Meiotic Studies in Some Species of Tribe Cichorieae (Asteraceae) from Western Himalayas

The present paper deals with meiotic studies in 15 species belonging to 6 genera of the tribe Cichorieae from various localities of Western Himalayas. The chromosome number has been reported for the first time in Hieracium crocatum (2n = 10) and Lactuca lessertiana (2n = 2x = 16). Further, intraspecific variability has been reported for the first time in H. umbellatum (2n = 2x = 10 and 2n = 6x = 54), Tragopogon dubius (2n = 2x = 14 and 2n = 4x = 28), and T. gracilis (2n = 2x = 14). The chromosome report of 2n = 2x = 10 in Youngia tenuifolia is made for the first time in India. Maximum numbers of the populations show laggards, chromosome stickiness, and cytomixis from early prophase to telophase-II, leading to the formation of aneuploid cells or meiocytes with double chromosome number. Such meiotic abnormalities produce unreduced pollen grains and the reduced pollen viability.


Introduction
The tribe Cichorieae (also known as Lactuceae) encompasses 95 genera and ca. 2500 species, primarily in temperate to subtropical zones of the Northern Hemisphere [1]. Members of the tribe are characterized by very important uniform characteristics, such as homogamous ligulate capitula and the presence of milky latex.
The presently investigated species are also known to have medicinal uses, such as Hieracium crocatum to cure gastric troubles, Lactuca dolichophylla to cure constipation, L. macrorhiza used as an ingredient to cure stomach ache, L. serriola to treat ailments of the urinary tract, and Taraxacum officinale used as diuretic and laxative [2][3][4].
Chromosome studies are valuable determinants in studying evolution. Many workers have studied the cytology of Indian Asteraceae, including members of tribe Cichorieae. Cytological studies on the family from Lahaul-Spiti area, an ecologically very fragile cold desert area of Western Himalayas, are almost lacking. As an attempt to fill this lacuna, the present investigations have been undertaken.

Plant Material.
Exploratory surveys were made during the years from 2009 to 2013 in selected localities (Table 1) of Himachal Pradesh (Kullu and Lahaul-Spiti Districts). The cytologically worked-out plants were identified using regional floras and compared with the specimens deposited at the Herbarium of Botanical Survey of India, Northern Circle, Dehra Dun. The voucher specimens (Table 1) were deposited in the Herbarium, Department of Botany, Punjabi University, Patiala (PUN).

Meiotic Studies.
For meiotic chromosome counts, unopened floral buds of suitable sizes were fixed in a freshly prepared Carnoy's fixative (mixture of alcohol, chloroform, and glacial acetic acid in a volume ratio 6 : 3 : 1) for 24 h. These were subsequently transferred to 70% alcohol and stored in refrigerator at 4 ∘ C until used for meiotic analysis. Meiocytes were prepared by squashing the developing anthers and stained with acetocarmine (1%). Chromosome number was determined at diakinesis/M-I/II/A-I/II from freshly prepared slides with light microscope Olympus. 500−600 pollen 2 The Scientific World Journal

Pollen Grain Analysis.
Pollen fertility was estimated through stainability tests using glycerol-acetocarmine (1 : 1) mixture and aniline blue (1%). Up to 450−800 pollen grains were examined for pollen fertility and size frequencies. Wellfilled pollen grains with stained nuclei were taken as apparently fertile while shriveled and unstained pollens were counted as sterile.

Photomicrographs.
Photomicrographs from the freshly prepared desirable slides having clear chromosome counts, dyads, triads, tetrads, and pollen grains were taken with a digital imaging system of Leica QWin.

Chromosome Number
Hieracium crocatum Bunge ex. Ledeb. The present species revealed the diploid cytotype (2 = 10, Figure 1(a)), which is a first ever chromosome report for the species. The Scientific World Journal H. umbellatum L. Both the cytotypes, 2 = 10 ( Figure 1(b)) and 2 = 54 ( Figure 1(c)), are the new records from the world, although the species is already known to have 2 = 18 [5] and 2 = 27 [6] from outside India. Polyploid cytotypes show some enlargement in vegetative and floral characters ( Table 2).
The present meiotic studies reveal a diploid cytotype (2 = 16, Figure 1(f)) which is a first ever chromosome report for the species.
L. macrorhiza (Royle) Hook. f. The present chromosome report of 2 = 16 ( Figure 1(g)) is already confirmed by many workers [8,12,13] from India. There is no chromosome record from abroad for the species.
L. sativa L. Meiotic analysis of the species reveals the diploid cytotype (2 = 18, Figure 1(h)), which is in conformity with the previous works of Chatterjee and Sharma [14] and Gupta and Gill [15].
T. gracilis D. Don. The present chromosome report (2 = 14, Figure 1(p)) is a varied chromosome count for the species at world level. Earlier, Mehra and Remanandan [12] reported diploid cytotype with 2 = 12 from the Western Himalayas.

Tribe Cichorieae.
The ancestral basic numbers in the tribe are = 4, 5, and 9, as suggested by Turner et al. [23]. But Stebbins et al. [24] proposed = 9 as the base number for Cichorieae (Asteraceae in general), which is supported by Tomb et al. [25], with the other numbers ( = 3-9) derived through phylogenetic reduction through chromosomal aberrations, particularly translocation.
Hieracium L. About 360 species are cytologically known with 94 species being diploid and 152 triploid and 149 species are tetraploid and rarely possess aneuploidy (7 spp.). The genus forms agamic complex and is considered monobasic on = 9. But, the present study reveals another cytotype (2 = 10), suggesting a new base number ( = 5) in the genus.   The Scientific World Journal    Prenanthes L. Twenty-two species in the genus are known cytotaxonomically, including 1 from India. The most common base number is = 8 represented with 19 species, including diploids (16 species) and tetraploids (3 species). However, the intraspecific polyploids are not available in the genus. Besides, = 9 is also present in 3 species that are diploid. Hence, the genus is proposed to be dibasic on = 8 and 9.

Meiotic
Abnormality. The phenomenon of inter-PMC migration of chromatin/chromosome between/among the contiguous meiocytes through cytomictic channels is termed as cytomixis (coined by Gates [26]). However, the phenomenon has been reported for the first time in gymnosperms by Arnoldy [27] and subsequently in angiosperms by Koernicke [28]. Since that time, cytomixis has been reported in a large number of plants [29]. Transfer of chromatin or chromosomes may take place through such inter-PMC cytomictic channels [30][31][32]. Some workers reported cytomixis to be more prevalent in polyploids than their diploid counterparts [33,34]. Occasionally, either hypoploid meiocytes [35][36][37] or enucleated meiocytes or meiocytes with a hyperploid number of chromosomes have been reported [30,[37][38][39]. It is very much clear that the enucleated meiocytes die, but hypo-and hyperploid meiocytes could lead to the formation of gametes with variable chromosome number and size. Cytomixis is considered as a process of evolutionary significance because it results in change in gametic chromosome numbers [30,40]. Chromosome stickiness also results in the formation of fragmented chromatin. This chromatin stickiness, late or nondisjuncting bivalents, and chromosomal laggards seem to be responsible for chromosomal bridges [41]. All these meiotic abnormalities consequently assert an effect on microsporogenesis, leading to the formation of monads, dyads, triads, or polyads with or without micronuclei, which ultimately produce heterogeneous sized (large and small) fertile pollen grains and reduced pollen fertility. The size difference may be due to the formation of unreduced gametes (2 ), which may produce plants with higher ploidal level through polyploidization (for review, see [42][43][44][45]). As observed in the presently investigated data, the chromatin rearrangement due to meiotic abnormalities is considered the base of inter-or intraspecific diversity. Further, it provides a catalogue for studying different evolutionary trends such as breeding system or polyploidy and hybridization.