UNIVERSITY OF CALIFORNIA PUBLICATIONS IN BOTANY Vol. 5, No. 17, pp. 457-582, 2 figures in text, plates 55-85 April 14, 1922 INHERITANCE IN NICOTIANA TABACUM A REPORT ON THE RESULTS OF CROSSING CERTAIN VARIETIES WILLIAM ALBERT SETCHELL THOMAS HARPER GOODSPEED AND ROY ELWOOD CLAUSEN UNIVERSITY OF CALIFORNIA PRESS BERKELEY, CALIFORNIA THE IIB»?«JY 0 C ™E i i 1949 UNIVERSITY OF CALIFORNIA PUBLICATIONS Note. — The University of California Publications are offered in exchange for the publl. cations of learned societies and institutions, universities and libraries. Complete lists of all the publications of the University will be sent upon request. For sample copies, lists of publications and other information, address the MANAGER OF THE UNIVERSITY PRESS, BERKELEY, CALIFORNIA, U. S. A. All matter sent in exchange should be addressed to THE EXCHANGE DEPARTMENT, UNIVERSITY LIBRARY, BERKELEY, CALIFORNIA, U. S. A. WILLIAM WESLEY & SONS, LONDON Agent for the series in American Archaeology and Ethnology, Botany, Geology, Physiology, and Zoology. BOTANY.— W. A. Setcheil, Editor. Price per volume, $3.50 (vol. 5, $6.00) ; beginning with volume 6, $5.00. Volumes I, n, IIL TV, and IX completed. Volumes VII, VTIL and X in progress. Cited as Univ. Calif. Publ. Bot. VOL 5. 1912-1922. 1. Studies in Nicotiana. L by William Albert Setcheil. Pp. 1-86. December, 1912 $1.25 2. Quantitative Studies of Inheritance in Nicotiana Hybrids. I, by Thomas Harper Goodspeed. Pp. 87-168, plates 1-28. December, 1912 „ 1.00 3. Quantitative Studies of Inheritance in Nicotian a Hybrids. II, by Thomas Harper Goodspeed. Pp. 169-188, plates 29-34. January, 1913 20 4. On the Partial Sterility of Nicotiana Hybrids made with N. sylvestris as a Parent, by Thomas Harper Goodspeed. Pp. 189-198. March, 1913 10 5. Notes on the Germination of Tobacco Seed. I, by Thomas Harper Good- speed. Pp. 199-222. May, 1913 - .25 6. Quantitative Studies of Inheritance in Nicotiana Hybrids, in, by Thomas Harper Goodspeed. Pp. 223-231. April, 1915 10 7. Notes on the Germination of Tobacco Seed. IL by Thomas* Harper Good- speed. Pp. 233-248. June, 1915 ~ — .15 8. Parthenogenesis, Parthenocarpy and Phenospenny in Nicotiana, by Thomaa Harper Goodspeed. Pp. 249-272, plate 35. July, 1915 - 25 . 9. On the Partial Sterility of Nicotiana Hybrids made with N. sylvestris as a Parent. II, by T. H. Goodspeed and A. H. Ayres. Pp. 273-292, plate 36. October, 1916 20 10. On the Partial Sterility of Nicotiana Hybrids made with N. sylvestris as a Parent. Ill, An Account of the Mode of Floral Abscission in the F, Species Hybrids, by T. H. Goodspeed and J. N. Kendall. Pp. 293-299. November, 1916 - - -°5 11. The Nature of the F, Species Hybrids between Nicotiana sylvestris and Varieties of Nicotiana Tabacum, with Special Reference to the Conception of Reaction System Contrasts in Heredity, by T. H. Goodspeed and R. E. Clausen. Pp. 301-346, plates 37-48. January, 1917 _ 45 12. Abscission of Flowers and Fruits in the Solanaceae, with Special Reference to Nicotiana, by John N. Kendall. Pp. 347-428, 10 text figures, plates 49- 53. March, 1918 ..... - 13. Controlled Pollination in Nicotiana, by Thomas Harper Goodspeed and Pirie Davidson. Pp. 429-434. August, 1918 14. An Apparatus for Flower Measurement, by T. H. Goodspeed and R. E. Clausen. Pp. 435-437, plate 54, 1 figure in text. September, 1918 _ 15. Note on the Effects of Illuminating Gas and Its Constituents in Causing Abscission of Flowers in Nicotiana and Citrus, by T. H. Goodspeed, J. M. McGee and R. W. Hodgson. Pp. 439-450. December, 1918 ..... - 16. Notes on the Germination of Tobacco Seed. HI, Note on the Relation of Light and Darkness to Germination, by T. Harper Goodspeed. Pp. 451- 455. April, 1919 - 17. Inheritance in Nicotiana Tabacum. I, A Report on the Results of Crossing Certain Varieties, by William Albert Setcheil, Thomas Harper Goodspeed, and Roy Elwood Clausen. Pp. 457-582, 2 figures in text, plates 55-85. April, 1922 - - Index in press. 680.5 UNIVERSITY OF CALIFORNIA PUBLICATIONS I N BOTANY Vol. 5, No. 17, pp. 457-582, 2 figures in text, plates 55-85 April 14, 1922 INHERITANCE IN NICOTIANA TABACUM I A REPORT ON THE RESULTS OF CROSSING CERTAIN VARIETIES BY WILLIAM ALBERT SETCHELL, THOMAS HARPEE GOODSPEED, «T — and ROY EL WOOD CLAUSEN — . CONTENTS page t: I. Introductory 458 M II. Plan of the work 460 y III. Angustifolia-macrophylla series M 462 1. Parents of the angustifolia-macrophylla series 463 > . 2. Fi of the angustifolia-macrophylla series 467 3. F 2 of the angustifolia-macrophylla series 469 4. F 3 and subsequent generations of the angustifolia-macrophylla series 472 a. Stenophylla derivatives 475 c< , b. Latifolia derivatives 476 ;j c. Lanceolata derivatives 478 '/. Loriifolia derivatives 478 e. Atjriculata derivatives 479 /. Sessilifolia derivatives 480 5. Summary of flower color observations in F 2 and subsequent generations 482 6. Later sowings of F 2 and F 3 of the angustifolia-macrophylla series 483 7. Crosses of derivatives with the parents 487 8. Discussion of results of the angustifolia-macrophylla series 490 3 IV. Calycina-virginica series 494 1. Parents of the calycina-virginica series 494 2. Fi of the calycina-virginica series 496 3. F 2 of the calycina-virginica series 497 4. F 3 and subsequent generations of the calycina-virginica series 499 5. Discussion of results of the calycina-virginica series 504 V. Alba-macrophylla series 504 1. Parents of the alba-macrophylla series 504 2. F t of the alba-macrophylla series 505 3. F 2 of the alba-macrophylla series 505 4. F 3 and subsequent generations of the alba-macrophylla series 507 5. Discussion of results of the alba-macrophylla series 510 458 University of California Publications in Botany [Vol. 5 PAGE VI. General Conclusions 510 1. Origin and interrelationships of varieties of Tabacum 510 2. Methodology of Mendelian analysis in Tabacum 513 3. Mendelian heredity in Tabacum 516 VII. Summary 520 Literature cited 520 Explanation of plates 522 I. INTRODUCTORY The inception of the work on the various species of Nicotian a grown and bred in the University of California Botanical Garden has already been sketched in a previous number of this series (cf. Setchell, 1912). As stated there, the original intention was to assemble a collection of tobacco plants simply as a portion of the outfit of the Botanical Garden for general instruction and display. So great was the variety and evident misapplication of the names under which the seeds were received, however, that it seemed advisable to attempt to determine, as definitely as possible, the status of each plant. In this connection, the work of Comes, in particular, came under consideration and especially his views as to the origin and interrela- tionships of the various cultivated forms belonging to the Tabacum group. Comes (1899, p. 4 and elsewhere) regards the numerous culti- vated forms of tobacco as having originated in various ways from certain fundamental varieties. He estimated that there are six of these fundamental varieties of Tabacum, and he supposed the large number of various and seemingly more or less intergrading forms to have arisen through the influence of the forces of acclimatization, adaptation, hybridization, and selection. Of these, undoubtedly, the greater variations have been produced and perpetuated, according to the ideas of Comes, through hybridization and selection. In his mono- graph (1899) and in his later more exhaustive treatise (1905), Comes has attempted to estimate just which of his six fundamental varieties of Tabacum have cooperated in producing each one of the cultivated "races" so far as known to him. The statements of Comes as regards the constitution of his various races seem to have been based on the results of morphological study rather than upon breeding analysis. The advisability occurred to the senior author of attempting to test Comes' hypothesis by selecting varieties seemingly fundamental in type, and through hybridization i^ 22 ] Setchell-Goodspeed-Clausen: Nicotiana Tabacum 459 and selection attempting to secure constant races exhibiting various recombinations of the parental characters. The work thus conceived has been carried out in detail in a certain few but seemingly charac- teristic cases. Several different crosses were made in 1909, the first filial generations were grown in 1910, and each year since that time has seen successive filial generations in the field. Although the Nicotiana investigations were originally designed to attack experimentally a comparatively simple and definite problem, they have since been greatly amplified in scope. At the present time three rather distinct lines of investigation are actively in progress, viz., 1. Mendelian inheritance in N. Tabacum. 2. Inheritance of quantitative characters. 3. Studies of interspecific hybrids. The recent appearance of bud variations in hybrid lines favorable for an analytic study of that phenomenon has resulted in the addition of another research project. Now it has been found that, although seemingly distinct, progress in these separate lines of investigation is more or less interdependent. In particular it has been found that certain of the phenomena exhibited in interspecific hybrid populations from crosses between N. sylvestris and varieties of N. Tabacum require for satisfactory analysis and explanation an accurate and detailed knowledge of the Mendelian differences which exist among the par- ticular varieties of N. Tabacum that have been used in those investi- gations. Accordingly in later years these studies of hybrids between varieties of N. Tabacum, originally designed merely to test experi- mentally the interrelationships existing among such varieties, have been directed toward a specific Mendelian analysis of the germinal differences existing in a selected set of varieties. With this change in emphasis has come a full appreciation of the difficulties of Mendelian studies in N. Tabacum. It has been very evident that, for the most part, the character differences among varie- ties of N. Tabacum do not rest upon a simple genetic basis; on the contrary, they often depend upon very complex and involved Men- delian differences; so that in segregating populations it is often im- possible to demonstrate the existence of definite, discontinuous char- acter classes. Not uncommonly the members of such populations may be arranged in series connecting by imperceptible differences the most extreme character expressions in the population. But although complex intergrading segregation has often been observed in F 2 , it has not been found that such complex segregation 460 University of California Publications in Botany [Vol.5 persists in subsequent generations in the hybrid lines. On the con- trary, it has been found, as will become evident in a study of the ex- perimental material communicated herewith, that a great simplification occurs in the segregation in F 3 and subsequent generations, and that continuous segregation gives way to discontinuous just as might be expected from Mendelian theory. By observing the segregation in the consecutive generations of hybrid lines which have become homo- zygous in most of their loci through self-fertilization, it is possible to obtain some idea of the Mendelian factor pairs involved in the char- acter contrasts and of their relations to one another. It has also proved possible by a few years of self-fertilization to establish stable lines representing recombinations of parental characters. By investigating the interrelations among such stable derivative lines, which obviously should differ in fewer factors from one another and from the original parental varieties than the parental varieties differ from each other, it would seem possible to develop an indirect mode of attack by which the Mendelian analysis could be refined to any desired extent. The original plan of the investigation, therefore, having as its purpose a demonstration of the possibility of securing by hybridization stable derivative lines representing recombinatons of characters contained in the parents and comparable to the numerous existing varieties of N. Tabacum, has been diverted into a detailed study of Mendelian differ- ences among a typical set of N. Tabacum varieties. II. PLAN OF THE WORK In the introductory paper the senior author has discussed the fun- damental types of N. Tabacum, and as indicated there, has expressed a preference .for selecting some five fundamental varieties, or species, as representing the basal morphological elements found, or seemingly to be detected, in cultivated races of N. Tabacum. There is no neces- sity for discussing further, at present, the reasons for preferring the particular types selected by us as against those of either Comes or Anastasia (1906), since the fundamental conceptions agree sufficiently well and the important thing has been to make a beginning in experi- mentation by using varieties which present seemingly fundamentally different character complexes in most characteristic form in plants breeding true to type in the pure line. Certain reasons for selecting a particular type or types will be discussed in connection with the 1(>L'2 | Setchell-Goodspccd-Clausen: Nicotiana Tabacum 461 consideration of the various crosses. Besides the ' ' fundamental ' ' types, there have been selected for crossing certain other types, possibly fundamental, or in some cases derivative, which have been employed for testing the inheritance of some particular character or group of characters. All of these have been described in the first paper of this volume. The taxonomic problems in N. Tabacum do not appear to differ from those presented by many other species of cultivated plants. Barley, maize, oats, rice, wheat, among others, exhibit a similar diver- sity of forms with more or less obvious class distinctions. In these as in N. Tabacum it appears to be an easy task to shuffle and recombine characters indefinitely. Clearly there can be no segregation of forms into distinct species on genetic grounds ; the basis of speciation, if any, must depend either upon convenience merely or what amounts to practically the same thing, upon elevation of certain Mendelian char- acter contrasts to a higher rank in classification than others. Since the taxonomic problem, therefore, is not strictly a genetic one, it seems best to follow general usage in this respect, referring all the poly- morphic assemblage of forms to the on\ species N. Tabacum, and re- garding the several races included thereunder as varieties of equal rank. The varieties employed in this series of investigations are : N. Tabacum var. alba, U. C. B. G. 30/06, previously described by Setchell as "White" Tobacco; N. Tabacum var. angustifolia, U. C. B. G. 68/07, previously described by Setchell as N. angustifolia; N. Tabacum var. calycina, U. C. B. G. 110/05 ; JV. Tabacum var. macrophylla, U. C. B. G. 22/07 ; and N. Tabacum var. virginica, U. C. B. G. 78/05, previously described by Setchell as N. Tabacum "Maryland." In each instance the University of California Botanical Garden (U. C. B. G.) number contains in the numerator the accession number of the year given in the denominator. The varieties have in the majority of cases been grown in pure lines from the date of their receipt. In order to avoid needlessly encumbering the text with scientific names, the varieties mentioned above will be referred to by their varietal designations only, and when reference is made to the whole group the species name Tabacum will be used alone. Three series of cultures are described in the present article : the angustifolia-macrophylla series, which has been derived from reciprocal crosses of angustifolia and macrophylla; the calycina-virginica series, derived in the same way from calycina and virginica; and the alba- 462 University of California Publications in Botany [Vol. 5 macrophylla series, from alba and macrophylla. In the course of the investigations other crosses were made between different varieties of Tabacum and to a limited extent between other species of Nicotiana; but the principal attention has been paid to the three crosses noted above, and they and their progenies alone will be considered in the present paper. It may be said at this point that the different varieties of Tabacum cross readily with one another, giving an abundance of good viable seed. The hybrids are uniformly self -fertile. The methods of hybridization used need not be considered here, because they have been described in detail by Goodspeed (1912) else- where in this series. The particular refinements of technique which must be employed in sowing the seed, on account of its very small size, have also been there described. It might be well to state, however, that the most refined methods doubtless will not prevent the occasional appearance of a stray plant in the cultures. The danger of contami- nation arises not only during the sowing of the seed, but also when the bags are placed over the unopened buds. It is very easy to include a few stray seeds under the bag, for their small size makes it almost impossible to detect them in the coarse, sticky indumentum of the plant. In spite of these obvious difficulties, however, the number of plants that have certainly been strays has been very small. Their rare occurrence indicates clearly that the technique employed has been very successful. III. ANGUSTIFOLIA-MACROPHYLLA SERIES This series has received the most attention since the parents are so distinctly different, and the results have consequently been more complex than those which have followed the crossing of any other pair of Tabacum varieties. As will be demonstrated below, F 2 seemed at first hopeless in its variety of segregation. Later generations, however, exhibited so much less, or so little variety in their segregation products that it was easy to obtain new permanent combinations of characters or "fixations." Certain of its segregants have been followed out to F 7 , and have also been crossed back on the parents which they most closely resembled. Six successful crosses were made. Of these H 2 and H 3 had macro- phylla for the male and angustifolia for the female parent, while H 4 . H 5 , H 15 , and H 16 were reciprocal crosses. As a matter of convenience I 922 ] Setchell-Goodspeed-Clausen: Nicotiana Tabacum 463 the generations later than F x were grown only from H„ and H 4 , the larger number from H 4 . The predominance of H 4 in the later families selected for the continuation of the work was not, however, due to any especially different behavior evidenced in that particular series. 1. PAEENTS OF THE ANGUSTIFOLIA-MACROPHYLLA SERIES By selecting angusti] 'alia and macrophylla for crossing, two varie- ties were obtained which resemble each other in height and general habit, but which differ strikingly in leaf and flower characters. The differences are sufficiently great to lead one to regard them as belong- ing to different species ; in fact, all five Tabacum varieties selected by us as possibly fundamental differ sufficiently among themselves to be regarded as species in the Tabacum section rather than as varieties. It is not our intention, however, to emphasize this point, since any discussion would of necessity lead to a general survey of all the known varieties and races at present included under Tabacum. If, however, these five, viz., angustifolia, macrophylla, " Cavala," Maryland, and "Brazilian" (cf. Setchell, loc. cit.) could be considered by themselves as wild plants, it seems to us that any taxonomist of the present day would certainly award to each of them the^pank of a separate species. These considerations should be borne in mind in estimating the signifi- cance of the results obtained through crossing. Angustifolia, U. C. B. Cr* 68/07, is a variety which has long been known and which is represented in our breeding experiments by a pure line very closely approximating the type. It has been figured and discussed by one of us (Setchell, loc. cit., p. 9, pi. 7). The photo- graph given there is of a young plant just coming into flower and consequently does not represent the habit of the plant in full blossom or in fruit, after the full number of laterals is developed. A plant in the height of its vigor is represented in plate 55, figure 1. In stature angustifolia belongs to the low corymbose group of Tabacum varieties, which also includes the forms bred in the University of Cali- fornia Botanical Garden under the names calycina and macrophylla, and which is in decided contrast to the tall, more "racemose" (al- though these may be "corymbose" at the top) forms such as alba and virginica. In height angustifolia varies from 75 to 120 cm. The central axis develops its corymbose panicle of short racemes first, but it is usually soon overtopped by the successive laterals developed basipetally, each 464 University of California Publications in Botany [Vol. 5 lateral, in turn, developing a corymbose cluster of racemes, rising more or less above its predecessors.- The result is that the whole plant has the short corymbose habit mentioned above. The stems and branches of angustifolia are comparatively slender, being much more slender than those of macrophylla, or those of any other of the Tabacum varieties except those of calycina, which are very similar. The leaves of angustifolia are alternate and distinctly and moder- ately long, petiolate. The blade of the lower leaves is ovate-lanceolate,- tapering above to a long, curved point, more or less conduplicate below and with the rounded bases unequal. Above, the leaves are less con- duplicate, more so even at the base, with the petiole shorter, while the uppermost (bracts) become almost sessile and narrowly lanceolate even to almost linear in outline. The normal petiole is naked at the base and in the middle portion, but the base of the blade is slightly and narrowly decurrent along the upper portion. Occasionally a petiole shows a narrow wing throughout its length and at t'imes the petioles of all the leaves on certain plants are more or less winged, but the majority of the plants have naked petioles (cf. also Good speed and Clausen, 1917, p. 306, pi. 46, right-hand figure). The leaves of angus- tifolia have also a very characteristic drooping habit, much more pro- nounced than in any other Tabacum variety except calycina. In older plants, after capsule formation lias well advanced, all the leaves are hanging obliquely downwards. The flower of angustifolia is distinctive and differs in details of shape and color from that of any other Tabacum variety, and especially from that of macrophylla. The general shape of the flower is that of all the Tabacum section, but the corolla is much more slender and more gradually infundibuliform than that of any of the other varieties re- ported here. The calyx is broadly campanulate, prolonged above into 5 long, but unequal, linear-lanceolate, pointed lobes, of which one is longer than the remaining four and gives the calyx a zygomorphic appearance. The corolla is narrow and tubular below the middle, expanding rather gradually and evenly above into a conical infundi- bulum which bears the spreading, deeply 5-lobed limb at its summit. The length of the tube of the corolla is about 6 cm. and its greatest diameter about 7 mm. The limb of the corolla, at first erect (opening bud), then horizontal, finally becomes somewhat deflexed and measures about 3 or 3.5 cm. across. It is divided almost to the tube into 5 lobes which are ovate-lanceolate with long, narrow, tapering tips. The lobes of angustifolia are much longer and have narrower tapering tips than 1922] Setchell-Goodspeed-Clausen : Nicqtiana Tabacum 465 those of any other Tabacum variety, and in this respect are in direct contrast to those of macrophylla. The lohes are unequal and give a slight suggestion of zygomorphism to the corolla. The stamens are inserted on the lower portion of the tube and are usually slightly ex- serted in anthesis. The pistil possesses the usual 2-celled ovary, long, slender style, and thick, slightly bilobed stigma, more or less exserted in late anthesis, characteristic of the genus Nicotiana. The color of •the corolla is a light, though lively, pink, much lighter than -the red of macrophylla. The capsule at maturity is slightly flattened longitud- inally, is broadly lanceolate in profile, tapers above into an acuminate apex, and is about 25 mm. high and 8 to 9 mm. thick. It is the most slender of all the capsules borne by the various Tabacum varieties and in decided contrast to the stout capsule of macrophylla. In plate 55, figure 1, is illustrated a plant of angustifolia at the height of its blooming period. Typical features of the plant are shown in the line drawings of plate 56. Photographs of typical leaves are shown in plate 58, where they may be compared with photographs of the leaves of macrophylla. Photographs of the flowers are reproduced in plate 60, where they may be compared with those of macrophylla and of the hybrids between these two varieties. Macrophylla, U. C. B. G. 22, 07, has already been discussed and figured by one of us (cf. Setchell, loc. cit.)". The original seed was obtained from Comes, but the plants do not correspond to his figures (cf. Comes, 1899, pi. VIII) either as to habit or sh^pe of leaf. They differ also from his description in these same respects. The flower, however, agrees, and it seems best to retain for it the name under which we have cultivated it. The habit and height of macrophytta are both very similar to those of angustifolia. The habit is low corymbose, the central axis bearing a panicle of corymbose racemes and the laterals arising one after the other bearing similar inflorescences and equaling or overtopping the central axis. The stems and branches are stouter than those of angus- tifolia, however, and this, together with the broader, more solid looking leaves which do not droop so much as those of angustifolia, give a mature plant of macrophylla a much more robust appearance than is the case with a mature plant of angustifolia. The plant figured in the first number of this volume (pi. 6) was young. An older plant shown herewith on plate 55, figure 2, is in full blossom and beginning to ripen its capsules, and gives a better idea of the habit of a well grown plant. 466 University of California Publications in Botany [Vol. 5 The leaves of macrophylla are sessile by a partially clasping base and possess two basal lobes partially clasping the stem. The general shape is obovate, the widest portion being above the middle. The leaves taper gradually to the broad clasping base below and abruptly to a narrow more or less acuminate tip above. The surfaces show the secondary veins branching at a more obtuse angle than do those of the leaves of an gusti folia. The color of the leaves is a dark green in macrophylla and more of a yellowish green in angustifolia. In every way, then, the leaves of the two parents differ from each other as much, in fact, as do the leaves of many species. The flowers of macrophylla, while of the same general type as those of angustifolia, differ in details of shape and color. The flowers of macrophylla are about 4 cm. long. The calyx is broadly ovate in pro- file, deeply cut into 5 broad and somewhat unequal lobes. The corolla tube is stout cylindrical (about 5 mm. in diameter) below, broaden- ing suddenly into a stout infundibulum above (about 10 mm. in diam- eter). The limb is at right angles to the tube, is about 23 mm. across, and is more or less pentagonal with 5 shallow sinuses. The color of the corolla is deep red fading to an almost lilac tint after anthesis. On the limb are 5 triangular lighter areas, one having the narrow apex at each sinus and the broad base at the top of the tube. In the much darker color, in the broader tube and stouter infundibulum, and in the barely appreciable lobing of the limb, the corolla of macrophylla is the antithesisJpf that of angustifolia. In stamens and pistil, the flower of macrophylla shows little variation from that of angustifolia. The capsule of macrophylla is broadly ovate, tapering abruptly to a mucronate tip. It is about 2 cm. high and about 1.5 cm. in diameter, contrasting very decidedly with the comparatively slender capsule of angustifolia. A typical plant of macrophylla is shown in plate 55, figure 2. Typical features of the plant are shown in line drawings in plate 57. Photographs of leaves are reproduced in plate 58, where they may be compared directly with those of angustifolia. In plate 60 its flowers may be compared directly with those of angustifolia and with those of the hybrids. It has seemed best to call attention to the characters of and differ- ences between these two varieties, parents in the first set of crosses to be discussed, in order that the behavior of their hybrid progeny may be clear. In height and habit there is a close agreement, but in leaf, flower, and fruit there are sufficient differences to mark them as sep- arate species. 1922] Setchell-Goodspecd-Clausen: Nicoiiana Tdbacum 467 2. F x OF THE ANGUSTIFOLIA-MACROPHYLLA SERIES In late July of 1909, some 7 crosses were made between angusti- folia and macrophylla, 6 of which, as stated above, were successful. H 1; H 2 , and H 3 , as they were designated, involved angustifolia as the female plant, while H 4 , H 5 , H 15 , and H 16 were reciprocals. No seed was obtained from H 1; but all the other 6 crosses gave a fair yield. The usual care (cf. Goodspeed, loc. cit., pp. 129-131) was taken in cleaning and sowing the seed. This was done in the spring of 1910, germination was good in all cases, and 337 plants, distributed as follows, came to maturity and seed bearing. The family of H 2 had 56, H 3 had 60, H 4 had 47, H 5 had 58, H 15 had 55, and H 16 had 61 plants. A survey of all these plants showed in general a remarkable uni- formity in habit. A certain amount of difference was to be detected on careful scrutiny, but little if any greater than that which is ex- hibited among a large number of individuals of one or the other parent. In height, F 1 showed exactly the same variation as the parent, the central axes varying from 65 to 145 cm., but largely varying from 90 to 120 cm., while the laterals rose to 150 cm. Some rows showed uni- formly higher, others uniformly lower plants, the differences probably being due to different soil and water conditions. The habit (see pi. 61) was low corymbose and the general appearan^ as to stoutness seemed more or less intermediate, between the two parents. The leaves in shape, size, etc., very closely resembled those of angustifolia. There was some appreciable variation in the leaves, however, aild often con- siderable variation on the same plant, a characteristic of angustifolia which has already been mentioned. Plate 59 reproduces photographs of different types of leaves obtained from F x plants. The blade is | broadly elliptical ovate with the lateral veins at an obtuse angle, much as in macrophylla. The base is rounded, or even slightly cordate in some leaves, while the tip is more blunt. These characters seem, at least, to indicate an influence of macrophylla. The leaf, however, is distinctly petiolate, but the petiole is not so long as in angustifolia. The petiole is definitely winged and the wings are expanded at the base into auricles, which are often triangularly decurrent along the internode of the stem. This wing is usually present in all the plants of F 1; but some leaf or leaves on a plant may lack it, and in some plants it is only slightly developed, or at least, is without auricles. The wing was from 5 to 7 'mm. wide on some leaves. The leaf of lOFiH^P, represented in plate 62 even more closely resembles the typical leaf of angustifolia. The wing along the mar- 468 University of California Publications in Botany I VoL - 5 gins (or edges) of the petiole is narrow and is prolonged as a slight ridge to the internode and is decurrent (?) or can be traced as it bends sharply downwards. Such wings are, at times, found in pure bred angustif olia plants. This leaf, then, resembles the angustif olia leaf fairly closely, but differs from its ordinary expression in the more tapering base, in being less distinctly conduplicate, in tapering more abruptly toward the tip, in having shorter petioles and in having more of a wing on the margins of the petiole. The flower of F x (see pi. 60) resembles that of angustifolia more than that of macrophylla. The color is deep pink, decidedly of a deeper shade than is the flower of angustifolia, yet far from the red of macrophylla and in a way intermediate between the two. There is no trace, on the limb of the corolla of F 1} of the 5 white triangle- shaped areas so characteristic of the limb of the corolla of macrophylla. The infundibulum, while possibly slightly stouter than that of the flower of angustifolia, is not so stout as that of the flower of macro- phylla. In length the flower of F x averages about 4 to 4.5 cm. as against an average of 6 cm. in angustifolia, and of 4 cm. in macro- phylla. The tube averages about 3.5 to 5 mm. in diameter below, as contrasted with 2.5 to 3 mm. as an average in angustifolia and 5 mm. in macrophylla. %The infundibulum in the corolla of F t , while neither abrupt nor so stout as tjnat of the flower of macrophylla, is noticeably more abruptly enlarged and stouter than that of angustifolia. The limb of the corolla in f\ averages 2.5 to 3.25 cm. in greatest diameter, while that of angustifolia averages 3 to 3.5 cm. and that of macrophylla averages about 2.3 cm. in greatest diameter. The lobes of the corolla in Fj are about half the width of the limb from tube margiflfto extreme tip of lobe, while in angustifolia they are about two-thirds of thi in macrophylla they are only one-third or even less. The lobes, are decidedly broad at the base, particularly so as compared with tneir length. In general, then, the corolla of F 1} while closer to that of angustifolia, shows by its stouter tube, more abrupt and more swollen infundibulum, intermediate spread of limb, less deep lobing, shorter and broader lobes, and deeper shade of pink, definite influences of macrophylla also. The capsule of F x is broader than that of angustifolia, but nar- rower than that of macrophylla. There is greater variability in hori- zontal diameter in the capsule of F 1 . The flower and fruit of F^ then, although no careful biometric study has been made, are inter- mediate between those of the two parents, yet incline more toward angustifolia than toward macrophylla. 1 922 1 Setchell-Goodspeed-Clausen : Nicotiana Tabacum 469 In general, then, a survey of F 1 shows throughout a series of ten different families a uniformity of individuals as great as that exhib- ited in either of the parents. Some few slight differences exist among individuals both of F x and of the parents which may possibly be re- ferred to lack of a completely homozygous conditon in the parents. In characters in which the two parents differ, whether in color of flower, quantitative corolla character complexes, capsule character com- plexes, or leaf character complexes, the Fj hybrid exhibited through- out a character expression intermediate between that of the two parents. 3. F 2 OF THE ANGUSTIFOLIA-MACKOPHYLLA SEEIES In 1911, there were selected as parents for the F, 13 plants from H 2 and 12 from H 4 . Twenty-one families of approximately 50 plants each were set out in the field. On account of the great diversity shown in these populations, it was found impossible to study individually each of the thousand plants grown ; consequently particular attention was paid to only 5 families from each hybrid. The other families were gone over carefully, but nothing notably different was found in their behavior. All fifty plants survived in each family except in the last, viz., 11F 2 H 4 P 43 , where only 48 came to maturity. There were then 498 plants of F 2 under more careful observation, representing both the cross and its reciprocal, with about 550 remaining for only casual examination. As might have been expected, there was a great variety of^ plants resulting and segregation as to differences in combination of characters of flower, fruit, and leaf was little short of bewildering. An attempt was made to study and Arrange these combinations, but it .was found to be impossible. A eareful survey, however, was aaade./of the popu- lations and a tabulation of characters was attempted. "^orae 16 fairly readily separable types, based on leaf characters, were distinguished, but between these closely approaching types others were to be found of intermediate and overlapping character. One each of the types selected was drawn, and these drawings are reproduced in plates 63 to 78. A glance at these plates, which were carefully drawn to scale, will show something of the nature of the combinations of characters of the two original parents. Type 1 (pi. 63) shows a close approximation, yet not an absolute reproduction, of angtistifolia, while type 16 (pi. 78) in a similar way is a close approximation to macrophylla. The other 470 University of California Publications in Botany [Vol.5 14 types (pis. 64 to 77) are clearly intermediates approaching one parent more than the other, but types 12, 13, and 14 (pis. 74 to 77, inclusive) are decidedly different from either as to leaf, at least, and type 10 (pi. 72) is of another altogether different form, although all of these leaf shapes are connected to a greater or less extent into one series of more or less gently intergrading forms. As to the shape and dimensions of the corolla there is to be found a similar series of intergrading forms from the slender corolla tube with gradually expanding and slightly swollen infundibulum and deeply lobed limb of type 1 (pi. 63) to the corolla with stout tube, abruptly and considerably swollen infundibulum with slightly lobed limb of type 16 (pi. 78). In color the corollas vary from the light pink of angustifolia to the red of macrophylla and three shades are at times fairly readily distinguishable, the light pink of angustifolia, the deep pink of F 1? and the several nuances of the red of macrophylla. The capsules also show various combinations from the slender gradually attenuated capsules of angustifolia to the stout, swollen, abruptly upwardly attenuated capsules of macrophylla. Both cap- sules and corollas approaching one parent may be found with leaves more closely approaching the other parent. In stature and habit the plants of all the 21 families were reasonably uniform and agreed in general in these respects$-wkh the parents and F u there certainly being no greater amplitude of'variation in thes^respects than was to be found in the parental types. Among the great variations, two characters seemed to stand out fairly V clearly for rough statistical enumeration, viz., color of the corolla and the possession, or lack, of a petiile. Numerical data for these characters are given in table 1. Some can 1 was taken to obtain a careful -rbnsus of the families as regards each of the characters. As regards colorsjudf was, as noted before, possible to distinguish three shades, or sets of shades, which were designated as light pink, pink, 4ud red. In practice, however, it was usually difficult to distinguish the two shades of pink from each other. The red gave very little trouble. In attempting to classify the plants of F 2 with respect to type of leaf base, more difficulty was experienced because of the variety of forms which were produced and the degree of intergradation which existed between forms. In judging the presence or absence of petiole, therefore, in these populations, the classification is faulty because of lack of knowledge of the genetic constitution of the various distinct forms and those which grade into them. In table 1 the plants are 1922] Setchell-Goodspeed-Clausen: Nicotiana Tabacum 471 thrown into the petiolate class if they were distinctly narrowed at the base, and whether naked or winged. In F 2 , then, there appears to be simple Mendelian inheritance in only one pair of the original character contrasts of the parents, namely, red versus pink corolla color. Here the hybrid is intermediate and F 2 segregates sharply into pink and red in the ratio 3 pink : 1 red. Within the pink class there is a more or less evident segregation into 2 pink : 1 light pink, but the shades intergrade so that no distinct line of demarcation exists between the classes. As respects leaf base char- acters, the segregation is so complex that no reasonable genetic analysis is possible. The numerical data for this latter character presented in table 1 are of value only in that they indicate a close agreement in segregation among F 2 families, thereby furnishing a rough statistical demonstration of the equivalence of the several families. The more definite data on leaf base characters are derived from generations subsequent to F 2 . TABLE 1 CLASSIFICATION OF F 2 PLANTS OF THE ANGUSTIFOLIA-MACROPHYLLA SERIES ACCORDING TO COROLLA COLOR AND LEAF BASE CHARACTERS. Family i Garden \ Corolla Color ^ Leaf Base Designations Numbers / red pink light pink petiolate non-petiolate A 11F 2 H 2 P, 12 23 14 42 7 B 11F 2 H 2 P 3 14 25 10 30 20 C 11F 2 H 2 P 6 13 21 15 34 14 D 11F 2 H 2 P 7 7 21 22 35 15 E 11F 2 H 2 P 13 15 20 14 30 20 F 11F 2 H 4 P 2 13 28 7 32 17 G 11F 2 H 4 P 3 5 12 29 8 ' 35 „_~/"l4 H 11F,H 31 31 17 K 6 27 15 38 10 Totals 113 240 137 339 152 472 University of California Publications in Botany [Vor,. 5 4. F 3 AND SUBSEQUENT GENERATIONS OF THE ANGUSTIFOLIA- MACROPHYLLA SERIES From F 2 of H 2 and H„ 20 plants were selected for further ex- perimentation and families of 25 were determined upon as the unit. In all except four, the families of 25 each were successfully raised. Of one of the four only 14 plants were obtained, which were all that germinated, while in each of the other three families 24 plants were reared to maturity. Altogether, then, 486 plants were raised of the F 3 during the season of 1912. It was the intention to grow from each of the selected types, as drawn for illustration. Fifteen of the families from the type parents were successfully reared, but, in some way or other, the seed of type 4 (10F 2 H,P 7 P 1S ) was not to be found, and, unfortunately, no note had been made as to whether or not any seed was produced. No complete sterility, however, was noticed in any members of F 2 of either H 2 or H 4 , and the presumption is that F, seed of type 4 must have been lost in harvesting. The variation within each family was decidedly less than that of the families of F 2 . Of the 20 families reared wholly or in part, 4 families were very^^arly uniform, varying in minor details only. Five families segregated only in corolla^olor, 4 segregated only in leaf base characters, and the remaining 7 segregated .both in corolla color and leaf base characters. In table 2 are summarized the data as to gross behavior of thlse families of F v In those families grown in F 4 and subsequent generations, a definite attempt was made to fix the original characters of the F 2 type selection in a pure line. The genealogical relation of these selected lines to each other is shown in the chart reproduced herewith. The letters, A, B, C, etc., correspond to thej<\ family designations noted in table 1, and the numbers refer to type selection numbers corresponding to the type illustrations in plate 63 to 787 or, in tne case of types 17 to 21, corresponding to those types as described in the succeeding accounts of the later generations. IF A 1922]* Setchell-Goodspeed-Clausen: Nicotiuna Tabacum 473 TABLE 2 F 3 FAMILIES OF THE ANG U S TI FOLIA-MA CROPH YLLA SERIES. Type Nos. Garden Numbers No. of plants in fam- ilies Results in F3 1 ^ ■• T "X XX X"» X"» IIF2H2P7P49 25 Segregated both as to leaf and flower color 2 1 i TP tt n n 1H , 2 H2P3P30 25 Segregated both as to leaf and flower color 3 1 1 TP TT Tl "Fl HF 2 H 2 P 3 Pi4 14 Segregated only as to leaf 5 -f 1 T71 tt xi n 11F2H4P41P14 24 Segregated both as to leaf and flower color 6 -< 1 r\ XT Tl T* IIF2H4P2PJ8 25 Uniform except as to length and development of wing of petiole 7 IIF2H2P13P48 25 Uniform both as to leaf and flower color 8 11F 2 H 2 P 3 P 4 1 25 Segregated as to leaf only 9 IIF2H4P41P8 25. Segregated both as to leaf and flower color 10 HF^PwPlT 24 Segregated only as to flower color 11 11F2H4P4!?!) 25 Segregated onlyfe to flower color 12 HF 2 H 4 P 4 lPl2 25 Segregated only as to flower color 13 HF2H2P3P44 25 Segregated only as to flower color 14 IIF.HP.Pm 25 Segregated both as to leaf and flower color 15 IIF2H2P3P10 25 Uniform, slight variation in tint and lobing of corolla 16 11F 2 H2P 3 P 8 24 Segregated only as to leaf 17 11F 2 H 4 P 36 P 2 7 25 Segregated only as to flower color 18 11F 2 H 4 P 35 P 38 25 Segregated both as to leaf and flower color 19 11F 2 H 4 P»P« 25 Segregated both as to leaf and flower color .^-^ 20 HF2H4P40P44 25 ^Uniform, close to macrophylla 21 HF2H4P41P29 25 Uniform, close to angustifolia 474 University of California Publications in Botany [Vol. 5 T In F, primary selection for parents of subsequent generations was based upon the type of leaf borne by the plant, flower color being followed as a secondary matter. In order to systematize the discussion concerning P 3 and subsequent generations, six general types have been selected and named and the discussion of the families has been grouped Pi 1909 Fi 1910 Fa 1911 Fa 1912 F* 1913 Fs 1914 Fs 1915 F? 1915 Fig. 1. — Chart showing the relationships of the various families of the angusti- folia-macrophylla series. The different F a hybrids are connected with their female parents; no seed was secured from H,. The F, family designations correspond to those given in table 1, and the numbers in later generations are the type numbers under which the populations are described in the text. F 4 and F 5 of type 12a were grown in 1914 and 1915 respectively. under these headings. The six general types selected and the names given them are as follows : a. Stenophylla derivatives. As a series these approximate very closely in leaf shape to angustifolia. The distinguishing feature of this series is the possession of a distinct, long petiole. b. Latifolia derivatives, which are characterized by the possession of a broad leaf with a petiole shorter than that of angustifolia. The petiole in these types is more or less winged. c. Lanceolata derivatives, which are characterized by the posses- sion of a lanceolate leaf like that of type 13, illustrated in plate 75. This is a non-petiolate form. d. Loriipolia derivatives, characterized by possession of long leaves with very narrow blades. The type specimen, type 12, is illustrated in plate 74. This also is a non-petiolate form resembling the lanceo- lata derivatives, from which it differs in the extreme narrowing of the blade. * 1922] Setchell-Goodspeecl-Clmisen: Nicotiana Tabacum 475 e. Auriculata derivatives. The typical form of leaf is that of type 10, illustrated in plate 72. The leaf blade of this form is char- acterized by an abrupt contraction of the blade at the base, nearly, if not quite, to the midrib. Clasping auricles, from which the name is derived, are usually present in this form. /. Sessilifolia derivatives, of which the leaf of macrophylla may be taken as the type. These derivatives are non-petiolate, as the name indicates. a. Stenophylla derivatives Type 1, as may be seen from the drawing of F 2 .(cf. pi. 63), seems very close to angustifolia, and had light pink flowers. There were 25 plants of 12F 3 H 2 P 7 P 49 , the leaves of which were carefully noted ; bujj one passed its flowering stage too early to be judged, so that the colors of the flowers of 24 only are known. Sixteen plants had stenophylla leaves very nearly of type 1, but the extent of the wing structure varied somewhat. The remaining 9 had lanceolata leaves of type 14. In flower color the segregation ratio noted was 5 red : 19 pink. No further generations of this line were grown. Type 17 was not selected for illustration in F,, but was a plant very close to angustifolia. It had, however, somewhat narrower leaves and deep pink flowers. The 25 plants of 12F„H 4 P :!5 P 27 were uniform and like the F 2 parent except as to flower color ; 3 were red and 22 were deeper or lighter pink. This line was not grown through further generations. Type 21, also, was not selected for illustration of the F 2 plant, but was chosen later for perpetuation because of its extremely close agree- ment with angustifolia. The F 3 , 12F 3 H 4 P 4 P 29 , consisted of 25 vigorous plants which seemed to be uniform to the finest detail and agreed in every respect with plants of angustifolia. There were noticed in the plants of this family peculiar fimbriae attached to the corolla, or split corollas, or, in one instance, a split hose-in-hose flower such as occurs at times also in pure angustifolia. This type seemed toHSe a pure recombination of the characters of angustifolia. Type 21 was continued through tf the individuals in the several families. In 1913, 94 plants of F 4 wfere grown ; in 1914, 85 plants of F, ; in 1915, 10 plants of F 0 ; and in 1916, 8 plants of F 7 . All these populations closely resembled one another as to individuals as well as those of the F 3 and the F 2 parent. They are all so close to angus- 476 University of California Publications in Botany [Vol.5 tifolia as to be practically indistinguishable from it. This line may be regarded, therefore, as a stable derivative very closely approxi- mating angustifoUa in all its characters. b. Latipolia derivatives Type 2 (cf. pi. 64) approached in F 2 fairly near to some of the variations of F x (cf. pi. 62). It might be regarded as a stenophylla derivative possessing an unusually luxuriant development of the wing of the petiole, but it seems more reasonable to classify it as a latifolia derivative exhibiting marked narrowing at the base of the lamina, such as is shown in lajsTceolata derivatives. The flower was designated as light pink in the notes taken at the time of flowering. The leaves of F 3 showed segregation through a considerable range, 6 being close to the latipolia type of F 1; 6 to the latipolia type of its F 2 parent (type^2), 8 were sessilipolia leaves approaching in type those of mac- rophylla, and 4 were auriculata leaves of type 10. In 13 the flowers were noted as light pink, in 11 as pink, and in 1 as red. No subsequent generations of this population were grown. Type 3, in F 2 , had (cf. pi. 65) a distinctly ovate lanceolate leaf with a short fairly broadly margined petiole. The flowers were light pink. F 3 , 12F 3 H 2 P 3 P 14 , amounted to 14 germinations,, all of which developed, although slowly, into healthy, normal appearing plants. The flowers were all the light pink color of the F 2 parent, agreeing with those of angustifoUa; but varying somewhat in shape and size. The leaves were of various shapes, 4 were distinctly petiolate, while 10 were sessile. Of the 4 petiolate plants the petiole of 1 was naked and of 3 more or less winged. Of the 10 sessilipolia plants, 9 were very similar to type 11, but 1 was rather longer and narrower, although otherwise approaching the same general shape. No further generations from this line were grown. Type 5 in F 2 (cf. pi. 67) resembled F 1 most nearly, but the winged petiole ^i&wghort and the corolla tube slightly stouter. The flower color was pink. Of 12F 3 H 4 P 41 P 14 , 24 plants were grown. Of these 23 had leaves alm\j|£ exactly like those of the F., parent, but 1 had leaves more nearly like aurictHlata o f 'J &££jLX<4- P L ^Q)- Of the 24 plants, 16 had flowers of various grades of pink while 8 had red flowers. This line was not grown in subsequent generations. Type 6 was represented in F 2 by a plant which resembled F, in having a winged petiole to the leaf and a pink flower. It is well fepre- 1922] Setchell-Goodspeed-Clausen: Nicotiana Tabaciim 477 sented on plate 68. In the 25 plants of 12F 3 H 4 P 2 P 18 , the height, habit, flower shape, and flower color were close to, if not identical with, those of the F 2 parent. As respects leaf base characters, 14 were latifolia of type 6, 4 had long, naked petioles, 3 had short, naked petioles, 1 had a long, winged petiole, and 4 were auriculata plants nearly of type 10. This seems like a considerable segregation, but the leaves are of only two generic types, viz., petiolate and non-petiolate. In subsequent generations selection was made in one line for latipolia leaves of type 6, and in the other for auriculata leaves of type 10, called type 6a to indicate its derivation. Of type 6 as thus established 100 plants of F 4 were grown in 1914 ; 100 plants of F 5 (50 each from 2 different parents) in 1915; 20 plants of F 6 in 1915; and 20 of F 7 in 1916. All the individuals thus grown were constant to latipolia of type 6 as originally selected. Of auriculata of type 6a, similarly, 100 plants of F 4 were grown in 1913 ; 94 of F 5 in 1914 ; 20 of F, in 1915; and 20 of F 7 in 1916. All these plants were uniform and true to auriculata of type 6a, very close to auriculata of type 10. In both th^ types we have definitely obtained stable recombinations of germinal elements exhibiting char- acters different from those or the parents. F 3 of type 7, 12F 3 H,P 13 P 48 (cf. pi. 69), consisted of a family of 25 plants, all vigorous except one (P 17 ), which was set out in the field later and developed into a "runt," as often happens with such later plantings. All the plants agreed well with one another in height and habit except the ' ' filler, ' ' and all agreed in inflorescence, flowers, and leaves. There were some variations in size and lobing of the limb of the corolla, indicating possibly minor segregation, but in all general characters there was uniformity to a large degree. The plants agreed well in all characters with the F 2 parent, and also with the F 4 parent. The color of the flower was light pink, the petioles of the leaves varied somewhat in length, were distinctly and more or less broadlya&iged, and the blade was heart-shaped, at least at the base in the lower leaves. In all respects these characters were no more variablejthan they were found to be in F 4 . Type 7 continued to breed true in subsequent generations. It was grown in 1913 (100 plants, F 4 ), 1914 (^families of 50 plants each, F s ), 1915 (10 plants, F 6 ), and 1916 (10 plants, F 7 ). All were uni- form as to leaf and flower color. Type 7 is very close to the type of F 4 and to type 6 described above. It, too, evidently represents a stable recombination of germinal elements derived from both parents. 478 University of California Publications in Botany [Vol. 5 Type 9 resembles type 5, but had iu F 2 a very short winged petiole and elliptical lanceolate blade. It also had pink flowers. F.,, 12F 3 H 4 P 41 P 8 , consisted of 25 plants, 18 of which showed latifolia leaves of type 9, but 7 had sessilifolia leaves of type 14 (cf. pi. 76). Twenty-one had pink (or light pink) flowers and 4 had red. No further generations of this line were grown. Type 19 was an F 2 plant of which no drawing was made, but it resembled ¥ 1 (cf. pi. 62), having broadly ovate leaves with a long and broadly winged petiole and pink flowers. F 3 , 12F 3 H 4 P 35 P 43 , con- sisted of 25 plants, 6 of which had sessilifolia leaves of type 16 (cf. pi. 78) or nearer, perhaps, to those of macrophylla, while 19 had latifolia leaves of type 19. In 5 plants the flowers were a somewhat darker red than they were in the other 20. This line was not followed further. c. Lanceolata derivatives Type 13 is similar to type 12 described below, but the leaves of the F 2 plant were more lanceolate and broader and the flowers were lighter pink. The 25 plants of 12F 3 H 2 P 3 P 44 were uniform and like F 2 except in flower color. Four were red, 19 decidedly pink, and 2 inclined to light pink. The line was not grown in subsequent generations. d. Lariifolia derivatives Type 12, as shown in plate 74, differed very decidedly in leaf shape from either parent. The long linear-lanceolate leaf had the long taper- ing curved tip of angustifolia, but the blade tapered below, making practically a new type. The flowers were like those of angustifolia in shape but were pink. The 25 plants of F 3 , IZFJIJ*^?^, were exact duplicates of F 2 as to habit, leaf, and flower shape, but 10 had red and 15 had pink flowers of various shades, mostly dark. None seemed as light pink as angustifolia. Thw*is the most interesting of the types carried through subse- quent generations, representing, apparently, a new combination of leaf characters. OT^^the pink flowering F 3 plants was chosen for seed and the designation, type 12, retained for this and its progeny, while the designation. 12a, was gjven to one of the red flowering F. plants also chosen for seed. Type 12, as thus limited to the pink flowered plant, gave scanty germination and few plants for F 4 in 1913. Apparently , it was still varying slightly in color within the pink shades, althouglwfairly uni- 1922 ] Setchell-Ooodspeed-Clausen: Nicotiana Tabacum 479 form except for one aberrant ( ?) plant of a decidedly lighter shade. Two "normal" parents of F 4 gave 88 and 100 plants of F 5 in 1914, which were uniform and of a bright pink color. In 1915 P 0 showed 10 plants, and in 1916 F 7 also showed 10 plants, still uniform and pink. Type 12a on being segregated in the second growing of F 3 in the season of 1913 yielded 100 plants of F 4 in 1914, uniform and of deep red flower color. F 5 , of 10 plants in 1915, and. also F 6 , of 10 plants in 1916, produced uniform individuals of deep red flower color. "We find, then, in types 12 and 12a definite fixations of the lorii- folia type, one with uniformly pink flowers and one with uniformly deep red flowers. e. Aubiculata derivatives Type 8 is represented in F 2 by a plant which had a leaf with an extremely constricted base (cf. pi. 70) and deep red flowers. It is not a typical auriculata derivative, but is included under this heading because it resembles the members of this class more closely tha n those of any other. F 3 , 12F 3 H 2 P 3 P 41 , consisted of 25 plan$& which were uniform in height, habit, and flower color, and in agreement with F 2 in these respects. The leaves, however, we^'of two distinct types, 16 auriculata of type 8 and 7 sessilifoliI of type 16 (cf. pi. 78), the latter being near to the type of macrophylla. No further generations of this line were grown. Type 10, as shown by the drawing (.pi. 72), had a peculiar leaf, near to the macrophylla type, yet deeply constricted at the base into a narrow and extremely abbreviated structure which may resemble a petiole or only a deeply constricted blade. There were, however, auricles partially clasping the stem and slightly decurrent. The leaf form was that characteristic of N. Tabacum var. macrophylla purpurea (cf. Setchell, loc. cit.). All 24 plants of F 3 had the same type of leaf as F 2 , but the flowers were of three fairly readily distinguishable shades ; 3 were red, 16 pink, and 5 light pink. F 2 had very dark pink flowers. The line was not grown in further generations. -^S* Type 6a is a true auriculata derivative which segregated in F 3 from an F 2 latipolia selection. Its occurrence and behavior are de- scribed in connection with the account of type 6, tttf latipolia type from which it segregated. Grown in the pure line for five generations it has remained constant for the auriculata type of leaf. 480 University of California Publications in Botany [Vol. 5 /. Sessilifolia derivatives Type 11 (cf. pi. 73) in F 2 gave 25 plants in 12F 3 H 4 P 41 P n , all vigorous except one, but that one showed the. same characters of leaf and flower as the others. All 25 plants possessed a sessilipolia type of leaf very close to the F 2 parent and uniform among themselves. There were two distinct shades of color of the flowers, 9 red and 16 pink. No further generations of this line were grown. Type 14 (cf. pi. 76), so far as F, is concerned, was one of those having sessile leaves of a broadly lanceolate type and pink flowers. There were 25 plants in 12F :! H 2 P 3 P 3S , 24 had sessilipolia leaves of type 14, while one (a "filler") had auriculata leaves like type 8; 19 had pink (or light pink) flowers, while 6 had red flowers. This line was not followed through subsequent generations. Type 15 (cf. pi. 77) was represented in F 3 , 12F 3 H 2 P :1 P 10 , by 25 vigorous plants which seemed surprisingly uniform and approached macrophylla very closely as to leaf and color of the flower. In the flower, however, the color seemed even darker than that of macrophylla, there were only slight traces of the white triangular markings on the limb, the limb Avas much more deeply lobed, and the tube less stout and with the infundibulum much less abruptly swollen. These differ- ences seem to indicate that type 15, which all the F 3 plants closely resemble, is not an exact recombination representing macrophylla. Type 15 was represented in 1913 by two families, F 3 of 10 plants and F 4 of 100 plants. Both families were uniform as to individuals, and agreed with the F 3 population grown in 1912 as well as with the F 2 ancestor of the season of 1911. As this line seemed to be constant and very close to, although not absolutely identical with, macrophylla, differing in flower shape and leaf shape to some extent, type 15 was considered to be a fixation and no further cultivation of it was made. Type 16 (cf. pi. 78), which in F 2 approached macrophylla very closerjin leaf, flower shape, and flower color, was represented in F 3 , 12F s IiS|gjBL by 25 plants. These were all alike and closely resembled the F 2 paren\in all respects except in leaf shape. Fifteen had sessili- . folia leaves ontype 16 while 8 had auriculata leaves approaching those of type Kl^efe-pl. 72). This line was not grown in further generations. Type 18 is the designation given to an F, plant, of which no draw- ing was made. It seemed close to macrophylla, but the flower color 1922] . Setchell-Goodspeed-Clausen : Nicotiana Tabacum 481 was pink and the leaves were more slightly attenuate at the base. F 3 , 12F 3 H 4 P 35 P 38 , gave 25 plants, 13 of which had the sessilipolia leaf of type 18 ; 7, auriculata of type 10 ; and 5, auriculata of type 8. In flower color. 17 were some shade of pink and 8 red. The line was not grown in further generations. Type 20 was not selected for illustration in F 2 , but was a plant chosen because of its very close resemblance to macrophylla, coming even closer than type 16. The F 3 , 12F.,H 4 P 40 P 44 , consisted of 25 vig- orous plants of remarkable uniformity. In height, habit, inflorescence, flower, color, shape, fruit, etc., the details follow those of macrophylla so closely as to be indistinguishable unless possibly by careful and labor- ious biometric study. This type may represent a practically pure recombination equivalent to macrophylla, and is to be compared and contrasted with type 15. In 1913 two families of F 4 , one of 21 plants and the other of 100 plants, were uniform, as were 3 families of 50 plants each of F, in 1914. In 1914, however, a surprising thing happened. ^A fourth family of F 5 , consisting of 50 plants, was uniform except one plant which had pink (instead of red) flowers and an auriculata leaf ap- proximating type 8 or 10. It seems certain that this plant must have been an intrudeV, but its seed was saved under bag and grown and is noted below and on the pedigree chart as type 20a. The other 3 plants of F 5 whose seed was sown in 1915 gave type 20 in F c in families of 10, 9, and 8 respectively, and in turn the seed of 4 individuals of "pure" type 20 gave, in 1916, uniformity in families of 10 each. Type 20a, which originated or intruded in 1914, in one plant of F 5 of type 20 gave in F„, in 1915, 10 plants segregating for flower color and probably also for leaf characters, although the notes taken are inconclusive on the latter point. In 1916 F 7 of 10 good plants showed uniformly red flowers, but 7 had sessilifolia leaves, 4 of which were decidedly contracted at the base and 3 had very short winged petioles (auriculata of type 8 or type 10). On the whole it seems most likely that the single plant in the F 5 family was an Intruder, since all other families of the line have been constant sinee F 2 . A stray seed somewhere along the processes of culture would explain it and its appearance is all the more incomprehensible asra matter of inclu- sion in the pedigree of type 20, as it is so close "to macrophylla as to seem practically identical with it. 482 University of California Publications in Botany [Vol. 5 5. SUMMAKY OF FLOWER COLOR OBSERVATIONS IN F 2 AND SUBSEQUENT GENERATIONS Iii tables 3 and 4 we have summarized the numerical data with respect to flower color inheritance in F 3 and in the subsequent popu- lations. In table 3 are assembled data with respect to the behavior of red flowering selections from populations segregating for red and pink. It will be noted that all the five selections which were made bred true for red flower color in the succeeding generations. In table TABLE 3 INHERITANCE OF RED FLOWER COLOR IN Fa, el seq. Type Numbers Garden Numbers Flower color of population 8 12F 3 H 2 P 3 P« 25 red 12a 14F 4 H 4 P 41 P 12 P, 100 red 15 12F 3 H 2 P 3 Pi 0 25 red 16 I2F3H2P3P8 24 red 20 12F 3 H 4 P 40 P 44 25 red TABLE 4 POPULATIONS FROM PINK FLOWERING SELECTIONS OF ALL SHADES IN F 3 et seq. Flower color classification Type Parent Num- Garden Numbers Color bers red pink light pink 3 I2F3H2P3P14 light pink 14 6 12F 3 H 4 P 2 P 1S pink 25 7 I2F3H2P13P.18 light pink 25 12 I3F4H4P41P12P8 pink 88 12 13F 4 H 4 P 41 P I2 P 9 pink 100 21 12FsH 4 P 4 P 2 9 light pink 25 1 12FsH 2 P7P49 light pink 5 19 (or light pink) 2 12F3ILP3P30 light pink 1 24 (13 light pink) 5 12F3H4P41P14 pink 8 16 (or light pink) 9 K 12F 2 H 4 P4,P 8 pink 4 21 (or light pink) 10 12F 3 H 4 P4iP 17 pink 3 21 (5 light pink) 11 ^gF3H 4 P4lP 9 pink 9 16 (0 light pink) 12 12F 3 li 1P41P1J pink 10 15 (0 light pink) 13 12F 3 i8^P 4 4 pink 4 21 (2 light pink) 14 12F 3 H 2 PXj)8 pink 6 19 17 12F 3 H,P 4 P|P i7 pink 3 22 18 12F3H 4 P 36 Pl> r> - pink 8 17 19 12F 3 H 4 P 35 P 4 3 pink 25 (5 darker red) Totals of segregating populations 60 187 (Types 2 and 19 excluded) I 922 ] Setchell-Goodspeed-Clausen: Nicotiana Tabacum 483 4 are assembled the data from ptnk flowering selections from popu- lations which showed segregation into red and pink. In this table the populations which bred true for pink are assembled in the upper portion of the table, and those which showed further segregation into red and pink are assembled in the lower portion. Of the 18 selections made, 7 bred true for pink (or light pink), and 10 gave segregation in the succeeding generation in about the ratio of 3 pink : 1 red. The total figures for the 10 populations — 187 pink : 60 red — are in very satisfactory agreement with the simple Mendelian ratio. The family of type 19 behaved in an anomalous fashion, which may indicate mis- classification of the F 2 parent ; and the family of type 2, which showed only one red plant has been included among those which bred true for pink. Strictly light pink selections should have given only light pink flowers in subsequent generations ; the pink ones should all have given I segregating populations. The evidence indicates that this result would be obtained if segregation -occurred for only one pair of allelomorphs. The difficulty, in part at least, appears to be the result of segregation of modifying factors in the populations. These factors apparently have an effect on flower color sufficient to obscure segregation into pink and light pijik, but not enough to obscure the segregation into red and pink. The actual results indicate an approximate agreement with expectation, but the breeding test clearly is necessary in order to determine the actual distribution of the pink individuals into their genetic classes. 6. LATER SOWINGS OF F 2 AND F 3 OF THE ANGUSTIFOLIA- MACEOPHYLLA SERIES In 1916 and 1917 certain families of F 2 and P 3 of H 2 were grown in order to reexamine them in the light of data previously collected and to determine whether or not any more definite classifications could be made than those stated in the preceding pages. The populations grown are described briefly below. 16F 2 H 2 P 0 , as the population number would indicate, was a sowing of seed of lOFjtpPg from the original F t population of EL. As in previous cases, the segregation as regards leaf shape (was so complex as to preclude definite classification. The types previously noted for second generation populations were all in evilfllnce and along with them practically every sort of intermediate. The height of plants and general habit likewise agreed with the description previously given. 484 University of California Publications in Botany [Vol.5 It was possible as in previous instances to segregate the plants into definite flower color classes. In order to make this segregation as accurate and free from bias as possible a special method of classifica- tion was adopted. At the height of the blooming season, single typical flowers were collected from each plant of a population and placed in vials correspondingly numbered. These specimens were then taken into the laboratory, where they could be classified under optimum light conditions. The specimens so collected could then be shifted around into their phenotypic classes and properly compared with each other and with the parent colors. The color classification thus obtained was individually recorded, and later the population was checked over in the field to insure correction of any errors of classification. The surprising feature of this population was a sharp, three-class segre- gation into red, pink, and light pink; the reds the shade of macro- phylla, the light pinks almost exactly that of angustifolia, and the pinks intermediate between the two. Within the classes there ap- peared to be no significant differences in depth of shade. Two plants bore no flowers. The ratio obtained was 15 red : 23 pink : 10 light pink. 16F„H„P 17 was likewise a sowing of the seed of one of the original F x plants, in this instance of 10P 1 H 2 P 17 . As respects habit, height, and leaf shape, there was a strict resemblance throughout of this population to the one described above. Flower color was studied in the same manner and with substantially the same results. However, in this population there was a shading off from pink to light pink, such that it was impossible to draw a sharp line between these two classes as was done in the previous population. The shading off was abrupt, but there were, nevertheless, a few plants on the border line. The observed ratio was 16 red : 34 pink and light pink. Hp 1917 six F 3 populations, each containing approximately 100 planra| were grown in order to make further studies of the inheritance of leaiAhape. It was impossible, however, to study these plants as thorougmpas might have been desired on account of conditons ob- taining duriHtt 1917. However, specimens of leaves from each plant were pressed preserved and these were studied and classified in the summer of 19l\ A brief account of each population follows: 17F s H 2 P 17 P e w3%erssowing from 16F„H 2 P 17 P 6 , a stenophylla selection. With respect tVj.eaf base characters the segregation was phylla class approximating type 1 in appearance, and an auriculata roughly but rather STENO- 1922] Setchcll-Goodspced-Clausen: Nicotiana Tabacum 485 class approximating type 10. Within the stenophylla class there was a variation in the amount of "wing" on the petiole and in the type of blade base, some having the abrupt base of type 1, whereas others had an attenuated type of blade which gradually drew in to the petiole. In the auriculata class there was also a variation from the strict form of type 10 to a type which lacked the flaring auricle typical for that form, and had a very short naked petiole. In addition to this variation in the amount of "wing" of the constricted class there was also a difference in the presence or absence of attenuation noted for the stenophylla class, some plants having leaves abruptly drawn in to the midrib, whereas others were very markedly attenu- ated. The difference in this respect appeared to be equivalent in the two distinct classes, i.e., it was independent of any difference in the ' ' petioled " or " constricted ' ' condition. "With respect to stenophylla? vs. auriculata the segregation was 66 stenophylla : 32 auriculata. 17F 3 H 2 P 17 P 8 was a sowing from 16P 2 H 2 P 17 P 8 , another steno- phylla selection.^ The leaf classes obtained here were two,' steno- phylla (type 1) and sessilifolia (type 15). The segregation into the two classes was distinct, but, as in other populations, there was a great deal of variability in each class. There was attenuation of the kind previously noted in both classes. Some of the petioled individuals had distinct wings, but the larger number were naked. Some few individuals had very short petioles. The segregation ratio was 76 stenophylla : 24 sessilifolia. 17F 3 H 2 P 17 P 19 was a sowing from 16F 2 H 2 P 17 P 10 , a stenophylla selection. The population was remarkably uniform in leaf shape, which closely approximated angustifolia with minor differences. The straplike leaves which are a characteristic feature of the upper por- tions of plants of angustifolia were lacking in this population, and the leaf tip and distal portion of the leaf blade did not narrow so gradually in this population as in angustifolia. Otherwise, the char- acters of the plants throughout were closely similar to angustifolia. 17F,H 2 P 17 P 22 was a sowing of seed of 16F 2 H 2 P 17 P 22 , a stenoph^la selection. Of the six F 3 populations studied, this one exhibit^ the greatest diversity in segregation. With respect to leaf base characters, there were two outstanding classes, stenophylla and SESSidfOLiA (type 15), which could be separated readily. Within the stenophylla class, however, most of the individuals exhibited a more cnfness winged con- dition. Within the sessilifolia class, on the other hand, most of the individuals exhibited more or less narrowing of the leaf base, like 486 University of California Publications in Botany [Vol.5 type 14. A few of the sessile individuals, instead of exhibiting gradual and uniform narrowing toward the base of the leaf, were constricted to a degree intermediate between auriculata of type 10 and sessili- polia of type 15. With respect to stenophylla versus sessiltpolia the observed segregation was 67 stenophylla : 32 sessilipolia. 17F 3 H 2 P 17 P 3 was a sowing of seed of 16F 2 H 2 P 17 P 3 , an F 2 sessili- polia selection. The leaves throughout had the sessile type of leaf base characteristic of macrophylla, but there were many modifications of it in the population. A rough classification with respect to these modifications of the macrophylla type of leaf base gave the following results : On 59 plants, the leaf bases were very nearly the form typical for macrophylla. On 22 plants, the leaf bases were gradually attenuated toward the base, resembling lanceolata of type 13 as a mean. This at- tenuated form of the sessile leaf was a very striking feature of this population. On 10 plants, the leaf bases were intermediate in type between lanceolata of type 13 and the typical macroph ylla form. On 2 plants, the base of the leaf immediately above the point of attachment was noticeably constricted, the leaf base thus formed being intermediate between the macrophylla type and auricu- lata of type 10. On 2 plants, the leaves were intermediate in constriction of the leaf base between the strict macrophylla type and that of the two plants described immediately above. The classification here given is presented only to show that the sessile type of leaf base characteristic of macrophylla is subject to a number of very definite modifications which probably account for some of the complex types of segregation observed in other populations. 17F 3 H,P 17 P 12 was a sowing from 16F 2 H 2 P 17 P 12 , a sessilipolia selection. With respect to leaf base segregation there were two dis- tinc»classes, sessilifolia (type 15) and auriculata (type 10). There was hWl also a marked degree of variation within the classes. Within the sessiT^plass the variation was in amount and kind of narrowing of the leaf blade toward the base. A few plants showed a condition approaching Ae auriculata type in this respect, whereas others showed a graduatt^Jtenuated form of narrowing such as has been noted before in other populations. Within the auriculata class most of the individuals instead of possessing the slight wing and flaring I 922 ] Setchell-Goodspeed-Clausen: Nicohana Tabacum 487 auricles of type 10 had short naked petioles. A few were strictly of type 10. The following segregation ratio was noted : 61 sessilifolia : 27 AURICULATA. 7. CROSSES OF DERIVATIVES WITH THE PARENTS In the preceding account we have pointed out that by growing definite hybrid selections in the pure line through a number of gener- ations it has been possible to establish a certain number of stable derivatives which represent more or less obvious recombinations of characters of the original parents. In a Mendelian sense, they repre- sent stable reorganized germinal complexes containing hereditary ele- ments that have been derived from both parents. Obviously such recombinations of Mendelian units must differ in fewer units from either parental type than did the parental types from each other. To test some of these derivatives we have crossed them with the original parents, usually with the one to which they bore the closest resem- blance, in order to observe how complex a type of segregation the hybrids thus obtained would exhibit as compared with that of the original angustifolia-macrophylla hybrids. In so far as they have been studied to date, a description of these hybrids and their progenies follows : SESSiLiFOLiAxmacrop7f ?/^«. P 5 sessilifolia of type 20 was crossed with macrophylla giving H 50 = type 20J x macrophylla,^ and H 5t = reciprocal thereof. The derivative parent here very closely resembles macrophylla throughout in flower color and shape, habit, leaf shape, etc. ISFiHjo and two families of 50 plants each, were equiv- alent in every respect. The plants were very close indeed to macro- phylla, as is also the sessilifolia parent. The only difference readily observed was some variation in the amplitude of the corolla. In H-,, a plant with a larger and one with a smaller corolla were selected for pure seed. In F 2 , grown in 1916, one family of H 50 and two fakiilies of H 51 , of 50 plants each were grown. The flower color in^the F 2 populations was throughout that of macrophylla and the le£f charac- ters also were those of macrophylla. All three families jrere remark- ably uniform, not only agreeing with one another butfiiniform as to indivduals. They all resembled closely the macrophj/Ua type and there was no definite segregation of any kind in themfi The three popula- tions appeared to be replicas of macrophytta throughout except that they were slightly more robust. 488 University of California Publications in Botany [Vol- 5 Latifolia x angustifolia. F 5 latipolia of type 6 was crossed with angustifolia giving H 52 = type 6$ x angustifolia^ and H 53 , its recip- rocal. The derivative parent possessed the short winged petiole char- acteristic of latifolia of type 6. In ¥ 1 50 plants of each cross were grown. They exhibited the long naked petiole characteristic of angus- tifolia. In F 2 two" populations of 50 plants each were grown. In color of flowers the two populations were light pink throughout, closely cor- responding in this respect to angustifolia. In leaf shape the segre- gation was sharply into two classes: the stenophylla type of leaf base (long, naked petiole) and the latifolia type (shorter, winged petiole). There was some variation in the stenophylla class sug- gesting intermediacy between angustifolia and latifolia, but the forms exhibiting it showed a graded series from strict stenophylla to inter- mediate. The latifolia class was very uniform and sharply set off from the other class. The segregation ratios observed were as follows : Stenophylla Latifolia 16F 2 H 62 Pi 6 42 8 16F 2 H 53 P 36 36 14 Totals 78 22 Auriculata x macrophylla. F 5 auriculata of type 6a was crossed with macrophylla giving H 54 and H 58 — F 5 type 6a§ x macrophylla^ and H,- and H 39 , their reciprocals. It should be observed that type 6a is an early segregant from the latifolia of type 6 of H 5 „ and H 53 . In F x 50 plants were grown of each of the four parents. All four populations were equivalent in every respect. All the plants had pink flowers, although one plant had flowers of a lighter shade than the others, and leaves of a shape somewhat intermediate between the two parents, i.e., they were more contacted at the base than macrophylla. but much less so than those of type 6a. One planl of B 55 , namely lSFjBwPm, showed larger corollas than any of the other F t plants of any^femily, and was selected for further breeding. In F||£our families were raised and they proved to be equivalent in all respelte. except as noted. There was sharp segregation for leaf shape into the|BjSSiLiFOLiA and the sharply constricted auriculata type. In the SESSiLiFom class there were a number of obvious intermedi- ates, as might be expected from the characters exhibited by F 1( but they formed a continuouV-series with the strict sessilifolia forms. The auriculata class did not intergrade with the dominant class. 1922 ] Setchell-Goodspeed-Clausen: Nicotiana Tabacum 489 Segregation for flower color was studied by the method described above. The color distinction between red and pink was sharp and easily drawn. In the pinks, however, there was a continuous series of shades from the deep" rose pink characteristic of F x to the light pink typical for angustifolia. Numerical data are given in table 5. TABLE 5 F 2 SEGREGATION OF PINK AURICULATA X RED SESSILIFOLIA. Garden Numbers Pink sessilifolia Red sessilifolia Pink auriculata Red auriculata 16F 2 H 54 P 7 32 6 9 3 16F 2 H 55 Pi6 23 11 11 5 16F 2 H i5 P 29 26 13 7 4 16F 2 H 5S P 26 26 11 7 2 16F 2 H Ma P 35 26 12 6 - 2 Totals 133 53 40 16 Expected 136 45 45 15 In 1918 in connection with flower size studies three more popula- tions of F„II 35 were grown. Leaves were collected from each plant and pressed, and leaf shape studies were made on these preserved specimens. The studies were not so satisfactory as those made in the field, where it is possible to examine all the leaves on a given plant ; nevertheless, the data derived from the studies agreed substantially with those obtained in 1916 from field studies. It was noted in these studies that there was a distinct class of "attenuated" leaves similar to those which have been described in previous populations. Both attenuation and constriction were observed to occur in the leaves of some individuals, and this gave rise to some difficulty in classification. Numerical data are given in table 6. TABLE 6 F 2 SEGREGATION OF SESSILIFOLIA X AURICULATA. Garden Numbers Sessilifolia AuRIC^nj.TA 18F 2 H65P40 67 18F 2 H 55 P 41 79 J* 18 18F 2 H 55 P<6 79 17 Totals 225 63 490 University of California Publications in Botany [Vol. 5 Stenophylla x an gusti folia. Reciprocal crosses were made between F 5 , stenophylla of type 21 and angustifolia; H, 0 = F 5 type 21$ x angustifolia,^ and H g7 , its reciprocal. Stenophylla of type 21 has been described previously as a stable derivative closely approximating angustifolia in all its characters. F 1 families of 50 plants of each hybrid were raised in 1915. They were uniform throughout and so close to angustifolia in all characters as to be indistinguishable from it. One plant seemed to be of a slightly darker pink corolla color. lSFoHggaPn was the only F, family raised. The flower color of this population was about the shade of angustifolia and uniform through- out the population. The family showed only a slight variation in the base of the blade such as is also seen in populations of angustifolia. S. DISCUSSION OF EESULTS OF THE ANGUSTIFOLIA-MACEOPHYLLA SEEIES OF INVESTIGATIONS Obviously the outstanding result of this series of investigations of hybrids between angustifolia and mucrophylla is a demonstration of the complexity of the germinal differences which exist between the two varieties with respect to practically every character contrast which may be made between them. Only in one instance, the contrast be- tween the light pink flower color of angustifolia and the red of macro- pkylla, is a simple Mendelian formulation possible. Here evidently the main flower color difference is dependent upon a simple allelo- morphic contrast. Red x light pink gives ¥ 1 intermediate pink, and F 2 1 red : 2 intermediate pink : 1 light pink. The red segregants breed true for red, the light pinks for light pink, and pink continues to segregate in the typical mono-hybrid fashion. Inasmuch as the inter- mediate pinks and light pinks form an intergrading series, it is con- venient to look upon red as the recessive color. Accordingly we give this pair of factors the designation, Rr, following the mnemonic system advocated by Morgan, and shall so refer to it in what follows. The difficulty among the pinks appears to be due not only to phenotypic variant but also to the existence of modifying factors which have a relativeryyjlight effect upon flower color expression. These less strik- ing modifica^ons of flower color we are seeking to analyze further. In the leafilshape investigations, the complexity of the results is plainly evident from an examination of the data presented in the fore- going pages. Although the behavior here is complex, in every feature it parallels the Mendelian expectation for complex factor relations. < 1922 J Setchell-Goodspeed-Clausen: Nicotiana Tabacum 491 In F 2 the variety of leaf shapes encountered was nothing short of bewildering and series could be built up from them showing complete intergradation from one type to another. Selection of phenotypes from F 2 , however, gave P 3 populations in which the complexity of segregation was usually reduced in a very definite fashion. Most of the populations exhibited fewer classes than F,, and the selection of F 2 phenotypes held the expression of F 3 within very definite limits. Thus selection of sessilifolia forms gave in F 3 either all sessilifolia or approximately 3 sessilifolia : 1 auriculata. In no case did such selections give F 3 populations with stenophylla or latifolia leaf types. A summary in detail of the type of populations produced is as follows. Stenophylla selections may segregate in a variety of ways. Thus, type 1 showed approximate segregation into 3 stenophylla : 1 sessi- lifolia. Type 21 bred true to the stenophylla characters. Among stenophylla selections grown in 1917, population 17F 3 H 2 P 17 P 6 , showed approximate segregation into 3 stenophylla : 1 aurtculata; 17F 3 H 2 P 17 P 8 , 3 stenophylla :1 sessilifolia; 17F 3 H 2 P 17 P 9 bred true for stenophylla, and 17F 3 H 2 P 17 P 22 gave a rather indefinite segrega- tion of approximately 3 stenophylla : 1 sessilifolia. Latifolia derivatives crossed with angustifolia gave ¥ 1 stenophylla and F 2 approximately 3 stenophylla : 1 latifolia. Latifolia selections also segregate in perplexing fashion. The F x population of angustifolia x macrophylla is typically latifolia in its characters. Latifolia under certain conditions therefore is a very complex hybrid expression. Recurrence of complex segregation of a latifolia selection is shown in F, of type 2. F a of type 3 exhibited a rather anomalous segregation ratio of petioled and sessile forms. Type 5 apparently bred true, although there was one anomalous plant in the population. Type 6 exhibited complex segregation, with an indication of a ratio of 3 latifolia :1 auriculata; with subsequent establishment of both latifolia and auriculata in constant races. Type 7 bred true for a type of leaf like F x ; and type 9 gave approxi- mate segregation of 3 latifolia : 1 sessilifolia. J± Loriifolia and lanceolata derivatives are really variations of the sessilifolia type. They were both produced in constant^aces. Their genetic relation to the other forms is, however, nojpwell established by this series of investigations. Although these Jswo are really quan- titative variations from the strict sessilifolia type, nevertheless, cer- tain of our data indicate discontinuous inheritance of these contrasts. ■±92 University of California Publications in Botany (Vol. 5 The same quantitative factors that differentiate the narrow-leaved forms of sessilipolia from the typical broad-leaved forms may apparently differentiate narrow-leaved stenophylla, latifolia, and auriculata forms from the more typical broad-leaved ones. It is of interest in this connection to note that loriifolia derivatives have much narrower leaves than either of the original parents. We have been especially interested in these loriipolia derivatives because they are somewhat like the narrow-leaved forms that Hassel- bring (1912) found among Cuban tobaccos, and which are so well recognized among Cuban growers as to have received the specific desig- nation of lengua de vaca or "cow's tongue." Our results indicate that it is possible for such forms to arise by segregation from crosses between broader leaved forms. The lengua de vaca of the Cuban growers is, therefore, probably a segregation product which could easily be eliminated by the adoption of proper pure line methods of breeding. Auriculata forms appear to breed true whenever segregated. The exception is type 8, which requires further investigation. It may be a leaf type similar to auriculata but of different genetic constitution. Auriculata of type 10 bred true in F 3 . The auriculata form 6a, which segregated from type 6 bred true thereafter. Auriculata crossed with macrophylla, H 54 , H 55 , H 58 , and H- 0 , gave sessilipolia in Fj and in F 2 3 sessilifolia : 1 auriculata. Sessilipolia forms have broad sessile leaves, the distinguishing feature being merely their sessile mode of attachment. Of such selec- tions from the original F 2 populations, four, with the exception of one anomalous plant, bred true for sessilipolia. Each of the other three populations segregated into sessilipolia and auriculata in about the ratio of 3 sessilipolia : 1 auriculata. Two sessilipolia selections were grown in 1917. One of these bred true to sessilifolia ; the other gafce 3 sessilifolia : 1 auriculata. The behavior of sessilifolia in relation to stenophylla and latifolia is explained above. On the basis of these results we may distinguish certain definite allelonwphic pairs of factors as follows Ss, sfjgropiiYLLA versus sessilifolia : SS being long petioled like angustifom^&nd ss broadly sessile like macrophylla. The heterozygote may possibly ap«roach an intermediate condition similar to latifolia. LI, STENOPHYnL* versus latifolia : LL being long petioled like angustifolia, and 11 short petioled like latifolia and with a distinct but not broad wing. Tne contrast is really one of SSLL, stenophylla i 922 ] Setchell-Ooodspeed-Clausen: Nicotiana Tdbacum 493 versus SS11, latifolia. Both ssLL and ssll are probably typical sessi- lifolia forms. Here again the heterozygote probably shows an indis- tinct type of intermediacy. Aa, sessilifolia versus auriculata : AA having the broad clasp- ing leaf base characteristic of macrophylla, and aa the deeply con- stricted leaf bases with flaring auricles characteristic of auriculata. The contrast here is really one of ssAA, sessilifolia versus ssaa, auri- culata, for these factors are evidently latent when in combination with SS or Ss. Fig. 2. Leaf base types of the angustifolia-macrophylla series. Left to right: STENOPHYLLA, LATIFOLIA, SESSILIFOLIA, and AURICULATA. Some of the possible genotypes, their phenotypic expression, and genetic behavior are included in table 7. Here only monohybrid segre- gation is considered because it is doubtful, on account of the various types of intermediacy shown by heterozygotes, whether it would be possible to classify dihybrid and trihybrid populations satisfactorily. TABLE 7 genetic behavior of various leaf type genotypes. Genotype Phenotype Genetic behavior SSLL A A STENOPHYLLA Breeds true SsLLAA STENOPHYLLA 3 STENOPHYLLA 1 SESSILIFOLIA SSL1AA STENOPHYLLA 3 STENOPHYLLA 1 LATIFOLIA SSLLAa STENOPHYLLA Breeds true SsLLaa STENOPHYLLA 3 STENOPHYLLA 1 AURICULATA SS1IAA LATIFOLIA Breeds true SsllAA LATIFOLIA 3 LATIFOLIA LlfesSILIFOLIA Ssllaa LATIFOLIA 3 LATIFOLIA ^ fl AURICULATA ssLLAA SESSILIFOLIA Breeds true r* ssllAA SESSILIFOLIA Breeds true ssllAa SESSILIFOLIA 3 SESSILIFOLIA 1 AURICULATA ssllaa AURICULATA Breeds true 494 University of California Publications in Botany [Vol..") Although intermediacy of the heterozygote appears to be the normal thing in these leaf shape contrasts, it is proper to state that this inter- mediacy may depend to some extent upon the effect of modifying factors rather than upon the heterozygous conditon of a pair of allelo- morphs. Thus the intermediate conditon between stenophylla and auriculata is met with in populations which do not contain auricu- lata segregation products. There are so many modifying factors in this series of investigations that it is probably impossible for us to declare definitely that in any one instance our observed segregation was wholly the result of segregation of one pair of allelomorphs. Further investigations are in progress, the purpose of which is to isolate and evaluate, if possible, certain of these subsidiary factors. For the present we can only state our certain knowledge of their existence, and our belief as to their various effects. IV. CALYCINA-VIRGINICA SERIES The calycina-virgimca series of hybrids and derivatives has received much less attention than has been given to the previous series ; partly because the differences between the parents are less striking and the diversity of segregation products was not so great. Two hybridizations were made : H 18 which had calycina for the female and virginica for the male parent and H 20 which was the reciprocal cross. 1. PAEENTS OP THE CALYCINA-VIRGINICA SERIES Elsewhere Setchell has given descriptions of calycina and virginica ("Maryland"). Like angustifolia and maerophylla, these two varie- ties possess distinct sets of characters which set them apart from the other Tabacum varieties that have been grown in the University of California Botanical Garden. Calycina is represented in our cultures by a variety, U. C. B. G. 110/05, which was originally received from the Botanical Gardens of CamT|ridge University. The figure previously published (cf. Setchell. loc. cWj^xA. 4) well represents the general habit and type of the plant. The para&ular features of the characteristic teratological flower of calycina arajbetter shown in plate 79, in which the leaf shape is also illustrated inTtpre characteristic fashion. For illustrations of some of the variationsrWuch occur in the expression of the split hose-in-hose flowers the reader is iWerred to Goodspeed and Clausen, 1917, plate 45. The legends to the twoHgures of this plate should be reversed as indi- cated in the references to the plate in the text of this earlier article. 1922] Setchell-Goodspeed-Clausen: Nicotiana Tabacum 495 In stature, as previously mentioned, calycina belongs to the low corymbose group of Tabacum forms. In height the central axis usually varies between 60 and 75 cm. The laterals, however, which develop later from the base, overtop the central axis and reach a height of 120 to 135 cm. Like macrophylla, central axis and laterals bear close panicles of corymbose racemes, the laterals developing successively from the base. The stems and branches are stouter than those of angustifolia and the leaves do not droop so considerably. In these respects calycina occupies an intermediate position between angusti- folia and macrophylla. The leaves of calycina, as plate 79 will show, are sessile, but they are distinctly different from those of either angustifolia or macro- phylla. Curiously enough, however, they do rather closely approxi- mate certain of the derivatives of the angustij ' olia-macrophylla series, as, for example, the lanceolata and loriifolia leaves of types 13 and 14 respectively, illustrated in plates 75 and 76. The leaves vary from broadly to narrowly lanceolate, tapering toward both base and apex, and usually with a long curved tip. The broader leaves are borne at the base of the plant, those above them becoming successively narrower in a continuous series until the linear leaves or straplike bracts of the inflorescence are reached. There are no auricles at the base of the leaf. The inflorescence is in the form of a very close panicle of racemes, the secondary axes of which are mostly patent, and more or less re- curved or bent back. The flower as a whole is of a very characteristic split hose-in-hose type. The corolla is usually split on one side, some- times twice split, and more or less curved. The characteristic split- ting of the corolla is seen even in very young buds and often the pistil protrudes from them. Typically the calyx has an elongated whitish green tube, with 3 to 5 of its tips more or less petaloid. Sometimes strips of petaloid tissue extend down the entire length of the calyx. The pod is ovoid oblong in shape. As it enlarges it splits the calyx, which then withers and drops off like the corolla, leaving a naked, whitish green capsule. The flower color is red fading to bluish purple, apparently the same as that of macrophylla. Virginica is represented by U. C. B. G. 78/05, a strah£received from the United States Department of Agriculture unde/rae identi- fication number "205-20-7." It is figured by Setchar^ plate 3, and by Goodspeed and Clausen, plate 41, figure 1. Thaftypical leaf and flower characters are well represented in plate SQJfeTewith. In stature virginica belongs to the modeKatfe pyramidal group of Tabacum varieties. It is conspicuously taller than calycina, the cen- 496 University of California Publicat ions in Botany |; Vol. 5 tral axis reaching a height of 150 to 175 cm. While strong laterals develop they do not originate at the base of the plant as in calycina, and they do not overtop the central axis. These characteristics to- gether with the broad spreading basal leaves give the plant its pyra- midal or conical shape. The leaves are very close in general shape to those of calycina, but they taper less abruptly to either end. The apex is prolonged into a fairly long point curved to one side, and the base is expanded into two broad, partially clasping auricles. The inflorescence consists of a more ample panicle than that of calycina. The flowers are light pink in color, identical in this respect with those of angustifolia. The tube and infundibulum are narrow, gradually increasing in diameter from below in a funnel-shaped fashion. The corolla lobes are broad at the base, but have long, slender incurved points. Capsule and calyx present no very charac- teristic features, although the calyx is persistent in contrast to the deciduous calyx of calycina. It will be seen from the foregoing descriptions that there are a number of distinct character contrasts between calycina and virginica, a brief note of which may well be made at this point. In flower color, red of calycina is contrasted with light pink of virginica, the same contrast which existed in the angustifolia-macrophylla series. Simi- larly the split hose-in-hose flower of calycina is contrasted with the normal one of virginica; low stature with tall ; and a less decided contrast in leaf shape exists, depending upon the presence or absence of auricles at the base of the leaf. 2. F 1 OF THE CALYCINA-VIRGINICA SERIES In the season of 1910, 55 plants of lOP^^ and 58 of lOF^,, were grown. In the season of 1911, 10 plants were grown of each of kFJI 18 and llF^. Like other hybrids which have been grown, these populations were uniform and equivalent throughout. It was thought that IOFjH.,,, showed a more distinctly pronounced trace of calycina characters than did lOFJI^, but the populations of the same seed grown in 1911 showed njLappreciable difference in this respect. In generliappearance the F x plants resembled virginica more than they did calycha. The plants were somewhat shorter than virginica, running up to 135 cm. In these plants it was noted that some of the laterals overtopped the central axis as they do in calycina. The in- florescence was in general of the ample type characteristic of calycina. 1922] Setchell-Goodspeed-Clausen : Nicotiana Tabacum 497 The flower color was a deep pink intermediate between the two parents. The flower shape was normal throughout save that on every plant there was a small percentage of calyces with one or more pink and somewhat broadened calyx tips, or with a streak of white on one side. Sometimes these partially petaloid calyces were partly decid- uous. In shape of flower the hybrid closely resembled virginica except that the corolla lobes were longer and more decidedly mucronate. Calyx and capsule were almost identical with those of virginica, the calyx being typically persistent and accrescent. The leaves were some- what broader proportionately than those of virginica, but they pos- sessed the pronounced auricles of that parent. The usual gradation in leaf shape on each plant from the broad basal leaves to the linear bracts of the inflorescence was in evidence. The main features of H 18 and H 20 are well illustrated in the draw- ings of 10F 1 H 18 P 5 shown in plate 81. The general habit and charac- ters are illustrated in the photograph of 10F 1 H 18 P 54 which is repro- duced in plate 83, figure 1. 3. F 2 OF THE CALYCINA T VIRGINTCA SERIES In the season of 1911 four F 2 families of the calycina-virginica series were grown, viz., 11F 2 H 18 P 2;; , 11F 2 H 18 P 4!1 , 11F 2 H 20 P 7 , and 11F 2 H 20 P 26 . As in the angusUfoUa-macrophylla series, the segregation exhibited in those four families, comprising 97 plants, was nothing short of be- wildering, and in most cases an intergrading series of forms connected one character expression with another. However, an attempt was made to classify the plants into categories suggested by the four pairs of character contrasts existing between the parents. The results of this classification are given in table 8. TABLE 8 NUMERICAL DATA FROM F 2 POPULATIONS OF THE CALYCINA-VIRGINICA SERIES. Corolla color Corolla shape Stature Leaf width pink in-hose alhose- se S a 'f Garden Numbers -a M a *j J3 V ■a o s h 0 •3 V -A « o o u H 'a .SP o ■a O a J3 00 a "3 /> 1 f-> x> 13 a 11F 2 H 18 P 25 6 11 6 6 8 9 17 2J * 5 17 7 11F 2 H I8 P 49 7 14 4 12 4 9 19 3 14 11 11F 2 H 20 P ? 5 9 11 7 8 10 U f *6 8 21 4 HF 2 H 2 oP 2 6 5 10 8 10 6 7 12 5 6 15 8 Totals 23 44 29 35 26 35 59 16 22 67 30 498 University of California Publications in Botany t VoL - 5 In this cross, corolla color behaved in exactly the same manner as it did in the angustifolia-macrophylla series. The same remarks as to sharpness of segregation apply here as in that series. Red was nearly always readily distinguishable, but pink and light pink formed a more or less completely intergrading series. Taking the results in this way, we obtain 23 red ; 73 pink and light pink, which is substan- tially in accord with Mendelian expectations. TABLE 9 F 2 SEGREGATION IN CALYCIN A-VIRGINICA SERIES. Pink Pink Red Red • Garden numbers normal hose-in-hose normal hose-in-hose 16F 2 H 18 P 25 24 14 8 3 16F 2 Hi 8 P 49 25 8 14 3 I6F2H20P7 29 6 10 4 16F 2 H 20 P 38 30 8 11 1 Totals 108 36 43 11 Expected 112 37 37 12 With respect to corolla form some difficulty was encountered be- cause the expression of the hose-in-hose character in the segregants did not seem to be so extreme as it was in the parent, and a large number of the plants showed slight traces of it, but sometimes in a more pronounced form than in the Y x hybrids. Accordingly the classi- fication of corolla form in table 8 is not a wholly satisfactory one. The classification for stature and leaf width is subject to similar remarks as to its definiteness. Here there was also a more or less completely intergrading series of forms and no accurate measurements were taken. However, there is no doubt that there was segregation with respect to these characters, and a range of forms was obtained \thich completely bridged the gap between the parents. The behavior oflhese characters is to be considered in the light of their segregation in subsequent generations. In 1916 four additional V, populations of the calycina-virginica series w&rf. grown in order to reexamine populations for the segrega- tion of norifcal versus hose-in-hose flowers, and red versus pink flower color. The method of classifying the flowers was that used in studies of 1916 populauo^previously mentioned. The results of these studies are given in table 9.V. 1922] Setchell-Goodspeed-Clausen: Nicotiana Tabacum 499 In the segregation the same grading as before of the pinks into two intergrading classes in the proportion of approximately 2 inter- mediate pink : 1 light pink was observed, but it was even more difficult to draw a line between light pink and intermediate pink because of the effect of the hose-in-hose conditon on flower color expression in those plants which bore teratological flowers. In the matter of segre- gation into normal and hose-in-hose flowers, some difficulty was ex- perienced because some otherwise normal flowering plants bore some flowers which showed a tendency for the calyx to become petaloid, and others bore flowers which showed a very slight hose-in-hose ten- dency. A correspondingly slight hose-in-hose tendency is also present in ~F 1 plants. These plants were classified as normal. Here again it can be seen that the segregation ratios of 144 pink : 54 red and 151 normal : 47 hose-in-hose are in substantial agreement with Mendelian expectation for contrasts in a single pair of allelomorphs in each case. Moreover, the dihybrid ratio is substantially in agreement with that expected for independent segregation of the members of these two pairs of allelomorphs. 4. F 3 AND SUBSEQUENT GENERATIONS OF THE CALYCINA-VIRGINICA SERIES In 1912 twelve F 3 families of H 18 and five of H 20 were grown. They will be grouped for consideration according to the characters which TABLE 10 F 3 BEHAVIOR OF RED SEGREGANTS. Garden numbers Red 12F 3 Hi S P25Pn 25 I2F3H18P25P19 25 I2F3H18P49P12 25 I2F3H18P49P24 25 I2F3H20P26P25 25 / the F 2 parent exhibited. In table 10 the data with respect toithe be- havior of F 3 populations from red flowering F 2 plants ar&fjbllected. Five such populations gave nothing but red flowering planm indicating clearly that red segregants breed true. In table 11 thefflata from pink flowering plants are similarly collected. The reader will not fail to notice that some pink flowering selections were not heterozygous for 500 University of California Publications in Botany [Vol. 5 red. This bears out our statements as to the difficulty of classifying pink and light pink. In the seven populations which produced red flowering plants 38 plants had red flowers and 134 pink or pinkish flowers ; again in substantial agreement with the behavior of flower color in the angustifolia-macrophylla series. The behavior of segre- gants classified as light pink is shown in table 12. Of the four popu- lations from which data were gathered only one bred true to light TABLE 11 F 3 behavior of pink segregants. Garden numbers Red Pink Light pink 12F 3 H IS P 25 P 7 5 16 4 12F 3 H 18 P 25 P 21 21 4 12F 3 H I8 P 25 P 2 5 6 19 12F 3 H 18 P 49 P 9 4 20 I2F3H18P49P10 11 13 I2F1H18P49P25 4 16 5 • 12F.,H 20 P 2 6P6 4 20 12F 3 H 20 P 2 6Pll 4 21 Totals for segregat- ing populations 38 134 TABLE 12 F.i BEHAVIOR OF LIGHT PINK SEGREGANTS. Garden numbers Red Pink Light pink 12F 3 H 18 P, 5 P 24 23 12F 3 H 18 P 49 P 22 8 12 5 12F 3 H 2 ,P 7 P 9 6 15 4 12F 3 H 20 P 26 P 6 25 pink, one of the others bred true for pink, possibly a slightly darker shade than true light pink, and two segregated for all three colors: they must therefore have been pink heterozygotes. In P 4 two populations each of H 1S and of H 20 were grown. Popu- lation^HSF^^P^PjjPg from an F., population breeding true for red gave i»14 100 plants all red flowering. Population lSF^^Po-P^P^ from the^pune F 3 population gave 97 plants all red flowering like calycina. Population 13F 4 H 20 P 26 P B P 8 , which bred true for pink in F ., gave in F 4 96 plants, all pink flowering. These three populations were grown to F 7 without showing further evident segregation. The 1922] Setchell-Goodspeed-Clausen: Nicotiana Tabacum 501 pink of the pink flowering derivative was at first considered somewhat darker in shade than the light pink of virginica, but this line also showed the hose-in-hose flower character, which sometimes makes it difficult to determine flower color accurately. In later generations of this line its color was noted as equivalent to the light pink of virginica. TABLE 13 F 3 BEHAVIOR OF HOSE-IN-HOSE SEGREGANTS. Garden numbers Hose-in-hose I2F3H18P25P21 25 *12F 3 H I8 P 25 P 24 23 I2F3H18P49P9 24 *12F 3 H 18 P 49 P 12 25 12F 3 H I8 P 49 P 24 25 I2F3H20P26P6 24 I2F3H20P26P11 25 * Apparently not so extreme as calycina. TABLE 14 F 3 BEHAVIOR OF NORMAL SEGREGANTS. Garden numbers Hose-in-hose Partial Normal 12F 3 Hl 8 P25P7 9 16 12F 3 HlsP25Pll 4 4 17 12F 3 H 18 P 26 Pi 9 5 20 12F 3 H 18 P 25 P25 3 22 *12F 3 H 18 P 49 P 10 4 20 12F 3 Hi 8 P 49 P 22 25 12F 3 H I8 P 49 P 25 4 21 12F 3 H2 0 P7P 9 5 20 12F 3 H 2 oP 2 6P 6 7 3 15 12F 3 H2oP26P25 10 15 Totals 47 153 Taking up corolla form next, we may deal with the different popu- lations in the same manner as was done in the case of flower color. F 3 populations from F 2 hose-in-hose segregants are recorded in table 13. Seven populations were grown, all of which bred true tj> the hose-in-hose character, although curiously enough two populations. 12F.,H ls P 2r ,P 24 and 12F 3 H 18 P 49 P 12 did not appear to exhibit so ex- treme character expressions as calycina. Only one partially hose-in-hose plant was grown in'F 3 . For the sake of economy of space it is included in table 14, where it is marked 502 University of California Publications in Botany [Vol. 5 with an asterisk. Strangely enough, it was one of the two in the table which did not throw hose-in-hose flowers. The other normal selections all threw hose-in-hose flowering plants in the proportion of about 3 normal to 1 hose-in-hose. In subsequent generations only the three families which were pre- viously considered under flower color were grown. Normal flower selections from 12F 3 H 18 P 25 P 11 gave two populations, one of 100 and one of 97 plants. The plants all bore normal flowers. In 13F 4 H 18 P 25 P 11 P 13 it was noted that some flowers were split, but there was not even a suggestion of approach to the true hose-in-hose condition. The other population 13F 4 H 20 P 26 P 5 P 8 was from a hose-in-hose selection in the TABLE 15 Fs BEHAVIOR OF TALL SEGREGANTS. Garden numbers Tall Short 12F 3 H 18 P 25 P 7 19 6 12F 3 H 18 P 25 P 24 1 24 I2F3H20P7P9 6 19 TABLE 16 F 3 BEHAVIOR OF MEDIUM SEGREGANTS. Garden numbers Tall Short 12F 3 H 18 P 49 P 12 23 I2F3H18P49P22 1 24 corresponding F 3 population. Ninety-four plants were grown to ma- turity, all of which were strictly hose-in-hose. In subsequent gener- ations these three populations bred true to type save for the sporadic appearance of hose-in-hose flowers on plants which otherwise bore nothing but normal flowers. This, however, is not an unusual phe- nomenon even in pure line cultures of normal flowering varieties of Tabacum, and it is extremely doubtful whether the hybrid derivation ofVhese plants had anything to do with the production of occasional split flowers. As respects height of plants the F 3 data are given in tables 15, 16, and 17,lfc|ich give the behavior of tall, medium, and short F 2 segre- gants respectively. The behavior here is not very convincing. Prob- ably the difficulty in judging the character and the influence of variation in soil condition had something to do with it. I 022 ] Setchell-Goodspeed-Clausen : Nicotiana Tabacum 503 In the subsequent generations the behavior was, however, more definite. 13F 4 H 18 P 2;i P 11 P 9 was grown from a tall F 3 plant. No defi- nite notes were taken as to height, but the population was noted as varying. In F 5 and subsequent generations the line bred true to tall. 13F 4 H 1S P 25 P 11 P 13 was grown from a short F 3 plant. The ninety-seven plants were all of low stature and in subsequent generations the line bred true for low stature. 13F 4 H 20 P 26 P 5 P 8 was grown from a tall F 3 plant. Ninety-four plants, although variable in height, all be- longed in the tall class and in subsequent generations the line bred true for tall stature. Nothing but a careful biometrical study under TABLE 17 Fs BEHAVIOR OF SHORT SEGREGANTS. Garden numbers Tall Short I2F3H18P25P11 10 15 I2F3H18P25P19 16 9 12F,Hi 8 P„Ph 25 12F3H18P25P25 5 20 12F3H18P49P9 24 12F3H1SP49PIO 24 12F3H18P49P24 7 18 12F3HI8P49P25 9 16 12F3H20P2GP5 8 16 12F3H20P26P6 5 19 12F 3 H 2 oP26Pll 25 Totals 68 129 well controlled cultural conditions, however, would yield results capa- ble of strict Mendelian analysis. However, it can be said that none of the results here recorded preclude the possibility of such an analysis, although it evidently can not be done in any simple qualitative manner. As respects leaf width it was found impossible to make even such a rough classification as was attempted in the case of stature. Here again nothing short of a strict biometrical analysis would furnish the basis for a Mendelian formulation. As has been indicated above, three separate lines of thisjSeries were carried out to the seventh hybrid generation. Of these^/me was a recombination of characters from both parents exhibiting the tall stature and normal flower of virginica with the red flojrer color of calycina. One exhibited a stature intermediate between that of caly- cina and virginica in combination with the normal flower shape of 504 ' University of California Publications in Botany [Vol. 5 virginica and the red flower color of calycina. The third had the tall stature of virginica and red flower color, in association with the hose- in-hose flower form of calycina. These three lines apparently bred true for all their characters. 5. DISCUSSION OP RESULTS OF THE CALYCTNA-VIKGINICA SERIES No extended discussion of results is indicated in connection with the calycina-virginica series of hybrids because particular attention was given to so few characters. Just as in the case of angustifolia- macrophylla, so in this series of hybrids the character differences proved to depend upon complex genotypic differences. Apparently the flower color contrast in these two varieties was the same as that in the angustifolia-macrophylla series, and the same relations with respect to dominance and segregation were found to hold for it. Without doubt we are dealing here with the Rr pair of allelomorphs as in the previous instance. The demonstration of the simple factor relations in the inheritance of the split hose-in-hose form of flower adds to our series another pair of allelomorphs which we may call Cc (calycine). In this case the dominance of normal over split hose-in-hose appears to be nearly, if not quite, complete. The sporadic appearance of split hose-in-hose flowers on otherwise normal plants does not even seem to be clearly associated with the heterozygous genotype, Cc. The data for height are not of sufficient accuracy or extent to warrant an at- tempt at Mendelian formulation. It was again found possible very easily to shuffle and recombine the characters occurring in the parent varieties and to establish recombination derivatives in pure lines. V. ALBA-MACROPHYLLA SERIES 1. PARENTS OF THE ALBA-MACROPHYLLA SERIES Alba, which is one of the parents of the alba-macrophylla series, is the "White" tobacco, U. C. B. G. 30/06, described by Setchell. It is one of the taller forms of Tabacum, ranging in height from 165 to 220 c^k Typically alba is unbranched below ; above, it has flowering branchesVorymbosely arranged in succession from above downward. The leavesl^-e sessile, more ample, more rugose, and more velvety than those of macro phylla. They are narrowed suddenly above the ex- panded, somewhat auricled and partially clasping base. The leaves 1922] Setchell-Goodspeed-Clausen: Nicoiiana Tabacum 505 resemble those of macrophylla in shape but differ from them particu- larly in the basal portion. The corollas are white with a yellowish tinge ; but in shape, size, and general proportions they are very similar to those of macrophylla. Line drawings of typical features of alba are reproduced in plate 82. The reproductions of photographs of the leaf of alba and of the F 4 hybrid of the alba-macrophylla series are. shown in plate 84. Macrophylla, U. C. B. G. 22/07, has been described above. In these two varieties there are definite character contrasts in color of flowers, macrophylla being red and alba white ; and in stature, macrophylla being low of stature and alba distinctly taller. Other contrasts also exist, although they are not so definite, in the style of branching and in the shape and texture of the leaves. Like those which have been considered above, this is a hybrid series in which the contrasts between the parent forms are of a distinctly complex character. 2. OF THE ALBA-MACEOPHYLLA SERIES The crosses between alba and macropyhlla were made in July, 1909. The cross was successful in both directions, and seed was secured from alba*^ x macrophylla^, which was given the number H 23 , and from the reciprocal which was given the number H 24 . When mature the F t plants were tall, 100 to 200 cm., averaging 130 to 160 cm. Habit and leaf shape were in general those of alba. The corolla was deep pink of about the same shade as that of the F 4 of the angustifolia-macrophylla series. The variation in height in these populations possibly indicates a lack of constancy in the alba parent in this respect. In plate 83, figure 2, is shown an F x plant of IOFjH^. 3. F 2 OF THE ALBA-MACEOPHYLLA SEEIES In 1911 four F 2 populations were grown, viz., 25 plants each of 11F 2 H 23 P 13 , 11F 2 H 23 P 31 , and 11F 2 H 24 P 0 , and 23 plants of 11F 2 H 24 P 34 . The four populations, although small, proved to be equivalent in every respect. The type of segregation was very complex. That of differ- ences in types of leaves, especially, presented such a series of inter- gradations as to defy any definite classification. Likewise in height, there was a continuous series of forms from the tallest to the shortest. A rough classification was, however, made for purposes of reference into tall, medium, and short. An excellent illustration Of the segre- gation for this character is shown in plate 85, figure 1, which shows 506 University of California Publications in Botany [Vol. 5 two adjacent plants of 11F 2 H 24 P.; 4 , one tall and of the general habit of alba, and the other short and of the general habit of macropliylla. The classification for height is given in table 18. Obviously no satis- factory Mendelian formulation can be deduced from these data. As regards flower color, however, the classification is more definite. Four more or less distinct shades were distinguishable, viz., red, pink, light pink, and white. The pink and light pink shades merged into each other, consequently they have not been separately recorded in table 18. Bearing in mind the previous behavior of red and pink, as shown in the angustif oKa-macrophylla and calycina-virginica series, TABLE 18 F 2 SEGREGATION IN THE A LBA-MA CROPH YLLA SERIES. Stature Flower Color Garden numbers tall medium short red pink white 11F 2 H 23 P 13 11 6 8 3 14 8 11F,H 23 P 31 13 6 6 4 12 9 11F 2 H 24 P 6 7 10 8 3 15 7 11F 2 H 24 P 34 6 8 9 6 13 3 Totals 37 39 31 16 54 27 it would appear that we are here dealing with dihybrid populations in which a pair of allelomorphs for color versus white is concerned in addition to that pair upon which the contrast of pink versus red was found to depend. The pair of allelomorphs for the pink versus red contrast has been represented by R and r, respectively. If we repre- sent the contrast of color versus white by W and w, respectively, the two parents in this series would possess the following genotypes : Alba = RRww Macropliylla = rrWW The light pinks of the previous series would then be RRWW, and the factor R might be regarded as a dominant diluter. According to this formulation, F 1 of the alba-macrophylla series would be RrWw, pink, and \~ should segregate in the ratio 3 red : 9 pink : 4 white. The expectajl result in the classification of ninety-seven plants in whole numbersVf 18 red : 55 pink : 24 white. Agreement is thus fairly close. A check on the results above noted for the 1911 sowings of the F 2 population was made by growing in 1916, five additional F„ popula- 1922] Setchell-Goodspeed-Clausen : Nicotiana Tabacum 507 tions of the same series, viz., 16F 2 H 23 P 5 ; 16F 2 H 23 P 32 ; 16F 2 H 23 P 34 ; 16F,H 24 P 2S ; and 16F 2 H 24 P 33 . The segregation in the resulting popu- lations is recorded, in table 19. The method of studying these flowers was the more accurate one previously described in connection with later generations of the angustifolia-macrophylla series. In the classification of flowers it was noted- that reds and whites were sharply distinguishable from pinks. The pinks were of many different shades ; some very light, others rela- tively dark, corresponding to the range obtained in the angustifolia- macropliyUa series. However, in these populations the range of vari- TABLE 19 F 2 SEGREGATION IN 1916 SOWINGS OF ALBA-MACROPHYLLA SERIES. Garden numbers Red Pink White Totals 16F 2 H 23 P 5 11 18 11 40 16F 2 H 23 P 32 12 19 11 42 16F 2 H 23 P 3 4 8 24 9 41 16F 2 H 24 P 28 5 34 11 50 16F 2 H 24 P 33 7 29 14 50 Observed 43 124 56 223 Expected 42 125 56 223 ation of pink appeared to be greater and the intergradations more gradual than in that series. In the whites there was also evidence of differentiation into classes depending upon the amount of yellow or creaminess in the flowers. Some of the whites appeared to belong to a clear white albino class, but most of them had a distinctly creamy tinge. The observed segregation in these populations was in almost exact agreement with the formulation advanced above. 4. F 3 AND SUBSEQUENT GENERATIONS OF THE ALBA-MACROPHYLLA SERIES In table 20 we have summarized the behavior of the F 3 populations as respects color of flowers and height of plant. Of the five red F 2 plants from which F 3 populations were grown, three proved to be homozygous for red and one proved to be a heterozygote of the^genetic constitution rrWw. Of this latter selection two sowings wpe made, one in 1912 and another in 1913. The combined results Jrom these two sowings, 35 red : 14 white, are in fair agreement with Mendelian 508 University of California Publications in Botany [ VoL - 5 expectation. The other population exhibited an anomalous type of segregation, and gave 2 red : 23 white. It is unfortunate that this line was not investigated further, but the results probably are due to an experimental error. TABLE 20 F 3 SOWINGS OF THE A LBA-MA CROPH YLLA SERIES. F2 Phenotypes Garden Numbers Flower Color Stature Flower Color Stature Red pink white short tall (or medium) Tall (orM) I2F3H03P13P3 19 6 25 Short 1 / H 1 H no t 11 r 1 22 22 Red Sh't(orM). 12FiH9iP<..Pn AArfA j A J. J 3 J. A\A. 17 25 4 21 Tall 12F3H23P31P22 24 4 20 Tall 1 / H 9 H .)■> \ 11 r.it: aa^a i)'-i-2o A 61*- 2o 2 23 1 24 Tall 16FiH,iP n P, A v -' A 1) 4 - 1 i J 1 Id 4 - J . 16 8 Tall 12FiH-nPnPn 2 14 6 8 10 lYXeclium ^FiHo^PnP,^ 0 1 9 7 99 Tall 12F 3 H 23 P 31 P 7 6 14 5 25 Tall 12F 3 H 23 P 31 P 19 10 9 4 23 Pink Medium Tall 12F 3 H2 3 P 3 lP20 12F 3 H 24 P C P 5 6 3 12 22 5 , 4 19 25 Short 12F 3 H 24 P 34 Pi 8 5 13 7 24 1 ' Tall 12F 3 H 24 P 3 4P 2 o 20 5 25 Tall 16F 3 H 24 P 3 4P 2 o 13 4 Tall 12F 3 H 23 P 13 P 14 25 25 Tall 12F 3 H 23 P I3 P 15 25 25 Short 12F 3 H 23 P 13 P 24 25 25 White Tall 12F 3 H 24 P 6 P 2 24 24 Short 12F 3 H 24 P 6 P 3 25 4 21 Tall 12F 3 H 24 P 6 P 4 23 23 Short 12F 3 Ho 4 P 34 P 23 1 23 25 Short 13F 3 H 23 Pi 3 P2 4 10 10 Short 13F 3 H 24 P 34 P 23 10 10 Eight families of F ;i plants were grown from pink F 2 's. Of these F 2 plants six proved to belong to the RrWw genotype. The totals from these six populations, viz., 35 red : 74 pink : 34 white, are in fair agreement with the dihybrid ratio 3 red : 9 pink : 4 white. One of the other populations gave 3 red : 22 pink. It was probably the result of sowing seed from an F„ plant of the genetic constitution RrWW which should give 3 pink : 1 red. The observed segregation ratio is not good\ but the numbers are small. Two sowings of F-jH^P^Pop gave totals of 33 pink : 9 white. The F 2 plants in this case must have been of the genetic constitution RRWw; in which case expectation would be 3 pink : 1 white. No selection was observed to breed true 1922] Setchell-Goodspeed-Clausen: Nicotiana Tabacum 509 for pink in F 2 . This, however, is not inexplicable, for only one in nine among the F, pinks should belong to the ERWW genotype. Sowings were made from seven white F 2 plants. Among 190 plants so produced there was one pink flowering individual. It surely rep- resents some kind of experimental error. We may say, therefore, that for flower color the formulation advanced to account for the F 2 segre- gation ratio, also accounts for the behavior observed in the various F 3 populations. "We have reported the data on height in table 20, largely in order to show that this character, although obviously dependent on factor differences, is so complex as not to permit of a simple qualitative treatment. Thirteen F 3 sowings from tall F 2 plants gave ten popu- lations showing only tall plants. Two of the remaining populations showed segregation into 31 tall (and medium) : 13 short. One popu- lation consisted entirely of short plants. The classification of the F, parent of this plant as "tall" was noted as doubtful at the time, the note "or medium" being appended. Two- populations were grown from F 2 plants of medium height. One of these populations was uni- formly of low stature; the other showed segregation into 19 tall (and medium) : 4 short. Six populations were grown from F 2 parents, four of which apparently bred true for low stature, the other two showed segregation into tall (and medium) or short in the ratio 42 : 8. It is interesting to note that at the time of classification the parents of these two later populations were classified as short (or medium), indicating a doubt as to proper classification. More definite data will be necessary before a satisfactory formulation of these height differ- ences can be made, but certain of our results seem to indicate that there is one allelomorphic pair which has a rather marked effect on stature, and that there are other subsidiary pairs of factors which have less marked effects. Only one line in this alba-macrophylla series was carried out to subsequent generations to demonstrate the possibility of fixing char- acter complexes from a hybrid. It was a low stature white flowering line. In F 4 , 100 plants of 13F 4 H 24 P 34 P 23 P 2 bred true to low stature and white flower color. The population was uniform, the plants ex- hibited the general habit of macrophylla rather than that of alba; and the leaves were the same shape as those of macrophylla, but they were slightly rugose, although not so much so as those of alba. In F ri , two populations of 25 plants each were grown, viz., 14F 5 H 24 P J fP 2:! P 2 P 47 and 14F,,H„ 4 P 34 P 23 P 2 P 83 . No differences were detectable between these two populations, and the characters exhibited were those we have noted U. OF ILL Li3. 510 University of California Publications in Botany [Vol. 5 for F 4 . In F 6 10 plants each of 15F G H 24 P :!4 P 23 P 2 P 47 P 5 and 15F 6 H 24 P34P 2 3P 2 P 83 Pi2 and in F 7 10 plants each of 16F 7 H 24 P 34 P 23 P 2 P„,P 12 P 8 and 16F 7 H 24 P 34 P 23 P 2 P 47 P 5 P 8 were grown. In both cases the parallel populations were equivalent and the characters exhibited and described in F 4 remained constant. Plate 85, figure 2, is a good illustration of the type of this family as fixed. A photograph of the original F 2 plant, from which the family descended, is reproduced in plate 85, figure 1. It will be noted that the derivative represents a fixation of the characters of the original F 2 selection, and that no important seg- regation occurred in it either in F 3 or in subsequent generations. 5. DISCUSSION OF RESULTS OF THE ALBA-MACROPHYLLA SERIES Here again, as in the calycina-virginica series, no extended discus- sion of results is necessary. Obviously the differences separating the two varieties are of a complex nature genetically as in the two previous cases. The series demonstrates the existence of another pair of allelo- morphs for flower color in this group of Tabacum varieties, viz., Ww, and the part played by it in the production of both red and light pink flower color has been determined. The height contrast again proves to be too complex for qualitative Mendelian formulation. As in the previous cases, the establishment of stable recombination derivatives proved to be a simple task. VI. GENERAL CONCLUSIONS We shall limit the discussion of these results to three main topics upon which these investigations seem to have thrown some light: (1) the origin and interrelationships of varieties of Tabacum; (2) the methodology of Mendelian analysis in Tabacum; and (3) Mendelian heredity in Tabacum. 1. ORIGIN AND INTERRELATIONSHIPS OF VARIETIES OF TABACUM As a result of extensive studies of a considerable assemblage of Tabacum varieties, Comes (1905) came to the conclusion that the s Tabacum could be subdivided into six fundamental varieties: a. var. fructicosa Hook. J), var. la mi folia (W.) Comes, fc. var. virginica (Agdh.) Comes, cf^-var. brasiliensis Comes. e. var. havanensis (Lag.) Comes. /. van macrophylla Shrank. 1922] Sctchell-Goodspeed-Clausen : Nicotiana Tabacum 511 Inasmuch as practically every Tabacum variety shows combinations of characters of two or more of these fundamental varieties, Comes assumed them to have been derived mostly through hybridization be- tween the fundamental varieties, and he proceeded from purely morph- ological studies to classify the different commercial varieties on the basis of their supposed hybrid derivation. Anastasia (1906), who has criticized this scheme of Comes very severely, reduced the number of fundamental varieties to four, striking out fructicosa and lancifolia from Comes' list, and substituting purpurea for macrophylla. Al- though disagreeing as to the fundamental varieties, Comes and Ana- stasia seem to agree in referring existing varieties to derivation, mostly through hybridization, from a relatively small number of fundamental varieties. The Howards (1912) object to the mode of classification of Comes and Anastasia, and point out as a result of their studies of types of Indian tobaccos that no attempt at classification based on derivation can be considered seriously unless supported by actual experimental studies. In her later paper in particular Miss Howard (1913) shows that segregation products may be obtained through hybridization which transcend the limits set by the parents. The Howards propose a scheme of classification based primarily upon leaf and habit char- acters, and they adopted this morphological system purely as a pro- visional means for facilitating identification and reference among the numerous forms of Indian tobaccos. Our results agree with those stated by the Howards, and we raise the same objection to schemes of classification such as Comes and Anastasia have advocated. Any scheme of classification based on morphological considerations alone cannot well meet with the approval of geneticists, for it does not take into account genotypic differences which exist among forms of similar morphological appearance. Thus it is possible, as Miss Howard points out, by crossing different mem- bers of a given group to obtain segregation products which belong in an entirely different morphological group in the scheme of classifica- tion. In particular she points out that "petiolate" forms have been produced as segregation products from two "sessile" parents, yet "petiolate" and "sessile" have been used as primary indexes for classification of tobaccos into groups. The difficulty from the genetic point of view with any classification of Tabacum varieties is the same as that which is met witll in the species as a whole and viewing the entire assemblage of its varieties, classification of varieties of other polymorphic species 512 University of California Publications in Botany [Vol.5 there is evidently in Tabacum, to those who accept current interpre- tations of heredity, a series of allelomorphic contrasts, the number of which cannot even be guessed, but which need not perhaps be more nu- merous or striking than those which have been discovered in Drosophila. But whereas in Drosophila the factors have been kept in stocks in- volving for the most part single factor differences from a common wild type, in Tabacum, and in other cultivated crop plants such as barley, maize, oats, rice, wheat, etc., these factor differences have been shuffled about through long periods of cultivation until existing varieties are no longer related clearly to a common form or to each other. In some instances in such groups certain factor differences have a more strik- ing visible effect than in others. In such instances we have an obvious mode of classification based not upon number of factor differences so much as upon the striking character differences which arise from certain factor contrasts. Thus in barley we have the classification of varieties advocated by Harlan (1918) based upon recognition of a number of major morphological distinctions, some of which at least have been clearly analyzed in Mendelian fashion ; and the same prin- ciple has been recognized in the classification of varieties of maize, where it has led to the absurdity of erection of a heterozygous form, podded maize (vide Collins), as one of the primary group distinctions. In some instances, doubtless, the sorting of factors may give rise to certain recombinations which are more favorable to life processes than others, as Muller has pointed out in another connection, and such genotypes may act as centers around which groups of varieties may be built up, thus giving rise to more or less obvious grouping of varie- ties. The attempt to base a system of classification upon reference to certain fundamental types does not, however, promise much simpli- fication of the difficulty; moreover, such an attempt rests upon the rather naive assumption that it is unnecessary to account for the fundamental types. From a genetic standpoint, therefore, it would appear that in attacking the problem of classification and interrelations of varieties in a polymorphic species the major premise should be a recognition of the fundamental equivalence of every homozygous genotype. Start- ing from this premise a system of dichotomy beginning with those factor contrasts which produce the most striking, visible effects and proceeding to those of lesser effect might be set in operation. Such a systentobviously would in certain eases separate some similar varie- ties intcfcseparate groups, and would lead to recognition of group differences without obvious morphological distinctions, but the system 1922] Setchell-Goodspeed-Clausen: Nicotiana Tabacum 513 would have a real significance, and the relationships indicated by it would be fundamental ones. It is, however, necessary to have a much more extensive knowledge of Mendelian heredity in Tabacum than we have at present before such a system can be formulated. 2. METHODOLOGY OF MENDELIAN ANALYSIS IN TABACUM Prom the Mendelian side there are certain obvious facts associated with Tabacum as a species. In the first place, as we have stated before, the species is highly polymorphic. A large and striking assemblage of varieties exists, the most extreme of which hybridize readily and give fully fertile hybrids and full fertility in their derivatives. A few teratological forms are known in which fertility is somewhat re- duced, but the above generalization does not far overstate the facts. The species is, moreover, so highly polymorphic that with respect to any given character a representative collection of varieties may be arranged in a series connecting the most extreme expressions of that character by imperceptible steps. Thus in flower color we have repre- sented in the collection of varieties of the University of California Botanical Garden dark red, red, light pink, pinkish, and white, and descriptions occur in the literature which indicate the existence of further shades of red connecting these. Now flower color is a rather definite character, comparatively speaking, for it appears to be little affected by ordinary environmental conditions. In many polymorphic forms, such for example as maize, there are a large number of such definite characters, and as a consequence studies of inheritance in these forms have resulted in definite Mendelian analysis of many char- acter differences. But in Tabacum unfortunately most of the char- acters involve quantitative elements, and these with few exceptions depend so largely for their particular expression upon environmental conditions that it becomes a difficult matter in a segregating population to distinguish between those differences which are inherent and refer- able to the genotype and those which have come about through the action of extrinsic forces. And yet our assemblage of tobacco varie- ties indicates clearly that there are genotypes which give rise to all possible expressions in these characters. Here we find the reason for the present backward state of knowledge of inheritance in Tabacum, for while there have been numerous investigations which indicate clearly that the Mendelian mode of transmission may be followed in all these character differences, yet there are very few investigations which have resulted in the precise type of factor analysis characteristic 514 University of California Publications in Botany [Vol. 5 of investigations with other forms, specific mention of which is un- necessary. The general features of inheritance in Tabacum varietal crosses are plain enough. The results of our investigations in this connection agree throughout with the conclusions which Miss Howard drew from her studies. "When we are dealing with complex differences, the F 1 is commonly intermediate in character expression between the two parents. Not only is this true as respects the F 1 plant as a whole but it is also true for individual characters. The F 2 commonly consists of a varied assemblage of forms covering the range between the two parents, or even not uncommonly presenting products not included in the range between the two parents. So many and of such variety are the forms obtained that accurate classification is entirely out of the question. But in F 3 and in subsequent generations segregation, even for characters commonly regarded as quantitative, sometimes occurs in distinct discontinuous classes in marked contrast to the inter- grading series of forms obtained in F 2 . This is shown particularly well in our analysis of leaf base factors, for in this case we have been able to adopt a qualitative mode of attack on one of the features which contributes to leaf shape. If such an analysis proves successful in one instance, there seems to be little reason why it should not be ex- tended to others. There is, therefore, additional evidence in this suc- cessful application of the mode of qualitative analysis to quantitative characters in support of the oft repeated contentions of East (1913), Hayes (1912), Hayes, East, and Beinhart (1913), Miss Howard (1913), and others that fundamentally the same mode of inheritance holds for quantitative characters in tobacco as for qualitative ones. The distinction between the two classes of characters is purely an artificial one erected for the purpose of convenience in formal treat- ment, and at most depending merely upon an increase in complexity of the factor relations involved and on the greater fluctuation of the characters in response to environmental differences. The question remains to be discussed whether semiquantitative characters admit of a qualitative mode of analysis, and if so, how? Miss Howard (1913) as a result of her extensive studies of inheritance in Indian tobaccos concludes that the easiest way to determine the principles underlying inheritance in these forms is to establish as many extracted homozygous intermediate forms as possible. The estab- lishment of such forms in themselves, however, is only a step in the Mendelian analysis of the differences. Such forms are, as might have been expected on theoretical grounds alone, less different from one 1922] Setchell-Goodspeed-Clausen: Nicotiana Tabacum 515 another and from the parents than the original parents are from each other. Moreover, our experiments show that as a result of simplifi- cation of the factor differences the derivative strains crossed with each other or with the parents give F 2 progenies which often exhibit clear- cut segregation in characters which showed intergrading series in the original P 2 population. In other populations, however, from crosses between derivatives, the populations still exhibit perplexing com- plexities which make classification difficult and uncertain. In such cases we could again resort to the method of establishing intermediate derivatives from them; but if the number of factors concerned in a given character is even moderately large, as is certainly the case with many of these quantitative characters, the number of genotypically different derivatives which may be secured becomes so great as to make the method impracticable. Our experience indicates that the successful factor analysis of these quantitative character differences depends not only upon getting what Castle (1919) has called the residual heredity equivalent throughout the population, but also in establishing the proper kind of residuum which will most emphasize the character differences associated with the pair of factors or pairs of factors under investigation. The prob- lem may be illustrated crudely by considering the pair of flower color factors Rr. If the residuum should contain PP, the effect of which is described below, segregation would give PPRR, PPRr, and PPrr. In character expressions these three different genotypes would doubt- less all be of various shades of dark red, difficult or impossible of ac- curate separation. With such a residuum, therefore, it would be im- possible to investigate satisfactorily inheritance in the factor pair Rr. But if we should substitute pp for PP in the residuum, the segregation products would be ppRR and ppRr, which would be pink, and pprr, which would be red. Here the segregation would be sharp and dis- tinct, and there would be practically no difficulty in classification. How complex such interrelations can be has been shown most clearly by Bridges (1919) in his account of specific modifiers of eosin in Drosophila. As Bridges shows it would easily be possible to obtain populations of Drosophila defying classification, but by keeping the factors separate and studying their character effects with known residual genotypes, it has been possible to determine and locate the factors involved. Doubtless much of the extraordinary success of Mendelian analysis in Drosophila has been due to the fact thatjractor differences arose under conditions such that the residual araotype gave no difficulty ; whereas in crop plants, the geneticist starts with 516 University of California Publications in Botany [Vol. 5 long established diverse types, evidently related to one another in fundamentally the same manner as are the various Drosophila mutants, but more complexly, and from these complex assemblages he must unravel the tangled skein of heredity. There are, however, other and perhaps quicker ways of establishing a uniform and favorable residual heredity than that of securing and testing homozygous extractives, and these may be employed in certain special cases. Thus, if it be desired to study the relationship of the pair of factors Ss for the petioled versus sessile condition, it should be possible to proceed by crossing back the F x of angustifolia x macro- phylla, for example, to macrophylla, selecting the petioled forms from the back cross for again crossing back to macrophylla, and continuing the process until clear-cut segregation was obtained. Such a mode of procedure should establish a residual genotype equivalent to that of macrophylla itself, and should thereby enable the student event- ually to. study the effect of substituting SS for ss in the macrophylla genotype. In tobaccos technical details make it particularly easy to adopt such a procedure, but it is useless to speculate further 'Upon its results until it shall have been attempted. 3. MENDELIAN HEREDITY IN TABACUM From the standpoint of factor analysis, we have demonstrated clearly in the foregoing pages, the existence of a number of distinct pairs of factors. Two of these affect flower color, one flower form, and three affect the character of the leaf base. The particular effects of the opposing members of these pairs of factors and the interrela- tions which they exhibit so far as these have been investigated have been set forth in the discussions which follow the description of each of the three series of hybrids. Although evidently many other factor differences were concerned in these studies, and remain for further investigation, the results which we have described make a beginning toward a more accurate knowledge of Mendelian heredity in Tabacum. So far as our results furnish any data on the question, the six pairs of factors isolated exhibit no linkage relations. The data here are far from complete, but the results are in accordance with theory. According to "White (1912), there are twenty-four pairs of chromo- somes in NicoHana. Assuming for the sake of discussion that each of these pairs of chromosomes bears a set of factors comparable in mimblis to any other pair, then the chances of finding linkage when only six pairs of factors are studied is very slight. This large number I 922 ] Setchell-Goodspced-Clausen: Nicotiana Tabacum 517 of pairs of chromosomes may account for the ease with which recom- bination pure lines were established. Even with a large number of factor differences, such as evidently distinguish these Tabacum varie- ties, the chances are slight with so many pairs of chromosomes that linkage will enter in as a factor to cause the continued preservation of a heterozygous condition as a consequence of selection for a certain set of characters. It remains to consider those portions of the Nicotiana literature which deal specifically with the Mendelian inheritance of the charac- ters which we have investigated, and to harmonize our results with those which have been reported previously. Unfortunately there have not been many investigations in Tabacum which have been prosecuted far enough to arrive at a definite factor analysis of the differences under consideration. The investigations of Miss Howard (1913), promise of the continuation of which has not thus far been fulfilled, in general confirm those which we have presented in this paper. On the strictly analytic side, however, Miss Howard did not carry her work very far. This doubtless was due to the difficulty of making a factor analysis of the characters which she selected for study, viz., (1) time of flowering, (2) height of stem, (3) arrangement of the leaves on the stem, (4) length of the decurrent portion of the lamina, (5) venation of the leaf, (6) leaf shape, and (7) undulation of the surface and margin of the leaf. For most of these characters she demonstrates, by the presentation of numerical data in some cases as far as F 4 , the probability of the character differences in ques- tion depending upon multiple factor differences. In the case of height certain of her cultures strongly suggest the existence of a pair of allelomorphs, which has a relatively great effect, for in some of her cultures there are definite discontinuous height differences. For the inheritance of length of the decurrent portion of the lamina Miss Howard postulates the existence of at least three or four distinct pairs of factors. As respects leaf base, she records the synthesis of petiolate types from sessile parents, observing in two cases a simple 1 :2 :1 segre- gation into petiolate : intermediate : sessile. As respects corolla color, she records one F 2 population from pink x very pale pink fading into white which consisted of 72 pinks of various shades to 45 whites, but some of the palest pinks were indistinguishable from white. She found evidence of grouping among the pinks, and postulates the existence of two factor differences to account for it. The investigations which we have reported do not throw light upon the factor constitution of the very pale pink varieties with which Miss Howard worked. Our 518 University of California Publications in Botany [Vol.5 varieties angustifolia and virginica have lively pink flowers. Of the paler pinks or "pinkish" forms we have a representative in our N. Tabacum var. Cavala, U. C. B. G. 72/05, which has flowers distinctly lighter in color than those of angustifolia or virginica. Our petiolate forms also seem to he of different constitution from those with which Miss Howard worked, for she presents evidence to show that hers are combinations of recessive factors and that they breed true whenever they occur as segregation products, whereas our petiolate forms often gave plants with sessile leaves as segregation products. We have, however, secured evidence that some distinctly short petiolate forms arise from sessile ones, perhaps by modifications of the aurictlata leaf type in the direction of stripping the auricle and lower portion of the lamina from such leaves, but our results are not yet definite enough to permit of rigid formulation. Further investigation of the relationships of the various petiolate forms is necessary. As respects flower color Allard (1919) has presented some inter- esting data which at first sight appear to contradict those which we have presented. Allard found that carmine x pink gave F t carmine and F 2 3 carmine : 1 pink. The back crosses gave consistent data. Thus Fj carmine x carmine parent gave all carmine, and F x carmine x pink parent gave 1 carmine : 1 pink. In F :! pink segregants bred true for pink, and carmine either bred true for carmine or gave again 3 carmine :1 pink. The difficulty here is that our red is not genetically identical with Allard 's carmine. Our flowers of macrophylla and calycina at full expansion show a color lying between rose red and pomegranate purple of the Ridgway color scale. This color, which we have called red for the sake of brevity, is very close to carmine, but we have an- other flower color, which we call dark red, represented by N. Tabacum var. macrophylla purpurea, which is probably identical with the Giant Red flowering tobacco which Allard used in his experiments. "We have made some preliminary tests of this dark red, and find that it behaves differently from red. Crossed with our white it gives dark red in F 1; instead of pink as was obtained from red x white. Since our white carries the factor R, which is responsible for the production of pink flower color, dark red must differ from pink in a dominant factor. If we call this factor pair Pp, then our various colors of tobacco would have the following genotypes: Dark red Red Pink White WWRRPP WWrrpp WWRRpp wwRRpp I 922 ] Setchell-Goodspeed-Clausen: Nicotiana Tabacum 519 Obviously this formulation would account for Allard's results with- out contradicting those which we have presented, but inasmuch as our experimental evidence is not yet complete we refrain from any further discussion of the consequences of this scheme save one. Allard presents certain data for a cross of carmine x white which gave in F 1 light carmine, and in P 2 3 colored : 1 white, the colored being various shades of carmine and pink. Allard's discussion of this case is somewhat mixed, but he evidently erroneously expected a simple monohybrid segregation of the 1:2:1 kind. That more than one factor is con- cerned in the cross is clearly shown by the results of crossing some of the extracted whites with pink varieties. The results of three such crosses gave : 1. Pink (Maryland Mammoth) x Extracted white 36 carmine : 18 pink 2. Extracted white x Pink (Maryland Mammoth) 20 carmine : 23 pink 3. Pink (Conn. Broadleaf) x Extracted white 12 carmine : 39 pink Totals 68 carmine : 70 pink In (1) above we have combined in the carmine class 17 carmine and 19 somewhat lighter than carmine. If we consider a cross of dark red x white according to the genetic formulation given above, the F x should be dark red, and F 2 should consist of 9 dark red : 3 pink : 4 white. Doubtless the pinks and the dark reds would exhibit various shades, but the three classes should be distinct. If we combine "carmine" and "lighter than carmine" to form a carmine class and dark and light pink to form a pink class, Allard 's P 2 data reduce to the following form : 149 carmine : 64 pink : 65 white. This ratio compares very favorably with a 9 : 3 : 4 expectation, viz. : 157 dark red : 52 pink : 69 white. No F 3 results from sowings from colored F 2 plants are given, but the single F 2 white, which gave when crossed with pink approximately equal numbers of carmine and pink flowering plants, is accountable for as of the genotype wwRRPp. Further investigations are in pro- gress for the purpose of determining precisely the relation of dark red and pinkish to the red, light pink, and white colors reported upon in this paper. There are other references in the literature to Mendelian inherit- ance in Tabacum, but inasmuch as these do not bear upon the characters which we have attempted to analyze it does not appear necessary to discuss them at this point. 520 University of California Publications in Botany [Vol. 5 VII. SUMMARY Studies of three intervarietal crosses in Tabacum demonstrate that : 1. All the differences between varieties of Tabacum can be analyzed in a Mendelian fashion, if sufficient refinement in methods be intro- duced. 2. Stable recombinations of parental characters can readily be obtained with three or four generations of self-fertilization. 3. Characters outside the range between the parents are sometimes produced following hybridization, and these may be readily established in stable lines by self-fertilization. ■4. The petioled leaf base of angustifolia and the sessile leaf base of macrophylla differ in at least three pairs of factors. 5. A single factor difference exists between normal and split hose- in-hose flowers. 6. Two pairs of factors account for the relation existing between red, light pink, and white flower color. A third pair of factors is necessary to account for dark red. On the theoretical side it has been pointed out that : 1. Derivation of relationships and erection of systems of classifi- cation after the manner of Comes and Anastasia cannot be relied upon unless supported by experimental evidence. 2. An adequate scheme of classification should be based upon iden- tities and dissimilarities in the genotypes, irrespective of the derivation of the forms in question. 3. Mendelian analysis in Tabacum requires that special attention be paid to residual portions of the genotype, so that the factor differ- ences under consideration act in a stable residuum most favorable for emphasis of the character differences under investigation. LITERATURE CITED Allard, H. A. 1919. Some studies in blossom color inheritance in tobacco, with special reference to N. sylvestris and N. Tabacum. Am. Nat., vol. 53, pp. 79-84. Anastasia, G. E. 1906. Le varieta tipiche della Nicotiana Tabacum L. Scafati. Bridges, C. B. 1919. Specific modifiers of eosin eye color in Drosophila melanogastcr. Jour. Exp. Zool., vol. 28, pp. 337-384. 1922] Setcliell-Goodspeed-Clausen: Nicotiana Tabacum 521 Castle, W. C. 1919. Piebald rats and the theory of genes. Proc. Nat. Acad. Sci., vol. 4, pp. 126-130. Collins, G. N. 1917. Hybrids of Zea ramosa and Zea tunicata. Jour. Agric. Ees., vol. 9, pp. 383-396. Comes, O. 1899. Monographie du genre Nicotiana comprenant le elassement botanique des tabacs industriels. Atti del E. Instituto d 'Incoraggiamento di Napoli, ser. V, vol. 1. 1905. Dello razze dei tabacchi filogenesi, qualita ed uso. Ibid., vol. 57. East, E. M. 1913. Inheritance of flower size in crosses between Nicotiana species. Bot. Gaz., vol. 55, pp. 177-188. GOODSPEED, T. H. 1912. Quantitative studies of inheritance in Nicotiana hybrids. Univ. Calif. Publ. Bot., vol. 5, pp. 87-168. Goodspeed, T. H., and Clausen, R. E. 1917. The nature of the Pj species hybrids between Nicotiana sylvestris and varieties of JV. Tabacum. Univ. Calif. Publ. Bot., vol. 5, pp. 301-346. Harlan, H. V. 1918. The identification of varieties of barley. U. S. Dept. of Agric, Bull. 622, pp. 1-32. Hayes, H. K. 1912. Correlation and inheritance in Nicotiana Tabacum. Conn. Agric. Exp. Station, Bull. 171, pp. 1-45. Hates, H. K., East, E. M., and Beinhaet, E. G. 1913. Tobacco breeding in Connecticut. Conn. Agric. Exp. Station, Bull. 176, pp. 1-68. Hasselbring, H. 1912. Types of Cuban tobacco. Bot. Gaz., vol. 53, pp. 113-126. Howard, A., and Howard, Gabrielle L. C. 1910. Studies in Indian tobaccos. No. 2, The types of Nicotiana Tabacum L. Mem. India Dept. Agric. Bot. Series, vol. 3, pp. 59-176. Howard, Gabrielle L. C. 1913. Studies in Indian tobaccos. No. 3, The inheritance of characters in Nicotiana Tabacum L. Ibid., vol. 6, pp. 25-114. MULLEH, H. J. 1918. Genetic variability, twin hybrids, and constant hybrids in a case of balanced lethal factors. Genetics, vol. 3, pp. 422-499. RlDGWAY, BOBERT 1912. Color standards and color nomenclature. Setchell, W. A. 1912. Studies in Nicotiana, I. Univ. Calif. Publ. Bot., vol. 5, pp. 1-86. White, O. E. 1913. Bearing of teratological development in Nicotiana on theories of heredity. Am. Nat., vol. 47, pp. 206-229. 1914. The history of Nicotiana, II. An account of the heredity and envi- ronment of a family of tobacco plants. Brooklyn Bot. Garden Leaflets, Series 2, No. 12. EXPLANATION OF PLATES A special note is due the illustrations in this paper. The line drawings were made by Miss Anna Hamilton and Miss Helen M. Gilkey. Special attention was paid to accuracy in proportions and details. No attempt, however, was made to represent the characteristic Nicotiana pubescence. The photographs require no special mention save that sometimes the garden number given in the legend does not correspond with that given on the label in the photograph. The dif- ference is due to a change in system of numbering used in F 3 and in subsequent populations. In this paper, in order to avoid confusion, garden numbers from the beginning have been made to conform to this change. The legends of all the plates have been made more complete than is usual in order to facilitate cross-reference and to enable the reader to grasp their essential significance more readily. PLATE 55 Fig. 1. Nicotiana Tabacum var. angustifolia, U. C. B. G. 68/07. A typical plant of angustifolia at the height of its blooming period. The laterals over- topping the central' axis and the long-petioled stcnophylla form of leaf are especially to be noted. The drooping of the leaves is very characteristic of this variety. Fig. 2. Nicotiana Tabacum var. macrophylla, U. C. B. G. 22/07. A typical plant of macrophylla at the height of its blooming period. Note especially the stout laterals overtopping the central axis and the sessilifolia type of leaf. [522] 1 1 I i PLATE 56 Nicotiana Tabacum var. angustifolia, U. C. B. G. 68/07. Line drawings of typical details of angustifolia. In the upper right-hand coiner the characteristic straplike sessile leaf or bract of the inflorescence. Upper left, details of bud, flower, and capsule. Lower right, details of pistil and stamens. Lower left, the typical long petioled stenophylla leaf of angustifolia. Leaves X % ; flowers and capsules natural size. UNIV. CALIF. PUBL. BOT. VOL. 5 [ SETCHELL-GOODSPEED-CLAUSEN J PLATE PLATE 57 Nicotiana Tabacum var. macrophylla, U. C. B. G. 22/07. Line drawings of typical details of a plant of macrophylla, showing floral details and the extreme variations in leaf size and shape on the plant. Leaves X % ; flowers and cap- sules natural size. [526] » UNIV. CALIF. PUBL. BOT. VOL. 5 [ S ETCH E LL-GOODS PE E D-C LAUSEN ] PLATE 57 PLATE 58 Fig. 1. Nicotiana Tabacum var. angustifolia, U. C. B. G. 68/07. Typical leaves of angustifolia of the stenophylla type showing the range of variation on a single plant. Fig. 2. Nicotiana Tabacum var. macrophylla, U. C. B. G. 22/07. Typical leaves of macrophylla of the sessilif olia type showing the range of variation on a single plant. [528] PLATE 59 Angustifolia-macropftylla series, F 1 leaves. Fig. 1. Typical leaf of IOF^jPs,,, an F 1 of the angustifolia-macrophylla series. Note the short, winged petiole and the clasping auricles. Fig. 2. Typical leaf of 10F,H 3 P M , a variation from the usual latifolia type of the F, leaf. Note the shorter petiole, less conspicuously winged condition, and the almost total lack of auricles. Fig. 3. Typical leaf of 10F,H 4 P i5 . The petiole is somewhat longer than that normal for the F,. Fig. 4. Typical leaf of lOF^P-,,. The petiole here is shorter than that normal for the F,. PLATE 60 Portions of inflorescences of N. Tabacum var. macrophylla, N. Tabacum var. angustifolia and the F, hybrid between them. Fig. 1. Left, portion of inflorescence of macrophylla, middle, of the F,, and right, of angustifolia. Fig. 2. Left, portion of the inflorescence of macrophylla, middle, two of the F„ and right, of angustifolia. | 532 | PLATE 61 August if olia-macrophylla series, Fj plants. Fig. 1. Photograph of 10F,HjP5 0 , the Fj plant from which the leaf shown in plate 59, figure 1, was taken. Fig. 2. Photograph of 10F,H 5 P M , the F t plant from which the leaf shown in plate 59, figure 4, was taken. [534] PLATE 62 Angustifolia-macropliyUa series, line drawings of F t . Line drawings showing morphological details of the typical Y 1 plant of the angustifolia-macrophylla series. The garden number of the plant was 10F,H 10 P,. Leaves X %; flowers and capsules natural size. [536] UNIV. CALIF. PUBL. BOT. VOL. 5 [ SETCH E LL-GOODS PE E D-C LAUSE N ] PLATE 62 PLATE 63 Angustifolia-macrophylla series, type 1. Line drawings of morphological details of F 2 of type 1. The garden number was IIF.HjPjPj,,. Note particularly the stenophylla type of leaf. Leaves X %; flowers and capsules natural size. The F, progeny of this plant consisted of 16 stenophylla of type 1 and 9 lance- olata of type 14. [538] UNIV. CALIF. PUBL. BOT. VOL. 5 [ S ETCH E LL-GOODSPEE D-CLAU SEN ] PLATE 63 PLATE 64 Angustifolia-macrophylla series, type 2. Line drawings of morphological details of F 2 of type 2. The garden number was 11F 2 H 2 P ; ,P :;0 . The leaf is of the latifoUa type. Leaves X %; flowers and capsules natural size. The F a progeny of this plant consisted of 12 latifoUa, 8 sessilifolia, and 4 auriculata. [540] UNIV. CALIF. PUBL. BOT. VOL. 5 [ SETCHELL-GOODSPEED-CLAUSEN ] PLATE 64 PLATE 65 Angustifolia-macrophylla series, type 3 Line drawings of morphological details of F 2 of type 3. The garden number was llF 2 H 2 P 3 Pn. The leaf is of the latifolia type. Leaves X %; flowers and capsules natural size. The F 3 progeny of this plant consisted of 4 latifolia and 10 sessilifolia. [542] UNIV. CALIF. PUBL. BOT. VOL. 5 [ SETCHELL-GOODSPEED-CLAUSEN ] PLATE 65 PLATE 66 Anguntifoliii-nuicrophyUa series, type 4. Line drawings of the morphological details of type 4. The garden number was lir 2 H;.PjP 18 . The leaf is an extreme form of the latifolia type. Leaves X % ; flowers and capsules natural size. No progeny was grown from this plant. [544] UNIV. CALIF. PUBL. BOT. VOL. 5 [ SETCHELL-GOODSPEED-CLAUSEN ] PLATE 66 PLATE 67 Angustifolia-maerophylla series, type 5. Line drawings of morphological details of type 5. The garden number was 11F 2 H J P. 1 ,P 14 . The leaf is of the latifolia type. Leaves X %; flowers and cap- sules natural size. > The F 3 progeny of this plant consisted of 24 latifolia and 1 auriculata. [546] PLATE 68 Angustif olia-macrophylla series, type 6. I Line drawings of morphological details of P 2 of type 6. The garden number was 11F 2 H 4 P 2 P ]8 . The leaf is of the lati folia type. Leaves X %; flowers and capsules natural size. The F 3 progeny, of this plant consisted of 5 stenophylla, 17 latifolia, and 4 auriculata plants, but the segregation was not distinct. [548] PLATE 69 Angustifolia-macrophyUa series, type 7. Line drawings of morphological details of F 2 of type 7. The garden number was 11F ; ,HJ > I1 P 1S . The leaf is of the latifolia type. Leaves X %; flowers and capsules natural size. The F„ progeny of this plant was uniformly of the same type as the parent, and the line bred true in subsequent generations. [550] UNIV. CALIF. PUBL. BOT. VOL. 5 [ SETCHELL-GOODSPEED-CLAUSEN ] PLATE 69 PLATE 70 Angustifolia-macrophyUa series, type 8. Line drawings of morphological details of I\ of type 8. The garden number was llFjELPsP,,!. The leaf approached the auriculata type. Leaves X % ; flowers and capsules natural size. The F :! progeny of this plant consisted of 16 sessilifolia and 8 auriculata, indicating that the plant was an extreme variant of the heterozygous sessilifolia- auriculata condition. UNIV. CALIF. PUBL.. BOT. VOL. 5 [ SETCHELL-GOODSPEED-CLAUSEN ] PLATE 70 PLATE 71 Angustifolia-macrophylla series, type 9. Line drawings of morphological details of F 2 of type 9. The garden number was 1 1F 2 H 4 P 41 P 8 . The leaf is of the latifoUa type. Leaves X %; flowers and capsules natural size. The F 3 progeny of this plant consisted of 18 latifolia and 7 sessilifolia. [554] UNIV. CALIF. PUBL, BOT. VOL. 5 [ SETCHELL-GOODSPEED-CLAUSEN ] PLATE 71 PLATE 72 August if olta-inacropltylla series, type 10. Line drawings of morphological details of F 2 of type 10. The garden number was lir 2 H t P 4 ,P n . The leaf was of the auriculata type. Leaves X %; flowers and capsules natural size. The F :i progeny of this plant consisted uniformly of auriculata plants. [556] UNIV. CALIF. PUBL. BOT. VOL. 5 [ SETCH E LL-GOODSPE E D-CLAU SE N ] PLATE 72 PLATE 73 Angustifolia-macrophylla series, type 11. Line drawings of morphological details of F 2 of type 11. The garden number was 11F 2 H 4 P 41 P 9 . The leaf is of the sessilifolia type. Leaves X %; flowers and capsules natural size. The F 3 progeny of this plant was uniformly of the same sessilifolia type. [558] UNIV. CALIF. PUBL. BOT. VOL, 5 [ SETC H E LL-GOODSPE E D-CLAUSE N ] PLATE 73 PLATE 74 Angustifolia-macrophyUa series, type 12. Line drawings of morphologicaj details of F 2 of type 12. The garden number was 11F 2 H 4 P 4 ,P 12 . The leaf is of the loriifolia type. Leaves X % ; flowers and capsules natural size. The F a progeny of this plant was uniformly of the same loriifolia type, and two constant races, one with red and one with light pink flowers, were obtained from it. [560] PLATE 75 Angustifolia-macrophyUa series, type 13. Line drawings of morphological details of F 2 of type 13. The garden number was 11F 2 H 2 P 3 P 44 . The leaf is of the lanceolata type. Leaves X %; flowers and capsules natural size. The F, progeny of this plant was uniformly of the same lanceolata type. [562] / UNIV. CALIF. PUBL. BOT. VOL. 5 [ SETCHELL-GOODSPEED-C'-AUSEN J PLATE PLATE 70 Angustifolia-macrophylla series, type 14. Line drawings of morphological details of F 2 of type 14. The garden number was 11F 2 H 2 P 3 P. )S . The leaf was classified as sessilifolia, although strictly it is intermediate between sessilifolia and lanceolate. Leaves X %; flowers and capsules natural size. The F 3 progeny of this plant consisted of 24 plants of the same sessilifolia type and 1 "filler," which had leaves more like auriculata of type 8. [564] UNIV. CALIF. PUBL. BOT. VOL. 5 [ SETCHELL-GOODSPEED-CLAUSEN •] PLATE 76 PLATE 77 Angustifolia-tnacrophyUa series, type 15. Line drawings of morphological details of F 2 of type 15. The garden number was lir 2 H 2 P 3 P 10 . The leaf is of the scssilifolia type. Leaves X %; flowers and capsules natural size. The F 8 progeny of this plant was uniformly of the same leaf type. [566] UNIV. CALIF. PUBL. BOT. VOL. 5 [ S ETCH E LL-GOODSPEE D-CLAU SE N ] PLATE 77 PLATE 78 Angustifolia-macrophylla scries, type 16. Line drawings of morphological details of F 2 of type 16. The garden number was 11F 2 H 2 P 3 P S0 . The leaf is of the sessilifolia type. Leaves X %; flowers and capsules natural size. The F 3 progeny of this plant consisted of 15 sessilifolia and 8 auriculata. [568] UNIV. CALIF. PUBL. BOT. VOL. 5 [ SETCH E LL-GOODS PE E D-C LAUSE N 1 PLATE 78 PLATE 79 Nicotiana Tabacum var. calyctna, U. C. B. G. 110/05. Line drawings of morphological details of leaf and flower of calycina. The leaf is of the lanceolata type, and the flowers are of the conspicuously teratolog- ical, split hose-in-hose form. Leaves X %; flowers and capsules natural size. L570] UNIV. CALIF. PUBL. BOT. VOL. 5 [ SETCHELL-GOODSPEED-CLAUSEN ] PLATE 79 PLATE 80 Nicotiana Tabacum var. virgmica, U. C. B. G. 78/05. Line drawings of morphological details of leaf and flower of virginica. Con- trast the normal flowers and auricled leaves with the corresponding details of calyeina, shown in plate 79. Leaves X % ; flowers and capsules natural size. [572] UNIV. CALIF, PUBL. BOT. VOL. 5 [ S ETCH E LL-GOODSPEE D-CLAU SE N ] PLATE 80 PLATE 81 Calycina-virginica series, an F, plant. Line drawings of morphological details of an Fj plant of the calycina-virginica series. Note the normal flowers and the slightly auricled leaves. Leaves X %; flowers and capsule natural size. [574] UNIV. CALIF, PUBL. BOT. VOL. 5 [ S ETCH ELL-GOODSPEE D-CLAU SE N ] PLATE 81 PLATE 82 Nicotiana Tabacum var. alba, U. C. B. G. 30/06. Line drawings of morphological details of flower and leaf of alba. Note especially the rugose leaf. Compare this drawing with those of macrophylhi shown in plates 57 and 58, figure 2. Leaves X % ; flowers and capsule natural size. [576] UNIV. CALIF. PUBL. BOX. VOL. 5 [ S ETCH E LL-GOODSPE ED-C LAU S E N ] PLATE 82 PLATE 83 Tig. 1. Calycina-virginica series, an F, plant. A typical F, plant of the calycinct-mrginica series. This plant is at the height of its blooming period. The garden number was 10F,H 18 P M . Fig. 2. Alba-macrophylla series, an F, plant. Photograph of a typical F, plant of the alba-macrophylla series. The garden number was 10F,H 2 ,P r>1 . The plant is at the height of its blooming period. [o78j PLATE 84 Alba-macrophylla series, I\ leaves. Fig. 1. Photograph of typical leaves of alba. Fig. 2. Photograph of a typical leaf- of lOF^^Pu, F, of the alba-marcopliylla series. Fig. 3. Photograph of a typical leaf of lOF^^P^, F t of the alba-macrophylla series. Compare these leaves with those of macrophylla, shown in plate 58, figure 2. The rugoseness of alba has been carried over, to a somewhat reduced extent, into the F, hybrid. [580] PLATE 85 Fig. 1. Alba-macrophyUa series, F 2 plants. Photograph of two adjacent plants, IIFoELjPjjP^ and 11F;,H 24 P M P =:1 , from the same F 2 population of the alba- macrophyUa series. An illustration of segregation for height in this population. Fig. 2. Alba-macrophyUa series, an F t plant. Photograph of a typical F, plant, 13F 4 H 24 P 34 P 23 P 2 P-„, of a dwarf line of the alba-macroph ylla series. The line here illustrated was derived from the dwarf F 2 plant shown in figure 1. This line has bred true for seven generations. [582] UNIVERSITY OF CALIFORNIA PUBLICATIONS — (Continued) VOL 6. 1914-1919. 1. Parasitic Florideae, by William Albert Setchell. Pp. 1-34, plates 1-6. April, 1914 „ ; .35 2. Phytomorula regularis, a Symmetrical Protophyte Related to 'Coelastrum, by Charles Atwood Kofoid. Pp. 35-40, plate 7. April, 1914 05 3. Variation in Oenothera ovata, by Katherine Layne Brandegee. Pp. 41-50, plates 8-9. June, 1914 10 4. Plantae Mexicanae Purpusianae. VL by Townshend Stith Brandegee. Pp. 51-77. July, 1914 .25 5. The Scinaia Assemblage, by William Albert Setchell. Pp. 79-152, plates 10-16. October, 1914 _ 75 6. Notes on Pacific Coast Algae. I, PylaieTla Tostelsiac. n. sp., a New Type in the Genus Pylaiella, by Carl Skottsberg. Pp. 153-164, Diates 17-19. May, 1915 „ _ .... .15 7. New and Noteworthy Califomian Plants. IL by Harvey Monroe Hall. Pp. 165-176, plate 20. October, 1915 „ _ „ 15 8. Plantae Mexicanae Purpusianae. VII, by Townshend Stith Brandegee. Pp. 177-197. October, 1915 _ 25 9. Floral Relations among the Galapagos Islands, by A. L. Kroeber. Pp, 199^220. March, 1916 20 10. The Comparative Histology of Certain Califorjiian Boletaceae, by Harry S. Yates. Pp. 221-274, plates 21-25. February, 1916 ." 50 11. A Revision of the Tuberales of California, by Helen Margaret Gilkey. Pp. 275-356, plates 26-30. March, 1916 , 80 12. Species Novae vel Minus Cognitae, by T. S. Brandegee. Pp. 357-361. May, 1916 :. , 05 13. Plantae Mexicanae Purpusianae. VIII, by Townshend Stith Brandegee. Pp. 263-375. March, 1917 _ 15 14. New Pacific Coast Marine Algae. I, by Nathaniel Lyon Gardner. Pp. 377- 416, plates 31-35. June, 1917 .40 15. An Account of the Mode of Foliar Abscission in Citrus, by Robert W, Hodgson. Pp. 417-428, 3 text figures. February, 1918 ' 10 16. New Pacific Coast Marine Algae. II, by Nathaniel Lyon Gardner. Pp. 429- 454, plates 36-37. July, 1918 25 17. New Pacific Coast Marine Algae. HI, by Nathaniel Lyon Gardner. Pp. 455-486, plates 38-41. December, 1918 35 18. New Pacific Coast Marine Algae. IV, by Nathaniel Lyon Gardner. Pp. 487-496, plate 42. January, 1919 15 19. Plantae Mexicanae Purpusianae. IX, by Townshend Stith Brandegee. Pp. 497-504. November, 1919 „ 05 Index in preparation. VoL 7. 1916-. 1. Notes on the Califomian Species of Trillium L. I, A Report of the General Results of Field and Garden Studies, 1911-1916, by Thomas Harper Good- speed and Robert Percy Brandt. Pp. 1-24, plates 1-4. October, 1916 .25 2. Notes on the Califomian Species of Trillium L. IL The Nature and Occur- rence of Undeveloped Flowers, by Thomas Harper Goodspeed and Robert Percy Brandt. Pp. 25-38, plates 5-6. October, 1916 ... .15 3. Notes on the Califomian Species of Trillium L. Til, Seasonal Changes in Trillium Species with Special Reference to the Reproductive Tissues, by Robert Percy Brandt. Pp. 39-68, plates 7-10. December, 1916 „. .30 4. Notes on the Califomian Species of Trillium L. IV, Teratological Varia- tions of Trillium sessile var. giganteum H. & A., by Thomas Harper Good- speed. Pp. 69-100, plates 11-17. January, 1917 - ~ .30 UNIVERSITY OF ILLINOIS-URBANA 3 0112 118457982 UNIVERSITY OF CALIFORNIA PUBLICATIONS — (Continued) 5. A Preliminary List of the Uredinales of California, by Walter C. Blasdale. Pp. 101-157. August, 1919 JO 6, 7, 8. A Rubber Plant Survey of Western North America. I. Chrysothamnus nauseosus and Its Varieties, by Harvey Monroe Hall. II. ChrysiL a New Bubber from Chrysothamnus nauseosus, by Harvey Monroe Hall and Thomas Harper Goodspeod. III. The Occurrence of Rubber in Certain West American Shrubs, by Harvey Monroe Hall and Thomas Harper Ooodspeed. Pp. 159-278, plates 18-20, 8 figures in text. November, 1919. 1.26 9. Phycological Contributions. I, by William Albert Setchell and Nathaniel Lyon Gardner. Pp. 279-324, plates 21-81. April, 1920 50 10. Plantae Mexican ae Purpusianae. X, by Townshend Stith Brandegee. Pp. 325-331. December, 1920 ... _ J.0 VoL 8. 1919-. 1. The Marine Algae of the Pacific Coast of North America. Part L Myxophyceae, by William Albert Setchell and Nathaniel Lyon Gardner. Pp. 1-138, plates 1-8. November, 1919 - _ 81.50 2. The Marine Algae of the Pacific Coast of North America. Part II. CMorophyceae, by William Albert Setchell and Nathaniel Lyon Gardner. Pp. 139-374, plates 9-33. July, 1920 „ 2.75 VoL 9. A Report upon the Boreal Flora of the Sierra Nevada of California, by Frank Jason Smiley. Pp. 1-423, plates 1-7. October, 1921 _ 5.00 VoL 10. 1922-. 1. The Genus Fucus on the Pacific Coast of North America, by Nathaniel Lyon Gardner „ (Jn press)