The Structure of the Somatic Chromosomes of Alstroemeria and Bomarea.1 By Frank W. Jane, Dept. of Botany, University College, London.

With Plates 2-4 and 2 Text-figures.

THE exceptionally large chromosomes of Alstroemeria and Bomarea furnish good material for study; an attempt has therefore been made to trace the changes which occur in these bodies during the mitotic cycle. Other genera of the same section of the Amaryllidaceae (Hypoxidoideae) have been ex- amined with the same end in view, but with less success owing to their chromosomes being smaller. The somatic nuclei of Alstroemeria and Bomarea are almost identical, but differ, as Whyte (43) has shown, in that those of the latter genus contain eighteen chromosomes, whereas there are sixteen in the somewhat larger nuclei of the former. Although no attempt has been made to follow the history of mitosis in any one species, this has been traced with tolerable completeness in Bomarea multiflora; a number of species of both genera has been examined. METHODS. Eoot-tips were used for the investigation, and were fixed mainly in Benda's fluid, using about three drops of glacial acetic acid to 19 c.cm. of the chrome-osmic mixture: the omission of acetic acid (Gatenby's modification of Flemming's mixtures) proved unsatisfactory, penetration being so slow that the cyto- plasm was badly contracted. Flemming's strong mixture, diluted with an equal quantity of water, proved useful, but was some- what capricious. In the later stages of the work fixation with Benda's fluid, diluted with an equal quantity of water, was tried, and results were very satisfactory; it did not cause so 1 Part of thesis approved for the Degree of Doctor of Philosophy in the University of London. NO. 305 E 50 FRANK W. JANE great a contraction as the stronger medium. In the early stages of the work Merkel's fluid was also used, but was given up, not owing to its fixing qualities, but because it was so difficult to obtain satisfactory staining subsequently. A suction pump was employed at the time of fixation, but was sometimes dis- pensed with, the material being dipped into absolute alcohol and then placed into the fixative. The results in the two cases appeared to be identical. Even the diluted Benda's fluid penetrates somewhat slowly; it gives, however, a very clean fixation of the chromosomes, which do not show the swollen appearance so often associated with fixation in a mixture containing a relatively large quantity of acetic acid. Chromosomes of pollen mother cells, just re- moved from the living anther, and those fixed in Benda's fluid are indistinguishable. Newton (28) found that in Galtonia, when fixed in this fluid, the chromosomes most nearly corre- sponded to those of living material. Three stains were tried out: (a) Iron Haematoxylin, which was not used extensively because it was so much a matter of chance whether good or mediocre preparations were obtained. (&) Arnold's Stain: this technique closely resembles the better-known Breinl stain. It was used in a somewhat modified form, as follows: sections were treated with iodine solution (1 gr. each of iodine and potassium iodide in 100 c.cm. 80 per cent, spirit for 5-15 minutes, washed in water, stained for at least 4 hours (preferably overnight) in safranin (equal parts saturated alcoholic solution and saturated aniline water solu- tion), washed in water, stained for 2-10 minutes in the poly- chrome methylene blue solution used by Arnold, washed in spirit, and stained in orange G in clove oil. Old solutions of safranin and polychrome methylene blue are to be preferred to freshly mixed stains. Well-stained preparations give prophase figures unsurpassed by any other method, although metaphase and anaphase chromosomes are rather densely stained. (c) Newton's Gentian Violet was most used, on ac- count of the transparency of the preparations. When the pre- parations were counterstained, orange G in clove oil was largely CHROMOSOMES OF ALSTEOEMBRIA AND BOMAREA 51 used, and later, light green in the same medium. The latter appears to constitute the ideal combination. Sections were found to stain best at 10 p., although satisfactory staining was obtained with sections 14r-16/u thick; it was not found possible to obtain critical staining with thicker sections.

DEFINITIONS. The original and modern meanings of cytological terms often differ. For this reason it is proposed to define certain terms, giving the sense in which they are used in this paper. Chromatid.—' A half chromosome between early prophase and metaphase of mitosis . . . after which stage, i.e. during an anaphase, it is known as a daughter chromosome . . .' (7). Chromomeres.—'The serially aligned granules of the spireme threads of chromosomes: equivalent to "Pfitzner's granules" . ..' (44). Chromonema.—The spiral or zigzag thread, visible at certain stages in the matrix of the chromosome, at other times, when there is no matrix present, forming, together with the chromomeres, the whole chromosome. Chromosome.—Usually denned as a chromatic nuclear structure, developed in early prophase, and persisting until telophase. It is defined here as a nuclear structure which, in its simplest terms, consists of chromomeres, which are permanent bodies, and chromonema or chromonemata, which are non- permanent in the sense that during the mitotic cycle a new series is produced; between the mid prophase and telophase these two components are embedded in a ground-work or matrix. Matrix of Chromosome.—The transient, less chromatic part of the chromosome, which appears in prophase and persists until early telophase. It is not known if it is chemically distinct from the chromomeres or if the difference is purely physical.

OBSERVATIONS. Anaphase. The structure of the fully formed chromosomes is best seen at anaphase: as they travel to the poles of the spindle they are seen to consist of a fairly chromatic ground- work, in which are embedded one or two more chromatic threads, 52 FRANK W. JANE the chromonemata. These threads have the appearance shown in figs. 1-4, PI. 2, and are never seen to take the form of straight lines, indicating that they pursue a spiral and not a zig- zag course. Complete chromonemata, running the length of the chromosome, were rarely observed; usually the threads were visible in parts of the chromosome only, sometimes single, sometimes in twos. Chromomeres were frequently visible in the anaphase chromosomes; they stained as deeply as the chromone- mata, and varied somewhat in size (fig. 1, a, fig. 2, PL 2); they were always very small and approximately paired. Densely staining, more or less transverse bars were sometimes observed in the chromosomes at this stage (fig. 2, a, and base of b, PI. 2), and probably represent imperfectly differentiated chromone- mata or chromomeres; evidence for this view is seen in chromo- some b, fig. 1, PL 2, in which the spiral single thread appears thick in parts, in such places resembling the bars in chromo- some a, fig. 2, PL 2. When chromomeres are seen they are generally connected with one another by the chromonema (figs. 1, a, and 2, b, PL 2), which in such chromosomes appears thinner than in those where no chromomeres are visible. In an anaphase figure the chromosomes do not all show the same structure. In some the chromonemata are complete, forming a single or double spiral, in others they are in the process of formation and the chromomeres are visible. Polar Concentration.—The chromaticity of the matrix of the chromosomes gradually decreases; this does not proceed uniformly, parts of the chromosomes becoming less avid for nuclear dyes earlier than other parts. The chromosomes reach the poles of the spindle, where they form a dense mass. This polar concentration, termed by Gregoire and Wygaerts the tassement polaire is very pronounced in both genera. At this time the chromosomes are very closely associated, and the diffuseness with which they stain suggests the possibility, put forward by several investigators, that at this stage chromatic material is passing out of the chromosomes. This polar con- centration has been attributed to faulty fixation, although de Litardiere (21) attempts to show that this view is fallacious, CHROMOSOMES OF ALSTEOEMERIA AND BOMARBA 53 and explains the phenomenon as due to the narrowing of the spindle near the poles. It is certain that the polar concentration cannot be regarded as a clumping of the chromosomes; such a conclusion might be reached from a study of lateral views only, but a similar clumping should be visible in polar views also, and this has not been noted in any nucleus; what is seen in polar view is a number of chromosomes, more or less in end- view, very close together, but not touching adjacent ones at any point; viewed from the side, together with the diffuseness already mentioned, this close aggregation of large chromosomes would produce an appearance of clumping. It seems probable that to some extent the figures are exaggerated by fixation, as the dense mass of chromosomes might well hinder rapid penetration of the fixative. It not infrequently happens that parts of certain chromosomes are excluded from the general mass seen at the polar concentration; it has been observed in several cases that these represent the ends of the satellite- bearing pair of chromosomes referred to by Taylor (39) and Whyte (43); this chromosome pair occurs in both Bo mare a and Alstroemeria; owing to their greater length, the ends of these chromosomes reach the poles after the beginning of the polar concentration (Text-fig. 1, a and h). It is rarely possible to make out details of chromosome structure at polar concentration, but in these excluded parts well-defined chromo- nemata are sometimes seen (figs. 5 and 6, PI. 2), and show all the irregularities of the chromonemata already noted. The matrix at this time appears to be less chromatic than in ana- phase, and this certainly applies to the excluded portions of the chromosomes. As the chromosomes pass into telophase, practi- cally all traces of the matrix have disappeared and it is only on occasion faintly visible. Telophase.—In Alstroemeria and Bomareaanasto- moses are rare in early telophase, although a few very fine strands of chromatic material can sometimes be seen between adjacent chromosomes; the general linking up of the chromo- somes by anastomoses does not take place until late telophase. During telophase the chromosomes, which now consist of chromatic material only, have the same appearance as the 54 FRANK W. JANE chromonema within the anaphase chromosomes; the matrices have disappeared. Sometimes this is seen very clearly (fig. 7, PI. 2), in which figure it will be noted that there are a number of chromatic dots on the chromosomes. Some of these may be nothing more than turns in the spiral, but it is difficult to put this interpretation on others, e.g. top of chromosome a or

TEXT-FIG. 1. A, lateral view of anaphase, Bomarea multiflora, showing two chromosomes, the ends of which are farther from the pole than are those of the other chromosomes. B, one of the satellite- bearing chromosomes of Bomarea multiflora, the end of which is sometimes excluded from the polar concentration. Arrow marks region of spindle-fibre attachment. middle of chromosome b, these are regarded as chromorneres. In chromosome b the beginnings of anastomoses are visible on the left-hand side; in fig. 10, PI. 2, they are more marked and the chromosomes have a reticulate appearance rather than one produced by a single or double spiral: in this figure chromomeres are visible; their appearance does not suggest that they are due to the ends of spirals. As telophase proceeds there appears to be a flattening of the spirals (figs. 8 and 9, PI. 2) producing the reticulate appearance of the chromosomes noted above. Anastomoses now become more frequent; their beginnings are seen in fig. 9, PI. 2. The chromonemata become more attenu- ated, indistinguishable from the anastomoses, and the nucleus in late telophase presents the appearance of a fine network, the knots being formed by the chromomeres, the meshes by the chromonemata and anastomoses; in this condition, judged by CHROMOSOMES OF ALSTBOEMERIA AND BOMAREA 55 fixed material, the nucleus passes into the resting condition. It is not always possible, however, to determine whether there are connecting strands between the net-knots; this may be due to their tenuity or lack of chromaticity, or may indicate that they do not exist. Besting Nucleus.—The network is not limited to the peripheral region of the resting nucleus, but extends right through it, a type of reticulum which is referred to in this paper as distributed. In two other members of the Hypoxidoideae, Anigozanthos rufa and Curculigo recurvata, the reticulum is peripheral and the chromosomes are much smaller than those of Bomarea and Alstroemeria. It may be suggested that, for some reason, it is possible for only a limited area of the periphery of the resting nucleus, relative to its surface area, to be occupied by chromosomes, and that in Alstroe- meria and Bomarea this area is too small to accommodate all the chromosomes, with the result that some of them, or parts of them, are situated within the peripheral zone. Evidence which tends to confirm this view was obtained by measuring the width and total length of the metaphase chromosomes, and the diameters of the resting nuclei in Alstroemeria auran- tiaca, Bomarea multiflora, Anigozanthos rufa, and Curculigo recurvata (Table 1). All measurements were obtained from cells of root-tips, fixed in Benda's fluid. Very accurate measurements were not possible in the case of the metaphase chromosomes of the two former species, since it is not often that chromosomes are encountered lying in one plane. How far the size and number of chromosomes serves to account for the difference between the two types of reticulum cannot be determined until nuclei of a larger number of species have been examined; moreover, it has been assumed above, since so few species were dealt with, that there are but two types of reticulum, the peripheral and the distributed. No information is available to show if these two types are as sharply separated as is here suggested, or whether the peripheral type varies in thickness in different species, so that there might exist every gradation between the peripheral and distributed reticulum. An additional factor would also demand attention when 56 FRANK W. JANE measurements were made of a large number of species; allow- ance should be made, if possible, for the contraction in length of the chromosomes during prophase. Nucleoli are not favourable for study in either Alstroe- meria or Bomarea. There may be one large one or several

TABLE 1. Relation between length and width of metaphase chromosomes and reticulum of resting nucleus.

Anigozanthos 43,* 04 ,1 8-7,1 238 n* 7-2 Peripheral rufa . Curculigo recur- 42 ,t 0-3 /x 64/* 92 p.* 13-7 Peripheral vata 8 Alstroemeria 163 ix 10 n 12-8/* 614 ii 31-7 Distributed . aurantiaca Bomarea multi- 137 p. 1-0 ii 11-5 p 415 ^ 33 0 Distributed flora rather smaller ones, not uncommonly they are small and rather numerous. In the resting nucleus, or more probably in those of very late telophase, bodies having the appearance of early telophase chromosomes are sometimes seen (fig. 16, PL 3). These are regarded as the ends of chromosomes which were excluded from the polar concentration; it is not unlikely that their subsequent development lags behind that of the shorter

Telophase in Tecophilea and Anigozanthos.— Apart from Kaufmann's (17) studies in the chromosomes of certain ferns, descriptions of chromonemata have been based on large chromosomes only. In two other genera of the Hy- poxidoideae, Tecophilea and Anigozanthos, evidence is CHEOMOSOMES OF ALSTEOEMERIA AND BOMAREA 57 available that small chromosomes possess a similar structure. In Anigozanthos rufa telophases are occasionally seen which show spiral chromosomes (fig. 14, PI. 3). In this figure chromomeres are visible, and some at least do not appear to represent turns of a spiral: the nuclei show both single and double spirals, and a chromosome in which the matrix is still fairly chromatic. Anastomoses have not yet appeared between the chromosomes. In fig. 15, PI. 3, a late telophase of Teco- philea violacea, the chromatin corresponds fairly well with that of Alstroemeria and Bomarea at this stage. Prophase.—The most characteristic early prophase stage is that in which the chromatin appears as a series of spiral bodies (fig. 20, PL 3), but one of the earliest recognizable stages is seen in fig. 17, PI. 3, in which, in parts, the reticulum is still visible, while in other parts, chromosomes, either straight or spirally coiled, are present. This condition appears to arise by the breakdown of parts of the reticulum of the resting nucleus, presumably those parts formed by the telophasic anastomoses. The rest of the network thickens up, in parts more rapidly than elsewhere; in some places the reticulum appears as ladder-like structures, elsewhere as single spirals. Chromomere-like bodies are often visible at this period (figs. 18 and 19, PI. 3). Fig. 20, PI. 3, shows the spiral stage at its height, and here in one or two cases, chromosomes are visible which are clearly composed of two chromatids side by side. The spirals of early prophase usually appear to be single, but it is occasionally possible to distinguish the two chromatids of which they are composed (fig. 19, top of chromosome a, fig. 18, chromosomes a and b, fig. 20, chromosome a, PI. 3). A localized loosening of the spiral may reveal the paired chromatids in certain places, and it is this, it is believed, which has been interpreted by a number of investigators as the first appearance of the longitudinal split in the spirals. When the chromosomes (as usually defined) first appear, they are double structures (fig. 24, PL 4); the two chromatids are well separated although lying parallel; chromomeres are usually prominent, and opposite chromomeres are frequently connected by very thin bridges of chromatic material. The chromosomes, 58 FRANK W. JANE evolved directly from the spiral stage, often retain traces of the spiral twisting until late prophase (fig. 27, PL 4). In fig. 22, PL 4, a chromosome, a, of the type just described shows traces of the original spiral at its lower end. Late Prophase and Metaphase.—In the later pro- phases (fig. 27, PL 4) the chromatids become more chromatic, the process appearing to start with the chromomeres: the chromatic bridges may still remain. Chromosomes of the late prophases are frequently considerably twisted and may not become straightened until after they are attached to the spindle fibres. This twisting has been interpreted as the remains of the spiral twisting of early prophase. In the late prophases and in metaphase, the chromatids have thickened, forming paired, densely chromatic, ribbon-like bodies. Prolonged differentiation of the stain shows that they are not homogeneous structures; they contain chromomeres more or less paired, and in the most favourable preparations fine connexions are visible between the chromomeres (figs. 28 and 30, PL 4). In fig. 30, PL 4, which represents the long chromosome of Alstroemeria aurea, parts show the double chromomeres in each chromatid, while chromonemata are visible in other parts, sometimes single, sometimes double spirals; similar evidences of chromomeres and chromonemata are to be seen in fig. 28, PL 4, which represents metaphase chromosomes of Alstroemeria aurantiaca. It will be noted that at one point, a, in fig. 30, PL 4, the sister chromatids are connected by an anastomosis. Anastomoses— the chromatic bridges referred to above—are also to be observed between the chromatids of the metaphase chromosomes of Bomarea multiflora (fig. 29, PL 4). Begion of Spindle Fibre Attachment.—Taylor (39) investigated the position of spindle-fibre attachment in Al- stroemeria brasiliensis, and this question has not been studied intensively in the present investigation. In both genera, however, the long chromosome pair was examined, as it could be recognized in all except the earliest prophases. Newton (28) found constrictions visible in the later prophases of Galtonia, and Taylor (40) succeeded in tracing them back to the spiral stage in Gasteria. In the genera under present consideration CHROMOSOMES OF ALSTROEMERIA AND BOMAREA 59 the long chromosome pair has a median attachment constriction (fig. 80, /, PI. 4). The constriction is visible in late prophase (fig. 27, PI. 4). In the late spiral stage shown in fig. 24, PI. 4, the attachment region of one of the smaller chromosomes is distinct (fig. 25,/, PI. 4). In earlier spiral stages it is less easy to identify the constriction, but there can be little doubt that the point/ represents this region in one of the long chromosomes in fig. 20, PI. 3, and that/' probably represents the same point in the homologous chromosome.

INTERPRETATION. The chromomeres are regarded as the essential and permanent part of the chromosomes, and the cycle of events in mitosis, at least as far as the chromosomes are concerned, to centre around their activities. It is held that the matrices of the chromosomes never split: the new chromatids are built up from the chromomeres, which are the only part of the chromosome to divide. While the matrix of the chromosome appears fairly early in prophase and disappears by telophase, the chromone- mata are held to persist through one mitotic cycle, and the chromomeres to be permanent, dividing once in each mitosis. In late prophase, at the time when the double chromatids become densely chromatic, each chromomere divides (Text-fig. 2, j); a possible instance of such a division is seen in fig. 28, PI. 4. The two daughter chromomeres separate, lying on opposite sides of the chromatid. The chromonemata are formed almost at the same time, and appear to represent anastomoses between the chromomeres: those parts of the chromonema which connect sister chromomeres may be chromatin which remained when the incompletely separated sister chromomeres moved apart. The chromonema becomes more easily demonstrable during anaphase, as the matrix of the chromosome decreases in chromaticity (Text-fig. 2, A). At this time the chromonema tends to become stouter, owing perhaps to the addition of material from the chromomeres or the matrix, and becomes as wide as the chromo- meres, which consequently merge, optically at least, into the chromonema. In figs. 1 and 2, PI. 2, it will be noted that in chromosomes showing chromomeres the chromonema is thin, in 60 FRANK W. JANE those where chromomeres are not visible it is coarser. The chromonemata of the metaphase chromosomes are very thin. The period of polar concentration (Text-fig. 2, B) seems to mark the time at which a great deal of chromatic material is got rid of from the matrices of the chromosomes, and this probably

TEXT-FIG. 2. Diagram of history of chromosomes during mitosis, based on interpretation put forward in this paper. A. Anaphase. a-c, types of structure in anaphase chromo- somes, a, single chromonema; 6, paired chromonemata; c, chromo- meres and chromonemata. (Matrix of chromosomes stippled.) B. Polar Concentration (end of anaphase). (Matrix of chromosomes stippled.) C. Telophase. D. Late Telophase. Anastomoses appearing between adjacent chromosomes, spirals becoming flattened. E. Resting Nucleus. F. Early Prophase. Formation of spirals has started, as and 6, formation of daughter chromatids. Adjacent chromo- meres connected by chromatin (shown by broken lines), to form the paired chromatids. Old chromonemata shown by continuous lines. The chromatin shown by broken lines represents the begin- ning of the matrix of the chromosome. G. Early Prophase, full Spiral Stage. Note the irregularity of the spirals as compared with that shown on left in C. H. Mid Prophase. Chromosomes have emerged from spiral stage: chromomeres are distinct: remains of old chromonemata seen as cross-connexions between sister chromatids. a, part of a chromosome at this stage, consisting of two chromatids each composed of chromomeres and matrix (stippled): connexions between chromomeres of sister chromatids represent remains of old chromonema. J. Late Prophase. Chromatids densely chromatic, a and b, parts of chromosomes at this stage and at early metaphase, after prolonged destaining. a, daughter chromomeres formed: at x a pair of daughter chromomeres is connected by a thin strip of chro- matin which may represent the incomplete separation of the chromomeres, or the appearance of the new chromonema; b, a slightly later stage, in which chromonemata have appeared. In both a and 6 the connexions between the two chromatids represent the remains of the old chromonema, which was formed in the pre- ceding late prophase or early metaphase. These diagrams are based on the following figures of Plates 2-4: A, Figs. 1-4. B, Figs. 5 and 6. C, Figs. 7 and 8. D, Figs. 9-13. E, Fig. 16. F, Figs. 17-19. G, Figs. 18-20. H, Figs. 21-5, and 27. J, Figs. 28-30. CHROMOSOMES OF ALSTROEMERIA AND BOMAREA 61 62 FRANK W. JANE accounts for the mistiness associated with this stage. The ultimate destination of this extruded chromatin is uncertain; it may pass into the nucleoli, which begin to appear at this stage, or perhaps into the cytoplasm, where it disappears. With the advent of telophase, the matrices have become invisible. During telophase the chromonemata become thinner, and with this increasing tenuity the chromomeres are more easily seen (Text-fig. 2, c, D). In the reticulum the chromomeres occupy the interstices of the network, i.e. they form the net-knots. In the resting nucleus (Text-fig. 2, B), whether the network is visible or not, minute lumps of chromatin, which are regarded as chromomeres, may always be seen. The earliest prophases are marked by a breaking down of the reticulum between adjacent chromosomes (Text-fig. 2, F): this does not occur simultaneously throughout the nucleus (fig. 17, PI. 8). It is possible that stresses are set up as the skeleton chromosomes are freed in parts, and that these result in the curling of the free parts, giving the prophase spirals (Text-fig. 2, G) ; this suggestion involves the assumption that the telophase and prophase spirals are not related, and this is thought to be true. In the early prophase the scalariform appearance of the chromatin is so similar to that of late telophase that it is difficult to avoid relating them, but the prophase spirals are less regular and somewhat coarser structures than the anaphase chromonemata. Further, it is by no means certain, in Bomarea at least, that every chromosome assumes a spiral form in prophase. The difficulty of obtaining details of the structure of the prophase spirals is enhanced by the increasing chromaticity, and this increases the difficulty of interpretation. It is thought that at this time adjacent chromomeres are connected, where this has not already been accomplished, by anastomoses between them (Text-fig. 2, F) ; in this way are formed the paired chromatids, which are spirally twisted. The coiling of the spiral may lead to so intimate an association between the two chromatids that their spiral appears as a single body; with a slight loosening of the parts, however, the two chromatids are seen, and this is what has been interpreted as the beginning of the longitudinal split (Darlington, 7). The fact that in early prophase adjacent CHEOMOSOMBS OF ALSTROEMBEIA AND BOMAREA 68 chromomeres are sometimes connected by fine bridges of chro- matin, and that occasionally a fine strand of chromatin runs from a chromomere without connecting with an adjacent one, affords some evidence that the chromatids are formed in the manner described. In fig. 21, PI. 4, which represents a later stage of prophase, the same thing would seem to be occurring in chromosome a. The appearance of chromomeres in later prophases, frequently recorded by cytologists, is probably nothing more than an increase in their size, in preparation for their division, causing them to stand out as bead-like swellings on the thinner regions of the chromatids (Text-fig. 2, H). Shortly after this there is a general increase in chromaticity, which results in the appearance of the strap-shaped chromatids ; thus the chromosomes now possess a matrix in which are em- bedded the chromomeres, at this stage differing but slightly in chromaticity from the matrix (Text-fig. 2, j). It is in these chromatids that the chromomeres divide, and that the chromo- nema is formed. It is suggested that the anastomoses seen between the chromatids, even at metaphase (figs. 29, 30, PI. 4), represent nothing more than the remains of the chromonemata laid down during the previous metaphase or late prophase. If this interpretation be correct, it is to be assumed that the chromomeres will keep the same order on the chromosomes in each generation. The keeping together of the chromomeres, after the disappearance of the chromosome matrix, is brought about by the chromonemata. On this view, then, the splitting in the chromosomes takes place in the preceding late prophase stages, and this splitting is the division of the chromomeres. There is no splitting at telophase, it is unnecessary, for the anaphase chromosome, con- sidered as a whole, is a transient structure. The new chromo- some, which develops in prophase, arises on the foundation of the chromomeres, held in position by the chromonemata; at its inception it is a double structure composed of two chromatids, joined in places by the old chromonema. As a double structure it persists until the discrete halves separate at metaphase: each daughter half persists, as a chromosome (as usually defined) until the end of anaphase, or very little later. 64 FRANK W. JANE

DISCUSSION. Investigations on chromosome structure have been sum- marized and discussed in several recent publications (8,12, 13, 26, 35, 40, 41); it would be superfluous, therefore, to review this work here. Certain features in the interpretation advanced above call for consideration. These are: (a) The importance assigned to the chromomeres. (b) The irregularity of the chromonemata. (c) The assumption that the reticulum of the resting nucleus is a significant artifact. (d) The alleged difference between the telophase and prophase spirals. (e) The early doubleness of the chromosome in relation to recent theory. (a) The Chromomeres.—Animal cells provide the most striking examples of chromomeres (1,10, 25, 42). Both Agar (1) and Wilson (44) discuss these bodies at length and are inclined to regard them as significant structures, while Chambers (5) has demonstrated the presence of chromomere-like bodies in the prophases of the male germ cells of Dissosteira Caro- lina, when the living spermatocyte is pricked. There has been a tendency among botanists to adopt a more sceptical attitude towards chromomeres (8, 12, 34), although they are shown in the figures of certain authors (9, 29, 30).x Kaufmann (15) noted chromomeres in the somatic prophases of Tradescantia pilosa, and regarded them as the centres from which the chromonemata arise: and later (17) as turns of the spiral chro- monemata; evidence against this opinion has already been brought forward. Both Heitz (14) and Geitler (9) are inclined to oppose the view that because the chromomeres stain more deeply than the rest of the chromosome they are essential con- stituents of the chromosome, but Geitler found that the number of chromomeres was more or less constant for each type of chromosome in Crepis virens. Belling (2-4), from studies of the chromosomes of a number of liliaceous genera, concluded that the chromomeres were significant bodies, and 1 Numerous cytologists who have noted ohromomeres in animals and plants are recorded by Reuter (31). CHEOMOSOMES OF ALSTROEMERIA AND BOMAREA 65 as a working hypothesis, held that they were the genes. He found that the chromomeres varied in size, and that pairing at zygotene occurred between chromomeres of the same dimensions. The evidence for regarding the chromomeres as significant parts of the chromosome is, therefore, by no means negligible. The fact that they alone, of all structures visible in the chromo- somes, have a linear arrangement, cannot be lightly set aside, nor can it be considered unimportant that in meiosis accurate pairing of the chromomeres of homologous chromosomes has been described; it would seem that such pairing is more likely to occur between permanent than between ephemeral bodies— bodies more prominent, perhaps, at one stage of the cycle than at others, but none the less, always present. It is not unlikely that their appearance in fixed material differs from that during life. Chromomeres are demonstrated better by some fixatives than by others: Hedayetullah (13) finds them clearly visible after fixation in Merkel's fluid, a mixture which both Newton (28) and the writer have found to yield apparently lifelike pictures of mitotic figures. Eichhorn (8) concluded that the chromosomes are homo- geneous structures, but Shigenaga (36) found that plant chromo- somes swell up and disappear in solutions of sodium glycocholate; part of the chromosome becomes disorganized first, leaving a more resistant spiral part which remains distinct for a time. (b) The Irregularity of the Chromonemata.—In Bom are a and Alstroemeria a continuous regular form of the chromonema does not always appear in the chromosomes. Sharp (35) appears to have made similar observations, for he refers to a ' zigzag or spirally coiled chromonema running more or less continuously throughout the length of the [anaphase] chromosome': Eobertson (32) shows anaphases (fig. 11) some- what resembling those figured in the present paper (figs. 1 and 2, PI. 2). Most investigators describe the chromonema as continuous, but differ in their opinions as to the nature of the telophase chromonemata. Thus Kaufmann (15), Telezynsky (41), and Smith (37) find double chromonemata at anaphase: Kuwada (19) regards Kaufmann's observation as due partly to poor fixation, partly to misinterpretation. Taylor (40), after NO. 305 F 66 FRANK W. JANE examination of Kaufmann's material, confirms this author's observations. Smith (37) claims that the double chromonemata in root-tips are so closely associated as to appear single, while in somatic cells of the anther the two are clearly visible. The failure of competent observers to agree on the particular form of the chromonema suggests that it is less regular than is generally supposed; it is not easy to assume that differences of interpreta- tion have arisen from misinterpretation of optical appearances, as suggested by Darlington (7). To some extent the observations in this paper agree with those of Martens (22,23), who finds irregularities in the chromo- nemata, although in Alstroemeria and Bomarea broken chromonemata in early prophase were not observed. The present interpretation is in agreement with Martens' view of bilateral repartition of the chromonemata in prophase, and also that the strands connecting sister chromatids are the remains of the old chromonemata; but it does not suggest, as does that of Martens, that the appearance of chromomeres in prophase is due to accumulation at the margins of the chromatids, of chromatin from these transverse bridges. Martens does not regard the chromomeres as significant bodies. The possibility that these chromatin bridges arise in some other way has to be considered. Assuming a prophase splitting of the chromosomes, the connexions may remain after the split has occurred. Assuming an earlier splitting, it is possible that the bridges represent points of adhesion of the viscous chromo- somes, brought into intimate contact at the spiral stage. The former alternative is rejected because it is not found possible to homologize the telophase and prophase spirals. The latter alternative seems untenable because some of the bridges are not transverse but oblique (figs. 26, 27, PL 4), and it seems inconceivable that oblique, non-parallel strands can arise by adhesion. It might be urged that adhesion, followed by relative change in position of the sister chromatids, may account for the obliquity of the bridges; if this be true it is to be expected that adjacent connexions would be approximately parallel; it does not seem possible to explain the bridges seen in chromo- some b, fig. 26, PI. 4, or at x in fig. 27, PI. 4, in this manner. CHROMOSOMES OF ALSTROEMERIA AND BOMAREA 67 Moreover, such oblique, non-parallel connexions would suggest that, if there be a prophase division of the chromosomes into the paired ehromatids, this division is by a process of vacuolation rather than by true longitudinal splitting. (c) The Eeticulum of the Besting Nucleus.—The views advanced above involve the assumption that the resting nucleus possesses some sort of retieulum. Some observers have found that the resting nucleus of plant cells is not optically homogeneous. Scarth (33), examining living tissues by micro- dissection, found an irregular network, ' an irregular framework of soft gel with interstitial sap ...', in nuclei of Tradescantia. A retieulum has been recorded in living nuclei several times, and this has been found to be unaltered by fixation: Chodat (6) noted a network in the nucleus of the living megasporocyte of Gymnadenia conopea, but this was only visible with oblique illumination, which suggests extreme tenuity of the mesh. Working with plant cells Nemec (27) succeeded, by use of a centrifuge, in causing the nucleolus to form a bulge in the nuclear membrane; subsequent fixation revealed the presence of a distorted retieulum; if the network be merely a coagulation product, it should have been as regular as in a normal nucleus. In animal cells there appears to be little or no evidence of a retieulum in the resting nucleus. Gray (11) discusses the problem and sees no reason to doubt the observations of Martens on the retieulum of the living nucleus. He argues that these observations imply that 'the coagulable part of the living nucleus is already in a relatively high state of aggregation'. In this respect it may perhaps be inferred that the nuclei of higher plants differ from those of Metazoa in which, quoting from Gray, 'we may suppose that the normal degree of aggregation of the colloidal constituents is very much less . . . '. In this connexion it is perhaps significant that while a retieulum has been observed in the nuclei of the two plants under consideration, no evidence of a similar struc- ture has been found in nuclei of the epidermal cells of the larval newt (Molge vulgaris). (d) Telophase and Prophase Spirals.—Attempts have been made by several workers to trace the chromosomes in DO FRANK W. JANE the reticulum of the resting nucleus; this was not found possible in the nuclei of Alstroemeria or Bomarea. Kuwada (18) held that telophasic vacuolation of the chromo- somes occurred and that a nuclear reticulum was formed; in prophase the anastomoses disappeared along parts of the walls of the old chromosomes, leaving zigzag or coiled threads which formed the prophase spirals. The same interpretation might be placed on some of the nuclei here figured (cf. fig. 17, a, PI. 8); in other places (cf. fig. 17, middle of b), the side strands appear to be equally prominent, and at one point, where they are most prominent, a twisting has taken place. Similarly, in fig. 21, a, PI. 4, there is a suggestion that the zigzag (or spiral) which is seen at the top of the chromosome, and which is regarded as the remains of the telophasic chromonema, is becoming bordered by two rods of chromatin, which are held to be the new chrorna- tids. Further, the doubleness of chromosome a, fig. 17, PI. 3, does not suggest that arising from a splitting spiral, but rather one arising in the manner suggested. It is therefore concluded that the prophase spiral is not homologous with the spiral of anaphase and telophase, a view which is strengthened by the irregularity of the former (cf. figs. 4, 7, chromosomes c and d, PI. 2, and fig. 20, PI. 3), and the possibility that every chromo- some of a nucleus does not necessarily pass through the spiral stage; at least, such stages as that shown in fig. 20, PI. 3 (the full spiral stage), are rare, which may imply that the stage is of short duration, or alternatively that it does not occur in every nucleus; nuclei such as are shown in figs. 17 and 18, PI. 3, are much more common. Information regarding the physical condition of the nucleus in the early prophases is scanty, and it is impossible, in conse- quence, to test the validity of the view advanced to explain the prophase spirals. It may be that the irregular breakdown of the reticulum is sufficient to bring about the formation of the spirals, but it is questionable whether the very fine con- nexions between the chromosomes would be sufficient to counter- balance such forces; it is more likely that the formation of the spirals is responsible, in part at least, for the breakdown of the reticulum. CHROMOSOMES OF ALSTROEMERIA AND BOMAREA 69 (e) The Early Doubleness of the Chromosome.— According to the interpretation put forward in this paper, the chromosomes (as usually denned) do not split: preparation for the separation of the daughter chromosomes at anaphase is made in the metaphase of the preceding division at latest by the division of the chromomeres; it is from these daughter chromomeres that the paired chromatids are built up during prophase. It is unnecessary to recall the earlier controversies relating to telophase or prophase splitting; Darlington (7) holds that the chromosomes cannot normally divide before the resting stage: his first reason is that 'spiral structure is found in ana- phase chromosomes . . . and the supposed split would then cut across the spiral'. Kuwada and Sugimoto (20) regard anaphase splitting as impossible, but admit that the split for the homo- type division has occurred in the preceding heterotype division. This objection to early splitting is based on the assumption that there is a single anaphase chromonema, and it assumes an actual longitudinal split of the old chromosome; such a split is held not to occur, a view which Hedayetullah (13) also holds. Darlington's second point is that ' a split spiral is found at the prophase, and there should be two independent spirals side by side if the chromosomes were already longitudinally split at the preceding division'. This difficulty does not arise in the interpretation here advanced; further, Taylor (40) has shown that doubleness at anaphase is not incompatible with the appear- ance seen at prophase. The third objection which Darlington advances is that 'the chromosomes are always single at the earliest prophase of meiosis. This follows a normal telophase.' McClung (25) and Eobertson (32) argue, from the apparent doubleness of the chromosomes of the premeiotic telophase, that the leptotene thread is double; this, as Darlington remarks, is not permissible. Nevertheless, these two cytologists figure telophase chromosomes which show what may be regarded as doubleness, and also paired chromomeres. Taylor (38) observed somatic anaphase chromosomes in Gasteria which appear to be longitudinally split; much more definite, however, is the more or less divided satellite of one of the chromosomes, with 70 FRANK W. JANE indications of a double attachment to the main body. These observations, if widely confirmed, will demand a revision of certain recent theories. SUMMARY. 1. Investigations have been made on the chromatin through- out mitosis in Alstroemeria and Bomarea, and an attempt has been made to interpret the observations. 2. Anaphase chromosomes contain single or double spirals and often chromomeres. The spiral chromonemata are held to arise from the chromomeres. 3. The resting reticulum is formed partly from the remains of the spirals, partly from the telophasic anastomoses between adjacent chromosomes. The chromomeres form the net-knots. 4. The spiral chromosomes seen in early prophase are not regarded as homologous with the anaphase chromonema. It is not certain that all the chromosomes of a nucleus assume the spiral form during prophase. 5. The paired chromatids arise during prophase by the con- necting up of adjacent daughter chromomeres into two strips of chromatin. The chromosomes do not split longitudinally at any stage. 6. Connexions between the sister chromatids are regarded as remnants of the chromonema of the previous anaphase. In this respect the interpretation agrees with Martens' theory of bilateral repartition. 7. Chromomeres appear in prophase as the chromatids emerge from the spiral stage. They cease to be visible in late prophase as the chromatids thicken and become densely chromatic. 8. Prolonged destaining of the early metaphase chromosomes shows that the chromomeres are still present. Each has divided to form two daughter chromomeres. Between the chromomeres on opposite sides of the chromatid appear connexions, the new chromonema. This investigation was begun at Birkbeck College, University of London, while I was the recipient of a maintenance grant from the Department of Scientific and Industrial Research. My thanks are due to the Trustees of the Dixon Fund for the loan CHROMOSOMES OF ALSTROEMERIA AND BOMAREA 71 of a suitable microscope; to the Directors of the Eoyal Botanic Gardens, Kew, the Chelsea Physic Garden, and the University Botanic Garden, Cambridge, for material; and especially to Professor Dame Helen Gwynne-Vaughan, under whose direction the work was carried out and who has encouraged me with friendly criticism and advice.

REFERENCES. 1. Agar, W. E. (1923).—"Male Meiotio Phase in two Genera of Mar- supials (Macropus and Petauroides)", 'Quart. Journ. Micr. Sci.', 67, 183-202. 2. Belling, J. (1928).—"Ultimate Chromomeres of Lilium and Aloe with regard to Numbers of Genes", 'XJniv. Calif. Pub. Bot.1, 14, 307-18. 3. (1928).—"Genes and Chromomeres in Flowering Plants", 'Nature', 121, 831. 4. (1931).—"Chromomeres of Liliaceous Plants", 'Univ. Calif. Pub. Bot.', 16, 153-70. 5. Chambers, R. (1925).—"Physical Structure of Protoplasm as deter- mined by Micro-dissection and Injection", Cowdry, 'General Cytology', Chicago, 235-309. 6. Chodat, R. (1924).—"Caryocinese et reduction chromatique observers sur le vivant", 'C.R. Soc. Phys. Hist. nat. Geneve', 41, 96-9. 7. Darlington, C. D. (1932).—'Recent Advances in Cytology.' London. 8. Eichhorn, A. (1931).—"Recherches caryologiques comparees chez les Angiospermes et les Gymnospermes", 'Arch. Bot.', v, m&n. 2. 9. Geitler, L. (1929/30).—"Der feinere Bau der Chromosomen von Crepis", 'Zeits. Zellforsch. und Mikros. Anat.', 10, 195-200. 10. Gelei, J. (1921).—"Weitere Studien iiber die Oogenese des Dendro- coelum lacteum. II. Die Langskonjugation der Chromosomen", 'Arch. Zellforsch.', 16, 88-169. 11. Gray, J. (1931).—'A Textbook of Experimental Cytology.' Cambridge. 12. Guilliermond, A., Mangenot, G., and Plantefol, L. (1933).—'Traite de Cytologie v6g6tale.' Paris. 13. Hedayetullah, S. (1931).—"Structure and Division of the Somatic Chromosomes in Narcissus", 'Journ. Roy. Micr. Soc.', 347-86. 14. Heitz, E. (1929/30).—"Heterochromatin, Chromocentren, Chromo- meren", 'Ber. deuts. bot. Ges.', 47, 274-84. 15. Kaufmann, B. P. (1926).—"Chromosome Structure and its Relation to the Chromosome Cycle. I. Somatic Mitosis in Tradescantia pilosa", 'Amer. Journ. Bot.', xiii. 59-80. 16. (1926).—Ditto. "II. Podophyllum peltatum", ibid., xiii. 355-63. 17. Kaufmann, B. P. (1931).—" Chromonemata in Somatic and Meiotic Mitosis", 'Amer. Nat.', lxv. 280-3. 72 FRANK W. JANE

18. Kuwada, Y. (1921).—"On the so-called Longitudinal Split of Chromo- somes in the Telophase", 'Bot. Mag. Tokyo', 35, 99-104. 19. (1927).—"On the Spiral Structure of Chromosomes", ibid., 41, 100-9. 20. Kuwada, K., and Sugimoto, T. (1926).—"Structure of the Chromo- somes in Tradescantia virginica", ibid., 40, 19-20. 21. de Litardiere, R. (1921).—"Recherches sur l'element chromosomique dans la caryocinese somatique des Filicinees", 'Cellule', xxxi. 253H173. 22. Martens, P. (1922).—"Cycle du Chromosome somatique dans les Phanerogames. I. Paris quadrifolia, L.", 'Cellule', xxxii. 331-428. 23. (1925).—Ditto. "II. Listera ovata", ibid., xxxvi. 125-214. 24. (1927).—Ditto. "III. Recherches experimentales sur la cinese dans la cellule vivante", ibid., xxxviii. 67-174. 25. McClung, C. E. (1927).—"Synapsis and related Phenomena in Meco- stethus and Leptysma (Orthoptera)", 'Journ. Morph. and Phys.', 43, 181-265. 26. Nebel, B. R. (1932).—"Chromosome Structure in Tradescantiae. I. Methods and Morphology", 'Zeits. Zellforsch. und Mikros. Anat.', 16, 251-84. 27. Nemec, B. (1929).—"Uber Struktur und Aggregatzustand des Zell- kernes", 'Protoplasma', 7, 423-43. 28. Newton, W. C. P. (1924).—•" Studies on Somatic Chromosomes. I. Pairing and Segmentation in Galtonia", 'Ann. Bot.', 38, 197-206. 29. (1926).—"Chromosome Studies in Tulipa, &c", 'Journ. Linn. Soc. Bot.', xlvii. 339-54. 30. Newton, W. C. P., and Darlington, C. D. (1929).—"Meiosis in Poly- ploids", 'Journ. Genet.', xxi. 1-56. 31. Reuter, E. (1930).—"Beitrage zu einer einheitlichen Auffassung gewisser Chromosomenfragen", 'Acta Zool. Fennica', 9, 1-487. 32. Robertson, W. R. B. (1931).—"Chromosome Studies. II. Synapsis in the Tettigidae", 'Journ. Morph. and Phys.', 51, 119-45. 33. Scarth, G. W. (1927).—"Structural Organization of Plant Protoplasm in the Light of Micrurgy", 'Protoplasma', 2, 189-205. 34. Sharp, L. W. (1926).—'An Introduction to Cytology.' New York. 35. (1929).—"Structure of Large Somatic Chromosomes", 'Bot. Gaz.', lxxxviii. 349-82. 36. Shigenaga, M. (1933).—"Action of Sodium Glycocholate on Nuceli and Chromosomes", 'Mem. Coll. Sci., Kyoto Imper. Univ.', Ser. B, viii. 217-31. 37. Smith, P. H. (1932).—"Structure of the Somatic and Meiotic Chromo- somes of Galtonia candicans", 'Cellule', xli. 243-63. 38. Taylor, W. R. (1925).—"Cytological Studies on Gasteria. II. A Com- parison of the Chromosomes of Gasteria, Aloe, and Haworthia", 'Amer. Journ. Bot.', 12, 219-23. CHROMOSOMES OF ALSTROEMERIA AND BOMARBA 73

39. Taylor, W. R. (1926).—"Chromosome Morphology in Fritillaria, Alstroemeria, Silphium, and other Genera", ibid., 13, 179-93. 40. •> (1931).—"Chromosome Studies on Gasteria. III. Microsporo- genesis and Poat-meiotic Mitoses", ibid., 18, 367-86. 41. Telezynsky, H. (1930).—"Le Cycle du Chromosome somatique. I. Observations vitales sur les Poils staminaux de Tradescantia vir- giniana, L.", 'Acta Soo. Bot. Poloneae', 7, 381-433. 42. Wenrich, D. H. (1916).—" Spermatogenesis of Phrynotettix magnus ... Synapsis, and the Individuality of the Chromosomes", 'Bull. Mus. Comp. Zool. Harv.', 60, 57-135. 43. Whyte, R. 0. (1929).—"Chromosome Studies. I. Relationship of the Genera Alstroemeria and Bomarea", 'New Phytol.', xxviii. 319-35. 44. Wilson, E. B. (1925).—'The Cell in Development and Heredity.' New York. EXPLANATION OF PLATES 2 TO 4. In drawing the figures the following optical equipment Avas used with critical illumination: Leitz Apochromatic Objective, 2 mm., N.A. 1-8, Zeiss 18 compensating ocular, Leitz Aplanatic Condenser, N.A. 1-4. Except for figs. 23, 25, 26, 28. xc. 4100. Figs. 23, 25, 26, and 28. xc. 2900. Figs. 8 and 21 are drawn from material fixed in Flemming's Strong Mixture diluted with an equal quantity of water. Fig. 27 from a root-tip fixed in Benda's Fluid diluted with an equal quantity of water: the remainder were all from root apices fixed in Benda's mixture. PLATE 2. Fig. 1.—Anaphase chromosomes of Bomarea patacocensis, showing chromonemata. Chromomeres are seen in chromosome a, and in 6, bars of chromatin, probably imperfectly differentiated chromonema. Fig. 2.—Anaphase chromosomes of Bomarea multiflora, showing chromonemata. Chromomeres are seen in chromosomes b and c, and chromatio bars in a and 6. Fig. 3.—Parts of anaphase chromosomes of Alstroemeria auran- t i a c a, showing chromonemata. The figure in which these chromosomes occurred was a late anaphase, just before polar concentration. Those in figs. 1 and 2 were from somewhat earlier anaphases. A constriction in one of the chromosomes is seen at c. Fig. 4.—Ends of chromosomes of Bomarea multiflora, at beginning of polar concentration: the irregularity of the chromonemata will be observed. Fig. 5.—Polar concentration, Bomarea multiflora, showing ex- cluded ends of chromosomes, in which the chromonemata are to be seen. 74 FRANK W. JANE

Fig. 6 a and 6.—Ends of chromosomes excluded from polar concentra- tion, Bomarea multiflora. Chromomeres and chromonemata are seen in these ends. Fig. 7.—Early telophase, Bomarea patacocensis. Chromomeres are visible in parts, especially in chromosomes a and 6. Some of the chromo- somes appear as spirals (c and d). Fig. 8.—Parts of telophase chromosomes of Alstroemeria auran- tiaca. Fig. 9.—Parts of telophase chromosomes of Alstroemeria auran- t i a c a. These chromosomes are at a later stage than those shown in fig. 8. The beginnings of anastomoses may be observed. Fig. 10.—Late telophase, Alstroemeria aurea. A few anastomoses are to be observed between adjacent chromosomes, and chromomeres may also be seen. In this figure, and in fig. 9, the chromosomes have a reticulate rather than a spiral appearance. Fig. 11.—Parts of late telophase chromosomes of Bomarea multi- flora. Anastomoses and chromomeres will be noted.

PLATE 3. Fig. 12.—Part of late telophase chromosome, Bomarea multiflora. Fig. 13.—Parts of late telophase chromosomes, Alstroemeria aurea. Chromomeres are well marked. Fig. 14.—Telophase nuclei of Anigozanthos rufa. In the lower nucleus there are clear indications of single spirals. In the upper nucleus, however, there are indications of a double spiral in part of chromosome a. Fig. 15.—Late telophase nuclei of Tecophilea violacea. Fig. 16.—Very late telophase, Alstroemeria aurea: most of the chromatin has reached the condition to be seen in the resting nucleus, but traces of chromonemata are to be seen at c. Note the numerous small nucleoli. (Owing to the extreme tenuity of the reticulum in certain parts it was not possible to copy all the network accurately; in a few places it is slightly diagrammatic.) Fig. 17.—Very early prophase, Bomarea multiflora. Traces of the reticulum are visible just below the centre of the nucleus. Chromosome a may be interpreted as a pair of spirally twisted chromatids, lying so close together that the doubleness is not apparent except at the upper end; alternatively, the central part of the chromosome may be regarded as the old chromonema, forming the spiral chromosome. The middle of chromo- some b shows the beginning of twisting, but the two chromatids are still well separated. Fig. 18.—Early prophase. Spiral stage, Bomarea multiflora. In parts, there are well marked spirals, but at the lower end the chromosomes are in an earlier stage. Fig. 19.—Spiral prophase chromosomes of Bomarea multiflora. Chromosome a has assumed the full spiral form, b and c are still partly reticulate. CHROMOSOMES OF ALSTROEMERIA AND BOMARBA 75

Kg. 20.—Early prophase. Spiral stage. Alstroemeria aurantiaca. /, region of spindle-fibre attachment in one of the very long chromosomes. /',? region of spindle-fibre attachment in the other very long chromosome. x, chromosomes which have passed, or almost passed, the spiral stage.

PLATE 4. Fig. 21.—Mid prophase chromosomes, Bomarea multiflora. In a the old chromonema is more conspicuous than the chromatids. Fig. 22.—Late prophase chromosomes, Bomarea multiflora: in chromosome a the remains of the spiral are to be seen at the lower end. Fig. 23.—Late prophase chromosomes of Bomarea multiflora. In chromosome a the oblique connexions between the chromatids will be noted. Fig. 24.—Late prophase, Alstroemeria aurantiaca: matrix of chromosome fairly well developed, chromomeres and connexions between chromatids conspicuous. Fig. 25.—Chromosome from nucleus shown in fig. 24, showing region of spindle-fibre attachment, /. Fig. 26.—Late prophase chromosomes of Anigozanthos rufa. Chromomeres and connexions between chromatids are to be seen: in b note oblique connexions at lower end. Fig. 27.—Late prophase chromosomes of Alstroemeria auran- tiaca: chromomeres and connexions between chromatids conspicuous. Note that certain of these connexions are more or less at right angles to the chromatids, but that others are oblique; both types may be in close proximity as at x. In this nucleus there is a large nucleolus, an unusual feature in the late prophase. Fig. 28.—Early metaphase chromosomes of Alstroemeria auran- tiaca: note chromomeres in each chromatid, and the connexions between them; these connexions are the beginnings of the new chromonema. Fig. 29.—Metaphase chromosomes of Bomarea multiflora, show- ing connexions between the chromatids or daughter chromosomes. Fig. 30.—Metaphase chromosome of Alstroemeria a urea, showing chromomeres and chromonema. Region of spindle-fibre attachment shown at /. Muyr: Set.. Vol. 17. MS.. 3k. Z

\

\

'si 6A * ... 6B

Vq ^'

F. W. Jane, del. 10 Quart. Sawn. MLc/r Set. Vol. 7 7, MS.,

. V

12

15

13

,4

16

19

* .-,•?:*<&

>> 20

18 , del. QJJAM. fowrn. Mar. Sd \U 77 MS., $(.•

Zl

11

•- 24

0 25 f i 26

23

28

29 B Z9A

EW.Jane, del.