Cytologia 49: 295-304, 1984 Cytological Studies of Some Helobiales of Kashmir Himalayas I. Family Alismataceae 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. Plants 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 fruit. 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, leaves petiolate with expanded blades, petals mostly caducous, ovules campylotropous, and seeds 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 inflorescences 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 leaf-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: Alisma 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.