Cytologia 37: 281-296, 1972

Cytology of Some W. Himalayan

P. N. Mehra and P. Remanandan Departmentof Botany,Panjab University, Chandigarh-14,

ReceivedSeptember 24, 1970

Introduction Ranunculaceae is a moderately large family, chiefly of the temperate regions of the Northern Hemisphere with 35 genera and about 1,500 species. In India it is represented by 115 species belonging to 19 genera (Hooker 1882), a majority of which are distributed in the alpine and sub-alpine belts of the . The family is of considerable importance because of its relatively primitive position in the hier archy of flowering and inclusion in it of a number of important ornamental and medicinal plants. On these scores the family deserves a greater attention on the part of cytogeneticists than it has so far received. The previous work on the family is rather scattered. The more important contributions in the field are those of Langlet (1927, 32, 36), Stebbins (1938), Gregory (1941), Zhukova (1961) and recently of Kurita (1959, 61, 62, 66, 67) who has studied the karyotypes of a number of taxa. Members of the family growing in India have hitherto attracted little atten tion. Barring a few reports of chromosome numbers by Sobti and Singh (1961) on Ranunculus sps. and Mehra and Sobti (1955) on Indian aconites no work of any significance has so far been attempted. The present paper gives a cytological account of 23 taxa met with in the Western Himalayas, based mostly on meiotic studies.

Material and method

The material for the present investigation was collected from the wild sources in Kashmir, Simla and Kumaon hills within an altitude range of 100-5,000m. The plants were identified in the field with the help of local floras and confirmed by comparing them at the Central National Herbarium, Calcutta. The voucher speci mens have been deposited in the Herbarium of the department of Botany, Panjab University, Changigarh-14. Cytological studies were made mainly from the pollen mother cells at meiosis.

Flower buds were fixed in carnoy's fluid. Acetocarmine technique was employed for staining the chromosomes. In order to detect the existence of intraspecific cytological races, same species was screened from a wide range of localities. All the photomicrographs were taken at a magnification of •~1,510 and the camera lucida drawings made at a uniform magnification of •~1,375. 282 P. N. Mehra and P. Remanandan Cytologia 37

Observations Table 1 summarises the data in respect of the taxa worked out and the specific localities from where they were collected. Species marked * are investigated for the first time, whereas those marked t represent new cytological races. Short notes in respect of each species are mentioned in the text below. 1. Anemone vitifolia Ham. It is a robust perennial herb with beautiful white flowers in decompound cymes, very much similar to the garden A. japonica. It is a good potential orna mental worth introduction. Diakinesis revealed 7 large bivalents which were of the rod, ring, or X type (Fig. 1). Their behaviour during meiosis was regular. However, nonsynchronous disjunction and laggards were observed in some cells and these later organised themselves into micronuclei (Fig. 2). The pollen fertility was 98 percent. This is a new report for the species. 2. A. obtusiloba Don. This beautiful perennial medicinal herb grows extensively on the open hill tops in the temperate and alpine Himalayas at an altitude of 2,400-2,500m. Hooker (1882) states that it is a variable species in respect of the size, hairiness and colour of flowers. He distinguishes a form which is quite glabrous with very many golden sepals as var. glabra. The typical species in which the leaves and peduncles are densely covered with silky hairs was collected from Simila Hills. It revealed the 2n number to be 26 from the root tip squashes. The chromosomes are fairly large in size and mostly metacentric. The karyotype is symmetric (Fig. 6). The var. glabra was collected from Gulmarg and other alpine regions in Ka shmir. At metaphase I, it showed 7 large sized bivalents. The meiosis was found to undergo a normal course. However, precocious disjunction of one or two bivalents was a common feature at anaphase I (Fig. 3). Occasionally laggards were observed which failed to be included into the telophase nuclei resulting in the formation of micronuclei (Fig. 5) of different sizes. The pollen fertility was 90 percent. Sobti and Singh (1961) reported the gametic number n=8 under the name of A. obtusiloba from Kashimir. Possibly it is the same taxon as the one studied presently under the name var. glabra. If this surmise is correct, then there is some error in the report made by these authors. In conclusion it may, however, be stated that the two taxa, A. obtusiloba pro per and the var. glabra are sufficiently distinct morphologically. Now it turns out that they are distinct cytologically too. In our opinion there is every justification in raising this variety to a specific rank. 3. A. rivularis Buch.-Ham. It is a perennial herb found throughout India, especially in meadows above an altitude of 1,500m. The species revealed 8 large sized bivalents at diakinesis (Fig. 7). Three bivalents showed two interstitial chiasmata each, two were with a single interstitial chiasma and of these one was associated with the nucleolus (Fig. 1972 Cytology of Some W. Himalayan Ranunculaceae 283

Table 1 284 P. N. Mehra and P. Remanandan Cytologia 37

Table 1 (Contd.)

* Species investigated for the first time . •õ Cytotypes investigated for the first time.

7). The course of meiosis was regular. Late disjunction of one of the bivalents was observed in a few cells. The pollen fertility was found to be 95 percent. The species is worked out for the first time. 4. Thalictrum reniforme Wall. The species is common in the woods at an altitude of 2,400-3,000m in the Naini tal Hills. The finding of n=7 (Fig. 8) is a new report for the species. Chromo somes are rather small in size. The behaviour of them at the first and second meiotic divisions was normal resulting in about 97 percent pollen stainability. 5. T. foliolosum DC. It is a common perennial herb of the temperate Himalayas. Being of medicinal value it is much sought for its rhizome. At diakinesis 7 bivalents were discernible. This confirms the earlier report by Langlet (1927) for the species. Meiosis was normal and pollen fertility 97 percent. 6. Clematis montana Ham. It is a gracious woody climber rambling over shrubs and with rich clusters of strikingly beautiful white flowers. It is common on open hill sides ascending to 3,600m. The species is widely cultivated as an ornamental. It is poisonous and its root is of medicinal value. The finding of n=8 from the pollen mother cells confirms the previous report of Meurman and Therman (1939). At diakinesis 8 medium sized bivalents were

Figs. 1 and 2. Anemone vitifolia, n=7. 1, M-I showing chiasmata formation. 2, abnormal cell showing micronucleus formed from lagging chromosomes at A-I.

Figs. 3-5. A. obtusiloba var. glabra n=7. 3, M-I displaying precocious separation of one pair

(arrow). 4, M-II, 7:7 chromosomes at each pole. 5, abnormal cell showing organisation of laggards into micronuclei at A-I.

Fig. 6. A. obtusiloba, 2n=26, root tip mitosis. All figs. •~1,510. 1972 Cytology of Same W. Himalayan Ranunculaceae 285 286 P. N. Mehra and P. Remanandan Cytologia 37

Figs. 7-15. 7, Anemone rivularis, n=8, diakinesis displaying the distribution of chiasmata and a nucleolar bivalent at arrow. •~1,510. 8, Thalictrum reniforme, n=7, diakinesis, •~1,375. 9,

Adonis chrysocyathus, n=16, A-I showing 16:16 separation, •~1,375. 10 and 11, Ranunculus hir tellus, n=8. 10, diakinesis displaying chiasmata distribution and two nucleolar bivalents, •~1,510. 11, abnormal cell with a micronucleus, •~1,510. 12, R. diffusus, n=16, diakinesis, •~1,375.

13, R. laetus, n=16, diakinesis, •~1,375. 14, R. laetus, n=14, diakinesis, •~1,510. 15, R. ar vensis, n=16, M-II, •~1,375. 1972 Cytology of Some W. Himalayan Ranunculaceae 287

clearly observed. Their arrangement at the equatorial plane was regular at meta phase I. Further stages of meiosis were normal and the pollen fertility was found to be 98 percent. 7. Adonis chrysocyathus Hook. f. and Thomas . This perennial herb of the alpine belt is dreaded by shepherds in Kashmir who believe it to cause frequent deaths among sheep and goats. It grows at an elevation of 3,000-3,900m. Because of its large golden yellow flowers and luxur ious fern-like leaves it would be a good potential ornamental worthy of cultivation . The species is a tetraploid on the base number x=8. At anaphase I 16:16 distribution was observed. However, 5 pairs of rather longish chromosomes dis junct later than the rest (Fig. 9). The course of meiosis was normal and 93 percent of the pollen was fertile. This is a new report for the species. 8. Ranunculus hirtellus Royle. It is a common perennial herb of the sub-alpine Western Himalayas which grows at an altitude of 800-3,000m. At diakinesis 8 bivalents and a nucleolus were seen (Fig. 10). Four of these were ring-bivalents held together by two terminal chiasmata, two bivalents possessed a single terminal chiasma and the remaining two had a single interstitial chiasma each. One of the rings was associated with the nucleolus. Meiosis was found to undergo a mildly disturbed course. Anaphase I was regular with 8:8 distribution. However, laggards were not uncommon. These got organised into micronuclei (Fig. 11). Tetrad formation was mostly normal, but rarely 5 nucleate cells were observed. 90 percent of the pollen was fertile. This is a new record for the species. 9. R. diffusus DC. The species is common in the temperate Himalayas on exposed hills at an altitude of 1,800-3,000m. The finding of n=16 (Fig. 12) is a new report for the species. It is a tetraploid on the base number x=8. The chromosomes are of medium size. Meiosis was regular and pollen fertility 97 percent. 10. R. laetus Wall. This perennial is common on the inner ranges of temperate Himalayas be tween 1,200-2,400m. The present study on the populations in Kashmir valley and Simla hills has revealed the existence of two intraspecific races. Both are at the tetraploid level but with different chromosome numbers, n=16 and 14 re spectively. The taxon with higher number grows in Simla hills. At diakinesis 16 bivalents of medium size were observed (Fig. 13). This confirms the earlier reports of Sobti and Singh (1961) and Arora (1961). Meiosis was normal resulting in about 96 percent pollen stainability. The aneuploid taxon with n=14 is found in the Kashmir valley. The plants are highly variable in size and morphology. Meiosis followed a mildly disturbed course. At diakinesis bivalent formation was complete (Fig. 14). Stickiness of chromosomes was noticed in the majority of metaphase plates. Anaphase I was regular with laggards observed occasionally. Pollen fertility was found to be 90 percent. 288 P. N. Mehra and P. Remanandan Cytologia 37

11. R. arvensis Linn. This is an annual, commonly found in corn fields at 1,200-2,100m. The species has some medicinal properties. It is a tetraploid with n=16 (Fig. 15). The chromosomes are of medium size and their behaviour during the two divisions of meiosis was normal. Ninety-six percent of the pollen was fertile. 12. Caltha palustris Linn. It is a perennial species which is commonly found on marshy grounds and near streams at an altitude of 2,400-3,000m. It is poisonous to cattle and horses. The species has been worked out earlier and is known to exist in numerous intra specific races (cf. Table I). The population at Gulmarg is found to be a new cytotype of the complex hitherto unreported, with n=20. The chromosomes are of medium size and the behaviour of them during the course of meiosis was normal. Pollen fertility was 97 percent. 13. Aquilegia vulgaris Linn. Few wild flowers have the airy grace and ornamental poise of this member of the butter-cup family. The erect branching stems reach a height of several feet, growing from a perennial root and producing delicately compound leaves with many rounded and lobed leaflets. At the summit of the leafy branches grow the nodding flowers. The petals have curved spurs which contain nectar. It is a plant worthy of introduction as an ornamental. The herb is commonly found on wet hills of the sub-alpine belt between 1,500-3,000m. Meiotic studies revealed it to be a diploid with n=7. This confirms the earlier reports by many workers (cf. Table 1). The course of meiosis was regular and the pollen fertility 97 percent. 14. A. canadensis Linn. It is an ornamental cultivated in hills for its unsurpassed beauty of flowers. At diakinesis 7 bivalents were discernible. This confirms the reports of Linnert (1961) and Lewis et al. (1962). Chromosomes are of small size. Meiosis was regular and 97 percent of pollen was fertile. 15. denudatum Wall. This perennial herb has beautiful spurred flowers and stately foliage and hence a worthy plant for introduction in the gardens. At diakinesis 8 bivalents were discernible (Fig. 16). Three of these were with two chiasmata in each and five with a single chiasma. Meiosis in majority of cases was found to undergo a regular course. However, laggards and late disjunction of 1 or 2 bivalents was occasionally seen (Fig. 17). An interesting feature observed was the non-homologous associa tion of several chromosomes at late Anaphase I (Fig. 18). Ninety-five percent of the pollen was stainable.

Figs. 16-18. Delphinium denudatum, n=8. 16, diakinesis. 17, A-I showing nonsynchronous

disjunction. 18, A-I showing delayed separation of one pair (arrow) and nonhomologous associa tions of several chromosomes.

Figs. 19-21. D. cardiopetallum, n=8. 19, diakinesis displaying structural heterozygosity. 20,

A-I showing 8:8 separation and belated disjunction of one pair. 21, abnormal cell with laggards at T-I. All figs. •~1,510. 1972 Cytology of Some W . Himalayan Ranunculaceae 289 290 P. N. Mehra and P. Remanandan Cytologia 37

16. D. ajacis Linn. It is commonly cultivated in hills as well as in the plains of India. It is a diploid which shows 8 bivalents at diakinesis. This confirms the previous report by Gregory (1941). Meiosis was found to undergo a partially disturbed course. Uni valents, laggards and 6-nucleate mother cells have been observed. Ninety-six percent of the pollen was fertile. 17. D. cardiopetallum DC. This is commonly cultivated as an ornamental in the plains of India. It is also a diploid with large chromosomes. Bivalent formation was complete at diakinesis. However, a single interchange heterozygote was noticed in many cells (Fig. 19). Nonsynchronous disjunction was found to be a constant feature and laggards were occasionally observed (Fig. 21). However, majority of the cells showed 8:8 distribution at anaphase I (Fig. 20). Pollen fertility was 92 percent. 18. Aconitum lycoctonum Linn. The tuberous roots of several of the aconites have long enjoyed considerable reputation in medicine and a number of them are now known to contain highly toxic alkaloids. A. lycoctonum is one of these. It grows at high altitudes between 2,100-3,000m. Eight bivalents were discernible at diakinesis of which the two larger ones possess heterochromatic arms which remain unusually extended (Fig. 22). The other bivalents are of medium size. Stickiness of chromosomes and delayed separa tion were common features (Fig. 23). Only 70 percent of the pollen was fertile. 19. A. heterophyllum Wall. It is also an important drug plant of the north-west Himalayas. The roots con tain a non-toxic amorphous alkaloid and are exported on an extensive scale to the plains of India. The plant is very variable in morphology. Numerous biotypes have been recorded (Mehra and Puri 1968). However, all of them have been found to be cytologically alike. Meiotic studies revealed 8 bivalents at diakinesis (Fig. 24). This confirms the earlier report by Mehra and Sobti (1955). Like the preceding species here too a pair of large bivalents was found to possess the peculiar extended heterochromatic arms. Late disjunction of varying number of bivalents has been observed. Fig. 25 shows one such bivalent at the equatorial plate. Pollen fertility was found to be 85 percent. 20. A. kashmiricum Stapf. In the alpine belt of Western Himalayas this species grows along with A. hete-

Figs. 22 and 23. Aconitum lycoctonum, n=8. 22, diakinesis showing stickiness and one pair of bivalents with heterochromatic segments (arrow). 23, A-I showing delayed separation.

Figs. 24 and 25. A. heterophyllum, n=8. 24, diakinesis showing one pair of bivalents with he terochromatic segments (arrow). 25. A-I showing 8:8 separation and late disjunction of one

bivalent.

Figs. 26-29. A. kashmiricum, n=8. 26, M-I. 27, A-I displaying 8:8 separation and a fragment

(arrow). 28, A-I showing chromatin bridge and fragment. 29, abnormal cell with a micronucleus formed from laggards at A-I. All figs. •~1,510. 1972 Cytology of Some W . Himalayan Ranunculaceae 291 292 P. N. Mehra and P. Remanandan Cytologia 37 rophyllum. It starts appearing from where A. heterophyllum begins vanishing and ascends upto 4,000m. However, in a narrow strip both the taxa are found to grow together. Cytological studies revealed it to be a diploid, as reported earlier by Mehra and Sobti (1955). Diakinesis showed 7 bivalents with two chiasmata and 1 bivalent with a single chiasma. Two large bivalents showed heterochromatic segments. Fig. 26 shows the M-I stage with 8 bivalents at the equator. The meiosis was found to undergo a partially disturbed course. Chain formation, occurrence of variable number of univalents, pseudobivalent formation between non-homologous chro mosomes, non-congression and non-orientation of bivalents, laggards and mic ronuclei (Fig. 29) have all been observed. In a mother cell at anaphase I, shown in Fig. 27, a fragment is present at the lower pole. Fig. 28 shows a bridge and fragment association indicating an inversion. All these aberrations are indicative of its hybrid nature. Eighty percent of pollen was apparently fertile on the basis of stainability. 21. Paeonia emodi Wall. This species forms lovely gregarious patches in the coniferous woods at 1500 3,000 m. The tubers are highly esteemed as medicine for a number of diseases. It is a diploid with n=5 (Fig. 30). This confirms the earlier report of Dark (1936). The chromosomes are large in size. Three bivalents were with two chias mata in each and two were rods with a single chiasma. Meiosis was found to un dergo an undisturbed course and 97 percent of the pollen was stainable.

Figs. 30. Paeonia emodi, n=5, diakinesis displaying chiasmata formation, •~1,510.

Discussion From the tribe Anemoneae, Anemone vitifolia (n=7), A. rivularis (n=8), Tha lictrum reniforme (n=7), Adonis chrysocyathus (n=16), Ranunculus hirtellus (n=8), R. diffusus (n=16) and R. arvensis (n=16) have been worked out for the first time. 1972 Cytology of Some W . Himalayan Ranunculaceae 293

All these reports are consistent with the base numbers already established for the respective genera. Two new cytotypes are presently discovered in Anemone obtusiloba which need some comments. Anemone is a large genus , though it is not well represented in India. Cytologically it has been extensively worked out . It comprises two groups of species, one with 8 as the basic chromosome number and the other with 7. Majority of the cytologically known species belong to the former group and a large number of taxa in this group are at various polyploid levels . A. nemorosa, however, exists in many aneuploid races (2n=30 , 37, 45, 46: Bernstrom, 1946, cf. Darlington and Wylie 1955). Presently two taxa with n=7 and 2n=26 have been discovered in A. abtusiloba. The former , described under var. glabra, is sufficiently distinct from the latter morphologically so as to merit a distinct specific status. R. laetus is found to exist in two intraspecific forms with n=14 and 16. The genus Ranunculus is reported to be dibasic on x=7 and 8. Numerous species, diploids as well as polyploids, are now known on these two base numbers. Majority of the species, however, have the base number 8. Triploids, tetraploids, hexaploids, octaploids and even 12-ploids are known (Darlington and Wylie 1955, Cave et al. 1964). There are many species like R. bulbosus, R. chius, R. melsoni, etc. which exist in two races with 2n=14 and 16. Thus the presently reported taxa of R. laetus (n=14, 16) are obviously tetraploids of a similar type. Similar report has been made by Mattick (1950 cf. Darlington and Wylie, 1955) in R. lanuginosus (2n=28, 32). A perusal of the literature reveals that polyploidy has played a very prominent role in the evolution of this genus. Among the 9 species belonging to the tribe Helleboreae Delphinium denudatum (n=8) is the only species newly worked out. The report on all the other species are in line with the previous reports on these taxa, excepting Caltha palustris. This species exists in numerous intraspecific races (see Table 1). The presently studied population with n=20 is a new one in the series, not yet reported. The generally accepted base numer for the genus is 8. However, the new race with n=20 and perfectly undisturbed course of meiosis suggests the possibility of the original base number for the genus to be x=4. Paeonieae is an unigeneric tribe and Paeonia emodi is the only species studied by us. The present report of n=5 is consistent with the earlier report by Dark (1936). The interrelationship of Paeonia needs some comments. Its recognition as belonging to a separate unigeneric family was advocated on anatomical grounds by Worsdell (1908). The gaudily coloured corolla and the bright red seeds of Paeonia were pointed to be comparable with those of Magnoliaceae rather than the Ran unculaceae. Kumazawa (1935) supported Worsdell's view on the basis of wood anatomy, and Eames (1953) recommended its removal from the Ranunculaceae considering evidences from floral vasculature. Maheshwari (1964) justified the removal of Paeonia from Ranunculaceae on the following embryological grounds. The exine of the pollen grains of Paeonia is reticulately pitted, whereas it is granular, papillate or smooth in other members of Ranunculaceae. The ovules are large and 294 P. N. Mehra and P. Remanandan Cytologia 37 borne on projections of the placenta, and the embryo sac is extremely long and narrow. The germination of the seed is of the hypogeal type. He considered that in all these features Paeonia stands much apart from other members of Ranuncu laceae. Yakolev and Yoffe (1957) described the embryo development in Paeonia as free nuclear which would be unique among the Angiosperms. A reinvestigation by Murgai (1959, 1962), however, revealed some errors in this account. The suspensor cell in this genus becomes free nuclear while the terminal cell, which in other genera behaves as the embryonal cell, degenerates. Then wall formation occurs in the free nucleate suspensor and some of the peripheral cells give rise to the embryos. This condition according to Maheshwari (1964) is not met in any member of the Ranunculaceae. The latter author concluded that Paeonia does not quite fit in with any of the other families and hence shared the views of Lawrence (1951) and Davezac (1957) for the erection of a new family Paeoniaceae for its reception. The pertinent question which arises now is whether the cytological evidence supports the removal of Paeonia from Ranunculaceae or its retention. Cytologically Paeonia consists of a remarkably homogeneous assemblage of species and has the basic chromosome number x=5 which no other genus of the family possesses. The most common base number in the family is 8. The other common number is 7. Morphological, anatomical (Worsdel 1908, Kumazawa 1935, Eames 1953), embryological (Maheshwari 1964) and the cytological evidences are all in favour of removal of Paeonia from the Ranunculaceae and the erection of a new family Paeoniaceae for the reception of this unique genus. If this is done the family would then be a very natural taxon. Kurita's (1959, 1963) contributions have thrown some light on the structural changes which the chromosomes have undergone during speciation in the family. It has now become evident that though a wide range of base numbers are not prevalent in the family, structural changes in the chromosomes and polyploidy have mainly contributed towards the evolutionary diversification of the family. Poly ploidy occurs in about 45 percent of species. Evolution has been of a higher order in Helleboreae than in Anemoneae. It is manifested morphologically in the complexity of floral organisation (zygomorphy) in Aconitum and Delphinium, formation of spurs and partial fusion of carpels in Nigella, chromosomally in the alteration of chromosome number in Nigella (x=6) in consonance with its floral advancement, and in the reduction of chromosome size coupled with alteration in number in Coptis (x=9), Glaucidium (x=10) and Hy drastis (x=13). In Anemoneae, however, apart from the reduction in the number of ovule to one in the carpel, there has been a relative stability in floral structure and also of chro mosome number (n=8, 7). There is also no such extreme variation in chromosome size such as is known in Helleboreae. The original basic chromosome number of the family appears to be x=4. This is suggested for Adonis by Kurita (1963) on the basis of occurrence of n=8, 12, 16, 20 in some of its species and for Caltha on the basis of a cytotype with n=20 1972 Cytology of Some W. Himalayan Ranunculaceae 295

discovered presently in C. palustris. This also corroborates the views of Sato (1962) arrived at on purely theoretical considerations. Furthermore , the occurrence of n=8 in majority of the members indicates that basically these genera are at the tetraploid level and this must have been attained rather early in the evolutionary history of the family. Undoubtedly alteration in chromosome number, karyotypic changes and gene mutations have all been responsible for the diversification of species and genera within the family.

Summary

Cytological account based on meiotic studies of 23 taxa belonging to Ra nunculaceae is presented. Eight species have been investigated for the first time and three intraspecific races have been discovered. Chromosomal aberrations are recorded in Anemone obtusioloba, Ranunculus hirtellus, R. laetus, Delphinium cardiopetallum and Aconitum kashimiricum. It is inferred that the original base number of the family is x=4 and early in its evolutionary history it became doubled so that the prevalent haploid number in the family is n=8. Evolutionary diversi fication in the family has involved alterations in chromosome number, karyotype changes, polyploidy and of course gene mutations.

Acknowledgement Financial assistance from U. S. Department of Agriculture under PL 480 Project (Grant No. FG-In-281) is gratefully acknowledged. The authors thank the Director, Botanical Survey of India for the facilities provided at the Central National Herbarium, Calcutta, and the Regional Head Quarters, Dehradun. Our thanks are also due to Atomic Energy Commission for the laboratory facilities provided at High Altitude Research Laboratory, Gulmarg.

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* Not consulted in original .