cytotaxonomic studies on anthoxanthum odoratum l. s. lat. : iv. karyotypes, meiosis and the origin...

Heretlilas 64: 153-176 (1970)

Cytotaxonomic studies on Anthoxmthuwl odoraturn L. s. lat. IV. Karyotypes, meiosis and the origin of tetraploid A . odoraturn

I N G A H E D B E R G Institirte of Systematic Botany, University of Uppsala, Sweden

(Received January 16, 1970)

The author has investigated the karyotypes occurring within Anthoxanthum odoraturn L. s. lat. from various parts of its distribution area. Whereas the diploid (2n = 10) seems to be very uniform there exist within the tetraploid (2n=20) two apparently rather distinct karyotypes. One (karyotype I) is found in the majority of the specimens studied whereas the other (karydype 11) seems to be very rare. This karyotype replicates that of the diploid. Deviations from the strict autotetraploid complement were found to a various extent in all specimens with karyotype I1 although those specimens must be descendants from recent autotetraploids. It is suggested that further structural changes could have transformed the autotetraploid karyotype into the more or less allopolyploid condition of karyotype I. The present study of meiosis shows that most diploids had regular bivalent formation but some showed univalents and also inversion bridges. In the tetraploids frequent quadrivalents were found as well as multivalents of higher order. In some specimens no irregularities were found in the subsequent stages whereas others showed inversion bridges, laggards, etc. I t was as a rule very difficult to obtain hybrids between diploids and tetraploids. A considerable number of hybrids were, however, obtained from a cross tetraploid with karyotype I1 ( 9 ) x diploids. I t is concluded that evidence from several sources indicates that autopolyploidy has played an important r6le in the origin of A. odoratum (4x) and furthermore that it is not possible to maintain the diploid as a separate species.

The Linnean species Anthnxanthum odoraturn contains diploid and tetraploid cytotypes with partly overlapping morphological variation and distribution (HEDBERG 1967, 1969). The cytology of this species has already been the subject of several investigations (cp. review in HEDBERG 1967). The first attempt to study the chromosome complement in some detail was made by HUNTER (1934), who reported three pairs of fragments in tetraploid specimens. Such fragments have not been reported in tetraploids by any other author, and they were probably only parts of ordinary chromosomes in which the secondary constriction had not

been stained (cp. e.g. MARKARIAN and SCHULZ- SCHAEFFER 1958; HEDBERG 1967, p. 13) . HUN- TER’S results would then indicate the presence of satellited chromosomes in the tetraploid, which has later been confirmed by other authors (cp. below), but no further information was given on the chromosome morphology. A detailed study of meiosis in tetraploid A. odoratum was carried out by KAITERMANN (1931), and by PARTHASARATHY (1939), who also studied the morphology of the mitotic chromosomes to some extent. The last-mentioned author concluded that the chromosome complement of tetraploid A. odoratum consists

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154 INGA HEDBERG

of four similar sets of chromosomes. The same conclusion was drawn by ROZMUS (1958), who also stated that the chromosome morphology of the tetraploid corresponds to that of the diploid. On the other hand JONES (1964) con- cludes “The analyses of chromosome form have clearly shown that Anthoxanthum odoratum [i.e. the tetraploid] does not possess a karyotype which replicates that of alpinum [i.e. the diploid], neither can it be said that its four chromosome sets are of comparable morphology”.

Also the studies of meiosis in tetraploid Anthoxanthum odoratum have given diverging results. KATTERMANN (1931), PARTHASARATHY (1939), OSTERGREN (1942), and JONES (1964) report frequent quadrivalents and presence of multivalent formations, whereas GUINOCHET (1942-1943) found regular bivalent formation in tetraploid material from France.

Each of the above studies seems to have been carried out in a rather limited material. Be- cause of the divergencies mentioned I found it desirable to study a larger material from different regions. The distribution, morphology and anatomy of the two cytotypes was earlier studied in a similar material (HEDBERG 1967, 1969).

Material and methods The material studied is listed below (Table 1 and 2). In order to avoid such variability as could be ascribed to Botanic Garden source (cp. JONES 1964, p. 257) only plants from natural populations, or raised from seeds from such populations have been used. Each population sample was given a five-figured number in which the first two figures indicate the year of collection, and each specimen within the sample was given a number after a colon (e.g. 66015: 2, 69010: 4). Plants obtained from spontaneous seed material were numbered by the year of sowing followed by an oblique line and the sample number and individual number (e.g. 681201: 6, 69/02: 8, etc.).

Routine chromosome counts were performed in root tip sections (cp. HEDBERG 1967, p. 8) and chromosome morphology was studied in root tip squashes. I have tried several methods,

combining various kinds of pretreatment with various fixatives, in order to obtain the best possible results (cp. p. 157). The treatment finally chosen is the method worked out by OSTERGREN and HENEEN (1962), slightly modified by the use of a-monobromnaphtalene instead of oxyquinolin for pretreatment. The method may be summarized as follows:

Excised roots (2-3 cm) were pretreated in sat- urated a-monobromnaphtalene for 2 hours and fixed over night in the fixative recommended by USTERGREN and HENEEN (1oc.cit.). After hydrolysis in 1-N HCI for 8 minutes at 62- 65” C they were stained in Feulgen for 2 hours. The roots were then softened in pectinase ( 5 % solution) for 2 hours. Finally the meriste- matic regions were squashed in 45 % acetic acid.

I consider the times given above as the minimum ones. As regards pretreatment, however, two hours seemed to be the optimum but after a cool night 1 1/2 hours might be sufficient. The times for staining and softening could be extended for one or two hours without any notable effect. Too long pectinase treatment, however, made the material too soft for easy handling.

Loss of material can often not be avoided during the process of making the slides permanent. Hence the preparations were sealed with a thin layer of nail polish immediately after squashing, and the slides stored in a freeze. In that way they kept for months, during which time they could be thoroughly studied and all microphotographs taken. The preparations were then made permanent by leaving them in acetone for 10-20 minutes, which dissolved the nail polish, removing the cover slip after freezing the slides for 1-2 minutes, passing them through absolute alcohol for 5 minutes, and mounting them in euparal. For karyotype investigation at least three

slides were prepared from each specimen and a minimum of 10 cells as a rule inspected from each slide, but owing to various difficulties (p. 157) all chromosomes in tetraploid specimens could not always be studied in each cell. Consequently the number of SAT:s, etc. as given in Table 2, is in some specimens based on combined observations from different cells. In other specimens it has been possible to obtain several cells in which all chromosomes are

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KARYOTYPES IN ANTHOXANTHUM OWRATUM 155

Table 1 . Survey of number and type of SAT chromosomes, medians and submedians observed in the diploid material examined

Ref. no. Origin Number Number Number of and type of small submedians of SAT:s medians

A itstria 66011: 3 Salzburg, Hohe Tauern, Gross Glockner, 2000 m 2 D 2 6

66018: 3

66028: 3

66046: 2 66046: 5

69001: 2

69005 69007: 1

69012: 3

69/01: I 69,'Ol: I I 69/02: 8

69/02: 15 69/02: 16 69/02: 19 69/02: 20 69/02: 22 69/03 : 4

Swit:erland Graubunden, Ober Engadin, between Ponte and Bevers, 1680 m Graubunden, Oberhalbstein, Lenzerheide, 1480 m Bern, Gantrisch, Leiterenpass, 1980 m ibid.

Sweden Lycksele Lappmark, Hemavan, Mortsfjlllet, lo00 m

Nor way

Nordland Fylke, Krutvassrodiken, 700 m Nordland Fylke, shore of the lake below the glacier Svartisen, 250 m Nordland Fylke, Nesna, 450 m

USSR E. Carpathians, Mt. Bliznitsa, 1600 -1700 m ibid. E. Carpathians. Lyuta (50 km N.E. of Uzhgorod) 1200--1300 m

ibid. ibid. ibid. ibid. ibid. E. Carpathians, Czornohora range, Mt. Pop Ivan. 1600 1800 m

2 D 2

2 D 2 2 D 2 2 D 2

2 D 2

2 D 2

2 D 2 2 D 2

2 D 2 2 D 2

1 D, 1 modified D I 2 D 2 2 D 2 2 D 2 2 D 2 2 D 2

2 D 2

6

6 6 6

6

6

6 6

6 6

7 6 6 6 6 6

6 681204: I Tuva Rep., (S. Siberia), Ergak Mt. range, 1700 m 2 D 2 6

well spread and show the same number of secondary constrictions from cell to cell. Kar- yotype analyses and chromosome measurements were carried out on enlarged microphotographs of the best metaphase plates (magnification x c. 2000). Because of a considerable variation in chromosome contraction between different plates (p. 157) relative length values have been used for the individual chromosomes. In order to make possible a direct comparison between complements of different ploidy the values given here cor- respond to a total length of 100 units in the diploid, 150 in the triploid, and 200 in the

tetraploid. The arm index is calculated as the ratio between the short and the long arm. In satellited chromosomes the length of the satellite is included in the arm length, while the length of the secondary constriction is not measured. This is due to the very variable length OE the constriction, and to the fact that the satellite may often become detached from the rest of the chromosome during the preparation (Fig. 4a). In the idiograms the various categories have been grouped together, from left to right SAT chromosomes, submedians, and medians (chromosomes with an arm index of 0.75 and higher). Within each category the

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156 INGA HEDBERG

Table 2. Survey of the tetraploid material examined In some specimens only the karyotype (based on the type of SAT:s) is given whereas in others a more detailed examina- tion has been performed (cp. text)

Ref. no. Origin Karyo- Maximum number type and types of of medians

Number and size

SAT:s observed

A B D large medium small

66005: 2

66008: 4

6601 1 : 2

66015: 2

66032: 1 66034: 2 66040: 4 66041: 1 66041: 2 66041: 3 66041: 6 66041: 8 66041 : 9 66041: 10 66043: 2 66044: 4 66045: 2 66045: 3

68001: 2 68002: 2 68004: 4 68008: 1 68008: 2

69009: 8 69009: 10 69010: 1 69010: 2 69010: 3 69010: 4 69010: 5

68/201: 6

681201 : 13 681201 : 15 68/201: 16 68/201: 20 681202: 12

68/202: 19 69/01 : 18

69/02: 10

69/02: 14

Austria Tirol, Rofan, Buchan, (near Kufstein), 1310 m I 2 2 Tirol, Kitzbiihler Alpen, Pass Thurn, 1270 m I 3 Salzburg, Hohe Tauern, Gross Glockner, 2000 m I 2 1 Tirol, Brenner Pass, 1300 m

Switzerland Uri, Oberalp Pass, 1840 m I 2 1 Bern, Oberland, W. of Sustenpass, 1370 m I Valais, E. of Morgins, 1250 m I 2 2 Valais, Morgins, 1800 m 11 4 ibid. I1 ibid. I 2 2 ibid. I1 4 ibid. I1 4 ibid. 11 4 ibid. I 2 2 Valais, Morgins, 1620 m I 2 2 Valais, Morgins, I500 m I Valais, Morgins, 1400 m I 2 2 ibid. I 2 2

Sweden

I

Uppland, Vasterlovsta I 2 2 Ostergotland, near Linkoping I Angermanland, near Sollefteh I Uppland, Oregrund, Kavaro I 2 2 ibid. I 2 2

Norway Nordland Fylke, Nesna I 1 2 Nordland Fylke, Nesna I 2 2 Nordland Fylke, island of Tomma I 2 2 ibid. I 2 ibid. I ibid. I 2 2 2 ibid. I 4 2

USSR Prov. of Moscow, Zvenigorod distr., Perkhushokovo I 2 ibid. I 2 1 ibid. I ibid. I ibid. I Prov. of Moscow, Solnechnogorsk distr., Chashnikovo I ibid. I E. Carpathians, Mt. Bliznitsa, 1600-1700 m I E. Carpathians, Lyuta (c. 50 km N.E. of of Uzugorod), 12 W 1300 m I 3 I ibid. I 3 1

2

2 2

1 2

1 2

2

2

2

2 2

2 2 2

2 2

2

2 2 1 2

2

2 4

2 4 4 4 2 2

2 2

2

2 2

2 2

1 2 2 1 2 2

KARYOTYPES IN ANTHOXANTHUM ODORATUM 157

chromosomes are as a rule represented in decreasing order of length, and those having the same length value are arranged in decreasing order of arm indices.

Meiotic studies were made after fixation in Carnoy (1 part glacial acetic acid: 3 parts absolute alcohol) and staining in aceto-carmine.

Observations 1. The somatic chromosomes

In spite of its relatively few and large chromosomes cytological analysis in A . odoraturn S.

lat. is rather difficult (cp. JONES 1964). Sections have proved very satisfactory for routine chromosome counts, but in my opinion they can not be used for studies of chromosome morphology since they do not give a clear picture of the individual chromosomes (cp. e.g. BJORK- QVIST 1968, p. 21). In good preparations it may be possible to recognize satellited chromosomes, since the secondary constrictions do not stain in crystal violet (Fig. 9 b, c and 20, and HEDBERG 1967, p. 13). The exact position of the centromere is, however, often impossible to find, which makes measurements performed in sections rather unreliable. Furthermore the chromosomes have often been cut. Hence considerable time has been devoted to find the most suitable method for root squash preparations (p. 154). During that work I have become convinced that owing to various technical difficulties great caution has to be taken in drawing conclusions concerning chromosome mar- phology. Such difficulties are encountered especially in the tetraploid, where the degree of contraction may differ not only between plates from the same individual (cp. HENEEN 1962, p. 482) but also between homologous chromosomes within the same cell. The same phenomenon was encountered by HENEEN and RUNEMARK (1962) in EIymus rechingeri (Run.) RUNEMARK. When such differences occur between chromosomes without satellite only a slight difference in chromosome length will be noted. In satellited chromosomes, however, two homologues may appear quite different even if there is only a relatively small difference in contraction. As an example one plant might be

I i i i j w

A B D Fig. 1. Diagrams of the type of SAT chromosomes occurring in Anthoxanihum odoraturn s. lat.

mentioned in which the first squash preparation presented several cells in which the chromosomes were well spread seemingly showing the secondary constrictions quite well. The number of SAT chromosomes varied, however, between two and four in the cells studied (about 20). Two types could be seen: one (A type, cp. Fig. 1 and p. 158) varied in number from one to three, whereas the other (B type, cp. Fig. 1 and p. 158) was represented only once in all cells studied. Because of the variation in the number of A SAT:s another slide had to be inspected, in spite of the apparently very good quality of the first one. The second root tip had been treated simultaneously with the first one through the whole procedure, but in most cells studied one pair of B type SAT:s were clearly seen besides the A type SAT:s. Apparently one of the B type SAT:s had appeared as an ordinary (non-satellited) chromosome with median centromere in all cells studied in the first slide. I n another plant the two A type SAT:s found seemed to differ slightly as regards the position of the secondary constriction, which at fiirst was thought to be due to differences in contraction. Further preparations showed, however, that the two SAT:s in fact belonged to two slightly different types, but that always only two A SAT:s were seen, viz. either two of the same type or one of each. Similar phenomena were encountered repeatedly during the course of my work. Obviously the SAT chromosomes are not so easily identified as supposed by JONES (1964, p. 250). The con- sequences of this will be discussed in connection with the tetraploid complement (p. 162). Evidently any detailed discussion of karyotype variation must be based on the study of a large number of plates of each specimen. Even such a cautious procedure will, however, in some

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158 INGA HEDBERG

a

l l l r l

I l l l l

b

C d

Fig. 2. Idiograms of the haploid set in diploid A. odoralum s. lat. ( A . alpinum L ~ V E and MvE); a: is taken from ROZMUS (1958), slightly modified in order to facilitate comparison; b: constructed by me from a photograph published by JONES (1964); c: my specimen 69012: 3; d: my specimen 681204: 1 . The variation in chromosome contraction in two plates from the same root is demonstrated by marking those from one plate by black, those from the other by hatching.

cases not solve the problem of identifying the various SAT chromosomes in the tetraploid. Hence I have refrained from any subdivision of the types of SAT:s found and only classified them according to their general appearance as follows (Fig. 1):

Type A: centromere submedian, secondary constriction in the short arm.

Type B: centromere median. Type D : centromere submedian, secondary

constriction in the long arm. The SAT:s of the latter type have a very conspicuous appearance: their primary and secondary constrictions divide them into two distal segments of equal size, embracing a third which is about half their length.

This classification corresponds on the whole to that of JONES (1964), though the last-mentioned type was not designated by him by any letter. Furthermore it should be noted that the arm index for type A is much more variable than the value (0.5) given by JONES (op. cit. p. 251). From his drawings it seems to range be-

tween 0.4 and 0.7, whereas the variation range in my material is slightly less, viz. 0.54.7. I have not recovered the rare C type of JONES’S which he describes as a submedian with a secondary constriction one third the distance from the distal end of the long arm.

A. The diploid

In his report on the occurrence of diploid A . odoratum (A. alpinum LOVE and LOVE) in Poland ROZMUS (1958) also briefly discussed the chromosome complement. The diploid set, studied in sections, is reported to comprise “one pair of nearly isobrachial chromosomes, three pairs of distinctly heterobrachial chromosomes and one pair of heterobrachial chromosomes with a secondary constriction”. The morphology of the chromosomes in the haploid set is illustrated by an idiogram (ROZMUS op. cit. Fig. 8). Later JONES (1964) described the complement, from squash preparations, as follows: one pair of small medians, three pairs of submedians, and a single pair of SAT chromosomes. This complement is illustrated by a microphotograph but no diagrammatic repre- sentation is given. In order to faciliate further discussion I have therefore prepared an idiogram based on JONES’S (op.cit. Fig. 4) microphotograph together with a slight modification of the idiogram drawn by ROZMW (op.cit.), cp. Fig. 2 a and b.

The descriptions quoted above seem at first to agree well, but a more detailed comparison between the karyotypes reveals striking differences between them. In ROZMUS’S material the “nearly isobrachial” chromosomes (medians) are two of the largest ones in the whole set, and in those with a secondary constriction this divides the shorter arm into two equal parts (A type according to the classification given above, Fig. 1). In JONES’S material, on the other hand, the medians are the smallest ones and in the SAT-chromosomes the primary and secondary constrictions divide them into two distal segments of equal size embracing a third one which is about half their length (D type according to the above classification, cp. Fig. 1). No comments on these discrepancies were made by JONES (op.cit.), probably because he considered observations made on sections less reliable (cp. also p. 161). Since, how-

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Table 3. Relative chromosome length (a) and arm index (b) in some diploid specimens of A . odoraturn s. lat. The values given refer to measurements in single plates.

Specimen Chromosome pair

I 11 111 IV V

a b a b a b a b a b

69005 10.5 0.75 11.0 0.57 10.0 0.54 10.0 0.43 8.5 0.89 69005 10.5 0.72 11.0 0.53 10.1 0.54 10.1 0.40 8.3 0.89 69012: 3 9.8 0.73 11.9 0.52 10.8 0.50 9.8 0.44 7.7 0.88 69/02: 20 10.8 0.69 11.3 0.55 9.8 0.56 9.8 0.44 8.3 0.89 681204: 1 11.3 0.73 11.0 0.57 10.3 0.50 8.9 0.46 8.5 0.80 68/204: 1 12.3 0.75 10.7 0.61 10.0 0.50 9.0 0.40 8.0 0.83

Jones (1961) Fig. 4 1 1 . 3 0.67 11.3 0.55 9.5 0.52 10.1 0.44 7.8 0.78 calculated from

ever, JONES investigated only four specimens, none of which came from the same region as those investigated by ROZMUS, it could not a priori be assumed that the apparent dissimi- larity between the karyotypes could be ascribed only to misinterpretation by ROZMUS.

In an attempt to clarify this situation I have studied the karyotype of diploids from various populations in Europe and also a few from Asia (Table 1). Since my main intention was to find out whether two distinctly different karyotypes exist within diploid A . odoraturn detailed measurements have been performed in some cases only.

With one exception (cp. below) I have found a very good agreement between the chromosome complements of the various diploids investigated. These complements are illustrated here by a few metaphase plates and idiograms (Fig. 3, 4, and 2c-d), and may in general terms be described as follows: One pair of D

Fig. 3. Somatic chromosomes of thc diploid plant 69/02 20. X2500.

type SAT-chromosomes (p. 158 and Fig. l), three pairs of submedians, and one pair of small medians. In spite of only slight differences between the submedians there are as B rule no difficulties in identifying the different pairs due to their relative lengths and arm indices. In some specimens decrease in length of the submedians is accompanied by a decrease in arm-index (Fig. 2 c), in others the two shottest submedians are of about the same length but are easily distinguished by their

b Fig. 4. Somatic chromosomes of the diploid plant 69005. a: photomicrograph of metaphase plate, X 1300. b: combined idiograms from two different plates in the same root tip showing slight differences in contraction. Chromosomes from onc plate marked by black, those from the other by hatching.

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160 INGA HEDBERG

b Fig. 5. Somatic chromosomes of the diploid plant 69/02: 8. a: photomicrograph of metaphase plate. One chromosome which was rather widely separated from the others has been moved in order to save space. The modified D SAT is indicated by an arrow, the new submedian by an x. X1700. b: idiogram of the whole complement indicating a translocation between one of the SAT:s and one of the medians (cp. text).

arm indices (Fig. 15 d and Table 3). If one chromosome is compared to a homologous chromosome in another plate from the same individual, small differences in length and arm index may be noted, but this does not affect the general pattern (Fig. 2 d and 4 b). The slight differences between the chromosome complements of the different diploid specimens studied are of minor importance as regards karyotype classification. The karyotype accounted for above is essentially the same as was described by JONES (opcit.) though the arm indices given by him for the submedians neither agree with those found in my material nor with those calculated by me from his microphotograph. The last mentioned do in fact agree quite well with my own results

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Fig. 6. Somatic chromosomes of the tetraploid specimen 66045: 3 (karyotype 1) (cp. the idiogram in Fig. 14 a). X1700.

(Table 3), whereas those given by JONES are too small, especially as regards the smallest submedians.

In view of the amount of material investigated here it seems allowable to assume that the karyotype described above is the typical karyotype in the diploid. In no case I have found a karyotype similar to that illustrated by ROZMUS (op. cit.). Since part of my material emanates from about the same area as Roz- MUS’S I am inclined to ascribe most of the divergencies to the inadequate methods used by him.

The only exception from the common karyotype was found in one plant from Poland (69/02: 8) . As shown in Fig. 5 the chromosome complement of this plant differs signifi- cantly from the typical one in having only one SAT-chromosome of the D type. The other SAT is shorter and has a much lower arm index. Furthermore only one small median is found whereas seven instead of six submedians are present. The only explanation of this deviating karyotype seems to be a trans-

KARYOTYPES IN ANTHOXANTHUM O W R A T U M 161

Fig. 7. Somatic chromosomes of the tetraploid specimen 69102: 10 (karyotype I ) (cp. the idiogram in Fig. 14 b). X2000.

location between one of the SAT-chromosomes and one of the small medians. Measurements confirm that if the part lost from one of the SAT:s is added to one of the medians we get a submedian of the same appearance as the extra one (Fig. 5 b). Since this plant was raised from seeds obtained last spring it has not yet been possible to study meiotic pairing, etc.

B. The tetraploid

I n connection with his report on the Occurrence of diploid Anthoxanthum in Poland ROZMUS (1958) also comments upon the chromosome morphology of the tetraploid. He states very briefly: “On the whole the chromosome morphology of the tetraploids corresponds to that of the diploids”, and refers to his diagram of the haploid set (op. cit. Fig. 8), cp. Fig. 2 a. Also PARTHASARATHY (1939) had found it possible to recognize five types of chromosomes, each represented four times.

The only more detailed karyotype studies were performed by JONES (1 964). According to him “the analyses of chromosome form have clearly shown that Anthoxanthum odoratum [i.e. the tetraploid] does not possess a karyotype which replicates that of alpinum [i.e. the diploid] neither can it be said that its four chromosome sets are of comparable morphology”.

Fig. 8. Somatic chromosomes of the tetraploid specimen 69010: 5 (karyotype I) (cp. the idiogram in Fig. 14 c). X1200.

JONES’S observations do obviously not at all agree with the earlier studies but he made no attempt to clarify the situation - apparently because both PARTHASARATHY and ROZMUS had used sections for their studies (cp. p. 158, and JONES op.cit. p. 267). Since, however, JONES investigated a few population samples from the British Isles only, whereas PARTHASA- RATHY probably had material of botanical garden source, and ROZMUS used material from Poland, I found it necessary to perform karyotype studies in material from various parts of Europe. These studies have been carried out with three different aims. The first was to find out if any major differences could be found between karyotypes within the tetraploid. “Major” here refers above all to the general morphology of the SAT-chromosomes and to the occurrence of medians of various sizes, since those are of particular diagnostic importance. This part includes a more general survey of the karyotypes in various populations, without measurements. The second was to perform more detailed studies in a few specimens in order to give more information on the type of variation found. The third was to combine the results of these two approaches in order to elucidate a possible mode of origin of the karyotype in tetraploid A . odoralum.

The material used for the actual survey is

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162 INGA HEDBERG

Fig. 9. Somatic metaphase plates showing the appearance of the D type SAT in a tetraploid and (for comparison) in a diploid; a: root quash preparation from the tetraploid specimen 69010: 4, all visible SAT:s have been marked, X1500. b: root tip section from the same plant, X1500; c: root tip section from the diploid specimen 69001: 4, X1500.

listed in Table 2. Based on the morphology of the satellite chromosomes, which are the most conspicuous feature, two general karyotypes have been distinguished. The S A T s charac- terizing the first one (karyotype I) are of the A and/or B type, whereas in the second (karyotype 11) only D type SAT:s occur.

Karyotype I, which is the most common in my material, is illustrated here by a few metaphase plates (Fig. 68). The fact that all secondary constrictions do not show up regu- larly (cp. p. 157) makes it very difficult to give a general description of this karyotype. Three pairs of medians occur: large, median and small. In most cases at least one of the large medians is, however, provided with a secondary constriction, which means that it should be grouped with the SATs. Strong evidence that both large medians a re in fact SAT:s is apparent from Table 2, which shows that if two SAT:s with median centromere are present only medium sized and small medians occur. If, on the other hand, only one SAT with median centromere has been found, one large ordinary median occurs together with the medium and small ones. Similar difficulties are encountered in classifying the other S A T s (A type) versus the submedians. If the secondary constriction

in an A type SAT does not show up, the actual chromosome appears as an ordinary submedian. Unawareness of those difficulties might make it tempting to indulge in detailed descriptions of single individuals (or even plates), paying undue attention to a variation which may in fact be due not t o absence of some of the SAT:s but only to the fact that their secondary constrictions d o not always show up. The reason for this variation will be discussed below (p. 174), but it is necessary to be aware of the phenomenon of concealed secondary constrictions when comparing the chromosome complements of various specimens. From my experience it may be possible that most of the specimens studied by me conform to the same general pattern in spite of the differences accounted for in Table 2. This general pattern may be described as follows: 4-6 SAT chromosomes, 12-10 submedians, and 4 medians. A few idiograms have been constructed (Fig. 14), from which it may be seen that most of the chromosomes occur in pairs.

This karyotype apparently comes rather close to that described by JONES (1964) and considered by him to be the only karyotype occurring in tetraploid A. odoraturn, the description of which reads as follows: “three to eight

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KARYOTYPES IN ANTHOXANTHlJM ODORATUM 163

SAT chromosomes, eight to ten submedians and six to eight medians”. Considerable inter- plant variation is reported and illustrated by two microphotographs and three drawings. Furthermore the variation in frequency and combination of SAT:s in various complements is illustrated by another 16 drawings. The same two types of SAT:s as found by me were reported by JONES (I.c.), but whereas I found secondary constrictions in the largest medians only, JONES reports such to occur also in the medium sized medians. Another discrepancy is the number of SATs . Whereas JONES reported up to eight in one complement I have never found more than six, and in most cases not more than four.

Though a considerable inter-plant variation occurs, especially as regards the length and arm index of the submedians (Fig. 14) the general pattern seems rather stable. Of the 35 specimens accounted for here (and many more have been inspected during my work) all but one show SAT:s of the A and B type only. The only aberrant specimen, 69010: 4 from Norway (Table 2), shows, however, besides the A and B SAT:s also two D type SAT:s, i.e. the typical diploid satellite chromosomes (Fig. 9 a-b). In other respects, however, this specimen apparently belongs to karyotype I. The occasional Occurrence of the D type SAT in a karyotype otherwise seemingly devoid of it offers very interesting aspects which will be discussed below (p. 173).

The second karyotype, which is extremely interesting from an evolutionary point of view, was found only in one population from Switz- erland close to the French border. It differs most conspicuously from karyotype I in the morphology of the SAT chromosomes and in the number of medians. Neither A and B type SAT:s nor any large or medium sized medrans are found in karyotype 11. Instead four SAT:s of the very conspicuous D type are present together with four small medians and, as a rule, twelve submedians (Fig. 10-13). Idio- grams for some of the specimens with this karyotype are shown in Fig. 13 b and 15. It has in no case been possible to classify the submedians in groups of four. Especially in some of the specimens (Fig. 10 and I I ) this possibility is, however, close at hand. Obviously all specimens showing karyotype I1 must be de-

Fig. 10. Somatic chromosomes of the tetraploid specimen 66041: 1 (karyotype 11). The SAT:s and the medians are indicated by D and m. respectively. Cp. the idiogram in Fig. 15 a. X1500.

scendants from recent autopolyploids whose chromosome complements have undergone modifications to various degrees.

The complement of one of those specimens, 66041: 9, consists of three groups of four, two groups of three and two single chromosomes (Fig. 12, 15 c). Both these single chromosomes are submedians, but whereas one deviates by being longer than all the others and having an unusually low arm index, the other one could as far as such features are concerned easily be grouped with three other submedians. On the other hand this chromosome is remarkable in having a secondary constriction (clearly visible in some cells) near the distal end of the long arm. I have not found such a type of SAT chromosome in any other specimen. In some cases the smaller differences (cp. the idiograms in Fig. 15) may perhaps be due to technical difficulties only, such as differences in contraction (p. 157), but in other cases structura1 changes must have been involved which, like in the specimen mentioned above, have resulted in new chromosome types. One of those types is shown in Fig. 13. The idiogram shows one chromosome which apparently must have orig-

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164 INCA HEDBERG

/” Fig. 11. Somatic chromosomes of the tetraploid specimen 66041: 6 (karyotype 11) . The SAT:s and the medians are indicated by D and m, respectively. Cp. the idiogram in Fig. 15 b. X1700.

inated through loss of the distal part of the short arm of a submedian. In another specimen (66041: 2) also a large median seems to be present besides the subterminal, but I have not so far managed to get plates sufficiently clear for safe analysis. A subterminal chromosome of the above type was also reported by JONES (1964) to occur exceptionally in karyotype I, but it is apparently very rare. Hence it seems probable that such chromosomes, like the very special SAT mentioned above, may represent only transitional stages in the repatterning process.

C . Comparison between the diploid and the Fig. 12. Somatic chromosomes of the tetraploid

The diploid karyotype (as found in all speci- specimen 66041: 9 (karyotype I I ) , the two new chromosomes are indicated by arrows. A more clear picture of the new satellited chromosome is inserted but One) and karyotype I in the tetraploid in the lower right corner. The S A T S and the medians Seemingly have little in common. Similarities are indicated by D and m. respectively. Cp. the might be found in the submedians but the

satellite chromosomes are of entirely different idiogram in Fig. 15 c. X1400.

tetraploid karyotypes

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. *

0

a

b Fig. 13. Somatic chromosomes of the tetraploid specimcn 66041: 8 (karyotype 11). a: Metaphase plate. The new chromosome with subterminal centromere is indicated by an arrow, the SAT:s and medians by D and m, respectively. X2500. b: Idiogram.

types, and whereas (large,) medium and small medians occur in the tetraploid only small medians are found in the diploid. These marked karyotype differences were pointed out already by JONES (op.cit.) and may be confirmed by comparing the idiograms of the relevant karyotypes, Fig. 14 and 15d.

If the diploid karyotype is instead compared to karyotype I1 of the tetraploid it is quite obvious that the latter replicates that of the diploid (Fig. 15). The satellite chromosomes are of the same, very characteristic type, the submedians of the tetraploid are easily matched to those of the diploid, and only small medians are found in both (see above), The existence of a tetraploid karyotype of this kind has not earlier been demonstrated and was emphat- ically denied by JONES (1964, p. 253).

In this connection also the results of Roz- MUS (1958) must be taken into consideration. According to him the chromosome morphology of the tetraploids corresponds to that of the diploids, which clearly indicates that he considers the chromosomes to occur in sets of four. As shown above (p. 160) I have, however, not found a haploid chromosome set similar to that diagrammatically illustrated by ROZMUS

in any diploid. Also the similar statement by PARTHASARATHY (1939) must be strongly doubt- ed since his drawing (op. cit. p. 48) in my opinion indicates that the plants investigated by him had the common tetraploid karyotype. The conclusions drawn by ROZMUS and PAR- THASARATHY demonstrate the disadvantage of using sections for studies of chromosome morphology.

2. Meiosis A considerable number of slides for meiotic studies have been prepared during the course of my work. Technical difficulties (cp. JONES 1964) resulting in a strong tendency for the chromosomes to clump together during metaphase make, however, analyses of the configurations extremely difficult in many cases. The most favourable stage for study seems to be early metaphase I, but this stage is obviously hard to hit. The difficulties in analysing the metaphase configurations could easily lead to an unintentional choice of those cells most suitable for analysis, which might bias the results to a considerable extent. Hence I have at present refrained from any statistics and will

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166 INCA HEDBERG

b

Fig. 14. Idiograms of the chromosome complements in three tetraploids with karyotype I. a: Plant no. 66045: 3. b: Plant no. 69/02: 10. A faint secondary constriction in one of the large medians is indicated by an arrow. c: Plant no. 69010: 5.

only summarize the results of my investigations as follows.

In the diploid about 15 individuals were examined, most of which showed bivalents only. In a few cases also univalents were found and sometimes inversion bridges (Fig. 16). which indicates structural changes already on diploid level (cp. p. 160) . Specimens from certain populations (e.g. 66039) seem to show such changes more frequently than others.

More than 20 tetraploids were inspected, all of which formed quadrivalents to a varying degree (Fig. 17). Also multivalents of higher order were found in most specimens. Some individuals seemed to have a higher frequency of bivalents than others, and some tended to show a more pronounced multivalent formation. Those multivalents were formed by up to 10 chromosomes, possibly sometimes still more.

In some individuals the subsequent stages passed without any notable disturbances. apparently due to the high degree of alternately oriented multivalents at metaphase I, whereas in others laggards, inversion bridges etc. were found to variable extent. Occasionally pollen cells with 1 1 or 9 chromosomes were seen.

There is possibly a difference between specimens with karyotype I and those with karyotype 11, since the latter Seem to have a much lower frequency of multivalents of higher order. In order t o prove this much more detailed studies are, however, needed. The general observations accounted for above agree w r y well with those of KATTERMANN (1931), who performed a very detailed analysis of meiosis in tetraploid A. odoraturn, and with those of PARTHASARATHY (1939), OSTERGREN (1942), and JONES (1964).

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KARYOTYPES IN ANTHOXANTHUM OWRATUM 167

a

b

11111

d Fig. 15. ldiograms of the chromosome complements in three tetraploids (a-c) with karyotype I 1 and of the haploid set of a diploid (d). a: Plant no. 66041: 1. b: Plant no. 66041: 6. c: Plant no. 66041: 9. d: Plant no. 69/02: 20.

Frequent formation of quadrivalents is gen- erally considered to be due to autopolyploidy, and figures of more than four in autotetraploid specimens indicate structural hybridity (cp. e.g. DARLINGTON 1937; PARTHASARATHY 1939; MUNTZING 1936; DAVIS and HEYWOOD 1963). Thus A. odoratum was considered by KATTER-

OSTERGREN (op. cit.) to have originated through autopolyploidy followed by structural changes. JONES (op. cit.), on the contrary, considers it to be of allopolyploid origin, stating that the frequent quadrivalent pairing must be the result

MANN (Op. Cit.), PARTHASARATHY (Op. cit.) and

of chiasmate association between heteromorphic chromosomes. This conclusion seems, however, to be based not so much on meiotic evidence as on his morphological studies of mitotic chromosomes, amongst which "only occasionally it is possible to find any chromosome type represented by four morphologically similar replicas" (JONES op. cit. p. 267). In view of the existence of a tetraploid karyotype which definitely replicates that of the diploid (cp. above) JONES'S conclusion seems hardly convincing, especially as there is nothing in the meiotic behaviour to contradict autopolyploidy.

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168 INCA HEDBERG

Fig. 16. Irregular meiosis in a diploid specimen (66039: 4). Bridges in anaphase (a) and telophase (b) x 1600.

Hybrids In connection with my cytological studies a number of crosses between diploids and tetraploids were attempted.

1. Diploid X tetraploid Since diploids as a rule flower earlier than tetraploids (HEDBERG 1967, p. 20) it was difficult to obtain enough pollen for crossing experiments, and so it became necessary to use pollen from more than one tetraploid in order to distribute a safe amount of pollen to each stigma. Of the 14 combinations involved only three yielded triploid progeny. Two of the mother plants (66016: 1 and 66038: 4) gave one triploid each whereas three triploid plants were obtained from the third one (66022: 1). When these crosses were performed the existence of two different karyotypes within the tetraploid was still unknown. Hence, unfortun- ately, a mixture of pollen from specimens with karyotype I and from those with karyotype I1 was used, which means that for some of the triploids it is impossible to tell whether they are hybrids or whether they have arisen by auto- gamy from one unreduced and one reduced

gamete (cp. Fig. 18). Only one (68/03: 1 ) of the five triploids concerned could be safely classified as a hybrid, since in root tip squashes the SAT chromosomes from the tetraploid karyotype I could easily be identified (Fig. 19). Interestingly enough the very characteristic D SAT from the diploid mother (Fig. 20 a) could not be observed. Also in one spontaneous triploid hybrid (66026: 3) only the A and B type S A T s could be seen (Fig. 20 b). Only root tip sections were available from the last-mentioned specimens, but from a careful inspection it has been possible to recognize the various types of SAT:s also in such preparations (p. 157).

In the first-mentioned case the presence of D S A T s in the mother plant has been estab- lished. Since the second hybrid mentioned was spontaneous the parental karyotypes were not available. It is, however, highly unlikely that the D type SAT was absent in the diploid parent plant, since the diploid karyotype seems to be very uniform as regards the SAT chromo-

The only plausible explanation to the fact that the typical diploid SAT chromosome does not appear in the above hybrids must then be that it is actually present, but that its secondary

somes (p. 159).

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Fig. 17. Mciosis in tetraploid specimens; a: diakinesis in a specimen with karyotype I1 (66041: 1) showing 41~211; b: diakinesis in a specimen with karyotype I (66007: 3) showing 1 ~ 1 1 1 ~ 5 1 1 ; c: metaphaw in a \@men with karyotype I (65004: 1 ) showing 4 1 ~ 2 , ~ . All X1600.

constriction does not show up. In that case it should be possible to recognize the actual chromosome by means of its relative length and arm index. An attempt at such an identification was made in the above mentioned artificial hybrid 68/03: 1 , an idiogram of which is shown in Fig. 19 c. A careful analysis of this idiogram reveals that one of the submedians can with great probability be classified as a concealed D type SAT, since it has the same arm index and relative length as such a chromosome.

The phenomenon that one or more secondary constrictions are suppressed in the presence

of other chromosomes with secondary constrictions was first reported by NAVASHIN (1934) in Crepis hybrids, (cp. also DARLINGTON 1937, p. 305; HENEEN 1962).

2. Tetraploid X diploid

Whereas crossing attempts within A . odornfurn s. lat. using the diploid as mother plant have on different occasions yielded at least a few hybrids such have never been reported from the reciprocal crossing (BORRILL 1963; H E D - BERG 1967). Quite a few attempts to obtain

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170 INGA HEDBERG

Fig. 18. Somatic chromosomes in an experimental triploid, 68/06: 6. The SAT:s and the medians are indicated by D and m, respectively. X1800.

hybrids by using the tetraploid as female had been performed in vain by myself in Scandi- navian material, hence it was considered im- probable that hybrids would be obtained from this combination in Swiss material. Since, however, I had found in Switzerland quite a few tetraploids which in all morphological features were undistinguishable from diploids it was thought that crosses in Swiss material might be more successful. Of the seven cross combinations involved all but one failed. This single combination gave, however, no less than 24 triploid hybrid plants. They were all vigorous and 16 of them flowered the second season (as d o usually both the diploid and the tetraploid). Pollen fertility (as tested in lactic blue) ranged from 8 % to 28 %, seed set was in all cases fairly poor. No offspring has yet been obtained from these hybrids since the seeds were harvested last season.

In view of the fact that no hybrids have earlier been reported from crossing attempts, using the tetraploid as female parent, the above result is rather remarkable. An investigation of the karyotypes of the plants involved did, however, provide an explanation. Of the tetraploid mother plants all but one had the common karyotype (I). From those plants no hybrids were obtained. The remaining tetraploid, however, had the deviating karyotype (11) and this single plant (66041: 8) yielded all the hybrids. A comparison between this karyotype and that

P

b

Fig. 19. Somatic chromosomes in an experimental triploid hybrid (68103: 1) obtained from crossing a diploid to a tetraploid with karyotype 1. a: Metaphase plate. The chromosome supposed to be a concealed D SAT is indicated by an arrow. X1800. b: Diagrammatic drawing of the same plate. c: Idiogram. The chromosomes supposed to be derived from the diploid parent are marked with an x, the one considered to represent the concealed D SAT with an x in a circle.

Heredilas 64. 1970


a b

Fig. 20. Somatic metaphases in root tip sections; a: the diploid specimen 66016: I ; b: the spontaneous triploid hybrid 66026: 3. X1800.

of the diploid shows a remarkable similarity (p. 165). If, as suggested earlier (p. 163) the plants with karyotype I1 have originated from recent autopolyploids, the plants obtained after crossing such a plant to a diploid should apparently have three more or less identical sets of chromosomes. This is also the case in some of the hybrids (Fig. 21 a). Since, however, changes from the regular autotetraploid set were encountered already in the mother plant, noticeable above all in the occurrence of a chromosome with a subterminal centromere (Fig. 13), such irregularities were also found in some of the hybrids (Fig. 21 b). The above results are of great interest for the following discussion and will be considered later (p. 173).

Discussion The present investigation of a large material of diploid and tetraploid Anrhoxanthurn odora- rum has revealed that whereas the karyotype of the diploid seems to be very uniform (p. 159) there exist within the tetraploid two distinctly different karyotypes. Such a possibility was not taken into consideration by JONES (1964) who in spite of the very wide distribution of the tetraploid (see e . g. HEDBERG 1967) restricted his study to material from the British Isles only. According to JONES (op. cit.) tetraploid A. odoraturn shows a remarkable inter-plant variation as regards the chromosome complement. All his material conforms, however, to the same general pattern, and the variation may possibly to some extent be ascribed to differences caused by technical difficulties (p. 157). The karyotype described

Fig. 21. Somatic chromosomes of hybrids A. odora/rrm 2n=20 (karyotype 11, plant no. 66041: 8)XA. odora- frtm 2n=10; a: plant 681381: 10 with a complement of 3 x 5 . X1500. b: plant 68/381: 16 which shows the new chromosome with subterminal centromere (indicated by an arrow) From the tetraploid mother plant (cp. Fig. 13 and p. 163). The SAT:s and the medians are indicated by D and m, respectively. XZOOO.

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172 INGA HEDBERG

by JONES and considered by him to be the only one occurring in the tetraploid seems to be by far the most common, since it was also found in most of the specimens investigated by me. Out of the 40 specimens from 21 samples accounted for here (Table 2) - and many more have been inspected during the course of my work - only 5 specimens, all from the same sample, had the deviating karyotype (11). see Fig. 10-13. Even within this sample ‘two specimens had the common karyo-

The occurrence of two so sharply distinct karyotypes within one species seems to be rather exceptional. As shown by the idiograms (Fig. 15) one of these karyotypes (11) obviously replicates that of the diploid and it seems beyond doubt that specimens showing this karyotype must be descendants of recent autopolyploids. Morphologically all those tetraploids are indistinguishable from diploids but this is also the case with some specimens with karyotype I both from the same sample and from others. Further support of an autotetraploid origin of A. ddoratum (4x) is obtained from studies of meiosis, which already before the discovery of a diploid cytotype within A. odoraturn s. lat. led some cytologists t o postu- late autopolyploidy because of the frequent Occurrence of quadrivalents (e. g. KATTERMANN 193 1; PARTHASARATHY 1939). More recent works by OSTERGREN (1942) and JONES (1964) as well as my own confirm the existence of such meiotic configurations, which are gener- ally considered to characterize autotetraploids (cp. p. 166). Despite those conditions JONES (op. cit.) concluded that “neither karyotype nor meiotic pairing contains any support for the supposition that odoraturn is an autotetraploid”. This statement is rather misleading since it intimates that the type of meiotic pairing occurring in the tetraploid indicates allopolyplaidy. No evidence in that direction is brought fourth by JONES. His statement re- garding meiosis is, however, easily explained since it is apparently based on his observations on mitotic chromosomes. Lacking evidence on autopolyploidy in the morphology of the mitotic chromosomes he ascribed the frequent quadrivalent pairing to “chiasmate association between heteromorphic chromosomes” (op. cit. p. 267).

type (1).

The hypothesis brought forth by JONES a n the origin of the tetraploid involves hybridisa- tion between diploid A. odoratum (A. alpinurn LOVE and LOVE) and A. ovaturn LAG. (also with 2n = 10) followed by doubling. An amphi- diploid of this kind was produced artificially through colchicine treatment by BORRILL (1963) and its meiosis was studied by JONES (op. cit.). I t showed regular and complete pairing with bivalents prevailing and rnulti- valents infrequent. JONES concluded that tetraploid odoratum could have evolved from such a plant. In order to explain the frequent quadrivalents occurring in the tetraploid cytotype (cp. above) he postulated selection for in- creased quadrivalent pairing in the above plant.

According to JONES his hypothesis is supported by the phenotypic and hybridization studies carried out by BORRILL (op.cit.). I t is difficult to evaluate the significance of the hybridization studies mentioned since the ovaturn plant involved was of unknown origin and apparently genotypically somewhat dubious (BORRILL op,cit. p. 187, 192). That the material used by this author was quite insufficient fur general conclusions as regards morphological features has earlier been pointed out (HEDBERG 1967, p. 66). Like TUTIN (1950) he considered diploid A. odoraturn to be devoid of pubescence contrary to the tetraploid. Whereas this distinction holds good in part of the distribution area it breaks down in other areas (HEDBERG 1967, 1969).

In view of these rather inconclusive argu- ments the above hypothesis seems less convincing. Though not excluding other possibili- ties the present investigation on the. contrary gives strong support t o another hypothesis, viz. an autotetraploid origin of the widespread tetraploid cytatype. This conclusion is based not only on a comparison between the chromosome complements of diploids and those of some orf the tetraploids (karyotype 11, Fig. 15) but also on meiotic studies revealing frequent quadrivalent pairing in all tetraploids examined (p. 166). Furthermore a considerable number of triploid hybrids was obtained from crossing a tetraploid with karyotype I1 ( 0 ) t o diploids (d). As far as I know hybrids have never before been obtained from crosses within A. odoraturn s . lat. using the tetraploid as mother plant. If the degree of compatibility

Hereditas 64. 1970


may be considered as a measure of the phyletic affinity of the specimens involved these hybrids certainly indicate a very close relationship between the tetraploid and the diploids involved (p. 170-171. The chromosome doubling may have come about through either somatic or gametic doubling. It seems at present impossible to tell with certainty in which of those ways the doubling has occurred but the second one seems most probable. Such a gametic doubling would in the first line result in the formation of triploids which would yield occasional tetraploids amongst their offspring. The existence of spontaneous autotriploids in the actual species has been proved earlier and support for the theory that such triploids have arisen through gametic doubling was given by the occurrence of a triploid amongst the offspring from a selfed diploid in experimental material (HEDBERG 1967). It has also been shown that triploids of A . odoraturn s. lat. in which irregularities at meiosis must cause a high degree of sterility, nevertheless could pro- duce tetraploid offspring (HEDBERG op. cit.).

The karyotype of tetraploids originated through the process described above would then evidently replicate that of the diploid. Such tetraploids have now been proved to occur spontaneously in the Swiss Alps. In terestingly enough no diploids were found in the same sample though my field notes indicate the impression of a mixed population. Since this sample was one amongst many taken in Austria and Switzerland in order to achieve more information about the cytotypes in this area (HEDBERG 1969) it was not very large. A more comprehensive sampling from the actual population would probably reveal the existence of diploids within it. Incidentally the diploid number was counted in one specimen taken in a sample a couple of hundred meters lower on the same slope (cp. HEDBERG, op. cit. p. 235) but since this plant perished during the first winter in cultivation this number could not be verified.

As shown above tetraploids which have a karyotype replicating that of the diploid are very rare, whereas the majority of specimens investigated have a karyotype deviating from the latter. In my opinion this fact is, however, not inconsistent with an autoploid origin of the tetraploid cytotype. Rather different as

the two karyotypes appear to be it would not be too difficult to visualize the derivation of karyotype I from karyotype 11. In the first place it is apparently possible to find in the first mentioned equivalents for some of the submedians and small medians occurring in karyotype I1 and the diploid. This is shown by comparison between the idiograms of the karyotypes involved (Fig. 14 and 15) and also by the chromosome complement of a hybrid between a diploid and a tetraploid with karyotype I (Fig. 19), cp. also JONES (1964 p. 254). Secondly it was shown in the discussion on the chromosome complements of the autotetraploids that they had all been subject to modifications to a variable degree. Changes in submedians resulting in modification of both length and arm index were noted, as well as Occurrence of new chromosome types supposed to be transitional stages in the repatterning process. In this way some of the submedians in the autotetraploid karyotype (11) could have been transformed into other types of submedians occurring in karyotype I. It would seem more difficult to explain the changes which would have given rise to the entirely different types of SAT chromosomes in karyotype I compared to those of karyotype 11. Trans- ference of a secondary constriction from one chromosome to another was suggested by JONES (1964) to be responsible for some of the karyotypic variation within the first mentioned karyotype. Such a transference would then also possibly account for some of the differences between the karyotypes as regards types of SATs. A similar event was suggested in Ni- gella doerfleri V I E R H . by STRID (1969). Another possible origin of the A type SAT is furnished by the deviating diploid specimen accounted for earlier (p. 160). Apart from illustrating the change of a median chromosome into a distinctly submedian the karyotype of this diploid showed a modified type of SAT chromosome apparently originated through loss of about half the short arm in one of the SATs. A translocation of a segment to the distal end of the long arm in such a chromosome would result in a SAT chromosome of the A type (Fig. 5 ) . The absence of the D type SAT in karyotype I could also to some extent be a question of suppression of the secondary constriction. It was shown above (p. 168) that in

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174 INCA HEDBERG

hybrids between diploids and tetraploids with karyotype I the D type SAT of the first mentioned did not show up. On the other hand two D type SAT:s were found in one of all the tetraplaids with karyotype I (p. 163 and Fig. 9). This may indicate that the showing up or dis- appearance of secondary constrictions could to some extent be attributed to physiological influence caused by differences in genotype (cp. HENEEN 1962).

Meiosis in a hybrid between a diploid and a tetraploid with karyotype I (p. 168) showed fairly frequent trivalents which also indicates rather a close relationship between them. One intriguing detail in this case is the dominance and relative uniformity in the general pattern of karyotype I in the tetraploid (p. 162). Ob- viously it must represent some sort of balanced condition, which is combined with a very successful gene combination.

More research especially including crosses between tetraploids with different karyotypes and studies of the hybrids and hybrid deriva- tives from the cross tetraploid (karyotype 11) to diploid (p. 170) is apparently needed to elucidate the origin of karyotype I. Since the existence of the two karyotypes within the tetraploid was only discovered in the last year, data from such investigations are not yet available. The above observations might, however, serve as examples of the events which could have transformed the autotetraploid karyotype into a more or less allopolyploid condition. A continuous process of chromosome changes in tetraploid A . odoraturn with extensive recom- bination of terminal segments was also suggested by JONES (1964) t o explain the great interplant variation in karyotype I. Further evidence of such events is provided by the formation of multivalents of higher order (more than 4) at meiosis, which must be the result of interchange heterozygosity (see p. 166). As indicated above the frequency of such multivalents seems to be higher in those tetraploids having karyotype I than in those with karyotype I1 - which however, remains to be checked. If this can be confirmed, it also points towards a transformation as outlined above.

According to STEBBINS (1959) very few successful autotetraploids have arisen from a single diploid population. Probably such p l y - ploids have mostly been derived through

Hereditas 64, 1970

doubling after hybridization between different- ly adapted diploids, such as interfertile eco- types within the same species. This conception does apparently not oppose the above discussion since despite their hybrid origin such tetraploids are cytogenetically to be regarded as autotetraploids.

Another possibility is that the tetraploid represents some kind of a “pillar complex” (STEBBINS 1950). Hybridization between autotetraploids arisen in cytologically slightly different populations would then account for the origin of the widespread tetraploid cytotype.

In my earlier papers (HEDBERG 1967, 1969) the difficulties in finding reliable morphological and anatomical differences between the two cytotypes of A . odoraturn s. lat. were pointed out. Differences which had been considered (e. g. by TUTIN 1950; BORRILL 1963; JONES and MELDERIS 1964) to be correlated to chromosome number were shown to break down when a more comprehensive material from various parts of the distribution area was investigated. The necessity of examining a large material before taxonomic conclusions are drawn have been repeatedly p in t ed out (see e. g. BELL 1954; DAVIS and HEYWOOD 1963). The present investigation strongly supports the same necessity as regards cytological studies. Apparently it is highly desirable to study not only the chromosome number but also the chromosome morphology within various populations. Such a painstaking verification has obviously not very often been performed, yet it may give very valuable information and a safer basis for discussions on phyletic relationship. The conclusion drawn by JONES (1964) in the case of A . odoraturn “since therefore evidence from several sources indicates hybridity whilst none supports autopolyploidy” is in view of the present results hardly justified.

Conclusions Conclusive interpretation of the genesis of a tetraploid within a group of diploids may often be unattainable - the larger the material available, the more difficult it seems to generalize. Speculation and hypotheses are necessary in order t o provide an outline of the probable


course of events in desirable that such cumulative evidence possible.

the past, but it is highly hypotheses are based on from as many sources as

In the case of Anthoxanthum odoratum L. s. lat. such evidence indicates a very close relationship between the common tetraploid and the diploid ( A . alpinurn LOVE and LOVE). I n part of the distribution area they a re morphologically indistinguishable. Within this area descendants f rom recent autotetraploids are found. T h e mode of morphological variation in the tetraploid points towards its origin geo- graphically close t o that area. Thus it seems highly probable that autoploidy has played an important role in the genesis of tetraploid A . odoraturn, though some details concerning its evolution still remain obscure.

From a taxonomical point of view my earlier investigations together with the present one clearly show that it is not possible t o maintain the diploid as a separate species. Subspecific status might seem possible in Scandinavia but not in the Alps where in several cases the only possibility t o recognize the diploid is t o perform chromosome counts. In a scheme of general classification it is therefore impractic- able t o give taxonomic distinction t o the cytotypes.

AcktroH*ledRer~ients. - The work has been carried out at the Institute of Systematic Botany. University of Uppsala. I am indebted to its director, Professor J. A. Nannfeldt. for comments and criticism of my manu- script. Financial support towards the costs of technical assistance has been obtained from the Swedish Natural Science Research Council and the field work has been supported by a grant for field work from the Faculty of Science, University of Uppsala. I wish to thank Dr. A. K. Skvortsov, Botanic Garden. University of Moscow. and his colleagues Drs. K. Kiscleva, N. Bela- nina, and 1. Koropachinski for providing me with seeds from the USSR. To my husband, Dr. 0. Hedberg, I wish to express my deepest gratitude for his continuous encouragement and stimulating criticism.

Literature cited BJ~RKQVIST, 1. 1968. Studies in Alisma L. 11. Chromo- some studies. crossing experiments and taxonomy. - Opera Bot. I Y : 1-138.

BORRILI.. M. 1963. Experimental studies of cvolution in Arirhoxarirhrtni (Gramineae). - Gerieticu 34: 18%- 210.

DARLINGTON, C. D. 1937. Recent advances in cytology. - J . arid A . Churchill, Ltd., Loridori. 671 p.

DAVIS, P. H. and HEYWOOD, V. H. 1963. Principles of angiosperm taxonomy. - Oliver arid Boyd, Editibrirgh arid London, 556 p.

GUINOCHET, M. 1942-1943. Recherches de taxinomie expkrimentale sur la flore des alpes ct la rtgion mtditerrantenne occidentale 1. - Notes caryologiques sur quelques Graminkes. - Rev. Cytol. Cyrophysiol.

HEDBERG, 1. 1967. Cytotaxonomic studies on AnrIioxari- r h r m odoratrim L. s. lat. 11. Invcstigations of some Swedish and of a few Swiss population samples. - Symbolae Bot. Upsalien. 18(5): 1-88, PI. 1-8. - 1969. Cytotaxonomic studies on Arirhoxarirhum odoratum L. s. lat. 111. Investigations of Swiss and Austrian population samples. - Sverisk Bor. Tidskr.

HENEEN, W. K. 1962. Karyotype studies in Agropyrori jioicectm, A. repem and their spontaneous hybrids. - Heredilas 48: 411-502.

HENEEN, W. K. and RUNEMARK, H. 1962. Chromosomal polymorphism and morphological diversity in Elymus rechingeri. - Ibid. 48: 545-564.

HUNTER, A. W. S. 1934. A karyosystematic investigation in the Gramineae. - Can. J. Res. 2: 213-241.

JONES, B. M. G. and MELDERIS, A. 1964. Anthoxari- rhitnt odorurirm L. and A. alpirirtm A. and D. L ~ v E . - Proc. Bot. SOC. Brit. Isles 5: 315-377.

JONES, K . 1964. Chromosomes and the nature and origin of Arirlioxari/hrrm odoratum L. - Chromosoma IS: 248-214.

KATTERMANN, G. 1931. Uber die Bildung polyvalenter Chromosomenverbande bei einigen Gramineen. - Plaiita 12: 132-174.

MARKARIAN, D. and SCHULZ-SCHAEFFER, J. 1958. A p o s sible origin of supernumerary fragment chromosomes. - J. Hered. 49: 3-1.

MUN~ZING, A. 1936. The evolutionary significance of autopolyploidy. - Hereditas 21: 263-378.

OSTERGREN, G. 1942. Chromosome numbers in Ari- /hoxari/hrim. - Ibid. 28: 242-243.

~STEUGREN, G. and HENEEN, W. K. 1962. A squash technique for chromosome morphological studies. -

PARTHASARATHY, N. 1939. Cytogenetical studies in Oryzeae and Phalarideae. - Arm. Bot. N . S . 111:

ROZMUS, M. 1958. Cytological investigations on An- thoxanthttm alpiriiim L. et L., a new species of the flora of Poland. - Acta Biol. Cracov. SPr. Bot. I :

SIRID, A. 1969. variation in the satellite chromozomes of Nigella doerjleri (Ranunculaceae). - Bo/. Notiser 122: 9-19,

TUTIN, T. C. 1950. A note on species pairs in the Gramineae. - Watsoniu 1 : 224-227.

V&. 6 : 209-220.

63: 233-250, PI. 1-111.

Ibid. 42: 332-341.

43-76.

11 1-1 84.

lnga Hedbcrg Institute of Systematic Botany S-751 04 Uppsala 1 , Sweden

Hereditus 64. 1970

176 INCA HEDBERG

Literature cited

ADDENDA

BELL, C. R. 1954. The Sanicula crassicaulis complex (Umbelliferae). - Llrtiv. Calif. Pub[. Bot. 27 (3): 133- 230.

NAVASHIN, M. 1954. Chromosome alterations caused by hybridization and their bearing upon certain general genetic problems. - Cyfologia 5: 169-203.

STEBBINS, G. L. 1950. Variation and evolution in plants. - Coliimbiu Utziv. Press, New York, 643 p. - 1959. Genes, chromosomes and evolution. - In: W. B. Turrill (ed.), Vistas in Botany (I): 258-290. Pergamon Press, Londort.

Hereditas 64, 1970

cytotaxonomic studies on anthoxanthum odoratum l. s. lat. : iv. karyotypes, meiosis and the origin...

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