First record of chromosome count and cytomixis in an endemic species of Clematis ladakhiana Grey-Wilson (Ranunculaceae) from cold deserts of Jammu and Kashmir

ABSTRACT

Meiotic investigations carried out from the cold desert regions of Ladakh (India) record the first ever chromosome count (2n = 32) and cytomixis in Clematis ladakhiana. The phenomenon of cytomixis involving chromatin transfer among 2–6 adjacent pollen mother cells (PMCs) through cytoplasmic channels was observed at different meiotic stages. The chromatin transfer resulted into hypo, hyperploid and enucleated PMCs. Although the frequency of meiocytes involved in chromatin transfer was low it induces abnormalities in chromatin and spindle activity, resulting into some pollen malformation in the form of variable sized fertile and sterile pollen grains. The Majority (86.60%) of the pollen grains observed were large sized, (86.60%) while (7.79%) are relatively small sized.

KEYWORDS:

• Clematis ladakhiana
• cytomixis
• chromatin transfer
• meiotic abnormalities
• pollen grains

Introduction

Clematis L. (family: Ranunculaceae), a genus with 250 species of woody climbers, possessing twinning leaf-stalks and showy flowers, is very widely distributed in the temperate and tropical regions of the world (Srivastava and Shukla 2015). Of the 32 Indian species, 6 are present in the cold deserts of Ladakh. As a part of the study to explore the cytomorphological diversity in the flowering plants from the high altitudinal and cold desert regions of Western Himalayas, present investigations have been focused to study the detailed meiotic course and pollen fertility in wild individuals of C. ladakhiana. The plant is a climbing or straggling shrub with 2.5 m long bearing leaves with 5–7 narrow lanceolate leaflets. The flowers are yellow, axillary, sometimes tinged with spotted red colour. The species also referred as “Old man’s beard”, is a native to the mountains of Kashmir and Tibet, and is one of the most drought-tolerant species of the genus (Srivastava and Shukla 2015). Tibetians traditionally burnt and the remains used to cure indigestion and dysentery. The species has been counted chromosomally for the first time at world level. Besides, the analyzed accession also showed the phenomenon of cytomixis involving chromatin transfer and meiotic irregularities and pollen malformation.

Material and methods

Sampling and collection of plant material

The material for male meiosis was collected from the wild plants present in the alpine and cold desert regions of Ladakh division of Jammu and Kashmir in the months of July–August. Young and unopened floral buds of suitable sizes were fixed in a freshly prepared Carnoy’s fixative (mixture of alcohol, chloroform, and acetic acid in a volume of 6:3:1) for 24 hours and preserved in 70% ethanol in a refrigerator. Cytologically worked out accession was identified by consulting the floras of Lahaul-Spiti- (A Cold Desert in North West Himalaya) (Aswal and Mehrotra (1994) and Srivastava and Shukla (2015). Besides, the specimens were also compared with the already deposited and well-identified voucher samples submitted by the taxonomists in the Herbaria, Botanical Survey of India, Northern Circle, Dehra Dun and Forest Research Institute, Dehra Dun. Identified specimens were deposited in the Herbarium, Department of Botany, Punjabi University, Patiala (PUN¹ 62,164).

Cytological studies

Meiocyte preparations were made by squashing the young and developing anthers in 1% acetocarmine by using the standard acetocarmine techinique. Meiocytes were examined for detailed meiotic course at different stages of prophase-I, metaphase-I, anaphase-I/II, telophase-I/II and sporads.

Pollen fertility and pollen grain size

Fertility of pollen grains was estimated through stainability tests by smearing the mature anthers in glycero-acetocarmine mixture (1:1). Well-filled pollen grains with uniformly stained cytoplasm and nuclei were scored as fertile. The shrivelled and flaccid pollen grains with unstained or poorly stained cytoplasm were counted as sterile. Pollen grain size was measured through micrometry.

Photomicrographs

Chromosome spreads, meiotic abnormalities, sporads and pollen grains (fertile/sterile) were photographed using Nikon Eclipse 80i Microscope (Melville, New York, USA).

Results

The meiotically analyzed wild accession collected at high altitudinal region around Panikhar, Ladakh (3250 m) revealed the presence of countable 16 large-sized bivalents at diakinesis (Fig 1(A)) and metaphase-I (Fig 1(B)). The Majority of the meiocytes showed perfectly normal meiotic course. However, some of the PMCs exhibited the phenomenon of cytomixis involving chromatin transfer among proximate meiocytes at different stages of meiosis (Figs 1(C–E)). Analysis of 324 PMCs (Pollen mother cells) revealed that 29 meiocytes (8.95%) were noticed to be involved in chromatin transfer (Table 1). In majority of the cases, 2–3 PMCs were observed to be involved in the process, but the number of PMCs goes up to 6 in some cases (Figs 1(F,G)). The chromatin transfer among meiocytes was noticed to be both partial and complete resulting into hypo-, hyper and enucleated PMCs (Fig 1(H)). The meiocytes involved in cytomixis also showed the presence of extra chromatin masses lying away from the main genome (Fig 1(I)). Besides, PMCs involved in chromatin transfer showed an aberration on spindle activity and chromatin material as reflected in the basis of stickiness in chromatin, presence of few bivalents away from the metaphase plate (Fig 1(J)), pycnotic chromatin (Fig 1(K)), laggards (Fig 1(L)), chromatin bridge (Fig 1(M)) and micronuclei (Fig 1(N)). Consequently, the accession showed pollen malformation in the form of sterile/unstained pollen grains (5.61%) and fertile pollen grains of two variable sizes (Fig 1(O)). A great majority of them (86.60%) are large (typical) sized measuring 28.20–29.80 µm × 27.40–29.60 µm while 7.79% are relatively small sized 21.20–23.40 µm × 20.30–22.25 µm.

Table 1. Cytomixis, meiotic aberrations and pollen grains in the analyzed individual of Clematis ladakhiana.

Meiotic stages	PMCs analysed	PMCs depicting chromatin transfer	Frequency (%)
I. Cytomixis
Prophase-I	122	14	11.47
Metaphase-I	88	08	9.18
Anaphase-I	72	05	6.94
Telophase-I	42	02	4.76
Total	324	29	8.95
II. Meiotic aberrations
a. Chromatin stickiness	114	10	8.77
b. Out of plate bivalents	92	04	4.34
c. Hypoploid PMCs	72	05	6.94
d. Hyperploid PMCs	78	08	10.25
e. Extrachromatin masses	88	04	4.54
f. Laggards	81	03	3.70
g. Chromatin bridges	163	07	4.29
III. Pollen grains
Fertile (stained)	Size	Number (%)	Pollen fertility
Small sized	(21.20–23.40 µm×20.30–22.25 µm)	25/321 (7.79)
Large sized (typical)	(28.20–29.80 µm×27.40–29.60 µm)	278/321 (86.60)	94.40%
Sterile (unstained)	(20.40–22.30 µm×20.20–21.60 µm)	18/321 (5.61)	5.60%

Figure 1. (A) A PMC showing 16 bivalents at diakinesis. (B) Metaphase-I. (C, D, E) PMCs showing chromatin migration at different stages of meiosis. (F, G) Chromatin migration among adjacent meiocytes (arrowed). (H) The partial or complete transfer of chromatin results into hypo- (arrowed) and hyperploid PMCs (arrowhead). (I) A PMC showing extra chromatin mass lying away from the main chromosome complement. (J) A PMC showing chromatin stickiness and out of plate bivalent (arrowed). (K) A PMC showing pycnotic chromatin (arrowed). (L) PMCs showing a laggard (arrowed). (M) A PMC with a chromatin bridge at A-I (arrowed). (N) A PMC with micronucleus at T-II. (O) A large sized (arrowhead), a small sized fertile pollen grain (arrowed) and a sterile pollen grain with unstained cytoplasm (stared). [Scale-10 µm].

Discussion

Clematis ladakhiana, the presently analyzed species which is endemic to the cold deserts of Ladakh, has been worked out for chromosome counts for the first time at world level. The studied individuals with a gametic chromosome count of n = 16 exist at 4x level (based on x = 8). The worked out plant species which harbour the harsh climatic conditions of cold deserts depicted the phenomenon of cytomixis involving chromatin transfer among neighbouring meiocytes. Cytomixis, the process involving the migration or transfer of chromatin material from the nucleus of one PMC into the cytoplasm of adjacent PMCs, is reported in a large number of flowering plants (Singhal and Gill 1985; Bedi 1990; Singhal and Kumar 2008; Mandal et al. 2013; Mursalimov et al. 2013; Mursalimov and Deinko 2017). This transfer of chromatin was observed more than a century ago by Kornicke in (1901) and was named cytomixis after a gap of 10 years by Gates (1911). The migration of chromatin during cytomixis was noticed to be both partial and complete, resulting into formation of hypo- and hyperploid PMCs. Besides, the meiocytes involved in chromatin transfer also showed various abnormalities in chromatin and spindle activity, resulting in fertile pollen grains of two sizes and sterile pollen grains. Similar findings regarding the impact of cytomixis on pollen size and fertility had been reported earlier in several flowering plants harbouring such harsh climatic conditions (Singhal et al. 2008; Singhal and Kumar 2008; Kumar et al. 2010, 2012, 2017).

Ever since the first report of cytomixis which appeared 117 years ago, the precise mechanism underlying the phenomenon of cytomixis is currently vague (Mursalimov and Deineko 2017). On the basis of meiotic analysis carried out on more than 1500 species of flowering plants by the cytologists from this laboratory, it has been inferred that the occurrence and frequency of meiocytes involved in cytomixis has no correlation with the ploidy level/genomic status of the plant. Rather, it is the genetic make up and prevailing environmental conditions which are responsible for the presence or absence of cytomixis (Stern 1946; Merwine and Bennet 1996; Singhal and Kumar 2008; Kumar and Srivastava 2009; Mason et al. 2011; Pecrix et al. 2011; Kumar et al. 2012; Malik et al. 2014). Earlier cytologists from this laboratory had reported the phenomenon of cytomixis in several species of the genus Clematis analyzed meiotically, viz. C. buchananiana (Kaur and Singhal 2017), C. flammula (Kumar et al. 2008), C. grata (Rani et al. 2010), C. graveoleus (Singhal and Kaur 2011), C. montana (Singhal et al. 2010) and C. orientalis (Kumar et al. 2010). It is thus apparent that the genus Clematis seems to be pliable to the phenomenon of cytomixis and chromatin and spindle related meiotic irregularities.

Acknowledgements

The authors are also highly thankful to the Head Department of Botany, Punjabi University, Patiala and Sophisticated Instrumentation Centre of the University for providing necessary lab facilities during the work.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

The authors wish to thank the University Grants Commission, New Delhi (India) for providing financial assistance under the Departmental Research Support Special Assistance Programme I, II and III, the assistance for strengthening of infrastructure for science and technology programme, Junior Research Fellowship to Nissar Ahmad Khan under DBT- I.P.L.S project [award letter number 12019/Research-29/01/2015].

Notes

1. Abbreviation used for Herbarium, Department of Botany, Punjabi University, Patiala in Index Herbariorum by Holmgren and Holmgren (1998).