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Phyletic and evolutionary relationships of Brachyscome lineariloba (Compositae).

Article in Plant Systematics and Evolution · January 1987

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Phyletic and evolutionary relationships of Brachyscome lineariloba (Compositae) 1

K. WATANABE and S. SMITH-WHITE

Received March 14, 1986

Key words: Angiosperms, Compositae, Asteroideae, Brachyscome (= Brachycome) lineari­ loba, B. breviscapis. - Life pattern, annual inbreeding, ascending dysploidy, amphidiploidy, karyotype, hybridization, meiotic pairing. - Flora of Australia. Abstract: A comparison of karyotypes of Brachyscome breviscapis (2 n = 8), B. lineariloba cytodemes E (2n = 10), B (2n = 12) and C (2n = 16) suggests that these species have a homoelogous basic set of four chromosome pairs, two large pairs and two small, and that the B. lineariloba cytodemes E, Band C are related to B. breviscapis by successive additions of small chromosomes. A pronounced asynchrony of chromosome condensation between these large and small chromosomes has been observed. In the artificial hybrids between B. dichromosomatica (2 n = 4) x B. breviscapis, and the B. lineariloba cytodemes, the B. dichromosomatica chromosomes are similar in size and condensation behaviour to the small chromosomes of B. breviscapis and of B. lineariloba cytodemes E, Band C. Meiotic pairing in these hybrids also demonstrates the strong affinities between these chromosomes. It is suggested that B. breviscapis may be of amphidiploid origin between a species with two large early condensing chromosome pairs and another, B. dichromosomatica-like species with two small late condensing pairs. It seems most likely that the additional small and late condensing chromosomes in B. lineariloba cytodemes E, Band C are derived from the B. dichromosomatica-like parent, and that each addition increases vigour, fecundity and drought tolerance, allowing these cytodemes to colonize more open and arid environments. Transmission of the univa1ents in the quasidip10id B. lineariloba cytodeme E was verified as being via the pollen, and not via the embryo sacs.

The Brachyscome lineariloba complex, recognized by DAVIS (1948) as a single species, has been found to include five "races", cytodemes or species, designated as A, B, C, D and E by SMITH-WHITE & CARTER (1970) and CARTER & al. (1974). Two of these, race A, with 2 n = 4, and race D, with 2 n = 8, have been raised to specific rank by CARTER (1978 a), as, respectively, B. dichromosomatica C. R. CARTER and B. breviscapis C. R. CARTER. The other three races which remain within B. lineariloba (DC.) DRUCE are the cytodemes B with 2n = 12, C with 2n = 16, and E with 2 n = 10. The latter is a quasidiploid with four chromosome pairs and two univalents. All these cytodemes and taxa A - E do not constitute a

1 The cytology of Brachyscome lineariloba (Compositae, Asteroidae), 10. 122 K. WATANABE & S. SMITH-WHITE: polyploid series. CARTER'S (1978 a) taxonomic revision, although fully justified, does not alter the close evolutionary and phyletic relationships of the members of the complex. For convenience in this paper, the "race" symbols A, B, C, D and E will be frequently used to refer to the several species and cytodemes. KYHOS & al. (1977) proposed a phyletic scheme of relationship for the members of the complex which was based on a number of criteria: plant size and vigour, growth form, (small) morphological differences, chromosome number and mor­ phology, chromosome association in putative hybrids and geographical distribu­ tion. Obviously, natural hybrids can only occur in regions of species overlap, and KYHOS & al. (1977) give a list of localities where mixed populations are found. Sympatry is not known for the important pairs A and E, A and D, C and E, C and D, and Band D. The species and cytodemes are not internally homogeneous. B. dichromosomatica exists in two geographically disjunct taxonomic varieties, var. alba C. R. CARTER formerly named A3, and var. dichromosomatica C. R. CARTER which is made up of three cytodemes, Ab A2 and A4. The four forms are related to one another by chromosome interchanges, by differences in chromatin condensation patterns at meiotic first prophase, and by suppression or loss of the nucleolar organizer (WA­ TANABE & al. 1975). Also, in about 10% of AI, A2 and A3 plants, one, two, or three B-chromosomes are present (CARTER & SMITH-WHITE 1972, CARTER 1978, SMITH-WHITE & CARTER 1981). Within cytodeme C two subdemes, CI and C2, occur, and even these show considerable heterogeneity for chromosomal variants (WATANABE & al. 1985). Chromosomal variants are also known to occur in cy­ todemes Band E, and in B. breviscapis. The study of artificially produced hybrids overcomes some of the difficulties which were imposed upon KYHOS & al. (1977). Such hybrid material has precisely known parentage, and the range of hybrid material is not geographically restricted. The present study comprises the artificial hybrids D x E, D X A3, D X Ab E X Ab Al X E, B X Ab B X A3 and C I x AI' Throughout this paper, crosses are always indicated with the pistillate or seed parent to be left. The hybridization E x D was attempted, but was unsuccessful. Cytodeme D, which is B. breviscapis, has minute flower heads and produces very little pollen.

Materials and methods The geographical sources of the species and cytodemes used in this hybridization work are:

B. dichromosomatica var. 15 km north of Simmondston, S. Australia dichromosomatica A I B. dichromosomatica 70 km south-east of Willcannia, adjacent to var. alba A3 the Barrier Highway, N. S. Wales B. breviscapis D Laura Bay, S. Australia B. lineariloba E Laura Bay, S. Australia B. lineariloba B Hookina, north of Hawker, S. Australia B. lineariloba CI 11.2km west of Broken Hill, N. S. Wales

The cytological, hybridization and germination techniques used were identical to those described by WATANABE & al. (1975). . Cytology of Brachyscome lineariloba 123

1•• 111 JIIIII

10 203040 10203040 1B 2B 3B 4B 5B 6B 1B2B3B4BSB 6B

1E 2E 3E 4E 5E 6E 1E 2E3E 4E 5E 6E 1C 2C 3C 4C 5C 6C 7C BC 1C 2C 3C 4C 5C 6C7C 8C M (3,41 (1,10) ~~ ~':I'.l ~5,11\l (.~ (Ill (1.01 (3,41 (1,10) ~~ (lIP) ~5,11\l (5.e) (\Ol

Figs. 1-4. Idiograms of the haploid chromosome set at metaphase (left) and prometaphase (right) of the Brachyscome lineariloba complex; early condensing chromatin in prometaphase dotted, late condensing white. - Fig. 1. B. breviscapis D; n = 4. - Fig. 2. B. lineariloba E; n = 5, chromosomes 5 E and 6 E are univalents. - Fig. 3. B. lineariloba B; n = 6. - Fig. 4. B. lineariloba C; n = 8. Idiograms are drawn from Figs. 5 - 11 and Fig. 4a in WATANABE & al. (1985). Scale = 1O)lm

Results

Parental karyotypes. Following KYHOS & al. (1977) the haploid set of chromosomes of B. breviscapis D have been arranged in order of size, and are numbered ac­ cordingly (Fig. I). The four chromosomes are all quite characteristic. The corre­ sponding chromosomes of B. lineariloba cytodemes E, Band C have been given the same numbers (Figs. 2 - 4). The implication is that they are homologous, or at least essential homeologous, in the four taxa. The univalent chromosomes of cy­ todeme E have been numbered 5 E and 6 E, and are comparable in size and form with 5 Band 6 B of cytodeme B, and with 5 C and 6 C of cytodeme C. Cytodeme C then has the additional pairs 7 C and 8 C. This numbering system is different from that adopted by WATANABE & al. (1985), where the complete somatic chro­ mosome set had to be ranked in order of size regardless of homeologies. For reference that former rank order for CI is given in parenthesis in Fig. 4. The homeologous ranking, size and form of the chromosomes of the cytodemes D, E, Band C is in general agreement with that given by KYHOS & al. (1977), with the difference that 5 Band 6 B, and 7 C and 8 C are distinctly larger. The chro­ mosomes of B. dichromosomatica Al and A3 have been described by WATANABE ,..... Table 1. Karyotype data of Brachyscome breviscapis D, B.lineariloba E, Band C 1 (length in /lm). L, S Long arm length, short arm length, Ttotal tv .j::>. length, L/ S arm ratio, long arm/short arm. Measurements from Figs. 5 - 8. Chromosomes of B. lineariloba E, B, C 1 are arranged in correspondence to the length and arm ratio of those of B. breviscapis. Relative lengths, normalized to 1.00 for chromosome 1 in parentheses. The numbers in the bottomline of C 1 give the chromosome arrangement as used by WATANABE & al. (1985)

Species or cytodeme Chromosome

1 2 3 4 5 6 7 8

D L,S 9.4, 7.5 7.6, 2.5 4.7, 3.3 3.7,2.3 T 16.9 (1.00) 11.1 (0.66) 8.0 (0.47) 6.0 (0.36) LIS 1.3 3.0 1.4 1.6 E L, S 8.2,6.3 7.1, 1.5 3.8,2.7 3.3,2.0 3.7,2.0 3.7,2.0 T 14.5 (1.00) 8.6 (0.59) 6.5 (0.45) 5.3 (0.37) 5.7 (0.39) 5.7 (0.39) LIS 1.3 4.7 1.4 1.7 1.9 1.9 B L, S 9.0, 6.8 8.0, 1.8 4.8,3.0 4.3,2.7 5.3, 3.2 5.1,2.7 T 15.8 (1.00) 9.8 (0.62) 7.8 (0.49) 7.0 (0.44) 8.5 (0.54) 7.9 (0.50) LIS 1.3 4.4 1.6 1.6 1.7 1.9 C] L, S 8.2,6.3 7.9, 1.5 3.8,2.7 3.3,2.2 3.7,2.2 3.5, 1.8 3.5, 3.4 4.8,2.0 T 14.5 (1.00) 9.4 (0.65) 6.5 (0.45) 5.5 (0.38) 5.9 (0.41) 5.3 (0.37) 6.9 (0.48) 6.8 (0.47) ~ LIS 1.3 5.3 1.4 1.5 1.7 1.9 1.0 2.4 ~ (1.2) (3.4) (9.10) (13.14) (11.12) (15.16) (5.6) (7.8) >-l )-

~ 1:0 tIl ~ en en 3: ::j :r: ~ :r: ::j tIl Cytology of Brachyscome lineariloba 125

Table 2. Results and interpretations from reciprocal crosses involving quasidiploid Bra­ chyscome lineariloba E. * Could include a few cases of failure of parental transmission of Qs; ** could include a few maternally transmitted Qs; S. t. seedlings tested; H. d. hybrids detected

Cross S.t. H.d. Constitution Presumed Constitution of of amphihaploids non-hybrids presumed selfs

DxE 52 2 (4+4+2Q) 50 (4 + 4)* ExD 50 0 (4 + 4) 50 (4 + 4 + 2Q)** E X AI 60 6 (4 + 2) 54 (4 + 4 + 2Q) AI X E 50 3 (2 + 4 + 2Q) 47 (2 + 2)

& al. (1975), and repetition is unnecessary. Chromosome measurements for D, E, Band C are given in Table 1, and the distribution of early and late condensing chromatin is illustrated in Figs. 1 - 4. B. breviscapis D (2n = 8) (Figs. 1, 5,9, 13). The large metacentric 1 D and the satellited near-telocentric 2 D are both uniformly early condensing at mitotic pro­ phase. The other two chromosomes have distinctive patterns of early and late condensing chromatin. Minor characteristics include the existence of small con­ strictions on the long arms of the 1 D and 2 D pairs, which are often hard to observe. B. lineariloba E (2n = 10) (Figs. 2, 7,10). The four chromosomes of the basic set, each represented in diploid dosage, are judged to be homeologous with the genome of D. Chromosomes 1 E and 3 E appear identical with 1 D and 3 D, but the short arm of 2 E is clearly shorter than that of 2 D. Chromosome 4 E appears to have suffered a change in the condensation pattern of its long arm, either by interchange or by conversion of the pattern. The univalent chromosomes 5 E and 6 E are similar in form and pattern, but they never pair in meiosis. The secondary constriction on chromosome 5 E observed by CARTER & al. (1974) as distinguishing it from 6 E, could not be identified in this study. B. lineariloba B (2n = 12) (Figs. 3, 6, 11, 14). The four chromosomes of the basic set are apparently identical to the corresponding chromosomes of cytodeme E, and have similar condensation patterns. The two extra pairs, 5 Band 6 B, differ from one another in condensation pattern: 6 B is similar to 5 E or 6 E, but 5 B has a longer segment of early condensing chromatin in its long arm.

B. lineariloba C (2n = 16) (Figs. 4, 8). Cytodeme C exists in two subdemes, C 1 and C2, which contain many variants (WATANABE & al. 1985). The form used in the hybridization work reported here was the standard C b and Fig. 4 shows the karyotype of this form. The chromosomes of the basic set are similar to those of cytodemes E and B, although the long arm of 3 C has less early condensing chro­ matin and more late condensing chromatin than 3 B. Chromosome 5 C is similar to 5 B. The short arm of chromosomes 6 C is totally early condensing and has a small intercalary constriction. Otherwise 6 C is similar to 6 B. Chromosomes 7 C and 8 C are larger chromosomes with different patterns onate condensing chromatin in both arms. Chromosome banding. C-banding patterns were investigated using the Giemsa technique of NOGUCHI & TANAKA (1981). In B. dichromosomatica var. dichromo- 120 K. WATANABI & S. SMITH-WHITE:

,. -:J.I rl'~ \ 7 8 _--

12 13

Figs.5-14. Somatic chromosomes at prometaphase and metaphase of Brac/z)'scome. - Figs. 5 - 11. Aceto-orcein. - Figs. 12 - 14. ASG (Giemsa) staining; arrows indicate the C­ bands. - Figs. 5, 9, 13. B. breviscapis D (2 n = 8). - Figs. 6, 11, 14. B. Iineariloba B (2 n = 12). - Figs. 7,10. B.lineariloha E (2 n = 10). - Fig. 8. B.lineariloha C (2 n = 16). - Fig. 12. B. dichromosomatica var. dichromosomatica A I (2 n = 4 + 2 B). Scale = 10 Ilm somatica AI collected from Wild Dog Glen (WATANABE & al. 1975), two C-bands are present terminally on the long arm of chromosome 1 and there is one terminal C-band on the short arm of chromosome 2 (Fig. 12). In B. breviscapis D, and in the B. lineariloba cytodemes E, Band C, the only C-band present in the genomes is on the short satellited arms of the several rep- Cytology of Brachyscome lineariloba 127 resentatives of chromosome 2, i.e. 2 E, 2 Band 2 C. Thus, C-banding supports the homology of chromosome 2 throughout the complex. Otherwise, the value of the technique is limited. Experimental hybridization. Hybrid studies were undertaken for two purposes, (1) to obtain data on meiotic pairing and so to extend the knowledge of interdeme relationships, and (2) to examine the inference made by CARTER & al. (1974) that the univalents of the quasidiploid cytodeme E (the Q univalents 5 E and 6 E) are in fact inherited patrilinearly. B. dichromosomatica is self-incompatible and outbreeding. B. breviscapis and the B. lineariloba cytodemes E, Band C are self-compatible and predominantly inbreeding. In all forms, the ray florets are unisexual pistillate and become receptive up to two days before the disc florets start to open. Thus, there is some opportunity for outcrossing. This behaviour permits the use of the method of hybridization described by WATANABE & al.(1975) which avoids emasculation, an extremely difficult operation. The limitation of the method is that hybrids make up only a small proportion of the seedlings obtained, and that the hybrids have to be rec­ ognized in early seedling stage by their chromosome constitution. For the purpose of the examination and interpretation of meiotic chromosome pairing this is of little consequence. It is more serious for the purpose (2) in crosses such as D x E and E x D. From the D x E crosses actual hybrids can only be detected mitotically by the presence of the Q univalents. Therefore, possible failure of transmission through pollen has not been examined. The E x D cross, which was attempted, gave, as expected, no recognizable hybrids, and thus, leaves the actual occurrence of hybrids unproven. This limitation does not arise for the A x E and E x A crosses, since hybrid seedlings can be recognized as amphihaploids from the "nor­ mal" chromosomes, without reference to the presence or absence of the Q univalents. The data available are given in Table 2. The results, although meagre, confirm the inference that the Q univalents are, at least usually, paternally inherited. The E x Al results shows that maternal transmission of the Q univalents must be at least infrequent. Mitotic chromosome measurements in the hybrids are given in Table 3. Chro­ mosome morphology and condensation pattern in the hybrids are the same as for the corresponding chromosomes in the parental material. It is recognized that the identification of particular chromosomes in meiotic association is much more dif­ ficult than at mitosis, and in the following discussion, where such identification has been virtually impossible, the chromosomes in question have been given the designation "chro". Crosses with B. breyiscapis and B. lineariloba include two combinations. B. breviscapis D x B. lineariloba E (Figs. 15,24-25). Amongst 52 seedlings from this attempted cross only two possessed the Q univalents and were definitely hybrid. Verification of hybridity for the others by precise karyotype analysis has not been made, although this could have been done by a comparison of the two represen­ tatives of chromosome 2, since 2 D has a longer short arm and shorter long arm than 2 E. This difference is clearly recognizable in the hybrid (Fig. 15). Chromo­ somes 2 D and 2 E may differ by a small pericentric invesion: the small satellite and the C-band on the short arm are identical in both. The same karyotype, i.e. with the two Q univalents, but with heterozygosity of chromosome 2, was found ...... Table 3. Karyotype data of artificial hybrids (length in )lm). Abbreviations as in Table 1. Chromosomes are arranged according to their length N and arm ratio in correspondence to those of the parental karyotypes 00

Cross Chromosome

1 (D-B) 2 (D-B) 1 A3 1AI 3 (D-B) 4 (D-B) 2AI 2A3 5 (E-B) 6 (E-B)

L, S 9.2,7.0 7.5,2.7 4.8,3.5 4.0,2.5 DT 16.2 (1.00) 10.2 (0.63) - 8.3 (0.51) 6.5 (0.40) DxE LIS 1.3 2.8 1.4 1.6 (2n = 10) L,S 9.3,6.7 8.3, 1.7 4.5,3.0 4.0,2.5 4.5,2.2 4.0,2.2 ET 16.0 (0.99) 10.0 (0.62) - 7.5 (0.46) 6.5 (0.40) 6.7 (0.41) 6.2 (0.38) LIS 1.4 4.9 1.5 1.6 2.0 1.8 D x A3 L, S 7.8,6.3 6.7,2.5 2.3, 2.5, 3.7 - 4.3,3.0 3.8,2.3 3.3, 1.1 (2n = 6) T 14.1 (1.00) 9.2 (0.65) 8.5 (0.60) 7.3 (0.52) 6.1 (0.43) 4.4 (0.31) LIS 1.2 2.7 1.3 1.4 1.7 3.0

D X Al L, S 6.5,5.0 5.2,2.0 2.2, 1.5, 2.7 3.7, 2.5 3.2,2.0 2.8,2.2 [2B chros 1.8, 1.3 (2n = 6 + 2B) T 11.5 (1.00) 7.2 (0.63) - 6.4 (0.56) 6.2 (0.54) 5.2 (0.45) 5.0 (0.43) 3.1 (0.27)1 LIS 1.3 2.6 1.4 1.5 1.6 1.3 1.4 E x Al L, S 9.5,7.7 8.8, 1.5 2.7, 1.7,4.0 5.1,4.0 4.5,2.8 4.2,2.8 (2n = 6) T 17.2 (1.00) 10.3 (0.60) - 8.4 (0.49) 9.1 (0.53) 7.3 (0.42) 7.0 (0.41) ~ LIS 1.2 5.9 1.1 1.3 1.6 1.5 ~ X >- Al E L, S 9.3,7.7 9.0, 1.7 4.0, 1.5, 3.3 4.7, 3.0 4.3,2.5 4.2,2.5 5.0,2.3 4.2,2.7 --l (2n = 8) T 17.0 (1.00) 10.7 (0.63) - 8.8 (0.52) 7.7 (0.45) 6.8 (0.40) 6.7 (0.39) 7.3 (0.43) 6.9 (0.41) >- Z LIS 1.2 5.3 1.2 1.6 1.7 1.7 2.2 1.6 >- mtx:I B X Al L, S 9.3,7.0 8.0, 1.8 2.7, 1.7,4.0 4.7, 3.7 4.2,2.3 3.7,2.7 5.3,2.7 4.2,3.0 (2n = 8) T 16.3 (1.00) 9.8 (0.60) - 8.4 (0.52) 8.4 (0.52) 6.5 (0.40) 6.4 (0.39) 8.0 (0.49) 7.2 (0.44) ?l> r:FJ. LIS 1.3 4.4 1.1 1.3 1.8 1.4 2.0 1.4 r:FJ. B x A3 L,S 7.7,5.7 7.7, 1.7 2.3,2.7, 3.8 - 3.8,2.7 3.0,2.3 3.8, 1.2 4.3, 1.8 3.7,2.0 ~ ::J (2n = 8) T 13.4 (1.00) 9.4 (0.70) 8.8 (0.66) 6.5 (0.49) 5.3 (0.40) 5.0 (0.37) 6.1 (0.46) 5.7 (0.43) ~ I LIS 1.4 4.5 1.3 1.4 1.3 3.2 2.4 1.9 ~ ~ ::J ~ Cytology of Brachyscome lineariloba 129 in one plant in the natural population at Laura Bay, where the D and E cytodemes grow in intimate mixture. The meiotic behaviour of the hybrids is practically identical with that of the pollen parent E. In 291 of 293 first metaphases there were four bivalents and two univalents (Fig. 25). In the remaining two it could not be decided whether either 5 E or 6 E is associated with the 2 D - 2 E pair or just overlies the 2 D - 2 E pair (Fig. 24, diakinesis). At first and second anaphases and in microspore development the behaviour of the univalents conforms with that described by CARTER & al. (1974) for cytodeme E: the univalents divide at the first division, lag at second anaphase, and form accessory nuclei. In a sample of 617 microspores, 46.8% possessed accessory nuclei, and there were 2.4% microcytes. CARTER & al. (1974) gave 45% and 2 - 5%, respectively, for natural cytodeme E plants. It is clear that B. breviscapis D and cytodeme E are very closely related. Where intimately mixed, they form natural hybrids, and gene transfer between the two forms could be sufficient to hold them together through generations. B. lineariloba E x B. breviscapis D. This cross was attempted but no hybrids were obtained. All the progeny examined (50) had the quasi diploid chromosome constitution, 2 n = 10 and were presumably selfs. Hybrids would be expected to have 2 n = 8. The very low pollen production of B. breviscapis was apparently responsible for the failure. Crosses with B. dichromosomatica have been based on two different varieties of this species, var. dichromosomatica Al and var. alba A3. These differ in respect to an interchange between chromosomes 1 and 2 (WATANABE & al. 1975), and in Al x A3 hybrids a single ring-of-four is formed. In all the F I hybrids of B. di­ chromosomatica with cytodemes C, B, E and D, meiotic breakdown leads to very low pollen and seed fertility. B. breviscapis D x B. dichromosomatica var. alba A3 (Figs. 16, 17,26- 31, Table 4.). Crosses ofD x A and A x D are likely to give the most easily analysable meiotic configurations, as compared with crosses involving A and the B. lineariloba cytodemes B, E and C. Five possible hybrid seedlings were examined, and four of these proved to be selfs, with 2 n = 8. One only had the amphihaploid complement, 2 n = 6 = 4 + 2. In growth form and general morphology this plant was inter­ mediate to some degree between the parents, with longer flower scapes, larger capitula, and larger ligulate florets than the maternal parent. Otherwise, in rather closely resembled B. lineariloba E. All six chromosomes can be clearly identified at mitotic metaphase (Figs. 16 - 17). At prophase, the early condensing chromosomes 1 D and 2 Dare conspicuous. At meiosis (Figs. 26 - 31) identification of chromosomes 3 D and 4 D is often difficult, and has been attempted only occasionally. Sometimes it is also difficult to distinguish chromosomes 1 A and 2 A from 3 D and 4 D. The interpretations of the meiotic configurations given in Table 4 were made directly at the microscope, with the advantage of three-dimensional focusing. Pho­ tographs, lacking depth, are more difficult to interpret. Amongst 119 PMCs at first metaphase and at diakinesis, the maximum association seen was in three cells, each with an association of five, and one univalent, the multiple association being of two sorts (Table 4). It is noteworthy that chromosome 1 A has a strong pairing affinity with the small late condensing chromosomes 3 D 130 K. WATANABE & S. SMITH-WHITI::

-40 -.. 1A T

16

3D

1A 1A

18 19

.? 2E 2E F

20 1E

2B ..,'a 2D ...... 1B

15 i;' 22 23

Figs. 15- 23 . Somatic chromosomes at prometaphase and metaphase in hybrids of BfiI­ chyscome.-Fig.15. FI-hybrid B. hreviscapis 0 x B. lineariloha E (2n = 10); note the difference of the short arm lengths between the 2 0 and 2 E chromosomes. - Figs. 16-17. FI-hybrid B. breviscapis 0 x B. dichromosomatica var. alha AJ (2 n = 6); note the different chromosome size and asynchronous condensation. - Figs. 18-19. FI-hybrid B. breviscapis o x B. dichromosomatica var. dichromosomatica AI (2 n = 6 + 2 B); note that the con­ densation behaviour of the B-chromosomes is identical with that of the 1 0 and 20 chromosomes of B. breviscapis D. - Fig. 20. FI-hybrid B. lineariloba E x B. dichromoso­ matica var. dichromosomatica AI.-Fig. 21. FI-hybrid between B. dichrol11osomatica var. dichromosomatica A I x B. lineariloba E (2 n = 8); note the reciprocal difference in chromo­ some number between the E x AI hybrids (Figs. 20- 21). - Figs. 22- 23. FI-hybrid be­ tween B. lineariloba B x B. dichromosol11atico var. dichrol11osol11l1tica A I (2 n = 8); Trabant of the 1 A chromosome. Scale = 10 11m Cytology of Brachyscome lineariloba 131 and 4 D, and that the early condensing chromosome 1 D can pair with 1 A and less often with 2 A, and autosyndetically with one or both of the small chromosomes 3 D and 4 D. Both subterminal chromosomes, 2 A and 2 D show low pairing affinity with all the other chromosomes. The two associations of four illustrated in Figs. 26 and 27, each with a triple chiasma, are different in that the first includes 2 D whereas 2 D is a univalent in the second. From the frequencies of association given in Table 4 the pairing affinities of the six chromosomes is estimated as either strong: =, medium: , or weak: ------, as follows: Strong: 3 D = 1 A = 4 D (i.e. both "chros") Medium: 1 AID chro Weak: 2 A ------chro, 2 A ------1 D, 1 A ------2 D, 2 D ------chro

From this interpretation, the maximum association possible, which was n~t seen, would be 20---11 II 1A====rr-10 ----,-CHRO. II i (40?) CHRO.-----2A (3D?)

From this maximum the two associations of five and all the lesser associations listed in Table 4 can be derived by the failure of particular chiasmata. B. breviscapis D x B. dichromosomatica var. dichromosomatica Al (Figs. 18 -19). The plant of Al used as male parent carried a B-chromosome. Of six possible hybrid seedlings only one proved to be hybrid, the others being maternal selfs. The amphihaploid hybrid possessed six ordinary chromosomes plus two B­ chromosomes. In B. dichromosomatica B-chromosomes are subject to· an accu­ mulation mechanism in pollen development (CARTER & SMITH-WHITE 1972, CARTER 1978 b, SMITH-WHITE & CARTER 1981) which tends to maintain them in local populations. The growth form and general morphology of this hybrid plant was essentially similar to that of the D x A3 hybrid. The full chromosome complement, consisting of 1 D, 2 D, 3 D, 4 D, 1 A, 2 A and the two B-chromosomes can be recognized (Fig. 19). Unlike the ordinary chromosomes 1 A and 2A, these B-chromosomes are totally early condensing (Fig. 19). They are the smallest chromosomes in the complement, and have submedian centromeres. - Meiosis has not been studied in this hybrid. B. lineariloba Ex B. dichromosomatica var. dichromosomatica Al (Figs. 20, 32 - 41, Table 5). 60 plants from this attempted cross were examined. 53 seedlings were maternal selfs, with 10 chromosomes. Seven had six chromosomes, with chromosome 1 A readily identifyable, and were hybrid. None of the seven possessed the quasidiploid univalents. In growth form and general morphology, these hybrid plants resembled E rather than A, but were intermediate to some degree, with longer flower scapes, larger capitula, and larger ligulate florets than E. In fact, they resembled B. lineariloba B rather closely. 132 K. WATANABE & S. SMITH-WHITE: Cytology of Brachyscome lineariloba

Table 4. Meiosis in the hybrid between B. breviscapis D x B. dichromosomatica var. alba A3 (2 n = 6).D refers to the chromosomes of B. breviscapis D, A to those of B. dichromosomatica var. alba A3; chro refers to 3 D or 4 D, where these can not be discriminated in meiotic configurations

Configurations No. of Interpretations Figs. PMCs (%)

1 V + 1 I 2 (1.7) V (chro-I A-I D-2A-chro) + 2D 1 (0.8) V(2DT1 A-I D-chro + 2A) chro lIV + 1 II 2 (1.7) IV CAili hro- 2 A ) + II (1 D;-chro) 26 lIV + 21 7 (5.9) IV (1 A-chro-I D-chro) + 2A + 2D, or IV (chro-I A-chro-1 D) + 2A + 2D 5 (4.2) IV (2A-chro-I A-chro) + 1 D + 2D, or IV (1 A-chro-2A-chro) + 1 D + 2D 4 (3.4) IV(I ATI D-chro) + 2A + 2D 27 chro 2 (1.7) IV (2D-1 A-I D-chro) + 2A + chro 1III+1II+1I 2 (1.7) III (3 D-I A-4D) + II (1 D-2A) + 2D 28 2 (1.7) III (3D-ID-4D) + II (IA-2D) + 2A 1 (0.8) III (1 D-chro-2A) + II (1 A-chro) + 2D 29 1 (0.8) III (1 A-I D-chro) + II (2A-chro) + 2D 1III+3I 34 (28.6) III (3D-1A-4D) + 2A + ID + 2D 12 (10.1) III (1 A-I D-chro) + 2A + 2D + chro 3 (2.5) III (1 A-I D-2A) + 2D + 3 D + 4D 1 (0.8) III (2D-1 A-chro) + 2A + 1 D + chro) 3II 1 (0.8) II (1 A-chro) + II (2 A-I D) + II (2D-chro) 2II + 21 5 (4.2) II (1 A-chro) + II (1 D-chro) + 2A + 2D 30 2 (1.7) II (1 A-chro) + II (1 D-2A) + 2D + chro 31 1 (0.8) II (1 A-chro) + II (2A-chro) + 1 D + 2D 1 II + 41 21 (17.6) II (1 A-chro) + 2A + 1 D + 2D + chro 3 (2.5) II (1 A-I D) + 2A + 2D + 3 D + 4D 1 (0.8) II (1 D-chro) + 1 A + 2A + 2D + chro 61 6 (5.0) Total 119 (99.8)

The four larger chromosomes (Fig. 20) are clearly I E, 2 E, I A and 3 E. In this figure, the trabant of I A has been broken off. In size and condensation pattern chromosomes I A and 2 A are like 3 E and 4 E, and are in part late condensing. Although this hybrid has the amphihaploid number 6, similar to that of the D x A3 hybrid, the interpretation of meiotic configurations is more difficult, as I A, 3 E, 4 E and 2 A are harder to discriminate in meiosis. Chromosome I E is easily distinguishable, and 2 E and I A can be recognized if they appear associated with the nucleolus, or if the secondary constriction of I A is visible. Critical ob­ servations can be made where heteromorphic associations are present. In Fig. 32 there is a ring-like association of I E and I A. The univalent 2 E overlies this, and

3 E, 4 E and 2 A are also univalent. The abbreviated description is In + 41, I E and I A can associate in both arms. In Fig. 33, I E and 2 E are univalents, and the two ?O¥O!~~A 26

?O 1A

2A

20 28 29 30

3E 4E • 2E· t~A 32 33

1E 3E

36 37 38 39 ~ . 40 42 43

Figs. 24- 43 . Meiosis in F,-hybrids of BrachY.I'come. - Figs. 24- 25 . F,-hybrid between B. breviscapi.l' 0 x B. lineariloba E (2 n = 10); - Fig. 24. 4 IT + 2 lor I II[ + 3 [J + I I; either 5 E or 6 E (long arrow) is associated with the 20-2 E pair or just overlies the 20-2 E pair; one free univalent (short arrow) overlies the bivalent 3 D-3 E pair. - Fig. 25 . 4 II + 2 I. - Figs. 26- 31. F,-hybrid between B. hr('vi.l'capi.l' 0 x B. dichromosol1wtim var. alha A3 (2 n = 6). - Fig. 26. I IV + III; - Fig. 27. I IV + 21; - Figs. 28-29. I III + I n + I I; note the chromosome constitution of the trivalent and bivalent, different in the two cells. - Fig. 30. 2 [J + 2 I. - Fig. 31. 211 + 21; N refers to the nucleolus; arrow indicates the small constriction on the long arm of the I D.-Figs. 32- 41. F,-hybrid between B. lineariloba E x B. dichromosomatica var. dichrol11osol11alica A, (2 n = 6). - Fig. 32. [II + 4 I; I A pairs with I E at the both arm ends. - Fig. 33 211 + 2 I. - Fig. 34. 3 II. - Fig. 35. I IV + 21; note the nucleolus (N) and the length of the tightly paired chromosomes ar the one end of the association of four. - Fig. 36. [ IV + I II. Figs. 37-39. Association of five, with one univalent, (I V + [ I); in each association there is one triple chiasma (arrow). - Figs. 40-41. Association of six, with a small chromosome hold in both by a triple chiasmata (arrows). - Fig. 42. F,-hybrid B. dichromosomatica var. dichrol11osol11alica A, x B. lineariloha E (2 n = 8). I VIII. - Fig. 43. F,-hybrid B. liJ1('ariloha B x B. dichrol11o.l'ol11alica var. alha A3 (2 n = 8). 1 VII I. Scale = 10!lm 134 K. WATANABE & S. SMITH-WHITE:

Table 5. Meiosis in the hybrid (2 n = 6) between B. lineariloba E x B. dichromosomatica var. dichro­ mosomatica AI. E refers to the chromosomes of B. lineariloba E, A to those of B. dichromosomatica var. dichromosomatica AI; chro refers to 3 E, 4 E and 2 A, where these can not be discriminated in meiotic configurations

Configurations No. of Interpretations (some examples) Figs. PMCs (%)

1 VI 10 (6.7) VI includes one or two triple chiasmata: 1 A--rchro--rchro-Chro) 40-41 ( 1 E 2E 1 V + II 15 (10.0) V frequently includes one triple chiasma: V(l AT 1 E-chro-chro + 2 E\ 39 chro ) VC A~hro-chro-chro + 2 E) 38 V CA~hro-chro-chro + 2 E) 37 I is frequently 2 E IIV + 1 II 27 (18.0) II are (1 A-I E), (1 E-chro), (2 E-chro) or (chro-chro) IV(l AT1 E-chro) + (chro-2E) 36 chro lIV + 21 10 (6.7) IV [1 E-(1 A or 2 A)-chro-(2 A or 1 A)] + 2 E + chro, or IV [1 E-chro-(1 A or 2A)-chro] + 2E + (2A or 1 A) I are (1 E, 2 E), (1 A, 2 E), (1 E, chro), (1 A, chro), 35 (2 E, chro) or (chro, chro) 2 III 27 (18.0) one of the III frequently involves on triple chiasma 1 III + 1II+ II 30 (20.0) II is (1 A-I E), (1 A-chro), (1 E-chro), (2E-chro) or (chro-chro) 1III+3I 9 (0.6) III either linear or with one triple chiasma 3II 5 (3.3) (1 A-I E) + (2E-chro) + (chro-chro) 34 2II + 21 7 (4.7) II are [(1 A-I E) + (chro-chro)], [(1 A-chro) + 33 (chro-chro)], [(1 E-chro + chro)-(chro)], [(1 A-chro) + (1 E-chro)] or [(1 A-chro) + (2A-chro)] 1 II + 41 9 (6.0) II are (1 A-I E), (1 A-chro), (1 E-chro) or (chro­ chro) (lA-1E)+2A+2E+3E+4E 32 61 1 (0.7)

Total 150 (100.1)

paired associations, which are almost homomorphic, may be 1 A - 3 E and 2 A - 4 E. In Fig. 34 there are three associated pairs, and at least one pair must involve autosyndetic association of two E chromosomes. In Fig. 35, 2 E is a univalent attached to the nucleolus, and 1 E is at one end of an association of four, whilst the other end Qf the association involves chromosomes with interstitial chiasmata. Cytology of Brachyscome lineariloba 135 Since there is no obvious association of (1 A-2 A) (1 E-2 E) or (3 E--4 E) in any of the other configurations studied, this association of four may be 1 E-(1 A or 2 A)-chro-(2 A or 1 A), or 1 E-chro-(1 A or 2A)-chro. In Fig. 36 the triple chiasma may involve 1 A, 1 E and 3 E, and the pair may be 2 E and chro. In Fig. 37-39 triple chiasmata may involve 1 A, 1 E and chro, since the smallest chromosome in each association is clearly shorter than the 3 E of Fig. 36 and may be 4 E. Thus, 1 A and 1 E may be able to form triple chiasmata, alternatively, either with 3 E or 4 E. Associations of six always involved one, and often two triple chiasmata. Where two triples are present, as in Figs. 40 and 41, the one "chro" associated with 2 E, is involved in both. From this interpretation, the maximum association possible, capable of giving all the configurations of Table 5, but not actually seen, would be

CHRO.I --Co1A}- CHRO. CHRO.I (3E?) E (4E?) T (2A?) 2E

B. dichromosomatica var. dichromosomatica Al x B. lineariloba E (Figs. 21, 42, Table 6). Of 50 possible hybrid seedlings examined, 47 were found to be maternal selfs, with 2 n = 4. Three only were hybrids, and all three had eight chromosomes, including the quasidiploid univalents and, recognizable, chromo­ somes 1 E, 2 E, 3 E and 1 A. In growth and general morphology the hybrids re­ sembled the E x A hybrids, despite the presence of the quasidiploid univalents. Meiotic conditions in the three hybrid plants were found to be complex. As the meiotic discrimination of chromosomes 2 A, 3 E, 4 E, 5 E and 6 E is extremely difficult and often impossible, reasonable interpretations of multivalent associations could not be made. Table 6 shows the results of observations and limited attempts at interpretation and analysis. All the chromosomes can be held in one association of eight (Fig. 42), but whether such associations are completely linked by chiasmata is not clear. The hybrid genome must include a large amount of segmental dupli­ cation. Where univalents can be distinguished they can often be identified as 1 E and 2E. B. lineariloba B x B. dichromosomatica var. dichromosomatica Al (Figs. 22 - 23, Table 7). Of 17 possible artificial hybrid seedlings examined, eight were maternal selfs. Nine were amphihaploid hybrids with the somatic chromosome number 8 = 6 + 2. In growth character these hybrids resembled B. lineariloba C, which has the somatic number 16. In the illustrated karyotype of the hybrids (Fig. 22), chromosomes 1 B, 2 Band 1 A are clearly recognizable. The size and condensation behaviour of the A chro­ mosomes is similar to the smaller chromosomes of cytodeme B. At meiotic first metaphase, the pairing behaviour of the chromosomes was found to be similar to that reported for natural hybrids by K YHOS & al. (1977) and for the artificial hybrid Al x E.1t is noteworthy that the artificial hybrid shows a slightly higher association than was reported for the natural hybrid (compare Table 7 with table 1 of K YHOS & al. 1977). 136 K. WATANABE & S. SMITH-WHITE:

Table 6. Meiosis in the hybrid (2 n = 8) between B. dichromosomatica var. dichromosomatica A 1 X B. lineariloba E. chro refers to the chromosomes 1 A, 2 A, 3 E, 4 E, 5 E, 6 E; 1 E or 2 E to the chromosomes 1 or 2 of B. lineariloba E

Configurations No. of Interpretations (No. of PMCs) PMCs (%)

1 VIII 3 (2.3) Fig. 42 1 VII + 11 3 (2.3) I is a medium-sized submedian chromosome IVI+III 12 (9.1) II are (1 E--chro) (7), (2 E--chro) (1), (chro--chro) (1) or undecided (3) 1 VI + 21 4 (3.0) 12 E and chro (4) IV+ 1 III 5 (3.8) IIV + 1 II + 11 8 (6.1) II are (2E--chro) (3), (chro--chro) (2), (1 E--chro) (1) or undecided (2) I is 1 E (3) or cbro (5) 21V 25 (18.9) IIV+ 1 III + 11 14 (10.6) I is chro (7), 2 E (6) or 1 E (1) IIV + 211 31 (23.5) two II are [(IE--chro) + (2E--chro)] (20), [(1 E- chro) + (chro--chro)] (9) or [(2 E--chro + chro--chro)] (2) 1 IV + 1 II + 21 13 (9.8) II are (1 E--chro) (9), (chro-chro) (2) or undecided (2), two I 2 E and chro (7), 1 E and 2 E (2), 1 E and chro (2) or 2 chros (2) 2 III + III 1 (0.8) 2III+21 I (0.8) I is 1 E and chro 1 III + 2II + 11 6 (4.5) two II are (l E + chro) (4) or undecided (2) I is IE (2), 2 E (2) or chro (2) IIII+III+31 I (0.8) 4II 2 (1.5) 3II+21 3 (2.3) two I are 2 E and chro (2) or I E and 2 E (1)

Total 132 (100.1)

B.lineariloba B x B. dichromosomatica var. alba A3 (Fig. 43, Table 8). Of 42 possible hybrid plants, five were found to be amphihaploids, with, somatically, 2 n = 8 = 6 + 2.These hybrids closely resembled the B x Al hybrids. In both mitosis and meiosis, chromosomes 1 B, 2 B, and 1 A, and also the smaller 2 A are recog­ nizable. The high proportion of multiple chromosome associations, in this hybrid (Table 8) as well as in the previous one (Table 7), suggest that there are strong intra~genomic as well as inter-genomic pairing affinities. It is again noteworthy that, where univalents are present, these are most often 1 B, 2 Band 2 A. B. lineariloba C x B. dichromosomatica var. dichromosomatica AI' 27 possible hybrids were examined. One only proved to be hybrid, with the unexpected

chromosome constitution 2 n = 12 = 8 + 2 + 2 of one set of cytodeme CI chro­ mosomes and two sets of Al chromosomes. It possibly resulted from the functioning of an unreduced Al pollen grain. This seedling was lost before maturity. Cytology of Brachyscome lineariloba 137

Table 7). Meiosis in the hybrid (2 n = 8) between B. lineariloba B x B. dichromosomatica var. di­ chromosomatica AI' 1 Band 2 B refer to the chromosomes 1 and 2 of B. lineariloba B; chro to the chromosomes 1 A, 2 A, 3 B, 4 B, 5 B, 6 B

Configurations No. of Interpretations PMCs (%)

IVII+11 2 (0.7) chain VII + 2 B 1 (0.3) chain VII + 1 B 1 VI + 21 4 (1.4) VI (including one triple chiasma and 1 B) + 2 B + chro 1 (0.3) chain VI + 2 B + chro 1 (0.3) VI (including one triple chiasma) + 1 B + 2 B 1 (0.3) chain VI (including 2 B) + 1 B + chro IV+lII1 3 (1.0) V (ring IV-2B) + III (1 B-chro-chro) IV+ 111+ 11 2 (0.7) V (ring IV-chro) + II (1 B-chro) + 2 B 1 (0.3) V (ring IV-2B) + II (1 B-chro) + chro 1 (0.3) chain V (including 1 B) + II (2 B-chro) + chro 1 V + 31 26 (8.8) chain V + 1 B + 2 B + chro 3 (1.0) V (including one triple chiasma) + 1 B + 2 B + chro 1 (0.3) V (ring IV-chro) + 1 B + 2B + chro 1 (0.3) chain V (including 1 B) + 2 B + 2 chros 1 (0.3) chain V (including 2 B) + 1 B + 2 chros IIV+ 1 III + 11 6 (2.0) ring IV + chain III (1 B-chro-2 B) + chro 2 (0.7) ring IV + chain III (1 B-chro-chro) + 2 B 2 (0.7) chain IV + chain III (1 B-chro-chro) + 2 B 1 (0.3) ring IV + chain III (2 B-chro-chro) + 1 B IIV+ 211 12 (4.1) ring IV + II (1 B-chro) + II (2 B-chro) 4 (1.4) chain IV + II (1 B-chro) + II (2 B-chro) lIV+ 111+21 37 (12.6) ring IV + II (1 B-chro) + 2 B + chro 27 (9.2) ring IV + II (2 B-chro) + 1 B + chro 12 (4.1) chain IV + II (1 B-chro) + 2 B + chro 4 (1.4) chain IV + II (2 B-chro) + 1 B + chro 2 (0.7) ring IV + II (chro-chro) + 1 B + 2 B 1 (0.3) chain IV (including 2 B) + 1 II (chro-chro) + 1 B + chro lIV + 41 100 (34.0) ring IV + 1 B + 2 B + 2 chros 26 (8.8) IV (including one triple chiasma) + 1 B + 2 B + 2 chros 1 (0.3) chain IV + 1 B + 2 B + 2 chros 2 III + III 1 (0.3) chain III + III (including one triple chiasma and 2 B) + II (1 B-chro) 2 III + 21 1 (0.3) chain III + chain III (including 1 B) + 2 B + chro lIII+211+11 3 (1.0) chain III + II (chro-chro) + II (2 B-chro) + 1 B lIII+ll1+31 1 (0.3) chain III + II (2 B-chro) + 1 B + 2 chros lII1+51 1 (0.3) chain III + 1 B + 2 B + 3 chros 311+21 1 (0.3) II (1 B-chro) + II (chro-chro) + 1 II (chro-chro) + 2B + chro Total 294 (99.4) 138 K. WATANABE & S. SMITH-WHITE:

Table 8. Meiosis in the hybrid (2 n = 8) between B. lineariloba B x B. dichromosomatica var. alba A3. I Band 2 B refer to the chromosomes I and 2 of B. lineariloba Band 2 A to chromosome 2 of B. dichromosomatica var. alba A3; chro to the chromosomes I A, 2 A, 3 B, 4 B, 5 B, 6 B

Configurations No. of Interpretations (No. of PMCs) PMCs (%)

1 VIII 3 (1.9) Fig. 43 IVII+II 7 (4.5) I 2A (4), I B (3), chro (3), or 2 B (1) VII including one triple chiasma IVI+lII 8 (5.l) 1 VI + 21 5 (3.2) I I Band chro (2),1 Band 2B (1), 2 chro (1), 2A and chro (1) IV+IIII 9 (5.8) IV+ III+ II 11 (7.1) I is 1 B (4), (2B (4), or chro (3) II is (l B-2A)(1), (2B---chro)(1), or undecided (9) 1 V + 31 11(7.1) I are 1 B, 2B and chro (5), 1 B, 2B and chro (4), or 2B, 2A and chro (2) 2IV 13 (8.3) IIV+ 1 III + II 10 (6.4) I chro (5), 2A (2), 1 B (2), or 2B (1) IIV+2II 21 (13.5) two II are [(1 B---chro) + (2 B---chro )](5) and undecided (16) IIV+III+2I 26 (16.7) I are 1 Band 2A (6),1 Band chro (6), 2B and 2A (5), 2B and chro (5), 2 A and chro (2) or 2 chros (2) 1 IV + 41 14 (9.0) I are 1 B, 2B, 2A and chro 2 III + III 6 (3.8) 2III+2I 3 (1.9) I are 1 Band chro (2), undecided (1) IIII+2II+II 3 (1.9) I are 2B (1), 2A (1) or chro (1) lIII+III+3I 1 (0.6) I are 2B, 2A or chro IIII+5I 1 (0.6) I is 1 B, 2 B, 2 A and chro 4II 2 (1.3) 3II+2I 2 (1.3) two I are 2 Band 2 A (1) or 2 Band chro (1) Total 156 (100.0)

Discussion The cytological evidence from the artificial hybrids reported in the present paper reinforces the inference made by K YHOS & al. (1977) that the phyletic relationship of the sequence from B. breviscapis, through B. lineariloba cytodemes E and B, to cytodeme C has been by successive chromosome addition, and does not involve an ordinary polyploid series. The addition chromosomes in B. lineariloba cytodemes E (2 n = 10), B (2 n = 12) and C (2 n = 16) are likely to have been introduced from a B. dichromosomatica­ like source for the following reasons, (1) the additions are in multiples of two, either in haploid or diploid dosage, (2) the added chromosomes have similar con­ densation patterns as have the chromosomes of B. dichromosomatica with respect to early and late condensing chromatin, (3) in hybrids with B. dichromosomatica there is strong pairing affinity between the additional chromosomes and chro­ mosomes 1 A and 2A, (4) amphihaploidy ofD, E or B with Al or A3 only modifies quantitative characters such as plant size, the length of flowering scapes, number of florets per capitulum, and the size of the florets, particularly the ligulate florets. Cytology of Brachyscome lineariloba 139

B.LlNEARILOBA C(2N=16) ENVIRONMENTS t INBREEDING MORE ARID AMPHI-DIPLOIDY MORE OPEN HARSHER

B. LlNEARILOBA B(2N=12) t INBREEDING AMPHI- DIPLOIDY

B. LlNEARILOBA E(2N=IO) t INBREEDING ADOPTION OF QUASIDIPLOID SYSTEM

B. BREVISCAPfS (2 N = 8 ) INBREEDING t DWARF AMPHI- DIPLOIDY I

,aSPECIES ~2N=4~ fe.sPEclEs \T2N"=4")1 ILATE CONDENSING I I EARLY CONDENSING I I CHROMOSOMES I I CHROMOSOMES : L_~~~E!~~__ J l_~~~R~~~~_.J B. DICHROMOSOMATICA B. CAMPYLOCARPA (2N=4,ANCESTOR ?) (2 N = 8, PRECURSOR ?)

Fig. 44. Phyletic scheme for the possible origin of Brachyscome breviscapis D and B. li­ neariloba E, B, C. B. breviscapis may be of amphidiploid origin between a species with two large, early condensing chromosome pairs and another with two small, late condensing chromosome pairs. B. campylocarpa has totally early condensing chromosomes, but 2 n = 8; it may close to the latter precursor. Ascending dysploidy is caused by successive hybridi­ zations with the B. dichromosomatica-like precursor (A) species with two small, late con­ densing chromosomes. B. lineariloba E is assumed to be derived from D x (D x A) hybridization and the adoption of a quasidiploid system. B. lineariloba B is assumed to be derived from amphi-diploidy after E x A [i.e., 4 (~) + 2 (6') = 6] hybridization. This implies that the two univalents of cytodeme E do not contribute to the origin of cytodeme B. The morphological similarities between chromosomes 5 E, 6 E and 6 B are due to the similar sources of additional chromosomes for cytodemes E and B. B. lineariloba C is assumed to be derived from amphidiploidy after B x A hybridization

The fruit, a cypsela, is similar in all the species and cytodemes, and is distinctive from that of all other species of the genus. This addition sequence has complications, and it is not suggested that it has involved the presently known forms of B. dichromosomatica. The apparent increase in drought resistance or water stress tolerance, from B. breviscapis to cytodeme C of B. lineariloba is certainly associated with an increase in chromosome number, but is not clearly due to that increase, and the change from compulsive outbreeding, characteristic of B. dichromosomatica, to permissive inbreeding must be due to selection acting on the breeding system. Considering present day geographical distributions, the known races of B. di- 140 K. WATANABE & S. SMITH-WHITE:

chromosomatica could not have been involved in hybridization with B. breviscapis. The obvious occurrence of intragenomic association in amphihaploid hybrids with B. dichromosomatica indicates that the additional chromosomes in the B. lineariloba cytodemes E, Band C have suffered interchanges or other structural alterations either before or after their addition. B. dichromosomatica obviously has been in­ volved in a great deal of karyotypic change and genome reorganization after this addition phenomenon. The C-banding segments present in chromosomes I A and 2 A of B. dichromosomatica Al from Wild Dog Glen might well have been lost in genomic reorganization, either before or after addition. Progress in our present understanding of the group beyond the phyletic scheme proposed by K YHOS & al. (1977) is illustratred in Fig. 44. It is suggested that B. breviscapis had an amphidiploid origin between two species, each with n = 2, one with two large early condensing chromosome pairs and the other, like B. dichro­ mosomatica, with two smaller, largely late condensing chromosome pairs. A com­ parable cytological condition is realized in the artificial hybrid between B. dichro­ mosomatica and B. campylocarpa cytodeme A. The latter is closely related to B. dichromosomatica (DAVIS 1948) but has four pairs of chromosomes, all showing early condensation (WATANABE & al. 1976). A combination of these two kinds of genomes, with chromosomal and genetical changes, could have been the source of B. breviscapis. The present day B. breviscapis is extremely dwarfed, which may be a response to severe selection in exposed coastal situations, largely on poor limestone soils. This extreme dwarfness is paralleled by a form of B. goniocarpa growing in the same situations, which is extremely dwarf, in contrast with taller forms found under better conditions (SMITH-WHITE & al. 1970). The inbreeding system, developed under severe selection by B. breviscapis with small inconspicuous flower, has been maintained through the cytodemes E, Band C of B. lineariloba. The inbreeding system may have played an important role in the development of it colonizing potential, whilst the restriction of its genetic recombination system due to inbreeding may have been partly compensated by the increase in chromosome number and considerable genetic duplication in the de­ rivative species. The development of the system apparently has permitted the fine adjustment of opposing regulatory factors as postulated by GRANT (1958). This research constitutes part of a major project in the study of chromosome evolution in the Australian Eremean flora, and has been financially supported by the Australian Research Grants Committee.

References CARTER, C. R., 1978 a: Taxonomy of the Brachycome lineariloba complex (Asteraceae). - Telopea 1, 387 - 393. 1978 b: The cytology of Brachycome 8. The inheritance, frequency and distribution of B chromosomes in B. dichromosomatica (n = 2), formerly included in B. lineariloba. - Chromosoma (Berlin) 67, 109 -121. SMITH-WHITE, S., 1972: The cytology of Brachycome lineariloba. 3. Accessory chro­ mosomes. - Chromo soma (Berlin) 39, 361- 379. - KVHOS, D. W., 1974: The cytology of Brachycome lineariloba. 4. The ten chromosome quasidiploid. - Chromosoma (Berlin) 44, 439 - 456. DAVIS, G. L., 1948: A revision of the genus Brachycome. - Proc. Linn. Soc. New South Wales 73,142-241. Cytology ,of Brachyscome lineariloba 141

GRANT, V., 1958: The regulation of recombination in plants. - Cold Spring Harb. Symp. Quant. BioI. 23, 337 - 363. KYHOS, D. W., CARTER, C. R., SMITH-WHITE, S., 1977: The cytology of Brachycome lineariloba 7. Meiosis in natural hybrid and race relationships. - Chromosoma (Berlin) 65,81-101. NOGUCHI, J., TANAKA, R., 1981: C-banding after Aceto-orcein staining for plant chro­ mosomes. - Japan J. Genet. 56, 529 - 532. SMITH-WHITE, S., CARTER, C. R., 1970: The cytology of Brachycome lineariloba 2. The chromosome species and their relationships. - Chromosoma (Berlin) 30, 129 - 153. - 1981: The maintenance of B chromosomes in Brachycome dichromosomatica. - In ATCHLEY, W. R., WOODRUFF, D., (Eds.): Evolution and Speciation. Essays in honor of M. J. D. WHITE, pp. 335 - 355. - Cambridge, London, New York, New Rochelle, Melbourne, Sydney: Cambridge University Press. WATANABE, K., CARTER, C. R., SMITH-WHITE, S., 1975: The cytology of Brachycome lineariloba 5. Chromosome relationships and phylogeny of the race A cytodemes (n = 2). - Chromosoma (Berlin) 52, 389 - 397. -- 1976: The cytology of Brachycome lineariloba 6. Asynchronous chromosome condensation and meiotic behaviour in B. lineariloba A (n = 2) x B. compylocarpa A (n = 4). - Chromosoma (Berlin) 57,319-331. -- 1985: The cytology of Brachycome lineariloba 9. Chromosomal heterogeneity in natural populations of cytodeme C (2 n = 16). - Canad. J. Genet. CytoI. 27, 410 - 420. Addresses of the authors: K. WATANABE, Biological Institute, Faculty of General Ed- ucation, Kobe University, Kobe, 657, Japan. - S. SMITH-WHITE, 25 Robin Ave, Turramura, N. S. W., 2074, Australia.

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