Meiosis in Polyploids the Number of Long Chromosomes with a Second Constriction (Darlington, 1926) Was Identified It Is Denoted by the Letter L I

C. ]). ])AlZLINGTON 17

PART II. ANEUPLOID HYACINTI~tSL

BY C. D. DARLINGTON.

CONTENTS. PAC:E 1. ] N'I:1l O1"~ UO'I'I 0I,~ 17 2. Mz'nmu8 18 3. M,vrsRIm~ . 19 4. SOMATI0 :DIviSiONS 19 5. POLLNN ~{O'I'HER-tJELL DIVISIONS IN TIIlSOMI0 VARIETIES 20 (i) Oeac.'~l 20 (ii) Nobe on the Diploid . 22 (iii) First MeUq~h~o 22 (iv) Anaphasc: Division of the Trivalo.t. 28 (v) Second J)ivislon 31 (J. I:~OLLEN 1%IOT]INIt-OBLL DIVISIONS IN T:I~TIIASOMIO VARIETIES 32 (i) Tho Tetrasomio Variotios . 32 (ii) First MOtal~hase 32 (iii) Second Division 37 7. l~ItOl'IthSE O~" 'mE POLLEN ~'[O'I'[LER-OI~SLDiViSIONS 38 (i) Triple ]?airing 38 (ii) Quadruple Pairing 39 8. DlsoussloN . 41 (i) Mult{p|c Association of Cl}l'OnlO8OlllO8 .. 41 (ii) lndlrc~;l~ .Evidence on the Origi. of Chlasmat~, 42 (iii) Gc.cl~ical Considerations . 43 (iv) Chromosome Matiug and Crossing Over 47 9. St~tMARY ,52 RE FERENCEH 54 EXPLANATION OF PLATES 56 NOTE ON 'HIE DIAGRA~I 56

I. INTRODUC"rION. DE MOL'S work revealed in the hyacinths maberial of pecldiar interest for fm'ther cytological study. It was evident that individuals having almost every possible chromosome combination between diploid and triploid and between triploid and tetraploid had given rise to the ctflMvated clones of hyacinths. Moreover the chromosomes were of three sharply differentiated types having, as was shown later (Belling, 1925; Darlington, 1926), definite and consbant constrictions. Finally, being derived, so far as we know, ~4thout any ga'oss hybl~disation from a diploid species, the homologous chromosomes might be expected to be x I,mludl.g triploids. Joum. of Oon. xxI 2 18 Meiosis in Poly2~loids indi'fferently attracted to one another in whatever numbers they occurred. Thus the hyacinths ofered an opportunity for the comparative study in the same cell of the structure and behaviour of bivalent, trivMent and quach'ivalent chroln.osomes of distinct types. Belling has already described the metaphase of the first pollen mother- cell division from aeeto-earmine preparations in both diploids and triploids. At the present moment I am not directly concerned with the diploids, my purpose being, first, to show the relationslfip between compound structures at metaphase and their antecedents at prophase, and, secondly, to describe the reduction of these structltres and the effect it may have on the general com'se of division. The difference in method and in point of view makes these studies complementary to Bellfllg's.

2. METtlODS. The methods were similar to those used with the tulips, but observations of early prophase were made fl'om sections as well as from smears. Hyacinths are less well suited for smearing than tlflips, ~specially in prophase of the pollen mother-cell division, on accmmt of the small size of the anthers. The prophase nucleus is apparently less resistant to the action of fixatives than is the case with the tulips, and a period occm's between the completio n of pairing at pachytene and diakinesis when fixation-- judging from the irregtdarity in the thiclmess of the chromosomes, particularly theft' g~'eater contraction at flee ends than when attached to nucleoli, their less even distribution and more angldar outline is usually less satisfactory than in the stages immediately preceding and following in the same preparations. At this stage the nuclei are evidently more seusitive to the action of fixatives. This sensitiveness occasionally takes the form of contraction to one side of the cell, and might be regarded as synizesis. For this reason the post-diplotene stages will not be described. A similar condition is sometimes fmmd in the tulips although by no means so pronmmeed, and it appears that fixation by our present methods cannot be comparable at all stages of prophase. Bgla~ has advanced the view that two processes go on side by side at prophase of division; first, a reduction of viscosity in the "Kern- grundsubstanz," secondly an increase in the viscosity of the chromosome owing perhaps to loss of water to the "sap." A point may then be reached when the viscosity of the supporting medium has been reduced without the rigidity of the chromosomes being sufficiently increased to enable them to stand the el~ects of treatment, especially with fixatives. In C. D. DARLINGTON 19 these circumstances more Of less pl'ommneed contraction will follow fixation. This theory aeeom]ts fox' the behaviom' of the prophase nucleus in Hyacinlhus, and acemmt,s also fox' the still greater difficulty of studying the critical stooges of pairing in most plants.

3. IVIATEIII A L. The probable origin of the triploid hyacinths has been referred to in the first part of this study. They are remarkable, probably exceptional, in one other way; they are highly fertile. As De Mol has shown--and his result,s have been confirmed with several varieties--they are self-sterile, but when crossed give a highproportion of good seed. This fertility extends even to the approximately tetraploid clones Totnla (2n = 30) and La Grandesse (2n = 28). A whole spike of Totnla selfed gave one seed; the flowel~ of another spike crossed with La Grandesse gave 160 seeds. Although self-sterility is absolute in the triploids, De Mol found, as here in Totula, that exceptional selfed seeds may occm: in Ax'entine Arendsen, which I find also has more than the triploid nmnber (2n = 28). Perhaps correlated with the ordinax T fexgility of these aneuploid hyacinths is the fact that hyacinths appear to be equally successful with every chromosome number between 16 and 30, so that gametes with vcry irregular chromosome numbers are no less likely to produce viable zygotes. The question as to whether IIyac6~thus o~ienlalis with the somatic mlmbcr of 16 can in any way be considered a tetraploid is of importance in relation to thane studies. My earlier opinion (1926) that the presence of a single long chromosome with a second constriction prevented our regarding the species as haxdng been derived by simple doubhng has been confirmed by the stndy of the pollen mother-cells. There was strong evidence of tetraploidy in one preparation of prophase in Grand Maitre which I attribute t~ the bulb being a misnamed tetrasomic form. One or two similar suggestions at metaphase in other triploid varieties I have been xmable to confirm. There is little doubt therefore that the species is a flmctional diploid.

L~. SOM:ATIODIVISIONS. As a preliminary to studying their pollen mother-cell divisions, root- tips Of a number of varieties, some of which lull been described by De Mol, were examined. The following table summarises the results, L, 11I and S being the long, medium and short chromosomes respectively. Where 2-2 20 Meiosis in Polyploids the number of long chromosomes with a second constriction (Darlington, 1926) was identified it is denoted by the letter L i. Nimrod (Texbfig. 19, 2~ + 3) ... 11 L (3 L i) 4. M 4. S City of Haarlem (3n - 1) ... 12 L 6 M 5 S Marconi (3n) ...... 12 L 6 M 6 B La Peyrouse (3n + 2) ...... 14. L 6 M 6 g Arcntine Arendsen (3n + 4.) ... 14. L (4. L i) 6 M 8 S The pollen mother-cells of the variety Totula, in whi.eh somatic variations in number had been found, disagreed with both De Mol's results and my own in regard to the proportions of the different types, having the constitution 15 L, 8 M, 7 N. The counts of Nimrod and City of Haarlem also disagree with De l~{ol's results, but are confirmed from the pollen mother-cell divisions.

5. rOLLEN i~{OTIIER-CELLDIVISIONS IN TRISOMIC VARIETIES. (i) General. At metaphase in diploids, and less regularly in aneuploids, the long chromosomes lie flattened in the plane of the equatorial plate and appear very little contracted as compared with somatic di~dsions. At anaphase each of the bodies passing to the pole is seen to be double, the two halves (which are to pass to opposite poles at the second division) being already entirely or almost entirely separated. This will be dismissed in the light of special observations later. At interphase the daughter-nuclei pass into a resting stage, and a cell wall divides the pollen mother-cell before the metaphase of the second division. At this point the chromosomes are only held together at the attachment constriction. It is a remarkable fact that although compound bodies are formed with great regularity at metaphase in the polyploid hyacinths there is yet no trace of any other kind of attraction between homologous chromosomes, whether paired or unpaired, before or after division. Chro- mosomes having once separated from one another in the division of a trivalent, at the first metaphase, show no affinity for one another at the second; nor does an odd chromosome, which has failed to form a ~rivalent with a pair homologous with it at the first metaphase, show any attraction towards this pair. Thus, although the division of the trivalents in the hyacinths is comparable, for example, with that in Prunus (Darlington, 1928), their later behaviour is distinct in the entire absence of secondary pairing. This question will be discussed later. Text-fig. 19. Somatic metaphase of the hyacinth variety Nimrod with 11 long chromosomes, 3 having ~ second ooush'ic~iou, 4 medium cliromosomesand 4: shor~, x 2~LO0. Text-fig. 20 ev-d. l~o,,r first metaphases in tile hyacinth va.riety Roi des Beiges. y. 2400. Text-fig. 21. Diakiuesis chromosomes of the h.il)lo]d variety Lady Derby; all trlva.leng oxeel)t one short bivalent a~ld univalent. The short h'iv~le.nt has throe ehi~smata, a number not found in umtal~hase, x 2400. Text-fig. 22 a and b. First metal~hase in the trilfloid varle~y King of the Blues: 8 trlvMents. a • 1600, b • 2400 redneed ~ ,~. Y~xt,.fig. 23 t~-c. l'~irs~ metaphase in the triploid variety Lady Derby: t~ 8 h4valents; b 7 ~rlv~lents, 1 short bivalent and nuiv,~leht; c 6 trlvaleuts, 2 short bivalents and llnl- wlents. • 2400 reduced to -~. Text-fig. 24. Flint metapha.se i,, fire h.iplold'v~fiety Queen of the Pinks : 6 trlvalents, 1 long blvaleu~ and nulv~leut, 1 shor~ biv,~lenb and uulvalent, x 1600. Text-fig. 25 a and b. ~irst met;,~plmse in the trisomle w~riety Nimrod: 5 bivaleuts, 3 long h'iwlents, x 2400 reduced to {.. l~ig; 26. l~irst umtal~lmse in the h'~somie vr~riety Nimrod: 5 blvMent,% 3 long h'ivaleuta. x 2400. 22 2]1eiosis in Polyploids I~ may be that this is to some extent accounted for by the unwieldy bulk of the chromosonms, which fills up the whole available space and certainly leaves no room for deploying in. In Tex~-fig. 36, as a consequence apparently of this crowding, we have the cutting off'. of a pro- truding piece of chromosome by the interphase cell wall, and it will be noticed that even the amlmtated fragment is passing through the condensed metaphase stage.

(ii) Note on the DilJloid. The diploid variety "t/.oi des Belges" was examined for comparison with the triploids, and Text-fig. 20 a-d shows m etaphases of the first division. These are comparable with Belling's illustrations, but his interpretation evidently ditters from mine because he represents bivalents which he considers as having only one chiasma where I should say that there were two, one being tenuinal or sub-terminal. The interpretation is of importance in so far as Belling has attempted to show (1925) that the frequency of chiasmata in the long chromosome of the diploid corre- sponds with the fi:er of crossing over in the first chromosome of Drosophila. The chiasmata in the 16 long bivalents ilIustra~ed in this variety, as well as in the aneuploids described later, vary in number between two and six or more, three being probably the commonest number. Their frequency is not therefore in direct agreement with the frequency of crossing over in this chromosome of Dq'osophila. Even in the medium chromosome., which is less than half the length of the long ~ype, two is the commonest nnmber of chiasmata--giving a very nmch condensed ring. A single chiasma seems to be the rnle with the short chromosome--a point that must be recalled in connection with the frequency of short trivalents--the homologous arms sometimes !ying parallel, sometimes opposite to one another to give the well-known cross seen in so many plants and animals. The longer bivalents lie Mth their greatest length in the equatorial plane, another point in which they are to be compared with trivalents.

(iii) triq'st Metaf3hase. The following varieties have been studied: Queen of the Pinks ...... 2n = 24 Lady Derby ...... 2n = 24 Lord Derby ...... 2n = 24 Grand Maitre ...... 2n = 24 C. D. DARL].NGTON 23 King of the Blues ...... 2n= 2~: Moreno ...... 2n = 2~t- h[areoni ...... 2n = 24 City of Haarlem ...... 2n = 23 Nimrod ...... 2n = 19 The triploid varieties and the last two in so far as they are trisomic in certain chromosomes are comparable in behaviour. No siguifieant change takes place between diakinesis and metaphase beyond the flattening out of the chromosomes on the equatorial plate, and a possible reduction in the nmnber of elfiasmata. Diakinesis (Text- fig. 2I) shows the same disting~fishing featm'es as metaphase. Belling has found at this stage the regular occurrence of eight trivalents in Lady Derby. I find on the contrary that in this variety as well as in the others the formation of a trivalent fails with a frequency that varies according to the type of chromosome. The observations may be summarised as follows : 4 long chromosomes--failure of trivalent very rare 1, 2 medium chromosomes--failure of trivalent observed once, 2 short chromosomes--failure of trivalent over 20 per cent. Althottgh this conch~ion depends only partly on recorded observations, the distinction between the small and the other types is obvimmly too great to be ignored. The difference, on the other hand, between long and medium is not significant because the nmnber of long examined is much greater. A large proportion of divisions in all varieties therefore have more f,han eight bodies at metaphase, the extra bodies being accmmted for by the presence of short bivalents and mfivalents instead of trivalents (P1. II, fig. 5 a; P1. IV, figs. 9 and 10f The exceptional case of the occltrrence of a long ~mivalent is ilhLstrated in Text-fig. 2~. In Nimrod (TeXt-figs. 25, 26), in which only long chromosomes are trisomic, I have never folmcl a univalent at metaphase, the three long trivalents appearing constantly. In City of Haarlem, lacking one short chromosome of the triploid complement, long and medium trivalents together with a short mfivalent with two short bivaients have been fmmd. Although most of the trivalents are intelligible in polar view--which is owi~lg perhaps largely to the transparency of the gentian violet stain-- they can scarcely be represented intelligibly in black and white. I have Observed ~wiee ,~ merci?base a.nd once a~ diakinesis. 24 Meiosis in Polyl~loids therefore ilh~strated a mlmber of trivalents taken singly 'from divisions in side views; it is not usnally possible to intei'pret with confidence the whole of a division from this aspect. The arrangements of.the short trivalents (Text-fig. 27 a-e) are of the simplest kind, one chromosome being joined by one chiasma to each of the other two which are not directly connected themselves. In a-c anaphase has already begml and the chromosomes are in a state of tension. If, as appears to be the. case, chromosomes of this type never undergo more than two exchanges (never form more than two ehiasmata), no more complicated arrangement is possible. In the medimn chromosome the same arrangement is most commolfly 'folmd (Text-figs. 23, 27f). A first complication may be introduced by two of the chromosomes being 1mired at two points and one of them a~tached at the third subterminal point with the third chromosome

Tex~-fig. 27. Tl~valents from side view of firs~ me~aphase: a-e short~ f-I medium, a~ b, f and 9 from Oiby of Haarlem; d and e from Grand Ma~bre. In a, b and c anaphase separation has already begun, g and l show iatercalary ehiasma~a, x 24.00 reduced to -~-. (Text-figs. 21, 27 k). The second complication is that the tldrd chromosome may be joined to its mate by an intercalary chiasma (Text-figs. 23 c, 2 o'clock, and 27 g and/,), a possibility of genetic importance that is introduced by the capacity (depending apparently on the length of the chromosome) of forming three chiasmata. The limiting number of chiasmuta in this type appears to be four (as in Text-fig. 27 1). Turning to the long chromosome we find a much greater oomple~ty, owing evidently to its greater length and consequent capacity to form as many as seven (Text-fig. 23 a) or eight (Text-fig. 23 b) chiasmata in trivalent combinations. The simplest form is where only three chiasmata occltr (Text-fig. 28f and g), a comparatively rare type; commoner are those with 4. or 5 cl~asmata, the odd chromosome being attached terminally. The most interesting feature of the long trivalent is the high Text~-ll.g. 28. Long, t~valell~s from side views of fii's~, me~aphase except~ laud. t,. wlfich, are from l~olar views: a-d, h and t from Graud Ma~re; e and r fi.om C~ty of ~aar]em; f-l~, m,o and qJ fi'om Lady Derby; ~, g and s ~rom Nimrod. n shows one loop in a long f,rlvalen~ ]'?assing ~hrc')ngh ~l{e leo]? of a medium h'lvalcn~, x 2~[:00 reduced to ~-. 26 Meiosis in Polyploids frequency of the form where one of the chromosomes is attached by a chiasma intercalated between two others. This, found also in the simplest form in the medium chromosomes, is illustrated in Text-figs. 26, 28 a, e, k (probably), n, q and t, and elsewhere. In Text-fig. 28 q the chiasma is directly demonstrable, but as a rtde the occm'rence of a chiasma at metaphase can only be inferred from the general arrangement of the configuration, and more particlflarly from the change in plane of the elements that contribute to it in passing through. A further complication is the occurrence of two adjacent intercalary chiasmata with one of the other two, and of a further one with the third. Text-fig. 21 at 11 o'clock shews a more symmetrical arrangement of the same type. Here also is a trivalent with seven chiasmata which showed very s~rikingly--morr so than in the illustration--the chain appearance due to alternate loops lying in planes at right angles to one another. Another inclication of the commmmess of the intercalary chiasma in the long trivalent is afforded by Text-fig. 23 b, in which three of the long tri- vMents have this form, one of them having, from the point of view of the least associated chromosome, two separate intercalary chiasmata. Arrangement of bivalents on the plate in the diploid is very regular, but the great irregularity of form of the trivalents would itself permit of no rule as to their arrangement. In fact bodies apparently identical in structure (as in Text-fig. 28 t, a polar view, or c, d, l~ and m, all side views) may arrange themselves on the pla~e at ditterent times in every conceivable way. Sometimes the division will be along the plane of the greatest association, sometimes along that of least association. The presence of the third chromosome evidently upsets the relations of the body with the spindle as much as it has upset the relations of the two chromosomes with one another. The maximum namber of ehiasmata formed is greater in the triploid than in the diploid when one considers particlflar strands, but the average mtmber is probably the same and the frequency of chiasma-formation over a given length of chromosome is not reduced, as one might expect, front the prophase observation that only two of the three ckromosomes can pair at any one point and at any one moment. In the short chromosome for example, where one chiasma seems to be the rule in the dipioid, the formation of a trivalent necessarily requires one of the three chromosomes to exceed the usual number. This maintenance of the frequency of chiasma-formation supports the view (see Part I of this paper) that a secondary process of zygotene pairing takes place, allowing each of the chromosomes to pdr in turn all along its length. Text.fig. 29. Chromosomes ~ ~he begimling of auaph~ue, from Niml~d : d, medium aud shor~ blvalell~s; the res~, ioug ~rivalou~s. Crosses mark ~he p~r~s where ~he ehroma~ids have no~ been pulled apar~ by ~he ~uaphaso separa~iou. For a schem~tle represe11~ ~ion of 29b see Tex~-fig. 30, I). 30. The ~wo ehronlosomes of 29 m fornled par~ of a trlva.leu~. 28 Meiosis in Pogyploids Among anomalous arrangements at metaphase may be mentioned an odd chromosome passing through a loop in the bivalent to which it belongs without association (Text-fig. 23 b). This is doubtless the result of a lapsed zygotene pairing: There is also the case of the two long chromosomes of a bivalent which pass through the loop of a medimn bivalent. This is reminiscent of accidental interlockfilg reporbd by Gelei, whicli I have also observed in Tulipa.

(iv) Anaphase: Division of the Tvivalent. The most stalking feature of anaphase is the freqnent lagging of chromosomes showing no trace of pabing. Although I have never seen more than two nnivalents at metaphase, and long univalents are extremely rare, at anaphase I have fonnd as many as four unpaired Chl'O- mosomes lagging, and among such laggards long chromosomes occur in the triploid with a frequency apparently proportional to their numbers in the whole complement. These laggards can therefore be only partially referred to the occm'rence of metaphase univalents, and are due rather to one of the members of a trivalent association becoming impaired as a result of the separation of the other two from it and from one another. Being unpaired and left on the equabr such a chromosome will divide at the first division in every way ~ though it had been a metaphase univalent. Sometimes however the unpaired chromosome shows signs of its earher association by lying across the equator (Text-fig. 32) instead of lying fiat, as an unpaired chromosome does before dividing equationally. Such a chromosome has probably been attached at opposite ends to chromosomes going to opposite poles, failing in consequence to go to either itself. In other cases a lagging chromosome may still be attached to one or both of the chromosomes going to the poles, as in Texbfigs. 29 n, 32, 33, 34. In the last of these the attached laggard is a long chromosome of the type with the second constriction of one arm. Apart from proving that a trivalent may divide to give eqnal products, this body also shows the effect of an arm of a divided chromosome being attached to an arm of an unclivided chromosome, i.e. the pulling back of the half-chromosome by the great weight of the other so that its attachment constriction no longer moves foremost to the pole. From the earlier stages we can ~mderstand why the method of division of the trivalent should be extremely variable, for it must depend on the orientation, as well as on the struch~re of the combination, both of which are variable. Sometimes separation takes place only between C. D. DA~LINGTON 29 two of the chromosomes, the third passing whole with one of these to the pole (Text-fig. 29 c). The actual separation in such a case is virtually that of a bivalent, and no lagging is to be expected. Probably, judging from both anaphase and second mctaphase, this is the most usual method of di~dsion of a trivalent. F,requently two whole chromosomes may go to one pole and the th6fl to the other, although at their point of attachment the two arc entirely separate (Text-fig. 29 g, k and l). One of these two may become dis- engaged and leave the other lagging behind still attached to the third (Text-fig. 29 m). This situation brings us back to that of the attached mfivalent (Text-figs. 32, 33, 34). Occasionally one of the chromosomes appears about to divide equationally at early anaphase (Text-fig. 29f). As regards division of trivalents, I have paid partic~flar attention to the variety Nimrod, in which only three long chromosomes are trivalent and where, in consequence, the chromosomes are less crowded on the mctaphase plate. An interesting feature at anaphase is the frequent appearance of a Y-shaped chromosome with the vertical arm double, pointing to the pole (Text-fig. 29 a and Iz). Comparing these chromosomes with others in which one d0uble arm or part of an arm can be found (marked with a cross in Text-fig. 29), we fred that the second division separation of the daughter halves of the chromosomes, which is characteristic of the first anaphase in the hyacinths, olfly occurs along those parts of the chromosomes which have been pulled out of metaphase --and prophase--association. It is in fact in the pulling out of the chiasmata that the half-chromosomes are separated. This is, as I see it, a proof that the original diplotene strands sel~arate rmbroken at anaphase, and that the lnetapllase pairing is not maintained at anaphase, as Chodat's interpretation (1925) wotfld demand, at the expense of the chiasmata. I have shown that division of the trivalent may take place into one- and-a-half and one-and-a-half, and into two and one. But not seldom a chromosome, lagging for one of the various reasons given, may be cut off from both cells, and carry on to divide alone (Text-figs. 39, 40, 43), passing through comparable stages along with the two main cells. Such losses no doubt also occur as a restflt of lagging at the second division, and both together probably account for the frequency of gametes with the various possible chromosome numbers found in the triploid hyacinths (Belling, 1925; Darlington, 1926) having a mean below 12. Lagging may lead to still more abnormal res~dts, such as aml)utation of part of a chromosome by the interphase cell wall (Text-fig. 36). This Text-fig. 30..Diagl.~m to show constituf~ion of divid/ng gl~va.leng ill Tex~-fig. 29 b. Text-figs. 31 ~nd 32. I~irst a.naphases ill Nimrod sho~_ug final s~ages of division of long ~l~valents ~vi~h simplo, ~l)]?~renfly unpaired, olnomosomes lef~ on the equator. • 2400. Te.x~-fig. 33. lrirst t~lophase in Nimrod; partial porsistonoo of ~tt~ehment of ~hird chromosome. >: 2400 reduced ~o r Text-fig. 34. gilzt anaphaso in King of the Bhms; ~ long ~nd a short unpaired chromosome divided; final s'~ago of division of a long trivalon~ of tlm type ~4~h ~ho socond cou- sfa'iction (ace ~xt). x 2400. Texi;-fig. 35..First ~olo]?hase in Lord Derby; ~ univalent on tho eqll~tor and a fragment of chromosome off ~he spindle, x 2400 reduced to ~-. O. D. DAI~LINGTO~ 31 may, as in Text-fig. 36, cut through one of a pair of chromosome.s that has not separated. Here the points of att~mhment of the two chromosomes are evidently on opposite sides, so that they have passed to the poles at the second anaphase although still held by the cell wall.

(v) Second division. At the second division the chromosomes retain the form they had at the first anaphase, though wifll the two halves connected only at the attachment constriction. Corresponding halves of the chromosomes th,~t divided at the first division are found on the metaphase plate in the two daughter-cells (Text-fig. 36) and lag behind at anaphase, without showing any signs of division, either to segregate at random or be lost. It is interesting to notice that with chromosomes dividhlg in separate cells the mechanism of division functions in the normal way and they pass through the same stages as tlle main bodie"s; at metaphasc they arc united at the attachment constriction; at anaphasc they separate at the constriction (Text-figs. 39, ,~0). At the second division the connections seen between homologous chromosomes at the first division have entirely disappeared, there being no "secondary pairing" as in Dahlia (Ishikawa, 1911) or Prunus (Darlington, 1928). This disappearance of tlle first division association may be due to tlle intervention of a re.sting st~uge after the first division, in which case it would appear that secondary pairing is indeed typical chromosome association obscured by the minuteness of the threads involved in the chiasmata, and not, as it seems to be in these plants with small chromosomes, a rather mysterious expression of affinity between homologous chromosomes.

r r e xt-fig. 36.' ~ Seeomli met~.~hase m Lt~ly Derby w1~h, one half of a medmm~ chromosome ent off by the cell wan; evidently one long uuivaleut clwomosome and one short oue h0,ve divided a,t tile first division, so flw, t we he,re on t~ho left,-hand .side 7~ Ioug, 3 medimu and 2 short, and on the right-lmnd side ,l~ long, 3 medium ~nd ~3~ short (hMf-eln'omosom~ marked), x 0,400 reduced to .~. Text-fig. 37. Second anaplmse in Queen of the Pilfl~s; r uuivalents lae~:inm x 1800. Text-fig. 38. Second auaphase iu City of Haarlem (2~ = 23); one long, one me~nm and one short daughter mlivaleut l~ggi g oH eac i side, x 2400 reduced t~) ~. Text-fig. 39. ]:nterphaso in Queen of (,he Pinks; ;~ lagging chromosome h,~s formed a separate dell. x 1800 t~duccd to ~:. Text-figs. 40 and 41. Early second met,aphase and aual)ha~c in Queen of bhe Pink~; los6 chromosome passes through the s~me 8tagc~ of diwsiou as the main mmlei. • 1600. Text-fig. 42. Second bclophase in Nimrod; ehrom(monm cut by cell wall has passed to the pole; probably the l~snlt of the condition shown in Tex~-iig. 33 of persistent attachment, x 2400 reduced to !~. Text-fig. 43. l n~gular ~e~rad in Queen of ~he Pinks with intc~ccted chromosome. • 2400 redneed to ~-. 32 Meiosis in Po~yp~oids

8. I)OLLEN I~{OTHEI%-O]~LL DIVISIONS IN TETRASO~HC I VARIETIES. (i) TI~ Tetrasomic Va'rieties. The hyacinth variety Totula has been examined in somatic (hvisio~ by I)e Mol and myself. Our rcslflts differ, and as I found various chromosome munbers I take it that aberrations occur in somatic divisions in this variety as in others I have studied. Observations on the pollen mother-cell divisions in tile material now trader consideration leave no doubt that here, at least, the number is constantly 30, made up 'of 15 long, 7 medium and 8 short chromosomes--thus dil~ering fl'om both previous acemm~s. Both Totlfla and La Grandesse maybe assmned to have arisen, through the triploids, from diploid hyacinths, without any clear hybridisation, merely by the formation of diploid gametes: and we have no reason to expect any marked differentiation between the four homologous chromosomes of each of the three types. Totlfla is therefore of peculiar interest because we have the possibility of the occurrence side by side of lmi- valents, bivalents, tl~valents and quadi~valenf~ in chromosomes dis- tinguished by well-marked size differences.

(ii) First Metaphase. In examining this stage I have fmmd relatively understained preparations most useflfl. The mass of chromatin associated in some of the bodies is too great to allow of any interpretation when heavily stained; and even ~dth light staining it is due to the transparency of gentian violet that the more complicated figm'es can be made out. Even so some of the figm'es illustrated are only partly clear to me, while others which are clear to me arc almost impossible to explain by a simple drawing of any kind. Text-figs. 44-.49 show polar views of divisions, the attached formulae indicating the manner in which long (L), medimn (M), and short (Lq) chromosomes are associated. In Text-fig. ~.4 we have the extreme case in which all the homologous chromosomes of each of the eight types are associated together; thus there are three long quach'ivalents, one loug trivalcnt, one medium quadrivalent, one medimn tl'ivalent and two short quadrivalents; an arm in a medinm quadrivalent is passing through a loop in a long one. In Text-fig..45 the two short quadrivalents are replaced by bivalents, while

1 TI1L'~ term is used here merely to indicate ~h~t certain chrolnosonle types t~l.O repro- sensed four times. Text;-figs; 44-49. :Poh~r views ol! first; mel~ph~se in To~;tll~ (15 long, 7 medium, 8 shorl~); t~lle ~rrbws indie~o where ~ chromosomehe,s been moved in drawing. Wrying fonna~iotl of:cluadriv~lolll~s ~nd t~rival0~fi;s (see t;ext~), x 2400. JoUnl. of Gen?'xxT 3 34 Meiosis in Polyploid8

a bivalent and a univalent take the place of the long trivalent in Text- fig. ~[.6 (the arrow shows the direction in which the long lmivalent has been shifted in order to show it separately). Text-figs. 47-49 show fllrther increases in the number of bodies as a result of the formation of bivalents aud nnivalents instead of quadrivalents and trivalents. It will be noticed however that the three long quadrivalents appear constantly, although their only association may, as in one case in Text-fig. 47, be merely a single terminal chiasma. All the other compound arrangements including the 9 trivalent are reduced at times to their simpler form, the short quadrivalents more often than not.

Text-fig. 50. ~ideviews of the cln'omosomesof a complete first men,phase in Totula; the le~ers hldlcate the chromosome type, tim Roman numerals tim number of chromosomes associated. • 2400. The metaphase plate is less easily studied in side views, and as a nile I have contented myself with drawing individual bodies (Text-figs. 51-55). In Text-fig. 50 I have attempted to illustrate the whole of one division, moving the camera-hleida ufirror after drawing each configuration. The long quadrivalents occur in almost innumerable variations, although certain simple forms predominate. In the first place we have that in which the members of diiterent bivalents merely appear to touch at the tip (Text-figs. 45, 12 o'clock, and ~7, 52 a, b, c). Their conneotion~ as in the case of bivaleuts--is probably of the type referred to (Part I) as a terminal ehiasma, and when in a state of tension its doubleness, showing it to be a change in association of chromatids, is plainly visible (cf. Text-fig. 50, MIll). Secondly we have cases where there is still only one chiasma at which the two bivalents are connected, but where the elements distal to the chiasma are clearly visible (Text-figs. ~1-6, 5 o'clock, and ,1-8, 1 o'clock). This form is less common than the third in which the pair left free in C. D. I)A]ALINGTON 35 the second type are also associated so that there is symmetrical exchange of partners renfiniscent of an ordinary chiasma but on double the scale. This occurs in all ~he plates illustrated (including Text-fig. 50); six examples are given in Text-fig. 54:: The rightmost long quadriva]ent of Text-fig. 50 illustrates the fourth and extreme form of this series in which all fotlr ends of the two bivalents arc engaged (cf. Diagram, ~l-O (ill)). An incomplete form of this is Text-fig. 53 b, where one pair of ends of the four lies free.

Texg-fig. 51. Side vimvs of quadrivaleng shorg and mecHumohromosomes from firsg megaphases in Togula. x 2400.

l~g. 52. Fig. 53. Tex~-figs. 52 and 53. Side views of long quidvivalontsin To~uh~. x 2"4:00,

In Text-fig. ~Pl we see one arm of a medium quadrivalent passing through a loop in a complex long cluadrivalent. Such interlockings have been referred to by Gelei in the diploid Dead,rocoelum, and are said to occur with definite regularity in certain Orthoptera. The method of pairing of fmtr chromosomes in the hyacinth however is of such a ldnd, as ~dll he seen later, that such interloekings should occur less .sehlom. The arrangement of the long quadrivalents on. the spindle is altogether haphazard on account either of theh' unwieldly bulk or Of th.eirattach- ments being median. Sometinles o~.~e may be found lying entirely off the plate but parallel to iC. Lying in the plate, as Text-ilg. 50 shows, 3-2 36 Meiosis in Polyploids their axes may lie parallel to the spindle or at right angles. Texbfig. 54. a-d shows side sdews of comparable forms, a and d being entirely opposite in orientation to the long quadrivalents in Text-fig. 4.6. On bhe whole the latter type of orientation is the more frequenL Medium quadrivalents are shown in polar view in Text-figs. 4.4., ~5, 4.7, and ~18, and hi side view in Texbfig. 51 d, e andf. The las~ ~orm, a symmetrical ring, is analogous ~o ~he ~hii'd ~ype of long quadrivalent. The shor~ quadrivalen~ which occnrs leas~ often, although of the simples~ form, is by no means constant. As Was pointed out in considering the diploid, ~he shape of short bivalents is very variable, depending on the position at which the chiasma occurs. The forms shown in Texbfig. 5I a, b and c show that, as in the long quadriva!euts, chiasma-formation is independent in the different parb of the association. The difference in the state o~ tension of the two associated bivalents in c is pl'obably due ~o one lying on the equator, the other to one side of it.

Texbfig. 54. LongquadrlvalelibS in side viewin Tobnla,; ~ll exceptg regain bhe cliarac~erisfie symmetry of ~he o~rly pmph~se forms. The direegion of the sl)hidle is up a.nd dowu.

The interpretation of ghese small quadrivalents is of some importance because they resemble so markedly those formed in plants with smaller chromosomes. The quadrivalent rings in Texbfigs. 44 and 51f, for exainple, recall the quadripartite ring found so generally in tetraploids such as P. lcewensis (Newton and Pellew), Solanum (Jm'gensen), Prunus ce,rasus (Darlington), Campanula persieifolia (Gairdner), Datura (Belling) and Primula sinensis (New~on, unpublished). The bodies illustrated are larger and consequently show the stmmtnre of the chiasmata more definitely. The body in Text-fig. 51/cleariy COIISLCJtsOf eight ternliual C. D. DAI%I]INGTON 37 elements as well as the four .main masses of clu'omosome, although it is not improbable that an analogous form composed of the much smaller c]n'omosomes of one of these species wolfld appear simply quadripartite. This quadrivalent is apparently of the type fo~md so commonly among long quadrivalents--the type described above where a double exchange of partner takes place. Thus the pairs of bodies at opposite cornel's a.pproxiinately correspond, while those at adjacent, corners are non- homologous. The division of tihe i'ing has not been observed.

Texbfig. 55, THwlen~u in Tobula; a medium chromosomes; b-f long chromosomes. a--e side views; f polar view.

Text-fig. 56. Sideq ..... view of quadnw%leut aud tnvaleu~ ul La CAI raudesse at fi*'s~anaphase; ~lie sep~%ra~h,g ehroma~ids are a~aehed a~ differen~ points, in oue pair sub~el'miual, iu the other terminal. • 2400.

(iii) Second Division. As in the triploids, the exceptional relationships of the inetaphase of the first division have entirely disappeared. In one pollen mother-ce/1 counted, 7 long, 4- medimn and ,t short chromosomes were found in one plate, 8 long, 3 medium and 4 short in the other--the most probable distribution with random segregation. 38 Meiosis in Poly~loids

7. PROPHASE OF TIIE POLLEN I~0T/IEI%-fJELLDIVISIONS. In a number of fixations preserved in paraffin the stages intervening between the comp!etion of pairing and diakiuesis have been entirely wanting, and in smear preparations where the fixation of the earlier and later stages has been satisfactory nnclci at diplotene have usually been collapsed and worthless for interpretation. I have therdore to omit any account of the opening out of the diplotene loops to form the bodies we see at metaphasc, evidently a relatively rapid process, and content myself for the present with describing the more critical stage of pairing (zygotcnc). I offer these fragmentary observations for two reasons. First, they corroborate, as far as they go, the account of prophase in the comparable tnlips. Secondly, they render intelligible the more det~ilcd description of mctaphase in the hyacinths concerned, showing how the chromosomes come to pair in. fours.

(i) T~'iple Pairing. Observation has been confined to the varieties Queen of the Pinks, Lady Derby, City of Haarlem and Nimrod, to which the following remarks apply. Pairing is characterised by the parallel arrangement of three threads. These either lie separately--presumably before pairing is complete--or are associated so that two he together and the third remains free. 0ccasional!y, for a short distance--P1. V, fig. 12 a-f shows several examples of this--all three appear to be associated. This in most cases is clearly due to a frequent exchange of partners, so that the odd chromosome which enters into combination at one point is not the same as the one that emerges farther on. As in the tlflips, although three corresponding chromosomes lie side by side at zygotenc, genuine association is Only between pairs, and what is, at any particular point, the odd chromosome is apparently lmaffected by the presence of the others when once the primary requirement has been fulfilled. An "amphitenc" condition is pel,~istent. As in the tulips, there is no polarisation and the threads probably come together at random. In general the appearance of the chromosomes at this stage is sinlilar to that in the tulips, the more detailed description of which will. suffice for the hyacinths. C. D. DAI~LINGTON 39

(ii) Quadruple Pairing. The varieties in which quadruple pah'ing has been studied are Totula (2n = 30) and La Grandesse (2n = 28). Comparable phenomena were seen in preparations of a bulb obtained as Grand Maitre, which from this fact !take to have been misnamed. The important featm'e of pairing in these appro~mately tetraploid varieties (at metaphase three of the four long chromosome types in each form quadrivalents, the fourth a trivalent) is that association is always between two chromosomes at a time but, apparently, never" between

Text-f~g. 57. Zygotene associationof foul'chromosomes hl Tot~fla; ahmomplete; b complete. • 3400. the same two chromosomes throughout their length. Thus we get certain characteristic forms, such as that shown with diagrammatic clearness in Text-fig. 57, where the two pairs of chromosomes exchange partners. Plate VI, figs. 14-17, shows in greatar detail this charactel~stic pemdiarity in the pairing of fm~r chromosomes apparently indifferently attracted to one another. Although I amsometimes able to follow the threads for great distances, it is only possible to guess what propol~ion of theh" length has been examined. It is nevertheless evident that many exchanges may take place in the length of a chromosome. For example in P1. VI, fig. 16 four exchanges take place along the length ilhmtl'ated. So great a number of exchanges of partner I'have not fmmd in a metaphase quadrivalent. If therefore our interpretation of pr0phase is correct, it follows that chromosomes paired for short lengths must frequently separate along the same plane as that along wlfich they came together, without the formation 40 Meiosis in Polyploids of chiasmata. ~len pairing is complete, pairs of paired threads are seen to be running parallel for great distances in the nucleus, touching fl'om time to time at points where the threads exchange partners (P1. u fig. 14.). The appearance of the cell thus recalls the common diplotene exchanges of partners in a diploid. It might be suggested that the similarity argues an error in interpretation, but thin is put out of the question by the fact that, apart fl'om more general considerations of size and structure, single exchanging threads never lie free for any distance in the diploid diplotene, although they are always to be seen in tile tetraploid zygotcne until pairing is complete. This superficial analogy of structure between the two stages in the two types, the diploid diplotene and the tetraploid zygotene, implies ~he same limitation on pairing at the two stages, namely, that not more than half the flu'cads present can associate at any one point. Thus two cords of fern' strands in the tetraploid at zygotene behave like two cords of two strands in the diploid. Here again it in plain tllat lnore or less mechanical conditions determine the behaviour of the living material. We shall retm'n to this point in considering the theory of diplotene looping. Another result of zygotene conditions is that, while the forms pro- duced in the triploid may be utterly irregular, sylmnetry is inevitable in the tetraploid; if one thread surrenders its partner to take on another, the two left out immediately combine. The resulting metaphase figures, it will be noticed, to a great extent preserve tlfis original quality in the regular form of the quadrivalents. The remarkably symmetrical arrangement of the multiple associations illustrated by Kihara in Rumex acetoseIIa are therefore strictly in accord with an interpretation of their origin on a basis of pairing in a polyploid. If pairing at zygotene is a condition of crossing over, as it is certainly the condition of assortment of chromosomes to opposite poles, then the interspecific segregation in such a tetraploid as P~'imula !~ewensis should be accompanied by interspecific crossing over, and this appears to be the case (Newton and Pellew, 1929). It has been suggested that the segregation of genetic factors in a tetraploid is an index of the relative frequencies of pairing of the chromosomes concerned (Crane and Darlington, 1928), but the present observations show that it in only posssible to consider the factors themselves, for different parts of the same pair of chromosomes may conjugate with different frequencies. These observations indicate that the sepa,ration of a quadrivalent C. D. DAltLINGTON 41 takes place more ustu~lly along the axis where most pairing has taken place. Thus if pairing is determined by difterential affinity, the pair of ctu,omosomes having the greatest affinity over the greater length will most fl'equently pass to opposite polas.

8. DISCUSSION. (i) Multiple Association of Oh~'omosomes. The observations of prophase in the trisomic hyacinths agree with those on the tulips: they show side by side conjugation at zygotene of. homologotm chromosomes which associate only in pairs at any particular point, but associate in different pairs at different points. We are therefore probably justified in assuming that these early associatiolm give rise to the diakinesis and metaphase associations in the same way as in the tlflips, although it has not been possible to follow the intervening stages. But in the t~rophase of the tetrasomic hyacinths we see an extension of the principles that operate in the trisomic forms; for while never more than two of the foltr homologolm chromosomes pair at any one point the threads are differently paired at different points and double exchanges of partners take place, and the combinations in this way come to have the symmetrical appearance of diplotene bivalents. The metaphase quadrivalent combinations that are e~ddently derived from these are also as a rule strikingly symmetrical in structm'e although of very varied shape. Although the whole process by which multivalent combinations arise has been followed only in the tulips, the hyacinths provide material for conclusions on the conditions of their origin. Thlm we attempted to show in the tlflips that the association of chromosomes at metaphase was due purely to exchanges of partners among clu'omatids, i.e. that the occasion of pairing was simply the random formation of chiasmata. In the hyacinths we have three types Of chromosomes, short, medium and long~ with an average of one, two, and between three and four chiasmata respectively in each bivalent in the diploid. If the conditions of chiasma- formation in the triploid are comparable with those in the diploid, no im- ped{ment to the association of three chromosomes sholfld normally arise in the long and medinm ones; but in the short, where only one chiasma is usually formed, we sholfld expect both the occurrence and the faihu'e of the trivalent; for where it failed the average woldd be less than the normal one chiasma per two chromosomes, and where it occun'ed the 4.2 Meiosis i% Potyploids average wotdd be greater than the normal. This is borne out by observation, for there is a regular failure of u considerable proportion (more than one-fifth) of the odd short chromosomes to form trivalents. A greater frequency of mlivalents among the smaller types was noticed in the tulips, but in the absence of marked size differences no such critical test as this was possible. Actually the freqnency of mHvalents in the tul{ps suggests that about 9 of the 12 chromosomes are comparable with the short hyacinth chromosomes. And it will be observed that although the 9 shortest chromosomes in the tulips are much longer in somatic divisions than the short hyacinth chromosomes, they are apparently comparable in size at the maturation metaphase as well as in the frequency of chiaslna-fornmtion. The same plinciple seems to govern the formation of qnadrivalents in the hyacinths. They are constant where the long chromosome is concerned, but subject to dissociation where the short chromosome is concerned. In fact the short quadrivalent fails in an even higher proportion of cases in the tetrasomic forms than the short trivalent does in the trisomie forms. Possibly this is due to the oecm'rence of slight movements in the chiasmata, these being relatively more important in 'the short elu'omosomes than in the long, where the trivalent is the less constant. Such movements could sometimes resolve a qnadrivalent into two bivalents but would leave a trivalent ~mdistm'be& If variations in pairing at metaphase are indeed the result of random variations in the formation of chiasmata, then short chromosomes newly arisen by fragmentation might be expected, in some cases, to show the fMhtre in forming bivalents that the short chromosomes of Hyacinthus show in forming trivalents. This is, in effect, the observation of Gotoh (1924) on 8ecaIe. Comparable observations on T,radescantia will be reported later. 0!) Indirect Eviclence on the Origin of ghiasmata. If we assume that chiasmata arise as Wem'ich and others believe by the random formation of loops in four associated threads, and not as a restflt of earlier breaks on the hypothesis advanced by Belling (1927), we see that certain mnltiple formations require special assumptions. The type oftrivalent with a single chiasma intercalated between ehiasmata joining one of the chi'omosomes concerned with a third [Diagram, fig. 3 B (ii)] involves the association of the two chromatids of one parent chromosome with chromatids of both the two other chromosomes at the same point in their length. In order to achieve this it is necessary to assume that at C. D. DAI%LINGTON 43 paehytene in the triploid two of the four threads not derived[ from the same parental chromosome dissociate and p~r with the third chromosome. Although it is necessarily impossible to prove this by direct observation, behaviom, at paehytene in both the tulips and the hyacinths strongly suggests that this is what occurs, viz. that random re-association takes place just as though all six threads were simultmleously paired. But it will be noticed[ that, in spite of the great variety of forms of quadrivalents seen., not one shows the simple intercalary type of ehiasma found where only three chromosomes are concerned. Two chiasmata always iutervene between two changes oi partner among the chromosomes, as is shown in Diagram, fig. 4 C (ii). Hence where two changes of partner have oecun'ed at zygotene, an even number of ehiasmat~ compensating for one another, or none at all, nmst be formed at diplotene, so that the ehromatids are left paired as they originally went into association. In the tetraploid therefore there is not any free re-association between more than four threads, which is what the observation of pairing would lead us to expect; four chromosomes are associated in pairs and no such re-associations can take place. Thus the essential differences between the triploid and the tetraploid in zygotene behaviour, and in the metaphase arrangements resulting, seem to be correlated, and, further, they do not requh'e the assumption that breaks occur in the ehromatids before diplotene. If, on the other hand, ehiasmata are supposed to arise from pre- diplotene breaks and re-associations in the way Belling suggests (see Part z), no difference can be expected between the arrangements of ehiasmata in triploid and tetraploid; a single ehiasma does not change the relations of the threads, so that a second one is not required to com- pensate for the fa'st.

(iii) @enetica~ Considerations. It will be seen from this account that the stage at which the conditions of the whole later conduct of division are determined and[ the stage which itself is governed directly, or at least more directly than any other, by the genetieal relatiogs of the chromosomes, is zygotene. At later stages the chromosomes seem to follow a mechanical course t0 a great extent determined by the conditions imposed at their original pairing. I propose to trace the variations folmd at metaphase to an ol:igin at this earlier stage ; for at metaphase it is frequentlyimagined that we are looldng at maternal and paternal chromosomes paired for reduction with a regu- 44 Meiosis i~z Polyploids larity dh'eetly determined by theft' genetic relationship. This is evidently far from being the case, for at metaphase we see the final stage in a process ~hat was originally set in motion under conditions governed no doubt by the genetic relations of the elu'omosomes; bus how far these are responsible for the final result we cannot tell without having some idea of the type of change that the chromosomes may undergo in the intervening pro'led. In the polyploid hyacinths and tulips we have every reason to believe that the homologous chromosomes are indifferently attracted to one another; they a~e, if not identical, at least equally dissimilar. For example, the three homologons chromosomes in the triploid hyacinth, or fom' of the tetraploid, of the long chromosome type with uumerous chiasmata, form trivalents, or quadrivalents as the case may be, with the utmost regularity. Similarly in tulips, where the chromosome types cannot be picked out with. such certainty, the two orthree longest chromosomes probably also form ti'ivalents with great reglflarity. It is probable therefore that these chromosomes are similarly attracted to one another as wholes, although theh' parts may have preferences in paidug. The evidence from zygotene points in the same dh'eetion. In spite of this the shorter chromosomes of the hyacinth, and nine or ten of the twelve tlfiip chromosomes with which these can be compared for the frequency of'ehiasma-formation, probably have a definite frequency of faihu'e to form a trivalent. This failm'e of pah'ing of a third chromosome can clearly be referred to the failure of a chiasma to be developed. Thus we have one important respect in which, in species of this kind, an adventitious mechanical process interferes with the expression of genetical relationships at metaphase. If in ,the hyacinths, for example, we had not the two tyl~es of chromosome together in the same individual, we might have been led s infer that failm'e of pairing in the short was the result of lack of affinity, indicating in fact a hybrid conclition. Evi- dently therefore metaphase observations may lead us astray if they are not interpreted ia terms of'prophase behaviour. Examples of variation within the chromosome complement probably not correlated with genetical variations inside the set are found in tetrap!oid Datum'a, Pvimsda 6inensis (Belling), P~im~da kewensis (Newton and Pellew), Pr~tn~ts cemssts (Darlington), Solanstm lycopersicson (gargensen), and Ca~npamda pe~'sicifolia (Gairdner). In the last five a single ring of fom' may be formed at dialdnesis, ~he other chromosomes assorting themselves in separate pail's. In tetraploid Datst~'a (Belling and Blakeslee, 1924-) we C. D. ])AI~LINGTON 45 have mffortmlately no detailed desm'iption of the form and frequency of quadrivalen~s available beyond the statement that "the chromosonles are, as a ru].e, arranged in connected sets of four each." On the other hand in a tetraploid Rztbus hybrid (Crane and Darlington), where we have genetical grounds for believing that the chromosomes are not indifferently attracted to one another, rings of ~our are frequently formed. Similarly, variations in hybrids in the number of pairs formed in adjoiniilg recta- phases, where the conditions are assmned to be identical, seem to be the natural result of the random formation of ciffasmata. Federley (1913, 1927) has pointed tiffs out as a difficulty in correlating metaphase behaviour with genetical relationships. If the formation of ciffasmata is correlated with crossing over as well as with pairing, it is interesting to recall that Karpeehenko (1927) retold a possible difference in the number of pairs on the male and female sides in Ral)han~s-Brassica hybrids, and I~osenberg (1926) ~n Hie~'acit~m fotmd changes with age. tIow far genetieM considerations actually contribute to the final resNt we are not yet in a position to decide. Where oMy two chromo- seines correspond to oneanother, as in a flmctional diploid, we know that only rarely are the differences great enot~gh in a hybrid to prevent them pNring. This is the case in the Ral)hanus-B,rassica hybrid (Kar- pechenko) and in the Pygae~'a pigrct,-curtula hybrids (Federley). In others that are none the less completely sterile as diploids, pairing never fails; such for example are Prim~da lsewensis (Newton and Pellew, 1929) and[ various Prunus hybrids (Darlington, 1republished). Silnilarly in polyploicL% both in those resulting from hybridisation between known species such as Priq~uda ]sewensis, and in those of unknown parentage such as T~"iticu,m vuhja,re and Arena sativa (Htmkins, 1928), a considerable genetic differentiation between their diploid aneestorg does not prevent the occurrence of cross-pairing between their chromosomes, shown by ~he occurrence of secondaq?l seg~'egation. Failure in a polyploid to fornl trivalcnts, quadrivalents and so forth at metaphase can thtm in some measm'e be attributed to inability of a chromosome ~o maintain a emmeetion with more than one other at a time, and not necessarily to lack of affinity for more than one of its homologues. There is another mechanical consideration that seems bmmd to in- fluence the formation of compound bodies, namely polarisatiofl of the nucleus. If pairing can only begin terminally and can only continue a,s it began, possible exchaflges along the length of the chromosome will be reduced to one, and the freqnency of trivalents and quadrivalents will necessarily be reduced. 46 Meiosis in Polypioids l~ings of smaller c]iroinosoines are probably formed on the same plan as the rings illustrated in Hyacinthus, although i;he attachment is usually terininal. Xihara (1927) has pointed out that this terminal attachment does not prove that pairing is telosynaptic; actually it is a very common end result of side by side pairing, and[ does not even indicate any modifica- tion of typical parasynapsis. In Prunus cerasus for example the method of ]?airing is parasynaptic, judging froin the ocmxrrence of diplotene chiasmata and their occasional persistence to inctaphase as interstitial exchanges. Chiasma-forination may come to have a certain genetieal significance in a tetraploid, for where genetically unlike chromosomes form a ring, as in Primula kewensis, they will certainly segregate at random; where no ring is formed owing to lack of ehiasmata, only paired chromosomes will segregate. Hence the constancy and, by implication, the fei%ility of a tetraploid hybrid may depend on a low frequency of chiasma-formation in the absence of a high genetical differentiation of pairs of chromosomes. Fui~her, high frequency of chiasma-formation, where thei'e is genetical affinity, will make for the formation of compmmd bodies in a polyploid; these, by their irregqflar segregation into three and one (the results of which have been shown in Datura and Primula kewensis) instead of two and two, will impair the fertility where gametes or zygotes with aberrant chromosome numbers are less viable than those with the uorinal or orthoploid number. There is another respect in which more or less mechanical accidents of prophase behaviom" may be held responsible for important variations at metaphase. In triploids, as in hybrid diploids, there is frequent irregularity in the stage of didsion reached by adjoining cells in a loculus that they have. This has been noticed in Hierasium (Rosenberg), Nisotiana hybrids (l%ybin), and in P~'u~us (Darlington, 1republished) to be associated with gross irregmlarities of division, and partimflarly with the formation of "restitution-mlclei." In the present study and in the work on tlfiips we have not been able to correlate these observations in any way, owing to our having used so little sectioned material; but it seems reasonable to suppose that pure triploids and hybrid diploids have this in common, that the process of accommodation of the pairing chromosoines at zygotene can never be a straightforward one, owing in the one case to an extra strand being at any given time necessarily unpaired, and in the other to the dissimilarities in linear arrangemeut that are known to occur between the homologous chromosoines in related forms. If therefore contraction is related to pairing, C. D. ])AI~LINGTON 47 as it appears to be, the subsequent stages of division are not likely to fall into any regular time table. I-Ienee I am incliued to attribute irregularities of this kind largely to abnormal conditions at zygotene.

(iv) Chromosome Mating and Crossing Over. The observations of meiosis in the polyl)loid tulips and ]iyacin~hs, taken in conjlmction with the earlier work on diploid tulips (Newton, 1927), provide a direct proof of the pairing of homologol~s chromosomes side by side at prophase of the mahlration division in these species; for while in the diploid two threads come together at this stage, where the chromosomes are ti{somic three are seen to "pair," and where tetrasomic, four. These threads must therefore correspond to whole chromosomes and not, an has been suggested in the cases of Osmunda (Digby, 1919) and .Tryxalis (Brlmelli, 1911), to half-chromosomes. Thus Digby (19].9) re-exainining Osmunda, in which Gr6goire had described the side-by-side conjugation of threads, arrives at the conclusion that "In the heterotype division prophasic stages, ordinarily included lmdei' 'synapsis,' do not consist in the lateral conjunction of two enti~'e somatic chromosomes but in the lateral re-association of the threacls in pairs which together make a single enti~'e somatic chromosome." It is impossible in Osmuuda or in Tu~ipa to count the threads pairing at zygotene as has been done in animals, so that a proof entirely independent of the interpretation of later stages can only be had in tdploid forms. The present studies provide this proof and t]llm show that Newton's earlier interpretation of the tulips was correct, while Digby's interpretation is inapplicable. But hu'therll in view of the close analogy between the course of prophase described by Gr6goire and others in Osm~mda, Allium, Liliu~ and other genera and that described in the tulips, there can be little doubt that the parasynaptic interpretation is substantially correct as ~ai' as these genera also are concerned, and that Digby's interpretation, so much quoted in snpport of the telosynaptic view (e.g. Gates, 1923), is due to a misunderstanding of the essential features of meiosis. Let us now consider what type of evidence has recently been brought forward in favoltr of the occurrence of "telosynaptic" pairing i. Latter (1926) has described the prophase association of the chromosomes in Lathy~.us in the following terms: "The later prophase stages show clearly

i Th~ is ~o s~y ~he associ~t,io~l of homologous ~nd non-homologous chromosomes end- ~o-eiid ~ prophase ~o form a single ~hread which usu~]iy segments ~o g~ve ~he me~aphase p~ir,~. 48 Meiosis in Poly2~loids that the method of chromosome p,Mring in Lathy~'us is typically telosynaptie." What does this exactly mean? It means, as I lmderstand it, ,that at the later prophase stages the arrangement of the chromosomes is such that it can only be supposed to result fl'om an earlier end-to-end association of all the chromosomes. How this association actually, takes place is not described here; indeed, as far as I am aware, it has never been described in any plant or animal in the way that parasynapsis has been described in so many. Nor in tiffs case is any evidence brought forward[ to show that all the chromosomes really are joined together in one con- tinuous thread. Such evidence would presmnably consist in faihu'e to observe any ends of chromosomes, but since at this stage the ends of chromosomes are frequently attached to nucleoli, an apparent continmty in the contracted nucleus is tmcomdneing. Because all the chromosomes are befieved to be tin[ted end-to-end at the later stages, it is suggested that they came together end-to-end. In other words the proof that pairing is "typically telosynaptie" depends on inference and nat on observation. The summary leaves l~s in no doubt as tohow the infel'enee is drawn: "The method of chromosome pairing in Lathy~'us is telo- synapgie; each of the seven loops represents a pair of h01nologous chromosomes joined end-to-end. The spireme is thus 1composed of the fore'teen somatic ehromosonles 1mired tandem, the homologous maternal and paternal elements alternating." There are several subordinate in- ferences involved here, but they hardly reqNre discussion. The ditfictflty with Lathy~'us, as ~dth so many other plants, is probably that in the critical stages the chromosomes are very much distorted by the ordinary methods of fixation, so that it is impossible at present to do more than infer either telosynapsis or parasynapsis. Actually at metaphase the behaviom' of the bivalents as illustrated in Lattel"S paper and as shown in microphotographs (especially Plate IV, fig. 4) by Naeda (1928) 0nly permits of a parasynaptie explanation. The arrangement of the bivalents shows the exchange of partners by interstitial ehias- lna.ta, the normal consequence of the formation of diplotene loops following parasynapsis. Evidence of parasynapsis accumulated in Lilium, All[urn, .Osmunda (G-rggoire), Tulips (Newton), T~'adescantia (l~{iyake), Godetia (Itfi.kansson), and Epilobium (Schwemnfle ). I11Rumex (Kihara ),Sohtnum (1VI.l~l. Lesley), Hyacinthus and other genera, it has been doubted on the grotmd that the supposed pahing threads were really split halves of the same chro- mOSOlne ', but the present study shows that where this intel'pret~tion can 1 The i~lics arc mine. C. ]). DAI~LINGTON ~t9 be tested it is fallacious. Apart, however, from this there is now indirect evidence of five kinds which points to the very general occurrence of some form of parasynaptic conjugation. First, we have shown that in the hyadinths and tl~ips metaphasc pairing depends on the fortuitmm formation of chiasmata at the separation of the chromatids of chromosomes paired side by side. ~Thcre aide by side c.onjugation is entirely absent we are asked to imagine what, is mechanically an entirely di'fferent type o:f: attraction joining the paired chromosomes at metaphase; whereas such fundamentally r methods would not be expected, ~dthin closely related groups, to apply to the whole chromosome complement. Secondly, we find in widely different genera forms of multiple association that arc stl~ctly analogous with the metaphase results of parasynaptie pairing in hyaeiuths and tulips. Such are the strings of chromosomes folmd in Arena and T~'iticu~n (Husldns) and the rings and figm'es of eight found in Dat~t~'a, P,ri~n~da, P~'un~ts, ,S'olanu~t, and in Dahlia (Lawrence, lmpublished). The symmetrical arrangements found by Kihara in Ru~nex acetosella are also very probably of ~his type, and have been shown to remflt from parasynapsis. Thirdly, there in the widespread occmTence at metaphase of bivalent chromosomes whose structm'e permihs of a parasynaptic interpretation only. This is particularly obvious in Gaste~'ia (Taylor) and in P~'unus (Darling~on) where, in spite of the minute size of the chromosomes, the change of partners amongst the four chromatic]~% characteristically the result of side by side pairing, is constantly observed in side views of metaphase. In the hyacinths we can see that the distinction between the cross form and an appa~'ently telosynaptic 1talon is a mechanical one of secondary importance. Where only a single chiasma oeeurs terminally, as in the typically short and medium types, the pulling apart of the chromosomes leaves them intact, and their diakinesis and metaphase association is "tclosynaptic"; but where a chiasma intervenes between the poin~ of at~achment and the ends of the c]n'omosomes their pltlling apart reqlfires the lmravellhlg of the chiasma, the separation of the chromatids, and the exposure of the tetrad nature of the bivalent. Where a third chromosome takes part and passes to the pole without mn'avelling, its ehl'omatids do not separate, and its doubleness is not revealed until the second division. In the hyacinths and tulips we know that the suppression of the appearance of duality at metaphase is just a momentary change in the apparent closeness of association of two distinct bodies. Journ. of Con. xxi 4 50 Meiosis in Polyploid8 Fomehly, it is no~T possible to show that Grossing over of the Droso- phila, type between corrcsponcling chromosomes demands a parasynaptic mechanism. ]bridges and[ Anderson (1925) have shown by experiment with trisomic X individuals that crossing over takes place in Dq'osophgla, first, between four strands and not between two clu:omosomes, and secondly, at different points--never at the same point--between Strands of all three pairing chronmsomes. The physical req.lfircments of this interchange are fidfilled by parasynapsis as it ocem's in the tltlips and hyacinths if we suppose that paMng of chromosomes is the condition of crossing over between their threads. The occurrence of exchanges of partner takes place (cf. Diagram) as ]Bridges and Anderson d.escribc: "two strands which have crossed over with each other are as free to cross over with a strand from the third chromosome as with a strand from the original two chromosomes." Crossing over has been demonstrated on the lines of the Droso- phila resnlts in a great number of species of the flowering plants from widely separated families, and it is increasingly difllcult to believe that in these families any other than a parasynal?tic system of pairing prevails. In fact there cannot be nmch doubt that the course of meiosis is governed by the same parasynaptic principle in plants as has been shown to apply so generally in animals, inch tding the Protozoa (B5la~, 1922). Finally, a hypothesis has been put forward ~Darlington, 1929) according to which ring-formation and the apparent association of non- homologous chromosomes (in Oenothera and other plants) is intelligible in terms of parasynapsis. This hypothesis appears to flflfill the genetical reqnirem.ents in Oenotheq'a, as telosynapsis does not, and one of the principal objections to regardfilg parasynapsis as the lmiversal mode of conjugation is therefore removed. To return to the problem of the basis of genetical crossing over: we do not wish to re-open the qncstion of the relation of chiasm.ata to crossing over in its widest aspects. The evidence on the genetical as well as on the cytological side is still altogether too scattered to enable one to put forward a working hypothesis with any possibility of its being generally applicable, and useflfl. It is impossible for us to say, for example, how widespread is the simple and complete form of side by side pairing described in the present study. It is equally impossible as yet for the geneticist to say how widespread is the simplest form of linkage pheno- menon found in plants, or how significant are'the differences between the sexes. Gowen's work (1922, 1928), in showing that a single factor will suppress crossing over, emphasises the impression that crossing over is C. D. ])A!~LINGTON 51 an unessential consequence of p~ring rather in the nature of an accident and not, like chiasmata, apparently characteristic of chromosome- 1)ah'ing. It will be seen that there is no evidence in this material for the occm'- renee of a regular physical interchange of the kind to be expected from the genetieal evidence in Drosophila. There are, nevertheless, as has been pointed out (Part I), special circumstances in which the behaviour of the chromosomes in polyploids answers to the special requirements of the genetieal evidence, whether we assume a correlation between pairing, or between dhanges of pairhlg, and crossing over. But the.re are two fm'ther respects in which tHS work provides evidence of possible value in connection with the crossing-over problem. The appearance of fragments of chromosome in polyploid tulips and hyacinths after diplotene is apparently correlated with t;he presence of odd chromosomes whose association is subject to special conditions. Now a characteristic of the association of chromosomes where an uneven munber is paired is that one is usually associated by a single, frequently sub-terminal, chiasma, of which many have been illush'ated here. If any strain at all is imposed on chiasmata in the stages between diplotene and diakinesis, an mmsual strain must be imposed on this single ehiasma; and if it breaks, a fi'agment is likely to be set fl'ee. This seems to make more plausible the view that crossing over is due to a reduction of ehiasmata by breaking. Actually such breaks are not easy to prove, and although many times in the present studiea ehiasmata have seemed to be undergoing a break, such as that described by Janssens in Stethophyma,we cannot ignore the possibility of such an appearance being due to an error in fixation or an accident in smearing. In any ease the occurrence of odd clu'omosomes seems likely to encourage any possible fragmentation of these chromosomes or their homologues, an important possibility from the genetical point of view. The second consideration bears on the correlation of the frequency of chiasmata ~dth the frequency of crossing over. It has commonly been assmned that at diplotene splits occur either only reductionally~ha~ is between maternal and paternal chromosomes, where the chiasmata are due to.earlier breaks and interchangas--or at least alternately reductionally and equat.ionally, which implies an alternate faflm'e and success of the threads in discriminating between one another.' On the other hand, there seems to us no less reason on cytological grounds for supposing that alternate loops might be more .frequently or entirely equational. This assmnption would lead to two different consequences according to ~-2 52 Meiosis i'u Polyploids the theory of crossing over adopted. Thus if it is held with Wilson that crossing over may be due to an orderly interchange between pMred threads, then it might take place throughout tlle whole length of the chromosomes, or not at M]; but if, on the other hand, crossing over is occasioned by the breMdng of chiasmata, its frequency may be similarly reduced to zero by the preferential pMring of unlike threads, and the occurrence of only e~.ational chiasma,ta, or to two-thirds on the basis of a random association of threads. The occurrence of u different degree of discrimination in the two sexes might on either view thus account for every variation in. crossing over between them. In view of thase considerations it does not seem too much to hope that later, when both genetieal and cytological observations have been extended over a wider field, we shall be able to find. a satisfactory basis for the correlation of the random formation, of ehiasmata in paired chromosomes with. the random distribution of crossing over between them. It is to the reduction of ehiasmata in the pro:fed between diplotene and dialdnesis that I am most inclined to look for the opportunity of crossing over taking place.

9. Su~,I~.~Y. 1. Pairing at prophase in the t~'iploid hyacinth reveals the same features that characterise the prophase of the triploid tulips already discussed in detail. 2. At pairing in prophase in approximately tet~'a21oid hyacinths the same rnles apply as in the triploid and are actually more easily demonstrated. 0nly two chromosomes associate a t any one point and frequent exchanges of partner take place amongst the four chromosomes; no part of a chromosome appears to be left Impaired. 3. In the triploids eight trivalents are usually formed. ~t. The frequency with which trivalents fail is high in the short chromosomes and very low in the longer chromosomes. 5. The forms of trivalentn are various and[ their arrangements haphazard in relation to the spindle. The division of greatest gh.eoreticM importance is between those in which each is associated at some point with both the others, and those in which two of them fail to meet. 6. The division of the trivalent at anaphase is nsuMly such that two chromosomes go to one pole and one to the other. Frequently however the triwdent divides into. equal products either directly, or by separation of one of its membels which lags to divide, C. D. DAt~LINGTON 53 7. From the fact that more t:han half of a chromosome belonging to a trivalent may be unpaired, we Can show in the triploids that separation of the chromosomes at anaphase is due to the lmravelling of the individ.ual chromatids that change partners at the chiasmata, so that between the attachnlent constriction and the first chiasma the chromatids remain attached, distal to this chiasma they are separated; no break occurs in separation. 8. At the second division the chromosomes associated at the first show no connection with, or attraction for, one another; the halves of rmivalenb that divided at the first division lag without dividing. 9. In the approximately tetral?loid varieties quadrivalents at lneta- phase are of the various complex forms that would be expected from their pairing at prophase; they are characteristically symmetricah 10. The frequency of the formation of quadrivalents, like that of trivalents, is highest in the long chromosomes. The lower proportion in the short chromosomes suggests that the low proportion of quadrivalents in tetraploid plants with sinai1 chromosomes is due to the low frequency of chiasmata at metaphase in short bivalent chromosomes, for it is shoml that the frequency of failure of trivalenb in the triploids and faihu'e of quadrivalents in the tetraploids is correlated with the number of chiasmata fmmd in the chromosomes concerned in the diploid. 11. It is clear fl'om thase observations that, while ch~'omatids at diakinesis and metaphase are held together in pairs, &~'omosomes are only held together through the exchanges of parbiers amongst these pairs, at the chiasmata. 12. The bearing of this work and the studies in T~di,l)a on chias- matypy and the theory of meiosis is discussed. Indirect evidence is adduced to show that no breaks occur, in the chromatids before diplotene, and direct evidence to show that no brea3~s occur in the chromatids at the anaphase separation. It is therefore thought desirable to look to the period between diplotene and dialdnesis for evidence of crossing over; in this connection the appearance of fragnients at the later stages in these hyacinths, as well as in the triploid and pentaploid tulips, is significant. 54= Meiosis in Polyploids

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EXPLANATION OF PLATES I--Vl.

:Pig. l. Palling of chromosomes taldng [}lace (zygotene) in l~'Fassenet, x 2250. Fig. 2. The same stage in Keizerskroon. x 2250. Fig. 3 a-o. Successive stages of association of particular threes of chromosomes inKeizers- kroon and 15/laSSellet. o, l~: a11d I X 3200, the rest x 2250. ~ig. 4:. Early dipIor in Koizerskroon. 0peniug out el loops between pairs of chromo- seines and complete separation of the third ekromosonm for long distances, x 3200 reduced to }. ]~ig. 5 a-d. Particular brivalents from early diplotene in KeizemkrOOll. x 3200 reduced to }. ~ig. 6. Three diplotelm t}~valents from Keizerskroon. x 3200 reduced to }. Fig. 7. Post-diploteno nucleus in Kcizerskroon; tl~valell% bivalelltS and nlliValeuts. x 2000. ~'ig. 8 a-n. Post-diplotene trivalellts in Keizerskroon. • 2250. Fig. 9 a-l. Post-diplotelle trivalellts in Keizerskroon and Massonet (e). x 2250. Fig. 10. Early diakinesis in Keizerskroon, 9 t}~valonts, 3 bivalent~s and 3 Ullivalents (one at 1 o'clock and two at 6 o'clock). • 3200. Fig. II. Post-diplotene figures from Tul@a Clusiana; a alld d, tlq_valelltS; b, ClUadiqvalent; c, quinqnovalont, x 3200. Fig. 12 ~-h. Zygotene palling of chromosomes in the tetrasomio hyaein~ La Gralldesse. • 3200. ~'igs. 13-17. Zygotene pairing in La Gr~ndesse. x 3200.

NOTE ON THE DIAGRAM,

Broken lhms indicate where one thread passes under another, 4 A and 4 B indicate the two possible in~rpretations of the method of division--reduetlonal and equational--in the absence of earlier crossing over; the method of division is assumed to be det~l~uiued by whether idelltieal or opposite threads are associated at the spindle attachmellt. 3 C' is hypothetical. In 4 G the c]n'omosomes ar6 represented as double tllreads for clearness. Compare 1 B witll Pl. I, fig. 31~. ,, 1 O ,, Pl. VI, fig. 16. ,, 2 B ,, P1. I, fig. 30. ,, 3 ]9 (i) ,, Z~I. TV, fig, 9 l~. ,, 3 B (ii) ,, Pl. IV, fig. 9 b. ,, 3 B (i~) ,, •[. m, fig. 8h. ,, 4 B (i) ,, Text-fig. 55 a, ,, 4- B (ii) ,, Text-fig. 28 m and 16 2;. ,, 4 O (i) ,, Text-fig. 51f. ,, 4 0 (ii) ,, Text-fig. 54. ,, 4: O (ifi) ,, Text-fig. 50. ,, 5 B (i) ,, Text-fig. 29 a and o. ,, 5 B (ii) ,, Text.fig. 29 g. JOURNAL OF GENETI08, VOL. XXI, NO. I. PLATE I JOURNAL OF GENETI08, VOL. XXI, NO. I. PLATE II JOURNAL OF GENETI08, VOL. XXI, NO. I. PLATE III JOURNAL OF OENETI08, VOL. XXI, NO. I. PLATE IV JOURNAL OF GENETI08, VOL. XXI, NO. I. PLATE V dOURNAL OF CIENETI08, VOL, XXI, NO. I. PLATE VI