Cytologia 38: 237-258, 1973
Cytological Investigations on W. Himalayan Pooideae
P. N. Mehra and P. Remanandan Departmentof Botany,Panjab University, Chandigarh,India ReceivedSeptember 9, 1971
Introduction
In recent years the agricultural scientists in India have started realising the severity of shortage of cattle feed and a number of exotics have been introduced and tried for fodder purposes. The results have not been much encouraging . What is perhaps needed is the exploitation of the indigenous material for which the Hima layas are exceptionally rich. The moist hills and valleys of these mountains possess excellent grass-lands which extend up to 4,500m. A very small proportion of these meadows is utilized for grazing by goats, sheep and yaks. With such a wealth of indigenous species which give excellent green stuff and can be made into palatable hay or silage it is worthwhile to make selection and effect systematic improvement of these by cytogenetic methods. The objective of the present study was to investigate the cytology and distribu tional pattern of Indian grasses to enrich the germplasm and also to delineate the evolutionary trends based on the meiotic behaviour of chromosomes. The first paper of this series (Mehra et al. 1968) presented the cytological data on 145 species belonging to 68 genera mostly distributed in Punjab plains and nearby hills. An attempt was made to trace the evolutionary trends and a scheme for the probable cytological evolution of the family was presented. The second paper (Mehra and Shyamsunder 1970) presented the cytological data on 63 species belonging to 23 genera, distributed mainly in the Kashmir hills. The present paper is a continua tion of these earlier works and cytological data of 33 taxa belonging to 28 species comprising 16 genera is presented.
Material and methods The material was collected from wild populations in the Kashmir and Kumaon hills of the northwest Himalayas. The grasses were identified in the field with the help of Bor's keys (Bor 1960) and confirmed by comparing them at the herbaria of Forest Research Institute, Dehradun, and Botanical Survey of India zonal head quarters, Dehradun. The voucher specimens and the permanent slides have been deposited in the herbarium of the department of Botany, Panjab University, Chan digarh. Cytological studies were made at meiotic divisions of microsporocytes. Flower buds were fixed in Carnoy's fluid and simple acetocarmine squashes were prepared 238 P. N. Mehra and P. Remanandan Cytologia 38
Table 1 1973 Cytological Investigations on W. Himalayan Pooideae 239
Table 1. (contd.) 240 P. N. Mehra and P. Remanandan Cytologia 38
Table 1. (contd.) 1973 Cytological Investigations on W. Himalayan Pooideae 241
Table 1. (contd.) 242 P. N. Mehra and P. Remanandan Cytologia 38
for the study of microsporogenesis. To detect intraspecific cytological races, same species was screened from a wide range of localities. All the photomicrographs
were taken at a magnification of •~1,130. Slides were made permanent following
the procedure of Bhaduri and Ghosh (1954).
Results
Table 1 summarizes the results of investigations on 28 species. Five species, marked * are worked out for the first time, whereas those marked t represent new
cytotypes. A+mark on the•eprevious authors'side indicates that this species has been extensively investigated and only the more significant reports are included. In the succeeding pages only the more interesting features, such as the meiotic
aberrations and comparative morphology of the intraspecific cytological races are briefly discussed.
Meiotic irregularities It has been noticed that most of the grasses of alpine regions, as a rule, do not set seeds, or if they do so, the seeds are mostly sterile. This is ascribed to a number of causes such as environmental factors, extensive vegetative method of multipli cation, browsing by animals, and genomic instability as reflected in the various meiotic aberrations and subsequent production of sterile pollen. Alopecurus Three species of this genus were investigated of which A. arundinaceus is a valu able constituent of the alpine pastures. In Kashmir A. himalaicus has been noticed only on hill tops of snowy ranges which are not easily accessible to cattle. How ever, it has been reported that cattle and yak do relish it (Bor 1960). Meiotic disturbances were observed in all the three species. A. aequalis is a diploid with 2n=14. In this species as many as 8 chromosomes were found to be involved in the formation of a complex structure with chiamata between nonhomologous chromosomes, representing reciprocal translocations. In majority of cases this structure did not form a complete ring and two free ends were noticeable (Fig. 1). It showed delayed separation of chromosomes at A, (Fig. 2). At late AI laggards were common (Fig. 3). Only 75% of pollen grains were stainable. A. arundinaceus is a tetraploid and 14 bivalents were observed in some cells at diakinesis (Fig. 4). However, at MI several pollen mother cells showed a large ring suggestive of translocations, obviously involving several chromosomes (Fig. 5). Further course of meiosis was regular and 90% of the pollen grains were fertile.
Figs. 1-9. 1-3. Alopecurus aequalis, n=7. 1, diakinesis showing 3 bivalents and a multivalent body consisting of 8 chromosomes indicating structural heterozygosity for reciprocal translocation. 2, late separation of multivalents at AI. 3, late AI showing laggards at the equator. 4-5. A. arundinaceus, n=14. 4, diakinesis showing 14 bivalents. Note the chromatin link between chro mosomes of two bivalents (arrow). 5, Mi showing a multivalent ring. 6-9. A. himalaicus, 2n= 45. Illustrations demonstrating meiotic breakdown. 6, nonorientation of univalents at MI. 7, AI showing 2n=45. 8, laggards at late AI. 9, Two chromatin bridges (arrows) and laggards at late AI. •~l,130. 1973 Cytological Investigations on W. Himalayan Pooideae 243 244 P. N. Mehra and P. Remanandan Cytologia 38
A. himalaicus is a newly investigated species with 2n=45 (Fig. 7). Meiotic studies revealed that the divisions undergo a highly disturbed course resulting in various aberrations which lead to a total breakdown of meiosis, and consequent production of sterile pollen. The following were the chief aberrations noticed: i) Nonhomologous associations, ii) loose pairing of chromosomes at diakinesis, iii) nonorientation of univalents at M, (Fig. 6), iv) nonsynchronised disjunction at AI, v) bridges (Fig. 9), laggards (Figs. 8, 9), vi) fragments and vii) unequal distribu tion of chromosomes at A, and AII. Although spindle apparatus was formed, the congression of chromosomes at metaphase plate was imperfect and a number of univalents were found scattered throughout the cytoplasm. This is the first report of a taxon in Alopecurus with 2n=45, all the other cytologically known species of the genus having been found to possess the basic number 7. Cytological evidence suggests that the species may probably be of hybrid origin and meiotic breakdown is due to its unbalanced genomic constitution. Calamagrostis C. pseudophragmites showed a regular cycle of meiosis with 14 bivalents at diakinesis. Thirteen were the ring bivalents with terminalised chiasmata and one bivalent had interstitial chiasma (Fig. 10). However, in C. emodensis meiosis was found to undergo a highly abnormal course leading to total meiotic breakdown. Well spread metaphase plates could not be obtained due to stickiness, secondary associations, and clumping of chromosomes. The taxon, however, appears to be a triploid with 2n=21, but this needs confirmation from mitotic preparations. Polypogon Two species, P. fugax and P. monspeliensis, which provide a rich feed for graz ing animals, have been investigated. The former is a hexaploid (n=21, Fig. 12) and the latter a tetraploid (n=14). These reports are in line with the previous records of many authors (cf. Table 1). However, Mehra et al, (1968) reported n=14, 21 in P. monspeliensis. Their voucher specimen for the taxon with n=21 probably is that of P. fugax. These two species are closely related and are dis tinguished by the relative length of the awn which is shorter in the latter species compared to the former. Avenafatua Linn. This species closely resembles the cultivated species A. sativa Linn. But it can be readily distinguished by its rhachilla which disarticulates between all the florets at maturity and the presence of a thickened callus at the base of all lemmas. The present studies revealed that the species exists in two cytological races, a diploid (n=7) and a tetraploid (n=14). The diploid race, which is investigated for the first time, was detected in Kashmir, while the tetraploid in Kumaon hills. A comparative morphological analysis of the taxa is presented in Table 2 and they are illustrated in Fig. 42. The diploid is evidently more sturdy than the tetraploid.
Figs. 10-19. 10, Calamagrostis pseudophragmites, n=14. Diakinesis displaying chiasmata dis tribution. 11, Phleum alpinum, n=14. Diplotene displaying formation of chiasmata. 12, Polypogon fugax, n=21. 13-14. Avena fatua, n=7. 13, diakinesis showing 7 rings. 14, early 1973 Cytological investigations on W . Himalayan Pooideae 245
disjunction at AI. 15-19. A. fatua, n=14. 15-16. early and late diakinesis displaying chiasmata distribution. 17, MI with 14II. 18, MI with 12II and 1VI (arrow). 19, 14: 14 separation at AI. •~1,130. 246 P. N. Mehra and P. Remanandan Cytologia 38
The diploid taxon showed 7 ring bivalents and a well differentiated nucleolus at diakinesis (Fig. 13). Mild disturbances were noticed at AI. Precocious separa tion of some chromosomes (Fig. 14) was occasionally observed. However, further stages were normal. In most of the cells at late AI 7:7 distribution was noticed. 95% pollen were viable. Figs. 15-16 display the chiasmata distribution in the tetraploid at diakinesis.
Table 2. Avena fatua*
* Average of 10 measurements in each case .
In the majority of cells 12 ring bivalents were observed and 2 bivalents showed a single chiasma in each. A well differentiated nucleolus was noticed at this stage. Some cells showed structural heterozygosity as illustrated by the formation of a ring of 4 chromosomes (Fig. 18). AI showed regular disjunction of 14:14 (Fig. 19). Further stages were normal with 95% fertile pollen.
Hierochloe laxa R. Br. Diakinsis revealed 14 bivalents (Fig. 20). Secondary associations were ob served occasionally at this stage. At AI nonsynchronised disjunction and laggards
Figs. 20-31. 20-21. Hierochloe laxa, n=14. 20, diakinesis. Note the chromatin link between chromosomes of different bivalents (arrow). 21, AI displaying 14:14 separation. 22, Agropyron sp., n=14. 23, Bromus japonicus, n=7. Diakinesis revealing chiasmata distribution (5rings +1 ring with an interstitial chiasma+1 rod). 24, Dactylis glomerata, n=7. 25, Festuca undata, n=21. 1973 Cytological Investigations on W. Himalayan Pooideae 247
Diakinesis showing secondary associations and stickiness. 26-27. F. valesiaca, n=14. 26, nonorientation of two bivalents at MI (arrows). 27, AI showing breakage due to stretching. 28, Lolium perenne, n=7. 29-31. L. temulentum, n=7. 29, MI showing 7II. 30, delayed separa tion at AI. 31, 7:7 separation at AI. •~l,130. 248 P. N. Mehra and P. Remanandan Cytologia 38 were noticed in some cells. However, majority of them showed 14:14 separation (Fig. 21). The species, which is a tetraploid on the base number 7, has been reported for the first time.
Koeleria cristata (Linn.) Pers. This is another species of high mountains found on rather inhospitable alpine slopes. It provides certain amount of fodder in these areas.
Table 3. Bromus japonicus*
* Average of 10 measurements in each case .
Diploid, tetraploid and aneuploid races in this species have been reported by previous authors (cf. Table 1). Some have also reported the occurrence of B chromosomes in the diploid race. The presently studied taxon is a diploid and at diakinesis 7 bivalents were clearly discernible. Six were rings with terminalised chiasmata and the seventh was a rod-bivalent. This population did not show any B chromosome. Meiosis was regular with 98% fertile pollen.
Bromus himalaicus Stapf. var. grandis Stapf. At diakinesis 14 bivalents were clearly discernible. AI was regular. However, in a few cells late separation and laggards were observed. Further stages were nor mal and 90% of pollen grains were fertile. The species has been reported for the 1973 Cytological Investigations on W. Himalayan Pooideae 249 first time.
B. japonicus Thunb. A very common grass in Kashmir and Kumaon hills which constitutes a signi ficant part of the pasture in these areas. Cytological survey of the populations from a number of localities revealed the existance of two races, diploid and hexa ploid. These cytotypes have already been reported by many workers (cf. Table 1). A morphological analysis of the races is presented in Table 3. It is worthy of note that the hexaploid is weaker than the diploid (Fig. 43). All the populations studied from Kashmir hills were diploid. Meiotic pre parations revealed 7 bivalents at diakinesis (Fig. 23). Five were ring bivalents with terminalised chiasmata. One bivalent showed one terminal and another interstitial chiasma. The seventh was a rod bivalent. A total of 13 chiasmata were formed in the majority of cells. Meiosis was regular. The hexaploid race occurs in Kumaon hills. Meiotic studies revealed 21 bi valents at diakinesis. At AI 21:21 distribution was observed. Further course of meiosis was regular. Dactylis glomerata Linn. The species is one of the most important pasture grasses and has been extensive ly worked out earlier. Numerous polyploid races and aneuploid derivatives have been reported (cf Table 1). Presently diploid (n=7, Fig. 24) and tetraploid (n=14) races were discovered from two distantly located areas in the Himalayas, the former from Kashmir hills and the latter from Kumaon hills. Meiosis was regular in both cases. There were no appreciable morphological differences between the two races to distinguish them in the field. Festuca Three species have been investigated. Bor (1960) recorded F. undata from Sikkim in the Eastern Himalayas. The grass has now been discovered in the Western Himalayas. It has been further found to exist in two intraspecific races, tetraploid and hexaploid. Meiosis was regular in the tetraploid taxon and 90 pollen grains were fertile. The hexaploid taxon was characterised by various chromosome aberrations such as secondary associations (Fig. 25) and univalents at diakinesis, stickiness of chromosomes and late separation at AI. Meiotic ir regularities were reflected in pollen fertility. Only 45% of the pollen grains were stainable. The species is investigated for the first time. F. valesiaca is an alpine grass of high mountains. It is grazed by yaks and sheep. Meiosis was found to undergo a partially disturbed course. At MI non orientation of some bivalents was noted (Fig. 26). Chromosome breakage due to stretching was occasional at AI (Fig. 27). However, 80% pollen were stainable. The present report of n=14 is in conformity with all the previous reports (cf. Table 1) except that of Mehra and Shyamsunder (1970). They reported n=21 for the species representing another cytological race. 250 P. N. Mehra and P. Remanandan Cytologia 38 1973 Cytological Investigations on W . Himalayan Pooideae 251
Lolium Three species of this genus have been studied. It was observed that the features distinguishing L. multiflorum and L. perenne are not well marked. Cytological studies gave the same result in both these species. Seven biva lents were observed at diakinesis and meiosis followed a regular course (Fig. 28 f or L. perenne). These are among the valuable grazing and hay grasses of hills. L. temulentum is met with at lower elevations where it is a weed in wheat fields. It is also a diploid with n=7 (Figs. 29, 31). Mild aberrations such as late disjunc tion (Fig. 30) and laggards were observed in some cells. Ninety-seven percent pollen grains were fertile. Poa Different species of Poa consistute a significant part of the sub-alpine and alpine meadows of the Himalayas. Many species are liked by cattle for grazing. Four species have been investigated presently. Meiosis was found to undergo a highly disturbed course in all of them. Poa alpina presented a case of total meiotic breakdown . Diakinesis was abnormal with univalents and multivalents. At MI the chromosomes were not oriented on the metaphase plate, instead they were found scattered . Clumping of chromosomes was common. AI was abnormal. However , some cells could be obtained in which 51 chromosomes were counted clearly. Meiotic aberrations were reflected in the pollen fertility which was only 40%. The species has been extensively worked earlier. Numerous races, mostly at high polyploid levels are known (cf. Table 1). Many authors have reported the occurrence of varying number of B chromosomes. A new intraspecific race of P. annua has been recorded with 2n=52. Meiosis was highly abnormal. Following aberrations were noticed: i) multivalents at MI, ii) univalents and laggards in varying number at AI, iii) associations at second meiotic division, iv) heteropycnosis of chromosomes and v) nonsynchronization of cytokinesis and chromosomal division. Only 45% pollen grains were stainable. P. bulbosa, another valuable fodder grass, was found to exist in two intra specific cytotypes in Kashmir, with n=21 and 2n=50. Meiosis was abnormal in both. In the hexaploid with n=21, a large number of chromosomes were involved in various associations (Fig. 32). AI was irregular with late separation and laggards (Fig. 33). However, 80% pollen grains were fertile. Total meiotic breakdown was observed in the taxon with 2n=50. At diakinesis and MI most of the chro mosomes were involved in complex types of associations (Figs. 34-35) indicating structural rearrangements. Univalents, fragments and clumping of chromosomes
Figs. 32-41. 32-33. Poa bulbosa, n=21. 32, diakinesis showing multivalents and associations. 33, aberrant cell with laggards. 34-37. P. bulbosa, 2n=50. Illustrations displaying meiotic breakdown. 34, diakinesis showing various associations and univalents. 35, Mi showing multi valents of various number of chromosomes, univalents, secondary associations and nonorientation of chromosomes. 36, early AI showing 2n=50. 37, aberrant cell with laggards. 38-40. P. nemoralis, 2n=c.70. Illustrations displaying meiotic breakdown. 38, diakinesis showing multi valent rings (arrows) and univalents. 39, AI showing 2n=c.70. Note the presence of bivalents
(arrow) at this stage. 40, aberrant cell with laggards. 41, Phalaris arundinacea, n= 14. •~1,130. 252 P. N. Mehra and P. Remanandan Cytologia 38 1973 Cytological Investigations on W. Himalayan Pooideae 253 were common. AI was highly irregular. Non-synchronised disjunction , laggards (Fig. 37) and unequal distribution of chromosomes were commonly observed. Only 40% pollen grains were stainable. A number of cytological races of P . bulbosa have been reported from various parts of the world by the previous workers (cf. Table 1). P. nemoralis is another high polyploid species which showed meiotic break down. Fig. 38 illustrates multivalent rings and many other associations at MI. Univalents and non-orientation of chromosomes were also noticed at this stage. Delayed separation of chromosomes and laggards (Fig. 40) were common at AI. The diploid complement, 2n=c.70, is presented Fig. 39. P. pratensis, an excellent pasture grass, presented yet another taxon with com pletely irregular meiotic cycle. Total asynapsis was observed at early stages of meiosis. All the above mentioned aberrations were also noticed in this species which resulted in the formtaion of a very high percentage of sterile pollen. Due to acute clumping of chromosomes and stickiness accurate count was not possible.
Discussion Our experience in grass cytology has shown that grasses are in an active state of evolution. Numerous chromosomal changes like polyploidy, aneuploidy, structural re-arrangements are exhibited in diverse species, many of which are eliminated during sex cell formation in the form of high pollen sterility. But such abnormal genotypes have a chance of limited propagation and survival through multiplication by vegetative means in case they are physiologically balanced in relation to environments. Numerous instances of aberrant genotypes occurring in nature are thus observed and are cited in the present paper. Such fluctuations in chromosome numbers are especially rampant in certain genera and species which have an agamospermic mode of reproduction. Instances of these are met in species of Poa and Festuca which show a wide variety of unbalanc ed chromosome numbers with consequential high percentage of pollen sterility. The reasons are obvious. Such apomicts bypass the sexual sieve which eliminates the unbalanced sex cells from becoming functionally operative in nature. Multi plication is carried through adventive embryony where mostly the nucellar tissue is involved. A pertinent question that needs some discussion is in regard to the taxonomic status of the polyploid taxa met within a species. Should they have distinct taxo nomic entity at the specific level? Different views have been expressed in this direction. Some authorities feel that such taxa should be given distinct specific status (Mayr 1942, Stebbins 1950, Love 1964, Love and Love 1967, Rogers and Appan 1969). There is lot of weight in this way of thinking, for logically, a species should have a biological entity of its own with a capacity of free exchange of genes amongst members of its population and there should exist reproductive isolation or barrier in gene exchange with such other populations. Polyploids thus do become entitled to a distinct specific status as against their diploid ancestors from which they may have been derived, since between them and the diploids there 254 P. N. Mehra and P. Remanandan Cytologia 38 is now a barrier in free exchange of genes because the offsprings would be sterile. But there is another way of looking at things. Many such polyploids are hardly distinguishable from their diploid ancestors, especially in the field, except for quantitative or minute qualitative differences which even may not be obvious under varying ecological conditions in which these taxa grow in nature. At best they could be regarded as sub-species or incipient species which are potentially capable of evolving into distinct taxonomic categories but not yet mature enough to merit species status. Botany is not merely chromosomal taxonomy in the limited sense. It has many facets. If a field ecologist, for example, has to analyse chromosome numbers before he can proceed further, such a procedure forfeits the purpose of his research. The same applies to a herbarium taxonomist, an anatomist and so on. Therefore, in our opinion a cytotype be given a distinct specific status only when such a taxon also shows sharp easily identifiable morphological features different from the diploids. Thus a polyploid chromosome number coupled with definite visible discontinuities in morphological characters should entitle a cytotype to merit the rank of a separate species. In our investigations of the families Grami neae, Orchidaceae and Compositae we have come across numerous instances of indistinguishable cytotypes at diploid and polypliod levels. In the present investi gation diploid and tetraploid Dactylis glomerata can hardly be distinguished from one another. Two other instances which deserve mention are 1) of Avena fatua in which in nature the diploid is grosser compared to the tetraploid in plant size as well as in the size of its various parts (Fig. 42) and 2) of Bromus japonicus in which the diploid is larger than the hexaploid (Fig. 43) but there are no noteworthy qualitative mor phological differences. These instances are in contrast to many others where the condition is just the reverse i.e. the polyploid races are larger than their diploid counterparts. Such cases are illustrated in our two previous publications dealing with Gramineae (Mehra et al. 1968, Mehra and Shyamsunder 1970).
Summary The paper deals with the cytology of 28 species of fodder grasses of the W. Himalayas based on meiotic studies. Five species have been investigated for the first time. Two new races have been discovered in the species already investigated. Intraspecific variation has been noticed in Avena fatua (n=7, 14), Bromus japonicus (n=7, 21), Dactylis glomerata (n=7, 14), Festuca undata (n=14, 21) and Poa bulbosa (n=21, 2n=50). Taxonomic status of the polyploid races is briefly dis cussed.
Acknowledgements
This work was supported by grant FG-in-281 from PL 480 funds in India to the senior author. Our sincere thanks are due to U.S. Government for financial assistance. We thank Dr. V. J. Nair, Botanical Survey of India, Dehradun and Miss Rajkumari Kaul, University of Kashmir for assistance in identification. 1973 Cytological Investigations on W. Himalayan Pooideae 255
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