2019 年 84 巻 1 号 p. 25-29
Meiotic analysis on 26 wild plants of Phacelurus speciosus collected from Kullu district, Himachal Pradesh recorded the existence of three intraspecific euploid cytotypes, the diploid (2n=20), tetraploid (2n=40) and hexaploid (2n=60). Besides chromosome number, cytotypes also differ in morphometric parameters such as plant height, number of internodes, length of uppermost internodes, length of leaf sheath, length and width of leaf lamina, length of spike/spikelet etc. The 4x and 6x plants grow much taller and robust compared to those of 2x. These cytotypes also differ in sizes of pollen grains and stomata. Plants of 2x, 4x and most of 6x showed normal meiotic course and high pollen fertility. On the other hand, three plants of 6x cytotype showed meiotic irregularities in the form of secondary chromosomal associations, non-synchronous disjunctions. Besides, one plant of 6x cytotype also showed the presence of 1-2 B chromosomes in PMCs.
Phacelurus speciosus (Steud.) C.E. Hubb. is an erect perennial grass with solid and hard rootstocks, is found on grassy slopes along roadsides and field margins between elevation 2000–3500 m. The species found commonly in Afghanistan, North-West India and Nepal, possessed linear leaves with acuminate apex, more or less hairy with scabrid margins, and cylindrical solitary racemes. To assess the evolutionary and taxonomic relationship in the species, field surveys were conducted to the different regions of Western Himalayas in Kullu district. In order to reveal the cytological and morphometric features of P. speciosus growing in this region the present study has been undertaken with following aims—to work out the exact chromosome numbers on individual plant basis, to know the distribution pattern of 2x, 4x and 6x individuals, to segregate the cytotypes through morphometric analysis, to study in detail the male meiotic course, and to work out the pollen fertility.
Materials were collected from the 26 wild populations of P. speciosus from different localities within elevation 1600–3223 m in the district Kullu, Himachal Pradesh, India (Table 1). Cytologically examined specimens were identified on the basis of study of floral and vegetative characters and consultation of various floras viz., Flora of Himachal Pradesh (Chowdhery and Wadhwa 1984), Flora of Kullu District, Himachal Pradesh (Dhaliwal and Sharma 1999) and Flora of Great Himalayan National Park, Himachal Pradesh (Singh and Rawat 2000). The identifications of specimens were revalidated by comparing with the samples lying in the Herbaria, Botanical Survey of India, Dehra Dun, Department of Botany, Punjabi University, Patiala and Forest Research Institute, Dehra Dun. Voucher specimens were deposited in the Herbarium, Department of Botany, Punjabi University, Patiala (PUN).
| S. No. | Characters | Cytotypes | ||
|---|---|---|---|---|
| Diploid (2x) | Tetraploid (4x) | Hexaploid (6x) | ||
| 1. | Site of collection (altitude, PUN number) | Barshani (2150 m, PUN 60305), Tosh (2400 m, PUN 60314), Malana Village (2652 m, PUN 61432) | Naggar (2047 m, PUN 61436), Kothi (2500 m, PUN 61429), | Jari (1600 m, PUN 61420), Shaat (1600 m, PUN 61271), Kasol (1640 m, PUN 61430), Khanag Village (2086 m, PUN 61435), Manikaran (2090 m, PUN 61434), Nehru Kund (2100 m, PUN 61422), Pulga (2100 m, PUN 61421), Barshani (2150 m, PUN 61423), Kanial Village (2280 m, PUN 61424), Jana (2336 m, PUN 61426), Palchan (2400 m, PUN 61437), Bijali Mahadev (2435 m, PUN 61427), Vashisht Village (2475 m, PUN 61428), Chauki (2500 m, PUN 61216), Solang Valley (2560 m, PUN 61431), Sheela Village (2650 m, PUN 61425), Malana Village (2652 m, PUN 60297), Satyam Tunnel (2750 m, PUN 61217), Jibhi (3100 m, PUN 61227), Shoja (3120 m, PUN 61225) and Jalori Pass (3223 m, 61226). |
| 2. | Distribution | Common | Less common | Frequent |
| 3. | Habit | Perennial | Perennial | Perennial |
| 4. | Habitat | Roadsides | Roadsides | Roadsides, grassy slopes |
| 5. | Plant height (cm) | 26.3–70.1 | 35.0–80.4 | 40.0–110.2 |
| Stem | ||||
| 6. | Number of internodes | 4–5 | 6–8 | 7–9 |
| 7. | Length of uppermost internode (cm) | 6.0–10.0 | 9.2–14.0 | 18.0–23.0 |
| 8. | Length of 2nd internode from top (cm) | 5.0–9.0 | 6.2–9.0 | 10.0–11.0 |
| Leaf | ||||
| 9. | Length of sheath (cm) | 6.0–7.1 | 7.3–8.0 | 9.0–13.1 |
| 10. | Length of lamina (cm) | 6.1–11.0 | 10.8–14.3 | 15.1–25.6 |
| 11. | Width of lamina (mm) | 3.2–6.5 | 4.4–8.0 | 6.0–10.1 |
| Inflorescence | ||||
| 12. | Length of spike (cm) | 7.0–10.1 | 8.0–12.2 | 8.5–20.0 |
| 13 | Length of spikelet (cm) | 8.2–9.0 | 9.2–10.4 | 10.8–12.2 |
| 14. | Pollen fertility (%) | 80–84 | 89–90 | 80–98 |
| 15. | Pollen size (µm) | 23.85×23.85–25.35×25.35 | 25.35×25.35–28.33×24.45 | 31.31×28.33–32.80×29.82 |
| 16. | Stomatal size (µm) | 29.50 ×13.48 | 30.10×11.16 | 48.05×22.92 |
| 17. | Stomatal index | 28.0 | 26.66 | 14.28 |
For investigating gametic chromosome numbers, meiotic course and pollen fertility, young and unopened spikes of each specimen were fixed in a freshly prepared Carnoy’s fixative (glacial acetic acid : chloroform : ethanol=1 : 3 : 6) for 24 h and subsequently stored in 70% ethanol in a refrigerator. A total of 50–100 meiocytes were examined for determining the chromosome number, while 35–70 slides were prepared from different anthers/florets for analysis of meiotic course. Pollen fertility was estimated through stainability test for which anthers from mature florets were squashed in glycerol–acetocarmine mixture (1 : 1). Well-filled pollen grains with uniformly stained cytoplasm were scored as fertile, while unstained or poorly stained were counted as sterile. Photomicrographs of meiotic chromosomes, sporads, and pollen grains were taken using a Nikon Eclipse 80i microscope.
Stomatal studiesAbaxial epidermal peels obtained from mature leaves through KOH treatment were stained in safranine red.
Plants of three populations gathered from Barshani (2150 m), Tosh (2400 m), and Malana Village (2652 m) showed the 10 bivalents in PMC at metaphase I (M I) (Fig. 1A) and 10 : 10 chromosomes at poles during anaphase I (A I) (Fig. 1B). Plants with chromosome number n=10 were named 2x cytotype. The plants from Kothi (2500 m) and Naggar (2047 m) depicted the presence of 20 bivalents at M I (Fig. 1D) indicating 4x cytotype.
Plants collected from different parts of the Kullu district (Table 1) showed the gametic chromosome number of n=30 as confirmed from the presence of 30 bivalents (Fig. 1F,G) and 30 : 30 chromosomal segregation during A I (Fig. 1H). Plants with chromosome number n=30 were considered 6x cytotype. Total of 18 populations analyzed depicted normal meiotic behavior and equal-sized fertile pollen grains. However, the three accessions collected from three localities at higher altitudes, Bijali Mahadev (2435 m), Jibhi (3100 m) and Jalori Pass (3233 m) showed some meiotic irregularities (Table 2) such as secondary chromosomal associations (Fig. 1I), non-synchronous disjunctions (Fig. 1J). Plants collected from Jibhi (3100 m) also showed the presence of 1-2 B chromosomes in some meiocytes (Fig. 1K). These cytotypes are randomly distributed in the area (Fig. 2).
| Meiotic abnormalities | Total PMCs observed | The locality of accessions with elevation and PUN No. | ||
|---|---|---|---|---|
| Bijali Mahadev (2435 m, PUN 61427) | Jibhi (3100 m, PUN 61227) | Jalori Pass(3223 m, PUN 61226) | ||
| PMCs with secondary chromosomal association | 154 | 2/31 (6.45%) | 6/48 (12.5%) | 13/75 (17.33%) |
| PMCs with asynchronous disjunction | 156 | 4/33 (12.12%) | 7/48 (14.58%) | 11/75 (14.66%) |
| Pollen fertility (%) | 90 | 88 | 80 | |
Plants of three cytotypes grow as perennial herbs along roadsides and grassy slopes at an elevation between 1600 to 3223 m at Jalori Pass in the district. Morphometric analysis involving plant height, length of uppermost and 2nd internode, leaf, spike and spikelet size were studied separately on three cytotypes (Table 1). Analysis revealed that 6x plants grow much taller (40.0–110.2 cm; Fig. 3C) compared to 4x (35.0–80.4 cm; Fig. 3B) and 2x (26.3–70.0 cm; Fig. 3A). Length of uppermost internode and 2nd internode from the top was also noticed to be significantly larger in the 6x (18.0–23.0 cm; 10.0–11.0 cm) compared to the 4x (9.2–14.0 cm; 6.0–9.0 cm) and 2x (6.0–10.0 cm; 5.0–9.0 cm). Leaf size (sheath and lamina), spike and spikelet were also observed to be much larger in 6x plants compared to 4x and 2x plants (Table 1). Microscopic parameters analyzed for ploidy-related differences included stomatal size (Fig. 3D–F), pollen diameter was also noticed to be larger in 4x and 6x cytotypes compared to 2x. Size of stomata varies from 29.50×13.48 µm in 2x to 30.10×11.16 µm in 4x and 48.05×22.92 µm in 6x (Table 1). However, the stomatal index was calculated to be significantly lower in the 6x (14.28) compared to the 4x (26.66) and 2x (28.0).
P. speciosus in India showed the existence of intraspecific euploid cytotypes, these are diploid with 2n=20 (Mehra and Sunder 1969, Mehra and Remanandan 1973, Mehra and Sharma 1975, 1977, Sharma and Sharma 1979), tetraploid with 2n=40 (Mubarik et al. 2017), hexaploid with 2n=60 (Mehra et al. 1968) and octoploid with 2n=80 (Mehra 1965). On the basis of meiotic analysis made on wild plants, we have detected the plants exhibiting three different chromosome numbers and ploidy levels (2n=20, 40, 60) from Kullu district, Himachal Pradesh, India. The chromosome number of 2n=22 recorded by Mehra and Sunder (1969) from North India indicates the existence of intraspecific heteroploidy. Besides, reporting the existence of three euploid cytotypes (2x, 4x, 6x) in the species from the area, we here record the presence of B-chromosomes in 6x cytotype. All examined plants of 2x and 4x cytotype and 18 plants of 6x cytotype showed regular meiosis and high pollen fertility. Polyploidy has played an important role in bringing out genetic diversity in a wide variety of plant lineages (Stebbins 1950, Grant 1981, Rieseberg and Wendel 1993, Masterson 1994, Barker et al. 2016). Polyploidy is quite widespread in grasses and is an evolutionary successful path chosen by most of the grasses from the beginning of their formation to the present and this is particularly true of P. speciosus as large number of other grass species.
Polyploids are categorized into auto- and allopolyploids based on the mode of their formation and genome constitution. The autopolyploid is originated by polyploidization within a species, and forms multivalents during meiosis to cause sterility. Whereas the allopolyploid is the result of hybridization between different species followed by chromosome doubling. The polyploids are characterized by diploid-like chromosome pairing and high pollen fertility. Owing to these facts, allopolyploids have been considered to be more common than auto-polyploids, mainly due to their adaptive superiority owing to high pollen fertility. In P. speciosus, the individuals of 4x and 6x cytotypes showed normal chromosome pairing, regular segregation and high pollen fertility as that of observed in a typical allopolyploid. There are numerous reports of intraspecific euploid cytotypes which show diploid-like meiotic behavior in grasses (Kaur et al. 2011, 2014, Singhal et al. 2014, Kumari and Singhal 2017).
It has often been observed that changes in morphological characters in polyploids having multiple sets of chromosomes are often associated with plant vigour and ability to grow under different environments. In P. speciosus, the 4x and 6x plants are more robust, grow much taller and also possessed larger sized leaves, internodes and panicles. Also, the polyploids possessed significantly larger sized stomata and pollen grains. All these factors might enable polyploids of the species to be more competitive and potentially more invasive in a new environment.
The authors are thankful for the financial assistance provided by UGC (SAP-III) and DBT (IPLS). Vandna Kumari is also grateful to Department of Science and Technology, Government of India, New Delhi for providing fellowship under WOS-A Scheme [award letter no.SR/WOS-A/LS-145/2014/(G)]. Thanks are also due to Director, Botanical Survey of India, Dehra Dun and Forest Research Institute, Dehra Dun for providing herbarium facilities for the identification of the plant species. Authors are also grateful to Head, Department of Botany, Punjabi University Patiala, for necessary laboratory and library facilities.
