Misopates orontium and Antirrhinum majus: Testing the boundaries of evolution: to what extent can the differences between closely related genera be bridged by mutagenesis?

Wolf-Ekkehard Lönnig and Heinz Saedler Dept of Molecular Plant Genetics, Max-Planck-Institute for Plant Breeding Research, Köln, Germany

Results continued Abstract Materials and Methods (8) The last point is in agreement with the following result: according to data repeated twice, in Antirrhinum, AmM1 is strongly expressed in the leaves, Mutagenesis: 310 000 Misopates plants have been investigated weakly in bracts, but there is no expression in the sepals. In Misopates, Of the seven features basically distinguishing however, expression is weak in the leaves, strong in the bracts and moderate (including 10 000 M families; treatment of seeds with fast neutrons or Misopates orontium from its closely related 2 in the sepals. Thus, at least a part of the leaf programme not expressed in A. gamma rays). Moreover, 1.5 million Antirrhinum plants (different majus sepals appears to be directly expressed in the sepals of Misopates. species Antirrhinum majus, five differences mutagens) have been studied during the last two decades. have phenotypically been clearly diminished or Fig.6: fully overcome by mutant genes, so that either Molecular work: Since locus identity test crosses between Misopates and Antirrhinum are not possible, the genes were identified by Misopates approaches, meets or even overlaps sequence analyses of the homologous Misopates wildtype genes and the characters of Antirrhinum or vice versa their mutant deviations. (aspects of the life cycle, leaf form, flower size, (9) Homeotic flower and inflorescence flower colour und mode of fertilization). Yet mutants of Misopates displayed features most of this phenotypic variation can also be Results never detected in Antirrhinum. Usually found on the subgeneric level within different these deviations were correlated with the loss of the differentiation Antirrhinum or Misopates species. However, to Misopates mutants approach, meet or even overlap the programmes for special bract- and Fig. 7: plena date the morphologically most distinguishing features of Antirrhinum and vice versa in the sepal morphology, i.e. the long sepal character between the two genera, the strongly following characters. elongated sepals in Misopates, could not be feature. The fimbriata mutant displayed some most unusual features, e.g. a reduced to that of the length of Antirrhinum nor (1) Life cycle: several mutants of Misopates (including a pedicel followed by a bush of sepals (but could the development of the short Antirrhinum female fertile one) display an extended life cycle (Fig. 2: no flowers) and internodes, pointing to sepals be extended to that of the length of from their normal 3-4 months to about a year and to time (possibly) further losses-of-functions. Fig. 8:fimbriata Misopates. This constancy of the most indefinite by cuttings). Also, some Antirrhinum lines are important generic difference addressed here is classified as annual. (10) Appearance of variegation pointing to TE activities in agreement with basic results of systematics (see Fig.9, below/left). and paleontology. (11) Jeong Hee Kim: Identifying the homeotic Misopates genes and mutants (see methods):

Gene Mutation Plena large deletion Squamosa large deletion Floricaula nu: 1 sub, 12 bp deletion aa: 1 change, 4 aa deletion Fimbriata nu: 1 sub, 2 bp deletion, aa: frame shift, andstop Deficiens (weak allele) nu: 21 bp deletion aa: 7 aa deletion Deficiens (strong allele) nu: 7 bp deletion aa: frame shift and stop Cycloidea nu: 6 bp deletion aa: 2 aa deletion

Discussion Figure 2 (above): Sister Fig. 3 (above): M. orontium plants of same age wildtype (left) and leaf mutant Unexpectedly the most important morphological diffe-rence (right); below: M. orontium leaf between the two species proved to be nearly invariable so mutant under white string,rest: far (except in syndromes in Misopates which were mostly Antirrhinum lines. highly disadvantageous). Among the possibilities buffering a feature against mutations (2) Leaf form: As shown above in Fig. 3, mutants of both belong: (a) characters due to losses of gene functions, (b) species overlap each other in this caracter, too. redundancy by functionally similar gene duplicates, (c) multiple functions including vital ones of one and the same Fig. 1: Antirrhinum (above) in comparison to Misopates (3) Flower size: M.orontium mutants gene. - In such cases mutations and selection are mostly (below). Note the enormously elongated sepals of Misopates. with larger flowers appeared regularly incapable to even out the differences found between closely in our field trials (Fig. 4). Also, in related species and genera. Antirrhinum, mutants with smaller The findings so far found for our pair of species are in flowers have repeatedly been agreement with the variation and constancy detected in Introduction described. In A. valentinum the systematics and paleontology (Lönnig and Saedler, 2002, flowers are as small as in M. and paper in preparation.) However, for a final answer the The synthetic theory (Neo-Darwinism) explains the origin orontium. Thus, the two genera material investigated is not extensive enough. of all differences in the organic world essentially by two overlap each other in this character Although our results do not yet falsify the synthetic theory, factors: random mutation and selection. To test the boundaries are hinted at which in the long run might show Fig.4: Left: larger as well. theory, a large mutagenesis programme with A. majus its limitations flower mutant of and M. orontium has been carried out focussing on the M. orontium (right: following questions (among others): wildtype). (1)Can the long Misopates sepals be reduced in one (or more) step(s) to that of the length of the Antirrhinum (4) Flower colour: Although M. orontium appears to be unable so far to approach the full red colour of A. majus, the mutants sepals or vice versa? Concluding Remarks of both species meet each other in their loss-of-function (2)To what extent can the rest of the different features be phenotypes (gradual reduction of anthocyanin synthesis until Many important questions are still open, for example: to what overcome by mutagenesis? pure white flowers are developed). extent are the mutant genes involved in the overlapping features of the two species also involved in the subgeneric (3)What do the homeotic flower- and inflorescence (5) Mode of fertilization: The wild Antirrhinum majus species is features of corresponding phenotypes of wild species of the mutants reveal about the genetic differences between a selfsterile outbreeder. In contrast, Misopates is selffertile two genera? What is the genetic basis of the constancy of the two genera? with a tendency to autogamy. Yet most of the Antirrhinum the long sepal feature of Misopates orontium? Would culture varieties (which are also mutants) are selffertile, too. mutagenesis by transposons produce qualitatively different In spite of “Dollo’s Law” several reversals have been results? reported for the history of the origin of species. At least quantitative morphological differences should not (6) The long Misopates sepals could not be reduced to that of the length of the sepals of Antirrhinum nor could the sepals of constitute an insurmountable barrier to test the Antirrhinum be extended to those of Misopates. In Misopates Acknowledgements possibilities and of the synthetic theory. any significant reduction was accompanied by some The help and advice of Britta Grosardt and Thomas Münster teratological feature. in methods of molecular genetics as well as that of Peter Huijser at the Binocular and photographer Maret Kalda is (7) Whenever the leaves of Misopates were affected by a gratefully acknowledged. mutant gene, the bracts and sepals were likewise affected. This does not necessarily happen in Antirrhinum (Fig. 5). Literature: Lönnig, W.-E. and H. Saedler (2002): Chromosome Rearrangements and Transposable Elements. Ann. Rev. Genet. 36: 398-410. Fig. 5: Width of sepals and leaves Lönnig, W.-E. and H.-A. Becker (2003): Natural Selection. (In Press.) are often not correlated in Antirrhinum. Left, above and (As well as 8 further refereed papers of 2001-2003.) below A. majus, ssp. majus, Fig. 9: First Right, A.m. ssp. tortuosum. recorded appearance of variegation in Misopates