_??_1992 The Japan Mendel Society Cytologia 57: 409-415, 1992

Karyological Studies on Some Indian Charophyta

B. V. S. Subrahmanyam and Y. B. K. Chowdary

Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi-221 005, India

Accepted January 16, 1992

The charophytes have been subjected to extensive cytological investigations because of their wide distribution coupled with easily available nuclear division stages and large sized chromosomes etc., and also controversial taxonomic treatments. Karling (1926) has given a detailed historical account of earlier investigations. Since then, several investigators (Tuttle 1926, Lindenbein 1927, Steil 1941) have made valuable contribution to the knowledge of the cytology of this division. Consequently, chromosome numbers of vast number of taxa have been brought to the lime light by various workers from India (Sundarlingam 1946, Sarma and Khan 1964, 1965a, b, c, Khan and Sarma 1967a, b, c, Sinha and Noor 1967, Chatterjee 1971, Ramjee and Sarma 1971, Ramjee and Bhatnagar 1978, Labh and Verma 1984, 1985a, b, Bhatnagar and Johri 1986, Ray and Chatterjee 1988a, b) and outside this country (Gillet 1959, Imahori and Kato 1961, Hotchkiss 1963, 1965, Tindall and Sawa 1964, Guerlesquin 1967). The cytological work has been reviewed from time to time by Sarma (1982, 1988). However, the number of investigated taxa are less as compared to the number of taxa known from India. Inview of this, an extensive collection of charophytes from south India has been made and the present communication deals with karyology of two taxa of Chara Linn. and a taxon of Nitella Ag. Leonh.

Materials and methods

The algal materials under investigation (C. vulgaris var. gymnophylla f. grovesii (Pa1) RDW, C. zeylanica var. sejuncta f. formosa (Roxb.) RDW and Nitella heterodactyla Fil. and G. O. A. em Fil.) were collected from the freshwater ponds and tanks of Kerala, India during October 1984. From the collected materials, branchlets bearing young antheridia (globules) were fixed in 1:3 glacial acetic acid absolute ethanol mixture (Carnoy's fluid) at the time of collection and some portions were also fixed during 7.00 P. M. to 10.00 P.M. at the camp site. The plants from which portions were fixed for karyological study were also preserved in the form of dried herbarium or pickled material in 4% formalin solution and were used for sys tematic identification. Materials were also identified following the monographs of Pal et al. (1962) and Wood and Imahori (1965). Godward's iron alum acetocarmine technique (Godward 1948)was followed for cytological studies of spermatogenous filaments. The chromosome numbers were determined from the actively dividing filaments. Photomicrographs were taken from temporary preparations, which were made permanent later following the method derived by Darlington and La Cour (1962). Chromosome morphology was designated according to Levan et al. (1964) on the basis of centromeric index values.

Observations

Karyological characteristics were made in the spermatogenous filaments of antheridia, since they are free from any pigments and food reserves. Nuclear divisions are very frequently 410 B. V. S. Subrahmanyam and Y. B. K. Chowdary Cytologia 57

Figs. 1a, b-3a, b. Photomicrographs of squashed metaphase of antheridial filament nuclei and respective outline drawings of chromosomes of Chara Linn. and Nitella Ag. Leonh species. 1. Chara vulgaris var. gymnophylla f. grovesii (n=35).

Figs. 2-3. 2. C. zeylanica var. sejuncta f. formosa (n=42), 3. Nitella heterodactyla (n=18). Bar=5ƒÊm.

Figs. 4-6. Karyotypes of Chara and Nitella species. Bar=10ƒÊm. 1992 Karyological Studies on Some Indian Charophyta 411

synchronous in young filaments . Interphase nucleus occupies half of the cell volume of the spermatogenous cell. Interphase nuclear diameter ranges from 11 .0ƒÊm in N. heterodactyla to 13.4ƒÊm in C. zeylanica var . sejuncta f. formosa. Interphase nucleus enlarges to 1.5 times

to the original sizes during prophase stage . There is a centrally placed nucleolus in all the taxa studied. Nucleolar size varies from 2 .0ƒÊm in C. vulgaris var. gymnophylla f. grovesii to 4.2ƒÊm in C. zeylanica var . sejuncta f. formosa. Single nucleouls per nucleus was observed in both the taxa of Chara while in the taxon of Nitella two nucleoli were noticed. At early

prophase, chromosomes appears as a few stained granules, later followed as thick pieces of chromatin threads. In all the three taxa, early prophase chromosomes exhibit discontinuous

staining. At metaphase the chromosomes condense and become short and thick . Chromosome counts recorded in the present study are n=35 for C . vulgaris var. gym nophylla f. grovesii (Fig. 1), n=42 for C . zeylanica var. sejuncta f. formosa (Fig. 2) and n=18 for N. heterodactyla (Fig. 3). The chromosomal length varied from 1 .12ƒÊm to 5.60ƒÊm in the taxa of Chara and in Nitella it is 3.66-8.80ƒÊm; thickness, it is from 0.8ƒÊm to 1.6ƒÊm in Chara and 1.6ƒÊm in Nitella. The longer chromosomes usually possessed median and sub median centromeres and the shorter chromosomes invariably possessed sub-terminal and terminal centromeres (Figs. 4-6). After anaphasic separation of chromosomes telophase was established and two daughter nuclei were formed.

Discussion

The karyological features of the three taxa studied have been summarized in the given table. The nuclear events encountered in these taxa are in conformity to the standard pat tern of mitosis met with in higher plants (Tuttle 1926, Khan and Sarma 1967c). Variation in the interphase nuclear diameter was quite evident in all the three taxa studied. The diameter ranged from 11.0ƒÊm to 13.4ƒÊm in the taxa of Chara and it was 11.0-12.3ƒÊm

in Nitella species. Variation in size of interphase nuclei in both the genera were also reported by Khan and Sarma (1967c) and Guerlesquin (1967). The interphase nuclear diameter is nearly

proportional to the level of ploidy in both the genera studied. This finding is also in conformity with the one reported by Khan and Sarma (1967c). They opined that 'lower the ploidy level smaller the size of interphase nucleus while higher the ploidy larger the size'. The presence of solitary, spherical nucleolus in the taxa of Chara was in conformity with the earlier findings of Khan and Sarma (1967c), Guerlesquin (1967) and Ramjee and Sarma (1971). However, in the taxon of Nitella two nucleoli was observed. This is indicative that this taxon may have originated due to hybridization between taxa of two different genomic constituents. The chromosome numbers determined in the present study are n=35 in C. vulgaris var. gymnophylla f. grovesii, n=42 in C. zeylanica var. sejuncta f. formosa and n=18 in N. hetero dactyla. These three counts represent intermediary chromosome complements between those reported by earlier workers, representing n=6 as the lowest number in several taxa of Nitella (Sarma 1982) and n=70 in C. zeylanica as highest number (Hotchkiss 1963) so far reported in charophytes. The present two counts (n=35, 42) have also been recorded for a number of other taxa belonging to the genus Chara (Hotchkiss 1963, Griffin and Proctor 1964, Sarma and Khan 1965b, Sinha and Verma 1970, Ramjee and Sarma 1971, Subramanian 1983, Ray and Chatterjee 1988a) and n=18 in various taxa of Nitella (Sarma 1982). It is evident therefore that the distribution of a particular count over a large number of morphologically distinct taxa clearly indicates a significant role of chromosomal differentiations in speciation besides role of polyploidy in both the genera. All the taxa worked out in the present study were investigated for the first time and their Table 1. Karyological characteristics of the investigated taxa

m=median; sm=sub-median; st=sub-terminal; t=terminal. 1992 Karyological Studies on Some Indian Charophyta 413 chromosome numbers determined are new to science (Table 1). From the chromosome num bers so far reported in both the genera, including the present ones suggest that euploidy appears to have played a decisive role in speciation and evolution in these two cosmpolitan genera of Charophyta. The chromosomes of the three taxa worked out showed variation in their chromosomal size at metaphase. Such type of chromosome variation in their sizes were also reported by

Guerlesquin (1967), Khan and Sarma (1967c) and Labh and Verma (1986) in the taxa of Chara and Nitella. Maximum variation in the length of chromosomes within the karyotype of the taxon, in the present study was noticed in N. heterodactyla, where in the size ranged from 3.66ƒÊ m to 8.80ƒÊm (Fig. 6). There is no inverse relationship between chromosome length and number of chromosomes (Table 1). Therefore, the present observations are not in agree ment with the findings of Khan and Sarma (1967a, b) and Sinha and Noor (1971). The chromosomes are comparatively large in size (Figs. 4-6) morphologically dis tinguishable N. O. chromosomes with satellites as are common in Oedogonium spp. (Hen ningsen 1963, Srivastava and Sarma 1979, Singh and Chaudhary 1990), have not been made out in the present taxa. In contrast to this observation, Bharati and Chennaveeraiah (1983) re ported 2 SAT chromosomes in Lychnothamnus barbatus var. gigantii var. nov. Similarly, Ray and Chatterjee (1988a) also observed one satellite bearing chromosome in C. fibrosa var. and f. hydropitys. Karyotypes of each taxon is characterised by distinctive chromosomal features. The taxa studied here when compared with the earlier reported ones having same chromsome numbers also showed differences in their karyotypic organization. Such type of variation in the karyo typic features may be due to chromsomal rearrangements in the taxa of Chara and Nitella. So far, 196 taxa of charophytes including the present ones have been subjected to kryolo gical investigations (Sarma 1982). The chromosome number recorded for the tribe Chareae is n=7 (Noor and Mukherjee 1977) could logically be concluded to represent the basic chromosome number for the tribe Charae as suggested by Moutschen et al. (1956). On the other hand, the chromosome count (n=18) ascertained to N. heterodactyla is in multiple of the basic chromosome number (x=3) as suggested by Sarma and Khan (1965c) to the tribe Nitel leae, although the lowest number in the taxa of Nitella investigated so far is n=6 (Sarma 1982).

Summary

Cytological investigations on two taxa of Chara Linn. (C. vulgaris var. gymnophylla f. grovesii and C. zeylanica var. sejuncta f. formosa) and a taxon of Nitella Ag. Leonh. (N. heter odactyla) were carried out. Nuclear events conformed to the standard pattern of mitosis described. The chromosome numbers determined are n=35 for C. vulgaris var. gymnophylla f. grovesii, n=42 for C. zeylanica var. sejuncta f. Formosa and n=18 for N. heterodactyla. All the three taxa were investigated for the first time and are new reports.

Acknowledgements

The authors wish to express their thanks to the Head, Department of Botany, Banaras Hindu University for providing laboratory facilities and University Grants Commission, New Delhi for financial assistance to BVS. 414 B. V. S. Subrahmanyam and Y. B. K. Chowdary Cytologia 57

References

Bharati, S. G. and Chennaveeraiah, M. S. 1983. Lychnothamnus barbatus var. gigantiii var. nov. (Anew variety) Cytologia 48: 843-846. Bhatnagar, S. K. and Johri, M. 1986. Some aneuploids in Indian Charophyta. Acta Bot. Neerl. 35: 377-381. Chatterjee, P. 1971. An analysis of the structure and behaviour of chromosomes as an aid in the taxonomy of certain higher and lower groups of plants and the cytological effects of certain chemical and physical agents in the latter. Ph. D. Thesis, University of Calcutta, Calcutta, India. Darlington, C. D. and La Cour, L. F. 1962. The Handling of Chromosomes. George Allen and Unwin Ltd., London. Gillet, C. 1959. Nomber chromosomique de plusiers especes de Charophycees (genres Nitella et Chara). Rev. Cytol. Biol. Veg. 20: 229-234. Godward, M. B. E. 1948. The iron-alum acetocarmine method for algae. Nature 161: 203. Griffin, D. G. and Proctor, V. W. 1964. A population study of Chara zeylanica in Texas, Oklahoma and New Mexico. Am. J. Bot. 51: 120-124. Guerlesquin, M. 1967. Recherches caryotypiques et cytotaxonomiques chez les Charophycees d'Europe oc cidentale et d'Afrique du Nord. Bull. Soc. Sci. Bretag. 41: 1-265. Henningsen, K. 1963. Chromosome numbers in five species of Oedogonium. Phycologia 3: 29-36. Hotchkiss, A. T. 1963. A first report of chromosome numbers in charophyte genus Lychnothamnus (Rupr.) Leonh., and comparison with the other charophyte genera. Proc. Linn. Soc. New South Wales 88: 368-372. - 1965. Chromosome numbers in Characeae from South Pacific. Pacific Sci. 19: 31-37. Imahori, and Kato, T. 1961. Notes on chromosome numbers of charophytes in Fukui Prefecture, Japan. Sci. Rep. Osaka Univ. 10: 39-48. Karling, J. S. 1926. Nuclear and cell division in Nitella and Chara. Bull. Torrey Bot. Club 53: 319-379. Khan, M. and Sarma, Y. S. R. K. 1967a. Studies on cytotaxonomy of Indian charophyta I. Chara. Phykos 6: 36-47. - and - 1967b. Studies on cytotaxonomy of Indian Charophyta II. Nitella. Phykos 6: 48-61.- and - 1967c. Some observation on the cytology of Indian charophyta. Phykos 6: 62-74. Labh, L. and Verma, B. N. 1984. Cytological studies in three species of Tolypella. Cytologia 49: 712-723. - and - 1985a. First report of chromosome number in four taxa belonging to the genus Chara Linn. Cy tologia 50: 21-24. - and - 1985b. New record of chromosome numbers for the charophytes. Cytologia 50: 55-58. - and - 1986. New counts for Chara fibrosa complex. Cytologia 51: 185-191. Levan, A., Fredga K. and Sandberg A. A. 1964. Nomencalture for centromeric position of chromosomes. Hereditas 52: 201-220. Lindenbein, W. 1927. Beitrag zur Cytologie der Charales. Planta 4: 436-466. Moutschen, J., Dahmen M. and Gillet, C. 1956. Sur les modifications induites par les hydrazides maleique et isonicotinique dans les antheridies de Chara vulgaris L. La Cellule 58: 65-78. Noor, M. N. and Mukherjee, S. 1977. Some new records of chromosome numbers in Indian Charophyta. Cytologia 42: 227-232. Pal, B. P., Kundu, B. C., Sundarlingam, V. S. and Venkatraman, G. S. 1962. Charophyta. ICAR, New Delhi. Ramjee and Bhatnagar, S. K. 1978. Studies on charophytes from Rohilkhand division I. Moradabad: Taxonomic enumeration and chromosome counts. Phykos 17: 87-92. - and Sarma, Y. S. R. K. 1971. Some obervations on the morphology and cytology of Indian Charophyta. Hydrobiologia, 37: 367-376. Ray, S. and Chatterjee, P. 1988a. Cytotaxonomical study of some members of Chara fibrosa complex. Cryp togamie: Algologie 9: 65-72. - and - 1988b. Cytotaxonomical study of West Bengal Charophyta: a new chromosome number count of Nitella stuartii and its karyotypes. Acta Bot. Neer. 37: 523-525. Sarma, Y. S. R. K. 1982. Chromosome numbers in algae. Nucleus 25: 66-108. - 1988. Chromosome numbers in algae. Final Technical Report submitted to Department of Environment, New Delhi, India. - and Khan, M. 1964. Chromosome numbers in some Indian species of Nitella. Chromosoma 15: 246-247. - and - 1965a. Chromosome numbers in some Indian species of Chara. Phycologia 4: 173-176. - and - 1965b. Some new observations on the karyology of Chara zeylanica Klein ex Willd. Curr. Sci. 34: 293-294. 1992 Karyological Studies on Some Indian Charophyta 415

- and - 1965c. A preliminary report on the survey of chromosome numbers of Indian Charophyta. The Nucleus 8: 33-38.- and Ramjee, 1971. Significance of chromosome numbers in Charophyta-A discussion. Caryologia 24: 391-401. Singh, H. V. and Chaudhary, B. R. 1990. Karyological investigations on the genus Oedogonium Link (Oed ogoniales, Chlorophyceae). Caryologia 43: 169-181. Sinha, J. P. and Noor, M. N. 1967. Chromosome numbers in some members of Chlorophyceae of Chotanagpur Plateau, India. Phykos 6: 106-109. - and - 1971. Studies on the karyology of some members of Charophyta of Bihar. Phykos 10: 112-117. - and Verma, B. N. 1970. Cytological analysis of charophytes of Bihar. Phykos 9: 92-99. Srivastava, S. and Sarma, Y. S. R. K. 1979. Karyological studies on the genus Oedogonium Link (Oedogoniales, Chlorophyceae). Phycologia 18: 228-236. Steil, W. N. 1941. Antherozoids of some of the Characeae. Bot. Gaz. 102: 623-628. Subramanian, D. 1983. Studies on charophytes from Tamil Nadu I. Taxonomic enumeration and chro mosome counts. Phykos 22: 18-22. Sundarlingam, V. S. 1946. The cytology and spermatogenesis in Chara zeylanica Willd. J. Ind. Bot. Soc. (M. O. P. Iyengar Commemoration Volume), pp. 289-303. Tindall, D. R. and Sawa, T. 1964. Chromosomes of the Characeae of the Woods Hole (Massachusetts) region. Am. J. Bot. 51: 943-949. Tuttle, A. H. 1926. The location of the reduction division in a charophyte. Univ. Calif. Publ. Bot. 13: 227-234. Wood, R. D. and Imahori, K. 1965. A Revision of the Characeae. Monograph and Iconograph. Verlag Von. J. Cramer Pub., New York.