata {Epipactis t. of Gray's Manual, ed. 7.) When Loddiges described this species in 1824, he stated it to have been received from "New York and Philadelphia," but these were merely shipping points. Four Pennsylvania stations for it are known, the southernmost in Huntingdon County, latitude 40° 40'. Habenaria dilatata. A specimen in the Porter herbarium, now at the Academy of Natural Sciences of Philadelphia, from Union IRREGULAR PAGINATION 44 PENNSYLVANIA ACADEMY OF SCIENCE City, Erie County, latitude 41" 55', represents a southern limit for this species. Habenaria hookeri. This is known from a number of north- eastern and central stations, the southernmost being at Bedford Springs, Bedford County, latitude 39" 50'. It probably also ranges into West Virginia. Habenaria hyperborea. Porter had specimens of this from three counties, the southernmost station represented being Mt. Pleasant, latitude 41" 44', in Wayne County. Malaxis brachypoda (formerly known as Microstylis mono- phyllos.) This occupant of cold calcareous swamps enters the State from the northeast, and extends down along the higher mountains. Its limit here is represented by a sheet in the Porter herbarium lab- elled "Summit of the Allegheniea, Blair County," which would place it at latitude 40" 30'. Spiranthes romanzoffiana. The southernmost record for this orchid is one in the Porter herbarium from Harmonsburg, Crawford County, latitude 41" 40'. 2. SouTHEJRN Species CoraUorrhiza wisteriana. In addition to having been discov- ered in Pennsylvania, this coral-root is of interest in reaching its northern limit here, near Oley Furnace, Berks County, latitude 40" 20'. Habenaria cristata. Although most abundant on the Coastal Plain, the Lesser Orange Fringe-orchid locally invades more north- ern or upland provinces. Its north westernmost station was in Fraser's Bog, Montgomery County, lat. 40" 5', long. 75" 5'. Isotria af finis (Pogonia a. of Gray's Manual.) Because of its erratic behavior, in appearing at any one station only at inter- vals of several years, the range of this species is but incompletely known; however in view of its greater abundance and more con- sistent reappearance in southeastern North Carolina and Virginia, it is regarded as a southern plant. Its westernmost record is that in Greene County, Pennsylvania {Trillia 7:44, 1923). Interest- ingly enough, Muhlenberg's herbarium contains a specimen of it, labelled as a variety of his "Arethusa verticillata.'* List era reniformis (L. smallii) . A southern Appalachian T way- blade, reaching its northern limit in Bear Meadows, southeastern Centre County, latitude 40" 45'. Malaxis bayardi. This rare relative of M, unifolia, distinguish- ed by its very short pedicels and correspondingly narrow raceme. PENNSYLVANIA ACADEMY OF SCIENCE 45 ►■-> T%-1 ->K •• ' r .-On has been known heretofore only from two stations in the mountains of North Carolina and from Nansemond County, near the coast of Virginia, from where it was described by Fernald in 1936 (Rhodora 38:402). In the University of Pennsylvania herbarium there are specimens of it from Schuylkill and Luzerne counties, identification of which has kindly been confirmed by Professor Fernald. Thorough examination of herbarium sheets here and elsewhere has failed to show any collections farther north. Spiranthes beckii. Though chiefly a Coastal Plain plant, this tiny Spiral-orchid occasionally enters the uplands, and has been collected in Pennsylvania as far northwest as Gibraltar, Berks County, lat. 40" 17', long. 75" 53'. 3. Midland Species Cypripedium candidum. The White Slipper-orchid is classed as a Midland plant, in that its principal range is from Kentucky and Missouri north to the Great Lakes. Locally, however, it ex- tends eastward across New York to northern New Jersey. The Lancaster County station where the species was discovered, already referred to above, represents a southeastern limiting occurrence, lat. 40° 0', long. 76° 22'. 1 vyy 1/ II !*■ ■♦ •^^ < •7' 4j <^ THE PHLOXES OF OREGON BY Edgar T. Wherry. For some years the writer has been colleetin^ data on the genus Phlox, but so complex are the in'oblems involved that there seems no hope of completing a monographic treatment in the near future. In order that certain taxonomic findings thus far made can be included in the Flora of Oregon which is under preparation by Professor ^lorton E. Peck, of AVillamette University, the following notes on species and subspecies (designated by simple trinomials) known to occur in that state are being l)ublished. The plants are taken up in three groui)s, respectively those with tall erect stems and short styles; with like stems and long styles; and with short branched stems yielding cespitose clumps. Erect-stemmed, Short-styled Group Phlox speciosa Piirsh. The variant of this northern species on which the name was based, P. speciosa euspeciosa Brand, is characterized by tall habit (average height 35 cm.), glabrate lower herbage, and thinnish long-acuminate leaves. It grows chiefly in Idaho and Washington, barely entering Oregon in the neighborhood of The Dalles. From eastern Washington southwestward it grades into a derivative with lower stature (average height 25 cm.) and slightly thickened, short-acuminate leaves, which has never received sub- species classification, so is here termed P. speciosa occidentalis (Durand) Wherry.^ The absence of this from northern and central Oregon is appar- ently due to its having been destroyed there by Tertiary volcanic activity, for it is frequent in the southwest corner of this state, and southward into California. Two variants of Phlox speciosa with thick, glandular leaves have de- veloped in central Washington, and extend a short distance into Oregon. One may be known as P. speciosa lanceolata (E. Nelson) Wherry ;=' it averages 28 cm. tall, with long internodes, and few lanceolate leaves up to 30 to 60 nun. in length. The other was termed by E. Nelson P. whitedii, but Brand's name P. speciosa lignosa is the earliest in subspecies status. It differs in averaging but 20 cm. tall, the internodes being short and the leaves 1 Stiitus noviis: P. divoricnta var. occulcntaUs Durand. J. Acad. Nat. Sci. Phila. 12] 3- 97 1855- P occkUntaUa Durand ex Torroy, Ropt- Expl. & Survey RR. 4: 125. 1857; P. speciosa . . . f. occidtntalis Brand, in Engler's Pflanzenrcich IV. 250: 74. 1907. 2 Status novus: P. lanrcohla E. Nelson, Rev. W. N. Am. Phlox: 29. 1899; P. speciosa . . . f. lanceolata Brand, in Engler's Pflanzenrcich IV. 250: 73. 1907. (133) IRREGULAR PAGINATION 134 PROCEEDINGS OF THE ACADEMY OF [Vol. XC I numerous, linear to lanceolate, and up to 15 or exceptionally 30 mm. long. Material from Tygh Valley, Oregon, is gradational between these, but typical specimens of the second were distributed from the state's " eastern prairies " by Howell. Another variation from the ancestral P. speciosa stock consists in loss of pubescence from the entire plant except the inner surfaces of sepals, resulting in P. speciosa nitida (Suksdorf) Wherry/ in southern Washington. This migrated southward into California, but as was the case with its immediate ancestor, P. s. occidentalis, it vanished from northern and central Oregon, and survives in this state only toward the southwest corner. Phlox colubrina Wherry and Constance. While similar to P. speciosa nitida in the limitation of pubescence to inner sepal-surfaces, this Phlox has leaves averaging only 1.5 mm. wide (as against 4 mm. in most subspecies of P. speciosa) ; moreover its corolla-lobes are unique in the group in being elongate and acutish to mucronate, those of P. speciosa being normally short, obtuse, and notched. It has accordingly been described recently as a new species.* It is endemic in Idaho and Oregon along the Snake River canyon, in the latter state also extending well up into the northeastern mountains. Erect-stemmed, Long-styled Group Phlox adsurgcns Torrey ex Gray. The type locality of this species was Canyon Pass, south of Canyonville, Douglas County, Oregon. A visit there in 1931 showed it to be abundant as a ground-cover in thin woodland, the diagnostic character given for it by Brand, "stolons lacking," being erroneous. It is closely related to the eastern P. stolonifera Sims, which also has well-developed creeping stems with broad leaves, the two having no doubt arisen from a connnon ancestor which grew in the far north in pre-Glacial times. Northern occurrences were destroyed, however, by the ice, and the limiting station at present known is 8 miles south of McKenzie Bridge, Lane County, Oregon. Phlox viscida E. Nelson. Diagnostic features of this Phlox comprise the presence of glandular pubescence nearly throughout the herbage, and flat intercostal membranes of the calyx. Brand associated it with P. stanshuryi, but it differs markedly from that southern species. It is endemic in the mountains of southeastern Washington and adjacent Oregon. 8 Status novus: P. speciosa var. yiitida Suksdorf, Deutsch. botan. Monatsb 18- 32 1900; P. speciosa . . . sub var. nitida Brand, in Engler's Pflanzenreich IV. 250: 74. 1907^ * Amer. Midi. Nat. 19: 433. 1938. r%,^y H- v^r 1938] NATURAL SCIENCES OF PHILADELPHIA 135 Phlox Icngifolia Nuttall. This widespread northern Phlox is characterized by the presence of conspicuous bulges in the intercostal membranes of the calyx, often termed replications, but more aptly described as carinae. Several of its variants have been classed as independent species, and the fact that they sometimes occur in pure stands lends some support to this procedure. In numerous colonies, however, two or more occur so intermingled that they can only be regarded as extremes in a variable series, so subspecific status is here assigned to five of them. A variant ranging from moderately dwarf to tall and slender, with the upper herbage densely glandular-pubescent and the largest leaves around 50 to 100 mm. long is regarded as the ancestral one. Recombining the eariiest name assigned to it leads to its being termed P. longifolia longipes (Jones) Wlierry.^ From this arose a usually dwarfer and consistently short-leaved extreme— maximum fertile-stem leaf-length 25 to 45 mm.,— which may be known as P. longifolia compacta (Brand) Wherry; « this has been supposed to have the calyx membranes only obscurely carinate, but the type specimen does not bear this out. At the type locality this grows intimately admixed with two or three other subspecies, all blooming simultaneously. Another direction in which variation occurs is in the nature of the pubescence; this may remain abundant, but become wholly eglandular, without change in the plant's stature or leaf-length. Such was the variant on w^hich the original species description was based; it was also represented in Hooker's P. speciosa var. linearifolia, and Brand named it P. longifolia linearifolia, which is here adopted. As in the glandular material, another variant has on the average lower stature and shorter leaves; this was first collected by Douglas, and may be known as P. longifolia humilis (Douglas ex Hooker) Wherry.' Finally, the pubescence may tend to thin out, ultimately yielding plants which are wholly glabrous except within the sepals. St. John* has recently regarded this as the original P. longifolia, overlooking Nuttall's use of the term puberuli in characterizing that species. The glabrous subspecies has never been assigned a name, so may be known as: 5 status ct comb, nova: P. linearijoUn var. longipes Jones. Contr. W. Botany 12: 53 19C8; P. lonyijolia var. filifolia A. Nelson, Bot. Gaz. 54: 143. 1912. 6 Comb, nova: P. vimia E. Nelson, Rev. W. N. Am. Phlox: 24. 1899; ^J^^^'M^ var. piiherula ibid. 26; P. stamburyi comparta Brand, in FfSlor s Pflanzenreich IV^^O: 67. 1907; P. puberuln A. Nelson, in Coulter's Man. Bot. C. Rocky Mts.: 397. 1909. 7 status novus: P. humilis Douglas ex Hooker, Flora Bor.-Amer. 2: 72 1838; P. speciosa 3 Hooker, loc. cit.; P. loimjolia . . var. /lurfjj Brand m Englers P^an- zenreich IV. 250: 66. 1907; P. cernua E. Nelson, Rev. W. N. Am. Phlox: 22. 1899. sTorreya 36: 94. 1936. 136 PROCEEDINX.S OF THE ACADEMY OF [Vol. XC Phlox Icngifoiia calva Wherry, subsp. nova. Differing from all other variants of the species in liaving the herbage wholly glabrous, except inside the sepals. Plant 10 to 50 cm. tall; largest leaves 45 to 90 nmi. long and 2 to 4 mm. wide. Text-fig. 1. Fig. 1. Phlox lonpifolia calva, subsp. nova. Hal)itat viow of the nlmt selected as the type. Planta tota glabra, sepalis interne exccptis, 10 ad 50 cm. alta- folia maxnna 45 ad 90 mm. longa et 2 ad 4 mm. lata. Type collected by Edgar T. Wherry June 21, 1931, 13 miles southwest of Darlington, Custer County, Idaho, in herbarium Academv Natural Sciences Philadelphia. Range, central Washington to western' Montana, south to Utah anrl Colorado. May occur mingled with one or more of the other representatives of the species, but locally forms jiure stands. All five subsi)ecies here recognized occur scattered over northeastern Oregon, as far west as Mono, Shaniko, and Tygh Valley. Manv colonies mclude two or more of them, with intermediates between extremes. Cespitose Group Phlox dcuglasii Hooker. Phototyi)e and clastotype material of this northern Phlox in American herbaria shows it to be an o])en-cespitose plant averaging 15 cm. high, with the herbage abundantly beset with long septate gland-tipped hairs.' Un- 1938] NATURAL SCIENCES OF PHILADELPHIA 137 ^ yr < f > (% y-"^ 4>^ * ^ s ^ familiar with its features, E. Nelson applied to an unusually larsc but otherwise tv,,ical specimen of it the name P. piperi. It occurs cluef^y .n the Columbia Plateau region of eastern Washington and Oregon with an alpine extreme in the Cascades. Reports from other regions are based on what are here regarded as wholly distinct species. . The subspecies on which the name douglasii was based has been named bv Brand ssp. eudouglasii; its type locality was the Blue Mountams In bleak or drv places this grades into a plant differing chiefly m bemg lower in stature-average height 5 cm.-which has stiffish leaves and was nan.ed bv Bentham P. rkii,la. Gray referred this to a variety of the R..(kv Mountain P. cacspitosa, but the glandularity of its eaves leads to is reclassification here as P. douglasii rigida (Bentham) \\ herry." Thi» occurs in several iilaces in the Columbia Plateau. Above tree-line in the Cascades further evolution occurs; the eaves become more appressed and somewhat shorter (average maximum length 7 5 mm.) and the styles have an average length of but 2.5 mm. Tins is the plant termed by E. Nelson P. condcnsata var. hcmkr.om. Its resemb ance to P condcmata mav well be due merely to their occupying similar high- alpine situations, and in pubescence it is more like the P. douglasr^ series Did it not intergrade with ssp. rigida it might be '''^^■gn^^'4'7"^V Vhen- - it is more safelv classed as P. donglasii henderEom (E. Nelson) \Men>. From the Pldoxes just discussed P. cacspitosa Nuttall differs in having the glan.lularitv limited to the inflorescence. Its various subspecies occur largely in the Rocky Mountains, but it may possibly enter eastern Oregon locally. Phlox covillci E. Nelson. . . , • Thi- species differs from the others in the Great Basin region in haying tinv thickish leaves of elliptic-lanceolate outline, only 3 to 5 times as long as -wide. Its herbage is pubescent, but only a port on of the hairs are gland-tipped. It has been known heretofore from Califorma am Ncva.la, but Professor Peck has collected what appears to be a variant of it above Ice Lake in the Wallowa Mountains of Oregon. Phlox lanata Piper. The tvpe localitv of this northern Phlox was Steins Mountain, Harney Countv Oregon; it is now known to extend also into Montana. It is pXina c in habit, with long prostrate leafy shoots. The herbage, except toward the tips of leaves an.l sepals, is covered by copious tomentose hairs, and even the corolla-limb may bear minute pubescence. -^^^;;;;;r^;;^.: p. r,,„la lV.n,h„m. in Da Fro,l.o,„us 9 : 3C6. 1845; /'. ™c.,„«,«« var m/,Wn C!nw, Pvoc. Amor. Acad. Arts feci. 8: 254. 1870. wVAm "^0S,a.us e. 00,,..,. nova: /'. .„.U..a,„ -- '-»*-; -.^t™oKrPfl''unz™,o"^ Phlox: 14. 1899; P. cmspdosa . . . var. hcudcnunii liraml. in r.n!,.ii> IV. 250: 84. 1907. ]36 THOCKKDIX'.S OF TIIK ACADKMV OF [Vol. XC Phlox Icngifoiia calva Wlurr}-, .sul)si). nova. J)iricrii),u from all other \-ai'iaiits of the sju'cics in having- the licrbagc wholly .ulahrous, except inside the sepals. Plant 10 to 50 em. tall; lariicst leaves 4o to 90 nun. lon the lypc. Planta tola 1I II.ADFI.I'II lA 137 -V -v ^ fnmiliar with its foaturos, E. Xol-n mp|'1.-1 to m, unusually 1 .. b otherwise tvpieal spc-inK.,. of it the name /'. /»;»n. It oe,.,,- .hiel > ,n the Culu.nhia PhUeau n-ion of eastern \Vasl,in,tnn an,l <)re.cn, wUh an alpine extreme in tl,e Casea-U's. Rep-rt^ i>""' '"I"''' '■''"■""^ ^"■'' ''^'^^"' "" what ar^. Iiere re-arde.l as wh..lly distinct s|.eeies. The Mihspeoies .m wliieh tlie na.ne p. <,ulo.,losu: >ts type l,..'al,ty was tl,e Hhu. .Monntan.s In bieak nr .Irv plaees tlds grades into a plant dilVerin, elnelly n. ben,, l.-ue in statnre-avera^e he„ht o .an.-whieh ha- .tiir.h leav,. an, wa-^ na,,a.d l,v Henthan, P. n<,»/<,. Cray referred this t„ a var.ety oi the 1 .m1^ Mountain I'. rac.,>itosa, l.nt the ,landulari.y oi n> eaves leads I., > reelassifieation here as P. ,l,vuilu."^'-"";;"! 'l'";; '' 7 5 nnn. I and the stvles have an averasre lenj^th ol but 2... nun. 1 ht, ., the ,,,„„ ,e,.n».d bv K. N-eU.n /'. ro,„l,„.,U, var. /.«-^'i hender.om t K. Nel^nn Wheuy. Fron, the Phloxes just diseusse.l P. n,.si.l,.s„ Xnttall differs m having ,l,e .landularitv limited to the inflorescence. Its variou. subspecies occur largely u. the Uo.-ky Mountains, but it may possibly enfr eastern Oregon locally. Phlox covillei K. Nelson. •• i • This species .liffers fn.m the others in the Great Basin region m liaymg tinv tliickisli leaves of elli,.tic-lanceolate outline, only 3 to 5 titne. a. long ,/wide Its herbage is pub..s.rnt. but only a portion ot the hairs arc gland-tinned. It has been known heretolore from California am Nevada, brProli.or Peck has eollecte.l what appears to be a variant of it above Ice Lake in the Wallowa Mountains of Oregon. Phlox lanata I'ipor. The tvne lo<-nlitv of this nortlu^rn Phlox was Stems Mountain, Harnev C-ountv Ore-cm; it is now known to extend also into Montana. It is pX lU babit, with long prostrate leafy shoots. The herbage, except iowanl the tips of leaves and sepals, is covercl by copiou^ tomentose bans, and even the ccn'olla-lin.b may bear minute inibes.'encc. - .Snuus n„v„s: ,-. ,•„;/,/„ ■^'■"'^■'"•, "' "^.'^-"-';^i;,"- ^"^"'^^^ ''' """"^^ var. ,i,il,l„ (iray. l'n>c. Mnov. Acm.1. Arts Na. >.. 2.-,4 l.s.O. ,0S,„us ,.1 c,„nl,. nova.; /' r.,..lr„.„„ var ':^":''^Z'-'^'^".^^^'^''^^ IV. 250: 84. 1907. INTENTIONAL SECOND EXPOSURE 138 PROCEEDINGS OF THE ACADEMY OF [Vol. XC Phlox diffusa Bentham. In describing this northern Phlox from near the southern end of its range in California in 1849, Bentham suggested it to be related to P. douglasii, but the characters he gave to distinguish them were not well chosen, no reference to their difference in pubescence being made. Gray proceeded to reduce diffusa to varietal status, and has been followed by many subsequent workers, leading to great confusion. Phototypes and clastotypes of P. diffusa show it to be a spreading plant, pubescent with long wholly eglandular hairs; its leaves are thinnish, linear but not sharp-pointed, and Fij?. 2. Phlox diffusa lonjris(ylis. siibsp. nova. Habitat view of the colony from which the type specimen was selected. reach a length of 15 mm. In the upstanding P. douglasii, on the other hand, the hairs are gland-ti])ped, and the leaves are thickish, subulate with a sharp point, and rarely as much as 10 mm. long. If any division of the cespitose Phloxes (which Gray considered " almost inextricable ") is to be made at all, species differentiation has to be based on characters such as these. P. douglasii and P. diffusa are accordingly here maintained as independent. Phlox diffusa is common from the hills of western British Columbia southward along the Cascades to the Sierras of California, where it is limited 1938] NATURAL SCIENCES OF PHILADELPHIA 139 .*- ^ „^, -^7^ ■y wyy to fairly high altitudes. Toward the northern end of this range its styles are 6 to 12 mm., toward the south 3 to 5 (rarely 7) mm. long, the boundary between the two variants lying in northern Oregon. The southern one was the original P. diffusa, and may accordingly be named P. diffusa typica Wherry.'' Nearly all specimens seen from western Oregon belong here. The northern extreme deserves a subspecies name, as follows: P. diffusa longistylis Wherry, subsp. nova. Differing from ssp. typica in somewhat lower stature and smaller leaves and flowers, and especially in having longer styles, 6 to 12 mm. in length. Text-fig. 2. A subspecies typica differt planta paulo minor et stylis 6 ad 12 mm. longis. Type collected by the writer July 30, 1931, at 7250 feet altitude on the south slope of Mt.' Adams, Yakima County, Washington, in herbarium Academy Natural Sciences Philadelphia. Range, British Columbia south to House :Mountain, Marion County, Oregon.'- Phlox hoodii Richardson. While this species w^as described from the plains of Saskatchewan, and is especially frequent in the high plains and eastern foothills of the Rockies, it also crosses these mountains in Idaho and spreads over the Great Basin region. The phase most developed in the latter area differs from the original in having a slightly longer corolla-tube, and was named P. canescens by Torrey and Gray. The differences between them are so slight and the intergradation so complete, however, that only subspecific independence seems justified, leading to the new combination: P. hoodii canescens (T. & G.) Wherry.'^ This subspecies occurs sporadically in the Columbia Plateau region of Oregon, extending up to the eastern foothills of the Cascades. It thus approaches the range of P. diffusa, from which it differs in its more compact habit, and smaller leaves and flowers. Phlox austromontana Coville. While originally described from southern Utah, this Phlox with its dis- tinctive acerose leaves and carinate calyx-membranes is wide-ranging over the Great Basin and adjoining physiographic provinces. In Oregon it has been collected as far north as Union County. A long-styled phase of it 11 Nomon novum: P. diffusa Bentham. Plant. Hartweg.: 325. 1849; P. douglasii var. di^ffusa Gray, Proc. Amer. Acad. Arts Sci. 8: 254. 1870. 12 When I first encountered this Phlox and ob.served its differences from the original P. diffusa, I supposed it might prove to be P. caespitosa Nuttall and so reported to St John and Warren, who have included it imder that name m The Plants of Mt. Rainier National Park, Washington". Amer. Midi. Nat. 18: 978. 1937. Actually the Mt. Rainier Phhx is this newly described P. diffusa longistylis. 13 Status novus: P. canescens Torrey & Gray, Rept. Bot. RR., Mo. Pac. 41% 2: 122. 1855. 138 PR(Ki:i:i)l\(iS OF THE ACADEMY OF [Vol. XC NATURAL SCIENCES OF PIIILADELPIIIA 139 Phlox diffusa Bentham. In (Icscribin^ this nortluTn Phlox from near the soutlicrn end of its range in California in 1849, Bentham suggested it to be rehited to P. domjkmi, but the eharaeters he gave to distinguisli them were not well ehosen, no reference to their difference in jjubescenee being made. CJray i)r()eeeded to reduce difjusa to varietal status, and has been followed by many subsequent workers, leading to great confusion. Phot()ty])es and clastotypcs of P. diffusa show it to be a spreading i)lant, pubescent with long wholly cglandular hairs; its leaves are thinnish^ linear but not shari)-pointed, and Ki^. 2. IMiloN (lilTusa loimistylis. sulxj.. nova. Hahilaf vi<'\v of Iho rolonv from which tlic type s{Kciiii(u was mIccIciI. reach a length of 15 nuiL In the upstanding P. doHijUisii, ou the other hand, the hairs are gland-tipped, and the leaves are thickish, subulate with a shari) point, and rarely as much as 10 nun. long. If any division of the cesj)itose Phloxes (which (iray considered "almost inextricable"! is to be made at all, species differentiation has to be based on characters such as these. P. douglasii and P. diffusa are accordingly here maintained as independent. Phlox diffusa is common from the hills of western Britisli Columbia southward along the Cascades to the Sierras of California, where it is limited i Pr •»^> \ f -Vr ^4 A' »• I 1938] to fairly high altitudes. Toward the northern end of this range its styles are 6 to 12 mm., toward the south 3 to 5 (rarely 7) mm. Umg, the ))i)undary between the two variants lying in northern Oregon. The southern one was the original P. diffusa, and may accordingly be named P. diffusa typica AVherry.'' Neai'ly all si)ecimens seen from western Oregon belong here. The northern extreme deserves a subspecies name, as follows: P. diffusa longistylis Wherry, subsp. nova. Differing from ssp. tiipica in somewhat lower stature and smaHer leaves and flowers, and especially in having longer styles, 6 to 12 nun. in length. Text-fig. 2. A su])species typica d it under that name in "The Plants ot Mt. Rainier National Park. Washingtcm ". A-ner. Midi. Xat. IS: 978. 1937. Actually the Ml. Rainier I'hlnx is tliis newly deserilx'd /'. tUlJusa Ion(/ist yhs. !•' Status novii.s: P. cancscins Torrey & Gray, Kei)t. Hot. RR.. Mo. Pae. 41°, 2: 122. 1855. i INTENTIONAL SECOND EXPOSURE 140 PROCEEDINGS OF THE ACADEMY OF [Vol. XC was described by Brand as P. rhnsa, but this is scarcely separable; his P. pinifolia, so far as can be ascertained, is another minor variant of the same species. Phlox pcckii Wherry, sp. nov. Plant a ])rostrate under-shriib; main root branching; above into several creeping woody stems, sending u]) erect branches 1 to 3 cm. tall, yielding pulvinate-cespitose clumps; internodes short, thea* surface laminated, more or less ])ubescent ; leaves spreading, thinnish, linear, 4 to 8 mm. long and 0.5 to 1.5 nun. wide datio of length to width 5 to 10), basally densely ciliate with septate hairs, superficially granulate to glabrous; the tip bearing a short cusp'; inflorescence 1 -flowered, the jiedicel 1 nun. long, quad- rate; sei)als 8 nun. long, united about i their length, the costa prominent, awn-tipped; intercostal mem- branes distinctly carinate; i)ubescent with long kinky septate eglandular hairs, on the outside near the base and on the inside of the lobes toward the tip; corolla- tube about 12 mm. long, somewhat dilated upward; lobes cuneate, 5 nun. long and 2.5 nun. wide, termin- ally ero.^e-mucronulate, their upper surface beset to a distance of 3 mm. from the orifice with fine kinky septate hairs up to 0.5 mm. long; u])per i^art of tube yellowish, limb cream-color; stamens extending rather high up the tube, one or two anthers slightly cxserted; styles 4.5 mm. long, united to i their length, the 3 stigmas tlnis 1 mm. long; ovules solitary. Text-fig. 3. Fruticulus i)rostratus; folia linearia, ad 8 mm. hmga et 1.5 mm. lata; calycis meml)ranae carinatae, tubo baso eglanduloso-pubescente; corollae lobi superne i)ubescentes, i)ilis ad 0.5 mm. longis; stvli 4.5 mm. longi ad * longitudinis conjuncti. ' > o Type collected by Professor Morton E. Peck of Willamette University, Salem, Oregon, in whose Inmor the plant is named: No. 19302, July 14, 1936,' dry slope, north rim of Crater Lake, Oregon. Type in Herbarium Academy Natural Sciences Philadelphia. This Phlox is obviously a derivative of P. (lijjusa, in which the flat to wrinkly calyx-meml)rane of the parent species has become rather definitely carinate (as it has in P. austromontarm also). In most Phloxes the corolla limb is glabrous, but in a few of the cespitose ones it is minutely granulate, and in P. lanata occasionally fine-puberulent. The i)resent species, how- ever, is the only one known in which the corolla-limb is definitely ])ubeseent. Of the 12 species discussed in the above pages, 6 are northern in geo- grai)hic relationship, being best develojied in the northern Rocky Mountains and enter Oregon from the northeast; 3 are southern, ranging over the Great Basin, and occupying southeastern Oregon; while the remaining 3 are endemic in Oregon, or in this state and adjacent portions of surrounding ones. ^ Viii. 3. Phlox pockii, species nova. Fh>wer X 2, showing unique pubescence. \ ^' Reprinted from American Fern Journal, Vol. 27, No. 1, January-March, 1937, pages 1-5. Southern Occurrences of Dryopteris Clintoniana Edoar T. Wherry^ On June 1, 1927, Mr. Harry W. Trudell and the writer, out on a camping trip, stopped to shave at a limpid, purling brook in the woods 1^ miles east of Forney, Cherokee County, Alabama. Most unexpectedly we found that just back of the spot we had selected there was growing a Dryopteris of the general aspect of D. cUn- toniana, though differing from the more northern mate- rial of this fern in certain respects. The photograph of it taken at the time is here reproduced in Plate one ; plants growing near-by in more open situations showed more erect fronds. Subsequent herbarium study showed that a similar plant had already been collected in the state : it was dis- covered about 1895 by the late Professor S. M. Tracy, in a needle-palm swamp said to be 4 miles south of Auburn in Lee County, and was referred by Mohr- to D. floridana; from this it differs, however, in numerous respects, notably in having the frond widest well below the middle, without any conspicuous, sudden change in degree of cutting where sterile pinnae give way to fertile ones, and in having the lower pinnae acuminate with a sharp tip 1 Contribution from the Botanical Laboratory and Morris Arbo- retum of the University of Pennsylvania. 2 Plant Life of Alabama: 317. 1901. and the marginal teetli sharp-pointed. I have been un- able to relocate this colony, but collections made there by Pollard and Maxon in 1900 are widely distributed in herbaria. More recently, ferns of more or less the same features have been found in Arkansas, Louisiana, and North Carolina. At intervals during the subsequent ten years I have repeatedly tried to find some feature which would sepa- rate this fern sharply from Dryopteris clintoniana, but have never succeeded. Only varietal status seems pos- sible for it, as follows : Dryopteris clintoniana (Eaton) Dowell, var. australis, var. nov. A varietate boreali differt soris plusminusve medialibus. Differing from the northern plant in having the sori more or less medial, instead of rather near the midvein. Sometimes, though by no means consistently, tending to develop fertile pinnae only above the middle of the blade, and sinuses in the upper one.s nearly as wide as the adjacent segments; the northern variety often showing fertile pinnae near the blade-base, and sinuses at most ^ as wide as segments. Type in herbarium Academy Natural Sciences Phila- delphia, collected by Edgar T. Wherry July 15, 1932, in humus-rich subacid woodland soil 1^ miles east of For- ney, Cherokee Co., Alabama. The northern plant, which now requires a varietal name, may become : Dryopteris clintoniana (Eaton) Dowell, var. genuina, nomen novum. Records of the southern varietv known to date are: Alabama. — The plants of both colonies above referred to show marked variation in rhizome scales, from pale brown and dull to nearly as dark brown and lustrous as in D, goldiana; the specimen selected as type exhibits the latter extreme, but a cotype sheet, bringing out the rootstock features (with attached sterile fronds), has i*i' I IRREGULAR PAGINATION -(Ml- I— CM O > < y. u o •-s y. oe bi y. OS *" u o to I— I H «3 t3 •< •< y o OS u: eu o >< fid tv pale scales. Pinnae may lie close together, as in the frond shown at the right of Plate 1, or again may be spaced as widely as is normal in var. genuina. The sinuses between fertile segments are usually very narrow, as in the frond pictured, and also the type specimen, but on occasional vigorous plants may be wider. Arkansas.— Specimens from rocky Avoods just north of Shirley, Van Buren Co., have been distributed by the discoverer, E. J. Palmer, and also by Demaree and Moore. Some of these have much the aspect of the Alabama mate- rial, and the sori lie well out, being at least medial or even slightly supra-medial. Here too the scales vary from the pale brown of D. clintoniana to the dark and shining ones of />. goldiana. The latter is approached also in one other respect— the basal pinnae may have their lowermost segments shortened, so that their outline is somewhat lanceolate. Sinuses between lobes vary from narrow to wide, the fronds in extreme cases becom- ing bipinnate. Louisiana.— Early in 1936 Dr. Clair A. Brown dis- covered a colony 6 miles southeast of Baton Rouge, .which I visited in September. Here the plants approach var. geiiuina in having sori beginning to appear not far above the base of the blade, and occupying a slightly infra- medial position. The sinuses between fertile lobes show, however, a marked tendency to widen, so the occurrence is classed as var. australis. North Carolina.— Material collected by Rev. Fred W. Gray 9 miles north of Charlotte, Mecklenburg Co., has been figured by Blomquist.^ This shows an approach to D. floridana in elliptic frond-outline, the lower 4 pairs of pinnae being progressively shortened, but i^ chii- toniana in every other respect. In the narrow sinuses 3 Ferns of N. C: 65. 1934. i # <'■ -.•V] y. y. y y. c < r. < y. *-^ y. y. Si a! c »^-- i-Wv* r \v pale scales. Pinnae may lie close too-otlior, as in the frond shown at the ri«,'ht ot* Plate 1, or a<»-ain may he spaced as widely as is normal in var. gcnuina. The sinuses between fertile se^-ments are usually very narrow, as in the frond pictured, and also the type specimen, but on occasional vi^^orous plants may be wider. Arkansas.— Specimens from rocky woods just north of Shirley, Van Buren Co., have been distributed by the discoverer, E. J. Palmer, and also by Dcmaree and :\Ioore. Some of these have much the aspect of the Alabama mate- rial, and the sori lie well out, bein<,- at least medial or even sli«,ditly supra-medial. Here too the scales vary from the pale brown of 1). clintoniana to the dark and shinino- ones of I), (joldiana. The latter is approached also in one other respect— the basal pinnae may liave their lowermost segments shortened, so that their outline is somewhat lanceolate. Sinuses between lobes vary from narrow to wide, the fronds in extreme cases becom- ing bipinnate. Louisiana.— P:arly in 193G Dr. Clair A. Brown dis- covered a colony 6 miles southeast of Baton Rouge, -which I visited in September. Here the plants approach var. gcnmna in having s.u'i beginning to appear not far above the base of the blade, and occupying a slightly infra- medial posit ion. The sinuses between fertile lobes show, however, a marked tendency to widen, so the occurrence is classed as var. austral is. North Carolina.— :Material collected by Rev. Fred AV. Gray 9 miles north of Charlotte, Mecklenburg Co., has been figured by Blomquist."^ This shows a.i approach to D. floridana in elliptic frond-outline, the lower 4 pairs of pinnae being ])r()gressively shortened, but is^ chn- toiiiana in every other respect. In the narrow sinuses sFernsof N. C: 05. 1934. INTENTIONAL SECOND EXPOSURE between fertile lobes it is like var. genuina, but the medial sori shown in the figure cited place it in var. australis. Variation occurs in the latter feature, however, for in fronds recently sent by Mr. Gray to the Philadelphia Academy the sori are distinctly infra-medial.* Two possible explanations of the remarkable variability • shown by the plants under discussion suggest themselves. One is that they represent the result of hybridization between several species. The occasional dark scales and lanceolate basal pinnae would indicate D. goldiana to have been one of these. Tendencies toward basally nar- rowed blade, development of fertile pinnae only ter- minally, and widening of sinuses between fertile seg- ments all point to D. floridana having been another. Movement of the sori out from the midvein suggests the entry into the complex of D. marginalis. Finally, the outlines and cutting of the pinnae correspond most often to D. clintoniana genuina. The alternative explanation, which is favored here, is that we are dealing with a stock which has already given rise to. several descendants, and is still undergoing evolu- tion. D. clintoniana genuina and 2>. goldiana represent, then, offshoots which migrated northeastward, while D. floridana is one which moved coastward. In these, features which appear in the ancestral stock as mere uncorrelated tendencies became so definitely combined and stabilized that we can recognize one distinct variety and two independent species. Philadelphia, Pa. * Although full discussion of more northern variants is beyond the scope of the present article, record should be made of the fact that material of D. clintoniana from as far north as Dauphin and Berks counties, Pennsylvania, shows an approach to var. australis m sorus-position. On the other hand, var. genuina is known down to Dade Co., Georgia (Pyron and McVaugh, 1936). Reprinted without change of pagination from Jour. So. App. Bot. Club Vol. II. 1937. THE JOURNAL of the Southern Appalachian Botanical Club Vol. 2. January, 1937 No. 1. Two Plant-geographic Notes' Edgar T. Wherry Thuja occklentalis in Pennsylvania and Maryland. — In a series of maps of ranges of trees published by Transeaur' dots are placed m counties of Ohio and adjacent states where the species are supposed to grow That these maps are incomplete as far as eastern Penn- sylvania is concerned has already been noted.^^ The map for Thuja is also unsatisfactory for the Appalachian re-ion. On map No. 1<, covering this plant, but one Pennsylvania county, Crawford, has re- ceived a dot. In the herbarium of the Carnegie Museum at Pitts- burgh however, specimens are preserved from two other counties in this state, namelv Armstrong and Westmoreland. No dots at all are allotted to Marvland, but in 1934 I collected Thuja on the limestone cliffs along the old canal 2% miles southwest of Sharpsburg, Wash- ington County;* a specimen has been placed in the herbarium of the Morris Arboretum. Thus, instead of there being a marked discon- tinuity between the northern range of this tree and its occurrences in the southern Appalachians, the two regions are connected by a chain of colonies. ^, ^_ Pseudotaenulia montana in Pennsylvania. - The Mountain- pimpernel was described by Mackenzie^' in 1903 from Kates Mountain, West Vir-inia, and about the same time was collected by Steele near Luray Vh-ginia," but for many years was known only from these two stalions Recent exploration in northwestern Virginia by Professor R. S. Freer and others has shown it to be rather widespread there. It T^ribution from the Botanical Laboratory and Morris Arboretum of the University of Pennsylvania. 2 Ecology 16: 429-35, 1935. sBartonia 17: 51, 1936. 4Amer. Fern Journ. 24: 99, 1934. r. Torreya 3 : 159, 1903. 0 Gray's Manual, ed. 7: 620, 1908. C V- grows on various formations, including sandstone and limestone, al- though it becomes most abundant on the barrens underlain by hard Middle Devonian shale rock. On following these shales northeast- ward, it was found on hills along the North Branch of the Potomac River three miles south of Ridgley, Mineral County, northeasternmost West Virginia. Search in Maryland then disclosed a small colony on a shale hill one mile north of Oldtown, Allegany County ; this has been noted in Claytonia.^ It was subsequently found at several points along the crest of Polish Mountain, in the same county. During August, 1936, an effort was made to find it still farther north. Some 7 miles southwest of Chancysville, in Bedford County, Pennsylvania, there are fairly well developed shale barrens, and the plant was found here at two places. Both lie within sight of the tele- phone wire marking the Mason and Dixon line, but unquestionably within the state. One occurrence consisted of a single plant on road bank on west slope of small hill east of Iron Ore Ridge; the other, of half a dozen plants, beside a little used road which runs along the west flank of Ragged Mountain, and south of the point marked Elbin^- ville on the topographic map ascends toward the crest of Polish Moun- tain. Search at more northern points has thus far disclose 1 only Taenidui inteyerrima, a far more widespread plant, identical in herb age, but with the fi-uit having the two carpels subcylindric instead of lenticular as in Pseudotacnidia. University of Pennsylvania, Philadelphia. •r <. x^ IRREGULAR PAGINATION Hepiinted from Phytopathology, March, 1938, Vol. 28, So. 'S, pp. 210-212. 1938] York : Inoculation of Thees with Rusts 211 INOCULATIONS WITH FOREST TREE RUSTS Harlan H. York In June, 1927, near Woo(lg:ate, N. Y., several species of pine and of the Scropliiilariaceae were inoculated with the aeciospores of the Pcridermivm kuown as the AVoodgate rust. The inoculations were kept moist by the use of a celluloid "iceless refrigerator," which is a modified form of the inoculation chamber described by Hubert.' The 1927 termiiud leaders, side shoots, and side branches of 5- to 20- year-old pines were moistened thoroughly and were smeared with aecio- sj)ores by means of a small artist's brush. A celluloid cone, which Avas 22 inches in length and resi)ectively 3 inches and l-l^^ inches in diameter at the ends, was slii)ped over the inoculated parts. It was anchored to the stem by means of a copper wire. A strii) of absorbent cotton, about 1 inch in width and 8-10 inches in length, which was saturated with water, was then pushed by means of a small rod through the smaller end, Avell down the inside of the cone. The entire outer surface of the cone was wrapped to a thickness of 1-1 M» inches with wet absorbent cotton. The larger end of the cone was left almost entirely open, the smaller almost entirely closed, leaving an opening sufficiently large for circulation of air through the cone. If the cone was on the terminal shoot of the main axis of the tree, it was sup- l)orted by one or two sticks, wired to tlie trunk of the tree; if on a side branch, it was supported from the ground by means of a forked stick (Fig. 1). The cones were left on the shoots for 24 to 36 hours. At the end of 36 hours the cotton on the outside of the cone was quite damp and it was al- ways possible to wring water from the stri]) of cotton inside the cone. The entire onter surfaces of the shoots were also quite damj). About 200 shoots of Pinus rcshwsa, P. sylvcstris, P. radiata, and P. pondcrosa were inoculated by this method. In all cases, infection spots ap- ])eared on the stems within four to six weeks, and in many cases, on Pinus stjlvcsfris, the spots were so abundant that the entire inoculated stem sur- face became reddish brown in color by the latter part of August. This was ecpially true of trees which had no galls on them at the time the inoculations were made and of those which had galls. At the time the shoots were inoculated, comparative germination tests of aeciosi)ores from collections of various ages were made in the inoculation chambers and in water cultures in the laboratory. The water cultures were made by dusting the spores on fresh well-water in wine glasses. In the inoculation chambers i)ieces of thoroughly clean, white China silk about 2 cm. in length and 1 cm. in width were laid on the pine stems and the aecio- 1 Hi BERT, Ernkst E. Colhiloid cylindpvs for inoculation chambers. Plivtoi)ath. 6: 447-450. 1916. 210 ^^. spores were dusted on them. The silks were examined under the microscope 24 to 36 hours later. The germ tubes were very much branched and aiuistomosed so much with one another that they formed very complicated mycelia. In the water cultures, the germ tubes of the same age were com- paratively unbranched and unanastcmiosed. In all of these experiments the percentage of germination in the inoculation chambers was far greater than that in the water cultures. For example, the germination of spores whieh had been kept in Syracuse watch glasses in the laboratory away from direct sunlight for about 10 days was approximately 25 per cent as compared with 75 per cent in the inoculation chambers. Fig. 1. Celluloid "icolcss rcf ligeiator " used in iuocul.'ition oxpeiimcnt.s with the aecio Hpoics of the Wood^'atc runt on Pinus siflvcstris and other species of hard pines. It is possible that Woodgate rnst may pi-ove to be one of the western gall rusts, Avhose alternate hosts are species of the Scrophulariaceae. Although several species of Scrophulariaceae occur in the region where the Woodgate rust has been found, none of them has ever been found infected with any rust. During the latter ])art of Jiuie, 1927, (luhnie (jlahm and /S'cro- phular'm leporclla were inocuhited with aecios])()res of the Woodgate I'ust. These two species Avei-e selected because of their size and because they occur nearby. The plants when inoculated wei'e about 26 inches in height. Both sides of the leaves were thoi-oughly moistened and smeared with aeciospores as mentioned above for pines. A stake about 4 inch in diameter, wrapped IRREGULAR PAGINATION Iic]iriiitf(l Iroiu I'll vroi'.\'lllOL()(i V. M;ircli, 1!>;1S, \'()1. L'S. No. ;>. ]»|». L'lU-lMl*. INOCULATIONS WITH FOKEST TIJEK lirSTS II A It LAX 11 . Y O K K 111 .Iiiiic, 1!)27, iwnv Wood^cilc, N. Y.. several species of i)iii(' jiiul ol' tlie Ser()j)liiihn'iai-e(l with T.") per cent in the inoculation chambers. I''i<;. 1. ('clliildid "i(il(s> icfiiocnitdc ' ' ii>(i| in iiHM-iil;it ioii cxitcrimciUs wiili llic aceio sjMHi's of till- \\'(H»(|y^atc nist (Ml /'iini.s si/l ri si ris :\\]i\ ntlicr s|i('('ics of liaiW pines. It is possible that Woodgate rust may proNc to be one of the western gall rusts, whose alternate hosts are species of the Sei<»phulariace;ie. Although nevei'al species (»f Scrophulariaceac occur in the re<_;ioii where the Woodgate mst has Ix'cii found, none of them has e\er been found infected with anv rust. During the latter part of .liiiie. 1!)27. Clnhnu oles in the secondary meristem have no general form nor are they derived from any characteristic primordia. They may be rod- shaped, thread-like or spherical. They may fuse into a single large central vacuole or fragment into many separate globules which later may become beaded chains or tangled skeins. In many cells, the vacuoles are carried along in the streaming cytoplasm and the form they assume is conditioned by the cytoplasmic activity. The vacuoles in the primary meristem are essentially like those in cambium (Zirkle 1932). All of the cells are vacuolate and the shape and size of the vacuoles seem to be conditioned primarily by the activity of the cytoplasm and the size of the cell. When the cytoplasm is quiescent, they tend to become spherical in the smaller cells ; when it streams actively, they either become rod-like or are drawn out into Holmgren canals, or they may even form a reticu- lar apparatus. Thus the shape of the vacuoles is of little impor- tance in itself, for it merely indicates the activity of the cytoplasm. As a rule, the larger the meristematic cell, the greater the relative amount of vacuolar material, and in the exceptionally large apical cell in the root of Osmunda they may be as large as they are in the cambium of Pinus during the growing season. There is no evidence that vacuoles in either the primary or sec- ondary meristem develop from tonoplasts, hydroleucites, mitochon- dria or any other cytoplasmic inclusion and, while the smaller vacuoles may often assume the forms ascribed to these primordia, no useful purpose is served by using any of these terms. All men- stematic cells which have been inspected adequately contain vacu- oles (Hof, 1898) which are constantly changing their shape, fusing and again fragmenting. The only mode of origin that could be dis- covered for any individual vacuole was the division of a pre-existing vacuole, as reported by Went (1888). Guilliermond (1923) has stated that vacuoles originate de novo in Saccharomyces and Sapro- legnia The fact that this has not been observed in any meri- stematic cells does not mean, of course, that it never occurs there, for it is quite possible that a new vacuole could be mistaken for a fragment of a dividing one in a cell whose contents are in rapid motion. The fact that new vacuoles can be made artificially (Klebs 1890 Pfeffer 1890, Mollendorf 1936, et al.) has no real bearing on this problem as it does not justify the inference that such vacuoles occur in nature. O THE BOTANICAL REVIEW THE PLANT VACUOLE AND THE ANIMAL GOLGI APPARATUS When Bensley (1910) found that the canals described by Holm- gren in cells of liver and kidney epithelium occurred also in root meristems, he suggested that these canals represented the Golgi apparatus in both animals and plants. As it was a very simple matter to demonstrate that these Holmgren canals developed into the large central vacuole of the mature plant cell and, even, that they themselves were smaller vacuoles drawn out into a canalicular form by the streaming cytoplasm, he inferred that the plant vacuole was the homologue of the animal Golgi apparatus. Guilliermond and Mangenot (1922), Dangeard (1923), Parat and Painleve (1924, 1925), Guilliermond (1927), Scott (1929), Bose (1931) and others have accumulated a great deal of evidence to support this view. On the other hand, Bowen (1926, 1927, 1928, 1929), Gatenby (1929) and Beams and King (1934, 1935) hold that vacuoles and the Golgi apparatus are separate structures and that the Golgi material in the plant cell constitutes the "osmophillic platelets," a distinct cell organ. Weier (1932, 1933), after study- ing Bowen's preparations, reported that the Golgi zone is similar in many respects to plastids. In spite of their differences of opinion as to what constitutes the Golgi apparatus in plants, these investigators really agree in all of their essential findings. There is little argument as to questions of fact and little question but that some of their inferences are reared on an insufficient factual basis. As Bowen (1926, 1927) started the controversy, it would be well to summarize his conclusions briefly and to cite the evidence upon which they are based. He stated that he was entering the field of plant cytology without any particular bias as to the possibilities in prospect and that he would attempt to apply to plant cells those methods which had long been familiar to animal cytology. At the outset he discarded Golgi's original technique of reducing silver in a reticular apparatus on the ground that silver nitrate will "impregnate" almost anything on occasion. He relied primarily for his identification of the Golgi material upon its ability to reduce osmium tetroxide when the osmium was protected by such oxidizing agents as chromic acid or potassium bichromate, although he recognized that plant vacuoles which contained tannin would also reduce the osmium. Bowen concluded that there was a special Golgi material, that the reticulum ♦••*'► rh A' ew ^"y>-- §4? 99-ill 1929 The intoke and accumulation of electrolytes by plant cells. Protoplasma 6 : 610-626. t> . i i c . Af.O-Am HoFLER K 1932. Zur Tonoplastenf rage. Protoplasma 15: 462-477. Hor A C. 1898. Histologische Studien an Vegetationspunkten. Bot. „oi-S^t^^1l|- Kllf ^,Bau der vegetablischen Ho.Sr^l:"W3"'*l5l^rJt^ I'SillK^^ u„d der tissues Chez Drosera inlermedia. Bull. Acad. Roy. Belg. t-1. =>«'•"'• tstM%Um. 1928. Modifications cytologiques au cours^u fonc- tionnement des organes secreteurs chez Drosera. I. Bull. Acad. Koy. How^LA^».''R''B.'^19^.'Ti;fpH of gastric vacuoles. Protoplasma 5: '-Sr^eilJiU'g^s'^S^^^^^^ T ^^^xi ,^„ ^(y>'\ The oenetration of dyes as influenced by hydrogen Irwin, Marion. 1923. ine peneirauon ui > The permea- ion concentration 1°"^^^/"^^ fc^^^^^ concentration, bility of living ceUsto dyes as ^f f f ^ ^^^Xb^^^^^ of brilliant cresyl '■«S.a:E 'S0"S:t' •;.ra^^-.£ K-^g'Su?"' iS '* di N«.r *. V...™.. Z«». Z.Ut MJ.. Anat. 15 : 731-761. :„:erttnn of certain salts into the protoplasm ''"and^ruofes If I rIo'f-hS'rforZ..loi:il spon,ia. Protoplasma 18: v,J^r^' 1913 The relative permeability of the surface and interior por- :^^^£^^geB;=r|r^^^^^^ K.Eisrj.'i.T"^¥„s^S:^«^^^^^ K0Bl^SA^:.rR0^?s.W^ K,:^ir%t ='S^r'"zeSfclasruid"Iiem^ran von BryoPsis. Ber. beut. Bot. Ges. 51 : 526-536. i THE PLANT VACUOLE 29 28 THE BOTANICAL REVIEW Lederer, Berta. 1934. Farbungs-. Fixierungs- tind mikrochirurgische Studien an Spirogyra tonoplasten. Protoplasma 22 : 405-430. LisoN L. 1935. Sur la determination du pH mtracellulaire par les colo- rants vitaux indicateurs. L'erreur metachromatique. Protoplasma 24: 453-465 rr T> Lister, M. * 1671. Some observations touching colours. Phil. Trans. Roy. Soc., London 6: 2132-2136. • • *u i: :«« /.-ti Lloyd F E. 1922. The mode of occurrence of tannm m the livmg cell. Jour. Amer. Leather Chem. Ass. 1926 Saturation and conjugatio^^^^ Spiropyra lingata. Trans. Roy. Con. Inst 15: 19^. The contractUe vacuole Biol Rev. 3: 329-358. 1928. Further observations on Uie behavior of gametes during maturation and conjugation of Spirogyra. LoEwT^ANrBoKot^TH. 1887. mer das Vorkommen von aktiven, Albumin S Zellsaft und dessen Ausscheidung in Komchen durch Basen. Lud?o°^,^b! L*^i924. Experiments on the impregnation of ^e Golgi ap^- ratus by means of osmium tetroxide. Jour. Roy Micr. Soc, 44 : 269-280 Mangenot, G. 1929. Sur^ la presence des vacuoles ^peciahsees dany|f cellules de certains v6g6taux. Compt. Rend. Soc. Biol., Pans 97 . 34^ 345 1922. Recherches sur les constituants morphologiques du cyto- plasm des algues. Arch. Morph. Gen. et Exp. 1929. Sur les ph€- SomTnes dits d^aggregation et la disposition des vacuoles dans les celhi- les conductrices. Compt. Rend. Acad, Sci., Pans 188 : 1431-1434 1929. Sur les phenom^nes de fragmentation vacuolant dits d aggregation. Arch. Anat. Micr. 25: 507-518. , ^. t, . a.u m a^. vuv^r Maschkle, O. 1858. Ueber den Bau und die Bestandtheile der Kleber- blaschen. Bot. Zeit. 17 : 409-417, 429^37 . .,,,,,,:„:„„ McCuTCHEON, M., AND LucKfe. B. 1924. The mechanism of vital staming with basic dyes. Jour. Gen. Physiol. 6: 501-50/. . -j^u^^ Mey^, F. J. F. 1827.' Uber die eigenthiimliche Saftbewegung m den Z^tn der Pflanzen. Nova. Acta Misc. Cur. C. L. C. 13 . 841-»/4. lo^/. Ueber die Cirkulation des Lebenssafts in den Pflaiizen. Linnaea 2 : 632- 670. 1835. Nouvelles observations sur la circulation du f^^^^^^^ dans les plantes. Amer. Sci. Nat. Bot. II 4: 257-262. 1838. Neucs System der Pflanzen-Physiologie. Miller, E. C. 1931. Plant Physiology. ^ ., . . , xt^u,^^ MiLOvi^v, P. E. 1930. Einfluss der Zentrifugierung auf das Vakuom. MoLrENDot,' W.^'voN^^^^ Experimcntelle Vakuolenbildung in Fibr^ zyten der Gewebekultur und deren Farbung durch Neutralrot. Zeit. Zcllf. Mikr. Anat. 23: 746-760. ^ . .u • r<;^ «,**»,nH MoLZ, F. J. 1926. A study of suction force by the simplified method. Amer. Jour. Bot. 13 : 433-502. „. , MCTTHES K. 1933. Der Tonoplast von Sphaeroplea. Zeits. Wiss. Biol. Abt! AE Planta 21 : 486-510. , . . • r.. A^^r ^d MoTTE, J. 1928. A la connaissance cytologique des muscinfecs. Amer. :5Ci. Mottier, D. M. ' 1899. The effect of centrifugal force upon cells. Amer. Bot. 13:32S-361. . ,,. ^, Naegeli, Carl. 1849. Gattungen einzelliger Algen Nirenstein, E. 1920. Uber das Wescn der Vitalfarbung. Arch. Gen. Noll, F!^°i899. * Die geformten Proteine im Zellsaft von Derbesia. Ber. Deut. Bot. Ges. 17 : 303. , . . .,., . ,^„. OSTERHOUT, W. J. V. 1919. 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Fatti e considerazioni a proposito di aluine formazioni neile cellule vegetali. Monitore Zool. Ital. 28 : 9-14. PFEFreR W 1889 Loew und Bokorny's Silberreduction m Pflanzenzellen. Flora 72 • ^54. 1890. Zur Kenntnis der Plasmahaut und der Vacu- olen Abh. Math.-Phys. Kl. Ges. Wiss. Leipniz. , ,^ „ , Plowe J. 1931 Membranes in the plant cells. I and II. Protoplasma Prat S 1932. The polarity of the vacuole. Protoplasma 15 : 612-615. PWESTLEY J H. im Cell growth and cell division in the shoot of the ScA^TrG%'!'%6.'^^^^^^^ of dyes in living SchS1L,'m"l''Ym2!- Gmndzuge der wissenschaftlichen Botanik. 1849. SchulTz^'karl" Ho^^^^^^^ '^?2T Die Natur der lebenden Pflanzen. Schwartz FS;NKl8k^ Die morphologische. und chemische Zuzammen- setzung des Protoplasmas. Cohn, Beitr. Biol. Pflanzen. 5 : 1-244. Sco^, F. M 1929. The occurrence of Golgi apparatus m the seedlmgs of Vicia Faba. Amer. Jour. Bot. 16: 59&-605. . • • ^« Sen-Gu^a, Jatts. 1935. Die osmotischen Verhaltmsse bei einigen Pflanzen in Bengal. Ber. Deut. Bot. Cies. 53 : 783-795. , Shapiro N N. 1927. The cycle of hydrogen ion concentration m the tood Vacuoles oi Paramecium Vorticilla and Stylinchia, Trans. Amer. Micr. Soc. 46 : 45-53. , . , ^ , Sharp L W. 1934. Introduction to cytology. . , i * ^ Sla^; Henry. 1834. Exposition des tissues elementaires de^ P^an ^^^.f f quelques examples de circulation vegetaux. Ann. Sci. Nat. Bot. II I . Smith^Emth^Ph^'ip. 1933. The calibration of flower color indicators. SMiT^n'TRnNDSfA.!. 1926. A begonia immune to crown-gall wilh observations on immune and semi-immune plants. Phytopath. 16: SPALLiNZ^Ni, Lazaro. 1768. Sopra gli animali della in^^^ioni Giornale d'ltaHa 3 • 12-13. 1776. Opuscoli di fisica animale e vegetabile. 1787. Oouscules de physique animale et v6getale. 1799. Tracts on the nature S^^lJnimls and vegetables. 1780. Tracts on the natural history of am- Ste^t, F^^c'^im^The absorption and accumulation of solutes by living plant cells. Protoplasma 18: 20&-242. ^ „, ^ ^ t u w r>«* *i . «;ii Strasburcer, E. 1898 Die pflanzlichen Zcllhaute. Jahrb. Bot. 31 . 511- Tre^^iS^nus L C. 1810. Beobachtungen iiber die Bewegung des kornigcn \?^seSs in einigen Conferven und einer Charen. Beitr. zur Naturkunde 2: 126-141. i 30 THE BOTANICAL REVIEW r Ursprung. a., and Blum, G. 1916. t)ber die Verteilmg des osmotischen Wertes in der Pflanze. Ber. Deut. Bot. Ges. 34: 88-104. Van TiEGHENS, Ph. 1888. Hydroleucites et grains d aleurene. Jour. Bot. 2* 429-432. ViREY, J. J. 1823. De I'organisation des tissues vegetaux dans les excrois- sances appelees galles. Jour, de Pharm. 9 : 314-316. VOLKONSKY, Michel. 1933. Digestion mtracellulaire et accumulation colorants acides. Bull. Biol. Franc, et Belg. 67: 136-275. DE Vries, Hugo. 1877. Untersuchungen uber die mechanischen Ursachen der Zellstreckung. 1884. Eine methode zur Analyse der Turgorkraft. Jahrb. Wiss. Bot. 14: 427-601. 1885. Plasmolytische Studien uber die Wand der Vakuolen. Jahrb. Bot. 16 : 465-598. 1886^ Ueber die Aggre- eation im Protoplasma von Drosera rotundtfolta. Bot. Zeit. 44: IZ-^O), 33-43 57-64. 1886. "Dber die Aggregation im Protoplasma von Drosera rotuttdifolia. Bot. Zeit. 44: 2-11, 18-26, 32-43, 56^3. Wakker. 1888. Studien iiber die Inhaltskorper der Pflanzenzellen. Jahrb. Wiss. Bot. 19 : 423-496. ^,^ ^ , ^ xt . i a Weber, Friedl, and Mohr, Daniel M. H. 1810. Beitrage zur Naturkunde. 1811. Beitrage zur Pflanzenphynalogie. „ , , c u-it.,«c Wray. J. 1670. Extract of a letter written to the publisher. Phil. Irans. Roy. Soc.. London 5 : 2063-2066. ZiRKLE. Conway. 1932. Vacuoles m primary meristems. Zeit. Zellf. Mikr. Anat. 16:26-47. 1933. Cytological fixation with the lower fatty acids, their compounds and derivatives. Protoplasma 18: 90-111. i|' >lr>-*