Chromosome count, meiotic abnormalities, pollen fertility and karyotype of Elymus semicostatus (Nees ex Steud.) Meld. (Family: Poaceae) from North-west Himalaya

ABSTRACT

The present study includes chromosome count, meiotic abnormalities, pollen fertility and karyotype of Elymus semicostatus from north-west Himalaya, India. This is the first attempt to present karyotype morphometric data of the species from India [2n = 4x = 28 = 22m(2sat) + 6sm]. At present 28 wild accessions of the species have been analysed, all of which shared the same tetraploid (4x) chromosome count of 2n = 28. In addition, seven accessions also showed the presence of 0–2 B-chromosomes in the meiocytes. Meiotic abnormalities were frequent and 17 accessions showed the phenomenon of cytomixis, involving chromatin transfer among meiocytes and associated meiotic irregularities in spindle activity and chromosomal segregation. Consequent to such meiotic abnormalities, these accessions showed pollen malformation in the form of sterile and variable sized pollen grains. Cytomixis in the species seems to be a natural phenomenon under the control of genetic factors and is responsible for inducing meiotic disturbance and pollen malformation.

KEYWORDS:

• Poaceae
• Triticeae
• karyotype
• male meiosis
• cytomixis
• unreduced gametes

Introduction

Elymus semicostatus (Nees ex Steud.) Meld. (=Agropyron semicostatum Nees ex Steud.), belongs to the economically important wheat tribe (tribe Triticeae Dumort; family Poaceae (R. Br.) Barnh.), and has been widely studied for interspecific hybridization. It is a perennial grass characterized by erect spike, solitary spikelets, palea round at tip, distinctly shorter than lemma, found growing in varied habitats, up to an elevation of 3600 m. This species is native to Afghanistan, India, Nepal and Pakistan, and has now been introduced in other regions of the world. The species is important as a fodder grass across the Himalayan belt (Sharma et al. 2017).

Nearly all taxa of genus Elymus are exclusively polyploid with high genetic and morphological variability (Dong et al. 2015). E. semicostatus is also a highly variable species showing a wide range of genetic and morphogenetic variations (Lu and Salomon 1993; Salomon 1994). The species is allotetraploid and belongs to “SY” genomic constitution group (Lu et al. 1991), which made it central genome “analyser species” to check the genomic constitution of other species through cytological analysis of artificially produced hybrids (Sakamoto and Muramatsu 1966; Lu et al. 1990, 1991; Lu and Bothmer 1990a; Salomon and Lu 1992, 1994a, 1994b; Lu and Salomon 1993). Salomon (1994) in his revisionary studies on genus Elymus has considered E. semicostatus as “centre species” and has ascribed nearly nine taxa in Elymus semicostatus group based on common SY genome, and morphological characters. With an established base number x = 7, the species unequivocally depicts the same tetraploid chromosome count of 2n = 28.

While exploring the cytomorphological diversity in the grasses of high altitudinal and phytogeographically unexplored regions of north-west Himalaya [Uttarkashi district in Uttarakhand and Pangi in Chamba district of Himachal Pradesh] we have recorded the presence of a considerable degree of morphogenetic variations in E. semicostatus in wild accessions. Although all the accessions studied shared the same gametic chromosome count of n = 14, the analysed plants also showed the phenomenon of cytomixis in the meiocytes and other meiotic abnormalities, consequent to which pollen malformation was observed. In the present paper the karyotype of the species has been given for the first time from the Indian Himalaya. Thorough analysis of the available literature on E. semicostatus reveals the importance of cytogenetic studies in discrimination of species and varieties, defining the species relationships and inters specific hybridization. We also try to correlate the occurrence of pollen sterility with meiotic abnormalities and cytomixis in the species.

Materials and methods

The wild accessions of E. semicostatus for the current study have been collected from different localities of north-west Himalaya. Details of collection localities, altitude and accession numbers are given in Table 1.

Table 1. Data on analysed wild accessions covering collection sites, vouchers (PUN and BSD), gametic chromosome numbers (n) and pollen fertility (%) of Elymus semicostatus.

Accession number	Collection sites with altitude	Gametic chromosome number (n)	Pollen fertility (%)
PUN 61948	Sangamchatti-Agora, Uttarkashi, 1900 m	14, 14 + 0–1B	99
PUN 61969	Sangamchatti-Agora, Uttarkashi, 1950 m	14	98
PUN 61968	Agora-I, Uttarkashi, 2100 m	14	95
PUN 61949	Agora-II, Uttarkashi, 2200 m	14	98
PUN 61938	Bebra, Uttarkashi, 2250 m	14	88
PUN 61950	Agora-III, Uttarkashi, 2250 m	14	98
PUN 61966	Barsu I, Uttarkashi, 2250 m	14	90
PUN 61967	Barsu-Barnala Lake, Uttarkashi, 2300 m	14	99
PUN 61951	Dharkot, Uttarkashi, 2350 m	14	99
PUN 61970	Gangarh-Osla, Uttarkashi, 2400 m	14	88
PUN 61952	Dharkot, Uttarkashi, 2450 m	14, 14 + 0–1B	94
BSD 118147	Luj, Pangi, Chamba, 2450 m	14	99
BSD 118148	Leu, Pangi, Chamba, 2600 m	14, 14 + 0–2B	96
PUN 61971	Seema Bridge, Uttarkashi, 2700 m	14	99
PUN 61972	Seema-Osla, Uttarkashi, 2750 m	14, 14 + 0–1B	98
PUN 61973	Seema-Renugaad, Uttarkashi, 2800 m	14	95
PUN 61947	Renugaad, Uttarkashi, 2950 m	14	73
PUN 61940	Gangotri, Uttarkashi, 3100 m	14	87
PUN 61941	Kankhu, Uttarkashi, 3100 m	14	86
PUN 61974	Kalkati Dhar, Uttarkashi, 3100 m	14	95
PUN 61942	Kankhu, Uttarkashi, 3150 m	14	90
PUN 61943	Devgaad, Uttarkashi, 3150 m	14	95
PUN 61944	Devgaad, Uttarkashi, 3200 m	14	99
PUN 61976	Kalkati Dhar-Har Ki Dun, Uttarkashi, 3250 m	14	96
PUN 61939	Har Ki Dun, Uttarkashi, 3300 m	14, 14 + 0–1B	98
PUN 61975	Har Ki Dun, Uttarkashi, 3450 m	14	99
PUN 61945	Chirwasa, Uttarkashi, 3450 m	14, 14 + 0–1B	95
PUN 61946	Chirwasa, Uttarkashi, 3600 m	14, 14 + 0–1B	87

For mitotic studies, mature seeds and young growing root tips were collected from different localities under study. However the seeds from different localities failed to germinate in standard laboratory conditions except from one locality (BSD 118148). The root tips were collected from the germinating seedlings and were subjected to pre-treatment with colchicine (0.2–0.5%) for 1–2 h. The tips were then transferred to freshly prepared Carnoy’s fixative (6 absolute alcohol: 3 chloroform: 1 glacial acetic acid) for the next 24 h and stored at 4°C until analysis. The tips were hydrolysed in acid water (1 N HCL) at 60°C for 10 min and then stained with acetocarmine following the standard squash technique. Ten dividing cells at metaphase were studied to determine the karyotype of the species.

Photomicrographs were taken using the Nikon 50i Eclipse microscope (Melville, NY, USA). For karyotype analysis, chromosome nomenclature of Levan et al. (1964) was followed. Ideogram and karyotype parameters were determined using karyotype software (Altınordu et al. 2016). The morphometric parameters calculated numerically (Table 2) are long arm length of chromosome (l), short arm length of chromosome (s), total chromosome length (c)], arm ratio of chromosome (r) and centromeric index (Ci) and type of chromosome. The chromosome pairs were arranged in order of decreasing length.

Table 2. Karyotype parameters of Elymus semicostatus.

Chromosome pair	l (μm)	s (μm)	c (μm)	r	Ci %	Type
1	7.11 ± 0.28	5.42 ± 0.35	12.52 ± 0.06	1.31	43.25	m
2	5.96 ± 0.01	4.83 ± 0.61	10.79 ± 0.59	1.23	44.79	m
3	5.12 ± 0.23	3.89 ± 0.13	9 ± 0.38	1.32	43.17	m^(sat)
4	4.85 ± 0.01	4.05 ± 0.74	8.9 ± 0.74	1.2	45.47	m
5	5.8 ± 0.25	2.89 ± 0.45	8.7 ± 0.70	2	33.29	sm
6	4.52 ± 0.13	4.13 ± 0.35	8.65 ± 0.47	1.09	47.76	m
7	3.97 ± 0.06	3.77 ± 0.18	7.74 ± 0.12	1.05	48.68	m
8	3.93 ± 0.02	3.48 ± 0.01	7.41 ± 0.01	1.13	46.92	m
9	3.83 ± 0.06	3.49 ± 0.30	7.32 ± 0.35	1.1	47.66	m
10	3.59 ± 0.26	3.12 ± 0.04	6.71 ± 0.31	1.15	46.51	m
11	4.53 ± 0.20	2.16 ± 0.01	6.69 ± 0.21	2.1	32.26	sm
12	3.71 ± 0.10	2.96 ± 0.63	6.67 ± 0.73	1.26	44.33	m
13	3.97 ± 0.18	2.19 ± 0.13	6.16 ± 0.31	1.81	35.57	sm
14	2.16 ± 0.00	1.66 ± 0.08	3.82 ± 0.07	1.31	43.38	m

Abbreviations: l, long arm; s, short arm; c, total length; r, arm ratio; Ci, centromeric index; m, metacentric; m^(sat) metacentric with satellite; sm, submetacentric.

Wild plant accessions were collected from the different sites for meiotic analysis and pollen fertility. Young and unopened spikes were fixed in Carnoy’s fixative. After 48 h the materials were transferred to 70% ethanol and stored in a refrigerator. Meiocyte preparations were made by squashing the developing anthers dissected out from appropriate sized florets using standard acetocarmine technique in 1% acetocarmine. Chromosome counts and meiotic course were studied from freshly prepared slides having pollen mother cells (90–100 PMCs) at diakinesis, metaphase-I (M-I), anaphase-I (A-I) and telophases (T-I, T-II). Apparent pollen viability was estimated through stainability tests by squashing the mature anthers in glycerol and acetocarmine (1:1) mixture. Around 400–500 pollen grains were studied. Well-filled pollen grains with completely stained nuclei and cytoplasm were scored as fertile/viable, and partially stained and shrivelled ones as sterile/non-viable. Good preparations of PMCs with spread chromosomes, meiotic irregularities and pollen grains were selected for photomicrographs using the Nikon 80i Eclipse microscope (Melville, NY, USA) and Leica Qwin Digital Imaging System (Wetzlar, Germany). Sizes of fertile and sterile pollen grains were measured through microscopy.

Meiotically analysed accessions were identified by consulting the following floras, viz. Flora of cold deserts of Western Himalaya, Monocotyledons (Murti 2001), Flora of Gangotri National Park, Western Himalaya (Pusalkar and Singh 2012) and a taxonomic article (Salomon 1994). Identifications were revalidated by comparing specimens with the vouchers already submitted by taxonomists in the Herbaria, Botanical Survey of India, Northern Centre, Dehradun and Department of Botany, Punjabi University Patiala. Vouchers of cytologically examined specimens were deposited in the Herbaria, Department of Botany, Punjabi University, Patiala (PUN) and Botanical Survey of India, Northern Centre, Dehradun (BSD).

Information regarding previous chromosome reports was gathered from Indexes to Plant Chromosome Numbers (Darlington and Wylie 1955; Fedorov 1969; Moore 1970, 1971, 1972, 1973, 1974, 1977; Goldblatt 1981, 1984, 1985, 1988; Kumar and Subramaniam 1989; Goldblatt and Johnson 1990, 1991, 1994, 1996, 1998, 2000, 2003, 2006; Khatoon and Ali 1993); databases (Rice et al. 2015; Tropicos 2017) and IAPT/IOPB Chromosome Number Reports in Taxon.

Results and discussion

Twenty-eight wild accessions of E. semicostatus, gathered from different phytogeographical diverse regions of north-west Himalaya (Uttarkashi district in Uttarakhand and Pangi in Chamba district of Himachal Pradesh), were analysed for morphological and cytological parameters. A considerable morphogenetic variation was encountered in vegetative (plant height, leaf length and width, size of internodes) and floral characters (spike length, glume size, lemma, awn), which seems to have been the result of its wide distribution in variable habitats and environments. Individuals inhabiting sub-alpine regions acquire dwarf habits possibly to survive harsh cold climatic conditions, while those growing under forest cover achieve taller size and acquire slender and erect habit. On the other extreme individuals growing on exposed sunny slopes develop white incrustations and/or sturdy vegetative and floral parts. Sun et al. (2014) suggested similar type of adaptation approaches in the plants growing at high altitudinal regions of Tibet Plateau.

Chromosome number and ploidy

All the accessions of E. semicostatus presently studied depicted the same tetraploid (4x) chromosome count of 2n = 28, which confirms the previous reports. The first ever chromosome count of 2n = 28 in the species was reported by Nielson and Humphrey (1937) on the basis of chromosomal analysis made on greenhouse samples grown in Iowa, USA. Since then, owing to its wider distribution and morphogenetic diversity the species has been examined cytologically by a number of cytologists from the Indian states of Jammu and Kashmir, Himachal Pradesh and Uttarakhand (Mehra and Sharma 1972, 1975, 1977; Sharma and Sharma 1979; Salomon 1994; Salomon and Lu 1994b; Singhal et al. 2014; Kumari and Saggoo 2016); and outside India i.e. Nepal, Pakistan and Afghanistan (Tateoka 1955; Matsumura et al. 1956; Lu et al. 1990, 1991, 1995; Lu and Bothmer 1990a, 1990b, 1992, 1993a, 1993b; Salomon and Lu 1992, 1994a, 1994b; Lu 1993; Lu and Salomon 1993; Moinuddin et al. 1994; Salomon 1994).

Mitotic study and karyotype

Mitotic study of somatic chromosomes of E. semicostatus, depicted a karyotype formula 2n = 4x = 22m(2sat) + 6sm, i.e. 22 metacentric and six sub-metacentric chromosomes at metaphase (Figure 1). The ideogram of the somatic chromosomes is shown in Figure 2. The karyotype asymmetry degree (Stebbins 1971) categorizes the species karyotype as 2B type. The karyotype morphometric data (Table 2) of E. semicostatus shows a gradual decrease in length of chromosomes from 12.52 ± 0.06 μm to 3.82 ± 0.07 μm (longest/shortest = 3.34 μm), with the total haploid length of the chromosome set (Peruzzi et al. 2009), THL = 111.07 μm. In general, the metacentric chromosomes (m) showed longer lengths. Based on the intra chromosomal asymmetry index (Romero 1986), A1 = 0.23, the species depicted a more symmetric karyotype. The third pair of chromosomes showed the presence of satellites on long arm.

Figure 1. Elymus semicostatus, mitosis 2n = 28. Scale bar = 10 μm.

Figure 2. Elymus semicostatus, ideogram. Scale bar = 10 μm.

Eleven species of Elymus have been worked out from China by Liu (1985), of which E. dahuricus, 2n = 6x = 42 = 36m(2sat) + 6sm(4sat) and E. nutans, 2n = 6x = 42 = 34m + 8sm (2sat) are also well distributed in Indian north-west Himalaya. Dubcovsky et al. (1989) worked out karyotype of six tetraploid species of Elymus from South America depicting symmetrical and uniform chromosomes. Chen et al. (2008) reported karyotype (1A or 2A type) of 10 StH genome tetraploid species of Elymus from Asia and North America. The presently studied species, E. semicostatus, also depicts the same pattern of karyotype formula as reported in the other species of the genus.

Meiotic study

Meiotic analysis of 28 accessions collected over a wide range of north-west Himalaya, shared the same gametic chromosome count of n = 14 as confirmed from the unequivocal presence of 14 bivalents at diakinesis (Figure 3(b)) and M-I (Figure 3(a)). The information regarding collection sites, altitude, vouchers (PUN and BSD), gametic chromosome number (n) and pollen fertility (%) is provided in Table 1. Only one third of all the accessions analysed meiotically showed perfectly normal meiotic course, sporads and uniform sized pollen grains and the rest showed various types of meiotic abnormalities. Various meiotic abnormalities such as B-chromosomes, cytomixis, laggards, chromatin bridges, non-synchronous dysjunction and chromatin stickiness were observed in 19 accessions. Table 3 shows the various meiotic abnormalities observed in the species. Consequent to such abnormal meiotic behaviour pollen malformation coupled with pollen sterility was observed.

Table 3. Data of PMCs depicting B chromosomes, cytomixis and other meiotic abnormalities at different stages of meiosis.

	Percentage of PMCs (observed number/total number) at different meiotic stages with
	B-chromosome	Cytomixis			Chromatin bridges, laggards and micronuclei
Accession number	Diakinesis	Prophase-I	Metaphase-I	Anaphase-I	A-I/T-I	A-II/T-II
PUN 61938	–	18.68 (17/91)	5.77 (6/104)	–	7.94 (5/63)	6.25 (4/64)
PUN 61939	1.98 (2/101)	2.97 (3/101)	–	–	–	–
PUN 61941	–	2.34 (6/256)	18.22 (39/214)	0.92 (1/109)	21.10 (23/109)	10.64 (5/47)
PUN 61945	13.46 (7/52)	–	–	–	12.90 (8/62)	–
PUN 61946	10.61 (14/132)	2.27 (3/132)	–	–	10 (6/60)	2.56 (1/39)
PUN 61947	–	31.30 (41/131)	7.74 (13/168)	2.70 (3/111)	14.41 (16/111)	7.69 (4/52)
PUN 61948	4.16 (2/48)	–	–	–	–	–
PUN 61949	–	4.88 (4/82)	–	–	1.75 (1/57)	–
PUN 61950	–	5.17 (9/174)	–	–	–	–
PUN 61952	4.79 (7/146)	13.01 (19/146)	–	–	3.30 (3/91)	–
PUN 61966	–	–	3.85 (3/78)	–	8.70 (8/92)	–
PUN 61968	–	–	8.85 (10/113)	–	9.60 (7/73)	–
PUN 61970	–	4.79 (14/292)	11.26 (33/293)	1.57 (2/127)	3.94 (5/127)	18.29 (15/82)
PUN 61972	4.25 (4/94)	2.13 (2/94)	–	–	–	–
PUN 61973	–	7.37 (7/95)	–	–	2.44 (1/41)	–
PUN 61974	–	4.30 (8/186)	–	–	–	–
PUN 61975	–	2.42 (3/124)	–	–	–	–
PUN 61976	–	7.41 (10/135)	–	–	2.60 (2/77)	–
BSD 118148	14.11 (12/85)	5.71 (4/70)	–	–	–	–

Figure 3. Male meiosis in Elymus semicostatus: (a) a PMC at M–I with 14 bivalents; (b) a PMC at diakinesis with 14 bivalents and 1B-chromosome (14_II + 1B, arrowed); (c) a PMC with secondary association of bivalents at M–I (arrow); (d) PMCs showing migration of intact metaphase bivalents (arrow); (e) prophase PMCs showing cytomixis (arrow); (f) PMCs depicting whole nucleus migration (arrow); (g) PMCs with migration of metaphase bivalents to adjacent PMC (arrows); (h) PMC at M–I with precocious dysjunction of two chromosomes (arrows); (i) PMC at A–I with late disjunction of chromosomes (arrow); (j) PMCs with intense chromatin stickiness and multiple thick chromatin bridges (arrow); (k) lagging chromosomes at A–I (arrow); (l) extra chromatin as third pole at early T–I (arrow); (m) prophase-II meiocytes with 1–3 micronuclei (arrows); (n) regular sized fertile/stained and sterile/unstained pollen grain; (o) heterogeneous sized pollen grains. Scale bars = 10 μm.

In addition to the basic set of 14 A-chromosomes, seven accessions (Table 3) showed the presence of 0–2 B-chromosomes in some meiocytes (Figure 3(b)). The presence of 0–4 B-chromosomes has been previously reported in Indian taxa analysed from Kasauli, Himachal Pradesh and upper Shillong hills, Meghalaya by Parkash (1979) and Mehra (1982). Singhal et al. (2014) reported 0–1B chromosomes in E. semicostatus from Parvati valley, Himachal Pradesh. B-chromosomes are supernumerary, showing non-Mendelian inheritance (Houben et al. 2014), and were first discovered about a century ago. The presence of B-chromosomes in low numbers has negligible harmful effects, however when frequency of occurrence is high, along with multiple copies, it results in phenotypic differences, reduced fertility and vigour (Randolph 1941; Jones and Houben 2003; Houben et al. 2014; Singh and Singhal 2018).

Cytomixis or chromatin transfer was observed in 17 accessions at early stages of meiosis (prophase-I to M-I). The percentage of cytomixis varied from 0.92% to 13.90% in different accessions. Migration of chromatin and other cellular contents were noticed to occur through cytoplasmic channels (CCs) (Figure 3(d–g)). During the latter stages of meiosis I, migration of chromatin was noticed to take place in form of intact and deformed bivalents (Figure 3(d, g)). The migration of chromatin along with nucleus both partial as well as complete resulted into hypoploid, hyperploid and enucleated meiocytes at different stages. It has also been noticed that migrated chromatin sometimes constitutes a separate group at T-I (Figure 3(l)). The meiocytes involved in cytomixis depicted various meiotic disturbances in spindle activity and chromosome behaviour during the subsequent stages.

Cytomixis is a poorly understood natural phenomenon (Mursalimov and Deineko 2017) involving transfer of chromatin/nuclei between plant cells through intercellular channels (Mursalimov et al. 2015), more frequently observed in plant male meiosis and microsporogenesis. The cause of cytomixis is still unknown in spite of its potential evolutionary significance. There are various explanations and conflicts regarding the causes of cytomixis and its role in evolution (Lone and Lone 2013; Mandal et al. 2013; Mursalimov et al. 2013; Mursalimov and Deineko 2017). However, it can be concluded based on cytological studies that high rate of cytomixis coupled with other meiotic abnormalities considerably affects pollen fertility. Such an impact of cytomixis on meiotic course, pollen fertility and size has been reported in number of flowering plants (Singhal and Kumar 2008; Singhal et al. 2008; Kumar et al. 2010, 2017; Guan et al. 2012; Kravets 2013; Kumar and Srivastava 2013; Rana et al. 2014; Mursalimov and Deineko 2015, 2017; Mandal and Nandi 2017). Singhal et al. (2014) reported cytomixis in E. semicostatus from Parvati valley, Himachal Pradesh along with chromatin stickiness and inter bivalent connections. Sidorchuk et al. (2016) studied cytomixis in relation to polyploid series in Triticum and Secale (tribe Triticeae). Their study demonstrated that the rate of cytomixis increases nearly by 2.4% with each transition to the next higher ploidy level and their view was that polyploidy series in family Poaceae could be serve as convenient model to study different factors affecting cytomixis.

In some cells it was also observed that the whole nucleus migrates (Figure 3(f)) towards the CCs and passes into the recipient cell which may possibly form a binucleate/polyploid cell with double/treble chromatin material, as reported earlier in Nicotiana tabacum (Sidorchuk et al. 2007; Mursalimov and Deineko 2015), Meconopsis aculeata (Singhal and Kumar 2008), Sorghum (Tsvetova and Elkonin 2013), Lindelofia longiflora (Rana et al. 2014) and Lippia alba (Reis et al. 2016). Such polyploid cells undergo normal meiotic course and produce unreduced 2n giant sized pollen grains. However, there have been no reports of any cytotype of higher ploidy level than 4x in E. semicostatus. Based on previous cytological literature it may also be inferred that such polyploid cells in this species may form viable pollen grains but have failed to establish themselves in nature.

Non-synchronous dysjunction was observed during segregation of chromosomes at A-I (Figure 3(h, i)). This may be attributed to interlocking of chiasmata and has been reported as a cause of pollen sterility in angiosperms (Kumar and Singhal 2008; Kumar et al. 2011). Consequent to non-synchronous behaviour of chromosome segregation, laggards (Figure 3(k)) were observed at anaphase and telophase in the PMCs. Formation of laggards due to non-synchronous dysjunction has been previously reported in Zea mays (dela Viña and Ramirez 1995) and many other plants. Intense chromatin stickiness along with broad chromatin bridges were observed towards the end of meiosis I (Figure 3(j)). The secondary chromosomal associations or bivalents/chromosomes having diffused connections existed in the species at diakinesis and pro-metaphase (Figure 3(c)). Such behaviour of bivalents is attributed to polyploid origin of the species (Lawrence 1931; Stebbins 1950; Malgwi et al. 1997). Chromatin in state of pycnosis was observed in some meiocytes. This condition may indicate PMC degradation (Figure 3(e, g)).

Lagging chromatin material was observed as variable number of micronuclei at prophase-II (Figure 3(m)). Consequent to meiotic disturbances the accessions showed reduced pollen fertility and heterogeneous sized fertile and sterile pollen grains (Figure 3(n, o)). Heterogeneous large fertile pollen grains measured 38.09 ± 4.30 μm, compared to 28.57 ± 1.27 μm for typical normal sized grains. Development of hyperploid, hypoploid and enucleated PMCs towards sporad formation was noticed as fully stained pollen along with distinguishable extra chromatin mass, partially stained pollen with little chromatin material (Figure 3(o)) and unstained empty pollen (Figure 3(n)). High rate of cytomixis (13.9%) in one accession from Renugaad (PUN 61947) showed a considerable decrease of 27% in pollen fertility. However, a low percentage of cytomixis along with B chromosomes and other meiotic abnormalities also resulted in reduced fertility (10– 20%) in some accessions. The hyperploid and hypoploid cells with variable amount of genetic material yield heterogeneous sized fertile and sterile pollen grains, possibly with different genetic constitution. On the other hand, empty cells are eliminated, thereby effecting the pollen productivity in the taxa.

The current paper presents a study of meiotic chromosomal behaviour and karyotype of E. semicostatus from phytogeographical diverse regions of north-west Himalaya. E. semicostatus is a complex species with high morphogenetic variability. Such cytogenetic studies can be beneficial in determining species relationships and discrimination of varieties and morpho-variants in the species. Cytomixis in the presently analysed wild accessions of the species is a natural process under the control of various genetic factors and appears to induce various meiotic irregularities, affecting the normal meiotic course and consequently producing heterogeneous sized pollen grains and reduced fertility.

Acknowledgements

The authors are very grateful to the head of the Department of Botany, Punjabi University, Patiala and Director, Botanical Survey of India, Kolkata and Head of Office, NRC, Dehradun, for necessary laboratory, Herbarium (PUN, BSD) and library facilities. .

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the University Grants Commission (UGC), New Delhi (UGC-BSR-Fellowship) [Award letter no. 15610/Research dated 3 June 2015]; Department of Biotechnology, New Delhi (DBT-IPLS project) [BT/PR4548/INF/22/146/2012]; and Science and Engineering Research Board-Department of Science and Technology (SERB-DST) Start Up Research Grant (Young Scientists) [vide SERB sanction No. SB/YS/LS-182/2014 dated 8 September, 2015].