C H g O M O S O M E V A I ~ I A T I O N A N ] ) B E H A V I O U I ~ , I N R A N U N U U L U S L.

BY L. N. H. LAtITEg.

(John l~mes Hort@zdtural Institution, Mutton.)

(With Forty4our Text-figures.)

CONTENTS. PAGF~

I. In~roduc~ion 255 II . l~Iat)erial trod methods 256

I I I . Observat~ions on root; ~ips 256 (1) Somatic chromosomes 256 (2) 5{itol~ic irregularities 259 (3) Comp~rat, ive chromosome morphology 261 (~i) ~Bch~viour of ~r~bants 265

IV. Observations on pollen mother ceils 266 (1) Genera l . 266 (2) Chiasm~ formation 268 (3) Termin~lisatfion 269 (4) Cln'omosome separ~lfion at~ an~phase 270 (5) Behaviom ~ of fragments in R. Ficarla (55/1) 273

V. Diseussion--~heore~ical considerations 275 (1) Termin~Iisa~ion 275 (2) Geno~ypic con~rol of bhe chromosome complemen~ 279

VI. Summary 281

VII. l%eference~ 282

I . I]NTgODUCTION.

PREVIOUS cytological work on Ranunoulus has revealed a number of features of unusual interest, added to this the comparatively large size of the chromosomes in most genera o~ the I%anunculaceae makes the family particularly favourable for cytological study.

The present work involves four main aspects, which are briefly: (a) a comparison of several species with a view to ascertaining their

c)4oIogical relationship ; (b)- the comparative morphology of trabal:tS; (c) the formation and behaviour of chiasmata at meiosis; (d) the fragments and their behaviour in relation to theres t of the

chromosomes. Of the more recent workers on this genus, Langlet has recorded

fragmentation in R. acris and has also published chromosome counts Journ. of Genetics x x w 17

256 Chromosome V ar ia t i on and Behav iour in B, a n u n c u l u s L.

from the root tips of thirty species, but has not touched on chromosome behaviour at meiosis. Sorokin and others, however, have dealt with tlfis aspect in R. acris, though somewhat inadequately. This investigation is therefore designed to probe more deeply into the comparative morphology and behaviour of the chromosomes of the genus and to interpre~ this behaviour in the light of more modern cytological theory.

II. I~IATEI~IAL AND h[ETKODS.

An examination has been made of the root-tip divisions of thirty-six species of RanuncuZus and of meiosis in the anthers of five of these. Root tips were fixed in 2BE (La Coat, 1931) and sections were cut 20/z thick. Flower buds were fixed for one minute in Carney and ~hen transferred to 2BD (La Cour, loc. cir.) a.nd cut at 25/,. Fixation in most species was good in the root tips, but variable in the anthers. The stain used in all cases was Newton's Iodine-gentian violet.

Drawings were made with a Zeiss Abbe camera lucida using a Zeiss 1.5 ram. apoehroma~ic objective in conjunction with a x 30 compensating eyepiece, giving a magnification at bench level of 6~I00 diameters. Draw- ings were subsequently reduced for reproduction to the scale indicated.

In drawing the somatic and meiotic figures some of the chromosomes have been spaced out for the sake of clearness. All drawings of side views of meiosis have been spread out laterally for ~he same reason.

I am indebted to Mr E. M. Marsden-Jones for much of the material and to the Director of the Royal Botanic Gardens, Kew, for the re- mainder.

III. O~SE~VaTmNS ON ~OOT TIPS.

(1) Somatic chromosomes.

The following is a list of counts made Dom polar views of metaphase plates in the rood-tips.

DIJ2LOID.

29~ 2n 7L acris L. ( =R. acer) 14 It. Yicaria L. Pl~nb 55/1 18" R. UMus DC. 14" B. Gouanii Willd. 16" It. Nelsouii A. Gr~y 14" 3?. ~ramineus L, 16" R. abortiwts L. 16 R. ophioglosslfolius Viii, 16 It. aconltifoliu~ L. 16 22. oxyspermus l~oss. 16" It. Broteri :~rcyn 16" R. l)olyanthemus L. 16' 22. bulbosus L. 16 It. ~ardous Cr, 16 (32)* It. 6'ymbalaria Pursch. 16 12. g13runerianus ]3oi,~s. 16* It, Ficaria, L. Plant~, 5 o~c. I6 (32)

L. iN. H. LAICTEI{ 257

TETRAPLOID. R. aurlitifolius 28* B. I,'icaria. L. JR. Kerneri Freyn 28* va.r. incu.mbcns Schultz 32* 12. l)arvijlor~s L. 28 JR. flabdlatus Desf. 32* JR. pcdat'us Walds. and KiL 28* R. Fhtmm'ula L. 32 R. rul)cstris Guss. 28* R. illyricus L. 32 JR. serbicus Vis. 28* R. Lcnormandii Schultz 32* JR. arvcnsis L. 32 B. psiloslachys Griseb. 32* JR. aslati~us L. R. pcllatus Schr~nk 32

var. Turban Gr~ndiflora, 32* var. lrwncatus Koch 32* JR. auricomus L. 32* R. re:pens L. 32 JR. I~icaria L, 32

H ~XAPLOID •

JR. conslantinopolilanus Urv. 4,2* JR. h'ilobus Desf. 48 JR. va~ricatus L. 48

OOTO~LO~ (?). JR. lincjua L. 56 or 64(~)

These numbers agree in general with those published in previous accounts (HoequeRe, 1922, Langle% 1927, Niyake, 1927, Senjaninova, 1926). The form of R. asiaticus which Langlet listed was, unlike mine, diploid with 16 somatic chromosomes; again, for R. serbicus he gave n = 12, whereas in the plant of this species examined by me the diploid nmnber was 28 (n = 14). Counts of those species marked with an asterisk in the above list have not been published previously. It is interesting to note that Hoequette (los. sit.) cites 2n = 16 for R. acris vat. boreanus; this remains uneonfu'med and I have not had the opportunRy of examining this variety.

The species of Ranunetdus apparently fall into two polyploid series founded on the basic numbers n = 7 and n = 8. ItRherto no hexaploids in the former series (2n = ~2) have been recorded, though in agreement with Langlet I find 48 chromosomes in both R. muricatus and R. trilobus (Text-fig. 21).

Of nine different wild forms of R. acris examined, including male, femMe, hermaphrodite and neuter types, all were cytologically similar and no tetraploid strains were found (cf. Senjaninova, los. sit.). The sex variation which has been demonstrated in this species (veianowsky, 1900, Marsden-Jones and Turrill, 1929) is due therefore to genie control and is not dependen~ on sex chromosomes.

Owing to the high number and crowded condition of the chromosomes of R. lingua it proved impossible to obtain even an approMmate cotmt from the roo~ tips. It is probable, however, that the species is at least oetoploid, with 56 or 64 chromosomes.

The phases of somatic nuclear division follow a normal course and[ require no special comment, Chromosome contraction continues tmtil

17-2

258 Chromosome Vai'iation and Behaviour in l ~ a n u n c u l u s L.

late met~phase, with the result that there is a slight but definite variation in the lengths of eori'esponding chromosomes on different p labs

Fig. 1 ~

H9.3

J l y , ' ~

Text-figs. I -6. Somatic mitoses, x3200. Fig. i . /P, acris, 2n,--14. Fig. 4. 2 .5 'prunerianus, 2.n= 16. :Fig. 2. /~. IYel~onii, 2n=14. Fig. 5. 2 . ophioglosslfolius, 2n=16. Fig. 3. ,R. Chius, 2n=14. Fig. 6. ~ . oxysl~crmus , 2n=18.

of one root (Text-fig. 1~) and the coral)arisen of t]~e complements Jn dit~erent forms is t]lereby rendered difficult. To some extent this variation

L. N. H . LAI%TEI:~ 259

may be due to slight differences of fixation ~nd staining. As is usually the ease, subterminally constricted chromosomes lie with their short arms in the equatorial plane and their long ones more or less erect in the spindle, while those with median constrictions frequently have only the region of the constriction actually on the equator. The long chromosomes, especially the asymmetrical ones, tend to lie near the periphery and the short ones near the centre of the spindle.

(2) Mitotic i,r~'egula, q'iges.

Somatic doubling in the rook tips is frequently encountered and is doubtless due to irregularities of either nuclear or cell division. In other- wise diploid roots of R. sa~'dous and R. _~ica~'ia isolated groups of tetra- ptoid cells, in which each chromosmne type is seen to be represented four times, were fmmd (Text-fig. 13). Such cells are easily recognised even when not dividing, by theh" greater diameter and the large size of their nucleoli. This behaviour has probably given rise to the tetraploid strains of R. I~ca~'ia (Text-fig. 11) listed above. Of the twenty-five wild varieties of this species which have been examined five were such tetra- ploids and showed strong evidence of being of the auto-polyptoid type. They had all the chromosome types of the diploid form including the satellited one, fourfold. I have been lmgble to stucly meiotic divisim~s in any of these, but iVIr Marsden-Jones informs me that attempts to self and cross them have so far resulted in failure to set seed, and presumably they are propagated under natural conditions in a purely vegetative manner. In this connection it is important to notice that of all the forms of R. Fica~'ia encountered the tetraploids alone produce bulbils.

Evidences of fragmentation, which appears to be so common and variable in wild plants of R. ate'is in Sweden (Langlet, loc. cit., Brunn, 1932), have not beenmet with in the British forms. In a male plant (55/1) of R. fica~'ia, however, two extra (H) chromosomes-with sub-terminal constrictions are present (Text-figs. 8, 10). These, unlike Langlet's "e-Chronaosomen," are about a third the length of one of the smaller normal chromosomes and are constantly present tlu~oughout the roots of the plant. As a rule, therefore, these H-chromosomes divide normally. Darlington (1932 b) has suggested, arguing on analogy with other ex- amples of fragmentation (e. 9. f~'itilIa~'ia i,mpe~'ialis, Darlington, 1930), that the variability in the numbers of fragments fmmd by Langlet in R. a, eris is due to their relatively small size rendering them less adapted to the mitotic mechanism. Should tkis be so, a stdlicient explanation for the comparative stability of the two fragments in R. fica~'ia is found in

260 Ch~'omosome Va~'ia~ion and Behaviou~' in Ranuncu]us L.

.B C E~ E.. F G D 7-[

i ' '

i . ' : ,

Fi9.8

~j

Fig.lO

Fig.1 1

Text-fig. 7. Somatic ehromosonles of R. PicaHa (5) and/~. JPicaria (55/]). x 2200. Text-fig. 8. Photograph of somatic mitosis in R. Ficaria (55/1) (2n=18) showing two

fragmentary chromosomes (H). × 1000. Tex~-fig. 9. Somatic mitosis in R. Fica:ria (5) (2n=t6) . x 3200. Text-fig. 10. Somatic mitosis in 12. Fica'ria (55/1), same cell as in Fig. 8. x 3200. Text-fig. 11, Somatic mitosis in 27. Y~icarih (9/3) (2'n =32,) x 3200.

L. N. H . LAIgTEI{ 261

their large size. Irregularities of somatic division must, however, have occurred occasionally, for in one anther of this plant four such chromo- seines were present in each cell The remainder of the complement is identical with that of plant 5 and other normal diploid Ficarias (of. Text-fig. 9), which suggests thatthe fragments are reduplicated segments (of. IV (5)). A similar case of fragmentation has been found in Vicia Cracca (Sweschnikova, 1927); in this case, however, both fragments of the parent chromosome have developed attachment constrictions in- dependently and have thus become perpetuated. In the animal world, fragmentation fix Phragmatobia fuliginosa (Seller, 1925) is a parallel example.

Other male strains of R. tziea,ria possess perfectly normal chromosome complements.

(3) Compararative chromosome morphology.

The diploid complement of R. Ficaria (Text-fig. 7) consists of the following types :

A, B Two pMrs medianly constricted 7.5/* long C One p~ir sub-medianly constricted 6t~ ,, E l , E 2 Two pairs with one shor t and one long arm 5-6t~ ,, D, F , G Three ]?airs sub-~erminally constricted 5-6/~ ,,

Each type, with the exception of the loilgest which rarely lie fiat, can be separately identified on most plates by the position of the constriction and by the length. The constriction of the E 2 type is nearer the median position than that of the E 1 . Of the three, D, F and G, F have the longest short arm whilst that of G is distinctly larger than that of D. Further- more, the short arm of D bears a small trabant terminally. The chromo- seines of/~. bulbosus and R. sa~'dous correspond exactly as regards the position of their constrictions and relative lengths with those of R. ficaria. Other species, R. Sl)rUnerianus, R. oxyspermus, etc. (Text-figs. 4, 6), however, differ in the position of the constriction in one chromosome pair. R. o2hioglossifolius stands alone in the genus in that it has the trabants attached to the long arm of a sub-terminally constricted pair (Text-fig. 5). This is evidently typical of the species, as it was fotmd in two plants, one growing wild in Gloucestershire, and the other obtained from Paris . Such a state of affairs has probably arisen during the evolu- tion of the species by a translocation of the segment carrying the trabant to the o~her end of the D or of a similar chromosome. In R. Sl)ru~erianus (Text-fig. 4) the arm to which the trabant is attached is considerably larger than that in other species.

262 Ch~'omosome Va ' r ia t io~ a n d Behaviou~' i ~ l ~ a n u n c u l u s L.

From the oolnp]elnent of I~. ac~'is (Text-fig. 1) the E I type is a]?- patently absent, and the renaaindcr eorresl)ond with those figured by Senjaninova, Langlet and ~{iyaki (lot. tit.), as far as can be judged; though these authors do not show the position of the constrictions clearly. The cytological account of this species, as found in the neighbourhood of Leningrad (Sorokin, ].927 b), shows remarkable differences from that given here; not only is the B-chromosome lacking (% = 12), hut the longest chromosmne is more than twice as long as the smallest. I t is possible therefore that Sorokin's A-chromosome is the product of fusion of two of the smaller types found in the British form of the species.

The only other two species found to have 1~i somatic chromosomes were R. Chius and R. Ndsonii . R. 6'hius (Text-fig. 3) has no representa- tive of the C type; R. Nelsonii (Text-fig. 2), on the other hand, has neither trabants nor D-chromosome, all other seven types are, however, present.

It is more difficult to correlate the tetrap]oid species with the diploid owing to the habit of the chromosomes of not lying flat on the equator. Distinct relationships are however seen. The A-, B- and C-chromosomes occur in all tetraploid forms and the G too is usually present. There are also five or six pa.irs of medium size, but it is rarely possible to distingmsh separately the E and P tyl~es. R. se~'bicus (Text-fig. 15) lacks the G- chromosome and R. pa~'viflo'rus (Text-fig. 16) the 6', though each of the remaining seven are present four times. A recently published account of the chromosomes of R. pa~'viflorus (Jane, 1932) records the occurrence Of two ":fragments "resemblingLanglet's "e-chromosomen" ; fromthe figares illustrated, however, it is apparent that these are the two trabants. This species, together with R. ~'epeg~s (Text-fig. 18), R. asiaticus and R. serbicus, has only one satellited pair, whilst no trabants have been seen in R. Ke~'- ~,e~'i, R. flabellaa~s, R. psilostachys, R. peltatus (Text-fig. 19), etc., though they are quite possibly present. R. a,9'vensis (Text-fig. 17) is the only one of the normal tetraploid species examined which possesses two pairs of D-chromosomes with trabants. In one cell of the hexaploid R. constant,i- ~,opolitagzus (Text-fig. 20) five trabants were seen, so it is highly probable that there are six in this species; i~ is, however, difficult to locate them owing to the crowding of the chromosomes.

R. Ke,rncri is remarkable in possessing two pairs of sub-medianly constricted chromosomes only 3tx long and R. a,~'vensis (Text-fig. 17) has four pairs, sub-terminally constricted, only 2/~ in length; the rest of the complement in both these species consists of typical forms.

These resulLs, considered together with the behavi0ur at meiosis of

L. N. H . LAP~TE]~ 263

such of the tetraploid species as have been examined, other than those forms of R. I~icaria mentioned above, lead to the conclusion tha t they are all allopo]yp]oids.

I t is noteworthy tha t the chromosomes of R. sa,rdo'us and R. 2a,'r~iflorus and other polyploids aye considerably smaller in bulk than those of either

A Text-fig. 12.

Fig. 1 4 ,Som~tie prophase in R. acris showing grabant (t). × 3200.

Text-fig. 13. Phot~ographs el somatic mitosis in B. 8a.rdous (2n=16) sim~dng diploid celt on left and tetraploid cell on righ% in one roo~ tip. x 1000.

Text-fig. 1,I. D-elu'omosomes of -~..Yicaria. f rom somatic divisions, showing varit~ion in size of chromosome and t rabant , x 3200.

R. Yicaria or R. acris, though in each case the chromosome morphology is otllerwise very similar. The most remarkable ditYerentiation of tllis type is seen in R. pelmt,us (Text-fig. 19), an aquatic species belonging't0 the sub-genus Batrad~,ium, where the chromosomes are less than a sixth the bulk of those of R. acris. In general the cells in ~shese po]yl)loids are the same size as tllose of the diploid species, whilst tlhe size of the chromo- som e,s varies inversely as their number ; whereas in the tetraploid forms

26~ Chromosome Varia t ion and Behaviour in l ~ a n u n c u l u s L.

o~ R. ]#ica.Ha the chromosomes ~re ~he s~me size ~s those in ~he diploid forms, while the cells a,re considerably l~rger. ]:t is shown later that such

l=ig 15

r~ 9. I /

Tcx~-figs. 15-18. Som~ttic mi toses . × 3200. Fig. 15. 2/?. scrbicus, 2~z.=28. Fig. 17. 1L arve~sis, 2 n = 3 2 . Fig . 16. It. parviflor~ts, 2 .n=28 . Fig. 18. J~. relJe~s, 2 n = 3 2 .

differentiation is probably ~n expression of ~he genotypic control of the chromosome complement.

This comparison of the chromosome types in ~he genus demonstrates that there are considerM)le eytologicM resemblances between all the

L. N. H . LAg ' rm~ 265

species studied. There has, however, been much chromosomal variation during the evolution of the genus. The probability is not so much tha t different chromosomes are lacking from different species, as tha~ changes of the nature of translocation and fragmentation have occurred in the complement of a basic parental type during the course of evolution, resulting in an alteration ot! the position of the at tachment constriction

f in 1 Q

Fig. 20 gig. 21 Text-figs. 19-21. Somatic mitoses, x 3200. :Fig. 19. R. peltatus v a r . lru..ncal~ts, 2n=32. Fig. 20. R. conslantinopolilanus, 2n=42. :Fig. 21. /~. t rilobus, 2n=,~8.

in particular chromosomes. Such changes, accompanied by gene muta- tions, have led to a differentiation between the species which inhibits the production of inter-specific hybrids. Crosses of this nature have been made by Mr Marsden-Jones but he has been unable to obtain viable seed.

(4) Beha,,viour o f t~'a, ba~zts.

The trabants, which appear only at somatic divisions, are observed some considerable time before the chronmsomes approach their maximum contraction, and in favourable cells they may be seen at tached to the

266 Chromosome Var ia t ion and Behaviour in ]~a~nunculus L.

chronmsomes in the prophase nucleus (Text-fig. 12). On no occasion were tlmy attached to the nucleolus and subsequently transferred there- from to the ends of the appropriate ctn'omosomes, as has been suggested (Senjaninova, 1926, S. G. Nawasehin, 1927, Sorokin, 1929). During the prophase the nucleolus develops gemmae which might readily be mistaken for trabants. These nueleolar stuctures, however, not only vary in size from very minute particles to half the size of the nueleolus but as many as six may be present in one cell of a diploid plant. Whether this behaviour is of the nature of an artefact resulting from fixation, or whether it represents actual nucleolar activity at division, remains undecided.

The trabants themselves in any one root exhibit a variation in size fl'om cell to cell (Text-Sg. 14). This is probably nmre apparent than real and is attributed to the amount of stain retained. Hence there appears to be no justifiable reason for departure from the view that a trabant is essentially an integrat part qf, and morphologically homologous with, the rest of the chromosome. The sole claim it has to be termed a specialised structure rests on the facts that it is a segment separated from the bulk of the chromosome by a marked constriction, and of so small a size that on contraction it is reduced to a sphere less in diameter than the normal chromosome width (cf. Darlington, 1926).

IV. OBSEUVAmIONS O~ ~'OLL~N ~mmm~R CELLS.

(I) Ge~ze~'al.

A study of meiosis has been made on the anthers of R. acris (plants 1 and 2), R. sardous, R. ~'el)ens , R. Fiearia (plants 5 and 55/1) and R. Ker- neri. In all these, fixation is good at metaphase I and anaphase I, but indifferent except in occasional cells at diplotene, early diakinesis and second division. Meiosis as a rule follows a normal course.

Parasynaptic pairing is inferred from a typical zygotene and the diplotene loops (Text-fig. 22) open out in the normal way after pachytene. The chromosomes at diplotene do not appear to be very twisted and where fixation is good enough, clfiasma cotmts are possible. No "synaptic knot" as seen by Senjaninova (lee. cir. Abb. 11) was noticed, and the criticism of poor :fixation which she levels at Sorokin's preparations should, judging from her figures, be applied be her own. In badly fixed material and particularly in cut cells the prophase chromosomes often collapse on to the nucteolus and give the appearance of chains of granNes issuing from that body (of. Sorokin, 1927 a). The nucleolus disappears at late diakinesis and the chromosomes arrange themselves on the recta-

L. N. H. LAi%TE~ 267

phase plate withou~ developing any marked secondary constrictions (el. Sorokin, 1927 a).

Metaphasc proceeds normally in typical forms of the five species, bivalcnts being lmiversally formed; no mMbiva.lents were ever seen, only excepting the fragment association in R. Fica~'ia (55/1) which is discussed below. Such behaviour in R. rel)e'ns and R. Kerne?'i indicates that these are allo~etraploids, and it is highly probable that obher polyploid species of this genus which set seed regularly are of ~he same t;ype. Failure of pairing is very rare, and only in one or two cells of the many hmldreds examined in R. ac,ris and R. Fica~'ie (5) have univalents been seen (Text-figs, 24-, 39).

(2) Chiasma formation.

A s~udy of the chiasma behaviour from mid-diplotene to metaphase has been made and the results are summarised in the tables.

TABLE I.

Percentage of bivalents with 1 to 5, chiasmata at ,metaphase. No. of Chiasmah~ per b iv~leng ( % )

Z. cells r 5'

P l a n t n e x a m i n e d 1 2 3 4

R. ¢t~?'is (1) 7 30 91.4 8.6 - - - - - - R. sardous 8 12 78.2 21.8 - - - - - - 17, Fiearia (5) 8 16 7.8 54.7 28.1 8.6 0.8 R, F iearla (55/1) 8 + 2 f 18 27,8 59.7 11,1 1.4 - - t¢. relgens 16 6 86.5 13.5 - - - - - - tt. Kerneri 14 7 61.2 38.8 - - - -

TABLE II.

Chiasmc~ freffuencies at metaphase. No. of T e r m i n a l Te rmina l i -

cells C h i a s m a t a c h i a s m a t a s~tion P l a n t n e x a m i n e d per b i w l e n ~ per b i v a l e n t eoefficien~

t~. acris (1) 7 30 1.09 0.23 0-22 1~. acris (2) 7 10 1.34 0.,I3 0 '33 1~. sardous 8 12 1.22 0.63 0.37 1L Fica, ria (5) 8 16 2.40 0.63 0-26 t~. l~'icaria (55/1) 8 + 2 f 18 1.83 0,52 0.29 .R. rel)ens 16 6 1.14 0.29 0.26 2~. Kerneri 14 7 1.37 0.52 0.38

These tables, I and II, are both compiled from observations of com- plete cells. In the case of R. Fica,~'ia (55/1) only the eight normal bivalents are considered here, the behaviour of the fragments is discussed later.

At metaphase the frequency of total and terminal chiasmata is very similar in both R. acris (Text-figs. 23-26) and R. ~'el)ens (Text-fig. 27), and a comparison shows that the predominant type is the cruciform bivalent

268 Chromosome Variation and°Behaviour in l%anunculus L.

with one ehiasma, in fact in many cells of both species all bivalonts are of this type (Text-fig. 26). The ring and rod-shaped cont~gurations, how- ever, are not uncommon in both these species.

In R. sardous chia.sma frequency is-slightly higher and though con- figurations are of ~,he same types, bivalent rings (with two chiasmata) are present in a higher proportion than in the preceding species. The main

Fig.22

.44 e F

Fig. 25

Fig.23

Fig, 22.

Fi9.24

Fig. 26 Text-figs, 22-20. Pollen mother-cell divisions in R, acris,

L~e diplo~ene, x 3200.

Figs. 23, 28. Polar view of me~aphase I sho~4ng loe~lised chiasmata. Note fiwo uni- vMents (I) in Fig. 24.

Figs. 25, 26. Side views of metaphase I. Fig. 25 shows precocious separation of the 2' and another chromosome pair.

difference; however, is that the metaphase chromosomes are considerably thinner, though no shorter (Text-fig. 28) than in the preceding species.

In R. Fioaria (5), the normal type, there is a considerable increase in the ehiasma frequency, which is double that of R. aoris, and consequently the bivalent configurations (Text-figs. 29-31) are unlike those of the preceding species. The simple bivalent ring is, however, frequently met with. R. ficaria (55/1) is intermediate between the normal Ficaria (5) and the acris type, both as regards the number of chiasmata and the

L. N. H. LA]~TEP. 269

form of bivalent, which of course are interdependent. Cross-shaped bivalents are frequent and both rods and rings are more common than in R. l~ica~'ia (5).

Fig, 27

Fig. 28

Fig 3t

Tex~-figs. 27-31. ~e~o,phase I . x 3200. Fig. 27. 12. r~pen,s, side view, Fig. 28. R. 8ardous, aide view. Figs. 29-31. ~. Ficaria (5), side a,nd polar views.

(3) Terminalisation.

Examination of R. acris incheates that ~bout 50 per cent. of the bivalents show a m~rked degree of loealisation of ohiasm~ta in the region. of the at~achmen~ constriction (Text-fig. 23; el. Fq'itit~aria Me~eagris, Newton and Darlington, 1930, and Mecostethus gracilis, MeClung, 1928). This may be seen in different chromosome pairs in different cells and is

270 Chromosome V a r i a t i o n a n d B e h a v i o u r i n R a n u n c u l u s L.

therefore not due to an interruption of the homology of a particular pair resulting in the arrest of germinalisation (Darlington, i929),

TABLE III.

~%'howing total chiasma frequency fi'om diplotene to ,metal)base. TermhlM

No, of ceils Chiasm~g~ ch i~sm~a Termina.li- or bivMen~s l~er per s~i)ion

Pla.ng Phase exa, mined bivalcnb bivMeng coett]cienC R. acris '5'[id-diplogene 50 biva.lenfs 3.22 0.96 0"30

n = 7 L~ge diplogene 50 bivMen~s 3.04 0.76 0.25 Diakinesis 8 cells 1.50 0.,18 0.32 h'Iet)~phase 30 cells 1.09 0.23 0.22

R,. Ficaric~ (5) Mid.diplo~ene 30 bivMen/)s 3"13 0.73 0.23 n = 8 L ~ o diplot~ene 20 bivMenfs 2.65 0.85 0.32

Men,phase 16 cells 2.40 0.63 0.26

Terminal ehiasmata, in R. ac~'is at least, are not abundant at recta- phase, yet a reference to Table I I I shows that in both this and R. t#ica,ria the chiasma frequency at mid-diplotene is over 3. Terminalisation, which is seen from the coefficient to occur in R. aerls mMnly between late diplotene and diakinesis, is shown by the fM1 in chiasma frequency to be considerably greater than in R. ~Vica,ria; but in both species there is simultaneously a fall in the number of terminal chiasmata per bivalent. This can only be explMned (5{offett, 1932) as a result of the chiasmata slipping off the ends of the chromosomes owing to a reduction in terminal affinity (Darlington, 1932 b). I t is remarkable that ~he only three plants in which such behaviour has been fmmd, Anemone (Moffett, los. cir.), Ranuneulus and Adonis (Mather, tmpublished), are all members of the A n e m o n e a e .

(4) Ch~'omosome sepa,ration at anaphase.

In R. acris (1) it was noticed in some cells that the smallest (/~) chro- mosome pair, which contracts at metaphase to a very short rod a~ld practically always involves a single terminal chiasma, separates as soon as the rest of the complement is arranged on the equator (Text-fig. 25). I t was at first thought that the two P-chromosomes had failed to pair, univatents, however, are never fotmd at diakinesis. Tile fact that these chromosomes always lie perpendicularly above one another at equal distances from and on opposite sides of the equator, and that their adjacent ends are often slightly attenuated, has led[ to the conclusion that these pseudo-univMents are the result of a precocious separation. In anaphase these chromosomes complete their separation and pass ~dth ~he rest normally to the poles. In this strMn (1) of R. ae,ris such precocious

L. N. H. LA~T~ 271

II

~F ig 32

Fig. 88

Fig. 8 4 Fig.a5

Tex~.figs. 32-37. Anaphase I.

:D~igs. 32-34. Jg. acris. Fig. 35. 7L rcl;ens.

Fig. 86 x 4300.

F|g, 3 7

Figs. 36, 37. 7~. 7~icaria.

Journ . of Gene~ics x x v I 18

272 Ghro~noso~ne Variation and Behaviour i~, P~anuneulus L .

separation was seen in 7-9 per cent. of the 850 cells examined, but in another plant (2) it was much rarer, occurring in only 0.8 per cent. of 500 cells, whilst in R. ,reports and R. sardous separation was apparently

'HI //

O /f

i:ig,38

Fig.40

Text-figs. 38~I0. Side views of meeaphase I in R. Ficaria (55/1). x 3200, sltowing be- haviour of the H-ehrmnosome.

Fig. 38. Tlu'ec tmivaleng H-chromosomes; the fourth in a~tached go a cruciform bivalent by a germinal chiasm~.

Fig. 39. Two tmivalents and one bivalent. The two large univalen~s are due to failure to form ~ eMasma in one of the larger pairs.

Fig. ~10. Two bivalent H.ehromosomes.

quite normal in all cells. A similar precocious separation is fmmd in the sex chromosonies bf Mus ~nusezdus (Painter, 1927).

During anaphase, separation of the chromosomes in one cell is not simultaneous (Text-figs. 32, 33), the time at which separation is completed

L. N. H . LA~T:Z~ 273

varying wirE the length of th.e free arms distM to the ehiasm.ata in a bivalen% wEich in turn depends upon the length of a chromosome and the position of chiasmata. As has..been shown above, the F-chromosome having a single terminal chiasma and being the shortest always separates fn'st, even before other larger terminally ~%ssociated pairs. The longer chromosomes, especially those having chiasmata near the attachment constriction, are always the last to become completely separate. The tension caused in the pMred arms of the chromatids involved in ohias- mata, during anaphase results in their becoming very attenuated, so that they may appear to have suffered fragmentation (Text-tigs, 36, 37) ; when separation is eventnMly complete gEe ehromatids contract and often a small eba'omatic bead appears temporarily a~a the end of one of them (Text-figs, 3¢, 35). It is probable that Senjaniuova (los. eft.) has mistaken such a bead in R. acris for a trabant, for in Eer figttre of anal)Ease I (Abb. 18) sEe shows a trabant apparently attached to the long arm of a chromosome, whereas in all species studied by me, excepting only R. ophioglossiJblius, the trabant is attached to a short arm. In no case have I seen trabants during meiosis, their non-appearance being doubtless due to the greater contraction of the chromosomes at this stage causing the trabants to become adpressed to the main body of the chromosome. This behaviour is parallel with the observations of M. Navashin (1927) on @topis. The derivative of the cross 6'. capillaris × C. tectorum, both of which have trabants on the D-ehi'omosomes, lacks a trabant and its absence is related to the fact that the chromosomes of the hybrid are more condensed than those of either of the parents. The apparent absence of trabants at somatic mitoses in forms ~dth relatively small and probably greatly contracted chromosomes, c. 9. R. 2)eItatus, is doubtless due to the same cause.

(5) Behaviour of fl'agments in R. Ficaria (55/1).

In one anther of R. Ficarga (55/1) in which metaphase I was studied, owing to duplication of the two fragments seen in the root tips mentioned above, there were 20 chromosomes in each cell. Of these, the 16 large chromosomes invariably paired to form eight bivalents and their be- haviour has been discussed. The four fragments in most oases formed one bivalent and two univalents, but other configurations were seen (Text- figs. 38-~1:3, and see Table IV).

It is noteworthy that unlike the sEort chromosome in Stcnobothrus .parallelus (Darlington and[ Dark, 1932) the fragments in R. F4cm'ia (55/1) have approximately the same chiasma feequency per unit length as the

18-2

274 Chromosome Var ia t ion and Behav iour in P~anuneulus L.

longer chromosomes in ~his s~rain; as a result therefore ~hese fragments are less highly adapted to the meioNe mechanism thal~ are the short chromosomes in Ntsnobothrus and cons,equently rafivalengs (Text-figs. 38,

TABLE IV.

Numbers of cells with,frag'm,c,ts associated in var,ious ways in }~. Ficgria 55/1. 41 l II + 2 t 2 H t m + 11 1TM Tol;al

Cells l l 26 9 5 2 53 Chia.sma~ 0 26 18 10 8 62

Chiasmggg per po~cnt.igl biw~,leng =0.59. UniwdenCs per 100 fragments =47.6 %.

°

Fig. 41

H

H g . 4 2

a b

Fig. 43

Text-figs. 41-43. Metaphase I ill R. Ficari(~ (55/]). Fig. 41. Side view showing H-}~'hromosonm qm~drivMml~. Fig. ~L2. :Pob~r view showing four univ,~len~ frt~gmen~s. Fig. 43. (c~) chMn ~rivMen~, (b) chMn qu~driwlent .

39, 42) are far more common. On tlle other hand, as has been pointed ou~ above, -Lhey are better adapted to the mechanism of somaNe mitosis than are ~hose found by Langlet in R. aerie.

L. N. H. LAl~a'E:a 275

In all eases associated fragments are united by terminM chiaasma.ta, at metaphase, as would be expected fl'om their shortness. Trivalents are Mwaays of the eha.in type and quadriva.lents were of the ring aand chMn types (Text-figs. 41, 43); had more materiM been a.vMlaable doubthss other configurations would have been found (eft Darlington, ]930).

At meta.pha.se these fragments, whether pMred or mlpMred, are dis- tributed at random in the spindle and, unlike the pseudo-univaalents in R. acris (1), are ra.rely found normally orientated in the plate (Text-figs. 4:0, ~[1). Usually a pair of fragments will be lying across the spindle some distance above the pla.te and very often call four lie towards the saame pole. It is therefore highly probable, though I hays not been able to obtain ma.tcrial of ana.pha.se I, that the fragments in the pollen grains would[ va.ry in nmnber fi'om 0 to 4. This supposition is substa.ntia.ted by the observa- tion in one cell of a. fragment lying undivided on the equator a.t a.na- phase II; as a rule, however, fixations of second meiotic and pollen-gra.in divisions are insufficiently clear for deta.iled study.

Very occasionally one fl'a.gment is found paired by a. terminal chiasma with the distal arm of one of the norma.1 chromosomes (Text-fig. 38). This suggests that these fragments, which a.rs known from their pairing to be homologous ~dth one another, are at least in part homologous with a segment of one of the chromosomes and therefore originally arose by segmenta.1 reduplication (cf. ])arlington, 1929, 1930); there is no evidence, however, to show how or when such reduplica.tion took place. The ra.ri~y of associations between fl'agments and whole chromosomes indicates tha.t the mntuM homology is confined to a smM1 segment, and that proba.bly a. certain amount of genie differentiation has occurred since the origin of the fragments. Early in development of either the anther or the flower (the other anthers werg not dividing), the pair of fra.gments have again re- duplicated at an aberrant somatic division.

V. DDISCUSSION--TtIEOR, ETICAL CONSIDERATIONS.

(1) Terminalisation.

In a recent paaper on the chromosome dyna.mies of 5'tenoboth,rus l)arallehts ])arlington and ])ark (1932) have modified the electrosta.tie theory of chromosome separation (Kuwada 1929) to include aa wider aspect of cytological phenomena than has hitl~erto been possible. These anthers postulate that the separation of the chromosomes at meiotic prophase and to some extent at early anaphaase is explicable on the

276 Chromosome V ar ia t i on c~nd Behceviour i~z I { a n u n c u l u s L .

assumption of two electrostatic charges, one locMised at the at tachment constriction and one uniformly dispersed over tlle surface of the chromo- some. These changes not only resMt in the mutnM repulsion of parts of tlhe coml)onent chromosomes of a bivalent or multivMent but also in rel~ulsion between the bivMents, etc., themselves. They further argue tha t whilst the generMised charge is constant, having the same e:ffect in all organisms, the locMised charge is specific to the organism, species or genus, etc., and the extent to which these charges are separately re- sponsible for chromosome movement, e.9.tm'minMisation,correspondingly varies. In a bivalent with severaI chiasmata the et~eet of either .force on terminalisation will be greater within a closed loop than when acting between ~wo free arms, owing to the closer proximity of the parts of a chromosome forming a loop.

Thus in Text-fig. 66 if G is the effective terminMising force of a chiasma within a closed loop due ~o the generalised charge, a smaller force g is the corresponding e:f~ect between free arms, and a similar argu- ment applies to the localised force L and 1. Then at the chiasma most distM go the constriction, G being greater than g, the chiasma will shift to a terminal position. Similarly at the chiasma nearest to the attach- merit constriction the terminalising force is g q- 1 - G, and where L a~zd 1 are sz~ciently large this too will terminalise. In an organism where oNy terminal chiasmata occur at metaphase g + 1 must be considerably greater than G; the process will then be rapid, so tha t all chiasmata will terminalise before metaphase, e.g. Primz&t, etc. As would be expected in organisms with a large looMised charge, where there is a c.hiasma on each side of the at tachment constriction the loop containing the con- striction will be larger than more distal ones. No such distinction is seen in the bivMcnts of Ra~au~zculus at any meiotic phase. On the other hand, in a plant such as T~d@a, where there is very little terminalisation, L is relatively small and 1 is so small compared with g tha t bivalen.ts with single chiasmata will show no terminalisation (Darlington and Janaki- Ammal, 1982). I t is therefore conceivable tha t where L is sufficiently weak, in a bivalent with one chiasma and with a subterminal constriction, terminMisation may even ta](e place tozva,~'ds the at tachment constriction (Text-fig. 4,1), and it is probable that this accounts for the occurrence of chiasmata localised near the constriction in R. acris (cf. ,v~'itillarict mele- ag'r'is, Newton an/[ Darlington, 1930), and for the preponderance of bivalents at metaphase with a single intcrstitiM chiasula. Such localisa- t ion would also be expected, owing to the additionM complications opera eive, in organisms where interlocking bivalents are common.

L. N. H. Lawi'~L~ 277

\ \

/

f:ig. 44 Text,-fig. ~-'L Diagram illusbr~l~ing ~ernfin~lis~l,ion ~nd "st ipphlg off'" of chiasma£~ dm'ing

diptotene in Ranu'~czd.~ls. ]?osiifion of ~t~l;~chmen~ constriction is nmrked by ,~ Lhin line anti is sub~ermim~l in all bu~ ~he bobl~om figure. ]3lack ~rrows lm~rk t,he direotion of movontenL of chiasnl~d~a. (oxpl~n~tion ill t;exl,).

278 Chromosome Variations, and Behaviour in I%anunculus L.

The conclusion seems not unjustified, therefore, that in Ranu~c'dus the locMised charge is relatively weak, and that terminMisation in the genus is mMnly brought about by the generalised charge and is operative principMly in bivMents with a chiasma on each sidle of the attachment constriction.

A consideration of terminal <~ffinity is necessary to throw further light on the anomalies of ternlinalisation in Ranuncuhts. This term implies a speeiM attraction of the ends of hMf-ehromosomes (chromatids) which alIows them to remain attached go one another when chiasmata termina- list. This idea was independently introduced into cytological thought by O'Mara (1.931) and Darlinggon (1932 b). The former author attributes it to a longitudinM affinity between the chromomeres lying along the ehromatid, which in the absence of terminal chiasmata remMns unsatis- fied at the ends of the chromosome. Whether this be so, such an MYcmity must e~s t in most organisms, and to explain the permanence of ~erminM chiasmata mast exceed[ the mutual repnlsion of neiglibouring interstitial chromosome segments.

In Ranunc~dus, as terminalisation proceeds, there is simNtaneously a reduction in the number of terminal chiasmata (Table III) instead of the expected increase; this can only be attributed, as in Anemone, to the terminal chiasmata slipping off the ends of the cln'omosomes as they arc formed (Moffett, 1932). ])arlington (1932 b) attribntes the "slipping off" to a reduction of terminal affinity of the chromatids in these plants. This hypothesis supplies an explanation for the apparent anomaly that though the localised charge is small in Ranunculus a, cris there is, as Table III shows, a high degree of terminalisation; for the bulk of the mechanical resistance to ehiasma movement imposed by the persistence of the first terminal chiasma is removed, so that a slight Iocalised charge is sufficient to move the chiasmata along the bivalent. The temporary accumulation of terminal ehiasmata during diplotene shown in the table is indicatb}e of a high rate of terminalisation.

It is apparent, however, that terminal affinity between ehromatids is not completely absent in Ranunculus, etc., for terminal chiasmata are seen after diakinesis, i.e. at metaphase, when chiasma movement has ceased. Furthermore, in no organism have terminal ehiasmata been observed at late paehytene, that is, they are not formed ab i'n~tio, when the chromosomes sp]it longitudinally, but are always due to terminalisa- glen of interstitia]s (])arlington, 1932 b) ; it is obvious therefore that there is no germinal affinity at this stage. The inference is that whilst in organisms with normal terminalisation, fermium affinity is developed at

L. IN-. I-I. LARTER 279

early diplotene, in Ranunculus the slipping off of chiasmata is due to a delayed onset of this attraction.

The same hypothesis may be applied to explain, the precocious separation of the/~-chromosome in/L acris (1) ; in this plant it is suggested that the onset of terminal affinity is slightly later than in other forms, e.g .R, ac'ris (2), with the result that the smallest chromosome pM{' (repulsion between whose constrictions is greatest owing to their closer proximity) separates before the rest and before the stage at which the corresponding pair in R. acris (2) separates. [['his is borne oat by the fact that the higher total ehiasma frequency of this latter plant as compared with R. ac'ris (1) is almost entirely due to the retention of a greater number of terminal chiasmata. The number of interstitials is approxi- mately the same in both strains (see Table II).

An alternative explanation, that this difference is due to an unusually large charge in the F-chromosome of strain (1), is not adopted, as it implies a difference in the localised charge on various bivalents of one organism, an additional assumption both mmecessary and incompatible with the electrostatic theory. The third hypothesis that ill the F-bivalent of strain (1) chiasmata are fl'equently formed between the short arms of the chromosomes, also supposes a differential behavionr for which there is no other evidence, this hypothesis is nevertheless quite tenable.

In the case of R. Ficarict the fignres in Table III suggest that fewer terminal chiasmata slip off the chromosomes in this species, and at the same time terminalisation is much less than in R. ac~'is. Observation on the latter plant indicates that in bivalents with more than one chiasma the generalised charge is alone sufficient to cause all but the most proxi- mal chiasma to terminalise in the absence of terminal affinity of the chromatids, and consequently the assmnption that the locMised charge is even smaller in R. Ficaria than in R. acris would not afford an explanao tion of the difference in terminalisation between these two plants.

Therefore it can only be inferred that the lack o~ more complete terminalisation in R. Ficaria is due to the resistance to the movement of chiasmata imposed by the retention of terminal chiasmata which is due to an earlier onset of terminal affinity in this species, In this respect, therefore, R. F,icaria approaches nearer to normality than does R. acris.

(2) Genoty.pic cont~'ol of the ch~'omosome compgement.

The suggestion has been put lorward by Lewitsky (1931) and Darling- ton (1932 a) that the chromosome complement, in common with the form and function of other parts of an organism, is subject to geno~bypic

280 C h r o m o s o m e Va~'iation a n d Behaviou~' i~ ~ , a n u n c u l u s L.

modification, and the present study of Ranuncuhts lends support to this doctrine. For instance, there seems little doubt that variation in the size of the chromosomes in the root tips of different species, e. 9. R. pclta- tus, R. sa'rdous, R. ac~'is, etc. mentioned above, is due to such a cause.

At meiosis the chromosomes are, however, subject to more varied influences than at somatic mitoses, and consequently differences in size are often of more obscure origin. In R. sa~'dous, for instance, at meiotic metaphase I the chromosomes are unusually attenuated, especially be- tween the constriction and the proximal chiasma. In this species the somatic chromosomes are both shorter and thinner than those of R. acris, wlfile the degree of contraction at meiosis is similar; the mechanical resistance being tess and the attachment constrictions being closer together, the distorting effect due to the repNsion between them is correspondingly greater. This attenuation is therefore dependent on a modifieatiou which is equally applicd)le to the soma. In Matthiolc~ incana var. Snowflake (Lesley and Frost, 1927) ~he chromosomes at meiosis are subject fie a special modification which does not affect somatic division; in this case ~hey are less contraceed than in the normal strain. Furthermore, there may be a differential effect even within the comple- ment, as in many animals where the sex chromosomes undergo a preco- cious contraction, e. 9. Nat,'ix, Nyete¢'eutes (Minouchi, 1929), StenobotN'us l)a~'allehts (Darlington and Dark, Ice. cir.). In ~qtenobotN'us there is another special modification, in that the short chromosome has a chiasma fre- quency per unit length over twice as great as that of the long chromosomes, wi~h the result that the short ones never fail to pair. Though, as these facts show, genotypic control may be very specialised in its application, generally the chromosome complement is affected as a whole.

In R. ~ica~'ia the high chiasma frequency at metaphase which is due $o a lower rate of terminaI~sation, a fimction of the chromosome repulsion and the terminal affinity, is evidence of the variation of that affinity within the genus as is shown above. Thus the localised charge which is responsible for the difference of chromosome repulsion is, together with the terminal aKhfity, instrumental in determining the configm'ation of the bivalent. The type of configuration, however, which is adopted by any one chromosome pair depends in the first place on the nmnber and position of chiasmata formed at pachy~ene, within limits a matter of chance; in view of this, doubt is east on the validity of the statement made by Sorokin (1927 a), that one pair of ctu'omosomes assumes a definite configuration, for example, that her A-chromosome in 7?. acris always forms a cruciform bivalent.

L. N. H. LA:[¢TEt~ 281

It is well known that the characteristics of the chromosomes, both individually and as a complement, are to a large extent constant ~dthin a species or larger group. I have endeavoured to show here that the variations which do occm: are of a definite and orderly kind[, that once having occurred they do not fluctuate, and that they are such as would be due to an alteration in the genetical constitution of the organism ra~her than to environmental changes. There appears no reason to doubt that in Ranunculus, as in fact in most other organisms, the chromosomes are subject to the genotypie control of such characteristics as chromo- some size, contraction, reptllsion, chromatid attraction and terminal affinity, in a manner that is in all but very few respects entirely com- parable with the control of the better known hereditary characters.

0hromosome counts from the root tips of thirty-six species of Ranun- cuIus reveal two polyploid series in the genus, founded on the basic numbers n = 7 and n = 8. Comparison of the chromosomes of different species shows marked[ resemblances throughout the genus. There is uo evidence of correlation between chromosome morphology and the occur- rence of intersexual forms of R. ac,ris and R. Iricaria.

Somatic doubling is common in root tips, and probably in other tissues of several species, and has possibly gNen rise to the tetraploid strains of R. Fica~'ia.

Observations on the behavionr of trabants during somatic mitosis shows that they are morphologically homologous with the chromosomes and cannot be regarded as distinct organs. No trabants are observable at meiosis.

Fragmentation has been discovered in/7. I~ica~'ia,, and evidence from the study of ehiasma formation of these fragments suggests that they arose as reduplicated chromosome segments.

A study of meiosis, and[ in particular of chiasma behavionr, shows that the terminatisation of chiasmata in Ranu~'~,culus is explicable on the electrostatic theory of chromosome separation as emmciated by Darling- ton and Dark (lee. cir.). T_erminalisation results in ehiasmata slipping og the ends of the chromosomes, and there is support for the assumption that this is due to a delayed onset of the terminal affinity of the chroma- rids. The same assumption may be employed to explain the anomalous separation of the smallest chromosomes in one strain of R. ac~'is.

Consideration of variation o~ the chromosomes within the genus finds support for the theory (Darlington, 1932 a,) that the morphology and

282 Ch,romosome Va~'iation a~,d Behaviou~' i~, .Ranunculus L.

be haviour of ~he chromosome complement is subjec t to genotypic control

in a, m a n n e r s imilar fo tha,t of other par~s of an organism.

I ~dsh to express m y si,~cere thanks to Dr C. D. Dar l iug ton for his

va luab le crRicism and advice t h r o u g h o u t the course of this inves t igat ion.

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