Genetic Study of Sex and Cultural Characters in Neurospora crassa Citation Lindegren, Carl Clarence (1931) Genetic Study of Sex and Cultural Characters in Neurospora crassa. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/HR2E-X838. https://resolver.caltech.edu/CaltechETD:etd-12202005-084117 Abstract No abstract. Item Type: Thesis (Dissertation (Ph.D.)) Subject Keywords: Biology Degree Grantor: California Institute of Technology Division: Biology Major Option: Biology Thesis Availability: Public (worldwide access) Research Advisor(s): Morgan, Thomas Hunt Defense Date: 1 January 1931 Record Number: CaltechETD:etd-12202005-084117 Persistent URL: https://resolver.caltech.edu/CaltechETD:etd-12202005-084117 DOI: 10.7907/HR2E-X838 Default Usage Policy: No commercial reproduction, distribution, display or performance rights in this work are provided. ID Code: 5085 Collection: CaltechTHESIS Deposited By: Imported from ETD-db Deposited On: 20 Dec 2005 Last Modified: 03 Oct 2019 23:40 Thesis Files Preview PDF - Final Version See Usage Policy. 3MB Repository Staff Only: item control page

Thesis by Oarl C. Lindegren Ill'l'RODUCTI 0)1 I. MlCHODlSSEOTION •lfD CULTUR~ liEfHon~ IX. HEAt .. 'l'ftEATMENT ICE'I'HOD3. l'• Experiment• to detellJline an opti.llwa heat-treatment+ !:?.. Bxpuihl.~• on lt11ltng con14U.. 3,. Experime:ita on 1nber1 tan cut ot reaponae to be•t-t.,.at. ment. 4. summar1 ot th• e~ri••nt• on heat• troatmerit of ..coaporea. L IV. IJBElllTAliCR OF CULTUR.t\L CllARACTERB. l. Description ot cultural oha1'8.otera • . a•. 'Inher1tanoe of tan. · "· Pl"Oot' ot nuo:J.oar uteroba.nge 'b•1i•••n tball1 ot oppoa1te Mx. · 4. Cftol.ogioal and genetic e~denoe or the mB.&ll,. ·double fWl10ll•. ln tr-. 5.. ~ rtmautla mi t.blJ Tmlerliioe ot tf.ti an<1 . •1ld-t1J)e, bf the uat\ at aeleoted oloM1 in \'IULt1nga am th• ~i».g •t theae data on the qUBat1on or a tt1t?"11pl.oid h#1Qn 1ri the per1theo1u 1n1Ua1 ot. N•.. oraaaa. 6•. Jteroent•g•• of fir1tdl*v!alon aegreg•tion ot taotora to.,. \fu>io1.1.a oul.t\Jl"a.1 ohanotcu•• and the be•ring ot tll.eae data on i'Ollr•a• Hnd Ol'Oaaing•OTSl' 1n N. Ol'&aaa •. 't. St'lDlllllry ot t:t.. exP,r1iiiiii'Ea on the 1nher1 t•noe ot oultUi'IU char.. oi•~ aa.1 an uple.nation or the genettc behavior ot l'l. orae111. wbioh beat ~ita the known tact•~ - V. SUJOU.RY. INTRODUCTION The importance of genetic study of the ascolllJ'Cetes results from the following considerations: Genetic study of higher plants and animals indicates (1) that a reassortment of genes takes place in meiosis and ( 2) that the change or linkage known as crossing-over results from an interchange between homologous chromosomes in meiosis. Cytological study shows that meiosis provides a mechanism for the reassortment of genes and indicates in an unconvincing way that crossing-over might be the result of a meiotic mechanism. The genetic evidence that meiosis effects reassortment and crossing-over is completely convincing despite the fact that it is purely inferential. It is based on the assumption that two classes of gametes result from meiosis of a single diploid nucleus heterozygous for one gene and are equally numerous. FUrthermore. it is as- sumed• that when crossovers occur. they are the result of a single diploid nucleus having produced two types -1- of exceptional gametes. However, the technical d.1ffi' culties of isolating the gametes resulting from meiosis of a single diploid nucleus in the higher plants and animals have prevented any experiments. In the ascolll'1cetes the eight ascospores usually produced in a sac are the result of meiosis of a single nucleus. Moreover, the sacs in some species are long and narrow and the products of division are not jumbled together but are arranged in a row so that their positions indicate their relationships. Micromanipulation and aseptic culture technique enable one to isols.te these ascospores in the proper order and to study the genetic characters of the myeelia resulting from their germination. If the histol"f of the chromosomes were known, the cl~ar cut genetic characters found in the fungi would soon settle the questions raised. But it is probable that no sex phenomenon has received so many contradictory interpretations at the hands of different writers as that of the ascOlll'1cetes. A single form has been described as being parthenogenetic, or having a single sexual fUsion, or a double sexual fusion. Scarcely less divergent are the interpretations of the divisions in the ascus. There seems to be a general agreement that the chromosomes undergo what resembles -2- a meiotic division but the smallness of the chromosomes and the difficulty of counting has led to a variety of interpretations. The results presented here show that in !• crassa a double sexual fusion best explains the data. An analytical knowledge of the genetics of an organism depends upon coordinating the nuclear history with the genetic behavior. This means that before the mechanism of meiosis can be studied by means of the arrangement of the ascospores in the long ascus of .!!• crassa, its nuclear histol"f and sex phenomena must be known accurately. Once they are understood, it will provide an excellent object for studying the immediate effect of meiosis. The object of this study is to investigate meiosis directly rather than inferentially, and to attain this object understandingly, the nuclear history of the organism must first be described. The present paper contains the results of a genetic and cytological study of Neurospora crassa. store are sufficiently accurate. A needle, inserted in a short glass rod, is stuck to the brass tube with modelling clay so that it just clears the surface of the microscope stage. Movement of the needle in an arc about the steel rod is obtained by a wooden lever pointing away from the operator. The base of the brass tube fits solidly in this wooden lever and the direction of motion is changed by a one-half centimeter wooden rod placed so that it is conveniently situated at the right hand of the operator. This rod is fitted solidly in an upright wooden handle which rests upon the table. The wooden rod must have enough spring so that it will allow the handle to rest on the table while the brass rod is being moved upward and downward. Sensitivity of the movement in the arc about the steel rod depends upon the length of the needle and the length of the wooden lever by the principle of similar triangles. For moderate magnifications one to five sensitivity is sufficient using a ten centimeter needle and a fifty centimeter lever. A large cork is fitted firml~ on the upper end of the brass tube. A two centimeter test tube which has been cut off about two centimeters from the base is fitted solidly in a hole in the base of the cork. -s- A fifty by two by two centimeter wooden bar slides in the grooved base. At its upper end a one centimeter piece of wooden rod about five centimeters long is tightly fitted. The tip of the rod is carefully rounded to rub against the cup formed by the base of the test tube. Up and down movement of the needle is obtained by sliding the fifty centimeter bar back and forth in the groove. To determine the sensitivity of this movement, let the length of the bar form the constant hypotenuse a of a variable right triangle. Let the horizontal side (groove) equal x and the vertical side (brass tube and steel rod) equal y. Then x2 By implicit differentiation with respect to x -x y The sensitivity depends upon the ratio of the variable horizontal side to the variable vertical side of the triangle. Therefore, the upper angle must be acute to minimize the movement. It is possible to work with a horizontal side of about one centimeter and a vertical side of about fifty centimeters, thus minimizing the manual movement about fi~y times. Chromel needles were used. They were made by hammering a piece of wire very thin and cutting to a fine point with a scissors. After the last cut the wire was banmered to produce a hoe-like end rather than a pointed one. The dissections were performed on clear three percent agar. Perithecia were crushed between flamed microscope slides and a drop of water was added from a sterile lip pipette. The asci emerge from the perithecia in groups and the clusters can be easily picked up with the pipette and placed on the agar. These asci still attached to the ascogenous hyphae spread out radially on the agar. Then individual asci are dissected as soon as the drop bas dried out. The location of the spores in the ascus is indicated by numbering from one to eight; the spore at the outer end of the ascus being number one. The thin but broad hoe-like end of the needle is dropped between the first and second spore and the plate pulled away by hand, thus breaking the ascus wall. A mechanical stage is not necessary to hold and move the plate although some individuals might desire it. Most people soon become skillful at manipulating by hand and in this way they can work more rapidly. All eight spores are dissected, "hoed" out, one by one and placed in a -7- row. Then they are transferred to a second plate set up under a second binocular so that there is no difficulty in returning to the row of ascospores in the first plate. The position which a spore occupied in the ascus is indicated by a number marked in the agar along side it. Heat treatment is used to germinate the spores and they are transferred to agar in small tubes by cutting out small pieces of agar containing the germinated spore under a binocular microscope. The heat treatment kills all of the conidia which might have been transferred with the spores into the agar plate. The transfer of the germinated spores to small tubes is made in a small glass chamber which has been liberally sprayed with dilute alcohol and wiped down with mercuric bichloride solution. The small tubes {9 by 100 mm. or 3/8" by 4") are kept in racks made of pressed cork strips. This pressed cork is three millimeters thick and is cut in strips two by thirty centimeters. About sixteen holes, each nine millimeters in diameter, are punched in the strips with a cork borer. The cultures are kept in the same position in which they occur in the ascus and are held tightly enough in out. place so that they cannot fall These strips of tubes are mounted in wooden racks. -s- II. Heat Treatment Methods Dodge ( /8 ) has shown that the ascospores of certain species of the Ascobolaceae, which do not ordinarily germinate in the laboratory, can be induced to germinate by heating. in petri He places the spores on agar plates and puts the plates in a gas oven, which is then raised to about 70°c. in about twenty to thirty minutes, and then removes the plates. In some species some spores fail to germinate even after this treatment and a few spores are always encountered which germinate without heating. He has since shown in other papers that this method may be employed successfully in inducing ascospores of certain species of Aleuria, Lachnea, and Neurospora to germinate. This method has also been employed by Ramlow( 55 ) , Betts ( :2. ) , Wilcox ( '1-.. ), and others. The writer has conducted a series of experiments on ascospore germination and describes a more refined but not necessarily infallible method of heat treating spores of Neurospora. This method and a discussion of the variability of ascospores of Neurospora in their response to heat treatment follows& l. Experimenta to determine an optimum heat treatment. In studying heat treatment spores of !• tetrasperma were placed on agar in small petri dishes. The depth of the agar varied from three to seven millimeters. The dishes were placed on a one-eighth inch copper plate set on top of a. Columbia paraffin oven. The copper was allowed to come to a stable temperature (73° to 77°0.) before the petri dishes were placed on it. A few drops of water under each dish insured a good contact. The temperature at the surface of the agar graduall7 rose to nearl7 70°c. Temperatures were determined b7 the melting points of various organic compounds. These were placed on thin cover glasses which were laid on agar in petri plates containing no spores. After man7 trials, the compounds shown in Table I were selected. The table also shows the time at which the compounds melted in two blank plates, giv1Dg a record of the approximate temperatures of the aacoapores on the surface of the agar in the other plates. Fig. 3 shows the smooth curve plotted among these values. The main errors of this method are: (l) raising the cover of the blank plate to wipe off the moisture film tor observation obviousl7 cools the plate, (2) the lowering of the melting point of the -10- Table I. COlllpounds used as thermometers. Kelt1ng Point 'rime of Melting in Minutes Plate I Plate ll Bemopheno:ae 47.5 -48.5 c. 6.5 - 9.0 a.o - u.o Betabromnaphthalene 53.00 ..55.00 c. 32.0 -34.0 38.0 - 42.5 Betachlornaphthalene 56.50 -57 .. 0 0 c. 39.0 -40.0 49.0 - 51.0 Pallid.tic acid 61.0 -61.5 c. 61 75 Stearic acid 69.0 -69.5 c. 0 0 0 0 0 0 -1.0a- - 100 240 - 105 240 organic compounds due to distillation into each other ' and water vapor, (3) the irregular heating of different plates due to differences in size, agar depth, position, poor contact, etc. It is questionable if a better method for determining the temperature would be worth the trouble because the variation among the several plates is probably considerable. A series of experiments were performed to determine the optimum time of heat treatment. Sixty- seven petri dishes containing spores of !.• tetrasperma were treated for different lengths of time on the copper plate. Blanks were used with the organic compounds as thermometers. The results corresponded roughly with those shown in Table I and Fig. 3, and were not inclUded in the data presented. When all the spores had germinated a count was made. Each percentage recorded is the final count from a single plate containing from forty to several hundred spores but rarely less than a hundred. Seven loadings of the copper plate were made and treatments of from eleven to eighty-six minutes were used. shows the data and Fig. Table II 4 is a graph showing the rela- tion between the time of heat treatment by the copper plate method and the percentage of germination. These are the total data from sixt7-seven petri dishes each containing on the average well -11- over a hundred ascospores. Table II. qerminat1on counts on sixt7-seven petri dishes eonta1ni:og Neurospora tetraaperma ascospores heat treated bi the copper pls te method for 'ftl"ious lengths o~ time. 'i'Iiit8 Per cent filii nn. cent ll. 2 0 0 7 l 44 46 98 98 48 50 52 54 54 99 nn. l2 13 14 15 16 17 18 18 19 1.9 20 21 22 23 24 25 25 26 26 27 28 29 30 30 31 32 33 34 3 0 2 4 56 58 59 PIP 9'7 85 93 98 56 78 30· 81 88 63 66 68 94 88 81 92 70 97 77 84 86 78 2 62 74 82 86 90 100 110 120 130 140 150 160 1'70 1.80 100 92 9'7 100 96 95 98 100 97 98 93 98 34 38 40 95 190 200 99 210 42 80 98 220 230 240 -lla- 93 97 93 97 95 97 92 93 82 94 '78 0 35 ll 8 0 l l 2 3 In-analyzing Fig. 4 it can be seen that the optimum heat treatment has the rather long range of from twenty-five to forty minutes. It can be con- cluded that a heat treatment of from twenty-five to forty minutes, with the temperature rising from 54o to 5s0 c., if the treatment takes twenty-five minutes to attain 54°c., will give very high percentages of germination. There is evidence to show that lower temperatures for longer times will give the same results but no effort has been made to determine a cri• tical temperature. -12- I, I \ i \\ ,,,,,,,; \\ '""'. ' \ \ \. '' I .. ·.... · """""""" i ' I .. ' loo •\ ·! 40 !• 1· I :--0 :~ !. ~ • ! i '~ . lo if1-+ \ti ~ ih ' I• '" 5V )fl fP ti ~Q 'I '1 '?;. • :, "' io lo a' M\..,, r . ....<_;. 10 " ' 2;0. i 30 'ft" "' ; 1 .. ! . l l"+g.3 ' 'i I ! 1 ! \ ! \ , ' I I l/'t. ,7, 4~ : I 1!> •1> " •( . I ''\ Ir' S-o. I I .fti I ! ' '. ' I I I ' fv t•4 /(o l'ifo. <f~ I I i : , 1' I'\ I I 1 I I l'(u f I a I l I 1;tt1 ' \ l rr..:. I ! ' II '+--JII\ , ! .... _ ; ~ ' ' I I I 0 2(f', I I t,f4 I. l>"o >fJ4 I i i-~o I I I >-?- •r ,,, '~ '" r,v •,'I ,,,.. 'r 'F "" • 1 •11 u· •r I ~ I I I I Ii II i I i .TV'"' !.1~ f/f1 1 ·~t~,! ~ Tt~rj y~+ r~ 1 ~,., 1 . I I I I I ' I 1 i I Gr~ph ehow~ng ~h' pero~.ntages f ~.erra1nat1j.o!l <lbtallned in ~7 dtfte ent 1agar ,r II 'i '\ I i I 1 I ! f . ! • I I II 4 ¢.._ I t I l plates' placed on the abppor plate 'for various 1len$ths of tima• T•• e tempera• tu-re of th~ agjir durinl; the.· tL~e ti.he a:.ish I.was jon t.he 4opp~r p~ate ! is also! sj,i8wn,,,, tNote ~re [lat maximum P,nd the trapi!d 1r or.oaiao beg1nn1n~ at i about I :.. mrnu es ';s we l aa' the· rapid fall after 140 minutes.. ' ' ' · · . , 1'),.. ! I, •(t '. I I. J.j'fo A .,,9 I 2. Experimen~s on killing of conidia. The peculiar advantage of using Neurospora for genetic studies lies in the fact that the conidia and mycelia, which are of parental origin and may be transferred with the ascospore, are killed by the heat treatment which induces the ascospore to germinate. After the ascospores have germinated, fifteen to eighteen minute heat treatments by the copper plate method are sUfficient to kill the mycelium of the germinated spores. In these experiments it required about eighteen minutes to raise the cultures from room temperature to 4a 0 c. After changing from the copper plate method to the thermostat for inducing germination, it was necessary to discover if this treatment was sUfficient to destroy the conidia. Fresh N. tetraspe~~ conidia were selected. These showed one hundred percent germination on cornmeal agar. Ten minutes in the thermostat at 58°c. de- layed germination for a few hours, while forty minutes delayed germination for as long as two days but practically all of the conidia germinated. From 45 to 70 minutes heating at 5a 0 c. killed all the conidia in thirteen of the small plates used and no later growth was pr~duced although they were held for ten days. -13- In two cases, however, (one petri plate held for sixty minutes and the other for sixty-five minutes) the plates were overgrown when examined five days later. It is probable that only a small number of conidia survived in these plates. In general the asci are carefully washed and no conida are carried over. The chances of any one which might accidently be carried over surviving the sixty minute treatment are evidently not great. Random single ascospore cultures of !• crassa were often made by sowing conidia and ascospores on an agar plate and allowing the conidia to germinate. Then the plate was heat treated and the ascospores transplanted to tubes. As a control many of the germinated and subsequently heat treated conidia were also transplanted but non& ever grew. -14- 3. Experiments on the inheritance of response to heat treatment. In the present method or heat-treating aacospores, the copper plate is no longer used. It was thought that lower temperatures with longer treatments gave greater reliabilit"f with less inju1"7 and hence fewer killed spores. A small thermostat was built for this low temperature treatment. In the early part of the work it was thought that cooling the petri plates containing the spores down to about 10° o. for 12 to 24. hours before heating increased the certainty or germi:nati on. Recent experiments show that cooling has no such beneficial ettect. It was determined that the optimum heat treatment for a cooled petri plate in the thermostat held at 58° c. was one hour. The thermostat is so small that its temperature drops to about 450 c. on introducing the cooled plates and does not attain 590 c. minutes later. until about 20 It may be that the aaooaporea on the sur- face of the agar never quite reach the temperature whioh the thermometer in the thermostat indicates. In the course of this study the spores from over 500 individual asci were isolated in order, one by one, -16- Fifty-two exceptional asci of N. crassa from a total of over 500 studied. 'Miese are exceptional in containing some ascospores which do not respond to heat treatment in the ordinary manner. Table III. As cospore Ascus 217 222 257 286 312 322 330 332 336 340 341 342 361 498 500 550 551 552 553 554 555 556 557 562 563 641 l 2 3 4 l l l 1 1 x 1 l l l l 0 0 l l x l 2 l l x l l 2 x l 2 l 1 2 2 1 x l 2 1 l l 2 2 5 1 l l l 1 1 2 2 2 l l, -l l 2 7 2 1 2 2 x l l l 2 x l l x x x x l l 0 2 1 1 2 l 3 2 l ab ab ab ab 1 x 1 0 l l x 1 l l 0 1 1 l 1 l 1 l 2 2 2 2 2 ~{ x l l 6 0 0 x 1 x 2 1 1 1 2 2 l x 0 x 0 1 0 l l l 2 l l 2 2 2 2 l l 2 2 2 l l "2 2 2 2 2 1 1 2 1 1 1 2 l l 0 2 ~ x 1 1 x l 2 3 3 0 x: 1 2 1 x l 2 3 8 Ascospore Aacus 642 643 l 644 l 646 l 650 1 651 l 654 0 655 2 657 2 659 2 676 1 677 2 678 x 683 1 688 l 715 l 718 l 724 l 726 2 730 2 731 1 732 l 741 1 744 l 745 2 690 3 2 1 2 3 4 5 6 l l 2 x l 3 l l 3 2 2 2 1 2 2 1 1 l 1 1 1 2 2 2 l l 2 l 2 l x x l 1 l x 1 2 2 l l 2 l l l l l l l 1 1 1 1 1 1,, l 1 l 1 1 1 2 1 1 1 1 2 l l l l l l 2 2 l 1 1 1 1 2 1 l x v A 2 2 l 2 2 l. l l l l l 2 l 2 2 l 2 l l 1 2 l l l l l l l l l 2 2 ~ 1 1 l l x , ~ l l l l l 2 2 3 2 2 3 l '.... l l l 3 3 l 1 l 2 1 l 1 l 3 2 1 2 l l l l 2 l l 2 l 1 1 7 8 2 1 1 2 l l l l 2 l l 2 l l 2 1 1 l 2 l l l l 2 l. 2 1 l 1 2: 2 1. 2 l l 2 2 x x: x 2 2 2 Legend l - spore germinated Rf'ter first hour heat treatment 2 - spore germinated after second hour heat treatment 3 - spore germinated after third hour heat treatment O - ap.>re serminated without heat treatment X - spore does not germinate ab - spore aborted -16a- so that their positions in the particular aaoi were known. Only asoi containing eight ripe spores were used. These spores were incubated on agar over night at room temperature to determine 1t any germinated without heat• ing, and it any co nidia had been carried over with them. At first all plates were cooled aa above described be• fore heat-treating, bu.t later the cooling was abandoned. The plates were then treated in the thermostat held at ss0 c. for one hour. About ten hours later the germinated spores were removed to culture tubes and those which had not germinated were treated for a second and sometimes for a third hour. Table III gives a record of the asci (from the 500 aaci of !• crassa) in which aaeospores were found that germinated without heating or that responded to heat treatment e~ :;~~er than one hour. Less than one percent of the total number of spores germinated without heat treatmentJ about fourfifths of the total number germinated following the first hour of treatment, about two percent of the spores yielded to the subsequent heat treatments and about one• fifth of the spores failed to germinate. The ratio in which the different types of spores occurred and their distribution in the aacus suggest genetic differences but these supposed differences have not been adequately -17- '!'able IV. As cos2ore Asous 202 l 203 204 Nine asci of Ii. sito§lila showing the position of t e ascospore and the manner in which they respond to heat treatment. 2 3 4 5 6 7 x x 2 2 2 2 2 2 2 2 2 2 x x ,, 2 l l l l l l l 1 8 . 205 1 l l l l 1 1 l 206 2 2 2 2 l l l 1 20'7 l l 1 1 2 2 2 2 208 3 x 3 x 2 2 x x 209 l 2 2 2 2 2 2 x 210 1 2 l 2 l 2 l 1 Legend v germinated after first hour heat treatment -- spore spore germinated after second hour heat treatment third hour heat treatment - spore .0. - 1 " ~ 3 germin~ted a~ter spore does not germinate -17a- tested. In order to test this point selection experiments and subsequent crosses would have to be made. Only one experiment was performed. A mating was made between the mycelia from two of the aacospores that germinated without heating. Fitty•nine ripe asco• spores were selected from the offspring. They were incubated on a.;ar at 27° c. for nearly two days, but none germinated. A one hour heat-tl'l!latment induced fifty-five or them to gerllli nate indionting that they were mature. At first this was supposed to indicate that variationafrom response to one hour heat-treatrnent were not inherited. BUt subsequent experiments with crosses or definitely inherited t7pes gave simi.lar first generation results. Further generations, which in this case were not raised, would be required to prove this point. It must also be noted that this group of asci were the inbred descendants from an ascua in which the spores germinated following a treatment of one hour and it is possible that other races of !• .£!!_S.!!, may exist whose spores require heat treatment for different lengths of time. Nine aaci of !!• sitophila, described in 'J'able IV, showed a striking contrast to the case of!• crassa. -18- It must be noted ~hat all the asci of !• ,!!tophila studied appear in Table IV, while Table III includes fifty-two tberrant asci selected from a total number of over 500. Therefore. 1n the case of ~· sitoph1la a.bout one half of the spol'ea required two hours heattreatment. while less than half of them germinated after one hour treatment. In the case of aso1 202. 206 and 207, the ar- rangement of the spores seems to point to a segregation of a factor determining the response to heat treatt!ll!lnt, but no fUrther breeding tests were 1!19.de. That the type of treatment necessary for germination depends on genetic factors 1s indicated clear• ly by the folloWing experiments, which were startedr for another purpose, namely, to test the nature of a supposedly hybrid strain produced by Dr. B. o. Dodge. By crossing a non•conidial mutant of !• sitophila with typical con1d1al .!!• tetrasperma and selecting and back crossing for several generations he had first secured a non•conidial 4•spored race. Then from this non-conidial, 4-spored type he grew a bisexual, he1'1!1aphrodit1c, noncon1d1al raycelium, which he designated 3C. re mated thia 1 with a um.sexual, conidial mycelium of ll• tetrasperma, producing thereby psr1thecia which were supposedly hybrid. -19- Table v. Ascospores d1seeeted fl'om sixteen asei obtained i'?>om hybrid peritheeia refe~d to in the text. It is also indicated whether or not the myeelia produce con1d1a and whether or not the spore needed heat treatment to induce germination. Spore RO. 1 Iseua t :a 402 + c A+B Iseus LH 406 lieus 4ll Ascus 412 lieus 413 lseus 404 Xseus 41.0 lieus 405 Ase us 407 Ascua 41.5 LH + c Ail:B t fO L tc \VO A+B B? +C A+B A+B t H +o A+B A+B t +C A+B LH +C A+B A+B 403 +C +c A+B A+B S t;JO -c A 408 L WO -c ASCUS S V'iO A licua 401 A+B sH -c tR +c A+B -c -c A+B LH t ii +c A+B t +fib -c A +C B? +c +C tli Lli sH A LI! LH A+B t \ID BT +c A+B LH +c A+B lseus 414 8li +C not ger- t-C +c A+B minated B? t H SH tH +c +c +c A+B B A+B t H 8 H LR +c +c +c A+B B A+B L WO t WO B fJO +c -c +c A+B A+B B? , LH SiWJ LH t li +c Ascus A sli +C +C A+B? Aseus 417 Ascus 416 s wo +C B? LR +C BT SV~'O -c s~vo A t It +c A+B? sR +c B s li +c B 5 ¥JO tli +c A+B LH tli +C A+B LR +c A S vVO -c A S VJO A -c A g H +c A s li +c A+B t li +C A+B t li A +C +C B? A+B? s wo s Ii +c B? s li -c sli +c tli tli +c A+B +c A tu +c B ? A .& +C A+B 8 Wo -c S WO -c Med.WO -<.: BT A tli +c -c do -c -c A+B S \Vo B? A+B SH +c B sli +C tied \VO -c B? -l9a- 7 6 -c +c A+B 8 iflO -c t 5 LH A+B tli +C +C I· R 4 8H B s H +C B sH sH A A +C +c s li +C ? Legend: L means large•) s means smal.l,) all rel!'er to the sises o:r the spores Med means medium) H means the spore needed heat treatlllent to induce germination WO means that the spore ge:rmi.nated without beating +c means the mycel1um produced conidia -c means that the mycellum. .tilled to produce conidia A and B mean sexes A and B A+B means hermaphroditic B? means sex B 1ncomps.t1bl.e A+B? means hermaphroditic but producing no perithecia The manner of producing these peritheoia is described in detail by Dodge in a paper that has recently appeared ( Dodge 1 31 ) • Proof that a hybrid had been produced between the hermaphroditic and the unisexual mycolium would be furnishen if both conidial and non•conidial asoospores were found in the same ascus. . The writer accordingly dissected sixteen exceptional 5-spored asci from this 4-spored hybrid. Since one parent was non•conidial and the other was conidial, 5-spcred asci were selected in the expection that, if a cross had been made, one of the two small unisexual spores from each of these asci would show the character of one pa.rent while the other would show the character of the second parent. In ea.eh of the three other standard, bisexual, hermaphroditic spores, containing two genetically different nuclei, one from each parent, Q&sking of the non•conidial character might be expected. As Table V shOi!'s, all ~ycleia from the bi· sexual ascospores did produce aonidia, indicating that the non•conidial character, if present, \Vas ::is.alto d. That the n'on-c"nidial characters were actuall;r present was shown by its manifestation in about half of the mycelia from the 3l!lall spores. 'l'he presence of lhe conidial an:! the non• conidial nuclei proves that the supposed h'fbrid had -19-e- actually been produced by the cross. The data 1n Table V are represented diagrama.tically in Fig. 4. The 5-spored a.sci are grouped in classes according to the arrangement ot the two small spores and the types of mycelia produced. The nuclei are shOll'n as white, black, and black with a white center. The white nuclei indicate sex A; the black, sex BJ the black nuclei with white centers were probably sex B but were incompatible with the tester strains used so cannot be definitely determined in every case. Incompati- .'ility is sterility with tester strains of opposite sex, a phenomenon resembling self sterility. The large, i.e., standard-sized, spores were assumed to be binucleate in origin and the small spores uninucleate. Table V shows that the nuclei in most of the large spores contained two compatible nuclei, their sexes being indicated by A+B. A few ot them carried incompatible nuclei, for the mycelia failed to produce perithecia. cases the sex is indicated by A+BT. In such The sign +C indi- cates that the mycelium from the spore produced conidia while -c indicates that it was non-conidial. The mycelia from all of the large spores (except from two in ascua 408, discussed in detail below) produced conidia. The two small spores in each a.sous were one conidial and one -20- Fig. 4. Diagram of thirteen 5-apored aaci dissected rrom a hybrid obtained by Dodge. 'l'he size, arrangement 1n the aacus, sex and incompatibility of the spores is shown. The presence or absence of con1d1a on the myoelium and whether or not the spores require heat treatment to induce germination ia also shown. 'llhite nuclei are sex A; Black nuclei are sex B; Black nuclei with white centers are lex B incompatible; Cross-lined spores germinate without heat treatment open spores require one hour heat treatment; -c • means that the myoelium develops no conid1a; +c means that it develops norm.al conidia. -2oa.- non•conidial (with the exception again of ascus 409 where both were tc). th~ r.~~1t.~ecia It is obvious f'rom Fig. 4 that were true hybrids since half ot the nuclei in each ascus contained the conidial factor and half the non•conidial factor. Moreover, when a non• conidial and a conidial nucleus were present in the same ascospore, the ll!J'.Celium produced conidia. The special interest of this hybrid tor the Wl'iter lay in the tact that Dodge had tound that a large proportion of the spores ge1'1'111nated without heat•treat• ment. Thia characteristic appeared clear-cut in the spores dissected from the asci just described. In Fig. 4 the diagonally lined spores are those which germinated without heating. 'l'he figure shows also that the spores " which germinated without hes.ting were all non-conidial.. In all bUt two asci (405 and 407) these non•oonidial spores that germinated without heating were moreover sex A. This three-character linkage was very striking. Ascus 408 was exceptional in two respectsJ first, the small spores were alike (B, +c, H), second, the three large spores were uniaexual instead of herma• phroditic, and were -c. This anomolous situation can be accounted for in terms of the known peculiarities -21- of the spindles leading to the formation of the spores, provided the segregation for these characters occurs at the second division. Dodge ( 23 ) has described in de- the arrangement of the spindles in !• tetraaperma, showing that firat-"division segregation of factors for sex would always produce four h6rmaphroditio spores in each ascus. Since Dodge found no binucleate unisexual spore• he was of the opinion thst segregation of sex always oc• curred at the first division, or that if segregation occurred at the second division the orientation of the nuclei was such that unlike sexes came together. With• out this peculiar orientation, second division ssgrega• tion would produce binucleate uniaexual spores. Asous 408 is ot particular interest, since all three of the binucleate spores were unisexual, and hence preau:nably lacked this special orientation of the nuclei. Follow- ing Dodge ts study of spindle arrangement, and assuming - that this hybrid has the same spindle mechanism as N. . tetrasperma, we can conclude that ascus 408 shows second-division segregation of the sex factors as well as those for eonidia and those for germinating without heating. Fig. 5 is a diagram ltlowing the type of a.sous be: a vi or necessary to produce this rare kind of ascua. The straight lines are the axes of the spindles as they would lie if' they followed the arrangements found -22- Fig. 5. Diagram showing how second division of the factors determining sex, response to heat-treatment and the presence or absence of conid1a couJ.d give rise to a 5•spored ascus such as ascus 408, containing no hermaphroditic spores. Diagonally lined nuclei contain the factors for sex A, absence of oonidia and ability to germinate w1 thout heat treatlllent. Black nuclei contain the factors determining sex B, production of oonidia and inability to germinate without heat treatment. -22a- c for !• tetrasperma. This differs from Dodge• s diagram for second-division segregation in onl7 one feature, namely, that the orientation of' one Of the nuclei 1a reversed in the binUcleate state of the ascus. (Fig.6). The failure to produce perithecia 1n the caae of the mycelia from spores from aacus 408 cannot be ascribed to incompatibility• since it was on17 sex B that showed incompatil1 lit7 in thia hybrid and in ascus 408 the sex B strain was teated and found compatible with the tester strain. That the failure to produce perithecia waa actually incompatibility in the caae of' some of' the m:ycelia of other a.sci was shown by the fact that the sex B strain from ascus 40'7, which failed to fruit with tester strains, fruited readily when mated with the sex A strain from ascus 406. This cross (406•5 x 40'7•3) was the mating of' two mycel1a both of' which were non•conid1al and were produced by asoospcres which germinated without heating. Untortunatel7 nothing is known of' the reaction of the pi.rents of' this hybrid to heat treatment. Cross- ing a clone derived from non-heating ascospores by another of the same type, since both were supposed to be haplonts and presumably pu.re tor all1 character which they show, should produce only offspring showing the character. TABLE VI. Twenty-one aaci dissected from a cross of two non-conidial ascospores that germinated without heating. The size and arrangement of the spores in the aseus are shown and whether or not germinated without heating. Abbreviations same as those used 1n Table v. - AsCo- soorea Ascua l Aacua l 2 3 L L s s 3 Ascua J,, 4 w.o 5 VJ .O 6 w.o L w.o Ascus Ascus Ascus 7 ASCUS J,, B 8 As cu a .i.. \'! .o J,, J,, J,, J,, ... L v J,, i.. ;v,. o i'/. 0 .:> J,, u s w.o J,, v; .o J,, w.o L J,, i.. J, w.e J,, Vi .O L L .i.. L 3 .i.. 3 L B w.o ::l w.o s J,, L L B w.o L B L s J,, J,, w.o w.o s L 3 w.o w.o L s L w.o J,, s B w.o w.o w.o L L w.o w.o J,, ... .. w.o J,, J,, L J,, L . J,, J,, w.o L w.e w.o .I.I w.o very large 6 s .o s s s ;;) L i'i w.o s w.o w.o w.o L w.o L B J,, 5 B VJ .O ll ASCUS 12 Ascua 13 Aseua 19 Ascus 21 L J,, 9 As cu a 10 Aacua 14 Ascua 15 ASCUs 17 ASCUS 18 ASCU8 20 ASCUS 16 Ascua 2 ASCUS "' w.o s w.o J,, w.o J,, w.o 4 w.o w.o J,, L w.o L L \V.O w.o L s w.o w.o -23a- To tea~ this point the spores from. twenty-one 4, 5 1 nnd 6•spored asei from the cross 406-5 x 407•3, were dissected out and incubated at room temperature for several days. Table VI shows t.he ascospores trom this group which germinated without heat-treatment. Porty-five percent of the large spores germinated without heating in this oross as contrasted with only twenty percent at the large spores in the parent hybrid. It is remarkable that 1n only one case ( 20) did both small spores germinate ?d.thout treat:nent. This :nay have been a ease like ascus 408. «Ve can conclude from this experiment that the factor causing ascospores to germinate without heating is hereditary and it is usually carried by nuclei of sex A. That half of the offspring, both binuoleate and uninucleate spores, failed to gerlll.1.nate without heattreatment negatives one of the assumptions made - namely, that the nuclei were haploid. This peculiarity of be• havior is typical of the hereditary strains and will be discussed in detail later. 4, Summary bf the experiments on the heattrea tment of asoospores. These experiments showed that it was possible to determine, by appropriate tests, the optimum heat-treatment which causes the largest percentage (in some cases 100 percent) of the ascospores of Neurospora to germinate. Furthermore, it was shown that the heat-treatment commonly used killed the asexual spores, thus simplifying the sepa• ration of sexual from asexual offspring. The ascospores from the race of !• tetraspeI'lll& seemed to be highly uniform in their response to heattreatment. ·There was evidence that part or the small percentage of variation from one hour heat-treatment in ~ crassa was due to genetic factors. Dodge's hybrid and the race of !!.• sitophila produced two types of ascospores in regard to their response to heat-treatment, In both races the differences were the result of genetic constitution, but there was evidence that the ability of a spore to respond to heat-treatment was varied by the environment. This was best shown by the fact that although all of t.~e large spores in the hybrid had one nucleus con- taining a factor for germinating without heat-treatment, and one containing a factor determining response to one hour heat-treatment, some germinated without heating while others did not. In Dodge's hybrid, the unisexual -25- aacoaporoe fell into 'two genetic groups; in one croup all ot the ascoeporee uerminated without heat-treatment. In the other grou.p the ascosporee required one hour heattreatment to initiate germination. The race ot N. sitophila contained two genetic groups as well. One kind ot asooai;:ore required one hour heat-treatment. and the other required two. Despite the evidence that response to heat• treatment is a genetic character, in these cases, it is improbable that it can be used extensivel7 in a study ot the genetics or Neurospora, because ot its variability. In a closely inbred race, such as the race or ~· crassa, which seemed to be rather uniform in its response to a one-hour heat-treatment, ascospores were encountered which germinated without heating and others which required a three-hour treatment. Although in a few cases, the order or the spores in the ascus indicated that factors datermining response to heat-treatment had been segregated, it was 0:-;-!0•:!! in mos;; cases that the identical twin asoosporos sometimes responded differently indicating that environmental factors were also effecting the variation. Faul.l (32) concluded, on the basis of some experiments on heat-treatment of the ascospores of a strain of N. crassa, that heat-treatment does not increase the percentage of gel'!!lination. ·The data 'Nhich she pre• sents do not justify this conclusion. ShB found that 4 to 25 percent of the ascospores in various random samples 3erminated without hea.t•treatment. Ascospores in various random samples which she heat-treated at s1.50 c. for one to four hours showed 5 to 94 percent germination. Obvi011sly she was workin'S with a mixed stra1r:. producin3 at Jaast two genetically different kinds of ascosi:ores. III. Sex and Arrange!llent ot Spor~s in the Ascus ot !• crassa. Dodge ( 25 ) has pointed out the advantage of the ascomycetes tor studying meiosis. The zygote nucleus is formed by the fusion ot two nuclei. The cell contain• ing this nucleus gives rise to the long, tu:'e•like ascus. This nucleus then divides three times to foM!I ei;~t nuclei. Each of the eight spores receives one of these ei;ht nuclei. In the species of Meurospora craaaa and N. sitophila, an ascoapo:re on germination gives rise to a onyoleium which may be either one ot the two sexes. ~ ~ycelium, such usually called the "haploid" mycelium, is sterile when 6rown alone or with another ot the same sex, but growing the mycelia of two opposite sexes together produces zygotes and ascospores again. In the species mentioned, the tube-like ascus is long and narrow. Wilcox ( 62 ) has studied the sp indles in the ascus of n. sitophlla and has found that in the first two divisions they are oriented parallel to the sides of the ascua and do not overlap. maintain their :relailllie order and positions. The nuclei This means that it is possilie, as shown in Fig. 6 1 to determine at which division segregation of factors has taken place. -28- Table VII. All Possible permutations or ascospores in the ascus permutations @/'70 c~ance Spore + .,. + + + + + + + + + + + + + + + + + + + 't + + + + + + + + - 3 4 + + - + + + ... -- - + + - -+ + - + + -- ----- -+ + + + + + + + -- + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + T + + 8 -- - --- - - -- - - -- - -- --- -- - -- -- -- + + + + + + + + + + + + + + - - --- - - -- - -- - - + + + + 7 + + + + + + + + + + + + + + + + + + + + - - clymm.etricai permutatlons f77o cnance -- - - - - - + + - + + + + +· + + + + + + + + + + + + -28a- + + Fig. 6. In group a, is, shown a diagram of the arrangement of spores of opposite sex in t.'ie ascus of' n. orassa that would resUlt from segregation of sex in the first division in the ascus, provided there was no passage of nuclei in the ascus. Black and white are used to designate the opposite sexes. l'he onl7 shift that occurs in this arrangement that has been detected is a shift in the fourth and fifth place, 3iving three black, one white, one black and three white nuclei, but this shift is rather infrequent. The second obvious possibility of a first-division segregation would result from the opposite orientation ot the tetrad in the first division. The second figure would then show a white nucleus above and a black one below and the final arrangement in the ascus would be reversed. In group b, is a diagram showing the arrangement of spares of opposite sex in the a.sous that '.'l'OUld result from a. segregation of the factor for sex in the se• cond division 1n the ascus and a failure to segregate in the first division. A second possibility would result from e..'langing the orientation of both nuclei 1n the nucleate stage. In this case the last figure would show, from top to bottom, two white, two black, two white and two black. Ascua 348 is an example of a shift in positions 2 and 3, followin;i; this type of segrega t1 on. In group o, is shown a diagram of another arrangement or the spores of diverse sex in the aacus of N. crassa which would result from a second division nesregation of the faotor for sex. If both nuclei in the binucleate stage were reversed, another possibility would be obtained with the four tlack nuclei in the cente~ and two white at each end. Ascus 301 shows the ~'lange in this arrangement resultin~ from a one place shift. -28b- ) ~ 0 ~o • o • ) e) ~000 0 • • • •) ~ ~ cl ) () () ~o • ) ·~ o ~--oo• • o o.• 9) ~ ~ ~- Cl ) () () ) •) ,~••oo o o • •) 0 -28c- 0 This is done by studying the characters of the cultures grown from the respective ascospores of a single ascus, since the position of the spores in the ascus is known. Fig. 6 shows three of the six possibilities for firstversus second•diVision segregation. 'l'he other three are represented by reversing the positions of the two vari• eties of nuclei. In the case or the figure (6a) showing first-division segregation, the upper tour nuclei would be ttwhite" and the lower fournblaok" in the other equally probable orientat :Soni!. Dodge ( ?:l>) and Wilcox { 62) have shown 1n .!• !!_tophila that the iactors for cultural characters and the factors determining the two sexes can be segregated at either the first or at the aecond•d1V1sion in the ascus. DOdge's ( 23 ) cytological evidence shows that in !• tetrasperma, where there is a very complex series of changes in spindle orientation, there is a greater probability of the sex factors bei.~g first-division than at the second. segregated at the The sex factors might be segregated at any one of the three diVisiona and still give rise to four hermaphroditic spores. He has also studied the segregation of the sex factors in !• erassa, and r.as inferred that they are segregated at the first division in this species. -29- By means ot the arrangepient of the spores in the long narrow ascus, the writer has studied the segregation of the factors for the two sexes and for various pa.ire or cultural characters in !!, craesa. Segregation was f'ound not to occur at the third diVision• since tor all characters studied the spores were found grouped as four pairs of identical twins. Dodge's study of the orientation of the spindle in this species shows that it is probably identical with that ot !• sitophila. All or the asci from Which ei~ t spores were germinated showed four spores of one sex and four ot the opposite sex. The a priori possible arrangements in an aaoue for eiA;ht spores, four of one sex and tour of the opposite sex, are seventy. This is given by the formula: 41 81, x 41. = 70 Table VII shows 32 ot the ;:;.symmetrical and the 6 symmetrical permutations. permut~tions The remaining 32 asymmetrical are obtained by an end to end reversion of the 32 asymmetrical ones shown. If th~ aTrangement of spores in the aseus is determined by pure chaB-Oe, there is one chance in 70 of eae.~ such permutation occurring. -30- A diagram ahowll:li how paaoing of' t!";e :t1eeond 1!u'ld tnirn mtc.lei in tlla i'OUP nuelea to stage ~t rllsul.t 1n 14n actual f1ra dirtsion aogregation produc1n;; tt.e a11T1wgemtult of spores aattib&d to a !!OCO."lfi d1v181on aegH>iat1on !:md vice versa. ".:'h$ cytolo '.ical oviclenos ~ wtlco.x: .. rd Do;:ge B'.OW this to be b~y improf.lable. -30a- Table rx. sex and arrangem.ent o:t: aaeospores 1n 275 a.sci. o:t: li• era.ass.= P3 Generation 11.SCUS 0 ~ - - - ~· + + + P2 -Gene~ation. --+++--+ (O-l x 0-8) (I se:t.t:ed) 6 ++-++---- ---e++++ (10} Q - - + + 0 - 0 --+++@-++..-©--e- 7 ®+++ ---- 4 I : 2 !I : 1 {?) (0-1 x 6-2) I x I aseus us -e+e++-- 114 - - - - + + + + 118 + + + 0} - - - - 116 120 + + - - - - + + ++--++-- - - - + + + + ----+++® 122 123 (124 @ ~r + + - - - (125 - - - - + + + + (126 + + ~- + - - - (12'7 + + + + - - - 124(128 + + + + - - - (129 + + + + - - - (130 + + ·r + - - - (131 - - - - + + + + (132 + + + + - - - ... (1z;s + + + + - - - (134 {136 e + + + + - - - + + - - - - + + 15 I : 4 II (O-l x 7•3) I x I a.scua 137(137 ----++®+ (139 ----++++ 141 142 144 e-e-'c+++ ----++++ ++-----1-@ 4I : l II (0-1x10-4) Ix II{?) aseus 106 107 (109) 110 112 +++·r----- ----++++ ---@- + + + - - + + - -+@ + + + + - --- 4I ; 1 II (0-1 x 1-~) I x II (?) ascus 100 G---+0+-r 101 --+-i-++eg 103 ----$++..,. 104 -;-@@+---3 I : l II P1 c~ne:ration (4-o x 6-o) I x I 299(!)+++---{501)+ - + - - - + + 502 + + + + 303 + + -r + - - - 306 - - - - + + + + 308++++---314 + + + @ - - - 316 - - - - + + + + 317 T + + + - - - - e- - :518 -f' + + + - - - 320 - - - - + + + + 322 - - - - + + + + 32:3 + ~;. - - - - ·t- T (324)- - - + - + + + 325 - - - - + T 1" T 329 - - - - + + + + 14 I : S II 71 Generation (Aacus 114 sel.f'ad) I selfed 114- 1.xB(I ael.fed) e- 158 ·c _, ... + - 159 7 + + + - -E>160 + + + + - - - 164 C!l + + - - - - e 4 I : 0 II -50b- F1 Generation (Ascus 114 sel£ed) I ae1£ad 114-1.X'l 216 - - - - + + ©@ 217 - - - - + + ~ + (218 + + + + - - - ( 21'.V r -~ + + - - - ((220)- - - + - + + + 21a , 221 '" + ~~ e (251 251{252 (253 254 e--- (222 + <:!78 .. + + - (223 - - - ... + ;- + -l· (225 + -~ -\· + - - - ({226 ) + ~- + ~ -:- - - - e-- e+ -e- - --++·r-+--e--++++ 2~7 ++·r+--e- 260(266 \26'7 (268 (230 - - - - + -~ + + + + + - + - - ... 2~3(236 - - - - + + + + (23'7 r + + + - - - (238 - - - - + + + + '1"' .,.. 1'" 'T - - - - ----~r@++ (275 275(276 - - - - + + + + + + + + ( 27'7 (280 ++++---+ T ,.. + - - - Ti"-e--++ 281(2Sl. ----+++-r (282 + + - - - ""'A. -----.:·+++ +" + + + - - - - ----+++~ -----1"'+++ ----@+1-+ ++++---- +++@--ee 2'7'7(278 + + - + + + + -l·+++ 271 273 274 + + + + -;.. T + + + - - - - 255 (263 (204 (265 230(231 - - + + + + - (232 - - - - i- © + + 242 'Y' ++++---- --++++ +-t----- ('2:35) ·i'- ++----+® -- 224 + ., 227®+ -1- + (2~3 - - - (2~4 + + - - - - - - .,. @ + (£) - - - - + + + + - - "F + + + ++++---- c284 e+ e- + + .-,. +- ~- e+ "'o-c28o 286 - 248 {248 - - - - +El:}++ 288 289 ( 249 - - - - ® + + + - - - t t t + - - + + + + ~~+----++ 200 e@--®+-- 291(291 (292 5S I : 9 !I -30c- - - + + + + + -~ .,. + - - - F 2 Generation (223-l :c 230-6) :t x r a.sous 331 ----+®++ 332 333 ++®+--e++®+-e-- (3:35 (336 ----+-t-++ ---++++e- {337 (338 335(339 +~r----++ (340 {341 (343 (160-1 x 8) I selfed alilOUS (493X5 - - + - + + + 494----•++® 496 - + + + 4988---+@++ 500 - - - - + + + + see 502++8'+---503 + + + + - - - - --++++-+ ,. ©& &+++---- - - -e (604 + + + + - - - - (505 - - - - + + -!· + 504(506 8 + - - + + - {507 - - - - + G:l + + (508 - - - - + + + + ++++---·@++...;., ___ _ 8 I : 3 II coo9++++--e+ + + - - (5118 - G - + ·r- + + ( 512 - - - - -~ ·!- + + (223-7 x 229-8) I x I a.sou.a 345 - - -- - T + + + 346 -----"-+++ 347 + + + + ((348) 349 e--- c( c049 l 350 + .. + - ·i- + - - + - "" + + +++·r----- 351 352 353 s54 + + - - - - + + - - - - + + + + - - - - + + + + + + + + 355 356 357 S58 359 360 362 ,. 509c~ Sl~ e + ·i- + + - - - - i314 -r + + + - - - - 515 T + T + - - - - e e,. -e ~ csis + + - vl.7(:519 - - - - + + + + 020----@+++ e++++--e - 21 I : 1 ll --6-++++ ----+·r++ {226-l x 6) I sel..f'ed @~r++----- ascus - - - - + + + + + + + + - ----+@+@ 450+@++---- 452++++ee-453 + + + + - - - 4os .,. + .,. (:!> e--e 14 I : 3 II 46oT+++-e-467 ,. + T + 6:J; : 0 !I -;,sod- F2 Generation (288-€1 x 290-7) (243 - 2~) I selfed) ascus + + + + (364 - {365 ( Z.66 - - -- -- .,.. + ... - '- .,. - (357 ;564(368 + + ·1'" .r- + + \ ~..:o..: ----+-r+·r- (370 -++---++ -1- + + + h (550 ·r .......... , (548 546(54;? '"51 (551. ;;) - -- ( 3'72 (::073 "'~"'(55:5 ~Jil~..r(~54 -++~r+ ++++---~ + + + 373(374 (37~ M - (500 555(500 (557 - ----- --- - + (3) + + + + + + 376 377 (378) + ( 552 (558 558(559 + + + 12 I : 2 ! I Ix !I -- - (!;) + ·r + + +@ -9-G + .,.. + + -ee+ .,. + + + + •j- ..... ---- - -- - + ·r + + + + + + - -- - - - t + + + + + + + { l:i61 e + + ++ + + +© + + + + -- ---,.. + + + 560(562 - - + + + + (564} (3) Go)+ - + 200-3} I .x. Il (569 569(570 ~11(071 v (572 (573 5'73(574 (5'75 - - - - - - ... - - -e-- -- 567 -@ + ® + .,. + .... + + + .,.. + + + 15 I + + ----+©®+ ee0+--.,..9 ®++Gee-- -- - - - -- -- -.,. -- - -581~:~ -576 + -;- + + (5'77 + + + + (~37J :577(579 (580 + + + + + + .,.. + + + + + + + + ++ + + {583 583(584 (585 -- -- -+ -+ -+ + +- + +- e+ - + + + + 15 I : 2 I I -30e- - - ·r + + + : l :! F.,, ., ' G€:nera.tion (337 - 6x7} S.SC'J.S ·+- + 379 (SS"l I I selfed .,. + -+ +- -+ -+ -331 - - - - + + + + + + --- - + + 383 + + + - - - -- - - + + + 380 382 ' 384 + 5 I (SSS - •• l 5 x 7) ascus (470 470(471 (476 (4rf9 - e -_,_ - .. 9 + + + ·!- -8++++ + +G+e--+ ·!- + + --e- + @®+ + -r@ -~ + - - -~ (483 + ·r- + + + + i' 17 I . 4 I • 2 II a.s@Zlll -e + + + .• 2 I I 3) I I self'ed -----·+ -- -- - -- - - (338-2~) -- -- - -+ + + - - ·- ·(480 - - - - + + + + (481 - - - + + + 480(482 - - + + + + - -- - - - + +8++ (484 - -e- + + + 486(486 - - - +@+ - - - ·(48'7 (488 - - + + + ++ 488(439 - - - et (490 - -+ ++ + (477 477(478 2 aaP - - + ++ +e386 + + + +e387 + + + 388 +++ + - 389 + + + + 390 +®+ + I I sel.fad -:- + + + (473 (474 473(475 n - x I I selfed - - -- - - -- - + + + + + + (525 525(526 (527 + + + + + +~ ++ + + (528 (529 528(530 (531 -- -- -- -- + ++ ++ ++ + +++ - -- - 532(532 + + - -+ + e+ + ++ ---·!- ++ + + - - - .. --(534 - - - - + + + + + + ++ - - - - -e- + ++ (533 .;. 534(535) + (536 -- -+ + +@ - - - 540(541 - - - - + + + + ~'7 + + + + (540 54:s 544 e-®+ + + + + + + . -e - --- 15 I • 3 I I -30f- The 6 arrangements ~ which 3 al"e shown in Fig. 6 coUld all be the result of either first-or se• cond-division segregation, provided the spindles in the second divi st.on were long enough and the ascus wide enough to permit the two middle nuclei to pass each otl:er in the four nucleate stage. Fig. 7 is a diagram showing m.ch a shift in position. With such a shi1't, a first-division would produce the arrangement usually ascribed to a second-division segregation and vice versa. rr such a shitting occurred, it might be objected that the data do not show evidenee 01' more than one type 01' segregation. But Wilcox ( 62 ) in her cytological study of a closely related species, !• sitophila, shows that no such shitting takes place. Moreover, ascus 114 (Fig.a) proves that both types ot segregation occur. A character which was cal.led tan, appeared in the mycelia grown from the ascospores of this ascus. The oonidia ot tan were pro- duced more sparsely than in the wild-type, and were white or yellow, while those ot the wild-type were orange or light orange. In '8.ble VIII it can be seen that the sex factors were segregated in the first division, while wild and tan were segregated in the second. Even if it were assumed that the second and third nuclei exchanged positions in the second diV1sion, the results show that both first·31- and second-division segregation 111:11st have taken place. fig. 8 shows a diagram of aacua 114. Table IX is a record or the sex and arran;ement of the ascosporea in 275 aaci or !• craaaa, comprising six genePations. single ascus. These asci are all descended from a In each column the numbers at the left are the munbers of the individual asci. Plus and minus signs are used to .indicate whether or not the myoleium of an individual ascospore produced fertile perithecia when mated to a tester strain. The p lus sign denotes one sex and the i:U.nus sign the other. The eight spaces from left to right correspond to the positions of the ascosporcs in the ascua. The first position indicates the aacospore located at the tip of the ascus and the eightth position indicates the ascospore located at the base. A bracket at the left or the numbers of the asci indicates that all of the asci bracketed come from one perithecium and the number at the left of the bracket is the number of the perithecium. Above each group is shown the ms.ting "hich produced the corresponding asci. ?or example, 0-1 x 5-2 :::ieana :natin.:; the mycelium from -3la- ;;·1g. a. A diagram o£ tho arrangement of spo?'Ds of opposite ';&:;t a:::d producing tan and Wild t7po ::IjOella 1 n llSOU$ 114. The a~nt of the spores P•-OVes ::hat even 11' a l'lh1tt as dcscr1~:ed 1n i?1g. 7 lmd tal;;,::m place., ootb t1rat-rand second-division se,;rcgat1on oecurred. -3lb- the first ascospore In ascus O to the mycelium from the second ascospore in aacus 6. The S1l!lbols r x I follow- ing indicate that ascus 0 was an ascus in which sex was segre3ated in the first division in the ascus ani asous 6 was an aacus in which sex was also segregated in the first division. Roman numberal II is the s'Ylllbol used to indicate second di vis ion segregation of sex. , ~T·y.; ,fr··1. , ; ,·' -tYt ,. When 6UPJ' spore;;did not germinate, the position /l of an ungerminated spore is indicated by a ring. When it 1a possible from the position and sex of the other spores to determine the sex of the missing spore a plua or minus sign is placed within the ring. From the f'aet that in every ease where all eight spores germinated, there were four of one sex and four of the opposite sex in the ascus, this determination is easy to make. The arrangement of the spores in the asci shaa' s that segregation of factors determining sex may occur either in the first or the second division of the ascus nucleus. Exceptional asci are marked by parentheses. These undoubtedly are all due to slipping of' adjacent nuclei past each other during or after the third division. Proor of this point rests on the correspon- dence between the arrangement of cUltural characters as shorn in Table X:V and sex as shown in Tatle IX in -3lo- the case of asei containing such shifted nuclei. Ascus ' 226 is a case in point. The arrangement of the ascospores of different sex shown in ~able IX (for ascus 226) indicates a shift in positions 4 and 5. 'l'he ar- rangement of the spores producing different types of mycelia as shown 1n 'fable X::V confirl!ls the fact that this is merely shifting of the nuclei. It is clear that tan and sex are independently segregated yet the same peculiar arrangement of the fourth and fifth spores in ascus 226 holds for both tan and sex as shown in Table x. Reversing th~ fo~rth and fifth spores gives a simple first division segregation,,.of sex and second division segregation ot tan and orange. Two other exceptions which cannot be readily interpreted on this basis are asci l and 10, btlt these were dissected before the writer became expert enough to be certain of his technique. To date no evidence has been obtained of segregation of any factors in the third division in the ascus. Fig. 9 is a diagram of a hypothetical ascus which would be evidence for a third division segregation if encountered. None such ascus has been dis- covered. -32- Fig. 9. Diagram of a 1ypothetical e.scue aheo.:ring or spores of opposite sex and different Cl.lltt1X'91 ehAlraoters that ll1l arrangel:l:!nt would prove tr.a~ t~1~d'd1v1a1on segregation had oecun>ed. :10 such aseus has been tOl.Uld. -32b- In order to 'facilitate discussion from this point on, some abbreviations will be used. In making me.tings, L'1.dividual ascoaporea are planted in separate tubes on nutrient agar until mycelia and conidia appear. 'l'he:-: some of the mycelia and conidia from planted into a third tube. two tubes is Strictly speaking matings are made between clones produced by ascospores and not between the ascospores. However, from now on, such me.tings as that which produced the Pz generation will be described as follows: Asoospore 0-l was mated to aaco• spore o-a. or more brietl.y; 0-l x o-e (I selfed}. The expression in parentheses is a second convention indicating that the mating was lllllde between mycelia produced by ascospores from the same ascus (selfed) and that segregation of sex in that aaeus occurred in the first division (I). Ascua 0 shown in Table IX is the parent ascus of all the !• crassa ascospores studied. From the Pz generation !ihich was produced by a I selfed ::-.ating (0-1 x 0•8), six asc1 were dissected. In four of these sex was segregated 1n the first division and 1n two of them it was segregated in the second division. Stated briefly: the,P2 generation produced by a I selfed mating contained four I and two II. This is 67 percent I. The four groups in the Pz x P3 ba.ek cross generation contained 79, so, ao and 75 percent I respectively. 'l'he first two groups were produced by a I x I mating and the second two groups by a I x II mating. It is evident in this and the succeeding genera- tions (perithecia 124, 216, 230, 233, 239, 251 and 277) that first-and second-division segregation of sex !ll.11.y occur in asei side by side in the same peritheoiuin. Table XI summarizes the data in Table IX. is apparent that It the three types of matings ( I x I, I x II, and II x II) give approximately the same per- centage of r. The !!lean of the total 275 asci is 85 percent I, and the percentages obtained in the II x II matin~s vary no more from this mean than those obtained by the I x I matings. Table XII illustrates this point. matin~s All v~ the are grouped into their three differeuG classes. I x I produced 85 percent I; I! x II produced 84 percent r; and I x I! produced 88 percent r. -34- Table xr. Generation Summ1u•y o:f Table IX shoW1ng the percentages o:f :first division segregation produced by twenty matings in seven generations. Total No. oI' A:seI'. !fating '~-I ~ P3 1 P2 6 ! selfed 67 P2x P3 19 Ix I 79 Pzx P;s 5 Ix I 80 P2x P3 5 I x II? 80 P:a:it P3 4 I x II? 75 pl 16 I :x. I 88 F1 62 I selfad 86 F1 4 I sel1'ed 100 1" -2 ll I x I 73 F2 17 Ix I 82 P2 22 I selfed 96 P2 14 I selfed 86 F2 6 I sel:fed 100 F2 16 I x II 94 F2 17 I x II 88 F3 6 II sel.t'ed 83 P3 19 II selfed 90 P3 6 II sel1'ed 67 1" 18 II selfed 83 -3 100 L!ean -Ma- 8'5% I Tabl.e Xll.' Data in Table XI arranged to ahow the percentage of first divia ion segregation produced by all the I.XI, !XII and I:rx:rI matings. Mating 11Ulliliiir of ase.1 sh01fing .f'1rst division segregation I x I I x II I I :x I ! loo lumber or aae1 show- 1ng seoond diviaililn segrega.t:ton 27 Percentage Of first division segrei;ation 8~ 88 a 84 The best test -for the possibility of genie factors regulating the type of segregation is selection and subsequent crossing. Table XIII gives the results of six generations of selection for first division segregation. It may be seen from the table that even after six generations of I x I matings. the percentage of first division segregation is not increased. Therefore, there is established this definitive numerical datum: Sex in Neurospora crassa is segregated in the first division in the ascus in 85 percent of the asei and in the second division in the ascus in 15 percent of the asci. These percentages are constant re- gardless of whether sex had been segregated in the first or second division in either or both parents. It is not possible by selection to increase or diminish these percentages. Thus the type of segregation obtained can- not be directly attributed to the effect of genie factors. On the basis or pure chance, four types of II should occur equally frequently. Table XIV shows the f.'requency or each one of the fCllr types encountered in the forty II obtained. The 26 eases in which four like nuclei are grouped in the center of the ascus as compared with the 14 cases in which the like nuclei are arranged by twos in the aseus indicates an attraction of like nuclei for each other, but the numbers obtained are too Blll&ll to be conclusive. -35- 'i'abl.e ;~:n:I.. Results of selection for first division segre,;;e.t.1on carried on t:'.J.rougb. six generations. NUlll.001' Generation ,, ·3 of asei :Percentage of first division aeg;regation Mating l P2 67 (0-1::.0-:3) (: selfed) ?2L) 19 79 (O•lx6-2)(I x I) ?1 l.S 88 (4-5%6-5) (I x. I) Fl 62 86 c:..i4-:U7) '.;.. sal.::"ed) F.- 17 82 (223-7x229-8)(I x I) "' -35a- Ta~1e XIV= frequency diatribution 0£ the four poaai"u1e c;rpes or second d1vi.e1on aegregation. Type of ! I Frequenc;,r + + + - - 16 ++----++ 10 r+--++-- 9 --++--++ 5 - - ..j- -35b- IV. INHERITANCE OF CULTURAL CHARACTERS 1. Description of euJ.tural characters. Among the asci produced in the first two genera- . tions, no deviationsfrom the wild•type cultural characters were noted. In a third generation from which over thirty asci were dissected, one ascus was different from the rest in that the Dcy"Celia produced by four of its ascospores were extreme variants from the characteristic wild type growth. '!'his ascus (114) was one of the 19 others obtained from a sin6le mating, but none of the other cultures from ascospores of the other asci showed any indication of this variation. This variant was called tan and. by using it as a parent. several subsequent variants were obtained. A description of the cultural characters of these variants as they appear on corn meal agar follows: a. Wild Type. The wild type culture of !• crassa contains a dense growth of bright orange eonidia in large clumps almost filling the diameter of the tube a short distance above the agar. The agar slope is covered with a fluffy growth or short grayish white ae~ial mycelium. At the upper rim of the agar. below the mass of conid!a, there is a crescent of dense cottony white fluff. The substrate rarely has any color other than that of the -36- agar itself, but oc'easionally a small light or dark brown spot appears. Numerous bulbils appear in most wild-type cultures. In some cultures these are color- less and can only be seen with the microscope, but in others they turn dark brown and give the cUlture a specked appearance. Occasionally a few of these enlarge to the size of normal perithecia but crushing them always discloses the fact that they contain no ascospores. Al- though these sterile perithecia are encountered in both kinds of unise:mal cUltures, they are more frequent in those of minus sex. The abbreviation w is used to de- signate this type of culture. b. Tan. The tan variant never produces the dense mass of conidia that characterizes the wild ::ype cultures. The color of the conidia may be white, yellow or light orange. The substrate is smooth with no aerial mycelium and has a tan color. It is unusual to find the specking characteristic of the wild type cultures in a tan culture. Five classes of the tan variant are recognized: dark tan (dt), tan (t), light tan (lt), very light tan (vt), and extremely light tan {xt). Dar~ tan cultures (dt) pro- duce extremely sessile conidiophores and white cont:lia. Extremely light tan cultures (xt) produce almost the same color and density of conidial growth as the wild-type -3?- cultures. The other classes fall between these two ex- tremes in the order named. ~hat is to say, there seems to be a definite correlation, with few exceptions, between the density of the substrate color and the amount and color of the conidia. c. Albinistic. Albinistic (a) cultures contain fluffy white mycelium growing two or three times higher in the tube than wild type conidiophores. The ends of the hyphae that touch the tube send out a radiate fingerlike cluster of short hyphae tightly applied to the glass that are suggestive of appressoria. The llllllle albinistic is used because of the similarity of this character to the albinistic strains developed by Dodge in the species N. sitophila. At the base of the white fluffy growth, conidia are produced in masses considerably less dense and lig..~ter in color than in the wild type. Tan sub- strate sometimes occurs in albinistic cultures (ta) and when this happens, the hei,sht to which the flUffy r!l'1celium grows in the tube is much reduced and conidiwn formation is almost entirely suppressed. d. Even. The even (e) character gets its name from the fact that an even velvety yellow 3rowth of myceliUlll fills the tube completely a short distance atove the agar. The even character of the growth is very pronounced. It spreads entirely around the inner -38- circle of the tube with no prominences or depressions. ' The outer edge of this circle is made up of yellow elavate appressoria tightly applied to the glass. Much reduced sessile bunches of conidia are prOdueed below the ring of even growth. e. Pale. The pale (p) cultures differ from the wild type in the extremely light orange color of the conidia and the perfuse sparse growth of the conidiophores. f. Black. Black (b) cultures resemble the wild type except for the appearance of an orange coloration of the substrate a week or so after the ascospore is planted which changes to dark brown in a few weeks. When this c~l~ration is very intenae the culture is called dark black (db) and when it is not very pronounced, the culture is called light black (lb). g. Opalescent. Opalescent cultures {o) are characterized by a perfuse growth of conidiophores bearing a few light orange eonidia and a pearly felted appearance of the substrate. This felted appearance of the substrate sometimes appears in tan cultures (to). h. Orange. Orange (or) resembles wild type ex- cept for an orange coloration of the substrate. -39- 2. Inheritance of tan. Following the appearance of the tan variant in ascus 114, a series of breeding experiments were performed• Table XV gives a detailed record or these experiments and Fig. ll is a condensed genealogical tree. Three matings were made from ascus 114: 1 x 7 (w x t), l x 8 (w x t), and 3 x a (t x t). The first mating was made in duplicate in two different tubes. Fifty-five asci were dissected from this cross and planted on corn meal agar. Table XV shows the position of the ascoaporee in the asous and the various characters developed by them in culture. A first glance at the table shows the scarcity or opalescent cul• tures obtained from the ma ting made in Tube A and the abundance or them obtained from the mating made in Tube B. This indicated that accidental selection of one sector in transferring the cultures had resulted in hereditary transmission of the selected character. The next thing that strikes the eye is that a single perithecium as, far example, perithecium 233 may produce (in this case) five different kinds of asci. Further inspection of the table reveals that four tan cultures were produced from almost every ascus, "JUt the other four cultures instead of being wild type were -40- Table X:V Cultural characters of five generations of ~~. crs.ssa and position of as co spores in asous. First GQners.tion 114 - 1 x 7 { TUbe A \V TUBE B or o~ or 251 (252 w w (253) t t (218 t t t t lb lb lb lb (219 t t t t or or db db 254 0 0 255 0 ((220} t t or t or t b b 257 t ~ (221 w @ t t @ t b b 218(222 b b t t b b t t (223 t t t t lb lb db db 258(258 0 0 (259 0 0 (225 t t lb lb or or t t ((226) or or t or t or t t (263 0 0 224 lb @t t t t 0 lb 217 t t t t (230 230(231 (232 (233 (234 0 0 0 0 0 0 0 ((235) 0 0 lt dt lt dt 0 233(236 t t 0 0 0 0 t (237 or or lb lb t t t (238 or or 1.b 1.b t t t (239 t t or or db db t 240 242 243(243 (244 249 t t t 0 0 0 t t 0 0 © 0 0 0 lt lt 0 0 lt lt 0 0 lt lt 0 0 t t 0 0 lt lt dt dt 0 dt dt lt lt 0 0 0 dt dt lt lt 0 0 lt lt lt lt 0 0 db db t t or or b b lt dt dt dt 0 0 ~265 o 0 0 lt lt dt dt lb lb ... Yf or or db db 260 266 o {267 lt lt 0 0 dt dt 0 0 268 0 dt 0 dt 1.t ~ 0 0 lb lb " w lt lt dt dt or or lb lb t t t t 271 dt dt dt dt 0 0 0 0 or or t t w © w t 274 lt lt 0 0 0 0 dt dt t t or or db db or or Tube Bj (264 228(228 (229 x t) or or t t or or t t t db db or or t 0 lt lt 0 0 0 0 0 0 0 0 w ..,, t t t t 0 0 0 0 0 0 0 t t t t t t 0 0 0 0 (277 0 277(278 0 (280 0 0 dt dt 0 0 lt lt dt lt lt 0 0 0 lt 0 dt dt 0 0 0 0 lt lt lt lt dt dt lt lt 275(275 0 (276 0 B (281 lt lt dt dt 0 0 0 0 0 2 81(282 lt lt 0 0 0 284(284 0 0 0 (285 0 lt 0 or or 0 0 dt dt lt@ 0 0 ® dt 286 0 0 0 289 lt lt 0 0 0 ~ dt 0 0 0 w t t 0 0 0 0 291(291)0 (292 ..,, -40a- dt dt dt lt lt t t t t 0 0 lt lt " w Table XV - First Generation - continued. 11,1 - 3 x 8 (t x t) .,, _) 'II' 0 0 0 0 @ J.to VI VI lto vto vto vto vto to to @ to ~ to to w 627 630 0 631 0 (632 w w ,, f332{ 633 w w ~ ( 634 lto lto 635 "' 636 to 637 xt 636 VI 639 VI' 640 'N 641(641 to ( 5,,,.,:: y; w to xt .. 11' w to to 11' w 'If "Ill to w w ~ vt ® to to w ~ " w w 114 - 1 x 8 156 159 w " 164 0 135 t .. w t w ,,to to VI w "' to to w w w w YI to to w w lto lto w w to to lt to to t to to 11' VI ® w w to w to x t) ( 11' t to to w t t t w 0 t w t w w t w 0 0 VI ~ w t t t 111' t Table X. - continued. Second Generation 22:S•l x 230-6 331 t t 332 tt t 333 t t 273-3 x 290-3 (ox o) (t:J\t) t t VI @lt w c t t @w w 569 (569 w w w w @ VI @w (570 w w w w w w w <!>. w " " ... w w w w w w 571(571 w {335 0 0 t ( 572 w w w t t lt 0 @ w w " (336 w lt dt ® dt ( 337 w "' • 573 w w ... w w@@ ... ... w"' t t t t (338 t t t t 0 576 w w w w w w w ... 0 335(339 t t t t w {577 ... ... ... w t ~~~ (340@ ... ...w (341 lt lt dt dt w w 577(578 w ... w ... " t t t t (579 w w w w w (342@ w © vt vt 'If 0 'If 0 "If 0 "If VI 'II VI 0 'If 0 228-5 x 290-7 {dt x dt) ® ® . ~o~o .,, ... ww ...w (553 to to to to w 553 (554 to to to to w w (555 w 555(556 w ( 557 Vil w w VI 'If w ... " w VI to to to to to to to@ to to to to (558 dtodtoto tow 558 (559 to to to to VI w w VI 'ii' ... w (561 w W' W VI to to to to 560(562 VI w w w to to to to ((564~@ tow to w w w ( 565 w w w to t l to @ ® 566 567 9w to ~ to w w w w w dtodtodtodto VI VI -400- VI 'If VI w w w w w w 581 (581 w w w w w (582 w w w w VI ( 546 to to to w w ~ ~ (548 w w ... w ~ t (583 w 546 (549 ..... w t t ~ (550 w w w w to to to o 583(584 w (585 .. to to vlt to 551(551 w ( ~, '::' w 586 to to w '-""""- .,, 'If (580 w w 'If VI . w w w w w w w w w w w w w w w w w w @ w w w w . .,, . .,, w w w w w w .,, 223-7 x 229-8 (db x db) 345 b 346 b 347 b (348)b (349)b 350 b 351 b 352 b 353 b 354 b 555 b b b b b b b b b b b b b b b b b @ b b b b b b b b b b b @ b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b @ b 357 b b b b 358@ b b b 359-b b b b 360 b b b b 361 b b @ b 362 b b b b 356 b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b@ @ b $ Table XV - continued. Second Generation - continued. Ottapring of selected clone 160-lt x 160-8t (493) ® w lt " lt lt lt ~.t 494 .. w lt lt w .. lt l!:9 243-2x5 (o x o) (364 w w db db w 'w b b (365 dbdbw w w w b@ (366 w i K b b b b w w (36'7 b b w w vb vb w w ( 495 @ l t 0 'if " w ,, ( 496 w w 0 lt lt lt lt 364(368 w w lb lb vb vb w w (369 b b w w w w b b 495(497 lt lt w w lt llill@) .. (498 0 lt'ilt lt'l w '9'" .. { 3'70 db db ~ w w w b b (372 db db db db \IJ .. 'fl ,., 499 ® w lt lt w .. 0 0 3'73(374 w \IJ db db .. .. b b (375 b b w w w w b b 500{500 lt lt lt lt .. w 'If w (501 0 0 .. 'If lt lt lt lt 376 b b w w db ((itl w \IJ 502 w .. lt lt lt lt 'If 'If (378) db w .. lb w 'ft w w 503 lt lt w w w w lt lt 226-lx6 (or x or) 160-l;v x l60-8w g 449 :~=~~~== 459 eew@@wee wewea@ww eewwwwa@ 450 452 e e e e O O w w 453 e e w w w w e . e 454 w @ w @ e e O O 466 467 228-7 x 216-4 545a p @ p 545b w w p 545c t v p ~ {lb x lb) p ® p w VI to to lt lt 0 0 p p w w ilandom ascosporea 1'7 t : 17 w : 31 p -40d- (504 lt lt lt lt w w w w (505 ~ w w w lt lt lt lt 504~ 506 1.!:9 lt w w lt ~.. .. 507 w w w w l t l t l t 508 w w w w lt lt lt (509ltltltltw w ® w 509(510 lt lt lt lt .. w Vf ® (511@ 'if® w l t l t l t l t (512 w w w w lt lt lt lt 513 t wf w' t w w w w 514 lt lt lt lt w w w w 515 lt lt lt lt w w w w 517(518 ~ lt lt lt w vr w ® (519 W \Y W lt lt lt t 520 ( 520 w ''1 w w VI/ "' w " (521@) w 0 0 ® w lt lt Table X!I - continued Third Generation 368-6 x 374-8 (lb x lb} (724 (725 (726 ('727 (728 726(729 (738 (739 (740 ( 741 730 731 732 '733 lb w YI w \'I w w w w w VI VI w @ w w w YI w VI w w w VI w w w lb lb VI w w w lb w w 'If VI w w w w w YI YI w w 0 YI YI w w w w w w w w VI w lb w VI w VI w w w w w w w w 'If w w w lb w w w w w w w .,, VI VI VI 0 w 0 lb w w qb VI VI w w w lb w w VI ( 734 734(735 w w w w 0w w VI w w w w w w 736 VI 'If IV w 73f/ w w w w w w 'If lb VI w w w w w w w w w w w 545c-4x5 (p x p) 776 - 25 single as cos pore cultures all p 453-1.xS ( e x e) 742 - 150 ascospores all e 355-lxS (b x b) 755 - 15 random ascospores all b -40 - Table XV - continued ?bird Generation -continued 337-6x7 {t x t) 379 380 381 382 383 384 t t t W VI t 337-2x3 t t w w w w t t w w w t t w w t w w t? t? w t t t t? t? w t W W w w w w t? t? t w w w t? t? 338-2x3 (t x t) 524 lt xt w 385 w 386 VI 387w 388 W 389 w 390 w w ~ w lt xt 470 w 471 w (473 w 4"3(474 w ( 534 W VI to to w w lt lt ' (475 w ( 476 w 534((555) vt w vt w lt lt w w (536 VI W fi lt lt xt w w ( 477 w 537 tovto to vt w w w w 477(478 w ( .479 ® 544 w w w w t lt vt 1 t ( 528 528( 529 ( 530 (531 (540 540 (541 543 (538 538 ( 539 w w W Yf W w w w w w w w w w w w w IV W w Vf w w w w w w @> w W W W W W W W W W W VI W W W w w w w w w w w w w w w w w w w w w @ w .,, 338-6x7 (ox o) 532(532 @ w lt lt lt lt w w (533 lto vt w w lt lt w w ( 525 525(526 (527 (w x w) W W W yr VI (480 w (481 w (482 W (483 W 480(484 W w w w w w w @> w w @ w w w • w ® w w w w "' ® w w w w w ® w @ w w w w w w w w @ w w w w w w w w w @> w w w w w w • @ w w w w w w w w w w w w w 1.'1 w w w W V'I W "•T W W W W YT W W @ N @ w W W W w w ·;7 @ w W ( 485 w \7 w @ ® w @> w w w w w w w w w w w W ' l f W W Vf ( 486 W W W W W \V W w w w w w w w w w 486 (487 w w ® 'Ii w '."! w w w w w w w w w w (488 w w w w w w w "CJIWWYfWWWW 488{ 489 w WW@'N''fWW@) SU SU SU SU W SU SU SU SU @) W W W W W W -40 - (490 w 7{ w w w w w .·1 'i'I Vf w w Table XV - continued Third generation continued 338- lx4 { t x t) All wild {674 w (675 vr 674(676 w (677 w (678 w (679 w VI VI VI all tan VI VI w w VI VI VI ® w w 696(696 J!D to llD to to ~ to to (697 vto vto "<to v1r> vt vt vt vt w VI w w w w w xt w w ~ 716 Jto llD vlD vlD Jto lio vlD vb:> xt xt w w w w w 71 717 vto vto vto ito ltJ lD :im l1D w w <i VI w VI w " w w w " .:.:xtremely light tan and wild '~ixed ( 663 .:rJ:o J!ID Jd:o J!ID W W VI VI ( 643 lio J!D Jto ]to w w w w ( 664 l!ll:l :ito 0 .:'Ito w w w w ( 644 xt w xt xt 0 0 VI w ( 665 W VI W 'If .:¢0 xto :xl:o Jfz 043(645 w w w w w w xt xt ( 666 w w w w :lllb .l!to :ito .ltD (646 w ~ w w w w w w ( 667 VI W W W :Ito .l!to ;to :itXJ ( 647 w @ ® w w w ® w 663( 668 W W W VI :i!l;O J!1D :to Xn ( 669 w w w w xto :ito :to :m (670 w w w w xt w xt xt (648 llD 1D lio lio lb to lt lt ( 671 .xto :xto :xlD ::d:D w w w w 648 ( 649 w w w w J!:o JlD :xtx:> lto ( 672 .:r.ID :xto a> .Jto w w w w (673 xt w xt xt w w w w 685 xt xt xt xt w w w w Light tan and wild { 650 w .,, ( 651 Vf w 650( 652 w w (653 €} vt ( 6 54 w w w Vf w w w vto :l!to vlD i.to -;to vto vto lJD llD lio 0 Vf Jto lto ]fl) Jt.o w vt vt w w w w w ( 655 w w Vf w lto llD Jtollo 655( 656 DD JJD ~ ('.!iQ w w w (657 w w dt'l:!:t vt dt w w { ,359 t t t lfD W VI W W -40 - Table XV - continued Third gene rat ion - continued 338-l.x4 (t x t) continued tan and wild (661 661(662 O 0 0 tow w w w to to to to w w w (680 (681 (682 680(683 (684 w w w w to vtn w VI w " VI w w VI VI VI " :u.oO o to ® to vto w w w to to 0 lto VI VI to to to to xt xtoxt lto (686 to to to tow ~ w w ( 687 lto to to vt w ® w w 686( 688 to to to to w (i) w w ( 600 w w w w to to to to ( 690 ® w to to w w to to 1691 691 (692 (693 693(694 ( 695 (698 to to to tow w w w to to 0 0 w w w w ... w w ® to to to 0 w w w t t t t 1lf VI @ w Q lto to to w w w w t 698( 699 vto llD l1o :xto w ( 700 lto llD 1.to 1.to w ( 701 w w t w to xt w w @ w w w w w :it.o xlX> :idD 1 to (702 w w w w llD 1lD lt> lto (703 xto .xto 'Vb:> 0 w w w w 702( 704 w J'.. w w lto UD ];o lto ( 705 ltO U XtO vto w W w W ( '706 t Q t Q W VI W VI -40 - ( 7fYl to to w @ w w to to 70'7(708 xt lJ:o J.to )to W W VI W (709 WW 'It VI llovtQ vt (71.0 t t t t w w w w ( 711 w w w w Jto JJ:o Jto lto ( 712vto "ilto w w w ( 713vto vto vt.o lto w 71.2( 714lto lto w w w ( 71.5 w w 1lD O w w 'Vix> vto w w w w llo lto w lio 1. to ( 718)l!o JJ:o ]to w .,, 1.to.,, 11' ( 719lto lf:o llo 1.to w w w w ( 720 w w w w JID :xto lb:> lto ( 721 1'D0 w w w w .xto lto 718( 722 ti) w • w J:f:o ito ltD lto ( 723 Th:> Q 1.to J:1:o W VI W VI TABL:Zi: XV - continued ' Third generation - continued Off spring of Selected Clones 338-l w a.elected x 338-4 w selected 835 t t 836 w VI 837 w w 838 'fl VI 839 .,, w 840 .. {84llw (j 842 w w 843 w ® 844 w w " (845 t 845(846 t t t t t w w w 'If w Vf w 'ii' @ t t . .. w w w w w w ® w t t w t w w w Vil Vil w w ® \V w w w w w w w w t t w YI VI 'If t t t w @ w w t t 'II 'II w w t t w w w w 1lf w t t w w w w 847 W' Vf w w 848 w w w VI t 849 VI w? t w 11' ® 850 " 851 t t w w 852 w t 11' t 353 w w w w 854@ VI w @ 855 w VI w w 856 w w VI w Total( 5 (ll ( 5 VI t t t t w ® w ® w w t t w w w w t t w VI t t w w w VI t t w w w w w w t t w w t t w w w VI w w w w w t w w t t t w t t c;!l.oh ascus. except 845 and 846 are from an individual perithecium -40 - TABLE XV - eontinued Fourth ' Generation 381-8 x 379-6 ( ( 593) 593 ( 594 598 600 GOe (w x w) a 'If a w :x:ta :x:ta lta a 0 a 0 a .,, 1f w w 595 w w w 0 a vta lta lta (598 ( 599 (600 a w w a w (t) Vf to w a t a w w 0 (603 w ((605} w w w (606 w (607 a ((608) a w 8. a <A> "aa w w a a w a I .., ,r ,.... \VV..; a w w 0 0 a a a a toa xta w w w ® a w a a ~ a a a a lt a w 0 a w ( 610 (611 (612 a w (613 613(614 w a ® 615 0 x.ta ® t --{ 616(616 ( 617 618 619 620 623 a toa tc w a to a w a e w a to a ta w 0 t to to ''If VI a w w a 0 Vf a a 0 w w a lt to 0 w a w VI' "' "' w :x:ta :x:ta a a w w w w a toa toa@ a w w w w ® a I\ l!' w w w toa toa w a a w ~ ~ a w w w e e e 8\7 o a a a: w w toa 8 w 742 -(e x e} (743 743 (744 (745 e e e e e e e e e e e e e e e ® @) ® e e -40 - e •rABLE XV - continued Fourth Generation - continued 775-l x 8 (b x b) 831 w w w w w w w w 832 @wwww@ww 833 w w w w w w w w 834 w w w w w w w w -40 - Table XV - continued Fifth Geru\ration dlO-l x 606-'7 ® " lt lt w 748(749 w 750(750 (751 w ( 753 753('754 ('755 w (756 756(757 ( 758 t lt w lt lt 760((764) w ( 765 t w w w lt w vt 'if lt w lt ~ 0 0 0 ... 'II' w w w "t ~ lt w t "w t 0 w 0w ~ w vt vt t w w 0 0 VI t w w ® w w w t t w t lt w I!} lt .. ~ ® ( t x t) w 'ii lt 0 \'f 593-1 x 6 lt w w (a x a) 766 a a a a a a vta vta. 76'7(767 (768 a. a a a a a a a a a a a ta a ta a 769 7'70 a a a a a a 771 a a a a a a a a a a a a a a a a a ta a a ta a ~ ata ata aa a a a a a a e e e e e e 7'72 (773 ta 773(774 @ a 743 -1 x 6 (778 (779 778(780 (781 e e e e e e e e e e e e (1 e e e (e x e) e e e e e e e e e e Also 28 random single ascoapore cultures - all e -40 - TABLE xv - continued Sixth Generation - continued 772-lx6 (a x a) (762 782(783 ta ta ta ta a a a a a a a @ ta ta ta ta 784(784 (785 a a 786 787 783 a a ta ta a ta ta a a a a a a a a a a a a a a @@ a lta lln a a a a a a a @ a a a a -40 - TABLE XV - continued ' x F;; F (a x b) 5 772-8 x 775-1 a a a a 826 @ a 809 816 a 822 a 812 VI w 814 w w 321 0 0 830 w w 813 815 819 820 826 w ~'I 'If w w w w 'If w Vi w w @ @ VI a a a a a a a a a a a a a w w a a a a a a a a 0 a a a a w w w w 'II VI 'If w ... VI w VI a a a a a a ... w a 0 w w a 0 w w w a 810 w 811 w 817 w 818 w 823 w t827) w 824 829 w 'If Vf a VI VI a a a a a a w w VI w w w a a a a a a a 7r \7 a w w VI w w w w -. a a a a w w 0 w Vf Vf a a a a a a a w a w a a a a a a a a w w 'if a a .,, ... a a -40 - mostly diverse variations from wild type. the 414 ratio was not obtained. In seven asci From four asci (226, 233, 243 1 and 254) there were produced two tan and six nontan cultures. From three asci (229, 234, and 244) no tan cultures were produced. In many of tbe asci two different kinda of tan, light and dark, were obtained. This indi- cated the effect of another factor modifying tan. Fig. 10 gives these data in condensed form. Instead of indicating the eight ascospores in an aacus, four symbols are used to indicate the four pairs ot aacospores. When individual a.sci are indicated, the first symbol to the left indicates l and 2, the second s;;rmbol indicates 3 and 4, the third symbol indicates 5 and 6 1 and the fourth symbol indicates 7 and s. When a number of a.sci are indicated, only t~:e ratio ia given by the symbols. All cultures other than tan are shOll'n as wild type, because all of the non-tan variants resembled wild type in most of their characters. From a second mating within the ascus, ll4-3x6 (t x t)J fourteen asoi were dissected. duced four tan and Nine asoi pro- tour variants resembling wild type, while five asci contained two tan producing and six nontan producing ascospores. It is obvious from this test -41- .r-- ---······---1J,ij__ ( w i) . ..\, > x g (i· xt) I x7 (w d) t t W w tJ/j 11/tUu f 1xi(w"'i) (-t <c vv~ '1 \.tw w"" 5 tw w vv If 55<1Sc1 l.y I: ~- 5 QSc1 { W W\A./ W 3 -->----~-------:----~-~~ :ZC)ovvvv*'+ -z..sso.,,,tt Z.<\O<.lott - y-ww -::c - / 2,7-) ~· ><.::::::~ Rs"'"'~" ' ly,,I H1 ,\ l1 ' ~ -I: Id(+<+) s;.. 338-\: -;,o&,~,,J /6lls1 'vvww wf6 r;,,-1 id- {-i w w IP // Q~c 1 { ·t- + t t ( t· 1"14><1 fu "'""'~ \. 0 I x 'I ( + <") ffsiJ"\ !/\ '''°"' 71 +-.+;<..+ ww b'I s "'! k <. ~" " " " /lSet "I+ "' t: ' t ;1, <;> L2. :LO lis ,, n Ct-"~ L x. 7 (o v. <) (w\Nwvv/V +i- w & t.t-H W w 39 <+xi w w l'I w Zo4,,;fwwvvw1-• I' S<. ' - W VII 1.. wwviw8 W-----y··· yr I lSel<d••) \N 6._Jc, vv /l_ t { ~i \N W \N l>T~d) '"'d{wwvvwl '•"J[Uww{, ~ -r r--' 1, ,-":' \ ,, II \'V vv vv t -I:: Fi 'l I o G-e..._ .... ._, 6 'liC<v\ Tv-..~ ct\"'"' (.l,: d<L!) w~·o./<cf<4) >.O(tN,>v) QSC<> ~ s· vv vv w v< ilr-w_:;;:·t= ":_~ 13 f<\"' d,.,""' 4(> ,' I ec \ , '))-"1 ', '-. ___ ,, _______,_,, 7<t<c1{f{w~;1 fJQS<if-lc-lc wvvlZ. ) vvwY..1wt Q 'I c __...."''.--* ~.. vv tf?.J.,).,/), -le €•/<o/ •4) ~-~------"""'"--------...:----··----- It: M /{,O {w"", i; 473i:o-t0 "\ + - t:c-1t wvv5 "'M \f\l,l~-l'yr"- that the tan charac:tter does not breed true. A third mating 6 ll4-lx8 ( w x t) • was made. Five asoi were dissected and these produced each four non-tan and four tan cUl. tures. It was noted at this time that when eon1d1a and l'Jl7oelium from tan cul turea were transplanted to other tubes, the tan character usually failed to appQ.r, the culture reverting to wild type. Two tan cultures (160-1 and 160-8), ottapring of this third na ting, were chosen to study this phen.,• menon. Conidia were sowed on plates and subcultured by cut ting ott hyphal tips and transplanting them to small tubes. 'l'he sUbcultures from each original culture were selected for tan and wild t'YPS• AbOut 400 subcultures were made in from three to six clonal generations and cUl.tures of wild type were obtained which rarely produced any tan offspring. The tan lines, however, always showed some reversion to wild type. One or t..~e tan cultures selected from 160-1, was then mated to one of the tan cUl.tures selected from 160-8 and seven of the resulting aaci ..ere analyzed. Each contained :f'our tan and tour wild type producing ascoapores. one or the wild type cultures selected from 160-l was mated to one of the wild -42- type cultures selected from 160-8, end thirteen or the asci analyzed. Twelve contained four tan prodUeing and four wild type produei:og ascospores. One of them oontaine~ eight wild type producing ascospores. As shown in Fig. 10, two more tan by tan matings we:roe :nade 1n the Fi. From the ma ting 223•1 ::t 230-6 ( t x t) , eleven asci were dissected. Ten oi' these produced asc1 containing four tan producing and four wUd type producing a.scospOI'es while one of' the asc1 produced eight tan From a second mating 28S.6 x 290•7 (t x t), cultures. nineteen asoi were dissected and each contained tour tan producing and four wild type producing aacospores. It is evident that the tan character does not breed true any more readily in the !'2 than in the b\ 1 and th.at the attempt to stabilize the c'-aracter by asexual selection had failed. Fig. 1.0 shows a third matinp; of' opalescent by opalescent amo:og ;he sister asci of this group. The asc1 from which tr.ese opalescent cultures had been obtained had also produced ascospores i':rom wniah tan mycelia grew. Sixteen asci were dissected from this mat- ing or 290-3 x 273•3 (o x o) and every ascospore produced the wild type character. -43- Opalescent resembles wild type closely so tbat it is difficult to state absolutely that this was a Cqse of reversion, but it is important to note that no tan appeared 1n the progeny of this opalescent x opalescent mating. Apparently tan was completely separated from its allelomorph in the second division in the ascus. A second mating of opalescent by opalescent was ll3ll.dfi as is shown in Table XV. The parent ascus (243) had produced two tan and six opalescent cultures • • From 111at1ng 243-2XS ( o x o), twelve asci were dissected. Ee.eh ascus produced four ;;lld type and i"our >clack cul "urea. It 1a clear that one pair of opalescent cultures differed ger.etically from another pair. The mating 823•7 x 229•8 (db x db) produced onl~- black cultures. The '-lack character bred true in this generation, b'lt on later inbreeding of this line, as shown in Fi,;;. l.l• only wild type offsprin.:' were ob- tained. It is interesting to note that variations of intensity of black were entirely different characters _;enetically althoUgh one looked like a dilution of t."ie other. This fact •_-raa shown by the ma.ting of the light ~lack and black culture (228-7 x 21C-4) (lb x b). Each ascus produced pale, wild•type, and tan cultures in the -44- ratio of 2ilt1. sut:y•flve l't.nd.Olll ascoapOl"e oulturea ver1f'ied thia ratio. 'lbia ahowed that non-tan aegl"e .. gatod trom tan may agd.a throw tan oftepnns. A se- cond example of the • .,.. kind 1• the inl>'l'ff'din.g of tho aJ.b1.n1etio 1Sne, enowu ill J'ig. 11. Although the parent• ot the albbiatio li?IO ••r• wlld.'!" t11"1. when alb1n1at1o 1 wae pro<S.ucec'l, taa 1'$&ppctare4. A atr1Jt1ng o:r.alllpl• of the taot f;bat the ott• 1p:rmg obtained by mating two like 11.yoelia ma1 ahow ent1re1r new charaoton 1 ta in the ma.ting of the two orange oul.t'03!'•• 126•1 z 226-6. Each 1tacu1 produced four wild•t;ype and fOUI' e•en cU.l tul'••• Aaol $3'1 and 338 W.1'• ae1oote4 troa ~ aecond generation tor f'W:tther ln"Hding test•. in.g 337•6lt'7 ( t x t), aix aaoi were diaaeoted, l'l'O!lt •t• Four tan and. to'lll" 11114 trpo Ollltvea •r-e produced from ea.eh asoua. 'file pa.rent•• grand•parenta and one of the gl"e&. t:..gran4parenta we:N tan tv.t atlll tn. llllltin.g three balt tan and half wild t1P0 otf'spr1ng. Two mati~a of' tho non•tan cult\U'al eh&raotora appearl.na ln th& aso1 :S37 1u1d 338 were made. FrO!ll ma,\.. in& ~7·2X3 (w ~ w), a1x aao1 were dissected. Only wildtype ot:f'opl'i.llg W$1'9 produoe4o •45- 'rwO Of these when 111$.ted Pig. 11. Genealogical tree showing the development of pure 'breeding lines by i.n.'brced1_ng..,._.___ - - - - - 226 or t or t Pi F2 337-w w t t P3 :579-t t w w •:581-t t w F4 L .....\ . / w ( '°"t with) 45:5-e w w • v ,....i . ._. v 229 228 d'b t or ,'b M5o•t p p w y b 742•Random 776-Random aaooaporea aaooapor•• all e •o•tlV v 7<>-'\/ • v 1''5 772-a a a a (•Ollle with t) 778-e e e e 11'5 783-a a a a ( aome with t) -45a- 216 216-4-l'b 776•Random aacoaporea all p 934-w w w w ' -" tl1nw wild t,.,e 1n the nen a-mratloa. Pl'• mats.aa Qa.sx7 ( • x •), twlnity uot were dia.11utetett. A.ll et the o1'tapPf.ng wen wild tJfe• !lila la the me 1'e1nll.t u that obtalne4 1n oJM ot the ••l1'inga ot opaleaoent 1n the 1'1nt ..-_.atton. A.pplllnnt.17 the w114•t7P1J . 1ud o,&1e1Mat '1U.tvea obtalud" ta aatl aarr n4 3a8 4o not ~,...., ·oae14aal& . . . .,. or tan ottapt'tng. It 1a 1ntoPeatll\g to no'h, en the othel' baa4, that J)Paottal.17 half ef the o:ftapnng ot the tan cllltUHa tHll\ the sa.1111 two aaol were wUt•t7Plf• ~· mtlag 131-bl (t z. t)• 4laae•te4• tw•nt'f &ael nn ft••• tell llbarply tnto three ellllaMI. 'L'ea aeol produtot oa17 "'114 tnel •icht aaol pt'Ollae4 halt wi.14 t;ype an4 halt tan, im4 wo uot p1'0!.luoot halt ril4 tne and halt etibmel."p<t. ( Sttbllel'pct ta ahown bf the h1Jlhlle h • tht 1•11'1Unated aatoepore powtng oat,- a tn oentd.iaewra *fflh the ap.r beton the tlllttb'tl 41••• Thla la a tl'tUt lethal ehal'aoter). .IOH lnteHa111ng ilhan the ta.t thilt thr" kinda of aaol \fe:N pt'oduoot, ta the faet thlltl all at the aael hma a alngle patth••l• we1'9 alike. s.tenln, to Table D, Pl!l2!'1thee1a &s&, us, am NO, ~- 01111 asot wl:d.Oh eoataf.ned el&bt w114 t;ype pt'Od•du,g ••••SJ>OH•• P•ri theota 631 an4 634 pro- d\loed oa17 aeel 10D11a1nlnc tO'IU' w114 type productnc and -46- t01U' tan pr«!1&oing aaoospol'ea. .Perltheoiua 538 oou• talud. onl1 aeci produ.0111& tour wild trpe prodU.olng IUld tol.U' au '-<lllOl"ged producing a.aoo8p<:ll'ea. A second 11u~tin& we.• made 1n aa011t ~a. two tan ail!tera ot 2 and a, that la, 1 and •, l':t'Oll tld.e mating, 331-lx4 (t .i; t), r.,, 'l'he ••N mated. 79 aacl ware diaaeoted. " Table XVl lhowa the ll'Wllbel"t ot pePt~oia whioh toll into the th~• dltfa1"8Jlt ol•••••• (1) all wild t1JM•, (8) tolll" aaooai»rea Wild t7pe and. tout" tan troa eacl:l aaoua, (a) all tau. 'table XV give• the detail• of ihia utbg sn4 lhowa tba t '21• eeoond claaa 01111 \le su'bdivid&d L~to ct lea.at th:ree othoP olaM••• In two perttheota, 643 and G•&, the aau were 111sed. It wlU \le l&tel' ahOWA that the 111.xed. penthecia oau " lo4i0&ll7 explained, ata1lai"1t7 'fh• ot the aeoi obtalntd tl'Oa the aue peritheo1WI wu doll gl'tatar tlml oan " mdioatctd la the table_ There waa I. atPiJdng \Ulit01"llit7 0 t the Oul tllJ"ea from t&ob pel'1tbeo1• in the col.or of the oonidia, the habit ot growth, the apdd.ng ot the aubtltrate, the pr-odllctioQ ot tlldt, etc. that; diatingu11hed. the• fl"Oll tM culture• tro• otbex- pe1'1th•o1a. spaee doea not perJdt a 1\1.ttiolent 4eeol"iptioa ot tbeae Mtte:renoea. It i• olear that the na tvr• ot the aaou.a ta deul"lll1ned at the origin ot the pel"itheo1u. 'fh1• i• a genet1e ®mout:rat1on e~ the lila.M taet that Haatpe ( 42 ) de•utrate4 e'J't•l•aieaU7. -48- 3. Proot ot zmolee intereha~• between tbal.11 ot oppeaite aez. An ex.pel"lment on the oroaa 'breeding ot P1ll'9 lin"a gives a elee pl'OOt of the tsot that noleal' intel"Ohange l'll'llat be efte•t•4 between tballl .ol opposite sex. Pig. 11 " ahowa the 4evelopmcmt and anoeat1"7 ot tour pli!'e llnea that were d.evelop$d. Table XV. The details ot the eta are given in Th• even line bred perteotl7 true. The a.lbinis- tie lines always threw a number ot tan e'llltures, 'but all these tan cultures were also al'bia1at1c.. Proa a matii.g 772•1 x "'3-1 (a x e) several asoi were obtained pred.ue- ing tour al'biaistio and tO\at" even eultwea. produced se11Ut wild tnJe• aoae a.sol Thia la expected .S.noe noh pure line ma7 eontaln the noPlllal alleloaorpb ot the other. The detail• ot the rea'lllt will be. published. la a later paper. It is iaportant to note that the re- eove17 trom a single aseua ot true breeding types 110 diftel'ent tl'om the wild type and ot euh d.ivel'gent ori• gin la ole&P proot that mioleal' interllhanp between the two different thalli D&t have taken plaee. -49- 4. Cytological and genetic evidence of the "double fusion" in N. Grtu111a. Harper ( 42) first proposed the idea that there were two seXWll fusions in the ascomycetes. He demonstrated cytolog!cally that one fusion takes place at the initiation of the perithJcoium. Dangeard ( 14) ", had previonsly shown that one fusion takes place at the hl1t1at1on of the ascus, after the psr1thecium i111 form8d. Cytolo31cal and genetic evidence will be pre- sented to show that both these fullions occur in N. orassa. Thie phenomenon is called the "double fusion". It will al- so be shown that the first fUBion in the case of N. craesa involves four nuclei, two from each parent. tha.t three 11eio11es mu.st occur. ~1eio11as ·- 'l'his means The places where the 1u1 occur will also be shown. liiat ings were •de on a thin la~er of agar sproad on slides and the hyphaa killed with Fle~~ng'a wit1.out acetic and stained with iron hael!!Otoxylin. Plate I,(figs. 1 1 2 1 and 3) show hyphae of!• .!!!~!.!. in the neighlorhood of young peritheeia. Each cell in tr.ese hyphae was a dicaryon except the initial of the peritheeium which contained. four nuclei. clei were large and st~ikingly paired. The nu- These myoelial threads could be traced back through anastomoses to -50- unisexual hyphae such as those shown 1n f1g. 5. These threads grew rapidly and the lllyeel1a 8.8 well Qc the per1thee1Wll in1t1al became mttltinueleate, as shown 1n fig. 4. The initials coiled as shown Lt figs. 4 and 6, and a gl"OWth of' hyphae around the ooil formed the peri- theoiu.m. Within the perithecium of N. oraasa, typiea crosiers were found as deseriN1d b,' the Koreau.x. shows five such aaeus initials. F'ig. '7 The ~oreau.x have shown that trie crosiers, like the,one shown 1n fi3. 7d, arises from a uninueleate coll. It is on this fact that the complete proof of the "double f'usionn rests. There 1s much objection on a priori grounds to the idea of a "double fusioan. On such a ~•sis it could very well be objected that the diearyons in the peritheeit1.111 initial were merely as11oeiated nuclei which divide conjngately until reaehin.1.5 the a&eua initial and t ere fuse. 'i'he finding or the More11:1Ut that the aecus crozier arose trom a mononucleate initial, combined with the genetic evidence collected by Dodge and the writer, firmly establish the tact that a "double fuaion" did take place. The genetic evidence is clinched by cross breedifi6 of the pure lines albinistic and even. -51- The genetic experiments already reviewed show that both a tan x tan and a tan x .,ild mati ng may produce tan and wild offspring. This is a strong argu- ment against the use of characters that have not been inbr• d until there oan be no q i»stion of their purity, in breeding experiments designedr;~o solve the question , of nuclear interchange betw en thalli of opposite sex. Furthermore, it is an argument agninst the use of wildtype in matings to study nuclear behavior because several of the variable types studied revert to wild type. How- ever, a certain definite percentage of wild type must be expected on the basis of the assumption that eaoh pure line contain the n·ormal. allelomorph of the other. Albinistic and even were both carefully tested for purity of t..1-ie two ohe.raoters involved. was 1!1!1de between them. A !!lilting The offspring in many asoi con- tained four al.binistio and tour even producing 11.scospores. This is proof of the interehange of nuole1 between two thalli of opposite sex. The!'e are two objections to the interchange of nuclei between thalli of opposite sex in Neurospora t:1111.t have b~en recently raised in the literature. 'l'he first 1s that of the Moreau.x.(52)Xhey point out that in -52- both homothallio tho peritheoia ori;inated from a coil suoh as that L~own in Plate I. fig. 4 1 and that ~here was no evidence at the organ thst they consider to be the triohogyne effecting a union with the coil. Pig. 6. shows a coil origina- tin5 near two anastomoses. CoiYs;always originate near anastomoses, but it is impossible as Koehler (49 ) has sl1own, to find a field i:i a total 1110unt ot a culture o:t fieurospora the t doos oo t contain some anastomoses. Furthermore, the septations in the larger hyphae a~e Lehfeldt ( 51 ) in the basidiomyeetes and should allow an easy e:irohange of nuclei. It is thus clear that an organ au.eh !ls a trichogyne 1s Ul'l.'lsoe1uary and t"ia.t if' it did exist, it would tis at some stage previous to the initials, arid not at a stage so lste as the ooil. Tr.a second o";Jeotion is that of Koehler ( 49 ) • Re pointed out that an.astomoses were eqWtlly numerous between the hyp.':lae of like and unlike sex. formed the .:'ollowin experiment. Re also per- He grew conidia of un- like sex together and re~oved and transplanted the hy• phal tips expecting to gst some of them to produce peritheo1a. He failed to obtain peritheoia in ever:r one or 150 trials. The following experiments show that this would -53- be e:iq;:ieeted. \'fnen ma tin :)l were made in test tubes, 1 t we s necessary to plant the two inoeula some distance fraa each other in order to obtain many peritheeia. If the two inooula were planted in the same spot the crop of peritheeia was emall. Dodge ( 29 ) has sh\JI' n that peri• y' theeia arise usually on only one "~ide of the plate when two inoeula of opposite sex are sides of petri diahe s. placed in the opposite The writer spread a very thiek suspension of mixed spores of opposite sex over the surface of a plate of corn-meal agar. No peritheeia developed. In a see td plate, half of the plate was eovered with the thick suspension used above and on the half of the plate inoculated, no peritheeia appeared while on the other half a few small peritheeia developed. In a third plate, inoeula from the same two eul tures we"9 placed e.t opposite ends of the plate and numerous perithecia were prOduced on one side after the m;·celia met and later a sprinkling of peritheeia appeared on the other side as well. This evidence showed that Koehler' s experill!.ent did not t:is t the question of whether or not nuclear interchange took place at anastomoses. There is ample proof' that the -ascus nucleus originates by the fusion of two nuclei. -54- Since the Moreaux have &hown that these two nuclei are derived from a single nucleus and since the aecospores produced from the esous nucleus the o in tr~ ~10W tho cr~r~oters of both parents 1 conclusion possible is that the sin~le nucleus asoogenous hyphae is a fusion nucleus. Since pairing in the aecogenous hyphae did not occur, it is reasonable to believe that the p:U..red nuclei found in the peritr.eoium initial fuse. This is the first fusion, and the crosier fusion is the second. -55- 5. riments on the inheritance of tan and wildt;rpe by the use of sel: eted clones in $ ting111 and the in~; of these data on the question of a tetraploid fusion in the peritheeiu.m initial of ?~. erassa. The unsuceessful. selection experiment cited in the ease of the tan cultures from asous 160 1 was '¥~ I repeated an the tan oul.turel! l and 4 from aseus 11138. Cc:tological study of pyphal tips, aftel' the first ex- periment w1u1 completed, l!lhowed that the hyphal tips were more crow4ed wttt. nuclei than any other part of the thallus, and since these hyp.~ae arose from an anastomosed myoelium originating from a polys~orous inoculation, it was decided to try aae.l!:llSl selection by single oonid1um ~ilturea. A single conidiwa some• times contained a single nueleua and often three nuclei, but in the majority of eaaea, two nuclei were found in each eonidiWll. Single conidia were isolated from the tan cultures 338-1 and 338-4. T'ne subcultures grown trCJm these conidia fell into three elasaes1 tan, wild type, and intermediate. Tan and wild- type were selected from both sources for fUrther aeleetion and at the end of the fourth aaeX1.Ull generation, all four clones were apparently pure. Selection waa made for a fifth generation 11-~d all twenty cultures from each clone -56- maintained their purity. Then two ot these purified wild-type olones were mated with the result shown in Table XV - Tni'"ti Generation,Offspring of Selectnd Clones. A sinc:;le litllcU.s wcu1 selected from each per1- thec1um since the previous results from unselected clones hlii.d s:1own that all of the.a11oi from one perir.' ; theoium ware alike. The table shows that three typelll of perith$eia were obt!l.inodJ one in which all eight spores trom the asou.s produced wild-type cultures; one in which six wlld•type and two tan 11>peres were obtained from each a.sous J and one in w'i 1ch four wild-type and four tan spores were obtained from eaoh aseus. There were no peritheeia fou.lld. containing all tan ascospores and a new type of peritheoium containin0 asci with a 3:1 ratio of wild to tan was discovered. Table XVI shows t.lie kinds of peritheeia obtained from the lli£iting of the Ullll&lected clones 338-lx4. Calculatin,~ the total percentage of wild- type found in all qt the various kind of perithecia ~not asci) 1 the table shows that 47 perru~nt wild•type were obtained. Table XVII shows oom.parable dat"- ob- tllined from the sting of selected clones. It is ap- parent thet the ability to throw tan hes not been lost entirely by selection, but the percentage of wild-type -57- Table XVI of p~ritheeia obtained from of unsc:lect'''i clones 338-1 x 3:38-4 {t :it t) Clae~es t:t1e ~ati 1 '. Classes of Perithecia ww t l t 14 t .ii,t t 2 per• centage of w asoospore 47 'l'otal Table XVII. Classes of peritheoia obtained from mating of wild t7Pe clones selected fJ'Om tan clones 338-1 x 338-4 through five oonidial ,i;enerat ions. w w w w w 'II! w t w w t t 5 ll 5 Total peroentage of w ascoap:'re 75 -57a- has been increased from 47 to 75 percent. The pereen- tage of tan r~s been decreased from 53 to 25 percent. The new type of per1theoium which was encountered, namely, one which contained asoi with six wild-t7pe and two tan asoospores, as well as the absence of the all tan asoi, were ftll'ther evidence of the positive effects of selection. ' The seleetions were made ju.st as soon as it was poss! ble to reeogniae the cultural characters, that is, two or three days after planting the single conidiu.m. No matings were made between the asexuall;r 9eleoted tan ou.ltures, but after they had ,;rown for a few weeks, forty polys"orous trmsfers were lllllde from them. All of these subcultures dc1veloped wild-type or intermediate cultural characters. ThB.t this can• not be considered evidence for mutation of tan back to wild-type is ~hown by the feet that when the hyphae of a sister of a tan a11coap:;re are 'l'.!Utilated 1n transferring the germinating asoospcre. the cultural character developed by the mycelium is '.zild-type. Th.a main reason why hyphal tip selection is so unsuccessful is because eilllilar phenotypie variations are prod:uced by the nr<l• tilation necessary to remove the hyphal tip. This is snOW'!l by the uniformity of hyph!i!.1 tip cultures~ as -58- c~upared with the diversity or single conidiU!ll cultures made from the same original e\llture. 'I'heae experiments show tr,at thallus developed from a ain3J.e a11oospore contained 1110re than one kind of nucleus. In this connection, it is important to recall - that the aseospore of E. eras a 1a a diearyon. -~- Cytolo- Y-; I g1eal investigation of Dodge 1 eonl'i'.M!led by the writer ahowed th as soon as the wall or the asoospore began to form the single nucleus 1nelosed within the ",all divided fol'l!ling two nuclei which took up their positions opposite each other on the short axis or the spore. Moreover, the writer has found that the single nucleus delimited within the aeoo11pore, followin; the third division in the '.:sous, was very different from any ot the other 11.aeus nuclei. long stringy spireme. It er.larged to tioally the These stages were very freqt1t1ntly found and apparently lasted longer than any of the other extremely infrequent. this diearyon, which resulted from a nucleus resembling one preparing for l!ll!l.turation, 9ontained different kinds of nuclei. -59- In the face of this evidence, it was unnecessary to assume mutation as the cause of these different kinds ot nuclei, but much more reasonable to suppose that the nucleus delill'lited in the ascospore was a diploid nucleus (homosygous for sex) preparing for a meiotic division. From this meiosis, a maximlll!! of four different kinds of nuclei could be formed by a combi?ilt ion o! pre- and postreduction of the various faoto~s. It has been shown that the ftT'st two divisions in the ascus we;re reduction divisions, which constituted a single meiosis, and that the third division waa mitotic. If the nucleus 'Mlsulting from this third division were diploid, the a.sous zygote nucleus woUld be tetraploid. If a single meiosis oc- curred in the croiHer, as was indicated by the variety of asci produced in some perithecia, the single nucleus in the crozier initial must have been tetrs.ploid. If this nucleus was derived by mitoses from a single fusion nucleus that was formed in the p rithecium initial, that fusicn must have been tetraploid and involved tour nuclei, presumably two th!!.ll1s. in the from one thallus and t•o from the other It is noteworthy that four nuelei were found peritheciUl!! initials. This line of reasoning indicates that all four of these nuclei t'Used to form a single perithecial fusion nucleus, but this fusion has not been observed. -60- It is obvious thti!.t meiosis of a diploid heterozygous nucleus (1f no other genes are involved) could not produce a Stl ratio. Moreover, a single meiosis of a tetraploid nucleus with one factor for tan and three for wild•type would produce four diploid nuclei, two of them heteroz;rgous tor tan and wild-t~. and two of them homozygous for wild-type. A second meiosis would result in a S:l ratio• with the subse~uent result that the reduced nuclei would be haploid. In order to obtain a Stl ratio with the production of diploid nuclei in the asoospore, double llleiosie in the first two diVisions in the asous of an octoploid 11.scus zygote nucleus would be re• quired. Another assumption, justified by the genetic data, is thti!.t a single meiosis of a tetraploid ascus zygote nucleus produces two nuclei heterozygous for tan and ~ild but one of them is phenctypically wild because ot the independent segregation of a ll!Odifier. The varia- tions found in the tan character, partictl!.rly in the 338-l x 4 mating, are evidence t<::Tr the existence of modifiers. The PI"oduotion by inbreeding a1b1n1st1c and by selfing light rolaek, {characters g:l.vL-i.g no evidence of tan) with the subsequent production of tllm in their progeny showed complete suppression of the expreasion ot tean in mycelia containing the factor. ·61- 6. Percentages of first-d1v1sion 11egre3ation of the factors fo· various cu.ltur&l. cha:re.eters and the bearing of these data on four-strand crossing-over in N. orssss. Fig. 10 &~ows that 1n the attempt to inbreed black, the character reverted to wild-type. Since black re11ambl•?d wild-type in every rel!lp0ct except substrate , color, these reverted cultures were used as pure inbred A mating with inbred alb1n1st1c is interest- wild-type. ing 1n its beal.'1ng on the segregation of cultural c..1-ie.rac- This mating, 772·8 x 775-1 (a x w), produced 21 ters. ase1 ccnta1n1ng four alb1n1st1c and four wild-type pro- ducing ascospoNs. In nineteen of the~ albinistio w«s segre6ated from wild-type in the second d1v1s1on 1n the asous. This, is therefore, segregation. 10 percent first division Table XVIII shows the percentages with which other oul tursl character" segregate in the first division. It has been sho"lm that sex 1s segregated 1n the fil"st division in 85 percent or the asei Ellld ~'lat is relaticn is consta.'1t. '!'his lllllSt mean th.at the dif'• other in this division in 85 .!II rceint of the asei. Two nisms are known which may effect this result. Carothers ( 8 ) has sr·own that segregation of different -62- TABLE XVIII. Mating Percentage of first division segregation of cultural eharaeters in several different T.atin::;s. No. of' A soi Percentage of I 1l4-lx7 48 35 228-5 x 290-7 15 93 243-2x5 12 8 86 772-8 x (axa) 775-l 21 -62a- 10 heteromorphio chromosoll!es rM£y occur always in the first reduction division, or al.Haya in the second reduotion division, or , in other caaes, a oerta1n definite per• eentage of first division segregation Bridges ( 3 ) h~~hown that ls fou :d. four strand crossing-over best EU11pla1na the production of ':,eq"Uational" exceptional ", indivld"ilals in Drosophila matin,;s. In four-strand cross- ing-over and reduction in Drosophila, segregation oocura at the oocnnd division for all genea lyin3 beyond a oro1u1- ing-over point and at the first division for all genes lying between the sp:I:: dle fiber and a orossin;~·over point. A definite percenta~e of crossing•over in the four-strand stage can explain the constant first-division segregation of f'aetO:t>s in the ueus, provided that the the spindle fiber and the locus of' the ;;1 ven character is not more than 50 percent. In the ease ot 10 percent of' first-division segregation obtained in the !l1atin0 of albiniatic by wild-type, this explanation fails over, there would be an even cl1anoe of an odd or 11.:1 even number of br 4 al!'.S occurring between the two factors in• volved and the p rcentage of first-division segregation -63- resu.l tin~~ ooUld not be !!!Ore tl:'>..an 50 percent. This de- monatrates that f'ou.r-strirnd o:roes in::;-over doo 11 not 110coo.nt f'or all the seoond-divi sion segre 3at ions obtained and, furthermore, that the spindle-'!'iber attachments of' the homologous pairs of' tetrads are not always seg:regated in the f'i~st division in !• -64- 7. sw1111i1u•7 of the experiments of the 1nhe ri t11.:nce of eultural eharaoters, and 11.n explanation of the genetic behavior of N. orasea which best fits the known facts. - 'l'he follorlng sohe11e for the geootio behavior of !• cr11.ss11. which best explains the data 11.coUllllll.11.ted ia proposed& {l) '!.'hat a tetraploid f'ua ion take a pla oe 1n the perithec1u.m initiel. (2) That two nuclei of opposite sex from each gamete are involved 1n this fusion. (3) from lhat these nuclei are selected 11.t random the nuclei in the two thalli of opposite sex. (4) That clooal selection is effective in sepa- rating these nuclei. (5) Thd;; this tetrsploid nucleus divides :nito- tioally until the mononucleate initial of the crosier is 11. tta ined. (6) That the two divisions 1n the crozier con- stitute 11. meiosis. ( ') '!.'hat reduction of a gene may take place either in the first or second division of the crozier meiosis. (8) That the reduced nuclei formed in the crozier are both diploid. -65- ( 9) That the tetraploid fusion nucleus in the aseUll reiuoes 1n two divisions. (lO)Tl:lat a gene 'f!Jlil.y be reduced in either the first or second division in the aacU11, the first two divisions constituting a meiosis. ( ll) T',at the factors for sex are segregated in the y; I first division in 85 percent of the asoi. (12} That the first two divisions in the asous result in the production of diploid nuclei,. homo:&':gous for sex. (13) T"nat the third division in the asoUll is mitotic. (14) That the diploid nucleus delimited in the asoospore is reduced in the next two divisions, which constitute a meiosis. {15) 'i'hat this reduction may result in a maximum of four kinds of haploid nuclei beir~s produced in the thsllus originating fl"Om a single asoospore. The first four considerations have been discussed 11.00 evidence for them has been presented. The fifth consideration, that is, that the fusion nucleus fro~ the peritheoitll!l. initial div-ides mitotioally until attaining the mononuoleate crozier initia1 1 involves no difficult a111su.'!lptions. Evidence for the fact that the crozier divisions ai "lduotional follows: Table XV shows that peritheeium -66- 233 produced five different ki'lds of &sci. This 1s easily e .:::plained provided the P' r1 thecilllll origin& ted froll1 a single tetraploid fusion and 11ecolld-division reduction took place frequently with the random fuaion of the non-sister' nuelei which were produced by the second division. Only 35 percent first-division segre- gation of cultural characters toak., place in the &scus as a result of this mating- or ~5 p$rcent second-division segregation of cultural characters took place. If the factors were segregated with the same frequency in both t~:e crozier and the ascus divisions, that is, if' a fac- tor that is segregated most of the time in the first division in the ascus, is also segregated ioo11t of the time in the first d1vis1cm. of the crozier nucleus, it is obvious that over 30 percent ot exceptional asci would be expected, if 65 percent aeoond-division reduction took place in the crozier. The ta.ct that the cultures at this stage of the investigation had not been inbred• e calculations impossible, u~t a large variety of asci are shown in Table xv., as resulting from the lll!l.t• ing ll4~lx7 (w x t). In the l!l!lting, 33S-lx4, whieh has been discussed at len?th, tan and wild-type factors were segregated in the first division of the asous in 85 percent -57- zier took place in the same percentasa of the oases, the recombinations lihould reproduce the original. fusion nucleus in a large nunlb"r of the caseP 1 and most of the asci from a perithecium should be alike. Only two mxed perithecia in 19 were encountered"''• We may conclude that if a high percentage of second-division segregation in the ascu.s occurs, a variety of asei from a single peri• theciU!ll can be expected, but if a high percentage of first-division segregation occurs, most perithecia will produce asci which are all alike. The only difficulty with this assumption lies in the fact that although sex is segregated in the firat division :tn So J;D roent of the oases, no ase1 have ever been encountered from which all of sex. the ascospores were of one This necessitates the assumption that homozygos1s in the tetrayloid al!!c•.ui initial is a zygote lethal. The fact is assumption easy, for only about 8 psrcent of the asci would be expected to be homozygous for sex. The eighth eoneide"ation, namely, that the re- duced nucleus in the crosier is diploid, follows on the basis of the assumption that only a slngle me:tosiR tskes place in the crozier, al though either pre- or post•reduction U!Jll.Y occur. -68- othor considerations have all ban oo.".l.sidered in detail. 'I'he variations encountered in this study a"e all explained as arising in an orderly way throug.~ reduction and fusion and the assumption of mu.tations is u.VL--ie<hesary. so far :!.n this study there is no evidence to show that imy po:!.nt !!l'!lta tions ~ve been found. -69- V. SUD.ARY. A llliorodissection apparatus was devised to remove single spores in order from the ascus in a study of Neurospon. The aseospores of Neurospora required heattreatment to initiate germination. A hybrid O.tween N. 11 tophila and N. cra11sa, and a race of N. 111 tophila stualed, were botn shown to produce two kinds 01' ascospores in regard to response to beat~treat!llent. It was shown that these differences were genetic. In the case of the hybrid, it was shown that tpe factors determining sex and absence or presence of'~onidia were linked to the factors determining response to heat-treatment. sex ll'lils shown to 0. segregated in the first division in ~. crnssa in 85 percent of the a.sci and in the second divislan in 15 percent of the a.soi. It was shown that genie factors do not influence this ratio. A cytological study shows that tour nuclei are delimited in the initial of the perithaoium of !• e:rassa. Cross-breeding of pure liIBS shows that nuclear interchange between thalli of opposite sex occurs. 'l'he genetic results obtained are explained• without assuminf the occurrence of mutations, by means of the 1'o lo'iing nuciear mecfom sm1 anr 1. Te•raploid f'u.~ion of two pairs of nuclei (one from each parent) in the perithecium initial. 2. Mitotic division of th.is nucleus until the crozier is attained and a single meiosis there, with the same frequency of first and second division aegregation as follows in the a111eus. 3. F«ll!lion of two of these reduced diploid nuclei to form a single tetre.ploid ascus zygote nucleus. 4. Red:uction of this tetraploid nucleus to a diploid nucleus in the first two divisions in the a.sous. 5. Mitotic division in the third division ot the ascus. e. ~eiosis in tr~ first two divisions of the aseospore nueleus w1 th the possibilit' of producing 1u1 many as four difterent kinds of haploid nuclei in the thallus formed. A scheme is prssented,(Pig. 12) to show that this nuclear macranism could aocotll:lt for the genetic results obtained. -70- Fi~. 12 • seheme oonforming to the a1111umptior111 !Ude in the text showing how it lllight be po11sible to Mte two tan m;rcelia ot:ta in half tan and halt wild type/ o'fsprin,:; in one per:l.theoiU!!! and 11.ll wild type in another peritl'u!loiura. Each letter indieates a gene and the nlllllber of letters indicates whether a nucleus is haploid, diploid, or tetraploid. 13. replaeing the w1 s by other letts?'11, as e (even), 1t is poasible to explain other geMt1e results obtained by this scheme. a (albinistic) or Ascospore nucleus Phenotype Peritheoial Fusion -- Crozier Reduction Fusing Crozier luclei Crozier P".ision AsoospOI"e Nucleus /,tt /' ,/ Aseospore Reduction Phenotype t / - t t t w w w 'Ill' tan -71- wild '\ // /Jill'fl - W W W VII W W W W wild wild The interest of Dr. T. H. Morgan in this problem hll.s been a great enoour,1gement. Durin: the s1ll!ll!l.er of 1930 1 the writer enJoyed the faoilities of the New York Botanical <klrdens through the gen• y;. erosity of the director, Dr. J. w. Merrill. While there, he was intimately associated with Dr. B. o. Dodge, and, through this association, he received many valuable suggestions which have grEntly facili-1;, tated the pursuit of the work. Dr. A. R. Sturftevant, Dr. K. J. Eelar, and Dr. Albert Tyler, have offered ms.ny valuable 11111ggestions. The writer is particu- larly grateful to Dr. Calvin B. Bridge• who has spent ,,-4ny hours peinatakingly revising the manuscript. -72• VI. BIBLIOGRAPHY Barnes, B. 1928. Variations in Eurotium herbariorum, (Wigg.) Link., induced by high temperatures. Ann. Bot. 42:783-812. Pl.15 (2) Betts, E. M. ~ 1926. Heterothallism in Aacobolua carbonarius. Amer. Jour. Bot. 131 427-432. (3) Bridges, Calvin B. ~ . 1916. Jon-disjunction as prOpt of the chromosome theory ot heredity. Genetics. l:l-52 and 107-183. (4) Brierley, w. B. 1929. Variation in tung! and bacteria. Proo. Int. Cong. Plant Sciences. 2&1629-1654. (5) Brown, F. M. and Hetfner, H. •; lendelism among ba.oteriaf Science 691198-200. (6) Buller, A. H.R. ~ 1930 .The biological significance ot conjugate nuclei in Copr1nus lagopus and other hyJ11.enOll'17cetea. Nature 126: 686-689. (7) Burgett, H. ~ 1928. 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