: ri eaten amtaake ernie easiecmaere “l SMa ttre soda tl heer te geen arc eigen i Anadis dohe te paw t bee PP malls ne PLR ONTAM. Rertooe, Ne aa re a ror i te Stee ee LIBRARY OF GHE FOR THE & s PEOPLE ~4 < FOR SZ , EDVCATION © yy oA FOR ©. SCIENCE aS S YS “Td CORNELL UNIVERSITY AGRICULTURAL EXPERIMENT STATION MEMO TR No e 3 9=66 except those of no interest in AMNH 1921 =23 R762 MAR 2 2 1955 / 7 of oe Cornell University Arricultural Experiment Station. The following Memoirs have been discarded = No.- 42 Bean anthracnose -1921- 45 Botrytis of tulips 46 Class ification of .. barley 48 Inheritance of salmon silk color in maize eee ewe eS ewe == ee Seem me ewe we ewe = Memoir " MARCH, 1921 MEMOIR 39 CORNELL UNIVERSITY AGRICULTURAL EXPERIMENT STATION _ THE GENETIC RELATIONS OF PLANT COLORS IN MAIZE R. A. EMERSON ITHACA, NEW YORK PUBLISHED BY THE UNIVERSITY MARCH, 1921 MEMOIR 39 CORNELL UNIVERSITY AGRICULTURAL EXPERIMENT STATION THE GENETIC RELATIONS OF PLANT COLORS IN MAIZE R. A. EMERSON ITHACA, NEW YORK PUBLISHED BY THE UNIVERSITY CONTENTS PAGE TEIREN TIONS) LEANER NTNTIOINENS a 6 wre ceeacosacstcas-o cece OSG OOO ROI Or Gio BiG Sic ROn Cin eee ye eo 8 Sourncerand description of materials used!2.. 22... hes sec eee cote nee cee ence nce 9 JPHUTROND, CHADS Le o' 6 ci: ciseser ner eH eae erate a er a 9 Sram rag). Giga Te 2 Sos Ae eae area a snr SN Pipi ek ae 2 ete 11 TD slkanthce panescralte,, (inp aeeU WE See ee caesar epee ec 11 DikieRsUMened smtyiOe wD Verrs tee cya eo us ccckdin wi renNarasrerrnset Mia aumanaa ci ura ee Gia ste Ses 12 IRE, THAOS Vevey ens ORS ee eee ane SPictoite oA it Seo ReneS AAA er ere a 14 Gireain, WS Wls: 5h cleats cuss eee cease eee eRe eo ce cera oR ec 14 Relationvoluplanticolorsito environment.)..2.-2+ 40 asses 04dene sess ns dees ss dests ees. 15 Sunlishigapactorunycolondevelopment. 1c... 2 = occ le esc ee esio oss one ee ee eee 16 Wiroistumemmenela GOmptOnCOlOnpsrcdtics. ce caceuers Saeed cha a Sine Eee 18 MemmeraiUnesnyrela tl On svOxCOlOn csp es, ae a aa ree OE Seen on eerie ie ss el ute soca 19 Solbterwlinvgandecolordevelopment.< {522-20 canes ales ease anos esos ees ns ee eels 21 Ric hacompane dewablupOor Soles. six peicwle evs esc dsc8 tee erelib ses Noses sl Ao Meese eee 21 lrackqoispanticularmutrient elements na. son ose oilers yds a st ee oes cde de 23 Relatonmolcanbobydratesstorcolore. «ca. \aeqae Go ace teria eee eas ch sees e 26 SUTTON 2 9 60.0. 0°66 ey SPU EEE CRT Rte ee Set ROR ee Sa CT aa a 28 GeneticrmalysistomcolonityPeS 4... ose save se aise leneiavee ne greih ictus! Walesa dee anes Goa as 29 Crossesnmyolvanesthe factor pairs Aa, Bibs Pl plies. 65k vecnls ov scs cee ee ee. 29 gong lewlcupx@omee nin Vil Cs erga et setn eo mndin ann at ian, sani s hin Uraleaeh ins ache Wl escnce esd arucdewters 29 IDalhavice een Travel TAYE S35 ov Roh ia wu eee as ator ace ee alee ete el 32 BACKCEOSSeS(Oiglapxaivilc andolViaex Vi with Vamarcdac see dy Sean yea ne 34 Behavior of F2 color types in later generations......................00 00000 eee eee 35 MiTHEREROSSESKOIME ouCOLOMNLY DES: c5)s.clsuce de cee aie le och te ee ssbb neki cistiat Heeb 48 Evidence from aleurone-color and linkage relations......................2-0-0-0-- 58 Summanvgotresultssnvolving Ava. Diol pl rn. sie ee otis der Aes cle ae ee ee 64 Crosses involving the multiple allelomorphs B, BY, b8,b.............0..0005-. A ye ews 65 Interrelations of sun red IIa, weak sun red IIb, and dilute sun red IVa............. 67 Relation of weak purple Ib to purple Ia, dilute purple IIIa, and weak sunred IIb...... 69 Crosses involving the multiple allelomorphs R*, RI, R79, 717, 79, 7% 222. ee 73 (CreerM IN Cexa LOW INI E moe a ricen te imcnare Sates ain oe mronthue dete Bilt Sig ene el neeele ald demi 74 Kitercrossest ole HorCOlonibypeS ain sirscirsrseaciistcisie shcesus a oon een eed eey wm iden ecw ena 86 iPurplevlaesreen-anthered' dilute/sum red’... 2.0.2. ....¢ eee: set ec see ee tee eee: 111 Summary of results involving the allelomorphic series R’, RI, R™9, 77, 19, 7%... 112 Relation of aleurone factors Cc and Prpr to plant color..................0000-2008- 113 Expression of plant-color and aleurone-color factors...........0.0.. 00000 cece cece eee 114 SHETTY 0. 6 0 bie 5c '5 BOE GAY AERIS eae oes EY Bree i Cae SUMS tea eOD OR AO a eG 118 Miner nulnemelbed manpraranr mera sac eure eee Namen coal al ait teibm rac avdwitleld eae a's 120 EMSHPHOUan (COM LAINIE CAD ICS) 2 naptgte ian Sata sis ie cele ee oe ae oe oe aie et eS deaa teebeces 121 THE GENETIC RELATIONS OF PLANT COLORS IN MAIZE THE GENETIC RELATIONS OF PLANT COLORS IN MAIZE! R. A. EMERSON Under the designation ‘ plant colors” are included the colors other than those related to chlorophyll, commonly seen in, but not limited to, such external plant parts of maize as the culm, the staminate inflorescence, the husks, the leaf sheaths, and to some extent the leaf blades. In con- trast to this group are colors and color patterns related to chlorophyll or associated with the pericarp and the cob, the silks, the endosperm, the aleurone. The colors included in the group considered here are due to water-soluble pigments, but the same is true of some of the other color groups named above. Moreover, colors of the chlorophyll group (Lind- strom, 1918) are found in the same plant parts as are the “ plant ” colors considered in this account. The plant colors as a whole are closely interrelated, but they are closely related also to aleurone colors and to certain of the silk and pericarp colors. It is obvious, therefore, that, while this classification is a more or less natural one, it is based primarily on convenience. The term “ genetic relations ”’ in the title to this memoir is to include not merely an account of the genetic analysis of the material at hand by means of hybridization experiments — tho that constitutes the greater part of the paper — but also some consideration of the variations of the several color types induced by or associated with environmental diver- sities. Some little attention to matters of this kind was made necessary by the fact that presumably homozygous material exhibited marked variations in extent and intensity of pigmentation when grown under diverse conditions. Since, as will be apparent later, the principal differences between certain of the color types under investigation are apparently quantitative ones, and since the materials at best exhibit no little complex- ity with respect to factorial interrelations of a genetic nature, little progress could have been made without some notion of the response of particular color types to certain factors of the environment. But this study has 1 Paper No. 78, Department of Plant Breeding, Cornell University, Ithaca, New York. 7 8 R. A. EMERSON been wholly subsidiary to the main purpose, namely, a genotypic analysis of the color types under observation. The writer’s realization of the superficial nature of the environmental studies reported in this account in no way weakens his belief in the importance of acquiring an accurate knowledge of the chemistry of the pigments concerned and of instituting fundamental investigations into the physiology of their development — problems that must await the interest and effort of other workers. The studies reported here were begun in a small way in 1909 and have been continued, along with other problems in the genetics of maize, to the present time. The work was conducted at the University of Nebraska and supported by funds of that institution from 1909 to 1914. During 1911 facilities for growing and studying a considerable part of the cultures then in hand were generously afforded the writer by the Bussey Institu- tion at Harvard University. Since 1914 the work has been conducted at Cornell University. During these years, the writer has been assisted by a number of persons, among whom he desires to mention particularly Dr. E. W. Lindstrom and Dr. E. G. Anderson. Some data from the records of students associated with the writer are included in this account. The cultures giving these borrowed data are indicated in the tables by initial letters preceding the pedigree numbers, as follows: A = E. G. Anderson, L = E. W. Lind- strom, and S = Sterling H. Emerson. The illustrations are from water-color drawings by C. W. Redwood, Miss Carrie M. Preston, and Miss Bernice M. Branson. PREVIOUS INVESTIGATIONS So far as the writer is aware, little work with the plant colors of maize has been reported previous to this time. Webber (1906) reported the results of studies of the interrelations of aleurone, silk, anther, and glume colors, with the conclusion that color in all these parts is closely correlated but that there are definite breaks in the correlation. This conclusion, in terms of present-day usage, is apparently equivalent to the idea of close linkage with some crossing-over. East and Hayes (1911) identified certain aleurone-color genes, which are shown in the present account to be related to plant colors as well as. to aleurone colors, and reported data concerning the inheritance of silk and anther colors. The writer (Emerson, 1918) added another aleurone-color pair also known to PLANT Cotors IN Maize 9 be concerned in plant-color development. . He had earlier (1911) announced some of the plant colors discussed in the present paper and placed on record some evidence as to their genetic behavior. Gernert (1912) described types of maize that differ widely in color of anthers, glumes, silks, sheaths, and husks, and reported simple mendelian behavior in F, and F, of certain crosses. With this exception, Gernert’s extensive investi- gation of plant-color types has not been reported, but the writer has been able, thru an exchange of material, to compare some of Gernert’s types with those in his own cultures. SOURCE AND DESCRIPTION OF MATERIALS USED The plant-color types discussed in this paper came in the main from the crossing of two little-known varieties, one of which was obtained at a national corn exposition and the other from an exhibit at a local agri- cultural fair. One of the color types produced by this cross is the same as that of the dent varieties generally grown thruout the Corn Belt; a second is not infrequently seen in certain pop, flint, and sweet corn varieties; and a third occurs in the fields of flour corn of certain Indian tribes of the Southwest. One of the color types produced by the cross had no existence, so far as the writer knows, until it appeared in his cultures. Modifications of several of the six color types noted above have been produced by crossing with a color type common in a few varieties of sweet corn and closely related to the type most common in field maize. The principal color types concerned in this account are discussed in some detail in the descriptive notes below. They are: I — Purple Il — Sun red III — Dilute purple IV — Dilute sun red V — Brown VI — Green PURPLE, TYPE I Material of the purple type was first obtained as a single ear from a local agricultural fair at Nehawka, Nebraska, in 1906. The varietal name is unknown. The uncrossed stock was a smooth-seeded pop corn 10 R. A. EMERSON of medium size. No other stock of purple has been used in the crosses described later in this account, and the writer has never seen this color type in cultivation outside his own cultures. A sample of dent corn of apparently the same color type was seen at a national corn exposition in 1909. A stock of purple was obtained from Dr. Gernert in 1914 but was not used in genetic studies. Another stock of purple was received more recently (1919) from Messrs. Collins and Kempton, the seed having come originally from Bolivia. Seedlings of the purple type are usually indistinguishable from those of types II, III, and IV (described more fully under type IVa, page 12), altho, unlike the other types, they develop some color when grown in darkness. Half-grown plants of type I usually have the lower sheaths prominently colored, in which respect they exceed type II plants in intensity of pig- mentation and are sharply differentiated from types III and IV. At the flowering stage, plants of type Ja have much purple color in nearly all parts, such as the culm, the brace roots, the leaf sheaths, the husks —even the inner ones — the cob, and the staminate inflorescence including the rachis, the spikelets, and the anthers (Plates I, 1, and V, 1). In some cases the color extends over the whole leaf, and it is always seen in the midrib. The purple pigment of type Ia develops in local darkness, as has been shown by covering various parts of growing plants with several thicknesses of heavy black paper (Plate VIII, 1). The color persists in mature plants with slight fading in the outer parts due to weathering (Plate VII, 1). The pericarp of type Ia is either colorless. red, or cherry, and the aleurone is either purple, red, or colorless. With red aleurone the anthers are reddish purple, and with cherry pericarp they are usually very dark purple, almost black (Plate I, 2 and 8). A subtype of purple known as weak purple, or type Ib, is similar to Ia but the pigmentation is less intense, particularly in the culm and the inner husks (Plate V, 2). In early stages of growth it is often difficult to distinguish Ib from Ila. The anthers of Ib are usually deep purple, as are those of Ia, and the pericarp is the same as for Ia. Another sub- class of purple, Ig, is like Ia except that the anthers are green (Plate I, 4) and the pericarp is red or colorless, never cherry. The aleurone color is the same as in Ia. PLANT CoLors IN MAIzE 11 SUN RED, TYPE II Sun red, tho not a common color type, is encountered in a few varieties of sweet corn and pop corn. It is always produced in F»2 of certain crosses, notably in purple x green. While this type is less highly colored than Ia, it has athe strong color that it is not easily distinguished from the latter in early stages of growth. At the flowering stage, type Ila is sharply differentiated from type Ia in several respects. The staminate inflorescence of Ila is lighter than that of Ia, and the anthers are deep pink instead of purple (Plate III, 1). In type Ila, pigmentation of the culm, the leaf sheaths, and the husks is limited almost wholly to parts exposed to sunlight, hence the name sun red. The inner husks are therefore without red color, and rarely does much color develop in any but the outer layer of husks (Plate V, 3) not- withstanding the fact that sufficient light penetrates to the inner husks to induce the development of some chlorophyll in them. A tassel inclosed in a black paper bag produces no red color in either glumes or anthers (Plate VIII, 4). Since the color of sun red plants is so largely superficial, it disappears almost wholly from mature plants thru weathering (Plate VII, 2). Sun red plants have either red or colorless, but never cherry, pericarp, and either purple, red, or colorless aleurone. Sun red of type Ilg differs from Ila merely in having green instead of pink anthers. Type IIb, known as weak sun red, differs from Ila in the lesser intensity and extent of its pigmentation. Particularly the leaf sheaths and the husks are less highly colored than in type Ila. Often the color of the husks develops in alternate dark and light bars parallel to the upper margins of the overlapping husks (Plate V, 4). Types Ib and Ilg have the same pericarp and aleurone colors as IIa. DILUTE PURPLE, TYPE III The dilute purple type, as well as the sun red, occurs regularly in F»2 of purple x green, and most of the dilute purple material in the writer’s cultures came originally from this and other crosses. It was first observed in the progeny of such crosses in 1909. Recently two stocks of this color type have been received from G. N. Collins, one obtained from the Hopi Indians of southwestern United States and the other from Bolivia. 12 | R. A. EMERSON Seedlings and young plants of type IIIa show no more color than do those of type IVa, and apparently do not develop color in darkness. As the plants approach the flowering stage, they usually show somewhat more color than do plants of type IVa, particularly at the base of the culm and in the brace roots, and sometimes in the leaf sheaths. The staminate inflorescence is usually, tho not always, somewhat more highly colored than that of type [Va. The anthers are deep purple, like those of type Ta (Plate II, 1). With red aleurone the anthers are usually reddish purple, and with cherry pericarp they are dark purple, sometimes appearing nearly black (Plate Ii, 2 and 3). The anther color develops fully in dark- ness, but the glumes are slightly if at all colored when protected from light by black paper bags (Plate VIII, 3). As the plants mature, considerable color develops in the inner husks (Plate VII, 3), on the leaf sheaths, and particularly in the culm even where it is protected from strong light by the sheaths. In some cases the culm and the sheaths ultimately become nearly as strongly pigmented as type Ia, but ordinarily the mature plant is considerably less highly colored than the purple type (Plate VII, 4). The color seen in mature plants develops well in local darkness, in which respect also type IIIa is like Ia. Dilute purple differs from purple, there- fore, mainly in a less intense pigmentation and in a delayed development of pigment. The pericarp of type IIla is either red, cherry, or colorless, and the aleurone is either purple, red, or colorless, Just as in type Ia. There exists a type of plant color which is closely related genetically to type IIIa, but which lacks red or purple color in culm, sheaths, silks, glumes, and anthers and is consequently known as Green, type IIlg (Plate II, 4). The aleurone of this type is either purple, red, or colorless, and the pericarp is either red or colorless, never cherry. With respect to aleurone and pericarp, therefore, type I1Ig is like type Ig. DILUTE SUN RED, TYPE IV Dilute sun red is the commonest color type of maize in cultivation. It is practically the only color type seen in the dent varieties grown in the Corn Belt of the United States, and is common in flint, flour, sweet, and pop corns. Like the sun red and the dilute purple types, it always appears in crosses of purple Ia with green VIc. The seedlings of type IVa usually show more or less sun red pigment in the coleoptile, the leaf sheath, and the leaf margins. The young PLANT Cotors IN MAaAIze 13 plants ordinarily have considerable color at the base of the lower sheaths, but little or no color except green in other parts except in the margins of the leaves (Plate IX, 1). When the plants are grown on infertile soil, much bright red color develops in all parts exposed to light except the youngest leaves (Plate IX, 2). The seedlings and the very young plants are not ordinarily distinguishable from those of types Ia, Ila, and IIIa. Some time before the flowering stage, the plants of this type are sharply differentiated from those of types Ia and Ila, and are usually somewhat less highly colored than those of type IIIa. In normally grown plants, the color is confined mostly to the brace roots, and to the sheaths and the exposed parts of the culm at the base of the plants. Even at the flowering stage almost no color is seen in the upper sheaths or the upper part of the culm, and very little in the husks (Plate VI, 1). The staminate inflores- cense is colored much as is that of the sun red type, tho the glumes are lighter than those of type Ila and the rachis is usually nearly devoid of color. The anthers show more or less pink, as do those of type IIa. There is much variation in the extent and intensity of pigmentation of glumes and anthers (Plate III, 2, 3, and 4), due in part to genetic differ- ences and in part probably to environmental influences. Late in the life of the plant, type [Va usually shows some color in the outer husks and also in exposed parts of the culm. Different strains show considerable variation in this respect (Plate VI, 1 and 2). Due to the slight develop- ment of pigment and because of weathering, the dry parts of mature plants show little red color (Plate VII, 6). Light is essential to the development of color in dilute sun red, IVa, Just as in sun red, Ila. The aleurone and pericarp colors of dilute sun red, IVa, are the same as those of sun red, ITa. A wholly green type, that is, one devoid of pigment other than green in the plant parts here under consideration, is closely related genetically to type IVa and is therefore known as type IVg (Plate II, 4). Phenotypi- cally it is the same as type IIIg. Just as in case of types Ig, I, IIIg, and IVa, the pericarp of IVg is either red or colorless, never cherry, and the aleurone is either purple, red, or colorless. Genotypic diversities in the amount of color are noted for type IVa above. The lightest types of dilute sun red show no color except mere traces of red in the staminate spikelets. This condition is found in most plants of at least two varieties of sweet corn, Black Mexican and Crosby. From these varieties there 14 . R. A. EMEerson have been isolated strains that lack even this minimum of color. These strains furnished the original stock of type IVg. In no environment as yet encountered has any red or purple plant color developed in type IVg. BROWN, TYPE V The brown type was first seen in 1912, when it occurred in F2 of the cross purple Ia x green VIc. So far as the writer has been able to learn, brown plant color had not been reported previously, and he is unaware of its existence outside of his own cultures or of stocks grown from them. Seedlings and young plants of type V are wholly green. Before the flowering period is reached, a brown pigment begins to appear in the lower sheaths. At the time of flowering, the culm, the sheaths, the husks (Plate VI, 3), and the staminate inflorescence (Plate IV, 1 and 2) are brown. The anthers are usually green. The brown color extends to the inner husks, to the culm beneath the leaf sheaths, and to the cob (Plate VII, 5). That hght is not essential to the development of brown is shown further by the fact that the color appears under several thick- nesses of black paper (Plate VIII, 2). It is not uncommon to find traces of purple associated with the brown in the brace roots and at the base of the inner husks (Plate VI, 3). Abnormally developed tassels, not infre- quently seen on plants grown in small pots in the greenhouse, in some— cases show a little purple (Plate XI). The aleurone of brown plants is always colorless, except for xenia grains, and the pericarp is either brown, brownish, or colorless, never red nor cherry. Brown pericarp color of type V corresponds to red of types I, II, III, and IV, and brownish to cherry of types I and III. GREEN, TYPE VI The writer’s stock of the green type originated from a single ear obtained at a national corn exposition held at Omaha in 1909. The corn was exhibited from southern Missouri, where it is grown locally. It is a large dent variety, rather late in season. Cultures of type VIe, derived from this stock, show no plant color other than green at any stage of development or under any environmental conditions to which they have as yet been subjected (Plates IV, 3, and VI, 4). PLant Coors IN MAIzE 15 Three subclasses of type VI are recognized. One of these, VIa, is like Vic in every respect except that a slight amount of brown is sometimes seen in the outer husks and sheaths (Plate VI, 5). The second, VIb. is green except for a slight tinge of brown in the spikelets of the staminate _ inflorescence (Plate IV, 4). Asa rule, the development of brown pigment in Vla and Vib is not sufficient to differentiate with certainty the one from the other, or either from Vic. The three subclasses, a, b, and ec, are therefore usually classed together as type VI. Both Vla and VIb have been isolated from crosses involving VIc. The aleurone of all type VI plants, just as in those of type V, is colorless, except for such color as may be due to xenia. The pericarp of Vla and VIc is either brown or colorless, never brownish, while that of VIb is brown, brownish, or color- less, as in the case of type V. With brownish pericarp, type VIb usually shows unmistakable brown color in the staminate spikelets. RELATION OF PLANT COLORS TO ENVIRONMENT From the preceding descriptive notes and accompanying illustrations, it is clear that many of the differences separating the six major color types and their several subclasses are quantitative. Purple plants are -more strongly colored than are sun red or dilute purple plants. Dilute sun ‘red plants have less color than sun red or purple plants. Weak purple plants have less color than purple ones, but more than dilute purple ones, and weak sun reds are intermediate between sun reds and dilute sun reds. Dilute sun red plants vary, from those showing considerable color to those which, except for green, are nearly colorless. Wholly green plants are classed as subgroups of both dilute. purple and dilute sun red. The subclasses of type VI differ so little with respect to color that they are ordinarily thrown together as one green type. Heterozygous brown plants are lighter than homozygous ones, and, since more than one factor pair is concerned, there is a fairly smooth gradation from the darkest to the lightest browns. Plants of types Vla and VIb, when they show any — brown, differ in the parts colored. The cclor of the staminate inflores- cence, and even of other parts, of purples, dilute purples, browns, and greens of type VIb is darker when the pericarp is cherry or brownish than when it is red, brown, or colorless. The natural intergrading of genetic types in this somewhat complex series is often made still more confusing by the variations accompanying 16 R. A. EMERSON environmental diversities. A prominent geneticist, on observing some of the writer’s cultures, was led to say that there were no sharply differ- entiating characteristics by which other than an arbitrary classification could be made, and asserted that he could select from a single progeny a series grading from the darkest to the lightest colors. The writer has some doubt that this could have been done, but the instance illustrates well the difficulties that confront one unacquainted with the materials. It is fortunate thaé some environmenta! influences which increase the difficulty of assorting certain color types make other types stand out more sharply than they otherwise would. Without some notion of these envi- ronmental effects, a genetic analysis of the material would indeed be difficult. SUNLIGHT A FACTOR IN COLOR DEVELOPMENT The relation of sunlight to the development of color has been noted briefly in the descriptions of some of the color types. The effects of sunlight or of local darkness, instead of adding to the confusion of color types, afford a means of sharp differentiation between certain types. So far as is known at present, no color develops in sun red or dilute sun red plants, or in the early stages of growth of dilute purple plants, except under the influence of fairly strong light. In the case of purple and of the later stages of growth of dilute purple, there is no doubt that the color develops more rapidly at first in light than in darkness, but ulti- mately color develops fully, or apparently so, even in local darkness (Plate VIII). The seedlings of purple plants develop some color when germinated and grown in a dark chamber where no part of the plant receives light. There is some, tho very little, evidence that the development of brown pigment of type V is hastened by the influence of light, and what little brown color ever develops in type Vla is confined to parts exposed to sunlight (Plate VI, 5). It would not be surprising to find that the pigments seen in the purple, dilute purple, sun red, and dilute sun red types are the same chemically. In fact they look alike in water solution and apparently react in the same way to simple chemical tests. If they prove to be identical, it would seem to follow that purple and dilute purple plants have some inherent mechanism, perhaps an organic catalyzer, capable of initiating or hasten- ing chemical reactions, and that this mechanism is lacking in sun red Puant Cotors IN Maize 17 and dilute sun red plants, in which the same reactions may possibly be brought about thru the action of sunlight. Usually a single thickness of black paper, such as is employed to pro- tect photographic plates from light, is sufficient to prevent the develop- ment of color in sun red plants (Plate VIII, 4). That more intense light is necessary for the production of sun red pigment than for the production of chlorophyll is shown by the almost entire absence of red color in all but the outer husks, while even the innermost husks are somewhat green (Plate V, 3). The pigments of purple and brown plants, on the contrary, develop well even when there is too little. light for the formation of chloro- phyll (Plate VIII, 1 and 2). That the effect of light on color development is a definitely local one is shown by the sharp line of demarcation between colored and colorless areas in culms, husks, and sheaths partly exposed and partly protected by overlapping sheaths or husks (Plate V, 3). Even a single piece of wrapping cord tied closely about a young ear, sheath, or culm of a sun red plant is sufficient to prevent the development of color beneath it. Evidently sun red pigment does not diffuse appreciably from the cells in which it forms. It is not meant to suggest by these observations that sunlight has no effect other than a local one on color development. On the contrary, there is evidence that the development of sun red color is influenced by the presence of an abundance of carbohydrates which in turn are dependent on sunlight for their formation. A striking example of the relation of sunlight to color development is afforded by the barred pattern seen in the husks of some weak sun red plants (Plate V, 4). The pattern consists of alternate bars of red and green parallel to the upper margin of the overlapping husk next below them. By tracing in pencil on each exposed husk of a rapidly growing ear the margin of the husk overlapping it, it has been ascertained with certainty that the red bars correspond to the areas that are pushed out from under the over- lapping husk between early morning and late afternoon, while the green bars correspond to the areas pushed out during the late afternoon and night. Why color develops in only those parts of the husk that receive the sunlight when first exposed to the air, and not in the parts exposed some hours previously, is not known. Another illustration of the effect of sunlight on freshly exposed husks was seen in a very light type of weak sun red (Plate V, 5). Of two ears on the same culm, both very lightly 7 18 : R. A. Emerson and about equally colored, the lower had its husks torn apart in the early forenoon so that the fresh inner husks were exposed at once to direct sun- light. In a few hours some red color began to show, and in a few days all the newly exposed husks were brilliantly colored, while the undisturbed upper ear remained only slightly colored. Similar results followed in repeated trials, and, in fact, failed only when the atmospheric conditions were such as to cause the newly exposed husks to wither during the first day. It is of interest to note also that similarly treated ears of dilute sun red plants, which rarely show any red color in the outer husks of young ears, failed to develop color when the husks were torn apart, even tho they remained fresh for some days. It is evident from all this, that, with respect to their relation to sunlight, there exists a series of color types varying more or less abruptly from dilute sun red, in which little or no sun red develops in even freshly exposed husks, thru weak sun red, in which color forms in only freshly exposed husks, and strong sun red, in which much color develops in all exposed parts of the husks but not in parts protected from light, to strong purple, in which, tho sunlight. may hasten color development, it is not essential to its formation. Tests of the influence on color development of light of different wave lengths have not been uniformly successful. Cramer photographie color screens were placed in partial contact with the uncolored inner husks of sun red plants, and the entrance of light otherwise than thru the screens was prevented by means of strips of black paper. These screens, by cutting out light of certain wave lengths, not only change the quality of light passing thru them but lessen the intensity of the light. While the results, therefore, can have little value, it may be of interest to physiolo- gists to note that considerable sun red formed under the orange and the bright red screens, and little or none under the green and the blue screens. MOISTURE IN RELATION TO COLOR It is well known that under field conditions maize does not grow well in wet soil. In such situations, not only are the plants small, with their ‘ leaves pale green, but they often develop much red pigment. The writer has repeatedly observed that young plants, in flooded parts of fields where the soil had been covered with water for some days, were brilliantly red in all parts except the youngest leaves, while near-by plants on slightly Z PLANT Coors IN MaIzeE 19 higher land showed only the slight red at the base of the culms character- istic of young dilute sun red plants. For a study of the effect of soil moisture on color development under controlled conditions, plants of well-known stocks of purple Ia, sun red IIa, dilute purple IIIa, dilute sun red [Va, brown V, and green VIec and IVe, were grown in rich soil in earthen jars in the greenhouse during the summer of 1914. When the plants had reached a height of from 10 to 15 centimeters, the jars were separated into three lots— one with dry soil, another with moist soil, and a third with wet soil. The dry-soil lot received only sufficient water to keep the plants growing slowly and not enough to prevent wilting during the hotter part of the day. The moist- soil lot received just sufficient water to insure normal growth. The wet- soil lot was kept constantly in saturated soil with some free water above the soil surface. The test was continued until the plants of all lots reached the flowering stage. The plants in moist soil made the most rapid growth and flowered some- what earlier than the plants of the other lots. Their leaves were of normal green color and they showed the colors characteristic of the several color types. The plants in dry soil were smaller and very dark green. The development of purple, red, and brown color was practically the same as with the plants in moist soil. The plants in wet soil grew less rapidly than those in moist soil, but more rapidly than those in dry soil. Their leaves were somewhat lighter green than those of the moist-soil lot, but they showed practically the same amount of purple, red, and brown color. In fact the only differences between the three lots with respect to color at any time during the test were such as might well be related to the stage of development of the plants. All color types show more color in the later stages of growth. The moist-soil lot developed somewhat more rapidly than did the others and for a time showed slightly more color, but ulti- mately all lots had practically the same amount of color. Evidently the reddening of plants in flooded fields is not due directly to the excess of soil moisture. TEMPERATURE IN RELATION TO COLOR Since moisture is not the direct cause of the reddening of maize plants in flooded fields, tho certainly connected with the phenomenon in some way, it follows that the effect must be produced by some indirect action 20 R. A. EMERSON of the excess of water. Wet soils in spring are cold soils, and if the wet areas are of considerable extent the air above them is doubtless somewhat cooler than that above drier soil. It has been frequently observed that young plants which show much color during a cold spring show considerably less in the leaves developed after the weather has become warmer. Young plants of early-planted maize sometimes have more color than plants that are started later. Moreover, full-grown plants from late plantings often develop more color in the cool weather of autumn than similar plants that mature in the warm weather of late summer. It seemed important, therefore, to study the effects of various tempera- tures on color development. The same color types and the same stocks—in one test the identical plants— used in the soil-moisture test were grown in the greenhouse under diverse temperatures. Altho both rich and poor soils of diverse water content were used, the comparisons noted here were made between plants in the same kind of soil and with practically the same soil-moisture con- ditions. Two lots were grown during the winter of 1913-14 and two dur- ing the following summer. During the winter, one lot was kept in a warm house at temperatures varying from about 18° to 26° C., and one was kept in a cool house at temperatures varying normally from about 7° to 15° C. but during a part of the test dropping at night to 1°or 2°C. Both lots were exposed to the full winter sunlight of the houses. During the summer test, one lot was kept as cool as possible by partial shading and free ventilation, the temperatures ranging from about 15° to 40° C. but occasionally exceeding these limits, and the other lot was kept in an unshaded house the ventilators of which were never opened. The night temperatures of the closed house averaged not more than one degree higher than those of the open house, but the maximum day tempera- tures in the closed house varied usually from about 44° to 50° C. and on three consecutive days reached 55° C. This extreme heat killed most of the plants grown in rich soil but did not seriously injure those in poor soil. Of course the relative humidity, as well as the intensity of the light, was materially different for the closed and the open house. As a result of these tests, no final differences in the development of color in any of the color types were observed between the lots grown at the very diverse temperatures. Of course differences were observed at certain times, but they are readily accounted for by the facts that the PLANT Coors IN Maize 21 plants developed less rapidly at both excessively high and excessively low temperatures than at more moderate temperatures, and that color shows less during the early stages of development than during later stages. It may be safely concluded, therefore, that color development in maize is not notably influenced, except perhaps indirectly, by diverse temper- atures. SOIL FERTILITY AND COLOR DEVELOPMENT There is still another way in which it was thought the excess of water might indireetly affect the development of color in maize plants in flooded fields. Not only may nutrient salts be removed in part by an excess of water, but certain of these salts — nitrates — are not formed normaily in very wet soils. Tests were made, therefore, of the relation of soil fertility to color development. Rich compared with poor soil The same plant-color types as were employed in the soil-moisture and temperature tests were included in these soil-fertility tests. In fact, for one of the tests the same plants were used as in the moisture and temperature studies. One lot of plants was grown in rich soil and a duplicate lot in poor soil. Field soil furnished the basis of both soils. To one lot was added about 50 per cent by measure of thoroly decayed stable manure, and to the other about 50 per cent of clean sand. The effect of soil fertility on color development of certain color types was strikingly apparent from the time the seedlings were two or three weeks old. At this age and for some time later, there was no appreciable difference in color between purples, sun reds, dilute purples, and dilute sun reds. In the rich soil all these color types had very little red color. There was some color in the coleoptile and the lower leaf sheath, but none in the leaf blades except for a slight amount in their margins. The same color types in poor soil had considerable color in the leaf blades and much color in the leaf sheaths. The plants in rich soil grew rapidly and were dark green, even the lower leaves remaining healthy. The plants in poor soil, on the contrary, grew less rapidly and were lighter green, and their lower leaves soon became yellow and died. In all cases the leaf blades became brilliantly red before they died. This is in strong contrast with the condition of the lower leaves of plants in dry, rich soil. When the 22 R. A. EMERSON death of the lower leaves is caused by drouth, there is no corresponding development of red color. At the age of six weeks, the plants in rich soil were beginning to show slightly the color differences that in later stages are characteristic of purples, sun reds, dilute purples, and dilute sun reds. In poor soil, on the contrary, no color differences were seen. All the four types were highly colored thruout except for the youngest leaves (Plate IX, 1 and 2). At the flowering period, the plants in rich soil exhibited all the peculiarities of color by which purples, sun reds, dilute purples, and dilute sun reds are normally differentiated. Even in the poor soil something of the same color differences were discernible between the purples and sun reds on the one hand and the dilute purples and dilute sun reds on the other, but it is doubtful whether these two groups could have been separated accurately from a mixed culture. It would have been very difficult also to separate with certainty the purples from the sun reds or the dilute purples from the dilute sun reds, except by differences in anther color and by an examination of the inner husks and other parts protected from sunlight. Differences between the plants in rich and in poor soil were still pronounced in the case of dilute purples and dilute sun reds, but were scarcely discernible in the case of purples and sun reds except that the leaf blades were somewhat more highly colored with poor than with rich soil and that thruout the plants the colors appeared brighter in the former case owing to the less intense green of the poor- soil lots. The seedlings of both brown and green color types showed no brown nor red color in either the rich or the poor soil.. At the age of two months, some brown pigment began to show in the lower sheaths of the brown type, and at the flowering stage the plants had the typical coloration of brown plants. The difference in the development of brown between rich and poor soil was at no time very noticeable. The color showed perhaps slightly earlier, and was perhaps slightly more intense, with the poor soil. Even this apparent difference, however, may have been due merely to the fact that the plants in poor soil were lighter and more yellowish green than those in rich soil. Dark green might readily mask the brown color somewhat. Green plants of both type Vie and type IVg exhibited no red nor brown color at any stage of development in either rich soil or poor soil. PLANT Coors IN MAIze 23 From these observations it is apparent that variations in soil fertility may effectively obscure genetic differences. A knowledge of the influence of soil fertility on color development is therefore essential to careful genetic work with the plant colors of maize. Moreover, since soil fertility is subject to control thru cultural methods, different degrees of fertility can be used as an aid to the sharp differentiation of certain genetic types. If, for instance, it is desired to separate, in the seedling stage, greens and browns on the one hand from the red-purple series on the other, this can be accomplished most readily in poor soil. In fact, the writer’s practice, in studies requiring this separation, is to grow the seedlings in pure sand. In this medium seedlings of the purple-red series of color types become highly colored at a very early age, while seedlings of the green and brown types show absolutely no red color. If, however, it is desired to distinguish sharply between purple and dilute purple or between sun red and dilute sun red, fairly fertile soil is essential, and, usually, the more fertile it is, the more easily can the separation be made. The stronger colors develop almost as well in rich as in poor soil, while the weaker colors develop much less intensely in rich soils than in poor ones. On very poor soils, it is difficult to separate sun reds from dilute sun reds, and almost if not quite impossible to distinguish with -certainty between sun reds and weak sun reds or between weak sun reds and dilute sun reds. Lack of particular nutrient elements It having been established that differences in soil fertility result in marked differences in the development of red color in maize plants, 1t seemed important to determine whether particular nutrient salts are more con- cerned than others. Accordingly, plants of all the color types included in the tests previously reported were grown in glazed earthen jars in clean quartz sand and watered with nutrient solutions. The quartz sand was obtained from the Department of Agronomy of the University of Nebraska, and was known to be practically free from nutrient elements except iron. The nutrient salts and distilled water were obtained from the Department of Agricultural Chemistry of the same institution. The nutrient solution employed was one that had given good results with maize in certain experiments conducted previously by the Department of Agronomy. The complete nutrient solution, 0.2 per cent strength, contained per liter of water the following salts: 1 gram Ca (NOs)2, 0.25 24 R. A. EMErRSon gram KNOs, 0.25 gram K2:HPO,, 0.25 gram MgSOu,, and 0.25 gram NaCl. Other solutions of approximately equivalent molecular strength, but each lacking one of the nutrient elements of the complete solution, were used. In the nitrogen-free solution, 0.7 gram CaCl, and 0.22 gram K.SO, were substituted for Ca(NO3)2 and KNOs, respectively; in the phosphorus-free solution, 0.25 gram K»SO, for K:HPO,; in the potassium-free solution, 0.2 gram NaNO; and 0.2 gram NaeHPO, for KNO; and KsHPO,, respectively; in the calcium-free solution, 1 gram NaNO; for Ca(NOs)s; in the magnesium-free solution, 0.3 gram NasSO. for MgSOu,; and in the sulfur-free solution, 0.2 gram MgCl: for MgSO,. A complete nutrient solution of four times the strength indicated above, 0.8 per cent, was also used, and one lot was given water without the addition of nutrients. After the first three weeks, the nutrient solutions were all used at double strength, 0.4 and 1.6 per cent, and clear water was occasionally given. This treatment, owing to considerable evaporation of water, doubtless resulted in a gradual increase in the strength of the solutions. The tests were carried on at the same time with one of the tests of rich and poor soil, so that the latter might serve as a check on the nutrient-solution tests. At first the seedlings given 0.2-per-cent complete nutrient solution reacted about as did those in poor soil, while those given 0.8-per-cent nutrient solution were no more highly colored than those in rich soil. At one month of age, the plants watered for three weeks with 0.2-per-cent and one week with 0.4-per-cent complete solution were growing rapidly and were no more highly colored than those in rich soil, while the plants in the very strong solutions (0.8 and 1.6 per cent) were beginning to wilt, perhaps from the toxic effect of the solutions. Thruout the remainder of the test, the plants given 0.4-per cent solution, alternated occasionally with clear water, were practically like those growing in rich soil both as respects vigor of growth and color development. In striking contrast to the plants given complete nutrient solution were the ones given clear water and those in nitrogen-free nutrient solution. Both these lots showed much color even at two weeks after germination, and soon thereafter the seedlings were red to the tips of their leaves. At the age of six weeks the plants of these two lots were much shorter and slenderer than those given complete nutrient solution. Their upper leaves were pale yellowish green, with much red, and the lower leaves were dead but still showing the red color that had developed earlier. - Puant Cotors In Matrze 25 Next in point of coloration to the seedlings given nitrogen-free nutrient solution and those given water alone, were the ones grown in phosphorus- free nutrient solution. The latter did not show red color so quickly as did the nitrogen-free lot, and at no time did they develop quite so much color. They showed, however, considerably more color at the age of one month than did seedlings in the complete nutrient solution. When six weeks old the plants of the phosphorus-free lot were relatively small, and had pale green upper leaves with little red color and dead lower leaves which still retained much red pigment. While somewhat larger than the plants in nitrogen-free solution and those in clear water, the phosphorus-free lot began wilting when about six weeks old and died considerably in advance of the nitrogen-free lot. Their roots showed early indications of injury, perhaps from toxic effects of the solution. Plants of all the other lots, in which one or another nutrient element had been omitted from the solution, exhibited little or no color reaction to the lack of a particular element. All of them were more vigorous in growth than the nitrogen-free and phosphorus-free lots, but much less so than the lot given complete nutrient solution. The sulfur-free lot for a time seemed to be developing more red, but later showed perhaps even less red, than the lot with complete nutrient solution. The mag- nesium-free lot showed prominent dark and light green stripes in the leaves similar to the green-striped chlorophyll pattern (Lindstrom, 1918). In some cases the tissue of the lighter stripes died and there was often some red coloration next to the dead tissue. The potassium-free lot had about the same amount of red color as the lot given complete nutrient solution, while the calcium-free lot showed less red color than any other lot in the test. It is perhaps noteworthy that in the nitrogen-free lot, and to some extent in the phosphorus-free lot, the new growth seemed to take place at the expense of the older leaves. The lower leaves first became light or yellowish green, then red, and finally died. That the development of red pigment is not necessarily connected, however, with the breaking down of the protoplasm, is seen in the failure of seedlings to develop red color in the older dying leaves of the lot in complete nutrient solution and of the potassium-free, magnesium-free, and calcium-free lots. In the calcium-free lot, growth was stopped by the death of the youngest parts, including the partly unrolled upper leaves, and yet these parts showed 26 R. A. EMERSON no red. Moreover, the dying of the lower leaves due to excessively dry soil, or of the upper leaves from intense heat, is not accompanied by the development of red pigment. In similar tests with cuttings of Tradescantia viridis and T. lockensis grown in distilled water, in complete nutrient solutions, and in solutions each lacking one nutrient element, namely, N, P, K, Ca, Mg, or S, Czartkowski (1914) found that after five weeks red color appeared in the newly developed leaves in the cases of only distilled water and nitrogen- free solutions. He states, however, that Susuki reported a similar effect on plants of Hordeum from a lack of phosphorus. It will be recalled that in the writer’s tests with maize, lack of nitrogen gave the most pro- nounced effect and lack of phosphorus induced considerable color develop- ment, while lack of sulfur seemed for a time to have an effect but no effect was apparent later. From the results of the tests reported above, it is apparent that the reddening of young plants in flooded fields, as well as the intensification of color in older plants grown on poorly drained heavy soils, is not due to any direct effect of the excess of water in the soil or to a direct effect of the somewhat lower temperatures accompanying such conditions, but rather, perhaps, to the lessened fertility of cold, wet soils or to inability of the plant to obtain adequate nutrients under such conditions. An excess of water not only may remove certain nutrient salts from the soil, but also may prevent or greatly check nitrification. Moreover, under these conditions the soil solution is probably less concentrated. The reddening of young plants in cold, wet soils in spring, the greater develop- ment of color in plants maturing in the cool weather of late autumn, and the excessive development of red in plants on very light sandy soils, are possibly all due to the plants’ inability to get from such soils an adequate supply of nutrient salts, particularly of nitrates. RELATION OF CARBOHYDRATES TO COLOR Several authors, notably Wheldale (1911), have discussed the relation of sugars to the production of anthocyanins in plants. Knudson (1916: 24, 62) found that maize and vetch grown in nutrient solutions containing certain sugars developed markedly more red color than did plants grown in sugar-free solutions. The writer has observed repeatedly an apparent relation between an excess of carbohydrates and the development of red PLANT Coors IN MaizE Di color in maize leaves. Of course the relation has been observed only in types that normally produce some red pigment. Neither brown, type V, nor green of either type 1Vg or type VI, has ever been observed with red color in the leaves, no matter what treatment has been given the plants. When leaves are folded at right angles to the midrib and the margin of the fold is creased sufficiently to break the softer tissues but not enough to break the water-conducting vessels, the part beyond the crease does not wilt, but within a few days it begins to lose some of its chlorophyll and within a week it becomes highly colored red (Plate X, 1). When leaves are similarly treated late in the afternoon of a bright day and the plants are kept in a dark room until the following day, the starch is, of course, found to have disappeared by translocation from the part of the leaves below the crease, while the cells of the bundle sheaths of the part beyond the crease are found to be packed with starch. There is so much starch in this part of a creased leaf that, on extraction of the chlorophyll with alcohol and treatment with iodin, the whole end of the leaf becomes almost black. While this does not prove a direct relation between an excess of carbohydrates and the development of red pigment, taken in connection with all the other observations it strongly suggests such a relation. It has been observed repeatedly that sweet-corn plants from which the ears have been removed in the edible stage develop within a week or two much more color than do neighboring plants that still retain their ears. Barren stalks also frequently show more color than do their ear- bearing neighbors. While no direct determination of the matter has been made it seems likely that barren plants, as well as plants from which the immature ears have been removed, may carry, in their leaves, husks, and culms, an excess of carbohydrates which would normally have been deposited in the developing seeds. The strong development of red pigment in the white, chlorophyll-free stripes of the japonica-striped type, when leaves are creased or when plants are grown in poor soil, may well be due to the passage of sugars from the green to the white parts. In some instances the red color seems to develop more quickly in the white stripes than in the green (Plate X, 2). Whether this difference is a real one, due perhaps to the readier access of light to the white parts, or is only an apparent difference due to the " 28 R. A. Emerson: masking effect of the green color, is not known. Certainly red pigments develop first in the chlorophyll-free epidermal cells.? Czartkowski (1914) suggested, in connection with the account of his study of the relation of nutrient elements to color development, that lack of nitrogen may check protein synthesis, thus leaving unused the carbohydrates that would otherwise be used in growth, and that the excess of carbohydrates may favor anthocyanin formation. He was unable to understand why a lack of phosphorus or of sulfur did not like- wise influence color development, since these elements also are necessary to protein synthesis. Lack of phosphorus does apparently bear some relation to color development in maize, but the writer’s tests afforded little or no evidence of such a relation between a lack of sulfur and pigment formation. If lack of nitrogen induces anthocyanin formation thru the checking of growth, thus allowing an accumulation of carbohydrates, it is not clear why other means of checking growth, such, for instance, as dry soil, do not also favor pigment formation, unless these other growth- checking factors at the same time limit photosynthetic activity. It is of interest to recall in this connection that plant colors of maize — brown no less than the red-purple series — develop first in the older parts where growth first ceases, such as the lower sheaths and the upper parts of the internodes of the culm. SUMMARY Whatever is the final outcome of studies of the relation of environmental factors to plant-color development in maize, enough has been noted to indicate a very complex relation. What is more complex than this chain of events — a chain that lacks many links in the way of particular chemical reactions: cold, wet soil checks or inhibits nitrification; lack of nitrogen in available form limits protein synthesis, which in turn allows an accumu- lation of carbohydrates; an excess of carbohydrates favors anthocyanin formation. The result is that young maize plants in cold, wet. soil become highly colored. But to all this must be added the factor of sunlight, without which no red color develops in the leaves of young plants. And not the least consideration is the important fact that only plants of certain genetic constitutions show this color reaction to wet soils. It is to be hoped that some day, thru the coordinated efforts of 2 The histology of color development of the several planiecolcn tvpes has been investigated by Dr. E. G. Anderson, but the observations have not been published. PLANT Coors IN Maize 29 biochemists, physiologists, and geneticists, it may be possible to reach conclusions in this field of quite as fundamental importance to biology as the recent results of similar efforts of cytologists and geneticists. GENETIC ANALYSIS OF COLOR TYPES In the preceding parts of this paper the several plant-color types of maize are described and the variations induced in them by diversities of environment are discussed. The remainder of the paper is devoted to a presentation of data of a moe distinctly genetic nature, and to an attempt at a factorial analysis of these data. The data are presented as if the F2 generation of the more complex crosses were the first which were obtained and on which hypotheses were formulated and appropriate tests made. As a matter of fact, this was not in all cases the actual procedure. In several instances the results of ‘some of the simpler crosses were at hand and were used as an aid to the interpretation of the more complex ones when the latter were obtained. Moreover, the hypothesis presented here was not the only one, nor indeed the first one, formulated. As is usual in such work, various hypotheses were devised, tested, and discarded, until finally a factorial interpretation was found that fitted fairly well all the facts known. Many results with a bearing on plant color were obtained in other studies extending over a period of some eight or nine years. Since the practice of the writer is to number his pedigrees consecutively from year to year, an inspection of the pedigree numbers, as listed in the tables, suggests at once that some of the data presented as checks on other results could not have been obtained after these other results. Any data applicable as a test have been so used whether obtained for that purpose or in connection with other studies. Whether this mode of presentation is the best one must be left to the judgment of others. This at any rate is certain: the data could not have been presented chronologically and discussed in relation to such hypotheses as happened to be under test at the time any particular results were obtained, without adding unnecessarily to the complexity of the paper. CROSSES INVOLVING THE FACTOR PAIRS Aa, Bb, Pl pl Purple Ia « green VIc Generations F; and F2— When purple plants with purple anthers (type Ia) are crossed with plants lacking all red, purple, or brown 30 R. A. EMERSON pigment, commonly known as green (type Vic), the F, offspring are full purple. Whether or not a quantitative determination of purple pigment might reveal a difference, no dilution of the purple color is apparent to the eye in the F; plants. Four crosses of this sort with a total F; progeny of 111 purple plants are listed in table 1 (appendix, page 121). Seven F2 progenies of the F; plants recorded in table 1 are listed in group 1 of table 2. Fourteen other similar F. progenies are shown in group 2 of the same table. The F, plants from which these fourteen F»2 progenies came are not recorded in table 1 because their purple parents were not homozygous. Some of the purple plants used as parents in these crosses were F*;’s of the original cross of purple with green. Others were from F,; or some later generation of other crosses having the purple type as one parent. In every case the other parent was a green plant of type VIc. Since the purple F, plants of these crosses were presumably the same genotypically as the F,’s shown in table 1, their F, progenies may well be included tentatively with those of group 1 of table 2. Each of the twenty-one F». lots exhibited six distinct classes of plants with respect to color. The 2117 plants were distributed among the six classes as follows: Parle Sun Dilute Dilute P red purple . sun red 952 305 275 91 278 216 2 ee Obviously no simple 3:1 mendelian behavior is in evidence here. More- over, only four classes are expected in dihybrids where dominance is exhibited. With dominance trihybrids ordinarily give eight classes in F»2 in the well-known numerical relation of 27:9:9:3:9:3:3:1, while only six classes were observed. Inspection of the distribution of the 2117 individuals given above, however, suggests the possibility of a 27:9:9: 3:9:7 relation, which should be realized in a trihybrid if the last three classes were indistinguishable. A comparison of observed numbers with those expected on this hypothesis follows: Sun Dilute Dilute Color types Purple red purple sun red Brown Green Total Brown Green Total Observed........ 952 305 275 91 278 ZIG a2 tla Calculated®...... 893 298 298 99 298 Day a AAMAS) Difference. ...... +59 +7 —23 —§ —20 —16 —1 3 In this and most of the following comparisons, the theoretical distributions are calculated to the nearest whole number, PLANT COLORS IN Maze 31 There are rather large differences between observed and expected numbers. The purples are considerably, and the sun reds slightly, in excess of expectation, while each of the other four classes has too few individuals. The probability that these deviations may be due to chance ‘is approximately 0.11. One might expect, therefore, to encounter chance deviations of the magnitude observed here about once in nine such trials. This, of course, does not substantiate the three-factor hypothesis, but merely indicates that it is not necessarily out of keeping with the observed facts. Backcrosses with green VIc—— A better criterion perhaps is afforded by the backcross of F; purples with the green parent type. Records of such crosses are shown in table 3. The backcrosses with F,’s of table 1 are listed in group 1, and backcrosses with similar F; purples of other lots in group 2. The same six phenotypes observed in the regular F, generation occurred here also. On the basis of the three-factor hypothesis and with the assumption that there are three sorts of greens indistinguishable from one another, the individuals of this backcross should be distributed equally to five classes with the sixth class containing three times as many indi- viduals as any other class. The observed distribution of the 1317 indi- viduals of the fourteen progenies is here compared with the expected distribution: Sun Dilute Dilute Color types mUrple 54 uel aon re" Brown Green Total Observed........ 170 160 176 160 172 AO amy led Calculated....... 165 165 165 165 165 AGS 1320 Ditterence: 2... . +5 —5 +11 —5 +7 —Il16- —3 While a few of the backcross progenies listed in table 3 exhibit con- siderable deviations from the expected distribution, the fourteen lots taken together approximate it closely. The probability that the observed deviations may be due to chance in random sampling is about 0.85. Deviations as great as these are to be expected thru chance alone, there- fore, in about six out of seven trials. Working hypothesis— To the three factor pairs used to interpret the results here reported, the symbols A a, B b, and Pl pl have been assigned. The gene A is an anthocyanin factor. In the presence of aa ordinarily no anthocyanic pigment develops, tho brownish, or flayonol (Sando and Bartlett, 1921), pigment may be formed. The pair J 6 is nanicd for its 2 32 R. A. EMERSonN connection with the development of brown pigment, tho when both A and B are present, sun red pigment is produced. The pair Pl pl is so termed because of its relation to purple pigment. The phenotypic formulae as- signed to the several classes of plant color under consideration here are as follows: : ABPI— fa, purple A B pl — Ila, sun red Ab Pl —IIla, dilute purple Abpl — IVa, dilute sun red aBPI— V, brown aBpl — Via) ab Pl — VIb} green abpl — Vice Obviously the hypothesis in accordance with which the above factorial assignments have been made is subject to several genetic tests. Naturally the first tests to suggest themselves are studies of the behavior of the several F. types in F; and later generations. Next in order are inter- crosses between the several classes. For reasons that will appear shortly, one of these intercrosses is here dealt with before consideration is given to F3 generations from the several F2 classes. Dilute sun red IVa x brown V From an examination of the factorial assignments listed above, it is evident that crosses of dilute sun red, A b pl, with brown, a B Pl, should produce purple F; plants, A B Pl. Moreover, these F; purples should be heterozygous for all three factors, Aa Bb Pl pl, just as was assumed for the original cross of purple, A B Pl, with green, ab pl. The F, and later behavior of this cross should also, barring linkage, be like that of the original cross, so that the two can most conveniently be considered together. Generations Fy and F>.— The F; generation of twenty-six crosses of dilute sun red with brown plants is given in table 4 (page 123). The dilute sun red parent plants were chosen from any convenient lots known to be homozygous with respect to A, b, and pl. The brown parent plants, on the other hand, were from the F, and later generations of the original cross of purple and green or from other crosses. It was to be expected, therefore, that some of the brown plants would be homozygous for both B and Pl, and some would be heterozygous for B, some for Pl, and some PLANT Coors IN MaIzE 33 for both B and PI. This expectation was fully realized. In group 1 of table 4 are recorded the progenies of nine crosses with a total of 263 indi- viduals. All but one plant of the lot were purple. The one dilute sun red plant was presumably due to accidental pollination of the dilute sun red mother plant. Since the dilute sun red parents of all these crosses were A A bb pl pl, the brown parents of the crosses listed in group 1 must presumably have been aa BB PI Pl. Similarly, the seven crosses listed in group 2 gave purple and sun red plants only, 143 of the former and 147 of the latter. Evidently the brown parents of these crosses were aa BB Pipl. Again, the six crosses shown in group 3 gave 105 purple, 123 dilute purple, and no other plants. The brown parents of the crosses were therefore, presumably, aa Bb PI Pl. Finally, the four crosses listed in group 4 gave 9 purple, 11 sun red, 19 dilute purple, and 17 dilute sun red. The brown parents of these four crosses are assumed, consequently, to have been aa B b Pl pl. hd The F, results from the purple F; plants of these crosses of dilute sun red with brown are recorded in table 5.~ Fourteen progenies of the F; plants listed in table 4 are shown in group 1 of table 5, and five progenies from similar F; plants not listed in table 4 are entered in group 2. Here, just as with the results of the cross of purple with green (table 2), fairly marked discrepancies between theory and observation appear when the several progenies are taken separately. When, however, the nineteen progenies are considered together, very close agreement is found between observation and expectation, as is shown by the comparison below. The probability that such deviations as are observed may be due to chance is approximately 0.88, which means that only about once in eight trials would as good a fit be expected. The comparison follows: E Sun Dilute Dilute Color types. Purple red purple sun red Ta Ila Illa IVa V Via; b;.¢ Brown Green Total Observed..... 847 282 281 94 267 233 2 004. Caleculated.... 845 282 282 04. 282 219 2 004 Difference... . +2 0 -—1 0 —15 +14 0 Backcrosses with green VIc.— In addition to the F»2 results noted above as derived from self-pollinated F, purple plants, a few F, purples were bac 34 R. A. EmMrErson crossed with the triple recessive green, type VIc. The records of these crosses, seven in all, are presented in table 6. The results are, as expected, in close agreement with the backcross data from the cross of purple with green. The comparison below indicates a good fit of calculated to observed frequencies for the lot as a whole. The probability that such deviations as are observed may be due to mere chance is about 0.82, indicating that as great departures from expectation as these might be expected about four times in five trials. The comparison follows: Dilute Dilute purple sun red la ita} Illa IVa VS Vilessie Color types Purple Sun red Brown Green Total Observed..... 84 72 78 72 79 249 634 Calculated.... 79 79 79 79 79 237 632 Difference. ... +5 —7 —1 —7 0 +12 +2 Backcrosses of Ia x VIc and IVa x V with IVa Purple plants of F; of the crosses purple x green and dilute sun red x brown were crossed with homozygous dilute sun red stocks. On the basis of the hypothesis used above, the F; plants are assumed to be A a Bb Pl pl and the dilute sun red plants A Abbpl pl. Four classes of plants, purple, sun red, dilute purple, and dilute sun red, should be pro- duced in equal numbers by this cross. The data are presented in table 7 (page 125). Progenies of F; plants from the cross purple x green are listed in group 1 and those from the cross dilute sun red x brown in group 2. As will be seen from the comparison below, the observed numbers are in fair agreement with the hypothesis. The probability that such deviations as occur may be due to chance is approximately 0.67. In other words, there are two chances in three that deviations of this sort are due to errors of random sampling alone. The comparison follows: Dilute Dilute Color types Purple Sun red purples ene Total la Ila IIIa IVa Observed ase ye ees 299 270 288 291 1,148 Galeulatedh 3-2 ieee 287 287 287 287 1 ,148 Witerence. a) ee ee +12 —17 +1] +4 0 PLANT Coors IN MAIzE 35 Behavior of F, color types in later generations From all the foregoing it appears that the results obtained are in close accord with the proposed three-factor hypothesis in the case of both the cross purple x green and the cross dilute sun red x brown, and not alone for the F; and F.2 generations but also for backcrosses with green and with dilute sun red. It is now in order to inquire into the behavior of these crosses in F3 and later generations. In the presentation of the additional data, the two crosses purple x green and dilute sun red x brown will be considered together. Later behavior of F2 purple Ia— Purple plants of the F2 generation of the crosses under consideration are expected to be of eight genotypes. The expected F2 genetic formulae and the F; color classes, together with the relative numbers of each, are as follows: F; color types F; genotypes Dilute | Dilute Purple Sun red purple | sun red Brown Green Ta Ila Illa IVa VI i == AWA BI JIE r 1 iad PO otetare DST al Fee h o 1 aN | De ee a (EES 2—AABB Pipl 3 LEN ener evel Der esitpre ec ee [poeta eee PEP ACAG DOP UBlel eh. feesn 2 Soles Sane ALFA np oay corned Mente moe > |ncep eee: 2—AaBB PI Pl B= | Sea S ces | eas crore 2 heer leet cee 4 = Al ALE) JEM iene arenes 9 3 3 Loy | tees teen seat ec eee Me SAN B oD RL DU. ee . << sais oe 9 eal liperceceatea| leseeres ot - 3 1 Aa= AV Gd} ILI ELS See ee eae OS Seer, Siler cars 3 1 SAL WBN THD Fay het eine taal 27 9 9 3 9 a If, instead of being selfed, the F. purple plants are backcrossed to green of type VIc, the same F; color classes are expected but the several classes should, of course, be equally frequent except in case of the F» triple heterozygotes, which should throw three times as many greens as of each of the other five types. The F; data from thirty-five F, plants are recorded in table 8 (page 125). In group | of the table are listed the progenies of eight selfed and one backerossed F, plants. From the backcross six color types appeared in frequencies of 4:4:11:4:4:18. The theoretical number for the first 36 R. A. EMERSON five classes is 5.6 and for the sixth class is 17. The probability that such deviations as occur are due to chance is approximately 0.35, or more than one in three. The eight self-pollinated plants gave together the six types in frequencies as follows: Dilute Dilute purple sun red Ta ita tla Ve V-. Viasbae Color types Purple Sun red Brown Green Total Observed =... 193 66 60 16 57 34 426 Calculated..... 180 60 60 20 60 46 426 Difference. .... +13 sine 0 —4 —3 —12 0 The probability that such deviations as occur may be due to errors of random sampling is practically 0.27. Similar deviations might there- fore be expected somewhat more than once in four trials. It will be noted that two progenies lacking class IV are included in this lot (group 1, table 8). The total number of plants in these progenies were 37 and 17, respectively, and they should therefore have had, respectively, two and one plants in class IV. Five F; purple plants (group 2, table 8) gave four color types (Ia, Ia, IIIa, and IVa) in F;, with total frequencies as shown below. Here the probability, P, equals 0.75, indicating that deviations of this magnitude might be expected thru chance in three out of four trials. The com- parison of observed with theoretical distributions follows: Dilute Dilute Color types Purple Sun red © : ; Total purple” sunied gap eos Ta Ila Illa IVa Observed sae = ss ee 102 36 29 13 180 Calculatedsr 2s. 2s 101 34 34 11 180 WDITeTENCE Aas Aceh es spl +2 —) +2 0 Progenies of seven other purple F2 plants (group 3, table 8) consisted of the four color types Ia, Ila, V, and Vla. Four of these F: plants were self-pollinated and gave a total of 164 F; plants. Four, including one that was also selfed, were backcrossed to green and yielded a total of 209 F; plants. For the progenies from selfed F: plants P=0.20, and for those from backcrossed plants P=0.57. There is, therefore, one chance in five PLANT Coors In Maize 37 in the one case and considerably more than an even chance in the other case that deviations of the kind noted may have been due to errors of ‘random sampling. The comparisons follow: Color types Purple Sunred Brown Green Total Ta IIa V Via URC i ate Ditierence ys. oe. ao ne = +5 0 ee { Observed. . .. 54 58 44 53 209 Calculated... 2h 52 52 52 208 Difference.... +2 +6 —8s +1 +] Seven self-pollinated F. purple plants gave progenies consisting of the four color types Ia, IIIa, V, and VIb (group 4, table 8). Here P=0.75, indicating that there are three chances in four that such deviations as are shown are due to chance. The comparison follows: ° ; Color types Purple eae Brown Green Total Ta IIIa V VIb ‘CSCIC li, ee ne ee 318 114 111 42 585 Walewmlabed anys eae ss 329 110 110 37 586 «(DINERS yoga eee eon —l1 +4 +] +5 —l] Five F. purple plants from self-pollination gave only two color types (Ia and IIa) in F; (group 5, table 8). The total number of F; individuals was 183, of which 139 were of color type Ia and 44 were of color type Ia, the expected numbers being, respectively, 137 and 46, and the deviation equaling 2+ 4. One of these F, plants was also backcrossed to two greens, resulting in 12 purple and 9 sun red F; plants where equality of the two classes was expected. The deviation here is 1.5 + 1.5. Finally, two self-pollinated F2 purple plants produced 217 F3 individuals (group 6, table 8) of color types Ia and IIIa. There were 168 purple 38 R. A. EMERSON and 49 dilute purple where the expected numbers were 163 and 54, respec- tively — a deviation of 5 + 4.3. It is seen, then, that in every case the F; progenies of F. purple plants were of color types expected on the basis of the three-factor hypothesis, and that the F; distributions within any group were in close agreement with expectation. It is particularly noteworthy, however, that not all types of F; behavior were observed, and that the distribution of the progenies of the thirty-five F2 plants tested was in rather imperfect agree- ment with expectation. Thus, no F2 purple plant bred true in F3 where one such plant was expected, and none gave progenies of purple and brown only where at least two with such behavior were expected.. It has already been pointed out (page 35) that eight classes of behavior of F, purples are looked for, and that any twenty-seven F, purple plants should be distributed with respect to their F; behavior in the relation 1:2:2:2:4:4:4:8. The actual and theoretical distributions are compared as follows: Observed ?su5.5.0: 0 5 2, 0 5 7 7 ‘9 35 C@alculated=s.s2< 3 2.6 2.6 2.6 OBZ 5B Ae iat 35.1 Ditterences seem oe —1.3 +2.4 0.6 2.6 0.2 4+1.8 +1.8 —1.4 —). 1 While mere inspection of the above comparison might suggest poor agree- ment between theory and observation, nevertheless P=0.36, indicat- ing that such deviations as occur might be expected in more than one out of three trials, which is not a bad fit. So far, therefore, the avail- able data are in fair accord with the three-factor hypothesis. - Before taking up a consideration of the F; behavior of other F»2 color types, 1t will be well to consider briefly the F, behavior of F; purple plants. Only one F; purple of the lot having all six color types (table 8, group 1), comparable to F, purples, was tested in Fi. This one plant gave an F4 with the four color types Ia, Ifa, V, and VIa. Only eight other F; purple plants were tested in F;. All these belonged to the lot consisting of color types Ia, Illa, V, and VIb (group 4, table 8). The F, purple plants giving rise to this group are assumed to have been of the genotype Aa@Bb PI Pl. The Fs; purple plants should therefore have been of four genotypes and should have given F, behavior as follows: PLANT Coors IN Maize 39 Fz color types otypes ; He Beno Purple Palit Brown Green Ta Ieee Vv b Illa LS AAIB IB IAPIE I ee eee ee IEA eo esac oran | ee cope tec |) Seen ee L=AAIBOIA TAS ee ee ee 3 Tigi pie cttsey Ih cos cosat ere 2 == ALGER IA) TA eae ee Bi Nak tenac Uae dbl |e cesta 2 == AL GID) JABIELS Soo eee ree eee 9 3 3 1 The data are presented in table 9. Four F,; progenies (group 1) were made up of the four color types Ia, IIIa, V, and VIb. The total numbers of plants of each of the four types, as seen below, were in close accord with expectation, P equaling 0.57. There is more than an even chance that such deviations as those observed may have been due to errors of random sampling. The comparison of observed with calculated results follows: : Dilute ; Color types Purple ale Brown Green Total la Ila V VIb Bserycde ee ose. 185 68 74. 20 347 Malewinnedet. 22... 6c. eS 195 65 65 22 347 MCKENCE Man... 2. ae oss ——10 +3 +9 —2 0 Three of the eight purple F;’s (group 2) gave in Fy only purple and dilute purple plants, 88 of the former and 28 of the latter. The expected numbers were 87 and 29, respectively, showing a deviation of 1 + 3.1. One of the eight F; purples (group 3) gave 67 purple and 21 brown plants in F;, while the expected numbers were 66 and 22, respectively. The deviation here is only 1 + 2.7. None of the eight F, purples bred true, but only one in nine was expected to do so. As already indicated, the theoretical distribution of nine Fs; purples of the sort here under consideration, with respect to the four kinds of behavior in Fy, is 1:2:2:4. The observed distribution was 0:3:1:4. There is more than an even chance that these deviations may have been due to errors of random sampling, P equaling 0.57. 40 R. A. EMERSON | It should not be forgotten that, while a very poor fit of observation to hypothesis, as measuted by values of P, throws doubt upon the correct- ness of the hypothesis, it does not follow that a good fit proves the hypothesis to be true. This is particularly true where small numbers are dealt with. It will be recalled in this connection that, owing probably to the small numbers tested, no F. purple has been found to breed true in F, and none has been found to give only purple and brown offspring. It has been shown, however, that purple plants of the genotype AaBB Pl Pl exist, since one F; purple threw only purple and brown plants in Fs. Moreover, one of these F; purples repeated this behavior in Fs. Similarly it can be said that purples of the genotype A A B B Pl Pl have been recovered from the crosses under consideration, for two F, purple plants of the lot composed of purples and dilute purples (group 2, table 9), when backcrossed to green, gave 18 purple plants and no other types in the next generation, and one of these two F,; purples, when crossed back to dilute sun red, gave 34 purple plants. Two other purples of the same F; lot, when similarly crossed, gave both purple and dilute purple, 23 of the former and 18 of the latter. Purple plants of all the expected genotypes have therefore been recovered in one or another generation from F: to F; from the original crosses of purple x green and dilute sun red x brown. Moreover, these genotypes have been found in numbers not far from what might reasonably be expected considering the relatively small numbers tested. It now remains to inquire into the F3 and later behavior of F2 color types other than purple. Later behavior of Fz sun red ITa—Sun red plants of Fo of the « crosses purple x green and dilute sun red x brown are expected, in accordance with the three-factor hypothesis, to be of four sorts with respect to their behavior in F3, as follows: F; color types F,2 genotypes Sun red ed Green Ila Sune Via, ¢ a i AA BiB pl pte eae sac ne Ee ee I Metricaut | sookooene 2 HAAS Bibl Divine See ceo ae So ee 3 1 iseees eee Di AG BiB pliplisssc': stipes oyse series isi is en On eee 3: [|G scleee eee 1 A ARGS BSD spl Dats no Aery Rin ose eo ee ee 9 3 4 PLANT Cotors IN MaIzE Al Only nine F, sun red plants were tested by their F; behavior, and no later generations were grown. All the available data are given in table 10 (page 128). Five F; plants, when self-pollinated (group 1 of the table), gave the expected three classes of progeny, sun red, dilute sun red, and green, with a distribution of the F; plants as given below, and in addition a single brown plant. To include this unexpected plant in the compari- son with the calculated distribution would give zero as the value of P, which is equivalent to saying that even in an infinite number of trials there is no chance of finding such a plant thru errors of random sampling. The single off-type plant is readily accounted for by supposing that a grain of foreign pollen was accidentally admitted in the pollination of the parent plant. Tho it is realized that, with such a convenient supposition always at hand, almost any result can be made to fit a theory, the reality of just such accidental pollinations will not be questioned by any one who has had experience in the technique of maize pollination. With the elimination of this one plant, the fit of observation to hypothesis is almost perfect. The comparison follows: Color types Sun red tule Green Total Ila IVa Via,‘e CIRGIRVEG! 6 & 2m ce ee eee ae 126 42 ae) 223 ‘CANICIUGRTE OL: = <2 1p Renee ae a ea 125 42 56 223 Difierentce 12 See ane ae +1 | 0 —] 0) = Three F, sun red plants, including one of the five in the former test, were crossed back to green (group 1, table 10). The same three color types were observed as in the self-pollinated plants, with the addition again of a single off-type plant, this time a purple one. Even if this plant is left out of consideration as due to an accidental pollination, the fit of observed with calculated numbers is not very good. Such deviations from theoretical behavior are to be expected thru chance alone only once in eight trials, P equaling 0.12. The comparison follows: Dilute ‘ Color types Sun red eure Green Total Ila IVa Via, ¢ Wpserved) 2 25 8.502: ail be cee oath 14 18 50 82 PACU ALCO sccm sc hccis iin BOS Ak aks 20.5 20.5 4] 82 we LAY SINCE = gee 2 —6§6.5 —2.5 +9 0 42 R. A. EMERSON A single F. sun red plant (group 2, table 10) gave, from self-pollination, 23 sun red and 9 dilute sun red F3; plants, a deviation from expectation of Ise WA A single F. sun red plant (group 3, table 10), when crossed with green VIc, gave 50 sun reds and 43 greens where equality was expected, a devia- tion of 3.5 + 3.3. By way of summary of the behavior of F. sun red plants, it must be . noted that, while four sorts of behavior were expected, only three sorts were observed. While any nine such F2 plants should be distributed with respect to the four kinds of behavior in the relation 1:2:2:4, the observed relation was 0:1:1:7. While mathematically this is not a very bad fit considering the small numbers involved, P equaling 0.24, it is inadequate for a determination of the possible genotypes of F. sun red plants. Fortunately, certain crosses considered later (page 51) involving the sun red type, with presumably the same genetic constitutions as the Fz sun reds of this cross, afford a more nearly adequate test of the matter. Later behavior of F', dilute purple I1[a.— F 2 dilute purple plants should present the same types of behavior in F; as F. sun reds, but, of course, with somewhat different color types appearing, as follows: F; color types Jee nova Dilute | Dilute | purple sun red Vib ae Ia Va ne PSA AM DOTA Lech eves teria kates ae ek eater Seog eat Ns Mears || ns G's oro DAMAGE EP lil one ac Warde oe, Recs cant eos 3 1 ie, a De SAL OLD REALL Ap esrb clot teehee g cae hapa oe ieee ae payee ease rere il AS AN AONE GEO Use eres cone Seana ener Tee TS Te leat ae 9 3 4 The available data from this test are given in table 11 (page 129). Four I’, dilute purples (group 1) yielded the three color types expected, dilute purple, dilute sun red, and green, in the numbers shown below. There is considerably more than an even chance that the deviations from expecta- tion may be due to errors of random sampling, P equaling 0.58. The comparison follows: PLANT Coors In MAIzEe 43 Dilute Dilute C purple sunred ~ Illa IVa Vib, ¢ Color types reen Total Olosarvecl. - . 25 2s eee ae 95 Si 50 176 Caleullaieel, 2 oS eae see eerie ne 99 33 44 176 IDMRIGTRNINGO S = ec a ee —4 —2 +6 0 One of the dilute purple F, plants used in this test was backcrossed with green VIc (group 1, table 11), with the result shown below. There is practically an even chance that the observed deviations may be due to errors of random sampling, P equaling 0.49. The comparison follows: Dilute Dilute Color types acimile gum nad Green Total Illa IVa Vib, ¢ Closeieredls. cic sake eee eee 21 25 57 103 CGAlculavedeere ts oct. ee ce ek 26 26 52 104 IDMHCTONGE 5 oe. BAER ee ee —5 —] +5 —] One F, dilute purple gave 57 dilute purple and 21 dilute sun red plants in F; (group 2, table 11). The expected numbers were 58.5 and 19.5, respectively, the deviation being 1.5 + 2.6. Three F, dilute purples gave a total of 85 dilute purple and 20 green plants (group 3, table 11), the theoretical numbers being 79 and 26, respectively. The deviation from expectation, 6 plants, is just twice the probable error. One F, dilute purple bred true in F3, producing 21 dilute purple plants and no other types (group 4, table 11). Thus, all the sorts of behavior expected of F, dilute purples were realized in F3.. The distribution of the F, plants with respect to the four sorts of behavior was 1:1:3:4, instead of the theoretical distribution 1:2:2:4. Differences of this sort might be expected thru chance in four out of five trials, P equaling 0.80. Only three plants of these lots were tested in Fy. One was a dilute sun red of the lot made up of dilute purples and dilute sun reds, and this one bred ‘true in F; as was expected of it, producing 34 dilute sun red plants. The other two plants tested further were dilute purples of the lot contain- ing the three color types III, IV, and VI. Both again gave these three 44 R. A. EmErson types, the total numbers of the respective classes being 29, 5, and 18. The expected numbers, 29, 10, and 13, show a deviation from expecta- tion which might result thru chance about once in nine trials, P equaling 0.11. Later behavior of F, dilute sun red IVa.— Dilute sun red plants of Fe should be of two sorts, AAbbplpl and Aabbplpl. Five such plants were tested, with results as shown in table 12 (page 129). Of these five, two bred true, producing a total of 92 dilute sun red plants (group 2). One of these two, when backcrossed with green,-gave 69 dilute sun red plants. Three of the five F2’s gave in F; dilute sun reds and greens, 62 of the former and 17 of the latter (group 1). The theoretical numbers were 59 and 20, respectively. The deviation of 3 plants is only a little ereater than the probable error, + 2.6. With two of the F, dilute sun red plants breeding true and three again throwing segregates, expectation was very nearly realized. Later behavior of F2 brown V.— Brown SRNE of F, are expected to be of four genotypes and to show consequent differences in behavior in Fs as follows: F; color types F, genotypes Brown. Green V6. BIB RR i ee aa ea ee ee 1 2010) BeBIRE Diss Sd eS CEA oe ee Se 3 DOC) BOGE UL Palace rm atcha ee ee PIO Occ ES 3 1 AE= CRG B VOL Dis ce Nat er ene Ia Ree Sn gM ee LS eater eee 9 Data for F; from fourteen Fs. brown plants are presented in table 13 (page 130). Five self-pollinated F, browns (group 1) gave, in addition to one sun red presumably due to accidental pollination, 96 browns and 74 greens in F;, which is almost exactly a 9:7 relation, the deviation being 0.4 + 4.4. ‘Nine other selfed F. browns (group 2) gave in F; a total of 354 brown and 104 green plants. An exact 3:1 ratio for the total of 458 - would be 343.5 and 114.5, respectively, the deviation being 10.5 + 6.3. Such a deviation might be expected thru chance alone about once in four PLANT Coors IN MatIzE 45 trials. One of the F, brown plants that, when selfed, gave a 3:1 ratio in F;, when crossed with green gave 34 brown and 41 green plants where equal numbers were expected, the deviation being 3.5 + 2.9. None of the fourteen F, brown plants bred true in F;. The fourteen plants should theoretically have given F; ratios of 1:0, 3:1, and 9:7 in approximately the respective numbers of 1.6, 6.2, 6.2, while the observed numbers were 0, 9, 5. Such deviations might occur by chance once in five trials, P equaling 0.22. It is often difficult and sometimes practically impossible from ordinary F; progenies to distinguish between the: two genotypes of brown which throw 3:1 progenies, namely, aa BB PlplandaaBb PI Pl. The green plants thrown by the former often show some brown pigment in the exposed parts of the sheaths and husks (type Vla), a condition not seen in the greens (VIb) thrown by the latter. In some lots the brown pigment is fairly conspicuous but in others it is very weak or is absent. Again, the greens of type VIb thrown by browns of the genotype aaBb Pl Pl show considerable brown in the glumes of the staminate flowers. This is particularly pronounced when r (a gene for cherry pericarp which is effective only in the presence of PI) is present, but when this factor is lacking the brown color is often so faint that it is impossible to distinguish between a green plant carrying Pl and one lacking it. If r-’ is present, the green plants carrying Pl develop a light brownish pericarp at maturity while those lacking Pl never show this pericarp color whether or not B is present. Here again, however, the lght brownish pericarp due to rk, Pl, and aa may be wholly masked if there happens to be present another pericarp color gene, P, which with aa brings about a strong brown color of the pericarp whether or not Pl or B is present. On the whole, therefore, it is difficult, and often impossible, to determine the genotype to which a brown plant belongs, by an inspection of the green plants occurring in its progeny. Because of this, the 3:1 lots of F; progenies of F, brown plants are lumped together in group 2 of table 13 without any attempt to separate them into the two classes expected. Fortunately, it is readily possible to distinguish between brown plants of the two genotypes under consideration here by means of appropriate crosses. 4 An account of these pericarp-color factors is to be published later by Dr. E. G. Anderson, who is making a study of the pericarp colors of maize. 46 R. A. EMERSON When brown plants of all the genotypes expected in F»2 of the crosses of purple x green or dilute sun red x brown are crossed with homozygous dilute sun red plants, the following behavior is expected in the next generation: F, x AA bbplpl F, genotypes Dilute Dilute Purple Sun red purple sun red Ta Ila Illa IVa NOS OTS ES SEU SAU cs 8 hy Pape Pacem pS RS Iss etre ae Beare S|? eae 2 NON BEE E Deore SAS te a ea ee 1 1 eres i ae Cea PAE OA UNA A hed el tree Bien aces Baker) I ee 1 ee ee es PeeRee a AS COE BRO Pe U Dies ticiek vac ae ee eee eee 1 1 1 1 A few such tests of F2 brown plants are recorded in table 14. Two plants (group 1), on being crossed with dilute sun reds, gave purples and sun reds only, 38 of the former to 45 of the latter, where equality was expected, the deviation being 3.5 + 3.1. One of these plants has progeny from self- pollination listed in table 13, in group 2, the 3:1 lot. This plant was expected, of course, to throw only two color types from the cross with dilute sun reds, for otherwise it should not have given a 3:1 progeny on being selfed. The two brown plants in group 1 of table 14 must have beenaaBB Pipl. Two other F2 brown plants (group 2) gave 32 purple and 38 dilute purple instead of the equal numbers expected, the deviation being 3.0 + 2.8. These plants are assumed to have been aaBb Pl Pl. A single F, brown plant (group 3) when crossed with dilute sun red gave 15 purple plants, and is therefore assumed to have been aa BB Pl Pl. The behavior of several F; brown plants when crossed with dilute sun reds is also shown in table 14. Three of these plants were from 9:7 F; lots and therefore are presumably comparable with Fs browns. One of these three (group 4) gave the four color types I to IV in the numbers 1:2:6:3. It was probably aa Bb Plpl and should have given a 9:7 progeny if it had been selfed. The other two F3; browns of the 9:7 lot gave 49 purple plants (group 7) and are consequently regarded as aaBBPlPli. All the other F; brown plants tested were from the PLANT Cotors IN MAIZE 47 3:1 lot listed in table 13, group 2. None of these should give more than two types when crossed with dilute sun red. One gave 46 purple and 1 dilute sun red (group 7), the latter doubtless from an accidental pollination of the dilute sun red mother plant. Two F; browns gave 22 purple and 24 sun red plants (group 5), and four produced 73 purple and 85 dilute purple plants (group 6). To summarize, all the theoretically possible genotypes of brown plants have been found either in F2. or in such F; lots as showed a 9:7 ratio of brown to green. Since these F3’s are comparable with F, browns, they may be added to the F.’s in this summary. Of the twenty-one brown plants thus grouped, the numbers found to belong to each geno- type are compared below with the calculated numbers. The deviations are such as might be expected to occur once in three trials, P equaling 0.34. The comparison follows: aaBB Pipl GO 153 Jas Jel eds or Gos bsbeRl! phe wloval GO OAEND JEN IPA, Observed....... 3 12 6 PA Calculated...... Dslel) 9.3(+) 9.3(+) 21 Difference...... +0.7(—) +2.7(—) —3.3(+) 0 Later behavior of F2 green VI.— All F, green plants should breed true phenotypically in F;. Data from eight such F3; progenies are given in table 15, group 1 (page 132). There were observed a total of 179 green plants, and no other types. Progenies of sixteen green plants of the F, lots listed in tables 3 and 6 (pages 122 and 124), produced by backcrossing F, purples to greens, are given in table 15, groups 2 to 5. The total number of green plants in these progenies is 311. A single brown plant found in one of these progenies is assumed to have been due to acci- dental pollination. Green plants are therefore found to breed true green as expected, but there is nothing in this fact to indicate that green plants of the crosses under consideration are genotypically alike. That the five genotypes expected on the basis of the three-factor hypothesis were present among the progenies listed in table 15 is demonstrated in the next section of this paper. 48 R. A. EMERSON Intercrosses of F. color types It has been shown in the preceding pages that all the six color types occurring in F, of a cross between purple and green behave in Fs and later generations as is expected on the basis of the three-factor hypothesis suggested to account for the F2 results. It remains to determine whether the several color types behave in accordance with the hypothesis when intercrossed one with another. Of the fifteen possible intercrosses between phenotypically different types, two have already been discussed. The cross of purple with green has formed the basis of the whole discussion. The cross of dilute sun red with brown, since it was expected to give the same results as the original cross of purple with green, was most conven- iently considered with that cross in generations later than Fy. The results of this second cross have been in accord with expectation. The other thirteen intercrosses are now to be considered, together with intercrosses of some types that are phenotypically alike. Dilute sun red IVa x green VIa, VIb, VIc.— The progenies of self- pollinated green plants were listed in table 15 in several groups in accordance with what was learned of their genotypic constitution by the crosses to be considered here. The regular Fs lots, from self-pollinated F, greens of self-pollinated F, purples, were put in group 1 of table 15. Only one of the same F» greens (table 16, group 2) was crossed with homo- zygous dilute sun red, A Abbplpl. The result was 67 dilute purple plants. Another green plant, an F; from a self-pollinated F. green, gave, when similarly crossed, 9 dilute purple plants (group 2). Evidently both these green plants were aabb Pl Pl. Four other F; green plants, when crossed with dilute sun red, gave a total of 148 sun red plants (group 1, table 16). One of these four belonged to an F; lot containing browns and greens in a 3:1 relation, and could not, theoretically, have done other than give all sun red or all dilute purple when crossed with dilute sun red. Two of the four were from greens of an F'; lot made up of purples, sun reds, browns, and greens, and were therefore assumed to be aaB B pl pl, as the crosses with dilute sun red showed them to be. One of the four green plants, however, belonged to an F3; lot of browns and greens in a 9:7 relation and was consequently comparable to an F», green. A sixth F; green also belonged to a 9:7 lot, comparable to an F. lot. When crossed with dilute sun red (group 3, table 16), it gave 24 dilute sun red plants, PLANT CoLors IN Maize 49 and is therefore assumed to have been aabbpl pl. All three of the theoretically possible homozygous genotypes have therefore been demon- strated among the F2 greens or among F';’s comparable to Fy’s. In addition to the green plants of the direct F. and F3; generations, noted above, fifteen other greens were crossed with dilute sun red. All these greens belonged to a single progeny, 2019, which was the result of a backeross of an F; purple with a green, aabb pl pl (table 3, group 1). All of them should therefore have been heterozygous for B or Pl, or have lacked these dominant genes. Seven of the fifteen, when crossed with dilute sun red, gave 110 sun red and 85 dilute sun red plants (group 4, table 16), a deviation from equality of 12.5 + 4.7. The green parent plants. are consequently regarded as aaBbplpl. Five others of the fifteen green plants (group 5) gave a total of 56 dilute purple and 65 dilute sun red, a deviation from equality of 4.5 + 3.7, and hence are assumed to have been aabb Pl pl. Three of the fifteen (group 6) gave a total of 106 dilute sun red plants. These three must, it is supposed, have been aabbpl pl. Naturally, in the course of the writer’s maize studies, many other crosses between green and dilute sun red have been observed. But no purpose can be served by presenting here all this mass of data. Much of it has accumulated in connection with a study of the interrelations of plant and aleurone color, and will find its appropriate place in a later publication on that topic. A few F: and backcross progenies of dilute sun red Fy’s of such crosses are, however, listed in table 17 (page 134), to serve as an indication of the behavior of all. Three F. progenies (group 1, table 17) contained 269 dilute sun reds and 99 greens, a deviation from the expected 3:1 ratio of 7+ 5.6. Five progenies of F, dilute sun reds back- crossed to green VIc (group 2) included 357 dilute sun reds and 358 greens, a deviation from the expected 1:1 ratio of only 0.5 + 9.0. The behavior of a number of the sun red and dilute purple plants listed in table 16 has been studied in F, and later generations. . Consideration of this later behavior is conveniently deferred to a later section of this paper (pages 51 and 53), where it is taken up with other crosses which should theoretically give similar results. Green x green, VIla, VIb, VIic— A number of green plants of progeny 2019, discussed above, were intercrossed. That these green plants bred true green when selfed was shown by the records of table 15 (groups 3 50 R. A. EMERSON to 5). That they were of three distinct genotypes was shown by the data recorded in table 16 (groups 4 to 6). The behavior of random intercrosses of the same green plants is now to be considered. The data are given in table 18. The green plants that served as parents of the crosses listed in group 6 of table 16, it was decided, must have been aabb pl pl. When such plants are crossed with green plants of any of the other genotypes, nothing but green plants should result. A single cross of one of these greens with a green of the constitution aa B b pl pl (table 16, group 4) gave 23 green plants (table 18, group 1) as expected. Another cross of one of these greens with a green of the genotype aabb Pl pl (table 16, group 5) gave 22 green plants (table 18, group 2). Crosses of green plants belonging to like genotypes should, of course, give only green plants. Three crosses of plants shown to be aa B b pl pl (table 16, group 4) gave 72 green plants (table 18, group 3). A single cross between plants shown to be aab b Pl pl (table 16, group 5) gave 24 green plants (table 18, group 4). Five crosses of plants of genotype aabbplpl with plants of genotype aabb Plpl gave a total of 40 brown and 105 green plants (table 18, group 5). Here a 1:3 ratio of brown to green is to be expected. The theoretical numbers are therefore 36 and 109, respectively, and the devia- tion is 4.0 + 3.5. The important fact here is that all these intercrosses of greens gave the color types expected on the basis of the results of crosses of the same individual green plants with dilute sun reds. The writer deems himself fortunate in having been able to obtain results approximat- ing so closely a complete demonstration of the several genotypes of green, since the selfing, the crossing with dilute sun reds, and the inter- crossing of greens, were made at the same time, with the green plants chosen wholly at random. ~ Brown V «x green VIc.— When brown plants are crossed with green plants of type VIc, the F, plants are brown, and browns and greens alone appear in F,. Since brown is supposed to be a B Pl and type Vie green ab pl, the F2 progenies should exhibit 9:7 ratios. Eleven F,: pregenies are listed in table 19 (page 135), with a total of 317 brown and 223 green plants. The theoretical numbers are 304 and 236, respectively, showing a deviation of 13+7.8. There is more than one chance in four that such _ a deviation is due to errors of random sampling, P equaling 0.27. PLant CoLors In Maize Rh Of any nine F, brown plants of this cross, theoretically one should breed true in F3, four should give a 3:1 ratio, and four should give a 9:7 ratio. Six F.’s were tested, with the results shown in table 20. Two bred true, with a total of 29 brown plants (group 1). Two gave ratios classed as 3:1, the totals (group 2) being 100 brown to 40 green, a devia- tion of 5.0 + 3.5. Two gave progenies interpreted as 9:7 (group 3), totaling 39 brown and 39 green, the deviation being 5.0 + 3.9. Of the 3:1 F; lot, two browns bred true in F:, producing 59 brown plants, and one green bred true, producing 56 green plants. The distribution of the F, brown plants with respect to their F; behavior — two breeding true, two throwing a 3:1 ratio, and two a 9:7 ratio — was as near expectation, 1:4:4 in nine, as could perhaps be expected from such small numbers. If these six Fz browns are combined with the four- teen F, browns of the original cross of purple x green noted earlier in this paper (page 44), a very good fit of the hypothesis and observation is found (2 = 0.88). Theoretically these two lots of Fz browns should be of the same genotypes, so that they may well be so combined. The comparison follows: F; ratios 1:0 sel 9:7 Total Winschycheree so. eS OES. 2 11 7 20 Solemiateate =e ee PS 2 9 9 20 Wiiherences 0 LS, ey xe, ats 0 +2 —2 0 Sun red Ila x green VIc.— When both parents are homozygous, the cross of green of type VIc with sun red results in sun red plants only. Three such crosses gave 112 sun red plants. Crosses with heterozygous sun red plants gave F, progenies of sun red together with dilute sun red or green or both, depending presumably upon whether one or the other or both of the factors A and B were heterozygous. F sun red plants of such crosses are presumed to have the formula AaB b pl pl, and should therefore produce in F, the three color types sun red, dilute sun red, and green, in the relation 9:3:4. Sixteen F, progenies of such crosses are listed in table 21, group 1 (page 136). It has already been shown (page 48) that crosses of some green plants, a B pl, with dilute sun reds, A 6 pil, give sun red F, offspring, which are also assumed to be AaB b pl pl. Five F, progenies of such crosses are, for convenience, considered here 52 R. A. EMERSON (group 2, table 21) with the crosses of sun red and green. While certain - of the individual progenies, due perhaps to the small numbers concerned, deviate considerably from the expected results, the twenty-one progenies (groups 1 and 2, table 21) taken together approach so closely to expectation that there is more than one chance in four that the observed deviations may be due to errors of random sampling, P equaling 0.28. The com- parison of observed with expected numbers follows: Dilute Color types Sun Ted ee Green Total Ila IVa Vila, ¢ Observed: Tle exe VIC ese eee eee 827 268 383 1,478 Vine SOV ila ia eee reese a ae 343 120 179 642 otal sac hier ee are sete 1,170 388 562 220) Calculated! = sec (on ees eee 1,193 398 530 U4 OA | Dilerencesya- wen Se ee eee —23 —10 +32 —] F, sun red plants, A a B b pl pl, were also backcrossed with green plants of type VIc, ab pl. Fifteen progenies of these backcrosses are listed in table 21, the progenies from the cross Ila x VIc in group 3 and those from the cross IVa x Via in group 4. The expected relation of 1:1:2 was realized fairly well in the results, the odds against the observed devia- tions’ being due to chance being about three to two, P equaling 0.39. The observed and expected results are compared as follows: Color types Sun red ae Green Total Ila IVa Vila; ¢ Observed: (illasxRVElc) exe VEG hen sn tre pert tcp 134 123 267 524 (TIGA SE AVA EN) SEN latin tierce SEN 442 A465 962 1 ,869 Ovale eee he ees ee 576 588. 14229 2 393 Calculatedhiavees eee. es 598 598 1,196 2 ,392 Difterence cre echt ee wc eee —22 —10 +33 +1 PLANT Cotors IN MaizE 53 Dilute purple IIIa x green VIc— Since dilute purple differs from sun red merely in having the dominant Pl factor instead of B, crosses of dilute purple with green of type VIc should behave just as did the crosses con- sidered in the preceding section, except that dilute purples take the place of sun reds in the progeny. Eight crosses of dilute purple with green of type VIc resulted in 91 dilute purple plants. The F. results of these crosses are given in table 22, group 1. Since the F, plants of these crosses are assumed to have been Aabb Pl pl, the F2 results should be the same as those expected from crosses of greens of type VIb with dilute sun reds. The Fy’s of the latter crosses have alreaidy been discussed (page 48). The F, results, six progenies, are for convenience considered here (group 2, table 22). While the expectation of a 9:3:4 relation was not very closely realized in the observed results, such deviations as those found might be expected thru chance about once in eight trials, P equaling 0.13. The comparison of observed and expected distributions follows: Dilute Dilute Color types anneal iin asl Green Total Illa IVa VIb, ec Observed: IU Dish 5° WAGs Senet ae een 416 149 ae 738 TINY eh 5% AV 0) 5 ua amar el a 274 102 107 483 WOU 6 oss Se Rr re - 690 251 280 22) Olculaned eres sos cw cc ce sk eles 687 229 305 1220 | DHIGIRSIONGS = satan eae iene +3 +22 —25 0 A single F, plant backcrossed with green gave the same three color types in the relation 26:20:56. The theoretical distribution is 25.5:25.5:51.0. Deviations of the observed order might be expected somewhat more than twice in five trials, P equaling 0.44. Seven F. greens bred true in F; with a total of 359 individuals. One dilute sun red F, plant bred true with a progeny of 156 dilute sun red plants. Of the F». dilute purples, some bred true, some threw the three types seen in F2, some gave only dilute purple and dilute sun red, and some gave only dilute purple and green. Notwithstanding the rather poor fit in F2, therefore, the fact that practically all the expected classes 54 R. A. EMERSON of behavior were exhibited in F3; makes it seem likely that the deviations in F, were due mainly to chance. Sun red Ila x brown V.— A single cross of brown with sun red gave purple plants only, as was expected. Since both parents were homozygous, all the F; plants should have been of the genotype Aa BB PIpl and should have produced in F, the four types purple, sun red, brown, and green, in the relation 9:3:3:1. The three F.2 progenies of this cross are recorded in table 23 (page 137). The expected color types were produced in approximately the expected numbers. The odds against the observed deviations’ being due to chance are three to two, P equaling 0.40. A comparison of observed with expected distributions follows: Color types Purple Sun red Brown Green Total a la Ila V Via @Observeds ac fesse ne see 120 29 37 10 196 Calculatede-ccqeaea ee eee 110 37 Si iP 196 PithereMnce Weak. es ees +10 —8 0 —2 0 Purple Ia x brown V.— Crosses of brown with purple gave purple ~ F,’s, and four Fs progenies gave a total of 116 purple and 38 brown plants, which is very near the 3:1 ratio expected from F, plants of the genotype AaBB PI Pl, the deviation being 0.5 + 3.6. Nine F,; purples backcrossed to browns gave progenies totaling 484 purple and 477 brown plants, a deviation from the expected equality of 3.5 + 10.5. Purple Ia x sun red IITa— Purples and sun reds should differ by a single factor pair, Pl pl. The F; purples backcrossed to sun red should give a 1:1 ratio of the parental types. Five such backcrosses gave 47 purple and 57 sun red plants, a deviation from expectation of 5 + 3.4. No progenies of selfed F;’s were observed. Purple Ia x dilute purple I[[a.— Purples are assumed to differ from dilute purples by the factor pair Bb. Six Fi purples backcrossed with dilute purple gave 40 purple and 52 dilute purple plants. This is a deviation from the expected equality of 6 + 3.2. No other tests of the cross of purple x dilute purple were made. Sun red IIa x dilute sun red IVa.—- Sun reds and dilute sun reds should differ in one factor pair, B b, and should therefore give a simple 3:1 result in F,. The F; generation of six crosses of these color types consisted of 135 sun red plants. Sixteen F2 progenies listed in group 1 of table 24 Prant CoLorRs IN Maze 55 (page 138) totaled 998 sun red and 314 dilute sun eel a deviation from the 3:1 ratio of 14 + 10.6. _ Fourteen backcrosses of F, sun red plants with dilute sun reds (group 2, table 24) resulted in 811 sun reds and 742 dilute sun reds, a deviation from the expected equality of 34.5 18+.3. Two F, dilute sun reds bred true in F3 as expected (table 25, group 1), with a total of 50 dilute sun red offspring. Two F, sun red plants (group 2) gave a total of 19 sun reds in F3, and a third F: plant, on backcrossing with dilute sun red, gave 101 sun reds. Four other F2 sun red plants gave both sun reds and dilute sun reds in their F3 progenies (group 3), the respective numbers being 373 and 127; the calculated numbers are 375 and 125, respectively, showing a deviation of 2+ 6.5. Of the seven F. sun reds tested, four were heterozygous and three apparently homozygous for the B factor. On the whole, therefore, the crosses of sun red with dilute sun red behaved approximately as expected. Dilute purple Illa x dilute sun red IVa.— Five crosses of dilute sun red with dilute purple gave a total of 344 F,; plants, all dilute purple. Since these F';’s are supposed to be heterozygous for the Pl factor only, a 3:1 F, distribution of color types should result. Seven F. progenies listed in group 1 of table 26 (page 139) had a total of 261 dilute purple and 87 dilute sun red plants, exactly a 3:1 relation. Five F, plants were backcrossed with dilute sun red (group 2) and resulted in 275 dilute purples and 263 dilute sun reds. The deviation from the theoretical 1:1 relation is 6 + 7.8. Only two F, dilute purples were tested by their F; behavior. Neither bred true, the total produced being 38 dilute purples to 17 dilute sun reds, a deviation from the 3:1 ratio of 3.3 +2.2. As far as they go, then, the results are in close agreement with what is expected of the crosses here under consideration. Sun red Ila x dilute purple IIla.— Theoretically, crosses of sun red, A B pl, with dilute purple, A b Pl, should give purple, A B Pl, in F,. Two crosses, as shown in group 1 of table 27 (page 140), gave a total of 24 purple and no other types. Here the parents were doubtless homozygous. If one or the other of the parents is heterozygous, two color types are to be expected in F;. A single cross (group 2, table 27) gave 74 purple and 75 sun red plants. Such a result is to be expected when the sun red parent is homozygous, A A B B pl pl, and the dilute purple parent is heterozygous, A A bb Pl pl. Two other crosses (group 3) gave 56 R. A. Emerson a total of 28 purple and 29 dilute purple plants. The parents are therefore assumed to have been AA Bbplpl and A Abb Pl Pl, tho the same results should have been obtained if one or the other, but not both, of the parents had been Aa. The important point here is that purple plants were produced in all crosses, showing that sun red and dilute purple carry complementary factors for purple. The factors are assumed, in keeping with the hypothesis under test, to be B and PI. In accordance with this hypothesis, the F; purple plants should be AABb Plpl and shouid throw four color types in Fs. No direct Fe progenies have been observed, but seven progenies from backcrosses of F; purples with dilute sun reds are recorded in table 28. While the deviations from the expected equality among the four classes are rather large, they are not greater than might occur by chance about once in four trials, P equaling 0.26. The comparison follows: ; Dilute Dilute Color types Purple Sun red purple annie Total la Ila Illa IVa @bserved 2s See ae 99 110 104 83 396 Calculatedor i i ie 99 99 99 99 396 Ditlerence os | he ee ee 0 +11 +5 —16 0 Purple Ia x dilute sun red [Va.— Crosses of purple with dilute sun red should give purple F; plants, A A Bb Pl pl, and 9:3:3:1 F.2 progenies. Four such crosses resulted in 65 purple plants in F,. The Fs. results are reported in table 29, group 1. The distribution of the individuals of the twenty-six progenies taken together is shown below in comparison with the calculated distribution. The four color types expected were observed in approximately the expected numbers. Deviations such as shown might be expected thru chance about twice in eleven times, P equaling 0.18. Dilute Dilute Color types Purple Sun red purple: Saeed Total Ta Ila Illa IVa Olosery cde) ee a eae 1,013 316 296 100 25 @alculated ent ae con 970 323 323 108 1 724 IDitierencen ae ee ee +43 —7 —27 —s +1 PLANT COLORS IN Maze 57 Some of the F; purple plants were crossed back to dilute sun red, with results as given in group 2 of table 29 and summarized below. The seventeen progenies together approached the expected equality of the four. color types so closely that the observed deviations might be expected thru chance more than twice in five trials, P equaling 0.44. Dilute Dilute Color types Purple Sun red nitadle avis Total Ta Ila IIIa IVa WOsenvedis es eo ess 323 306 320 289 1 243 PP emlaueds sa tian bse 311 dll dll 311 1 ,244 DM SRSGS i oe ee +12 = Sd =. Sixteen F, purple plants were tested by their F3; progenies (table 30). Seven F; purples (group 1) gave again the four color types purple, sun red, dilute purple, and dilute sun red, the several classes being represented by 268, 105, 78, and 28 individuals, respectively, while the calculated numbers were 269, 90, 90, and 30. The odds against such deviations being due to chance are about three to one, P equaling 0.24. One of the seven F, purple plants was crossed with green aabb pl pl and gave the same four classes of progeny, represented by 26, 25, 24, and 21 plants, respectively. Evidently these F: purples were like the F;’s, AA Bb Pl pl. Four other F, purples (group 2, table 30) gave only purple and sun red progenies. Three of these when selfed gave 60 purple and 22 sun red. Two of these three and one other, when backcrossed with dilute sun red or green, gave 32 purples and 31 sun reds. The four F,’s are therefore regarded asA A BB PI pl. Five F, purples (group 3) gave purples and dilute purples only. Four of these, which were selfed, gave 162 purples and 48 dilute purples, while the fifth, which was backcressed to dilute sun red, gave 17 purples and 15 dilute purples. These five F.2’s are consequently regarded as AABb Pl Pl. None of the sixteen F, purples tested bred true in F;, A A BB Pl Pl. A single F; purple (group 6), however, which occurred in the F; lot showing the four color types (group 1) and which was therefore comparable to the F, purples, bred true in Fy, producing 69 purples on being selfed and 18 on being backcrossed to green. Of three other F; purples of the same 58 R. A. EMERson F; lot, two (group 4) gave only purples and sun reds, and one (group 5) gave only purples and dilute purples. The twenty .F, and F3; purples tested, therefore, were distributed with respect to the four kinds of behavior in the relation 7:6:6:1, in contrast to the calculated distribution of approximately 8.9:4.4:4.4:2.2. There is more than an even chance that such a difference may be due to errors of random sampling, P equaling 0.53. On the whole, therefore, the F» purples of this cross behaved in later generations as was expected of them. F, sun red plants of the cross purple x dilute sun red showed two types of behavior in F; (table 31, group 1). Three F2’s bred true, with 53 sun red plants in F;. Four gave a total of 70 sun red and 24 dilute sun red plants. Where an expected ratio of one true breeding to two segregating progenies was expected, the observed relation of three to four is not a bad fit. F, dilute purples also showed the two types of behavior expected in F; (group 2, table 31). Three bred true, with a total of 97 dilute purple plants, and six gave a total of 217 dilute purple and 86 dilute sun red plants. The 1:2 ratio was therefore exactly realized. Three F, dilute sun reds bred true in F; (group 8) as was expected of them, producing a total of 72 dilute sun red plants. Numerous F; plants of the several color types of the cross under con- sideration here were tested by Fy and F; progenies, with results wholly consistent with expectation. It is deemed unnecessary to give the records of these later generations in detail. Evidence from aleurone-color and linkage relations The evidence presented up to this point in support of the three- Factee hypothesis, involving Aa, Bb, Pl pl, has had to do ,with the behavior of the several F, color types in later generations and in intercrosses. There remain to be discussed some bits of evidence which, while less direct, are perhaps no less trustworthy. This evidence deals with (1) the relation of aleurone color to plant-color types, (2) the linkage of certain plant-color types with endosperm color, and (8) the linkage of other color types with the liguleless leaf. Relation of aleurone color to plant color.— Of the alm olor factors considered in this section of the paper, the pair A a is concerned also in the development of aleurone color. It has been shown by the writer PLANT CoLors IN MaIzE 59 i a previous paper (Emerson, 1918) that the presence of three dominant factors, A, C, and R, is necessary for the development of aleurone color. It is assumed that the factor pair A.a for aleurone color is identical with the pair Aa for plant color. Some of the evidence on which this assumption is based may well be considered at this point in order to justify the use of the same symbols for both plant and aleurone color. After the identity of Aa has been established, certain relations of aleurone color to plant color can be used to check up some of the conclusions previously drawn with respect to the genetic interrelations of the several plant-color vypes. , 3 It will be recalled that dilute sun red crossed with green gave dilute sun red in F,; and a 3:1 ratio of the two types in Fe (table 17, group 1, page 134), and that backcrosses of F; with green gave a 1:1 ratio (group 2). The F, seeds of these F; plants also exhibited a 3:1 relation — 424 colored and 127 colorless, deviation 10.8 + 6.9— thus showing that only one factor pair, Aa, Cc, or Rr, was heterozygous. The colorless seeds produced 98 green plants, and the colored ones produced 269 dilute sun reds and 1 weak plant, recorded as green, which died in the seedling stage. Obviously the factor that differentiates dilute sun red from green is the same as the one that in these cases differentiated the colored from the colorless seeds, or some factor very closely linked with it. Fortunately, F, plants closely related to the ones which when selfed showed the behavior noted above, were backcrossed with green, colorless-seeded A _ testers (Emerson, 1918). Of the resulting seeds 632 were colored and 590 were colorless, evidently a 1:1 relation — the deviation being 21 + 11.8 — showing that the I; plants were, with respect to aleurone color, Aa CCRR. The colored seeds gave rise to 357 dilute sun red plants and the colorless seeds to 358 green plants. Evidently, therefore, it is the Aa pair that differentiates dilute sun red from green. This is in support of the assumed genotypes A b pl and ab pl for dilute sun red and green, respectively. - The single progeny recorded in group 3 of table 9 (page 127) came from a plant known to be A a with respect to aleurone color and pro- ducing 130 colored and:41 colorless seeds. The 3:1 aleurone-color relation shows it to have been heterozygous in only one aleurone-color factor, and therefore AaCCRR. The colored seeds, ACR, produced 67 purple plants, and the colorless ones, aC R, produced 21 brown plants. 60 R. A. EMeRSonN Evidently, purples are differentiated from browns by the Aa pair alone, just as dilute sun reds are differentiated from greens. This is quite in keeping with the assumed genotypes, A B Pl and a BPI, for purple and brown, respectively. Two of the progenies recorded in group 3 of table 8 (page 126) involved both aleurone and plant color. The heterozygous parents were back- crossed with green A testers and produced 125 colored and 127 colorless seeds. The factor pair differentiating these two seed classes was therefore Aa. The colored seeds, A C R, produced 15 purple and 14 sun red plants, while the colorless seeds, a C R, gave 9 brown and 14 green plants. Since it is shown in the preceding paragraph that purples and browns differ with respect to the pair Aaalone, it may be inferred that the sun reds and the greens of these lots also differed with respect to Aa alone. The assumption heretofore made with respect to the genotypes of these color classes, A B Pl, AB pl, a BPl, and aB pl, for purple, sun red, brown, and green, respectively, is given support by this relation of aleurone color to plant color. Two of the progenies recorded in group 1 of table 9 (page 127); and one in group 4 of table 8 (page 126), were grown from self-pollinated plants known to be Aa with respect to aleurone color and found to have 644 colored and 228 colorless seeds. The 3:1 seed-color relation shows them to have been AaCCRR. The colored seeds, A C R, gave 294 purples and 113 dilute purples, while the colorless seeds, a C R, gave 119 browns and 40 greens. If purples and browns differ with respect to Aa alone, as they have been shown to do, presumably the dilute purples and the greens of these lots also differ in the same way. This is in keeping with the assumption that the genotypes of the color classes are A B Pl, A b Pl, a B Pl, and ab Pl, for purple, dilute purple, brown, and green, respectively. These comparisons of the relations of aleurone color to plant color have confirmed definitely the supposition that purples, sun reds, dilute purples, and dilute sun reds have the dominant factor A, and browns and greens the recessive factor a. The comparisons have also afforded some support for the assumed genetic constitution of the several color types with regard to Bb and Pipl. More definite evidence for the latter, however, is afforded by the linkage relations now to be discussed. Linkage of plant color with endosperm color.— It has been known since 1912 that a linkage exists between the factor pair Pl pl and endosperm PLANT CoLors IN Maize 61 color. The data suggest irregularities or complexities which cannot be straightened out until more definite. information is at hand with regard to the two or more factor pairs concerned in the development of yellow endosperm.® Only such data are presented here as are necessary to show the relations of the several plant-color types to endosperm color. * A single progeny recorded in table 27, group 2 (page 140), was made up of 74 purple and 75 sun red plants. The lot resulted from a cross of a white-seeded sun red plant with a dilute purple plant which was heterozygous with respect to both yellow endosperm and plant color. The yellow seeds produced 58 purple and 20 sun red plants, and the white seeds produced 16 purple and 55 sun red plants. The yellow-seeded sun reds and the white-seeded dilute purples are known to be the crossover classes. The ratio of non-crossovers to crossovers is 113:36, and the percentage of crossing-over, therefore, is 24.2. Evidently a factor pair for yellow endosperm, Y y, is linked with the factor pair that differentiates purple from sun red. In accordance with the hypothesis under test, this plant-color factor pair is Pl pl— purple=A B Pl, and sun red=A B pl. Two other progenies (table 26, group 1, page 139) had a total of 116 dilute purple and 42 dilute sun red plants. The selfed parent plants were heterozygous tor yellow endosperm as well as for plant color. The yeliow seeds gave 99 dilute purple and 17 dilute sun red plants, and the white seeds gave 17 dilute purple and 25 dilute sun red plants. This F, distribution, as shown below, is very close to expectation ( x? = 0.26) on the basis of 25 per cent of crossing-over between the factor pair Yy and the pair that differentiates dilute purple from dilute sun red. It seems likely, therefore, that the same plant-color factors, Pl pl, are concerned here as in the progeny consisting of purples and sun reds. This is in keeping with the theoretical genotypes, A b Pl and A b pl, assumed for dilute purple and dilute sun red, respectively. The comparison between the observed F 2 distribution and that calculated on the basis of 25 per cent of crossing-over follows: MNSCEVEC eee. oes oe 99 iL7/ 17 Dy eels Malenlated 2. ko ci ahs 102 17 17 Da thee ad 90) BDinherence <.. 285 2S ec ees —3 0 0 +2 —] 5 This problem is being investigated by Dr. E. G. Anderson. 62 R. A. Emerson A single progeny (table 8, group 3, page 126) from a selfed parent heterozygous for yellow endosperm, contained purple, sun red, brown, and green plants, totaling 63, in the relation 35:15:6:7. These four color types are expected to occur in a total of 64 in the relation 36:12:12:4 from a selfed plant of the genotype AaB B Pl pl. The observed deviation from expectation might occur by chance once in nine trials, P equaling 0.11. Theoretically, the green plants of this lot, a B pl, are differentiated from the browns, a B Pl, by the same factor pair, Pl pl, that differentiates the sun reds, A B pl, from the purples, A B Pl. If this is true, the same linkage relations should exist for yellow endosperm with the brown-green lot as with the purple-sun-red lot. From yellow seeds there came 29 purples and 8 sun reds, and from white seeds 6 purples and 7 sun reds. Such a distribution should be very closely realized (2? = 0.97) from 30 per cent crossing-over between Y y and Pl pl. The yellow seeds produced also 5 brown and 3 green plants, and the white seeds 1 brown and 4 green plants. While the number of individuals is too small to give a reliable indication, it is of interest to note that the coefficient of asso- ciation (Collins, 1912) calculated from the series 5:3:1:4, or 0.739, is practically that calculated from 26 per cent of crossing-over. In so far as these records go, therefore, they support the assumption that brown and green in this lot are differentiated by the same factor pair as are purple and sun red, and thereby support the hypothesis under test. A plant heterozygous for the three plant-color pairs Aa, Bb, Pl pl, and for Yy, backcrossed with a white-seeded green plant of type VIc, ab ply, gave the six color types, purple, sun red, dilute purple, dilute sun red, brown, and green, in the numerical relation 10:13:17:11:9:33 (table 6, page 124), which is a close fit (P = 0.61) to the expected relation, 1:1:1:1:1:3. From yellow seeds the resulting series was 8:6:13:2:7:17, and from white seeds it was 2:7:4:9:2:16. When the classes having A Pl, purple and dilute purple, were lumped together, and similarly those having A pl, sun red and dilute sun red, the yellow seeds gave 21 plants with Pl and 8 with pl, while the white seeds gave 6 with Pl and 16 with pl. Of these 51 plants, there were 14 in the crossover classes, or a percentage of crossing-over of about 27.5 + 4.1, approximately the same as in the cases cited above. In this lot there are theoretically three kinds of greens, a B pl, ab Pl, and ab pl, one of which has Pl and two of which have pl, while all the browns, a BPI, have Pl. If there be Puant Cotors In Maize 63 assumed 25 per cent of crossing-over between Yy and PI pl, equivalent to a 3:1:1:3 gametic series, yellow seeds should give 3 brown to 5 green, and white seeds 1 brown to 7 green, as shown below: Yellow White LEVRONWHOL 0) JERR El hie os SA Bia Gn ORE Re A Te ee 3 1 SE RECT Bog DU eos te) ap asics. ts 3 aly ac sy eck asa 2 1 3 Gieen; C012 ee ne een 3 il CURE, GD FOCI ate etre aaa en Oe 1 3 5 7 The yellow seeds actually gave 7 brown to 17 green and the white seeds 2 brown to 16 green, which is a close fit to the calculated relation, 3:5: 1:7 (P=0.59). In this case as in the others, then, the linkage relations between Y y and PI pl afford additional support for the belief that the several color types actually bear to one another the relation assumed in the assignment of hypothetical genetic formulae (page 32). Linkage of plant color with leaf type-—It has been known for some years that a leaf type termed liguleless (Emerson, 1912) is linked with the factor pair that differentiates sun red from dilute sun red. As an illustration of this, two backcross progenies, 8250 and 8253, with a total of 145 sun red and 147 dilute sun red plants, may be cited. These progenies came from a cross of normal-leaved sun red, A B pl Lg, with liguleless- leaved dilute sun red, A 6 pl lg, backerossed with liguleless dilute sun red. Of the normal-leaved plants 104 were sun red and 41 were dilute sun red, while of the liguleless-leaved plants 48 were sun red and 99 were dilute sun red. The non-crossovers were to the crossovers as 203:89, or a per- centage of crossing-over of 30.5. Since the factor pair that differentiates sun red from dilute sun red has been assigned the symbol B b, the linkage noted here is evidently between B b and Lg lg. Six progenies from backcrosses of heterozygous nermal-leaved purples with liguleless dilute sun reds gave purples, sun reds, dilute purples, and dilute sun reds in the relation 197:177:178:167, which is not far from the equality expected, P equaling 0.46. Among the normal-leaved plants, the four color types occurred in the relation 123:117:47:55, and among the liguleless-leaved plants in the relation 74:60:131:112. Evidently the purples bear the same relation to the dilute purples as the sun reds do to 3 64 R. A. EmMerson the dilute sun reds. For sun reds and dilute sun reds, the non-crossovers are to the crossovers as 229:115, or a crossover percentage of 33.4 + 1.7. For purples and dilute purples, the relation is 254:121, or a crossover percentage of 32.3 + 1.5. It follows from this that the factor pair, B b, which differentiates sun red, A B pl, from dilute sun red, A 6 pl, is the same as that which differentiates purple from dilute purple. And this is in keeping with the hypothesis under test, in accordance with which purple and dilute purple have been assigned the genotypes A B Pl and A b Pl, respectively. In a single progeny resulting from a backcross of a heterozygous normal- leaved purple plant with a liguleless-leaved green plant, greens occurred, as expected, with about three times the frequency of the average of the other five color classes. The progeny included 14 browns and 49 greens. Of the normal-leaved plants there were 10 browns and 19 greens, and of the liguleless-leaved plants 4 browns and 30 greens. On the basis of the hypothetical genotypes assigned to browns and greens, and with the assumption of 33 per cent of crossing-over between Bb and Lg lg, the four classes, normal brown, normal green, liguleless brown, and liguleless green, should bear the relation 2:4:1:5. For a total of 638 plants, the relation would be approximately 11:21:5:26, whereas the observed relation was 10:19:4:30. The deviations from expectation are such as might occur by chance in more than three out of four trials, P equaling 0.78. In this case, as in the others reported, the linkage relations between Bb and Lg lg afford support for the view that the several color types bear the relation to one another inferred from the hypothetical genotypes assigned them. Summary of results involving A a, Bb, Pl pl The results of the cross of purple with green — which gave in F» six color types, namely, purple, sun red, dilute purple, dilute sun red, brown, and green, with a numerical relation of approximately 27:9:9:3:9:7 from selfed Fy’s and about 1:1:1:1:1:3 from F;,’s backcrossed to green — have been interpreted on the basis of the interaction of three factor pairs, Aa, Bb, and Pipl. This hypothesis has been subjected to practically every genetic test available, as summarized below. Each of the six F, color types has in turn been tested by its behavior in F'3, and in several cases behavior in Fy and even in later generations PLANT Coors IN Matzr 65 has been noted. All the possible combinations of intercrosses between the several types have been studied, except dilute purple x brown. In most cases these intercrosses have been carried to the F.2 generation, and in several instances to F3; and Fy. Thruout the tests, the results have been in close agreement with those expected from the hypothesis. In almost every instance all the color types expected in each generation of the several crosses, and no others, have appeared. Moreover, the numerical relations found to exist between the several color types and also between the several classes of behavior, have been reasonably close to expectation. It is true that in some instances the fit of observation to hypothesis has not been particularly good, but even here the observed deviations have been of such an order as might be expected to occur occasionally thru the chance errors of random sampling. In addition to the tests afforded by the behavior of the several Fs color types in later generations and in intercrosses, the relations of aleurone color involving the factor pair A a to the several plant colors, and the linkage relations of the plant-color factors Pl pl with the endosperm-color factors Y y and of the plant-color factors Bb with the leaf-type factors Lg lg, have been included in the investigation. These tests have shown that the several color types bear to one another the relations to be deduced from the hypothetical genotypes assigned them. The conclusion seems justified, therefore, that the three-factor hypoth- esis proposed as an interpretation of the F. results obtained in crosses of purple with green has been substantiated, in so far as it is possible to substantiate any hypothesis. CROSSES INVOLVING THE MULTIPLE ALLELOMORPHS B, B”, 6’, b In the preceding section of this account, six color phenotypes of maize have been discussed, namely, purple, sun red, dilute purple, dilute sun red, brown, and green. In addition to these six phenotypes, green plants have been shown to consist of three genotypes, which in some instances are slightly different phenotypically. Besides these six sharply separable phenotypes, there exist certain intermediate forms. The constancy of these types from year to year, under fairly uniform environmental conditions, leaves no doubt that they are genotypically as well as pheno- typically distinct from the types considered heretofore. 66 R. A. EMERSON One of these forms, known as weak purple, type Ib, is intermediate in certain respects between purple and sun red, and in other respects between purple and dilute purple. Plants of this type, prior to the flowering stage, frequently resemble sun reds more than purples. The pigmentation of the sheaths is less intense than with purples, and in some instances less than with strong sun reds. There is, however, sooner or later a tendency for pigment to develop on the stalk beneath the sheaths (Plate V, 2). In this respect weak purples resemble dilute purples as the latter often appear in a late stage of their development. The anthers of weak purples are usually full purple, like those of purples and dilute purples, in which respect they show no resemblance to sun reds. A second intermediate form, known as weak sun red, type IIb, stands between sun red and dilute sun red. The sheaths and husks are less extensively and less intensely pigmented than is true of full sun red, and yet exhibit much more color than in dilute sun red (Plate V, 4). The anther color of weak sun red is like that of both sun red and dilute sun red. While the difference between the extreme sun-color types, sun red and dilute sun red, is probably only a quantitative one — as is also presumably true of the difference between purple and dilute purple — little difficulty is experienced in separating sun red from dilute sun red plants on the one hand, or purple from dilute purple plants on the other. Frequently, however, it is difficult, or even impossible, at early stages of plant growth, to separate sun reds from purples. The existence of such intermediate forms as weak purple and weak sun red adds materially to the difficulties of classification. In fact, correct classification of all these types by inspec- tion alone is possible only at the flowering stage. For certainty in classi- fication, even at the flowering stage, environmental conditions, particularly soil fertility, must have been favorable thruout the growing period of the plants. While infertile soil exaggerates the difference between dilute sun red and green, by bringing about an excessive development of red pigment in the one type while no color develops in the other, on fertile soil only are revealed the finer distinctions between sun red, weak sun red, and dilute sun red. It is perhaps fortunate that the genetic relations off these several types are such that ordinarily not all of them occur in a single progeny. PLANT Coors IN MAIZE 67 Interrelations of sun red IIa, weak sun red IIb, and dilute sun red IVa Numerous crosses of weak sun reds, IIb, with dilute sun reds, IVa, have given weak sun reds in F; and approximately three weak sun reds to one dilute sun red in F», just as crosses of strong sun red with dilute sun red give three strong to one dilute sun red (table 24, group 1, page 138). Records of such crosses are given in table 32 (page 144). Twelve Fy, progenies, totaling 1729 individuals, showed the two types in the relation 1300:429, almost exactly a 3:1 ratio, the deviation being 3.3 + 12.1. The data for F; of these crosses are like those for crosses of strong sun red with dilute sun red (table 25). One weak sun red F:, bred true in F3 with a total of 77 weak sun red offspring (table 33, group 1). Four others gave both weak and dilute sun reds (group 2), in the relation 128:54, a_ deviation of 8.5 + 3.9 from a 3:1 ratio. One dilute sun red bred true (group 3), with 95 dilute sun red plants in F;. A cross of weak sun red, IIb, with strong sun red, Ila, gave strong sun red in F, and the two parent types in F2 in the relation 71:16, a deviation from the 3:1 ratio of 5.75 + 2.72. There is, therefore, nearly one chance in six that the cbserved deviation may be due to errors of random sampling, P equaling 0.16. : In none of these crosses, strong with weak, weak with dilute, and strong with dilute sun red, have other than the parent types appeared in F%. If weak sun red is due to the action of some additional modifying factor, not heretofore considered, types other than those of the parents should have occurred in some of the crosses. The natural conclusion, therefore, is that weak sun red, IIb, is due to an allelomorph of B and b, the pair concerned with the difference between sun red, Ila, and dilute sun red, IVa. This third allelomorph, responsible for weak sun red, may well be designated B”. Further evidence in support of the assumption that an allelomorph of B and 6 is concerned with weak sun red is afforded by linkage studies involving strong, weak, and dilute sun red with leaf type. Evidence has been offered (page 63) to show that Bb and Lg lg are linked with about 30 to 33 per cent of crossing-over. A single progeny, 8252, from a sun red plant heterozygous for leaf type and plant color backcrossed to liguleless weak sun red, contained 108 sun red and 109 weak sun red plants. Of the normal-leaved plants 80 68 R. A. EMERSON were sun red and 38 were weak sun red, while of the liguleless-leaved plants 28 were sun red and 71 were weak sun red. The ratio of non-crossovers to crossovers is 151:66, or 30.4 + 2.1 per cent of crossing-over. The percentage of crossing-over between Lg ig and the factor pair differentiating sun red and weak sun red, B B”, is, therefore, ee the same as the linkage between Lg lg and B b. Four backcross progenies, 8246-8249, involving sun red, contained 469 -weak sun red and 396 dilute sun red plants. Of the normal-leaved plants 153 were weak sun red and 261 were dilute sun red, while of the liguleless- leaved plants 316 were weak sun red and 135 were dilute sun red. The non-crossovers are to the crossovers as 577:288, or 33.5 + 1.1 per cent of crossing-over. Here again, therefore, the linkage between Lg lg and the factor pair differentiating weak sun red from dilute sun red, B” b, is practically the same as that between Lg lg and B b or between Lg lg and BeBe From the facts (1) that in crosses between any two of the three types sun red, weak sun red, and dilute sun red, the third type is not produced, and (2) that the linkage value between Lg lg and the factor pairs differen- tiating weak sun red from sun red and from dilute sun red is approxi- mately the same as that between Lg ly and B b, it seems evident that weak sun red is due to a factor BY” belonging to the triple allelomorphie series ‘By Bz, b: it seems probable that this series of allelomorphs contains other members in addition to the three listed above, but there is at present little conclu- sive evidence in support of the idea. There are certainly several forms, commonly classed as dilute sun red, that differ considerably in the amount of red pigment developed, and certainly some of these differences are genetic. As is shown in the next section of this account, some of these differences, particularly with respect to silk, anther, and leaf-blade color, are due to the effect of the aleurone-color factors Rr. Environmental conditions, particularly soil fertility, influence the development of this pigment so greatly that the problem becomes a difficult one. There is, however, some evidence that at least two forms of dilute sun red are differentiated by a factor pair belonging to the series B, BY’, b. These forms differ principally in the amount of color in the fresh husks (Plate VI, 1 and 2), and to some extent in the sheaths, which are the plant parts most strikingly different in sun red, weak sun red, and dilute sun red. PLANT CoLors IN MaIzE 69 A type of dilute sun red with stronger husk pigmentation than ordinary dilute sun red shows was crossed with an ordinary dilute purple. Leaf type also was involved in the cross. The F, plants were dilute purples with somewhat more pigment in the husks of young ears than is usual with that type. A single progeny, grown from an F, backcrossed with liguleless dilute sun red of a light type, consisted of 25 dilute purples and 18 dilute sun reds. Each of these classes was sorted with some difficulty into light and more strongly colored subclasses, in accordance with the amount of color on the husks of the young ears. Of the more strongly pigmented dilute sun reds 4 had normal and 6 had liguleless leaves, while of the lighter dilute sun reds 6 had normal and 2 had liguleless leaves. Of the more strongly colored dilute purples 4 had normal and 13 had liguleless leaves, while of the lighter ones 4 had normal and 4 had liguleless leaves. While these numbers are small and the behavior was somewhat irregular, it is perhaps noteworthy that the factor pair differentiating the lighter from the more strongly colored plants, of both the dilute sun red and the dilute purple classes, exhibited an apparent linkage with Lg lg of a value not far from that observed between Lglg and Bb, BB’, and BY b. The observed percentages of crossing-over were 32.0 for the dilute purples, 33.3 for the dilute sun reds, and 32.6 for the entire lot. This evidence, slight as it is, plainly suggests a fourth member, b*, of the B series of allelomorphs, which may be stated tentatively as B, BY, b’, b. Relation of weak purple Ib to purple Ia, dilute purple IIIa, and weak sun red IIb By methods similar in the main to those outlined above, Dr. E. G. Anderson has been able to show that weak purple is differentiated from purple on the one hand and from dilute purple on the other by the same factor, B”, that differentiates weak sun red from sun red and from dilute sun red. At the time when Dr. Anderson undertook to determine the genetic relations of weak purple, nothing was known of the relation of weak sun red to sun red and dilute sun red as presented above. Further- more, there was no indication as to whether weak purple was differentiated from purple and dilute purple by an allelomorph of B 6 or of Pl pl, or by some distinct factor pair that might modify the ordinary result of the interaction of the pairs Aa, Bb, and Pl pl then known to be concerned in the production of plant colors. The evidence to be presented here 70 R. A. Emerson is taken almost wholly from Dr. Anderson’s records, and the conclusions derived from it are his. It is with Dr. Anderson’s permission and at his suggestion that, for the sake of completeness of this account of the inheritance of plant. colors, his results are here presented. A cross of a weak purple Ib with a homozygous dilute purple IIIa resulted in 25 weak purples only, while a cross of another weak purple with a homozygous dilute purple, a sib of the plant used in the first cross, gave 63 weak purples and 53 dilute purples. Two of the F; weak purples were backcrossed to dilute purples, and a third to dilute sun red. The result (table 34, group 1, page 145) was 141 weak purples and 163 dilute purples, a deviation of 11 + 5.9 from equality. .Five crosses of weak purples with dilute sun reds gave a total of 32 weak purples and 25 dilute purples, a deviation from equality of 3.5 + 2.5, while two other such crosses gave 29 weak purples only. Evidently these weak purple plants differed from dilute purples by a single factor pair. This pair could not have been Pl pl, for the crosses of weak purple with dilute purple, A b Pl, gave the same results as those with dilute sun red, A b pl. This leaves the possibility that Bb or some unknown factor pair was concerned. Three crosses of weak purple Ib with purple Ia resulted in 52 purple plants. A single cross of weak purple with sun red Ila gave 18 purples. Evidently both purple and sun red carry some factor that acts to change weak purple to purple. Unfortunately, no later generations of any of these crosses were grown, but it 1s evident from the F,; results and from what is known of the interrelations of purple, sun red, and dilute purple that the dominant factor B, common to both purple and sun red, is con- cerned in the change from weak purple to purple. Since the crosses of weak purple with dilute purple, A b Pl, and with dilute sun red, A 6 pl, gave no purples, while crosses of weak purple with purple, A B Pl, and with sun red, A B pl, gave purple, the Pl pl pair is not concerned in the difference between weak purple and purple any more than in that between weak purple and dilute purple. These results, however, do not exclude the possibility that weak purple may be A b Pl, like dilute purple, with the addition of some unknown dominant modifying factor. A single weak purple plant, which was, so far as known, unrelated to the weak purples considered above, when crossed with two unrelated dilute sun reds gave progenies consisting of 15 weak purples and 13 weak sun reds. Seven progenies of these F; weak purple plants backcrossed PLANT Contors IN MaAtIzE 71 with dilute sun reds are listed in table 34, group 2. These progenies consisted of four color types, weak purple, weak sun red, dilute purple, and dilute sun red, in the numerical relations given below: Weak Weak Dilute Dilute Color types _ purple sun red purple sun red Total. Ib IIb IIIa IVa Mosenvedtrty sc: tcl csc. 8 481 526 460 5a 2,004 Malema tediy tas soa.co, fo. a 501 501 501 501 2,004 WeKENCEf.. 2. ke... es —20 +25 —41 +36 0 The deviations from equality of the four classes expected of a dihybrid are so great that they would not occur by chance alone more than once in twenty trials, P equaling 0.05. Dr. Anderson’s notes indicate that there was considerable difficulty, in the case of two of the cultures, in dis- tinguishing dilute purple from dilute sun red. Whether this difficulty may account in part for the poor fit is not known. The outstanding fact, however, is the appearance of the four classes and no others. Since weak sun red is known to differ from dilute sun red by the factor pair B” 6, the inference is clear that weak purple differs from dilute purple by the same pair and by no others. The formulae assumed for the four color types are, therefore, A BY” Pl, A B” pl, Ab Pl, and A 6 pl, respectively. | | If the foregoing conclusion is correct, crosses of weak sun reds with dilute purples should give weak purples in F,; and the same four color classes in F2 as are noted above for crosses of weak purple with dilute sun red. A single cross of a dilute purple with a homozygous weak sun red resulted in 18 weak purple plants. Two crosses of dilute purples with weak sun reds heterozygous for B” 6 gave 12 weak purples and 11 dilute purples. That the production of weak purples in these crosses was not due to the 6 or Pl factors of the dilute purple parents is evidenced by the fact that crosses of the same dilute purple individuals with sun reds gave full purples in F;. One of the F; weak purples, A A BY 6 Pl pl, of the above crosses was backcrossed with dilute sun red, A b pl, with the result (table 34, group 3) shown below. The expected equality of the U2, R. A. EMERSON - four color types was closely approached in the results, x2 equaling 0.80. The comparison of observed with expected results follows: Bente types Weak Weak Dilute Dilute purple sun red purple sun red Total Ib IIb Illa 1Va Observediwsn wrasse, 21 28 22 27 98 Calculated. i wes 24.5 24.5 24AN5 24.5 98 Ditterences icy ee 3.55 3-5)" —==2.5 2h 0 The progeny of a purple plant heterozygous for BB”, Pl pl, and the endosperm color pair Y y, backcrossed with a white-seeded weak sun red plant, A BY ply, affords evidence of another kind with respect to the interrelations of strong and weak purple and of strong and weak sun red. It has been noted previously (page 60) that Pl pl and Yy are linked, with a somewhat irregular percentage of crossing-over. The backcross gave the four color types purple, weak purple, sun red, and weak sun red, in the numerical relation 60:48:59:62. The observed deviations from the equality expected of a dihybrid are such as might occur by chance more than once in two trials, P equaling 0.54. The distribution of these 229 plants to the four color types when the progeny of yellow seeds and that of white seeds are considered separately is as follows: Color types Purple lace Sun red Were Total asc lil Ila IIb Miellowascedspyiy eee 48 36 8 ire 109 Wiknibevseeds 28 13 11 3 U 3 16 711 92 Calculated........ 29 10 10 3 10 3 13 414 92 Difference........ -—| +3 +1 0 —3 0 +3 —3 0 The third purple-anthered F.2 purple tested, 2961-3, gave in F; all the color types except dilute purple, Ila, and dilute sun red, [Va. A purple plant of the genotype AaBBPIplR’r’ should give the color types observed. The observed deviations from expectation might occur by chance about once in seven trials, P equaling 0.15. The comparison follows: Plant color Purple Purple Sun red Sun red Brown Green Total Anther color Purple Green Pink Green Green Green Ta Ig Ila IIg \ VI Observed........ 37 11 5 2 12 3 79 Caleculated....... 30 10 10 3 10 8 a Difference....... +7 +1 —5 ——I +2 -—5d5 —] A single green-anthered F2 purple, 2960-7, gave four F; color types, purple, sun red, brown, and green, all with green anthers. This behavior is to be expected from an F; genotype Aa BB Pl pl R’ R’. One of the F; purples, 4956-1, repeated this behavior in F,. The F; and F; progenies are shown together in the following comparison, for which P = 0.60: Color types - Purple Sunred Brown Green Total Ig Ilg Vv VI Meseiveds >. .4..:......0.- 84 Ail 35 7 153 Mememated@ ........ 55. 86 29 29 9 153 _ GIGGING) re —2 —2 +6 —2 0 It is of interest to note in this connection that a plant of the genotype AaBB Pipl R’ R’ could not exhibit a 27:37 fatio of colored to color- less aleurone, as was the case for some of the plants dealt with earlier. 84 R. A. EMEeRSoNn For Aa R?’ R’ the aleurone-color ratio must be either 9:7 or 3:1, depending on whether the third aleurone-factor pair is Cc or CC. The Fs: purple plant 2960-7 showed a 9:7 aleurone-color ratio with 86 colored and 74 colorless seeds, AaCcR R, while the F3; plant 4956-1 exhibited a 3:1 ratio with 213 colored and 67 colorless seeds, AaCCRR. Another purple plant of the same F; progeny, 4956-32, exhibited a 3:1 aleurone- color ratio and threw only green-anthered purple and sun red plants. Its genotype must have been AABBCc PlplkR’ R’. Thus it is often possible, from behavior in the following generation, to know the genotype not only with respect to plant color but for aleurone color as well. This is particularly true when the B factor is present. Of the twenty-four sorts of behavior possible, according to hypothesis, for F, purples of the cross under consideration, four sorts have been exhibited in F3; and a fifth shown in Fy. This is far from an adequate study of the F2 purples. All that can be claimed, therefore, is that, so far as they go, the results are in accord with the hypothesis. Behavior of other F: color types.— Only cne F2 sun red plant with pink anthers, 2961-4, was tested in F;.. It produced sun reds with pink and sun reds with green anthers, dilute sun reds, and greens. Since anther color was noted for only a part of the plants, it has to be disregarded in classifying the F; progeny. The color types sun red, dilute sun red, and green occurred in the numerical relation 114:23:57. Of the eight possible genotypes of pink-anthered sun red, only three could throw these three color classes—AaBbr'r’, AABDbR’r, and AaBb Rr. From the first genotype a 9:3:4, from the second a 12:3:1, and from the third a 36:9:19, relation should exist between the Fs; classes. The poor fit of observed numbers to the 9:3:4 relation makes it improbable that the first genotype is concerned, there being only about one chance in twenty-two that the observed deviations are due to errors of random sampling, P equaling 0.045. The comparison follows: Color types Sun red pie Green Total Ila IVa Via, ¢ Observed. eles A Raa era ata 114 23 57 194 Calculated! ssc 1 wae Wii Rees eerie 109 36 49 194 DitlerenCer:. hai cecs = see eee +5 —13 +8 0 PLANT Cotors IN Maize 85 A more conclusive reason for throwing out the first genotype is the fact that the plant had some seeds with colored aleurone, which would have been impossible if it were rr. The second genotype is discarded because of the extremely poor fit of observed numbers to the 12:3:1 ‘relation. There is an almost inconceivably small chance that the observed deviations may be due to errors of random sampling, z* equaling 180. (When n’=3 and x =29, P=0.000001. Higher values of z? when n’=3 are not listed in Pearson’s tables.) The comparison follows: _ 5 Dilute. Color types Sun red aun ed Green ‘Total avo lV; IVg MDccinucdmemer tk eet 114 23 57 194. Seeley ied) ee 146 36 12 194 Ti#enoiies, ee Bae Sea es, 0 The elimination of the first two genotypes leaves the third genotype as the only one that can be concerned here. The fit of observed numbers to the 36:9:19 relation is very close, x* equaling 0.84. (Values of P are not listed in Pearson’s tables for values of ~ less than 1; when #=1 and n’ =3, P=0.61.) The comparison follows: ; Dilute Color types Sun red Saree Green Total Ila, g War Vee Vala, ic CIISGINTEGLE ok Sas eee 114 23 SS 194 Palemiated ae tk oe Pa ee 109 Zi 58 194 MOI ERCUCOM iis fhe ce yee oe +5 —4 —1 0 This comparison leaves little doubt that the genotype of the F2 plant concerned is AaBbR’r’. There are, moreover, other considerations which go far toward identifying the genotype as given here. The fact that some sun red plants of F; had green and others pink anthers is evidence for the constitution R’r’. Since dilute sun red plants appeared in Fs, there can be no question as to Bb. The F, plant showed a 9:7 aleurone- color segregation, and therefore, in addition to Rr, it must have been 86 R. A. EMERSON either A a or Cc. An Fs sun red plant with green anthers, R’ R’, had§j 97 colored and 20 colorless seeds, again indicating either A a or Cc. If it was AA BbCcR! R’, both colored and colorless seeds should have given sun red and green plants in a 3:1 ratio; if it was Aa BBCC R'R’,§ the colored seeds should have given sun red and the colorless ones green) plants only, the plant-color ratio again being 3:1; but if it was A a B bff CC R® R’, the colored seeds should have produced sun red and greenf plants in a 3:1 ratio and the colorless seeds green plants only, the ratioff of sun reds to greens in the two lots together being 9:7. Actually the colored seeds resulted in 23 sun red and 10 green plants and the colorlessi seeds in 10 green plants only, the ratio of sun reds to greens being 23: 20,8) thus approaching 9:7. There is, therefore, considerable assurance that the F3; plant was Aa BbCC R’ R’, that the F. plant was Aa BbCC§ R’ 7", and that the F3 numerical relation of plant colors was 36:9:19, as originally suggested by the closeness-of-fit test. A single dilute purple plant of F2, 2960-4, was tested in F; and found to give 38 dilute purple and 39 green plants. Of the eight possible geno- types for F, dilute purples, the only ones that could give only dilutelj purples and greens in F; are A Abb Pl PIUR’?’, Aabb Pl Plr'r’, and AabbPlIPIR’r. The first two should give a 3:1, and the third a 9:7, F3 ratio. The plant had colored aleurone, which throws out of consideration the second genotype with rr. The F; plant-color ratio fits fairly well a 9:7 but not at all a 3:1 expectation, the observed numbers being 38:39 and the calculated numbers 43:34 and 58:19, with deviations] of 5 and 20, and probable errors of 2.6 and 2.9, respectively. The deviation from a 9:7 ratio might occur by chance once in five trials, P equaling 0.20, but that from a 3:1 ratio not more than twice in about a million trials, P equaling 0.000002. The genotype AabbPIPIR’r’ is thereforefl| decidedly favored by these results. The aleurone-color record shows that) this genotype is possible, since there were 57 colored and 56 colorlessi seeds, a relation about halfway between the 9:7 and the 27:37 ratiofj due to AaCCRr and AaC cRr, respectively. Intercrosses of F'2 color types It is realized that the tests of F2 types by studies of their behavior inf later generations as reported above, are markedly inadequate to servell PLantT Coors In Maize 87 as a demonstration of the hypothesis suggested to account for the Fe behavior of the cross of brown, type V, with green, type IVg. It is note- worthy, however, that no results have been found that do not agree with the hypothesis. Fortunately, several intercrosses of the types found in F, afford additional evidence. Purple Ig x green VIc.— Green-anthered purples, A B Pl R’, crossed with greens of type VIc, ab plr’, should give Fy, results identical with those found from the original cross of brown, a B Plr’, with green of type IVg, A b pl R’, since F; in either case should be AaB b PI pl R’7’. Two such crosses are recorded in table 37, group 1 (page 146). The F, plants were both purple, with purple anthers. In F.2 the same eight types were noted as in F» of the cross of brown with green IVg (table 36). The anther color was not recorded, however, for many of the plants, so that only six color classes are shown, as in table 35. While all the expected color types are present, the fit of observed to calculated numbers is so poor that the observed deviations should not occur by chance more than once in thirty trials, P equaling 0.033. The comparison follows: Dilute Dilute Brown Green Total purple sun red laces eta or Tian. Ve Ve clic. al VieenVil Observed.... 80 13 ao 9 20 Zi 158 Calculated... 66 22 We 6 22 25 158 Color types Purple Sun red Difference... +14 —9 —8 +3 —2 +2 0 If, notwithstanding the poor fit shown above, the F; was AaBb Pl pl R’r’, a backcross of F; with green of type VIc, a6 pl7’, should result in the same six major plant-color types, but no green-anthered purples or sun reds should occur. Such crosses are listed in group 2 of table 37. All the purple plants had purple anthers and all the sun red plants had pink anthers. Moreover, the six color classes appeared in so very hearly the expected relation of 1:1:1:1:1:3 that deviations as great as those observed might be expected to occur by chance perhaps ninety-nine times in one hundred trials, ~ equaling 0.85 (when x?=1 and n’=6, P=0.96). The comparison follows: 88 R. A. EMERSON ' Dilute Dilute Color types Purple Sun.red suiaal itn EOE Brown Green la Ila Hla IVa Wp VI Observed: 2.2). 36 29 Bill al ols Ne Calculated 4e = 31 6° 3126") SEG 31.6 23 o ote Difference. ..... +4.4 —2.6 —0.6 —0.6 —0.6 +0.1 If an F, supposedly AaBbFilpl Rr’, be backcrossed to sun red, type IVa, A b plr’, color types Ia, Ila, Illa, and IVa should appear, none of them with green anthers. Such crosses are presented in group 3 of table 37. The anthers thruout were purple or pink, and the several color types appeared in approximately equal numbers, as expected, there being more than two chances in five that the observed deviations may have been due to errors of random sampling, P equaling 0.42. Th comparison follows: : Dilute Dilute ~ Color types Purple Sun red purple sun red Total la Ila Itla IVa @bservied 25 soi eee 1S 97 95 total A418 Calculateden ee 104.5 104.5 104.5 10425 418 Ditherencenr es see +10.5 —7.5 —9.5 +6.5 0 If the same F, genotype, AaBb Pl pl R’r", be backcrossed with green of type [Vg, A b pl R’, there should occur five major color types, brown not appearing, and both green and colored anthers should be found in both the purple and the sun red plants. The records of such a cross are given in group 4 of table 37. The seven expected color types occurred in numbers near enough to expectation so that there are nearly three chances in ten that the deviations may have been due to errors of random sampling, P equaling 0.29. The most pronounced deviations are the excess of dilute sun reds and the deficiency of greens. The comparison follows: Plant color Purple Purple Sunred Sun red Dilute Dilute Green Tota! purple sun red Anther color Purple Green Pink Green Purple Pink Green Ta Ig Ila Ilg Illa IVa IIIg, 1Vg Observede eer were 10 13 U Sie all) 15 13 @alculatedheessee cee 9.5 9.5 9.5 9.5 9.5 9.5 19 Dinerencesi aoe eee ee +0.5 +3. —2.5 —1.5 40.5 +5.5 —6 PLANT Coors In MAIzE 89 In conclusien it seems safe to say that the cross of green-anthered purple, Ig, with green of type VIc, has given results similar to those yielded by the cross of brown, V, with green of type IVg. Since this was to have been expected from the hypothesis suggested by the F. generation of the latter cross, the results just discussed lend support to that hypothesis. Purple Ig x dilute sun red TVa—JIn accordance with the hypothesis under consideration, green-anthered purple is A B Pl R’ and dilute sun red is Abplr’. F, of the cross should be A A Bb Pl pl R’ 7’, and Fe should consist of the five major color types, purple, sun red, dilute purple, dilute sun red, and green of types IIIg and IVg, with both green-anthered and colored-anthered subclasses of purples and sun reds. The I; plants were purple-anthered purples, as expected. Three F: progenies are recorded in table 38, group 1. Anther color could not -be recorded in all cases, but in each of the three F. progenies both green and colored anthers were noted for both purple and sun red plants. In one progeny, 5042-5045, of a total of 57 purples and sun reds, 41 had colored and 16 had green anthers, which is not far from the expected 3:1 relation. The 415 F, plants were so distributed among the five color classes that the chances are nearly three in five that the deviations observed may have been due to errors of random sampling, P equaling 0.58. A comparison of observed and theoretical distributions follows: Dilute Dilute Color types Purple Sun red single Gam veel Green Total Wavgos eel or aia ae Via ers Ilo lic: Misenved ens 8 in ek 243 Gl 59 22 20 415 malewlateds o.45.4)>. 122s 234 78 58 19 26 A415 Minerence. .2...5..-... +9 —7 +1 +3 —6 0 An F, of the cross here considered, 6557-12, A A Bb Pl pl R’ 7’, was backcrossed to a dilute sun red, Ab plr’. Four color types occurred in the progeny, as expected, and all the plants had colored anthers. The deviations from expectation were suchas might occur by chancein consider- ably more than one out of any two such trials, P equaling 0.56. The comparison follows: 90 R. A. EMERSON Al Dilute Dilute Color types Purple Sun re Shile Som res Total la Ila IIla IVa @bserviedit == wae eee 43 43 35 48 169 Caleulated=see. = eee 42 42 42 42 168 Ditterences ee ee eee +] +] —7 +6 +1 Purple Ia x green IVg.— The cross between purple Ia and green IVg should have given results identical with those expected from the cross of green-anthered purple with dilute sun red. Tho parents are supposed to have been A B Pl?’ and A b pl R’, and the Fi, therefore, A A Bb Fl pl R’ 7’. The Fy’s were purple-anthered purples. Two F2 progenies are listed in table 38, group 2. All the expected color types occurred, but the observed frequency distribution was such as might be expected to occur by chance only about once in eleven trials, P equaling 0.09. If these progenies are grouped into five classes, anther color being disregarded, the fit is somewhat better, P equaling 0.16. The comparison of observed and theoretical frequencies follows: Plant color Purple Purple Sunred Sunred zee pee Green Total Anther color Purple Green Pink Green Purple Pink Green Ta Ig Ila Ilg Illa IVa IlIg,1Vg Observed eto) eres or. 26 14 17 3 9 2 1 72 Calculated’ tate ape 31 10 10 3) 10 3 5 72 Differences wasaceeei ee =i) +4 oil 0 1 1 4 0 The F» of this cross exhibited, as expected, practically the same results as were obtained from the cross of green-anthered purple with dilute sun red. Unlike that cross, the one under consideration here was checked by the behavior of some of its F2 types in later generations. A single F, purple-anthered purple produced in F; 16 plants (table 39, group 1), including only purple, sun red, and dilute purple in the relation 9:4:3. Of both the purples and the sun reds, some plants had colored and some had green anthers. Obviously two other types, dilute sun red and green, should occur in such an F; and doubtless would have been found had a larger number of plants been grown, for the F»2 plant, in order to have produced the color types recorded, must have been A A Bb Pl pi R’7’. Puant Coiors IN MaIze 91 nly one plant of each of the missing classes was to have been expected, and the distribution as a whole was not far from expectation, P equaling 0.59. Both the types lacking in F; occurred in Fy, a pink-anthered sun red F; producing sun reds and dilute sun reds, while green-anthered purples produced in one instance purples, sun reds, and greens, and in another instance purples and greens only, all with green anthers. This F; lot may consequently be regarded as A A Bb Pl pl R’7’, and therefore equivalent to the F2 lot from which it came, and its Fs progenies equivalent to F3 progenies. A second F,2 purple-anthered purple was backcrossed to green plants of types [Vg and VIc (group 1, table 39). From the backcross with green of type IVg, A b pl R’, five major color types appeared and both the purple and the sun red types contained subtypes with colored and with green anthers. While all the classes expected from an F»2 of the genotype AABbPlplkR’r’ occurred, the frequency distribution was so far from expectation that there is only one chance in five hundred that the observed deviations may have been due to errors of random sampling, P equaling 0.002. The expected and observed distributions are as follows: Color types Purple Sun red sans ae Green Total lato iia ome la Va) blo aig. MPSeLVeEd=s ees se. U5 15 5 1 9 45 alculateds ee... ee. 9 9 9 9 9 45 Whether the discrepancy is genetically significant or was due to some acci- dent of pollination cannot now be determined. A backcross of the same F»2 plant with green of type VIc, ab plr’, yielded only four color types, as expected (group 1, table 39), the anthers being colored in all cases. The excess of purples and deficiency in two other classes makes the deviations from expectation fairly great, so that there is only about one chance in seven that they may have been due to errors of random sampling, P equaling 0.14. The comparison follows: 92 R. A. EMERSON d Dilute Dilute Color types Purple Sun re purple oneal Total Ia Ila Illa Va @Observiedeares re eee 27 19 15 14 75 Calculated. a, 19 19 19 19 76 Difterencen see ee +8 | Oo - —-4 —5 —] A third purple-anthered purple, an F; plant of the lot regarded as equivalent to F.’s, gave in the next generation purple-anthered purples and pink-anthered sun reds in the relation 31:7 (group 2, table 39). From the genotype A ABB Plplr's’, these two phenotypes should appear in a 3:1 ratio. The deviation from expectation was 2.5 + 1.8, or only such as might be expected about once in three trials, P equaling 0.34. Two green-anthered purples of F2 and two of the equivalent F; lot noted above were tested by a later generation. Two of the four yielded three color types, purple, sun red, and green, all with green anthers (group 3, table 39). Such behavior is expected from the genotype A A Bb Pl pl R’ R’. The 9:3:4 relation is approached so closely that the value of P cannot be determined from Pearson’s tables, 2? equaling 0.36. The comparison follows: Color types Purple Sun red Green Total Ig Ilg IIlg, IVg Observed ree rene 37 aL 14 62 Calculated: s334-) ek 36 12 16 64 Ditierences nso. 4a +1 —] —2 —2 The same two .green-anthered purples were backcrossed with green of type IVg, and one of them and a sib of the other with green of type VIc, with results as shown in group 3 of table 39. The crosses with type IVg, 45 pl R’, gave the same three classes as did the self-pollinations, and the frequency distribution differed from expectation by values that might occur by chance about once in two trials, P equaling 0.49. The comparison follows: PLANT Cotors IN Maize 93 Color types Purple Sunred Green Total Ig Ilg Illg, [Vg BeseIViCd ee eases... el 34 32 53 119 | SLIGUIEW He I Ae ene 30 30 60 120 Difference..........0005- +4 +2 ff —1 |The backcrosses of these green-anthered purples with green of type VIc, ‘ab plr’, as was to be expected, gave very different results. There were ) produced four instead of three phenotypes, all with colored (purple or /pink) instead of green anthers. The deviations from the theoretical /frequency distribution are such as might be expected about once in five trials, P equaling 0.21. The comparison follows: Dilute Dilute | Color types Purple Sun red purple sun red Total | la IIa Illa IVa MDServied oe eer. es... 44 48 33 52 177 | Calculated oi Ace 3h rire 44 44 44 44 176 Difference «6 bik ee 0 +4 ——1] +8 +1] The other two green-anthered purples that were tested yielded only ‘two phenotypes, green-anthered purple and green, in the relation 56:18 ‘(group 4, table 39). The genotype A A.Bb Pl PIR’ R’ should give these two phenotypes in a 3:1 ratio. The deviation from expectation was therefore 0.5 + 2.5. One of the same plants backcrossed to green of type [Vg gave 28 green-anthered purples and 27 greens where equality was expected. . Of the twelve kinds of behavior expected of F2 purples of the cross of purple-anthered purple with green IVg, only four have been demonstrated. So far as they go, however, the results are quite in accord with the hypoth- esis under test. In addition to the F: purples, sun reds and dilute purples also were tested by later generations, as detailed below. Three pink-anthered sun reds gave sun reds and dilute sun reds only, all with pink anthers (table 40, group 1). These three plants are therefore Tegarded as AA Bboplpir'r’. The ratio observed was 97:26. The ‘deviation from the expected 3:1 ratio was 4.75 + 3.24, or such as might 94 R. A. EMERSON occur by chance once in three trials, P equaling 0.32. One of these three — sun reds, when crossed with a dilute purple, Ab Pl7’, gave 71 purples © and 77 dilute purples, all with purple anthers, where equal numbers were expected. . Three other F2 pink-anthered sun reds produced nothing but sun red~ plants in F;, 228 in all (group 2, table 40). Some plants of each progeny — had pink and some had green anthers. Small plantings of each lot were made in the garden and larger plantings in the field. Anther color was noted in the case of the garden plants only. The records show 44 with pink and 16 with green anthers, a deviation from a 3:1 ratio of only 1.0 + 2.3._ The F, sun reds are therefore assumed to have been A A BB pl pl R’7’. One of these F2 plants was backcrossed to green, both of type IVg and | of type VIc, resulting in a total of 108 sun red plants (group 2). Altho | no counts were made for anther color, it was noted that the cross with green IVg, A b pl R’, gave both pink- and green-anthered plants, while the cross with green VIc, ab pl7’, gave pink anthers alone. Only two of the | six possible genotypes of F2 sun reds were demonstrated. | Only one dilute purple F2 plant was tested further (group 3, table 40). | From self-pollination it yielded 46 dilute purple and 9 dilute sun val plants, all with colored (purple or pink) anthers. The deviation from | a 3:1 ratio, 4.75 + 2.17, is such as might be expected by chance about once in seven trials, P equaling 0.14. The same F, plant when back- | crossed to green of types [Vg and VIc (group 3) gave 85 dilute purples and 82 dilute sun reds where equality was expected. Evidently this F, was AAbbPlolr'r’. No F, dilute sun red or green plants were tested further. One F; dilute sun red, however, was found to breed true, producing an F, of 30 pink-anthered dilute sun reds. Likewise, eight F; and Fy, greens gave a total of 126 green plants in the next generation. In so far as tests have been made, therefore, the cross of purple-anthered purple with green IVg has behaved as expected on the basis of the hypo- thetical genotype assigned to Fi, namely, A A Bb Pl pl R%7’. Purple Ig x green IVg.— Green-anthered purples are assumed to be A B PIR’, and green IVg to be A b pl R’. ~The F, genotype is therefore, theoretically, A A Bb Pl pl R’ R’, and F, should consist of the three color types purple, sun red, and green, all with green anthers. Eight such F; progenies are recorded in table 41, group 1. The three types PLant Coxtors In Maize 95 occurred in so nearly the expected relation of 9:3:4 that the observed deviations might be expected by chance considerably more than once in three trials, P equaling 0.37. The comparison follows: Color types Purple Sun red Green Total Ig Ilg IIlg, [Vg _lugeinealss = G8 See 293 105 150 548 Brlemittedan 22... 6... ss 308 103 137 548 _ JIGS CO eal a a5 +2 +13 0 The F: greens of this cross are assumed to consist of the genotypes Ab PIR’ and Ab pl R’, which, if r’ had been present instead of R’, would have been dilute purples and dilute sun reds, respectively. In substantiation of this assumption, crosses of F,’s, all green-anthered purples, with dilute sun red, A 6 pl7r’, and with green VIc, ab plr’, are recorded in group 2 of table 41. As expected, the result was the four classes purple, sun red, dilute purple, and dilute sun red, all with colored anthers. The expected numerical equality of the four classes was so closely approached that deviations such as those observed might be expected by chance in nearly three out of four trials, P equaling 0.74. The comparison follows: - Dilute Dilute Total Color types ‘Purple Sun red aime. gun a la Ila Illa IVa | OSSIRVGS bo eee 58 61 62 70 251 | LALGUOIB TEC lc ek ek eer 63 63 63 63 252 Minicrences. se. 26s. se. —) —2 —l +7 —l Still another F; was crossed with a pink-anthered sun red, A B pl7’, and gave 68 purples and 67 sun reds, all with colored anthers, where equal numbers were expected. So far as tested, therefore, the cross of green-anthered purple with green [Vg has given the results expected on the basis of the hypothesis under test. Purple Ig x brown V.— A cross of green-anthered purple, A B Pl FR’, with brown, a B Pl 7’, gave in F, 49 purple-anthered purples, presumably 4 96 R. A. Emerson AaBBPIPIR‘%r’. An F2 progeny was grown from only one F, plant, 6653-6, resulting in two major color types, purple and brown, in approxi- mately a 3:1 ratio. The purples were, as expected, of two subtypes, one with purple and the other with green anthers. The theoretical rela- tion of 9:3:4 was realized so closely that the observed deviations might be expected by chance in at least two out of three trials, z* equaling 0.76 (when z2=1 and n’=3, P=0.61). The comparison follows: Purple, Purple, Colors hypess purple anthers green anthers =aonas stot la Ig V Observed? 0222 238 23 5 9 37 Calculated ae"2 see 21 2 9 37 Ditterence 4 centers +2 — 0 | 0 A second F, plant, 6653-2, was backcrossed with green IVg, A b pl R’, resulting in 39 purple plants, 21 with purple and 18 with green anthers, where equal numbers were expected, the deviation from expectation being } 1.5+2.1. The same F,; plant was crossed with a heterozygous dilute } sun red, Aabbplpl7r’7’, resulting in 45 purple-anthered purples and 18 browns, the deviation from the expected 3:1 ratio being 2.25 + 2.32. Purple Ig x dilute purple IlIa.— Crosses of green-anthered purple, AB PIR’, with dilute purple, Ab Plr’, gave in F; purple-anthered } purple, AA Bb PI PIR’r’. The F, should consist of purple-anthered } and green-anthered purples, dilute purples, and greens, the three major color types appearing in the relation 12:3:1. In F, from a single Fy plant, 5263-8, both purple-anthered and green-anthered purples were noted, but detailed counts based on anther color were not made. The deviations from the expected numbers for the three major types were such as might occur by chance in nine out of twenty such trials, P equaling 0.45. The comparison follows: Dilute Color types Purple sails Green Total Ta, g Illa Illg Observed :4. eee sr. sree 36 11 5 52 | Calculated 22. ae andeee ce 39 10 3 52 Differences<¢ pha ee —3 +1 +2 0} Puant Couors IN MAIZE 97 A second F, plant backcrossed with green IVg, A b pl R’, gave the expected four types. The deviations from the equal frequency expected for the several types was such as might occur by chance somewhat more than once in four trials, P equaling 0.27. The comparison follows: _ Color Purple, Purple, Dilute types purple anthers green anthers purple Green Total Ta Ig Illa IllIg | Observed... 59 67 80 7a 283 Calculated. . 71 71 7 71 284 Difference.. —12 yh “+49 +6 1 Dilute purple IIIa x green IVg.—A single cross of dilute purple, AbPlir’, with green IVg, Ab pl R’, gave dilute purple, A A 6b Pl pl R’7’, in F;, and three phenotypes, dilute purple, dilute sun red, and green, in F. (table 42, group 1, page 150). ‘The observed frequencies were 23:8: 10, which is the nearest possible approach to the expected 9:3:4 relation for ‘a total of 41 individuals. One F, dilute purple gave similar results in Fs, ‘indicating the same genotype as the F; dilute purples. The F; progenies ‘of this F3; lot may be regarded as equivalent to F3’s, and are therefore ‘grouped with the F; in table 43. Three F; and Fs progenies (table 43, group 1A) approached the 9:3:4 relation so closely that the observed deviations might occur by chance in nearly three out of five trials, P equaling 0.59. The comparison follows: Dilute Dilute Color types anal a oe Green — Total IIIa IVa IIIg, 1Vg . _ OSSIAVGG| Se Rese 143 48 We 264. eM wmlarede ete. hn is. ee 149 50 66 265 OMMETEMCOM mah 6 6.5/50086 6 5 as —6 —2 +7 —1 Cc The green plants of these F; and F, lots, as well as those of the Ws lot listed in group 1 of table 42, are assumed to be Ab PIR’ and A bplR’, and consequently to differ from the dilute purples and dilute sun reds only in having R’ R’ in place of R’r’ or rr’. That the Rr pair is thus con- cerned in these results can be shown by a comparison between the plant- 98 R. A. EMERSON color phenotypes resulting from seeds with colored aleurone and those | from seeds with colorless aleurone. The F2 progeny came from a plant | that produced from self-pollination colored and colorless seeds in the | relation 60:24. This close approach to a 3:1 ratio indicates that the. F, plant could have been heterozygous for only one of the aleurone-factor | pairs A a, Cc, or Rr (Emerson, 1918). A cross with a C tester, Ac R, | resulted in 43 colored and no colorless seeds, while a cross with an R tester, A Cr, gave 46 colored and 32 colorless seeds, thus indicating Rr as the factor pair concerned. The colorless seeds must therefore have been rr, presumably 7’ r’, and in accordance with the hypothesis under test should have produced no green plants. Some of the colored seeds, on the contrary, should have been RR, supposedly Rk’ R’, and these should have given green plants. For the most part, the colored and the colorless seeds were planted separately. The 9:3:4 relation of the three plant-color types is theoretically made up of a 6:2:4 relation from colored seeds and a 3:1:0 relation from colorless seeds. Actually, from colorless seeds there appeared dilute purple and dilute sun red plants in the ratio 69:15. The deviation from expectation, 6.0 + 2.7, might be expected to occur about once in seven trials, P equaling 0.14. From colored seeds the deviation from the theoretical distribution was such as might occur | thru errors of random sampling almost once in four trials, P equaling | 0.23. The comparison follows: | Dilute Dilute Color types Green Total | purple sun red Illa IVa Illg, IVg Observedic se se eee 92 42 70 204 @alculatedterc Geren one ee 102 34 68 204 Ditkerence: =a es oe ee —10 +8 +2 0 Aleurone is in some cases self-colored and in some cases mottled. Mottled aleurone ordinarily occurs only when the R factor is heterozygous, but not all heterozygous individuals are mottled (Emerson, 1918). Mottled seeds of the cross under discussion, just as colorless ones, since they are presumably R’7", should produce no green plants. In the case of some of the progenies noted above, the colored seeds were sorted into self-colored, mottled, and colorless. Since usually about one-third | PLuant Coors IN Maize 99 of the colored seeds are mottled, the 9:3:4 relation of plant-color types observed in this cross should be made up of a 3:1:0 relation from color- less seeds, 3:1:0 from mottled seeds, and 3:1:4 from self-colored seeds. Of the progenies for which the seeds were sorted in this way, the color- less seeds produced dilute purple and dilute sun red plants in the relation 60:14, with a deviation from 3:1 of 4.5 + 2.5, the mottled seeds gave the same plant-color types in the relation 30:12, with a deviation of 1.5 + 1.9, and the self-colored seeds yielded dilute purple, dilute sun red, and green in the relation 48:19:64 (the theoretical distribution for a total of 131 ‘Individuals is 49:16:66), the deviations being such as might occur by chance perhaps three times in four trials, x? equaling 0.64. On the whole, | therefore, these crosses, and particularly the interrelations of aleurone and plant colors, afford strong evidence in support of the hypothesis under test. Before presenting further F; results from these crosses, it may be well to consider other crosses of dilute purple with green IVg which, so far as plant color alone is concerned, have given results quite like those pre- sented above but which exhibit a wholly different relation between plant color and aleurone color. The green plants concerned in these other crosses were C testers for aleurone color (Emerson, 1918), and were there- ‘fore known to be Ac R, presumably Ack’. The dilute purple plants concerned were homozygous for aleurone color, and were consequently ACR, presumably Ach’. These crosses differ, then, from the ones discussed above in having R’ in place of r” and c in place of C. Since the Cc pair is supposed not to have any relation to plant color, the results for plant color should be quite like those for the other cross and there should be no relation between plant color and aleurone color. The results for F, are presented in table 42, group 2, and the F; results in table 43, group 1B. The three plant-color types appeared in F2 in the relation 328:113:148, and in F; in the relation 40:14:23. Considered together these lots deviated very slightly from expectation, x? equaling 0.31. The comparison follows: Dilute Dilute Color types purple sun red Cuces ees Ia Iva Illg, IVg Ste Le re eee 368 127 171 666 Calculated...... CSR aoe 375 125 166 666 PIETCMC Cr cc. se ee « * =i ag pat 0 100 R. A. EMERSON The seeds from which these plants were grown consisted of colored and | colorless in approximately a 3:1 ratio, as is expected when the C factor alone is heterozygous. The deviations from the expected 9:3:4 relation for plants from colored seeds was such as might occur by chance more than | once in three trials, P equaling 0.36, and for plants from colorless seeds such as might occur once in six trials, P equaling 0.17. The comparisons follow: Dilute Dilute Plant-color types Green Total purple sun red Ila IVa IIIg, 1Vg Colored seeds: @bsenviedsercta ae oer oe 215 58 89 362 @alculatediee see ae se 204. 68 90 362 Differences= ee +11 —10 —l 0 Colorless seeds: Observed et. eee 65 Eo 304 129 Calculated 73 24 32 129 IDitterenceas eee —§ +8 0 0 The results presented for plant color alone and in relation to aleurone color in these crosses are therefore quite in keeping with the hypothetical constitution assigned to the F, plants, namely, A AbbPIplR’R’Ce, just as the results from the other crosses were in keeping with the assumed genotype AAbbPI pl R’r’CC for their F, plants. A single F, plant was backcrossed with green IVg, A b pl R’, with results as shown in table 42, group 3. The three color types dilute purple, dilute sun red, and green, occurred in the relation 46:45:86. The expected distribution for a total of 177 individuals is 44:44:89, showing almost a perfect fit, x? equaling 0.21. For both the lots of crosses under discussion, further tests are afforded by the behavior in F; and Fy. As already shown, some of the F2 dilute purples had the same genetic constitution as the Fy plants (table 43, groups 1A and 1B). The progenies of two other dilute purples, one of F, and the other of an equivalent F3;, produced dilute purple and dilute sun red plants only (group 2, table 43), in the relation 82:23. The devia- PLANT Coors In Maize 101 tion from a 3:1 ratio is 3.25 + 2.99. From their behavior and in view of the crosses in which they occurred, one of these plants is assumed to have been A Abb Plplr'r’ and the other A Abb PlplR R’. A single dilute purple of an F; lot equivalent to an F2 gave dilute purple and green plants only (group 3, table 43). The two color types appeared in the ratio 62:16, a deviation from 3:1 of 3.5 + 2.6. The F; plant is therefore assumed to have been AAbUb PI PIR’r’. Colorless and mottled seeds produced dilute purple plants only, as was expected. From self-colored seeds there resulted dilute purple and green plants in the relation 26:16, a deviation of 2.0 + 2.0 from the expected 2:1 ratio. Two dilute sun red plants gave progenies of dilute sun reds and greens in the relation 63:22, a deviation from a 3:1 ratio of 0.75 + 2.69 (group 4, table 43). Presumably these plants were AAbbplplkR’r’ and AAbbpl pl R’R*. Four other dilute sun red plants bred true in the next generation (group 5, table 43), producing a total of 197 dilute sun red plants. These plants are therefore assigned the genotype A A bb pl pb’ x. Seven green plants likewise bred true (group 6, table 43), producing a total of 130 green plants. These plants were presumably A 6 pl R’ and AND UPL Res To summarize, all types of behavior were observed in F; and equivalent F, generations of the cross of dilute purple with green IVg except true- breeding dilute purples. Only eight dilute purples were tested, and only one in nine is expected to breed true. Sun red ITg and IIa and dilute sun red IVa x green IIIg and IVg.— Two crosses of green-anthered sun red with green IVg gave green-anthered sun red plants in F;, theoretically A ABbpl pl R’ R’. The parent types only appeared in F2 (table 44, group 1). The observed numbers of green-anthered sun reds and greens were, respectively, 216 and 77. The deviation from the expected 3:1 ratio was 3.7 5+ 5.00. A cross of pink-anthered sun red with green IVg gave pink-anthered sun red in Fj, theoretically A A Bbplpl R’r’. Fy; plants backcrossed with green IVg, A 6 pl R’, gave three major plant-color types (group 2, table 44) — sun red, dilute sun red, and green — with the sun reds appear- ing in two subtypes, one pink-anthered and the other green-anthered. Theoretically the four types should have been represented by an equal number of individuals. The deviations from this expectation were such 102 R. A. EMERSON that there is considerably more than an even chance that they might have been due to errors of random sampling, P equaling 0.56. The comparison follows: Sun red, Sun red, Dilute Color types pink anthers green anthers sun red Green Total Ila Ilg IVa IVg Observedtsees-< 105 90 105 109 409 Calculated. s9-2 102 102 102 102 408 Difference........ +3 Sea : +3 aL ae Crosses of dilute sun red with green IVg gave 54 dilute sun red plants in F;, AA bbpl pl R’7’. In Fe (group 3, table 44) there resulted from a self-pollinated F;, dilute sun red and green plants in the relation 55:22, a deviation from the expected 3:1 ratio of 2.75 + 2.56. An F, back- crossed with green IVg gave the same two color types in equal numbers, 30 each, exactly as expected. Numerous other crosses of this sort have been observed in connection with studies of the interrelations of aleurone- color and plant-color factors. Since these data are to be presented in a later paper and since they are wholly in accord with the data given in group 3 of table 44, they are not discussed here. In an earlier section of this paper dealing with the factor pairs A a, Bb, and Pl pl only (page 29), it was shown that the green plants there noted are of three kinds, namely, ab pl, a B pl, and ab Pl. Thruout the present section of the paper, which deals with the relation of the multiple-allelomorph series containing R’, 7’, R’, r’, it has been assumed that plants which in the presence of 7” or R’ are dilute purple or dilute sun red, are green in the presence of homozygous R’. The data presented are wholly in accord with this interpretation, thereby giving considerable assurance of the probable correctness of the hypothesis. The reported interrelations of plant color and aleurone color when the latter was known to involve the Rr pair, have still further strengthened this assurance. It remains now to present even more direct evidence, namely, that obtained from crosses of green plants encountered in this study, with sun red and dilute sun red plants. These green plants are assumed to be A b Pl R’, type IIIg, and A b pl R’, type IVg. Certain F; and F, progenies consisting of green-anthered purples and greens in a 3:1 relation are listed in table 39, group 4. These green plants PLant Cotors IN MaAIzE 103 were all, presumably, Ab PIR’. Green plants of a later generation, grown from these greens, when crossed with sun red plants, type Ila, gave 64 purple-anthered purples and no other types (table 45, group 1). Another green crossed with dilute sun red resulted in 4 dilute purples. Obviously the same results would have been obtained had the green plants used in these crosses been a b Pl 7’, instead of A b Pl R’ as they are sup- posed to have been. As a matter of fact, however, one of these green plants had homozygous colored aleurone, and therefore must have been ACR. The other two greens, while they had colorless aleurone, came from lots known, from their 3:1 aleurone-color ratios and from crosses with aleurone testers, to be heterozygous for C alone, and therefore A c R. Moreover, the green plants from lots consisting of purples and greens in a 3:1 relation could not have been aa, for the parents of such lots, if hetero- zygous for A, must have produced purples and browns rather than purples and greens. The green plants could therefore have been nothing other than A b Pl R’. Similarly, progenies consisting of green-anthered purples and sun reds, and greens, in a 9:3:4 relation, are listed in table 39, group 3. Green plants of these lots and their green descendants might be either A b Pl R? or A b pl R’, or might be heterozygous for Pl. Six such green plants were crossed with dilute sun reds (table 45, group 2). None of these greens could have been of the types discussed in the earlier section of this paper, namely, ab Plr’ and the like, for they were shown by appropriate tests (Emerson, 1918) to be A c¢R and some of them have even been used as C testers for aleurone color. Two of these green plants crossed with dilute sun reds gave dilute sun reds only, 59 in all, and are consequently regarded as being A b pl R’. Two others by similar crosses gave dilute purples and dilute sun reds in the relation 20:30, a deviation of 5.0 +2.4 from the expected equality from plants of the genotype A Abb Plpl R’ R’. Two other greens were crossed with heterozygous dilute sun reds, AAbbpl pl R’7’, and gave dilute purples, dilute sun reds, and greens in the relation 69:54:106. The theoretical distribution among these three classes for a total of 229 individuals, based on the assumption that the green parent plants were A Abb Pl pl R’ R’, is 57:57:115, a devia- tion that might occur by chance about. once in five trials, P equaling 0.19. Progenies consisting of dilute purples, dilute sun reds, and greens in a 9:3:4 relation are listed in table 43, group 1A. Descendants of one of 104 R. A. EMERSON these green plants were crossed with dilute sun reds which were F,’s of crosses between dilute sun red and green IVg. The results were dilute purple and green plants in the relation 328:338 (table 45, group 3), a deviation from a 1:1 ratio of 5.0 + 8.7. Since the heterozygous dilute sun red plants were A A 6 b pl pl R’ 7", the green plants crossed with them are assumed to have been Ab PIR’. That this assumption is correct appears the more evident from the fact that the green plants were homo- zygous for colored aleurone, and hence A C R. Green IVg x green VIc.— Twelve crosses between green plants of type IVg and green plants of type VIc gave a total of 159 F, plants, all dilute sun red. With respect to aleurone color, all the type IVg plants concerned in these crosses were known to be AcR, and, in fact, were in general use as C testers for aleurone color. With respect to plant color, therefore, they are assigned the constitution Ab pl R’. Of the type VIc greens, four were known to be A testers for aleurone color, and were therefore, with respect to aleurone color, aC R. Their plant-color constitution is accordingly set down as ab pl R’. Six of the type VIe greens had an aleurone-color constitution of aC yr, their plant-color genotype being accordingly ab plr’. The other two VIc greens were certainly a b pl, but whether they were R’ or r” is unknown. In F,, dilute sun red and green plants were present in the ratio 420: 291 (table 46, group 1, page 154). From an F; of the genotype A ab b pl pl plus: R’ r" or R’ Rk’, a 9:7 ratio of dilute sun red to green is to be expected in F2, since both A and 7’ or R" are assumed to be necessary for the pro- duction of anthocyanic pigment, which distinguishes dilute sun red from green. The theoretical ratio for a total of 711 individuals is 400:311. The observed deviation from this ratio, 20.0 + 8.9, is such as might occur by chance about once in eight trials, P equaling 0.18. Two F, plants backcrossed to green VIc, ab pl R’, gave 66 dilute sun red and 58 green plants, and two backcrosses with green IVg, A b pl’, gave 96 dilute sun reds and 96 greens, equality of the two classes being expected in the case of both crosses (group 2, table 46). That the two parent types of green occurred in F», is shown by their relations to aleurone and pericarp color. In the case of every cross, green plants were produced from both colored and colorless seeds. Those from colored seeds could have been only Ab pl R’. Since some seeds were colorless because of a a and some because of cc, both parent types of green should have been present in the lots grown from colorless seeds. PLANT CoLors IN MaIzE 105 In one cross there was present the pericarp factor P, which with A gives a red and with aa a brown pericarp. All the F2 green plants from colored seeds had red pericarp, and of those from colorless seeds the majority had brown pericarp. From the colorless seeds there should have occurred also a combination type of green, ab pl R’, but no tests were made for the identification of this type. _ Ten dilute sun reds of F; were tested by their F; behavior. Three of these (table 47, group 1) gave dilute sun red and green plants in the relation 108:77, a deviation from a 9:7 ratio of 4.0 + 4.6. Five other F, plants (group 2) gave the two color types in the relation 187: 66, a devia- tion from a 3:1 ratio of 3.0 + 4.6. Two F.’s (group 3) bred true dilute sun red, producing 78 dilute sun red and no green offspring. Theoretically, of 9 F». dilute sun reds, there should occur in F3, true-breeding, 3:1, and 9:7 progenies in the numerical relation 1:4:4. The observed rela- tion between these three sorts of behavior for the ten F».’s tested was 2:5:3. Deviations such as these might occur by chance about once in two trials, P equaling 0.49. Green IVg x green VIa.— Certain crosses of green IVg with green VI have given sun red plants in F;. The type VI greens belonged to families in which the B factor was known to be present. They were therefore doubtless a B pl plus 7’ or R’, and the Fy’s were probably AaB b pl pl plus r’ R’ or R’ R’. — F 2 consisted of the three major color types sun red, dilute sun red, and green (table’48, group 1) in the relation 586: 161:348. Obviously this is not a 9:3:4 relation, for the deviations from such expecta- tion, —30, -44,+74, could not be expected to occur thru errors of random sampling once in a million such trials, ~° equaling 30.9 and P equaling .000000+. As a matter of fact, an F, of the genotype suggested above should give in F, the three color types observed in the relation 36:9: 19. The observed frequencies of the several classes fit this expectation so closely that the deviations from it might occur by chance in about one out of five trials, P equaling 0.19. The comparison of observed and expected frequencies follows: Color types Sun red pune Green Total Ila, g Va TVg, Via; c ESET 727g ia ig 586 eli 348 1,095 eoriea: a 616 154 325 1,095 WREETENCE. 2... oe coc solos —30 +7 +23 0 106 R. A. Emerson z Not only were the frequencies of the major color types fairly close to expectation, as indicated above, but the expected subclasses of sun red with | pink anthers and with green anthers were observed. Counts of anther. color were made in the case of only 65 individuals. These plants were distributed to the four color classes, pink-anthered sun red, green-anthered sun red, dilute sun red, and green, in the order 24:9:10:22. The theoretical distribution of 64 individuals being 27:9:9:19, the deviations are such as might occur by chance perhaps twice in three trials, x? equaling | 0:91 (when: x2 =1-and n’=3)P'= 0:61): Only three F. sun reds were tested in F;. One of them (group 2, table 48) ‘ bred true sun red, but segregated with respect to anther color. It was there- | fore presumably A A BB pl plr’ R’. Two other F2 sun reds (group 3) | gave sun red and green offspring in the ratio 229:71, a deviation of only | 4.0 + 5.1 from a 3:1 ratio. One of these two F, plants was crossed with a dilute sun red, resulting in 55 sun red plants. The two F, plants, | therefore, were presumably AaB B pl pl. Anther color was not deter- | mined, but the fact that the green plants of F; all came from colorless | seeds is conclusive evidence for the presence of A a and against the pres- © t ence of r’ R’. The genotype of the Fy. plants is accordingly set down as AaBB pl plr' rr’. Green III g x green VIc.— Green plants known to be of type VIe, a 6 pl 7’, were crossed with greens which were known to be R’ R% and which from | their parentage might have had Pl. The result in F; was dilute purple, - supposedly Aabb Pl plr’ R’. Two F-» lots (table 49, group 1) consisted of dilute purples, dilute sun reds, and greens in the relation 109:37:135. From the assumed genotype of F;, there should occur in F2 the observed color types in the relation 27:9:28. The observed frequencies deviated from the theoretical ones by amounts such as might occur by chance | once in three trials. P equaling 0.33. The comparison follows: Color types Se ae Green Total — Illa IVa ile TVesWaloge Observed 222.038 ce W 109 37 135 281 Calculated.7. 4a sa 119 40) 123 282 Difference... Goce —10 —3 +12 —1] — PLANT Coors In Maize 107 _ The dilute purples of F, were presumably all A b Pl7r’ and the dilute sun reds all A bplr’. Of the F, greens there should theoretically have been six types, namely, AbDPIR’, Abpl R’, abPlr’, abplr’, abPl R’, and abplR’. The relation of these plant colors to aleurone color and to a pericarp color known as cherry, present in these families, affords an opportunity of checking some of these hypothetical formulae. Cherry pericarp is a bright reddish purple, somewhat variable in intensity. In the parent of one of these F2 progenies it was sufficiently light to make possible the determination of the underlying aleurone color. The F2 seeds consisted of colored and colorless aleurone in the ratio 140:171, a devia- tion from a 27:37 ratio of 9.0 + 5.9, or such a deviation as might occur by chance three times in ten trials, P equaling 0.30. The F; plants were known to be Aaf#r, and in order to give a 27:37 ratio with respect to aleurone color they must have been also Cc. Cherry pericarp is of such a nature that it never develops except in the presence of Pl. With A and Pl it is cherry, but with a and Plit is brownish. It had been regarded by the writer as due to a factor, Ch, but recently Dr. E. G. Anderson has shown (by unpublished data) that the writer’s Ch is apparently another allelomorph of R, and at present it is known to exist only in the form r™. Since all dilute purples of the lots under consideration here are assumed to be A b Pl r®, they should all have cherry pericarp. Again, since dilute sun reds are pl pl, they should all have colorless pericarp. Furthermore, since all green plants from colored seeds are supposed to be R! R%, their pericarp should likewise be colorless. Finally, since the colorless seeds may lack color because of either aa, rr, or cc alone, or because of both aa and rr, some green plants from colorless seeds should have color- less pericarp, a R’ or Ack’, and some should have brownish pericarp, aPlr”. Of course all green plants with pl pl also must have colorless pericarp. The observed results are wholly in accord with these suppositions. In one I’, progeny, pericarp color was determined for all except a few plants. From seeds with colored aleurone, all the dilute purples had cherry peri- carp and all the dilute sun reds and greens had colorless pericarp. These three classes of plant and pericarp color showed frequencies deviating from the theoretical 27:9:18 relation by quantities such as might occur by chance almost once in four trials, P equaling 0.23. From seeds with colorless aleurone, all dilute purples had cherry pericarp, all dilute sun —S— 108 R. A. EMERSON reds had colorless pericarp, and greens had in part brownish and in part ) colorless pericarp. The deviations from the expected 27:9:18:20 relation | of these four color classes were such as might occur thru errors of random sampling in more than seven out of any ten such trials, P equaling 0.72. The comparisons follow: Dilute Dilute Plant color Green Green Total purple — sun red Pericarp color Cherry Colorless Brownish Colorless Illa IVa Vib_—_—siII lg, IVg, Vic r Colored aleurone: : Observed... . 43 10 0 39 88 | Calculated. . . 44 15 0 297 88 |, Difference. . . il ==) 0 +6 0 | Colorless aleurone: . Observed.... 38 11 32 28 109 | Calculated... 40 els 27 29 109 Difference. . . =h yy, +5 ==. 0 Further tests of the factorial composition, with respect to Pl, of some F, green plants of this cross are afforded by crosses between them and sun red and dilute sun red plants. One F2 green crossed with sun red gave 27 purple plants (table 49, group 2). Since the green parent plant came from a colored seed, it is assumed to have been Pl Pl R?’ R?’ plus A A or Aa. Two other greens crossed with dilute sun red gave 39 dilute purple plants, and were therefore Pl Pl (group 2, table 49). Since one of these green plants had brownish and the other had colorless pericarp, they are assumed to have been also 7 and R! R’, respectively. A fourth Fs green crossed with sun red gave purple and sun red plants, and a fifth green crossed with dilute sun red gave dilute purple and dilute sun red plants, indicating Pl pl (group 3, table 49). The first of these two had brownish and the second had colorless pericarp. They must therefore have been r™ and R? R’, respectively. A sixth F, green crossed with dilute sun red gave only dilute sun red plants, and so must have been pl pl (group 4). PLANT Coors IN MAIzE 109 Green IIIg x green VIa.— In the sections immediately preceding this, it has been shown that intercrosses of greens may give dilute sun reds (page 104), dilute purples (page 106), or sun reds (page 105) in Fy, the particular color type depending on the genotypes of the greens chosen for crossing. It remains to be shown that purple Ia can be produced by intercrosses of greens. A cross of green Vla, aBplr’, with green IIIg, Ab PIR’, should give this result, F; beng AaBb Pl pl R’r’. Such a cross has been made, with results as expected. A stock of green plants was isolated from a cross of brown V, a B Pl7’, with green VIc, ab plr’, and was shown, by crosses with aleurone testers and with dilute sun red IVa, to be type VIa, a B pl7’. Another lot of greens arose from a cross of purple Ig with green IVg. The purple Ig parent was from a lot consisting of purple Ia, purple Ig, dilute purple Illa, and green IIIg, coming from a cross of purple Ig with dilute purple Illa heterozygous for R’r’. It was therefore AABb Pl PIR’ R’. The green IVg plant with which it was crossed was known to be A b pl R’. The F; of this cross consisted, as was expected, of purples and greens only. The purples were type Ig and must have been heterozygous for B 6b and Pl pl, and the greens must have been type IIIg and heterozygous for Pl pl, or AAbb Pl pl R’ R’. Two of these F; greens were crossed with one of the greens of type VIa mentioned above. The two crosses, 9659 and 9660, resulted as expected in purple-anthered purples, type Ia, and pink-anthered sun reds, type Ila, in the relation 18:20. It has been demonstrated, therefore, that by crossing wholly green plants of appro- priate genotypes it 1s possible to produce purple-anthered purples, the most highly colored type known, a type that is dominant to all other types. Green IITg x purple Ia.— A green plant with homozygous purple aleurone and belonging to a family (table 39, group 4) consisting of green-anthered purples and greens only, and therefore theoretically being A 6 Pl R’, was crossed with a purple-anthered purple, A B Plr’. A purple-anthered purple F,, A A Bb Pl Pl7’ R’, 5350-9, was backcrossed with green IVg of the genotype A 6 plr’, with the result that in the next generation there appeared four color types, purple-anthered purple, green-anthered purple, dilute purple, and green, in the relation 28:22:21:29. The deviations from the expected equal distribution of the 100 individuals were such as might occur by chance in considerably more than half of 110 R. A. Emerson such trials, P equaling 0.57. It will be recalled that results like these were obtained from a cross of green-anthered purple with dilute purple (page 96), and of course the same results were to be expected since the F; in both cases is supposed to have been A A Bb Pl Plr’ R’. The cross now under consideration has interest from the standpoint of the relation of aleurone color to plant color, and also for certain linkage relations. The F,; was known to be, with respect to aleurone color, AARr. Whether it was CC or Cc was not known, since a strong red pericarp made aleurone counts impracticable. The green plant on which the F, was backcrossed, was determined by appropriate tests to be C C, so that the relation of the F; purple to C is immaterial. The backcross resulted in approximately equal numbers of seeds with and without aleurone color, there being 109 colored and 110 colorless seeds. The colorless seeds must have been ABC Plr'r’? and AbC Plr'r’, and should therefore have produced purple-anthered purples and dilute purples only; while the colored seeds must have been A BC PI R’r’ and AbC PIR’ r’, and should correspondingly have produced green-anthered ~ purples and greens only. The results were quite in accord with expecta- tion, as is shown in the following comparison: Purple, Purple, Dilute Color types purple aie ereen Tes purple Green Total Ta Ig MMOGs LUE be Colored seeds. .... 0 22 0 29° 51 Colorless seeds... . 28 0 Dilt 0 49 It has been shown earlier in this paper (page 63) that a linkage exists between the factor pair B b and a factor pair, Lg lg, for normal or ligule- less leaf, the percentage of crossing-over being about 30. It happens that the F, of this cross was Lg lg as well as Bb, B lg having come from one parent and b Lg from the other, and that the green plant used in the backcross was blg. There is no question here that the purple-anthered purples and dilute purples produced from colorless seeds differed with respect to the 6b pair only. Their linkage with liguleless leaf, as indi- cated by the percentage of crossing-over, was 29.4, or a deviation from 30 of 0.6 + 2.0. Practically the same linkage relation was found for the plants from colored seeds, green-anthered purples and greens. In this case the percentage of crossing-over was 27.5, a deviation from 30 of PLANT Cotors In Maize 111 2.5 + 2.1, or such as might occur by chance about twice in five trials, -P equaling 0.42. It is to be assumed, therefore, that the same difference exists between green-anthered purples and greens as between purple- anthered purples and dilute purples, namely, a difference with respect to the factor pair Bb. This in turn is merely additional evidence that plants which in the presence of 7” are dilute purples, A b Pl, appear as greens in the presence of R’ r’, which is the hypothesis under test thruout this section of the paper. Purple Ia x green-anthered dilute sun red A purple-anthered purple, known from appropriate aleurone-color tests to be R R and hence A B PI R’, was crossed with a dilute sun red which differed from most dilute sun reds in showing much less anthocyanic | pigment, particularly in early stages of growth, than is usual in plants of | that type, and in having little, if any, color in its anthers. The Fy’s, | 2975, were purple-anthered purples. 2 was expected to show the four _ color types, purple, sun red, dilute purple, and dilute sun red, commonly found in crosses of purple Ia with dilute sun red IVa. As a matter of fact, the single F2 progeny grown was found to consist of these four color types as major classes, but each class was found to have colored-anthered (purple or pink) and green-anthered subclasses. The difference between the two subclasses for purple and sun red was sharp, just as is the case in crosses of purple Ia with green IVg, but it was often difficult. to separate green-anthered dilute purples from green-anthered dilute sun _ reds. _ Ordinarily, anther color (purple or pink) is the surest means of distinguish- ing between dilute purple and dilute sun red. When both have green anthers the separation must be based on the relative amount of pigment in other plant parts — a difference that is usually not very marked until late in the life of the plants, when dilute purples usually show materially -more pigment, especially in parts not exposed to the sun, than do dilute sun reds. It will be recalled that in crosses of purple Ia with green IVg, both colored and green-anthered purples and sun reds appear, but that all the dilute purples and dilute sun reds have colored anthers, the green- anthered individuals appearing as wholly green in all plant parts except perhaps the pericarp. But in the cross here considered, no wholly green plants were found. 1h R. A. EMERSON The natural supposition is that there is here concerned still another form of the R factor, such that, while it does not allow color to develop in the anthers, does nevertheless result in the development of some antho- cyanic pigment in other parts of the plant. The dilute sun red plant used as one parent of this cross was found to be A ¢ R with respect to aleurone. The factor particularly concerned in the behavior here reported is there- fore assigned the designation R”. The F, plants are accordingly assumed to have been A A Bb PI plR' R”®. The frequency distribution for the eight color types observed in F2 approached the theoretical distribution so closely that deviations of the magnitude observed might occur by chance nearly three times in any ten such trials, P equaling 0.72. The com- parison follows: Plant color Purple Purple Sunred Sunred eae ee Pe ae Total Anther color Purple Green Pink Green Purple Green Pink Green Observed... 212 77 66 22, 66 23 22 3 491 Calculated. 207 69 69 23 69 23 23 8 491 Difference... +5 +8 —3 —l] —3 0 —] —5 0 One F2, a green-anthered purple, was tested in F;. This pliant bred true, producing 128 green-anthered purples and no other types. It is unfortunate that the relation of aleurone color to plant color in this cross afforded no check on the assumption that the observed behavior with respect to anther color of dilute purples and reds was due to a factor belonging to the allelomorphic series R’, R%, 7’, 7°. - True, the Fy plant tested was heterozygous with respect to aleurone color, but this was known to be due to Cc. Since no further tests have been made, the only evidence in support of the assumption of a factor R” is the very close fit of observed with theoretical frequency distributions, the fact that colored and green anthers in purple and sun red types of many other crosses have been found to be due to the R factor, and the demonstrated presence of R in the green-anthered sun red plant used in the cross. Summary of results involving the allelomorphic series R’, R’, R™, 1’, r?, r™ Crosses of brown with green of type [Vg have been shown to result in purple Fy’s, and in eight color types in F2 in a numerical relation approxi- mating 81:27:27:9:27:9:36:40, or in six major color types, anther color being disregarded, in approximately the relation 108:36:27:9:36:40. PLANT Coors IN MaAIzE 113 It has been noted that these results are wholly unlike those for crosses of brown with green reported in an earlier section of this paper, and are similar in general, tho with marked differences in detail, to previously discussed crosses of brown with dilute sun red. As an interpretation of these results, it has been assumed that, in addition to the three pairs Aa, Bb, Plpl, a fourth pair—members of a multiple-allelomorph series, such as R’ R’, r’ R’, or R’ r? — is concerned. It has been assumed further that R* or 7’ is necessary ordinarily for the development of dilute purple and dilute sun red and for the appearance of purple and pink anthers in purples and sun reds, respectively, while R’ R’ or r’r* is necessary for green anthers of purples and sun reds and for the con- version of dilute purples and dilute sun reds into wholly green plants. Similarly, the appearance of green-anthered dilute purples and dilute sun reds in a single cross has been ascribed to R” R”. The relation of the R allelomorph to both aleurone color and plant color has afforded reliable tests of the hypothesis. Other tests have consisted of the behavior in later generations of the several F, color types and the results of intercrosses between these types. Neither of these tests has been carried to the point of exhausting all the possibilities, but in all a considerable number of tests have been made and all have given results in support of the hypothesis. A single linkage test, involving the Bb pair with leaf type, Lg lg, has afforded added support. On the whole, therefore, the hypothesis has been, if not substantiated, at least rendered highly probable. RELATION OF ALEURONE FACTORS C’c AND Pr pr TO PLANT COLOR The relations of the aleurone factors A and R to plant color have been noted repeatedly in this account. A single observation suggests a rela- tion between the aleurone-factor pair Cc and leaf color. Culture 2909 came from colored seeds of a selfed ear showing a 3:1 ratio of colored to white seeds, and therefore heterozygous for a single pair of aleurone-color factors. Several ears in the resulting progeny also gave 3:1 ratios. Tests of four plants with aleurone testers gave conclusive evidence that the Cc pair was the one concerned. One selfed plant of the lot, 2909-32, had 318 colored and 105 white seeds. Both the colored and the white seeds produced only sun red plants, some with green and some with pink anthers, indicating the genotype A A BBC cpl pl k’ Rk’. All the plants showed strong sun red pigment in the sheaths and the outer husks, but — 114 R. A. EMERSON there was distinctly more red color in the leaves of the plants from colored seeds than in the leaves of the plants from white seeds. Particular atten- tion has not been given to a possible effect of the C factor on mature plant colors of other color types. Many cultures of dilute sun reds and greens have afforded opportunities for observing any effect of C and ¢ on red color in the leaves of seedlings, but no effects have been noted. No particular attention was paid to the matter at the time when the seedlings were under observation, but if the C ¢ pair had exerted any marked influence it would probably have been noted. Numerous cultures of dilute sun red seedlings have been noted with respect to possible effects of the aleurone-factor pair Pr pr, but no effect has been observed, the purple and the red seeds having produced seedlings with apparently the same intensity of red color. Likewise, no influence of Pr pr on mature plant color has ever been observed in the case of either sun red or diiute sun red. With purple and dilute purple plants, however, a distinct effect is noticeable. Purple and dilute purple plants from seeds with purple aleurone have purple anthers, while those from seeds with red aleurone have reddish purple anthers (Plate I, 1 and 3, and Plate II, 1 and 3). A similar effect is often seen also in the color of the inner husks. In neither the anthers nor the husks is the effect always suffi- ciently distinct to make possible an accurate separation of plants from purpleeand from red seeds if they are growing in mixed cultures. In some cases, however, the difference is very distinct. And when the seeds are separated with respect to purple and red aleurone, the two lots of plants resulting usually show fairly distinct differences in anther color and often in husk color as well. EXPRESSION OF PLANT-COLOR AND ALEURONE-COLOR FACTORS The mode of expression of the several plant-color factors has been dis- cussed in detail in this paper, and similar discussions of aleurone-color factors are available in numerous other papers. Since aleurone colors and certain plant colors —the purple-red series — are doubtless antho- cyanins, it seems natural to expect close interrelations between them. Many such relations have been noted in this account. There are certain matters, however, which need to be brought together in a summary discussion. oa PLANT CoLors IN MatzE 131 It will be recalled (Emerson, 1918) that for the development of any aleurone color, the presence of three dominant factors, A, C, and R, and also of a duplex recessive factor pair, 77, is necessary. The Pr pr pair has no visible expression except when associated with this combination of the other factors, and then it determines whether the color shall be purple or red. So far as is now known, the plant-color situation with respect to complementary factors is not quite so complex. Something of the same sort is seen, however, in the fact that no anthocyanic pigment ordinarily develops except either in the presence of A and R’, 7”, or r™, or in the presence of A, B, and R’ R’ or r’r’. With the first of these combinations, the pairs Bb and Pl pl determine the particular color type of the purple-red series. Two of these types, purple and dilute purple, are modified further by Pr pr, and the intensity of their color depends also on the member of the R series present, r” exerting a more pronounced effect than R’ or 7’. One type at least, sun red, is influenced somewhat by Cc. With the second combination, A, B, and R’ R’ or r’ rv’, the pair Pl pl determines whether the type shall be purple or sun red. For the formation of the non-anthocyanic (flavonol) pigment, brown, the interaction of aa with either B or Pl is essential, and usually very little color develops except when both B and Pl are present. Brown is made more intense by the presence of r™. Of the factors concerned with plant colors of maize, the Aa pair is one of the most fundamental, since it differentiates sharply the antho- eyanins of the purple-red series, A B Pl, A Bpl, Ab Pl, Abpl, from the non-anthocyanic brown, aBPl, and the slightly brown or green aBplandab Pl and the wholly green ab pl. Without A no anthocyanin shows in either the aleurone or the other parts of the plant. A second fundamental pair is Pl pl, which differentiates the sun colors from those that develop in local darkness. Purple (A B Pl), dilute purple (A b Pl), and brown (a B Pl) are all able to .develop in darkness; while sun red (A B pl), dilute sun red (A b pl), and the slight brown sometimes seen in a B pl, do not develop except in the presence of light. Whether or not the slight brown sometimes present in ab Pl forms in darkness has not been determined. To the Pr pr pair is due a definite qualitative difference in the colors formed which is presumably associated with a difference in chemical composition of the pigments. In the presence of Pr aleurone color is purple, and with pr it is red, and a similar difference, tho not always 116 R. A. Emerson so sharp a one, is seen in the effects of Pr pr on the anther and husk color of purples and dilute purples. The factors R’ and r’ on the one hand, both recessive with respect to plant color, and R’ and r’ on the other hand, both dominant for plant color, apparently always differentiate between colored and colorless anthers and silks in the purple-red series of plant colors, and, when B is absent, determine whether or not antho- cyanin forms in any part of the plant. The pair B b influences mainly the intensity of pigmentation. Thus, purple, A B Pl, is more strongly colored than is weak purple, A B” Pl, which in turn is more strongly colored than is dilute purple, A 6b Pl. The same relation holds between sun red, A B pl, weak sun red, A B” pl, and dilute sun red, A 6 pl. Brown color shows very little in ab Pl but is strongly developed in a B PI. A similar difference, however, exists between the slight brown of a B pl and the full brown of aB Pl. In the one case in which an effect of Cc has been noted, C acted as an intensifier of color. There are somewhat marked differences between the several factor pairs with respect to the stage of plant development at which their influence is expressed. Seedlings of purple, sun red, dilute purple, and dilute sun red normally exhibit no characteristic differences in intensity or extent of pigmentation. The Bb and Pl pl pairs, which differentiate these color types so sharply at a later stage of growth, do not, therefore, come into expression early. All of these types are more highly colored late in their growth period than they are as seedlings, but the later changes are much more pronounced, for instance, in dilute purple than in dilute sun red, and somewhat more so in purple than in sun red. Apparently, Pl exerts its influence comparatively late, but under the intensifying influence of B, even PI expresses itself fairly early. The several factor pairs differ more or less with respect to the particular plant parts affected. Differences in the expression of B, B”, and b are more apparent in the husks and the sheaths, particularly the upper sheaths, than elsewhere. When plants of the genotype a B pl, commonly classed as green, show any brown, the color is limited to the sheaths and the outer husks. The difference between purple (A B Pl) and sun red (A B pl) on the one hand, and dilute purple (A b Pl) and dilute sun red (A b pl) on the other, is more pronounced in the husks and the sheaths than elsewhere. Little difference is apparent between the two groups with respect to the color of anthers, glumes, silks, and the like. The pair PLANT Coors In Maize 2 117 Fl pl is perhaps expressed most definitely in the color of anthers, tho the expression is by no means limited to them. Purple (A B Pl) and dilute purple (A b Pl) differ from sun red (A B pl) and dilute sun red (A b pl), not merely in having purple rather than pink anthers, but also in the coloration of their inner husks, their culms, and the like. What little brown color is seen in a b Pl is limited almost wholly to the staminate inflorescence. The staminate inflorescence of purples, A B Pl, and of browns, a B Pl, is strongly colored, but that of dilute purple, A b Pl, except for anther color, is not very different from what is seen in dilute sun red, Abpl. The Pl factor, when associated with ro", is expressed in the pericarp as cherry in purple and in dilute purple, and as brownish in brown and in green of the genotype ab PI. Factors Bb and Pl pl are not known to be concerned with aleurone color. All the other factors affecting plant color are aleurone-color factors also. Of these the pair Pr pr influences anther color of purple and dilute purple, and to some degree the husk color as well. The pair Cc has been observed to affect the leaf color of mature plants of the sun red type. The pair A a is expressed to some degree in all such parts as culms, sheaths, husks, glumes, anthers, and silks. The pericarp, if a pericarp factor P is present, is red with A and brown with a, or if r” and PI are present, it is cherry with A and brownish with a. The R series of factors influences many plant parts. With duplex R’ or r’, no color develops in any part of the plant, except the aleurone, provided B is absent. With B these factors give colorless anthers and silks merely. Factors R’ and 7’, if A also is present, affect practically all plant parts in which anthocyanic pigments ever develop, but are not |known to have any influence on the color of the pericarp. The factor r” is, how- ever, concerned with pericarp color provided PI also is present. This factor has a marked influence on the amount of color that forms in the leaves, particularly of dilute purple and dilute sun red. It is of no little interest that the R series of factors, which’ behaves as a group of multiple allelomorphs with regard to plant color, usually acts as a simple pair in respect to aleurone color.6 Moreover, some of these factors act as dominants with respect to aleurone color and as recessives with respect to plant color, while the dominance of others is 6 There isTsome evidence that at least one aleurone-color pattern is dependent on an allelomorph of Rr, the three thus constituting a group of triple allelomorphs affecting aleurone-color development. 118 R. A. EMERSON the reverse of this. For example, 7” and r™ are recessive for aleurone and dominant for plant color, and R’ is dominant for aleurone and recessive for plant color, while R’ is dominant and 7% recessive for both | aleurone and plant colors. SUMMARY In this account, six major plant-color types of maize, purple, sun red, dilute purple, dilute sun red, brown, and green (colorless), together with | the subtypes, weak purple, weak sun red, green-anthered purple, green- | anthered sun red, and five genotypes of green, are described and illustrated, and their environmental and genetic relations are discussed. The sun red and dilute sun red types are shown to be dependent on | light for the development of their color, while the purple, dilute purple, and brown types develop their characteristic’ colors in darkness. Diversities of temperature and of soil moisture are shown to have no direct effect on the formation of maize plant colors but to have an indirect relation to them thru their influence on soil fertility, which in turn bears a definite relation to the development of the purple-red series of plant color, anthocyanins, but little or no relation to brown. Sun colors particularly are shown to be markedly intensified by infertile soil. It is | noted that the several types of the purple-red series are sharply differentiated when grown on fertile soil, but that their characteristic differences are largely masked by growth on infertile soil, while the | brown-green series is most readily distinguished from the purple-red series, especially in the seedling stage, if grown on infertile soil. It is suggested that the effect of infertile soil may be due to a deficiency of nitrogen, and perhaps of phosphorus. Observations indicating a close connection between the accumulation of carbohydrates and strong colora- | tion are reported, and the inference that the effect of infertile soil is brought about thru checking growth without inhibiting photosynthesis, | thus allowing an accumulation of carbohydrates, is discussed. In an attempt at a genetic analysis of the several plant-color types, data accumulated during a period of some ten years, and involving an | examination of approximately 680 progenies and not less than 48,000 | individual plants, are reported. As an interpretation of the results! obtained from the more complex crosses, the allelomorphic pairs A a and Pl pl, and the multiple allelomorphs B, B”, b*, b, and R’, R’, R™, PLANT Cotors In MAIZE 119 r’, 77, r*, are assumed and genetic formulae are assigned to the several color types as follows: purple, A B Pl; sun red, A B pl; dilute purple, A 6b Pl; dilute sun red, A b pl; brown, a B Pl; green, a B pl, ab Pl, ab pl; all these having in addition R’, r’, or r™ The factor R™ is assumed to be the causal factor for green anthers and silks in purple, sun red, dilute purple, and dilute sun red types, and R’ and r’ are assumed to have the same effect on purple and sun red and to insure colorlessness (green type) thruout in what would otherwise be dilute purple and dilute sun red, the R series having no effect on brown, except for r, which intensifies brown as well as purple and dilute purple. . Of the R series, R’ is dominant _and 7’ is recessive for both plant and aleurone color, r’ and r” are dominant for plant and recessive for aleurone color, R’ is recessive for plant and dominant for aleurone color, and R” is dominant for aleurone color and also for plant color except of the anthers and the silks, for which it is recessive. The A a pair is concerned with both aleurone and plant color, and the aleurone pairs Cc and Pr pr are assumed to exert a modifying effect on certain plant colors. | The principal hypotheses involved are shown to be in keeping with observed facts when subjected to practically all the available genetic tests, such as backcrosses of F; with multiple recessives, behavior of F» types in later generations, intercrosses of the several F. types, relation of aleurone color to plant color, linkage of certain plant-color types with ‘normal- and liguleless-leaf types and of other plant-color types with yellow and white endosperm. Approximately 32 per cent of crossing- over is reported between B b and Lg lg and about 20 to 30 per cent between Pl pl and Y y. 120 R. A. Emerson LITERATURE CITED Coturns, G. N. Gametic coupling as a cause of correlations. Amer. | nat. 46:569-590. 1912. CzarTKowskI, Apam. Anthocyanbildung und Aschenbestandteile. | Deut. bot. Gesell. Ber. 32:407-410. 1914. Fast, E. M., anp Hayes, H. K. Inheritance in maize. Connecticut Agr. Exp. Sta. Bul. 167:1-142. 1911. Emerson, R.A. Genetic correlation andspurious allelomorphism in maize. | ' Nebraska Agr. Exp. Sta. Ann. rept. 24:58-90. 1911. The inheritance of the ligule and auricles of corn leaves. Nebraska Agr. Exp. Sta. Ann. rept. 25:81-88. 1912. A fifth pair of factors, A a, for aleurone color in maize, and its relation to the Cc and Rr pairs. Cornell Univ. Agr. Exp. Sta. Memoir 16: 225-289. 1918. GERNERT, W. B. The analysis of characters in corn and their behavior in transmission, p. 1-58. (Published by the author, Champaign, Illinois.) 1912. Knupson, Lewis. Influence of certain carbohydrates on green plants. | Cornell Univ. Agr. Exp. Sta. Memoir 9:1-75. 1916. Linpstrom, E. W. Chlorophyll inheritance in maize. Cornell Univ. Agr. Exp. Sta. Memoir 138:1-68. 1918. Sanpo, CHARLES E., AnD BartutEerT, H. H. The occurrence of quercetin in Emerson’s brown-husked type of maize. Journ. agr. research. 1921. (In press.) Weesser, Hersert J. Correlation of characters in plant breeding. | Amer. Breeders’ Assoc. 2:73-83. 1906. Gu. WueELpALE, M. On the formation of anthocyanin. Journ. genetics | pd loo. SOIT: Memoir 37, A Modified Babcock Method for Pa an angD sat in Butter, the second preceding number in this series of publications, was mailed on December 10, 121 PLANT CoLors IN Maize APPENDIX TABLE 1. F, Procenres oF Puree Ia x Green Vic Pedigree nos. iNqmmibaron F, plants Pi F, (Purple Ia) “DEEN sre TPRIET e ae ae aio ae a eterlt igd ENN, arti cea han ea ten paar ae 18 IES SNE Se ae ee IED) WHR, HOR = scoscacne ceases 40 = xe Dee eee Se ee be a AMO Fa SUM ye ots eee Pee te Re ioc eee 36 LSA ox ICD ee eee DOD OR on eRe EOP RE eT 17 Taal, 4b (ROUEES ccteiS aerate Dic eee cece Sioa seee CO Sams nie eres caer 111 TABLE 2. FF, PRoGENtIES OF PURPLE Ia x Green VIc Pedigree nos. Number of F, plants Group Dilute | Dilute F, FE Earle an rd ail || crane ee ee a IIIa IVa pon ate, 1419- 1 IRR Sc con 94 22, 26 12 20 23 1511- 1 ZOISE ae 61 19 13 4 13 9 2a 2020.25. 54 16 23 7 21 u 2022— 3..} 4012, 4013. 7 6 6 3 4 1 2056— 6. .| 2415, 2416, AD SA en 39 13 17 i al 16 10 1 —11..| 2417, 2418, 2553-2559, 4001-4007. 96 22 24 3 26 8 —16..} 2412, 4066 ANGT. 2. 17 3 11 1 8 7 Total, 7 progenies... . 368 101 120 o4 108 65 1514 74S 2054. =. 20 UO 8 1 5 2 =aill, sl] BOs S one 22 4 4 2 2 6 2000— 8. .| 2419, 4065. 92 29 All 8 19 25 ZOLG-2Sr ea |VAQZS I. oo 24 8 4 2 4 6 =O || CORY cece 21 6 4 4 Wl. 4 POO G—elee 5303" ..5.-. 17 7 5 2, 6 3 2907— 1. .] 5290-5293, 7050, 7051 93 26 34 uf. 34 23 2 — 7..| 5299, 5300, e 7054, 7055 105 46 30 10 38 31 2981— 2..}) 5056, 5067. 17 4 5 1 8 3 — 5..| 5068, 5069. 20 6 2 1 2 3 4020— 7..| 5712, 6810. 109 44 26 12 31 33 AQ32— (We 739): . 16 5 3 2 3 2 = Bo || Mee, Soe o. 15 a 5 4 4 3 = Gell Ge ace aiee 3 5 4: 1 a 7 Total, 14 progenies.... 584 204 155 SY 170 151 Total, 21 progenies.... 952 305 275 91 78 216 122 R. A. EMERSON TABLE 3. F, PRoGENIES OF PURPLE X GREEN BACKCROSSED WITH GREEN (Ia x VIc) x VIe Pedigree nos. Number of F, plants Group Sun | Dilute | Dil G Purple un uute ute Brom reen F, x VIc F, ia red | purple | sunred | y Via, Ila Illa IVa b, ¢ 1420- 1x 1430- 3.] 1514..... 12 19 15 16 14 1511— 1x 1516— 1.) 2019..... 18 8 12 8 18 1 1512-12 x —14.} 2021..... 23 18 16 105213 2056-16 x 1995— 6, 2413, 4068 4 10 8 6 8 Total, 4 progenies............ 57 55 51 40 53 2867-69 x 4082- 1:] 5740..... u 4 6 3 4 2906- 1 x 2887-10.| 5305..... 7 5 2 8 3 2907- 1x —22.| 5296,7052, 7053... 10 11 10 11 9 — 7x 4032-41 .} 5301, 5302 16 16 16 19 25 4020- 7 x 2888-13.] 5714..... 2 9 9 4 4 2 4032-— 2x 2921- 4.} 50%..... 8 6 18 12 15 3 x 2888- 5.| 5086..... 19 16 21 12 18 3 x 2922-16.| 5085..... 14 10 22 16 8 4x 2888- 1.| 5089..... 5 15 12 17 18 4x 2921— 4.| 5090-5092 25 13 19 18 15 Total, 10 progenies........... 113 105 125 120 119 Total, 14 progenies........... 170 160 176 160 172 PLANT CoLors IN Maize | 123 TABLE 4. F, Procenres or Ditute Sun Rep [Va x Brown V Pedigree nos. Number of F, plants proup Dilute | Dilut ilute ilute Py Fy Burp’ Pune purple | sun red Illa IVa 2025-23 x 2192-14. | 2333, 4314.......... Da eae re | ee weeekira | Wee nite 2029- 8x 1945-11..| 2304, 3596.......... OR PeeePret Wer seestemalin dy tani 6 = S >. PONS) eh PR ee boda oanee ALG [Dette es esl fee ea oe an ers — 8x 2014- 8..| 2310, 4034......... : Dial eeaies ae | Ms cates lb ara ny ae 2031-10 x 1945-10..] 2309............... LOSS ieetrese tener creole ce 1 aye 6 US AN al) PRY Pe eo ne tian ome PALIN Meets SAA linetinct es eal | enters ee 2948-16 x 4042— 2..| 5168, A108, A120.... TAGES leiparss tan tonaral | egies. al | mn lagaa ae 4253- 2x 4299- 2..} 5528, 6748A........ PO eee etal een oes 1 4305- 5x 4042— 2..| 5198, 5194.......... OATS) [ia tahoe Coes otal lee ia sterl | SP Satie oa mRotaleeOhprogenles<.)-).0S. et one oh amten ZOD Ese ec leery ees 1 2018-69 x 2192-18..] 2386, 4801.......... 30 |. Day [feealncerenne ities etree a 2030-13 x SAM AS Onesie aa hie a. 7 Ob eae errata tees oleae 2031-20 x 2012— 1..] 2325, 2326, 2548, 2544, 2950, 2951. . 55 Eh Ga ste nd [ecrneecenta co 2 2043-— 2 x 2026-17..| 2347, 4326.......... 15 AUS lee sees eH ition tO 2049-14 x 2192-14. .] 2336, 4827.......... 24 DA Wie RIN ey dae DAS ROE ZO4 lier | A029) sees oe ano or 4 POG afc ie a |e ere at ee 4370-— 5 x 8000— 2..| 4746, 4747.......... 8 Que Gere OOS ES ome a MG tale LOLEMICS 2... sist en ceeie Sawer nns 143 UA (ed eho ceed Sk se 2023-19 x 2192-12. .| 2332, 4311.......... ii eee Pie | ae ae —23 x HP, 5|| PEED, FNOs shes boos el ecleraerae Ge arte ee 2027- 9x HAL, al CBRE ARNG choo beans S| aigia Gos US ialies sateen 3 2410— 4x 2417— 2..) 2993, 2994.......... OF eta GaSe — 6x — 1..} 2995-2998.......... Dal ates Paella he aa 5500- 5x 5130- 1..| A6d................ DHSS ere ea PASay latest tot Ae MOT MOE PrOweMIESHe ich syria pores ee OS eect Sh eee seer 2025-10 x 2192-14. .| 4315............... 1 2 6 3 2029-27 x 2012— 1..| 2319, 4055.......... 4 3 5 5 4 —32 x = Meal FRG aoe ec bowen. 3 5 6 3 —34 x 2014— 8..} 2814, 4054.......... 1 1 2 6 Motaleaprogenies 22 sibel Sd aces 9 11 19 17 124 R. A. EMERSON TABLE 5. F, Proaentes of Ditute Sun Rep IVa x Brown V Pedigree nos. Number of F; plants Group Dilute | Dilute Green Fy F, He ple au Ted purple | sun red Brow Via, ee 3 IIa IVa b,c 2310- 2..}) 4036, 4037. 15 a 6 3 3 a 2332- 1..} 2999, 3000. Sl 9 8 1 15 O 2950- 1..}) 5036, 5037. 36 12 12 2 10 9 — 4..| 50380, 5031. 37 15 13 3 8 13 -17..| 5034, 5035. 32 5 14 6 13 9 —19..| 5032, 50383. 39 12 |, 10 3 12 5 2995- 7..| 5000-5007. 75 24 20 5 21 iy} 1 2996- 1..} 5008, 5009. 150 50 58 20 48 45 AQ 29 2 EN DO9D seas « 61 23 11 5 22 11 4034— 1..] 5098, 5099 46 12 19 v ile 7 = 12) OA 42 20 17 8 13 21 5193-— 1 PNB S peat 20 5 4 3 4 1 5194— 5 DRO rene a 10 3 12 il 4 Us 5528- 8 6748B...... 49 11 14 4 12 18 Total, i4 progenies.... 643 208 218 71 202 177 2973--5..| 5056-5062. 55 23 21 6 17 17 2974—.9..| 5068-5065. 75 “24 23 10 18 22 4046--3..| 5157, 5158. 20 cellu 6 5 i 4 2 5I738--4..1 Al28...... 19 5 8 1 9 9 SI7219 2 7760 35 11 5 1 14 4 Total, 5 progenies .... 204 74 63 23 65 56 Total, 19 progenies.... 847 282 281 94 267 233 | TABLE 6. F, Progentes or Dinute SuN Rep x Brown BAcCKCROSSED WITH GREEN (IVa x V) x VIe Pedigree nos. Number of F, plants } Dilute | Dilute Green Purple | Sun red Brown F, x VI EF purple | sun red Via, ak Wr : Ja sane, Ib ive Vv b, ¢ 2310 =x 24 TG AOSD ees ae 3 4 3 6 2 17 2922-13 x 4029- 2..) 5652, 5653... 22 18 19 24 27 75 4029- 2x 2921-10..| 5096....... 9 18 12 8 13 51 4034— 1 x 2922-16. .| 5100-5103. . 10 13 17 11 9 33 — 2x 2921-68..) 5105....... 12 5 5 4 4 16 5813-25 x 5528- 8..| 6749....... 3 0 2 4 1 9 A129-12 x A108-6..| A248, A244. 25 19 20 15 23 48 To’al, 7 progenies..... eS 84 72 78 2) 79 249 PLANT Cotors IN Maize 125 BLE 7. F, ProGenres oF PurPLE x GREEN AND DitutTe Sun Rep x Brown Back- CROSSED WITH DILUTE SUN RED (Ila x Vic) x IVa, anp (IVa x V) x IVa Pedigree nos. Number of F, plants oup Parole, lined Dilute | Dilute F, x IVa Fy I Pp "Ir purple | sun red a = IIa IVa 2056-16 x 1992-13..... 2414, 4069, 4070... 18 16 21 15 1 2889-54 x 4032-1.....] 5741-5744........ 24 27 21 24 Motalee2 prosentesy oc... na. cos ashes 2 ole 42 43 42 39 6730 — 9 x 6748A— 5..| 7467, 7828........ 87 79 75 71 GSA Gp G70 222 17229. 2 yh. ene 40 32 42 41 -18x Sel DOO. tre See ars, Ses 28 28 26 35 -19x Sr LEM | S72 hows ieee eee bese 40 33 30 36 > —20 x =i LS | ay Pps te SR Soe 30 25 32 20 A121— 6x A108— 8..| A241, A242, A461, NAGI EP. Pee 28 25 38 45 L188- 1 x 5528 — 8..] 6786, S2.......... 4 5 3 4 MO fale PPROSENIES auon aces 12 4 — 98x =I Vices WAV emeoann oes mess NL estates 12 104) x FOP ball PaiSioaamenumiocsude - Sol Pein 12 —106 x 2192-18....| 2359, 4351.......... 7 fe baer amare 15 Total, 7 progenies........... Jo Ae ois Sears Sues U0) ere corer 85 2019-33 x 2192-18..... Dan Dea SAD ata fo Nie dee sie 5 4 = ilexel 94AG— 4-2 2)| 28615 438470 Joe se. ll Soon. 19 15 —57 x 2192-18..... 2369, 2370, 4845.....] .......- 8 14 5 -73 x 1945-11..... FEV Pa isacd pce cot i | Mees eee 2 6 -84 x —10..... DATA BIE Fas meee mais aan c 22 26 Mota oMpPROP NICS «os eisa-c overs oo-erersiee oie he eg etd Ses acieae 56 65 2019-17 x 1945-11..... PB GR iss oe plodbid| | tan moe ee en | Doneenene 43 -19x Si aeete Foe bid GIRS OSG EGGLD | NER ORE el OSI eA 19 6 -25 x =) tl ane DS IA RAAT meee || aes eee sie al lig nook teaches 44 Ataiell. G3 yatoy xa weno Aneiieenis cha Ged OOOO! ec aon | mene oes 106 134 R. A. EMERSON TABLE 17. Fs anp Backcross Proceniss oF DitutTE Sun Rep IVa x Green Vie Group Pedigree nos. Number of F; plants F, F, TC Tos ka be ae er a oer ee I Tale er an 4502, 4503.......... Da lear tt ot ot eee cuen ee tua AGTI=40 19% nok eee oe 010d IS esince 5 rece ee rae ie aca oe 6470, 6472 03 winner Total 3 progenies ss 2.2.2.5 h eee hence ere Ae ee F, x Vic 28DAH13 xe 2881—OOte se ee eee 6325316326552 eer -16x OO ie See ce See OSLO _OS2IeS eee 2861- 1x AN re et teat 4686-4688.......... 286 6—= exe 28S = a2 es pele eee 5748-5750, 6485-6487 AN i=S2) xX 4685— ieee otek ale 6533-0530) = 95. =e Notal-s5yprogenies4..-7.c2 esse coe eee eae Green sun red IVa Vie 27 11 199 73 43 15 269 99 87 96 42 45 93 100 90 74 45 43 357 358 TABLE 18. F, ProGenies oF INTERCROSSES BETWEEN GREEN PLaNnts, Vla, VIb, anp VIe Group | Pedigree nos. Number of F, plants Brown Green Pi Fi V VI 2019-25 xo 2019-106... ee ce DBAS a cert te Sree a le scence 23 2O01G= 25a 20G=3 35 ee eee | 2350; 4343... 22 2019= 40x 2019= 632... . eee 2360.00.20. woke tee ee eee 25 — 98x =A (ieee: Be er cee DEY (GR eR i EDs 25 -104 x =(OGa nee 2362. on eh en eee 22 Motal <3 prokeniesi, = cea nad seh kai AOE eee [aa ee 72 | 2019-57 x 2019-51. es. A ae | 2371, 4346... 24 20193 2OL9=63ee eee ee oe DODO Mie hee Nee ae 6 19 —- 40x Se Ot aria arr e 2360434 8 28 — 57x = OS tir ieee. Mie ee 2324350) eee 14 26 — 73x A) ty oto metic 2380.63 ie eee 7 16 -106 x =e ae cea Q3G0)h eae gee 5 16 PxLant Cotors In Maize TABLE 19. F. Procrenies oF Crosses BETWEEN BRowN V Pedigree nos. F, F, Pi4eDen 6 bo oo bac OUR cee Eeeoee DA UPA) Rea Selene ts eset Seo noe: 2B 3 5G Siclth creo OR eae T a DOS Tee eihe ot cetera ata ciie es =S13)000 00.0 00 Cea aE Sree e QUZS atic: MORE eau s ee 2YUEE= Ts 0's Soba e eee cone SOMO OM2 a ot hoes one oe SE Mer ee ae ae NUD Reatetin 345 so cas Dees PO 2UNG= 426 Gabope eee nL ene DOSE Honea aan chute = ce ies = O65 cepiScle GORE eee EAU AL PP este t Arche ty chp ec ce HIBH=35). 0 6 bo Cod COREE eee ESTO erie e cits tee Opices c ROOT Oe ite= Zech Ses eGua as Ge Eee SPPAS ery teil cis oy eave OREO FOL =l0). 36565 Up eS aoe O22 ee eee Ee a ee =llllso0 6 ¢3'o od COR ae Ree SPDR OSIRIS 2s FA eee Se eerie Movalewllgnroceniesiiy 5... icc. 6 om oe. 5 RNG Ui OD AOE Bee 135 AND GREEN VIc Number of F, plants Brown Green V Via, b, ¢ 19 16 22 13 25 16 40 44 15 11 21 8 46 36 14 6 53 23 24 20 38 30 317 223 TABLE 20. F3 PROoGENIES FROM F2 BRowN PLANTS OF THE Cross Brown V xX GREEN Vie Pedigree nos. Group F, DIDI ~D acts n ee cen ae ems ABTS rte, Sener =) a0 eg ee = ee TET SIRES SE RG tale eOrOTEMIES! 32) cer. Fie eo tae beh erate aS teuciist stead e Magis) PAE) cio nb oc Sea Vales cis ces oleieras elk viet she the he 2324, 2541, 2542, PRYL PRIA BAGO BORO Eo SoDUDQOOMIOC MH OMA SOA OaS 2320 4S21. comes A OGHG OSS AIS eae BUS CY 46 5 oti Oe Ge s = Number of F; plants Brown Green V VI LOM [paws ae LOM |Past ees DOME eae ek 82 34 18 6 100 40 20 22 19 39 39 136 R. A. EMERSON TABLE 21. F2 ProGenres oF THE Crosses Sun Rep Ila x Green Vic anp Ditute Sun Rep IVa x GREEN Vla Pedigree nos. Number of F; plants Group Dilute Sun red Green Fy F, Ila an a wine N14 32) ns shea ee 2040\:4204 55 ae net 53 | 16 19 =1Oss2 hee waren 204 ADO DWAE ee ene 40 14 23 DOSS Vise Wis wits one tates A330 43002 soe see oe 24 10 11 ee Di aerate Racial bowedetere CREE CBR ey euobac 26 4 11 QOS TB a Si cieieseie ere A992 "4903. 525.3 Steck 91 42 45 SA aie iae ener teks 4994-4996.......... 203 55 84 AQUA RM ere cA ihettele 554 DOO Meee aces ae 83 33 33 =n te eas ere 5059-5503) o 2 occnes & 47 13 13 1 AIG =O Fee tees. ies ont 569155692. 28 6 18 A te ia atis soos 5685, 0080.0. ere 20 2 12 AQ20 =e a eetrcs seston SIOS Misa eae eee 28 12 10 AQ 3 aoe caren rawe chin HL 51S. ae eee eae 49 21 24 AQ AQ = OD" aye% en Aims reer oe D148 SLAG i ee 14 5 13 C661 29 ees Sees cite IRA Ree roar ore eee a 35 16 21 666222 a Ae eee TOOLBAR ae cs ater 44 9 29 SO Nes Saath ee CBSO Re eae eee et 49 10 17 hotalsdiGiprozeniesse reer acer oe ete 827 268 383 DOS ODE Ee hanes ek BADG AADT a eae 28 9 12 AQQ9 Sls oop. tat a tect BOOS oes eae 31 18 21 2 ee he. a pe sepa G951=695 dees 127 38 2 gag Al 2 AT30= 98 c: o b en Sects 6960;;6961. 5. a: 92 25 42 it Loewe er ene 6954-6956.......... 65 30 33 Lotaleosprogenies ae see er eee 343 120 179 TAG 1 x 1430— Ae. |) 1494, 20745) ee ee 39 39 92 2888-22) ADIO= Dae. 5694B, 5695A....... 16 14 22 2922-18 x 4014- 3..... 5DOS—O0G0% ne =e a: 30 26 68 4014- 1x 2922-1..... BOOS AMR cise ee 3 3 10 3 4019— 2x 2888- 1..... 5697, 5698s. 8 eas. 15 14 22 -4x = iene DOS9 5090 re were 24 13 31 4020- 1 x 2887-69..... LEV AU1! ie estates a aces Uf 14 22 Motale/progentess. paca swe ee one 134 123 267 2921-15 x 4029- 1..... 5604—5650n-ae eee 28 37 80 4774— 1 x 4710-45..... 6945,:6946. 82 78 71 151 4781- 2x 4707-35..... 6967169682 cere ae 54 76 132 4782- 5x SAS tre! 697256973 ee ae 103 88 195 4 -13 x = Gee 6974-6978, 7667, 7668 80 101 191 4789- 4x SLO Re ec 69896990 nes see es 50 43 108 6661- 9 x 6690-17..... (S28, (820 oes 17 17 38 6790- 5 x 6809-18..... U0). Cee ee 32 32 67 Totali Sjprogenies. ccs hecho oe nee eee 442 465 962 PLANT COLORS IN MarzE 137 TABLE 22. F, PRoGENIES oF THE Crosses Ditute PurpPLE IIJa x GREEN VIc AND Dizute Sun Rep IVa x Green VIb Pedigree nos. | Number of F, plants pro Dilute Dilute KF, KF purple sun red Green Illa IVa Vib, c ASO ee 2044, 2560, 2561.... 44 14 16 QOIGZNO Meek es ot. 2425, 2931, 2932.... 19 3 6 DTZ miliaris 4333, 43884.....0°0.. 38 12 10 Oy a eee Sree AOE eyed 22 13 7 1 2D OO Sracper ene 28 4899-4904.......... 153 58 73 AQB35-88). a. 5065. ce ees NO Peers alata ein 50 16 24 AQO8= Oe. heise SYP chs eaten Gee's 46 18 26 INTs serosa eee DDD aM eae 44 15 11 Totals-S) progenies... 5... Soe te a ns 416 149 173 DO Ne eae ae eee 4494, 4425.......... 14 4 4 AOS (Oe nas ae eens D280, O2ZO0 ne 4 eae oie eh: 133 51 52 SIlibs case ceeene eee PB WWE. ad abo dae 62 21 19 SYAOO > Glo ue eae 6696, 6697.......... 30 11 15 2 NO GU csr Nears ogous A416, A417......... 15 6 4 ING DO eh iste. sainos A407, A408......... 30 9 13 MotaliGmprogenies .......... 0.080... cess 274 102 107 6790-1 x 6809-8....... QOD Reese ina: peta 26 20 56 TABLE 23. F. PRoGENIES OF THE CRoss Sun Rep IJa x Brown V Pedigree nos. Number of F, plants Purple Sun red Brown | Green Bi Ee Ta Ila Vv Via PA ce coos Seen NOD enema 14 4 5 1 Vise othe) cus doeeE AO Cera eae teehee! 37 5 9 2 =Be6 fold sist ieee CUTS, SVBigeec abe 69 20 23 7 138 R. A. Emerson TABLE 24. F, PrRoGENIES oF THE Cross SuN Rep Ila x Dinute Sun Rep IVa Pedigree nos. Number of F; plants “roup ; Dilute F, F, Sau red sree 2 IVa AT ien ie es crea ia. cad er rene ly IQ98h a sa ee eae 40 13 GSU een eis Sten te eupe ee 12358 Soon See eee 15 4 PS QOS ON pen SIC .. iBlar ele PAO IU ates Ae mene Ma 31 8 ZOGS = A Ge Cae ie SES HS ieee as A330) ees se ae 14 5 Ea SRP as An aiee Sev aati 2ASIE 4331. eee 48 15 DAV AT Dik ct Pees sk, Ae Rear aetoR ee 2981-29922 eee 36 9 D9 i AR ore Ae nal nin meus 4983-4986, 7001, 7002 373 123 ADS bee dss pape es Mahan p eee aes 56438-5646.......... 22 7 1 AQAQE OS Or A ey Bites aime Snes ae tens OOO MOIS eee 41 8 ABBE O Ohm: kiN eis alee ae a eaunce \..-} 6491-6493... 2.22. 55 16 ATS (As ie iota en eh ents oe ere O779-6782 = see 166 50 DO Bie ee ae OM ene cortage re ia dntrets ALTA ooo. eae 55 20 DDR A PEGS acy aen re aan Ane tee ae se SITS 8119 oe 43 12 TODO NEON ROR IS Chea PENTA S/O 2S arse 9 3 CoN ge Pee DES Negi diet et hier 8094, 8095.......... 35 15 INGE Are a eee ane eins Wacteyeveers emanrese VND PATERS Ss ME Nene see a 3. 15 6 TRotsltG proeeniesry.<. arisae cera yao kee een eae 998 314 F, x IVa 2065— ll x = -2048= 2 ee ces AS 2 cee a eeraees 27 30 —-2x =D eae cre eeS 2432 A382 eee 6 13 Ar (VAS A ian all ere ae carne 6943, 6944, 7676, 7677 173 133 (22A— OX A220) Mine scare Sis) SINGH yee 196 180 (eis allio een (aie ct os cats dis ae 825078251 eee ee 86 96 GO MNGX MAGS Whee avers cere Sol eae ee 16 14 - 5x pal IPAs aL amen ig SIS20 sa ates 17 11 2 Al40-14.x Al05— 6.....205.2. A252, A468... ...5..5 92 87 LUG(3-=15.x L2049- Weg n: pelea DR Cte | 46 37 —20 x Pelee cso ol Ame ERT ET S.C), 41 39 L1844-14 x L2048- 24........... STAB Soe see 37 41 L2063- 5x =e Omen ena een SAG aa i ea Ree 56 48 =26 x L2049= 33)... sean. S14 Sait ae cua eee 7 7 TE20G4A—82 x 2043 a2 2 ene 814A ie aaa 11 6 Total 14 progenies 4. esd as mses cee ee 811 742 Piant Coors In MaizE 139 TABLE 25. F; Procentes or F; Sun Rep AnD DitutTEe Sun Rep PLANTS OF THE Cross Sun Rep Ila x DitutTe Sun Rep IVa Pedigree nos. Number of F; plants Group ; San red Dilute F, F; Tita sun red IVa 2 OD Maen as Ok SIs Gibesanats DICH OMESe x. sce aos oa 10 1 ANBS—OXG) 5 tia 6 Be aCe ne ee BLO rahe Bate has eee foal ements aan 40 MOtaleeAnPrOGeNIES).. 2.0: ccc cus s)he eco A iess sree teach ene ot 50 PRO. 55 a ese eee 1 BY), Bs cos condo. TI ERS he eee oa ie Ud EBs eee ee ere eae OVS Tey ares ead Opa Re iate weeccs 2 PNG talPeOBORO MEMES nee ke re ese SIO eo Se a OF eat ae on HOO (exe (O02RIN o.oo acne [EGS4eeGSomann wae 2 LO Wel Sareea 1D. 1 soe bee Ree Ree ae 1633-1635, 2009. . 23 10 DO SUA i cinema veda tis DOU rgectn oon tess 14 2 3 DSI Bacio ois Caer ae ee 4997, 4998, 6999, 7000 324 111 ae Dee Ee Se Aer eae age a AQO9 Lee ae 4844-4860... 59 21 8 1 14 18 LEE Laon pas 4861-4871... 43 15 7 4 11 15 STATS sigte 4872-4884... 42 12 9 2 15 16 SP SB aoe 4885-4898... 62 23 12 3 20 17 2953-10he5 se. 4822-4829... 84 24 25 8 23 30 Total, 6 progenies........... 309 100 67 19 88 98 146 R. A. EMERSON TABLE 36. F. PrRogeNIES OF THE Cross GREEN IVg x Brown V SS — ee Number of F, plants Dilute | Dilute : Purple Sun red purple |sun red Brown | Green Pedigree nos. F: ? f ? 0 Green Purple | Green | ,,- | Pink | Green| gn- | Purple} Pink | Green | anthers anthers] anthers] thers anthers| anthers} thers | anthers] anthers} anthers Illg Ta Ig I IIa Ilg IL Illa IVa V IVzg, VI 2958-2961........ 14 neue 20 1 1 3 6 1 5 2 4822-4829........ 61 12 11 10 4 10 25 8 23 30 4844-4860........ 42 16 1 10 7 4 8 1 14 18 Total, 3 progenies. 117 33 12 21 12 17 39 10 42 50 TABLE 37. F, Proceniss oF THE Cross Purpie Ig x Green VIc Pedigree nos. Number of F2 plants Group Dilute | Dilute ; Brown F, F: purple | sun red Green 4 Purple | Sun red Illa TVa V (Ia, g) | (IIa, g) (IIIg, IVg, VI) 1 5584-39. .......... 6795, 6796.. 65 11 6 7 15 26 6655= Give Sais cece ose CE Sei oe 15 2 3 2 5 1 Total, 2 progenies............... 80 13 9 9 20 27 F, x Vic (Ia) (IIa) (VI) 6655— 6 x 6690-17..] 7349....... 6 13 13 15 17 46 2 6808-13 x 6790— 8..| 7290....... 30 16 18 16 14 49 Total, 2 progenies............... 36 29 31 31 31 95 Fi x [Va 6779-2 x 6790-8... .| 7299, 7300. . 27. 25 30 iba (ema on ||| ooosooOoe 3 6792-2 x =O spent SOM ceuseeneae oie 29 29 19 CY ia asad. || scao-cannoo -8x —S nel M296 ea. 59 43 46 4S zee a erence ete vons Total, 3 progenies............... 115 97 95 niet easiness Yl oro ae aes Fi x IVg (Ia, Ig) |(IIa, Ig) (IIIg, 1Vg) 4 6656-9 x 6652-6....] 7344....... 23 15 10 DRG! WecteiotacG:o 13 PLANT CoLors IN MAIZE 147 TABLE 38. F) PROGENIES OF THE Crosses PurPLE Ig x DinutE Sun Rep IVa AND PuRPLE Ia x GREEN [Vg Pedigree nos. Number of F» plants Group 3 y Purple Sun red Diletee DH Green Fy Fy, purple sun red Ta, g Ila, g nine TA IlIg, [Vg 2954-3. .| 5042-5045. 43 14 13 5 7 2956-3. .| 4905-4914. 144 42 D5 14 7 1 —4..| 4915-4929. 56 ll) 21 3 6 Total, 3 progenies... . 243 71 59 22 20 2421-1. .| 2910, 2911. 14 7 5 1 1 5 —2..| 2908, 2909. 26 : 13 4 1 0 Total, 2 progenies... . 40 20 9 2 1 TABLE 39. Fs; anp Fy PROGENIES FROM Fy AND EQUIVALENT F3; PuRPLES OF THE Cross Purp ie la x Green IVg Pedigree nos. Number of F; and F, plants G ‘ hee Dilute | Dilute F, and F; F; and F,| Purple | Sun red! purple | sun red Green Illa IVa (Ia, g) | (Ia, g) PRIOO=NG.0 cis os One 5251, 5252 9 4 3 0 0 1 F, x IVg (IIIg, IVg) 2909-4 x 2884-21) 5255..... 15 15 5 9 F, x VIe (la (ITa) 2909-4 x 2887-38] 5256, A94. LY 19 15 PA ere se Y SARI-O. Signore aioe GMOSte eee 31 AMI NGe ean ease cee | een Mics oe (Ig) (Ilg) XN co bo 000D we 5257, 5258 14 S| Sone wl Veal Ls cate va 5 3 AAI 5s le ie bee 6652..... 23 Ol ake Tae aa lie estate ie 9 Total, 2 progenies............ 37 SL Fak Peet obahy [bacae ates aa 14 148 R. A. EMERSON TABLE 39 (concluded) ey Pedigree nos. Number of F; and F, plants eeeue Dilute | Dilute F, and F3 F; and F, | Purple | Sun red | purple | sun red Green IIIa IVa F., Fs x [Vg (Ig) (IIg) (IIIg, IVg) 2909- 9 x 2884-21) 5259, 5260, 7007, 7008, 7060, 7061. 19 Aleta) Meena Lea 34 A717-71 x 5252- 1] 6654A..... 11 B55 5. Seeaieal | ere 13 3 5252- 1 x 5669- 3] 6654B..... 4 Go| c.c ata (eres 6 (con- tin- | Total, 3 progenies.:........... 34 Pa V4is eerie oll s5's a0 5,0 53 ued) F., F3 x Vic (Ia) (IIa) 2909- 9 x 2887-38] 5261, 5262. 14 11 7 JED ek eee 4057— 1 x 2909- 9) 5534, 6790. 16 10 13 ISS Se aes: 5251- 1 x 5813-18] 6655...... 9 16 7 1 hse Ne 5813-18 x 5251- 1|- 6656...... 5 11 6 OSS ee uae Total, 4 progenies............. 44 48 33 SQM NA sare ae (Ig) (IIIg) 2909-34. ........- 5253, 5254, (O90E Ra DOP ocak |B Sa eae 6 Pace ese 6658, 7015. BOG eo8nc | ee | eee 12 4 Total, 2 progenies............. 56 [ince 2 ae ee eee 18 F; x IVg 4717-20 x 5251-7...) 6659, 7014. 284). col ieee 27 PLantT Cotors IN Maize 149 “TABLE 40. F; AND EQUIVALENT Fy PROGENIES FROM F) AND F3; SUN REDS AND DILUTE PURPLES OF THE Cross PuRPLE Ia x GREEN IVg Pedigree nos. Number of F3 and F, plants Boon Dilute Dilute F, and F; F; and F, Sun red purple sun red Illa IVa (IIa) POSE 20 ERE ioes sce oe DZISS se ee SOR eee 8 = Oo. Coo eee Ona: 66485502 mele: Sle lige re er 10 1 Qo 233 Bose eee 6109S eras SO Ss ait 8 MROtAlOMPEOPEMMES =. \ess¢ oc: ts isis oe Cea Se OEE ese sei 25 (IIa, g) RUUD Sa sce Aas eee EPA (VASP E5s Se 55s PAY | Sapna era eter Sore em DOMME TT secs eI: 5280-5283...... (GY cao ee eh [et See BY) sci bia AO eae 5274-5277... .. 1 OATS aS eecar ad Spice | ieee eae 5 MOvAlouPLOLENICS «25. cs ee cles eee aes see D285 Ooo: F, x IVg 2909-26 x 2884-35.........| 5284-5287, 7137. ANTS | Res ss ar |e oe, F, x VIe (IIa) 2909-26 x 2887-38.........| 5288, 5289...... Wii |S S6ceoem ||hesaoecee Motale2sprogenieS sec. 2st s oe ecss see Sowa TOSI Weegee Sa asec La oi aes 46 9 F, x IVg 3 2909-21 x 2884-35......... PIV, BYR coun! dooasoce 44 31 F, x Vie Poi MexeZ 00-215. 2 oc 6. -[P DLO9. ccc. ere take ste. 4] 51 Mintel zsPLOPENIER sh. fess cee eers cdl asset as 85 82 150 R. A. EMERSON TABLE 41. F, ProGenies oF THE Cross PurpLeE Ig x Green IVg Pedigree nos. | Number of F» plants Group Dilute | Dilute Fi Fr, Purple | Sun red | purple } sun red Illa IVa 5200 =e Ohana: 7094, 7095, (ig) (IIg) 7701, 7702 80 240) oe eee 5259 P= VON wae oe: 7010, 7011. 54 22.|) 5 see 4 eee 6654B— 3.2.55. - 22. TID ves Ge 23 Ven recs |\ a cc's - 6659U 1b ee MB0DEr Eee 28 DP) omtonitces || ico. 66's 1 DD ee ee CODE Voce 11 Boo, 22 eee ON (ee Ga is 7368, 8491 43 142]. sae Se eee 66605 = OE een (BXksee ace 21 (ha eres |e Soa c =D a aes WOU as Soak 33 130 ee | eee Total, 8 progenies............. 293 105} .n sees | Sees 150 F, x IVa Ta) (IIa) 6659-19 x 6691-8. .| 7339...... 14 9 14 Aan Seas: - IL 6660- 3 x —8..| 7340.:.... 9 11 12 Mia | eee 2a F, x VIec 2 | 6654A-2 x 6690-.9] 7335...... 20 15 13 LO ec B-1 x SUA (RBOs Sacic 15 26 23 Uta ene. Total, 4 progenies............. 58 61 62 (Olean. ae | TABLE 42. F: PRoGENIES OF THE Cross DiLtutTE PurRpLE IilJa x Green IVg Pedigree nos. Number of F; plants Group Dilute Dilute Cron IT Fy F, purple sun red | Illa roe | ae | il DADS ese ehe es aA sstpgehcas 2962-2966...... 23 8 10 DA 20 aileron cases ceene acne ey 2904, 2905 17 5 9 nO A Ae nk Sp ene ar 50388-5041...... 63 26 34 DIG (soe ee ec aa 6826, 6827 36 8 17 sl Bs Caen ich es Ceres 6828, 6829...... 78 32 31 2 D263 Ay teen saat clan eee el eyals 6669, 6670... 31 16 8 BD67— De. Mace ee ce 6675-6678...... 41 14 27) SDR VO dis? 6679-6682...... 62 12 22 a | Motales/\ progenies say nena eee eee ce 328 113 148 Fi x IVg 3 1322-3 x 7317-4...........}| 8210-8213. ..... 46 45 86 i | Group 1A Puant Coxtors In Maize 151 Pedigree nos. [TABLE 43. F; anp F; Procentes From F; anp EquivaLent F; Dinuts Purpues, DinuTe Sun Reps, AND GREENS, OF THE Cross DituTeE Purpue Illa x Green IVg Number of F; and F, plants Dilute Dilute F, and F3 F; and Fy, purple sun red Green IIIa IVa (Ig, IVg) 2966- 7...... S04G- 5050 Re. eee 5.05 ade ess 50 i) 26 5049-25...... GSIG=6818; (44s 5. ssa r. 52 13 20 5050- 6......] 6822-6824, 7058, 7059........ 41 10 27 Motalosprozenlesen. its. ccs ses) 1 eae e: 143° 48 73 6676-12...... ioCOb ae rec ae 26 10 16 6828-12. ...:. UGH ATGE De eneeecneed § oa 14 4 7 Motale2sprogeniesia.. icloacciec csc cect et ate 40 14 23 KU SP7 so ccocee 6825, 7323, 7442............. 27 CO) es ee reat 6676-8....... [SSD NZ OOhet oar cscs Ocehe 55 18) Hae 8 Oe Motales2sprozenlessi.. s.autaack take es bs ewes 82 PABA Ne tener che (Ig) 5049-37...... GSIOEG Vlas a cecsaoteeae tak ODF Maree tar eae 16 . (IVg) 2905-22). - 5... DIAN D5 OUR aS NG SE ee oe | ena ee eae 21 9 KOR = Io pogee Cava, GOI Go eden to dbdavsl: concdace 42 13 ARO tal PB LOR NICS. he cx via ia etd ciae eyesn sien || veisiats nie et 63 22 D050 ls a (CoE ck Bia tater HOS oro eee el (Uae ANC We Werciiee ate ¢ 5054-10...... GWA G24 OvBoococasso esse sels gos. OSH iayeteens as 0050> 2). 555. GSR SUD ese ieae etsy seer a ve, al| ester tte toasts Oey a pie ce, =o Dace TiA US Sea em sy fee gee ee cee Uieoneta ee erat QA sate eek kd mG tealleRAy TOP CNICS sc. csr scsi: aie sushoue wan a seeeoeen oll soie bec cohe IES eaten oar (IlIg, IVg) 2905- 5...... DAS i DAA atic Apis ia a patente ues itil lla. iee re 5 =19. 22.5. DAD MOA Ol cetemeay hucralerstennteecal le hema ancia eel remiss 13 5049-13...... GOS COMA reefs wees clare tre i|lhcheecypucees. cialis seeucetianaee 11 D052= 93)... OSSOtR Etat Oe ter miote: germane A, coat 24 =. Ge aes PLY Bek BS A ca IS CITES) alc PRR Ecker et ann Le e-aes aoe 15 21/7 (LoS ios 231 gs alah OHSS eBictiesea Enel Merete es veer a manne 32 6829- 9....:. MOSS ATG OTT inci aa e ay legen |NO2. acannon ph lea teen ae 30 SRO Cale MLOPENICS ashok ens tcc rena) elucesl coeeat hanks Lal] eau Sets or eel aR IE uM 130 152 R. A. EMERSON TABLE 44. F, PRoGENIES OF THE CRrOossES SUN Rep IIg x Green IVg, Sun Rep Ila x GREEN IVg, AND DituTE Sun Rep IVa x GREEN IVg Pedigree nos. Number of F plants Dilute Gasp Sun red suinet Green Fy FL EEE eee Pink Green Pink Green anthers | anthers | anthers anthers ; Ila Ilg IVa IVg AIST AG Ee G83) 60848 ol, eee 129-|" ee ; 52 1 DISA eta Ape eae (003—0065 252 52 |hee ee 94 Ae eee 25 MotalisD progenies isc eteat es ae 2167) Se eee 77 Fi x IVg 7317-6 x 7318-4..... 8214-8217...... 22 31 24 32 2 7318-1 x 7317-4.....|.8222-8225...... 38 25 34 26 4x =Gitae ae | 8218-8221...... 45 34 47 51 Motalsouprogeniesece soa ae escr irae 105 90 105 109 520 (owe eee eee 66 71=66 745 eas | ee en eee 55 22 3 F, x IVg PLANT Coors IN Maize 153 TABLE 45. F, ProGcentes oF Crosses oF SuN Rep I]a AND DinutTEe Sun Rep [Va witTH GREEN IIIg anp IVg Pedigree nos. Number of F, plants Group ve ie Purpl ilute ilute Py F, Te i purple sun red Green Illa IVa IIa x IIIg O09 bx AT59—25,...|- 0010... e022 Baal ae ea imal atic | cA Ba 7357-3 x 7356—- 1...] 8151, 8152...... Ray Fortes St esl Le A uate rl (te Geman 1 —_ — ——_——_ Total, 2 progenies.........02.+2.----- GAR ite cee ae A ecco ae tet IVa x Illg | A9-22 x 7097-1..... TETRO eeeet te Recerca a CT a eeeatgeed Geeoie | iets eee ee IVa xIVg . GSGOSRSexaGSG9 = NTS sco aa a eee) Ma DSal merece nats A9-14 x 7060-1... .| 7708, A283, A284) ...... ] ...... GML Wh sais Pointe MO talmeAwOrOTENIeS alse er heise wer ueeoes sees | [iG ams SOREN sterner: IVa x Illg 6860-13 x 6871-39...) 7714...........| ...... 11 UP) ass See cmer eats 2 GSGlSeZexeOol— de. ile eer. es || aa cae 9 Tks | Sess ese emer NO tala nprobeniesi.c. 52:05: can-ae Sass ae oi) stds oe 20 550: sey berets eae IVa x Illg | (IlIg, 1Vg) 6861-4 x 6882=5.....| 7512, 7513, 7716.) ...... 25 11 34 MSI SPOOL Ne (20 M20. es | a ee 44 43 72 MotaleeA progenies: a..c... 0.8 ascend es eek 69 54 106 IVa x IlIg (IIIg) 7312-8 x 7313-2..... SUISSE e cena sll ucesser ae Sir eer create 92 7313-2 x 7314-1..... CS evs Ee wid lolhaatcnsl Mnniiavenete UN rate Seats 19 3 7314-1 x 7313-1..... SZ00RSZ0 Ere allen : 2 Giitieer aes s.sto 129 —6 x oar SIS presare nar pra ltccas eewe Shuler ear 98 154 R. A. EMERSON TABLE 46. Fs PROGENIES OF THE Cross GREEN IVg x GREEN VIc Pedigree nos. Number of F, plants Group Dilute F, bee sun red Green IVa By GABA Me Se retoh loc ine crePet te esoueere soy Se asvede G/9OUNCTOZES ee 35 GHSOSN Ss eee sheer seis chete munsloneysi he aici TUCO TAR so ose 51 Pee se ey enh OPEN ee CAMS TAME Se 5 32 Gis Aat ne eh ean S eacrone pono b oT OG Wi Saad sc: 64 1 POO ele eae San sore eRe OWS: TA Dis so bo 63 MOSQUE seen ceniece =, soasverstfeyeteeiste arerctoheiere TASS (ANGE oe oe 52 MOBGH OMe See Stace ens BOE SCTE TAOON Altace a 60 COSTE DIET OR es Ci Snkas ciara ee CATA TAPS oe 5.5: 63 Rp Siisierovence ol ee ee 420 F, x VIe LOB 22TORGS 18-42 ao cece THO U1eWscanc « 24 7034-5 x SAD a eee a TUN, CUO Sco ac 42 otal“ Deprowenies so. sai wciecaeie res a iostiers Wea Pace ee 66 2 ——— eee F, x Vg FURY es <7(0) AS Aree A oa clolceakaat aicemeS TAUB TA eke ss Bs 48 MOAQE exe OB THAN pos stiches suai een CURE CAWAS dis 33 48 Totaly?) PrOgeniess 25.04 asc s cok ee ee ee 96 TABLE 47. F; Procentes oF F, Ditute Sun Rep PLaNnTs OF THE Cross GREEN iVg xX GREEN VIc Pedigree nos. Number of F; plants Grou ae | Dilute | Green F, Bs sunred | [Vg, VIc IVa Gi QS sSie ce eee een eR ee 7148, 7149 23 19 GLOZ=HO MA ety cites ene eee arenes | 7154, 7155 69 45 1 ST Meas Se oh spels rats alla eenenl een me oh FAS! Naeem > 16 13 Motals3 sprOgenies: essa eit ee ee OLE ee 108 77 (QTD PMP rae nes SS Se ee (1505 (Slee 65 23 SO ee. EemeNeS t eRMEER arteetenea: (AURA Alay 49 18 SY AYO FOR. eae ee Re oe nS et Aa LOT Aen Sots 38 12 2 =O RSE ee aoe LBS sent ar eee 23 10 Pe a ues ia sonnet cee nn ere | AGO sae eee 12 3 Total, '5 progenies. «665s. Se ee eae ee een ee 187 66 CoE) 5 ee aa an ene re SO Ls GH56.8 = Boe AS ee ei 3 SD Gah ut Skee ae a en ae HUGS ase eee 30)5| Reece Totals 2s progenies). 00st 5.0) a ss Be A ee 78 IS, cl i PLANT Cotors IN MAIzEe 155 TABLE 48. F, aAnp F; PROGENIES OF THE Cross GREEN IVg x GREEN Vla Pedigree nos. Number of F, and F3 plants G eee Dilute F, F, Sun red sun red Green IVa (IIa, g) (IVg,VIa,c) WVU = D. ke a Ae eee 2902, 2908...... DV 15) sola Seas a ee 4838-4843 ....:. 36 3 18 DP} s SOG Eee 4830-4837...... 99 15 57 1 OA MRR GO ois setae 4810-4813...... 88 32 59 = O55 Coa 4814-4817...... 111 20 62 Ol 3s Gale sae re een 4818-4821...... 92 30 45 DOP ora tri ceeENe e 4930, 4931...... 153 58 102 Motaleemproveniesiss 2. sorta. ca qos nelah 586 161 348 F, F; 2 AGB Qeopur ean ae Ai oi a nse 6991, 6992...... IU) S| reas ae ery hel [neers aeons (IIa) (VIa) DAUR +O sale oA ee ee 4783-4786...... GOR Rite years 32 ACEO OP). s 5 Sts eo 6993, 6994...... LSS | eee eters 39 3 Motale2nproremles|:: 2.45 ose atrksek aes bale DD OF iaeaena: are: 71 Fy x IVa 2903-2 x 2889-38.......... ; | 156 R. A. Emerson | | | TABLE 49. F> PRoGENIES OF THE Cross GREEN IIIg x GREEN VIc, AND F, PROGENIES OF Crosses OF F; GREENS WITH SUN RED Ila anp DituTE Sun Rep IVa Pedigree nos. Number of F plants | CRDi PucolellSu aed Dilute | Dilute Green iD F, ie 7 purple | sun red | IIIg, IVg, a a Illa IVa Vib-c 4 D90ES eee hee 52915-52987 4c een | bese 28 11 38 D202 = OM cere separ 7085, 7086, 1 CPPAINGS| scnsed || see Ss 81 26 97 Motal2 prozenless. +e. sko ate |e eee ele 109 37 135 12 F, Number of F; plants IlIg x Ila 7085-10 x A159-24.| 7717....... VA a ree te (Se csccs || occoeasos 2 IlIg x IVa (UVslterrs< Ales kal (Aisa sescaiiesaoes lledesce 14° |3 2 a ee os -3 x So ere te al )eichaeemisteres I gotchas ee eee maa 25 =| 22 ee ae Mo taile2 progenies cic ae ciaeecue sen | eee ele 39: | = =e ee ae Iilg x Ila 7086-6 x A159-17..) 7718, A298, i 3 A209 Tea: 28 4l | 2... 0 ee eee Ig x IVa 1086-4110 2E Sea lO ee a ests | eee 11 Oe ase sae: IVg x IVa 4 CAtsles eA anal PASS Sasol) Genes ll aewe: PP | cis egret | MEMOIR 39 PLATE I A. C.W. Redwood ANTHER, GLUME, AND RACHIS COLOR OF PURPLE 1, Purple, type Ia, typical, anthers purple; 2, type Ia with r¢%, anthers near- black; 3, type Ia with pr, anthers reddish; 4, type lg, with RY or r¥, anthers green (Drawings by C. W. Redwood, somewhat diagrammatic) MEMOIR 39 PLATE II A CW.Red vrood ‘ ANTHER, GLUME, AND RACHIS COLOR OF DILUTE PURPLE AND GREEN 1, Dilute purple, type IIIa, typical, anthers purple; 2, type IITa with 7°”, anthers near-black ; 3, type IIIa with pr, anthers reddish 4, Green, types IIIg and IVg with RI or r¥, green thruout (Drawings by C. W. Redwood, somewhat diagrammatic) MEMOIR 39 PLATE III ANTHER, GLUME, AND RACHIS COLOR OF SUN RED AND DILUTE SUN RED 1, Sun red, type Ila, intensely pigmented form 2, Dilute sun red, type IVa, intensely pigmented form; 8 and 4, near-green forms, little color in glumes, anthers green with reddish stippling as shown in enlarged anther (Drawings by C. W. Redwood, somewhat diagrammatic) MeEmorr 39 PLATE JV CW -Radwood ANTHER, GLUME, AND RACHIS COLOR OF BROWN AND GREEN 1, Brown, type V, intensely pigmented, homozygous form; 2, type V, less intensely pigmented form, heterozygous for B or PI or both 3, Green, type VIc; 4, type VIb, green with tinge of brown due to Pl and r‘/ (Drawings by C. W. Redwood, somewnat diagrammatic) MEMOIR 39 PLATE V CULM, HUSK, AND SHEATH COLOR OF PURPLE AND SUN RED 1, Purple, type Ia; 2, weak purple, type Ib 3, Sun red, type Ila; 4, weak sun red, type IIb; 5, type IIb, inner husks of lower ear highly colored from exposure to sunligkt directly after being torn apart (Drawings 1 and 3 by C. W. Redwood; 2, 4, and 5 by Bernice M. Branson) ee PLATE VI MeEmorR 39 CM:Redwood HUSK, AND SHEATH COLOR OF DILUTE PURPLE, DILUTE SUN RED, BROWN, AND GREEN 1, Dilute purple and dilute sun red, types IIIa and IVa; 2, more highly colored form of types IIIa and IVa CULM, 3, Brown, type V 4, Green, types VIb and VIc; 5, type VIa, with some brown in outer husks due to B (Drawings by C. W. Redwood) PLATE VIL Memoir 39 Xj em image he MATURE CULM, HUSK, AND COB COLOR 3, dilute purple, type IIIa: 4, 1, Purple, type Ia; 2, sun red, type Ila; 3, brown, type V; 6, intensely pigmented form of type Illa; type IVa (Drawings by Carrie M. Preston) more dilute sun red, oo, Mermorr 39 PLATE VIII DEVELOPMENT OF COLOR IN DARKNESS Tassels and sheaths developed under black paper bags: 1, purple, type Ia; 2, brown, type V; 38, dilute purple, type Illa; 4, sun red, type Ila, no red color (Drawings by Carrie M. Preston) ‘W ootudog Aq 1 SUIMVACT) JOS offptoyur Ur UMOLS Jue ‘Z quyyd ‘fy : BAT ods} ‘por uns aynTTp Jo sqyurypd sunox (U0}SoId TW Oltny Aq | + WOSTBUT {IOS o[Taoy UL UAOLS TNINAOTIATAG WOTOO OL ALITILAAL IWOS LO NOILV TU XI divid 6 WIONWATL MeEmorr 39 PLATE X COLOR DEVELOPMENT IN BROKEN LEAVES 1. Dilute sun red, type IVa, about one week after the leaf was creased: 2, dilute purple, type IIIa with japonica white stripes, about three days after the leaf was creased (Drawings by Carrie M. Preston) ae i nt Ate io eae a Nes Evy Memoir 39 PLATE XI ABERRANT COLORATION OF BROWN TASSEL Poorly developed tassels of brown, type V, sometimes exhibit purple in abnormally developed parts (Drawing by Carrie M. Preston) JLY, 1921 MEMOIR 40 CORNELL UNIVERSITY AGRICULTURAL EXPERIMENT STATION [BERATION OF ORGANIC MATTER BY ROOTS | OF GROWING PLANTS T. L. LYON AND J. K. WILSON ITHACA, NEW YORK PUBLISHED BY THE UNIVERSITY CONTENTS PAGE Review of literature concerning loss of nitrogen by growing plants... 7 meee ol thespresent experiments. .... 006... 6. eee Se ec eee 14 | _LETLAOG! WSSGLS . 5. 5 cae IRE Bem ee em 14 re etlans metal lite el Onlemp aye see sire sc haces ssa ec cic sh ene 16) ERC ananione@iethe TASKS .:. ..% incase 2 de aod nue Gielen bo ee es 16 Ree TRGOMOTOM 5 so osc fake hc ak ol a ge Ry Ok eee 16 Seunugecotathe plants. ..........<..8 Nie Date tee ar rece ne et A ne i Rieiwyestmmenune plants 6-80.05. sss wes ele nae peso eb betas 17 Analysis of nutrient solutions and residues in bottom of flasks.... 17 Tests for the presence of organic nitrogen in nutrient solutions in which miamcromseveraiukinds had grown. -). 006 000.¢. 06052. 18 Organic nitrogen present in nutrient solutions at successive stages in STMT OUAMOMIMMNALZOR 562). 2 sss s- 5 Ss eects Sa ae ae es ee ets D8) Total organic matter present in nutrient solutions in which maize Pernice ar bOmO wi Mr et) eo an eee ie ie ee eee oie 31 Reducing and oxidizing substances liberated by plant roots. ........ 34 BePivenemmotmliccratune <..% << 8) s.cs asec ences cn cede es 35 Tests for reducing and oxidizing substances liberated by plant TROOUESIs o 9.0" DRO Ga IBG EON: or ESTAR Shearer ae ar dem eee 36 ved M@elmeesUSlAN CES east or eos sence sti see ee ee AX VeEGUCHOMVOL MILTATES. a Wa. ine ee Ns ak eee: OM Oxtaizmorsulstances eco ps Aen ence ek alas jaa 39 Possible coexistence of oxidizing and reducing substances... 40 Nature of oxidizing and reducing substances.............. A() LLELOSEP Sw oo 8 cach] eS ae ee ee ec reer cae ing org 40 —._LEVESRE CLANS 5g eae tp Pec Ne A ge ee 43 aie YOM A a & uh { f % a7 © BH Tom tase ana ; Pee NE tee LIBERATION OF ORGANIC MATTER BY ROOTS OF GROWING PLANTS LIBERATION OF ORGANIC MATTER BY ROOTS OF GROWING PLANTS AR IB Lyon AND J. K. WILSON In the course of an investigation under way at this station, it became desirable to know whether organic matter, particularly that of a nitrog- enous nature, is liberated by the roots of growing plants, at least of the plants commonly raised on farms in-this region. Numerous investi- gations conducted elsewhere have shown that nitrogen is lost by certain plants during the late stages of growth, particularly about the ripening period. Theré seemed to be a question, however, whether this nitrogen escaped from the leaves or the roots, and it had never been shown to be in the form of organic matter. The previous investigations had, indeed, not touched on the question of the loss of organic matter by growing plants, and aside from its bearing on the investigation in hand this appeared to be a matter of some scientific interest. A closely related question is the possible liberation of reducing or oxidiz- ing substances by the roots of growing plants. There had previously been some investigation of this subject, but since the experiments on the liberation of organic matter furnished exceptionally good opportunities for making the tests for catalysts it was decided to try to obtain some information to supplement the previous work. REVIEW OF LITERATURE CONCERNING LOSS OF NITROGEN FROM GROWING PLANTS A large amount of work has been reported showing the percentage of the various nutritive elements found in crops at certain stages of growth. Less work has been done, however, on the actual weight of nutrients in plants during their growth and maturity. The work of Wilfarth, Romer, and Wimmer (1906) was concerned with the assimilation of the elements of nutrition by plants during different periods of their growth. This investigation extended over a period of about eight years. The experiments included both field and pot work, the former with barley, spring wheat, and potatoes, and the latter with 7 8 T. L. Lyon anp J. K. Wixtson barley, peas, potatoes, and mustard. The plants were harvested at | different stages of growth. These plants were carefully divided into | their component parts — roots, stems, ears, and so on — and were dried, | weighed, and analyzed. The results as stated below are based on pounds — of nitrogen in the crop. | The barley was planted on March, 30 and the cuttings were made on _ May 29, June 17, July 3, and July 27. The plant parts were separated _ and grouped into aboveground parts and underground parts. Separate | analyses were made of stems, green leaves, yellow leaves, ear stalks, awns, | straw, grain, roots, and stubble. Total weights of nitrogen showed that this nutrient was present in its greatest amount on June 17 (presumably when the plants were in bloom) and that the mature cutting of July za | had lost 25 per cent of the total nitrogen. The wheat was planted on April 23 and was harvested on June 22, | July 14, August 5, and August 28. The methods used were the same as | for barley. In the case of the wheat, the nitrogen was found in its greatest, | amount in the third cutting, on August 5, and the mature crop showed a | loss of about 20 per cent of the total nitrogen. The potatoes were planted on April 28 and analyses were made of the | various parts of the plant. Four different harvests were gathered as the | crop was maturing. The results were quite different from those with — barley or with wheat. In this case the greatest amount of nitrogen was © found in the last harvest, which represents the crop gathered in October. The barley was planted on April 20 in sand in pots, and was watered | with nutrient solutions. Quadruplicate cultures were grown in the green- house. On May 11 stems had commenced to show. The first harvest was made on May 24, the second on June 1, the third on June 12, the fourth on June 25, the fifth on July 20. The harvest consisted of both | roots and tops. In one series the greatest weight of nitrogen was found on June 25, in the other three on June 12. From these dates on to maturity there was a loss of total nitrogen ranging from 9 to 26 per cent. | The peas were planted and tended in a similar manner to the barley, with the same dates for planting and harvesting. The weight of nitrogen’ in the harvested crops was greatest on June 25 in three cases, and in the: fourth case at the last harvest. The decrease toward maturity in the three cases ranged from 9 to 30 per cent. LIBERATION oF OrGANIC Marrer By Roots oF GROWING PLANTS 9 The potatoes were grown in turf and sand in the greenhouse. Applica- tions of fertilizer were made so that plant nutrients would not be deficient. Harvests were made on June 12, June 30, August 7, and September 14. The plant parts were divided into foliage and tubers. The figures for weight of nitrogen show that there was a constant decrease of this con- stituent in the foliage after the first harvest, altho there was a constant increase in the weight of the tubers. The total plant, however, had its greatest amount of nitrogen on August 7, with a decrease of 6.46 per cent thirty-eight days later. Pots containing 5.3 kilograms of dry earth received fertilizers to stimulate growth of plants and to furnish an abundant supply of nutrients. The seeding of mustard was done on May 7, and subsequently all pots con- tained six plants. The experiment was run in duplicate. The mustard was harvested, first, on the appearance of the first pods, second, when the formation of seed was complete, and third, at maturity. Analysis of the total plant only was made. The total nitrogen was greatest when the formation of seed was complete. The loss at the third harvest was about 10 per cent of the total nitrogen. With the intention of verifying the results obtained by Wilfarth, Romer, and Wimmer, André (1912) cut barley at five different stages of growth from equal areas of land and analyzed the dry harvest. The cuttings were made (1) when the heads began to show, (2) when the barley was in bloom, (3) when seed began to form, (4) at the mature stage, and (5) beyond the ripe stage. The figures for total nitrogen show 7.023 grams at the first stage, 8.693 at blooming, 10.422 when the fruit was forming, 12.589 at maturity, and 10.360 at the last harvest. The object of an experiment by Ramsay and Robertson (1918) was to determine the relative proportions of each of the principal nutrient elements contained in the plant at various stages of growth. They grew potatoes in soil in boxes containing about 130 pounds of well-drained and fertilized soil. Approximately the same weight of seed was put in each box. The first harvest was gathered on January 29, thirty-three days after brairding, the second on February 25, the third on March 26, and the last on April 30. At the first three harvests complete recovery of tops and roots was |made. The last harvest was more difficult and about 30 per cent of the roots were lost. Cropping, harvesting, and analyzing were done in dupli- eate in each case. The total nitrogen contained in a 20-ton crop of 10 T. L. Lyon anv J. K. Witson potato plants at various stages of development was 69 pounds at the first harvest (thirty-three days), 241.3 pounds at the second harvest (fifty-eight days), 306.7 pounds at the third harvest (eighty-nine days), and 319 pounds at the fourth harvest (one hundred and twenty-four days). There was a constant assimilation of this important element. Hay was cut by Crowther and Ruston (1911-12) from uniform areas of a crop of grass which had been seeded the previous spring. The grass seed consisted of a mixture of perennial rye grass, Italian rye grass, white clover, trefoil, alsike, English single-cut cow grass, Chilean red clover, and rib grass. The first cutting was made when the rye grass was in full flower. A good growth of leguminous plants showed under-. neath. The second cutting was made when the rye grass was forming | seed and the clovers were beginning to flower. At the third cutting the | grasses were ripening and the clovers were in full bloom, while the fourth | and last cutting was made when the crop was decidedly ripe. Analysis | of the crops showed the greatest total weight of nitrogen to be present, at the third cutting, or when the grasses were ripening and the clovers were | in full bloom. In the last cutting there was a loss of 25 pounds of nitrogen | to the acre. The work was repeated the following year, but with barley as the crop. The seed was drilled on May 12. Cuttings were made on June 9, June 23, | July 7, and July 21, but the stages of growth reached on these dates were not noted. The changes with advancing age as to nitrogen were similar to those observed in the preceding year with grasses. The changes in chemical composition of the timothy plant during growth and ripening, with comparative studies of the wheat plant, are. recorded by Trowbridge, Haigh, and Moulton (1915). Timothy plants, cut from uniform areas, represented the following stages of growth: (1) about one foot high in rapid growth; (2) no heads showing; (3) no stalks! in bloom but beginning to head; (4) in full bloom; (5) just out of bloom and seed formed; (6) seed in dough; (7) seed fully ripe; (8) growth the following spring not yet started but leaves green. The plant parts of the samples thus collected were divided into heads, stalks and leaves, hay, and stubble and bulbs. The amounts of the various nutrients deter- mined were recorded in total pounds to the acre. Data from these plants were collected for one year only. The figures for weight of nitrogen showed that there was a gradual increase in this constituent in heads from re LIBERATION OF OrGANIC MaTrer By Roots oF GROWING PuLANtTs' 11 all plots. In the stalks and leaves, the nitrogen was found in greatest amounts in two cases when the plants were just out of bloom and the seed was formed, and in the third case when no stalks were in bloom but the plants were beginning to head. In the hay the greatest weight of nitrogen was found when the seed was all in dough. In the stubble and bulbs the greatest amount of nitrogen was present when the seed was fully ripe. The comparative studies with wheat represent two areas from the same field. The stages of development were: (1) plants green and in bloom; (2) seed formed and in milk; (3) seed in dough; (4) seed fully ripe.- The plant parts of the samples were divided into heads, stalks and leaves, plants above ground, and roots and stubble. The figures for nitrogen found in the heads showed a steady increase in this nutrient thruout all the stages. With stalks and leaves there was a constant loss of nitrogen after the first stage as the plant approached maturity. In the plants above ground there was an increase of nitrogen to the seed-in-dough stage and a considerable loss in the fully-ripe stage. Roots and stubble con- tained their greatest weight of nitrogen at the seed-in-milk stage. If the ‘total plant is considered, the nitrogen reached its greatest amount when the seed was in the dough stage. _ The influence of advancing maturity on the composition of timothy ‘was reported by Waters (1915). Results were obtained for five years of investigation, but the results are complete for only three of these years. Uniform areas were harvested at five different stages of growth: (1) when the plants were in full head; (2) when the plants were in bloom; (3) when the seed had formed; (4) when the seed was in dough; (5) when the seed was ripe but not shattered. The greatest weight of nitrogen was found in three of the trials when the plants were in full bloom, and in the other two trials when the seed had formed. The fluctuation in loss of nitrogen between the stages when it was at the maximum and when it was fully ripe ranged from 12 to 38 per cent. Schulze (1904) cut 100 plants of rye and collected the roots for examina- tion. The cuttings were made forty days after drilling, which was on September 20. On April 22 a second cutting was made. The plants at \this stage were very green. On May 20 a third cutting was made. The plants now were in the boot stage. The fourth cutting was made on June 16 and the plants were just thru blooming. The weight of nitrogen 12 T. L. Lyon anp J. K. Witson in these stages was as follows: first cutting, 0.153 gram; second cutting, 1.225 grams; third cutting, 2.723 grams; fourth cutting, 2.713 grams. With wheat only three harvests were made: the first was thirty-seven days after drilling, which took place on September 23; the second was on April 22, and no heads were showing at this date; the third was on June 16, when the plants were thru blooming. The nitrogen in 100 plants was as follows: first period, 0.132 gram; second period, 0.596 gram; third period, 1.802 grams. No later analyses are given, and thus it is not clear whether there was a loss in weight of nitrogen after the blooming period. Le Clere and Breazeale (1909) state that the loss of nutrients from plants may be explained in one of three ways: (1) by the backward flow of the salts of the plant juices thru the stems and roots to the soil; (2) by the decay or dying and falling off of leaves; or (8) by the action of rain, dew, wind, and other climatic agencies. Or there may be a combination of all these causes to a limited extent. In support of the third possibility, these investigators conducted experiments designed to imitate these climatic agencies. Barley plants were grown in soil contained in Wagner pots, and no water was allowed to come in touch with the aboveground parts during the growing period. Just at the heading period the whole plant was harvested, placed in a large evaporating dish, and soaked with water for several minutes. After drying, this operation was repeated. The plants were then dried and analyzed. The results show that 1.6 per cent of the entire content of nitrogen was lost on washing. Wheat plants were harvested at three periods of growth — bloom, early ripeness, and full ripeness. The plant parts were divided into stems and straw, and heads. They were separately washed or soaked for from five to ten minutes. The wash water was analyzed, as were also the dried stems and straw and the heads. The results show that at bloom 1.4 per cent of the nitrogen was washed out, while at full maturity 7 per cent was found in the wash water. Results were obtained also from apple twigs. Two twigs containing green leaves were gathered and washed for a few minutes with distilled water. They were then set aside, with their butt ends immersed in water, until the leaves were unquestionably dead, when they were again washed and analyses were made of both washings and residues. The | results of the analyses showed that thru the action of water about 3 per | cent of nitrogen had been washed out, LIBERATION OF ORGANIC MATTER BY Roots oF GROWING PLANTS 13 Two pots of wheat were kept in the greenhouse until the wheat was completely ripe. They were then placed out of doors, where they were subjected to four rainfalls on separate days. The pots were so arranged that the washings were caught in a tray. These washings, equivalent to about one inch of rainfall, dissolved from the plant 27 per cent of the nitrogen as well as other nutrients. Two oat pots were placed out of doors as were the wheat pots. The plants were about eight inches high. They were allowed to grow in this position until they were ripe. They were exposed to three rains during this time. The leachings contained 2 per cent of the nitrogen, as well as other nutrients. In two pots of potatoes, the aboveground parts were washed with 2.5 quarts of water in a very fine spray. This was done when the plants were twenty-four inches high, when they were beginning to ripen, and when they were completely ripe. The leachings and the plant parts above ground were analyzed. The results show that, due to the action of washing, 7.5 per cent of the nitrogen was washed out. Jones and Huston (1914) report the composition of the maize crop at stages corresponding in the main with those at which the crop would be used for practical purposes, such as soiling, ensiling, and grain pro- duction. Conditions of uniformity were maintained as nearly ideal as it was possible to have them. In order to insure adequate moisture, the field was irrigated when necessary so as to provide not less than one inch of water every week. The soil was in a good state of fertility. Analyses were made as follows: (1) when the plants had six leaves, June 16; (2) when the plants were about four feet high, July 24; (3) when tassels began to form, August 6; (4) when the maize was fully pollinized, August 28; (5) when the plants were in the medium milk stage, with the pollen all shed and the silks brown, September 10; (6) when the kernels were glazing, September 24; (7) when the kernels were hardening, this being the ensiling stage, October 1; (8) when the maize was ready to put into shock, October 8; (9) when the maize was fully cured and ready to be stored, November 12. The samples represented the crop cut at the soil level. Data for weight of nitrogen showed a gradual increase from the first analysis to October 8, the amount at first being 0.28 pounds an acre and increasing to 110.6 pounds on the last-named date. At the last analysis, on November 12, 14 T. L. Lyon anv J. K. WiLson which represented samples left in the field and in the shock, for the former a loss of about 23 pounds was shown, while for the latter a slight gain was reported. This loss of nitrogen when the plants were left in the field after October 8 was from both ears and stalks. In the former there was a loss of 9.2 pounds in the field and a gain of 7.6 pounds in the shock; in the latter there was a loss of 15 pounds in the field and a loss of an 2.1 pounds in the shock. Taking the results of these investigations as a whole, there appears to be in nearly all cases a loss of nitrogen from grass and small grains at some time between the period of full bloom and complete maturity. In maize this occurs if the plants are allowed to ripen when connected with the roots, but potatoes showed no loss of nitrogen in the Bernburg and Australia experiments, and only a small loss in those of Le Clere and Breazeale. OBJECT OF THE PRESENT EXPERIMENTS The experiments herein described had two more or less distinct objects’ The first was to ascertain whether growing plants liberate organic matter’ and, if they do, to determine at what stage of growth this takes place and what relation it bears to the absorption of nitrate nitrogen by the plant. The second object was to detect, if possible, the presence of reducing and oxidizing ferments in the nutrient solutions in which the plants used for the first investigation were grown. METHODS USED The plants used in these experiments were grown in water culture. This was done in order that an intimate study might be made of the plant as it grew and the solution as it was being drawn upon by the plant for various nutrients, especially nitrogen. Since a number of organic bodies may be given off by the developing plant, these may not be present when the nutrient solution is analyzed if it is allowed to become infected with molds or bacteria. Therefore, in order that this solution should represent the action of the plant alone, a method for growing the root system in contact with the nutrient solution without infection was used. This method has been published, together with data showing its reliability (Wilson, 1920). A description of the method follows, SSS LIBERATION OF OrGANIC MaTrer BY Roots of GRowinG Puants 15 SEED STERILIZATION Seeds were rendered sterile by the calcium hypochlorite method as employed by Wilson (1915). After disinfection the seeds were planted on a sterile medium, from which, after germination, the plants were transferred to the perma- nent position. The solid medium for the germination was usually composed of the same ingredients as were used in the large con- tainers, from which the plants eventually drew their nutrients, with from 1 to 1.5 per cent of agar. The agar was used in order that contaminations might be detected before the plants were transferred to their perma- nent position. This medium was made in sufficient quantity to meet the requirements and was distributed into large test tubes. Since the roots of most plant- lets spread out in a lateral direc- tion, thus making it difficult to transplant them quickly and con- veniently, some device was needed which would direct the root growth in a vertical direc- tion. To accomplish this there was placed in each test tube a short piece of glass tubing, 25 by 50 millimeters in size (fig. 1, e). A sufficient quantity of the medium was put into the tube to cover all but about 15 millimeters of this glass tubing. After ster- \ (4 Rd (thts | Se tk ole cl le e@ eel Vi a 7 Fig. 1. DEVICE FOR GROWING LARGE PLANTS IN STERILE MEDIA a, Cheesecloth; 6, cotton wool; c, outside tube into which e slides; d, agar carried over from test tube with plantlet; e, tube in which seed germinates ilization of the medium the sterile seeds were dropped onto it, where they germinated and produced rootlets for subsequent use. When the roots 16 T. L. Lyon anv J. K. WILson had developed and passed thru the agar to the bottom of the glass tube, the plantlets were transferred. The tube (fig. 1, e), with agar and plant- lets, was lifted out of the test tube and set into the mouth of the prepared flask. This was accomplished with sterile forceps. PREPARATION OF THE FLASKS The flasks used were in most cases of 8-liter capacity, altho some held 12 liters. As mu¢h of the nutrient solution was put into each flask as could be sterilized. A piece of cloth was stretched over the mouth, and a hole just large enough to permit the insertion of a piece of tubing from 28 to 30 millimeters long (fig. 1, c) was cut in the cloth. This tubing (c) was just large enough to allow the plant and its tubing (e), described above, to telescope into it. The lower end of the tube (c) was slightly constricted, to prevent the inner tube (e) with the plant from going too far into the flask. Enough cotton (6) was wrapped around the tube to hold it firmly in place when it was pushed into the mouth of the flask. The upper end of the tubing was adjusted so that it came about even with the top of the flask. The lower end extended below the cotton and the cloth (a), so that when the flask was full of liquid the liquid would just touch the tubing but not the packing. Each flask was then covered with a large beaker. The inverted — beaker was large enough to leave some space for the growing plant. | Since there was need for more solution in the flask than could be sterilized — in it, an extra amount was prepared which could be poured thru the opening that was left. | | NUTRIENT SOLUTION The nutrient solution had the following composition: Calcium nitrate, Ca(NO3)2.+4H,0............ 2.70 grams Magnesium sulfate, MgSO.1+7H.2O........... 0.60 gram Pocassiumarc iil orien © lise een ee 0.75 gram Potassium dihydrogen phosphate, KH:PO,... . 1.50 grams Rerricysullate: Shen(S@s)oe erate en a ee 0.05 gram Distilled water topmakey- 39 eae 1000 cubic centimeters This was designated as the full nutrient solution. In most cases of actual use it was diluted to ten times this volume. In earlier work it had been } noted that certain plants, especially maize, tend to become chlorotic | LIBERATION OF ORGANIC MatTrEerR BY Roots oF GRowinG PLANTS’ 17 before maturity. By painting the blades with a ferric chloride solution, it was found that this chlorosis was due to a deficiency of available iron. Therefore about 0.5 gram of ferric phosphate was added to nga container before sterilization. SETTING OF THE PLANTS Before the plantlets were set into the mouth of the flasks they were thoroly examined for contamination. Only the very best plantlets were used, and if properly handled they suffered no setback. In case the plantlets were large enough when placed in the flasks, they were wrapped in sterile cotton and the beaker was removed. In other cases several days were required for the plants to become large enough, and in this event the beaker was left on. HARVESTING THE PLANTS The plants were harvested in all stages of growth, as is shown by figures 5 to 9. The harvesting process consisted in bringing flask and plant to the laboratory, and, in most cases, first making a photograph and subsequently opening the flask by removing the plant and the cotton packing. When this was done the solution was examined for infection, which was determined by plating about 10, 5, 1/10, and 1/100 cubic centi- meters. The medium used in the plates contained, in addition to peptone and beef extract, some form of sugar, usually glucose. The plates were incubated at from 20° to 25° C. for a week, or longer in case no infection was noted. The results reported in the subsequent data herein were obtained from the platings in which no infection was found, and these flasks were considered to be sterile. ANALYSIS OF NUTRIENT SOLUTIONS AND RESIDUES IN BOTTOM OF FLASKS The presence of nitrite nitrogen was determined by Ilosvay’s modifica- tion of Griess’ test, described by Treadwell (1915). A qualitative test for the presence of nitrate nitrogen was made by the diphenylamine method (Withers and Ray, 1911). When it was present, ie total quantity was determined by the phenoldisulfonic-acid method, ‘slightly modified by the addition of 5 drops of a 0.5-per-cent solution of ‘Na:CO; before evaporating to avoid any loss of nitrogen which might be 18 -'T. L. Lyron ann J. K. Witson Fic. 2. OAT PLANT GROWN IN STERILE SOLUTION FOR 105 DAYS (Table 1, serial no. 1272) present in the form of HNO;. (The quantity of soluble organic matter in the plant solutions was not great enough to interfere with the proper nitration of the phenoldisulfonic acid.) Ammonia was determined by direct ness- lerization. Organic nitrogen in the plant solutions was determined according to the method described by the American Public Health Association (1905). The total organic matter in the solutions was determined by evaporating 100 cubic centi- meters of the filtered solution and igniting the residue thus obtained at dull red heat. The loss on ignition was reported as organic matter. The dry weight of the deposit in the bottom of each flask was ascertained by transferring it to a filter, drying it at 110° C., and weighing it, the weight of the dry filter being subtracted from the total weight. The amount of organic nitrogen in the deposit in the bottom of each flask was found — by transferring the filter and contents from the previous determination to a Kjeldahl flask, digesting by the Gunning method, neutraliz- ing the excess acid with ammonia-free Na,COs, and - distilling off the ammonia, which was nesslerized. . TESTS FOR THE PRESENCE OF ORGANIC NI- TROGEN IN NUTRIENT SOLUTIONS IN WHICH PLANTS OF SEVERAL KINDS HAD GROWN Several different kinds of seeds were germi- nated under aseptic conditions, and were trans- planted, in the manner described, to flasks containing the usual nutrient solution of one- tenth strength. The manner of growth of the plants in these flasks is shown in figures 2 to 4. a LIBERATION OF ORGANIC MATTER BY Roots oF GROWING PLANTS 19 Fig. 3. MAIZE PLANTS GROWN IN Fic. 4. PEA PLANT GROWN IN STERILE SOLUTION FoR 189 Days STERILE SOLUTION FoR 139 (Table 1, serial no, 1303) WEES, (Table 1, serial no. 1280) 20 T. L. Lyon anv J. K. WILson In one flask the usual nutrient solution was not employed, as it was desired to ascertain whether a leguminous plant grown in a solution containing no nitrogen would liberate nitrogenous matter in the solution in appre- ciable quantities. The flask used for this purpose was pea flask no. 1302, in which the nutrient solution was composed of 1.5 grams KH2POu, 1 gram CaCl, 0.07 gram NazSO., and 0.5 gram Fe,(POx)2 + 8H2O; the flask was then filled with sterile tap water, the Fe:(PO:)2 + 8H2O remaining largely undissolved. After the contents of the flasks were sterilized and the young plants transferred, the cultures were taken to the greenhouse, where they remained for various periods. It was not intended to make any systematic study of the relation of the stage of growth to the quantity of organic nitrogen in the solution at harvest. This would have been impossible under the circumstances, for the plants were set out at different times and, since conditions affecting plant growth vary greatly in the greenhouse at different seasons of the year, no comparison of this kind could be attempted. The same difficulty would obtain in case a comparison of different plants was desired, except in the case of such plants as were placed in the green- house at about the same time. | When it was decided to harvest a plant, the flask was brought to the laboratory, and, after a photograph had been taken, the plant was removed from the nutrient solution and the dry matter and nitrogen were determined in the entire plant including the roots. A plating of the nutrient solution was made to determine whether the solution was sterile. The presence of any molds or bacteria thus detected excluded from the experiment the flask so contaminated. -The volume of liquid remaining in the flask was measured, and determina- tions were made of the nitrate nitrogen remaining in the solution, the ammonia nitrogen, if any, and the organic nitrogen present in soluble form. The deposit at the bottom of the flask was collected and a determi- nation was made of the organic nitrogen contained in it. The reason for ascertaining the quantity of nitrate nitrogen remaining in the solution was merely to observe whether the presence or absence of this form of _ nitrogen affected the liberation of organic nitrogen by the plant. A small quantity of nitrogen was contained in the germinated seed and plantlet placed in the flask, but there was no way by which this nitrogen LIBERATION OF ORGANIC MaTTEeR BY Roots oF GROWING PLANTS 21 ‘qUSTUTIOdx9 9Y} JO SUIUUIZEq 94} 4% USFOIZIU pouTquUIOD OU poUTe{UOD UMOIS sem JuLld vod sty} YOIYM UT UOTINIOS JusTIyNU OY, x —— ————————————— OT 9'0 20 ZO G0 80 0010-0". 050500 0G DO 0010.0 O86 (SUrBast] tur) ysey JO W10}}0q 38 yisodep Ur UasOI}IU OLURSIC) bh r'¢ 0% O's ST STE me” ae Inet hata (SUIBIA[OL) 10948UI DTUSIO OUON £°9 Oz aoeL I, a0BL T, DEVE, cise jerce nemo ete leiielelenenelieusliieiceie (SUI'B.IST]] TUL ) ON ouoN, asuON ouoN 9°68 2° R61 7 a (sureasqyrar) 89 N ; JO WLIO} Ul JSOAIBY 4B UOTINJOS UL WIdOIZINE SEEMS SUES LAE UES ES SLB EATS 9]I49}$ SILLS S aay mel ere erat ae ee ae OTA ES 249) OEY OECD) 0 '062‘Z 0'0&6‘9 0° SI8‘9 0 018 ‘F 0 028'2 0'0¢8‘6 0 0s0 0° O'O-0. 0100! 06.0 o-Gn0-G800r0 56.0 0-0 (Saoqyouml1yU89 a1qnd) ysaarey 48 UOTyNTOS jo ouUMjOA 6 FFE f CLP 9 &F G 18 01ST GIS |e ae SUA T[ UT) ued Wl USZOIIN OOSO'SIE | OWRR 7 22° 0 €8"9 0 680'F A Ee. ee (SuIvasy[{ur) yuBd uy 109938 AT gal 681 6&1 681 col GOL ees ap [ik ae ak Ses (skep) porred Sutmo.ry Zl eune Z oune gg Judy 9¢ Tudy IL [udy ic ueahee fom ese Ysey wor yued Sursoured jo o3ecqy 0% ‘uUer I °9q 6 99d 6 99d 62 29d VeedasY a heme nee cae Ces ysey ut juevyd Suryyes jo 93%q S0ET SOT GOET OS growth of maize. As the periods of growth varied from a rather ‘ly stage, before the nitrate nitrogen was all removed from the solution, maturity, it would have been a very interesting experiment had it not on for the fact that the last two flasks opened were found to be con- ninated. The data, however, are all tabulated in table 4, altho there of course, no assurance that the results were not materially affected the organisms that had gained access to the solutions. he sterile flasks in this experiment agree with those in the experiment orded in table 3 in showing the presence of a large amount of organic tter in the solution in which the plants grew. The amount was less 34 T. L. Lyon anp J. K. WiItson for the data shown in table 4, but the growth of the maize plants in thel sterile flasks also was less. The quantity of inorganic matter and of organic matter appears to decrease and increase at the same periods if) credence is to be given to the data from the last two flasks opened. The TABLE 4. Torat Orcanic Matter AND NITROGEN PRESENT IN SOLUTIONS AT SUCCESSIVE Sraces IN THE GRowTH OF Maize iKandlofaplantivs eto e eee eee Maize Maize Maize Maize Serialinoie ys een eae eee ee ane 1 2 3 Ls Date of setting plant in flask.......... June 28 | June 28 June 28 June 28} Date of removing plant from flask. .... Aug. 3 Aug. 16 Aug. 30 Sept. 2( Growing period (days)..... meet ek see 36 49 63 84 | Dry matter in plant (milligrams).......| 10,200.0 | 24,100.0 32 ,200.0 65 ,000.( Volume of solution at harvest (cubic Centimeters eee eee cee 6,200.0 | 6,950.0 2,850.0 2,790.4 Condition*of solutions ..9m--< so ee Sterile Sterile | Contaminated Mold) Nitrogen as nitrates in solution (milli- j STAINS) ite percent ean as eee fe ees 32.4 Trace 0.0 0.6 Inorganic matter in solution (milligrams)| 1,333.0 | 1,056.4 310.6 460 <) Organic matter in solution (milligrams) 291.4 284.9 76.9 2518 data for inorganic matter were probably not influenced by the infectio1) of flask no. 3, and this shows a gradual decrease in amount up to th sixty-third day of growth and then an increase at maturity, indicating ¢ liberation of salts from the plant. Such liberation must, of course, hay) been by way of the roots. Admitting, then, that inorganic matter may be removed by the action of rainfall on the leaves, of which the investigation! of Le Clerc and Breazeale leave little doubt, there appear to be at leag| two means by which this material may be liberated by the plant. REDUCING AND OXIDIZING SUBSTANCES LIBERATED BY PLANT ROOT q While many investigations have been made of the oxidizing and reducin), enzymes in plants, very few have been undertaken for the purpose ( ascertaining whether any of these bodies appear in the substratum j which the roots of plants are immersed. It was thought that the steri) different kinds and of different stages of growth were produced, woul}, permit of a series of tests for these substances under conditions the} would exclude contamination from the seed or from outside source) LIBERATION OF ORGANIC Matrer BY Roots oF Growinea Puants 35 nd that these tests might possibly afford some information regarding he relation of the stage of growth of the plants to the presence of oxidizing r reducing substances. REVIEW OF LITERATURE Apparently the only examinations designed to detect the presence of educing substances in media in which plants grew were those conducted yy Schreiner and Sullivan (1910), who placed the roots, and in some ases the seeds also, of wheat seedlings in solutions of various reagents ommonly used for detecting the presence of reducing substances. Con- idering only the tests in which roots alone were introduced into the zagent solution, these authors obtained reactions with starch-iodide olution, sodium selenite, and sodium tellurite. Tests for reduction of itrates appear to have been made only where the seeds were present, nd under these conditions nitrites were found by means of the Griess saction. The seeds from which the seedlings were germinated had reviously been treated with a 0.1-per-cent solution of mercuric chloride. ‘he solutions in which the plants grew were not shown to be sterile. It is well known that oxidizing enzymes occur within plant tissues nd they are believed to play an important part in physiological processes. ‘hey have been found also in soils, but this does not furnish any proof iat they are liberated by plant roots altho it suggests such a possibility. ‘he presence in soils of large numbers of bacteria many of which are nown to secrete enzymes, may well account for the appearance in soils ' oxidizing enzymes without any contribution from roots of higher plants. ; 1s equally true that the occurrence of enzymes within the plant would ot necessarily lead to the conclusion that they are thus conveyed to the il. Not many investigators have taken up studies concerning the liberation ' oxidizing substances by plant roots. The work of Molisch, Czapek, aid Raciborski has been reviewed by Schreiner and Reed (1909), and ence it is not necessary to review it here. Schreiner and Reed, in the yper referred to, state that they have been able to detect the presence oxidizing enzymes on certain parts of the surface of wheat roots. For us purpose they used alpha-naphthylamine, benzidine, vanillin, vanillic id, phenolphthalin, aloin, and leuco-rosolic acid. As in their experiments r the detection of reducing substances, there was no evidence offered 36 T. L. Lyon anp J. K. WILson to show that the solutions were sterile, but the authors state that it improbable that the enzymes were produced by bacteria. Summarizing the experiments to determine the presence of oxidiziz enzymes outside of the growing roots of plants, it may be said that Molise Raciborski, and Schreiner and Reed report the finding of these enzym and consider them to have been excreted by the roots as a normal co dition of their growth. Evidence of the liberation of reducing substane is less conclusive, but if oxidizing enzymes are liberated by plant roo it is easily conceivable that reducing substances would be also, as boi are well known to occur within the plant tissues. TESTS FOR REDUCING AND OXIDIZING SUBSTANCES LIBERATED BY PLANT ROOTS The data already presented show that a comparatively large amou of soluble organic matter may be given off by plant roots during tl growing period. ‘This organic matter may be derived in part from ro caps and root hairs that are sloughed off by plant roots as developme! proceeds. The roots or detached cells may give up to the surroundi medium certain specific compounds, some of which may be enzymic — character. In order to obtain information on this subject a number tests were made to detect the presence of certain substances that mid influence reduction or oxidation. Reducing substances A number of reagents that had been proposed for the detection of t, presence of reducing substances were used for testing the solutions | which maize plants had grown, and at the same time for testing che solutions consisting of the nutrient solution made up as it was for t growth of the maize plants. In some cases the solutions in which the plar had grown were boiled before being tested, but an unboiled portion w always tested at the same time. Some of the reagents failed to gi a reaction with any of the solutions tested and were discarded. The were methylene blue, methyl violet, gentian violet, sodium selenite, a sodium tellurite. These failures may mean merely that the conditic under which their reactions occur did not obtain altho reducing substan may have been present. i i aaiaaiasaie LIBERATION OF OrGANIC Marrer By Roots oF GROWING PLANTS 37 Tests for reducing substances were made with prussian blue in solutions ym. six sterile flasks in which maize plants had grown, using both the boiled and the boiled samples of the solutions as well as check nutrient lutions. These tests were made in solutions taken from plants at ferent stages of growth. Ten cubic centimeters of each solution was ed, and each received two drops of a 0.5-per-cent solution of phenol. ter the prussian blue was added, the tubes containing the solutions were owed to stand for twenty-four hours, at the end of which time notes re taken on the results. In each of the six tests the unboiled solution which the plant had grown gave a distinct reaction for reducing sub- ices. The boiled solution gave no reaction in three tests, while in > other three the result was rather uncertain. The check solution ve no definite test for reducing substances. Reduction of nitrates.— Since traces of ammonia were found in the rile solution which had surrounded the maize roots, it was thought ssible that thru the action of enzymes liberated by the plant this tmonia might have been formed from the nitrates in the nutrient solution. sts for nitrates were made, using sulfanilic acid and naphthylamine state. No positive results were obtained. As a further test the yhenylamine reagent was employed. ‘This reagent is considered to be sitive to nitrite 1 part in 32,000,000 and to nitrate 1 part in 44,000,000. e tests were made with a series of sixteen flasks and no positive results re obtained. Chis does not necessarily show, however, that reducing enzymes might ; have been present, for the maize removed all the nitrates from the srient solution rather early in its development, and the liberation of ucing substances may not occur until after the plant has taken up most the nutrients necessary for its development, or the nitrites may be orbed by the plant. “he nitrites might not have been present because there were no nitrates n which they could be formed. The problem of supplying the nitrates | making the nitrite test was conducted as follows: Check flasks, lve in number, were prepared with the same nutrient solution that 7; used for growing plants. A like number of test flasks were used ch contained solution from around the plant roots. One hundred ic centimeters was used in each flask, and the nitrate content was le equal in both check and test flasks. The flasks received phenol 38 : T. L. Lyon anp J. K. Witson to make the concentration 1 to 500, and were placed in the incubat at 23° CG. The next morning tests were made for nitrites, using the Grie reagent. No visible differences were apparent. A second test for nitrit was made after forty-eight hours. The result was positive in eve case. This test was repeated with the solution from another flask — which a maize plant had grown. While not so striking, the results. this case were also positive. Some tests were negative. A differen of NO; readings was not detectable in a colorimeter. Another series of tests for nitrate reduction was conducted, using eubie centimeters of the solutions in which maize plants had grown f periods of varying lengths and adding to this a small quantity of calen nitrate solution, at the same time introducing two drops of a 0.5-per-ce solution of phenol. Alpha-napthylamine and sulfanilic acid were us to test for the presence of nitrites, the solutions being allowed to sta for certain lengths of time varying from two hours to three days. St tests were made of the sterile solutions from eight flasks in which pla‘ had been grown, the solutions being taken for the tests within a few minu| after the flasks were opened. A sample of the solution from each of} flasks except one was boiled before being tested, and another sample ¥ not so treated. The check nutrient solution was tested in each ce Of the eight flasks tested, the unboiled solutions showed the presence nitrites in every case but one, the check solution showed the absence nitrites in six cases out of eight, and the boiled solution gave a react for nitrites in six tests but did not color so rapidly as did the unboi Apparently nitrate-reducing substances were usually. present in unboiled solutions in which the maize plants had grown, and boiling ; solutions failed to render these substances incapable of bringing ab) reduction of nitrates in the presence of phenol. t While the process of boiling did not completely prevent the lic surrounding the plant roots from effecting reduction of nitrates, it seelp to curtail its activity, as is indicated by the slower coloration of the rea } with the alpha-napthylamine and sulfanilic acid. The tests for reduiy substances by means of prussian blue, on the other hand, did not } any more reaction with the boiled solutions than with the checks. WI therefore, the operation of boiling produced some effects correspont to what might be expected from enzymes, there is some question af a} LIBERATION oF ORGANIC MatTTEeR BY Roots oF GROWING PLANTS 39 rhether the reducing substances were of that nature in view of the results ith reduction of nitrates. Oxidizing substances Peroxidases were detected in the culture solution in which the roots { maize, vetch, oats, peas, soybeans, alfalfa, and timothy had grown. rom 8 to 10 cubic centimeters of each solution was placed in a dry test abe together with a few drops of hydrogen peroxide. This was allowed ) stand for from two to three minutes, and then from two to three drops f a 5-per-cent phenol solution were added.’ The latter was then followed y an alcoholic solution of guaiac. It was considered that a positive test as recorded if the color became blue in thirty minutes. With this test o difficulty was experienced in obtaining a reaction with solutions from ll of the flasks tested. Boiled solutions treated ikewise gave no reactions. In working with the solution in which vetch roots had grown, a very rong reaction was obtained by the use of hydrogen peroxide and phenol. ‘he solution was placed in dry test tubes and a few drops of hydrogen eroxide were added. This was allowed to stand at room temperature. fter from three to four minutes a few drops of a 5-per-cent phenol solution ere run down the side of the test tube. Shortly after contact of the laterials, a growing yellow color developed, which gradually increased od on long standing settled out. . This test was negative when the same ution was boiled. An extract of macerated vetch roots and nodules ave the same test. If this material was boiled, however, no reaction as obtained. Phenolpthalin was used as a reagent for indicating the presence of sidizing substances in the solutions in which maize plants had grown. or these tests both boiled and unboiled samples of the solutions were sed. Checks consisting of the nutrient solution in which no plants ad grown were also included. Phenol was added, as in the previous ists. Results from these tests were usually negative. Experiments in which guaiac were used without a peroxide indicated le presence of an oxidase in solutions in which maize plants had grown aly when the plants were very young. Agar in which timothy plants ere grown always gave a reaction with guaiac. Tests with pyrogallol included only one sterile flask. No phenol was sed. At the end of one-half hour the boiled solution was clear, the 40 T. L. Lyon anp J. K. Witson unboiled was brown, and the check was a light yellow. On the whole the tests for oxidizing substances cannot be said to have offered very strong evidence of their presence except in the case of peroxidases. Possible coexistence of oxidizing and reducing substances Altho the number of tests for oxidizing enzymes were rather few, reac- tions indicating their presence were confined mainly to the solutions from flasks which gave no marked response to tests for reducing substances It seems likely that both classes of substances are coexistent in solutions in which plants are growing, and that at one time the oxidizing substance; may be dominant and at another the reducing substances. Difference; in the intensities of the reactions at various times may thus be accountec for. Apparently with the maize plant the predominating reaction wai for reducing substances thruout most of the stages of growth. It would appear from these experiments that some form of reducing substance is always present in the solutions in which plants are growing Whether oxidizing substances are always present, it is more difficult t say. They were found in some of the solutions taken from flasks in whicl the plants had reached only an early stage of growth, and unless they ar destroyed later on they must be present thruout the entire life of the plant In the natural soil solution, enzymes might be destroyed and thus remover from active operation except when freshly liberated. Nature of oxidizing and reducing substances Boiling the solutions in which plants had grown completely terminate: the activities of the oxidases, as was to be expected. It did not alway! have that effect on the reducing substances, especially the nitrate reducers There would thus seem to be considerable doubt as to whether the reducin substances were enzymic in character, but they at least had the power ¢ bringing about reduction of nitrates in the absence of bacteria. SUMMARY Plants were grown with their roots in large flasks (8 or 12 liters capacity containing a nutrient solution, the entire contents of the flasks bein sterile. At various stages in the growth of the plants they were remove from the flasks and analyzed for nitrogen, and the nutrient solution we’ Z LIBERATION OF ORGANIC MATTER BY Roots oF GROWING PLAaNts 41 ested for sterility and analyzed for nitrates, nitrites, ammonia, and rganic nitrogen, and in some cases for total organic matter. A determi- ation of organic nitrogen in the deposit at the bottom of the flasks was lso made. . The plants grown were maize, oats, peas, and vetch. The nutrient olutions in which each of these plants grew contained nitrogen only ithe form of nitrate when the plants were set in the flasks, but when the lants had grown for several weeks there was always organic nitrogen resent. Even before the nitrate nitrogen had all been absorbed by the lants, organic nitrogen appeared in the solutions. The deposit at the bottom of each flask contained a small quantity f organic nitrogen, which was probably derived from sloughing off of the »0t cells as indicated by the presence of plates of cells in the deposit. here was no direct evidence that the organic nitrogen in solution was berated in any other way, but it is questionable whether such a large aantity could all come from these cells, especially during the early stages * growth. A pea plant which grew in a solution without the addition of combined trogen liberated organic nitrogen into the solution in which it grew. he growth was by no means as vigorous as that of another pea plant hich was furnished with nitrate nitrogen, and the plant itself contained ily about one-fifth as much nitrogen as did the latter plant, but it erated more than half as much organic nitrogen. A series of flasks in which maize was growing were harvested at successive ages in the growth of that plant, and the plant and the flask contents are examined in the manner already described. Organic nitrogen ypeared in the solutions at all stages in the growth of the plants, but Pere seemed to be a tendency for it to decrease in amount with pro- essive stages in the life of the plant and especially as the plant closely proached maturity. The organic nitrogen in the deposit at the bottom the flasks did not show any decided tendency to vary in amount with e successive stages of plant growth. Determinations of total organic matter were made in the solutions 4#m some of the flasks. This constituent was very large in amount 4 compared with the nitrogenous organic matter present. Apparently dere was much non-nitrogenous organic matter in the solutions. Calcu- ing the nitrogenous organic matter at 6.25 times the nitrogen present, 42 T. L. Lyon anp J. K. WILson this material would constitute only a small part of the total organic matter. | The presence of reducing substances in solutions in which plants hac grown was indicated by certain tests, but a number of other reagent failed to give reactions. Nitrates were nearly always reduced in thesi solutions in the presence of an antiseptic. Boiling the solution did no entirely prevent nitrate reduction, but it greatly decreased the rate a which reduction proceeded. | Peroxidases were always present in solutions in which plants had growr Boiling these solutions caused them to give no reaction for peroxidases Tests for other oxidizing substances were not sufficiently satisfactory t warrant the conclusion that they were present. LIBERATION OF ORGANIC MATTER By Roots oF GROWING Puants 43 BIBLIOGRAPHY \meRIcCAN Pupiic HEALTH AssoctaATION. Report of committee on stand- ard methods of water analysis, p. 1-141. (Reference on p. 37.) 1908. Awpreé, G. Sur l’évolution de l’azote, du phosphore, et du soufre au cours de la végétation de l’orge. Acad. Sci. [Paris]. Compt. rend. 154: 1627-1630. 1912. SROWTHER, CHARLES, AND Ruston, ArtHUR G. The influence of time of cutting upon the yield and composition of hay. Journ. agr. sci. 4:305- 317. 1911-12. : ionEs, W. J., Jr., AND Huston, H. A. Composition of maize at various stages of its growth. Indiana (Purdue Univ.) Agr. Exp. Sta. Bul. 175:595-630. 1914. kNupson, L. Viability of detached root-cap cells. Amer. journ. bot. 6:309-310. 1919. ——— The secretion of invertase by plant roots. Amer. journ. bot. 7:371-379. 1920. 4E CLERC, J. A., AND BREAZEALE, J. F. Plant food removed from growing plants by rain or dew. U. 8. Agr. Dept. Yearbook 1908:389-402. 1909. tAMSAY, J. T., AND Ropertson, W. C. The composition of the potato plant at various stages of development. Victoria (Australia) Agr. Dept. Journ. 15 (1917) :641-655. 1918. : SCHREINER, OSWALD, AND Reep, Howarp S. The role of oxidation in soil fertility. U.S. Soils Bur. Bul. 56:1-52. 1909. SCHREINER, OSWALD, AND SuLLivaAn, M. X. Studies in soil oxidation. U.S. Soils Bur. Bul. 73:1-57. 1910. SCHULZE, B. Studien iiber die Entwickelung der Roggen- und Weizen- pflanze. Landw. Jahrb. 33:405-441. 1904. [READWELL, F. P. Reactions in the wet way. Jn Analytical chemistry. (Translated from the German by William T. Hall.) 4th English ed., feeo2. 1915. TRowpripcE, P. F., Haicu, L. D., anp Movutron, C. R. Studies of the timothy plant. Part II. The changes in the chemical compo- 44 T. L. Lyon anp J. K. WILson sition of the timothy plant during growth and ripening, with a com | parative study of the wheat plant. Missouri Agr. Exp. Sta. Research | bul. 20:1-67. 1915. Warers, H. J. Studies of the timothy plant. Part I. The influence of maturity upon the yield, composition, digestibility, palatability, and feeding value of timothy hay. Missouri Agr. Exp. Sta. Research bul. “19: 1-68. 1915. Witrartu, H., Romer, H., anp Wier, G. On the assimilation of the elements of nutrition by plants during different periods of their growth. (English translation of Ucber die Nahrstoffaufnahme der Pflanzen in verschiedenen Zeiten ihres Wachstums. Landw. Vers. Stat, 63:1-70.) 1906. Wison, James K. Calcium hypochlorite as a seed sterilizer. Amer. journ. bot. 2:420-#27. 1915. Device for growing large plants in sterile media. Phytopath 10:425-429. 1920. Wrruprs, W. A., AND Ray, B. J. A modification of the diphenylamine test for nitrous and nitric acids. Amer. Chem.Soc. Journ. 33 :708-711 1911: Memoir 38, The Crane-Plies of New York. Part II. Biology and Phylogeny, the second precedin; 19: number in this series of publications, was mailed on July 18, 1921. \ ! ; Memoir 39, The Genetic Relations of Plant Colors in Maize, the next preceding number in this series © publications, was mailed on July 19, 1921. ULY, 1921 MEMOIR 41 CORNELL UNIVERSITY AGRICULTURAL EXPERIMENT STATION LYSIMETER EXPERIMENTS - II RECORDS FOR TANKS 13 TO 16 DURING THE YEARS 1913 TO 1917 INCLUSIVE T. LYTTLETON LYON AND JAMES A. BIZZELL ITHACA, NEW YORK PUBLISHED BY THE UNIVERSITY CONTENTS PAGE The soil used ........ 05 COG CO CRG Greer 8 eee le ch i Rg 51 Wrolumenmweren Grier cotracs crosses cere sie scree ai elena es cuate weiuel aides als gacee bluse 52 Mechanical composition.............. ar BBV TERED i SABRE He HORS EROS oe 52 Chemical GanniestiOnvceGen amen acne os coe Ocoee Oe once Ore en ee 53 MiamunevanuprentilizenswUsed! 0%)... 215s ie kee eh yee ere nue Geo hoe a egal oa els ceca 54 Methods for chemical and mechanical analyses................0.0 000 cece ese eee 54 Poltreatment and cropping. system. -.. 22.50... .:- 62s ete cect eee eee bes ene eee 54 Quantity and rate of percolation........... ss Sco Sect ery La cet tae a es Ce are Ge 56 Percentage percolation of rainfall............. A tees irene syvraa pene na ee Maree cee eae eee 56 ikec tor liming on percolation: : 22. dessa 86-4 os hee ees es tele detec scuuemes 57 MF IDEERUUTITZ a GlONMDVRCEOPShes i Men. lls Wiseicsp nie s.feaie dele Gs csg e wiareule gaclsdale Que wieces 58 | EA ADORtLAMS PIAL OMILAL Onan he teusrayt os eok biel ave sade vain Ane othe Ges Bde wlbwelels oles 58 | MiOIShUEe Re ations Ole ChODS= i pst vce io sce) Aen ole ele awe tone 60: Removal of nitrogen from the soil in drainage water and in crops.................... 61 | Effect of liming on removal of nitrogen in crops.......................-0-0-0-. 61 Effect of liming on removal of nitrogen in drainage water....................... 2 Effect of liming on removal of nitrogen in both drainage water and crops......... 62 Relation of different crops to formation of nitrates....................0.0.0 20.0000. 63 REIT OWA IMO LEC ALG TUTTE Mie reer er cena gn wR bee eta ent a a Sst cctee cyl las) Aumnieceealet44 soe ios 64 Effect of plant growth on removal of calcium................0000 00 ec eee eee 64 Biectroflimmexonremoval of caletum....... 2.25.2 -6-2+-+00ses sees ess esse sse 66 Liming to maintain the calcium Content etek sree ee gscos ere en ay waa eae 67 ReMONAMOisMACNeSIUMM Er sles plana Nanda cvakl iene sa ulalitcrs oleh cee lehersls 68 Effect of plant growth on removal of magnesium.....................000-0e00e 68 Effect of liming on removal of magnesium.................... 0c c eee ee cee eee 69 SPE In OWA MOBO LASSIUMIP Ne oe cares a tie clone Seb se tae wel cue Se dwt e dacdies cae § 69 | Effect of plant growth on removal of potassium................ 2000.00 c eee eens 70 ) Effect of liming on removal of potassium... . 1.0.2.0... 0... c cece eee 71 Hern o 2 molas liter ier whe icles eas: ald aretelle, Goeibiv wait Sea ao tice s Soniyw a tvg ate war aiive. cin gels 72 Effect of plant growth on removal of sulfur.............0 0.0.00. 0 cece eee eee 2, Effect of liming on removal of sulfur...........0..0... 00.0 cee ce cece eee eens 73 PEern OV ARO MphOsphonustemaiana ne ne Nee whe G ancdhe os Tike shel bh Be adios une ote wane ees 73 maaermeniseitectsoilimineg the two soils... 2 s.<. 5c sleek ono d eee wees ee ewes 74 | DEDEDE 0.0 6 6 0 0 8 016 6 Hid 5 OH UES rE ECE GRESSION 76 ee ecncix (Comnbarminewtalloles) Pyne ener eo ce wee th UE IL Galena Sails oie m wcae atest Vataes See aT Re ema age 0.50 100 Phosphoricianhydrideyy.j acne see ee i eee 0.37 74 Potassium’ Oxd d erse sey ery ops e tae ecar neh eee ane aay Suen) eR 0.36 72 Galeiummo xd Oh earn rakes eh cose eae a eon eae cee Oe aerate 0.63 126 | Macon esium oxide ttiane Slik o monnak esti netennn un Ney anraaleen, 0.27 54 The burnt lime, of which one application was made, contained 91.95 per cent of CaO and a trace only of MgO. METHODS FOR CHEMICAL AND MECHANICAL ANALYSES The methods used for the chemical analyses of the soil, crops, drainage water, rain water, manure, lime, and potassium sulfate, and for the mechanical analysis of the soil, are described in the appendix to Memoir 12, pages 85 to 91, inclusive. SOIL TREATMENT AND CROPPING SYSTEM 5 The four tanks used in this experiment were all filled in the late summer of 1910. From that time until the spring of 1913 they were not treated in any way, but the drainage was collected, measured, and analyzed in LYSIMETER EXPERIMENTS =aar II 55 order to have a complete record of all constituents removed in the drainage water from the time when the soil samples were taken for analysis. Since the present report does not attempt to show the difference in the com- position of the soil at a later period, but is rather a discussion of the effect of the application of burnt lime on the composition of the drainage water and the plant ash, the drainage for the period previous to May 1 1913, is not considered. Farm manure was applied to each of the four tanks in the spring of 1913, the application being at the rate of 10 tons to the acre. Burnt lime was applied to tanks 15 and 16 in the spune of 1913 at the rate of 3000 pounds to the acre. Tanks 14 and 16 were never planted to any crop, and growth of vege- tation on them was prevented by hoeing. In the year 1915, when maize was growing on tanks 13 and 15, the unplanted tanks were hoed at the same time and in the same way as were the tanks planted to maize; when other crops were growing on the planted tanks, the unplanted ones were merely scraped with a hoe. In each tank planted to maize there were aighteen maize plants. Seven rows of oats and the same number of rows of barley were sown in each tank planted to those crops. The barley was used to replace wheat, which had been sown in the previous autumn and had winterkilled. Canada peas were planted broadeast. All crops zrew to maturity and produced seed, but it was evident that the soil was not a very productive one. The manure, lime, and crop treatments are ‘shown in table 5: TABLE 5. Sor, TREATMENT AND Crops RAISED ON LYSIMETER TANKS 13 To 16 DURING THE PERIOD FROM 1913 To 1917 | Soil treatment | Crops raised H Tank Fertilizer Lime 1913 1914 1915 1916 1917 3 .| Farm manure.| None....| Oats...| Canada peas| Maize..| Oats...| Barley )4........| Farm manure.| None....! None..{ None...... None. .| None...| None 15........| Farm manure.| Burnt Oats...} Canada peas| Maize..| Oats...| Barley lime 6 Farm manure.| Burnt | None...| None.......| None...| None...| None lime | 56 T. Lyrrteron Lyon anp James A. [1zzELL The rainfall that percolated thru the soil was measured by monthly}! periods, and a record of each tank by months is therefore available. It}; is thus possible to ascertain the effect of certain treatments, such as liming}t and cropping, on the proportion of the rainfall passing thru the soil, and even to compare the relative permeability of the two soils. © QUANTITY AND RATE OF PERCOLATION The figures for the flow of drainage water from each of the lysimete tanks 13 to 16, expressed in liters for each month from May 1, 1913, t ) April 30, 1918, are given in table 2 of the appendix (pages 78 to 80). The flow calculated to acre inches annually for the same pericd is given in table 3 of the appendix (page 80). These tables furnish the data from which the average annual percolation in inches from the unplanted soil); may be found, and also that from the soil on which crops grew. This, |, together with the percentage percolation, is given in table 6. The rain-), fall during the five-years period averaged 32.97 inches annually. i I Percentage percolation of rainfall TABLE 6. AvpraGe ANNUAL PERCOLATION OF RAINFALL FROM UNPLANTED AND FROM) PLANTED SOIL DURING FIvE-YEARS PERIOD Average annual percolation | Tanks Crop treatment | Per cent | Inches of rainfall eh WOgedeoricosy ou Se sbiecca sex No plants allowed to grow...... ZiAS 82.) 11S Foie Hts) Sees cape ae aie cr a ce a Plants raised every year........ 20.62 62.é Difference in percolation....... 6.51 19. On the basis of these figures, about four-fifths of the rainfall percolated thru the bare soil and three-fifths thru the cropped soil. Somewhat more than one-fifth of the rainfall may therefore be considered as being used by the crops, since the evaporation from the surface of the unplanted soil may be assumed to be greater than that from the surface of the planted soil. This, however, is only a small proportion of the total rainfall and would account for only about six and one-half to seven inches of the annual precipitation. as LysIMETER EXPERIMENTS —II 57 Comparing the figures for percolation thru the Volusia soil with those thru the Dunkirk, given in the earlier publication already referred to, it will be seen that the former soil is more permeable than the latter and that the crops on the former used about an inch more rainfall. In table 4 of the appendix (pages 81 to 83) are given the weather records at Ithaca by months, including the rainfall, the mean maximum, mean minimum, and mean temperatures, the hours of sunshine, the average hourly velocity of the wind, and the mean humidity of the air, for the period from May 1, 1913, to April 30, 1918.2. These data were obtained at a distance of somewhat less than a mile from the lysimeters. Effect of liming on percolation As already stated, tanks 15 and 16 received an application of burnt lime at the rate of 3000 pounds to the acre. It has frequently been said that liming, especially with burnt lime, has a tendency to make a heavy soil more permeable to water. If such has been the effect of the lime on this soil, it may possibly be shown by comparing the volume of drain- age water from the limed and the unlimed tanks. There may be some question, however, whether any flocculating action which the lime may have had on the upper six or seven inches of soil with which it was incor- porated would increase the amount of percolation thru the entire four feet of soil. Comparisons would best be made between limed and unlimed tanks that have otherwise received similar treatment, and this is done in table 7. It is evident from this table that the application of lime has TABLE 7. Avrerace ANNUAL PERCOLATION OF RAINFALL THRU LIMED AND UNLIMED SOIL DURING FIVE-YEARS PERIOD Soil not limed Soil limed Crop treatment Fertilization Flow Flow Tank (acre Tank (acre inches) inches) No plants allowed to grow...... Farm manure. 14 | 28 .60 16 25.66 Oats, peas, corn, oats, barley. ..| Farm manure. 13 21.28 15 19.96 PR CER DOOD eo 2 Es one ay 24.94 22.81 2 The authors are indebted to Dr. W. M. Wilson for the weather records at Ithaca. 58 T. LytTLeton Lyon AND JAMES A. BizzELL not caused-a greater percolation of water. The same was true of the application of lime to the Dunkirk soil in tanks 1 to 12, in the earlier experi- ments. While this may not mean that the lime did not flocculate the upper layer of soil with which it was incorporated, it has some significance so far as drainage is concerned, since it indicates that liming a soil of this” kind would not result in facilitating the removal of water thru tile drains. | WATER UTILIZATION BY CROPS The water utilization by crops on this soil was large for the amount of | dry matter produced, both when calculated to the minimum transpiration ratio and by the evapo-transpiration ratio. The former was calculated by subtracting the drainage from the planted tanks from the drainage from the unplanted ones, and amounts to 451 pounds of water for every pound of dry matter in the crops raised during the five-years period. This appears in tabular form in table 8: ; TABLE 8. Minimum TRANSPIRATION FOR ALL Crops RAISED DURING FIVE-YEARS PERIOD ; Average annual panel ates Tania Cropping percolation Sica mae | treatment per tank per tank (pounds) (pounds) p A ANG Sse yee eten ae HARE IRE ER ogee eels Unplanted. . 24621 Se ee ae Ie Ua Yel eee eau Ur recta ee eee Sener pure oe Cropped.... 1,871 1.31 Minimum transpiration: mc cas ei svete eineaiie eee 5O LS ee ieee ae Minimum transpiration ratio. ..................0..-. gee ay beret tn aS an 5c Evapo-transpiration ratio The evapo-transpiration ratio was calculated by subtracting the average percolation thru the planted tanks for the five-years period from the rain- fall on the same area for the same period, and dividing this by the number of grams of dry matter per tank in the crops produced. This ratio is given in table 9: rr” LYSIMETER EXPERIMENTs —II 59 TABLE 9. Evapro-TRANsPIRATION FoR ALL Crops RAISED DURING FIvE-YEARS PERIOD | | Liters ; per tank 2 pieafinlll (arming ain DE ce ea ra a 1,388 .7 Percolation from planted tanks (average annual)..................2....--- 848 .7 Transpiration and evaporation from planted tanks........................ 540.0 ; Phy ApOxtlANs Diba tlONELAa tO sors c45 sects Acids ats ee cs iaidiare ele Srsbi@ eaclebs sie ote Sela 1:908 Neither the minimum transpiration ratio nor the evapo-transpiration tatlo is necessarily the same as the actual transpiration ratio. The former is likely to be less because the evaporation from the unplanted soil is almost always greater than the evaporation from the planted soil, and the latter is almost sure to be greater because it includes the water that evaporates from the surface of the soil as well as that which is transpired by the plants. The actual transpiration ratio therefore lies between 1:451 and 1:908. It is significant that both transpiration ratios for the Volusia soil are so much wider than those for the Dunkirk soil, the former being about 56 per cent wider than the latter in both cases. It seems fair to assume that the actual transpiration ratio is correspondingly wider for the Volusia soil. Such differences cannot be attributed to conditions other than the soil, and probably arise from a difference in the concentration of the soil solution. The transpiration ratios are inversely proportional to the con- centration of the drainage water and the crop yields from these two soils, as may be seen in table 10, the data in which are for the five-years periods already reported with the exception of the crop yields, which are for 1915, 1916, and 1917 only, since those were the only years in which the same crops were grown on both sets of tanks. 60 T. LytTTLETON LYON AND JAMES A. BizZELL TABLE 10. RELATION OF TRANSPIRATION Ratios, ToTat Sotips In Dratnace WATER, AND Crop YIELDS Total solids Crop Minimum | Evapo- | in drainage yields in trans- trans- of un- 1915-1917 piration piration |planted tanks (grams ratio ratio (parts per per million) tank) Wolusiaysiltsloamsasseee eer ee Se 1:451 1:908 297 .7 3,359.0 Munkarkiclaysloameess eer eee ee 1: 290 1:580 401.5 6,478.3 | It would appear from the data presented in table 10 that an economic — utilization of water by the plant accompanies a high concentration of the | water passing thru the soil, and that there is, moreover, a relation between the concentration of this water and the productive ability of the soil. Moisture relations of crops In table 11 is given a statement of the moisture relation of each crop grown on these tanks during the five years of the experiment. The TABLE 11. Moisture Rewations oF Crops on Tanks 13 AND 15 DURING THE PERIOD FROM 1913 To 1917 Trans- irati D Rainfall Minimum | Evapo- ere iene in Year Tanks Crop trans- Gees evapo- in crops | percolate | piration Pees ration (pounds (per ratio (acre per acre) cent) inches) LOTS eel ose ee eOatse see 1:373 | 1:690 10.13 3,542 1 OAs 13, 15..| Canada 1:525 | 1:1005 14.08 3,316 peas 1915 eee 13, 15..| Maize 133325) 4 1613 12.68 4,977 64.49 9 1OTG see ee S15 ee Oaterease 1:366 | 1:1108 11.85 2,546 54.75 LOVES. 13, 15..| Barley 1755) |e 61S 13.06 1,858 69.80) Wt Eee minimum transpiration ratios for all these plants are highef than fort the same plants grown on the Dunkirk soil, and the evapo-transpiration | ratios also are higher. These discrepancies emphasize the futility of - trying to determine a standard for the water requirements of plants when } soil is used as the medium in which the plants are grown, f LYSIMETER EXPERIMENTS —II 61 REMOVAL OF NITROGEN FROM THE SOIL IN DRAINAGE WATER AND IN CROPS There are large differences between the planted and the unplanted tanks in respect to the nitrogen removed in the drainage water, the amount from the unplanted tanks being in all cases larger. Nitrogen in the drainage water was all in the form of nitrates. There are considerable differences in the quantities of nitrogen removed from any one tank from year to year. In the unplanted tanks these differences do not appear to be associated with any weather conditions shown in the monthly records. It is possible that conditions extending over shorter periods may have influenced the production of nitrates. In the case of the tanks filled with Dunkirk soil in the earlier experiments, the removal of nitrogen appeared to be influenced by the total yearly rainfall for the year beginning May 1, but this does not account so satisfactorily for the removal of nitrogen from the Volusia tanks. Future experiments may throw more light on these relations. Effect of liming on removal of nitrogen in crops One of the beneficial effects which lime frequently exerts on soil showing a lime requirement is to promote the formation of nitrates from organic nitrogenous compounds. Such action might be reflected in the nitrogen content of the*drainage water or in the nitrogen removed in the crops. The amount of nitrogen removed in the crops produced each year of the experiment is shown in table 12: TABLE 12. Nuirrocen 1x Crops, CALCULATED TO PouNDS PER ACRE BY YEARLY PERIODS Nitrogen in crops Burnt (pounds per acre) Tank lime Oats Peas Maize Oats | Barley 1913 | 1914 | 1915 | 1916 | 1917 | Lota —. ee None....| 34.98] 68.75 | 28.35 | 21.74| 18.79] 172.61 ea 3,000....| 36.24] 94.86] 37.41| 24.93| 23.74] 217.18 It is apparent from the data presented in table 12 that in every year the crops on the limed soil contained more nitrogen than did the crops on the soil that was not limed. This, of course, is not positive proof that lime increases the production of nitrates in this soil. as nitrogen may not be 62 T. LytTrLeTon Lyon AND JAMES A. BiIzzELL an important factor in crop production; on the other hand, it may be significant, especially if it is supported by evidence drawn from the removal of nitrogen in the drainage water. Effect of liming on removal of nitrogen in drainage water The quantities of nitrate nitrogen removed in the drainage water of unplanted soil afford a better means of ascertaining whether liming in- creases nitrate formation than does the removal of nitrogen in crops. The data by years for the nitrogen in drainage water from unplanted soil are presented in table 13: TABLE 13. Nirrocen In DRAINAGE WATER OF UNPLANTED TANKS, CALCULATED TO Pounps PER AcRE BY YEARLY PrERtops (May 1 To Apri 30) Nitrogen in drainage water Burnt ~ (pounds per acre) Tank Fertilizer lime (pounds) |__| 1913 1914 1915 1916 1917 Total WAN Ne rete Manure..| None | 73.41 | 34.21 | 49.31 | 34.47] 38.71} 230.11 NG Seer tere Manure..| 3,000 | 88.45 | 49.56] 55.79 | 50.80] 46.19 | 290.79 In each year the removal of nitrogen in the drainage water from the limed soil was greater than from the unlimed, which is very good evidence that the lime produced a condition more favorable to the production of nitrates. It may be remarked that the application of lime to the Dunkirk soil was not attended by any increase in the removal of nitrogen by the | drainage water or by the crops. This difference in effect of lime is all the more striking inasmuch as the lime requirement of the surface foot | of the Dunkirk soil is very little less than that of the Volusia. The per- | centage of calcium, however, is about one-third less in the surface foot of | the Volusia. In this case the relative calcium content of the soil is a better | guide to its need of lime for nitrification than is the lime requirement as | determined by the Veitch method. t | Effect of liming on removal of nitrogen in both drainage water and crops While the nitrogen in the crops alone may not be an adequate a | to the effect of lime on the soil, the nitrogen in the crops added to that in the drainage water from the same tanks is perhaps somewhat more com- LysIMETER EXPERIMENTS — II 63 prehensive. These data are given in table 14. It is apparent from this table that the application of lime to this soil results in an increased TABLE 14. Nirrocen In BotoH DratnaGeE WATER AND Crops, CALCULATED TO PounDs PER ACRE IN YEARLY PERIODS Nitrogen in both drainage water and crops Burnt (pounds per acre) Tank lime Oats Peas Maize Oats Barley Total 1913 1914 1915 1916 1917 oe. 2 None | 46.03 | 74.47 | 44.781] 26.96 | 23.51 | 215.75 “=e 3,000 | 57.46 | 114.16 | 51.67 | 28.81 | 28.10 | 280.20 removal of nitrogen in the combined crop and drainage water. There would seem to be little doubt, in view of the data presented in the last three tables, that the effect of lime on this soil is to increase nitrate formation. RELATION OF DIFFERENT CROPS TO FORMATION OF NITRATES It has been noted that the experiments with Dunkirk soil ‘indicated certain rather definite relationships between certain kinds of plants and the formation of-nitrates. A similar relationship appears to exist in the experiments with Volusia soil, as may be seen in the data given in table 15: TABLE 15. AvatLaBLe NITROGEN IN Sor, PropuciING DIFFERENT Crops, AS MEASURED BY THE NITROGEN OF THE CROP AND OF THE DRAINAGE WATER (In pounds per acre) Nitrogen in planted tanks Nitrogen in Excess (++) (average of tanks 13 and 15) drainage water |. deficiency Cr in bare tanks Sin e (average lanted = In In Eas 7 tanks ee ions Paatat Total 14 and 16) ts (LOUS) esses 2. 16 35 12 63 81 a8 Beas (1914)0 o.oo S.... 12 81 27 120 42 +78 Maize (1915)......... 15 32 11 58 52 + 6 Mratse(@ONG) ea... 5: 4 23 8 35 42 — 7 Barley (1917)... ...-. t 21 7 32 42 —10 * Estimated at one-third the quantity in tops. 64 T. LytTLEToN Lyon AND JAMES A. BizzELL Estimating the nitrogen in the roots of each crop to amount to one- third the quantity in the above-ground part, it will be seen that the nitrogen in the oat crop added to the nitrogen in the drainage water from the tanks on which that crop grew was less in amount than the nitrogen in the drain- age water from the bare tanks for the same period. ‘The same was true of barley, but it was not true of maize or, of peas. The excess of nitrogen from the pea tanks can doubtless be ascribed to the nitrogen-fixing prop- erties of Bacillus radicicola in the nodules of the pea roots. The excess nitrogen from the maize tanks must be ascribed to some different phenom- enon. It has been suggested? that some plants have the property of depressing the formation of nitrates, and that certain plants possess this property to a greater degree than do others. The data here presented are in line with such an hypothesis. REMOVAL OF CALCIUM Calcium was removed in the drainage water to a much greater extent than was any other of the bases determined, but in relatively small amounts by the plants. A comparison of the calcium in the drainage water of the Dunkirk and Volusia soils shows that the latter lost more calcium by leaching than did the former, in spite of the fact that this soil contained only about two-thirds as much of that element as did the Dunkirk soil. On the other hand, the crops grown on the Volusia soil contained less calcium but the yield of crops was much smaller. The total removal of calcium from the Volusia soil, from both planted and unplanted tanks, was greater than that from the Dunkirk. Effect of plant growth on removal of calcium In the experiments with Dunkirk soil it was found that less calcium was removed from the planted soil in crop and drainage water combined than was found in the drainage alone from the unplanted soil. This is true also of the Volusia soil, as may be seen from table 16: 3 Lyon, T. Lyttleton, and Bizzell, James A. Some relations of certain poe plants to the formation of nitrates in soils. Cornell Univ. Agr. Exp. Sta. Memoir 1:1-111. 1913. 4 LysIMETER EXPERIMENTS —II] 65 TABLE 16. Averace ANNUAL REMOVAL OF CALCIUM FROM PLANTED AND FROM UNPLANTED TANKS (In pounds per acre) Calcium removed in T otal Tanks Soil treatment | calcium : removed Drainage Crops water HS ios cod oattos ) ones See one Rlanted separ 256.4 8.7 265.1 14. 1G. s ee pieeibdicto Bec ae ae eee Barer: eshte Sole AT ete ete 351.4 @alcinumuconsenved aby Cropping. 256 2... cee eles see eee oh sate seek 86.3 The process of cropping conserves the calcium in the soil even when the entire crop is removed. The reason for the greater removal of calcium from the uncropped soil may be found, in part at least, in the large for- mation and leaching of nitrates when plants are not present. In table 17 are shown the average quantities of nitrates found annually in the drainage water of the planted and the unplanted tanks. TABLE 17. Average ANNUAL REMOVAL OF NITRATES IN DRAINAGE WATER FROM PLANTED AND FROM UNPLANTED TANKS Nitrates Tanks Soil treatment removed (pounds per acre) 13 WS 5.6.6 oc.0':6 ee Cc eS Ie ee Cane Planted Ser ne ain oe AT eal GPP Ty ie hat cose seis shes oes IBALG eee te yanwin cee see ieee 231.7 The nitrates in the drainage water from the cropped soil would account for only about 11.5 pounds of calcium, while the nitrates from the unplanted soil correspond to about 56.5 pounds of calcium which might be removed in the form of nitrate. This would still leave about 245 pounds of calcium that had been removed in the drainage water from the planted soil in some form other than nitrate, and about 285 pounds from the unplanted soil. 66 T. LyrrLeton Lyon ANp JAMES A. BizzELL The concentration of calcium in the drainage water from the planted and from the unplanted soil shows little difference, but this is in the same order as its total removal. This may be seen in table 18, in which is stated in parts per million the average calcium content for the five-years period. TABLE 18. Average Catcium ConTENT OF DRAINAGE WATER FROM PLANTED AND FROM UNPLANTED TANKS : Calcium Soil treatment (parts per million) Tank : Average Crop Lime For for crop : each tank treatment 1 aA CAN N77 rca bee aM ce Planted..;... .<-: Not limed...... 52.3 5A A NS Ee SSecacbeek ht cctrontatrie eer. Plantedene ae Mimed=. 5 sees 56.6 . 1 i a nS NG aN Ree NR cats be IBarere cae iann es Not limed...... 49.9 58.9 LG eee ph ene eee ee Say Bares sari lee Lhimed' #2; 68.0 : The greater loss of calcium from the unplanted soil was not due entirely to the greater percolation of water thru that soil, since in that case the concentration would not be greater. It would appear that the presence of a large amount of a strong acid, such as nitric acid, in the unplanted soil would explain the greater concentration of the calcium in the drainage water of that soil as compared with the weaker carbonic acid in the planted soul. Effect of liming on removal of eclcium The application of burnt lime to the Dunkirk soil at the rate of 3000 pounds to the acre in the earlier experiments did not result in increasing the quantity of calcium in the drainage water or in the ash of the crops produced. A similar application to the Volusia soil in this «experiment appears to have decreased the amount removed in both of these ways, as may be judged from the data presented in table 19: LyYsIMETER EXPERIMENTS — II 67 TABLE 19. Canctum In DraINnaAGE WATER AND IN Crops (In pounds per acre, annual average) Burnt Calcium | Calcium Tank lime in in Total (pounds) | drainage crops calcium water Bie g 6, 6:0.0-0 a:b .e) 00 Ota Se era aa mr ne None 257 .6 7.46 265.1 AN ERO ati aces Speci scoiso vane ¢ c's spsiets Wiis None Be Shs coe ae 319.4 (Be gc052000.04 3 ae Conn ter enna irae ane 3,000 255.1 10.09 265.2 1G .5'S 6:0 ¢ 8 ol 0:0 00 CRO Rte aa 3,000 OSS AW Sandie coy. 383 .4 fy The figures for average annual calcium removal for the entire five-years period, as given in table 19, show a very large increase in the quantity of calcium leached out of the bare limed soil as compared with that from the bare soil unlimed; they show also a moderate increase in the calcium contained in the crops, but they do not indicate any effect from the liming on the calcium leached from the cropped soil. The evidence, however, is in favor of the conclusion that liming increases the amount of soluble calcium in Volusia soil, while it has no such effect-on Dunkirk soil. This is hardly to be accounted for by the absorbent properties of the soil for lime, since Volusia soil has a somewhat higher lime requirement than Dunkirk as determined by the Veitch method. The concentration of calcium was appreciably greater in the drainage from the limed soil than in that from the unlimed soil, both when planted and when kept free of vegetation, as may be seen in table 18. Liming to maintain the calcium content The Volusia soil, altho low in calcium, is annually losing a large quantity in the drainage water, particularly from the unplanted soil. The removal of calcium in the ash of crops has been small as compared with that in the drainage water. If the loss of calcium from the limed soil were to be replaced, it would require an annual application of 536 pounds of pure burnt lime, or 957 pounds of pure limestone, to supply the uncropped soil, and 371 pounds of burnt lime, or 662 pounds of limestone, to supply the planted soil with calcium to the amount removed in the crops and in the drainage water. 68 T. LytTrLeton Lyon AnD JAMES A. BizzELUL REMOVAL OF MAGNESIUM Magnesium was removed in much smaller quantity than was calcium, both in the drainage water and in the crops. In both ways the removal was less from the Volusia soil than from the Dunkirk, altho the removal of calcium was greater. Effect of plant growth on removal of magnesium The effect of plant growth on the removal of magnesium is brought out by table 20. It will be seen that there is a greater loss of magnesium in TABLE 20. AverRAGE ANNUAL REMOVAL OF MAGNESIUM FROM PLANTED AND FROM UNPLANTED TANKS (In pounds per acre) Magnesium removed in ; Total Soil : Tanks t magnesium reatment De rainage Grops removed water LSE Se costae eee ee Planted. c.-:. 30.6 3.5 34.1 AEST G2 hla siesta ee mee: fore) ce Barents eee A5 <4)'\" oo eee 45.4 Magnesium conserved by cropping\55 =... asc-.02 5s oe ke oe eee 11.3 the drainage water of the uncropped soil than in both the drainage and the crops of the planted soil. The large quantity of magnesium leached from the bare soil is apparently caused mainly by the solvent action of the nitric acid, as was the case with calcium. Not only is the total removal of magnesium greater from the bare than © from the planted soil, but its concentration is greater in the water from | the uncropped soil, as may be seen from table 21: LYSIMETER EXPERIMENTS — II 69 TABLE 21. Average Macnestum ConteNT oF DRAINAGE WATER FROM PLANTED AND FROM UNPLANTED TANKS Soil treatment Magnesium (parts per million) Tank | For Average Crop Lime each for crop tank treatment 1D. oo coe Cone Re ORG ee Sea Planted...... Not limed.... 6.1 6.3 Bo 9 066 660 nde Ue ens BEa eae Planted...... himed eee 6.6 : Id, 5 ooo pee eR Re Oe oe eee Bare. hear Not limed.... 7.3 82 GMP aa eeu Abie ee eee es Bare. wise: Limed....... Om : Effect of liming on removal of magnesium The effect of liming the soil was to increase the removal of magnesium both in the leachings and in the crops, as may be seen in table 22. There TABLE 22. Average ANNUAL REMOVAL OF MAGNESIUM FROM LIMED AND FROM UNLIMED TANKS (In pounds per acre) Magnesium removed from Magnesium planted tanks leached Soil treatment Tank Tank from cor- In re responding drainage unplanted water crops Hoes tanks INotilimed........ Be Yee 13 29.6 3.06 | 32.66 14 39.30 imed ee mateete a fs ass 15 31.7 3.93 | 35.63 16 51.60 appears to be a basic exchange, similar to that which occurred in the Dunkirk soil, by which magnesium was liberated and dissolved by the soil water. The concentration of magnesium also was greater in the drain- age water from the limed soil than in that from the unlimed, as may be seen in table 21. REMOVAL OF POTASSIUM Potassium differs from the other bases that were determined in the Dunkirk soil in that it was removed in greater quantities by the crops than by the drainage water. This was not true of the removal of potas- sium from the Volusia soil, 70 T. LyTTLETON LYON AND JAMES A. BizZELL Effect of plant growth on removal of potassium In spite of the fact that less potassium was removed by crops than by drainage water in these experiments, the total removal of potassium was greater from the planted than from the bare tanks. This is entirely contrary to the removal of calcium from the same tanks, as may be seen in table 23: TABLE 23. AvEeRAGE ANNUAL REMOVAL OF POTASSIUM FROM PLANTED AND FROM UNPLANTED TANKS (In pounds per acre) Potassium removed in Total Tanks Soil treatment =|—HHH-—________|_ potassium Drainage Grows removed water P IR ee aR erate aan hI ee eae tal Planted)... .. v2: 73.2 34.1 107.3 VARGA Gpaian eS ener Siege Bi ees Barence eta tee 84 bu Sie 84.5 Potassium conserved by not cropping. .... 0.0.52... 0c. c cece een eee eee 22.8 While the growth of crops conserved the calcium in the soil, the same operation increased the loss of potassium. There was little difference in the concentration of potassium in the drainage water from the planted and from the bare tanks, as is shown in table 24: TABEE 24. Avrerace Porasstum Content oF DRAINAGE WATER FROM PLANTED AND FROM UNPLANTED TANKS Potassium in drainage water Soil treatment (oartelpeseallion) Tank : For Average Crop Lime each for crop tank treatment [Soke eto ee ae Planted. ....... Not limed...... 18.3 |% ice CARGO NORA NIG Planted........ imed =) eae 13.6 ee LA ie oe a he ull a BE NKeo gale eais-o or Not limed...... 15.5 14 0 IU Cirroe Wea erent area cnn Ny. Barer Limed......... 1D) : LYSIMETER EXPERIMENTS — II 71 In respect to the concentration of potassium in the drainage water from the bare and from the planted tanks, the Volusia and the Dunkirk soils are in accord. It is probable that this is to be accounted for, in part at least, by the greater volume of percolate from the bare soil, but it seems possible that the plant growth effects a solvent action on the soil potas- -sium which is indicated by the fact that the total removal of potassium in the crops and in the drainage combined is greater than that in the drain- age from the bare soil. Effect of liming on removal of potassium The application of lime to this soil resulted in a decrease in the quantities of potassium contained in the drainage water and in the crops. This is shown in table 25: TABLE 25. Average ANNUAL REMOVAL OF POTASSIUM FROM LIMED AND FROM UNLIMED ; TANKS (In pounds per acre) Potassium removed from Potassium planted tanks leached : ; from cor- Soil treatment Tank a e Tank eqn’ drainage | crops Total unplanted water tanks Motwlimed ee acs... os Se. 13 88.6 | 35.08 | 123.68 14 99 LUNE icles OO eee eae 15 57.8 | 33.12 | 90.92 16 69.9 There is nothing in this experiment to indicate that the application of lime caused the liberation of potassium. The same was true of the ex- periment with Dunkirk soil. It may be remarked, however, that if the application of lime did liberate any potassium from the surface soil, it may have been absorbed by the lower layers of soil and thus have been removed from the drainage water. The concentration of the drainage water from the limed and from the unlimed soil does not give any more indication of the liberation of potassium than do the quantities removed. The concentration of potassium is stated in table 26: 72 T. LyTrueton Lyon AND JAMES A. BizzELL TABLE 26. Porasstum ConTENT OF DRAINAGE WATER FROM LIMED AND FROM UNLIMED TANKS Soil treatment h : Potassium Tank mn mam oT | (SENS) joere Crop Lime million) PST A Ss cheno tain 5 orn Rave nih frereme tenant Planted eo Not limed...... 18.3 i Uae Ree Meter Ream ape ama E sera te et, Planted) on: Limed!: =. / 23 ere 13.6 1 2 Uinta here rae eae ae ALPEN SL Reet tid 3 Bare. sos )405 Not limed...... 15.5 1 UG Soe sea ara mR EES. Fp Pere Ria EATON Barey sexy e imedie ae 12.5 REMOVAL OF SULFUR Sulfur was recovered in the drainage water as sulfate, and it is significant that the years in which the content of sulfur in the drainage water was large were the years in which the removal of nitrogen by leach- ing was large. Drainage water from the Volusia soil contained somewhat less sulfur than did that from the Dunkirk, but the crops on the former soil contained as much sulfur as did those on the latter altho the yields were much smaller. Effect of plant growth on removal of sulfur There is one respect in which nitrogen and sulfur differ radically in this experiment, and that isin the proportion removed by crops and by drainage water, respectively. Nitrogen is removed most largely by the crops on planted soil, while sulfur is carried off mainly by the drainage water. The figures for sulfur in crops and in drainage water during the period of the experiment are given in table 27. The total quantity of TABLE 27. Suntrur IN DRAINAGE WATER AND IN CROPS (In pounds per acre, annual average) Sulfur in Tank Lime treatment oes note UL Sia pica care een iren AME tephra tg Not limed...... OMe 44.8 (el eens Ree Reet IS: Pane ten Ae at Not limed...... 43.3 43.3 FT ees Neh at tL pasLyy eae AMIN RN aie ey retina imed) 4 fost BB 5 44.4 NG eit ee epe cence OEE aac tne himedines ss eae 39.0 39.0 LYSIMETER EXPERIMENTS — II 73 sulfur removed from the planted tanks is not materially different from that removed from the bare tanks. Effect of liming on removal of sulfur In the experiments with Dunkirk soil the application of lime was ac- companied by an increase in the quantity of sulfur in the drainage water. In the present experiments this was not the case, as may be seen in table 28: TABLE 28. Average ANNUAL REMOVAL OF SULFUR FROM LIMED AND FROM UNLIMED TANKS (In pounds per acre) Sulfur removed from Sulfur planted tanks leached : from cor- Soil treatment . Tank 1h Tank responding drainage Geis Total unplanted water oP tanks INGtalimed ee Geaa jase ces 13 35.2 9.6 44.8 14 43.3 Weirme cd ereretse keeles eta: 15 380 10.7 44.4 16 39.0 Liming the Dunkirk soil did not result in an increased formation of nitrates but apparently favored sulfofication. Application of lime to the Volusia soil was accompanied by increased nitrification but had no effect on the production of sulfates. This would perhaps indicate that the con- ditions favorable to one of these fermentations are not always favorable to the other. : REMOVAL OF PHOSPHORUS The Volusia soil, like the Dunkirk, has never furnished more than a trace of phosphorus in the drainage water. The data on removal of this element are therefore confined to the ash analyses of the crops. The average annual removal of phosphorus (calculated to the element P) is shown in table 29: 74 T. LytTrLeton Lyon AND JAMEs A. BizzELL TABLE 29. PHospHoRus IN Crops (In pounds per acre, annual average) Phosphorus Tank Soil treatment in crops ASE Pees RR Sree ate Pea SCE Gab tcom eaten ata Not-limed'ss 3. eee eee 9.36 1 Hea es ee he eas ie a tego Ab Ue ag Timed 232) 0s ae eee 11.12 There is a larger annual removal of phosphorus in the crops grown on the limed soil than in those from the unlimed soil. This was borne out by the data for each year, which are given in table 7 of the appendix (page 92). The year 1913 was the only one in which more phosphorus did not appear in the limed crops. In this respect there was no similar- ity between the Volusia and the Dunkirk soil, the latter having shown no increase in the quantity of phosphorus in the crops grown on the limed soil. DIVERGENT EFFECTS OF LIMING THE TWO SOILS Comparison of the results of applications of lime to the Dunkirk soil with those obtained from the Volusia soil shows some striking differences. It will be remembered that the Dunkirk soil contained about 50 per cent more calcium in the surface foot than does the Volusia soil, and that this ratio gradually increased with the depth, the fourth foot of the Dunkirk soil containing 319 per cent more than the corresponding layer of the Volusia. The lime requirement of the two soils by the Veitch method was about the same when averaged for the four feet, altho it was slightly greater in the surface foot of the Volusia. It is evident that the lime requirement as determined is not a measure of the calcium content of these soils. In the light of this information it is interesting to observe the effect of liming in order to ascertain whether the calcium content or the lime require- ment is the better guide to the need of the soils for lime as expressed by their response in crop yield. The records for the Dunkirk soil show that there was no larger yield on the limed tanks than on the unlimed. On the Volusia soil there was a consistently larger yield on the limed soil each LYSIMETER EXPERIMENTS —II 75 year except the first, and this increase averaged somewhat more than 12 per cent for the five-years period. The calcium content therefore appears to be a better guide to the need of these soils for lime than does the lime requirement as determined by the Veitch method. The data at hand are too limited to admit of generalization, but they my be worth further consideration. Greater crop yield on the limed Volusia soil was accompanied by more nitrogen in the drainage water and also by more calcium. On the Dunkirk soil neither of these constituents was found in greater quantity in the drainage water from the limed tanks than from the unlimed. It may be remarked also that analyses of the soil air aspirated from the tanks, as reported in a previous publication, showed no appreciable difference between the limed and the unlimed Dunkirk soil, but in the Volusia soil the carbon-dioxide content of the soil air was much increased by liming. The fact that nitrate nitrogen in the drainage water and carbon dioxide in the soil air were present in larger amounts in the limed Volusia than in the unlimed gives evidence that decomposition of the organic matter pro- ceeded more rapidly when lime was applied to that soil. This, however, was not the case with the Dunkirk soil, and there is presented the rather unlooked-for situation in which lime increased decomposition of organic matter in one soil but did not do so in the other soil. A possible explanation for this divergent effect of lime on the two soils is suggested by the quantity of calcium in their respective drainage waters. As before stated, the application of lime had no effect on the > removal of calcium in the drainage water from the Dunkirk soil, but it increased markedly the quantity of calcium removed from the Volusia soil. It seems probable that by increasing the concentration of calcium in the soil water, the ammonifying, nitrifying, and other bacteria concerned in decomposition of organic matter were afforded a more congenial en- vironment. If liming did not increase the concentration of calcium in the soil water; as was -the case with the Dunkirk soil, there was no acceler- ation of decomposition. This experiment would seem to demonstrate one way in which liming may benefit soils. Certainly a larger amount of nitrate nitrogen was placed at the disposal of the plants, and the increased decomposition 4 Bizzell, J. A.,and Lyon, T. L. The effect of certain factors on the carbon-dioxide content of soil air. Amer. Soc. ‘Agron. Journ, 10:97-112. 1918. 76 T. Lyrrueton Lyon anv JAmEs A. BizzELu doubtless rendered other plant nutrients more available by breaking down the compounds in which they were held, as, for instance, the phos- phorus of organic matter. SUMMARY The object of the experiments here described was to observe the re- moval, by drainage water and by crops, of calcium and certain other soil constituents from Volusia silt loam. This soil is a rather unproductive type widely distributed over the hills of southern New York. The ex- periments continued thru a period of five years. The average annual rainfall for the five years was 32.97 inches. Of the annual rainfall, 27.13 inches, or 82.3 per cent, percolated thru the unplanted soil, and 20.62 inches, or 62.5 per cent, percolated thru the cropped soil. About two-fifths of the rainfall bamed into the | air from the surface of the soil and thru the plants growing on it. Application of burnt lime had no appreciable effect on the proportion of rainfall that percolated thru the soil. Similar experiments with Dun- kirk soil reported elsewhere gave similar results. Liming either of these soils would probably not facilitate the removal of water thru tile drains. The average evapo-transpiration ratio for the cropped soils was 1:908, the crops being maize, field peas, oats two years, and barley. The average minimum transpiration ratio for the same crops was 1:451. Both of these ratios were much wider for the Volusia soil of these experiments than for the Dunkirk soil in the experiments previously reported. In this com- parison the soil having the greater production of dry matter in crops per unit of water used was the one that had the greater concentration of total solids in the drainage water. The application of lime apparently favored the production of nitrates in the Volusia soil used in these experiments, while it had no such effect on the Dunkirk soil. The lime requirement of the Dunkirk soil as deter- mined by the Veitch method is very little less than that of the Volusia. The percentage of calcium is about one-third less in the surface foot of the Volusia. In this case the relative calcium content of the soil is a better guide to the need of the soil for lime than is the lime requirement as deter- mined. The amount of nitrogen in the maize, allowing for that in the roots, added to that in the drainage water from the same tanks, was greater than the amount in the drainage water from the corresponding bare tanks; while é LysIMETER EXPERIMENTS — II 77 in the case of oats the amount of nitrogen in the crop and in the drainage water was less than in the drainage water from bare soil. The same relation held with the Dunkirk soil. The quantity of calcium in the drainage water of the unplanted soil was greater than that in the crops and the drainage water combined from the cropped soil. Therefore the process of cropping conserves the calcium in the soil even when the cropsare removed. This may be accounted for, in part at least, by the large formation and leaching of nitrates from bare soil. Apparently the application of burnt lime to the Volusia soil increased the amount of soluble calcium in that soil, but this was not the case with the Dunkirk soil. The Volusia soil has a greater lime requirement and a lower calcium content than has the Dunkirk soil. To keep the soil supply of calcium up to its present amount would re- quire an annual application of 536 pounds of pure burnt lime, or 957 pounds of pure limestone, to supply the bare soil, and 371 pounds of burnt lime, or 662 pounds of limestone, to supply the planted soil. Magnesium was present in the drainage water in much smaller quantity than was calcium. Application of lime to the soil increased the quantity of magnesium in the drainage water. Cropping decreased the removal of magnesium from the soil. These relations were the same as for the Dunkirk soil. Potassium was removed in larger quantity in the drainage water than in the crops, in which respect the Volusia soil differed from the Dunkirk soil. It agreed with the latter, however, in that the application of lime did not increase the quantity cf potassium in the drainage water nor in the crops. Cropping did not materially affect the total removal of sulfur from the soil. Applications of lime resulted in a slight decrease in the sulfur re- moved in the drainage water. With the Dunkirk soil, applications of lime increased the amount of sulfur removed in the drainage water. Phosphorus was present in the drainage water only in amounts too small to be determined. Applications of lime increased the removal of phos- phorus in the crops. With the Dunkirk soil, applications of lime did not increase the removal of phosphorus in the crops. Memoir 38, The Crane-Flies of New York. Part II. Biology and’ Phylogeny, the third preceding number in this series of publications, was mailed on July 18, 1921. j ‘ ieee: Memoir 39, The Genetic Relations of Plant Colors in Maize, the second preceding number in this serie: of publications, was mailed on July 19, 1921. 78 T. Lyttriteton Lyon anp JAMEs A. BizzELL APPENDIX 1913 to 1917, ExpressEpD as Dry MATTER . TABLE 1. Crop Yreups rrom LysimMeTeR TANKS 13 AND 15 DURING THE PERIOD FROM Per tank Per acre Year Tank Crop Straw, Straw, Grain Cob stover, Grain Cob stover, (grams) | (grams) | or vines |(bushels)| (tons) | or vines (grams) (tons) ee LONGER re IBS OES Gad c SOAS iene ce 302.3 62545 eee 0.83 T5nl Oatssa ss: Bey a) i cas eon 285.5 51-842 ae 0.78 O14 ae: Sel Reastar.s- 5" Gu llassee 452.2 828) |Eaneeee 1.24 jm |ePeastrm- AD? O's |e ie 486.5 SAO cl Sanne 1.34 LOTS See eee 13 | Maize.... Sie? 220 746.3 3.0 0.06 2.05 15 | Maize 121.6 68.4 821.8 ile 0.18 2.26 1 LOUGH oe Oatse = SAR SE! eee ete. 266.7 31:262|i eee 0.72 5M Oatse sees SO eee 293.5 3115: OF Peer 0.80 AOU evis Igo Barley/s=<|| VASr4anin sa. 161.2 PSO |e eee 0.44 15s) eBarley:. s2| al OIROu le aac 212.0 Qi. BF) Ss aa 0.58 TABLE 2. Frow oF DRAINAGE WATER FROM LYSIMETER TANKS 13 TO 16 FRom May Il, 1913, ro Aprit 30, 1918 : (In liters) : Tank Year and month 13 | 14 15 16 1QUS= Mayas ost e eden a SP ee ees 56.8 alee, 70.4 72.0 UNO a. Seas pe tic eee renee er eR ees 2.4 24.0 3.2 Doe TU yeas coke stoi eae Oe a 0.0 5.6 0.8 4.8 PATIO USE Kosi ern pee feelers ee eae. 0.4 0.8 0.0 0.0 September as. cengeeryces a ee ictae aa 17.6 72.0 0.8 67 .2 October ees Saar Sere a nese 37.6 124.0 26.4 92.0 Novem bersnnn ecm cee 91.6 115.2 79.2 89.6 December ee rae ct eee 49 2 54.0 44.8 41.6 19]4—Januanye oc ee ee ee eee 87.6 90.8 116.0 156.4 HeDrUALY Salk Se cota eee eel Dee, 5Hle2 38.0 40.4 Mir chivas = Sts Cees terete cies ee 159.2 211.6 206.8 188.0 Aprilsien tk 4c Reet ae cee mee Moc REe Seen 220KS 288.4 F5ae 149.2 Totals Sea. fetes ae, oe 750.4 | 1,108.8 | 801.6 924.4 LYSIMETER EXPERIMENTS —II - 79 TABLE 2 (continued) Tank Year and month Bc 14 15 16 HOA Maver en eR en. 102.4 130.0 98 .4 100.0 JUNC osetia ee ey A: 24.8 59.2 22.4 65.6 IUIRS 3.6.0 3 acct O RIE ee ae tea te ae 0.0 23.2 0.4 16.8 INUUB is 23 0 6 be Ee Ge Ee ee 5.6 220.0 0.0 152.0 SUGGS. 6 Too Ga eee Soe eae 17.6 64.0 3.2 87 .2 Ochoberpewnnmrr stn aoc ose: 0.0 2.4 0.0 0.0 Novem Deletyrirst oe ake cbse Geesat 0.4 19.2 0.0 4.0 ID AGGHT IGE. < See See eee a eee 28.4 50.2 15.6 57 .2 ELS SAMURTY a5 6 o Gece nae ene ee 221.6 278.0 246.8 285.2 INGLSTRIETATS 6 4. bois Dire eee aoe ee 224.0 179.2 339.6 304.0 ING TREN S 3.5 on craspiches Sean eee eee 9:2 2.4 12.0 3.2 A\p yall, <5 0.6 6.0000 SR Oe 5.6 1.6 Doh 1.6 Motalsergat 5th. os tes ek vhs cok 635 .6 1,034.4 741.6 1,076.8 LOIS E576 6 60b.0.o Oe nee eee 18.0 ASD 3 14.4 31.2 iMiGs55 ob bee ke bb eee lien, 85.6 63.2 66.8 AICI cia 6b 8 Ges bo Sener an 242.0 292.0 183.6 234.0 AN ORRT Roo 6 60.6 oars ee ce ee 26.8 114.8 10.0 94.4 MEDHEIMIMOCLP MT gets fice tanh cen aes ee 14.4 75.6 6.0 52.8 WeboWeire wie eee i ee ie aor 146.8 178.0 ghey 144.4 INoOvembenshre 0. eee 29.2 43.6 26.4 30.8 Wecempenprtee chine eens ees 69.2 67 .2 56.0 66.4 IONGATEIITRINY.S Scc-5 ole circle Soria aee e 90.4 86.4 72.8 73.6 ebnuamy eer esha es os 4.8 8.0 6.4 5.2 IMB CHROME Reet oh Gudectue. 96.8 143.6 109.6 137.6 ANOHIIS 5 6 9.606 oe Ra ae ane 238.8 268 . 4 187.6 178.4 Motalsertircs sees. whe. cas 1,054.4 1,388.4 847 .2 111526 LS LISLE 0d I 166.0 165.6 100.4 114.8 JUGS Gad 4 odie athe SOAS: See ee VaLay 6 6 acs Oats a ER ae eer INGRIGE. 639 RE See eee 241.6 454.8 224.0 366.0 EDLC CLs tiers. oh ieloccenck een cinco ODOT? 5.6 dg SCAR Eee IN@WGITLNES oo Ge ee eee Wecrembperer niet ae ce ae hee eat 76.8 2S 85.2 65.6 “Sl ET See MERI AN yael Ope etc ee gales sets IPQ OOBIAY a 9 aise eat oce pene ene ee ance Wien ns o 6p Ss RG e geo ee ae 162.4 142.8 130.0 132.8 AN Oral. oe eee gone a ee Osan Oreo eee TOs cos Sebo re bb oe Ore 646.8 836.0 539.6 679.2 80 T. LyTrLeton LYON AND JAMES A. BizzELL TABLE 2 (concluded) Tank Year and month 13 ey? ie 16 IteMag Se ee ae 178.8 | 194.8 [ezine 172.4 SULA CEM Oe MS Ae alae aN a SE CO AS ee ny 282.0 345.6 228 .0 330.0 uly. sieea essa eee ke ee a 46.8 80.8 |. 39.6 83.2 INVA S ke Reema sh aon ety Waly tea 268 .4 296.4 214.0 318.4 Septembers ss -cieey se es ae e 48.8 62.4 57.6 61.6 Octoberseacs ko sess neces see cea cee 203 .2 219.6 186.0 216.0 INovemberse ce sehen ti Tennent 24.0 20.0 22.4 a2 WMecember iste eee ee cee pane 15.2 17.6 24.8 24.8 IOS Tan vary aye tae eee oe etcey ee & 0.4 0.0 1:2 0.0 He brUany eee as ee eee eee 0.0 64.4 50.8 52.8 Miarchystisen get tie ee at ae ee aS Nad 32.0 73.6 29.2 66.8 Aor es Bowes pare rae earn ie we eae eed 192.8 143.2 152.8 142.8 Ray a elt tere ag each chore nage A ze 1,292.4 | 1,518:479)2 Ta7sno 1,486.0 Granditotals@. sateen eeu ae cease 4,379.6 | 5,886.0 | 4,108.0 5,282.0 TABLE 3. Fiow or DrainaGk WATER FROM LYSIMETER TaAnxs 13 To 16 FRom May 1, 1913, ro Aprin 30, 1918 (In acre inches) Tank Period 13 14 15 16 Mayall toill3 sto April’ 30; 194i Wien eee 18.23 | 26.94} 19.48 22.46 Mayale oid: tozApril SOLO seek Setesny ee acta sic: 15.44 | 25.13 | 18.02 26.17 May il. 1915:stozApriliSOP 0916. en. ie oes oe 25.62 | 33.74] 20.59 27.11 Mayol. 196 stovAprill SOW 19S yy ere 15.72 | 20.31 13.11 16.50 May 1, 1917, to April 80, 1918...........3......... 31.40 | 36.90 | 28.62 36.11 Average annual percolation................... 21.28 |} 28.60 19.96 25.67 LYSIMETER EXPERIMENTS — II : 81 TABLE 4. MerroronocicaL Recorps at IraHaca, May 1, 1913, ro Aprin 30, 1918 Year and month TWINS 6 otis cere INTERVEN s oo one decne September........ October.......... November........ December........ 1914-January.......... February......... AURUSGs oodcc os aes September....... October.......... November........ December........ 1915—-January.........- February......... September....... October.......... November........ December........ 1916—January.......... PMUICUSte esis: =e Octoberse- +): November........ December........ Data by months Rainfall (inches) on Soo RETR E WRN NNONE RN WRAWNOORTN OR RFORP WBE EEN WWRHNW oo bo — S pay Temperature (degrees Fahrenheit) Mean Mean maxi- mini- | Mean mum mum 66.2 44.5 | 55.4 78.6 51.4 | 65.0 82.5 68.2 | 70.4 82.0 57.2-| 69.6 73.0 49.2 | 61.1 61.3 Ab 4 | 53.4 51.5 35.8 | 43.6 39.8 Ze daloOnG 33.7 19.3 | 26.5 27.0 8.5 | 17.8 39.1 Aw Smale lter(G Mall Solem algal 47.5 | 59.3 76.5 HANS OD 5) 81.2 59.2 | 70.2 80.2 58.2 | 69.2 71.1 48.2 | 59.6 63.6 43.9 | 53.8 46.4 31.1 | 38.8 32.9 19.1 | 26.0 33.4 19.7 | 26.6 37.8 22.7 | 30.2 Sid 21.9 | 29.7 62.7 39.6 | 51.2 61 6 41.6 | 51.6 C0 51.6 | 63.6 80.2 58.2 | 69.2 75.0 Dat) || Koea4 75.6 54.1 | 64.8 59.9 AB at ols 47.7 Soule P40R4 33.6 23.3 | 28.4 40.0 Dre (lone 30.1 13-0) |) PALS SRO 127-0 53.7 37.0 | 45.4 68.4 46.9 | 57.6 70.9 51.9 | 61.4 85.8 63.5 | 74.6 83.7 Oils anil 2 72.8 50.9 | 61.8 63.1 39.6 | 51.4 46.7 31.4 | 39.0 35.9 22.0 | 29.0 Hours of sunshine wb © OO Nd 304. DUNT NOW RMONADOSCHRODRUNMHOWOEN BE WY NONE NOOO Ny © Average hourly velocity of wind (miles) 10. = ONIWOO S MW NI WHODRAROH NOH RODWARRODORRANRDOOUHAWANDOOHHE NAO — a Noo Mean humidity of air at 8 a. m. (per cent) 82 : TABLE 4 (continued) T. Lyrrieron Lyon anv James A. B1zzELL Year and month 171.2 Temperature (degrees Fahrenheit) Mean Mean maxi- mini- | Mean mum mum 34.1 17.7 | 25.9 29.6 10.4 | 20.0 41.9 DO MESono 52.4 35.5 | 44.0 56.7 40.0 | 48.4 74.1 53.6 | 63.8 81.4 61.6 | 71.5 79.9 58.5 | 69.2 69.9 47.2 | 58.6 53.6 36.7 | 45.2 43.3 26.4 | 34.8 26.9 10.7 | 18.8 22.0 6.8 | 14.4 35.0 13.7 | 24.4 48 .2 25.3 | 36.8 56.3 35.2 | 45.8 Mean humidity of air at 8a. m. (per cent) Average of each month 1917-January.......... 1.82 February......... 0.70 IM IRA NS 5 aga oc 1.59 iMprile pie 4-9) |37e2 11356 9.9 (mie Miaizenses|e otal ssl a24Zeouie One| 5.001 40.0) 1428 12.4 DUG peas ge ake. smleOstses.-.| Grainia.: SBS le Ose lh Mak ll 7s) | 20 4.8 mOatsem ce Straws... WSs Aen ILS |loakO IL |} Zhe 2.8 130 | Oatsia Motaleessien 1 5IeSeler4e3 te 2et4329 0 Ges 7.6 15m Oatse =o. Grains =>: Roy 4) Osi 0.4 6.7 1.8 522 yn Oats ee Stra wee | el 26ele ll eeoeo: |(mro- on noose tans 3.8 en eOatseea-+: otaleewae |e LGAsOn lors. || sedate ele Sal G26 9.0 3 rn ise pareve |sGrain’ ee \s ote OF2.|° OF2)) 4:4.) 1.8 4.3 13 | Barley...| Straw... 2G | Bet Woe) ADeak |e zal 1.8 13 | Barley ...| Total. ... 90.3 DEAD | ROSON lS 25m |e ono 6.1 15 | Barley...| Grain.... RO | OL Wray 1] sit Zeal 5:3 15) || Barley... .| Straw... - 72.9 | 3.9 1 (in| le OF | e2e9 2.9 15) |) Barley...) Total’....) 103.6) 4:1 DA) |) a¢/aal DON enee ——$<$<<§@2Jai_i i JULY, 1921 : MEMOIR 43 CORNELL UNIVERSITY AGRICULTURAL EXPERIMENT STATION VARIATIONS IN BACTERIA COUNTS FROM MILK AS AFFECTED BY MEDIA AND INCUBATION TEMPERATURE G. C. SUPPLEE, W. A. WHITING, AND P. A. DOWNS ITHACA, NEW YORK PUBLISHED BY THE UNIVERSITY = ; ¥' v7, ea 4 att + +a ees a SRS AAS SARS AAS SS i YATE ARE eee Abe 10 © PVUROTR BM tae 5 Sail VARIATIONS IN BACTERIA COUNTS FROM MILK AS AFFECTED BY MEDIA AND INCUBATION TEMPERATURE VARIATIONS IN BACTERIA COUNTS FROM MILK AS AFFEC- TED BY MEDIA AND INCUBATION TEMPERATURE G. C. Supeitee!, W. A. WaiItinGc, anp P. A. Downs The increasing importance of the liquid-milk supply for large centers of population and the greater demand for a better quality of raw mater- jal for manufacturing purposes have necessitated ‘further knowledge of the methods used in determining the quality of milk, and particularly have emphasized the significance of bacteriological analyses. Examination of milk for the purpose of determining numbers or types of bacteria seems to constitute the highest ideal in milk grading. This aspect is doubtless important and the respect for this method of procedure in judging milk quality should not be endangered. Obviously, the sani- tary aspects of the milk problem must involve determinations of this kind. The significance of bacteria in the economic phases of the milk supply also becomes quite clear when it is remembered that the entire business of supplying milk to the urban population is founded on modern dairy - science, of which the aims are maximum wholesomeness and maximum _ keeping quality. These two considerations have been responsible for the stress laid on the importance of bacteria counts in the milk industry at the present time. That the methods of enumerating bacteria in milk have many short- comings is well recognized by dairy bacteriologists. Bacteria counts, as now obtained, can be interpreted only on a comparative basis, and in no sense do they indicate the mathematical accuracy which their expression in numbers implies. Such comparative interpretations can be used only as indications of degrees of success in handling and of the variations of keeping quality. Undeniably this information is valuable in safeguarding the interests of consumers of milk in large cities, and its importance is shown by the report of the Committee on Statistics of Milk and Cream Regulations of the Official Dairy Instructors’ Association (1917). This committee obtained the complete milk regulations from 409 cities and 1At present Director of Research Department of the Dry MilkjCompany, New York. *Dates in parentheses refer to Literature Cited, page 247. 221 222 G. C. SupPpLer, W. A. WHITING, AND P. A. Downs towns in the United States, and found that 189 of these provided for a legal limit for bacteria in milk sold within the municipality. The limits allowed by these cities ranged from 50,000 to 5,000,000 to the cubic centi- meter, with approximately one-half of the cities permitting a limit of 500,000. The necessity of fixing legal limits for bacteria in cream seems to have been regarded as much less important, since only 30 of the 409 cities had established legal limits for this product. The bacteria allowed in the latter case varied from 50,000 to 1,000,000 to the cubic centimeter. These municipai regulations must of necessity imply provisions for their enforcement and for penalties for failures in their observance. Such provisions immediately bring into prominence the difficulty of application and enforcement of numerical bacterial standards. Unfortunately, the inherent inaccuracies of present methods of enumerating bacteria are too great to permit their results to be relied upon with the certainty of exact- ness which their fixed numerical standards would seem to warrant. REVIEW OF PREVIOUS INVESTIGATIONS The American Public Health Association (1915), recognizing the wide variations obtained by the ordinary, plating technique, have formulated, through their Laboratory Section, the following uniform method for deter- mining bacteria in milk. This procedure, known as the “ Standard Methods of Bacterial Analyses of Milk,’ has been of considerable value in securing uniform technique in different laboratories, and the results are comparable, since a uniform interpretation can be given to them. That the purpose of the Standard Methods is for securing uniform results rather than accurate counts, in the minimum length of time, is evident from the fact that 37° C. for forty-eight hours on plain agar is the only incubation temperature and medium recognized. In the routine exami- nation of milk samples, the short incubation period has certain distinct advantages. Conn (1915) compiled the results obtained from an exhaustive series of comparative determinations made from the same milk by four labor- atories. This work, involving many thousand platings made under uni- form procedure, nevertheless failed to give uniform and consistent results under those particular conditions. Viewed from the standpoint of abso- lutely accurate determinations of all bacteria present, there are numerous. VARIATIONS IN BAcTERIA COUNTS 223 reasons why the plate method gives widely discrepant results. Among the most important causes are: (1) the failure of certain species to produce visible colonies on the medium and in the incubation temperature used; (2) the tendency of many species to exist In groups of two or more individ- uals, which groups are broken apart with varying degrees of completeness during the plating operation; (3) too few or too many colonies to the plate; (4) the inhibiting or beneficial effect of diffused by-products from the growth of certain species on other species within the radius of diffusion; (5) the personal element involved in carrying out the method. Widely varying, results from the same sample of milk under the same conditions of incubation temperature and medium would still be caused by the clumping tendency, by the number of colonies on each plate, and by the personal element entering into the manipulations. Hill and Ellms (1897) early called attention to the unreliable results obtained from over-crowded plates used in water analysis. The Standard Methods stipulate that there shall be not less than 30, and not more than 200, colonies to the plate, altho Breed and Dotterrer (1916) conclude that limits of 30 and 400 are nearly as satisfactory. Altho the Standard Methods call for plain agar incubated at 37° C. for forty-eight hours, comparative counts published from time to time have shown that a carbohydrate medium and a longer incubation period at a lower temperature have many advantages. Heinemann and Glenn (1908), from their work on the effect of incubation temperatures and media, reached the following conclusions: 1. Since pathogenic bacteria are always difficult, and in most cases impossible, to find in milk, a high temperature of incubation has no advan- tage over room temperature from this viewpoint. 2. Incubation at 20° C. is superior to incubation at 37° C. because both a higher count and a better differential count are obtained. 3. Dextrose is preferable to lactose as an addition to the medium. 4. Miik is usually consumed before the results of bacterial examinations are available. Accordingly bacteriological «nd chemical examinations should have as their principal objects the improvement and control of the general supply; and accuracy being of greater importance than quick results, the loss of a day in its interest is irrelevant. 224 G. C. SuppLEE, W. A. WHITING, AND P. A. Downs Sherman (1916) points out the higher counts obtainable by the use of lactose agar in place of plain agar, and also the increase in the size of the colonies and the better differentiation of the types. Breed and Stocking (1917) published a preliminary report on a series of comparative determinations, in which they conclude that the plate method, when used by careful workers, will give more reliable results than those reported by Conn, which had been obtained under routine conditions and possibly, in some instances, by inexperienced operators. Obviously, inexperience and carelessness are factors to be avoided in any method of enumerating bacteria, especially when the results are for the determination of municipal regulations. The same authors (1920), reporting a similar but more extensive investigation, found the plate method and the micro- scopic method (Breed method) productive of reasonably uniform and accurate results for the total number of bacteria present, all factors known - to introduce inaccuracies having been first reduced to a minimum. For the plate method they report an average coefficient of variability of 8.3; for the microscopic determination of groups of bacteria, consisting of one or more individuals, 11.7; and for the microscopic determination of individ- aal bacteria, 13.4. Altho these results are remarkably uniform, it must be remembered that they are obtained from samples which were artificially inoculated in order to reduce the clumping tendency to a minimum, and that the time and labor necessary to obtain this degree of accuracy by the- microscopic method could not be expected in regular, routine examinations. . PRESENT EXPERIMENTS Comparison of media and incubation temperatures The experimental work reported herein was for the purpose of demon- strating the variations in counts obtained by plain and carbohydrate media at different incubation temperatures. It may indicate a further reason why the count at 37° C. for forty-eight hours, as used in routine work, may be more subject to discrepancies than counts obtained from longer incubation periods at lower temperatures. The samples. used for this work were selected at random from the ordinary market milk of the Ithaca city supply, at intervals extending over a period of one and one-half years.. Twenty-seven plates were made from the same dilution of each sample. Nine of the plates were VARIATIONS IN BACTERIA CoUNTS 225 poured with standard plain agar; nine with nutrient agar containing 1 per cent of dextrose; and nine with nutrient agar containing 1 per cent of lactose. The different agars were all made from single, large-quantity batches of plain, nutrient agar. These were subdivided, and the definite percentage of the particular carbohydrate desired was added to each. Three of the nine plates containing the different agars were incubated at 37° C. for forty-eight hours; three at 30° C. for five days; and three at 20° C. for five days. With the exceptions noted, the technique given in the Standard Methods was carefully followed. It was necessary, however, to include counts from plates containing fewer than 30 colonies and more than 200 colonies, altho in all cases the dilution was designed to give colonies between these limits from the forty-eight hour count at 37°. In table i are shown the counts obtained from 100 different samples of milk from each of the nine combinations of incubation temperatures and media. The individual counts appearing in this table are the averages of triplicate plates. Each plate of the series of three checked with the other plates of the series as closely as would be expected from duplicate or triplicate plates from the same dilution of any sample of normal milk. In order to indicate in a comprehensive manner the variations obtained, the 37° count was taken as the standard. Any variation above or below this count 1s indicated by a plus or a minus sign. The variations are shown also as. percentages, the 37° count being accepted as 100 per cent, and counts higher or lower being indicated by figures above or below 100. G. C. Supper, W. A. WuitinGc, AND P. A. Downs 226 £08 +006 ‘8% 002 ‘SE ZOE +008 ‘8% | 009 ‘Se 9ge +008‘ 22 009‘ 68 0G LOE +00P V6 006‘ 98 068 +000' 92 008" LE €g¢ +006°6@ | 00L'TP 08 SGI +0099 008 ‘ST SST +0089 OOL ‘ST OO Tie), Wiles Fe ee 008‘ TT Shee eae ‘8 £21 +000° 09¢ 000‘ 096% OST +000 062 000'0ZT's LZ +000 099 000'090'¢ 00% EST +000' 062 000‘ 061 g ISI +000' 02 000‘ 0ST’ E21 +a00‘0¢¢ | 000‘096'% | 08 16 —000‘O1T 000‘06z‘Z CL —000‘099 000° OFL‘T OO DER tle rae 000‘00F‘Z | LE ppasuhte5)): OTT +0005 QOL‘ OT TIT +009°T 00L°S1 821 +006'§ 000°ST 0G PIL +002 ‘2 008‘ OT SIT +008 ‘T 006‘ ST Ze +006 ‘PF 009 ‘ST o08 $11 +008 ‘T 006 ‘ST. G01 -+00F 009 FT OOM al aea ra EPPO OO TAL Ls gs crt) GPT +000‘0¢¢ 000‘ 090‘ T. LET +000‘ 09% 000/026 O81 +000‘01Z 000° 026 0G 801 +000‘09 000‘ O22 SIT +000 ' OST. 000‘ OFS SEI +000‘02z 000/086 08 ZG —000‘ OFS 000028 Z9 —(000‘ 02 000 ‘OPP OOM, ata ea aig 000‘ OTL aks soe 2G, 68T +000‘'F 008 ‘FT kil +002‘Z 00G°S1 OPT +001 'F OOF al 00 LET +008 '% OOT ‘ST OET +002‘ 000' FT GPT +009‘F 006° FI o08 06 —000‘T - 008'6 69 —002‘S OOT 2 OOlSe ea eae BOOGROL Sie ae ote P 86 —00F 002‘ 9T 60T +009'T 002 ‘ST CET +000‘9 OOT 16 002 OT +002‘T 008 ‘ST. 66 Oe, 000° 21 val +001 ‘P 006516 008 ¥6 —000‘T OOT‘9T 66 —00T ‘T 000‘ 9T OO mule oe oe OO TAT fs shia NaS £6 —00L'° 008 ‘8S G8 —009'9 009° VS 101 +00 009" TF 0G ¥6 —00P‘Z 009 ‘88 £6 —009‘Z OOP 88 101 +008 008" Th 08 68 —00F'F 00998 G9 —(09 ‘FT OOF 9% OT seis aes fmm OOOnTT, LG ee ail aoe be SPI +000'062°€ | 000'O60'OT — | TIT +000'0¢2 000‘ 09S" L 88 —000'0€8 | 000°026°S | 02 OCT +000‘082‘°8 | 000‘08S ‘OT ELI +000‘066‘F | 000‘'062‘TT POL +000‘08z 000°080'2 | 08 ELT +000‘0FT'S | COO‘OF6 ‘TT OLT +000‘018‘F | 000‘0T9‘TT OO Ten Sieg aera ne 000‘008°9 | Lg ek quod rod yuoo rod yuoo rod (CO) 9) Bul dIUIIO JIC, 9801} X90 94 Bul 9dUOIO FIC 980498] 9y Bul 9oUdIO ICT Iese ureyqg | eanyvaod) opdureg -1xo1ddy -ixoiddy| -1xoiddy -W9 J, STUALVATINAT, NOMVANONT INGUMITIG: LV UVOY GDLVUGAHOGUVY GNV NIVIG AG GANIVLAG SLNNOD NI SNOMVINVA “T WIAVL 227 VARIATIONS IN BACTERIA COUNTS —00F —002 —006'T +000' 006° T +000‘ 004 *€ —000‘OO0T —000' OT +000‘F +000 ‘6z +000 ‘098 +000‘ 09F +000‘ 0 +002‘ +00L'S —00F +000! 0L¢ +000' 079 —000° O21 006‘E —002 ‘6 Hye; 2 +000/ 091 +000‘ 064 —000‘ 00 —000‘s —008'T —007 009‘ TT 008 ‘TT OOT ‘OT 000‘ 00F ‘ OT 000‘ 006‘ TT 000‘ 00F ‘8 000‘ 89z 000‘Z8z 000‘ 208 000‘ O6T ‘T 000‘ 062 ‘T 000‘098 002‘'SE 00L° 28 009‘ TE 000‘ 022 ‘% 000! 066° 000‘ O8F ‘T 008‘Z8 00° LOT 000‘ 601 000‘ 0gh ‘9 000‘090‘ 4 000‘026‘'¢ 009‘ €z 008 ‘Sz 002 ‘9z —00F —002 ‘T —001 +000‘000‘T +000‘ 002 —000‘009 +000‘099 +000‘ 019 +000‘ OFT —008 ‘98 —=-00 2 +00F'S +000'02 +000‘ OTT —000‘ 082 +006 +000‘'¢ +008‘8 009‘ TT 008‘ OT 006 ‘TT 000‘ 006 ‘6 000‘ 06258 000‘ 006‘ 2 000‘ £92 000‘ 9Sz 000‘ PLZ 000' 086 000‘ 096 000‘ 082 00S ‘Se 002 ‘9 008 ‘Sz 000‘ 01g'z 000! 092‘ 000° 062 'T 00F ‘08 009‘ 68 OOT ‘Za 000‘ OFS ‘9 000‘088'9 000‘ 06F‘S 009‘ 1z 009‘ 6Z OOF ‘Sg 06 LOT OOT SIT GGL OOT LIT IIT OOT 6&T Tél OOT T&T 66 OOT OFT €ST OOT 63 G6 OOT vol 08 OOT 9&1 vit OOT —008‘T +006 +000‘ OOT ‘T +000‘006‘T +000 ° 9F +000‘TS +000‘ 02 +000‘099 +000‘028 +000‘ 08PT —000' 06 'T +002‘6 002‘ OI 006°ZI 000 °ZI 000‘009‘6 000‘ OOF ‘OT 000‘00¢‘8 000‘FZE 000‘ 60 000‘82z 000‘ OST'T 000 060 I 000‘0&8 0) Bei g OOF‘ 6z 000‘ cE 000‘ OTE ‘z 000'0zS‘Z 000'0¢9‘T ial ETS ra EE Heese ecoe G. C. SuppLEE, W. A. WHITING, AND P. A. Downs 228 LOT | +000' £08 86 —000‘61 96 —000‘0¢ 02 —000‘8¢ 06 —000‘ 2 SP —000‘S88 0¢ —000‘T OST +000‘T 0S —000‘T OSP +000‘8z LEP +000‘ 22 GZS +000‘ OT cor 008 *T SIT +008 SOL +001 OST +000‘ 00z ial +000‘ 09T EC +000‘ OST CIT +006 ‘82 SL — 009 ‘FE 96 —00L'9 6C1 +000‘ 08z 6c +000‘ 08z S01 +000‘08 quedo Jad 9} BUI soUsIO NIC, -rxouddy 000‘ £00'% 000° TST 'T 000° OST ‘T 000‘ ST 000° 99 000‘ ¢¢ 000‘T 000! 000‘T 000‘9 000‘SE 000‘S8T OOF Ss 00F ‘Z 002 ‘z 000‘098 000‘0z8 000‘0I8 00L ‘SLT 008‘ OZT OOT ‘SFT 000‘ OFZ ‘T 000‘ OFZ ‘T 000‘066 a801}X9(T G —000' 181 'T 000° 61 i lz —000‘¢28 | 000‘SzE ZI +000‘ FOI ‘T 000° F08*% 061 +000‘TS80‘T} 000‘ 18z°% OFT +000‘ LSP 000° 289‘T OOT EEE SELL NOOO OOS AL CT —000'29 000° TT OF —000‘ FF 000‘ 6z SI +000 02 000 £6 FOr +000°E 000/92 1s —000‘F1 000° 6S OOT ee O00. ez 00 ++000'% 000°F 002 +000'% 000°F COOL 4 Gees se OD) 00g +000‘'F 000°9 OST +000‘T O00RS= OOT D cwemeaceael| 01010) 16 OOF +000 ‘Fz 000‘ZE 00z +000‘8 000‘9T OOF +000° G 000‘Z GCP +000‘9z 000'FS OST. +000‘F 000‘ZT OT 1s ie ea as 000°8 621 +009 002°% 6IT +00F 00¢‘Z QOTe eae ys OOT ‘Z 7a +00¢ 009‘Z 6IT +00F 00¢‘z OOM AR vere Yaa OO‘ ital +000 ‘OFT 000‘008 CEI +000° 01Z 000/028 16 —000‘09 000‘ 009 GOT +000 ‘09 000! 0cL OOT BWawelictieveciaene cere 000‘099 OOL 0°60. 0 .0rD 0 & Ost 000 099 6L —000‘ze 008‘ ZZT SIT +008 ‘22 009 ‘2ST Z6 —006 ‘GT 009 ‘PT OZT +009 ‘08 OOF ‘SST 88 — 002‘ 6T 009‘ SET OOT:. sree 008 ‘FST. GPT +000‘ OS 000‘ 068 ‘T PIT +000 ST 000° 060'T LG +000‘ 092 000‘0zz‘T CIT +000'0FT | 000‘00E'T eel +000 ‘0z¢ 000‘ 0S8z‘T (010) Gere (cea ee 000‘ 096 queso rod quad Jed 07 eUL 9000.10 AIC, 98070B'T oyeUul QoUa1O BIC Iv3se UIT -rxoiddy -rxolddy (panuruod) [ AIAV], 006 06 oL& o0G 08 oL& o0G o0E oL€ (0) oan4vaod -W9 J, “sree sge ajdureg 229 VARIATIONS IN BACTERIA COUNTS IGI LET 66 LbT 8ST 1G POT 806 OFT 61€ VEG G0G iS) SOT GL TZI 906 8&6 68T c9l Té1 e1g 896 SL 0g Go0'% Z6L‘T +0006 21 +000‘ 08 —000‘9 +0006 +000‘ TI —000‘ST +000‘ 09T +000‘ 022 +000‘ 00 +000 OFT 'T +000 ‘092 ‘Z +000‘ 0g¢ —000‘008 ‘8 +000‘ 0zz —000‘060‘T +000‘82 +000‘ SOT +000‘ TFT +000‘TZ +000‘0¢ +000‘ ¢z +000‘ ¢sz +000‘ 18z —000 ‘ST —000' To » +000 90% ‘T +000‘81z‘T 000‘ 26 000‘ OTT 000‘ F2 000‘ 8z 000‘ 08 000‘F 000! OTF 000' 0z¢ 0000¢8 000'099'T 000! 08L°% 000° 0¢0'T 000‘0¢9 000‘ O21 F 000‘ 098°z 000‘ SLT 000‘ O01 000 ‘8% 000 ‘TST 000/081 000‘ SOT 000 ‘cre 000‘ TPE 000‘ LF 000‘ 12 : 000‘ 8ZF‘T 000 ‘062 ‘T +000‘ ¢F +000‘ 2 +000‘SI +000'8 +000'F% —000°E +000‘02z +000 ‘088 —000‘08 +000‘ 09T‘T +000‘ 082 ‘T +000‘ OST + 000/019 °% +000‘ 088 ‘Z —000‘099 +000'28 +000‘72 +000'29 +000‘ ST +000‘ SF —000‘'F +000 ‘Zt +000 118 —000‘8T —000‘ TF +000‘ 62 +000‘¢29 000‘ SzT 000‘ Z2T 00086 000‘ 22 000 ‘SF 000‘9T 000‘ 06S 000‘ 089 000° OT 000‘ 089 *T 000° 008 ‘T 000‘029 000‘09F ‘9 000‘0S8z ‘9 000 ‘062 ‘8 000 ‘FST . 000‘ FLT 000 ‘F9T 000‘S86 000! Sc 000‘92, 000‘Z0¢ 000‘ TLE 000 ‘cP 000‘TS 000‘ T¢9 000‘ 2FL 901 aa OOT OST G06 OOT 4 GGG GES OOT &GE I8é OOT PST OLT OOT Ila LST OOT 6ST LOT OOL Z6$ cP OOT Z8 86 OOT +000°¢ +000‘F +000‘ ZT +000 ‘08¢ +000' O9T “I +000 ‘09F ‘T +000‘ 018 '¢ +000‘062°2 +000‘8¢ +000 ‘FS +000‘ 862 +000‘228. eee ee 000‘¢s 000‘ FIT 000‘08 000‘98 000° 68 000 ‘6T 000 ‘089 000‘08¢ 000‘ 0Sz 000‘09z‘ 2 000 0FL'9 000‘0¢6‘§ 000‘&1Z 000‘ 09T. 000‘ZOT 000‘ 221 000‘ F&I 000‘08 000‘S8Ss 000‘ 288 00009 000/29 00°04 000‘22, o0G o0§ oL& o0G o0& oL& 0G o0E | oL& 0G o0& oL& o0G o0& oL& 0G 308 hs 0G VE oLG o0G o0E oL& 0G OE oL& CEERI og) G G. C. Suppuer, W. A. WHITING, AND P. A. Downs 230 queo sod ayeur -1xoiddy +000‘ 06z +000 OFT —000' 06 —000‘T +000‘ OLT +000‘ 08T +000 ‘08 +000‘ 0F9 . +000‘0¢ +000‘ 08z +000‘ OFZ +000‘ 0ZT. +000‘098 +000‘ 02S +000 0&z +000‘009 +000‘064 +000‘09 +000‘ 06z +000‘ 06¢ +000‘ 01Z aouUaIO HI 000‘008 000‘0¢9 000‘ 0zr 000‘ 61 000‘ 06T 000‘ OST. 000‘ OF 000‘0¢S9 000'09 000 008 000‘ 092 000‘ OFT 000‘ 088 000 ‘06¢ 000 ‘02 000‘0T9 000‘ 008 000‘02 000‘ 0zE 000‘ 029 000‘ OFZ 006 ‘6 006' FIT 008 ‘TST 9s0.1x0(T ques red oyBUl -rxoiddy +000‘08 +000‘ OF —000 ‘09% +000‘ OT +000‘ 09 aoudIo ICL 000 ‘OFS 000‘0S¢ 000‘ 0Sz 000‘ 0z 000‘ 0z 000‘0z 000‘ OT 000‘ OT 000‘ OT 000‘ OF 000‘0z 000‘ OF 000‘ 0 000‘0% 000‘ 0z 000‘0¢ 000‘ OT 000‘OT 000‘ 08 000‘ OF 000‘ 068 OOF OTT 002‘ SFT 006‘ ZT as0j0V'T | v6 ¥6 OOT 0g OST OOT OOT OOr OOT OOT 00€ OOT OST 20S OOT OOF 00¢ OOT 006 008 OOT Gol O&T OOL quad ad 9} eul -rxoilddy +008 ‘92 +00s‘T¢ aoudII IC 000‘ 0SF 000‘ 08F 000‘ OTS 000‘ OT 000‘ 0 000‘ 0z 000 ‘OT 000‘ OT 600‘ OT 000‘ 0z 000‘09 000‘'0z 000‘ 08 000‘0& 000‘ 0z 000‘ OF 000‘0¢ 000‘ OT 000‘09 000‘06 000‘ 08 OOT ‘EST OOL ‘SST COS‘ 90T IV’SE Ule 0G oS oh& o0G 06 olf 0G VE oL& 006 o0E oL& o0G o0E oL& o0G o0E oL€ o0G o0& oL& o0G o0E oL€ CO) aimyeied -Ul0 T, SEER co a]duieg (panuyuod) T ATAV J, I 231 VARIATIONS IN BACTERIA COUNTS +000‘'zz +000° 2 +000‘9 +C90'9 —000‘ OF —000‘ 6FT +000‘ZT +000‘ TT +000‘Z8 +000‘ 2 +000‘T +000‘'Z +000'3¢ +000‘ € +000‘ 0¢ +000‘ 901 +000 01 +000‘Z9 +000‘ &F +000‘ OF +000‘ 0F +000 ‘08¢ +000 0¢¢ +000‘ 08z +000‘ 0z¢ + 000/008 +000 ‘08s 000° 8z 000‘ 81 000‘ZT 000‘ZEe 000‘ 6zz 000‘ 0ZT. 000‘zE 000‘ TE 000‘ZOT 000‘ LT 000‘ TT 000 ‘ZT 000‘TL 000‘Z¢ 000‘ 69 000‘ eer 000,081 000/68 000‘F9 000‘ 19 000‘T9 000‘ 00% 000 O28 000‘0.z 000‘0SS 000° OTS 000 ‘OFS 00c OST 19 GIT 66 Té G8 O9T OGT OOT OFT 06 OOF €9P 891 Str FOV 9ET 8ES 916 Lg O0& 006 OST OOT OOF 09 +000‘9 +000‘¢ —000‘Z +000‘ TE —000°Z —000° E81 +000 ' 62 +000° 68 —000'6 +00! 0E —(00'F 000 ‘ZT 000‘6 000‘F 000‘ 008 C00‘ 19 000 ‘8 000‘ 2T 000‘ZE 000 ‘Fz 000‘ OT 000‘ FT 000'6 000°92 000‘ 88 000'ZE 000‘ T2T . 000‘ 60T 000‘FE 000‘ 0¢ 00009 000 ‘ZT 000‘ 09 000‘ OF 000 ‘0 000‘ OT 000‘ OF 000°9 996 SSG OOT 66 vIT OOT OOT OOT OOT OOT OOT OOT 616 GET OOT SIP ple OOT Srl GIG OOT 006 OST OOT OOT 006 OOT | 000° 000° LT 000°9 000‘99z 000‘ 20 000‘ 69z 000 ‘0z 000 ‘0z 000 ‘0z 000‘ OT 000‘ OT 000‘ OT 000‘s¢ 000‘ ¢z 000‘ 61 000‘'Z1T 000‘ TOT 000° 2z 000‘ TE 000'¢¢ 000‘ Tz 000‘ OF 000‘ 0¢ 00008 000‘ OT 000‘ 0z 000‘ OT ney 7 sees egy Heese yy Does esos EE = 232 G. C. Suppter, W. A. WuiTinG, anp P. A. Downs v9l Och LE9 OOT 8ST S&T 029‘ €89 89 G16‘ T 18¢‘G 08Z eT Oog - L9G 096 OFF OSP Ist OFT 002 £96 &ES 161 ques 9d 9} BUL -1xoiddy +0009 +000 641 +000 108 +000‘ 1¢¢'T +000‘ S6¢ —000‘E¢ +000‘ TL¥ + 000° +9 +000° LP +000°Z +000‘ZT +000‘OT +000‘ TST +000‘98z +000‘ 28z +000‘ 22 +000‘F2 +000‘98 +000‘Z2E +000‘ 00T +000‘89 aouaIO IC, 000‘Z6 000‘ Sz 000‘ 268 000‘ OLE 000‘ FLI 000‘ 9FT 000‘8¢9'T 000‘ 269 00069 000‘ 267 000‘ 129 000° EL 000'8 . 000‘ 8T 900‘ 9T. 000‘ SIZ 000‘S9E 000‘ 698 000 ‘SIT 000‘ OZ 000‘ ZLT 000‘ 26T 000‘ E21 000‘ €F1. as01}X9CT “616 6cP VOS GOT OLS 666 see‘T St6 GY v09°S G6I‘S LG ZU OST Lt LOT S8P IGP reveal Tvl SOL LCG 8GG G8 yueo rod oy BUL -1xolddy +000‘ 00T +000‘ FST +000 ‘FTE +000‘89 +000‘ 182 +900 ‘FT 000° 893 ‘T +000‘ 298 —000‘6¢ +000‘ T¢9 +060 ‘F08 +000‘ SF +000‘T +000‘S —000'¢ +000‘¢¢ +000‘F1E +000‘ 89% +000‘ 0z +000‘ S¢ +0002 +000‘&6 +000‘96 —000‘ TT adUdLO PIC, 000‘ 9°T 000‘ OFZ 000‘ OL1 000‘ SLT 000‘ Tre 000‘ ZEz 000‘ S98 ‘T 000‘ 296 000‘ SF 000‘ 229 000‘ 0gs 000‘ TL 00042 0006 000‘T 000‘ LET 000 ‘968 000 SPS 000° 90T 000! 1ZE 000‘ 6 000‘ OLE 000‘ TLE 000‘F9 9S0j0B'T GGT IVI OOT 9LT SPI OOT 9ST ‘T 008 OOT GOL‘ T 8G9‘Z OOT eét SEV GOL &6 v8 OO 9ST SST OOT 66T FOG O0T queso tod Oo} BUI -yxoiddy +000" ES 000° 601 0G +000‘ &z 000‘ 62 008 Ot OO Getesy 5a 0 000‘9¢ ~lk® = PAE AG. +000‘'F8 000‘ F6E 0G +000‘8¢ 000‘S9T 008 OT0-0 0/0°O10-0-0 000‘ OLT fo OG +000‘2S0‘T] 000‘6ST‘T | .02 +000‘FIZ | 000‘9T8 o08 abet alletishiecaiists. te 000‘ ZOT ole 2 ESASS ie +000'ZEF | 000! Scr 006 +000°¢99 | 000‘T69 o0E wbetietiettoriahemerelt® 000 ‘9% ae === QC +000‘% 0008 o0G +000‘ 02 000‘ 9% 008 ai Tenfot elteitagiatss) faite 000‘9 aks ° GG —000‘'9 00092 0G —000‘ST 000 ‘69 0S efinvielie nelle falleie ‘6, 000'Z8 jee TROBE OG +000'8F — | 000‘ FET 00% +000‘ LF 000‘ €81 008 TN sl 00°98 18 hes pg +000‘F2 000‘ 6FT 0G +000‘82 000‘ SeT o08 Sipe eiveie) ens eltesie: 000 ‘ Gc) ak . 6S (‘O) aoUaII IC, IVS ult[qd jein}vied ajduieg -UlD T, (panuyuor) [T aTaV, 235 VARIATIONS IN BACTERIA COUNTS +000 ‘F9F +000‘ FLT —000‘'8 —000‘9z +000‘ 0z +000‘2 —Z69 ‘SSF —000‘TS +000‘ S08 +000 ‘FT —000‘¢ —000‘T —000'6 +000 Sz +000‘S +000‘0¢ +000 86 +000‘ 09T +000! Fz +000‘ 8¢ +0008 000° OLF 000‘ OST. 000s 000‘0¢ 000‘96 000‘8 808 ‘T 000! 69h 000‘ S62 00098 000‘ AT 000‘ 1z 000‘98 000‘02 000‘0¢ 000 ‘Zz 000 ‘TF 000‘8z 000‘6 0008 000‘9 000‘8¢ 000901 000‘ S91 000‘ 28 000‘ TL 000‘ 1S -000‘9 999 ‘T 0g 6L PIT 96T oO LOT 92 cS 9ET Gg LOT OFT 18 OL +000 ‘#48 +000‘F6 —000°8 —000‘9T +000‘ TT +000 ‘61 18" SSP +000'S8 —000‘61T —000'T +000‘8 —000‘OT +000‘¢ +000‘ 8T —000‘9 000 ‘098 000‘ OOT 000 'S 000‘09 000‘ 28 000‘ &6 SZL‘T 000‘ Sz¢ 000‘ TLE 000‘ST 000‘ 08 000 ‘ZT 000‘ SF 000‘ 89 000‘ 68 000 ‘TE 000 ‘Sz 000‘ ST. 00g ‘¢ SE8'P OOT rea 16 OOT 10 986 OOT SOT OST OOT 19 66 OOT TZ IIT OOL OTT 006 OOL OST GGG OOT PST 006 OOL +000 ‘#28 +000 ‘F8z —€8S "68h +000‘ ¢16 000‘0e¢ 000 ‘062 000‘9 000‘98 000 ‘69 006 ‘92 192 000‘ SOF ‘T 000‘ 06F 000‘ z 000‘¢ 000‘ 000‘ 08 000‘ ZF 000‘ SF 000‘ 0z 000‘TE 000‘ 8z 000‘2 000‘ZT 000‘9 000'21 000‘ ST. 0008 000/02 000' 9% 000° 1 o4G VE oL& o0G oL& oL& o0& oL€ oS of Bea ees itt <1°) “99 G9 SOE.) eT G. C. SuppLEE, W. A. WHITING, AND P. A. Downs 234 VES 666 68 S& OOT €&1 aia IcT 2g 28 OOT GL TZ OOT 66 Il? OLE OST VEG 967 O[e]=[ = [ees © tie = S i > 3 S 3 CHARACTER 8 Seon SS o é 2 SuMMarRy CHartT afe2els ts] 5 Ss] 2] 8 01 RS ES S| aS CS iesan lease 3 Sn eee) a ie Se pcsees BS GS SN Sa §/ SF] 8 SSeS Salas I Shes Ss] | 38 & 3 > > & =) ES a& | -s ees sl sees js Saecalee SH SY eas [ls 2/s/s Key Tay —— + Yes — No ‘ ? Uncertain g o}] sas] 8 g ae lp | erp ce) ies SA cy fehl oS On ies a TS) feats aes By WS Gel teh | aly Ry Ss he fy |) at 2, rm ‘SI = 8 & 2 fo) Ons Sy Il -cal nos, ° 5 2 a 2 BIElA(SIalél 8 | lel 2 SRS OM Ene Ouaesaleate lig Mesothoracic pleural suture makiy + Spe (fl ts |p seth 1 forward in its course from coxa eer eal | Rael pe Up uF a + Tongue-like structure present j + SEH) ae 9 mesothoracic coxa (cx2)...... RD RS eg) | : az |e ? Metathoracic pleurotrochantin (q + 6 eee chitinized sclerite............ er af eeu) aE Thoracic and abdominal scleri + se ear si entisted ays eee nc bea | LAN Late SEP (ear ae || uelens Adventitious suture present iq + ft ' CErzilLenere ec bea + aly ae (ore | eo Suture between first and second | — MrLeEsentientiresy sr. |) | ie (eset [i First abdominal tergite subdivj — | STET GE) (Gr) 5 oe ee aa | Pon Second abdominal tergite subdi! — pranous)(m) sen. swe... Peat | ore | arg First abdominal sternite subdiy— branousl (im) eee. |. ie || Ze em Second abdominal sternite subd — branous) (m)pee ns ee 8. La tees Tae eee || + First abdominal spiracle in mem Te iar eee a iia peer + Second abdominal spiracle in m amteaP|ae . 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Puate XVI r=] g g Z oa i 0 J 3 a & 22, Hirmoneura sp., female (Nemistrinidae). 23, Oncodes incultus, female (Cyrtidae). 24, Anthrax alternata, female (Bombylidae) 290 Memoir 44 Puate XVII 25, Thereva fucata, male (Therevidae). 26, Scenopinus fenes- tralis, female (Scenopinidae). 27, Midas clavatus, female (Midaidae) 291 Memorr 44 Puate XVIII 28, Leptogaster loewi, female (Asilidae). 29, Dolichopus cuprinus, male (Dolichopodidae). 30, Rhamphomyia sp., female (Empididae) 292 7. Memorr 44 Prate XIX 31, Lonchoptera sp., female (Lonchopteridae). 32, Phora coneinna female (Phoridae). 33, Platypeza velutina, female (Platypezidae) 293 Memoir 44 Puate XX } 34, Pipunculus atlanticus, female (Pipunculidae). 35, Syrphus americanus, male (Syrphidae). 36, Myopa vesiculosa, male (Cono- pidae) 294 Menor 44 Puate XXI 37, Gastrophilus intestinalis, female (Oestridae). 38, Tachina mella, zfemale (Tachinidae). 39, Thelaira nigripes, male (Dexiidae) 295 Memoir 44 Puate XXII ' 40, Sarcophaga communis, male (Sarcophagidae). 41, Muscina stabulans, female (Muscidae). 42, Macrorchis ausoba, male (Antho- myiidae) 296 Memoir 44 Prats XXIII 43, Chortophila cilicrura, male (Anthomyiidae). 44, Hylephila paludis, female (Anthomyiidae). 45, Schoenomyza dorsalis, female (Anthomyiidae). 46, Ophyra leucostoma, male (Anthomyiidae). 47, Lispa sociabilis, female (Anthomyiidae). 48, Limnophora aequifrons, male (Anthomyiidae) 297 Memorr 44 Puats XXIV 49, Eremomyia cylindrica, female (Anthomyiidae). 50, Pegomyia affinis, female (Anthomyiidae). 51, Hylemyia lipsia, female (Anthomyiidae). 52, Anthomyia radicum, male (Anthomyiidae). 53, Hebecnema wmbratica, female (Anthomyiidae) | 298 ——< Memorr 44 Prath XXV 54, Scatophaga stercoraria, male (Scatophagidae). 55, Clusia later- alis, female (Heteroneuridae). 56, Leria serrata, male (Helomyzidae) 299 Memorr 44 Puate XXVI 57, Borborus equinus, female (Borboridae). 58, Dictya wmbrarum, female (Sciomyzidae). 59, Sapromyza lwpulina, male (Sapromyzidae) 300 Memor 44 Piate. XXVII {60, Rivellia viridulans, female (Ortalidae). 61, Rhopalomera flaviceps, ‘emale (Rhopalomeridae). 62, Huaresta festiva, female (Trypetidae) 301 Memorr 44 Pirate XXVIII 63, Calobata albiceps, female (Micropezidae). 64, Sepsis violacea, female (Sepsidae). 65, Piophila casei, female (Piophilidae) 302 Memorr 44 Pirate XXIX 66, Loxocera pleuritica, male (Psilidae). 67, Sphyracephala brevi- cornis, female (Diopsidae). 68, Parydra limpidipennis, female (Ephydridae) , 303 PuatTe XXX Menmorr 44 : : f 70, Drosophila melanogaster (?) 69, Chlorops sp., male (Oscinidae). female (Drosophilidae). 71, Anthomyza gracilis, female (Geomyzidae). 304 Memoir 44 Pratt XXXI 72, Agromyza lateralis, male (Agromyzidae). 73, Melophagus ovinus, female (Hippoboscidae). 74, Olfersia americana, female (Hippobos- cidae) 305 Memorr 44. Prats XXXII 75, Olfersia americana, female (Hippoboscidae); ventral view. 76, Panorpa venosa, female (Panorpidae) 306 a OCTOBER, 1921 MEMOIR 47 CORNELL UNIVERSITY AGRICULTURAL EXPERIMENT STATION TYPHA INSECTS: THEIR ECOLOGICAL RELATIONSHIPS P. W. CLAASSEN ITHACA, NEW YORK PUBLISHED BY THE UNIVERSI?¥ CONTENTS PAGE BiecolormcalastmoleswOl My pha. a4 255.8 el ale acl Goo se be os ce cs ee @ 463 herswaniprareasol the United States; 22.0 .......2...500....". 463 IPI BYERS: OL SIELUCKY cs ee os an ee Pn Pea Ae, 464 IMDS SORGUSS Oi IOS 7 0) ee eect les ener ee ce st ene 465 PemGistuiloMmlomsOle dy phan ens ose. Sk sys occle fm ale Gene ws evetas 465 Crowehehabitvand reproductions... 2..0:- 2. ...02. oo eo wie ee eo 466 Genminagtonmlj pnd (GHMfOUAs ee esis ae Soe eel ns ae oe 467 sey on aAgAsEagcOMMECrClal ASSEbs..-c..2 ccc sect oe sk ve cere 470 whe insect fauna of Typha.:..........: 2) Bie ae ean cage 471 BC POI GTC AMER ONS ac nats frase ath Saltic wes otnels Gb te aceaie wwioiaes 472 PAU EET ROOLLG ILO NN Actes cette: An eyo, hh ccemle din hae eee Se 472 NOM OGEUGROOLOMGANGLOLE cope. ese © 2 «scsi Soe is oss ge 8s loa as 479 WAU SLOMCNGE CLOOVENOSG, GOCLE. 0. Shen cd oe bee en 483 PAUECI TP) SHOUSOLCLOMOMNN Al Kevan elects ait Aco so apa Hie 8 Bs He he wee 485 WEMUNACCVOR PNTAGMILCLLG LANG oe. Se on ose ee oe tee 487 Drcnmolomiaeulanalis Walk. sx. s 2.65 hn sect ss elas 490 COLE OIDIESIR «oo thet Re ee a ee a a 493 Walendraepeninax Olver sis Sota 8a eee one oe Phe Boe 493 PRTOUOMUSMINECLUCOUUSDILC Capt rh ern wat aie Pe eh id clelele a ors, « 497 LEIGIECHOUGI.. 6 o o clk Beg te eoeeRe NS eR 497 HISCIULOGIILU ICI US NCSEAOE DAM Zo. es 8 aoe esos bn dee anes 497 UPROCORUME NUMpnNGeae WANN ..5. 0022 6s. neces eevee ee ee 500 FAUT IL ESROL CTL DD eee ate een wie he PR es Ge Sunes cite on iesalte ce «Se 500 ivkonalosuphum dvanth Schrank... 2. 02...) 2 cee ee 501 ihonmlosiphium persicae SUlZa....-5...260.--.--+-2+--+-- 501 AGUS GOSSTACHEAGNO Sexe oe eee Sc i ee 501 IVIGCEGSU DIUM GRONOTvuny WATDY Gs. 56 0 hese ses ee 501 CLO PLCRUS ORUNALIUSEH ADI. sats os 8cbt eh he te le ee 501 E SOT (INO) OUSTD 5. Eis oh Ora Ma Caco UE ane ae 501 PAllevodeswintenmeadvus CreSS.. 2. os. see ve le ce ce oe ew once 501 ADO EMLESCUNCHIONINUSY NACKs eee eae ee ele ole les ele 502 [BIOWARE WOES OSS © Bose Oo the aoc ne 502 POGUE CGR OS WEIR e oe aeRO eno Ae oO In ae 502 IPanADIG, CG ASHLOTUIO Dy I eo pe Aaa Me En bp elas cece 502 OPO ORS « 's. c.d: bid Bip OUD RE SERIO aCe ae er 502 JPG GUTUS GUOMKGIS SEN Go don po oHO bee ooo oeoo ore ode 502 VT CROSOMGIUSTCLOUUSEIVELL Uemura ere rinewe a icheostc-s = =) s)clae yee chee « 502 GITOC LOM SUSIOCIIEC EMAC Omer ness fe. Sol: ot ass lace eee = 502 SID DHE IOP los conn hoe AOC URE OB OOD DOOD DOED OD DOr 503 460 CoNTENTS Résumé 25 53.205 6s ahs eee see bd we ie eo RN a eee Insect inhabitants of the head of Typha... .- =... .-_ eee 503 Insect inhabitants of the leaf of Typha. --] -. 2 = ee eee 505% Insect inhabitants of the stalk of ‘Typha... .-).222555- eee 506 Insect inhabitants of the rhizome of Typha.............. pce 507 Literature cited «: 2035.0 oss Se ee eee 508 TYPHA INSECTS: THEIR ECOLOGICAL RELATIONSHIPS IME wINSHeihs: THEIR ECOLOGICAL RELATIONSHIPS P. W. CLAASSEN In order that the ecological relationships of the insect fauna of the cat-tail plant may be better understood, the first part of this paper deals with the ecology of the cat-tail plant. In the second part, the life history and biology of the insect inhabitants of the plant are discussed. This part of the paper has been treated from a systematic point of view, considering the insects under their respective orders, rather than grouping them according to their life habits. In the résumé, composing the third part, an attempt has been made to bring out the true ecological relation- ships, grouping the insects with reference to the parts of the plant they affect, their relative importance, and their interrelations. ECOLOGICAL STUDIES OF TYPHA THE SWAMP AREA OF THE UNITED STATES According to Davis (1911)! there are 139,855 square miles of swamp area in the United States, exclusive of Alaska. This includes bogs, marshes, muck lands, and the more typical swamps. The vegetation in these wet lands varies from the semi-floating forms to the wooded plants of the more solid areas. Needham and Lloyd (1916) state that the bogs, marshes, and swamps ‘‘ occupy a superficial area larger by far than that covered by lakes and rivers of every sort. They cover in all probably [sic] more than a hundred million acres in the United States.” Much of the vegetation of this wet area consists of cat-tail (Typha). Many of the marshes contain an almost pure growth of cat-tail, as, for example, the Montezuma Marsh at the foot of Cayuga Lake. This marsh covers an area of approximately 36 square miles. Although it is impossible to give definite figures on the size of the area occupied by eat-tails in the United States, it is safe to say that there are thousands of square miles of wet lands which are covered by either a pure growth of cat-tail or plant associations in which the cat-tail is the dominant form, AUTHOR'S ACKNOWLEDGMENT. The author is indebted to Professor James G. Needham for his helpful suggestions and criticism in this work. ; 1 Dates in parenthesis refer to Literature cited, page 508. 463 464 : P. W. CLAASSEN PLACES OF STUDY These studies have been made largely from material gathered in the swamps and marshes around Ithaca, New York, especially from Renwick Marsh, at the head of Cayuga Lake. Other collecting places around Ithaca were Michigan Hollow, Mud Creek Swamp, near Freeville, the McLean Bogs, Vanishing Brook, north of the Cornell University campus, Caseadilia Creek, and various other places, wherever cat-tails were found growing. Observations were made also on the extensive cat-tail marshes of Lake Ontario, at North Fairhaven. During the season of 1916-1917, studies on cat-tails were made also in the vicinity of Lawrence, Kansas. The Renwick Marsh comprises a field of many acres, of which a large proportion is covered with cat-tail. In some places, this plant grows so thickly that all other vegetation is excluded, and especially is this true of the central part of the marsh, where the soil is much wetter. As one approaches the outer margin, other plants mingle with the cat-tails; and at the border, where conditions are drier, the cat-tail growth is sparse. All the cat-tail patches along the Inlet Valley, up to the Buttermilk “alls region, are referred to as the Renwick Marsh. Michigan Hollow is a swamp located about six miles from Ithaca, in the Inlet: Valley. Cat-tails grow here only scatteringly, in small but rather dense patches. This swamp was visited several times to make collections and observations. In the McLean Bogs, cat-tails are not found in large numbers, but since these bogs were visited every Saturday throughout the spring and summer of 1916, the author was enabled to make more careful and complete observations of the conditions and life histories of the insects of the cat-tail plants there found. Moreover, where cat-tails are not so abundant, a higher percentage of infestation usually occurs, which renders it much easier to obtain material and to make comparative studies. The Mud Creek Swamp is an old swamp extending along Mud Creek, near Freeville. Here, also, the cat-tails grow but sparingly, but it was found easy and convenient to make frequent observations and collections — of material. Along Vanishing Brook, north of the Cornell University campus, there are a number of small patches of swampy ground, on which cat-tail plants TypHa Insects: THEIR ECOLOGICAL RELATIONSHIPS 465 may always be found. On account of its nearness to the laboratory, this was found to be a convenient place to make some of the observations. Occasional cat-tail plants are also found in Bool’s Back Water and in Cascadilla Creek, two places which were likewise chosen for study because their close proximity to the laboratory made daily observations possible. THE SPECIES OF TYPHA According to Britton and Brown (1913) there are about ten species of Typha in the temperate and tropical regions of the world. In the United States there are at least two species represented, Typha latifolia L. (type species) and T. angustifolia L. Dudley (1886) lists 7. latifolia L., var. elongata, n. var., as a variety occurring in New York. Hedescribes it as follows: ‘‘ Leaves very numerous, dark green, elongated (2-34m.) and fruiting spike elongated, often 30 cm.” Typha latifolia has broad leaves. The staminate and pistillate parts of the flower spike are contiguous. The stigmas are spatulate or rhomboid. Pollen grains occur in fours. Pistillate flowers are without bractlets. Typha angustifolia has narrower leaves than Typha latifolia. The staminate and pistillate flowers of the flower spike are usually separated by a short interval. The stigmas are linear or oblong-linear. Pollen occurs in simple grains. ~ The pistillate flowers have bracts. Typha is known by the following common names: cat-tail, great reed mace, cat-o’=nine-tail, cat-tail flag, cat-tail rush, flax tail, blackamoor, blackeap, bullsegg, bulrush, watertorch, and candlewick. The names “marsh beetle’ and “‘ marsh hog” have also been applied to the Typha plant. “THE DISTRIBUTION OF TYPHA Cat-tails are common in the temperate and tropical regions of America, South America, Europe, and Asia. Wherever favorable soil conditions occur, cat-tails will be found growing; even a spring on the hillside or the outlet of a drain pipe will sometimes support a few of the plants. Large patches of them grow in the Rocky Mountains, at an altitude of 7500-8500 feet. Typha latifolia, the commonest of all the species, occurs throughout the United States in any favorable location. Typha latifolia grows abundantly throughout North America, except in the extreme ‘466 P. W. CLAASSEN North. Typha angustifolia grows abundantly in the marshes along the Atlantic Coast from Nova Scotia to Florida, as well as inland and even in California. GROWTH HABIT AND REPRODUCTION Cat-tails are marsh or aquatic plants, with creeping rootstocks, fibrous™ roots, and glabrous, erect stems. They are perennial plants, the rootstocks remaining alive while the stem dies down to the ground every year. The following spring these rootstocks, or rhizomes, send up the new plants. Plants which attain only partial growth during the season keep the center alive that winter and probably reach maturity the following summer. The rhizomes spread in every direction and within a few years a large group of cat-tails results from the offsets of a single plant. It is really very difficult to define the limits of a single plant, since they are so linked together by these underground rhizomes (Plate XLV, 56). s-sieec|-s2e-s0-- | Re Bere Ayre 74 Having thus ascertained the approximate percentage of fertility in the seeds, experiments were conducted to determine the percentage of germination of the fertile seeds. The seeds were placed in covered watch glasses, and, though kept in the light, were protected from direct sunlight. Germination commenced within a few days after the seeds had been placed in the water. Careful counts were made of the number of seeds 470 P. W. CLAASSEN that germinated in each of the watch glasses. The results are shown in table 4. These figures would indicate that not more than two-thirds of the fertile seeds germinate under experimental conditions. Calculating, then, from the percentage of fertility and the percentage of germination, as given in tables 3 and 4, a head containing 250,000 seeds might actually give rise to 125,000 new plants — a 50-per-cent efficiency in reproduction. TABLE 4. DETERMINATION OF THE PERCENTAGE OF GERMINATION OF FERTILE SEEDS . Number Nabe Percent- Head Number | of seeds ree es age of of seeds germi- g ere germi- nated 8 nation I atest SMa Pe PCa ed eh le A HEN cine Ce aD 20 12 8 60.0 DEE i TSOP CCR AE at gh ete te elec way) aoe 50 34 16 68.0 Bia AR Ie ane SG Aa eS fiat tem ey Bee ener 18 12 6 66.6 oI aoe Bete Rage ek CASS ar AR res ey Soe a rep 73 37 36 50.6 MR Ota lisesi anaes ee eet ee eure ae em 161 95 6655 S| Ree: : Average per cent:germinated.. 5.:..5---.-2--|a0- 6-2 a |e oe ee ee | eee 60.5 TYPHA AS A COMMERCIAL ASSET The vast areas 6f cat-tail have as yet been little utilized. The plant is rich in starch and other food values and grows in situations now regarded as waste lands. It would yield great quantities of supplies, if only a definite use for it could be found. The Indians and a few other races have used cat-tail products for various purposes. Hooker (1876) says: The starchy rhizome of Typha possesses slightly astringent and diuretic properties, which led to its use in East Asia for the cure of dysentary, urethritis, and aphthae. The stems and leaves are used for thatching cottages. It has been vainly tried to utilize the bristles of the spike in the manufacture of a sort of velvet. [The pollen of Typha is made into bread by the natives of Scind and New Zealand.] Engler and Prantl (1889) say: ‘“‘ The rhizome, rich in starch, may serve as food material; the leaves of several species are used for weaving. The pollen, which is easily recognized by the occasional tetrads, serves at times as surrogate for lycopodium powder.’” 2 Translation from the original German, Typua Insects: THEIR EcoLoGicaL RELATIONSHIPS 471 Parker (1910) speaking of the plants used for food by the Indians, says: ‘“‘ The roots of the cat-tail were often used. Dried and pulverized the roots made a sweet white flour useful for bread or pudding. Bruised and boiled fresh, syrupy gluten was obtained in which corn meal pudding was mixed.” Muskrats are very fond of the rhizomes of the cat-tail, and in the cat-tail swamps the muskrats are accordingly found in large numbers. The leaves of the cat-tail are used to some extent in the manufacture of barrels. On account of their spongy structurg, the dried leaves, placed between the staves, expand greatly when moistened, thus making the barrels water-tight. The leaves are .also used for chair bottoms. (Dudley, 1886). ; The rich starch content of the plant is especially concentrated in the rhizomes, where the cells of the rhizome core (Plate XLV, 57 and 60) are completely filled with small starch granules. This is true of the rhizomes in their dormant or winter conditions (Plate XL, 13). If, however, one examines the cells later in the season, after the plant has attained a growth of several feet, the cells are found to be only partially filled with starch granules, much of the starch having been used up in the rapid early growth of the plant (Plate XL, 15). This fact, showing that they have a much greater concentration of starch during the dormant season than during the growing season, has a direct bearing upon the possible uses of the rhizomes, and any attempts made for the utilization of the starch should be made on the dormant rhizomes. The possibility of utilizing the Typha plant as a source of food has been discussed by the author in another paper (Claassen, 1919). THE INSECT FAUNA OF TYPHA The insects which are found on cat-tail have not hitherto been studied asa group. Most of them have been recorded as inhabiting cat-tail, but very little has been published on their detailed life histories and ecological interrelations. ; The following discussion includes only those insects which have been found on cat-tail and studied during the course of this investigation. It includes six species of Lepidoptera, two of Coleoptera, eight of Hemiptera, five of parasitic Hymenoptera, and four of Diptera. 472 iIPASWe Chancenie LEPIDOPTERA? \ Arzama obliqua Walk. Arzama obliqua Waik., a moth of the family Noctuidae, has been known in the adult stage for more than half a century. The species occurs throughout eastern United States and Canada, its host plant being Typha latifolia. In New York, near Ithaca, the writer has taken specimens from the following places: Michigan Hollow, Renwick Marsh, Bool’s Back Water, McLean Bogs, Cascadilla Creek, and Ringwood Hollow. Life history and habits The life history of this insect is unusual and interesting from an ecological point of view. There is only one generation a year. The full-grown larva passes the winter in its burrow in the plant. Eqg-laying.— The eggs are laid on the surface of one of the first-formed leaves of 7’. latifolia, from six to fifteen inches below the tip. This later becomes one of the outer leaves of the plant. The eggs are placed on the leaf several layers deep. The lower layer covers the largest area and contains from twenty-five to forty eggs, while the upper layers cover only the central part of this bottom layer, forming a gradually sloping mass and containing from ten to twenty eggs in the two or more upper layers. The total number of eggs in one egg mass varies from thirty-five to sixty. The whole egg mass is covered with a thick layer composed of a mixture of froth, hairs, and scales from the body of the female. The egg mass greatly resembles a mass of spider’s eggs (Plates XLI, 24 and 25, and XLVI, 65). It is of a dirty, yellowish-white color. It measures from twelve to fifteen millimeters in length, from seven to ten millimeters in width, and from three to four millimeters in height at the center. In shape it is oblong and convex, the edges gradually thinning out and adhering closely to the surface of the leaf. The long axis of the egg mass corresponds to the long axis of the leaf. One female apparently lays several egg masses. In dissecting out the ovary of one female, 225 eggs were found, all fully formed and developed; and since only thirty-five to sixty eggs occur in a single mass, this indicates that one female may deposit about half a dozen egg masses. ° 3 The Lepidoptera mentioned in this paper have all been determined by Dr. W. T. M. Forbes, TypHa Insects: THEIR EcoLoGicAL RELATIONSHIPS 473 Usually only one egg mass occurs on the same leaf, but sometimes two, and in one instance three, masses were found on a single leaf. It is not uncommon to find two or three leaves of the same plant with an egg mass on each of them. In the spring of 1918, careful observations were made for the appearance of the adults or the egg masses on the plants. In the laboratory, where moths had been bred, they failed to mate or to deposit eggs in the char- acteristic manner. A few females did lay infertile eggs on the stems and leaves of Typha. Egg masses were first noticed in the field on May 26. After this date new egg masses were constantly found until June 8. The height of egg-laying was between May.26 and June 2. At the McLean Bogs the egg masses appeared about six days later than those at the places around Ithaca. The larva=— On turning the egg mass over, after the larvae have hatched, the empty egg shells are disclosed. The hatching process does not disturb the egg mass in the least. Without devouring the egg shell, the embryo breaks through it and bores directly into the leaf of the cat-tail, where it works as a leaf miner. This manner of hatching seems to be an excellent protective adaptation against egg parasites and other enemies. The mass is practically impervious to water. Thus from the time the egg is laid to the time when the larva hatches and enters the leaf to become a leaf miner, it is not once exposed to the direct dangers of enemies or of weather conditions. Once the larvae enter the leaf, they begin their work as typical leaf miners. The structure of the leaf of the cat-tail plant is rather peculiar. The fibro-vascular bundles are found mainly in longitudinal, I-like partitions. This produces a loose inner structure with many large air spaces (Plate XL, 11). The longitudinal partitions are again traversed by transverse partitions which also are composed of parenchyma. A leaf of Typha with the epidermis removed to show this inner structure appears in Plate XLV, 58. When the larvae have entered the leaf, they begin to mine, mostly downward, scraping off the chlorophyll from the upper and lower epidermis of the leaf. They eat out the transverse par- titions, leaving the longitudinal partitions and the fibro-vascular bundles undisturbed except when occasional larvae cut through to get in other channels. A few of the larvae may first mine upward toward the tip of the leaf, but soon they all proceed downward, moving abreast along the 474 P. W. CLAASSEN channels. As many as eight larvae have been found together in one channel. It is probably due to such a crowded condition that a larva occasionally crosses over into another channel. After the larvae have mined down for a distance of twenty to twenty- four inches, they molt in the mines and immediately afterward leave the mines through a little exit hole which is usually made on the inner side of the leaf. As soon as the larvae appear on the surface of the leaf they at once seek shelter, usually continuing down the stem of the plant and crawling behind the sheath of one of the outer leaves. Since the larvae later. become true stem borers, the question arises why they should come out of the mines of the leaf ten or fifteen inches away from the stem instead of remaining in the mines and working down the leaf until they reach the stem and can enter it directly. There are two plausible reasons against such behavior: first, the larvae later become solitary borers and after coming out of the leaf they separate and indi-| vidually enter the stems of different plants; second, the width of the head of the second-instar larva exceeds the width of the average longi- tudinal channels in the cat-tail leaf. Careful measurements of the molted heads of the first-instar larvae and measurements of the width of the average channel of the leaf showed that the width of the head during the first instar was only slightly less than the width of the channel. The width of the heads of the second instar was considerably wider than the width of the channels of the leaf. Following are the average measurements: Width of head of first-instar larva........... 0.597 millimeter Width of head of second-instar larva......... 0.90 millimeter Width otchannelintleaia- 2 0.62 to 0.72 millimeter It would therefore be impossible for the larvae to remain in the leaf after the first molt unless they widened the leaf by taking out the longitudinal fibrous partitions. Although the larvae ultimately become solitary stem borers, they do not always scatter immediately after emergence from the mines and bore directly into the stems of the plant. In one instance, on June 29, 1916, it was found that a whole contingent of larvae had migrated to the head of the plant, where they found shelter behind the leaves that were sheathing the flower spike. Here they were feeding on the staminate flowers. A few days later they had all descended and scattered to different TypHa Insects: THEIR EcoLoGicaAL RELATIONSHIPS 475 plants. It is probable that such a migration to the flower spike was accidental; on the other hand, it may have been because the. plant had a central stalk with a flowering spike that the larvae could not or would not enter it, for the writer has never found that they bored into a plant which had a flower stalk, nor has he ever found a piant in which the stem borers occurred producing a flower stalk. - The larvae enter the stalk from behind the sheath after they leave the leaf, and there they feed for some time. Only once were two larvae found in the same burrow. They normally become solitary borers, tunneling through the center of the stem, going downward to the crown, and some- times even advancing for a short distance into the rhizome. This tunneling causes the central leaves of the plant to die, and consequently no flower spike is formed. The affected plants are easily recognized by the presence of the dead central leaves. The larvae grow rapidly and by late fall have attained a length of nearly two inches. ‘They leave the burrow full of the frass and the shreds of the fibrous tissue torn loose by their passage. In the fall, before the larvae go into hibernation, they eat out an exit hole in the stem, four to six inches above the ground, which they loosely plug up with frass and fibrous material. They then make a little compartment, or cell, by closing the burrow above and below with a mass of frass and fibrous material, as shown in Plate XLI, 27 and 28, and thus pass the winter. If one visits the marshes in winter and opens the plants, the larvae are found in the burrow, completely surrounded by ice. Larvae taken to the laboratory during September and October and placed in metal salve boxes on moist, sterilized sand, pupated in February and March. Adults emerged from sixteen to twenty days later. Larvae brought into the laboratory in the spring pupated much later, as is shown by table 5. In the laboratory, several days before the larva transforms to the pupal stage, it begins to spin a thin, irregular layer of fine thread all over the surface of the sand in the salve box. In the field, one finds these loose webs lining the burrows in the stalks. The larva then becomes very sluggish and gradually shortens until it seems only about half of its normal length. The shiny, almost black, larval skin becomes much lighter in color. This is a sign that pupation will occur within twenty-four to 476 PW. thirty-six hours. . CLAASSEN When the larva is ready to pupate, the larval head splits along the epicranial suture and the skin breaks open on the median dorsal line, along the first two thoracic segments, extending also about three-fourths of the way across the third thoracic segment. the skin slips off backward until the newly formed pupa is free. Gradually The pupa is at first entirely white except the cremaster, which is dark brown. TABLE 5. Lrenetu or Pupat PERIoD oF ARZAMA OBLIQUA : Date Date ae Specimen of of Sex 0 ae : stage pupation emergence (days) i ise ek a ireee Caen py BARN eA cu bees aap February 21 | March 11 | Female 18 Da ee bri aee ee cep seein ae Re en eot ence ayaa March 4 | March 22 Male 18 PB i eae ls ean aa ee Ae ace a TN ee March 10 | March 29 | Female 19 Avr deel auntie ae hsaa prem ACH pees rinwetee gees March 21 April 7 Male ies aaNet nein lun atten Sen Crate aa March 25 April 11 | Female 17 (Cake eRe eta Ore Reno aia erie ng etal March 25 April 12 | Female 18 fea Tene Neg Ay aI ARNT Og Wea Sige eel a el ale March 25 April 11 | Female 17 SYRUP Mba ene NG iNet tg e ere cen ai March 26 April 12 } Female 17 LOE ae eG AN eile heat crs Ree ec tae aE March 28 April 14 | Female 17 Oe pe ae ae eh Fee ces ee Ae It March 29 April 14 Male Gy Average length of pupal stage... s.|e 065 onl ees le onion ee eee 17.6 TABLE 6. MerasuREMENTS OF Pupar oF ARZAMA OBLIQUA* Females Males Specimen Length Width Length Width (millimeters) | (millimeters) | (millimeters) | (millimeters) . fs es Bae coe cae ee pes we eg SER 0 7.0 29.0 7.0 YEP cod epee No Va e hs Rar rte 35.0 8.5 28.5 6.5 Pais Art Aire ark cs ee Ral ety 31.5 6.5 28.0 6.5 LUT ie ie Then msi oa a Fait 30.5 Ae enna i oo wabala ce Sia AaVe is find ae AHR oat Te a 31.5 6:80 3) See eee Oe eae enn ao ee OY) 505) 8.10. ea ee eee TEER ARE Wl SC URSe Rae suet oa 33.0 (en amie meee ere CLS. 5 ws oo Re a = AV ELA SOR 25. Sones ee 32.85 7225 28.5 6.7 *The measurements of the pupae of Arzama obliqua were taken as follows: length, from the anterior end to the tip of the cremaster; width, the greatest lateral width of the pupa. TypHa Insects: THEerR EcoLtocgicaL RELATIONSHIPS 477 The first color of the body appears on the dorsal surface of the meso- and metathorax and on the first and second abdominal segments. After about ten minutes more the entire abdomen begins to assume a reddish eolor, the thorax, head, and wings still remaining nearly white, however. The pulsation of the dorsal vessel is very noticeable at this time. At the end of another twelve minutes the color is darkest on the sixth, seventh, and eighth abdominal segments, being more pronounced on the dorsal surface. Twelve minutes later the head begins to show color. In another half hour the entire pupa, except the wings, has become a reddish brown in color. The wings, which remain white the longest, now begin to show a little color. Later the pupa turns very dark brown, almost black. Description of the stages The egg Light yellowish in color; round, though somewhat flattened, with the micropyle on the upper side, away from the surface of the leaf (Plate XLI, 21). 1 to 1:2 mm. in diameter and 0.8 mm. in height. Sculpturing very fine but quite characteristic; micropyle represented by a small dot surrounded by a rosette of about twelve elongate cells. This surrounded in turn by two other rings of more or less elongate cells; a reticulation following, with cells more or less regularly hexagonal; and, finally, the outside cells slightly elongated transversely. Entire reticulate area around the micropyle covering about two-thirds of the upper surface of the egg. Remainder cf egg sculptured with a number of small tubercles, some occurring in lines so as to suggest circumferential bands of tubercles. The first-instar larva (Plate XLI, 29) Length 3.43 mm., width 0.58 mm. across the head. Head light brown, labrum and eyes darker. General colcr white, with a median purplish stripe. Spiracles on the eighth abdom- inal segment very large and dorsally located, as in the full-grown larva. After larva has been feeding for a few days, general color yellowish green. The full-grown larva (Plates XLI, 22, and XLVI, 66) Length 50 to 60 mm., width 6 to 7 mm. General color shiny muddy black. Head very dark brown. Lower half of clypeus light yellow. Basal knobs of antennae light grayish yellow. Labium light gray. Thoracic shield the same color as the head. A light median line along the length of the prothorax. Individual segments of the body darker postericrly. A dark median line along the dorsal surface of the entire larva. Ventral surface of the larva much lighter, being whitish gray in color. The larva appears very much like a typical noctuid larva except for the position of the spiracles of the eighth abdominal segment. The spiracles on the other segments of the body occur in the natural place, 478 P. W. CLAASSEN ~but the spiracles of the eighth segment have migrated from the lateral margin to a position on the posterior margin of the dorsal surface, as shown in Plate XLI, 30. The ninth abdominal segment conse- quently is much smaller, being only about half as thick dorso-ventrally as the other segments. This better adapts the larva to live in its. burrow in a plant where an excess of moisture occurs. It is not at all uncommon to find a larva entirely submerged in the water with the exception of these large spiracles, which protrude above the surface of the water. These two spiracles are more than twice as large as the other abdominal spiracles. The arrangement of the tracheal system in the larva is shown in Plate XLI, 31. It consists of. two main longitudinal tubes, which originate from the spiracles of the eighth abdominal segment and extend as far forward as the first thoracic segment, where they are united by a transverse trunk. From this transverse trunk arise the tracheal tubes of the head. Paired spiracles are present on the meso- and metathorax and on the first eight abdominal segments. All of these spiracles are functional except those of the metathorax, which are much reduced and seem almost vestigial. Each of the spiracles, except those on the eighth abdominal segment, are connected with the main tracheal trunk of the body by small tubes, the tubes on the metathoracic segment being reduced to mere threads. Most of the tracheal branches of the body take their origin from the longitudinal trunk, near its Junctions with the spiracles. From the thoracic spiracles, only small, branching tubes originate. From. the first abdominal spiracle a large tracheal tube originates from the tube joining the spiracles to the longitudinal trunk, and smaller branches spring from the trunk. Segments 2, 3, 4, 5, and 6 of the abdomen each have a pair of large tubes arising from the main trunk just above the spiracles. These tubes branch out into two parts, as shown in Plate XLI, 31. Segment 7 of the abdomen has a number of smaller branches, and segment 8 has a number of still smaller branches in front of the large spiracles. The pupa (Plate XLI, 23) Female, average length 32.85 mm., width 7.25 mm., male, length 28.5 mm., width 6.7 mm. Head, thorax, and appendages black. Abdomen dark brown. Frontak prominence very weak. Wings extending back over two-thirds of the fourth abdominal segment. Prothorax about two-fifths of the length of the metathorax. Surface of the head and thorax rugose. Clypeo-labral suture very distinct. Labium distinct, the labial palpi nearly twice as long as labium. Maxillae extending to the posterior margin of Typua Insects: THER EcoLtogicaL RELATIONSHIPS 479 the third abdominal segment. Femur of prothoracic leg not visible. Prothoracic tibia and tarsi prominent, reaching down two-thirds the length of the maxillae. Mesothoracic legs — extending to the tips of the maxillae. Metathoracie legs invisible. Antennae reaching almost to the tips of the maxillae. Segments 4, 5, and 6 of the abdomen crossed dorsally by a transverse line of tubercles; the surface in front of the ridge coarsely punctate, but the part of the segment caudad of the ridge very finely punctate. On the ventral surface these transverse ridges occurring on segments 5, 6, and 7. Cremaster about as wide as it is long, somewhat flattened dorso-ventrally, very rugose, and bearing four short setae of equal length. Spiracles on the eighth abdominal segment dorsad. Female with two genital orifices. The peculiar sculpturing of the larva carried over and showing somewhat in the pupa. The adult (Plate XLI, 26) ‘Length of body of female, 26 mm. Expanse of wings 54mm. The original description by Walker (1865 :438) is as follows: Cinereous brown....Antennae moderately pectinated in the male, slightly pectinated in the female... .Femora and tibia fringed; spurs moderately long. Fore wings with a dark brown oblique stripe, which extends from the base of the interior border to the tip of the wing, and is very diffuse on the outer side; an oblique fusiform pale cinereous ringlet; another ringlet of like shape and hue, longitudinal, nearer the base, much smaller than the first and often obsolete; two submarginal oblique lines of blackish lunules: exterior border almost straight, rather oblique. Hind wings with a black oblique spot in the disk beneath. Nonagria oblonga Grote Nonagria oblonga Grote, a moth which also belongs to the family Noctuidae, has been reported by various authors as boring in the stems of Typha latifolia. Walton (1908) has described to some extent the habits of the later larval stages and has figured the full-grown larva, the pupa, and the adult. The writer has found the species to be common on Typha latifolia near Lawrence, Kansas, and in the following places around Ithaca, New York: Vanishing Brook, Cascadilla Creek, Bool’s Back Water, Renwick Marsh, and Michigan Hollow. Life history and habits Nonagria oblonga Grote apparently produces only one generation a year. Egg-laying.— The writer has been unable to find the eggs of this species, although the work of the larvae, from the first instar on, has been observed for three seasons, two seasons around Ithaca and one season in Kansas. The young larvae may be found just as soon as the cat-tail leaves appear above the surface of the ground. In Kansas, on April 20, 1917, when the leaves of the cat-tail were not more than four inches above the surface of the ground, the larvae were found at work in the tips of the leaves. 480 . P. W: CLAASSEN . _ Although the larvae had evidently just started their work, and more larvae continued to appear during the following days, no eggs could be found on the plants. Again, in Ithaca, on May 20, 1918, first-instar larvae were discovered at work in the tips of the leaves, but no trace of the eggs could be discovered. This suggests the possibility that the females deposit their eggs in the fall on some of the old plants or other objects in the field, and that the species overwinters in the egg stage. The larva— The larvae enter the leaf of the cat-tail near the tip and at once begin to work as leaf miners. In their mining they do not restrict their work to the longitudinal channels, as do the larvae of Arzama obliqua, but they zigzag back and forth in the leaf, cutting through both the longitudinal and the transverse ‘partitions. They feed on the chlorophyll and on the spongy parenchyma of the plant. The larvae are strictly solitary in their habits; only occasionally do two, or sometimes three, larvae occur in the same mine or even in the same leaf. The charac- teristic mine produced by the larva is shown in Plate XLII, 37. It is easily distinguished from the mine made by Arzama obliqua. When the larvae are ready for the first molt, they suddenly widen their mine to the outer margins of the leaf, thus producing a narrow transverse mine extending nearly the entire width of the leaf but not severing it completely. This causes the leaf to wither from this point outward to the tip. In this withered part of the leaf the larvae molt, after which they mine downward through the lower, uninjured part. It seems that the natural condition of the leaf, which is very moist, is unfavorable to the molting of these larvae, and it is in order to overcome this excess of moisture that they sever the conducting tissues, thus causing the leaf to dry quickly. In this manner they obtain the required dryness in which to shed their first coat. This allows the larvae to remain under cover, where they are more protected than they would be in the open. The characteristic appearance of such a leaf and the cast skin of one larva in the severed part of the leaf, just above the transverse cut, aze shown in Plate XLII, 37. The larvae of this species do not cease mining after their first molt, and come out of the leaf, as do the larvae of Arzama obliqua, but continue a miners in the leaf for some time, often remaining in the leaf through th second, and even through part of the third, instar. Then, however, th larvae crawl away from the upper part of the leaf and seek protectio Typua Insects: THEIR EcoLocicaAL RELATIONSHIPS 481 behind the sheath of the outer leaf, where they feed for a time before they enter the stem and become true stem borers. If the larvae emerge from the leaf soon after the first molt, they usually go down to the sheath of one of the first-formed leaves and there mine in the sheath for some time before entering the stem. Occasionally they feed for a while between two contiguous leaves. The effect of Nonagria oblonga Grote is easily recognized on the plant, since the work of the first-instar larva always causes the leaf at first to bend over and wither, and later, after the severed portion has become dry, to break off and fall to the ground. The leaf thus broken at the end, and the presence of the mine, are indicative of the work of these larvae. The writer once found a plant in which five leaves had been cut off by these larvae (Plate XLII, 36). On entering the stem, the larvae work toward the center, where their borings materially hinder the further growth and development of the plant (Plate XLVII, 70). The presence of the larvae in the stems is in- dicated by the dried and withered central leaves of the leaf bundle. A plant so affected never heads, because the larva keeps the center tunneled out. The habits of the later larval stages of N. oblonga and Arzama obliqua, and their effect on the plant, are very similar. Walton (1908) says of the habits of the larvae: ‘‘ From all appearances the larva feeds for a time on the sheath of the stem, . . . As it increases in size it bores directly into the succulent central shoot, where it afterward remains until emerging as a mature insect.” When the larvae become full-grown, they transform to the pupal stage in the burrow of the plant. The larva lies with its head upward in the burrow. The exit hole is from two to four inches above the pupa and is carefully plugged up with a combination of frass and plant fibers. Bird (1902) states that Nonagria has an extremely short pupal stage, from seven to nine days being the record of one brood. At Ithaca, New York, the writer first found pupae on August 2, 1916. In Kansas the insects mature much earlier. There one larva pupated on June 24, 1917, and emerged on July 5, 1917, the pupal stage covering only eleven days. The adult—— Around Ithaca the first adults were noticed on August 8, 1916, while in Kansas they were beginning to emerge by the 30th of June. Whether there is a second generation, especially in Kansas where the adults emerge so early, the writer has not been able to ascertain. The 482 “P. W. CLAASSEN adults failed to mate and lay eggs in captivity. However, no larvae were observed at work on the cat-tails later in the season. Therefore it is likely that if there is a later generation, it occurs on another plant. Description of the stages The larva.— The color markings of the larvae (Plate XLVII, 67) vary somewhat in different individuals, but mainly only in the degree of intensity of the colors. They may be described as follows: General ground color light brown with a slight tinge of flesh color. Head light brown, mottled or speckled with darker brown. Epicranial suture, mandibles, and area just above the clypeus and laterad, darker brown. Six longitudinal, flesh-colored to brownish stripes along the entire length of the body. On each side of the median dorsal line two broad stripes; and laterad to these stripes narrow stripes, located on the lateral margin of the body. Above the spiracles another broad stripe. Below the spiracles, often, another more or less broken line, especially noticeable in the young larvae. Prothoracic shield light brown. At the base of the hairs on the body a dark brown spot. Dorsal surface of the last abdominal 38 segment light brown, speckled with darker spots. Ventral side of the body of a light yellowish | color. Length of full-grown larva, from 40 to 50 mm.; width, from 4.5 to5 mm. Larva — cylindrical in shape and not as muck flattened as the larva of Arzama obliqua. The pupa (Plates XLII, 33, and XLVII, 71) Average length 27 mm.; width 7 mm. Color reddish brown, with head, thorax, and cremaster darker brown. Head with a conical projection about 2 mm. long. Wings extending backward over three-fourths of the fourth abdominal segment. Prothorax half : as long as the mesothorax. Surfaces of the head and thorax nearly smooth. Anterior — margin of labrum sinuate. Labial palpi about two and one-half times as long as the labrum. Maxillae extending about one-sixth of the distance along the fourth abdominal segment. Maxillary palpi present as small triangular pieces. Prothoracie femur visible. Prothoracic legs extending two-thirds the length of the maxillae. Mesothoracic legs reaching a little beyond the tips of the maxillae. Antennae reaching a point half-way between the tips of the mesothoracic legs and the tips of the maxillae. Metathoracic tarsi visible. Abdominal segments 2 to 7, inclusive, dorsally roughened with tubercles, especially prominent on segments 5, 6, and 7. Cremaster somewhat bilobed, with a rough margin bearing four straight setae, two originating underneath, and the other two originating above, laterad to the median line. All these setae equidistant from each other, the middle ones being longer _ than the outer ones. The adult—— The adult (Plate XLYVII, 68) is of a pale reddish or yellow- ish color, measuring about 35 millimeters across the extended wings. The original description of the adult, as found in Grote (1882), is as follows: Male. Pale reddish or yellowish gray, something the color of Mythimna, Pseudargyria, Guen. Primaries somewhat oblong, internal angle rounded away; apices softened, costa Typua Insects: THetrr HcoLtocicAL RELATIONSHIPS 483 a little arched. Eyes naked. Clypeus mucronate. Palpi prominent, concolorous. Mark- ings obsolete. The fine dark linear denticulate t: p. line barely discernable. Stigmata |very vaguely indicated by paler shades. Hind wings with a faint mesial black shade band; centrally stained with b!ackish; fringe and external edge like abdomen and very little paler jthan the rest of the insect. Beneath pale, with the disk of fore wings blackish; a common blackish extra-mesial shaded line. Minute black discal points. Smaller than Typhae. Arsilonche albovenosa Goeze . | Arsilonche albovenosa Goeze is another member of the family Noctuidae. It is found in Canada and in the northern, eastern, and central parts of the United States. It is a general feeder and has been reported on willow, smartweed, buttonbush, grass, and other plants. Typha latifolia, the writer believes, is here reported for the first time as a food plant of this |species. Life history and habits This moth is reported to have two generations a year. The writer, |however, has followed it through only one generation at Ithaca, where |two adults emerged on August 15. Egg-laying.— The eggs are deposited in long patches on the surface jof the cat-tail leaf, usually ten to fifteen inches from the tip. The eggs overlap one another as shingles do. They lie on the leaf in rows, the number of rows varying from three to seven, and the rows overlapping one another as well as the individual eggs in each row. ‘The number of |eges in one patch ranges from 60 to 161. As the egg develops, it becomes much darker, turning very dark just |\before the larva emerges. The larva.— Immediately after hatching, the larva devours the empty shell and then begins to feed on the surface of the Typha leaf, where it serapes off the chlorophyll. As thelarva grows and feeds more voraciously, it usually migrates to the end of the leaf, where it eats off the tip of the leaf or devours chunks out of the edge of the leaf, as shown in Plate XLVI, 62. When the larva has attained its full growth, it ties two cat-tail leaves together, and between them spins a tough cocoon in which it pupates. Two larvae pupated im the laboratory, under the author’s observation, jon July 23, 1916, and two others on July 25, 1916. The former both emerged on August 15, 1916. This apparently indicates the length of the pupal stage to be nineteen days. 484 P. W. CLAASSEN Description of the stages” The egg (Plate XLVI, 61) Flat; saucer-like or shell-like in shape, and grayish white in color. Diameter, as seen — from the top, varying from 0.89 to 1 mm.; thickness of egg about 0.2. Sculpturing very — pretty, micropyle in center of dorsal surface consisting of a dot with a rosette of elongate | cells around it. Radiating from the rosette to the margin of the egg, about 45 small ridges, indented transversely by small, rounded depressions. The larva— The first-instar larva, within twenty-four hours after hatching, is described as follows: Length 2mm. Entire head jet black; thoracic shield dark brown; meso- and metathorax — light gray with dark tubercles. Segments 1, 4, 5, and 8 of the abdomen dark brown, with gray tubercles. The other segments of the abdomen light yellow, with gray tubercles. From these gray tubercles originate long hairs. Beutenmiiller (1901) gives the following description of the full-grown larva (Plate XLVI, 62): Head black, with an inverted V mark on the face, two white stripes on top, and mottled with white at the sides. Body black, two yellow lines on each side of the back and one on each side below the spiracles. The body is also mottled with confluent striae, but less so on the dorsum. Warts orange with light and dark bristles; along the extreme sides a row of orange spots. Underside pale whitish. Length 40-45 mm. The pupa (Plate XLVI, 63) Length 18 mm., width, 5 mm. General color dark brown. Wings extending as far back as the fourth abdominal segment. Front of the head with two rounded, rugose ridges running — up and down. Clypeo-labral suture very distinct. The front margin of the labrum rounded. Labial palpi three times as long as the undivided labium. Maxillae extending down to the beginning of the third abdominal segment. Prothoracic femur visible and the prothoracic tibia and tarsi extending down almost to the tips of the maxillae. Mesothoracic legs extend- ing to the middle of the fourth abdominal segment. Antennae just failing to reach the tips of the mesothoracic legs. _Metathoracic tarsi plainly visible. Ventral surface of segments 5, 6, and 7 finely granulate anteriorly, posteriorly finely punctate. The other segments smooth. On the dorsal surface, the metathorax and the first seven abdominal segments very roughly tuberculate. Cremaster broader than it is long and bearing from 40 to 50 short, straight spines. The adult.— The adult (Plate XLVI, 64) is described as follows by. Beutenmiiller (1901): ‘‘ Fore wings white, and more or less heavily marked with fawn brown streaks between the veins, giving the insect a very characteristic appearance. Hind wings and body white. Expanse, 34-45 mm.”’ ~ TypHa Insects: THEIR EcoLoGicaAL RELATIONSHIPS 485 Archips obsoletana Walk. Archips obsoletana Walk. is a moth belonging to the family Tortricidae. This insect has been reported from the Atlantic states and from Illinois. The author has found it in Kansas and in New York. Slingerland (1901) suggested ‘‘ the obsolete banded strawberry leaf-roller”’ as a common name for the insect. Archips obsoletana Walk., although it lives on various host plants, prefers those which grow in moist situations, . and is here reported on Typha latifolza. ; Life history and habits The habits of this insect as a leaf-roller on strawberry have been studied rather carefully by Slingerland (1901). According to his report, there are three generations a year in New York. It is not known in what stage the insect passes the winter. Egg-laying. —The eggs have not been observed in nature. In the labora- tory, they were deposited in a large mass on the side of the glass cage. The larvae— The writer’s observations on the habits of the larvae of this species have been restricted to those specimens sound on Typha latifolia. The larvae and their work on cat-tail were first noticed on some cat-tail heads from Lawrencé, Kansas, sent to the author by Dr. H. B. Hungerford and received at Ithaca on July 17, 1916. A number of the heads showed the effects of the work of the larvae of Archips obsoletana. On August 12, 1916, larvae of this species were also found at work on the heads of cat-tail plants in the McLean Bogs. One pupa was also discovered at this time. The larva works on the immature heads of the cat-tail, feeding on the vender styles of the pistillate flowers and sometimes eating off the tops of the developing ovules (Plate XLVII, 69 and 72). The stigmas are not eaten; instead they are lined underneath with a thin, but closely woven, layer of silk. This silk layer, together with the stigmas on top, forms a protective covering over the larva. When this covering is torn loose, the larva quickly repairs it. In the laboratory, the larvae at times left the heads and fed on the leaves of the cat-tail; but they always provided themselves with a protected place, either by tying two leaves together or by spinning a silken tube between a leaf and the side of the glass cage. In this tube the larva “remained, never leaving it entirely but always keeping the tip of tke 486 P. W. CLAASSEN abdomen covered and protruding the head to feed. As the larva fed downward, it lengthened this silken tube. In the field, the author has not observed the larvae feeding anywhere on the plant except on the head. When the larva becomes full-grown, it goes to the top of the head, to which it then ties a leaf in order to form a place in which to spin its cocoon” for pupation. The silk used to tie the leaf to the head is covered with a mixture of frass and the remains of the staminate flowers. If a leaf ‘is not within reach of the larva, the cocoon is made on top of the head, hear the rachis, and covered with the remains of the staminate flowers. After pupation, the wind and rain soon tear off the covering made by the larva and the head has then the appearance of having been shaved in patches (Plate XLVII, 73). Description of the stages The egg.— Slingerland (1901) describes the egg somewhat as follows: Thin, oval, light lemon yellow, overlapping each other not unlike the shingles of a house. Shell is finely reticulated, the micropyle showing plainly at one end. The larva.— The larva may be described as follows: Olive green, with a light brown head and thoracic shield, both marked with black; the body sparsely clothed with light-colored hairs arising from pale, roughened tubercles. The newly hatched caterpillar light yellow, with a brown head. Length of full-grown larva, about 17 mm. (Plate XLVII, 76). The pupa.— The pupa is shown in Plate XLVII, 74. The following description applies to the female. Length, including cremaster, 12 mm.; width, measured across the wings, 3.8mm. General color reddish brown, the wings being somewhat lighter-colored than the rest of the body. Wings reaching back as far as the middle of the fourth abdominal segment. Thoracic region much enlarged, the appendages forming a distinct salient. Front of the head with an inverted Y ridge, as viewed from the cephalic aspect. A transverse ridge at the base of this Y, Clypeus and labrum prominent, and the clypeo-labral suture distinct. Labium clearly visible. Labial palpi one-fourth as long as maxillae. Manillae extending half way to the tips of the wings. Maxillary palpi elongate and triangular, reaching to the pro-lateral angles of the maxillae. Coxae ef mesothoracic legs visible below the maxillae and the femora. Femur and tibia of prothoracic legs large. Mesothoracic legs extending below the tips of the antennae. Antennae shorter than the wings by 0.8 mm. Metathoracic tarsi visible beyond the tips of metathoracic legs and antennae. Genital orifice double. Cremaster slender, tapering, longer than it is broad, and bearing eight stout, curved setae, four extending from the apex and two on each side. Setae of body long and prominent. On the dorsal surface, the first abdominal segment smooth. Segments 2 to 8, inclusive, each having two Typua Insects: THEIR EcoLoGicaAL RELATIONSHIPS 487 transverse rows of strong spines, most prominent on segments 4, 5, 6, and 7. A few spines present on the ninth abdominal segment. The adult.— The following description of the adult (Plate XLVII, 75) is quoted from Slingerland (1901): General color varies from a wood-brown through cinnamon to russet; the hind wings and all four wings beneath are of a lighter yellowish-brown color. Many fine, wavy, trans- verse, dark brown lines occur on the front wings, showing more distinctly in the male. And extending obliquely across these wings is a broad, dark brown band, more or less obsolete in the middle, and there is a subapical spot of the same color on each front wing. Lymnaecia phragmitella Staint. Lymnaecia phragmitella Staint. is a little moth belonging to the family Tineidae. Without question, this is the most common and the most abundant of the insects infesting the cat-tail. In distribution it is world- wide. It is found in England, central and southern Europe, northern Africa, Australia, New Zealand, and the United States. Its host plants are Typha latifolia and T. angustifolia. The writer has invariably found that the majority of Typha plants in any patch are infested by this insect. Life history and habits The larva.— Regarding the larvae of this species, Stainton (1870) wrote: If we visit a boggy piece of ground where Typha latifolia grows, we shall find that some of the thick club-like heads of that plant exhibit a curious, tattered and frayed appearance; if the period of our observation be autumn, we shall find on examining amongst the soft downy interior of the fertile catkin some small larvae; if we seek at the end of winter or in early spring, we shall find the same larvae, nearly ful! fed, about five lines long; and if these larvae are rather broad and flat, of a yellowish-white, with broad darker lines, we need not hesitate to pronounce them the larvae of Laverna phragmitella. The .larvae restrict their work to the head of the plant, except occasionally when they bore into the stem to transform (Plate XLII, 35). The young larvae feed on the tender styles of the pistillate flowers, but as these grow larger and become dry, the larvae move farther inward and eat the seeds of the plant. As cold weather approaches, they migrate still farther inward, and finally locate near the rachis of the flower spike, where they often eat away the basal part of the little stalks which bear the seeds. The larvae spin an abundance of silk with which they tie the down, or pappus, together, thus keeping it from being torn off or blown away. The cat-tail heads which are infested by these larvae present a striking appearance. The silk spun by the larvae holds’ the downy material together and does not allow the seeds to escape, but the heads fluff out 488 P. W. CLAASSEN greatly and become twice or three times their natural diameter. Two heads, one heavily infested with the larvae of L. phragmitella, and the _ other uninfested, are shown in Plate XLV, 59. This is the appearance of the heads in the fall. During the winter and spring the uninfested heads lose all their seeds so that only the rachis remains, but the in- fested heads retain their seeds in the fluffy condition just described till the following summer, when the heads finally drop to the ground. A field in which the majority of the heads are heavily infested is shown in Plate | XLIX, 84. This photograph was taken in July, just about the time that | the new heads were forming. In the old heads, as well as in the newly | formed ones, these larvae were present. | The larvae overwinter in the half-grown stage in the head of the jet, the fluffy material of the fruiting spike being their protection. In the latter part of May or early June the larvae attain their full growth. Then, in the midst of the downy material, the larvae spin their. thin, tough, white cocoons and transform to the pupal stage. Many of the | larvae, leaving the heads, go down and bore into the stem of the cat-tail | plant, forming burrows which they line with silk; and there they pupate. In the laboratory, larvae were placed in vials containing little bunches of seeds from the head of Typha. The larvae spun cocoons in the vials’ and pupated. Some of the larvae bored into the cole lined the tunnels with a little silk, and then transformed. The average pupal stage lasts 29.4 days. This was ascertained from data on five individuals in the laboratory, as shown in table 7. Cocoons TABLE 7. DertTeRMINATION OF THE PupATION PrRIOD OF LYMNAECIA PHRAGMITELLA STAIN. Length Specimen Date of Date of of pupal pupation | emergence | period (days) Dero ee. A SN URIS MOLD Sn Acoma et At ee RPE June 4 July 1 27 DCE BRAR RR eea Cre Mata pe IRR GRE eater RCL TE |e gen A June 63 |e seen 5% | OYA ene OA RTT 5 en Me orate nba ae A SLL ie ait May 30 July 1 32 2 AP ie ol eA ome Me eat tated Ra ney aeRO Hiren eA cA ee May 31 July 3 33 ye (2. 2 oe Soe SOMmMs SDESSE ree patch cet in resis Val ORT oe ae June 5 July 6 31 Cee 5 Sapa ReE tre =, AMEMEn OIA R anlar totes tguel fy 1ae ene [a Be ca) aadhbiar) 4! June 28 24 PNG) 41a Ro nen as Soe ene CSR Meme the, Mouse thee aicic Goa OT Gaia lded boo avo. 29.4 » = TyeHa Insects: THerrR EcoLoGicAL RELATIONSHIPS 489 and pupae, as they were removed from the pappus of the head of a Typha plant, are shown in Plate XLVIII, 82. The adult.— The first adults were observed to emerge in the laboratory on June 8, 1916, and the maximum emergence occurred between June 25 ‘and June 30. In the spring of 1918, the moths first appeared in the laboratory on June 10. Immediately after emergence the adult moths often rest on the cat-tail heads, as shown in Plate XLIX, 85. Stainton (1870) speaks of the adult as follows: If in July we visit a locality in which the Typha latifolia grows we may probably find towards evening some small grayish-ochreous moths, with the anterior wings rather streaked with brownish towards the apex, and with two dark brown spots ringed with white on the disc; these would no doubt be the perfect insects of Laverna Phragmitella. 1 Description of the stages The larva (Plate XLII, 32) Length from 10 to 12 mm., width 2.5 mm. General ground color yellowish white. Ventral side entirely white, with the exception of the brownish, chitinized legs and prolegs. Dorsal surface with 5 longitudinal brown stripes. The median stripe rather narrow; the next stripe, on each side of the median line, wide and somewhat lighter in color; the stripes on the lateral margin, above the spiracles, more or less broken into blotches. Head light yellow, blotched with brown. LEpicranial suture dark brown. Posterior part of the head dark brown. Mandibles and labrum dark. Prothorax mottled with dark brown. The last abdominal segment dotted with dark brown spots, as shown in Plate XLII, 32. The pupa (Plate XLII, 34) Length 9-10 mm., width 2.1-2.3 mm. General color yellowish brown. Head with ‘a blunt, rounded projection. Wings reaching to the middle of the sixth abdominal segment. Front of clypeal suture faint. Labrum with outer margin rounded. Labium not visible. Mavxillae broad at the base and much narrower at the proximal half. Maxillary palpi present as small triangular pieces. Prothoracic legs extending two-thirds the length of the maxillae. Mesothoracic legs not reaching quite to the tips of the maxillae. Antennae very slender, reaching the tips of the wings, and contiguous all the way to the tip. Meta- thoracic legs invisible. Rudimentary prolegs visible on the sixth segment. No definite sculpturing on the body. On the dorso-caudad surface of the last abdominal segment, eight hooked setae, arranged in groups of four. In each group three setae in a straight trans- verse line, but the fourth seta just cephalad to the middle one of the group. Cremaster undeveloped. The adult.— The length of the adult, with wings folded, is from 10 to 12mm. Stainton (1870) gives the following description of the adult: Head pale brownish-ochreous, face paler. . . Antennae pale grayish-ochreous, spotted with dark fuscous. . . Anterior wings pale brownish-ochreous, the costa beyond the 490 P. W. CLAASSEN middle paler; on the disc, nearly in the middle,is an elongate dark brown spot surrounded by white, and in a line with it at the end of the discoidal cell is another similar spot: a brownish — streak frequently connects the two, and the entire apical portion of the wing is more or less streaked with brown. . . Posterior wings pale gray, with grayish- ochreous cilia. Dicymolomia pate Walk. Dicymolomia julianalis Walk. is a member of the family Pyralidae. : It is found throughout the southern part of the United States. Ithaca is probably near its northern limit. Its host plant is Typha latzfolia. Life history and habits Dicymolomia julianalis Walk. has but one generation a year. It passes the winter in the half-grown larval stage. The habits of this} insect are very similar to those of Lymnaecia phragmitella. Egg-laying.— The eggs of D. julianalis are placed in the heads of the }; cat-tail, being inserted singly in the down, or pappus, of the-seed. They }} common. It was not at all difficult to locate them, once it had been } discovered where to look for them. The period a eo. has not i the middle of Tale and none after hae 1O¥-ihe ee are ein in fj the heads of the cat-tail with the blunt, or anterior, end outward. Several eggs are shown in the cross section of a head of Typha latifolia, in Plate XLIII, 45. On July 25, 1916, the writer watched one of these eggs hatch. The? larva lay in the egg, stretched to its full length, and could be seen moving § back and forth inside the egg shell. It ate its way through the egg shell} and escaped (Plate X LILI, 46). When about half of its body was out of thet shell, the larva gained a foothold on the pappus and pulled itself the rest} of the way out of the shell. The larva does not devour the empty shell, but at once buries itself in the head of the plant, where it eats the tender) styles of the pistillate flowers. The empty shell remains attached to the pappus. The time from the moment that the larva actually began to eat through the egg-shell till it was freed was about twenty-fivel minutes. The larva D. julianalis spends its entire larval period in the head}: of the cat-tail, obtaining its food, first from the styles of the pistillate™ TypHa Insects: THerrR EcotocicaL RELATIONSHIPS 491 flowers, and later from the seeds and the dried-up parts of the flower. As soon as hatched, the larva begins to feed on the styles, leaving the {stigmas to form a sort of covering over itself. These severed stigmas are spun together with a little silk and thus held in place. The larval habits fof both Lymnaecia phragmitella and D. julianalis are very similar in their early stages. As the cat-tail heads become more mature; and the larvae grow larger, they enter deeper into the head, and their presence is not {so readily detected as when they are working near the outer surface where the little raised patches of fluffy material they produce are easily seen. |The appearance of a head of Typha within a week after the larvae had hatched and entered the head is shown in Plate XLIX,86. As inthe case of Lymnaeciaphragmitella, the seeds are kept from scattering, by being tied together with silk woven by the larva. Neither wind nor rain is able to {tear apart the heads so protected. Accordingly they form a good shelter for the larvae during the winter. The larvae of D. julianalis bore into the jaxis of the flower spike and there spend the winter in the half or two-thirds- grown stage. The rachis, with the characteristic tunneling of the larvae, jis shown in Plate XLIII, 42 and 43. These tunnels are later lined with a jlittle silk and in themthelarvae construct tightly-woven cocoons in which they transform to the pupal stage. Many of the larvae, however, remain in the fluffy material in the heads to spin their cocoons and pupate. Pupation begins about the first of June. The adults emerge during the latter part of June and the first part of July. } During the spring of 1916 the author did not find any dead larvae in the heads of the cat-tail; but in the spring of 1918 all the larvae of this |species which he observed were dead, evidently having been killed by \the severe cold of that winter. At that time even the larvae in tunnels of the axes of the heads were dead. ‘This was not true of the larvae of |Lymnaecia phragmitella, however: Description of the stages The egg Elongate oval, tapering considerably toward the posterior end and rather blunt at the | anterior end (Plate XLIII, 38). Very long in proportion to its width, measuring 1 mm. in length and 0.219 mm. at its greatest diameter. Color of egg white, with a slight bluish | tinge in refracted light. Sculpturing rather-faint, consisting of fine, more or less hexagonal reticulations (Plate XLIII, 39, drawn with the camera lucida). ) } 492 P. W. CLAASSEN The larva.— The larva of the first instar is shown in Plate XLIII, 40. The following description is taken from a larva about thirty minutes after its emergence from the egg: Length 1.19 mm., greatest width 0.28mm. General color light pinkish or flesh color. Head and thoracic shield mottled with darker brown, restricted in the thoracic shield to the posterior part. A dark mottled area on the dorsal surface of the last abdominal segment also. The full-grown larva (Plate XLIII, 41) is described thus: Length from 7 to 10 mm.; about 2 mm. at its greatest width. Much flattened, and in general shape much like Lymnaecia phragmitella. General color flesh color. No special markings on the body except, as in the first instar, on the dorsal surface of the first thoracic segment and on the last abdominal segment. Head dark brown, with darker blotches near the outer margin. Epicranial suture very dark brown. Prothoracic shield dark brown, slightly lighter than the head, with two oblique oval spots near the lateral margin, the shield being mottled near the posterior margin. Dorsal surface of the last abdominal seg- ment mottled with brownish patches or spots, as shown in Plate XLIII, 41. Larva easily distinguished from that of Lymnaecia phragmitella in that it does not possess the five longi- tudinal stripes on the dorsal surface of the body. The pupa (Plate XLIII, 44) Length 7-8 mm., width 2.9-3 mm. General color yellowish brown to dark brown. Front of head not visible from the ventral aspect. Clypeo-labral suture distinct. Labrum with an emargination and very small, appearing somewhat like an arrow head. Two long hairs on the clypeus. Wings extending two-thirds across the fourth abdominal segment. Maxillae extending to the wing tips. Maxillary palpi absent. Prothoracic femora visible. Pro- thoracic leg extending two-thirds the length of the maxillae. Mesothoracie legs reaching to the tips of the wings. Antennae reaching to a point halfway between the tip of the prothoracic leg and the tips of the maxillae. Metathoracic legs not visible. Cremaster subquadrate, nearly smooth, with six cqually long, hooked spines arranged in groups of threes on the outer angle of the cremaster. Rudiments of prolegs on segments 5 and 6 of the abdomen. General surface of the body smooth. The adult— The adult female, shown in Plate XLVIII, 81, measures 6 mm. in length and has a wing expanse of 18 mm. Walker (1859) describes the adult as follows: Whitish, slightly marked with ferruginous. . . Antennae stout, submoniliform. Abdomen with brownish speckles. . . Legsstout. . . Fore wings with two reddish bands; the first exterior; the second marginal; the intermediate part with blackish speckles, which are somewhat confluent by the bands. Typua Insects: THEIR EcotoGicAL RELATIONSHIPS 493 COLEOPTERA Calendra pertinax Oliv.‘ Calendra pertinax Oliv. is a beetle belonging to the family Calandridae. Blatchley and Leng (1916) state that Calendra pertinax “ranges from ) New England and Canada to Michigan and Utah, south to Florida.” Satterthwait (1920) reports this species from the following states: Indiana, Missouri, Maryland, and New York. The author has collected and reared it in Lawrence, Kansas, and in Ithaca, New York. A variety of pertinax, called typhae Chittendon, has been reared from the roots of | Typha latifolia in California. The known host plants of C. pertinax are Typha latifolia, Acorus calamus, corn (Zea mays), and Sparganium sp. | The writer has found C. pertinax in Typha latifolia and in Sparganium sp. Life history and habits The weevil is found to be most abundant in Typha patches where the plants grow in sod or grassy soil. This has been found to be true in New York as well as in Kansas. In the wet, grassy places along the railroad tracks south of Ithaca, where Typha grows intermingled with | various species of grasses, the larvae were found to be most numerous. In some of these patches nearly every plant was infested. However, the weevil was found also in the larger cat-tail patches of Renwick Marsh around the biological field station. Egg-laying— The eggs are inserted into the outer sheath at the base of the plant, very near the surface of the ground. No females were actually observed in the act of ovipositing, but the newly laid eggs were _ always found with the end protruding from a little slit in the sheath (Plate XL, 17). In very wet places it is likely that the eggs are placed above the surface of the water, but the writer observed them only on cat-tails growing in a rather dry situation. The period of egg-laying has not been fully determined. Eggs were first found in Kansas on June 28, 1917. At that time, however, first- and second-instar larvae also were found in the plants, so that egg-laying must have started some time before, probably as early as the latter part of May. Eges were found in the stems as late as July 17, when the 4 Determined by Dr, E. C. Van Dyke, 494 P. W. CLAASSEN observations had to be discontinued. ‘The period of egg-laying is therefore spread over a number of weeks. The larva.— In the laboratory, the larvae were placed in tin salve boxes which had been partly filled with sterilized sand and moistened with boiled water. A little excavation was made at one end of a fresh piece of the rhizome of cat-tail, and in this the larva was placed and left to feed. Such pieces of rhizome, two or three inches long, remained fresh from three to five days. As the larva became older and needed more food, these pieces of rhizome had to be replaced more often. By splitting the plece open, excavating a little hole in the center for the larva, and then binding the pieces together again with rubber bands, observations could be made from day to day without unduly disturbing the larva. As soon as the larvae are hatched, they begin to bore directly into the stem, working toward the center, and thence downward toward the crown, and from there into the central axis of the rhizome (Plate XL, 19). Like the other borers of the cat-tail, this larva at once seeks the central part of the plant, where the tissue is most tender and succulent. However, the weevils seem to have a special preference for starchy food, and for this reason they work downward to the rhizome, the core of which is:composed mainly of starch (Plate XLV, 60). In rearing the larvae, it was found that they would not eat any other part of the rhizome except the starchy core. As many as seven larvae have been found in a single plant. In one instance they were all working at the crown and as a result had nearly cut off the plant. The affected plants present a somewhat stunted appearance. Some- times the central leaves die and the plant fails to head out. The tunneled rhizomes shrivel up considerably and often darken decidedly. | The larvae grow very rapidly, and the time from hatching to the pupal | stage averages about three weeks. When the larva has become fully grown, it prepares an oblong pupal cell in the stalkof the plant, from one to three inches above the ground (Plate XLVIII, 80). The pupal cell is made of partly masticated pieces of the stalk, with which the burrow is plugged above and below. In the laboratory some of the larvae pupated in their burrows in the rhizome, while others, that were reared in plants growing in flower pots, tunneled through the soil to the bottom of the pot and there made a smooth, oblong, unlined, earthen cell in which they trans- formed. The reason for their going down into the soil appeared to be a TypHa Insects: THEIR EcoLtocicaL RELATIONSHIPS 495 desire for a moist place in which to pupate. The plants in the pots had dried up completely, and the soil, too, was quite dry, so that in order to find a moist place the larvae were forced to go to the bottom of the pot. The length of the pupal stage seems to vary considerably. Eight pupae were kept under observation, and the results are given in table 8: TABLE 8. LenetH or Pupat Stace oF CALENDRA PERTINAX OLIV.* | Length : Date of Date of Specimen . a of stage pupation emergence (days) Liscege ce umole ee eee July 15, 1917 July 24, 1917 9 ecco CSc bu EO be Sot eE Soe July 15, 1917 July 22, 1917 7 Veacencck eek e6d Reel eee eee duly UG) OU, | coe cdancewcc Died thong 2 clo 6 Be OSU OEE Eee ea ee July 21, 1917 July. 27, 1917 6 SN mers eee ce oceadY s Cok aaa ara ems db A UO NN chee cn amococes Put in : preservative Dc cial A eRAA Ts Aue bo0 Lec ESSER en eee July 26, 1917 | August 8, 1917 13 Che cee noe C6 nd bODs eae Sane July 29, 1917 | August 11, 1917 13 Secgocsobeedo Une Ct eae eee July 29, 1917 | August 11, 1917 13 SOETEIRE «2 0 2.66, CSRS NCSC Oe cat enone eee aie ene 10.16 * The data on the first six pupae were determined for the writer by Dr. P. B. Lawson, of Lawrence, Kansas. The last two pupae were observed by the author himself. Description of the stages The egg (Plate XL, 16) Average length 2.15 mm., average greatest width 0.85 mm. Elongate oval, scarcely subreniform-elliptical. Color almost pure white. No distinct marking or sculpturing. As the time of hatching approaches, turning yellowish, and becoming quite dark just before the larva emerges. The larva (Plate XLYVIII, 77) Color dirty white. Head yellowish brown. Epicranial suture distinct. On each side of the epicranial suture a light line starting indistinctly at the vertex and running obliquely to the frontal suture. Mandibles very dark brown, almost black. Front of head darker near the fronto-clypeal suture. Clypeus light brown. Labrum with two curving sulci which divide it into three subequal parts. On the labrum four prominent hairs and a number of marginal hairs. Thoracic segment distinct. Prothorax with a yellowish, chitinized shield. Spiracles of prothorax large, oblong, and nearly twice as large as the other spiracles. Seg- ments 4, 5, and 6 of the abdomen greatly enlarged. Spiracles located on the dorsal surface. Length of larva, in its curled-up position, about 13 mm.; when straightened out, about 16-17 mm.; greatest diameter 7 mm. 496 P. W. CLAASSEN The pupa.— The pupa is shown in Plate XLVIII, 79. The size of the ~ pupae varies considerably. The average of the measurements of six pupae (table 9) showed the average length to be 14.2 millimeters and the width, taken across the prothorax, 5.76 millimeters. TABLE 9. MeraAsuREMENTS OF THE PUPA OF CALENDRA PERTINAX OLIV. Specimen | Length - Width (millimeters) | (millimeters) HU STD ee tes Bali NEE ear ee Manor Lees ae a re Ee Mt ee 15.8 7.0 OD Nie eee ped we Bee ape hGH: aa Celt aa en ra an 2 Ben eee I ed Bi OBZ, Be een oe arene am eo Run cers Sime gainer Nn ee Me he to I2E5 48 fs Died Aria ee Pacemae AN tert CaN eh five naam ir) VR) Sheet the pees Lad 15.4 6.3 ya tae ete GE SAO SAE gma sts ter, SOMITU SL OSV eae Sau eng pee eek ee CN 14.8 5.8 Ge Os IBS aes Sotere ek ceo ape me KE: eee ny, Nea Rete LCRA nce ae aan Beh 55) IAC E) es Nae hsm Sime NUM MTS ML TPO te gn Bards NMC dare Seite oe 14.2 5.76 | The pupa is large, naked, and dirty white in color. It may be described as follows: From the dorsal view: Head almost or entirely concealed by the prothorax. Prothorax a little longer than the meso- and metathorax combined. Eight spines on the surface of the prothorax, arranged in pairs, near the four corners of the subrectangular dorsum. Meso- thorax terminating in a triangular lobe, without spines or setae. Metathorax with two prominent setae. On each of first six abdominal segments a transverse row of setae arranged as follows: segment 1, with a group of three setae on each side of the median line and one laterally just above the spiracle; segments 2 to 6, inclusive, with the same arrangement except that the groups laterad of the median line have four. setae; segment 7 with one seta on the lateral-margin; segment 8 with stout spines, arranged in groups of fours, on the posterior margin. From the ventral view: Rostrum stout, reaching to the prothoracie tarsi. One pair of spines at the base of rostrum and another pair in line with the base of the antennae. Antennae elbowed and reaching almost to the tips of the femora. Each femur with a stout spine near the distal end. Wings reaching to the ends of the hind femora. On the eight abdominal segments, in each of the outer two apices, eight spines, arranged in groups of fours. The adult.— Blatchley and Leng (1916) describe the-adult (Plate XLVIII, 78) as follows: Flongate-oval. Black or reddish-black, shining, the interspaces of thorax and flat alternate intervals of elytra covered with a dirty white coating. Beak as in key, three-fourths the length of the thorax, finely and sparsely punctate, foveate and finely grooved above at base. Thorax longer than wide, foveate and finely constricted; vittae entire, the median one widest at middle, narrowed before and behind; lateral ones with edges sinuous, branched as described Typua Insects: THEIR EicotocicaAL RELATIONSHIPS 497 above; interspaces and sides of dise coarsely punctate. Elytra broadest at humeri, sides feebly converging to apical fourth, then more strongly to the rounded apex; striae with rather coarse, regular punctures; the broader and more convex intervals somewhat interrupted, minutely and sparsely punctate. Length 11-15 mm. Notaris puncticollis Lec.* _ According to Blatchley and Leng (1916), Notaris punceticollis Lec. (Plate XLVIII, 83) ranges from Newfoundland and Quebec to Minnesota and as far south as the Ohio River. The host plants reported for this species are cabbage, Peltandra virginica, and Typha latifolia. Webster (1893), writing of Notaris puncticollis, says: In Wayne County, Ohio, a field of this swamp land was underdrained last year, and last January was plowed; no further cultivation being given it until late spring, when it was prepared and planted to cabbage, about 50,000 in number, set late in June. These have been attacked and many of them destroyed by the adults of two species of Rhynchophora (Listro- notus appendiculatus Boh, and Erycus puncticollis Lec.).. The former is supposed to be the chief depredator, though I myself saw the latter attacking the plants. First, great cavities are gouged out of the stems of the young plants, and later the base of the larger leaves are attacked from beneath....... It is not unlikely that one and perhaps both of these species breed in Sagittaria, though I have some reasons for suspecting that the Erycus may breed in the common Typha latifolia or cat-tail. On August 19, 1915, at the field station in Renwick Marsh, W. A. Hoffman found the adult of Notaris puncticollis Lec. in the burrow in the stem of Typha. The burrow appeared very much the same as that of Calendra pertinax. ‘The writer, however, has not been able to find this species during the course of his studies. HEMIPTERA Ischnorrhynchus resedae Panz.$ Ischnorrhynchus resedae Panz. is an insect belonging to the family Lygaeidae. It is of general distribution, being reported from Europe, Asia, Central America, Mexico, Canada, and the United States. Among its host plants are included birch, conifer, heath, arbutus, Typha latifolia, and 7’. angustifolia. ‘The two species of Typha are here reported for the first time. Life history and habits Egg-laying.— The eggs are laid in the spring, during May and June. They are deposited singly in the pappus of the old cat-tail heads of the * Determined by C. W. Leng. 6 Determined by Dr. H. H. Knight. 498 P. W. CLAASSEN previous year. They are attached either to the seeds or to the pappus. When the egg hatches, the nymph either opens the cap or breaks through ~ the egg shell, bursting it near the top. {| The nymphs.— The various nymphal stages and the adults were first observed on the overwintering cat-tail heads in the summer of 1916. It was at first assumed that they were merely accidentally present on the cat-tail heads, but closer examination revealed that the bugs were feeding on the dry seeds of the heads. The nymphs obtain their nourishment by thrusting the stylets of their beaks into the dry seeds (Plate XLIV, 53 and 55). During feeding, the long labium is often folded back under the body. In just what manner the bugs are able to extract nourishment from the dry seeds the author has not been able to determine. When crushed on the slide and examined under the microscope, the seeds show very little moisture. It is very probable that the insects secrete a fluid which dissolves or predigests the dry food material before it is taken into the body. The author has succeeded in rearing nymphs to the adult stage on these dry heads of cat-tail alone with no other food available. When placed on the green leaves of the cat-tail, the nymphs insert their beaks and feed. Theyare easily disturbed while feeding on the seeds in the laboratory. At the slightest provocation they rise up on their hind legs, quickly extract their stylets, and, by means of their front legs, stroke the stylets back into the labium. The labium is then folded into place and the nymph retreats to some sheltered place. The adult—— Adults were found mating in May and June. The female inserts her ovipositor into the male and copulation lasts from six to nine minutes. Mating is repeated a number of times at intervals of from five to ten minutes. Description of the stages The egg (Plate XLIV, 47) Length 0.93 mm. to 1 mm., greatest diameter 0.29-0.80 mm. Egg elongate oval in shape, tapering considerably at the posterior end, and closed by a cap at the anterior end. This cap with a cone-shaped protuberance in the center and surrounded by a circle of hooked ~ spines. The upper two-fifths of the egg finely reticulated; the lower three-fifths with longi- tudinal wavy and branching ridges. Color lemon yellow at first, turning bright red before the nymph emerges. Empty egg shell white. The egg ciosely resembling the seed of cat- tail, both possessing caps and very similar markings on the surface. TypHa Insects: THEIR EcoLoGicaAL RELATIONSHIPS 499 The first-stage nymph (Plate XLIV, 48) Length 0.857 mm.; greatest width, across wing pads, 0.280 mm. Length of antenna “0.428 mm. When first emerging from the egg, the general color of the nymph bright red. Eyes carmine red. Abdomen, vertex of head, and lateral margins of the-body, of a darker color than the rest of the body. Thorax, front of head, antennae, and legs, of a light yellowish color. Several hours after hatching, nymph of a different appearance: Head, thorax, and tip of the abdomen very dark red, almost brown. Abdomen carmine, mottled with yellow. Legs and antennae greenish yellow, the antennae lighter at the joints. The epicranial suture and the median dorsal thoracic line lighter in color. The second-stage nymph (Piate XLIV, 51) Length 1.5 mm.; greatest width, across wing pads, 0.368 mm. Length of antenna 0.575 mm. General color carmine red. Head and thorax dark reddish brown. Intermixed with the red color of the abdomen, many yellowish blotches. Antennae dark red, lighter at the joints. Rostrum somewhat lighter than the rest of the head. Epicranial suture and median thoracic line pale. First thoracic segment uniformly dark; in the second segment the dark color restricted to two rectangular patches; in the third segment the darker color present in two transverse lines. The dorsal glands showing as short, brown, transverse lines between the abdominal segments 3 and 4, 4 and 5, and 5 and 6. The third-stage nymph (Plate XLIV, 50) Length 2.08 mm.; greatest width, across wing pads, 0.598 mm. Length of antennae 0.69 mm. General color a little darker than in the preceding stage. Head uniformly dark brown, except for the lighter epicranial suture and a lighter spot on the rostrum. Head and thorax covered with faint white pile. In this stage the pro- and mesothorax uniformly dark brown, with the dark patches on the metathorax a little wider than in the preceding stage. The light median line on the thorax present as in the previous stages. The mottled appearance of the red and yellow color of the abdomen more pronounced in this stage. Dorsal glands more plainly visible. Wing pads just beginning to show. Entire body more hairy than in preceding stages. ‘ The fourth-stage nymph (Plate XLIV, 49) Length 2.71 mm.; greatest width, across wing pads, 0.989 mm. Length of antennae 1.04 mm. Color of head and thorax dark brown. Epicranial suture and median dorsal line of thorax light red. Rostrum of head with a short black longitudinal line on each side. Eyes carmine red. Antennae slightly lighter than head and thorax, much lighter at the joints. Dorsum of prothorax on each side with a blackish, triangular, transverse spot, as shown in Plate XLIV, 49. Wing pads extending 5 mm. beyond the posterior margin of the mesothorax. The mottled color of the abdomen much as in the preceding stage. The white pile on the head and thorax thicker and more plainly visible than in preceding stages. Dorsal glands as in third stage. 500 P. W. CLAASSEN The fifth-stage nymph (Plate XLIV, 54) Length 3.35 mm.; greatest width, across wing pads, 1.61 mm. Length of antenna 1.38 mm. The general color similar to that of the previous stage, but the head and thorax now distinctly patterned. Epicranial suture as in previous stages. The part of the head back of the epicranial suture uniformly dark red. Rostrum yellowish with brown lines on each | side, which meet behind the rostrum and then diverge outward until they join the brownish | border inside the epicranial suture, thus producing on the head four yellowish patches separated by the brown lines in the shapeof the letter X. Prothorax dark brown, punctate with circular yellow spots. From these spots, short white hairs arising. Transverse dark bands on the prothorax, as indicated in Plate XLIV, 54. Rest of thorax, including wing | pads, dark brown. The surfaces of meso- and metathorax and the wing pads punctate with yellowish spots, less numerous than those on the prothorax, however. The bases of the | wing pads indicated by light-colored, diagonal lines. The margin of the entire thorax and wng pads of a blackish brown color. Wing pads reaching to about the middle of the third | abdominal segment. Abdomen colored much as in the preceding stage. The adult (Plate XLIV, 52) Female, length 5.4 mm.; greatest width, across the prothorax, 1.5 to 1.6mm. Length of antenna 1.75 to 1.85 mm. General color dark brownish red. Posterior margin of head and area around eyes and ocelli black. Sides of rostrum black. Basal segment of antennae black, second and third segments of antennae yellowish brown with fuscous at the bases and apices, and the fourth segment dark red. Head and thorax thickly covered with dark punctures. Pronotum with two wavy transverse dark bands near the anterior margin. Corium pale yellowish brown with two black spots on the disk and four black spots on the inner lower margin. Legs reddish brown. Apical segments of tarsi black. Body covered with a very fine white pile. Male slightly smaller than female. Stphocoryne nymphaeae Linn.* Siphocoryne nymphaeae Linn., the reddish brown plum aphis, is found in numbers on cat-tail during the spring and summer. This species | also uses other water plants as its summer hosts, such as Nymphaea, Potamogeton, and others. The aphids are found on the surfaces of the | leaves from the sheath out to the tip of the leaf. The writer observed | this species on T'ypha latzfolia at Ithaca in 1915, 1916, and 1918. | Aphis avenae Fab.® The author found Aphis avenae Fab., the oat aphis, in large numbers, feeding on cat-tail, during the spring and summer of 1917, at Lawrence, 7 Determined by Dr. Edith M. Patch. 8 Determined by J. J. Davis. TypHa Insects: THEIR EcotocicaL RELATIONSHIPS 501 | Kansas. Frequently the young aphids were found behind the sheaths ‘of the leaves, in the gelatinous material below the surface of the water ‘in which the plants were growing. Rhopalosiphum diantht Schrank Rhopalosiphum dianthi Schrank was reported on cat-tail by Sanborn (1906). | Rhopalosiphum persicae Sulz. Rhopalosiphum persicae Sul. was reported on Typha latifolia and on 'T. angustifolia by Wilson and Vickery (1918). Aphis gossypwt Glov. Aphis gossypii Glov. is found in small numbers on Typha latifolia during the spring and fall, according to Davidson (1917:65). Macrosiphum granarium Kirby Macrosiphum granarium Kirby, the grain aphis, is found in great numbers on Typha during the summer and fall, according to Davidson (1917:65). Hyalopterus arundinis Fab. Hyalopterus arundinis Fab., according to Davidson (1917:65), is found from April to June. The infestation on cat-tail is never large. There are four to ten generations. Aphids settle mainly on both sides of the blades, locating in colonies, usually not far from the tips. HYMENOPTERA? Five species of parasitic Hymenoptera were reared on insects which were found on cat-tails. Aleiodes intermedius Cress Aleiodes intermedius Cress was reared on larvae of Arsilonche albovenosa. On August 12, 1916, six specimens emerged from one larva, 9 Determined by C. F. W. Muesebeck, : 502 P. W. CLAASSEN Apantales cinctiformis Vier. Apantales cinctiformis Vier. was reared on larvae of Nonagria oblonga. A number of specimens emerged on August 8, 1916. Elachertinae sp. Five specimens of Elachertinae sp. were reared from a larva of Lymnaecia phragmitella. These emerged on June 15, 1916. Pimpla indagatriz Walsh On June 8, 1916, several specimens of Pzmpla indagatrix Walsh emerged from the heads of cat-tails which had been kept in a covered jar in the laboratory. Pimpla inquisitorielia D. T. Several specimens of Pimpla inquisitoriella D. T. were reared from pupae of Arsilonche albovenosa. DIPTERA !° The following flies were reared from cat-tail. Platychirus quadratus Say Platychirus quadratus Say was reared from the heads of cat-tail. The larvae were noticed in early spring in the overwintering cat-tail heads. Many adults emerged between May 21 and June 10. Macrosargus clavis Will. The larvae of Macrosargus clavis Will. live in the burrows made by the larvae of Arzama obliqua Walk. or of Nonagria oblonga Grote. They winter over in the larval stage, and the adults emerge in May and in early June. Chaetopsis aeneae Wied. The larvae of Chaetopsis aeneae Wied. also are found in the burrows of Arzama obliqua Walk. and of Nonagria oblonga Grote. Adults emerged on August 8. 10 The first three species were determined by Dr. O. A. Johannsen, the last one by Dr. J. D. Tothill. TypHa Insects: THEIR EcotocicaL RELATIONSHIPS 503 Sturmia nigrita Town. Sturmia nigrita Town. is a parasite which was found living in the larva of Arzama obliqua Walk. In each of the two instances observed, there was only one parasitic larva present in each of the larvae of Arzama obliqua. Both dipterous larvae emerged from their host on March 25, 1918, through an opening which was made on the ventral side of the first thoracic segment. They pupated on the following day, and one adult emerged on April 9 and the other on April 10. RESUME From an ecological point of view, the insect inhabitants of Typha may best be considered with respect to the part of the plant they affect. Accordingly they are thus classified in the following pages. INSECT INHABITANTS OF THE HEAD OF TYPHA The insects inhabiting the head of Typha include, among the Lepidoptera, Lymnaecia phragmitella Staint., Dicymolonia julianalis Walk., Archips obsoletana Walk.; and among the Hemiptera, Jschnor- rhynchus resedae Panz. The work of L. phragmitella and D. julianalis is very similar. Each has one generation a year. Their early larval habits are almost identical. They feed first on the tender styles of the pistillate flowers of the cat-tail plant, leaving the stigmas to form a covering over themselves. Later, they advance deeper into the head and feed on the seeds and other parts of the fruiting spike. Both overwinter in the half-grown larval stage. In the spring before pupation, however, their habits become somewhat different. Many of the larvae of D. julianalis bore into the rachis of the head, where they transform. The majority of the larvae of L. phragmitella, on the contrary, remain in the pappus of the cat-tail, where they pupate in closely woven cocoons. A few of the L. phragmitella larvae migrate down to the stalk of the plant, where they bore into the stems and transform. The adults of both species emerge at about the same time. — L. phragmitella is a species of world-wide distribution, while D. julianalis is generally restricted to the Southern States, though it is found as far north as New York. Of L. phragmitella the writer has found as many as 76 pupae in asingle head, while of D. julianalis he has never observed more than six or eight individuals in one head. 504 P. W. CLAASSEN Both of these insects are well adapted to live in the heads of cat-tail. Both spin an abundance of silk whereby they tie the pappus together and keep the head from being torn and the seeds from being scattered. This process of tying the pappus together assures the larvae of retaining their food supply and also furnishes them a protected and sheltered place for passing the winter. D. julianalis, however, being a less hardy southern form, was unable to stand the severe temperature during the winter of 1917-18, and all the larvae found in the Typha heads that spring were dead. Archips obsoletana should probably be classified as an incidental feeder on cat-tail. It is a typical leaf-roller, occurring chiefly on strawberry plants. However, once the larvae locate on the head of the cat-tail, they spend the entire larval period there and transform to the pupal stage on the plant. Since there are three generations a year, it is very probable that never more than one generation is passed on cat-tail; for these insects feed only on the tender styles of the pistillate flowers, and as these soon dry up, the later generations would not be able to find the tender food they relish. When living on the strawberry plant, these larvae roll themselves up in a leaf for protection. On the head of eat-tails they protect themselves by tying the stigmas together underneath with a lining of silk, thus forming a cover under which they live while feeding on the styles of the flowers. When placed in a cage with cat-tail leaves, the larvae prepare a covering for themselves by tying two leaves together and crawling between them. At the time of pupation they tie a leaf to the head of the plant and thus obtain the protection necessary during their transformation. In the spring, the females of Ischnorrhynchus resedae deposit their eggs in the old, downy heads of the cat-tail. The eggs closely resemble the seeds of cat-tail and thus are well protected from enemies. Immediately after hatching, the nymphs begin to feed on the seeds of the plant. They thrust their beaks into the dry seeds and apparently obtain their nourish- ment by injecting saliva into the seeds, which dissolves the solid material there so that they can suck it up into the body. The entire nymphal stage is spent in feeding on the dry seeds, a very remarkable and interesting adaptation. Due to the work of L. phragmitella.and D. julianalis, the seeds of many of the old heads are kept from being scattered by the winter storms, and Ischnorrhynchus resedae siniply takes advantage of these TypHa Insects: THrEetrrR EcoLoGicaAL RELATIONSHIPS 505 conditions. It inserts its eggs into the pappus, where they are hidden from all enemies and where the nymphs find an abundance of food at hand which is not contested by any close relatives and which, indeed, is used by few other insects. INSECT INHABITANTS OF THE LEAF OF TYPHA The inhabitants of the leaf comprise two classes, the surface feeders and the leaf miners. The surface feeders include, among the Lepidoptera, Arsilonche albovenosa, and among the Hemiptera, the Aphidae enumerated on page 501. The most common of the surface feeders is the noctuid caterpillar, A. albovenosa. It is a general feeder but is very commonly found on eat-tail. The eggs are placed on the upper part of the leaf, and the larvae, as soon as hatched, feed on the leaf. A leaf thus infested has the appearance of having been skeletonized. After they grow larger, the larvae begin feeding on the edge of the leaf, where they eat out large sections. The species of aphids mentioned on pages 500-501 may be classed as feeders on the leaf, although they occasionally feed lower down on the stem and sheaths of the plant. The leaf miners include Arzama obliqua Walk. and Nonagria oblonga Grote. These twonoctuid larvaedo not restrict themselves entirely to leaf mining but they begin their larval life as leaf miners, later becoming true stem borers. Although the two species are related, their habits differ ereatly. A. obliqua overwinters as a larva in its burrow in the cat-tail plant, whereas N. oblonga apparently passes the winter in the egg stage. The eggs of A. obliqua are laid in the spring, while those of N. oblonga are apparently laid in the fall. The young larvae of A. obliqua burrow gregariously, but the larvae of N. oblonga are solitary miners. The nature of their mines, too, is very different. A. obliqua advances down the channels of the leaf, leaving the longitudinal partitions of the leaf intact and-only destroying the cross partitions, while N. oblonga produces a sort of blotch mine by zigzageing back and forth in the leaf and destroying both the longitudinal and the transverse partitions. Both species feed mainly on the chlorophyll of the leaf. When ready for the first molt, A. obliqua sheds its skin at once, right in the mine, near the healthy, undisturbed, succulent tissue of the leaf; but N. oblonga, when ready for its first molt, first severs the connecting tissue of the leaf in order to 506 P. W. CLAASSEN produce a drier situation in which to cast off its coat. This variation indicates that A. obliqua is better adapted than WN. oblonga to live in moist or wet situations. A comparison of the tracheal systems of the two larvae shows this yet more clearly. A. obliqua has the spiracles of the eighth abdominal segment located on the dorsal surface and they are more than twice the size of the other spiracles of the body. Directly attached to these spiracles are the two longitudinal tracheal © trunks of the body. Segment 9 of the abdomen is flattened dorsally so as to be only half as thick dorso-ventrally as the other abdominal segments, thus making room for the large spiracles on the eighth segment. ‘This allows the body of the larva to be almost entirely submerged in the water, for as long as these spiracles remain above the surface it suffers no harm. The tracheal system of N. oblonga has not undergone any such modifica- tions, however. The spiracles on- its eighth abdominal segment are located in the natural position and are the same size as the other abdominal spiracles. Consequently the larva is likely to suffer harm if much water gathers in the burrow, as often occurs in wet situations. The larvae of A. obliqua remain in the leaf of Typha only through the first instar, while the larvae of N. oblonga often remain in the leaf through the second and even the third instar. The nature of their mining habits may have much to do with the difference. A. obliqua does not destroy the longi- tudinal partitions of the cat-tail leaf, and consequently must get out after its first molt on account of its increased size in the second stage. N. oblonga, however, cuts through the partitions in any direction and so is able to remain in the leaf for a longer period. After leaving the leaf, both larvae become solitary borers in the stalks of cat-tail. INSECT INHABITANTS OF THE STALK OF TYPHA The msects which work in the stalks of the cat-tail include two species of the Lepidoptera, Arzama obliqua Walk. and Nonagria oblonga Grote, and the Coleoptera, Calendra pertinax Oliv. and Notaris puncticollis Lee. After the larvae of A. obliqua and N. oblonga leave the mines of the leaf, they become stem borers. Their methods of entering the stem are very similar. Both are frequently found feeding for a time behind the sheaths of the outer leaves of the plant. From the sheath they either bore directly into the stem or enter from between the leaves of the leaf bundle. Both work their way to the center of the plant and locate at TypHa Insects: THEIR EcoLoGicaAL RELATIONSHIPS 507 the point where the tender new tissue is forming. The effect of their work on the plant is very similar: the central leaves of the leaf bundle die and the plant fails to produce a fruiting stalk. _ C. pertinax, the weevil, begins its larval life as a stem borer, later becoming a borer in the rhizome of the plant. The eggs of C. pertinax are inserted into the sheaths of the plant, near the ground. The newly hatched larvae bore to the center of the stalk and hollow it out just above the crown, thus arresting the further growth of the plant. After feeding on the tender tissue at the center of the stem for some time, the larvae enter the rhizome and there feed on the more substantial starchy food. When full-grown, the larvae return to the stalk and there form a pupal chamber in which the transformation takes place. There is only one generation. The larvae are ordinarily solitary borers, although as many as seven larvae have been found in one plant. INSECT INHABITANTS OF THE RHIZOME OF TYPHA The inhabitants of the rhizome are the Coleoptera, Calendra pertinax Oliv. and probably Notaris puncticollis Lec. The larvae of C. pertinax feed during the major part of their larval period on the starchy core in the rhizome of the plant. By first tunneling out the center of the stalk at the crown of the plant, they prevent the formation of new leaves, and in this way the larvae cause the starch to remain in the rhizome for their nutriment which would otherwise be used up in the growth of the plant. The leaves already formed are left undistrubed to manufacture and send down more starch to the rhizome. Very likely the habits of N. pwnctzcollis are similar to those of C. pertinaz. 508 P. W. CLAASSEN LITERATURE CITED BEUTENMULLER, WILLIAM. Arszlonche albovenosa (Goeze). In Descrip- tive catalogue of the Noctuidae found within fifty miles of New York City. Amer. Mus. Nat. Hist. 14:article 20:261. 1901. Brrp, Henry. Boring noctuid larvae. New York Ent. Soe. Journ. 10:214-216. 1902. BuatcHury, W. 8., and Lene, C. W. Sphenophorus pertinax Oliv. In Rhynchophora or weevils of north eastern America, p. 556. 1916. Britton, NATHANIEL L., AND Brown, Appison. An illustrated flora of the northern United States, Canada, and the British possessions, 1:1-680. (References on p. 68, 69.) 1913... CuLaassEN, P. W. A possible new source of food supply. Sci. mo. 9: 179-185. 1919. Davipson, W. M. The cat-tail rush, Typha latifolia, as a summer host of injurious insects. California Com. Hort. Mo. bul. 6:64-65. 1917. Davis, CHartes A. The uses of peat for fuel and other purposes. U.S. Dept. Int., Bur. Mines. Bul. 16:1-214. (Reference on p. 12.) 1911. Duprey, WititiAM R. The Cayuga flora. Cornell Univ. (Science). Bul. 2:1-132. (Reference on p. 102.) 1886. ENGLER, A., AND PrantuL, K. Die natiirlichen Pflanzenfamilien nebst ihren Gattungen und wichtigeren Arten insbesondere den Nutzpflanzen, 2':1-262. (Reference on p. 186.) 1889. FarMER, J. BreTuaNp (editor). The book of nature study, 5:1-224. (Reference on p. 44-45.) 1902-10. ; Grotr, Aucustus R. The North American species of Nonagria. New York Ent. Club. Papilio 2:94-99. (Reference on p. 96.) 1882. Hooker, JosppH Datron. [Title not given.] Jn A general system of botany descriptive and analytical, by Emmanual Le Maout. (Reference on p. 827.) 1876. NerepuHaM, J. G., and Luoyp, J. T. The life of inland waters, p. 1-488. (Reference on p. 90.) 1916. Parker, ArTHUR C. Troquois uses of maize and other food plants. New York State Museum. Bul. 144:5-119, (Reference on p. 108.) 1910, Typua Insects: THEerr EcotocicaL RELATIONSHIPS 509 SANBORN, CHARLES EMERSON. Kansas Aphididae, with catalogue of North American Aphididae, and with host-plant and plant-host list, Part II. Kansas Univ. Science Bul. 3:225-274. (Reference on p. 252.) 1906. SaTTerRTHWAIT, A. F. Notes on the habits of Calendra pertinax Olivier. Journ. econ. ent. 13:280-295. 1920. _SLINGERLAND, M.V. The obsolete-banded strawberry leaf-roller. Cornell Univ. Agr. Exp. Sta. Bul. 190:145-149. 1901. Stainton, H. T. The natural history of the Tineina, 1I1:1-330. (References on p. 150, 154.) 1870. WaLkeER, FRANcIS. Cataclysta? julianalis. In List of the specimens of lepidopterous insects in the collection of the British Museum, part 17: 255-508. (Reference on p. 438.) 1859. Edema? obliqua. In List of the specimens of lepidopterous insects in the collection of the British Museum, part 32:323-706. (Reference on p. 428.) 1865. Watton, W. R. Notes on the life history of Nonagria oblonga Gr. Ent. news 19:295-299. 1908. Wesster, F. M. Insects of the year. Insect life] 7:202-207. (Refer- ence on p. 205—-205.). 1893. Witson, H. F., anp Vickery, R. A. A species list of the Aphididae of the world and their recorded food plants. Wisconsin Acad. Sci., Arts, and Letters. Trans. 19:25-355. (Reference on p. 347.) 1918. Memoir 41, Lysimeter Hie enum? —IJT, the sixth preceding number in this series of publications, was mailed on November 16, 1921. PLATE XXXIX TYPHA LATIFOLIA 1, Sterile seed, showing large size of pericarp. 2, Fertile seed. (The pericarp fits closely over the kernel.) 3, Seed, or kernel, removed from pericarp. 4, Embryo protruding through pericarp. 5, Same as 4, with pericarp removed. 6, Growing embryo as it appears when removed from seed. 7, Embryo pushing open cap of seed. 8, Beginning of formation of root. (The arrow indicates the developing leaf.) 9, Further development of young plant. (The leaf has protruded and root hairs have developed on the root.) 10, Young plant with three leaves and three roots, showing disintegration of tip of first leaf, whereby it frees itself from the seed 510 CTX PLATE Memoir 47 Sit PLATE XL TYPHA LATIFOLIA AND CALENDRA PERTINAX Typha latifolia: 11, Cross section of leaf. 12, Cross section of a small part of leaf, show- ing more detail. 13, Cells of rhizome filled with starch grains. (Dormant season.) 14, Part of 12 enlarged to show structure of epidermis, chlorophyll, supporting tissue, and vascular bundles. 15, Cells of rhizome partly filled with starch grains. (Growing season.) 18, New offsets Calendra pertinax: 16, Egg. 17, Egg inserted in sheath of cat-tail. 19, Rhizome of cat-tail cut open to show larval work. 20, Newly hatched larva 512 ; Memo 47 513 PLATE XLI ARZAMA OBLIQUA 21, Egg. 22, Full-grown larva. 23, Pupa. 24, Egg mass on cat-tail leaf. (The larvae have emerged and mined along the middle part of the leaf, thus causing the center to die.) 25, Cat-tail leaf with egg mass, showing mine and exit holes of larvae. 26, Adult female. 27 and 28, Full-grown overwintering larvae in stalks of cat-tail. 29, Newly hatched larva. 30, Dorsal view of caudal segments of larva, showing position of spiracles on eighth abdominal segment. 31, Tracheal system of full-grown larva. (Only the first one of the five branched tracheal tubes is shown in its entire length) Memoir 47 PuaTte XLI 515 PLATE XLII LYMNAECIA PHRAGMITELLA AND NONAGRIA OBLONGA Lymnaecia phragmitella: 32, Full-grown larva. 34, Pupa. 35, Stalk of cat-tail with leaf turned aside to show where larvae have tunneled in, preparatory to pupation Nonagria oblonga: 33, Pupa. 36, Cat-tail plant showing work of larvae. (The first-stage larvae have cut the leaf. A mine appears also in the outer sheath.) 37, Cat-tail leaf show- ing Be! larval work. (The arrow points to the cast skin of the first molt in the transverse mine 516 Memoir 47 | Pirate XLIT SL? PLATE XLIII DICYMOLOMIA JULIANALIS 38, Egg. 39, Reticulations on surface of egg. 40, Newly hatched larva. 41, Full-grown larva. 42, Axis of cat-tail head cut open to show larval work. 43, Axis of cat-tail head, showing opening of larval tunnels.’ 44, Pupa. 45, Cross section of head of cat-tail, showing location of eggs (indicated by a). 46, Empty egg shell after emergence of larva FAIS Puats XLII 519 Memoir 47 PLATE XLIV ISCHNORRHYNCHUS RESEDAE _ 47, Egg. 48, First-stage nymph. 49, Fourth-stage nymph. 50, Third-stage nymph. 51, Second-stage nymph. 52, Adult female. 53, Enlarged drawing of beak of nymph in- serted in seed of cat-tail. 54, First-stage nymph. 55, Fifth-stage nymph feeding on seed of cat-tail . : AON Memorr 47 521 Puate XLIV PLATE XLV TYPHA LATIFOLIA 56, Two plants connected by underground rhizome, showing also new offsets at bases o old plants. 57, Two pieces of rhizome with outer covering removed to show the relative size of the central starchy core. 58, Leaf with part of the upper epidermis removed to show the structure. 59, Cat-tail heads as they appear in late fall. (The one on the left is infested with the larvae of Lymnaecia phragmitella; the one on the right is uninfested.) 60, Cross| section of a rhizome on bo no Memorr 47 Puats XLV 523 PLATE XLVI ARSILONCHE ALBOVENOSA AND ARZAMA OBLIQUA Arsilonche albovenosa: 61, Eggs. 62, Larva feeding on cat-tail leaf. 63, Pupa. 64, Adult Arzama obliqua: 65, Egg mass. 66, Full-grown larva Memoir 47 PLATE XLVI On bo on PLATE XLVII NONAGRIA OBLONGA AND ARCHIPS OBSOLETANA Nonagria oblonga: 67, Full-grown larva. 68, Adult. 70, Larva in tunnel in stalk of cat-tail. 71, Pupa Archips obsoletana: 69, Young cat-tail head showing larval work. (The covering is pulled aside, revealing the head of the larva underneath.) 72, Young cat-tail head showing larval work. (The stigmas of the pistillate flowers are tied together to form a covering for the larva.) 73, Appearance of cat-tail head after wind has torn off covering made by larvae. 74, Pupa. 75, Adult. 76, Larva 526 Puate XLVII Memorr 47 PLATE XLVIII CALENDRA PERTINAX, DICYMOLOMIA JULIANALIS, LYMNAECIA PHRAGMITELLA, AND NOTARIS PUNCTICOLLIS Calendra pertinax: 77, Larvae. 78, Adult. 79, Pupa. 80, Pupa in burrow in stalk of cat-tail Dicymolomia julianalis: 81, Adult Lymnaecia phragmitella: 82, Cocoons removed from ecat-tail head. (One is cut open to show pupa inside) Notaris puncticolus: 83, Adult Memoir 47 Puate XLVIII PLATE XLIX TYPHA LATIFOLIA 84, A cat-tail patch in July, showing the large number of fluffy heads which are infested with the larvae of Lymnaecia phragmitella. 85, Laboratory cat-tail head on which adults of L. phragmitella are resting. 86, Nearly mature head of cat-tail, showing evidence of work of young larvae of L. phragmitella and Dicymolomia julianalis 530 Memoir 47 Puate XLIX NOVEMBER, 1921 MEMOIR 49 CORNELL UNIVERSITY AGRICULTURAL EXPERIMENT STATION THE BIOLOGY OF EPHYDRA SUBOPACA LOEW CHIH PING ITHACA, NEW YORK PUBLISHED BY THE UNIVERSITY CONTENTS PAGE PI SRORMmOMIHCESTECIOS Seay ec anton 2 tine yams coal oS ole cress sratahecal cal orataoaea ee oon oa es 561 Distribution and range in Ithaca and vicinity...............0 00. e ccc eee 561 ehysicaleceavures:olesalt POOlS-csren cian al ie eckson cic ee ees ube 563 Sle co.cc oad ARES ee oe Ios eee AR RR a ee Me A At aah oe eee 563 Wie Lenape sense cer eis NPCS en Beanie, Ree Re ate Eid ok St oe 563 DinlaptcatecontentiOlsalt: POOR s, <6. < Giatese ss renee i 565 Life history........ #00 0 ai SOOO Gan Ome ee SU cee coche ke, PED TER eGR ade op ee 567 TINS: LEIA. oc 6 26-0) Hie eer ere i i ere Fie hese cnc ee eee 567 NUGTTEI NG) CRY 6 & So oe SIRO eee I eee Ae ee ae ON Be peer 567 Bxtermalestructures: ne: ois scone se Se oe or ee ee 567 Generalefeaturessnssi eee ee eee 567 Wbhewintegumentss. sc Nae see Seen Oe Bas RR a ieee LN ede 568 Phe RAPE agesn tice: Mewes ice teren eee ceric solic tn nL rence gm Toate 568 AMNLEENAIEStRUCL ULES ar meer fey Bin, Mates atte patient em means ee ae eRe 569 pluhegtrachealisy stem ters. wc sore cai Sie ee Ee ee 569 PERNT V.OUSISY SLE ter pian ope ie oo eee Re eee eee 572 pihesmuscularisysStemman swe ccrcincer hem ees aoe eee 573 pRhegalimentaryasystem core. 2c ene cies era dela eee eto eee 575 wthemvascularisystemien sacg--s conte ees oe ok imei nr ee 577 uihe;reproductiversystems.\5.= 2.45.65. oes ace ec is ee oe 578 hhepimacinaltdiskstee acct tin eee heme eco a weer eet ny caer a 578 (GERD. 6.a-c-0 nse Behe Cope Die SMAI rio fierce eear co a SP ae 579 Wioltingmpwetre eer eenttct ns dapsone eager s Oe she RE ae oes 579 TSEDIED 5 6 a1 0G Bis Rents RE ER RO ene Stat oe Ca Pe a ree apd 580 Observations on growth in salt and fresh water......................-. 581 TS EPID. 6 6:c:0:G Ba OSD ER Ee Me UTR Rete ET ite ena tw Tor a etree 582 GO CONVO GOT PTE Sse Pake ee CATES Oe eer EE oe nee 582 TREBO TINE, 5's a eles eee OG CER nS Bicitot eel Bei ne iairen Shine mu ent irre ee ore 584 TERES PL evil OT ee eeceetm yer Steen Me aca = a caw coin. ce eh eB gly meen Sn in fa cae 586 Preference for stagnant and shallow water........................--.- 588 IBreferences OMSalls wate nc on fe on toa ree Os ees 589 iHactorssiniuencin sha bits . OV (Ju) : Bradford, Letter Book (None Hist. Soe., Collections, 1st . Winthrop, New England,-vol. 1, appendix, p. 368. . Same reference, vol. 1, p. 29. 24. Calendar of State Papers, Col. Ser., 1574-1660, p. 141. 25. Winthrop, New England, vol. 1, p. 111. . Same reference, vol. 1, p. 104. . Same reference, vol. 1, p. 161. . Same reference, vol. 1, p. 183. . Same reference, vol. 1, p. 134. . Prothero, English Farming, p. 183. . Massachusetts Col. Records, vol. 1, p. 403. lections, Ist ser., vol. 1, p. 124). . Josselyn, Voyages to New England (Massachusetts Hist. Soc., Col- lections, 3d ser., vol. 3, p. 240-241). . Eliiot, Essays upon Field Husbandry, 3d, p. 57. . Douglass, Summary, vol. 2, p. 209 An account of the difficulties of one such journey, made in 1632, is given by Winthrop, History of New England, vol. 1, p. 91-93. . Johnson, Wonder-working Providence, book 3, ch. 1 (Jameson, Original Narratives, p. 234). . Plymouth Col. Records, vol. 10, p. 158. . Massachusetts Col. Records, vol. 3, p. 398. 10. . Plymouth Col. Records, vol. 4, p. 93. . Massachusetts Col. Records, vol. 2, p. 195. . Pynchon, Diary, p. 126. . Updike, The Narragansett Church, p. 514. . Josselyn, Voyages to New England (Massachusetts Hist. Soc., Col- Connecticut Col. Records, vol. 1, p. 284. lections, 3d ser., vol. 3, p. 338). ser., vol. ayu]Oy any) | 7. Winthrop, New England, vol. 1, p. 91-92. . Smith, New Englands Trials (Force, Tracts, vol. 2, no. 2, p. 17). : Bradford, Plymouth Plantation, p. 302. . Oldmixon, British Empire in America, vol. 1, 5B: . Massachusetts Col. Records, vol. 1, p. 36, 54, 108. Wallace (The Horse of America, p. 128) states that twenty of this first importa- tion were mares and stallions, but he does not give the source of his information. ’ Horse RAIsInc IN CoLONIAL New ENGLAND 931 . Prothero, English Farming, p. 36, 183. . Wallace, The Horse of America, p. 128. . Calendar of State Papers, Ireland, 1633-1647, p. 38. . Prothero, English Farming, p. 137. Calendar of State Papers, Treland, 1625-1632, p. 536. . Winthrop, Letters (Massachusetts Hist. Soc., Collections, 4th ser., vol. 6, p. 149). . Massachusetts Col. Records, vol. 1, p. 221. . Same reference, vol. 2, p. 14. . Same reference, vol. 4, part 2, p. 367, 552. . Plymouth Col. Records, vol. 11, p. 225. . Connecticut Col. Records, vol. 2, p. 244. . Massachusetts Col. Records, vol. 2, p. 225. . New Haven Col. Records, vol. 2, p. 590. . Connecticut Col. Records, vol. 2, p. 28. . Rhode Island Col. Records, vol. 1, p. 150. . Arnold, History of Rhode Island, vol. 1, p. 486. . Plymouth Col. Records, vol. 3, p. 222. . Winthrop, New England, vol. 1, p. 116. . Same reference, vol. 1, p. 161. . Bradford, Plymouth Plantation, p. 366. . Winthrop, New England, vol. 2, p. 18, 21. 2. Winthrop, Letters (Massachusetts Hist. Soe., Collecticns, 4th ser., vol. 6, p. 149). . Massachusetts Col. Records, vol. 3, p. 298. . Connecticut Col. Records, vol. 2, p. 61. 55. Massachusetts Col. Records, vol. 4, part 2, p. 367. . Same reference, vol. 5, p. 138. . Same reference, vol. 2, p. 190. . Maverick, Discription of New England (Massachusetts Hist. Soe., Proc., 2d ser., vol. 1, p. 247). . Winthrop, Papers (Massachusetts Hist. Soc., Collections, 5th ser., vol. 8, p. 65). . Calendar of State Papers, Col. Ser., 1669-1674, p. 232. . Same reference, 1675-1676, addenda, p. 213. . Winthrop, New England, vol. 2, p. 72. 3. New York Docs. Relative to Col. Hist., vol. 1, p. 362. . Description of Virginia (Force, Tracts, vol. 3, no. 8, p.1). Bruce, Economie History of Virginia, vol. 1, p. 298. . New York Does. Relative to Col. Hist., vol. 1, p. 385, 455, 503. . Plantagenet, Description of New Albion (Force, Tracts, vol. 2, OMe: 0). . Campbell, Considerations on Sugar Trade, p. 6. Anderson, Origin of Commerce, vol. 2, p. 28, 331. DEANE PHILLIPS . Oldmixon, British Empire in America, vol. 2, p. 2-4. -Anderson, Origin of Commerce, vol. 2, p. 28, 72, 331. Campbell, Considera- tions on Sugar Trade, p. 6-8. . Savary Desbrulons, Dictionary of Trade and Commerce, vol. 2, p. 766-767. . Anderson, Origin of Commerce, vol. 2, p. 72, 146. . Calendar of State Papers, Col. Ser., 1574-1660, p. 451. . Same reference; p. 329, 379, 382, 385, 390, 392, 393, 395, 401, 402, 404, 409, 411, 417, 420-426, 428, 431, 432, 436, 438, 451, 452, 461. Same reference, 1661-1668, p. 441, par. 1382. . Same reference, 1574-1660, p. 414. 3. Winthrop, New England, vol. 2, p. 312. . Same reference, vol. 2, p. 327. . Wallace, The Horse of America, p. 130. . Plantagenet, Description of New Albion (Force, Tracts, vol. 2, NOS MA kD -O) . Massachusetts Col. Records, vol. 3, p. 168. . New Haven Col. Records, vol. 2, p. 3. . Calendar of State Papers, Col. Ser., 1661-1668, p. 346. . Same reference, 1669-1674, p. 475, par. 1059. . Same reference, 1675-1676, p. 221. . Same reference, 1677-1680, p. 577. . Winthrop Papers (Massachusetts Hist. Soc., Collections, 5th ser., vol. 8, p. 386, 432, 445, 495, 532). . Some contemporary opinions regarding the special advantages of these regions are to be found in the following references: Calendar of State Papers, Col. Ser., 1661-1668, p. 348; same reference, 1675-1676, p. 221; Description of Rhode Island by Daniel Neal (cited by Field, State of Rhode Island at the End of the Century, p. 565). . Rhode Island Col. Records, vol. 1, p. 337. . Hull, Diaries (Amer. Antiquarian Soe., Collections, vol. 3, p. 127). . Same reference. . Quoted from original in British State Papers Office, N. Eng. Papers, B. T., vol. 8, no. 121, by Arnold, History of Rhode Island, vol. 1, p. 488. (The copy given in Calendar of State Papers, Col. Ser., 1677-1680, p. 524, is apparently incomplete.) | . Calendar of State Papers, Col. Ser., 1677-1680, p. 577. . Caulkins, New London, p. 236. . Rhode Island Col. Records, vol. 1, p. 150. . Connecticut Col. Records, vol. 2, p. 28. . Caulkins, New London, p. 254-255. . Rhode Island Col. Records, vol. 1, p. 337. . Massachusetts Col. Records, vol. 4, part 2, p. 394. . Caulkins, New London, p. 2538. 116. H17. 118. #19. Horse RAISING IN COLONIAL New ENGLAND 933 . Same reference. . Massachusetts Acts and Resolves, vol. 1, p. 444. . Edwards, History of the British Colonies in the West Indies, Ol, Gy 10. Pee . Caulkins, New London, p. 236. . Savary Desbrulons, Dictionary of Trade and Commerce, vol. 1, p. 853. Campbell, Considerations on Sugar Trade, p. 6. . Oldmixon, British Empire in America, vol. 2, p. 163. Ashley, British Colonies in America, vol. 2, Di 6: . Anderson, Origin of Commerce, vol. 2, p. 72, 386-887. Oldmixon, British Empire in America, vol. 7a Oy 160. . Hall, Importance of British Plantations in America, p. 44. _ Same reference, p. 45, 50. . Rhode Island Gol. Records, vol. 6, p. 60. New York Does. Relative ton Colwrist: vol. 5: p. 056, and vol. 6, p. 127, 393: . Bennett, Letters and Calculations on Sugar Colonies and Trade, no. 1, p. 63. . New York Does. Relative to Col. Hist., vol. 5, p. 597. . Ashley, British Colonies in America, vol. 2, p. 70. . Letter from Governor Robert Lowther to the Board of Trade, October 25, 1715. Colonial Office Papers 28:15 — T 101 (quoted by Pitman, Development of British West Indies, p. 202). . Anderson, Origin of Commerce, vol. 2, p. 335-338. . This part of the memoir follows the general account of the effects and enforcement of the Molasses Act as given by Beer, British Colonial Policy, chapter 3, and eee 9, p. 280-231. . New England Papers, B. T., vol. 3, p. 121, in British State Papers Office (quoted by Arnold, Taster, of Rhode Island, vol. 1, p. 488). Rhode Island Col. Records, vol. 4, p. 60. Report of Gover- nor Jenks to the Lords of Trade in 1731 (cited by Arnold, His- tory of Rhode Island, vol. 2, p. 106). The general importance of the export trade of New England in horses is emphasized by the following writers: Hall, Importance of the British Plantations in America, p. 104; Bennett, Letters and Calculations on the Sugar Colonies and Trade, letter 4, p. 5; Little, State of Trade in the Northern Colonies, p. 35; Savary Desbrulons, Dictionary of Trade and Commerce, vol. 1, p. 344, 367; Ashley, British Colonies in America, vol. 2, p. 99. A summary of this controversy is given in Anderson, Origin of Commerce, vol. 2, p. 335-3838. New York Does. Relative to Col. Hist., vol. 5, p. 556, and vol. 6, JO UAT eee An account of this contraband trade and the measures adopted to check it is given in Beer, British Colonial Policy, chapters 6 and 7. 934 120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 133. 134. 135. 136. 137. 138. 139. 140. 141. 142. 143. 144. 145. 146. DEANE PHILLIPS New York Does. Relative to Col. Hist., vol. 6, p. 226, and vol. 7, p. 164. Wm. Popple to the Lords of Admiralty (Adm. Sec. In Letters, 3819, Eng. Pub. Ree. Office), quoted by Beer, British Colonial iPolicyz pa ailale Edwards, History of the British Colonies in the West Indies, vol. 3, p. 258. Beer, British Colonial Policy, chapter 13. Calendar of State Papers, Col. Ser., 1661-1668, p. 144. Same reference, 1675-1676, p. 314. Savary Desbrulons, Dictionary of Trade and Commerce, vol. 1, p. 826. Oldmixon, British Empire in America, vol. 2, p. 396. Same reference. Same reference, vol. 2, p. 147. Rhode Island @onmience (Massachusetts Hist. Soc., Collections, 7th Ser viOle9 en0. 169 peel So. 2lom ako eo IONE New York Does. Relative to Col. Hist., vol. 5, p. 556, and vol. 6, p. 127, 393. Elliot, Essays upon Field Husbandry, 3d, p. 57. Governor Sanford to the Lords of Trade, British State Papers Office, New England Papers, B. T., vol. 3, p. 121 (cited by Arnold, History of Rhode Island, vol. 1, p. 488). Rhode Island Col. Records, vol. 4, p. 59, 60. Governor Jenks to the Lords of Trade (cited by Arnold, History of Rhode Island, vol. 2, p. 106). Rhode Island Col. Records, vol. 5, p. 18. Douglass, Summary, vol. 2, p. 99. MacSparran, America Dissected (Updike, History of the Narra- gansett Church, p. 514). Same reference, p. 515. Moses Brown, ms. letter on commerce (cited by Weeden, Economic and Social History of New England, vol. 2, p. 658). Rhode Island Commerce (Massachusetts Hist. Soe., Collections, ith ser, volt-95 no. 69; p.. 14. 16,183) 271 33io 390). Stiles, History of Windsor, p. 481-489. Caulkins, New London, p. 245, note. Douglass, Summary, vol. 2, p. 162. MS. Journal in Hartford Courant, April 25, 1881 (cited by Weeden, Economie and Social History of. New England, vol. 2, Deion): Caulkins, New London, p. 578. Cooper, Last of the Mohicans, eno: Caulkins, New London, p. 241. Same reference. Horse RAIsInc IN COLONIAL New ENGLAND 935) Rhode Island Commerce (Massachusetts Hist. Soc., Collections, uheserenviolk 9, 10:69; p..16)): Caulkins, New London, p. 254-255. Winthrop Papers (Massachusetts Hist. Soc., Collections, 5th ser., vol. 8, p. 3886, 482, 445, 495, 532). Stiles, History of Windsor, p. 481-484. Channing, The Narragansett Planters, p. 5. Potter, History of Narragansett (Rhode Island Hist. Soe., Col- lections, vol. 3, p. 16). Same reference, p. 275. Same reference, p. 58. Douglass, Summary, vol. 2, p. 101. Hull, Diaries (Amer. Antiquarian Soce., Collections, vol. 3, p. 127). Douglass, Summary, vol. 2, p. 101. Same reference, p. 107. Johnston, Slavery in Rhode Island (Rhode Island Hist. Soe., Pub- lications, n. s., vol. 2, no. 2, p. 165). Livingston, Edinburgh Encyclopedia, vol. 1, p. 336. Cooper, Last of the Mohicans, p. 14, footnote. Updike, History of the Narragansett Church, p. 515. Wallace, The Horse of America, p. 174. Winthrop Papers (Massachusetts Hist. Soe., Collections, 5th ser., vol. 8, p. 446). Updike, History of the Narragansett Church, p. 514. Savary Desbrulons, Dictionary of Trade and Commerce, p. 348. Calendar of State Papers, Ireland, 1625-1632, p. 536. Updike, History of the Narragansett Church, p. 514. MacSparran, America Dissected (Updike, History of the Narra- gansett Church, p. 490). Same reference. Watson, Annals of Philadelphia, p. 209. Updike, History of the Narragansett Church, p. 515. Caulkins, History of Norwich, p. 478-479. Caulkins, New London, p. 578. Same reference, p. 640. Edwards, History of the British Colonies in the West Indies, vol. 3, p. 273. Same reference. Elliot, Essays upon Field Husbandry, 2d, p. 21. Updike, History of the Narragansett Church, p. 519. Same reference (letter of I. P. Hazard). Watson, Annals of Philadelphia, vol. 1, p. 210. Wallace, The Horse of America, p. 182. Wallace, The Horse of America, p. 95, 144, 183. Craig, Pacing Horse, Standardbred (Cyclopedia of Agriculture, vol. 3, p. 476). 936 _ DEANE PHILLIPS SOURCES OF INFORMATION AMERICAN ANTIQUARIAN Socrery. Archaeologia Americana: transac- tions and collections. 7 vols. Worcester, Mass. 1820-85. AnpersoN, ApAM. An historical and chronological deduction of the origin of commerce, from the earliest accounts. 2 vols. London. 1764. ANDERSON, JAMES. The interest of Great Britain with regard to her American colonies considered. London. 1782. ARNOLD, JAMES N. The records of the proprietors of the Narragansett, otherwise called the Fones record. Rhode Island colonial gleanings, vol. 1. Providence. 1894. ARNOLD, S. G. History of the State of Rhode Island and Providence Plantations, 1636-1790. 2 vols. New York. 1859-78. ASHLEY, JOHN. Memoirs and considerations concerning the trade and revenues of the British colonies in America, with proposals for ren- — dering those colonies more beneficial to Great Britain. 2 vols. London. 1740-43. 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Economic history of Virginia in the seventeenth eentury. 2 vols. New York. 1896. Horse RAISING IN COLONIAL New ENGLAND 937 BurnaBy, ANDREW. ‘Travels through the middle settlements in North America, in the years 1759 and 1760; with observations upon the state of the colonies. 3d ed. London. 1798. CALENDAR of [British] state papers, Colonial series, vols. 1, 5, 7, 9-23. America and West Indies, 1574-1708. London. 1860-1916. CALENDAR of [British] state papers relating to Ireland. 33 vols. _ (1509-1670). London. 1860-1910. CALLENDER, JOHN. An historical discourse, on the civil and religious affairs of the colony of Rhode-Island [1638-1738]. (Reprinted in Rhode Island Hist. Soe., Collections, vol. 4. 1838.) CAMPBELL, JOHN. Candid and impartial considerations on the nature _ of the sugar trade. London. 1763. CauLKins, Frances M. History of Norwich, Connecticut, from its settlement in 1660, to January 1845. 2d ed. Hartford. 1874. History of New London. New London. 1899. CuHannine, Epwarp. 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State of Rhode Island and Providence Plan- tations at the end of the century; a history. 3 vols. Boston. 1902. 938 DEANE PHILLIPS Force, Peter (collector). Tracts and other papers relating principally to the origin, settlement, and progress of the colonies in North Amer-— ica, from the discovery of the country to 1776. 4 vols. Washington.) 1836—46. GoopwWIN, DANIEL (editor). The MacSparran diary [written 1748-51 by Reverend James MacSparran, rector of St. Paul’s Church, Nar- ragansett, Rhode Island.| Boston. 1899. Hau, F. The importance of the British plantations in America; with the state of their trade, and a description of the colonies. London, 1781. _Hazarp, Carouine: Thomas Hazard, son of Robt., eall’d College Tom; a study of life in Narragansett in the xviith century. Boston. 1893. HIGGINSOoN, New England’s plantation. (Reprinted im Force’s Tracts, vol. 1, no. 2, p: 6. Hut, Joun. The diaries of John Hull, mint-master and treasurer of the colony of Massachusetts Bay. Amer. 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Memoir 50, The Relative Growth- -Promoting Value of the Protein of Coconut Oil Meal, and of Combinations of It with Protein from Various Other Feeding Stuffs, the fourth preceding number in this series of publications, was mailed on March 9, 1922. Memoir 51, The Hog Louse, Haemotoninis suis Linné: Its Biology, Anatomy, and His- tology, was mailed on March 9, 1922. Memoir 52, etic? im Pollen, with Special Reference to Longevity, was mailed on ‘March 9, 1922 F . a Fi ae 4 ' 7 " ; beet Rey kaye ge Sie Pu eh PRT ECT | WS sone 4 Lave van ee lit) pages vt i. . ites Na ‘ My 9 tien wa Vy ‘ t) Year , eat eth 1 j : é stilt arene a Rave ‘ | : Pans OAs lie rf ; A : ; AY t | y } ‘ t } é ht ny ‘ MAY, 1922 MEMOIR 55 CORNELL UNIVERSITY AGRICULTURAL EXPERIMENT STATION INSECTS AND OTHER ANIMAL PESTS INJURIOUS TO FIELD BEANS IN NEW YORK I. M. HAWLEY ITHACA, NEW YORK PUBLISHED BY THE UNIVERSITY SES ee ek 4 WUSEUM MORE he : Pea NAC TELL A IU LU Oe fF ; Zs vi i OS . y 2 axel LPNS Ssst Sane Shiasssk + Ss } x i re ] ) ‘ ; = hf “ a) Fi : ; G F oe . | Dor can: ; | me i < i ; ‘ | ; ; L ‘ ) ; ¢ ; : \ et ; ‘ a . ‘_ LE ‘ | \ ie 7 s&s fi ; . is CONTENTS PAGE ithe seed-corn mareot (Hylemyza cilicrura Rond.)............:..0-0.-c0ce sees e ees 949 SVSLC UTA CRD OSIULOMES ried u emit Aine Phi sreyrapehe mS nik Wilendts colons Mrs (ers syoh wities, oan. out 950 Common names........ sg is SE BIS GEN EO PORE RTL Ue Md pate ie LR REE Pale 950 TBISTIOIEY cs a0 0:6 0'6:8. 005) Axe Bie Vasc Sy GCL Rca ENA ME ch Pca en cots Noe oe Ap aN ROA 950 ID TRY] QUVTGTD,» © 6.0.5 Goa Se eehe HN OD aE eee AIC RRA FU eM Pate Run RC ade Reem Rie Oe 951 ID@OG! TETAS. 6-0 no ido DLS OE TOE RRL ES SII tomy SEIN, Ste One ere ona Nest 951 INGIEUREROM@IT WEY BLOPOCADS hater ae ny atne cleaves: ee. 5c cena neni areca oe teas MAE 952 Tm Oneyy toy CHAS (0) Maes ee feos Pe es Ae oP NRT ee Wem ey ee a 952 i MV ACOMUDCECOLY LCC ODS Aig ties aie eek teehee Rew re ean aay Bete 955 I TIT RCOMU CRT ACI CLO R tes taciaa sy ore ees nyc ee As GIN Ayling Sa.ce PORT antsy ans 955 IL@ES GRVOSCGL «6 5 craig Sets eel eS AEN eS ouSI ICO rte Seer EEE PTET vet rts ee De PT 955 IDESCrUD ILONMOISLAP ES Er try cos: af A sie tptee bier ontyaiz A aoeaieNate in ease ooolae Vee Ven 956 ING®. HE « 2 6 86,5 6:8 OES ON SIE EERE Oe TE oe EPR Sore nee Ae 956 are TIPE) 5c ob 0 a:d.5 Soars re eae he CERO De ne et ney EE eee Re 956 INES SCY. 5 758 6 Ree Eee cee ae a ee ERE Die URE Ira. ee Se nee 4 956 TL lnvyioray Bins) Tne TSI aeTe ated ete eer eh ancy bt Re rir gD eo ROI Cn 958 TENDS: CELE o.oo ideo BSc NO ate REESE ES ETE HE re Ste PCY FU ea MII Rae UO 958 err oduoteine batt Om cus sr ces ..g teres ot seer teri ok eee iene teem SENS Opens BIER 958 alate CROMMO VA DOSLELOMY. ss euaz rere aah causuctacieys hat d Sati nicola ates as eee eed anepses 959 EEN CTROMMED DSpace bore nasties eis, techn ince & ctetusin ecu lca RC .. 960 PIC LOMOVADOSITION Ey ane mee sis AS kee Nh. dee eae ee anole OR oe ae, aCe 960 “TPE: TEETER 9 0c eso CORLORG CRP EH ON ERE AE Se oP ER SNORT 961 encthwot@larvalisiar erate c-2. 0. a el Shere eine wivting aaa Naes RGR 961 ETN EL SHO MGA CHL ATAU Eye ei pce cuis. ctee M teces ap eecye: coo acta conl stain, Scdaadene ceeedeane hes 961 TEIN® OUIOD 0.0'0:6 b Gb hoe Ea ee aoe sl Oa ea a re ral pn EL At nh ree Oe 962 MITTENS ET ate DATUM oes a erence otk ieee errr Ne a hed Nol Se sueops Ao PTS 962 EAR CCAOIBDUP A GLON RAN Le eats | A ay ase ea scp yn REI ise Fe ERT Ne 962 TNO EXCH «0 0:0 a0 cr SIS CSRS PRS ae Eo SPA Ce NCAT ar ee a ree er 962 THER RRC RE Ie Pus oe en RS eta iy Be Peak fate ata ee oe ac teers 962 APE ATe EROMMERT CR rae ee irae 2. IRE soc sia he Seen ia, Shoyh een paay he, heats 964 LEIBV OVE 5 ow aialdce EPR Aces apc RRR vg Rina SE ana ge rang kaeewe Ur de 964 STESEPUTIOR . 4 2 655 CSS eS ae ee HO a ERI DCR REO ret ee EL 965 Sved-coeel] TNISIOINT .. 5 ola d Simcoe ee ete ee ae ee ee A 966 “SUDDO), co.00000 6 040 SRS EEE ORE ane a ane ern eee ane eee 966 PRTC IR BITIE LOGS YOInCONLOl ee cee eles iis dveshus si orsieb bovcliecle « Qk ROME Soe © 966 Naturaland cultural methods of control. ..........0.....-..s4s.0se0--+3- 97C Moisture and temperature as factors in the life of the seed-corn maggot... 970 Relation of time of bean planting to time of oviposition................ 972 Relation of kind and condition of soil to maggot infestation............. 974 Influence of preceding crop and time of fitting a field, on maggot attack.... 974 Relation of depth of planting to injury by maggots..................... 975 SunimeanyeOlsprev.entive MEASULES 40. eee cj. a ee oe eee ee ss seus eee Oe ee . 976 he imported field gray slug (Agriolimaz agrestis L.).....-......-..00e cece eeee eee, Drivin ee... (CURRIE RO GCap ener Qu NU inn AER NER eM Wt San Ene 5 EP 977 ee ay MCAS ETL ODN oes hye) eee CEs GLAS pays ohn aie ts, S08 isons husee Guseerenete PAM 977 ~~ PSIEBITESD URE [ECO SUIT OTE ee paean e AB em Se oe te nO Aee e ae e eo Soe 978 General description of the slugs of the family Limacidae................. AN Sekt 978 MA CCMESUDPetStItLONSiCONCELNING SLUGS 20 em olen cleo yetns Speiaa a Sete eee eae = 979 RAC MAUSROMSIUL SH seen Sens ne MRT Oh es per, etext ts ae Ne ay h lege Royal cakes eRe Me 979 HICOMOMICMMLPOLEANCE-..c\- 6% s< 1h fees elota min sons SARIS, oe eo BAe SraSe, rc ysty aye 980 NiGHTS OF aS TT AOM Eom extosnty Green Oe On Rene ERO e nor sanoan senor 980 eMTEyAbOND ants Ouner Goan) DEANS! stern sic yayeteievie ws el Sheva Pie Bie deetel> Myedneraye sane 982 MS TSEAnclepossible TOO SUppliess® — = Lee aa wets Wsespeyo0< nysyert colder crs sepeese elem listen es 982 | CREDITOR, OO? CURRTSESRENS Oa Bea oe RE cos ts ek ec ee Eater ico mie ho ae 983 ING GEG. 5 5. So Gales in ae Be ee De ee nd EGE a Dae iene O ene apes 983 TMi {ROWE GIlbtee ONO Gate des oleae ee Meo Coe Dene OOO ara Onion pomn coos ar 983 Tine inillaaron may his SR ee Ai, a eee ete eb nun en Orb einai 983 945 946 CONTENTS The imported field gray slug (Agriolimaz agrestis L.) (continued) PAGI Life history and habits. . 2... 00.00... ee ee oe ood oto eee ee 98 The C22... 02. sleek eee tee nese ue besstdie sss uthitE—— .. 98: The young slug... 000. cc gece eee dele eos ale 6 er 98 The full-grown. slug. ... 0. 025.2502 ede eee bs oe be eee eee 98 Method of feeding. ......... 0022.0 00.+.0.+ 0) oe 987 Mating and oviposition..............0.++.. +020 987 Time required to reach maturity...........-. 2.25352 ee 987 Nature of outbreaks... 00.0... eee bee eee le cee) er 98% Relation of Agriolimaz agrestis to moisture.........2...... see eee 98: Relation of Agriolimax agrestis to extremes of temperature...................... 9g! Seasonal history... 620s cece cee cee cee epee ne ee 99( Predatory and parasitic enemies.....:......:. 2.220302 se eee 99( Control. wn lc cc eee ce get emias tues paseo 99( Experimental work... 2 00055062 0h ejes coe bw ee ee 99) Summary of control suggestions.................... PORE ets isis 4 cena ¢ 997 How to distinguish the various species of slugs found in bean fields............... ei Agriolimaz campesiris Binney. ......... 2.2. 1. 2 ee 99) The spotted garden slug, Limax maximus L..................--- Nae on eae ] ( Arion cireumscriptus Johnson... ...... 0.0... 50. ee 1 oe 996 The pale-striped flea beetle (Systena taeniata Say).............. 2.00 cece ec ee tee eee 100( Description of stages......... eo kbc dos 100! The 626... oe acl we entre coe eeln ot oe ct Se rr 100) SS Cf: a oa Soncec: 100) Phe pupae... oie oe cee Pe oe oie an SEE SRI eee 100) The adult... 2.20.2... ees e ee sta bee 100: Life history and habits... 00.0000. occa oe ee bene ee 100! The egg... oe eee ceeded eee bideeste tet te 100: The larva... 0... vie ce cee levee eves sun: J 100 The pupa... . 2... bee ee eee ewes oe 100 The adult... 0... ec elec cee eee dees os ee 100 Seasonal history... 2.0.0.0... 00. 0ce cece cnt ends let 100 Control... oe ice teed cae eee lute’ vetet lie een 100 The red-headed flea beetle (Systena frontalis Fab.).... 2.0.00. .0 000 cc eeecueceseveees 100) Description of stages... 0.2... ce ccc cee cece ec lec cet 100 The egg... cece ccc ve ote eens sateen 100 Phe larva... 0.0 so. ec e eee ee bus ecko see 100 Theadult....... 2.0... 0.ccl ecu a e 100 Life history and habits.......20....00.05.. 02.) 4 32 10¢ he eg8. 2 eee eee center, 1 100) The larva and the pupa......00..0.000...50.0. 0 2 ee 100 __Thevadult.-.. 0 se 1003 Seasonal history...... 00.0... c cel ek epee eres 1 Control... 0... ecient ee cre oo ied The green clover worm (Plathypena scubra F ab.) oo. ee eee 101 The bean weevil (Acanthoscelides [Bruchus] obtectus Say)... 2. 5. Sie 1014 The blue-banded millepede (Julus caeruleocinctus Wood) :.... 0.052) sae 101 The wheat wireworm (Agriotes mancus Say)............................4........ 4) 10 The red spider (Tetranychus telarius L.)......................). no 10 : irasshoppers (M elanoplus atlantis Riley, M. femur-rubrum DeGeer, and M. bivittatus Say) 10 njuries to beans in the pod, caused by hemipterous insects ( Adelphocorus rapidus Say, am E uschist us variolarius Palisot de Beauvois, Lygus pratensis L.) 1 Phe réle of insects in the transmission of bean diseases........................, 1. 10 Bibliography and literature cited SOS chem carne a ..10 INSECTS AND OTHER ANIMAL PESTS INJURIOUS TO FIELD BEANS IN NEW YORK INSECTS AND OTHER ANIMAL PESTS INJURIOUS TO FIELD BEANS IN NEW YORK I. M. Haw.uery In June, 1917, a laboratory was established at Perry, in the bean-growing section of western New York, for an investigation of the diseases and the insects that had been causing much injury to field beans. In this work the Departments of Entomology, Plant Breeding, and Plant Pathology at Cornell University were each represented by one member. This investi- gation has been carried on for four years, and the results, on the whole, have been satisfactory. The entomological work, however, has been hin- dered by one unavoidable circumstance: in some summers the insect pests under investigation were very scarce, and field experiments for their control were thus impossible. As a result, the recommendations in some cases are based on fewer data than the writer had wished. The more important part of this investigation is the part concerning the seed-corn maggot (Hylemyia cilicrura Rond.). The field gray slug (Agriolimax agrestis L.), a mollusk of the family Limacidae, also has been studied in detail, and some attention has been given to the green clover worm (Plathypena scabra Fab.), the red-headed flea beetle (Systena frontalis Fab.), the pale-striped flea beetle (Systena taeniata Say), the blue-banded millepede, or thousand-legged worm (Julus caeruleocinctus Wood), and the bean weevil (Acanthoscelides obtectus Say). Observations were made also on the habits of some insects of lesser importance, in particular those that produce the pitting of the bean known as dimpling. THE SEED-CORN MAGGOT (Hylemyia cilicrura Rond.) It is difficult to obtain exact data concerning Hylemyia cilicrura,' for it is an erratic insect that may occur in a field in great numbers in one season, and not reappear in, or even near, that field the following year. The flies usually disappear in late summer and the hosts of the larvae during that part of the year are not definitely known. Reared flies apparently do not mate in captivity. Infestations of the insect in cultivated crops are not usually found until considerable damage has been caused. By that time the maggots are full-grown and it is too late for control experiments with that brood. The writer realizes the many gaps in the present work, but, as the insect is scarce at the present time, it seems desirable to record the results thus far obtained. . . . . U6 . . . 1This species is more commonly known in American literature on economic entomology as Phorbia fusciceps Zett. 949 950 I. M. HAawneEy SYSTEMATIC POSITION The parent insect of the seed-corn maggot (Hylemyia cilicrura, Plate LXIX, 1) is a fly of the order Diptera and the family Anthomyiidae. The insect was first described by Rondani (1866)? as Chortophila cilicrura. Until recently, however, cilicrura has been considered synonymous with fusciceps of Zetterstedt (1845), and, since Zetterstedt’s description precedes that of Rondani, fusciceps has been accepted as the specific name of the insect. Stein (1916) finds that fusciceps is a distinct species and not the cilicrura of Rondani. Malloch (1920) accepts the separation of the two species | made by Stein. The species fusciceps of Zetterstedt occurs in Lapland | and other parts of northern Europe, and recently Malloch (1920) has | recorded it from North America. The species cilicrura, in addition to a | wide European distribution, is present in most parts of North America, | and is the pest known as the seed-corn maggot. The fusciceps described by Slngerland (1894) is not the fusciceps of Zetterstedt but is czlicrura Rond. Stein (1916) places czlicrura in the genus Chortophila, but Malloch (1920) unites the genera Chortophila, Phorbia, and Hylemyia in the strict — sense, in the genus Hylemyia. If we follow this latest paper on the subject, the seed-corn maggot must be called Hylemyza cilicrura Rond. — In the recent European writings of Reh (1913) and Oberstein (1916), the specific name Chortophila cilicrura is applied to this insect; but in older works, such as that of Ritzema Bos (1890), mention is often made of Anthomyia platura. The species platura is a composite of cilicrura and trichodactyla, and often it is impossible to determine definitely which species was blamable for the work these authors have described. COMMON NAMES The common names given to Hylemyza cilicrura include the following: | deceiving wheat fly, locust-egg anthomyian, Anthomyia egg parasite, seed-corn maggot, corn Anthomyia, seed-corn flower-fly, bean maggot, | bean fly, fringed anthomyian. Of these, the name seed-corn maggot is — the best known and is the one retained in this paper. HISTORY Hylemyia cilicrura is probably of European origin. In North America — the first record was that of Fitch (1856), who found the fly on wheat heads and described it under the name Hylemyia deceptiva. Riley (1869) dis- | covered the larva attacking corn in New Jersey and named the fly Antho- mya Zeae, but nine years later (Riley, 1878) he called it Anthomyia angusti- ~ frons Meigen when he found the maggots feeding on locust eggs in Kansas © and other western States. It was reported that ten per cent or more of 2 Dates in parenthesis refer to Bibliography and Literature Cite?, pages 1025 to 1037. INSEcTs AND OTHER ANIMAL Pests I[NsuRIoUS TO FIELD BEANS 951 these locust eggs were destroyed in this way. Later, Jack (1886) found the maggots destroying field beans in Canada. At intervals since that time the pest has suddenly appeared, destroying bean seedlings and injuring many other crops both in the United States and in Canada. During the last few years H. cilicrwra has once more become active in the New York bean fields, after a period of scarcity covering many years. Since 1914 moist weather conditions have tended to augment the normal number of flies. The injuries caused by the maggots of this species reached a maximum in 1917, but since that time there has been a gradual decrease in the amount of damage, and in 1919 and 1920 the loss due to the insect was hardly noticeable. DISTRIBUTION The seed-corn maggot has been found in many parts of the United States and Canada. It has been reported from nearly every State, from Maine to Florida and westward to the Pacific. In Europe, reports of its presence in Austria, Germany, Italy, England, and France may be found. It has been reported also from Hawai. Chittenden (1916) states that the species cilicrura causes much of the loss in the States south of New Jersey which is credited to the cabbage maggot, Hylemyia (Phorbia) brassicae Bousché, and to the onion maggot, Hylemyza antiqua Meigen (Phorbia ceparum Meade). Chittenden believes also (1909) that some of the work on the Pacific Coast attributed to Hylemyia planipalpis Stein may be due to H. cilicrura. FOOD PLANTS Hylemyva cilicrura has a wide range of food plants, according to Ch tten- den (1902) and other writers. Among the commoner of these may be mentioned beans, peas, lettuce, corn, cabbage, cauliflower, beets, turnips, radishes, seed potatoes, sweet potatoes, domestic garlic, crimson clover, onions, and hedge mustard. Whelan (1916) reports the maggot as breed- ing in fresh manure, in clover and alfalfa sod, and in rotting clover stems. Tucker (1917) reports cilicrura injury on tomatoes and cauliflower, and says that the larvae were found developing in decomposed cotton seed. Garman (1904) found the insect in young hemp plants. Pettit (1910) mentions pumpkin, cotton, orange, artichoke, and strawberry as hosts. as bred czlicrura from maggots in the “bulbs” of wheat. Howard (1900) states that the fly has been bred from human excrement. Riley (1878) found the maggots feeding on locust eggs. The attraction of the insect for decaying matter has been recognized by many writers. Chittenden (1902) cites, as an example of this, the finding of the larvae in tineid galls on poplar trees. Quaintance and Jenne (1912) found the flies appearing in cages where decaying plums 3 As stated in a general discussion reported in the Journal of: Economic Entomology, vol. 9, p, 133, 1916, 952 I. M. Hawuey were used in rearing the plum curculio. Johannsen (1911) thinks the species is attracted by decaying matter in the soil. Berger (1908) found the insect working in cut surfaces of seed potatoes that showed decay. Schoene (1916) has often bred the insect on cabbage, and believes the species is attracted to that plant by decomposition in parts of it. Black- man and Stage (1918) bred the species on a decaying root of larch. The insect has been reported also in Europe. It was found on sea kale in England, and Ritzema Bos (1890) reported finding the species platura (which, as already noted, is a composite of cilicrura and tricho- dactyla) on human excrement, on asparagus, on leek (Allium porrum), and on shallot (A. ascalonicum). More recently this species has been dis- cussed, under the name Chortophila cilicrura, as a pest of rye and corn in Silesia (Oberstein, 1916). Kornauth (1916) reports trichodactyla as injurious to beans in Moravia. ' Under field conditions in western New York during the progress of the present study, larvae of Hylemyia cilicrura have been found in beans, peas, corn, seed potatoes, and alfalfa roots. Baits of decaying materials were placed near the laboratory, and later examination showed the fol- lowing to contain maggots: cabbage, bean pods, bean vines, grass stems, clover roots, and clover stems. Two larvae have been found in mustard growing near a bean field, and two flies were bred from larvae taken in late summer in the roots of quack grass (Agropyron repens). The species has been reared also from pupae found in a pile of rich soil that had been taken from beneath decaying stumps. The writer has never bred the fly from manure. : From these data it may be seen that the list of known hosts is both large and varied, including not only healthy and decaying vegetable tissue, but also animal tissue. It is probable that this list is far from complete. The first flies taken each spring have been found by sweeping old wheat fields, and the writer believes that wheat, oats, and possibly other grains, may constitute important late-season hosts; but as yet sufficient data are not available for proof of this. Mature females of the second brood, taken in July, were numerous near sod and quack grass, and these also may be common winter hosts of the insects. NATURE OF INJURY TO BEANS The larvae of the seed-corn maggot may feed on three parts of a bean seedling—the plumule, the cotyledons, and the radicle. The injury to each part of the plant is here discussed separately. Injury to the plumule When the small larva locates a source of food in a sprouting bean, it usually crawls between the cotyledons, or seed leaves, and feeds on the two leaflets of the plumule and on the small bud of the growing tip between INSECTS AND OTHER ANIMAL Pests INsJuRIOoUS TO Firrtp BEANS 953 them (Plate LXIX, 4, and fig. 86, A). This vegetative part of the plant may be entirely eaten away so that when the seedling comes above ground Fic. 86. INJURY BY HYLEMYIA CILICRURA A, Types of injury in bean seedlings. B, Injured bean plants known as snake- heads, showing the result of feeding by the seed-corn maggot only the cotyledons remain. This stunted form of plant is known to bean growers as a snakehead, or baldhead (fig. 86, B). Usually a snakehead shrivels up and dies, but occasionally one succeeds in producing accessory 954 f I. M. HaAwLey buds and in developing leaves and a few flowers (fig. 87, A). At harvest | time a plant of this type is found to bear few if any pods and is still a dwarf plant (fig. 87, B). The formation of snakeheads is the severest form Eee aor Fic. 87. RESULT OF WORK OF HYLEMYIA CILICRURA, AND EGGS AND ADULT OF AGRI- | OLIMAX AGRESTIS A, Snakeheads putting out anew growth of small leaves. B, Two bean plants in late summer; the | one on the left came from a snakehead, while the one on the right is a normal plant C, Eggs deposited in the soil by Agriolimax agrestis, X 5. D, The field gray slug on a cabbage leaf, slightly reduced INSECTS AND OTHER ANIMAL Pests INJURIOUS To FreELD Beans) 955 of injury to the bean caused by the seed-corn maggot, and in some fields the writer has found 75 per cent of the plants to be thus deformed. If the maggot feeds on the leaf tissue of the plumule but does not destroy the growing tip, a thrifty plant may still result. The first two leaves may be misshapen and ragged, but new leaves are soon produced to take their places. Injury to the cotyledons Often a larva does not injure the cotyledons until it has fed on the plumule. Its entrance into a cotyledon is thru a hole made in the side, and the maggot usually hollows out the fleshy interior until little more than a shell remains. The maggots are often carried above the ground concealed in the cotyledons, and a single plant may have eight or even more hidden in these two seed-leaves. Damage to the cotyledons alone is not a serious handicap, as these are of little use to the plant after the true leaves have been formed. Injury to the radicle When a seed germinates so quickly that the cotyledons are pushed above the ground before any maggots locate the plant, the radicle may be at- tacked. The larva makes a small hole for its entrance and then mines upward thru the fleshy tissue of the stem. This injury is not serious, as the course of the maggot is thru the pith and it seldom disturbs the vascular tissue. In 1917 the writer observed a field near Batavia in which the beans were planted very deep. Soon after planting, a period of dry weather baked the top soil solid. The beans grew until they reached this upper impenetrable surface layer, and then they were bent over. Many maggots were found in the stem of each plant. LOSS CAUSED The year 1917 was a serious one for New York bean growers, because of the continued rains and the prevalence of maggots during the planting time, in June. In five townships of Genesee County the loss of seed at- tributed to Hylemyia cilicrura was estimated at $15,000. In Erie County the loss on 10,478 acres was said to be 40 per cent. In Monroe County from 50 to 75 per cent of the beans on 16,000 acres were destroyed, while in Orleans County one-fourth of $96,000: worth of seed was wasted. Many growers had to plant their beans two or three times, and one grower, who reseeded twice before getting a stand, estimated his loss for seed at $300. Similar injuries to bean crops were reported from New Jersey, Pennsyl- vania, Michigan, and Canada. At intervals in the past this insect has appeared thus suddenly and unexpectedly, has seriously damaged beans, corn, and other crops for a few subsequent years, and has then gradually disappeared for a time. 956 : I. M. Hawiey DESCRIPTION OF STAGES The egg The chorion, or outer covering, of the egg (figs. 88 and 89, C) is white, | glistening, and marked with longitudinal furrows. Similar cross-furrows | connect the longitudinal ones, cutting off irregular | areas about twice as long as their width. One end | = of the egg is rounded and the other is rather bluntly | flattened. Two prominent ridges, starting at either Fic. 88. = 2 ; oe neo Sah end of the flattened part, meet at a point about | one-fourth the length of the egg. When the larva | emerges, the chorion splits near these ridges. The length of the egg is about 1 millimeter (1/25 inch). = = 5 Zi eae opie The larva The full-grown larva (Plate LX IX, 2,and fig. 89, D) is white, and is largest | at the caudal end, tapering anteriorly. In the early stages it is slender and © almost conical, but as it nears the time for pupation it becomes shorter | and almost elliptical in form. The first segment bears a pair of black, | hooked jaws which may be extended and retracted. The anterior spirac- ular process is heavily chitinized and bears six or seven lobes. The | posterior spiracles are small and consist of three slitlike openngs with | toothed edges. These spiracles, which are the external openings of tracheae running lengthwise thru the body, may be found on the flattened | caudal end of the larva. This flattened, almost truncate, segment bears | seven pairs of fleshy tubercles. The length of the larva is from 6 to 7 | millimeters (2 inch). | The puparium.—The puparium (figs. 89, A, and 90) is brown in color | and elongate-oval in outline. The puparium is the cast skin of the last molt of the larva, and so shows many larval characters. The anterior spiracles are present on the anterior part of the puparium and still show six or seven lobes. The fleshy tubercles on the caudal end of the body also remain but are less prominent. The length of the puparium is about | 4 to 5 millimeters (§ to 4 inch). oO The adult . The male (Plate LXIX, 1, and fig. 89, B)—The body color of the} adult male is greenish gray, with the legs darker and the antennae black. | The entire body bears many black bristles. Faint dark lines run length- | wise on the dorsum of some specimens, and a prominent black line runs |) along the middle of the dorsal side of the abdomen. The main distinguish- |) ing character of the species is a row of regularly arranged bristles on the | tibia of the hind leg (Plate LXIX, 3). This separates the species czlicrura | INSECTS AND OTHER ANIMAL Pests INJURIOUS TO FIELD BEANS 957 E Fic. 89. THE SEED-CORN MAGGOT, HYLEMYIA CILICRURA A, Puparium, X 8. B, Parent fly, male, X 10. C, Eggs, on dirt, X 6. D, Larva, X 5. E, Parent fly, female, X 10 from brassicae and antiqua (ceparum), with which it is often found asso- ciated in the field. In brassicae there is a tuft of fine setae at the base of the femur, which is lacking in cilzcrura. In trichodactyla the middle metatarsus bears long hairs on the upper side, which are lacking in cilicrura. The length of the adult male of cilzcrura is about 5 millimeters (4 inch). aid The female (fig. 89, E)— The female 4,4. 90. -pupartum oF SEED-CORN MAG- of cilicrura is similar to the male, but - gor, X 53 958 I. M. Hawiery the abdomen is pointed instead of rounded and the bristles on the body are fewer and shorter. The femaie lacks the prominent fringe of hairs found | on the tibia of the male, and is harder to distinguish from related species. In brassicae the pre-alar bristle is as strong and as long as the first dorso-central one, while in cilicrura and antiqua it is only about half as long. In antiqua there are two nearly equal setae on the anterior (outer) side of the middle tibia, while in c7licrura there is usually but one. The species antiqua is ordinarily larger than cilicrura. The length of the female of celicrura is 5 millimeters (4 inch). LIFE HISTORY AND HABITS The egg There is little in the literature regarding the egg-laying of Hylemyia cilicrura. Whelan (1916) reports that the fly usually places its eggs either on the stems of plants just as they come thru the soil, or on decaying | vegetable matter. Howitt (1911) states that the eggs are deposited on decaying matter in the soil. Lugger (1896) was able to bring about the deposition of eggs by flies in captivity, but he believed the eggs to be sterile for he failed in his attempts to rear flies from them. Chittenden (1909) mentions an instance in which decomposing crimson clover that had been plowed under attracted flies as a place for oviposition. Period of incubation In July, 1917, at Perry, New York, the length of the egg stage under very moist conditions was between 24 and 48 hours. Very few eggs were found and the exact time of oviposition was in doubt. In 1918 eleven first-brood eggs under observation hatched in an average of 66 hours, as shown in table 1. There was a wide variation, from 41 to 91 hours, and many eggs, the exact hatching time of which could not be noted, hatched within these limits. Eggs were kept in petri dishes on moist blotting-paper or damp earth. TABLE 1. Leneru or tHe Hae Srace in 1918 Number Time of ; : Length of stage of eggs deposition Time of hatching (hours) Dis eres May 23; 3\p.m....- May 27, 9.30, 10, 10, a.m...... 96.5, 91, 91 A ere leave 20 apse May 27, 9, 10, 10, 11.30, a. m...| 41, 42, 42, 43.5 4.........| May 25, 3 p.m.....) May 28, 9.30, 10, 10.30, 10.30, BONN ihe t as cae See (OYA, Al, Too Ales: Average, 66 hours. ; INSECTS AND OTHER ANIMAL Pessts INJURIOUS TO FIELD BEANS 959 In 1919 notes were taken on the period of incubation of 28 eggs on moist earth. The average for these was 2.8 days, with a range from one to five days. Four eggs kept on dry blotting-paper in a petri dish required between three and four days. One egg, deposited on June 4, did not hatch until June 17, but this is the only instance of so long a period of incubation. Place of oviposition The insect is strongly attracted to moist and decaying material as a place for oviposition, but under some conditions the flies will place their eggs on dry material, as the following experiment shows. On June 4, 1919, many flies, taken by sweeping, were placed in a cage with a flower-pot containing dry bean stems and dry soil. This pot was kept dry until the flies died, after which it was moistened in order to see whether a new lot of flies would develop. On July 3 one fly was found in the cage, and on July 8 two more were found. In another cage the same conditions pre- vailed except that the jar was moist during the entire experiment. Four flies were found in this cage on July 3, and eight more on July 8. In the field, eggs have been found around decaying bean pods and vines and around rotting cabbage. The writer has spent a great deal of time in looking for eggs on freshly plowed ground where mature flies were seen in large numbers, but has succeeded in finding only a few in such locations. Two eggs were found on newly turned soil and one was discovered in a ‘erevice in a recently disked field. Egg-laying was induced by throwing water on the parched and cracking ground near the laboratory at a time when the flies were numerous. Eight eggs were found on top of the ground in one of these spots within two hours after it was thus moistened, and, in all, about one hundred eggs were obtained in this way. Whelan (1916) says that the maggots of H. cilicrura are sometimes found in fresh manure. The writer, however, has not seen the larvae in manure, nor has he been able to bring about oviposition on manure. On June 5, 1919, flower-pots containing fresh cow, horse, hog, and hen manure were placed in a cage containing many adults of cilicrura that had been taken in the field. When these pots were examined later, no eggs could be found, and no flies ever emerged in the cage. On June 3, 1919, flies were placed in a large cage containing one flower-pot of manure of different kinds and another filled with decaying bean vines and grass sod. After the flies were all dead, the pot of manure was moved to another cage. No flies emerged from this pot. In the cage containing the pot with the decayed beans and grass sod, thirteen flies emerged. This apparently shows the insects’ preference for decaying beans and clover rather than for animal manure, as a place for oviposition. Many times fresh manure found in fields where the flies were abundant has been placed in cages, but no flies have ever emerged. Furthermore, the flies have not been found in abnormal num- bers around either manure piles or fresh manure dropped along the road. 960 I. M. HawiEry Is it possible that manure has an attraction for the maggots developing from eggs deposited in the soil, which leads them to use It as a secondary host ? In cages, eggs of the first brood have been found singly as a rule, tho they have been found occasionally in groups of from two to five, in some cases side by side and in other cases piled on one another irregularly. When first-brood flies taken in the field were placed in cages, eggs were deposited on decaying mustard stems, on stems and roots of grass, on old bean pods and vines, and on cabbage stumps. Some eggs were found a so on the sur- face of the ground, and some in the top inch of dirt. few were attached to the side of the flower-pot, both above and below the soil line. Eggs of the second brood were deposited by flies in captivity on top of the soil and in the dirt itself. Number of eggs The writer has not been able to bring about oviposition by flies reared in captivity, and accordingly he could not determine the exact number of eggs deposited by a single female. The following may be mentioned as typical examples of many dissected specimens. On May 27, 1918, 87 eggs, of which 25 were almost fully developed, were found in a dissected fly. On May 15, 1919, two flies were found with nearly mature eggs, one containing 30 and the other 48. In 1920 two:flies were found containing 83 and 64 eggs, respectively, on May 7; and on May 19, five flies contained, respectively, 56, 25, 37, 85, and 72 eggs. It is believed that all the eggs in an insect mature at about the same time and that oviposition is completed within a few days. From these data it seems possible that a female may deposit 80 or more eggs, but the number may often be much smaller. / Time of oviposition Since it is from the eggs of the first brood of flies that the maggots so_ injurious to growing crops are produced, special study has been given to | this generation. Very little is known of conditions in 1917, as life-history | studies did not begin until June 22. In 1918 adults were found containing | well-developed eggs on May 9, and, tho some eggs were no doubt deposited | by May 15, most of the eggs of the first brood were deposited between | May 23 and June 1. Eggs of the second brood were deposited during the | first half of July. In 1919 eggs of the first generation were mostly deposited from May 20 | to June 15, with the maximum deposition occurring about June 4. Second- | brood flies were ready to oviposit between June 25 and July 4. Since a | few flies were captured on September 13 and 15 whose abdomens contained | immature eggs, 1t seems possible that a few eggs may have been deposited | as late as October. However, these flies may hibernate, which would | explain why a few flies with well-developed eggs are found in early May, | several weeks ahead of most of the first brood. INSECTS AND OTHER ANIMAL Pests InsuRIOUS To FIELD BEANS 961 The season of 1920 varied little from the two preceding years. The first flies were found on May 7, some containing eggs that were partly developed. Most of the eggs of the first brood were deposited during the first week of June. Eggs of second-brood flies were deposited early in July. The relationship between the time of oviposition and the dates of bean planting is discussed later (page 972). The larva Length of larval stage The average length of the larval stage for eight specimens of Hylemyia cilicrura, which had been bred in vials in a field cage at the Perry laboratory in 1917, was 10.4 days, with a variation from 8 to 12 days. The data are given in table 2: TABLE 2. Breepinc Data For Eraut Fires Rearep In 1917 Length Date of Length Date of Time : of larval | beginning | of pupal | emergence | from egg Date of hatching stage of pupal stage of to adult (days) stage (days) adult (days) Illy 176053 5 12 July 29 20 | August 18 32 JURA 364 6 Soe Bee eee oe ee ila July 28 14 August 11 25 Jal? USS 3.c.5s 6 Seale tol roe eee eee 12 July 30 11 August 10 23 Telly 2sis ob tonn sos See 8 August 2 10 August 12 | IZ Talk, 2, oo ce oc dee oe eee 10 August 4 12 August 16 22 TROUT AD ions cals Siva d Oo ae eee 9 August 4 12 August 16 21 Uwlhy AO va bic eocen eRe 9 August 4 16 August 20 25 JRaP AGS sio's 5 ue ota eae ee | 12 August 6 15 August 21 27 A STCTEIGD) doda.d gn ese ere ee TCO Y3e: Sra aaa ae Ree 1 SReie |e 24 In June, 1919, the average larval period in the case of twenty-one first-brood maggots bred in the laboratory at Perry, was 9.4 days, with a range from 7 to 12 days. Specimens bred in the field cages required about two days more. Habits of the larva The larvae of Hylemyia cilicrura are instinctively internal feeders. When they hatch, the small larvae crawl thru the crevices of the soil in search of food. In this search they show a preference for material that is beginning to rot, and they are always most numerous in decaying beans. Seventeen maggots were once found in a slightly decomposed bean, and seven to ten is not an uncommon range. They may be found in sound 962 . I. M. Hawuery beans also, tho a sound seed rarely contains more than two or three. | Beans that are entirely decomposed have no attraction for the insect. | In order to test the ability of a maggot to find its bean host, ten newly | hatched larvae were placed on top of the soil in large vials, in July, 1917, and an unsoaked bean seed was placed at the bottom of each vial. Eight of the ten maggots found the bean and were reared to adults. The pupa Time spent in puparium In the summer of 1917, the length of the pupal stage of eight specimens was found to be 13.7 days, as shown in table 2. The average length of the pupal stage of seventeen additional specimens, carried thru in the outdoor cage in July, was 12.8 days, the time varying from 10 to 14 days. In June, 1919, the average time required for nineteen pupae was 10.2 days, witha range from 8 to 14 days. The time required in a field cage, from the hatching of the egg until the emergence of the adult fly, was found to be 22.7 days for forty-five specimens, with a range from 16 to 27 days. Allowing 10 days for the larval stage, this would make the overage pupal period 12.7 days. Cages have been examined for puparia tending to show a lengthened pupal period, but cases of retardation, such as those noted by Schoene for the closely related cabbage maggot (Hylemyia [Phorbia\ brassicae), have not been observed. In 1918, however, the rain- fall at Perry during July and August was far below normal (table 6, page 971), and in the fall of that year only one fly of a possible third brood was seen. In both 1917 and 1919, third-brood flies were numerous. The absence of a third brood in 1918 might have been due to a lengthening of the pupal period of the second brood caused by the high temperature and low rainfall. Place of pupation Puparia of Hylemyza cilicrura are usually found near the surface of the ground, a short distance from the place of larval feeding. They are occasionally found as deep as six inches below the surface, but ordinarily not more than three inches below. In bean fields they are often seen within a few inches from the plant food of the larva. The adult Emergence The emergence of what are believed to have been second-brood flies reached its height at Perry, New York, on July 18 and 14, 1917. The flies continued to appear in the rearing cages until about August 1. Flies reared from eggs deposited, no doubt, by this brood and hatching about July 25 and 26, produced, late in August and September, what was pos- sibly a third brood. It is believed that an entire brood of flies was INSECTS AND OTHER ANIMAL Pests INJURIOUS To FIELD BEANS 963 missed in that year, owing to the delay in opening the laboratory. In that year the spring was very late and the rainfall was heavy thruout June and July. In 1918 a few females with well-developed eggs were found on May 9. It is not definitely known whether these flies hibernated as adults or were very early specimens of a main brood which came later. Judging from the number of flies taken in the field, the maximum emergence of first-brood flies occurred between May 23 and 26. Second-brood flies appeared in the cages from June 26 to July 5. After July 23 only one fly was found. This was a male, taken on August 26. In that year there were apparently only two broods. The adults came out early in the spring, but, as a result of the hot, dry weather at Perry in late July. ether the flies died or the development of the stages of the following brood was retarded. In 1919 a few flies were found after May 15, but it was not until June 2 that they were at all abundant. They could stil be found easily on June 9, and occasionally until June 17. Most of the females deposited their eggs during the first week of June. Second-brood flies appeared ‘n the cages from June 18 to July 3, with the maximum emergence about June 25. The hot weather of mid-July must have proved fatal to most of the flies, for they disappeared entirely and no more were seen until September 9. Then, for a few days, flies of the third brood were taken. In 1919 there were apparently three broods, or at any rate two and a partial third. The data leading to this conclusion are given in table 3: TABLE 3. Summary or Recorps TENDING TO SHOW THE NuMBER OF Broops In 1919 Date Observations May 15 | 307, 69, taken by sweeping; 22 containing well-developed eggs 20 | A few flies taken in an old wheat field 27 | Flies still scarce 29 | 27, 39, taken; eggs small; few flies seen; weather cold June 2 | Flies plentiful; eggs in females ranging from partly developed to mature 4 | Eggs found on decaying material in cages; females in field containing a few mature eggs or no eggs 9 | Fewer flies; abdomens empty Small maggots found in beans planted June 2 11 | 39 taken containing no eggs; abdomens collapsed 19 | First of second-brood flies emerged in cages 20 | 13 second-brood 2 from field examined; eggs undeveloped 25 | Many second-brood flies coming out July 3 | A few second-brood flies still coming out 15 | Weather hot; few flies 26 | No flies seen; flies in cages dead, probably owing to hot weather Sept. 13 | 1c’, 39, taken on ground plowed for wheat; eggs immature 15 | 1o taken 964 I. M. Hawiey In 1920 the first flies were found on May 7, and during periods of warm weather they continued to emerge in numbers until: the first of June. Most of the eggs of this brood were deposited between May 25 and June 5. Weather conditions in July of that year were more favorable than in 1919, and flies lived in the cages until the first of August. Dates of emergence vary greatly at different altitudes. Flies have been found in abundance, a week or more before the Perry emergence, at places ten miles from Perry where the elevation is much less.. The bean labora- tory was located on what is said to be one of the highest points between Lake Erie and the Genesee River, its elevation being 1400 feet. Data obtained at Perry, therefore, while holding true for much of the western New York bean-growing section, are probably later than for most parts of the State. The opening of spring at Perry is at least a week later than it is at Ithaca. The time of emergence of Hylemyia cilicrura is dependent largely on temperature. If there are several warm days early in May, some flies will appear. If such a warm period is followed by colder weather, addi- tional flies may not be found for a week or two, or until the temperature has again moderated. Length of life Adults of Hylemyza cilicrura have lived in cages for from 2 to 44 days. In 1920 nine flies under observation lived for an average of 26 days. Without food, life ‘s short, but in cool weather the flies will live for many. days in moist cages if they are supplied with sweetened water. The time in this period when eggs are deposited is not well known; but from the fact that adults with immature ovaries are normally found for many days before mature specimens appear, it is probably toward the end of the adult life. Many flies were taken by sweeping in the field on May 20, 1920, at which time most of the females dissected contained immature eggs. Flies taken at this time were placed in cages, and deposited eggs. between June 1 and June 6. Apparently, then, the length of the pre- oviposition period is about two weeks. Habits . The author’s inability to obtain eggs from flies reared in captivity nt already been mentioned. Molasses, sugar water, and decaying material were placed in cages, but all failed to supply suitable conditions. A large cage, six feet square, in which beans, cabbage, and mustard were growing) and which contained decaying material also, failed to give any better results. Flies were attracted in large numbers by sugar water and molasses. In the field many flies were found in bait pans contain’‘ng a mixture of molasses, water, and sodium arsenite. In the spring the flies are attracted to’ moist, newly plowed ground, They crawl deep into the crevices of the soil, stopping occasionally to lap INSECTS AND OTHER ANIMAL Pests INJuURIOUS TO FIELD Beans 965 up a drop of moisture as they find it. When the flies are moving about in this manner, the wings are overlapped on the back and are thus out of the way. Twelve flies have been counted in three feet of furrow, and forty-two were seen in a square yard. A count of the flies taken on new soil on May 27, 1918, showed fourteen mature females and one male. At this period or a little later, many flies may be taken by sweeping along the edges of fields and roadways. On June 18, 1918, many were caught in the tall grass at the edge of a field, a count showing about four males to every female. On July 8 there were forty-one males in a lot of forty-five flies taken by a roadside. It is apparent, then, that while the females are searching for places suitable for oviposition, the males may be found sunning themselves in grassy and weedy places. On sunny days, adults have been seen resting on mustard; and in the evening they are found on onion tops, kale, potato vines, daisy, and ragweed, and more rarely on other plants. Flies have also been observed hovering about a dead earthworm lying on the surface of the ground. On warm days, or during the hotter part of the day, the flies are very active, crawling restlessly near the top of breeding cages; but in cold weather they move slowly over the dirt on the floor of the cage, or remain quiet in the cracks of the soil. Tt was observed in the spring of 1919 that flies might be taken in the grass | along roadsides, and in wheat and oat fields, before they made their appearance around plowed ground. It would seem, then, that as the egg-laying period approaches, flies have a tendency to come into the open and seek loose, moist soil. This is especially true if such soil contains, as an additional attraction, the decomposing roots of clover or quack grass. HIBERNATION The writer has little data on the hibernation of Hylemyia cilicrura. Cages have been placed in fields and meadows early in the spring, but no flies have emerged in them. LEarly in May a few flies with ovaries partly mature have been found. It is probable that these were early specimens of the subsequent first brood, which had wintered in puparia; but they may have been flies that had emerged late in the previous summer and had hibernated as adults. It has not been possible to keep flies that are taken in late summer, alive in cages thru the winter. The writer believes that the insects more commonly pass the winter hibernating as second-gen- eration pupae, and emerge as flies from May 15 to June 1 of the next year. Such flies, taken in late May, had only partially developed ovaries and were fresh-looking specimens. In support of this pupal-hibernation theory, N. F. Howard? found czlicrwra hibernating in the pupal stage in onions in Wis- consin, and Dickerson (1910) showed that from pupae of czlicrura placed in cages in November, flies would emerge early in May of the next year. 4 As stated in a general discussion reported in the Journal of Economic Entomology. vol. 9, p. 133, 1916. 966 I. M. Hawtiry SEASONAL HISTORY Adult flies of the first brood are found in the fields from early in May | until the middle of June, and deposit their eggs on decaying material or | on moist soil about bean-planting time, during the last of May or early | in June. The maggots work in beans, corn, potatoes, or rotting vegeta- tion, and emerge as second-brood flies in July. These flies soon disappear in normal hot, dry summers, but apparently they deposit eggs about | decaying vegetable matter before they die. A few third-brood flies may — appear in August and September, some of which may hibernate; but most - of the flies taken in May of the next year are believed to come from the | midsummer brood of pupae which overwinter. CONTROL Control measures for Hylemyia cilicrura may be classed either as arti- : ficial, such as seed treatment and the use of baits, or as cultural. The | cultural methods involve studies of influential factors in the environment - of the insect, and of practices used in growing the crop which may affect | the extent of infestation by the maggots. The artificial methods are recorded first. | Artificial methods of control The studies in artificial control that have been directed against Hylemyza | cilicrura in the past have been concerned mainly with seed treatments and | with the application to the soil of such materials as would either kill the | maggots or act as repellents for the adult flies and thus prevent the depo- sition of eggs. Lintner (1882a) suggests soaking the seed in gas tar or > copperas to keep the maggots away, and Lugger (1896) says these. materials work well on a small scale. Headlee (1913) tried solutions of : corrosive sublimate, sulfocide, and potassium cyanide, in an effort to kill” the maggots and prevent oviposition by the flies, but his results were unsatisfactory. Later (1918) he tells of trying strips of tar, and also of. the application of sand treated with carbolic acid to the surface of the soil just after beans were planted. As a result of this treatment, a few more plants came up in the treated plots than in the checks. Still later” (1920), Headlee found that a repellent effect on the maggots resulted from treating lima beans with coal tar and dusting them with ashes, lime, or tobacco dust. Chittenden (1909) suggests that carbolic acid might act as- a repellent to the adult flies, and both he and Bruner (1910) advise the application of kainit, nitrate of soda, or sulfate or chloride of potash to the soil as a top dressing. In addition to discouraging oviposition, this practice is said to have the added advantage of stimulating plant gr owth. | While seed treatment and the application of insecticides to the soil may: be of value when used in a small way, these practices are of doubtful: importance as control measures on a field scale. An infestation of the’ - INSECTS AND OTHER ANIMAL Pests INJURIOUS TO FIELD BEANS 967 G &6 9T OO i Se a = RENT). fe F £6 9T OO Tes | earnest cee : = PEED) Go =|] me uaoe ez 91 QOL ee ek |p pees ae yooyg [occ Tne | ee ez 91 OL ioe Sag oaae as yooyy [occ jae |) omnes 61 ZI Noe Roeser ee ee as ‘yooys [vos s 00° S| Beers 61 ZL. Clie (eae ae ‘yooyg [occ te Spa |pennone 61 ZI OTe eae Gee ‘yooyg [occ oa faucson 61 ZI COIs aes ee ‘yoor.) 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Weg) |] e288 88k 259109] [9 ET Bend hit Loin o Peo MPO ee vane e Uiokd catenin D 0z a 91 0¢ . |occcccur pam |-°- exo qeTTaET i ahaa TG eae! Se es a Pn) Sy Ai en Aine cr Ame) 0003) see es Pella NSE NIEN ST sprey ake pourme peels id : zi -103 wee ajyueyd spoes uorzeot dds ; JUOUT ESPERO) | “OxBUS JO! spaas jo 2) Perea rand jo TEU Mee SR -MOdXx oy TOQWNN |roquny | °%C -UINN LODOVIAL NUOO-daaO AHL YOK LNUWLYAUT, TOG GNY aaMTG NO ‘Y{G@] NI ‘“NNOX MAN ‘ANNA LV GALOAGNOD SENAWINGEXY “b WTAWT 968 I. M. Hawiry seed-corn maggot cannot easily be predicted; its first indication to the grower is usually the discovery of the maggots in beans that have failed to germinate. Since this is true, an efficient repellent or seed treatment would have to be used every wet year, which would make the cost for material and labor very high. Furthermore, it is difficult to find a material which does not injure the seed and yet has a killing or repellent effect on the insect. Maggots usually enter the bean between the cotyledons, and therefore, after the seed coat, which bears the treatment, is broken or cast off by the swelling of the seed, the tender plumule is again left unprotected. The writer has tried various materials as control measures, on a small scale, in the hope of finding something suitable for larger tests. The results of these experiments are recorded in table 4. In the experiments reported in table 4, the materials were placed either on the seed coat, on top of the soil, or in the row with the planted seed. These experiments were located in fields where bad infestations of maggots had just been found and where the flies were very numerous. Infestations were not large, however, as the plantings came between broods. Dry bordeaux mixture seemed the most promising of the materials tested, but on the whole the results of experiments in 1917 were not encouraging. In 1920, seed- and soil-treatment experiments were again conducted. A part of the experimental field which had not been under cultivation for several years, and which was covered with quack grass, was plowed, and the experiments were started here on June 4. At that time many females of H. cilicrura were mature and had been depositing eggs for several days. Beans were planted, following a rain of 0.25 inch, on June 3. It was noted that the flies had been more numerous on this part of the field than on the other parts which had been previously under cultivation, showing the attraction of the species to turned-under quack grass as a place for oviposition. Snakeheads, the evidence of maggot attack, were much more abundant here than in the main part of the field when counts were made on June 22. The results of the experiments of 1920 are given in table 5. None of the materials tested in 1920 gave promise of success in practical use. While in a few cases the number of injured plants was reduced, in no case did the seedlings entirely escape harm. Taking into consideration the difficulty of predicting infestations of the seed-corn maggot, it seems to be unwise to rely on control measures of this type in New York. The bait of sodium arsenite, water, and molasses, which was tried against the onion maggot by Sanders,’ was tested against H. cilicrura. On June 21, 1917, the material was sprinkled on the soil with a whisk broom, and pie tins containing it were placed in a row across the field. Flies were very numerous in this field and the second planting of beans had just been made, the first seeding having been destroyed. On the morning of June 22, twenty-nine flies were found in the pans, together with many beneficial 5 As stated in a general discussion reported in the Journal of Economic Entomology, vol. 8, p. 89, 1915, 969 To FirLtp BEANS INSECTS AND OTHER ANIMAL Pests INJURIOU poypnsear Arnluy z OL 0 z 61 OOT ‘* MOL UT sess OUT] puB dpPOSOO.IL) nN Sane ee Veen L 8 i 9 09 OO “MOL UT a OUT] PUB OTTOqIBD) L 6 a! b G G9 OOT ae ey ee: page Lae Stee oe IO ected L 6 I 9 g9 OOT " poos ud | °°" * + (@B=T) Ny[Ns-owrT 8 uae P 9 j j 99 OOT “poss ud |" (OG-P-P) 998Rd xnvOp10 g U 8 ra g g 99 OOT "+ pases uO |'***"(OG-Z) PBI JO o7BUOSIV @) poynsor Auntuy G G z ) IP OOT OSU OAs eee i PUGe pOLTe yy d sis Pico ae ans imeem ¢ OL G : OS OOT "* MOL uy (OG -T) UWOTS| NUL OTTOGABe) (@) podvpop YIMOTL) 2 Z ( ? 6e OOT ¥ *[t0s UC) : aa “OuUll] pus IUISOLI N Baie a ees, a oe Meee bn 9G a od ial SQ OOT een sX\ C) iL uy ade ape pe otal “gsnp OD0 30 T, IN aes oH G Or Ge 0¢ Chern ay She nak pay aches GG ( XS OF Og “* MOL uy : a eer “XNBIPLOG ALT oT as ee G 4 89 OOL “*" MOT uy tie 6 ae a ak “uunsdAry M eg LG I OL OF OOT ** MOL UT eee! ee DOOTG spol, c slam oo navoa 06 0 8 68 OS eens ay Saas aie Hee Ors soy MOJS IFAD) oy) or 0 Z OOL “MOL UT meee ss strseqod JO 07BIFTN I a ee eae Ol PL € je OOT “MOL UT OUOSOUNTT PUNOLY) H poypnser Arnfuy G FI 0 6 OOL “MOL UT OPBITNS VUNTWO UU %) cron ee Seow ees LI GG g ral OOT “MOL UY] eee S$YVOT ST ul ere SI Ig z OL OOT MOL UY] “ oyeydsoyd ploy a a 08 I Z 0g teen pe Nii Gara a IO poypnset Surling, ge 09 | f OOL { "MOL UT srs BpOS JO 07°R.19TN ad poqpnsot Sulu gy, G ine I ~ OOL “MOL UT ‘pruaeuBdd TUNDRA) 9)! "Seca teh eg 0G se 18) FL OOT “* MOL UT oS Bess ysvyod JO O7YBI[NY tL IZ 9 G 91 OOL “MOL UT Sie etiam, eee eO IS Vv ieee rT 08 2 G 0¢ Seiten erates Sr Rea areis a sy eee poy poyurd = |pozeuturiod asoyy OF oso} OF UOLZBlAI UT | UOL}B]aL Uy pointut spBoy poywurunioes| poyueyd uoly Bo quo SYAVUOY, —— ———| Aq ysI|s -oyvUsS jo Spoes jo Spoos jo -17dde jo [BL00} BIT =o dX pountut TO QUT AT LOQUIN AY 1OQUIN NY 19 {UD AY LOUUBT © : be suRoq Jo yuo. Jog LODOVIN NUOO-GIAG THL NOM SUMASVAT TOUINOD NO QZGT NI SUNAWINTaXTy > 970 I: M. Haw tery carabid beetles. No dead flies were seen in the field outside of the pans, and later, when the beans came up, there was no reduction in the number of snakeheads near the pans. Pans were placed in other fields, and, tho many flies were captured, no great benefit was noticeable when the beans were examined two weeks later. Before planting was done in this field, which was very wet, the seed was drenched with kerosene. Eight days after planting, the counts showed 49 maggot-infested seeds out of 110 that were examined. When the seed was treated overnight with carbon disulfid, 17 out of 57 beans contained larvae of H. cilicrura. A check had 28 infested seeds in 50. Altho neither material injured the germination of the seed, there was little if any repellent effect produced. Beans were treated also with arsenate of lead in the form of a strong paste. This was allowed to dry and the seed was then placed in the ground with a bean planter. The poison so injured the seed that only about 15 per cent germinated. A check showed an 85-per-cent stand. Neither seed treatment nor other artificial control measures have given promise of success. No satisfactory material has been found, and nothing that looks promising for tests on a large scale has been discovered. Natural and cultural methods of control There are many factors bearing on the presence or the absence of Hylemyta cilicrura in a field. Some of these are discussed in the following pages, and practices which are in the nature of preventives are pointed out. Moisture and temperature as factors in the life of the seed-corn maggot Hylemyia cilicrura has been found to be a serious pest in. New York when the early summer is rainy. This increased injury in moist seasons is apparently due to the tendency of the cultivated hosts to decay in the wet soil, thus becoming attractive to the flies as a place for oviposition. It seems probable, also, that maggots already in the soil feeding on other decaying vegetation, are attracted to the beans when they begin to decay. In June of 1916 and also of 1917, the rainfall far exceeded the normal. In 1917 the June rainfall (6.4 inches) was more than twice the monthly average for the previous twenty years, and the damage from H. cilicrura was severe 1n all the bean-growing sections of the State. July of that year was rainy also, and thruout that month flies could be taken easily, altho normally they are scarce at that season. The years 1918, 1919, and 1920 were nearly normal, and the loss was slight. In table 6 are given data from the United States Weather Bureau at Rochester, New York. In periods of drought such as prevailed at Perry during July and August of 1918, flies are difficult to find. By July 22, 1918, only six flies of many hundreds were still alive in the cages, and on the following day not a fly INSECTS AND OTHER ANIMAL Pests Insgurtous To Fietp Beans 971 could be found in the field. On August 26 one male of H. cilicrwra was taken, and this was the only fly seen in 1918 after July 22. The con- ditions at Perry in that year were abnormal, for this region suffered much more for want of rain than did the surrounding places. The reduction in the number of flies appearing at Perry in 1919 is possibly due to this pro- longed dry period. TABLE 6. TrmMpERATURE AND Morsture Recorps OF THE UNITED STATES WEATHER Bureau at Rocuester, New YORK, DURING THE SUMMERS OF 1917 To 1920, INCLUSIVE ‘(Records of marked variations from the Rochester records found at Perry are given in bold- faced type) Mean temperature Rainfall (Fahrenheit) (inches) Month 1917 1918 1919 1920 Normal 1917 1918 1919 1920 | Normal INI WA Bes Sees AON 228 | 6ily ae 56 .4° 55.8° 56 .7° 3.16 1.75 | 5.20 0.78 2.94 2.01 SUNG Mes senor: 62.8° | 62.6° | 72.8° | 65.7° | 66.1° 6.40 2.40 2.96 1.15 | 3.13 LIVIN yoo eibe oo eral face Latte “OR 22 W222) 160 82 70.4° 4.23 2.70 3.40 2.93 3.09 1.03 1.21 : PAI PUSGA seers = 69 .0° ileroe 67.8° 70.3° 68. 3° 2.51 1.83 3.60 ilepyl 2.96 1.34 5.70 In July of 1919, the rainfall at Perry was again below normal, and the flies in the cages died rapidly after July 1. Between July 1 and July 20, the rainfall was only 0.67 inch, and the maximum temperatures for this period ranged from 65° to 96° F. After July 15 no flies were seen for two months and the specimens in cages all died. August of that year had nearly twice the normal rainfall. The temperature early in the summer appears to have an influence on the abundance of H. cilicrura. In 1917 the mean temperature for May was 49.2°, which is several degrees below the average for the month and is the lowest recorded since 1871. The maggots and flies during that season were the most numerous in the history of the insect. June tem- peratures in 1917 also were lower than the normal, and had been each year since 1913. H. cilicrura is an insect which in the past has been injurious for one year, or for a few successive years, and has then become of negligible importance for an indefinite period. This variation is undoubtedly connected to some extent with the moisture and temperature conditions of the early summer. Moderately low temperatures with an abundant rainfall in the spring appear to be favorable to the insect, while dry, warm weather during the summer is detrimental to its successful development. A succession of cold, wet years brings forth the insects in greatest numbers. 972 I. M. Haw ey _ Relation of time of bean planting to time of oviposition Eges may be deposited by Hylemyza cilicrura near decaying vegetable matter in newly plowed or recently fitted soil before the beans are planted, while they are being planted, or even after planting. In rare instances a combination of these two possibilities may be found. When the eggs are deposited previous to planting, the maggots feed on decaying matter in the vicinity for a while, becoming nearly full-grown before they enter the bean seed; but when the eggs are deposited subsequent to planting, small larvae will be found in the beans within a few days after they are planted. As examples of oviposition in the soil before the beans are planted, the following instances may be mentioned. In the experimental field at Perry, in 1919, one piece was plowed on May 14 and planted on May 28. When it was examined on June 5, many full-grown maggots were found. Allow- ing ten days for the larval period and two days for the egg stage, the eggs were probably deposited about May 24. A field that had been in alfalfa for several years was plowed for beans in May. Planting took place on June 6 and 7, and when the field was examined on June 12 the maggots present were ready to pupate. These maggots were hatched from eggs deposited probably about June 1. Maggots of about the same size were found in the old alfalfa roots. As illustrations of egg-laying either at the time of planting or subsequent to it, the following examples are given. In 1919 a field of beans was planted on June 2. When it was examined on June 9, maggots a few days old were found. [lies were very common in the field on June 2 when the seed was drilled in. Beans were planted in a test plot on the experimental field on June 5. On June 10, when these were examined, some contained newly hatched maggots. Ina field in Niagara County beans were planted on June 9. Small maggots were found feeding on the plumule leaves on June 14. In the rainy year of 1917, when H. cilicrura could be found everywhere, fields were inspected late in June. Small and large maggots were found in the beans, pupae were in the soil, and flies with eggs in all stages of develop- ment were hovering over the ground. It is only late in the spring of very wet years that all stages can thus. be found simultaneously. In more normal years the flies seem to appear in roughly defined broods, and the heavy oviposition of each brood does not extend over a period of more than a week. From the foregoing data it is evident that a grower cannot be sure that maggots are not already working on other matter in the soil at the time when he plants his beans. Moreover, if mature flies are numerous at planting time, his beans may be infested with maggots from eggs deposited at that time. In 1919 an attempt was made to connect the time of fly emergence and the time of oviposition with some of the more obvious occurrences in INSECTS AND OTHER ANIMAL Pests INsuRIOUS TO Fir~p Beans 973 nature. In that year, and also in 1920, the first flies were taken in small numbers early in May, about the time when cherries were in bloom. Mature flies were out in numbers near plowed ground on June 2, 1919, at which time the last of the petals had fallen from late apple trees at Perry. Flies with fully developed eggs had not been found in quantities on plowed ground before this, altho a few females with immature eggs had been collected in winter-wheat and oat fields. Many eggs were deposited in cages about June 4. Dissected females showed that some eggs were mature on June 2 and many on June 4. It is evident, therefore, that under Perry conditions in 1919, beans planted between June 2 and 12, or within ten days after the last of the petals had fallen from the late apple trees, were open to maggot attack. Serious infestations of maggots were very few in 1919, but in two bean fields which did show cilicrura injury the seed was planted on June 5 and June 6, respectively. On May 28, 1920, when the petals had partly fallen from the late apple trees, flies nearly mature were found in numbers on moist, newly turned ground, especially where the field had previously been in sod. After May 7, when the first specimens of the year were taken, adult females were captured almost daily and their abdomens examined for eggs. Between May 7 and May 28, a few flies with ripe eggs were occasionally found, showing that eggs were doubtless deposited in small numbers during that period. A cornfield planted on May 22 and examined on May 29 showed typieal cilicrura injury in a few seeds. However, this examination of females taken thruout May showed that most of the first brood of flies were not mature before May 28. At that time there were many more females than males present on plowed ground. Many eggs were de- posited between May 28 and June 7 by flies in the cages. Most of these eggs were deposited after June 1, when the nights as well as the days were warm and the flies showed unusual activity. Many fields in which beans were just appearing above ground were examined on June 7, 1920, and only six infested plants were found. Good weather had made it possible to plant some of these beans by May 21, and all were in before June 1. Therefore, in these fields, the seed was in the ground before most of the flies were mature, with the result that there were but few maggots in the soil. Beans planted in the experimental field on June 4, 1920, were heavily infested, and potatoes and corn planted in neighboring fields about the same time contained many maggots. This infestation is probably due to the fact that the seed was put into the ground at the time whin the flies were mature and eggs were being deposited. Beans planted in the middle of June were uninjured. From these data it would seem that beans planted just after the last apple blossoms have fallen, which under the conditions at Perry in 1919 and 1920 was from May 28 to June 7, stand a greater chance of being infested by the maggots of H. cilicrura than do those planted 974 I. M. Hawiey before or after these dates. Since the larvae already in the soil may attack the newly planted seed, it is wise to delay planting for a few days after all the eggs have been deposited. This would extend the time for probable infestation in 1919 and 1920 from May 28 to about June 15. Unfortunately, it is not always possible to choose the time of planting as suggested above. Weather conditions may prevent planting in May, before the flies are mature, and if beans are put in too late in June they may not ripen before they are killed by frost. Seasons vary so much from year to year that no absolute rule can be given; but, if weather permits, it is best to plant before the oviposition period of the flies, when the last of the petals have fallen from late apple trees. Relation of kind and condition of soil to maggot infestation Abundant moisture provides favorable conditions for the development of Hylemyza cilicrura, as is shown on page 970. Beans on heavy soil, which holds moisture, grow more slowly, decay, and furnish conditions attractive to the flies for oviposition. In 1917 one side of a bean field near Perry was badly infested, while the remainder, which was planted on the same day, was free from injury. An examination of the soil in the infested part showed it to be heavy and sticky, while the unattacked beans were growing in lighter soil of a sandy constituency, which was relatively dry. The division between the two types of soil was very marked, and the good and the poor beans followed this line closely. Well-drained fields are not attacked as often as are those where the drainage is poor. Low and wet spots, where water may collect in otherwise good fields, often yield poor beans. ‘This is in part the result of maggot work, but it may often be due to the decay caused by the excess of moisture in the soil. Warm, dry soil that is well fitted furnishes ideal conditions for the growth of beans. In soil of this kind they will germinate quickly, and when once above ground there is little chance of serious injury from maggots. In wet seasons it is best to delay planting until the soil can be well fitted. A field should be dragged and rolled, and the top layer of earth allowed to dry out and become warmed by the sun. Influence of preceding crop and time of fitting a field, on maggot attack Heavy infestations of Hylemyia cilicrura have been found on land that had previously been in potatoes, corn, tomatoes, wheat, oats, and beans, as well as in clover and alfalfa sod. Many infestations have followed sod, since the upturned roots of decaying clover and alfalfa furnish good breed- ing condifions for maggots, and since clover forms a part of a regular rotation of beans, wheat, and clover which is practiced in western New York. Just as serious outbreaks have been found, however, where the preceding crop was beans, especially if the field had an. abundance of quack grass and weeds. In the writer’s garden there was a patch of quack grass. This was turned under, and beans and peas planted on INSECTS AND OTHER ANIMAL Pxsts INJURIOUS TO FIELD BEANS 975 this spot were infested with maggots, tho in other parts of the garden there was no injury. Whelan (1916) has found maggots in fresh manure, and he says: Furthermore, it appears that while beans were apt to suffer when planted on freshly turned clover sod, especially if recently fertilized with undecomposed manure, they stood a much better chance of escape if the field was prepared early in the season and the maggots given a chance to develop and disappear before the beans were planted. Tho the writer has been unable to find evidence of flies breeding in manure, he has found many maggot-infested fields which had been covered with manure just before plowing. It must be said, however, that serious out- breaks have been found where manure was not used. If a field is fitted early and is allowed to dry out before the mature flies seek places to oviposit, it appears to be less attractive for oviposition than newly turned soil. In 1920 the laboratory field was plowed at a time when flies with well-developed eggs were numerous, and many bean seed- lings were infested. Fields near by that were plowed earlier and allowed to stand were free from maggots. Relation of depth of planting to injury by maggots Beans planted deep in the ground take longer to reach the surface and are thus exposed for a longer period of time to maggot attack. It has been observed many times that beans planted deep in wet ground suffer more from Hylemyza cilicrura than those that are planted less deep. For example, in 1917 it was often noticed that the headlands yielded better beans than the remainder of a field. This is attributed to the more shal- low planting, for the soil was not so loose at the edges of the field and therefore the drill did not sink so deep. In 1917 a field under observation had nearly every bean attacked by maggots. The seed had been planted in wet soil at a depth of from three to five inches. After this first planting was destroyed by maggots, the field was reseeded at once, and the beans were dropped as near the surface of the ground as possible. Some of the seed was even left on top of the soil, and a boy with a, hoe followed the machine to cover the beans left exposed. A 95-per-cent stand resulted. In another case a grower started to make a very shallow planting of beans. When he had gone part of the way across the field, he decided that he was not getting the seed in deep enough, and so he planted the remainder of the seed much deeper. At harvest time the beans planted first, the shallow-planted ones, were the only ones worth harvesting. If the beans are planted too deep, many will decay because of the excessive moisture, and the maggots will destroy a large proportion of the remainder Experiments were conducted in 1917 to test the effect of the depth of planting on the time required for the beans to break thru the soil. Beans were planted on good, tho very wet, soil on July 12. When the field was 976 I. M. Hawtey examined on July 19, the beans that were planted about one inch deep | were nearly all up, those planted three inches deep were about half thru | the soil, and none of those planted five inches deep were yet above ground. — In another wet field 100 seeds were planted at depths of one, three, and | five inches, on July 16. On July 23 there were, in the one-inch planting, 57 plants; of which 4 were snakeheads; in the three-inch planting there were 35 plants, of which 5 were snakeheads; and none of the beans planted five inches deep had appeared above ground. In the laboratory field, under rather warm. dry conditions in July, 1918, it was found that beans planted three inches deep came up nearly as quickly as those planted more shallow. Summary of preventive measures The results of experiments on artificial control measures, such as coating the seed before planting and treating the soil with materials of a repellent nature, afford small hope for their future successful development. As a result of a study of some of the factors governing infestations, the possible preventive measures that have been discussed in the foregoing pages are summarized in the following paragraphs. The seed-corn maggot is more serious as a pest when the months of May and June are rainy, and the ground is cold and wet at bean-planting time, than under other conditions. The greatest injury results when several unfavorable years occur in succession. Hylemyza cilicrura thrives when oviposition takes place under wet conditions; and therefore it is wise to plant when the soil is dry and the earth is warm. The soil should srst be well fitted with a disk or a harrow, and then rolled, and finally, after a few warm days have dried out the top soil, the beans should be planted. if the field is fertilized in order to hasten the germination of the seed, there is a still better chance of getting a stand. However, fields fertilized with fresh manure just before plowing often show a heavy infestation of mag- gots, and so this condition should be guarded against in wet years. As maggots developing from eggs deposited on newly tilled ground are often found in decaying matter in the soil, it is sometimes wise to fit a field early, before most of the flies are sexually mature. The ground will then be dried and less attractive to flies for oviposition by the time they come out. The presence of many flies of this species crawling over newly turned soil between plowing and planting time is a good indication that seed planted there will probably be infested. In 1918, 1919, and 1920, sexually mature flies were most numerous in the fields at Perry from May 25 to June 10. In 1919 the greatest number were present about June 4, and in 1920 about June 2. If the weather permits, it is better to plant ahead of this brood. If this is impossible, planting should be delayed until the fltes are less common. It is extremely important that beans should not be planted too deep in wet soil, If they are, some of the seed will rot and the maggots will destroy INSECTS AND OTHER ANIMAL PEsts INJURIOUS TO FIELD BEANS 977 most of the remainder. Not only is the soil three or four inches below the surface much colder and more moist than the top inch, but also the deeply planted seed germinates more slowly in wet years. It is wise to force seed to germinate and grow as rapidly as possible, since it will have escaped serious injury when it is once above ground. If in shallow planting some of the beans are left on top of the ground, they may easily be covered with a hoe. A bean planter or a corn planter usually will give better service than a drill in wet years, for either is lighter and will not sink so deep in wet places. If a drill is used, it should be adjusted to make a shallow planting. A grower who plants his seed deep in wet soil at a time when the sexually mature flies are numerous, is sure to have a heavy infestation of maggots on his beans. THE IMPORTED FIELD GRAY SLUG (Agriolimax agrestis L.) ORIGIN The field gray slug, or garden slug (Agriolimaz agrestis, Plate LXIX, 6), is an imported species which, with two other foreign forms (Limaz maximus L. and L. flavus L.), does more damage and attracts more attention than all of the other twenty-nine species of slugs reported from the United States (White, 1918). A. agrestis is an old res dent of Europe, having been listed in England as early as 1674. Taylor (1907) reports it in the fossil rocks of the Pliocene and Pleistocene periods from many places in the British Isles, as well as from Germany and France. It apparently came to this continent from Europe early in the eighteenth century. HISTORY AND DISTRIBUTION Theobald (1905) states that Agriolimax agrestis is found in near'y every garden in England, and also on the continent of Europe and in Siberia, Madeira, and Algeria. Taylor (1907) states that it occurs also in Turkes- tan, China, Japan, Asia Minor, Morocco, Cape Colony, Zanzibar, Aus- tralia, and New Zealand, as well as in Brazil, Jamaica, and parts of Canada, on this continent. In the United States it was first reported near the seaports of Boston, New York, and Philadelphia. De Kay (1848) states that Binney knew it before 1843, tho Binney (1851) still had trouble in separating A. agrestis from the native species A. campestris, which it very much resembles. Since 1851 A. agrestis has spread gradually westward, and it is now found locally in many States. Its presence is reported in the literature of Maine, Massachusetts, New York, Pennsylvania, Michigan, New Jersey, Ulinois, Wisconsin, Ohio, Colorado, Washington, and California, but it is probably present also in many other parts of the country. Slingertand discovered the slug at work on the college farm at Ithaca in 1891. Baker 978 I. M. Haw try (1902) did not find it around Chicago in 1902, tho it had been reported from Michigan in 1899. Cockerel!® found agrestis in Colorado in 1890, and he states that it was brought there from New Jersey. He reports it also in Oregon in 1891 and in California in 1892. In western New York the localities infested by A. agrestis are increasing, and in wet seasons many beans, as well as other field and garden crops, are injured. The insect is apparently not a pest in all sections of the bean- growing counties, but appears to be limited to a few farms and gardens in each district. Some places seem to be entirely free from it. It is often abundant on small truck farms, and around the shrubbery and the gardens n city lots. It has, no doubt, been carried into its present habitats in the straw or moss packing of bulbs, shrubs, or nursery stock. Dissemi- nated in this way, it seems to thrive, and it apparently prefers cultivated crops to woods and pasture land. As it becomes better established the species may be expected to spread from the present centers of infestation until it is of almost general distribution. The long, cold winters often experienced in New York, however, should tend to prevent the serious damage that it causes nearly every year where the climatic conditions are milder and more uniformly moist. SYSTEMATIC POSITION The field gray slug belongs to the phylum Mollusca and the class Gas- tropoda, which includes the slugs and the snails. Agriolimax agrestis is placed in the family Limacidae, the members of which have no external shell. This family, according to Pratt (1916), is represented in America by only six species. The large spotted slugs of the family are now placed in the genus Limax L., while smaller forms, such as agrestis, belong in Agriolimax Morch. Because of its varied coloration, this slug has been described under many specific names. Taylor (1907) gives a complete synonymy for the species, and lists ten varieties, with the localities from which each has been reported. GENERAL DESCRIPTION OF THE SLUGS OF THE FAMILY LIMACIDAE The field gray slug belongs to the same group of Mollusca as the snails, and differs from them but little except in the size and form of the shell. In slugs of the family Limacidae no shell is visible on the outside of the body, but there is a thin calcareous plate (Plate LXIX, 5) concealed in the mantle — the fleshy shield over the front part of the slug. The body is elongate-subcylindrical, and bears a more or less prominent dorsal keel. On the retractile head are two pairs of tentacles; the anterior pair aids in feed- ing, while the upper, or posterior, pair bears the eyes. The eyes have the . form of rounded knobs on filament-like stalks. When the slug is disturbed, 6 As cited by Taylor (1907:120). INSECTS AND OTHER ANIMAL Pests INJURIOUS TO FIELD BEANS 979 the eyes may be withdrawn down the tentacle, and in young, transparent specimens they may be readily seen even after they have been retracted into the head. Slugs have the power of secreting from pores in the body, and especially from the anterior ventral surface of the foot, a slimy sub- stance known as mucus. The shell-concealing mantle has, near its pos- terior lower border on the right side, a circular breathing pore which opens into a respiratory chamber beneath. This is lined with a richly vascular epithelium subserving the function of a respiratory organ. Just behind the right eye-stalk is the opening of the genital organs, thru which eggs are extruded. Slugs of this group are hermaphroditic, both male and female genital organs being present in a single individual. ANCIENT SUPERSTITIONS CONCERNING SLUGS Slugs have been known in Europe for many years, and the older writings in regard to them contain many interesting notes. They have been used as food in Europe, and it is said that as late as 1863 they were prescribed by French physicians, to be taken in the form of a sirup. A slug distillate was considered good for the complexion (Kingsley, 1885). A plaster of slugs with the heads removed, bound on the forehead, was believed to cure a headache, and slugs eaten alive in milk were thought to cure con- sumption (Rogers, 1908). As slugs always appear after a rain, they were believed by English farmers in the eighteenth century to come from heaven in a rain cloud (Theobald, 1895). Since toads gather in infested fields to feed on the slugs, it used to happen that gardeners and farmers of a hundred years ago, finding that their plants had been destroyed during the night, would blame and kill the toads, while the real culprit was concealed in a safe retreat under ground (Ritzema Bos, 1890). Later, the value of toads as enemies of the slugs was appreciated and three francs a dozen was paid for them (Guénaux, 1904). Years ago the small shell of the slugs was regarded as an amulet, which, worn on a string around the neck, was believed to protect the wearer from harm. When the slugs suddenly appeared in large numbers in the gardens of European countries, it was customary to invoke the power of the Church against them, in the hope that they might be thus removed (Kingsley, 1885); and Taylor (1907) states that the Ritual of Paris, A. D. 1712, contains definite exorcisms for this purpose. PECULIAR HABITS OF SLUGS Some slugs are said to have a partiality for moist newspaper, dead fish, earthworms, dead clams, dead slugs, meal, flour, cream, butter, Pears’ soap, sponge cake, and book bindings, as food (Cooke, 1895). They have been known to eat out the corks of wine bottles, to crawl into nearly empty beer bottles and bathe themselves in the contents, and even to attach their small mouths to the dripping faucets of containers of 980 I. M. Hawuey ; alcoholic beverages. They will crawl into beehives and feed on the honey, apparently immune to the stings of their enraged hosts (Reh, 1913). ECONOMIC IMPORTANCE In wet years the field gray slug is one of the two most destructive animal pests of field beans in New York. During the rainy summers of 1916 and 1917, nearly all of the plants in some fields were atta¢ked and many were entirely destroyed. Estimates of the losses on twenty-one farms in Orleans County in 1917 varied between 5 and 70 per cent. In 1918 the writer saw a bean field in Monroe County in which about: one-third of a ten-acre field was so badly attacked that not a trace of a plant was left above ground. ~ In addition to its attacks on field beans, the slug often causes ntieh injury to garden beans, lettuce, cabbage, peas, potatoes, radishes, and strawberries. As the species becomes better established in the farms and gardens in new localities thruout New York, it is probable that more widespread attacks may be expected on crops during wet seasons. NATURE OF THE. INJURY TO BEANS ; Immature specimens of Agriolimax agrestis eat the tender tissue between the veins and the veinlets of the leaves, thus giving them a skeletonized Fia. 91. INJURIES CAUSED BY AGRIOLIMAX AGRESTIS : A, A bean pod showing a hole made by the slug in feeding. _B, Bean plants injured by slugs. (Photo- graphed in midsummer.) , Bean plants that were injured by slugs soon after they appeared above ground, photographed at harvest time. INSECTS AND OTHER ANIMAL Pests INsguRIOUS To FIELD BEANS QS81 appearance. Older slugs, however, eat parts from the edges of the leaves, and frequently continue to feed until every leaf is devoured (fig. 91, B): Fie. 92. RADULA OF AGRIOLIMAX AGRESTIS, AND RESULTS OF WORK OF SLUG A, Radula, or lingual ribbon, of slug, made up of many hundred small, sharp teeth, X 25. B, Young bean plants showing result of feeding by the slug. C, A bean field in Monroe County, New York, in parts of which all the plants were destroyed by slugs; bare areas shown. Sometimes the petiole and a few of the main veins are left, but oftener the entire plant above the surface of the ground is destroyed. During 982 Oe TONG eee the daytime and in dry weather the slugs feed beneath the ground, eating large parts of the stems of plants (Plate LX_X, 2, and fig. 92, B). Such plants may be so severely injured that a wilting of the parts above ground results. When a slug crawls up the stem of a young bean plant, it usually devours the budlike growing tip before it moves on to the leaves. Plants of this kind are always stunted and show an abnormal, useless growth of small leaves (fig. 91, C). Such plants commonly die, and even when they survive they fail to produce mature seeds. If the slugs are numerous at the time of pod formation, it is not unusual for them to eat holes into the sides of the pods (fig. 91, A) and feed on the soft beans within. Often several holes of this kind are found in a single pod, and sometimes a slug after destroying one bean will move along inside the pod and feed in turn on all the remaining seeds. INJURY TO PLANTS OTHER THAN BEANS In its nocturnal feedings above ground, Agriolimax agrestis seeks the tender leaves of lettuce, corn, cabbage, and cauliflower. The injury which the slug does to these plants is similar to that which it produces on large bean leaves, and causes the plants to become unhealthy and unmarketable. When the weather is such that the slugs are active in late summer, they frequently eat large holes in the sides of ripe tomatoes and fall strawberries, in addition to eating the leaves. The pest feeds underground on potatoes, and, together with the millepede Julus caeruleocinctus Wood, causes considerable damage in western New York by eating out large cavities in the tubers. Carrots, turnips, radishes, and beets suffer similar attacks on their fleshy roots. HOSTS AND POSSIBLE FOOD SUPPLIES Agriolimax agrestis has such a wide range of food plants that it is classed as almost omnivorous. Among the plants which it feeds upon are cabbage, potatoes, eggplant, lettuce, beans, lima beans, peas, corn, strawberries, gooseberries, cucumbers, melons, cauliflower, wheat, turnips, beets, carrots, radishes, celery, clover, oats, dahlia, dandelion, dock, chicory, tobacco, hops, and tomatoes. There are also many weed hosts on which this mollusk may be found, such as burdock, ragweed, lamb’s-quarters, and mustard. It finds palatable several species of mushrooms also, and many ornamental shrubs and vines, and it finds abundant food in sod land and in lawns © Overgrown places in fence corners and along the edges of fields often harbor many of the slugs. Manure is acceptable to them as food, and their eggs, as well as young slugs, have been observed in large numbers where manure had been scattered in piles around a field. Cooke (1895) mentions as among the possible foods of agrestis, may-flies, beetles, and dead INSEcTS AND OTHER ANIMAL Pests INsguRIOoUS To FreLD BEANS 983 slugs; Lovett and Black (1920) add sow bugs, earthworms, and aphids to this list; Taylor (1907) records a case in which A. agrestis killed and ate slugs of the species A. campestris when the two were placed in the same box; and Lebour (1914-15) finds that they eagerly devour the proglottides of Moniezia, a tapeworm of sheep. It would seem, therefore, that, while the slugs usually prefer a vegetable diet, under some conditions they relish animal food. DESCRIPTION OF STAGES The egg The egg of Agriolimax agrestis (fig. 87, C, page 954) is elliptical or spher- ical in shape, and is translucent, jelly-like, bluish white, and iridescent. Under magnification the thin outer covering is found to be slightly roughened “with regular raised and depressed areas. The eggs are found either singly or in masses, in the latter case being held together by a trans- parent secretion. On one end the small, projecting micropyle is visible, especially in newly deposited eggs. The eggs vary from 1.6 to 3 milli- meters (1/16 to 1/8 inch) in length. ~ The young slug A newly hatched specimen of Agriolimax agrestis is without definite form at first, but 1t soon assumes much the same appearance as its parent except that its tentacles are relatively larger and its body is transparent, permitting the black nerve cord running to the eye to be easily seen thru the body covering. Young slugs have a pinkish tint at first, but later turn to darker hues as they begin feeding. The full-grown slug The slug (Plate LXTX, 6, and fig. 87, D) is described by Taylor (1907 :105) as follows: Animal limaciform, with large but flattened tubercles; of a somewhat uniform whitish or pale ochreous ground colour, but sometimes dull lavender or other tint, often mottled, speckled or reticulated with brown or black, and at times totally suffused with black; body somewhat compressed and keeled towards the tail; tentacles dark coloured; shield more than one-third the total length of the animal, rounded in front and behind, concentric striae not deep, with the nucleus on the right side and towards the rear; respiratory orifice with a broad usually unpigmented raised ring, which is cut anteriorly by the anal cleft; sole pale and’ longi- tudinally tripartite, the side areas sometimes darker, especially towards the tail; sole-fringe separated as usual from the body by a furrow, containing a row of elongate tubercles, upon which the body tubercles rest unconformably. Mucus plentiful and viscous, often clear when crawling, but becoming milky-white on irritation, due to innumerable particles of car- _bonate of lime. Length usually about 35 mill, 984 I. M Hawtey LIFE HISTORY AND HABITS The egg Under New York conditions, some of the eggs of Agriolimax agrestis are deposited in the fall, from August until December, and many of the mature slugs die soon afterward from the cold. If full-grown slugs live thru the winter, they may deposit eggs during May and June of the follow- ing spring. Slugs developing from these overwintering and spring eggs will mature and in turn deposit eggs in the fall of that year or in the follow- ing spring. In the summer of 1917, which was very wet, eggs were deposited more or less continuously from May to December, being especially numerous in September and October. Theobald (1905) reports this to be the normal condition in England. Lovett and Black (1920) state that in Oregon egg-laying occurs at all seasons of the year, and that the greatest number of eggs are deposited in the spring and early summer. In 1918, which was a drier and more nearly normal season for New York than the preceding years had been, eggs were deposited by the slugs in cages during May and June, and not again until September 23. In that year, as well as in 1919, most of the eggs were deposited late in October and during November. Moisture is a factor of great influence. A few of the eggs deposited in July and August of 1917 hatched that fall, but wether these young slugs survived the cold winter that followed is not