Chromosome Botany・6・3・85

Exploration of cytomorphological diversity in the Scrophulariaceae from Kashmir Himalaya, India

Reyaz Ahmad Malik1, Raghbir Chand Gupta and Santosh Kumari

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

1Author for correspondence: ([email protected]) Received May 31, 2011; accepted August 11, 2011

ABSTRACT. Cytomorphological exploration has been undertaken in 14 species of the Scrophulariaceae from Kashmir, Himalaya. The varied gametophtyic chromosome counts of n=23 for Veronica cana and diploid chromosome number i.e. n=7 for V. persica are reported here for the fi rst time on global basis. Besides this 0 to 2 B-chromosomes have been observed in Verbascum thapsus for the fi rst time. Likewise Digitalis grandifl ora (n=28) and Scrophularia scopolii (n=13) are fi rst chromosome reports from India. The meiotic course observed in these scrophs is normal except some irregularities like bridge formation in Mazus japonicus and Pedicularis rhinanthoides, interbivalent connections in P. pectinata and precocious disjunction in Euphrasia offi cinalis. Morphologically the different accessions within a species showed no conspicuous differences except Digitalis purpurea that has been seen to exist as three morphotypes on fl ower colour basis.

KEYWORDS: Chromosome, Digitalis, Kashmir Himalaya, Veronica

Scrophulariaceae is commonly called the fi gwort family. MATERIALS AND METHODS Most of the species included under this family are annual Appropriate sized fl ower buds of the plants growing in or perennial herbs with usually zygomorphic and rarely their natural habitats were collected and fi xed in Carnoy’s actinomorphic fl owers. It is a large family comprising fi xative. After 24 hrs. the buds were transferred in rectifi ed around 280 genera and 3000 species (Watson and Dalwitz alcohol for longer storage purpose. The meiotic studies 1992) which are mostly distributed to temperate regions were carried out in young fl oral buds by standard aceto- of the world. From Kashmir Himalaya the family is carmine smearing technique. Photomicrographs of pollen reported to cover 19 genera and 77 species (Dar et al. mother cells (PMC’s) and pollen grains were taken using 2002). There is ambiguity in the reports regarding the Nikon 80i Digital Imaging System. Pollen fertility was number of taxa under this family due to some taxonomic estimated by their stainability in 1% glycero-acetocarmine revisions made from time to time and thereby transferring (1:1). Well fi lled and well stained pollen grains were some taxa from one family to other e.g. Digitalis to the regarded as fertile while as the shrunken and unstained Plantaginaceae (Olmstead et al. 2001). pollen grains were considered as sterile. For morphological Kashmir Himalaya (1600 to 4500 m) is a land of hubs of studies simply metric scale and hand lenses were used. many important medicinal and aromatic plants including species from the Scrophulariaceae. The examples are RESULTS AND DISCUSSION Digitalis spp. reported to be benefi cial in heart diseases The meiotic studies in 14 species of the Scrophulariaceae (Goldthorp 2009), Picrorhiza kurroa in dysentry, stom- have been carried out on population basis covering achache and fever (Ballabh and Chaurasia 2009), different altitudinal ranges of Kashmir Himalaya. All the Verbascum thapsus for mental relaxation and wounds individuals worked out are either diploids (2x) or (Kumar et al. 2009), etc. This family which is in the list of tetraploids (4x) and reveal normal meiosis except some top ten families of Kashmir Himalaya as far as species irregularity in Euphrasia offi cinalis, Mazus japonicus, composition is concerned (Dar et al. 2002) has not been Pedicularis pectinata and P. rhinanthoides. The informa- comparatively explored on cytological basis. The reason tion regarding the observed chromosome number, location behind this is the combined hindrances posed by with altitude, ploidy level, pollen fertility and pollen size topography of far fl ung inaccessible areas and political of each species is presented in Table. challenges of the valley. Keeping in view the above said importance of the family along with its unsatisfactory Digitalis grandifl ora Mill. (Common name: Big-fl owered cytological exploration it is imperative to make more Foxglove) The plant is usually distributed to subalpine attempts for further cytological contributions. This will Kashmir Himalaya and growing from 2200-2900 m. It is help in framing chromosome number data base and a perennial plant, 1-1.5 m tall; leaves simple, lanceolate & understanding meiotic behaviour and meiotic irregularities serrate. Corolla tubular and yellow. Flowering period: if any caused to these species growing there in cold and June-July. harsh climatic conditions. The gametophytic chromosome number of n=28 (Fig. 1a) for this species is fi rst report from India. This is in accordance with the previous report of Dobes and Hahn 86 MALIK ET AL.

Table 1. Data regarding the name of the species with accession number, collection site & its altitude, haploid chromosome number (n), ploidy level, pollen fertility and pollen size Taxa/ Locality Altitude Chromosome Ploidy level Pollen fertility Pollen size Accession (PUN) (metre) No. (n) (%age) (μm) Digitalis grandiﬂ ora Mill.* 54378 Gulmarg 2,600 28 4x 99.5 21.19x16.65 D. lanata Ehrh. 54374 Gulmarg 2,600 28 4x 100 13.72x12.91 D. purpurea L. 54372x Gulmarg 2,600 28 4x 99.8 16.59x14.45 54373y Gulmarg 2,600 28 4x 99.6 55468z Gulmarg 2,600 28 4x 99.9 Euphrasia ofﬁ cinalis L. 55171 Aharbal 2,200 22 4x 46.6 28.40x23.69 55467 Thajwas 3,300 22 4x 48.5 Mazus japonicus (Thunb.) Kuntze 54350 Pulwama 1,750 20 4x 100 18.70x16.85 Pedicularis pectinata Wall. ex Benth. 55170 Aharbal 2,200 8 2x 79.2 26.55x24.90 55466 Dhara 2,400 8 2x 76.4 P. rhinanthoides Schrenk. 54366 Aharbal 2,250 8 2x 95.8 (37.21x30.80 55465 Gulmarg 2,400 8 2x 95.5 & 55464 Kellar 2,150 8 2x 97.0 28.82x24.31) Scrophularia himalensis Royle 55586 Aharbal 2,300 12 2x 92.8 19.85x17.37 S. scopolii Hoppe* 55192 Aharbal 2,300 13 2x 58.4 26.26x24.98 Verbascum thapsus L. 54376 Pahalgam 2,200 17+0-2B** 2x 89.5 17.46x15.92 55469 Pakharpora 1,900 17 2x 97.6 Veronica beccabunga L. 35.70x33.68 54377 Thajwas 3,100 9 2x 90.3 & 24.90x21.70 V. cana Wall. ex Benth. 55172 Aharbal 2,250 23˚ 2x 62.0 23.50x21.80 55462 Yusmarg 2,400 23˚ 2x 61.2 V. persica Poir. 55463 Pulwama 1750 7˚˚ 2x 81.2 07.92x07.38 55470 Srinagar 1650 7˚˚ 2x 82.3 V. serpyllifolia L. 54371 Thajwas 3100 7 2x 63.9 23.04x21.86 * First chromosome report for India; **First B-chromosome report for the species; °First aneuploid chromosome report for the species; °°First euploid (diploid) chromosome report for the species; X, Y and Z: White, lilac/light pink and rose/bright pink morphotypes respectively. PUN=Herbarium Code of Botany Dept. Punjabi Univ. Patiala.

(1997). Flowering period: June-July. The present chromosome count of n=28 (Fig. 1b) further conﬁ rms the base number Digitalis lanata Ehrh. (Common name: Wooly Foxglove/ 7 for the genus and is in accordance with its previous Grecian Foxglove) The species is mainly distributed to cytological reports of n=28 by Gill (1972) and Gupta and subalpine Kashmir Himalaya from 1900-2600 m. It is a Molnar (1979). perennial up to 0.8 m high, unbranched; leaves elongated, wooly and hairy on underside. Corolla tubular, white or Digitalis purpurea L. (Common name: Purple Foxglove/ lilac and ﬂ owers in spikes. Flowers and stem also wooly. Common Foxglove/Deas Men’s Bells) CYTOMORPHOLOGY IN THE SCROPHULARIACEAE 87

The species is distributed to the Himalayan range from also Eastern Himalaya. In Kashmir it is found growing 2000 to 2600 m. It is a biennial herb; leaves spirally between 2400-3500 m. It is a perennial herb up to 25-80 arranger, simple, 10-35 cm long and 5-12 cm broad, cm tall with cauline pinnatifi d and opposite leaves. compound and hairy and forming a rosette at the ground Flowers are rose-pink. Flowering Period: June-August. level in the fi rst year. Flowering shoot typically 1-2 m tall. The present chromosome count of n=8 (Fig. 1h) in both The fl owers in a terminal cluster, pendant with beautiful the populations confi rms its diploid nature. Except some spots on the inner surface of corolla; corolla tubular. sort of interbivalent connections (Fig. 1i) in Aharbal Flowering period: June-July. population, the meiosis has been found to be normal. The three morphotypes have been found in this species from the same site growing in thin associations. One Pedicularis rhinanthoides Schrenk. (Common name: Rattle morphotype bearing white colored fl owers is shorter in Lousewort) The species is distributed to subalpine and height than the other bearing light pink. The third one that mostly alpine Western Himalaya and Eastern Himalaya. bears bright pink fl owers is tallest among all. It seems In Kashmir it is found between 2400 to 5000 m. It is 5-25 here that there might be a multiple allelic gene with cm high. Leaves linear-oblong; the radical leaves form a quantitative inheritance responsible for this variation. rosette and are petiolate. Flowers pedicellate, axillary and The chromosome report of n=28 (Fig. 1c) presented pink or purple. Flowering period: June-August Three here has been already assigned to this species by Verma populations of this species have been worked out. and Dhillon (1967) and Chandan and Bhavananda (1987). Specimens collected from Aharbal and Gulmarg show some irregularity in the form of bridges (Fig. 1k). Euphrasia offi cinalis L. (Common name: Eyebright) The Gemetophytic chromosome number has been observed to species is distributed to subalpine and alpine Himalaya be 8 (Fig. 1j) in all the accessions, confi rming its diploid from Kashmir to Kumaon. In Kashmir Himalaya it grows ploidy level. This is in accordance with the earlier reports above 2400 m. It is an annual, small, sparsely pubescent of n=8 (Mehra and Vasudevan 1972). and branched herb reaching up to 10-30 cm; leaves sessile, ovate and deeply toothed. Flowers white to lilac, usually Scrophularia himalensis Royle. (Common name: Himala- tinged with yellow; bracts foliaceous and corolla bi- yan Figwort) Distributed to temperate Western Himalaya lipped. Flowering period: June-August. The present chro- from Kashmir to Kumaon between 1900-3200 m. It is an mosome count of n=22 (Fig. 1d) has been already assigned annual herb reaching up to 1-1.5 m with branched stem; to this species by Vasudevan (1975 b) which confi rms its leaves egg shaped and usually sharp pointed; fl owers in tetraploid nature as per the base number 11 (Darlington cymes or sometimes panicles, greenish; stamens far pro- and Wylie 1955). In both the worked out populations truding. Flowering period: June-September. The present meiosis has been found to be normal except some early gametophytic chromosome count for the species has been disjunction (Fig. 1e) in one of the bivalents at metaphase reported to be 12 (Fig. 1l). Its previous cytological report I, which is ascribed to short crossover segments. Thajwas is 2n=24+2-4 B (Dhillon 1979). The species has been population shows more frequency of PMC’s with such worked out for the fi rst time from the study area. precocious disjunction which may be ascribed to cold harsh climatic conditions prevailing there (Malik et al Scrophularia scopolii Hoppe. (Common name: Scopoli’s 2010). Microsporogenesis in both the accessions shows Figwort) Distributed throughout subalpine Western Hi- some irregularity in the form of triads (Fig. 1t). malaya from 2500-3300 m. It is a biennial/ perennial plant with stems up to 0.5-1 m high, and glabrous; leaves Mazus japonicus Thunb. (Common name: Japanese Mazus) petiolate, oval-oblong and coarsely serrate; fl owers short The species is distributed from temperate to nearly pedicellate in cymose panicles, greenish; stamens not subalpine Himalaya from 2400-3000 m in Kashmir. It is protruding out. Flowering period: June-July. Presently an annual herb up to 25 cm high, decumbent, with opposite the species has been cytologically worked out for the fi rst phyllotaxy; above leaves sessile and below leaves stalked. time from India. The gametophytic chromosome number Calyx tubular, corolla bilobate and purplish. reported is n=13 (Fig. 1m) showing its diploid nature and Flowering period: May- June. Our chromosome count of which coincides with its already assigned sporophytic n=20 (Fig. 1f) for the species is as such for the fi rst time report of 2n=26 (Gadnidze et al 1998). from the study area and confi rms its earlier reports of Bir et al (1976), and Hoshimoto (1986). At late Anaphase I, Verbascum thapsus L. (Common name: Great Mullein or some of the PMC’s have been observed with Bridge Aaron’s Rod) Distributed to entire Himalayan range formation (Fig. 1g) from Kashmir to Sikkim. In Kashmir Himalaya it occurs between 1600-3500 m. It’s a biennial herb with a height of Pedicularis pectinata Wall. ex Benth. (Common name: 0.5-2 m, hairy; leaves wooly on both the surfaces, radical Pectinate-leaved lousewort) The species is distributed leaves petiolate and stem leaves becoming gradually throughout western subalpine and alpine Himalaya and smaller and sessile, with alternate phyllotaxy; fl owers 88 MALIK ET AL.

Fig. 1. Meiotic chromosomes of Kashmir Himalaya’s Scrophulariaceae. a. Digitalis grandifl ora, PMC at anaphase I showing 56 chromosomes. B. D. lanata, PMC at anaphase I with 28 chromosomes at each pole. c. D. purpurea, PMC at metaphase I with 28 bivalents. d, e and t. Euphrasia offi cinalis. d. PMC with 22 bivalents at metaphase I. e.PMC with early disjuncted bivalent (arrow). t. triad, f and g. Mazus japonicas. f. PMC at anaphase I showing 20 chromosomes at each pole. g. Bridge at late anaphase I. h and i. Pedicularis pectinata. h.PMC at diakinesis with 8 bivalents. i. Interbivalent connections (arrows). j and k. P. rhinanthoides. J. PMC at anaphase II showing 8 chromosomes at each pole. k. PMC at anaphase II with a bridge (arrow). l. Scrophularia himalensis, PMC at metaphase I with 12 bivalents; m. S. scopolii, PMC at metaphase I showing 13 bivalents; n and o. Verbascum thapsus, n-PMC with 17 bivalents at M-I, o-PMC showing 2 B-chromosomes (arrows); p-Veronica beccabunga, PMC at early anaphase I. q. V. cana, PMC at anaphase I showing 23 chromosomes at each pole. r. V. persica, PMC at diakinesis with 7 bivalents. s. V. serpyllifolia, PMC at metaphase I with 7 bivalents. PMC= Pollen Mother Cell; A-I= anaphase I; a-II= anaphase II. Bar= 10μm CYTOMORPHOLOGY IN THE SCROPHULARIACEAE 89 yellow in terminal spikes. Flowering period: April- Flowering period: May-August. August. The accession from Pahalgam is cytologically The gametophytic chromosome count of n=7 (Fig. 1r) found to bear gametophytic chromosome number of is here reported for the fi rst time on worldwide basis n=17+0-2 B (Figs. 1n &o). Such a report has been already which confi rms the base number of Veronica as 7 assigned to the species by Subramanian and Pondmudi (Darlington and Wylie 1955). This is the fi rst diploid (1987) except reporting B-chromosomes. From Kashmir cytotype report for the species, since its previous report is its earlier chromosome report is n=18 (Gohil et al 1981). n=14 (Vasudevan 1975b). It seems here that presence of B-chromosomes may be responsible for apparently low pollen fertility (89.5%) for Veronica serpyllifolia L. (Common name: Thyme-leafed Pahalgam population, but perusal of the literature reveals Speedwell) Distributed to temperate and subalpine Hi- that B-chromosomes have neutral effect when only a few malaya from Kashmir to Kumaon. In Kashmir it is found of them are present (Jones 1975; Jones and Rees 1982; between 2200-3200 m. The species is a perennial herb Jones 1995). The Pakharpora population showing no about 5-15 cm tall; upper leaves sessile, lower leaves occurrence of B-chromosomes has comparatively higher short petiolate, oblong-egg shaped and opposite; fl owers reproductive potential (97.6%). small, solitary axillary, blue and short pedicellate. Flow- ering period: May-June. The meiosis was seen to be Veronica beccabunga L. (Common name: Water Pimpernel normal with haploid chromosome number of n=7 (Fig. 1s) or Brooklime) The species is distributed to Western which is similar to its previous reports (Bhattacharyya Himalaya from Kashmir to Kunawer between 1700- 1969a; Vasudevan 1975b). 3100 m. It is a semi-aquatic fl eshy plant, sparsely hairy with hollow stem and pigmented branches; leaves sessile, CONCLUSION oval, blunt and opposite. Flowers on axillary racemes and Out of the 14 species of the Scrophulariaceae cytologically blue coloured. Flowering period: May-September. The worked out species, chromosome reports of fi ve species meiosis has been found to be normal showing 9 bivalents are new, varied or fi rst from India. In addition to this, three at M-I (Fig. 1p) which is in conformity with its earlier species have been studied cytologically for the fi rst time reports (Vasudevan 1975b; Gomal 1991). The species from the study area excluding those reported for the fi rst shows heterogeneous sized pollen grains in both the time from India. Some cases such as Euphrasia offi cinalis, populations (Table 1) Pedicularis pectinata, P. rhinanthoides different populations differ in meiotic behavior, one or more being normal Veronica cana Wall. ex Benth. (Common names: Hui mao while other/s being abnormal as has been already found po po na, source-China; kuwagataso, source- Japan) out in many plant species of North-west Himalaya. Such a Distributed to Himalayan range from Kashmir to Sikkim. kind of information is of colossal signifi cance for Perennial herb, reaching up to 20-60 cm, erect, with grey chromosomal database cataloguing of plants of this hairs; lower leaves petiolate, cordate to deltoid-orbicular important biodiversity region of Himalayas, besides and upper leaves subsessile, and ovat-orbicular; hairy; throwing light on the nature of variable chromosome/ fl owers in racemes of 3-6 cm and blue. Flowering period: meiotic behavior among the populations of the same May- July. species. Two populations of the species were cytologically examined. The present chromosome count of n=23 (Fig. ACKNOWLEDGEMENTS. The authors are highly grateful to Joint Director, Deputy Director and other taxonomic experts particularly Dr. 1q) was given for the fi rst time on worldwide basis. Prashant of Botanical Survey of India, Dehradun for providing help in the Veronica is a polybasic genus with the base numbers of 7, identifi cation of the plant species. 8 and 9 (Darlington and Wylie 1955). The earlier chromosome reports for the species were n=25 (Mehra and LITERATURE CITED Vasudevan 1972) and n=26 (Vasudevan 1975b). The Ballabh, B. and Chaurasia, O. P. 2009. Medicinal plants of cold desert Ladakh used in the treatment of stomach gametophytic chromosome number of 23 has been earlier disorders. Ind. J. Trdnl. Knowledge. 8: 185-190. reported in one species of Veronica viz. V. melissifolia by Bhattacharyya, N. K. 1969a. Cytological studies of a few more Mehra and Gill (1968a) and Vasudevan (1975b). The taxa of Scrophulariaceae. Proc. 56th Indian Sci. Congr. Part 3: 375. species was cytologically worked out for the fi rst time Bir, S. S., Kumari, S., Shoree, S. P. and Saggoo, M. S. 1978. from the study area. 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