Male Meiotic Studies in 29 Species of Lamiaceae from Sirmaur District of Himachal Pradesh, India

Vijay Singh1, Raghbir Chand Gupta2, Kusum Sharma3, Vikas Sharma4, Manjul Sharma1 and Kuljit Kaur1*

1 Department of Botany, Mata Gujri College, Fatehgarh Sahib, Punjab140407, India 2 Department of Botany, Punjabi University, Patiala, Punjab 147002, India 3 Department of Botany, Shoolini Institute of Life Sciences and Business Management, Solan, Himachal Pradesh 173212, India 4 Department of Botany, DAV University, Jalandhar144012, India

Received January 5, 2018; accepted March 2, 2018

Summary Lamiaceae is a requisite depository of many important medicinal herbs, distributed all over the world. The detailed meiotic studies, firstly are helpful in solving taxonomic and evolutionary enigmas and sec- ondly provide best chemotypes for further exploration and conservatory purposes. Male meiosis in 29 species of 54 populations belonging to the 18 genera of Lamiaceae has been undertaken for the first time from Sirmaur district of Himachal Pradesh falling in Western Himalayas. Euploid cytotypes are reported in Ajuga parviflora (n=8x=32), Nepeta eryostachys (n=4x=18) and N. graciliflora (n=4x=18). Besides, B-chromosomes in previously known cytotypes of five species, A. bracteosa (n=32+0-2B), Leucas lanata (n=11+0-1B), Lycopus europeus (n=11+0-1B), Phlomis bracteosa (n=11+0-1B) and Vitex negundo (n=16+0-1B), are reported for the first time. A substantial amount of meiotic abnormalities in the form of chromatin stickiness, cytomixis, chromatin bridges and laggards are reported in the presently worked out population that leads to the high variability in the microsporogenesis, pollen grain size heterogeneity and ultimately, reduce pollen viability. Only, the populations with B-chromosome had a low or negligible frequency of meiotic abnormalities.

Key words Chromosome number, Lamiaceae, Meiotic abnormality, Sirmaur district.

The family Lamiaceae commonly called “Mint fam- ried out (Harley et al. 2004, Bendiksby et al. 2011) to ily” is cosmopolitan in its distribution with Mediter- solve this taxonomic confusion in the family, but seems ranean regions as its center. The family is represented not enough. by about 7200 species in 236 genera overall (Heywood The cytotaxonomy is an important tool in unravel- et al. 2007) and about 335 species in 55 genera in India ing the taxonomic confusions. Moreover, the meiotic (Suthar and Patel 2014), mainly distributed in mountain- abnormalities are also imperative in studying evolution- ous regions. The district Sirmaur is located in the West- ary processes (Gupta et al. 2017, Singh et al. 2017). A ern Himalaya with an altitude ranges from Paunta Sahib significant amount of meiotic studies had been carried (398 m) to high mountains of Churpeaks (3650 m) and out in north Indian plains from south India, western comprises 42 species in 23 genera of the family (Kaur Himalayas and Kashmir Himalayas (Malik et al. 2016). and Sharma 2004). The district is a hub of wild me- However, cytological work on the Lamiaceae is still dicinal plant species that are widely used traditionally as lacking in this part of the western Himalayas. To fill this well as in modern systems of medicine and horticulture. gap, the present meiotic study is an addition to the chro- In pharmaceutical perspective, Lamiaceae are reported mosome database and also helps to solve such taxonomic with antibacterial activity (Maksimovic et al. 2008), confusions. anticancerous activity (Ozkan and Erdoğan 2011), anti- inflammatory anti-tumor activity (Lee et al. 2004) and Materials and methods antioxidant activity (Hussain 2009). However, the family seems taxonomic scrapyard as inter/-or intra tribal and The plant materials have been collected from vari- generic relationships among its members are much com- ous localities of Sirmaur districts (Table 1) of Himachal plex, confusing and poorly understood owing to both Pradesh. Fifty four populations of 29 species belonging homologous and homoplasious morphological characters to 18 genera and ten tribes have been studied cytologi- of its members. A number of molecular studies were car- cally. The voucher specimens are deposited in the Her- barium, Department of Botany, Punjabi University, Pa- * Corresponding author, e-mail: [email protected] tiala (PUN). The specimens are submitted after a proper DOI: 10.1508/cytologia.83.235 identification by consulting various floras of Himachal 236 V. Singh et al. Cytologia 83(3)

Table 1. Table showing details regarding name of taxon, locality with altitude, chromosome number (n), ploidy level (x) and size of pollen grains.

Locality (altitude in meters), Chromosome Ploidy Pollen grain Pollen viability Name of taxon/Accession number (PUP) Geographical Coordinates number (n) level size (µm) (%)

Ajuga bracteosa Wall. P-1 60056 Bagthan (2100), 30°41′N 77°12′E 32+0-2B 8x 31.9×33.5 85 A. parviflora Benth. P-1 58659 Sangrah (1400), 30°46′N 77°33′E 16 4x 20.6×21.0 96 P-2 60451 Haripurdhar (2400), 30°46′N 77°31′E 32* 8x 19.5×22.0 88 35.4×38.3 76 Leonurus cardiaca L. P-1 60453 Churdhar (3600), 30°52′N 77°28′E 9 2x 19.71×18.43 56 Leucas lanata Benth. P-1 60084 Nauradhar (1800), 30°48′N 77°25′E 11+0-1B** 2x 30.5×31.6 76 Calamintha umbrosa (M. Bieb.) Fisch. & Mey. P-1 60064 Haripurdhar (2400), 30°46′N 77°31′E 18 2x 34.3×35.0 88 P-2 60067 Haripurdhar (2400), 30°46′N 77°31′E 18 2x 33.5×36.1 83 C. vulgaris L. P-1 60081 Nauradhar (1400), 30°48′N 77°25′E 20 2x 35.7×36.9 84 Lycopus europeus L. P-1 60058 Chapdhar (2200), 30°49′N 77°25′E 11+0-1B** 2x 29.6×31.5 96 Mentha longifolia L. P-1 58612 Bagthan (2200), 30°41′N 77°12′E 12 2x 20.3×23.0 82 P-2 58613 Sangrah (1400), 30°46′N 77°33′E 12 2x 21.6×22.9 80 Micromeria biflora (Buch.-Ham.) Benth. P-1 58638 Jamnala (2600), 30°50′N 77°26′E 15 2x 30.1×24.5 78 P-2 60062 Nauradhar (1800), 30°48′N 77°25′E 15 2x 29.5×26.5 73 Nepeta hindostana (Roth) Haines. P-1 60078 Chapdhar (2200), 30°49′N 77°25′E 18 4x 19.2×20.9 92 N. eryostachys Benth. P-1 59785 Chapdhar (2400), 30°49′N 77°25′E 18* 4x 20.9× 22.5 78 P-2 60066 Haripurdhar (2400), 30°46′N 77°31′E 18* 4x 20.1×23.4 73 N. govaniana Benth. P-1 58646 Churdhar (3650), 30°52′N 77°28′E 9 2x 19.8×20.1 95 P-2 59788 Tisri (3100), 30°51′N 77°27′E 9 2x 18.9×19.5 93 P-3 60070 Tisri (3100), 30°51′N 77°27′E 9 2x 18.3×20.5 98 P-4 60074 Haripurdhar (2400), 30°46′N 77°31′E 9 2x 18.9× 20.5 N. graciliflora Benth. P-1 58621 Bhangiani mata (2600), 30°46′N 77°33′E 18* 4x 16.2×19.2, 65 N. leucophylla Benth. P-1 58634 Shilai (2100), 30°40′N 77°42′E 18 4x 28.4×29.3 79 P-2 58670 Haripurdhar (2400), 30°46′N 77°31′E 18 4x 27.3×29.8 83 P-3 60060 Haripurdhar (2400), 30°46′N 77°31′E 18 4x 27.9×28.0 89 P-4 60072 Haripurdhar (2400), 30°46′N 77°31′E 18 4x 25.9×28.6 84 N. spicata Benth. P-1 60057 Haripurdhar (2400), 30°46′N 77°31′E 9 2x 16.5×19.5 63 P-2 59509 Haripurdhar (2400), 30°46′N 77°31′E 9 2x 16.0×17.9 71 Origanum vulgare L. P-1 58618 Sangrah (1400), 30°46′N 77°33′E 15 2x 32.6×33.5 96 P-2 60068 Sangrah (1400), 30°46′N 77°33′E 15 2x 33.5×36.4 91 P-3 60080 Haripurdhar (2400) 15 2x 31.0×34.2 89 Prunella vulgaris L. P-1 60075 Haripurdhar (2400), 30°46′N 77°31′E 14 2x 39.0×40.6 89 P-2 60077 Haripurdhar (2400), 30°46′N 77°31′E 14 2x 38.9×41.2 82 Salvia lanata Roxb. P-1 58687 Sangrah (1400), 30°46′N 77°33′E 11 2x 35.2×38.7 86 P-2 60059 Sangrah (1400), 30°46′N 77°33′E 11 2x 36.0×39.8 89 S. moorcroftiana Wall. ex Benth. P-1 58688 Sangrah (1400), 30°46′N 77°33′E 11 2x 34.8×35.9 70 Thymus linearis Benth. P-I 58674 Haripurdhar (2400), 30°46′N 77°31′E 13 2x 15.3×16.8 69 P-2 60069 Sangrah (1400), 30°46′N 77°33′E 13 2x 14.9×17.5 67 T. serpyllum L. P-1 60063 Nauradhar (1800), 30°48′N 77°25′E 13 2x 16.5×19.5 58 Plectranthus mollis P-1 60083 Haripurdhar (2400), 30°46′N 77°31′E 14 2x 29.3×31.5 90 P. rugosus (Ainton) Spreng. P-1 58631 Sangrah (1400), 30°46′N 77°33′E 12 2x 32.4×34.6 86 P-2 58684 Sataun (1080), 30°33′N 77°38′E 12 2x 33.0×35.6 82 P-3 60071 Tisri (3100), 30°51′N 77°27′E 12 2x 31.9×32.8 84 P-4 60082 Nauradhar (1800), 30°48′N 77°25′E 12 2x 33.0×35.0 88 Phlomis bracteosa Wall. ex Benth. P-1 60061 Churdhar (3650), 30°52′N 77°28′E 11+0-1B** 2x 24.6×25.9 93 P-2 60076 Churdhar (3650), 30°52′N 77°28′E 11+0-1B** 2x 25.0×26.3 97 Pogostemon benghalensis Royle ex Benth. P-1 60079 Kala Umb (650), 30°29′N 77°12′E 32 4x 20.3×23.6 76 Stachys sericea (Burm.f.) Kuntze P-1 55493 Churdhar (3650), 30°52′N 77°28′E 15 2x 18.6×19.0 65 Scutellaria repens Wall. P-1 58629 Nauradhar (1800), 30°48′N 77°25′E 10 2x 26.5×27.0 76 P-2 60073 Sangrah (1400), 30°46′N 77°33′E 10 2x 27.0×28.3 79 S. scandens D. Don P-1 58633 Nauradhar (1400), 30°48′N 77°25′E 11 2x 26.4×27.5 86 P-2 58679 Sangrah (1400), 30°46′N 77°33′E 11 2x 25.9×27.3 91 P-3 60065 Nauradhar (1400), 30°48′N 77°25′E 11 2x 27.0×29.3 83 Vitex negundo L. P-1 58648 Renuka Ji (670), 30°36′N 77°27′E 16+0-1B** 2x 19.6×2 0.5 93 P-2 60088 Renuka Ji (670), 30°36′N 77°27′E 16+0-1B** 2x 18.9× 21.5 92

* New euploid cytotype at world level, ** New report of B-chromosomes. 2018 Cytology in Several Species of Lamiaceae from District of Himachal Pradesh 237

Pradesh (Chowdhery and Wadhwa 1984, Aswal and chromosome report of n=20 (Fig. 1ag) is in conformity Mehrotra 1994, Dhaliwal and Sharma 1999, Kaur and with the previous reports (Vij and Kashyap 1976). The Sharma 2004), and finally confirmed from Botanical species is also reported with another cytotype (2n=20) Survey of India (BSI), Dehradun. For meiotic studies from India (Mehra and Gill 1968). young floral buds were fixed in a freshly prepared Car- Lycopus europeus L.: The present report of n=22+0- noy’s fixative (ethanol, chloroform, glacial acetic acid in 1B (Fig. 1ah) is the first-ever report of B-chromosome a volume ratio of 6 : 3 : 1) for 24 h and then transferred for the species. Previously, the species is known to exist to 70% alcohol and stored in a refrigerator at 4°C, sub- at diploid level with 2n=22 (Malik et al. 2016). sequently. The slides of meiocytes were prepared by a Mentha longifolia L.: The present chromosome count squashing the developing anthers in 1% acetocarmine. of n=12 (Fig. 1ai) is in line with the previous reports The chromosome number was determined by analyzing from India (Sobti 1965) and abroad (Parfenov and Dmi- 500–600 pollen mother cells (PMCs) at different count- trieva 1988). Besides this, the species is also known as able stages of meiosis [metaphase I, II (M I, II)/anaphase 2n=18 (Heimans 1938), 2n=27 (Sobti 1962), 2n=36 I, II (A I, II)] under a microscope. Pollen viability was (Sobti 1965), and 2n=48 (Khatoon and Ali 1993). estimated through gently heating mature pollens in glyc- Micromeria biflora (Buch.-Ham.) Benth.: Meiotic erol–acetocarmine (1 : 1) mixture for 24 h. At least, 450 study in the species reveals a diploid cytotype with pollen grains were studied for pollen viability and size n=15 (Fig. 1aj), which is in line with the previous report frequencies. Well-filled pollen grains with stained nuclei (Mehra and Gill 1968, Khatoon and Ali 1993). were taken as fertile, while shriveled and unstained pol- Nepeta hindostana (Roth) Haines.: The meiotic analy- len were counted as sterile. Photomicrographs from the sis in the species reveals a tetraploid cytotype with freshly prepared slides having various stages of meiotic n=18 (Fig. 1ak) which is previously confirmed by Vij course and microsporogenesis were taken with a digital and Kashyap (1975). The species is also known to exist imaging system of Leica QWin. at diploid level with 2n=18 (Saggoo and Bir 1982). N. eryostachys Benth.: The chromosome number of n=18 Results and discussion (Fig. 1al) is a new euploid report in the species at world level. Previously, the species is known to exist at diploid Chromosome numbers level with 2n=18 (Gill 1969). N. govaniana Benth.: The Ajuga bracteosa Wall.: The present chromosome present chromosome count of n=9 (Fig. 1am) is in con- count of n=32+0-2B (Fig. 1aa) is a first ever report of B- formity to the previous report (Gill 1969). N. graciliflora chromosomes in the octoploid cytotype. Earlier, the spe- Benth.: The present chromosome count of n=18 (Fig. cies is reported with 2n=16, 32 (Bir and Saggoo 1981) 1an) is a new euploid cytotype for the species at world and 2n=64 (Bala and Gupta 2011). A. parviflora Benth.: level. Previously, the species is known with the chromo- Meiotic study in the species reveals a tetraploid cytotype some count of 2n=18 (Bir and Saggoo 1984). N. leuco- with n=16 (Fig. 1ab), and an octaploid cytotype with phylla Benth.: The meiotic study in the species reveals n=32 (Fig. 1ac). The octoploid cytotype with 2n=64 is the chromosome count of n=18 (Fig. 1ao), which is in reported for the first time in the species. Prior to this, the conformity to the previous work (Gill 1969). N spicata species is reported to exist as tetraploid level 2n=4x=32 Benth.: Meiotic studies in the species reveal a diploid (Bir and Saggoo 1982) and with B- chromosomes (Bala cytotype with n=9 (Fig. 1ap) that confirms the previous and Gupta 2011). report (Khatoon and Ali 1993). Leonurus cardiaca L.: Meiotic study in the species Origanum vulgare L.: The chromosome number of reveals a diploid cytotype with n=9 (Fig. 1ad), which is n=15 (Fig. 1aq) and other chromosome counts of 2n=24, in conformity with the previous report from India (Gill 32 and 40 have already been confirmed previously by 1970). many workers (Khatoon and Ali 1993). Leucas lanata Benth.: The B-chromosome with Prunella vulgaris L.: Meiotic studies in the species n=11+0-1B (Fig. 1ae), is the first-ever chromosome reveals n=14 (Fig. 1ar) that confirms previous reports report in the species. Though, the species is previously (Malik et al. 2016). Beside tetraploid cytotype, the spe- known with the chromosome numbers of 2n=22 (Vembu cies is also reported with variable chromosome counts and Sampathkumar 1998), 2n=28 (Gill 1970) and 2n=30 like 2n=14, 21 and even 2n=28-30 (Malik et al. 2016). (Bhat et al. 1975). Salvia lanata Roxb.: The present meiotic study reveals Calamintha umbrosa (M. Bieb.) Fisch. & Mey.: The n=11 (Fig. 1as), which is already known in the species present chromosome count of n=18 (Fig. 1af) is in (Mehra and Gill 1968) and along with another chromo- conformity with the previous report of Mehra and Gill some count of 2n=24 (Sarkar et al. 1975). S. moorcrofti- (1968). Besides, the chromosome counts of 2n=10 (Gill ana Wall. ex Benth.: Meiotic study in the species reveals 1971a), 2n=36 (Saggoo and Bir 1981), 2n=38 (Krish- the present chromosome report of n=11 (Fig. 1at) that nappa and Basavaraj 1982) and 2n=40 (Vij and Kashyap confirms the previous report of 2n=22 (Podlech and 1976) are also reported. C. vulgaris L.: The present Bader 1974). The species is also known to exist with 238 V. Singh et al. Cytologia 83(3)

Fig. 1. Meiotic chromosome numbers observed in PMCs of presently investigated aa Ajuga bracteosa, at M I with 32II+2B (ar- rowed) configuration of chromosomes (2n=64). ab–ac; A. parviflora, ab (Cytotype-I) at M I with 16 bivalents (2n=32), ac (Cytotype-II), at diakinesis showing 32 bivalents (2n=64). ad; Leonurus cardiaca, at diakinesis showing 9 bivalents (2n=18). ae; Leucas lanata, 11+1B chromosomal configuration at M I (2n=22). af; Calamintha umbrosa, showing 18 : 18 equal distribution of chromosomes at A I (2n=36). ag; C. vulgaris, showing 20 : 20 equal distribution of chromosomes

at A I (2n=40). ah; Lycopus europeus, showing 11II+1B configuration at M I (2n=22). ai; Mentha longifolia, A I showing 12 : 12 distribution of chromosomes (2n=24). aj; Micromeria biflora, showing 15 bivalents at diakinesis (2n=30). ak; Nepeta hindostana, showing 18 : 18 chromosome at A I (2n=36). al; N. eryostachys, showing 18 bivalents at M I (2n=36). am; N. govaniana, showing 9 bivalents at M I (2n=18). an; N. graciliflora, showing 18 bivalents at diakinesis (2n-36). ao; N. leucophylla, showing 18 : 18 distribution of chromosomes at A I (2n=36). ap; N. spicata, at diakinesis with 9 bivalents (2n=18). aq; Origanum vulgare, showing 15 bivalents at M I (2n=30). ar: Prunella vulgaris, showing 14 chromosomes at A I (2n=28). as; Salvia lanata, showing 11 : 11 distribution of chromosomes at each pole (2n=22). at; S. moorcroftiana showing 11 chromosomes at A I (2n=22). au; Thymus linearis, showing 13 bivalents at diakinesis (2n=26). av; T. serpyllum, with 13 bivalents at diakinesis (2n=26). aw; Plectranthus mollis, A I showing 14 : 14 distribution of chromosomes at each pole (2n=28). ax; P. rugosus, with 12 bivalents at M I (2n=24). ay; Phlomis bracteosa, showing 11 bivalnets at diakinesis (2n=22). az; Pogostemon benghalensis, showing 32 bivalents at M I (2n=64). ba; Stachys sericea, with 15 bivalents at diakinesis (2n=30). bb; Scutellaria repens, at A I with 10 : 10 equal distribution of chromosomes (2n=20). bc;

S. scandens, at A I showing 10 : 10 chromosomes at each pole (2n=20). bd; Vitex negundo showing 32II+1B conﬁguration at diakinesis (2n=64). Scale bars=10 µm. 2018 Cytology in Several Species of Lamiaceae from District of Himachal Pradesh 239

Table 2. Data on cytomixis and other meiotic abnormalities in the various populations of twenty nine species belonging to the family Lamiaceae from Sirmaur district (H. P.).

Cytomixis Meiotic course Accession No. PMCs involved No. of PMCs Involved PMCs with chromosomal PMCs with un-oriented PMC with bridges (%) PMCs with laggards (%) (%) (%) stickiness at M I (%) bivalents (%) at M I at A I, II/T I, II at A I, II/ T I, II

Ajuga bracteosa P-1 60056 -/- -/- 0.1 (1/101) 5.4 (6/110) -/- -/- A. parviflora P-1 58659 24.6 (31/126) 2–6 5.5 (6/109) 2.3 (3/130) 4.3 (6/139) 1.5 (2/129) P-2 60451 19.8 (22/111) 2–4 11.5 (14/121) 13.5 (16/118) 26.9 (38/141) 10.4 (14/134) Leonurus cardiaca P1- 60453 31.2 (35/112) 4–6 24.6 (31/126) 19.1 (25/131) 5.4 (6/110) -/- Leucas lanata P-1 60084 -/- -/- -/- -/- -/- -/- Calamintha umbrosa P-1 60064 5.5 (6/109) 1–2 22.1 (29/131) 0.1 (1/101) 5.4 (6/110) -/- P-2 60067 0.9 (1/113) 1–2 3.5 (4/113) 1.7 (2/113) -/- 1.8 (2/113) C. vulgaris P-1 60081 -/- -/- 0.1 (1/101) 5.4 (6/110) -/- -/- Lycopus europeus P-1 60058 -/- -/- -/- 0.1 (1/101) 5.4 (6/110) -/- Mentha longifolia P-1 58612 5.4 (6/111) 1–3 3.1 (4/126) 2.7 (3/111) -/- -/- P-2 58613 1.8 (2/113) 1–2 7.1 (9/126) 2.7 (3/111) -/- -/- Micromeria biflora P-1 58638 3.4 (5/144) 2–3 5.5 (6/109) 8.3 (11/131) 6.1 (8/130) 10.0 (12/114) P-2 60062 24.6 (31/126) 2–6 5.5 (6/109) 2.3 (3/130) 4.3 (6/139) 1.5 (2/129) Nepeta hindostana P-1 60078 -/- -/- -/- -/- 0.1 (1/101) 5.4 (6/110) N. eryostachys P-1 59785 12.8 (15/117) 2–3 13.9 (18/129) 13.7 (16/116) 12.5 (17/136) 10.4 (14/134) P-2 60066 12.8 (15/117) 2–3 13.9 (18/129) 13.7 (16/116) 0.1 (1/101) 5.4 (6/110) N. govaniana P-1 58646 9.27 (9/97) 1–2 11.3 (1/97) 5.5 (5/90) 7.2 (7/97) 2.1 (2/97) P-2 59788 5.5 (5/90) 1–2 -/- -/- -/- -/- P-3 60070 6.1 (6/97) 1–2 11.3 (11/97) 9.3 (9/97) 7.2 (7/97) 2.1 (2/97) P-4 60074 8.1 (8/98) 1–2 11.3 (11/97) 6.1 (6/97) 7.2 (7/97) -/- N. graciliflora P-1 58621 2.4 (3/125) 1–2 4.0 (5/125) -/- -/- -/- N. leucophylla P-1 58634 -/- -/- 0.1 (1/101) 5.4 (6/110) -/- -/- P-2 58670 -/- -/- -/- -/- -/- -/- P-3 60060 2.5 (3/119) 1–2 15.1 (18/119) 4.7 (5/107) 9.7 (1/103) 7.9 (6/76) P-4 60072 -/- -/- -/- -/- 2.8 (3/107) 2.7 (3/109) N. spicata P-1 60057 22.7 (25/110) 2–3 2.8 (3/105) 4.1 (5/123) 9.3 (11/118) 16.1 (21/130) P-2 59509 22.7 (25/110) 2–3 2.8 (3/105) 4.1 (5/123) 9.3 (11/118) 16.1 (21/130) Origanum vulgare P-1 58618 2.0 (2/100) 1–2 -/- -/- 2.8 (3/107) 2.7 (3/109) P-2 60068 2.5 (3/119) 1–2 15.1 (18/119) 4.7 (5/107) 9.7 (1/103) 7.9 (6/76) P-3 60080 2.0 (2/100) 1–2 -/- -/- 2.8 (3/107) 2.7 (3/109) Prunella vulgaris P-1 60075 -/- -/- -/- -/- 2.8 (3/107) 2.7 (3/109) P-2 60077 -/- -/- -/- 2.8 (3/107) 2.7 (3/109) -/- Salvia lanata P-1 58687 -/- -/- -/- -/- 0.1 (1/101) 5.4 (6/110) P-2 60059 2.5 (3/119) 1–2 15.1 (18/119) 4.7 (5/107) 9.7 (1/103) 7.9 (6/76) S. moorcroftiana P-1 58688 14.5 (19/131) 2–4 11.1 (12/108) -/- -/- -/- Thymus linearis P-1 58674 -/- -/- -/- 2.8 (3/107) 2.7 (3/109) -/- P-2 60069 2.5 (3/119) 1–2 15.1 (18/119) 2.8 (3/107) 2.7 (3/109) 7.9 (6/76) T. serpyllum P-1 60063 2.5 (3/119) 1–2 15.1 (18/119) 4.7 (5/107) 9.7 (1/103) 7.9 (6/76) Plectranthus mollis P-1 60083 2.5 (3/119) 1–2 15.1 (18/119) 4.7 (5/107) 2.8 (3/107) 2.7 (3/109) P. rugosus P-1 58631 -/- -/- -/- 2.8 (3/107) 2.7 (3/109 -/- P-2 58684 -/- -/- -/- -/- -/- -/- P-3 60071 2.0 (2/100) 1–2 -/- -/- 2.8 (3/107) 2.7 (3/109) P-4 60082 -/- -/- -/- -/- -/- -/- Phlomis bracteosa P-1 60061 2.0 (2/100) 1–2 -/- -/- 2.8 (3/107) 2.7 (3/109) P-2 60076 2.0 (2/100) 1–2 -/- -/- 2.8 (3/107) 2.7 (3/109) Pogostemon benghalensis P-1 60079 2.0 (2/100) 1–2 -/- -/- 2.8 (3/107) 2.7 (3/109) Stachys sericea P-1 55493 8.4 (9/107) 1–4 4.1 (5/123) 11.4 (15/132) 8.7 (9/103) 10.1 (13/128) Scutellaria repens P-1 58629 2.0 (2/100) 1–2 -/- -/- 5.6 (6/107) 2.5 (3/119) P-2 60073 2.0 (2/100) 1–2 0.9 (1/107) -/- 0.9 (1/107) 4.6 (5/109) S. scandens P-1 58633 2.0 (2/100) 1–2 -/- -/- 3.0 (3/100) 1.8 (2/109) P-2 58679 2.1 (2/95) 1–2 -/- -/- 2.7 (3/110) 3.0 (3/100) P-3 60065 1.8 (2/110) 1–2 -/- 0.9 (1/107) 2.8 (3/107) 2.7 (3/109) Vitex negundo P-1 58648 2.0 (2/100) 1–2 0.9 (1/107) -/- 3.6 (4/111) 2.7 (3/109) P-2 60088 2.7 (3/110) 1–2 -/- -/- 2.8 (3/107) 2.7 (3/109)

Figures in parenthesis denote observed number of abnormal PMCs in the numerator and total number of PMCs observed in denominator. 240 V. Singh et al. Cytologia 83(3)

Table 3. Data on abnormal microsporogenesis in different populations of 29 species of family Lamiaceae from Sirmaur district (H. P).

Taxon/Accession Monads Dyads Triads Tetrads Meiotic index % No. WMN (%) WM (%) WMN (%) WM (%) WMN (%) WM (%)

Ajuga bracteosa P-1 60056 -/- -/- -/- -/- -/- -/- 100 (103/103) A. parviflora P-1 58659 -/- -/- 11.9 (13/109) -/- -/- -/- 77.0 (84/109) P-2 60451 2.7 (3/109) 0.9 (1/109) 11.9 (13/109) -/- 7.3 (8/109) -/- 77.0 (84/109) Leonurus cardiaca P-1 60453 2.9 (3/103) 2.7 (3/109) 10.0 (11/109) 2.7 (3/109) 11.9 (13/109) 4.5 (5/109) 65.1 (71/109) L. lanata P-1 60084 -/- -/- -/- -/- -/- -/- 100 (111/111) Calamintha umbrosa P-1 60064 1.5 (2/130) 0.76 (1/130) 2.3 (3/130) 3.07 (4/130) 2.3 (3/130) 3.8 (5/130) 86.1 (112/130) P-2 60067 -/- -/- 1.6 (2/120) -/- -/- -/- 98.3 (118/120) C. vulgaris P-1 60081 -/- -/- 3.1 (4/129) 0.8 (1/129) 7.0 (9/129) 8.5 (11/129) 80.6 (104/117) Lycopus europeus P-1 60058 -/- -/- -/- -/- -/- -/- 100 (115/115) Mentha longifolia P-1 58612 -/- 0.9 (1/115) 2.6 (3/115) -/- 2.6 (3/115) 1.7 (2/115) 92.2 (106/115) P-2 58613 -/- -/- 0.8 (1/125) -/- 1.6 (2/125) 0.8 (1/125) 96.8 (121/125) Micromeria biflora P-1 58638 -/- -/- -/- -/- -/- -/- 100 (121/121) P-2 60062 -/- -/- 1.7 (2/121) -/- 4.1 (5/121) -/- 95.0 (115/121) Nepeta hindostana P-1 60078 -/- 0.9 (1/105) -/- 1.9 (2/105) 2.8 (3/105) 1.1 (2/105 92.4 (97/105) N. eryostachys P-1 59785 0.8 (1/124) 1.6 (2/124) 11.3 (14/124) -/- 5.6 (7/124) 1.6 (2/124) 79.0 (98/124) P-2 60066 0.8 (1/124) 3.2 (4/124) 9.6 (12/124) -/- 4.0 (5/124) 2.4 (3/124) 79.8 (99/124) N. govaniana P-1 58646 -/- 1.0 (1/95) -/- -/- -/- -/- 99.2 (139/140) P-2 59788 -/- 1.7 (2/116) 4.3 (5/116) 2.6 (3/116) 4.3 (5/116) 5.2 (6/116) 81.8 (95/116) P-3 60070 -/- 1.5 (2/130) 0.7 (1/130) 0.8 (1/130) 2.3 (3/130) 1.5 (2/130) 93.1 (121/130) P-4 60074 -/- -/- 0.8 (1/125) 2.4 (3/125) 0.8 (1/125) 1.6 (2/125) 94.4 (118/125) N. graciliflora P-1 58621 2.3 (2/87) 4.6 (4/87) 1.14 (1/87) 2.3 (2/87) 3.4 (3/87) 11.5 (10/87) 72.4 (63/87) N. leucophylla P-1 58634 -/- 1.8 (2/117) 7.7 (9/117) -/- 11.9 (14/117) 5.1 (6/117) 73.5 (86/117) P-2 58670 2.9 (4/139) 3.6 (5/139) 5.0 (7/139) 2.9 (4/139) 5.7 (8/139) 7.9 (11/139) 71.9 (100/139) P-3 60060 -/- 1.7 (2/116) 4.3 (5/116) 2.6 (3/116) 4.3 (5/116) 5.2 (6/116) 81.9 (95/116) P-4 60072 2.2 (3/133) 1.50 (2/133) 3.7 (5/133) 2.2 (3/133) 8.3 (11/133) 6.8 (9/133) 75.2 (100/133) N. spicata P-1 60057 -/- 1.1 (1/95) -/- -/- -/- -/- 99.2 (139/140) P-2 59509 -/- 1.7 (2/116) 4.3 (5/116) 2.6 (3/116) 4.3 (5/116) 5.2 (6/116) 81.9 (95/116) Origanum vulgare P-1 58618 0 1.8 (2/117) 7.7 (9/117) 0 11.9 (14/117) 5.1 (6/117) 73.5 (86/117) P-2 60068 2.9 (4/139) 3.6 (5/139) 5.0 (7/139) 2.9 (4/139) 5.7 (8/139) 7.9 (11/139) 71.9 (100/139) P-3 60080 2.2 (3/133) 1.5 (2/133) 3.7 (5/133) 2.2 (3/133) 8.2 (11/133) 6.8 (9/133) 75.2 (100/133) Prunella vulgaris P-1 60075 -/- 1.0 (1/95) -/- -/- -/- -/- 99.2 (139/140) P-2 60077 -/- 1.7 (2/116) 4.3 (5/116) 2.6 (3/116) 4.3 (5/116) 5.2 (6/116) 81.9 (95/116) Salvia lanata P-1 58687 2.5 (3/121) 0.8 (1/121) 4.9 (6/121) 0 6.6 (8/121) 2.5 (3/121) 82.6 (100/121) P-2 60059 2.5 (3/121) 0.8 (1/121) 4.9 (6/121) 0 6.6 (8/121) 2.5 (3/121) 82.6 (100/121) S. moorcroftiana P-1 58688 2.2 (3/133) 1.5 (2/133) 3.7 (5/133) 2.2 (3/133) 8.3 (11/133) 6.8 (9/133) 75.2 (100/133) Thymus linearis P-I 58674 1.5 (2/130) 0.8 (1/130) 2.3 (3/130) 3.1 (4/130) 2.3 (3/130) 3.8 (5/130) 86.2 (112/130) P-2 60069 -/- -/- 1.6 (2/120) -/- -/- -/- 98.3 (118/120) T. serpyllum P-1 60063 -/- -/- 3.1 (4/129) 0.7 (1/129) 7.0 (9/129) 8.5 (11/129) 80.9 (104/117) Plectranthus mollis P-1 60083 1.6 (2/121) 4.1 (5/121) 0.8 (1/121) 4.1 (5/121) 0.8 (1/121) 0.8 (1/121) 87.6 (106/121) P. rugosus P-1 58631 -/- -/- -/- -/- -/- -/- 100 (121/121) P-2 58684 1.7 (2/121) 4.1 (5/121) 0.8 (1/121) 4.1 (5/121) 0.8 (1/121) 0.8 (1/121) 87.6 (106/121) P-3 60071 -/- 0.9 (1/115) 2.6 (3/115) -/- 2.6 (3/115) 1.7 (2/115) 92.2 (106/115) P-4 60082 2.7 (3/109) 0.9 (1/109) 2.7 (3/109) -/- 2.7 (3/103) -/- 90.8 (99/109) Phlomis bracteosa P-1 60061 -/- -/- -/- -/- -/- -/- 100 (105/105) P-2 60076 -/- -/- -/- -/- -/- -/- 100 (115/115) Pogostemon benghalensis P-1 60079 -/- 0.9 (1/105) -/- 1.9 (2/105) 2.8 (3/105) 1.1 (2/105 92.4 (97/105) Stachys sericea P-1 55493 -/- 0.8 (1/124) 1.6 (2/124) -/- 4.0 (5/124) 1.6 (2/124) 91.9 (114/124) Scutellaria repens P-1 58629 0.9 (1/105) -/- 1.9 (2/105) 2.8 (3/105) 1.1 (2/105 -/- 92.4 (97/105) P-2 60073 -/- 0.8 (1/124) 1.6 (2/124) -/- 4.0 (5/124) 1.6 (2/124) 91.9 (114/124) S. scandens P-1 58633 -/- 0.8 (1/124) 1.6 (2/124) -/- 4.0 (5/124) 1.6 (2/124) 91.9 (114/124) P-2 58679 -/- -/- 3.1 (4/129) 0.8 (1/129) 7.0 (9/129) 8.5 (11/129) 80.9 (104/117) P-3 60065 1.5 (2/130) 0.8 (1/130) 2.3 (3/130) 3.1 (4/130) 2.3 (3/130) 3.8 (5/130) 86.1 (112/130) Vitex negundo P-1 58648 2.7 (3/109) 0.9 (1/109) 2.7 (3/109) -/- 2.9 (3/103) -/- 90.8 (99/109) P-2 60088 1.5 (2/130) 0.8 (1/130) 2.3 (3/130) 3.07 (4/130) 2.3 (3/130) 3.8 (5/130) 86.1 (112/130)

Figures in parenthesis denote observed number of abnormal PMCs in the numerator and total number of PMCs observed in denominator, WMN, without micronuclei; WM with micronuclei. Meiotic index=Number of normal tetrad/ Total number s of tetrads observed. 2018 Cytology in Several Species of Lamiaceae from District of Himachal Pradesh 241

2n=16 (Gill 1971b) and 20 (Koul and Wakhlu 1976). stickiness was observed in 24.6% and un-oriented Thymus linearis Benth.: Meiotic analysis in the spe- bivalents in 19.08% PMCs of L. caridiaca. Besides, cies reveals a chromosome count of n=13 (Fig. 1au), interbivalent connections were also observed in N. gova- which is in conformity with the previous report (Malik naina. The chromosomal laggards and bridges are also et al. 2016). The species also exists with a chromosome reported to be maximum in Salvia moorcroftiana and count of 2n=24 (Khatoon and Ali 1993). T. serpyllum N. eryostachys, respectively. Such meiotic abnormalities L.: The chromosomes number of n=13 (Fig. 1av) is in lead to the abnormal microsporogenesis and the forma- conformity with the previous works from India (Bir and tion of heterogeneous-sized pollen grains (Table 3) and Saggoo 1980). Besides this, the species is also reported also affect pollen viability (Table 1). with 2n=24 (Gregor and Hand 2006) and 50 (Krogulev- In Lamiaceae, the Salvia is reported with the most ich 1976). primitive and highest base number, x=11 (Fujita 1970). Plectranthus mollis (Ainton) Spreng.: The species But, the genus is also based on x=6, 7, 8 and 9, of which reveals a diploid cytotype with n=14 (Fig. 1aw), which x=6 and 9 are derived from x=7 and 8 (Ranjbar et al. is in line with the previous reports (Morton 1962). P. ru- 2015), possibly due to irregular cytological behavior and gosus Wall. ex Benth.: The present chromosome count of asymmetrical karyotype (Bhattacharya 1978). Hence, n=12 (Fig. 1ax) confirms the previous work (Mehra and the chromosome number evolution is not a descending Gill 1972). dysploidy (Enke and Gemeinholzer 2008), but a bidirec- Phlomis bracteosa Royle ex Benth.: The present study tional process (Blöch et al. 2009), which cause specia- reveals B-chromosome with n=11+0-1B (Fig. 1ay), tion and diversification (Stebbins 1971, Levin 2002). which is a first ever report in this cytotype. The species In the present meiotic study, four species are reported is known to exist with 2n=22 (Gill 1970), 2n=28 (Raina with small, enigmatic B-chromosome along larger A- and Ashruf 1981) and 2n=42 (Jee et al. 1985). chromosomes. The exact origin of these is still con- Pogostemon benghalensis (Burm. f.) Kuntze: The fusing, but are believed to be originated from the A’s present chromosome count of n=32 (Fig. 1az) reveals the (Houben et al 2014), or as a consequence of structural previous report (Cherian and Kuriachan 1993). The spe- reshuffling of chromosomes (Jones and Houben 2003). cies is also reported with 2n=34 (Mehra and Gill 1968) B-chromosomes are known to suppress homoelogous and 2n=42 (Vembu and Sampathkumar 1999) in Indian pairing and promote cytological diplodization (Kausaka populations. and Endo 2012), if present in fewer numbers or reduce Stachys sericea Wall.: The present chromosome count pollen viability, if present in large number. The popula- of n=15 (Fig. 1ba), has already been confirmed by Gill tions with B-chromosomes reveal low meiotic abnor- (1970). malities (Table 3), which resemble the previous studies Scutellaria repens Buch.-Ham. ex D. Don: Meiotic of many workers (Jones et al. 2008, Houben et al. 2013). study in the species reveals diploid cytotype with n=10 (Fig. 1bb) that was earlier reported by Mehra and Gill Acknowledgements (1968). B-chromosome (2n=20+0-1B) has also been reported in the species from India (Gill 1970). S. scandens The study is supported by the financial grant D. Don: The present meiotic study reveals a diploid cy- under IPLS-DBT (Reference number: BT/PR totype with n=11 (Fig. 1bc) that was reported earlier by 4548/INF/22/146/2012). Thanks are also due to Late Sh. Saggoo and Bir (1981). The species is also reported with Kishan Lal, who assisted the author with their intense 2n=26 from India (Gill 1970). field knowledge during the collection tours. Vitex negundo L.: The present study reveals the pres- ence of B-chromosome (Fig. 1bd) for the first time in References previously reported tetraploid (2n=32) cytotype (Sandhu and Mann 1988). Apart from this, the species is also Aswal, B. S. and Mehrotra, B. N. 1994. Flora of Lahaul Spiti̶A Cold known to exist with 2n=26 (Bir and Chattha 1983) and Desert in North-West Himalaya. Bishen Singh Mahendra Pal Singh, Dehra Dun. 2n=34 (Ma et al. 1990, Khatoon and Ali 1993). Bala, S. and Gupta, R. C. 2011. 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