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Polyploidy and Evolution in Wild and CultivatedDahliaSpecies
Annals of Botany 81 : 647–656, 1998
Polyploidy and Evolution in Wild and Cultivated Dahlia Species M E L A N I E G A T T*, H O N G D I N G*, K E I T H H A M M E T T† and B R I A N M U R R AY*‡ * School of Biological Sciences, The Uniersity of Auckland, Priate Bag 92019, Auckland, New Zealand and † 488C Don Buck Road, Massey, Auckland 8, New Zealand Received : 4 November 1997
Accepted : 29 January 1998
Observations on the chromosomes of nine species of Dahlia Cav. (Asteraceae, Heliantheae—Coreopsidinae) show that some have 2n ¯ 32, others 2n ¯ 64, with a third group having both chromosome numbers in the same taxon. Karyotype investigations showed that the chromosomes can be divided into groups of 14 metacentrics plus two submetacentrics per set of 16 chromosomes. In situ hybridization using an rRNA gene probe indicated that the 2n ¯ 32 species have eight hybridization sites whilst the 2n ¯ 64 species have 16 sites. Silver nitrate staining of these regions showed that not all of these nucleolar organizers are active. Meiotic analysis at metaphase I and pachytene, by synaptonemal complex spreading, shows that the 2n ¯ 32 species have exclusive bivalent formation whereas the 2n ¯ 64 species have small numbers of univalents plus quadrivalents in addition to bivalents. This study proposes that Dahlia species with 2n ¯ 32 are allotetraploids whereas those species and chromosome races with 2n ¯ 64 are their autopolyploid derivatives. We suggest that a bivalent-promoting mechanism in the 2n ¯ 32 species may account for their meiotic behaviour as their component genomes appear so similar, and that this mechanism is also responsible for the low number of quadrivalents in the 2n ¯ 64 taxa. # 1998 Annals of Botany Company Key words : Chromosome pairing, Dahlia, in situ hybridization, karyotype analysis, polyploidy, synaptonemal complex analysis.
INTRODUCTION The garden dahlia (Dahlia ariabilis hort. non (Willd.) Desf.) is a relatively new taxon which is believed to have arisen following the introduction into Spain from Central America of wild species of Dahlia in 1789. With the exception of the garden dahlia, Dahlia species can be divided into two groups depending on flower colour : Group I with magenta or ivory flowers and Group II with orangescarlet or yellow flowers (Lawrence, 1929, 1931 a ; Lawrence and Scott-Moncrieff, 1935). Since the garden dahlia combines both colour series, these authors concluded that the garden dahlia, with 2n ¯ 64, was a hybrid derived from the crossing of two species (2n ¯ 32), one belonging to Group I, the other to Group II, followed by chromosome doubling. At that time the 2n ¯ 64 race of D. coccinea was unknown, all their material having 2n ¯ 32, and they did not have material of D. pinnata. To date, the exact origin of the garden dahlia remains unclear although the most widely accepted idea is that it is the product of hybridization between D. pinnata Cav. (flower colour Group I) and D. coccinea Cav. (flower colour Group II) (Sorensen, 1969). Early observations of the chromosomes of D. coccinea showed 2n ¯ 32 (as D. coronata in Ishikawa, 1911 ; Lawrence, 1929). However, subsequent studies have found an additional chromosome race with 2n ¯ 64 (Turner, Ellison and King, 1961). These chromosome races have not
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been segregated into separate taxa. D. pinnata has a chromosome number of 2n ¯ 64 (Sorensen, 1969), and garden dahlias all have 2n ¯ 64. Several lines of evidence summarized by Lawrence (1970) led him to propose that the 2n ¯ 32 plants were tetraploids and that consequently the 2n ¯ 64 plants must be octoploids. The first of these was that the basic number of x ¯ 16 in Dahlia is very high for the Asteraceae. More recent studies have confirmed x ¯ 9 as the most common number in the family, being found in 21 % of all taxa examined (Solbrig, 1977). Secondly, four factors were identified which governed flower colour in D. ariabilis. Each of these four factors is tetrasomic and gives the characteristic ratios arising from the random pairing of four homologous chromosomes (Lawrence and Scott-Moncrieff, 1935). Thirdly, at diakinesis and metaphase I there were secondary associations of a non-chiasmate nature (Darlington, 1928) between the chromosomes (Lawrence 1929, 1931 b). Lawrence (1929, 1931 a) and Lawrence and Scott-Moncrieff (1935) ultimately proposed that the garden dahlia was an autoallopolyploid, an octoploid that contains two different autotetraploid genomes. They also proposed that species with 2n ¯ 32 are allotetraploids which have descended from diploid ancestral forms, and that during the evolution of these tetraploid species differentiation occurred giving rise to the two flowercolour groups (Lawrence, 1931 a). Darlington (1973) also suggested that the wild species from which D. ariabilis (2n ¯ 64) arose are allotetraploids (2n ¯ 32) and that there are no known extant diploid (2n ¯ 16) species. To date, no Dahlia species has been found with 2n ¯ 16. Lawrence (1970) suggested that a probable explanation is that the
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# Annals of Botany Company
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Gatt et al.—Polyploidy in Dahlia T 1. Plant species used in this study and their origin Species
Chromosome number
Source
Origin
D. D. D. D. D. D.
apiculata HJ7343 australis HJ7250 australis HJ7346 coccinea HJ7310 coccinea HJ7357 coccinea IB52
2n ¯ 32 2n ¯ 32 2n ¯ 64 2n ¯ 32 2n ¯ 32 2n ¯ 32
Hjerting}Hansen, GIC Hjerting}Hansen, GIC Hjerting, wild collection Hjerting, wild collection Hjerting, wild collection Hjerting}Hansen, GIC
D. D. D. D. D. D. D. D. D. D. D. D.
coccinea HJ7000 coccinea HJ7337 coccinea HJ7382 coccinea HJ7128 coccinea HJ7051 imperialis HJ7319 pinnata Sor6519 rudis HJ7352 sherffii HJ7266 sherffii Spooner tenuicaulis G&K5090 ariabilis ‘ Cardas ’
2n ¯ 32 2n ¯ 64 2n ¯ 64 2n ¯ 64 2n ¯ 64 2n ¯ 32 2n ¯ 64 2n ¯ 32 2n ¯ 32 2n ¯ 64 2n ¯ 32 2n ¯ 64
D. D. D. D. D. D.
ariabilis ariabilis ariabilis ariabilis ariabilis ariabilis
2n ¯ 64 2n ¯ 64 2n ¯ 64 2n ¯ 64 2n ¯ 64 2n ¯ 64
Hjerting}Hansen, GIC Hjerting, wild collection Hjerting, wild collection Hjerting}Hansen, GIC Hjerting, wild collection Hjerting, wild collection Hjerting}Hansen, GIC Hjerting, wild collection Hjerting}Hansen, GIC Spooner, GIC Knees, wild collection Commercial cultivar, topmix type from Czechoslovakia Commercial cultivar Commercial cultivar Commercial cultivar Commercial cultivar Bonne Esperance¬Park Princess Commercial cultivar
‘ De 12 ’ ‘ Fern Irene ’ ‘ Pineholt Princess ’ ‘ Bishop of Llandaff ’ ‘ 45}1 ’ ‘ Elizabeth Hammett ’
Puebla State, Mexico. Alt 2250 m Oaxaco State, Mexico. Alt. 2300 m Puebla State, Mexico. Alt. 2500 m Mexico State, near Coatepec, Mexico. Alt. 2200 m Mexico State, Mexico. Alt. 1860 m Federal District, Mexico, via the Mexican Institute of Biology. Alt. unknown Durango State, Mexico. Alt. 2490 m Chiapas State, Mexico. Alt. 2200 m San Luis State, Mexico. Alt. 1600 m Morelos State, Mexico, Alt. 2600 m Guerrero State, Mexico. Alt. 2400 m Oaxaco State, Mexico. Alt. 2520 m Hybrid of intraspecific cross Mexico State, Mexico. Alt. 2700 m Chihuahua State, Mexico. Alt. 2870 m Chihuahua State, Mexico. Alt. unknown Mexico
HJ, Hjerting, J. P. ; IB, Mexican Institute of Biology ; G&K, Knees, S ; Spooner, Spooner, D ; GIC, grown in cultivation or possibly of hybridization.
original ancestors of today’s Dahlia species were diploids which became extinct because of competition with the more successful tetraploids derived from them. With the development of surface spreading techniques for the observation of synaptonemal complexes (SCs) in plants (Albini, Jones and Wallace, 1984), it is now possible to analyse pachytene pairing patterns in species with relatively high chromosome numbers. At pachytene, if we are dealing with autotetraploids, we would expect to observe 66 % of the chromosomes associated as quadrivalents if there are two autonomous terminal pairing initiation sites per chromosome (Sved, 1966). At metaphase I, multivalents should be observed at frequencies predicted by models based on random pairing of homologous arms (Jackson and Hauber, 1982). However, recent work on autotetraploid Crepis capillaris has shown that the frequency of pachytene quadrivalents can reach 96 % for specific tetrasomes, and that this is correlated with a correspondingly high frequency of pairing partner switches (Jones and Vincent, 1994). The converse, with fewer pachytene quadrivalents than expected, has been correlated with genome size}SC length (Jones and Vincent, 1994) and may reflect a low number of autonomous pairing sites. In addition, as Jones and Vincent (1994) also point out, a low quadrivalent frequency could also indicate that there is preferential pairing of homologues or homoeologues. As part of a larger study on wild species and cultivars of Dahlia, we have examined their karyotypes and pachytene and metaphase pairing to investigate the nature of polyploidy in Dahlia.
MATERIALS AND METHODS The plant species used in this study are listed in Table 1. Karyotypes were constructed from root tip cells at metaphase. The root tips were pretreated with 8-hydroxyquinoline for 2 h at room temperature in the dark followed by 2 h at 4 °C then fixed in ethanol : acetic acid (3 : 1). Chromosome spreads were prepared as follows : root tips were rehydrated in citrate buffer for at least 2¬15 min, the terminal 3 mm of the root tip was removed and placed on a slide in 20 µl of enzyme mix (400 units ml−" cellulase, ‘ Onozuka R-10 ’ Serva, 30 units ml−" pectolyase, Sigma P3026, in distilled water) and incubated in a moisture chamber for 1–1±5 h at 37 °C. Excess enzyme was carefully removed with filter paper and a drop of water added. This was removed with filter paper and another drop of water added, left for 5 min, then removed. A drop of fixative, ethanol : acetic acid (3 : 1), was added and the digested root tip gently stirred with a brass rod to separate cells. Slides were left to air dry, then stored under desiccation at room temperature until required. Slides used for in situ hybridization were 1–2 weeks old. For silver staining, the method outlined in Murray, Bennett and Hammett (1992) was followed. In situ hybridization followed the protocol in Leitch et al. (1994). Slides with good metaphase spreads were pretreated with DNase-free RNase (100 µg ml−"), then pepsin (5 µg ml−") and refixed with paraformaldehyde (4 %). The probe, pTIP6, containing the 26S rRNA gene from asparagus (King and Davies, 1992), was labelled with digoxigenin by
Gatt et al.—Polyploidy in Dahlia
649
A
B
F. 1. Karyotypes of D. coccinea HJ 7357 (2n ¯ 32) (A) and D. ariabilis ‘ Pineholt Princess ’ (2n ¯ 64) (B). Bar ¯ 10 µm.
F. 2. Somatic chromosomes of D. tenuicaulis (2n ¯ 32) (A) and D. australis (2n ¯ 64) (B) after in situ hybridization with the rRNA gene probe. Bar ¯ 10 µm.
nick translation following manufacturer’s instructions (Boehringer Mannheim). Digoxigenin-labelled probes were detected with a HNPP fluorescence detection kit (Boehringer
Mannheim) and the slides observed with a Zeiss epifluorescence microscope and filter set 02. For meiotic metaphase I (MI) analysis, immature capitula
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Gatt et al.—Polyploidy in Dahlia were fixed in ethanol : chloroform : glacial acetic acid (6 : 3 : 1) and squashed in orcein. Ten to thirty cells at metaphase I were analysed for each taxon. The SC spreading technique for pachytene analysis was that of Loidl (1989), with minor modification to suit the material. Observations were made using a Phillips CM12 transmission electron microscope and suitable cells photographed using Copex PET 10 film. Five cells at pachytene were analysed in detail for D. coccinea (2n ¯ 32 and 2n ¯ 64), D. pinnata (2n ¯ 64), D. australis (2n ¯ 64) and D. ariabilis (2n ¯ 64). Measurements of SC lengths were made from photographs of known magnification using a Summagraphics FX digitizer tablet connected to a PowerMac 6100}60 computer. RESULTS
F. 3. A silver nitrate stained mitotic metaphase of D. australis (2n ¯ 32). Arrows indicate active NOR sites. Bar ¯ 10 µm.
Karyotype analysis shows that the chromosomes of these Dahlia species are relatively small, from 2–3±5 µm in D. coccinea HJ7357 and 1±5–3 µm in D. ariabilis ‘ Pineholt Princess ’ (Fig. 1). The chromosomes can be divided into two groups : 14 metacentrics plus two submetacentrics per set of 16 chromosomes, but it is very difficult to distinguish many different chromosomes within these groups. This
T . 2. Mean meiotic metaphase I chromosome configurations in Dahlia species Mean number of configurations per cell* Species
Number of cells
D. apiculata HJ7343 2n ¯ 32 D. australis HJ7250 2n ¯ 32 D. australis HJ7346 2n ¯ 64
20 17 16
HJ7310 2n ¯ 32 IB52 2n ¯ 32 HJ7000 2n ¯ 32 HJ7337 2n ¯ 64
20 15 18 15
D. coccinea HJ7382 2n ¯ 64
14
D. coccinea HJ7128 2n ¯ 64
12
D. coccinea HJ7051 2n ¯ 64
11
D. imperialis HJ7319 2n ¯ 32 D. pinnata Sor6519 2n ¯ 64
20 17
D. rudis HJ7352 2n ¯ 32 D. sherffii HJ7266 2n ¯ 32 D. sherffii Spooner 2n ¯ 64
20 30 11
D. tenuicaulis G&K5090 2n ¯ 32 D. ariabilis ‘ Cardas ’ 2n ¯ 64
20 10
D. ariabilis ‘ De12 ’ 2n ¯ 64
14
D. ariabilis ‘ Fern Irene ’ 2n ¯ 64
12
D. ariabilis ‘ Bishop of Llandaff ’ 2n ¯ 64
10
D. D. D. D.
coccinea coccinea coccinea coccinea
I
cII
oII
cIV
oIV
0 0 1 0† 0 0 0 0±92 0 0 0 0±2 0 1 0 0 0±5 0 0 0±06 0±4 0 0 0 0 0 0 0±5 0 0±4 0
12±7 9±8 23±43 21±7 10±3 10±4 8±4 25±3 24±1 24 21±2 21±6 18±2 15 11±7 6±4 22±11 19±3 5 1±37 3±3 1±6 3±4 22±3 19±2 24±5 21±9 23±08 20±6 21 18±1
3±3 6±2 7±81 2±5 5±7 5±6 7±6 5±7 1±9 6 2±6 8±1 3±4 13±3 5±3 9±6 7±8 3±1 11 14±6 25±5 9±4 12±6 8±7 3±2 6±1 2±5 7±17 2±8 9±8 3±5
0 0 0 2±8 0 0 0 0 2±2 0±1 2±9 0 3±5 0±2 4±3 0 0±1 3±3 0 0 0 2±1 0 0 3±3 0 2±7 0±08 3±0 0 3±5
0 0 0±13 1±1 0 0 0 0±27 0±8 0±9 1±2 1±1 1±7 1±4 3±2 0 0±82 1±5 0 0 1±5 8±4 0 0±5 1±5 0±7 1±1 0±67 1±3 0±5 1±7
* I, Univalents ; cII, chain bivalents ; oII, ring bivalents ; cIV, chain quadrivalents ; oIV, ring quadrivalents. † Values in italics are expected values derived from the formulae of Jackson and Hauber (1982).
Mean chiasma frequency per cell 19±3 22±2 39±6 21±8 21±6 23±6 37±7 39±9 42±3 47±9 25±6 41±4 27 30±6 60±3 28±7 41±7 39±5 40±4 42±6
Gatt et al.—Polyploidy in Dahlia
F. 4. Meiotic metaphase I in D. coccinea (2n ¯ 32) (A) and D. australis (2n ¯ 64) (B). Bar ¯ 10 µm.
F. 5. Light micrograph of a surface-spread synaptonemal complex of D. australis (2n ¯ 64) showing the diffuse spherical structures associated with the centromeres and the association of these structures at pachytene (arrow). Bar ¯ 10 µm.
basic karyotype was seen in all species with 2n ¯ 32 (D. coccinea, D. sherffii, D. australis, D. rudis and D. tenuicaulis) and also in those with 2n ¯ 64 (D. coccinea, D. sherffii, D. australis, D. pinnata and D. ariabilis). No variation in karyotype was observed in three different accessions of D. coccinea (2n ¯ 32). The metacentric chromosomes can be grouped into fours in the 2n ¯ 32 taxa, but this is not
651
possible for the submetacentric chromosomes which are morphologically quite distinct. Alternatively, the chromosomes can all be arranged in pairs in the 2n ¯ 32 taxa. However, in either arrangement it would appear that there is considerable similarity between the component genomes of these species. In all the 2n ¯ 64 taxa, similar arguments can be made for arranging the karyotype into comparable groups of eight or four chromosomes. Chromosomes with secondary constrictions or nucleolar organizer regions (NORs) provided the most useful markers for chromosome identification, but specific pairs of NOR chromosomes could not be distinguished. Using a combination of silver nitrate staining and in situ hybridization with an rRNA gene probe, a maximum of eight NORs} hybridization sites could be identified in D. coccinea (2n ¯ 32), D. australis (2n ¯ 32), D. sherffii (2n ¯ 32), D. tenuicaulis (2n ¯ 32) (Fig. 2 A) and D. rudis (2n ¯ 32), and 16 sites were seen in D. coccinea (2n ¯ 64), D. australis (2n ¯ 64) (Fig. 2 B), D. sherffii (2n ¯ 64), D. pinnata (2n ¯ 64) and D. ariabilis ‘ Elizabeth Hammett ’. Although the NOR chromosomes have a secondary constriction, hybridization sites appeared to be located at the end of the chromosome arm, as the small terminal region beyond the secondary constriction was not observed. Silver nitrate staining did not identify the maximum number of NOR chromosomes in all species. Deposition of the silver occurred at the secondary constriction of chromosomes although it was often difficult to see the terminal region of the chromosome due to its small size. The NOR chromosomes identified by in situ hybridization in D. tenuicaulis could be divided into two groups : two pairs of chromosomes with rRNA sites were larger than the other two pairs. This pattern was also observed in silver-stained karyotypes of D. australis (2n ¯ 32) (Fig. 3), D. coccinea (2n ¯ 32) and D. rudis (2n ¯ 32). Furthermore, this pattern was also observed in D. ariabilis ‘ Pineholt Princess ’ with eight chromosomes occurring in each group instead of four. In addition to staining at the NORs, silver stained bands were also observed at the centromeres and telomeres of all chromosomes in D. australis (2n ¯ 32) (Fig. 3), D. coccinea (2n ¯ 32 and 2n ¯ 64), D. rudis (2n ¯ 32), D. sherffii (2n ¯ 64) and D. ariabilis ‘ Pineholt Princess ’ (2n ¯ 64), and consequently were not useful in the differentiation of specific chromosome pairs. At this stage it is not realistic to draw detailed conclusions about karyotypic relationships between the species of the same ploidy level, but karyotype observations do suggest that the 2n ¯ 64 species and races have the doubled karyotype of 2n ¯ 32 species. Analysis of the MI pairing patterns of nine species are given in Table 2. No univalents were observed in most 2n ¯ 32 taxa and they regularly formed a mixture of rod and ring bivalents in all cells (Fig. 4 A). At the 2n ¯ 32 level, the mean number of chiasmata per bivalent was very similar in D. coccinea, D. australis and D. apiculata and ranged from 1±2 to 1±5, whereas the mean number of chiasmata per bivalent was higher in D. rudis, D. imperialis, D. sherffii and D. tenuicaulis and ranged from 1±6 to 1±9. Many of the 2n ¯ 64 taxa formed small numbers of univalents and all formed some quadrivalents in addition to rod and ring bivalents (Fig. 4 B). The mean number of chiasmata per bivalent in
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Gatt et al.—Polyploidy in Dahlia
F. 6. Electron micrograph of a surface-spread synaptonemal complex of D. pinnata (2n ¯ 64). Bar ¯ 10 µm.
this group was very similar in most taxa ranging from 1±2 to 1±5 with the exception of D. sherffii, which had a mean number of chiasmata per bivalent of 1±9. Using the model developed by Jackson and Hauber (1982), based on the random pairing of chromosome arms in autotetraploids, it is clear that none of the 2n ¯ 64 species give an acceptable fit (Table 2). In all cases there is a large deficit in the number of quadrivalents and an excess in the number of bivalents. At pachytene, SCs were readily observed under both light and electron microscopes (Figs 5, 6 and 7). A single nucleolus was seen in most cells analysed and up to four pairs of chromosomes appeared to be associated with it in some cells. The preservation of centromeres was variable but appeared to be species-specific as they were seen only in D. australis and D. pinnata. Under the light microscope they appeared as darkly-staining granular regions roughly 1–2 µm in diameter associated with the SC (Fig. 5). In electron micrographs, two slightly different forms of centromere were observed. The first appears as a thickening
of the lateral elements associated with small amounts of fibrillar material (Fig. 8 A) whereas the second is a spherical to elongated, diffuse structure 0±7–2±6 µm in diameter (Fig. 8 B). An interesting feature of these cells was the frequent aggregation of the centromere structures into two to four clusters (Figs 6 and 8 C). In many cells the telomeres were clustered and were characterized by the association of a darkly-staining, more or less spherical granule 0±15–0±2 µm in diameter associated with the ends of the lateral elements (Fig. 6). Table 3 gives the results of an analysis of a sample of cells from the five taxa. In D. coccinea (2n ¯ 32), 16 bivalents were seen with no evidence of multivalents in any of the cells examined. The 2n ¯ 64 race of D. coccinea and the other three 2n ¯ 64 taxa all formed quadrivalents at low frequencies, (five being the maximum number seen), in addition to bivalents (Table 3). Only a single pairing partner exchange was seen in these quadrivalents and in all cases the mean frequency of pachytene quadrivalents was higher than at MI. There appear to be large differences in lateral element
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653
F. 7. Electron micrograph of a surface-spread synaptonemal complex of D. coccinea (2n ¯ 64). Bar ¯ 10 µm.
length between the species : D. pinnata, for example, has lateral elements that are twice as long as those of D. coccinea. However, because only five cells per species were analysed in detail and the staging of the cells (within pachytene) is not possible, the significance of these length variations must be treated with caution. DISCUSSION There has been uncertainty over the ploidy status of Dahlia species. Lawrence (1929) suggested that the 2n ¯ 32 species were allotetraploids, arising from hybrids between nowextinct diploids with 2n ¯ 16. Furthermore, he proposed that the garden dahlia (D. ariabilis) with 2n ¯ 64 was also a hybrid that combined the genomes of two species, one from each of his Groups I and II, that subsequently underwent polyploidization. Lawrence (1970) did not specify whether the species in these two groups (I and II) were genomically differentiated, but the fact that he calls the
2n ¯ 64 garden dahlia an autoallopolyploid, rather than an allooctoploid, would imply that he thought not. Sorensen (1969), on the other hand, proposed 2n ¯ 32 species were diploid and 2n ¯ 64 species tetraploid. With both these propositions, we would expect to see groups of four homologous chromosomes in the karyotypes and the frequent association of chromosomes as quadrivalents at pachytene and metaphase I of meiosis in the 64-chromosome plants. Although the present study has been unable to unambiguously confirm the ploidy status of Dahlia species, we suggest that the 2n ¯ 32 taxa are not diploids. The karyotypes of the 2n ¯ 32 species show that all the chromosomes cannot be grouped in fours. This is particularly evident with the submetacentric members of the complement, thus suggesting that they are allotetraploids. The karyotypes of the 2n ¯ 64 taxa appear to be double those of the 2n ¯ 32 taxa, suggesting that they are their autoploid derivatives. The high basic chromosome number coupled with the large number of NOR chromosomes also
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Gatt et al.—Polyploidy in Dahlia T 3. Lateral element length (µm), extent (%) of pairing and the numbers of quadrialents and bialents in fie Dahlia taxa
Species D. coccinea HJ7310 2n ¯ 32 Mean D. coccinea HJ7337 2n ¯ 64 Mean D. pinnata Sor6519 2n ¯ 64 Mean D. ariabilis ‘ Cardas45}1 ’ 2n ¯ 64 Mean D. australis HJ7346 2n ¯ 64 Mean
F. 8. Electron micrographs of surface-spread synaptonemal complexes of Dahlia showing the thickened structures associated with centromeres in D. australis (2n ¯ 64) (A), the diffuse centromere associated structures in D. pinnata (2n ¯ 64) (B) and the association of centromeres in D. pinnata (2n ¯ 64) (C). Bar ¯ 5 µm.
suggests that the 2n ¯ 32 chromosome plants are not diploid. Although the development of SC spreading techniques has greatly increased our ability to observe the patterns of chromosome pairing at pachytene, particularly for plants such as Dahlia with high chromosome numbers, the analysis of chromosome pairing at pachytene and metaphase I does little to immediately clarify the origin and evolution of Dahlia species. The 2n ¯ 32 D. coccinea shows exclusive bivalent formation from which we can conclude, equally validly, that it is either a diploid or an allotetraploid. The 2n ¯ 64 taxa, including the polyploid races of D. coccinea, D. australis and D. sherffii, are all characterized by low frequencies of quadrivalents at both pachytene and MI.
Cell 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3
Lateral element length 406±9 364±8 358±4 315±3 296±8 355±6 933±6 894±3 879±0 875±0 590±6 834±5 1886±5 1760±3 1711±8 1584±57 1425±6 1673±8 1081±7 991±4 950±6 769±0 483±0 855±1 1230±8 977±5 906±8 1038±4
% pairing No. IV No. II 95±1 82±2 97±9 93±1 97±9 93±2 98±4 98±7 92±2 99±4 71±4 92±0 99±6 100±0 98±1 98±1 99±3 99±0 99±3 98±9 100±0 71±0 80±6 90±0 98±9 76±5 87±6 87±7
0 0 0 0 0 0±0 5 2 3 2 0 2±4 2 0 2 2 2 1±6 3 3 0 3 0 1±8 2 3 3 2±7
16 16 16 16 16 16±0 22 28 26 28 32 27±2 28 32 28 28 28 28±8 26 26 32 26 32 28±4 28 26 26 26±7
They do not fit the expected pachytene quadrivalent frequency of 66±6 % for autotetraploids (Sved, 1966) nor do they fit the random pairing models of Jackson and Hauber (1982). At face value, this chromosome behaviour lends little support to the proposal that D. ariabilis is the autoploid derivative of a hybrid between parents from Group I and II (Lawrence, 1970 ; Darlington, 1973). It is also surprising that the 2n ¯ 64 races of D. coccinea, D. australis and D. sherffii, which are morphologically so similar to their 2n ¯ 32 races and therefore appear autoploid in origin, also form quadrivalents at very low frequencies. If the 2n ¯ 32 plants are allotetraploids, as Lawrence (1970) proposes, then their doubled derivatives with 64 chromosomes would be expected to form numerous quadrivalents. There are two possible explanations for the diploid-like behaviour of the 2n ¯ 32 plants. One requires the now extinct parental diploids (2n ¯ 16) to have had sufficiently distinct genomes so that when hybrids occurred there was no intergenomic pairing. Karyotype observations presented here suggest that this is not the case since there is so much similarity within the 2n ¯ 32 genomes. An alternative hypothesis is that they have a bivalent-promoting mechanism, analogous to the Ph gene in wheat (Riley and Chapman, 1958 ; Sears and Okamoto, 1958), to prevent multivalent formation amongst homoeologues. If the latter suggestion is correct, then it is possible that the low quadrivalent
Gatt et al.—Polyploidy in Dahlia frequency observed in the 2n ¯ 64 plants may be a consequence or byproduct of such a bivalent promoting mechanism. The mechanism of any bivalent-promoting system remains unknown. Clearly, in Dahlia the low quadrivalent frequency in the 2n ¯ 64 plants is not due solely to low chiasma frequency since ring bivalents are relatively common in these plants. It is possible that Dahlia shows the converse to the situation described in Crepis (Jones and Vincent, 1994) where a high frequency of autonomous pairing sites and pairing partner switches results in very high multivalent frequencies at pachytene. In Dahlia there may be a low frequency of autonomous pairing sites and, since only a single pairing partner switch was observed in any of the quadrivalents, these together could lead to a correspondingly low multivalent frequency. Another interesting feature of pachytene in Dahlia is the pairing or association of centromeres from several different bivalents. A similar phenomenon has been described in other plants such as maize (Gillies, 1973, 1981) with the suggestion that it represents the association of centromeric heterochromatin. Recent molecular mapping studies strongly suggest that maize, which has generally been accepted to be diploid, is in fact tetraploid (Moore et al., 1995). It is tempting to speculate that in both maize and Dahlia these could involve homologues}homoeologues and that this sort of association may influence the positioning of bivalents on the spindle, thus giving rise to the secondary associations described by Lawrence (1931 b). There is evidence that bivalents are not placed at random on the metaphase plate (Murray, 1986 ; Bennett and Bennett, 1992) but the mechanism of this selective placement remains unknown. The SCs of Dahlia also demonstrate the clustering of telomeres into a characteristic bouquet arrangement of the paired chromosomes at pachytene and apparent differences in total lateral element length. How significant these length differences are is unclear at present but in other plants SC lengths have been linked to genome size variation (Anderson et al., 1985). Since D. pinnata has been proposed as a putative ancestor of the cultivated dahlia (Sorensen, 1969) it will be important to measure genome size in the genus and establish whether it is correlated to total SC length. At present, on the basis of SC length, D. pinnata does not appear a likely ancestor of the cultivated dahlia. In conclusion, we suggest that Dahlia species have evolved through a number of polyploidization events from diploids with 2n ¯ 16. A definitive answer to the question of the polyploid status of Dahlia species may result from the application of genomic in situ hybridization to the 2n ¯ 64 plants. This approach has been successful with other polyploids (Bennett, Kenton and Bennett, 1992 ; Bailey et al., 1993) and such experiments are currently under way. Alternatively, the segregation patterns of molecular markers as used by McGrath, Hickok and Pichersky (1994) may also be informative. A C K N O W L E D G E M E N TS M. K. G. thanks the Agricultural and Marketing Research and Development Trust of New Zealand for a postgraduate
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scholarship. Grants from The University of Auckland Graduate Research Fund and The University of Auckland Research Fund are also gratefully acknowledged. LITERATURE CITED Albini SM, Jones GH, Wallace BMN. 1984. A method for preparing two-dimensional surface-spreads of synaptonemal complexes from plant meiocytes for light and electron microscopy. Experimental Cell Research 152 : 280–285. Anderson LK, Stack SM, Fox MH, Chuanshan Z. 1985. The relationship between genome size and synaptonemal complex length in higher plants. Experimental Cell Research 156 : 367–378. Bailey ST, Bennett ST, Bennett MD, Stace CA. 1993. Genomic in situ hybridization identifies parental chromosomes in the wild grass hybrid¬Festulpia hubbardii. Heredity 71 : 413–420. Bennett ST, Bennett MD. 1992. Spatial separation of ancestral genomes in the wild grass Milium montianum Parl. Annals of Botany 70 : 111–118. Bennett ST, Kenton A, Bennett MD. 1992. Genomic in situ hybridization reveals the allopolyploid nature of Milium montianum (Gramineae). Chromosoma 101 : 420–424. Darlington CD. 1928. Studies in Prunus, I and II. Journal of Genetics 19 : 213–256. Darlington CD. 1973. Chromosome botany and the origin of cultiated plants. London : George Allen & Unwin Ltd. Gillies C. 1973. Ultrastructural analysis of maize pachytene karyotypes by three dimensional reconstruction of the synaptonemal complex. Chromosoma 43 : 145–176. Gillies C. 1981. Electron microscopy of spread maize pachytene synaptonemal complexes. Chromosoma 83 : 575–591. Ishikawa M. 1911. Cytologische Studien von Dahlien. The Botanical Magazine Tokyo 25 : 1–8. Jackson RC, Hauber DP. 1982. Autotriploid and autotetraploid cytogenetic analyses : Correction coefficients for proposed binomial models. American Journal of Botany 69 : 644–646. Jones GH, Vincent JE. 1994. Meiosis in autopolyploid Crepis capillaris. II. Autotetraploids. Genome 37 : 497–505. King GA, Davies KM. 1992. Identification, cDNA cloning and analysis of mRNAs having altered expression in tips of harvested asparagus spears. Plant Physiology 100 : 1661–1669. Lawrence WJC. 1929. The genetics and cytology of Dahlia species. Journal of Genetics 21 : 125–159. Lawrence WJC. 1931 a. The genetics and cytology of Dahlia ariabilis. Journal of Genetics 24 : 257–306. Lawrence WJC. 1931 b. The secondary association of chromosomes. Cytologia 2 : 352–384. Lawrence WJC. 1970. The evolution and history of the Dahlia. The Dahlia Annual. Leamington : National Dahlia Society, 16–24. Lawrence WJC, Scott-Moncrieff R. 1935. The genetics and chemistry of flower colour in Dahlia : A new theory of specific pigmentation. Journal of Genetics 30 : 155–228. Leitch AR, Schwarzacher T, Jackson D, Leitch IJ. 1994. In situ hybridization : a practical guide. Oxford : BIOS Scientific Publishers Limited. Loidl J. 1989. Effects of elevated temperature on meiotic chromosome synapsis in Allium ursinum. Chromosoma 97 : 449–458. McGrath JM, Hickok LG, Pichersky E. 1994. Assessment of gene copy number in the homosporous fern Ceratopteris thalictroides and C. richardii (Parkeraceae) by restriction fragment length polymorphism. Plant Systematics and Eolution 189 : 203–210. Moore G, Devos KM, Wang Z, Gale MD. 1995. Grasses, line up and form a circle. Current Biology 5 : 737–739. Murray BG. 1986. Interchange quadrivalents and chromosome order at meiotic metaphase I in Briza L. (Gramineae). Chromosoma 94 : 293–296. Murray BG, Bennett MD, Hammett KRW. 1992. Secondary constrictions and NORs of Lathyrus investigated by silver staining and in situ hybridization. Heredity 68 : 473–478. Riley R, Chapman V. 1958. Genetic control of the cytologically diploid behaviour of hexaploid wheat. Nature 182 : 713–715.
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Sears ER, Okamoto M. 1958. Intergenomic chromosome relationships in hexaploid wheat. Proceedings of the 10th International Congress of Genetics 2 : 258–259. Solbrig OT. 1977. Chromosomal cytology and evolution in the family Compositae. In : Heywood VH, Harborne JB, Turner BL, eds. The biology and chemistry of the Compositae. Volume I. London : Academic Press, 267–281.
Sorensen PD. 1969. Revision of the genus Dahlia (Compositae, Heliantheae—Coreopsidinae). Rhodora 71 : 309–416. Sved J. 1966. Telomere attachment of chromosomes. Some genetical and cytological consequences. Genetics 53 : 747–756. Turner BL, Ellison WL, King RM. 1961. Chromosome numbers in the Compositae IV. North American species, with phyletic interpretations. American Journal of Botany 48 : 216–223.
Polyploidy and Evolution in Wild and Cultivated Dahlia Species M E L A N I E G A T T*, H O N G D I N G*, K E I T H H A M M E T T† and B R I A N M U R R AY*‡ * School of Biological Sciences, The Uniersity of Auckland, Priate Bag 92019, Auckland, New Zealand and † 488C Don Buck Road, Massey, Auckland 8, New Zealand Received : 4 November 1997
Accepted : 29 January 1998
Observations on the chromosomes of nine species of Dahlia Cav. (Asteraceae, Heliantheae—Coreopsidinae) show that some have 2n ¯ 32, others 2n ¯ 64, with a third group having both chromosome numbers in the same taxon. Karyotype investigations showed that the chromosomes can be divided into groups of 14 metacentrics plus two submetacentrics per set of 16 chromosomes. In situ hybridization using an rRNA gene probe indicated that the 2n ¯ 32 species have eight hybridization sites whilst the 2n ¯ 64 species have 16 sites. Silver nitrate staining of these regions showed that not all of these nucleolar organizers are active. Meiotic analysis at metaphase I and pachytene, by synaptonemal complex spreading, shows that the 2n ¯ 32 species have exclusive bivalent formation whereas the 2n ¯ 64 species have small numbers of univalents plus quadrivalents in addition to bivalents. This study proposes that Dahlia species with 2n ¯ 32 are allotetraploids whereas those species and chromosome races with 2n ¯ 64 are their autopolyploid derivatives. We suggest that a bivalent-promoting mechanism in the 2n ¯ 32 species may account for their meiotic behaviour as their component genomes appear so similar, and that this mechanism is also responsible for the low number of quadrivalents in the 2n ¯ 64 taxa. # 1998 Annals of Botany Company Key words : Chromosome pairing, Dahlia, in situ hybridization, karyotype analysis, polyploidy, synaptonemal complex analysis.
INTRODUCTION The garden dahlia (Dahlia ariabilis hort. non (Willd.) Desf.) is a relatively new taxon which is believed to have arisen following the introduction into Spain from Central America of wild species of Dahlia in 1789. With the exception of the garden dahlia, Dahlia species can be divided into two groups depending on flower colour : Group I with magenta or ivory flowers and Group II with orangescarlet or yellow flowers (Lawrence, 1929, 1931 a ; Lawrence and Scott-Moncrieff, 1935). Since the garden dahlia combines both colour series, these authors concluded that the garden dahlia, with 2n ¯ 64, was a hybrid derived from the crossing of two species (2n ¯ 32), one belonging to Group I, the other to Group II, followed by chromosome doubling. At that time the 2n ¯ 64 race of D. coccinea was unknown, all their material having 2n ¯ 32, and they did not have material of D. pinnata. To date, the exact origin of the garden dahlia remains unclear although the most widely accepted idea is that it is the product of hybridization between D. pinnata Cav. (flower colour Group I) and D. coccinea Cav. (flower colour Group II) (Sorensen, 1969). Early observations of the chromosomes of D. coccinea showed 2n ¯ 32 (as D. coronata in Ishikawa, 1911 ; Lawrence, 1929). However, subsequent studies have found an additional chromosome race with 2n ¯ 64 (Turner, Ellison and King, 1961). These chromosome races have not
‡ For correspondence. b.murray!auckland.ac.nz
Fax
0305-7364}98}05064710 $25.00}0
64 9 373 7416,
been segregated into separate taxa. D. pinnata has a chromosome number of 2n ¯ 64 (Sorensen, 1969), and garden dahlias all have 2n ¯ 64. Several lines of evidence summarized by Lawrence (1970) led him to propose that the 2n ¯ 32 plants were tetraploids and that consequently the 2n ¯ 64 plants must be octoploids. The first of these was that the basic number of x ¯ 16 in Dahlia is very high for the Asteraceae. More recent studies have confirmed x ¯ 9 as the most common number in the family, being found in 21 % of all taxa examined (Solbrig, 1977). Secondly, four factors were identified which governed flower colour in D. ariabilis. Each of these four factors is tetrasomic and gives the characteristic ratios arising from the random pairing of four homologous chromosomes (Lawrence and Scott-Moncrieff, 1935). Thirdly, at diakinesis and metaphase I there were secondary associations of a non-chiasmate nature (Darlington, 1928) between the chromosomes (Lawrence 1929, 1931 b). Lawrence (1929, 1931 a) and Lawrence and Scott-Moncrieff (1935) ultimately proposed that the garden dahlia was an autoallopolyploid, an octoploid that contains two different autotetraploid genomes. They also proposed that species with 2n ¯ 32 are allotetraploids which have descended from diploid ancestral forms, and that during the evolution of these tetraploid species differentiation occurred giving rise to the two flowercolour groups (Lawrence, 1931 a). Darlington (1973) also suggested that the wild species from which D. ariabilis (2n ¯ 64) arose are allotetraploids (2n ¯ 32) and that there are no known extant diploid (2n ¯ 16) species. To date, no Dahlia species has been found with 2n ¯ 16. Lawrence (1970) suggested that a probable explanation is that the
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# Annals of Botany Company
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Gatt et al.—Polyploidy in Dahlia T 1. Plant species used in this study and their origin Species
Chromosome number
Source
Origin
D. D. D. D. D. D.
apiculata HJ7343 australis HJ7250 australis HJ7346 coccinea HJ7310 coccinea HJ7357 coccinea IB52
2n ¯ 32 2n ¯ 32 2n ¯ 64 2n ¯ 32 2n ¯ 32 2n ¯ 32
Hjerting}Hansen, GIC Hjerting}Hansen, GIC Hjerting, wild collection Hjerting, wild collection Hjerting, wild collection Hjerting}Hansen, GIC
D. D. D. D. D. D. D. D. D. D. D. D.
coccinea HJ7000 coccinea HJ7337 coccinea HJ7382 coccinea HJ7128 coccinea HJ7051 imperialis HJ7319 pinnata Sor6519 rudis HJ7352 sherffii HJ7266 sherffii Spooner tenuicaulis G&K5090 ariabilis ‘ Cardas ’
2n ¯ 32 2n ¯ 64 2n ¯ 64 2n ¯ 64 2n ¯ 64 2n ¯ 32 2n ¯ 64 2n ¯ 32 2n ¯ 32 2n ¯ 64 2n ¯ 32 2n ¯ 64
D. D. D. D. D. D.
ariabilis ariabilis ariabilis ariabilis ariabilis ariabilis
2n ¯ 64 2n ¯ 64 2n ¯ 64 2n ¯ 64 2n ¯ 64 2n ¯ 64
Hjerting}Hansen, GIC Hjerting, wild collection Hjerting, wild collection Hjerting}Hansen, GIC Hjerting, wild collection Hjerting, wild collection Hjerting}Hansen, GIC Hjerting, wild collection Hjerting}Hansen, GIC Spooner, GIC Knees, wild collection Commercial cultivar, topmix type from Czechoslovakia Commercial cultivar Commercial cultivar Commercial cultivar Commercial cultivar Bonne Esperance¬Park Princess Commercial cultivar
‘ De 12 ’ ‘ Fern Irene ’ ‘ Pineholt Princess ’ ‘ Bishop of Llandaff ’ ‘ 45}1 ’ ‘ Elizabeth Hammett ’
Puebla State, Mexico. Alt 2250 m Oaxaco State, Mexico. Alt. 2300 m Puebla State, Mexico. Alt. 2500 m Mexico State, near Coatepec, Mexico. Alt. 2200 m Mexico State, Mexico. Alt. 1860 m Federal District, Mexico, via the Mexican Institute of Biology. Alt. unknown Durango State, Mexico. Alt. 2490 m Chiapas State, Mexico. Alt. 2200 m San Luis State, Mexico. Alt. 1600 m Morelos State, Mexico, Alt. 2600 m Guerrero State, Mexico. Alt. 2400 m Oaxaco State, Mexico. Alt. 2520 m Hybrid of intraspecific cross Mexico State, Mexico. Alt. 2700 m Chihuahua State, Mexico. Alt. 2870 m Chihuahua State, Mexico. Alt. unknown Mexico
HJ, Hjerting, J. P. ; IB, Mexican Institute of Biology ; G&K, Knees, S ; Spooner, Spooner, D ; GIC, grown in cultivation or possibly of hybridization.
original ancestors of today’s Dahlia species were diploids which became extinct because of competition with the more successful tetraploids derived from them. With the development of surface spreading techniques for the observation of synaptonemal complexes (SCs) in plants (Albini, Jones and Wallace, 1984), it is now possible to analyse pachytene pairing patterns in species with relatively high chromosome numbers. At pachytene, if we are dealing with autotetraploids, we would expect to observe 66 % of the chromosomes associated as quadrivalents if there are two autonomous terminal pairing initiation sites per chromosome (Sved, 1966). At metaphase I, multivalents should be observed at frequencies predicted by models based on random pairing of homologous arms (Jackson and Hauber, 1982). However, recent work on autotetraploid Crepis capillaris has shown that the frequency of pachytene quadrivalents can reach 96 % for specific tetrasomes, and that this is correlated with a correspondingly high frequency of pairing partner switches (Jones and Vincent, 1994). The converse, with fewer pachytene quadrivalents than expected, has been correlated with genome size}SC length (Jones and Vincent, 1994) and may reflect a low number of autonomous pairing sites. In addition, as Jones and Vincent (1994) also point out, a low quadrivalent frequency could also indicate that there is preferential pairing of homologues or homoeologues. As part of a larger study on wild species and cultivars of Dahlia, we have examined their karyotypes and pachytene and metaphase pairing to investigate the nature of polyploidy in Dahlia.
MATERIALS AND METHODS The plant species used in this study are listed in Table 1. Karyotypes were constructed from root tip cells at metaphase. The root tips were pretreated with 8-hydroxyquinoline for 2 h at room temperature in the dark followed by 2 h at 4 °C then fixed in ethanol : acetic acid (3 : 1). Chromosome spreads were prepared as follows : root tips were rehydrated in citrate buffer for at least 2¬15 min, the terminal 3 mm of the root tip was removed and placed on a slide in 20 µl of enzyme mix (400 units ml−" cellulase, ‘ Onozuka R-10 ’ Serva, 30 units ml−" pectolyase, Sigma P3026, in distilled water) and incubated in a moisture chamber for 1–1±5 h at 37 °C. Excess enzyme was carefully removed with filter paper and a drop of water added. This was removed with filter paper and another drop of water added, left for 5 min, then removed. A drop of fixative, ethanol : acetic acid (3 : 1), was added and the digested root tip gently stirred with a brass rod to separate cells. Slides were left to air dry, then stored under desiccation at room temperature until required. Slides used for in situ hybridization were 1–2 weeks old. For silver staining, the method outlined in Murray, Bennett and Hammett (1992) was followed. In situ hybridization followed the protocol in Leitch et al. (1994). Slides with good metaphase spreads were pretreated with DNase-free RNase (100 µg ml−"), then pepsin (5 µg ml−") and refixed with paraformaldehyde (4 %). The probe, pTIP6, containing the 26S rRNA gene from asparagus (King and Davies, 1992), was labelled with digoxigenin by
Gatt et al.—Polyploidy in Dahlia
649
A
B
F. 1. Karyotypes of D. coccinea HJ 7357 (2n ¯ 32) (A) and D. ariabilis ‘ Pineholt Princess ’ (2n ¯ 64) (B). Bar ¯ 10 µm.
F. 2. Somatic chromosomes of D. tenuicaulis (2n ¯ 32) (A) and D. australis (2n ¯ 64) (B) after in situ hybridization with the rRNA gene probe. Bar ¯ 10 µm.
nick translation following manufacturer’s instructions (Boehringer Mannheim). Digoxigenin-labelled probes were detected with a HNPP fluorescence detection kit (Boehringer
Mannheim) and the slides observed with a Zeiss epifluorescence microscope and filter set 02. For meiotic metaphase I (MI) analysis, immature capitula
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Gatt et al.—Polyploidy in Dahlia were fixed in ethanol : chloroform : glacial acetic acid (6 : 3 : 1) and squashed in orcein. Ten to thirty cells at metaphase I were analysed for each taxon. The SC spreading technique for pachytene analysis was that of Loidl (1989), with minor modification to suit the material. Observations were made using a Phillips CM12 transmission electron microscope and suitable cells photographed using Copex PET 10 film. Five cells at pachytene were analysed in detail for D. coccinea (2n ¯ 32 and 2n ¯ 64), D. pinnata (2n ¯ 64), D. australis (2n ¯ 64) and D. ariabilis (2n ¯ 64). Measurements of SC lengths were made from photographs of known magnification using a Summagraphics FX digitizer tablet connected to a PowerMac 6100}60 computer. RESULTS
F. 3. A silver nitrate stained mitotic metaphase of D. australis (2n ¯ 32). Arrows indicate active NOR sites. Bar ¯ 10 µm.
Karyotype analysis shows that the chromosomes of these Dahlia species are relatively small, from 2–3±5 µm in D. coccinea HJ7357 and 1±5–3 µm in D. ariabilis ‘ Pineholt Princess ’ (Fig. 1). The chromosomes can be divided into two groups : 14 metacentrics plus two submetacentrics per set of 16 chromosomes, but it is very difficult to distinguish many different chromosomes within these groups. This
T . 2. Mean meiotic metaphase I chromosome configurations in Dahlia species Mean number of configurations per cell* Species
Number of cells
D. apiculata HJ7343 2n ¯ 32 D. australis HJ7250 2n ¯ 32 D. australis HJ7346 2n ¯ 64
20 17 16
HJ7310 2n ¯ 32 IB52 2n ¯ 32 HJ7000 2n ¯ 32 HJ7337 2n ¯ 64
20 15 18 15
D. coccinea HJ7382 2n ¯ 64
14
D. coccinea HJ7128 2n ¯ 64
12
D. coccinea HJ7051 2n ¯ 64
11
D. imperialis HJ7319 2n ¯ 32 D. pinnata Sor6519 2n ¯ 64
20 17
D. rudis HJ7352 2n ¯ 32 D. sherffii HJ7266 2n ¯ 32 D. sherffii Spooner 2n ¯ 64
20 30 11
D. tenuicaulis G&K5090 2n ¯ 32 D. ariabilis ‘ Cardas ’ 2n ¯ 64
20 10
D. ariabilis ‘ De12 ’ 2n ¯ 64
14
D. ariabilis ‘ Fern Irene ’ 2n ¯ 64
12
D. ariabilis ‘ Bishop of Llandaff ’ 2n ¯ 64
10
D. D. D. D.
coccinea coccinea coccinea coccinea
I
cII
oII
cIV
oIV
0 0 1 0† 0 0 0 0±92 0 0 0 0±2 0 1 0 0 0±5 0 0 0±06 0±4 0 0 0 0 0 0 0±5 0 0±4 0
12±7 9±8 23±43 21±7 10±3 10±4 8±4 25±3 24±1 24 21±2 21±6 18±2 15 11±7 6±4 22±11 19±3 5 1±37 3±3 1±6 3±4 22±3 19±2 24±5 21±9 23±08 20±6 21 18±1
3±3 6±2 7±81 2±5 5±7 5±6 7±6 5±7 1±9 6 2±6 8±1 3±4 13±3 5±3 9±6 7±8 3±1 11 14±6 25±5 9±4 12±6 8±7 3±2 6±1 2±5 7±17 2±8 9±8 3±5
0 0 0 2±8 0 0 0 0 2±2 0±1 2±9 0 3±5 0±2 4±3 0 0±1 3±3 0 0 0 2±1 0 0 3±3 0 2±7 0±08 3±0 0 3±5
0 0 0±13 1±1 0 0 0 0±27 0±8 0±9 1±2 1±1 1±7 1±4 3±2 0 0±82 1±5 0 0 1±5 8±4 0 0±5 1±5 0±7 1±1 0±67 1±3 0±5 1±7
* I, Univalents ; cII, chain bivalents ; oII, ring bivalents ; cIV, chain quadrivalents ; oIV, ring quadrivalents. † Values in italics are expected values derived from the formulae of Jackson and Hauber (1982).
Mean chiasma frequency per cell 19±3 22±2 39±6 21±8 21±6 23±6 37±7 39±9 42±3 47±9 25±6 41±4 27 30±6 60±3 28±7 41±7 39±5 40±4 42±6
Gatt et al.—Polyploidy in Dahlia
F. 4. Meiotic metaphase I in D. coccinea (2n ¯ 32) (A) and D. australis (2n ¯ 64) (B). Bar ¯ 10 µm.
F. 5. Light micrograph of a surface-spread synaptonemal complex of D. australis (2n ¯ 64) showing the diffuse spherical structures associated with the centromeres and the association of these structures at pachytene (arrow). Bar ¯ 10 µm.
basic karyotype was seen in all species with 2n ¯ 32 (D. coccinea, D. sherffii, D. australis, D. rudis and D. tenuicaulis) and also in those with 2n ¯ 64 (D. coccinea, D. sherffii, D. australis, D. pinnata and D. ariabilis). No variation in karyotype was observed in three different accessions of D. coccinea (2n ¯ 32). The metacentric chromosomes can be grouped into fours in the 2n ¯ 32 taxa, but this is not
651
possible for the submetacentric chromosomes which are morphologically quite distinct. Alternatively, the chromosomes can all be arranged in pairs in the 2n ¯ 32 taxa. However, in either arrangement it would appear that there is considerable similarity between the component genomes of these species. In all the 2n ¯ 64 taxa, similar arguments can be made for arranging the karyotype into comparable groups of eight or four chromosomes. Chromosomes with secondary constrictions or nucleolar organizer regions (NORs) provided the most useful markers for chromosome identification, but specific pairs of NOR chromosomes could not be distinguished. Using a combination of silver nitrate staining and in situ hybridization with an rRNA gene probe, a maximum of eight NORs} hybridization sites could be identified in D. coccinea (2n ¯ 32), D. australis (2n ¯ 32), D. sherffii (2n ¯ 32), D. tenuicaulis (2n ¯ 32) (Fig. 2 A) and D. rudis (2n ¯ 32), and 16 sites were seen in D. coccinea (2n ¯ 64), D. australis (2n ¯ 64) (Fig. 2 B), D. sherffii (2n ¯ 64), D. pinnata (2n ¯ 64) and D. ariabilis ‘ Elizabeth Hammett ’. Although the NOR chromosomes have a secondary constriction, hybridization sites appeared to be located at the end of the chromosome arm, as the small terminal region beyond the secondary constriction was not observed. Silver nitrate staining did not identify the maximum number of NOR chromosomes in all species. Deposition of the silver occurred at the secondary constriction of chromosomes although it was often difficult to see the terminal region of the chromosome due to its small size. The NOR chromosomes identified by in situ hybridization in D. tenuicaulis could be divided into two groups : two pairs of chromosomes with rRNA sites were larger than the other two pairs. This pattern was also observed in silver-stained karyotypes of D. australis (2n ¯ 32) (Fig. 3), D. coccinea (2n ¯ 32) and D. rudis (2n ¯ 32). Furthermore, this pattern was also observed in D. ariabilis ‘ Pineholt Princess ’ with eight chromosomes occurring in each group instead of four. In addition to staining at the NORs, silver stained bands were also observed at the centromeres and telomeres of all chromosomes in D. australis (2n ¯ 32) (Fig. 3), D. coccinea (2n ¯ 32 and 2n ¯ 64), D. rudis (2n ¯ 32), D. sherffii (2n ¯ 64) and D. ariabilis ‘ Pineholt Princess ’ (2n ¯ 64), and consequently were not useful in the differentiation of specific chromosome pairs. At this stage it is not realistic to draw detailed conclusions about karyotypic relationships between the species of the same ploidy level, but karyotype observations do suggest that the 2n ¯ 64 species and races have the doubled karyotype of 2n ¯ 32 species. Analysis of the MI pairing patterns of nine species are given in Table 2. No univalents were observed in most 2n ¯ 32 taxa and they regularly formed a mixture of rod and ring bivalents in all cells (Fig. 4 A). At the 2n ¯ 32 level, the mean number of chiasmata per bivalent was very similar in D. coccinea, D. australis and D. apiculata and ranged from 1±2 to 1±5, whereas the mean number of chiasmata per bivalent was higher in D. rudis, D. imperialis, D. sherffii and D. tenuicaulis and ranged from 1±6 to 1±9. Many of the 2n ¯ 64 taxa formed small numbers of univalents and all formed some quadrivalents in addition to rod and ring bivalents (Fig. 4 B). The mean number of chiasmata per bivalent in
652
Gatt et al.—Polyploidy in Dahlia
F. 6. Electron micrograph of a surface-spread synaptonemal complex of D. pinnata (2n ¯ 64). Bar ¯ 10 µm.
this group was very similar in most taxa ranging from 1±2 to 1±5 with the exception of D. sherffii, which had a mean number of chiasmata per bivalent of 1±9. Using the model developed by Jackson and Hauber (1982), based on the random pairing of chromosome arms in autotetraploids, it is clear that none of the 2n ¯ 64 species give an acceptable fit (Table 2). In all cases there is a large deficit in the number of quadrivalents and an excess in the number of bivalents. At pachytene, SCs were readily observed under both light and electron microscopes (Figs 5, 6 and 7). A single nucleolus was seen in most cells analysed and up to four pairs of chromosomes appeared to be associated with it in some cells. The preservation of centromeres was variable but appeared to be species-specific as they were seen only in D. australis and D. pinnata. Under the light microscope they appeared as darkly-staining granular regions roughly 1–2 µm in diameter associated with the SC (Fig. 5). In electron micrographs, two slightly different forms of centromere were observed. The first appears as a thickening
of the lateral elements associated with small amounts of fibrillar material (Fig. 8 A) whereas the second is a spherical to elongated, diffuse structure 0±7–2±6 µm in diameter (Fig. 8 B). An interesting feature of these cells was the frequent aggregation of the centromere structures into two to four clusters (Figs 6 and 8 C). In many cells the telomeres were clustered and were characterized by the association of a darkly-staining, more or less spherical granule 0±15–0±2 µm in diameter associated with the ends of the lateral elements (Fig. 6). Table 3 gives the results of an analysis of a sample of cells from the five taxa. In D. coccinea (2n ¯ 32), 16 bivalents were seen with no evidence of multivalents in any of the cells examined. The 2n ¯ 64 race of D. coccinea and the other three 2n ¯ 64 taxa all formed quadrivalents at low frequencies, (five being the maximum number seen), in addition to bivalents (Table 3). Only a single pairing partner exchange was seen in these quadrivalents and in all cases the mean frequency of pachytene quadrivalents was higher than at MI. There appear to be large differences in lateral element
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F. 7. Electron micrograph of a surface-spread synaptonemal complex of D. coccinea (2n ¯ 64). Bar ¯ 10 µm.
length between the species : D. pinnata, for example, has lateral elements that are twice as long as those of D. coccinea. However, because only five cells per species were analysed in detail and the staging of the cells (within pachytene) is not possible, the significance of these length variations must be treated with caution. DISCUSSION There has been uncertainty over the ploidy status of Dahlia species. Lawrence (1929) suggested that the 2n ¯ 32 species were allotetraploids, arising from hybrids between nowextinct diploids with 2n ¯ 16. Furthermore, he proposed that the garden dahlia (D. ariabilis) with 2n ¯ 64 was also a hybrid that combined the genomes of two species, one from each of his Groups I and II, that subsequently underwent polyploidization. Lawrence (1970) did not specify whether the species in these two groups (I and II) were genomically differentiated, but the fact that he calls the
2n ¯ 64 garden dahlia an autoallopolyploid, rather than an allooctoploid, would imply that he thought not. Sorensen (1969), on the other hand, proposed 2n ¯ 32 species were diploid and 2n ¯ 64 species tetraploid. With both these propositions, we would expect to see groups of four homologous chromosomes in the karyotypes and the frequent association of chromosomes as quadrivalents at pachytene and metaphase I of meiosis in the 64-chromosome plants. Although the present study has been unable to unambiguously confirm the ploidy status of Dahlia species, we suggest that the 2n ¯ 32 taxa are not diploids. The karyotypes of the 2n ¯ 32 species show that all the chromosomes cannot be grouped in fours. This is particularly evident with the submetacentric members of the complement, thus suggesting that they are allotetraploids. The karyotypes of the 2n ¯ 64 taxa appear to be double those of the 2n ¯ 32 taxa, suggesting that they are their autoploid derivatives. The high basic chromosome number coupled with the large number of NOR chromosomes also
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Gatt et al.—Polyploidy in Dahlia T 3. Lateral element length (µm), extent (%) of pairing and the numbers of quadrialents and bialents in fie Dahlia taxa
Species D. coccinea HJ7310 2n ¯ 32 Mean D. coccinea HJ7337 2n ¯ 64 Mean D. pinnata Sor6519 2n ¯ 64 Mean D. ariabilis ‘ Cardas45}1 ’ 2n ¯ 64 Mean D. australis HJ7346 2n ¯ 64 Mean
F. 8. Electron micrographs of surface-spread synaptonemal complexes of Dahlia showing the thickened structures associated with centromeres in D. australis (2n ¯ 64) (A), the diffuse centromere associated structures in D. pinnata (2n ¯ 64) (B) and the association of centromeres in D. pinnata (2n ¯ 64) (C). Bar ¯ 5 µm.
suggests that the 2n ¯ 32 chromosome plants are not diploid. Although the development of SC spreading techniques has greatly increased our ability to observe the patterns of chromosome pairing at pachytene, particularly for plants such as Dahlia with high chromosome numbers, the analysis of chromosome pairing at pachytene and metaphase I does little to immediately clarify the origin and evolution of Dahlia species. The 2n ¯ 32 D. coccinea shows exclusive bivalent formation from which we can conclude, equally validly, that it is either a diploid or an allotetraploid. The 2n ¯ 64 taxa, including the polyploid races of D. coccinea, D. australis and D. sherffii, are all characterized by low frequencies of quadrivalents at both pachytene and MI.
Cell 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3
Lateral element length 406±9 364±8 358±4 315±3 296±8 355±6 933±6 894±3 879±0 875±0 590±6 834±5 1886±5 1760±3 1711±8 1584±57 1425±6 1673±8 1081±7 991±4 950±6 769±0 483±0 855±1 1230±8 977±5 906±8 1038±4
% pairing No. IV No. II 95±1 82±2 97±9 93±1 97±9 93±2 98±4 98±7 92±2 99±4 71±4 92±0 99±6 100±0 98±1 98±1 99±3 99±0 99±3 98±9 100±0 71±0 80±6 90±0 98±9 76±5 87±6 87±7
0 0 0 0 0 0±0 5 2 3 2 0 2±4 2 0 2 2 2 1±6 3 3 0 3 0 1±8 2 3 3 2±7
16 16 16 16 16 16±0 22 28 26 28 32 27±2 28 32 28 28 28 28±8 26 26 32 26 32 28±4 28 26 26 26±7
They do not fit the expected pachytene quadrivalent frequency of 66±6 % for autotetraploids (Sved, 1966) nor do they fit the random pairing models of Jackson and Hauber (1982). At face value, this chromosome behaviour lends little support to the proposal that D. ariabilis is the autoploid derivative of a hybrid between parents from Group I and II (Lawrence, 1970 ; Darlington, 1973). It is also surprising that the 2n ¯ 64 races of D. coccinea, D. australis and D. sherffii, which are morphologically so similar to their 2n ¯ 32 races and therefore appear autoploid in origin, also form quadrivalents at very low frequencies. If the 2n ¯ 32 plants are allotetraploids, as Lawrence (1970) proposes, then their doubled derivatives with 64 chromosomes would be expected to form numerous quadrivalents. There are two possible explanations for the diploid-like behaviour of the 2n ¯ 32 plants. One requires the now extinct parental diploids (2n ¯ 16) to have had sufficiently distinct genomes so that when hybrids occurred there was no intergenomic pairing. Karyotype observations presented here suggest that this is not the case since there is so much similarity within the 2n ¯ 32 genomes. An alternative hypothesis is that they have a bivalent-promoting mechanism, analogous to the Ph gene in wheat (Riley and Chapman, 1958 ; Sears and Okamoto, 1958), to prevent multivalent formation amongst homoeologues. If the latter suggestion is correct, then it is possible that the low quadrivalent
Gatt et al.—Polyploidy in Dahlia frequency observed in the 2n ¯ 64 plants may be a consequence or byproduct of such a bivalent promoting mechanism. The mechanism of any bivalent-promoting system remains unknown. Clearly, in Dahlia the low quadrivalent frequency in the 2n ¯ 64 plants is not due solely to low chiasma frequency since ring bivalents are relatively common in these plants. It is possible that Dahlia shows the converse to the situation described in Crepis (Jones and Vincent, 1994) where a high frequency of autonomous pairing sites and pairing partner switches results in very high multivalent frequencies at pachytene. In Dahlia there may be a low frequency of autonomous pairing sites and, since only a single pairing partner switch was observed in any of the quadrivalents, these together could lead to a correspondingly low multivalent frequency. Another interesting feature of pachytene in Dahlia is the pairing or association of centromeres from several different bivalents. A similar phenomenon has been described in other plants such as maize (Gillies, 1973, 1981) with the suggestion that it represents the association of centromeric heterochromatin. Recent molecular mapping studies strongly suggest that maize, which has generally been accepted to be diploid, is in fact tetraploid (Moore et al., 1995). It is tempting to speculate that in both maize and Dahlia these could involve homologues}homoeologues and that this sort of association may influence the positioning of bivalents on the spindle, thus giving rise to the secondary associations described by Lawrence (1931 b). There is evidence that bivalents are not placed at random on the metaphase plate (Murray, 1986 ; Bennett and Bennett, 1992) but the mechanism of this selective placement remains unknown. The SCs of Dahlia also demonstrate the clustering of telomeres into a characteristic bouquet arrangement of the paired chromosomes at pachytene and apparent differences in total lateral element length. How significant these length differences are is unclear at present but in other plants SC lengths have been linked to genome size variation (Anderson et al., 1985). Since D. pinnata has been proposed as a putative ancestor of the cultivated dahlia (Sorensen, 1969) it will be important to measure genome size in the genus and establish whether it is correlated to total SC length. At present, on the basis of SC length, D. pinnata does not appear a likely ancestor of the cultivated dahlia. In conclusion, we suggest that Dahlia species have evolved through a number of polyploidization events from diploids with 2n ¯ 16. A definitive answer to the question of the polyploid status of Dahlia species may result from the application of genomic in situ hybridization to the 2n ¯ 64 plants. This approach has been successful with other polyploids (Bennett, Kenton and Bennett, 1992 ; Bailey et al., 1993) and such experiments are currently under way. Alternatively, the segregation patterns of molecular markers as used by McGrath, Hickok and Pichersky (1994) may also be informative. A C K N O W L E D G E M E N TS M. K. G. thanks the Agricultural and Marketing Research and Development Trust of New Zealand for a postgraduate
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scholarship. Grants from The University of Auckland Graduate Research Fund and The University of Auckland Research Fund are also gratefully acknowledged. LITERATURE CITED Albini SM, Jones GH, Wallace BMN. 1984. A method for preparing two-dimensional surface-spreads of synaptonemal complexes from plant meiocytes for light and electron microscopy. Experimental Cell Research 152 : 280–285. Anderson LK, Stack SM, Fox MH, Chuanshan Z. 1985. The relationship between genome size and synaptonemal complex length in higher plants. Experimental Cell Research 156 : 367–378. Bailey ST, Bennett ST, Bennett MD, Stace CA. 1993. Genomic in situ hybridization identifies parental chromosomes in the wild grass hybrid¬Festulpia hubbardii. Heredity 71 : 413–420. Bennett ST, Bennett MD. 1992. Spatial separation of ancestral genomes in the wild grass Milium montianum Parl. Annals of Botany 70 : 111–118. Bennett ST, Kenton A, Bennett MD. 1992. Genomic in situ hybridization reveals the allopolyploid nature of Milium montianum (Gramineae). Chromosoma 101 : 420–424. Darlington CD. 1928. Studies in Prunus, I and II. Journal of Genetics 19 : 213–256. Darlington CD. 1973. Chromosome botany and the origin of cultiated plants. London : George Allen & Unwin Ltd. Gillies C. 1973. Ultrastructural analysis of maize pachytene karyotypes by three dimensional reconstruction of the synaptonemal complex. Chromosoma 43 : 145–176. Gillies C. 1981. Electron microscopy of spread maize pachytene synaptonemal complexes. Chromosoma 83 : 575–591. Ishikawa M. 1911. Cytologische Studien von Dahlien. The Botanical Magazine Tokyo 25 : 1–8. Jackson RC, Hauber DP. 1982. Autotriploid and autotetraploid cytogenetic analyses : Correction coefficients for proposed binomial models. American Journal of Botany 69 : 644–646. Jones GH, Vincent JE. 1994. Meiosis in autopolyploid Crepis capillaris. II. Autotetraploids. Genome 37 : 497–505. King GA, Davies KM. 1992. Identification, cDNA cloning and analysis of mRNAs having altered expression in tips of harvested asparagus spears. Plant Physiology 100 : 1661–1669. Lawrence WJC. 1929. The genetics and cytology of Dahlia species. Journal of Genetics 21 : 125–159. Lawrence WJC. 1931 a. The genetics and cytology of Dahlia ariabilis. Journal of Genetics 24 : 257–306. Lawrence WJC. 1931 b. The secondary association of chromosomes. Cytologia 2 : 352–384. Lawrence WJC. 1970. The evolution and history of the Dahlia. The Dahlia Annual. Leamington : National Dahlia Society, 16–24. Lawrence WJC, Scott-Moncrieff R. 1935. The genetics and chemistry of flower colour in Dahlia : A new theory of specific pigmentation. Journal of Genetics 30 : 155–228. Leitch AR, Schwarzacher T, Jackson D, Leitch IJ. 1994. In situ hybridization : a practical guide. Oxford : BIOS Scientific Publishers Limited. Loidl J. 1989. Effects of elevated temperature on meiotic chromosome synapsis in Allium ursinum. Chromosoma 97 : 449–458. McGrath JM, Hickok LG, Pichersky E. 1994. Assessment of gene copy number in the homosporous fern Ceratopteris thalictroides and C. richardii (Parkeraceae) by restriction fragment length polymorphism. Plant Systematics and Eolution 189 : 203–210. Moore G, Devos KM, Wang Z, Gale MD. 1995. Grasses, line up and form a circle. Current Biology 5 : 737–739. Murray BG. 1986. Interchange quadrivalents and chromosome order at meiotic metaphase I in Briza L. (Gramineae). Chromosoma 94 : 293–296. Murray BG, Bennett MD, Hammett KRW. 1992. Secondary constrictions and NORs of Lathyrus investigated by silver staining and in situ hybridization. Heredity 68 : 473–478. Riley R, Chapman V. 1958. Genetic control of the cytologically diploid behaviour of hexaploid wheat. Nature 182 : 713–715.
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