Cytologia 46: 623-633, 1981

Chromosomal Indications of Evolutionary Trends in the Genus Delphinium L.

Rehab K. Al-Kelidar1and A. J. Richards Universityof Newcastleupon Tyne,U. K.

Received February 7, 1980

The genus Delphinium (Ranunculaceae) contains some 250 species which are distributed circumboreally in temperate and mediterranean regions. All species are herbaceous annuals or perennials with showy zygomorphic flowers which are habitually cross-pollinated, and a number of species, hybrids and cultivars have become popular garden plants. All parts of the plants are toxic due to alkaloids, and deaths to stock from Delphinium poisoning are regularly reported. Although several partial or complete monographs of the genus have been published (Nuth 1895, Nevskii 1937, Ewan 1945, Pawlowski 1964), these are based on morphological comparisons alone, and subgeneric taxa are confused, with a complex history (Al-Kelidar 1978). Although, prior to this work, the chromosome numbers of no less than 74 species had been reported (Bolkhovskikh et al. 1969), no karyological work is known, and little attempt has been made to relate cyto logical features to evolutionary relationships in this large and complex genus. Considerable controversy has arisen over many years as to the status of subge nus Consolida DC, which in Chater (1964) is given generic rank. This status is ac cepted in this paper and from here on, the genera are treated separately. This paper presents some data on the size, shape, number, DNA content and rate of uptake of tritiated thymidine of the chromosomes of 14 species of Delphinium and 2 of Consolida, with additional chromosome counts on 5 species of Delphinium and one of Consolida; 22 species were studied in all.

Materials and methods

Seeds were obtained from a number of botanic gardens, germinated, and grown in pots under glass. On flowering, attempts were made to determine this material correctly; in the majority of cases the name given by the source was clearly incor rect, and a number that proved through morphological or cytological criteria to be hybrid or cultivars were disregarded. Voucher herbarium specimens were col lected and remain in Newcastle. The following taxa were examined cytologically (source names in brackets). It has not been possible to verify source names given here in quotes.

1 Present address: Hay Al-Muhamin, 10/14/23, Al-Havathiya, Baghdad, Iraq. 624 Rehab K. Al-Kelidar and A. J. Richards Cytologia 46

Delphinium present work Bolkhovskikh et al. (1969) section Delphinium D. verdunense Balbis (D. verdunense, D. 2n=16 cinereum) section Staphisagria DC D. requienii DC (D. requienii, D. exaltatum) 2n=16 section Diedropetalia Huth series Fissa Nevskii D. "leiocarpum" Nevskii 2n=32 D. geyeri Greene (D. decorum) 2n=32 series Semibarbata Nevskii D. semibarbatum Bienert (=D. zalil) 2n=16 2n=16 section Kolobopetala Muth D. grandiflorum L. (D. grandiflorum) 2n=16 2n=16 D. coeruleum Cambess. (D. tatsienense) 2n=16 section Delphinastrum DC series Moschata Nevskii D. brunonianum Royle (D. brunonianum) 2n=32 2n=16 D. "cashmerianum" Royle 2n=32 2n=16 series Foetida Nevskii D. caucasicum C.A.M. 2n=32 series Flexuosa Nevskii D. flexuosum M. B. (D. flexuosum, D. 2n=32 2n=32 cheilanthum, D. dictyocarpum) series Inconspicua Nevskii D. inconspicuum Serg. (D. speciosum) 2n=32 2n=32 series Elata Nevskii D. elatum L. (D. elatum, D. alpinum, 2n=32 2n=32 D. azureum, D. californicum, D. exaltatum, D. intermedium, D. maackianum, D. mosquensis, D. oxysepalum, D. przewalskii) D. denudatum Wallich 2n=32 2n=32 D. "glaucum" Wats 2n=32 2n=16 D. "formosum" Boiss. et Huet 2n=32 2n=32 series Crassifolia Nevskii D. "corymbosum" Regl. 2n=32 series Dissecta Nevskii D. triste Fisch. ex DC (D. triste) 2n=32 species incerta sedis (? section Delphinastrum) D. "bulleyanum" Forrest 2n=32 2n=32 Consolida C. regalis S. F. Gray (=D. consolida) 2n=16 2n=16 C. ambiguum (L.) P. W. Ball and V. H. 2n=16 2n=16 Heywood (D. azureum, D. ajacis) 1981 Chromosomal Indications in the Genus Delphinium L . 625

C. orientalis (Gay) Schrodinger (=D . 2n=16 2n= 16, 24 orientale)

Cytological technique

Root-tips were taken from young plants in plastic pots and pretreated in 0 .25% colchicine in water solution for 3 hours at room temperature before fixation in

3 parts absolute ethanol to 1 part glacial acetic acid; they were stored in a deep

freeze. Before staining they were washed in distilled water and hydrolysed in 1N

HCl at 60•Ž for 11 minutes. After another wash, they were stained in Feulgen

reagent for at least 1 hour. Root-tips were excised and squashed in acetocarmine,

and made permanent in 'euparal'. Measurements were made with a calibrated eye

piece graticule, using a •~100 objective. Chromosome lengths were standardised by comparison with known chromosome types on reference slides. In a number

of cases, cells were photographed and karyograms constructed. A minimum of

4 cells from 2 plants were compared karyologically. For microdensitometry, pretreatment was omitted, and the root-tips were fixed for 5 hours. Hydrolysis was achieved in 5N HCl at room temperature for 1 hour, and after staining for 1 hour in Feulgen reagent, followed by two changes in SO, water, were squashed in 45% acetic acid. The relative amount of DNA per nucleus was determined through the intensity of Feulgen staining using a Vickers M85 scanning microdensitometer at a wavelength of 570nm. A sample of at least 30 prophase nuclei and 30 half-telophase nuclei were used to achieve estimates of the mean 4C and 2C levels of DNA respectively in each species. For autoradiography, actively growing root-tips of 1cm were transferred to

a solution of tritiated thymidine, 3H-TdR, at 37 Kilobecquerels/ml in distilled water, for 1 hour. They were then fixed, hydrolysed and stained as for microdensitometry.

Cover-slips were removed after freezing on solid CO,, and after air-drying, slides were dipped in Ilford K2 nuclear emulsion dissolved in an equal volume of distilled water at 40•Ž in the dark, and exposed in a light-proof container at 4•Ž for 14 days.

The slides were then immersed in half-strength Kodak D-19 developer (5 minutes), washed, fixed in full-strength Kodak unifix (5 minutes) and washed again (Mac

Leod 1972). After dehydration in ethanol they were mounted in 'euparal' to give permanent autoradiographs. Any nucleus showing 5 or more developed silver halide grains was considered 'labelled'. The real grain count was established by comparing grain counts per nucleus with similar areas away from nuclei.

Results

Somewhat over 20 different types of chromosomes could be identified in the 15 species examined karyologically (Table 1). These ranged from 12.5 microns to 1.5 microns in length, and centromere positions varied from median to telocentric. Satellites were identified on both large and small chromosomes and satellited chromosomes varied from 4 to 12 in number per tetraploid (2n=32) cell. Acces sory (B) chromosomes were not identified and the level of casual aneuploidy was low. No aneuploid individuals were detected. A few triploids were discovered, Table l . Karyotypes of Delphinium and Consolida species. Numbers on x axis (along top) refer to chromosome length. All measurements

in microns (ƒÊm). Numbers in bulk of table refer to number of chromosomes. The total number of cells examined for each sample,

and the number of (sibling) plants from which these cells were taken are given on the right-hand side. Letters refer to centromere

position: m=median (arm ratio 1 to 1.9), sm=submedian (arm ratio 2-2.9), a=acrocentric (arm ratio 3 or more), T=telo centric (only one arm detectable), *=satellite.

(to be cont.) Table 1. (cont.) Fig. 1. Karyotype similarity indices between 15 species of Delphinium and Consolida (Table 1) arranged according to overall karyotype similarity. •¡ KSI 11-14, •¡ KSI 9-10, _??_ KSI 6-8, and • KSI O-5. A=section Delphinastrum, B=section Diedropetalia, Al=series Moschata, C=section Staphisagria. 1981 Chromosomal Indications in the Genus Delphinium L . 629 but these were evident hybrids and are discounted from this analysis. Species were compared karyologically by means of a karyotype similarity index (KSI) (Richards 1972, Richards and Booth 1976) and karyological relation ships are displayed in Fig. 1. Two diploid species, Consolida ambigua and Delphi nium requienii each show low similarity to the other species. Both are annual in breeding weeds of crops in the Mediterranean. The third diploid inbreeder from this area, C. regalis, also shows a low similarity among the tetraploid species, although it has the same overall scores as D. formosum, D. corymbosum and D. triste, Russian species each from different series of section Delphinastrum. The two North American species in section Diedropetalia, D. leicarpum and D. geyeri show a high similarity between themselves. D. elatum seems to take a linking position between these Diedropetalia and the remainder of section Delphinastrum. It is also closely similar to D. bulleyanum, of uncertain affinity, which might there fore be placed in series Elata. In the remainder of section Delphinastrum, two groups emerge; the closely related dwarf Himalayan species D. brunonianum and D. cashmerianum (series Moschata); and a geographically disparate group of phenotypically similar species, D. caucasicum, D. denudatum and D. glaucum. Thus, at least to a limited degree, karyological studies have borne out taxonomic groupings, emphasising the relationships of species within section Delphinastrum and within section Diedropetalia, and underlining the distinctness of section Staphisagria (D. requienii) and of the genus Consolida. The mean chromosome length of two of the three diploids studied, C. ambigua (7.0 microns) and D. requienii (5.0 microns) is longer than that of any tetraploid. There is therefore a suggestion that chromosome material has been lost from tetraploids subsequent to polyploidy. A comparison of mean chromosome length with DNA content (Fig. 2) shows that there is a positive relationship in diploids, suggesting that reduction in chromosome size has resulted in the loss of DNA-rich areas. Tetraploids have scarcely more DNA than the diploid C. ambigua, but more than twice the amount of C. regalis. C. regalis, a diploid with small chromosomes (mean 3.50 microns) also shows the greatest variation of any species in chromosome size, as expressed as a coefficient of variation (0.59) (Table 1). This suggests that extensive chromosomal reorganisation has occurred in this diploid, involving the loss of DNA-rich areas. Tetraploids in contrast display relatively slight variation in mean chromosome size (4.50 to 3.12 microns), in chromosome variability (co efficient of variation 0.50 to 0.33) and in DNA content (Fig. 2). Although the great variation in chromosome types found in tetraploids suggests that extensive chromosomal reorganisation has occurred, this has apparently resulted in relatively little loss of DNA. A comparison of mean chromosome lengths of chromosome types with differ ing centromere positions gives some indication as to how chromosomal reorganisa tion has proceeded (Table 2). Metacentric chromosomes average 3 times the length of telocentric chromosomes, and there is a progressive reduction in chromo some size with increasing inequality of chromosome arm length. Clearly, the loss of DNA in plants with smaller chromosomes has occurred through unilateral breakage of chromosome arms, with subsequent loss of DNA-rich acentric frag 630 Rehab K. Al-Kelidar and A. J. Richards Cytologia 46

Fig. 2. The relationship between chromosome size and DNA content for four diploid species and four tetraploid species. x=mean chromosome size (microns), y=DNA content (arbitrary units ,of absorbance) (sc). 1=D. caucasicum, 2=D. cashmerianum, 3=D. elatum, 4=D. triste, 5=Con solida ambigua, 6=D. requienii, 7=Consolida regalis, 8=D. grandiflorum, 1, 2, 3, 4=tetraploid, 5, 6, 7, 8=diploid.

Table 2. Mean length of chromosomes with different centromere positions

ments. In the absence of any evidence for Robertsonian translocation, it is thus reasonable to regard species with small chromosomes, low DNA content and a high proportion of acrocentric and telocentric chromosomes as being karyologi cally derivative to those with many long, metacentric chromosomes, both within the diploids and within the tetraploids. Thus C. regalis can usefully be regarded as derivative with respect to C. ambigua, and section Delphinastrum as derivative in comparison with section Diedropetalia, and the anomalous species D. formosum, Figs. 3-4. 3, the relationship between the mean number of silver grains (labelling) with 2•~ standard error of some D elphinium species, and their chromosome number. A=D. grandiflorum (2n=16) , B=D. elatum (2n=32), C=D. brunonianum (2n=32), D=D. caucasicum (2n=32), E=D. triste (2n=32), F=D. geyeri. (2n=32). 4, the relationship between the relative amount of DNA/nucleus (arbitrary units of absorbance) (4c) and mean number of silver grains (labelling) of nucleus , each with 2x standard error. A=D. grandiflorum (2n =16), B=D. elatum (2n=32) , C=D. caucasicum (2n=32), D=D. triste (2n=32). 632 Rehab K. Al-Kelidar and A. J. Richards Cytologia 46

D. corymbosum and D. triste. The latter perhaps represent karyologically primi tive forerunners of derived Delphinastrum species such as D. elatum. Although only 6 species were investigated autoradiographically, it is note worthy that the diploid D. grandorum showed a higher level of label per nucleus than did the 5 tetraploid species (Fig. 3). If it is assumed that the amount of label acquired in 1 hour's exposure to triated thymidine is proportional to the rate of DNA synthesis during the S-phase in the mitotic cycle, this suggests that the rate of synthesis, and thus potentially at least, the rate of tissue growth, is greater in the diploid. There is a suggestion (Fig. 4) that the rate of DNA synthesis is inversely proportional to the amount of DNA per nucleus, although D. triste does not agree well with this relationship in a sample of only 4 species. If this were the case, it is the reverse of what might be expected, nuclei with more DNA requiring a faster rate of DNA synthesis in order to complete a mitotic cycle in standard time.

Acknowledgements

We would like to thank R. D. MacLeod of this Department for assistance with the microdensitometry and autoradiography. This work was grant-aided by an Iraqi Government Research Studentship.

Abstract

Delphinium has about 250 species circumboreally. Of these, 22 are examined cytologically here; 8 are diploid (2n=16) and 14 tetraploid (2n=32). Most ac cessions from Botanic Gardens were wrongly named. Over 20 types of chromo some were identified; karyological relationships were calculated and show a rough correspondence with taxonomic groupings. Chromosomes with a median centro mere had an average length three times that of telocentrics. In diploids, there is a relationship between mean chromosome length and the amount of DNA in the nucleus, suggesting that DNA-rich areas are lost in chromosome re-organisation. It is suggested that species with smaller mean chromosome length can be considered to be karyologically derivative. Karyological trends are seen in both diploids and tetraploids, Consolida regalis being derivative with respect to C. ambigua, and Delphinium section Delphinastrum being derivative with respect to sections Staphi sagnia and Diedropetalia. DNA synthesis appears to be faster in nuclei with less DNA, contrary to expectations.

References

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