170 Cytologia 28

A Cytotaxonomic Study of the Genus Cyrtanthus

R. Wilsenach

Department of Botany, University of the Witwatersrand, Johannesburg, Republic of South Africa

Received November 19, 1962

The members of the family Amaryllidaceae have relatively large chromo somes and this facilitates the computing of accurate idiograms which are a help in the understanding of the phylogeny of plants. Many workers, e.g. Taylor (1925), Heitz (1926), Sato (1938, 1942), Gouws (1949), Mookerjea (1955), Sharma and Bhattacharya (1956), and Sharma and Bal (1956), have studied the karyology of amaryllids in order to obtain a clearer picture of their relationships. The phylogenetic relationships of the amaryllid groups as proposed by Gouws (1949), for example, must be considered as a great im provement on the earlier systems which were based only on morphological characters. Unfortunately the karyological data are still far from complete, and serious discrepancies still exist as regards the interpretation of results. This is especially true of the genera Cyrtanthus, Vallota and Anoiganthus. Taylor (1925), Sato (1938, 1942), Gouws (1949), Mookerjea (1955) and Ising (1962), have studied several species belonging to these genera. These authors differ concerning the relationships of these plants and also as to their basic chromo some numbers, the diploid numbers of 16, 18, 20 and 22 having been reported. The present investigation attempts to throw further light on this problem by an extended study of the genus Cyrtanthus, which has South Africa as its main centre of distribution. Intention was, firstly, to find out more about the variation in chromosome number reported by other workers, and secondly, to correlate the karyological observations with morphological data to obtain a better understanding of the phylogeny of the genus.

Material and methods

Almost all the plants were collected in their natural habitat and replanted in a sandy loam in 4 "and 6" pots. The meiotic divisions of the pollen mother cells were found to take place before the inflorescences appear, with the result that preparations of meiotic stages would result in the destruction of numerous bulbs, and this could not be afforded. For this reason only mitotic stages in the root tips were recorded. The root tips were fixed in Craf (Randolph 1935) for 24 hours , except in the case of C. carneus in which case the root tips were fixed for about 1 hour in 3 alcohol: 1 propionic acid. The root tips of C. herrei were fixed in both fixing fluids. After fixation in Craf the root tips were transferred 1963 A Cytotaxonomic Study of the Genus Cyrtanthus 171 to 70% ethyl alcohol and dehydrated in the ethyl alcohol-normal butyl alcohol series according to Randolph (1935), embedded in paraffin wax, and cut 16 18 microns thick. Sections were stained in a 1% aqueous solution of crystal violet, following the method described by Smith (1934), and mounted in canada balsam. Six year old preparations show very little sign of bleaching. After fixation in 3 alcohol: 1 propionic acid the root tips were squashed and stained according to Warmke's (1935) prescription, but propionic acid was substituted for acetic acid. In order to make these slides permanent the methods of McClintock (1929), Conger and Fairchild (1953) and Cave and Pocock (1951) were tried. None of these proved to be without drawbacks, with the result that the slides were made semipermanent by sealing off the sides of the coverslip with "nail varnish".

Table 1. Species investigated

Description of somatic chromosomes

In computing the idiograms of each species several metaphase plates were compared. The chromosomes of the species investigated are relatively long, with the result that the chromosome arms in polar view twist both towards and away from the observer, and this had to be taken into account. This was done by determining how far the chromosome arm twists towards the pole, and this distance is often as much as 6 microns. In the idiograms the chromosomes are generally arranged from the longest chromosome to the shortest. This is, however, not true of all the idiograms 172 R. Wilsenach Cytologia 28 e.g. C. parviflorus chromosome G is longer than chromosome F (Idiog. 9). These exceptions occur only to facilitate comparison of the different idiograms, and it infers a homology between e.g. chromosome G of C. par viflorus and the G chromosomes of the other species. The chromosomes were arbitrarily divided into long, medium and short,

Table 2. Genome formulae and chromosome numbers of species investigated 1963 A Cytotaxonomic Study of the Genus Cyrtanthus 173

Figs. 1-18. Somatic chromosomes (2n) of Cyrtanthus species. 1, C. erubescens, 16. 2, C. thorncroftii, 16. 3, C. huttonii, 16. 4, C. species (from Northern Transvaal), 16. 5, C. mackenii, 16. 6, C. mackenii var. cooperi, 16. 7, C. brachyscyphus, 16. 8, C. parviflorus, 16. 9. C. ochroleucus, 16. 10, C. smithiae, 16. 11, C. helictus, 16. 12, C. obliquus, 16. 13, C. carneus (fixed in 3 alcohol: 1 propionic acid), 16. 14a, C. herrei (Craf fixation), 16. 14b, C. herrei (fixed in 3 alcohol: 1 propionic acid), 16. 15, C. contractus, 16. 16, C. sanguineus, 16. 17, C. galpinii, 16. 18, C. balenii, 16. 174 R. Wilsenach Cytologia 28

for which the letters 1 (longum), m (medium) and b (brevis) are used. In addition the letters V, L and f are used to indicate the position of the primary (kinetic) constriction: V for a median or near median constriction; L for sub median; and f for subterminal, i.e. when the proximal arm is 1/3 or less of the length of the distal arm.

Discussion The karyology of the genus Cyrtanthus. Sato (1938) recorded 2n=22 as the somatic chromosome number of Cyrtanthus obliquus. According to Taylor (1925) the diploid number of C. parviflorus is 16, and Gouws (1949) reported the same number for C. tuckii var. transvaalensis. Flory (1955) investigated six species, viz. C. angustifolius, C. falcatus, C. ochroleucus (=C. lutescens), C. mackenii, C. O'Brieni and C. sanguineus, and quoted the diploid number 16 for all. Mookerjea (1955) studied C. sanguineus and a horticultural variety called Cyrtanthus "ifafa lily", and reported the diploid chromosome numbers as 18 and 20 respectively, and stated that it was not possible to make any sug gestions regarding the basic number for the genus. In the present work the three species, namely C. obliquus, C. parvi florus, and C. sanguineus were re-investigated and 16 chromosomes were regularly present. This is also true of all the other species investigated here, with result that the number 16 can be considered as the normal diploid number of this genus. Ising (private communication) made the following remark: "The number 2n=22 reported for C . obliquus... I think is wrong, as I found 2n=16 in my plants". The counts of 22, 20 and 18 reported by Sato (1938) and Mookerjea (1955) are difficult to interpret, unless they resulted from incorrect technique. It is apparently the first time that C. helictus, C. huttonii, C. mackenii var. cooperi, C. contractus, C. balenii, C. galpinii, C. brachyscyphus, C. smithiae, C. herrei, C. carneus, C. thorncroftii and C. erubescens have been investi gated cytologically. The idiograms of the different species show a fair degree of resemblance but vary from very asymmetrical ones to more symmetrical ones (e.g. C. brachyscyphus and C. obliquus respectively). Chromosome A is V-shaped or almost so in most species and varies in length between 15-19 microns. Chromo some B and C are often similar, are usually fairly long (10-18 microns) and usually have sub-median kinetic constrictions. Chromosome D is the most variable one in the complex and varies in length between 8 and 13 microns. Chromosomes E and F are often very similar and usually have sub-terminal kinetic constrictions, their length varies between 6-12 microns. Chromosome G is usually of mV type, the most striking exception being C. brachyscy phus, where this chromosome has a sub-terminal kinetic constriction. Its length varies between 5 and 10 microns. Chromosome H is always of the bf 1963 A Cytotaxonomic Study of the Genus Cyrtanthus 175

Idiograms 1-22. Idiograms of Cyrtanthus, Anoiganthus and Vallota species. 1, Anoigan thus breviforus (according to Gouws, 1949). 2, Cyrtanthus erubescens. 3, C. thorncroftii. 4, C. huttonii. 5, C. species (from Northern Transvaal). 5, C. mackenii. 7, C. mackenii var. cooperi. 8, C. brachyscyphus. 9, C. parviflorus. 10, C. ochroleucus. 11, C. smithiae. 12, C. helictus. 13, C. obliquus. 14, C. carneus (fixed in 3 alcohol: 1 propionic acid). 15, C. herrei (Craf fixation). 16, C. herrei (fixed in 3 alcohol: 1 propionic acid). 17, C. con tractus. 18, C. sanguineus. 19, C. galpinii. 20, C. balenii. 21, Vallota purpurea (accord ing to Gouws, 1949). 22, Cryptostephanus vansonii (according to Gouws, 1949). 176 R. Wilsenach Cytologia 28 type or nearly so, it varies in length between 5 and 10 microns. (The lengths quoted refer only to the determinations based on Craf-fixed material). Phylogeny. The genus Cyrtanthus has a rather interesting history. The first two species recorded were described under the generic name Crinum by Linnaeus in 1781. Eight years later the genus Cyrtanthus was created by Aiton (1789) to accommodate these species, which had curved flowers. Herbert originally separated the species which are now considered as belonging to Cyrtanthus into three genera viz. Monella, Cyrtanthus and Gastronema. When C. carneus became known to him he realised that it represented a link between Cyrtanthus and Monella, with the result that he combined these two genera. Baker in 1888 combined Gastronema with Cyrtanthus and the genus was composed of three sub-genera, Cyrtanthus proper, Monella and Gastr onema. Twenty species were listed. Dyer (1939) reviewed the genus for Herbertia, listing 44 species of which 3 were new descriptions. Amongst these is also C. vittatus, which, as Dyer pointed out, is most probably not a Cyrtanthus species at all. Dyer (1939) did not divide the genus into sub-genera, and remarked on the morphological similarity between Cyrtanthus and Vallota. When the ideograms of the different species are compared no support for the earlier sub-divisions of the genus into sub-genera can be found. A variety in the genus Cyrtanthus, called cooperi, which was originally associated with C. ochroleucus (=C. lutescens), has been combined with C. mackenii by Dyer (1939), although some doubt remained as to whether this was justified. When one compares the three idiograms concerned (Idiog. 6, 7 and 10) it becomes evident that the idiograms of these plants do not bring us much closer to a final solution of this problem. The idiograms of C. mackenii and C. ochroleucus are very similar, both differing from the variety cooperi in respect of the D chromosome. The variety is therefore more distinct from the two species than the latter are from each other. Dyer (1959) came to the conclusion that Cryptostephanus herrei repre sented a species of the genus Cyrtanthus. The feature which this plant has in common with Cryptostephanus is a so-called "corona", but Dyer (1959) stated that the "corona" was not an outgrowth of the perianth, but of the filaments. Cyrtanthus carneus also has such lateral appendages to the fila ments, and it is interesting to note that this fact has been overlooked for more than a hundred years. Idiogram 22 represents an idiogram of Cryptostephanus vansonii, as computed by Gouws (1949). When the idiogram of C. herrei is compared to those of other Cyrtanthus species and to that of Cryptostephanus vansonii it becomes clear that it is karyologically almost identical to some Cyrtanthus species, and very different from Cryptostephanus. There is also a difference in chromosome number. Dyer's new combination is thus fully supported by cytological evidence. The three genera Anoiganthus, Vallota and Cyrtanthus . Idiograms of 1963 A Cytotaxodomic Study of the Genus Cyrtanthus 177

Anoiganthus breviflorus and Vallota purpurea were prepared by Gouws (1949), and are reproduced here in Idiograms 1 and 21 respectively. The genus Anoiganthus, according to Baker (1888 and 1896) differs from Cyrtanthus in that the anthers of Anoiganthus are basifixed, whereas those of Cyrtanthus are dorsifixed and versatile. All the Anoiganthus flowers I have studied have dorsifixed versatile flowers. The stamens of Anoiganthus luteus are illustrated in Flowering Plants of Africa on Plate 539 of volume XIV, and are clearly versatile. Killick (1960) also stated that the stamens of Anoigan thus are versatile. It is important to notice that Baker originally described Anoiganthus as having versatile anthers, and it is difficult to understand why he used an anther difference (which according to his description of Anoigan thus does not exist) to separate the two genera. The only other criterion which has been used to separate the two genera is based on the length of the perianth tube. The tube is longer than the segments in Cyrtanthus but shorter than the segments in Anoiganthus ac cording to Phillips (1951) and Pax and Hoffmann (1930). That this criterion cannot be used to separate these genera can be illustrated by the following: In C. clavatus and C. brachyscyphus the perianth tube is often as long as the perianth segments, and in C. thorncroftii the tube is sometimes shorter than the segments. In C. erubescens the tube is much shorter than the seg ments. Killick (1960) has suggested that the description of the genus Cyr tanthus should be altered to include species with shorter tubes, such as C. erubescens. When the idiogram of Anoiganthus is compared to those of some short tubed Cyrtanthus species one notices that the idiograms are practically identical (compare Figs. 1 and 3, Plate 2). A cross between C. parviflorus and Anoiganthus breviflorus made by the present author, resulted in normal seed setting and 80% of the seed con sequently germinated. From the morphological, cytological and genetical data given above it would appear that the separation of species in two genera is artificial, and that the genus Anoiganthus should be merged into Cyrtanthus. This needs no further modification of our concept of the genus Cyrtanthus than has been suggested by Killick (1960). The morphological differences between the genera Cyrtanthus and Vallota are mainly based on the shape and the structure of the perianth tube. It must be stressed, however, that the genus Cyrtanthus shows great variation as regards the shape of the perianth tube. Some species, e.g. C. mackenii and C. parviflorus have long, narrow tubes, whereas some, e.g. C. balenii, C. galpinii, C. sanguineus and C. guthrieae, have tubes which approach very closely the type found in Vallota. When Bolus (1921) described C. guthrieae she stated that the spread of the perianth and the relatively short tube were more characteristic of Vallota than of Cyrtanthus, and that C.

Cytologia 28, 1963 12 178 R. Wilsenach Cytologia 28 guthrieae could be considered as a connecting link between the two genera. (This species was unfortunately not available for the present investigation). The species with long, narrow perianth tubes have asymmetrical karyotypes, and those with the broadly funnel-shaped tubes have the more symmetrical karyotypes and resemble closely that of Vallota. C. smithiae and C. helictus are intermediate forms as regards both the perianth tube and the karyotype. From the above it becomes clear that within the genus Cyrtanthus there is a graduation in a perianth shape ranging from the typical Cyrtanthus form, to that of Vallota, and this is accompanied by gradual change in karyotype from asymmetrical to more symmetrical. The other difference between Cyrtanthus and Vallota is the so-called "pulvinate callus" which is present at the base of the perianth segments in Vallota, which is a criterion used by Hutchinson (1934) to separate the two genera. This structure is a very small "flap" at the base of the perianth segments and it is difficult to realise why this structure has been called a "pulvinate callus" because it is membrane -like and suggests a rudimentary corona. It is doubtful whether this structure could be used as a criterion to maintain a genus. Herbert (1837, p. 134) made the following remark about Vallota: "This beautiful plant is so closely allied to Cyrtanthus, that I have even entertained doubts of its being distinct, and should wish to see it ascertained by further experiments whether it is incapable of mingling with that genus." That C. sanguineus has often given rise to intergeneric hybrids with Vallota speciosa has been observed by Dyer (1939). Had this information been available to Herbert, there is no doubt that he, the author of the genus Vallota, would have combined it with Cyrtanthus. From the above it is clear that the barriers between the genera Cyrtanthus and Vallota are also artificial and that our concept of the genus should further more be modified as to include also the genus Vallota. The modified concept of the genus Cyrtanthus. (the new genus will in corporate the genera Vallota and Anoiganthus). Rootstock a tunicated bulb. Leaves contemporary with or produced later than the inflorescences. Peduncle hollow or rarely solid and does not contain any sclerenchyma in the ground tissue. Flowers umbellate, subtended by 2-4 bracts , erect or sub-erect, nodding or pendulous. Perianth usually tubular for more than half its length , some times shorter; lobes sub-equal, usually shorter than the tube , 3 outer lobes furnished within the apex with an incurved point or tuft of hairs; small membrane-like flap present at the base of the segments in one species . Stamens usually inserted in the perianth tube, anthers oblong, dorsified and versatile; filaments may possess small lateral appendages at the point of insertion on the perianth. Ovary three celled, ovules numerous, crowded, superposed. Style long, filiform, indistinctly or distinctly three lobed at the stigmatic apex . Capsule mostly oblong, loculicidally three-valved, seeds flattened, somewhat 1963 A Cytotaxonomic Study of the Genus Cyrtanthus 179 winged, testa black. This genus is restricted to Africa, and the greatest number of species occurs in Natal and the Eastern Cape Province.

Summary Seventeen species and one variety of the genus Cyrtanthus were inves tigated cytologically, chromosome numbers were determined, and ideograms were prepared. Twelve of these have been studied for the first time. The diploid chromosome number is sixteen for all the species investigated. The idiograms vary from asymmetric to more symmetric ones and this varia tion is accompanied by a change in flower shape. Both as regards karyotype and flower structure, some Cyrtanthus species are extremely similar to species of Vallota, others are karyologically almost identical to species of Anoiganthus and have perianth tubes as short of those of Anoiganthus. It is suggested that the three genera are congeneric and a new concept, which involves only minor changes, is proposed for Cyrtanthus to combine these genera. The combination Cyrtanthus herrei by Dyer (1959) is supported by cytological evidence.

Literature cited

Aiton, W. 1789. Hort. Kew. Ed. I. 1: 414. Baker, J. G. 1888. Handbook of Amaryllidaceae. George Bell & Sons. 1896. Flora Capensis IV: 171-246. Bolus, L. 1921. Cyrtanthus guthrieae. Ann. Bolus Herb. 3: 79. Cave, M. S. and Pocock, M. A. 1951. The aceto-carmine technique applied to the colonial Volvocales. Stain Technol. 26: 173-174. Conger, A. D. and Fairchild, L. M. 1953. A quick-freeze method for making smear slides permanent. Stain Technol. 28: 281. Dyer, R. A. 1939. A review of the genus Cyrtanthus. Herbertia 6: 65-103. - 1959. Cyrtanthus herrei. Fl. Plts. of Afr. 33: 1281. Flory, W. S. (Jnr.). 1955. Chromosome complements of some Cyrtanthus species. Virginia Jour. Sci. (N. S.) 6: 250. Gouws, J. B. 1949. Karyology of some South African Amaryllidaceae. Plant Life 5: 54-81. Heitz, E. 1926. Der Nachweis der Chromosomen. Z. Bot. 18: 625-681. Herbert, W. 1837. Amaryllidaceae. James Ridgeway & Sons, Picadilly. Hutchinson, J. 1934. Families of Flowering Plants 2: 81-141. MacMillan & Co., London. Ising, G. 1962. Chromosome balance in Cyrtanthus. Plant Life 18: 95-128. Killick, D. J. B. 1960. Notes and new records of African flowering plants. Bothalia 7: 412 - 414. McClintock, B. 1929. A method for making aceto-carmine slides permanent. Stain Technol. 4: 2. Mookerjea, A. 1955. Cytology of amaryllids as an aid to the understanding of evolution. Caryologia 7: 1-71. Pax, F. and Hoffmann, K. 1930. Amaryllidaceae. Engler and Prantl. Nat. Pflzfam. 2nd Ed. 15a: 391-430. Phillips, E. P. 1951. The Genera of South African Flowering Plants. 2nd Ed. Govern ment Printer, Pretoria. Randolph, L. F. 1935. A new fixing fluid and a revised schedule for the paraffin method

12* 180 H. Wilsenach Cytologia 28

in plant cytology. Stain Technol. 10: 95-96. Sato, D. 1938. Karyotype alternation and phylogeny. IV. Karyotypes in Amaryllidaceae with special reference to SAT-chromosome. Cytologia 9: 208-242. - 1942. Karyotype alternation and phylogeny in Liliaceae and allied families. Jap. Jour. Bot. 12: 57-161. Sharma, A. K. and Ball, A. K. 1956. A cytological study of a few genera of Amaryllidaceae with a view to finding out the basis of their phylogeny. Cytologia 21: 329-352. - and Bhattacharya, N. K. 1956. An investigation on the karyotype of the genus Crinum and its phylogeny. Genetics 28: 263-296. Smith, F. S. 1934. The use of picric acid with gram stain in plant cytology. Stain Technol. 9: 95-96. Taylor, W. R. 1925. The chromosome morphology of Veltheimia, Allium and Cyrtanthus. Amer. Jour. Bot. 12: 104-115. Warmke, H. E. 1935. A permanent root tip smear method. Stain Technol. 10: 101-103.