Cytological Studies of Some Dicots from the Hills of Mandi District (Himachal Pradesh) in Northwest Indian Himalayas

Vijay Kumar Singhal,* Puja Garg and Puneet Kumar

Department of Botany, Punjabi University, Patiala-147002, Punjab, India

Received August 28,2012; accepted March 1,2013

Summary Details of male meiotic course and pollen fertility have been investigated in 45 species of dicotyledonous plants from the chromosomally unexplored area of the Mandi district, between the altitudinal ranges of 800 to 3360 m. All the species have been worked out cytologically for the first time from the study area. The meiotic chromosome count of n=9 for Impatiens micranthemum is the first ever chromosome report for that species. A new intraspecific 12x cytotype (n=42) has been reported in Potentilla gerardiana, supplementing the earlier reported cytotypes (2x, 8x). A new cytotype has been reported in Gentiana argentea (n=9), supplementing the earlier Indian report of n=10. Out of a total 45 species, 31 species (68.88%) showed normal meiotic course and high pollen fertility (91–100%), while 14 species (31.11%) depicted various meiotic irregularities and some pollen sterility (4–40%). The phenomenon of cytomixis involving inter-pollen mother cells (PMCs) migra- tion of chromatin material has been reported in 3 species, Clematis grata (n=8), Gentiana aprica (n=10) and Lamium album (n=9). In these species, cytomixis induces various meiotic abnormalities (enucleated, hypo-, hyperploid, tripolar and multipolar PMCs, chromatin stickiness, laggards and chromatin bridges at anaphases and telophases and micronuclei) and consequently some pollen sterility and variable sized pollen grains. Besides, non-synchronous disjunctions of bivalents at anaphases, abnormal spindles, and interbivalent connections during diakinesis/metaphase-I, univalents and multivalents have also been reported in certain cases. The chromosome counts in the remainder of the species substantiate previous countes by other researchers.

Key words Chromosome number, Cytomixis, Male meiosis, Meiotic abnormalities, Pollen fertility, pollen sterility.

Mandi district, a gateway to Himachal Pradeshʼs most famous places, including Kullu, Manali, and Lahaul-Spiti, lies between 31°13′50″ and 32°04′30″N latitude and between 76°37′20″ and 77°23′15″E longitude and is situated on the left bank of the river Beas in the foot hills of the Shivalik range. The vegetation of hills around Mandi is varied and the geographical location, cli- mate, and topography of the district have contributed to characteristic vegetation and flora. The forests here support and preserve a variety of flora and fauna. The region is primarily dominated by deciduous trees. It is sub-tropical in the valley and tends to be temperate on the hill tops. The vegetation constituting the forests falls under four main categories: (1) Himalayan Sub-Tropical Pine Forests, (2) Himalayan Moist Temperate Forests, (3) Sub-Alpine Forests, and (4) Alpine Forests. For many decades, the Himalayas have attracted a galaxy of researchers from various disci- plines on a global scale. So far, studies undertaken pertain either to floristic diversity or ethnobot- any (Chowdhary and Wadhwa 1984, Dhaliwal and Sharma 1999, Singh and Rawat 2000, Singh 2004; Sharma et al. 2005, Chauhan 2006, Boktapa and Sharma 2010). The perusal of existing cytological literature reveals that vast areas of the Indian Himalayas, especially the plants from the

*Corresponding author, e-mail: [email protected] DOI 2 V. K. Singhal et al. Cytologia 78(1)

Mandi district of Himachal Pradesh, are yet to be explored chromosomally. Keeping these facts in view and in consideration of the importance of cytomorphological studies, an attempt has been made to explore the cytomorphological diversity in those plants of Mandi district which have not been covered so far. Such studies can be useful in germplasm evaluation, chromosomal database cataloguing, and understanding the nature of intraspeciﬁc chromosomal variability/meiotic behavior, and also can provide an insight to chromosomal evolution in Himalayan plant species. The pur- pose of the present studys has been to record original chromosome counts and, details of meiotic course, microsporogenesis, and pollen fertility.

Materials and methods

Materials for male meiotic studies were collected from wild plants growing in various localities of Mandi district (Himachal Pradesh, India) falling in the altitudinal range of 800–3360 m. The localities were visited two to four times during the months of March to September 2011. The exact locality with altitude for each taxon is given in Table 1. For meiotic studies, unopened and young floral buds/inflorescences from healthy plants were fixed in freshly prepared Carnoyʼs fixative (1 part glacial acetic acid: 3 parts chloroform: 6 parts absolute alcohol) for 24 h. The materials were subsequently transferred to 70% alcohol and kept at 4°C in the refrigerator until analysis. Meiotic studies were carried out through standard acetocarmine squash technique for which developing anthers were squashed in 1% acetocarmine. A number of freshly prepared slides were examined for meiotic analysis in each accession and chromosome counts were made and abnormalities like cytomixis, laggards and bridges were recorded. Pollen fertility in each species/taxa was estimated through stainability tests for which the mature anthers from different flowers were squashed in glyceroacetocarmine (1 : 1) mixture and aniline blue dye (1%). Well-filled pollen grains with stained nuclei and cytoplasm were scored as apparently fertile while grains that were shrivelled and showed unstained/poorly stained cytoplasm as sterile. Pollen grain size was measured using an occulo- micrometre. Photomicrographs of well-spread meiotic chromosome counts, sporads, and pollen grains were made from the freshly prepared/temporary slides using a Nikon Eclipse 80i microscope fitted with a Digital Imaging System. Voucher specimens of the meiotically worked out specimens were deposited in the Herbarium, Department of Botany, Punjabi University (PUN), Patiala and accession numbers are given in the Table 1. The arrangement of families in the text as well in Table 1 is as per Flora of British India (Hooker 1872–1897). Within a family, the various genera and species are arranged alphabetically.

Results and discussion

Cytological investigations were made on 45 species belonging to 37 genera and 16 families of Dicots. Sixteen species with interesting cytological features and new information are dealt here- with. For the rest of the species, information regarding meiotic chromosome number, source of materials, plant accession numbers, ploidy level, and pollen fertility percentage is provided in Table 1. All the species have been worked out cytologically for the ﬁrst time from the study area. New or varied chromosome reports are based on the information gathered from what here are termed as Standard Cytological Indices viz., Darlington and Wylie (1955), Fedorov (1969), Moore (1970, 1971, 1972, 1973, 1974, 1977), Goldblatt (1981, 1984, 1985, 1988), Goldblatt and Johnson (1990, 1991, 1994, 1996, 1998, 2000, 2003, 2006), Kumar and Subramanian (1986), Khatoon and Ali (1993), IAPT/IOPB and SOCGI Chromosome Reports and journals concerned with chromosome reports as well as Internet sites (Tropicos.org., Missouri Botanical Garden. 11 Aug. 2011 ). The results on species of cytological interest are discussed in the following sections. 2013 Cytological Studies of Some Dicots from the Hills of Mandi District 3

Table 1. Information on locality with altitude, accession number (PUN*), meiotic chromosome number (ʻnʼ), ploidy level and pollen fertility (%) from previous reports on the presently studied species.

Locality Pollen Ploidy Taxon with altitude PUN* ʻnʼ fertility level (m) (%)

Family: Ranunculaceae Clematis grata Wall. Ropa, 1800 56858 8 2x 60 Ranunculus laetus Wall. ex Chachyot, 1580 56873 14 4x 93 D. Don. Rahla, 1900 56874 14 4x 96 R. muricatus Linn. Ner Chowk, 800 56875 24 6x 91 Family: Berberidaceae Berberis glaucocarpa Stapf. (=B. aristata auct. non DC.) Rahla,1900 56847 14 2x 99 Family: Hypericaceae Hypericum elodeoides Choisy Prashar Lake, 2730 56856 9 2x 100 Family: Balsaminaceae Impatiens amphorata Edgew. (=I. bicolor Hook. f.) Prashar Lake, 2730 56877 7 2x 99 I. laxiflora Edgew. (=I. micranthum Edgew.) Bagi, 2300 56876 7 2x 100 I. micranthemum Edgew. Shikari Devi 56882 9 2x 98 Temple, 3359 I. scabrida DC. Barot, 2200 56878 7 2x 100 Family: Papilionaceae Vicia hirsuta Koch. Ropa,1800 56868 6 2x 99 Family: Rosaceae Potentilla gerardiana Lindl. (=Potentilla fragarioides auct. non Linn.) Bagi, 2300 56844 42 12x 99 Family: Punicaceae Punica granatum Linn. Darang, 1220 56851 8 2x 100 Family: Compositae Achillea millefolium Linn. Janjehli, 3300 56836 9 2x 99 Ageratum conyzoides Linn. Bagi, 2300 56861 10 2x 100 Artemisia parviflora Buch.-Ham. ex Roxb. Ropa, 1800 56869 9 2x 100 Aster peduncularis Wall. ex Nees Bagi, 2300 56854 27 6x 100 Bidens pilosa Linn. Rewalsar Lake, 1360 56849 36 6x 100 Chrysanthemum leucanthemum Linn. Bagi, 2300 56837 18 4x 100 Conyza stricta Willd. Thunag, 3100 56866 9 2x 100 Erigeron annuus (L.) Pers. Thunag, 3100 56865 2n=27 3x 88 Galinsoga parviflora Cav. Shikari Devi 56864 8 2x 100 Temple, 3359 Gerbera lanuginosa (Wall. ex DC.) Benth. Darang, 1220 56880 24 2x 100 Ropa, 1800 56881 24 2x 100 Inula cappa DC. Barot, 2200 56883 10 2x 93 Lactuca dissecta D. Don. Ropa, 1800 56884 8 2x 100 Prenanthes brunoniana Wall. ex DC. Thunag, 3100 56879 8 2x 100 Saussurea candicans (DC.) Sch. Bip. Ropa, 1800 56840 17 2x 92 Senecio chrysanthemoides DC. Barot, 2200 56838 20 8x 100 S. rufinervis DC. Prashar Lake, 2730 56859 20 8x 100 Solidago virga – aurea Linn. Bagi, 2300 56862 9 2x 99 Sonchus asper Linn. Rahla, 1900 56870 9 2x 95 S. brachyotus DC. Hateshwari 56871 9 2x 99 Temple, 1400 Ropa, 1800 56872 9 2x 99 Taraxacum officinale Wigg. Barot, 2200 56843 16 4x 88 Xanthium strumarium Linn. Bagi, 2300 56855 18 2x 99 Family: Primulaceae Androsace rotundifolia Hardw. Barot, 2200 56850 10 2x 100 Family: Apocynaceae Carissa spinarum Linn. Darang, 1220 56853 11 2x 55 Family: Gentianaceae Gentiana aprica Decne. Ropa, 1800 56845 10 2x 74 G. argentea (D. Don.) Griseb. Jhatingiri, 2012 56852 9 2x 100 4 V. K. Singhal et al. Cytologia 78(1)

Table 1. Continued. Locality Pollen Ploidy Taxon with altitude PUN* ʻnʼ fertility level (m) (%) Family: Cuscutaceae Cuscuta reﬂexa Roxb. Thunag, 3100 56860 16 2x 100 Family: Solanaceae Physalis angulata Linn. Rewalsar Lake, 1360 56867 24 4x 100 Solanum nigrum Linn. Barot, 2200 56839 24 4x 100 Solanum viarum Dunal Bagi, 2300 56863 12 2x 100 Family: Lamiaceae Lamium album Linn. Multhan, 2300 56846 9 2x 93 Nepeta elliptica Royle ex Benth. Rahla, 1900 56857 9 2x 100 Family: Plantaginaceae Plantago lanceolata Linn. Jhatingiri, 2012 56841 6 2x 99 Deyot, 2200 56842 6 2x 100 Family: Euphorbiaceae Emblica ofﬁcinalis Gaertn. Prashar Lake, 2730 56848 52 8x 100 (=Phyllanthus emblica Linn.)

* PUN is the abbreviation used in “Index Herbariorum” by Holmgren and Holmgren (1998).

Clematis grata Wall. Ropa, 1800 m (56858PUN) It grows as a woody climber with a deeply furrowed stem, pinnate leaves and cup shaped creamish ﬂowers in axillary panicles. The species is commonly found in the sub-tropical and temperate Himalayas at altitudes of 1800–2500 m. During male meiosis, all the PMCs uniformly showed the same meiotic chromosome number, n=8, as conﬁrmed from the presence of eight large sized bivalents at diakinesis (Fig. 1a). Besides regular bivalent formation, individuals also showed the phenomenon of cytomixis, involving chromatin transfer among adjacent PMCs at various stages of meiosis. The maximum frequency of cytomixis was observed in PMCs at anaphase-I/II (A-I/II) (21.78%, Figs. 1b and 1c). Cytomixis induced various meiotic abnormalities in the PMCs, which include interbivalent connections (Fig. 1d), chromatin stickiness (Fig. 1e), extra chromatin material (Figs. 1f and 1g), and chromatin bridges (Fig. 1h). Bridges at anaphases/telophases, which resulted from sticky chromatin material, were recorded in 9.07% PMCs. As a result of partial chromatin transfer between PMCs, one to three extra chromatin material units were noticed in 11.11% PMCs, mostly at anaphases/telophases, which failed to get included in any of the poles and later on organized into micronuclei during telophases (Figs. 1i and 1j). The products of such meiocytes resulted in abnormal sporads (tetrads with micronuclei, Fig. 1k), high levels of pollen sterility (40%), and both heterogeneous-sized apparently fertile and sterile pollen grains and micro-pollen (Figs. 1l and 1m). The apparently fertile pollen grains were categorized as large (15.76⊗14.22 μm) or small (15.16⊗13.18 μm) and the sterile/unstained grains as large (14.50⊗14.28 μm) or micro-pollen grains (10.30⊗9.67 μm). Other populations of the species, studied by Rani et al. (2010a), from the hills of Kangra district shared the same meiotic chromosome number n=8 and also showed the phenomena of cytomixis, associated meiotic irregularities, and pollen malformation. The present diploid chromosome count of n=8 in the species agrees with the earlier chromosome reports of 2n=16 from Gobind Ghat in Garhwal Himalayas (Bir and Thakur 1980, Bir et al. 1987), Kashmir Himalayas (Koul and Wakhlu 1976) and Himachal Pradesh in North Western Himalayas (Rani et al. 2010a, Kumar 2011, Kumar and Singhal 2011, Kumar and Singhal 2012a).

Ranunculus laetus Wall. ex D. Don. Chachyot, 1580 m (56873 PUN), Rahla, 1900 m (56874PUN) Also known as ʻCheerful buttercupʼ, it is a perennial herb with woody root-stock and an erect, branched, hairy stem. It is found along roads, streams, and in waste places at the altitudes of 1500– 3000 m. It is a very variable and polymorphic species. The meiotic studies carried out on the plants 2013 Cytological Studies of Some Dicots from the Hills of Mandi District 5

Fig. 1. Male meiosis and pollen grains (1a–x): Clematis grata (a–m). a) Diakinesis with 8II. b) PMCs showing chromatin transfer at A-I (arrowed). c) Two PMCs showing chromatin transfer at A-II (arrowed). d) A PMC showing interbivalent connection at diakinesis (arrowed). e) PMCs with chromatin stickiness at M-I. f) A PMC showing extra chromatin material A-I (arrowed). g) A PMC showing extra chromatin material A-II (arrowed). h) A PMC showing a chromatin bridge at A-II (arrowed). i) Micronucleus at T-I (arrowed). j) Micronucleus at T-II (arrowed). k) Tetrad with micronucleus (arrowed). l) Apparently fertile/stained heterogeneous sized pollen grains. m) Sterile/unstained (arrowhead), micro- (arrowed) and apparently fertile/stained pollen grains. Ranunculus lae-

tus (n–q). n) M-I with 14II. o) A PMC showing out of plate two bivalents at M-I (arrowed). p) A PMC showing early disjunction of a bivalent (arrowed). q) Late disjunction of a bivalent at A-I (ar-

rowed). r) R. muricatus, M-I with 24II. s) A PMC showing laggards at T-II (arrowed). t) Impatiens micranthemum, M-I with 9II. u) Vicia hirsuta, M-I with 6II. v) Potentilla gerardiana, M-I with 42II. w) Ageratum conyzoides, M-I with 10II. x) A PMC showing early disjunction of two bivalents at M-I (arrowed). Scale bar=10 μm collected from two well separated localities, Chachyot and Rahla showed the same tetraploid chromosome count of n=14 as confirmed from the presence of 14 large sized bivalents at metaphase-I (M-I) (Fig. 1n). In a few PMCs, one to two bivalents remained out of the spindle plate (Fig. 1o). The early and late disjunction of bivalents was also recorded in some PMCs (Figs. 1p and 1q). Consequent to these spindle abnormalities, lagging chromosomes were observed in PMCs during A-I. These spindle abnormalities resulted in some pollen sterility (7%) and heterogeneous sized fer- 6 V. K. Singhal et al. Cytologia 78(1) tile and sterile pollen grains. The fertile/stained pollen grains were categorized as large (32.28⊗30.08 μm), medium (30.27⊗29.67 μm), and small sized (28.49⊗26.71 μm) whereas sterile/ unstained grains were categorized as large (26.97⊗23.94 μm) and small sized (23.83⊗21.90 μm). In previous communications on the species studies, carried out by the authors on the basis of collec- tions made from the cold regions of northwest Himalayas (Kumar and Singhal 2011, 2012a, Kumar et al. 2011), it showed abnormal meiotic behaviour in the form of asynaptic mutants resulting in 2n pollen grains formation and other meiotic irregularities, causing pollen steriliry. The present tetraploid chromosome count of n=14 in the species (based on x=7) is in agreement with the earlier chromosome records of 2n=28 from India (Mehra and Kaur 1963, Roy and Sharma 1971, Bhat et al. 1972, Mehra and Remanandan 1972, Bir and Thakur 1984, Bir et al. 1987, Singhal and Kumar 2010, Kumar 2011, Kumar et al. 2011, Kumar and Singhal 2012b) and outside of India (Goepfert 1974, Khatoon 1991). The other Himalayan populations of the species is also known to have intraspecific hexaploids, 2n=42 (Roy and Sharma 1971), and octoploid cytotypes, 2n=56 (Kabu et al. 1988). Further, the species is also known to have intraspecific diploids, 2n=16 (Kabu et al., 1988, Vaidya and Joshi, 2003), and tetraploids, 2n=32 cytotypes (based on x=8), reported by other researchers from India and Pakistan (cf. Standard Cytological Indices). The presence of B-chromosomes in the tetraploid cytotype (2n=28) has been reported in the plants collected from Dalhousie hills (Kaur et al. 2010). It is thus apparent that the species exhibits a considerable amount of chromosomal diversity and dynamism involving intraspecific polyploidy (based on two basic numbers, x=7 and x=8), abnormal meiotic behavior, and the presence of B-chromosomes.

R. muricatus Linn. Ner Chowk, 800 m (56875PUN) It is an annual herb with a glabrous stem and deeply lobed leaves and is native to Europe. It is common in fields in wet places at altitudes of 800–1500 m. Male meiotic studies in the species have been carried out on plants collected growing at the lower altitudes. The meiotic chromosome count of n=24 has been confirmed from the presence of 24 large sized bivalents in the PMCs at M-I (Fig. 1r). A few PMCs show the presence of laggards at telophase-II (T-II) (Fig. 1s) which resulted in some pollen sterility (9%) and heterogeneous sizes pollen grains. Based on x=8, the presently studied individuals of the species existed at 6x level. The majority of other researchers who have also examined this species have recorded the same hexaploid chromosome count of 2n=48 inside India (Mehra and Kaur 1963, Bir and Thakur 1981, 1984) and outside of India (cf. Standard Cytological Indices). However, the species is also known to have other chromosome counts of 2n=32, 40, 42, 64 which indicate the existence of intraspecific chromosomal variation based on x=7 and 8.

Impatiens micranthemum Edgew. Shikari Devi Temple, 3360 m (56882PUN) It is a glabrous herb with a slender stem, opposite, elliptic-ovate leaves and white flowers with slender straight spur. The species is commonly found at higher altitudinal ranges between 2500– 4000 m. The meiotic studies carried out on the individuals collected from the forests around Shikari Devi temple revealed a chromosome count of n=9 as confirmed from the presence of nine bivalents at M-I (Fig. 1t). Further meiotic course in the species is also normal resulting in regular microsporogenesis and high pollen fertility (98%). The species, which has been worked out chromosomally for the first time, exists at the diploid level.

Vicia hirsuta Koch. Ropa, 1800 m (56868PUN) The species grows as an annual climbing herb having white flowers tinged with blue at the apex in March-April and is found at altitudes of 1800–3000 m. The present meiotic chromosome count of n=6 in the species is confirmed from the presence of six bivalents at M-I (Fig. 1u). A few PMCs indicated interbivalent connections at M-I. However, further meiotic course was perfectly normal resulting in very high pollen fertility (99%). The present diploid chromosome count of n=6 2013 Cytological Studies of Some Dicots from the Hills of Mandi District 7 agrees with the earlier reports of 2n=12 by researchers from other parts of India (Sareen and Trehan 1976, 1977, Bairiganjan and Patnaik 1989) and outside of India (cf. Standard Cytological Indices). The species is also known to have chromosome counts of 2n=14, 28 from India and outside of India (cf. Standard Cytological Indices) indicating the existence of intraspecific aneuploidy and polyploidy.

Potentilla gerardiana Lindl. ex Lehm. Bagi, 2300 m (56844PUN) Also described under P. fragarioides auct. non Linn., it grows as a perennial herb with pin- nately compound leaves and yellow ﬂowers. The species is commonly found at altitudes of 2000– 2500 m in shady places. Meiotic studies performed on the plants collected from Barot revealed the presence of 42 bivalents at M-I (Fig. 1v). Further meiotic course was perfectly normal resulting in nearly 100% pollen fertility (99%). The present chromosome count of n=42 added a new 12x cytotype to the previously recorded diploid (2n=14) and octoploid (2n=56) cytotypes from India (Malik 1965, Mehra and Dhawan 1966, Kumar 2011, Kumar and Singhal 2011) and outside of India (Shimotomai 1929, Krogulevich 1978, Probatova and Sokolovskaya 1981, Iwatsubo and Naruhashi 1991).

Ageratum conyzoides Linn. Bagi, 2300 m (56861PUN) It is an erect annual herb with a hairy stem and pale-blue coloured ﬂowers. The species is commonly found on moist places at the altitudes of 1000–2500 m. Meiotic studies performed on plants collected from Bagi revealed the presence of 10 bivalents at M-I (Fig. 1w). Although in a few PMCs one to two bivalents showed early disjunction (Fig. 1x), the chromosomal distribution during anaphase-I is perfectly regular leading to 10 : 10 chromosomes at two poles. Further meiotic course was regular resulting in 100% pollen fertility. The meiotic chromosome count of n=10 recorded presently in the species agrees with the earlier reports of 2n=20 by a large number of researchers from India and outside of India (c.f. Standard Cytological Indices). Gill and Gupta (1971) have reported the presence of up to three B-chromosomes in the plants collected from India. The species is also known to have chromosome counts of 2n=36, 38, 40 reported from India and outside of India (cf. Standard Cytological Indices) indicating the existence of intraspeciﬁc aneuploidy and polyploidy.

Chrysanthemum leucanthemum Linn.Bagi, 2300 m (56837PUN) Also referred as ʻOx-eye daisy,ʼ it is an erect perennial herb with a glabrous stem, alternate, sessile, lanceolate leaves with toothed margins. The species grows in waste places in hilly areas at altitudes of 2000–3000 m. During the study of male meiosis from the individuals collected from Barot, 18 countable bivalents were clearly observed at M-I (Fig. 2a). The meiotic chromosome count of n=18 was also conﬁrmed at A-II (Fig. 2b). However, in a few PMCs, meiosis is characterized by the presence of one to two quadrivalents (Fig. 2c) and various meiotic irregularities, such as laggards and chromatin bridges during anaphases/telophases. Analysis of 46 PMCs revealed that 34.78% of the observed PMCs indicated the presence of one to two ring or chain type quadrivalents (Table 2). The percentage of chromosomes involved in the quadrivalent formation works to be 5.07% (Table 2). Consequent to these meiotic irregularities, heterogeneous sized pollen grains, both large and small sized, resulted. The species, which has been invesetigated quite extensively for chromosome studies by a large number of researchers from India and outside of India (cf. Standard Cytological Indices), is also known to exhibit intraspeciﬁc diploid and polyploid cytotypes at different levels (4x, 6x, 8x). Besides, Dorward and Malloch (1967) and Favarger (1963) have reported the presence of up to three B-chromosomes in the diploid and hexaploid cytotypes, respectively. 8 V. K. Singhal et al. Cytologia 78(1)

Fig. 2. Male meiosis and pollen grains (1a–x): Chrysanthemum leucanthemum (a–c). a) M-I with 18II. b) 18 : 18 : 18 : 18 chromosome distributions at A-II. c) 2IV (ring, arrowhead; chain, arrowed)+14II. d) Erigeron annuus, PMC showing 27I scattered in cytoplasm (2n=3x=27). e) Inula cappa, M-I with 10II. Taraxacum ofﬁcinale (f-J). f) 16 : 16 chromosome distributions at A-I (arrowed). g) Univalents scattered in the cytoplasm during M-I. h) Non-synchronously disjuncted bivalents during A-I. i) Dyad. j) Tetrad with micronucleus (arrowed). k) Gentiana aprica, 10 : 10 chromosome distributions

at A-I. l) G. argentea, 9 : 9 chromosome distributions at A-I. m) Cuscuta reﬂexa, M-I with 16II. n) Solanum nigrum, M-I with 24II. o) Apparently fertile/stained heterogeneous sized pollen grains. Lamium album (p-t). p) M-I with 9II. q) Cytoplasmic channels with (arrowed) and without (arrowhead) chromatin transfer. r) A hypo- (arrowed), hyperploid (double arrowed). and enucleated (arrowhead) PMC during chromatin transfer. s) Tripolar PMC. t) Multipolar PMC. Scale bar=10 μm. 2013 Cytological Studies of Some Dicots from the Hills of Mandi District 9

Table 2. Data on PMCs with bivalents and multivalents in Chrysanthemum leucanthemum

Meiotic conﬁgurations

PMCs observed II IV

46 776 5

Total number of bivalents/chromosomes → 828/1656

Number of chromosomes involved in → 1572 (94.93) 104 (5.07) meiotic conﬁgurations (%)

Erigeron annuus (Linn.) Pers Thunag, 3100 m (56865PUN) An annual herb with alternate leaves commonly found in moist shady places in forests and on rocky slopes at altitudes of 2000–3100 m. The meiotic studies carried out on the plants collected from Thunag revealed that the plants showed a chromosome count of 2n=27. The accession showed highly abnormal meiosis characterized by the presence of 27 univalents at M-I (Fig. 2d), laggards at A–I (25.92%), and micronuclei in sporads (44.82%). Consequent to these meiotic irregularities was pollen sterility. The present meiotic chromosome count of 2n=27 and all the earlier reports by researchers from other parts of Himalayas in India (cf. Standard Cytological Indices) suggests that the species uniformly exists at triploid level. However, from outside of India the species is also known to have diploid, 2n=18 (Hong and Zhang 1990), and hexaploid, 2n=54 (Nesom 1978, Carr et al. 1999) cytotypes. However, the record of up to nine B-chromosomes, as reported by Rudyka (1988), needs conﬁrmation as the author might have wrongly interpreted the univalents with B-chromosomes. The aneuploid chromosome reports of 2n=26, 28 (Chojnacki et al. 1982), also need further conﬁrmation.

Inula cappa DC. Barot, 2200 m (56883PUN) Common in forests, shrubberies and in waste places between altitudes of 1000–2400 m, where it grows as an undershrub or shrub with a tall erect woody stem. The meiotic studies carried out on the individuals collected from Barot showed a chromosome count of n=10 as conﬁrmed from the presence of 10 bivalents at M-I (Fig. 2e), and 10 : 10 chromosomes at two poles at M-II. Out of 10 bivalents, two bivalents are noticed to be relatively large in size. Meiotic course in these individuals is observed to be abnormal due to the presence of out of plate bivalents during M-I, chromosome stickiness, and interbivalent connections during diakinesis. Consequent to these abnormalities, micronuclei were observed in tetrads which resulted in some pollen sterility (7–8%) and unequal sized pollen grains. The present diploid chromosome count of n=10 in the species is in agreement with earlier reports of 2n=20 by workers from other regions of India (Mehra et al. 1965, Subramanyam and Kamble 1967). Mehra and Remanandan (1975) have earlier reported a tetraploid cytotypes (2n=40) from India. Banerjee and Sharma (1974) have reported an aneuploid chromosome count of 2n=18 in the species from India. It thus indicates that the species exhibits the existence of intraspe- ciﬁc aneuploidy (2n=18, 20) and polyploidy (2n=20, 40).

Taraxacum ofﬁcinale Wigg. Barot, 2200 m (56843PUN) Referred as a ʻdandelion weed,ʼ it is very widely distributed in the Himalayas up to an altitudinal range of 4000 m. It grows as a perennial herb with radical and sessile leaves. The male meiotic studies carried out on the plants collected from Barot revealed a meiotic chromosome count of n=16 at 16 : 16 chromosomes at A-I (Fig. 2f). Based on x=8 the species existed at the 4x level. The meiotic course in these tetraploid individuals is observed to be highly abnormal due to the presence of univalents which remain scattered in the cytoplasm during M-I (Fig. 2g) and non synchronously 10 V. K. Singhal et al. Cytologia 78(1) disjuncted bivalents during anaphases (Fig. 2h). Consequently, such PMCs yielded abnormal sporads which include dyads (Fig. 2i) and presence of micronuclei in the tetrads (Fig. 2j). Consequent to these meiotic abnormalities, the individuals showed some amount of pollen malformation (12%). The species, which has been worked out quite extensively from samples from India and elsewhere, exhibits an array of chromosome numbers (2n=8, 16, 18, 24, 26, 27, 32, 34, 38, 40) indicating the existence of aneuploidy and polyploidy. Besides, Dmitrieva and Parfenov (1985) and Krasnikov (1990) have reported the presence of one to two B-chromosomes in triploid individuals from outside of India.

Gentiana aprica Decne. Ropa, 1800 m (56845PUN) Commonly grows as a glabrous or nearly glabrous herb with silvery shinning leaves and in found in cultivated areas, shrubberies, and grazing grounds at altitudes of 1000–3000 m. The meiotic studies carried out in the species scored from Ropa revealed the chromosome number of n=10. During meiosis, 10 : 10 chromosomes are clearly observed during A-I (Fig. 2k). Besides, these individuals also showed the phenomenon of cytomixis through broad cytoplasmic channels involving two to three proximate PMCs. Consequently, some pollen sterility (26%) existed in the species. The same meiotic chromosome number of n=10 in the species has been reported earlier by Bala and Gupta (2011) in the populations collected from the Kangra district of Himachal Pradesh in India.

G. argentea (D. Don.) Griseb. Jhatingiri, 2012 m (56852PUN) It grows in alpine meadows as an annual herb with a branched simple stem and lanceolate basal leaves with finely pointed and recurved tips. The species is very common on open slopes, shrubberies, fields, and grasslands at altitudes of 2000–4000 m. The accession collected from Jhatingiri showed a meiotic chromosome count of n=9 which has been confirmed from the presence of 9 : 9 chromosomes at poles during A-I (Fig. 2l). Further meiotic course is normal, resulting in regular microsporogenesis and 100% pollen fertility. The present meiotic chromosome number of n=9 added a new intraspecific cytotype in the species, supplementing the earlier report of 2n=20 by Mehra and Vasudevan (1972) from plants from other parts of India.

Cuscuta reﬂexa Roxb. Thunag, 3100 m (56860PUN) The species, which grows as an annual herb, is commonly found as a parasitic climber on hedges around gardens and on bushes of Ziziphus and Berberis at altitudes of 1000–3200 m. Meiotic studies revealed the presence of 16 large sized bivalents at diakinesis (Fig. 2m). A few PMCs were observed to show interbivalent connections. Further meiotic course is normal resulting in normal microsporogenesis and 100% pollen fertility. Based on x=16, the species exists at diploid level and the same diploid chromosome count of n=16 has been reported by several researchers from India (Raghavan 1957, Kaul and Bhan 1974, Bir et al. 1978). Besides, the researchers have also reported several aneuploid chromosome counts of 2n=28, 36, 42, 48 in the species (cf. Standard Cytological Indices). Vij et al. (1974) and Bir et al. (1990) have also reported the presence of 1–3 chromosome fragments in species from Northern India.

Solanum nigrum Linn. Barot, 2200 m (56839PUN), Bagi, 2300 m (56863PUN) It is an erect, divaricately branched, unarmed, suffrutescent annual herb with white flowers. The species is quite common in waste places, along roadsides, gardens, hedges, and in cultivated fields at altitudes of 1000–2400 m. Meiotic studies carried out presently on the accession scored from Barot revealed the presence of 24 bivalents at M-I (Fig. 2n). Meiotic course is normal resulting in normal tetrad formation and 100% pollen fertility. In spite of the regular meiotic course, the fertile/stained pollen grains are of variable size, including as large (27.95⊗26.53 μm) and small sized (25.18⊗22.56 μm) (Fig. 2o). The presently studied individuals exist at tetraploid level, based 2013 Cytological Studies of Some Dicots from the Hills of Mandi District 11 on x=12. The species has been worked out quite extensively for meiotic studies by researchers from India and elsewhere and is already known to possess intraspecific diploid (2n=24), triploid (2n=36), tetraploid (2n=48), hexaploid (2n=72), ootoploid (2n=96) and 12-ploid (2n=144) cytotypes (cf. Standard Cytological Indices).

Lamium album Linn. Multhan, 2300 m (56846PUN) It is a tall perennial herb found in damp places in forests and shrubberies. The cytological investigations showed a meiotic chromosome count of n=9 confirmed from the presence of nine bivalents at M-I (Fig. 2p). These individuals also showed the phenomenon of cytomixis among proximate PMCs. 3% of PMCs have been observed with cytoplasmic channels, either with or without chromatin transfer (Fig. 2q). The phenomenon results in cytomixis, enucleated, hypo- and hyperploid (Fig. 2r), tripolar (Fig. 2s), and multipolar (Fig. 2t) PMCs. A few PMCs depict chromosome stickiness, early disjunction at M-I, late disjunction of 2 bivalents at A-I, and out of plate bivalents/ chromosomes during M-I, M-II. These meiotic irregularities in the PMCs results in some pollen sterility (7%) and heterogeneous sized pollen grains. The same diploid chromosome number (based on x=9) has been reported by researchers from India and elsewhere (cf. Standard Cytological Indices). Gill (1970) has reported the presence of one B chromosome in the species. The chromosome count of 2n=16 reported by Marchal (1920) seems to be erroneous and needs confirmation. Resultant to the present meiotic studies from the hills of Mandi district, which had not been explored cytologically, one species, namely Impatiens micranthemum (2n=18), has been counted chromosomally for the first time. A new intraspecific 12x cytotype has been reported in Potentilla gerardiana (2n=84) supplementing the earlier reported cytotypes (2x, 8x). A variable cytotypes has been reported in Gentiana argentea (2n=18), supplementing the earlier Indian report of 2n=20. In addition to this, a thorough chromosomal survey from the Standard Cytological Indices, together with analysis of presently studied species from the area, revealed the existence of chromosomal variation encompassing polyploidy and aneuploidy. Intraspecific polyploid cytotypes at different levels are existent in 23 species viz. Ranunculus laetus (4x, 6x, 8x on x=8; 2x, 4x on x=7), R. muricatus (4x, 5x, 6x, 8x on x=8; 6x on x=7), Hypericum elodeoides (2x, 4x on x=8), Vicia hirsuta (2x on x=6; 2x, 4x on x=7), Potentilla gerardiana (2x, 8x, 12x), Achillea millefolium (2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x), Ageratum conyzoides (2x, 4x), Artemisia parviflora (2x, 4x), Bidens pilosa (2x, 3x, 4x, 6x, 8x), Chrysanthemum leucanthemum (2x, 4x, 6x, 8x), Conyza stricta (2x, 4x), Erigeron annuus (2x, 3x, 6x), Galinsoga parviflora (2x, 4x), Inula cappa (2x, 4x), Senecio chrysanthemoides (8x), S. rufinervis (4x, 8x), Sonchus asper (2x, 4x), Taraxacum officinale (2x, 3x, 4x, 5x), Cuscuta reflexa (2x, 3x), Physalis angulata (2x, 4x), Solanum nigrum (2x, 3x, 4x, 6x, 8x, 12x), Plantago lanceolata (2x, 4x, 16x) and Emblica officinalis (4x, 8x on x=13; 14x, 28x on x=7). Aneuploidy causing chromosomal variation is equally common in the presently worked out species. As many as 20 species show intraspecific chromosomal variation at the diploid/polyploid level viz. Impatiens laxiflora (2n=12, 14, 16), I. scabrida (2n=12, 14, 16, 18, 20), Punica granatum (2n=14, 15, 18, 19), Achillea millefolium (2n=34, 53–56, 60–66, 74), Ageratum conyzoides (2n=36, 38), Bidens pilosa (2n=46, 70, 76), Erigeron annuus (2n=26, 28), Inula cappa (2n=18, 20), Saussurea candicans (2n=26, 32, 34), Senecio rufinervis (2n=20, 36), Taraxacum officinale (2n=18, 21–23, 22–24, 26, 27, 34, 36, 37), Carissa spinarum (2n=22, 28), Gentiana argentea (2n=18, 20), Cuscuta reflexa (2n=28, 30, 36, 42), Physalis angulata (2n=48, 50), Solanum nigrum (2n=36, 40), Lamium album (2n=16, 18), Plantago lanceolata (2n=13, 14). Achillea millefolium (2n=18, 27, 34, 36, 45, 53–56, 54, 60–66, 63, 72, 74, 81), Bidens pilosa (2n=24, 36, 46, 48, 70, 72, 76), Chrysanthemum leucanthemum (2n=9, 18, 36, 54, 72), and Taraxacum officinale (2n=8, 16, 21–23, 22–24, 24, 26, 27, 32, 34, 36, 37, 40). These species exhibited considerable amount of chromosomal variation constituting species complexes. 12 V. K. Singhal et al. Cytologia 78(1)

Acknowledgments

The authors wish to thank the University Grants Commission (UGC), New Delhi, for provid- ing ﬁnancial assistance under the DRS SAP I and II, ASIST programme, and the Dr. D.S. Kothari Post-Doctoral Fellowship Scheme [Award Letter No. F.4-2/2006 (BSR)/13-427/2011(BSR)] to Dr. Puneet Kumar. Thanks are also due to the Head, Department of Botany, Punjabi University, Patiala, for the necessary laboratory and Internet facilities.

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