Cytologia 39: 419-427, 1974

Cytological Studies in Ottelia alismoides Pers.1

M. P. Misra

Post-Graduate Department of Botany, Magadh University, Bodh Gaya, Bihar, India

Received November 20, 1972

The systematic status of Helobiae of Engler and Prantl (1936) to which Ottelia alismoidesPers. belongs has been subjected to considerable debate by taxonomists. In the two phylogenetic systems of classification, namely that of Engler and Prantl and that of Hutchinson, the position of this order varies significantly. Engler and Prantl considers Pandanales to represent the starting point of monocotyledons, me mbers of the order Helobiae being considered as advanced genera. On the other hand, Hutchinson, a champion of the reduction theory con siders the apparent simplicity in the floral structures of Pandanales of monocoty ledons is not due to primitiveness but due to extreme specialization and reduc tion. According to him, the two principal orders of Helobiales namely Alismatales and Butomales have been regarded as the two starting points of monocotyledons from which other orders of monocotyledons have been derived. He further stressed the origin of these two orders from Ranales of dicotyledon. In such case of taxonomic disputes, cytological evidences often provide with a good indication of phylogenetic relationship. Several workers have no doubt tried to deal with the cytology of the different members of Helobiae, Harada (1956), Sharmaand Bhattacharya (1956),Pogan (1961, 1962), Larsen (1963), Gadella and Kliphius (1963) practically no work has been done to study the karyotypes of Otteliaalismoides in relation to its different habitate. In this case high incidence of different degree of polyploidy have been obtained. No attempt has either been made to correlate such polyploidy with their ecological conditions or to find out the extent to which structural alterations too are associated with this polyploidy. In view of the possibilities mentioned above the present investigation was taken up. In the present scheme of work not only a detailed karyotype study has been made but on th basis of present and previous records, an attempt has been made to present a cyto-taxonomical picture of this group with special emphasis on the lines of evolution.

Materials and methods

Plant materials were collected from two different localities of Bihar and eight different localities of Bengal as detailed below:

I. Plants collectedfrom Bihar

1 This work was undertaken for the Ph . D. thesis. Part of the work was carried on in the Cytogenetics Laboratory of Calcutta University, Calcutta, India. 420 M. P. Misra Cytologia 39

1. Plant type A collected from Sahjangi Pond at Bhagalpur Plants with elliptica leaves without spines on the margin of the petioles. 2. Plant type B collected from a Pond at Patna Plants with cordate leaves with spines on the margin of petioles. II. Plants collected from Bengal 1. Kalyani lakes near Calcutta type Kl Plants with more or less lanceolate type of leaves, wavy in outline. 2. Kalyani lakes near Calcutta type K2 Normal type with cordate leaves, spines on the margin of petioles. 3. Kalyani lakes near Calcutta type K3 Plants with Vallisneria like leaves without spines, showing meiotic irregularities 4. Kalyani lakes near Calcutta type K4 Plants with screwpine like leaves. 5. Kalyani lakes near Calcutta type K5 Plants with more or less reniform leaves at the top and lanceolate leaves below, with spines on the margin of petioles. 6. Indian Botanical Gardens at Shibpur type G1 Plants with linear leaves with spines on the margin of petioles. 7. Indian Botanical Gardens at Shibpur type G2 Plants with large more or less reniform leaves with spines on the margin of petioles. 8. Indian Botanical Gardens at Shibpur type G3 Plants with elliptical leaves without spines on the margin of petioles.

Somatic preparations were made of healthy roots. They were first pretreated in different pretreatment chemicals such as 0.5% colchicine solution, 0.002M oxyquinoline solution, saturated para-dichlorobenzene and aosculine solution and

various combination of above chemicals. It was however found that 0.002M oxyquinoline saturated with aesculine was found to be most suitable pretreatment

chemical in this case. Healthy root tips were cut and pretreated with a mixture of 0.002M oxyquinoline saturated with aesculine at 0•K-5•Ž for 3-5 minutes and then transferred to and kept in cold (10•K-12•Ž) up to 3 hours. They were fixed in acetic alcohol (1:3) mixture for one hour and then kept in 45% acetic acid solution for

10 minutes followed by staining and squashing as usual. The magnification of the figure drawn was•~2100. The chromosome with secondary constrictions were drawn in out-lines. Photographs were taken from temporary preparation and were suitably enlarged.

Observations

The somatic chromosome numbers of the different plant types so far investi

gated have been found to be 2n=44, 2n=66 and 2n=Ca 132. A detailed karyo type analysis of different plant types reveals a difference in the position of the

secondary constriction. Though the chromosomes are of well graded series, yet the size difference is well marked, ranging from 12 .5ƒÊ to 2.75ƒÊ. On the basis of 1974 Cytological Studies in Ottelia alismoides Pers. 421

Figs. 1-6. la, Ottelia alismoides (type A) . Somatic metaphase plate showing 2n=66 chromosomes (•~2100) and its idiogram respectively . 2, O. alismoides (type B). Showing 33 bivalents in the metaphase 1. •~2100 . 3, O. alismoides (type CK1). Showing 22 bivalents at meiotic met aphase 1. •~2100 . 4, O. alismoides (type KC2). Somatic metaphase plate showing 2n=66 chromosomes . •~2100. 5, O. alismoides (CK2). Showing 33 bivalents in meiotic metaphase 1. •~2100 . 6, O. alismoides (type CK4) showing 32 bivalents and two univalents at meiotic meta

phase 1. •~2100. 422 M. P. Misra Cytologia 39

relative length of the chromosomes, they can be divided into five different groups viz. very long, long, medium, short and very short types. The main chromosome types which have been observed here are as follows: Type A: Very long to long chromosomes with median to nearly median primary constrictions.

Figs. 7-9. 7, O. alismoides (type CK5). Somatic metaphase plate showing 2n=Ca 132 chromo somes. •~2100. 8-8a, O. alismoides (type DG1). Somatic metaphase plate showing 2n=44 chro mosomes (•~2100) and its idiogram respectively. 9-9a , O. alismoides (tyoe DG2). Somatic meta phase plate showing 2n=66 chromosomes (•~2100) and its idiogram respectively.

Type B: Long to medium chromosomes ranging from 9 .5ƒÊ to 3.0ƒÊ with subter minal primary constrictions. Type C: Long to medium chromosomes each two constrictions , primary and secondary, one located at submedian region , the other at subterminal 1974 Cytological Studies in Ottelia alismoides Pers. 423

at the distal end of the short arm.

Type D: Medium sized chromosomes, ranging from 7.0ƒÊ to 2.5ƒÊ, with median primary constrictions.

Type E: Comparatively long chromosomes each with a submedian primary constriction and a satellite at the distal end of one arm.

Type F: Medium sized chromosomes, ranging from 6.25ƒÊ to 2.4ƒÊ with subtermin - al primary constrictions.

Type G: Medium sized chromosomes, each with two constrictions, primary and secondary, one situated at the submedian region, the other at the

subterminal position at the distal end of the short arm.

Type H: Short to very short chromosomes ranging from 4.25ƒÊ to 2.25ƒÊ, with nearly median to subterminal primary constrictions.

Type I: Very short chromosomes with subterminal primary constrictions.

The different plant types differ in having different combinations of the above types. A detailed description of the somatic components of the different plant types studied here is given below:

I. Plant type A collected from Bihar

2n=66 C8+D10+E4+F12+H24+I8 (Figs. 1 and la). 2. Pond at Patna Only meiotic preparation could be made. Meiotic metaphase I shows 33 bivalents with regular separation. The size of the PMC is 98.1ƒÊ in length and 63.7ƒÊ in breadth. Thus it is the largest PMC recorded here (Fig. 2).

II. Plant types collcted from Bengal

1. Kalyani lakes (type K1) Only the meiotic preparation could be made, The PMC shows n=22 at metaphase I. The size of the PMC is 64.47ƒÊ in length and 32.3ƒÊ in breath

(Fig. 3). 2. Kalyani lakes (type K2)

2n=66=A2+B2+C4+D8+F28+G2+H14+I8 (Fig. 4) The meiotic preparation shows a large PMC 63.75ƒÊ in length and 56.1ƒÊ in breadth, with a large prominent nucleous, slightly away from the centre, with n=33, one pair of bivalents are in contact with the nucleolus (Fig. 5).

3. Kalyani lakes (type K3) Only meiotic preparations could be made. Meiosis shows various irregu larities such as early separation and laggers of bivalents as well as univalents

(Figs. 14, 15 and 16). 4. Kalyani lakes (type K4)

Only meiotic preparation could be made . The PMC shows n=33, composed of 32 bivalents and two univalents . The size of the PMC is 71.06ƒÊ in length and 64.47ƒÊ in breadth (Fig . 6). 5. Kalyani lakes (type K5)

2n=Ca 132 (Fig. 7)

Only the somatic number could be counted , which is 2n=Ca 132. The 424 M. P. Misra Cytologia 39 chromosomes are very small in comparison to other types. A variation number of 2n=66 has also been observed in this case. 6. Indian Botanical Garden, Shibpur (Bengal) (type G1) 2n=44=A10+B16+D6+F12 (Figs. 8 and 8a). 7. Indian Botanical Garden, Shibpur (type G2) 2n=66=A10+B4+C4+D10+F26+H6+I6 (Figs. 9 and 9a)

The meiotic metaphase shows n=33 with regular separation. The size of the PMC is 70.125ƒÊ in length and 52.225ƒÊ in breadth (Fig. 10).

Figs. 10-13. 10, O. alismoides (type DG2) . Showing 33 bivalents at meiotic metaphase I. •~2100. 11-11a, O. alismoides (type DG3). Somatic metaphase plate showing 2n=66 chromosomes (•~2100) and its idiogram respectively. 12, O . alismoides (type DG3). Showing 33 bivalents at meiotic metaphase I.•~2100. 13, O. alismoides (type E) . Showing 33 bivalents during meiotic meta

phase I. •~2100.

8. Indian Botanical Garden Shibpur (type G 3)2 n=66=A14+B14+C2+D2+E2+F16+H10+I6 (Figs . 11 and 11a). The meiotic preparations shows a large PMC at metaphase with pointed ends. 1974 Cytological Studies in Ottelia alismoides Pers. 425

It is 76.67ƒÊ in length and 53.76ƒÊ in breadth. There are 33 bivalents. (Fig. 12). 9. Pond at Champahati Only meiotic preparation could be made. The meiotic metaphase I shows

33 bivalents while other irregularities as such as early separation and laggers

Figs. 14-18. 14, O. alismoides (type CK3). Microophtograph showing early separation in anaphase

I. •~2100. 15, O. alismoides (type CK3). Microphotograph showing early separation in ana

phase II. •~2100. 16, O. alismoides (type CK3). Microphotograph showing laggers of bivalents as well as Univalents. •~2100. 17, O. alismoides (type E). Microphotograph showing early sepa

ration and laggers. •~2100. 18, O. alismoides histogram showing length of haploid compliments

of different localities. 426 M. P. Misra Cytologia 39

are present in other PMC (Fig. 13) and (Fig. 17). The size of the cell is 49.725 ƒÊ in length and 34.425ƒÊ in breadth.

Discussion

Species of Ottelia are remarkable in the sense that the populations differ with respect to the external morphology of the plant. The differences of the external morphology mainly involve the character of the leaf including shape and size, exhibiting greater or lesser photosynthetic surface. As may be noted from the plants studied here and previous investigations, that polyploidy is of frequent occurrence in these species (Murthy 1933, Islam 1950, Rao 1950, Darlington and Wylie 1955, Harada 1956, Sharma and Bhattacharya 1956). Out of the six popula tions studied here, four have been noted to have 2n=66 chromosomes whereas of the rest two populations, one is characterised by 44 chromosomes (G1) and the other by 132 chromosomes (K5). In Botanical Garden alone, out of the three popula tions 2n=66 chromosomes have been found in two and 44 chromosomes in the other. In what way such polyploidy are related to the minute ecological detailsis yet to be investigated. Broadly, however, the ecological conditions do not appear to be different. The analysis of water of the different populations is under pro gress which may reveal facts indicating such precise correlation. However, the difference in external morphology in different populations contrast strongly with that of Hydrilla and Vallisneria where external morphological characters were apparently found to be identical in different populations. Though the degree of polyploidy is very high, reaching the number 2n=132 chromosomes, clear bivalents have been found in all of them. Such a condition evidently indicates interspecific hybridization before or after duplication leading to allopolyploidy. The absence of multivalents could otherwise have been explained on the basis of chromosomes length provided Ottelia would have been short. A feature worth noticing in the idiogram of this species is the comparatively longer chromosomes in type G1 with 2n=44 chromosomes, where as shorter chromosomes occur in populations with higher number of chromosome. The diminution in chromatic matter along with polyploidy has been an associat ed feature of evolution in different families of flowering plants. It was found that hetrochromatin elimination, appears to be more probable explanation for diminu tion in chromatic content, in the light of evidence so far obtained.

Status of the taxon: Ottelia falls under the family Hydrochantaceae included under Butomalesof Hutchinson. Even though the position of Butomales as a primitive order, be accept ed the position of Hydrochaitaceae within this primitive group is hardly justi fiable. It may be noted that in Ottelia asymmetry in chromosome, together with size difference amongst member of the complement, is remarkable. Coupled with these considerations polyploidy in this genus suggests an evolutionary advance. Ottelia comes under the tribe ottelieae under Vallisnarioideae of Hutchinson. On the basis of chromosomal characteristics it is not possible to comment on the 1974 Cytological Studies in Ottelia alismoides Pers. 427 justification of such a separation. On the basis of the external morphological consideration it is better at present to consider the position of the genus under the tribe as reasonable.

Summary

Ottelia from four different localities belonging to Nothern India, have been studied in the present work with the aid of improve method. Detailed study of karyotypes have been included within the scope of investigation. Special emphasis has been laid on the study of different populations of the same species to assess the role of structural alternations in the chromosomes in the evolution of different populations. Species of Ottelia are remarkable in the sense that the populations differ with respect to the external morphology of the plant. The diminution in chromatic matter along with polyploidy has been an associated feature of evolution in different families of flowering plants. The same is true in case of Ottelia. Hetrochromatic elimination appears to be more probable explanation for diminution in chromatic content, in this light of evidences so far obtained.

Acknowledgement

The author wishes to express his deep indebtedness to Prof. A. K. Sharma, D. Sc., Head of the University department of Botany, University of Calcutta, for regular guidance during the course of the investigation and facilities provided. The author is thankful to the University Grants Commission, for providing necessary funds to carry on his work.

References Darlington, C. D. and Wylie 1955. Chromosome Atlas of Flowering Plants. George Allen and Unwin Ltd. London. *Englerand Prantl 1936. Die naturlichen Pflanzenfamilien. *Gadella, Th. W. J. and Kliphy, E. 1963. Chromosome number of flowering plants in the Nether lands. Acta Botanica Neerlandica 12: 195-230. Harada, I. 1956. Cytological studies in Helobiae I. Chromosomes idiograms and a list of chro mosome number in seven families. Cytologia 21: 306-328. Islam, A. S. 1950. A contribution to the life history of Ottelia alismoides. Pres. Jour. Indian. Bot. Soc. 29: 79-91. Larsen, K. 1963. Studies in the flora of Thailand. Dansk Bot. Arkiv Bind. 20, 3: 211-275. Murthy, S. N. K. 1933. J. Mysore Univ. 7: 1, taken from Darlington and Wylie, 1955. Pogan, E. 1961. Odrebnose gatumkow i proba wyjasnieia genezy alisma lanceolatum with Acta Soc. Dot. Pol. 30: 667-718. - 1962. Badania Kariologicznenad Alisma L. Pochodzenie iewolucja knolypu, A. lancaolatum with Acta Biologica Cracoviesia 5. Rao, Y. S. 1950. Proc. Indian Sci. Cong. 1950. Pt. 3: 67, taken from Bot Monograph No. 3 by Subranyam. 11962 (Council of Scientific and Industrial Research, India). Sharma, A. K. and Bhattacharyya, B. 1956. A study of the cytology of the four members of the Hydrocharitaceae as an aid to trace the lines of evolution. 6: 123-134. * Original not consulted .