Cytologia 49: 295-304, 1984

Cytological Studies of Some Helobiales of Kashmir Himalayas

I. Family

P. N. Mehra and T. K. Pandita

Department of Botany, Panjab University, Chandigarh-160 014, India

Received June 16, 198

The family 2 Alismataceae belongs to the order Helobiales, and according to

Lawrence (1951) comprises 14 genera and 50-60 species distributed mainly in the

temperate and tropical regions of the northern hemisphere. Airy Shaw (1973) estimated 13 genera and 90 species in this family. grow mostly in fresh-

water swamps and streams.

Hooker (1894) divided the family Alismaceae into two tribes, Alismae and

Butomeae, on the basis of nature of the . In the former the fruit is of 3 or more achenes, while in the latter it is always of follicles. Pichon (1946) proposed family

ranks for the two tribes and stated that Alismataceae be treated as constituting mem-

bers having latiferous ducts, petiolate with expanded blades, petals mostly

caducous, ovules campylotropous, and with curved embryos. This emenda-

tion resulted in the transfer to Alismataceae of all members of Alismaceae except Butomus. Phylogenetically the family Alismataceae represents one of the most

primitive of the extant monocots. The objective presently was to investigate the cytology of members of this family with a view to trace the evolutionary trends in

their karyotypes and also to study the meiotic behaviour of chromosomes.

Material and methods

Collections were made from nature. Meiotic studies were carried out from pollen mother-cells (PMC's). Young were fixed in 1:3 acetic al- cohol with a few drops of chloroform for 24 hours and preserved in 70% alcohol.

Squashes were made in 1% aceto-carmine. Mitotic studies were done from root- tips. These were pretreated with 0.25% aqueous colchicine solution for 3 hours at room temperature and fixed in 1:3 acetic-alcohol for 24 hours. The tips were then washed in water, hydrolysed in I N-HCl for 8-11 minutes at 60•Ž, followed by Feu

gen staining, and squashed in 1% aceto-carmine or 45% acetic acid. Slidesl were made permanent by separating the cover glass from glass slide in 1:3 acetic acid-butanol and passing them through various grades of acetic acid-butanol i.e.

1:6 and 1:9, pure butanol 1, pure butanol 11. Euparol was used as the mount- ing medium for making slides permanent.

For a study of the position of chiasmata arbitrary classification of Frankel (1940) into three groups, distal (D), median (M) and proximal (P) is used. The nomen- clature for centromeric position on chromosomes was that of Levan et al. (1964).

For calculating the correct size of chromosomes at least ten cells with well spread- Table 1. cf. Darlington and Wylie (1955). Fedorov (1969). Moore (1973). Love and Love (In IOPB chromosome number reports, upto 1979) an some other references cited in bibliography. 298 P. N. Mehra and T. K. Pandita Cytologia 49

out chromosomes at mitotic metaphase were drawn with the help of camera-lucida

and then measured. One of these cells has been photomicrographed. Photomi. crographs were taken at a constant magnification of •~ 1360. Karyograms were

made from the photomicrographs. Total chromatin length was determined for

each species.

Table 1 summarises the cytological data of the taxa studied together with the

figure numbers, areas of collection, altitude and the previous reports.

Observations

Alisma L.

The genus comprises 10 species which are cosmopolitan in distribution (Airy

Shaw 1973). Some of the plants are grown as ornamentals. The following three species have been worked out.

A. plantago-aquatica Linn.: Pollen mother-cells revealed 7 bivalents at MI

(Fig. 1). The smallest bivalent usually disjuncted early. Chiasmata number per bivalent was calculated to be 2. 1. All the three types of chiasmata D, P and M were noticed. Sticky bridges were seen at A, but ultimately all the 7 chromosomes

reached the respective poles (Fig. 2). Pollen fertility was 95 %. Root-tip cells showed 2n=14 (Fig. 3). Karyotypes consists of 4M+6m+4T (Fig. 3a). Total

chromatin length is 85.14ƒÊ. Chromosome size ranges form 8.89 to 2.94ƒÊ.

A. gramineum Lej.: This species approaches A. lanceolatum and A. plantago-

aquatica in -shape, but differs from the two in having anthers suborbicular or isodiametric. Style is recurved and shorter than the ovary. The species is rare

in Kashmir. PMC's revealed 7 bivalents at MI (Fig. 4) which follow a normal ana-

phasic distribution (Fig. 5). Chromosomes showed stickiness. Pollen fertility was 100%. Root-tip mitosis revealed 2n=14 (Fig. 6). Karyotype consists of

6M+4m+4T (Fig. 6a). Chromosomes of the 1st pair of karyogram are slightly heteromorphic. Total chromatin length is 84.19ƒÊ. Chromosome size ranges

from 9.14 to 2.94y.

A. lanceolatum With.: This species is found throughout the valley. PMC's revealed 13 bivalents at MT (Fig. 7). The smallest pair showed late disjunction

(Fig. 8). Bridges and laggards were common. Pollen fertility was 92%. Root- tip cells revealed 26 chromosomes (Fig. 9) which could be arranged into 13 pairs

(Fig. 9a). Karyotype consists of 12M+10m+2t+2T (Fig. 9a). Twelfth pair pos-

sesses a satellite in its short arm. Total chromatin length is 196 .32ƒÊ. Chromo- some size ranges from 10.29 to 3.88ƒÊ.

Sagittaria L.

The genus comprises 20 species which are cosmopolitan in distribution (Airy

Shaw 1973). Two species occur in the Western Himalayas . S. sagittifolia L.: Chromosome number was determined both from PMC's and root-tips. PMC's revealed 11 bivalents at M, (Fig . 10). Chiasmata were of non-localised type and numbered two per bivalent . Pollen fertility was 97%.

Root-tip mitosis revealed 2n=22 chromosomes (Fig . 11). The karyotype consists of 2M+2sm+18t chromosomes (Fig. 11a) . Total chromatin length is 131.94ƒÊ. 1984 Cytological Studies of Some Helobiales of Kashmir Himalayas I 299

Figs. 1-6. 1-3a: plantago-aquatica. 1, PMC at MI showing 6 large bivalents, 1 small early disjuncted bivalent and general stickiness of chromosomes. 2, PMC at AI showing 7 chro- mosomes at each pole. 3, somatic cell at metaphase showing 14 chromosomes. 3a, karyogram of Fig. 3. 4-6a: A. gramineum. 4, PMC at MI showing 7 bivalents with complete synapsis and the general stickiness of the chromosomes . 5, normal chromosome distribution at AI. 6, somatic cell at metaphase showing 14 chromosomes. 6a, karyogram of Fig. 6. 300 P. N. Mehra and T. K. Pandita Cytologia 49

Figs. 7-11. 7-9a.: Alisma lanceolatum. 7, PMC at MI showing 13 bivalents. 8, laggards at AI. 9, somatic cell at metaphase showing 26 chromosomes. 9a, karyogram of Fig. 9. 10-11: Sagittaria sagittifolia. 10, PMC at MI showing 11 bivalents. 11, somatic cell at metaphase with 22 chromosomes. 1984 Cytological Studies of Some Helobiales of Kashmir Himalayas I 301

Chromosome size ranges from 10.26 to 3.08ƒÊ. S. guayanensis Kunth: This species is rare in Kashmir. PMC's revealed n=

11 (Fig. 12) and the bivalents showed general stickiness. Chromosome distribu- tion at AIr was normal (Fig. 13). Pollen fertility was 98%. Root-tip mitosis re- vealed 2n=22 (Fig. 14). Karyotype consists of 2M+2sm+16t+2T (Fig. 14a).

Figs. Ila-14a. Ila, Sagittaria sagittifolia; karyogram of Fig. 11. 12-14a: S. guayanensis. 12, PMC at MI showing 11 bivalents and the general stickiness of chromosomes. 13, normal distribution of chromosome at A11. 14, somatic cell at metaphase showing 22 chromosomes. 14a, karyogram of Fig. 14. 302 P. N. Mehra and T. K. Pandita Cytologia 49

Total chromatin length is 101.02y. and chromosome size ranges from 8.60 to

2.20 ƒÊ.

Discussion

Five species, Alisma plantago-aquatica (n=7, 2n=14), A. gramineum (n=7, 2n=14), A. lanceolatum (n=13, 2n=26), Sagittaria sagittifolia (n=11, 2n=22)

and S. guayanensis (n=11, 2n=22) of the family Alismataceae have been worked out.

Their chromosome number and total chromatin length is given in the tabular form

below:

Alisma: Except Baquar and Askari (1970) who observed n=6 for A. plantgo - aquatica, the present count is in agreement with the reports of earlier investigators as mentioned in Table 1. Chromosome count of 2n=14 for A. gramineum too con firms the reports of earlier workers (cf. Table 1), but differs from that of Packer (1968) who found 2n=16 for this species. In A. lanceolatum the three common numbers reported by earlier workers are 2n=24, 26, 28 (cf. Table 1). Only the cytotype with 2n=26 was found in Kashmir. Aleksandrova (1967, cf. Fedorov 1969) also reported 2n=14 for A. lanceolatum. According to Bjorkgvist (1967, 1968) the basic number of the genus is x=7 and the genus includes diploids, tetra ploids and hexaploid species. Furthermore there is one aneuploid number 2n=26 at the tetraploid level. The karyotype of A. plantago-aquatica with 4M+6m+4T chromosomes is at variance with the findings of Oleson (1941) who observed 2 median, 2 submedian and 10 subterminal chromosomes, but resembles very much with the findings of Bjor kqvist (1968) except that there is no satellite in any chromosome pair in the present preparations. Karyograms of the two diploid species Alisma plantago-aquatica and A. gramineum with n=7 is rather primitive. It seems to have been derived from the progenitors with n=6 with median-submedian type of chromosomes and the chromo somes 6 and 7 have evolved after a break at the centromere of a submedian chromo some. It may be recalled that Baquar and Askari (1970) have reported n=6 chro mosome in A. plantago-aquatica. Furthermore A. lanceolatum with n=13 chromo somes is at teraploid level in which only one pair has undergone break at the centro- 1984 CytologicalStudies of Some Helobialesof Kashmir HimalayasI 303 mere region so that instead of 12 pairs there are 13 pairs, or alternatively it is an am- phidiploid between the cytotypes with x=6 and 7 chromosomes. The size difference of the corresponding chromosome is a matter of degree of condensation in the diplo- ids and the tetraploid. Sagittaria: Present count of n=11 and 2n=22 for Sagittaria sagittifolia con- firms some of the previous reports, but is at variance with others (cf. Table 1). Pre- sent count of 2n=22 for S. guayanensis confirms the previous reports (cf. Table 1). Both the species have similar karyotypes, conforming to the observations of Sharma and Chatterjee (1967). The chromosome pairs 2-9 with slightly subterminal centro mere have probably been derived from Alisma-type chromosomes through brea- kage at centromeres of chromosome pairs 2, 3, 4, 5 with submedian constriction, thus giving rise to chromosomes 2, 3, 4, 5, 6, 7, 8, 9 of Sagittaria. This obviously has been followed by slight growth of short arms in the latter. Chromosomes of 1st

Fig. 15. Diagramatic.For explanationsee text.

pair of karyogram of Sagittaria with median constriction are comparable to chro- mosomes of 1st pair of Alisma. Eleventh pair of Sagittaria seems to be comparable to 7th pair of Alisma possibly after slight chromosomal repatterning, since there is no size difference. The species have stabilized themselves at diploid level with a consistent base number x=11. The authors believe that the original base number of Alismataceae was x=6 and the complement consisted of all median-submedian chromosomes. The number n=7 is the result of fission of one of these at the centromere region resulting in 2 term- inal chromosomes. Alisma lanceolatum (n=13, 2n=26) is a tetraploid in which only one median-submedian chromosome may have broken at the centromere, or as stated earlier may be an amphidiploid between n=6 and n=7. The genus Sagit- 304 P. N. Mehra and T. K. Pandita Cytologia49

taria with n=11 owes its origin through fission at the centromere region of all the chromosomes but one. But chromosome 11 has undergone some repatterning as stated above. The broad course of karyological evolution is graphically represent- ed in Fig. 15 which is based on karyotypic analysis of the two genera. Alismataceae being a very primitive family of monocots, this perhaps implies that the original base number of monocots was 6 chromosomes, all with median submedian centromere. -

Summary

The present communication deals with male meiosis and karyotypic analysis in five species of the two genera, Alisma and Sagittaria. The course of meiosis was investigated with great difficulty because of stickiness of chromosomes. Alisma plantago-aquatica and A. gramineum posssess n=7 chromosomes with 5 chromosome pairs belonging to median-submedian category and 2 pairs to terminal category, while A. lanceolatum is at the tetraploid level with n=13. Both the species of Sagittaria sagittifolia and S. guayanensis possess a haploid number of n=11 chro- mosomes of which only 1 chromosome is of the median category whereas the other 10 belong to subterminal category. Karyotype evolution in the different taxa is discussed.

Literature cited

Airy Shaw, H. K. 1973, A Dictionary of the Flowering Plants and Ferns. Cambridge Univ. Press. Baquar, S. R. and Askari Abid, S. H. 1970. Chromosome numbers in some flowering plants of West Pakistan. Genet. Iber. 22: 1-11. Bjorkgvist, I. 1967. Studies in Alisma L. I. Distribution, variation and germination. Opera Botanica 17: 1-128. - 1968. Studies in Alisma L. II. Chromosome studies , crossing experiments and . Opera Botanica. 19: 1-138. Castro, D. De and Wagner, N. M. 1950. Preliminary observations on the cytology of the genus Alisma L. in Portugal. Genetica Iberica 2: 75-82. Darlington, C. D. and Wylie, A. P. 1955. Chromosome Atlas of Flowering Plants. George Allen and Unwin Ltd. London. Fedorov, A. An. (ed.) 1969. Chromosome Numbers of Flowering Plants. Z. Bolkhovskikh et al. Leningrad. Komarov Botanical Institute. Hagerup, O. 1944. Notes on some boreal polyploids. Hereditas 30: 152-160. Hooker, J. D. 1894. Flora of British India Vol. VI. L. Reeve and Co., Ltd., London. Lawrence, G. H. M. 1951. Taxonomy of Vascular Plants. MacMillan Co . New York. Love, A. and Love, D. (upto 1979). In IOPB chromosome number reports. Taxon . 28. Moore, R. J. 1973. Chromosome numbers (1967-1971). Regnum Vegetabile 90: 313, Oleson, E. M. 1941. Chromosomes of some Alismaceae. Bot. Gaz . 103 (1): 198-202. Packer, J. G. 1968. In IOPB chromosome number reports XVII. Taxon 17: 285-288 . Pichon, M. 1946. Sur les Alismatacdes et les Butomacees (includes Albidella, gen. nov., key to genera of redefined Alismaceae). Not. Syst. (Paris) 12: 170-813. Sharma, A. K. and Chatterjee, T. 1967. Cytotaxonomy of Helobiae with special reference to the mode of evolution. Cytologia 32: 186-207 . Tschermak-Woess, E. 1948. Zytologische Untersuchungen an den Alisma-Arten der Umgebung Wiens, Osterreich. Bot. Zeitschr. 95: 270-276.