MEIOSIS IN HELLEBORUS CORSICUS X H . FOETIDUS M . J . HARVEY

Department of Biology, Dnlboz~sie University, Halifax, Nova Scotia

Introduction Helleborz~s, a genus of the Ranunculaceae, is comprised of about 20 species

of ~erennial. woodland herbs distributed across E u r o ~ e and Asia Minor with the griatest concentration of species in central and sohthern Europe. Only two species reach northwards to England although several others are commonly culti- vated in gardens. The delimitation of several of the species is not clear and hybrids are ltnown to occur, especially among the species which are cultivated for their early spring flowers.

Cytologically, all the published counts of chromosome numbers, for about 11 species, uniformly give 271 = 32 (Darlington and Wylie, 1955; Cave 1958, 1960), so on this basis cytology gives no guidance to the taxonomists. Most or all of these chromosome number determinations have been done on garden material and there is a need for an investination of wild ~ lan ts .

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In 1960 hopes of producing horticulturally desiiable specimens led t o attempts being made to make hybrids between five species (H. ntrorz~bens Waldst. and Kit., H. corsicz~s Willd., H. foetidz~s L., H . 7ziger L. and H. viridis L.) but for various reasons the onlv mature hvbrid ~ l a n t s obtained were H.

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corsicz~s x foetidus. An account of meiosis in this hybrid forms the basis for this paper.

Materials and Methods Both H. corsicz~s and H. foetidz~s were growing in the botanic garden at

Durham University and were of unltnown wild origin. T h e natural distribution of H . co~s iczu is Corsica and Sardinia and H. foetidzss is widelv distributed over western and southern Europe but the specimens used were ai'most certainly of English origin, probably from near Cambridge.

Cross pollii~ation was carried out outside, some flowers of H. corsiczu being emasculated before they opened and ltept covered with paper bags until the stig- mas matured. Pollell was transferred from H. foetidz~s and the flowers kept bagged until the seeds were ripe. The seeds were sown in pots in the autumn and kept moist in an unheated greenhouse where they germinated the following spring. Stratification in this manner seems essential at least for some species; a sample of H . foetidz~s seed did not germinate until it had been in soil for two winters. T h e plants flowered in the spring of their third year of growth.

Meiosis occurs shortly after the start of elongation of thc inflorescence in spring. Whole flowers were fixed in acetic-alcohol, anthers dissected out and squashed in iron aceto-carmine. Slides were made permanent by floating the cover glass in 45% acetic acid, dehydrating in alcohol and mounting in Euparal.

Results Both species and their hybrid have 2 71 = 32. In the species meiosis is regu-

lar, reproduction is sexual and the plants are self-compatible. I11 the hybrid, meiosis is irregular, the pollen is badly formed and apparently non-functional. N o seeds were obtained when the plants were left in the open. T h e hybrids were morphologically intermediate but grew vigorously and produced large, tall

Manuscript received April 27, 1966. Can. J. Genet. Cytol. 8: 516-519. 1966.

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MEIOSIS IN HELLEBORUS HYBRID

plants which unfortunately were not as decorative as the H. corsiczis parent. The few mid-late prophasgstages seen showed only 1 or 2 chiasmata per bivalent. Meiosis was not easy to analyse despite the large size of the chromosomes, mainly bccause the great variation in chromosome length occasionally made it difficult to distinguish between bivalents and univalents. Also, secondary associations, sometimes with two univalents lying very close together, increased difficulties of interpretation. Despite these difficulties it is thought that the figures in Table I givc a good idea of the range of bivalent formation seen. Figure 1 shows the appearance of some of the cells.

TABI.E 1

Distribution of bivalents in 97 pollen mother-cells examined a t metaphase 1

Discussion Table I shows a range of bivalent formation from 5-12 per cell with an

average of about 9. If, following Darlington and Wylie (1955) the basic chromosome number of the genus is talten as x = 8, the arrangement of 8 bival- ents plus 16 univalents is very close to the average found. This lends itself to the interpretation that the species used were allotetraploids, having genomes of the types AABB and BBCC respectively. The hybrid thus being of the consti- tutioh ABBC and deriving its 8 bivalents from the B genomes and the 16 unival- ents from the A and C genomes. Failure of chiasma formation and pairing between A and C chromosomes may have variously produced the other pairings observed.

This cytological interpretation throws light on the confused taxonomic situation. The most recent account of the European members of the genus is by Tutin in Flora Europaea (Tutin et nl., 1964) where only 11 species are recognized. This represents an ultra-cautious classification with many hitherto rccognized species lumped together as subspecies but in the absence of a thor- ough cytotaxonomic survey this is perhaps the wisest course to talte. H. foetidzis is rccognized, but the H. corsiczis of this papcr is reduced by Tutin to the ranlt of subspecies, as H. lividzis Aiton subsp. corsiczis (Willd.) Tutin. This treatment is not adopted here as it is expected that further cytotaxonomic work will justify the reinstatement of subsp. corsiczls to specific rank.

As noted earlier most of the published chromosome counts have been from garden material and natural selection acts particularly severely on botanic garden plants such that only those of a hardy nature usually survive. Tetra- ploids frcquently exhibit better survival than diploids and one of the purposes of this paper is to predict that a search of wild material from southern Europe may well turn up several diploid cytotypes (2 12 = 16). It is possible that the taxono- mic picture is confused because of the presence of a few diploid taxa together with scveral convergent tetraploids derived from them in a reticulate fashion, as in the case of the two species investigated here. H. lividzis Aiton is another tetraploid which probably differs from H. corsicz~s in its genomies structure and the two may well merit retaining specific ranlt. Only a cytogenetic investiga- tion can provide the answer.

No. of bimlents

No. of cells

10 --

18

7

1

11

14

12 -- 2

8 -

19

9 -

19

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A,I. J. HARVEY

Fig. 1. Stages in meiosis in Helleborzls cmsic~ls x H . foetid7~s. A Metaphase I, 9 11 plus 11 I. B. Late anaphase I showing univalents dividing late and lagging. C. Telophase I showing remains of t w o bridges. D. and E. Late anaphase I1 showing bridges and lagging chromosomes. F. Second telophase sho\ving unequal-sized nuclei. C

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MEIOSIS IN HELLEBORUS HYBRID 5 19

Summary

An analysis of meiotic chromosome associations in the hybrid between Helleborus corsicz~s and H . foetidus (both, 2 12 = 32) (Ranunculaceae) was shown to consist most frequently of 8 bivalents and 16 univalents. I t is sug- gested that the parents could be regarded as allotetraploids with the genomic relationship between them expressed as AABB and BBCC respectively. It is expected that further cytogenetic work will improve the presently confused taxonomy of the genus. The presence of diploid taxa (2 ~z = 16) in southern Europe is predicted.

Acknowledgements

This work was started at Durham University, continued at Birmingham University, England and completed in Canada. I would like to thanlc Professors D. H. Valentine and J. Heslop-Harrison for the use of their respective experi- mental facilities.

References Cave, M. S. 1958. Index to plant chromosome numbers for 1957. University of North

Carolina Press, Chapel Hill, N.C. Care, M. S. 1960. Index to plant chrornoson~e numbers for 1959. University of North Car-

olina Press, Chapel Hill, N.C. Darlington, C. D., and Wylie, A. P. 1955. Chromosome atlas of flowering plants. Allen

and Unwin, London. Tutin, T. G., Heywood, V. H., Burges, N . A., Valentine, D. H., Walters, S. M., and Webb,

D. A. 1961. Flora Europaea. Cambridge University Press, Cambridge.

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