1970 13

A Cytological Study of the Zingiberales with Special Reference to Their Taxonomy1

H. K. Mahanty

Sir John Cass College, London E. C. 3., Great Britain2

Received September 16, 1968

Abstract The chromosome numbers have been determined for sixty four species spread over all the families of Scitamineae. In about eighteen of these species, meiosis has been studied. The findings are correlated with the presumed evolution of the various groups. The evolution of the chromosome complements in the various families is discussed in the light of the past and present findings. In Musaceae (broad sense) three distinct groups: (a) Musa-Ensete, (b) Heliconia and (c) Ravenala-Strelitzia-Phenakospermum are recognised and sub-familial status proposed for these. According to the cytological data it is concluded that the genus Zingiber should be included in the tribe Hedychieae. From the evidence of flower morphology, geographical distribution and cytologi cal differences it is proposed that the African representative of Kaempferia should be restored to the status of a separate genus, Cienkowskya of Solms Laubach. It is suggested that haploid number 11 is the original basic number for the whole of the Scitamineae, and other numbers such as, 9, 10, 11, 12, 13, 14, 16 and 17 are secondary in origin. Of these, 9 occurs exclusively or predominantly in Ensete. Costus, Canna, and Orchidantha, 12 in the sub-family Zingiberoideae and 13 in Marantaceae.

The names of the species which have been studied in the present investigation are given below:-

MUSACEAE 1. Ensete sp., 2. Musa sp., 3. M. sapienoum Linn., 4. M. cavendishii lamb, 5. Heliconia caribea Lam., 6. H. bihai Lin., 7. H. brasiliensis Hook., 8. H. aurantiaca, Ghiesbreght., 9. Strelitzia reginae. Banks., 10. S. parvifolia. Dryand 11. S. parvifolia var juncea Bot. Reg. LO WIACEAE 2. Orchidantha longiflora H. Winkl., 13. O. maxillaroides (Ridl.) K. Schum. ZINGIBERACEAE 14. Tapeinochilus ananassae Hasak., 15. Costus macrostrobilus K. Schum., 16. C. albus A, Cheval., 17. C. niveus G. F. W. Mey., 18. C. sp. (Nigeria); 19. C. sp. (Costa Rica), 20. C. schlechteri H. Winkler., 21. C. englerianus K. Schum, 22. Hedychium thyrsiforme, Ham., 23. Kaempferia angustifolia Roscoe., 24. K. elegans Wall., 25. K. pulchra Ridl., 26. K. gilbertii. Hort., 27. K. rotunda. Linn., 28. K. carsonii. Bak., 29. K. sp. (African); 30. K. kirkii (Hook) Witt et per, 31. 1 This paper is compiled from two theses submitted for M. Sc. and Ph. D. of London University. 2 Present address: Cawthron Institute, Nelson, New Zealand. 14 H. K. Mahanty Cytologia 35

K. aethiopica (solms) Benth., 32. K. rosea. Schweinf; 33. K. sp. (African); 34. K. brachystemon K. Schum., 35. "K. ethleae". 36. Zingiber spectabile Griff., 37. Z. cylindricum Moon. 38. Globba winitii Wright., 39. G. heterobractea K. Schum., 40. G. albiflora var. aurea (Ridl) Holtt., 41. Alpinia mutica Roxb., 42. A. formosama K. Schum., 43. Roscoeae purpurea Smith., 44. R. alpina Royle; 45. R. cautleoides Gagnep., 46. R. Lumeaua Balf. et Sm., N. R. sp.

MARANTACEAE 54. Calathea albertii fide Roy. Bot. Gdn. Edin., 55. C. cylinerica (Roscoe) K. Schum., 56 C. nigricans Gang., 57. C. picturata (Lind.) K. Kochet Lind., 58. C. leucostachys Hook., 59. C. Wiotii (Morren) Reg.. 60. C. argyrophylla Hort. ex Kew Land List., 61. Stromanthe sanguinea (Hook) Lond., 62. Ctenanthe kummeriana (Morren) Eichler., 63. C. lubbersiana (Morren Eichler; and 64. Thalia geniculata Linn.

Introduction Taxonomic position: According to Bentham and Hooker's General Plantarrum (1883) the Zingibereles as a whole were considered to be one family among the Mono cotyledones, under the series Epigyneae. In Engler and Prantl's Die Naturlichen Pflanzen-familien (1889) the Scitamineae were divided into four families: Musaceae, Zingiberaceae, Cannaceae and Marantaceae. Endlicher (1836-40) (after Richard 1831) divided the family into two tribes; Heliconieae and Uranieae. Schumann (1900) divided the Musaceae into three subfamilies Musoideae, Strelitzioideae and Lowioideae, and further subdivided the Strelitzioideae into two tribes; Strelitzieae and Heliconieae. Winkler (1930) extended this division by the formation of another tribe, Ravenaleae. Hutchinson (1934) raised the status of Schumann's three sub-families to those of families. Nakai (1941) recognised another family, Heliconiaceae in addition to Hutchinson's three families. Lane (1955), however, recognised Lowiaceae as a distinct family from Musaceae and this is also agreed by various workers, but divided Musaceae into two subfamilies; Musoideae and Heliconiodeae and dividing Musoideae again into three tribes; Museae, Ravenaleae and Strelit zieae. Schumann (1904) separated the family Zingiberaceae into two sub families; Zingiberoideae and Costoideae and the Zingiberoideae were further sub-divided into three tribes; Globbeae, Hedychieae and Zingibereae. Hutchin son (1934) on the other hand divided the family directly into four tribes; Costeae, Hedychieae, Globbeae and Zingibereae. Holttum (1950) made an alteration to this scheme by transferring the genus Zingiber from the tribe Zingibereae to the Hedychieae and thus the former had to be renamed as Alpineae which is, in fact, Schumann's Zingibereae without Zingiber . This view is also shared by Tomlinson (1956). 1970 A Cytological Study of the Zingiberales 15

Cytological information about the whole order was sparse except for Musaceae which had been thoroughly investigated by various workers (Agharkar and Bhaduri 1935, Cheeseman 1935-36, Cheeseman and Larter 1935, Dodds 1943, 1945, Charkravorti 1948, 1946, and Simmonds 1962). Quite a number of workers have reported on the chromosome numbers of other families (Morinaga 1929, Boehm 1931, Sugiura 1931, Gregory 1936, Benerji 1940, Janakiamal 1945, Holzer 1952 and Malik 1961). In 1943 Raghavan and Venkatasubban first published a paper on cytology of the Zingiberaceae in relation to their taxonomy. They determined the chromosome numbers of 24 species but since their technique was based mainly on the sectioning method, very little data regarding the chromosome morphology was given. Nevertheless, this work laid the foundation for later workers. Ven katasubban (1946) made a preliminary survey of chromosome numbers in 38 species of various families of the Zingiberales. Later, Chakravorti (1948, 1952) and Sato (1948) made a considerable contribution to the karyological knowledge of this group. In 1957 Sharma and Bhattacharyya attacked this problem with modern squash techniques and reported on the cytology of 9 species of Calatheae and Maranta. In 1959 they made an extensive contri bution to our knowledge of the karyology of the Zingiberaceae. One of their main conclusions was the very widespread occurrence of inconstancy in chromosome numbers in individual species. Sato (1960) made another im pressive study on the karyotype analysis with special reference to proto karyotype and stable karyotype in the Zingiberales: but the main drawback in his investigation was the use of the sectioning method which is virtually useless for studying the chromosome morphology in order to make reliable idiogram. Information about meiotic divisions is sparse, reliable information being restricted to only a few species except in Musaceae (sensu stricto) have been considerably investigated by a number of workers (loc. cit.). It is worth noting that Tomilson (1956, 1959, 1960, 1961a, 1961b) has made a thorough investigation into the anatomy of the Zingiberales and by combining his findings with the morphological characters he has brought into light many interesting and hitherto unknown facts which will prove of great assistance in classifying this group. It will be obvious from the above survey of literature that an extensive study of cytology with modern techniques is extremely desirable. The, value of not only chromosome number but also chromosome morphology as an aid to taxonomy is becoming increasingly clear and it was with the aim of employing this powerful weapon to attack this problem the present investi gation is undertaken. Preliminary studies (Spearing and Mahanty 1964) have already clearly shown the value of this method. 16 H. K. Mahanty Cytologia 35

Whilst a lot of information can be obtained from idiograms of the somatic chromosomes, but essential information about the homologies of the chromo some complements can be obtained only from the study of meiosis which, for this reason has been investigated in quite a number of species. An attempt has been made to include representatives of not only the families, but also of the sub-families, tribes and smaller sections as was possible, despite the difficulty in obtaining the living material of many forms. Materials have been obtained from Botanical Gardens all round the world and an effort has been made to obtain specimens that would shed light on karyotypic changes related to geographical distribution.

Methods and materials The study of the somatic chromosomes of the Zingiberales is a matter of great difficulty because of their low stainability . After a long trial of the techniques commonly applied in plant cytology the one described below based on those of Tjio and Levan (1950) and La Cour (1941) has proved so far to be the best and has been extensively used in this investigation . Clean excised root tips were pretreated with a saturated solution of monobromonaphthalene in water for two hours . Next they were transferred to a watch-glass containing 9 drops of 2% orcein in 45% propionic acid and the watch glass was gently heated over a spirit lamp for 2-6 seconds , one d rop of NHCI was added , heating was resumed again for a few seconds and the root tips left in the mixture for one and a half hours . A root tip was then placed in a small drop of 1% orcein in 45% propionic acid on a slide , the extreme tip cut off (the proximal part being discarded) covered with a coverslip, and given a gentle tap with a blunt pencil in order to squash the growing point. Gentle tapping with a needle greatly facilitated the spreading of the cells. Uniform manual pressure was applied through a few thick ness of blotting paper, which helps to spread the chromosomes and also absorbs the excess stain. After preliminary observations , for which the edge of the coverslip was sealed with rubber solution , the slides were made permanents by freezing in CO2 vapour escaping from a cylinder , separating coverslip from slide with a scalpel, passing both through absolute alcohol and mounting each separ ately in 'Euparal'. Meiotic divisions in pollen-mother-cells were studied after fixation of young anthers in 3:1 alcohol acetic acid solution for one hour or more; squash preparations were then made essentially in the same way as f or somatic chromosomes. 1970 A Cytological Study of the Zingiberales 17

Observations

Family Musaceae

Ensete sp. (Figs. 1A, 2A)

The somatic number is found to be 18 and the length of the chromo somes ranges from 2.3ƒÊ for the longest to 1.4ƒÊ for the shortest. There are five pairs of comparatively long chromosomes (A, B, C, D, E) and four pairs of shorter one (F, G, H, I). Of the long ones only one (B) has a median primary constriction: in addition, this pair also bears a secondary constriction in a more or less sub-terminal position. Of the other long chromosomes, two

pairs (A, E) have submedian primary constrictions, whilst two pairs (C, D) have median ones. Of the four pairs of short chromosomes, two (F, G) have

submedian primary constrictions, and in the other two (H, I) this occupies a median position. The interesting feature in this genus is that the lengths of the chromosomes do not fall sharply into long and short, which is in contrast to those of Musa or Fig. 1. A, B, C. Idiograms showing the types of chromosomes Heliconia where the in Ensete sp., Musa sp., and Heliconia bihai respectively. distinction is remark ably clear, in spite of their overall small size.

Genus Musa

Unidentified Musa sp. (Fig. 1B)

The somatic number is found to be 22 (Fig. 4). There is a considerable range of size, varying from 2.9ƒÊ to 1.2ƒÊ long in the metaphase plates of cells treated with monobromonaphthol. There are six pairs of long chromosomes

(A, B, C, D, E, F) and five pairs of short ones (G, H, I, J, K). Of the long ones only three pairs (A, C, F) have a median primary constriction and of these only one (A) has a sub-terminal secondary constriction. Of the remaining long chromosomes (B, D, E) the primary constriction occupies a sub-median position. Two pairs of short chromosome (H and I) have sub-terminal primary constriction whilst in the other two (J and K) this is median.

Musa sapientum (Fig. 6A) Musa cavendishii (Fig. 6B) The somatic number has been found to be 33 in each case, as has been recorded by various workers (loc. cit). A reliable idiogram could not be

Cytologia 35, 1970 2 H. K. Mahanty Cytologia 35 18 prepared because of the technical difficulty resulting from the higher numbers and tendency of the chromosomes to become entangled, which hinder the chromosomes from spreading during slide preparation. It will be noticed, however, from the photomicrographs that the overall morphology is very similar to that of the unidentified Musa species.

Genus Heliconia Four species of Heliconia have been investigated in the course of the present study. Of these, three species, namely H. bihai, H. aurantiaca and H. brasiliensis have been studied before by other authors (Agharkar and Bhaduri 1935, Cheesman and Larter 1935, Venkatasubban 1946 and Chakra vorti 1949). The somatic number is found to be 24 in each case.

Heliconia bihai (Fig. 1C, Fig. 6C)

The somatic complement is found to be 24 and the same number is also recorded by Cheesman and Larter (1935).

Here again, as in Musa, the chromosomes form a closely graded series ranging in length from 4.5ƒÊ to 1.4ƒÊ. There are seven pairs of relatively long chromosomes (Fig. 7A, B, C, D, E, F, G) and five pairs of short chromo somes (H, I, J, K, L). Of the long chromosomes, only in one pair (F) is the primary constriction sub-median, whilst in the other six pairs it is more or less sub-terminal. One pair (D) shows a very clear secondary constriction in the median region. Of the short chromosomes, two (I and L) have a median primary constriction, but in the other three this ranges in position from sub-median (H and L) to sub-terminal (J). A feature of particular interest observed in this species is that the chromosomes with sub-terminal primary constriction are very numerous.

Heliconia caribea (Fig. 2B) The somatic chromosome number, now recorded for the first time, is again 24. The morphology of the chromosomes is very similar to that of H. bihai. Heliconia aurantiaca (Fig. 2C) Heliconia brasiliensis (Fig. 2D) In the present investigation, only the meiotic behaviour of pollen-mother cells has been studied. In both cases the pairing is regular and 12 bivalents are observed.

Family Strelitziaceae Genus Strelitzia In the present investigation, three members of this genus, namely S. reginae (Fig. 6E), S. parvifolia (Fig. 2E) and S. parvifolia var. juncea (Fig. 2F) have been studied. Only the meiotic behaviour of pollen-mother-cells has 1970 A Cytological Study of the Zingiberalei 19

Fig. 2. A, Ensete sp.: Drawing of a somatic metaphase showing 18 chromosomes; B. Heliconia caribea, 2n=24; C. H. aurantiaca, first metaphase showing 12 bivalents; D, H. brasiliensis 12 bivalents; E, Strelitzia parvifolia 7 bivalents, F, S. parvifolia var. juncea -7 bivalents and G, Orchidantha maxillaroides-somatic metaphase showing 18 chromo

somes. been examined. In each case there are clearly seven bivalents, showing the very regular pairing at the first meiotic division. An interesting feature observed in this group is the extreme range of endopolyploidy in the tapetal cells (Fig. 6D). From 14 to as high as approxi mately 224 chromosomes are observed in different tapetal cells. So far this feature has not been observed in any other members of Zingiberales.

Family Lowiaceae Previously, no cytological investigation has been made on this family. In the present investigation, two species, namely Orchidantha longiflora and O . maxillarioides, have been studied. In both cases the somatic number is

2* 20 H. K. Mahanty Cytologia 35

found to be 18. The length of the chromosomes ranges more or less con tinuously from 6.6ƒÊ to 4.3ƒÊ. It may be remarked, in passing, that this genus probably has the longest chromosomes to be found in the Zingiberales.

Orchidantha longiflora (Fig. 3A and Fig. 6F) In this species, three pairs of longer chromosomes (A, C, F) have their primary constriction in a sub-median position and in one (E), this is approach ing a sub-terminal position, whilst a secondary constric tion occupies the median position. There is also a secondary constric tion in the pair (D) at sub-terminal posi tion. Two pairs of relatively short chro mosomes (G and H) have a sub-median primary constriction and the third pair has this in a median position (I).

Fig. 3, A, B, C, -Idiograms showing the types of chromosomes Orchidanta maxil in Orchidantha longiflora (2n=18), Costus macrostrobilus (2n= larioides (Fig. 2G) 18) and Tapeinochilus ananassae (2n=18) respectively. Here again the somatic complement is made up of six pairs of long and three pairs of medium size ones. The chromosome morphology is virtually the same as that of O. longiflora, suggesting very little difference between the two species. A clearly stainable 'centromere' is observed in one of the long chromosomes.

Family Zingiberaceae Sub-family Costoideae Tapeinochilus ananassae (Fig. 3C) The somatic number is found to be 18. There are three pairs of long chromosomes (A, B, C), three pairs of medium length chromosomes (D, E, F) and three pairs of short chromosomes (G, H, I). Of the long pairs, two (B and C) show the primary constriction in a sub-median position, and one (A) in a median position. Of the medium length chromosomes, two pairs (D and F) have a median primary constriction and in one (E) it is further 1970 A Cytological Study of the Zingiberales 21 removed from the median position. Of the short chromosomes, two pairs (G and I) have a sub-median primary constriction whilst the third (H) has a median primary constriction.

Genus Costus 7 species of Costus have been studied. Of these only two, namely C. afer and C. speciosus have been investigated previously.

Costus macrostrobilus (Fig. 3B and Fig. 4A)

The somatic number is found to be 18 ranging in length from 3.7ƒÊ to

2.3ƒÊ. An analysis of the chromosome complement has been made. There are three pairs of long (A, B, C), three pairs of medium length (D, E, F) and three pairs of short chromosomes (G, H, I). Of the long pairs, only one (B) shows a clearly median primary constriction, whilst in the other two (A and C) its position ranges from a more or less sub median position (A) to a sub-terminal one (C). Only one pair of medium length chromosomes (D) has a median primary constriction, whilst in the other two (E and F) this is sub median. Of the short chromosomes, two pairs (G and H) have a sub-median primary constriction, whilst in the third one (I) this is median. Fig. 4. A, Costus macrostrobilus-drawing of a somatic meta The overall mor phase, 2n=18; D. Costus sp. (Nigeria), 2n=18: E, Costus sp. phology of the chro (Costa Rica), 2n=18; F, C. albus (Uganda), 2n=36; G, C. mosomes is very schlechteri 2n=27; and H, C. englerianus 2n=36. 22 H. K. Mahanty Cytologia 35 similar to that of Tapeinochilus. The chromosome numbers of the following species have been determined as given in the table below. It will be noticed from the drawings and photomicrographs that the gross morphological difference between the chromo somes of the species is very little.

Table 1

* These unidentified species are growing in our greenhouse and will be determined as soon as they flower.

Svb-family Zingiberaceae Tribe: Hedychieae Genus Kaempferia (sensu lato) Fourteen species of Kaemferia, comprising both African and Asiatic members, have been selected for the present study. K. angustifolia (Fig. 5A) In the present investigation the 2n number is found to be 22. There are three pairs of long chromosomes (A, B, C), five pairs of medium length chromosomes (D, E, F, G, H) and three pairs of short chromosomes (I , J, K). All the long chromosomes have their primary constriction in a sub-median position. Of the medium length chromosomes only one pair (D) shows a sub-terminal position of the primary constriction, two pairs show a median position (E, G) and in the other two the primary constriction occupies a sub median position (F, H). All the short chromosomes show sub-median primary constrictions.

K. sp. (The unidentified African species of Kaemferia , Fig. 5B and Fig. 6H) The 2n number is found to be 42. An analysis of the karyotype shows that there are three basic sets of four chromosomes each. Each basic set is composed of three long chromosomes (A, B, C), nine medium length chromo somes (D, E, F, G, H, I, J, K, L) and two short chromosomes (M , N). All the long chromosomes (A, B,C) show a sub-median primary constriction . Of the nine medium length chromosomes, only one (G) shows a sub-terminal position of its primary constriction, four (E, F, H, K) show this in a sub 1970 A Cytological Study of the Zingiberales 23

Fig. 5. A, Kaempferia augustifolia, (2n=22). Idiogram showing the chromosomal types; B, K. sp. (African)-chromosomal types (2n=42); C, K. carsonii (2n=28) D, K. elegans first metaphase showing 11 bivalents, E, K. gilbertii-somatic metaphase showing 33 chromosomes. F, K, pulchra-11 bivalents in first metaphase; G, K. Kirkii-somatic metaphase, 2n=28; I, K. aethiopica-somatic plate showing 28 chromosomes and J, "K . ethleae" -somatic metaphase showing 26 chromosomes. 24 H. K. Mahanty Cytologia 35

median position, three (I, J, L) have a median constriction and in one (D) the primary constriction is sub-median and also bears a secondary constriction in a sub-terminal position. There are only two small chromosomes (M, N) in each basic set of which one (M) is characterised by the median and the other (N) by the sub-median position of primary constriction. K. brachystemon (Fig. 6G) Only preliminary studies have been made on this species, in which the

Fig. 6. A, Musa sapientum, 2n=33; B, M. cavendishii , 2n=33; C, Heliconia bihai, 2n= 24; D, Strelitzia parvifolia-photomicrograph of a tapetal cell showing endopoly ploidy ( about 112 chromosomes), E, S. reginae-7 bivalents in the first metaphase of pollen mother cells; F, Orchidantha longiflora, 2n=18; G, Tapeinochilus ananasse , 2n=18; H, Kaempferia sp., 2n=28; I, K. rotunda, 2n=33; J, K. brachystemon-2n=26 and K , K. rosea-2n=28. 1970 A Cytological Study of the Zingiberales 25

somatic number is found to be 26. This species is specially included because of its difference in floral morphology from that of any other African species of Kaempferia. The labellum is more or less deeply bifid and is also separated by deep sinuses from the two lateral petaloid staminodes , so that the flower, although showing the general features of the Cienkowskya , resembles those of the Asiatic species of Kaempferia to a considerable degree.

"K . ethelae" (Fig. 5J) Regarding the identity of this plant Dr. Hoog of C. G. van Tubergen's, Holland ui literis says, "I am not sure about the correct name of Kaempferia ethelai. I received under it this name from South Africa, but it does not agree with the illustration in the Gardener's Chronicle of 1898. We have received it from North Transvaal."

Table 2

This is the second species of Kaempferia in which the somatic number is found to be 26. It is particularly noteworthy that the 26 chromosomes of the somatic complement appear to consist of 2 sets of 13, which number is intermediate between the basic number shown by the Asiatic species (namely 11) and that of the other African species so far studied (namely 14). Cytologically, the interesting feature noted is that the pair of chromo somes with a clear secondary constriction which is outstanding in most of the African Kaempferias is not recognisable in this species. It is tempting to suggest that the lack of this particular pair of chromosomes possibly indicates the intermediate position karyologically between Asiatic and African Kaempferias. All the species of Kaempferia studied are given in the table below with the respective chromosome numbers and source of origin. 26 H. K. Mahanty Cytologia 35

Genus Hedychium Hedychium thyrsiforme (Fig. 8B) One plant under this name has been studied previously by Sharma and Bhattacharyya (1959) who found 24 chromosomes in the somatic complement. In the present investigation, the meiotic behavior of pollen-mother-cells has been studied. There are 17 bivalents found at the first metaphase and the pairing seems to be regular. Genus Zingiber Only two species namely; Z. spectable (Fig. 8A and Fig. 9C) and Z. cylindricum (Fig. 9D) have been studied and the 2n number is found to be 22 in each species.

Table 3

Z. spectabile (Fig. 8A) This species is characterized by having two pairs of long chromosomes (A, B), seven pairs of medium length chromosomes (C, D, E, F, G, H, I) and only two pairs of short chromosomes (J, K) . Both pairs of long chromosomes (A, B) show the primary constriction in a sub-median position. Of the seven pairs of medium length chromosomes, four pairs (C, D, E, I) have a sub median primary constriction and the other three (F , G, H) a median one. The short chromosomes (J, K) both have an approximately median primary constriction. In this species the medium length chromosomes constitute the major part of the complement.

Tribe Globbeae Genus Globba G. atrosanguinea (Fig. 7F) The 2n is found to be 48. Only the meiotic division of pollen-mother cells have been studied. Various numbers of multivalent configurations are observed in the first metaphase plates. For example in the Fig. 7E there are 1970 A Cytological Study of the Zingiberales 27

6 trivalents, 12 bivalents and 6 univalents. Data of the various configurations found in different plates are given in the Table 3:

G. heterobractea (Fig. 7B) This species shows 32 bivalents (2n=64) in the first meiotic division of pollen-mother-cells. No multivalent formation has been observed, but in view of the difficulty in studying the metaphase plates of most of the cells, resulting from the overcrowding of the spindle equator by numerous highly contracted chromosomes it is impossible to be sure such never occurs. The interesting

Fig. 7. B, Globba heterobractea-first metaphase of pollen mother cells showing 32 bi valents. C, G. winitii-first metaphase showing 16 bivalents; D, G. albifera var. aurea - first anaphase showing 32 daughter bivalents. E, G. atrosanguinea, (2n=48). Various configurations of first metaphase in a pollen mother cell. F, Roscoea humeana (2n=24) -idiogram showing the types of chromosomes; G, R. Cautleoides-2n=24; H, R. alpina - first metaphase of pollen mother cell showing 12 bivalents; and R. purpurea-somatic metaphase showing 26 chromosomes. 28 H. K. Mahanty Cytologia 35

feature observed in the close spatial association of many of the bivalents in pairs (Fig. 7B). This is interpreted as due to secondary pairing, indicating a considerable degree of residual homology between the numbers of each haploid set, such as would result from hybrid origin. G. albiflora var. aurea (Fig. 7D) and G. winitii (Fig. 7C): In both of these species 16 bivalents (2n=32) were observed and the pairing seemed to be regular. Genus Roscoea Four species of this genus have been studied in this investigation. R. humeana (Fig. 7F) The somatic number is found to be 24. There are seven pairs (A, B, C, D, E, F, G) of medium length and five pairs (H, I, J, K, L) of short chromo somes in this species. Of the medium length chromosomes, two pairs (A, C) have a median primary constriction, in three pairs (B, E, G) it is sub-median in position, whilst in the remaining two pairs (D, F) it is somewhat further removed from the median position. Of the short chromosomes, three pairs (H, I, K) are characterised by a submedian primary constriction, and other two pairs (J, L) by a median one. R. Cautleoides (Fig. 7G) In this species also the somatic number is found to be 24. R. alpina (Fig. 7H) Meiosis in pollen-mother-cells has been studied for the first time, where 12 bivalents are observed in the first meiotic division. In the particular metaphase illustrated (Fig. 7I) there are six ring bivalents and six rod type ones, of course, variation in the configuration is found in different cells but the pairing is apparently always complete, which is in agreement with the fact that this species sets good seeds. R. sp. (Fig. 9B) The somatic number of this unidentified species is found to be 48. This plant could be a tetraploid one, but the study of meiosis and detailed analysis of, the somatic chromosomes are necessary to confirm this statement.

Tribe Alpineae Genus Alpinia A preliminary study of the somatic chromosomes of only two species, namely A. formosana (Fig. 9E) and A. mutica (Fig. 9F) has been made. In both cases the 2n number is found to be 48.

Family Cannaceae In the present investigation, 6 species of Canna have been studied. All 1970 A Cytological Study of the Zingiberales 29

of them showed 18 chromosomes in the somatic complement except only one,

Canna generalis, which was received from Jamaica and showed 27 chromo somes in the somatic complement.

Canna lutea (Fig. 8C)

The chromosomes, which range in length from 3.4ƒÊ to 2.1ƒÊ do not fall sharply into long and short ones. There are six pairs of relatively long chromosomes, (A, B, C, D, E, F) and three pairs of shorter ones (G, H, I).

Only two pairs of long chromosomes (D and E) have a truly median primary

Fig. 8. A, Zingiber spectabile, (2n=22)-idiogram showing the types of chromosomes; B, Hedychium thyrsiforme-first metaphase showing 17 bivalents; C, Canna lutea, (2n=18) -idiogram showing the chromosomal types; D, C. generalis-metaphase plate, 2n=27; E, C. limbata, 2n=18; F, C. orchioides, 2n=18; G, C. coceinea, 2n=18; H, Calathea albertii, (2n=18) first metaphase of a pollen mother cell showing 9 bivalents; I, C. nigricans (2n =22) first metaphase showing 11 bivalents, J, C. wiotii-somatic metaphase, 2n=26; K, C. argyrophylla 2n=27; L, C. leucostachys, 2n=26; and M, C. picturata 2n=24. 30 H. K. Mahanty Cytologia 35 constriction, whilst in the others this ranges through sub-median (A, B, C) to a position slightly nearer sub-terminal (E). Of the short chromosomes, one pair (G) shows a median primary constriction, one pair (H) a sub-median one and in the third pair (I) the constriction is very nearly terminal (Fig. 52, arrowed). This particular pair (I) forms a very characteristic feature in this genus, as with its markedly sub-terminal constriction it can easily be recognised in the complements of the other species. For the rest of the species, Table 4 idiograms have not been prepared individually at this stage. The variation in length of chromo some "I" has been noted and its position indicated in each plate and drawing. The general morphology of the karyotype seems similar, suggesting that the species of this genus form a very homogeneous group. The chromosome numbers for the rest of the species are given in the Table 4.

Family Marantaceae Tribe Phrynieae Genus Calathea

Table 5 7 species of Calathea have been studied and their various chromosome numbers are given in the Table 5: Calathea nigricans: Only a small amount of material for the study of the meiosis in pollen-mother-cells has been available so far. Only a very few cells were observed at the first metaphase and they all showed 11 bivalents . Chromosomes were found to be very small, and all formed ring bivalents with completely terminalised chiasmata. Calathea albertii: Here again, only fixed flower buds for the study of meiosis have been available to me so far. 9 bivalents are observed in the first metaphase of pollen-mother-cells. Again, as in the last species , the chromosomes are small and highly contracted at this stage. In all, the cells studied pairing is found to be regular. This is the first time 2n=18 has been recorded. Calathea argyrophylla: ("Garden variety") from Royal Botanic Gardens , Kew). This was found to show 27 chromosomes in the somatic root-tip cells . 1970 A Cytological Study of the Zingiberales 31

Chromosomes are small; analysis of the chromosome complements is proceed ing and it is not clear yet whether this is going to show the triploid nature of the plant. For the rest of the species of Calathea, the detailed morphology has not been studied at this stage because of their low stainability and adequately clear plates are rare; but, nevertheless, the determination of the chromosome numbers has been carried out with great care and the results can be accepted

Fig. 9. (Plate 2) A, Hedychium thyrsiforme-showing 17 bivalents; B, Roscoeae sp., 2n 48; C, Zingiber spectabile-somatic metaphase showing 22 chromosomes; D, Z. cylindricum, 2n=22; E, Alpinia formosana, 2n=48; F, A. mutica, 2n=48; G, Canna bidentata, 2n=18; H, Calathea cylindrica-first metaphase of 8 bivalents; I, Ctenanthe kummeriana, 2n= 20+2B; J, C. lubbersiana, 20n=20; K, Thalia geniculata-first metaphase of a pollen mother cell showing 13 bivalents; and Stromanthe sanguinea-drawing of a first metaphase show ing 18 bivalents. 32 H. K. Mahanty Cytologia 35

with confidence, although the technical difficulties have so far prevented the construction of detailed idiograms.

Tribe Maranteae Genus Ctenanthe As far as can be discovered, there is no previous cytological data of this genus. Two species, namely C. lubbersiana and C. kummeriana have been studied in the present investigation. In C. lubbersiana the root-tip cells are found to have 20 chromosomes (Fig. 97). Whilst in C. kummeriana, (Fig. 9I) in addi tion to the 20 chromosomes, two small supernumerary ones (possible 'B' chromosomes) have been observed. In the meiosis of the pollen-mother-cells of C. kummeriana we have also seen lagging chromosomes which gives positive evidence of incomplete pairing and possibly connected with the presence of supernumerary chromosome. Thalia geniculata: In the present investigation, only the preliminary study has been made on the somatic cells of the root-tip which show 26 chromosomes. Meiosis in the pollen-mother-cells has also been studied and 13 bivalents are regularly found (Fig. 9K). Regularity of the pairing is in full agreement with the fact that this species produces seed abundantly.

Stromanthe sanguinea: This species was studied previous by Sato (1960) who found 36 chromo somes in the somatic complement. In the present investigation, only the meiosis in the pollen-mother-cells have been studied and found to have 18 bivalents (Fig. 9L). The chromosomes are very small and the pairing is regular.

Discussion Musaceae (sensu lato) The various treatments of the classification of this family by the taxono mists have been reviewed in the introduction. It will be noticed that the Musaceae of Bentham and Hooker (1893) are now in four distinct families, Lowiaceae (Hutchinson 1934), Musaceae (sensu stricto i.e. of Nakai 1941, Heliconiaceae (Nakai loc. cit.) and Strelitziaceae. Excepting the case of Lowiaceae which is nowadays universally accepted, the only question now debated is the claim of the other three groups to familial status. Recently, a very admirable account has been given by Lane (1955) and this has been supported to a certain extent by Tomilson (1959) on the basis of his ana tomical findings. The Lowiaceae are distinct from the rest on their:- 1970 A Cytological Study of the Zingiberales 33

a) pecularity of venation-not found in any other members of Scita mineae, b) very zygomorphic flowers, c) inflorescence in a regular branched cyme (cf. Lane 1955), and d) geographical restriction to the Malayan Peninsula and Borneo. In the present work, two species have been investigated and they both show 9 as the basic number. The chromosomes are longer than those of any other member of the Scitamineae so far observed. The length as well as the morphology of the chromosomes suggest very strongly that this is a distinct group and the familial status, as recognised by the taxonomists, is highly justified, Morphologically, it is unique in its extremely zygomorphic flower, which is a derived character, and at the same time retaining 5 stamens, which is comparatively primitive. Consequently, it is suggested that the basic number 9, which is here definitely established, is a secondary basic number in the family derived by reduction from an ancestral type having 11.

Strelitziaceae (Nakai, loc. cit.) In this family the genus Ravenala is generally regarded as primitive (Stebbins 1951, Sporne 1959) on the basis of its:- a) caulescent habit, b) the nearly regular flower, c) the presence of six stamens, d) distichous arrangement of leaves, and e) the anatomical characters (Tomlinson, lot. cit.). It would be rash to be dogmatic as to the place of origin of Zingiberales as a whole. The majority of present-day species being native to Asia may indicate the centre of origin of the order. Alternatively, a good case could be made out, in view of the present distribution of Ravenala-Strelitzia - Phenakospermum, for an African origin of the Zingiberales. In any case, migration in either direction would have been much more feasible in the past before the great deserts of North Africa, Arabia and Northern India developed. Probably Ravenala-type ancestral forms had a much wider distribution in Central Africa in times past. From here, geographical migration (with con comitant evolution of new species and genera) may be assumed to have taken place in various directions:- i) towards Southern Africa where their descendants are represented by the present-day Strelitzia species, ii) through Western African and across into the tropical rain forests of eastern South American where Phenakospermum is now found, and iii) into Madagascar which has proved a refuge for Ravenala itself. Presumably competition and possibly changes in climate may have

Cytologia 35, 1970 3 34 H. K. Mahanty Cytologia 35

eliminated Ravenala from the mainland. The main changes involved in the evolution of two more advanced genera, Strelitzia and Phenakospermum, have been: -a) The loss of a stamen, b) the development of a straight embryo, and c) the development of differences in the structure of arile. In addition, in Strelitzia a highly modified flower structure has been evolved in connection with ornithophily. It is interesting to note that Lane (1955) has suggested that Phenako spermum is more closely related to Strelitzia than to Ravenala, whereas Tomlinson (1959) on anatomical grounds reported that Strelitzia and Ravenala are more closely related to each other than they are to any other genus. At least one fact is clear from the above conclusions; that these three genera, Ravenala, Strelitzia and Phenakospermum are a closely related group, and the two last-named genera have probably derived from the Ravenala-type ancestor and retain, in varying degrees, certain of the ancestral characters. Cytologically, Ravenala shows 11 chromosomes as the basic number (Cheesman and Larter 1955, Sharma and Bhattacharyya 1959) whilst in Strelitzia most of the species show 7 as the basic number, except one, S. augusta (Cheesman and Larter 1935, Sato 1948 and Simmonds 1954) where this is 11. Simmonds (1954) studied the meiotic pairing in the pollen-mother cells and found nine bivalents and one quadrivalent in about 96% of the cells studied, which indicates an irregularity in the pairing of the chromosomes. In the present investigation, the meiotic division of pollen-mother-cells in three species has been studied and they all show seven clear bivalents and perfect seeds have been recorded in all these instances. It is tempting to suggest that the basic number 7, as in most of the Strelitzias, is a derived one from a. Ravenala-type parent with 11 chromosomes by reduction and that the species like S. augusta take up an intermediate position in the path of this reduction.. Unfortunately, no cytological data have been recorded for Phenakospermum and the chromosome number of this species would have illuminated the above hypothesis to a great extent; but at this stage of our knowledge there is no, doubt about the existence of a number of parallel reduction series in the Zingiberales as a whole.

Musa (sense lato) This genus was divided into three sub-genera, Physocaulis, Eumusa and. Rhodochlamys by Baker (1893). This classification was recognised by tax onomists until Cheesman (1947) removed all species of the sub-genus Physo caulis to a separate genus Ensete Horan, which was originally described by Horaninow in 1862. The rest of the genus Musa he has classified mainly on the basis of cytological characteristics, but taking other facts into consider ation, into the sub-genera Eumusa and Rhodochlamys with a basic number 1970 A Cytological Study of the Zingiberales 35 of 11 and Callimusa and Australimusa with a basic number of 10. This view is now more or less widely accepted. Recently , Shepherd (1959) has found two exceptional forms for which the classification , he suggests, needs to be elaborated. Separation of Ensete from the rest of Musa is still not free from objec tions. For instance, Chakravorti (1951) is inclined to regard Physocaulis as related to Eumusa on cytological grounds and has gone further and attempted to show how the basic number 11, characteristic of most members of Eumusa , has been derived from the basic number 9. According to him Eumusa is a direct descendant of the Physocaulis (Ensete of Cheesman 1947). On the other hand, Cheesman, when considering a phylogenetic origin of the two genera, Musa (sensu stricto) and Ensete, concluded that neither of them is derivable from the other. However, he suggested, on the basis of their close affinities, that they both originated from a common ancestral stock, the genus Ensete probably retaining more of the primitive characters of the ancestral form than did Musa itself. Lane (1955) accepts this separation with hesitation and only on the grounds of:- a) monocarpic habit, b) pollen granulose-papillose, and c) embryo 'T' shaped, in case of Ensete. The size of the seed, to which Cheesman attached quite a lot of im portance, does not form a good distinctive character, since large-sized seeds are also common in Musa (Simmonds 1962). Cytologically, Musa (including Ensete) has the basic number 9, 10 and 11. It is interesting to note that Chakravorti (1951) claims to have shown the origin of basic numbers such as 10 and 11 from 9 by breakage of chromo somes at the region of a secondary constriction. However, increase of basic number in this way has not been accepted by various cytologists (loc. cit.) and, moreover, there are grave theoretical difficulties in the way of accepting it in that it postulates the origin de novo of kinetochore function in each fragment. Furthermore, even if his hypothesis is accepted in spite of these difficulties, it will involve the derivation of the rest of Musa from Ensete, whereas Ensete and Musa seem to be best regarded as two divergent groups from a common ancestral stock (Cheesman 1947). As an alternative, we would suggest in the case of Musa-Ensete, which may plausibly be con sidered to have originated from a common ancestral stock with a basic number 11, that the number 10 found in Musa sensu stricto and the number 9 in Ensete have originated through progressive reduction of one chromosome in each case. It is not suggested that this was a simple loss of a chromosome, but rather a reduction of number following complex structural changes (Darlington 1937 and Sinnot, Dunn and Dobzhansky 1958). Cytologically in Heliconia the basic number is predominantly 12, except in two cases, namely H. metallica and H. seemanii were it is 11. In the

3* 36 H. K. Mahanty Cytologia 35

present investigation meiotic pairing, as well as the somatic chromosomes, has been studied in various species. Taking into account the facts of vegetative and floral morphology, which indicate a high possibility that Heliconia has been derived from ancestors with a general resemblance to the Museae and Ravenaleae, it seems probable that the basic number 12 is a secondary basic number derived from 11, which is also present in some species of the genus. Very little data about the morphology of the chromosomes have been obtained by the earlier workers except for Shepherd (1959), who used the squash technique for preparation of somatic plates. It is interesting to note from his drawings that the previous workers (Cheesman and Larter 1935 and Chakravorti 1951) have certainly exaggerated the frequency of trabants and secondary constrictions. In the present investigation an idiogram has been prepared for both Ensete and Musa and by comparing these two it is found that there is only one pair of chromosomes in each case showing a median primary constriction and, in addition, a secondary constriction in more or less sub-terminal position. Simmonds (1962) may have referred to this when he said "one pair of trabants is usually (perhaps always) present". In Heliconia, however, there is a much higher frequency of sub-terminal primary constrictions among the long chromosomes than in Musa, in fact no less than 8 out of the 12 chromosomes have their centromere in approxi mately sub-terminal position. Here again secondary constriction do not appear to be as frequent as the earlier workers have reported, a conclusion already reached by Simmonds (1962). On comparing the general morphology of the idiograms of Ensete, Musa and Heleconia, it will be noticed that Ensete has a complement of more nearly uniform length, whilst in Musa and Heliconia the chromosomes show a much greater range in length: moreover, in the latter genus, the long chromosomes have a higher proportion of sub-terminal constrictions than in Musa, as noted above, and this character certainly separates the genus from the rest of the Musaceae.

Taxonomic considerations Hutchinson (1934) regarded Heliconia as closely related to Ravenala - Strelitzia group and consequently gave to the whole assemblage the status of a family under the name Strelitziaceae. Lane (1955), on the basis of the flower morphology as worked out by Eichler (1875) and others, the reduced number of ovules, the basal placentation and the root anatomy, suggested that this is a distinct group by giving it a sub-familial status. Although it would seem that this separation is justified, is should be pointed out that by dividing his sub-family Musoideae into three tribes; Ravenaleae, Strelitzieae and Museae, fails to emphasise that the members of the Ravenala-Strelitzia-Phenakospermum group are closer to each other 1970 A Cytological Study of the Zingiberales 37 than they are to Musa or to Heliconia . Despite the disagreement amongst taxonomists as to status, it seems quite clear , on both morphological and anatomical (Tomilson 1959, 1962) and combined with present cytological findings, that there is a need for three equivalent groups , namely; Musa Ensete, Ravenala-Strelitzia-Phenakospermum and Heliconia . This view is already expressed by Simmonds (1962), but by giving a familial status to each of these sections, as suggested by Nakai (1948), one would split them quite apart without showing their close inter-relationship. A better solution might be to recognise the distinctiveness of Lowiaceae whilst the rest of Musaceae could be divided into three sub-families; Musoideae, Strelitzioideae and Heliconiaideae which would be a slight modification of Lane's suggestion. Strelitzioideae could be further subdivided into two or three tribes, viz. 1) Ravenaleae and 2) Strelitzieae, whilst the genus Phenakaspermum could either form a separate tribe or it could be considered part of Ravenaleae. The latter seems very likely morphologically, but because of the lack of cytological data is would be rash to postulate anything definitely at this stage.

Family Zingiberaceae Sub-family Costoideae. So far 15 species of the genus Costus have been studied by various workers (loc. cit.). Of these, ten species have (2n) 18, four have 36 and only one (Simmonds 1954) has 27 chromosomes in the somatic complement. Two exceptional records have been published, namely Boehm (1931) found (2n) 16 in C. cylindricus and Charkravorti (1948) (2n) 44 in an unidentified species. In the present investigation 8 species of Costus and one species of Tapeinochilus have been studied, of which 5 have 2n=18, 2 have 2n=36 and in one 2n=27. It will be noticed that, with the two above-mentioned exceptions, all the species of Costus show somatic complements which are multiples of 9. It should be noted, however, that Simmonds (1954) has more recently reported C. cylindricus as having 18 chromosomes in its somatic complement, which renders Boehm's (1931) statement rather doubtful. Charkra vorti's (1948) report (without figure) or 44 chromosomes in an unidentified Costus may either be a miscount or a case of hypo-pentaploidy (2n=5x-1). The data presented on Tapeinochilus ananassae is also in agreement with a basic number (x) of 9 in this group. C. specious, studied by various workers, has been found to have either 18 or 36 chromosomes. The plant of C. speciosus studied in the present investigation was received from Hong Kong and also shows 18 chromosomes, Chromosomal biotypes were found too in C. afer, which was reported to have 2n=36 by Venkatasubban (1946), whilst in the present investigation 9 clear bivalents have been observed in the pollen-mother-cells. This species, inter alia, is known to produce viable seeds. If Venkatasubban's (1946) plant was C. afer, a genuine members of this species and not a hybrid, it will indicate 38 H. K. Mahanty Cytologia 35

that autopolyploidy has arisen, which is a very common feature in vegetatively propagated plants. Therefore, the present and past findings tend to corroborate the homoge neity of this group with a basic chromosome number of 9. The diploid condition is still predominant, whilst triploids and tetraploids are not un

common.

Tribe: Hedychieae Genus: Kaempferia 16 species of Kaempferia have been the subject of previous work (Raghavan and Venkatasubban 1943), Venkatasubban 1946, Chakravorti 1948 and Sharma and Bhattacharyya 1959) and varying somatic numbers from 22 to 54 have been found. 13 species are studied in this investigation. Raghavan and Venkatasubban (1943) and Venkatassubban (1946) suggested that, since the somatic numbers 24, 36 and 54 are all multiples of 6, there is a regular polyploidy series arising from the basic number of 6. On the other hand, Chakravorti (1948) suggested two distinct lines of polyploid based on the basic numbers of 11 and 14. Sharma and Bhattacharyya (1959), however, made the assumption of a single line of evolution, but with the basic number of 11. The genus Kaempferia is widely distributed in Asia and Africa. The probable origin of the genus is Asiatic, perhaps, in Burma where it is thought, from comparative evidence, to have grown as an evergreen in shady forest conditions (Holttum 1950). From thence it appears to have migrated across most of tropical Asia and right across Africa. With the geographical migration, an adaption to seasonal climates has occurred. Associated with this, new species have arisen showing modified morphological and cytological characters. A careful comparison of the literature (Hooker 1872, Baker 1894, Holttum 1950) and specimens of Asiatic and African species shows a marked difference of flower morphology between the species of the two different geographical ranges. In the flowers of the Asiatic members, the two lateral petaloid staminodes are so separate from the labellum and the latter so deeply 2-lobed as to give the appearance of a four-petalled flower . On the other hand, in the African species the two lateral staminodes and the two lobes of the labellum are strongly united and the compound structure consequently gives the appearance of a single 3 or 4 lobed petal. This difference led to Hooker's acceptance (Bot. Mag. 5994) of Solms-Laubach's (1867) distinct genus Cienkowskya, but later taxonomists have usually regarded Cienkowskya as a sub-genus of Kaempferia (Schumann 1904). K. brachystemon , an African species, is more like the Asiatic ones in general appearance of its flowers (Schumann loc. cit.) and stands, therefore, somewhere in the middle of these two extreme modifications of flower morphology . 1970 A Cytological Study of the Zingiberales 39

It is most interesting to note that a difference of chromosome number is found between the species of Cienkowskya and those of the remaining species of Kaempferia. In the African species, all the somatic complements are multiples of 14, except in K. brachystemon and K. (?) ethelae where only 26 chromosomes have been observed in the root-tip cells, whilst the Asiatic species all show somatic complements which are multiples of 11. On the basis of the morphological, cytological and geographical distribution it has been suggested that African Kaempferias the generic name of Cienkowskya should be resuscitated (Spearing and Mahanty 1964). Assuming the correct ness of Holttum's suggesting about the origin of Kaempferias in South East Asia, it would appear that the original basic chromosome number in this genus was 11, as is still found in the majority of species in this region, and that the new basic number of 14, characteristic of Cienkowskya, arose in connec tion with evolution of the African species. It is tempting to suggest that K. brachystemon and K. (?) ethelae with their different chromosome numbers stand perhaps as an intermediate stage of evolution between the Asiatic Kaempferias and the African Cienkowskyas. The plant of K. (?) ethelae, from which the chromosome count was obtained, has not yet flowered in our greenhouse and so no personal obser vations have been made on its floral morphology, in particular, with respect to its resemblance or otherwise to K. brachystemon, but presumably it is a typical Cienkowskya from the fact that it has been determined (although, possibly, erroneously) as K. (?) ethelae.

Tribe Hedychieae Genus Hedychium Varied chromosome numbers have been found in this genus by previous authors, (Gregory 1936, Raghavan and Venkatasubban 1943, Venkatasubban 1946, Sato 1948 and Sharma and Bhattacharyya 1959). Although Sharma and Bhattacharyya (1959) have reported H. thyrsiforme as having (2n) 24 chromosomes which would indicate a haploid number (presumed x) of 12, it will be seen from the previous data (loc. cit.) that most of the species have 17 as their haploid number, and a polyploid series based on this number exists in the genus. However, they have also found 34 chromo somes in the somatic complement of both H. coronarium and H. gardner ianum instead of the 54 reported by Raghavan and Venkatasubban (1943). In the present study, only one species, namely H. thyrsiforme, has been studied. This shows very regular pairing with 17 bivalents clearly visible; but Sharma and Bhattacharyya (loc. cit.) report the presence of 24 chromosomes in another plant of this species from the Himalayas. Evidentally, either chromosomal biotypes exist in the species or the plants studied in two cases were not conspecific. In any case, the number (x) 17, found in most species of Hedychium, would appear to have originated as a secondary basic 40 H. K. Mahanty Cytologia 35

number from 12 which is the suggested basic number for the whole sub family (Mahanty 1963).

Genus: Roscoea Only one species of this genus, R. alpina has been studied by Sharma and Bhattacharyya (1951) and Malik (1961) and is shown to have 24 chromo somes in the somatic complement. In the present investigation three species, including R. alpina, have been studied and they all show 24 as the diploid (2n) number. Genus: Zingiber Only six species of this genus have been studied previously by various workers (loc. cit.). In the present investigation three species are studied and they all have 22 chromosomes in their somatic complements. 22 is the somatic chromosome number in most of the species of this genus. Exceptions are Z. mioga where Sato (1948) has recorded 55 chromo somes and two forms of Z. officinale, where Janaki Amal (1945) has recorded two "B" chromosomes in addition to its 22 normal ones, and Takahashi (1948) has recorded 24 chromosomes as the somatic complement. It has been suggested by Chakravorti (1948) that the somatic number 55 of Z. mioga indicates pentaploidy with a basic number of 11. It was mentioned in the introduction that in a revision of the classifi cation by Holttum (1950) this genus has been transferred to the tribe Hedy chieae on the basis of their morphological similarities, and anatomical support for this conclusion has been put forward by Tomilson (1956). So far, most of the species studied in this tribe Zingibereae (sensu Schumann) show 48 chromosomes in their somatic complements except the genus Zingiber. Furthermore, the chromosomes of all the species, apart from Zingiber are small to minute in marked contrast to those of Zingiber. From these facts it can be concluded that the remainder of the tribe Zingibereae excluding Zingiber (i.e. Alpineae Holttum), forms a naturally established uniform group. On the other hand, in the tribe Hedychieae it has been found by previous workers and confirmed and extended by my own observations that polyploid, either aneuploid or euploid, occurs in practically all the genera. The lowest somatic numbers found in this tribe, are 22 in Kaempferia and 24 in Roscoeae. Thus on the basis of the number, size (particularly length) and morpho logy of the chromosomes, Zingiber appears to be much more correctly placed in the Hedychieae than in the Zingibereae.

Tribe Globbeae

Genus Globba So far three species of Globba have been studied cytologically by different 1970 A Cytological Study of the Zingiberales 41 workers (loc. cit.). In the present investigation, the meiotic behaviour of pollen-mother-cells in 4 species, namely G. heterobractea, G. winitii, G. albiflora var. aurea, and G. atrosanguinea has been studied. In G. bulbifera , Raghavan and Venkatasubban (1943) suggested that the chromosome number, namely 48, might have come from the basic number of 12, and on this assumption, it is tetraploid. Chakravorti (1948), on the other hand, described this species as an autotriploid arising from the basic number of 16. Sharma and Bhattacharyya (1959), however, recorded 44 chromosomes in G. bulbifera and 22 and 24 for two other species. They have suggested that chromosomal biotypes exist in G. bulbifera. According to them, (2n) 22 is possibly the original number from which 24 and 44 have originated. Alternatively, they suggested that (2n) 24 might be the original number and 22 and 44 have come through loss of chromosomes. G. atrosanguinea was found to have (2n) 48 chromosomes. Only the meiotic pairing in the pollen-mother-cells was studied. In this species a correlation diagram of the number of trivalents and univalents in 10 first metaphase plates showed a strong correlation be tween the number of trivalents and univalents, a fact which was inexplicable if this species was a triploid: but it was to be expected if, on the contrary, it was a tetraploid. This view was further substantiated by the presence of occasional quadrivalents. In the light of these findings, it was suggested that G. atrosanguinea might be a tetraploid plant with a basic number of 12. However, the other three members of this genus showing 16 bivalents in two species and 32 bivalents in another, suggests strongly that 16 may be the basic number in these forms; but the findings in G. atrosanguinea cannot be explained on the basis of 16 being the original number, and, consequently, it seems reasonable to suggest that two basic numbers are in existence in this group. The genus, although Asiatic in origin, has rather a wide range of distribution; from the Eastern Himalayas and Southern China to Borneo and Java. Holttum (1950) has pointed out that different species grow side by side, and it is possible that hybridization may occur, although there is no definite evidence of this. From our cytological findings of G. atrosanguinea it can be concluded, in view of the irregularity of the chromosome pairing, that this species is of hybrid origin, since if it were a simple autotetraploid the maximum associ ations expected would be quadrivalents. G. heterobractea also gives evidence of hybrid origin from the fact that it has 32 bivalents. It is clear from comparison with other species that its chromosome complement is derived from a basic number of 16 and so the plant must be a tetraploid (2n=4x =64). The pairing behaviour, however, is not consistent with its being an autotetraploid, whereas its observed behaviour would be expected on the assumption that it is an allotetraploid (amphidiploid). On the basis of the above facts indicating:- 42 H. K. Mahanty Cytologia 35

a) the wide range of climatic distribution of the genus Globba, b) the greatly overlapping distribution of many of its species, c) combined with the cytological findings of various authors who suggest either 12 or 16 as the basic number -it is suggested that 12 is the original basic number in this genus too and that 16 has arisen as secondary basic number and this has became well established.

Cannaceae No objection has been raised to the familial status of this group which dates from as early as 1889. Canna is the only genus of the family Cannaceae and includes about fifty species. Cytologically, Canna has 9 chromosomes as the basic number, and apart from the triploid species, the pairing is regular and the plants set highly fertile seeds. One characteristic feature is the presence of a pair of small chromosomes 'I' with markedly sub-terminal primary constriction. The American tropics are the natural home of this group, but species are now distributed throughout the tropical regions of the world. The phylogeny of this family presents a great problem. It is a very distinct group and the curious morphology of its flowers is related only to that of Marantaceae and these two families are more closely related to each other than to any other member of the Scitamineae (Holttum 1951). It is possible that the basic number 9 is a derived secondary number from an ancestral type with a basic number of 11, as proposed for other families of Scitamineae, but in the absence of any positive evidence whatsoever, this is tentatively hypothetical. An analogus reduction of chromosome number is, however, to be inferred in Marantaceae in the general Calathea and Maranta.

Marantaceae All earlier workers dealing with the sub-division of this family (cf. Petersen 1889, Schumann 1902, Loesner 1930 and Hutchinson 1934) have agreed that the family falls into two natural sub-units: Phrynieae, on the basis of its three celled and three-ovuled ovary (sometimes, however, two cells undeveloped) and Maranteae with one-celled and one-ovuled ovary. Except Calatheae, which is predominantly distributed in the American tropics, the rest of the genera in the tribe Phrynieae are spread over the tropics of the Old World. All of the genera in the tribe Maranteae are tropical American except Thalia which is also distributed in Africa. Marantaceae show a certain resemblance to Cannaceae in flower morphology (Costerus 1918, Holttum 1951), but Marantaceae can be considered a more advanced group on the basis of the reduction in the number of ovules per chamber to one and, further in the more advanced forms (Marantaceae) reduction of the ovary to a unilocular condition. 1970 A Cytological Study of the Zingiberales 43

Whilst, cytologically, Cannaceae seem to be a very stable group with 9 as the only basic number, Marantaceae, on the other hand , have extremely variable basic numbers ranging from as low as 4 up to 13 being recorded by various workers (loc. cit.). Venkatasubban (1945) suggested evolution along two lines. According to him, those with the basic number 4 have been directly derived from ancestral types with the same basic number: those with x=12 or 13 being derived from a basic number of 6 through a process of duplication. Moreover, he considers the basic number 11 has originated from a secondary basic type, having 12, by the fusion of two chromosomes. Sharma and Bhattacharyya (1958), however, criticized all the possibilities put forward by Venkatasubban (1945) and suggested a single line of evolution from an ancestral type with x=4. According to them, the chromosomal biotypes, as well as the other basic numbers which have arisen through speciation, are propagated by vegetative means only, since flowering in this group is very rare and they have never observed viable seeds. Sato (1960) suggested that Calathea veitchiana has got the most primitive karyotype in the Zingiberales, the basic number being 4 and that the other numbers, such as 6, 11, 12, 13 have been derived from the protokaryotype of 4. From the above survey of the literature, it is clear that most of the authors believe that the basic numbers 9, 11, 12, 13 are the derivatives of 4, which is present in one or two species only, and these particular species also show higher numbers recorded by other workers. Another interesting feature is that the observations of these authors have frequently been restricted to only a couple of genera, namely Calathea and Maranta, which are pre dominantly tropical American. In the present investigation, about 12 species, spread over 6 genera, have been studied. Observations have been made on meiotic pairing in pollen mother-cells as well as on somatic complements. It is thought that geographical distribution may perhapr have played a great part in the evolution of this group and their different basic numbers such as 4, 6, 9, 10, 11, 12 and 13. In this connection, it may be noted that Stachyphryium griffithsii, which is a Malayan genus, has a basic number of 13. Similarly, Marantochloa flexuosa (unpublished data) also has 13 chromosomes in the haploid complement and is restricted to Africa and Thalia geniculata which has a wide distribution in the New World and in Africa also shows x=13. At the moment there is far too little information available about the cytology of the Old World species for any reliable conclusions to be drawn about their phyletic relationship. It has been suggested that the original home of this family is in tropical America (Holttum 1951). If this is the case, then geographical migration to the Far East and African tropics must have occurred, and it is tempting to suggest (although the data at the moment is insufficient to warrant this) that the secondary basic number 13 has become well established in Old World genera. On the other hand, in the American tropics, this group is in an active stage 44 H. K. Mahanty Cytologia 35

of evolution, so that various basic numbers are still to be found. The number 11 which is found in several species of Calathea may be the original basic number in this family from which the other numbers have derived by struc tural changes in the chromosomes combined with gain or loss of chromosomes. It should also be mentioned that vegetative method of propagation may be an important factor in the evolution of this group, since it enables the perpetuation of the chromosomal biotypes which would otherwise die out, but production of flowers is not at all rare (e.g. Thahlia, Calathea, Ctenanthe, Maranta), and seeds are also often produced.

Evolution of Chromosome Number in Scitamineae There have been two different views regarding the evolution of higher chromosome numbers in different families of Scitamineae. On the one hand, Raghavan and Venkatasubban (1943), Venkatasubban (1946), Sharma and Bhattacharyya (1958, 1959) and Sato (1960) suggested that polyploidy and aneuploidy combined with structural changes of the chromosomes are the main causes of higher numbers. Most of these authors agree that lower numbers such as 4, 5 and 6 are the original basic numbers and secondary basic numbers such as 9, 10, 11, 12 and 13 have come through further evolution. Sato (1960) suggested that the most primitive karyotype in Zingiberales is to be found in Calathea veitchiana, which shows 2n=8, and various stable karyotypes such as 9, 11 and 12 have derived from this. In the light of morphological characteristics, Marantaceae cannot be regarded as primitive, and so the occasional records of 4 or 6 in the members of this family are more plausibly regarded as reductions from higher numbers than as true original basic numbers for the whole of the Zingiberales. On the other hand, Chakravorti (1948, 1951) suggested a completely different view. According to him, the increase in number has been brought about by fragmentation at the region of "supernumerary" (i.e. secondary) constrictions. His hypothesis has been described and criticized in detail previ ously (Mahanty 1963). In short, two changes are necessary for Chakravorti's hypothesis to be true: i) a wholesale fragmentation of the chromosomes at secondary con strictions must have repeatedly taken in some genera; and ii) the simultaneous development of centromeric activity at the points previously occupied by these secondary constrictions. The first supposition is, however, unacceptable in the light of the findings in the present investigation, because the higher number of secondary con strictions, as found by Chakravorti, are not observed. The second supposition , namely the development of centromeric activity at the region of the secondary constrictions, has yet to be demonstrated in the case of any other plant or animal and, consequently, particularly weighty evidence in support of it would be necessary before it would be acceptable. 1970 A Cytological Study of the Zingiberales 45

On morphological and anatomical grounds, Ravenala is regarded as the most primitive genus of the Zingiberales now living. It seems possible that the whole of the Scitamineae may have originated from Ravenala-type ancestral forms of which the only relict now left is in Madagascar, where perhaps competition is not as high as in the mainland. Ravenala has 22 chromosomes in the somatic complement, and in one of its allied genera, Strelitzia, the somatic number is predominantly (2n) 14, but in one species, namely S. augusta, the number is 22. In Heliconia only two species are known to show 11 as their haploid number, whereas the other species show 12. In Musa the subgenera Eumusa and Rhodochlamys have predominantly x=11. This number also occurs in all the tribes of Zingiberoideae and in Marantaceae, although, admittedly in some cases, only in one or two species of a genus. On these observations, it is suggested that 11 may be considered as the most probable original basic number and the other numbers as derived from this. Thus, in Musaceae (sensu stricto) there are two secondary basic numbers which are well established: in the genus Ensete the secondary basic number 9 is very stable, the number 10 as is in Callimusa and Australimusa. On the other hand, in Heliconia the predominant karyotype is 12, although 11 is found in certain species. In Lowiaceae and Cannaceae the basic number is exclusively 9, and this number is also found in most of the sub-family Costoideae. It is very interesting to note that these groups, which have 9 as their basic number, show a particular stability in this respect, and it would seem that further speciation in these groups, although it may involve poly ploidy, has not affected the basic number. The numbers are very variable in the sub-family Zingiberoideae and the family Marantaceae. On the basis of cytological observations it has been suggested that 12 is the original basic number in the Zingiberoideae: this must then be regarded as a secondary basic number, if 11 is considered to be the original number for Zingiberales. Since this group shows evidence of being in a stage of active evolution (Holttum 1950), establishment of secondary number in different directions is only to be expected, for example, 11 in Zingiber and Asiatic Kaempferias, 12 in the tribe Alpineae, 13 and 14 in Cienkowskya, 16 in Globba and 17 in Hedychium. Marantaceae present a particularly difficult problem because of the small size and very low stainability of the chromosomes and, in addition, the numbers recorded show a great range as well as variability in individual species. It is consequently very difficult to suggest with confidence the ancestral haploid number for this family. Various basic numbers (4, 6, 9, 10, 11, 12 and 13), most of which at least must be secondary, occur in both the tribes. It is interesting to note that 3 Old World species investigated by us show 13 as their haploid number: two of these species, Stachyphrynium griffithsii and Marantochloa flexuosa belong to the Phrynieae, the more primitive type from the point of view of floral morphology. Number (x) 13 also occurs in 46 H. K. Mahanty Cytologia 35

high proportion of the species of Calathea and Maranta investigated by various workers. It thus looks possible that 13 is the ancestral basic number in this family. Here again, it is presumed that this must have come from 11. A great deal of morphological and cytological work is needed in Marantaceae in order to confirm this hypothesis and work out the cyto-taxonomic relation ships. Geographical distribution has played a great part in the formation of these secondary basic numbers accompanied by morphological changes. This has been clearly demonstrated in the case of the genus Kaempferia. The increase or decrease of chromosome numbers from the original basic number of 11 has not evolved through simple loss or gain of chromosomes, but through complex structural changes in the chromosomes Part of the problem in considering this evolution of chromosome numbers arises from uncertainty as to the trustworthiness of the numbers recorded by various workers, particularly when different numbers are recorded for the same species. This could, at least in some cases, well be due to the technical difficulties arising from the small size and low stainability of the chromosomes frequently combined with their high numbers, which results in miscounting. Finally, in the course of this work it has become abundantly clear that if cytology is to be of real service to taxonomy, there is an urgent need for:- a) preservation of cytological slides in national collections where they can be consulted by other workers, and b) the preservation of herbarium and spirit material of the plants in vestigated. In this way, it would be possible for an unprejudiced observer to check whether unusual chromosome counts are due to an error or are genuine , and whether the plants studied really belong to the species alleged.

Acknowledgments

Most part of this work was carried out during the tenure of a Research Assistantship in the Department of Botany and Zoology at Sir John Cass College, London, and I would like to express my gratefull thanks to Dr . J. K. Spearing for his supervision and valuable criticisms during this investi gation. I am also greatly indebted to Mr. B. L. Burt and Dr. J. Ratter of the Royal Botanic Gardens, Edinburgh, and Dr. K. Jones, Royal Botanic Gardens , Kew, for their assistance in supplying material and technical advice.

Summary

1. The chromosome numbers of 64 species have been studied on which 56 are investigated for the first time. 1970 A Cytological Study of the Zingiberales 47

2. Morphological, anatomical and cytological findings on the Musaceae in the broad sense are considered from a phylogenetical standpoint and three distinct groups: a) Musa-Ensete, b) Ravenala-Strelitzia-Phenakospermum and c) Holiconia are recognised as suggested by some authors . For these groups sub-familial status is proposed. 3. Lowiaceae have been studied for the first time cytologically and their chromosome morphology, distinct from that of any other group of Zingiberales , confirms their claim to familial status. 4. Consideration of the conflicting theories proposed to account for the higher chromosome number in genera like Globba, Alpinia, and Phaemeria , etc. leads to rejection of Chakravorti's hypothesis of wholesale fragmentation of chromosomes and the acceptance of the opposed view of Raghavan and Venkatasubban and others. 5. Holttum's transference of the genus Zingiber to the tribe Hedychieae has been given cytological support on the following points: a) the basic number in the genus Zingiber correlates with that of Kaempferia. b) the new tribe Alpinieae (which is infact Zingibereae without Zingiber) have consistently 48 chromosomes in their somatic complements. 6. It is suggested that the African representatives of Kaempferia should be given the status of genus; Cienkowskya on the following points: a) the difference in floral morphology b) geographical separateness and c) the difference in the number and morphology of the chromosomes. By comparing the present and past findings, various lines of evolution of the chromosome complements within each group are discussed. 7. The basic number 11 is considered to be probably the original one for the Zingiberales as a whole, being present in Ravenala which is the most primitive member in the order. From this secondary basic numbers have arisen through evolution.

References

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