Cytomorphological Studies on Some North Indian Members of the Family Verbenaceae

Santosh Bala* and Raghbir C. Gupta

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

Received October 7, 2011; accepted January 31, 2012

Summary This paper presents cytomorphological diversity covering the chromosome number, meiotic behavior and pollen study of 5 species on an accession basis under the family Verbenaceae from North India. Six new chromosome reports have been made for the ﬁrst time which involve 2 varied chromosome counts; n=18 for Caryopteris odorata and n=6 for Verbena officinalis; 3 new euploid cytotypes of diploid (2n=2x=14) for V. officinalis and hexaploid (2n=6x=42) and octaploid (2n=8x=56) for Verbena bonariensis; and B-chromosome for Vitex negundo with 2n=32+0- 2B. Morphotypes are identiﬁed for 2 species; Caryopteris odorata and Lantana camara, and cyto- morphotype for Verbena officinalis. All the investigated species show abnormal meiotic behavior.

Key words Chromosome number, Cytotypes, Morphotypes, Micronuclei, Verbenaceae.

Verbenaceae, commonly known as verbena or the vervain family, are represented by 86 genera (Brummitt 1992) which is reduced to 41 by removal of many genera to Lamiaceae, the mint family of flowering dicots. Recently, Heywood et al. (2007) estimated that there were 35 genera and 1200 species, mostly distributed in all parts of the world and abundant in tropical and subtropical regions. In India, 105 species in 19 genera have been reported. Members of the family have tu- bular flowers in clusters, spikes or racemes. Cultivars and hybrids of some vervian species are used as ornamentals. Verbena officinalis is a relaxant herb used as remedy for asthma, migraine, insomnia and nervous coughing. It is also known to have anti-inflammatory (Deepak and Handa 2000); antioxidant and antifungal (Casanova et al. 2008); and analgesic (Calve 2006) properties. Vitex negundo has anti-inflammatory (Dharmasiri et al. 2003); antibacterial (Perumal Samy et al. 1998); antifungal (Sathiamoorthy et al. 2007, Damayanti et al. 1996); and analgesic (Gupta and Tandon 2005) properties. The chromosome data on such medicinally important members of the Verbenaceae family from India in general and from this region in particularly is very scarce. Cytomorphological variation is expected from the area and the present study is a part of research intended to fill this lacuna.

Materials and methods

Collection and preservation Plant materials of wild taxa of the Verbenaceae family were collected throughout the year in 2009–2010 from different localities of Kangra and Himachal Pradesh of North India (Table 1). Voucher specimens were identiﬁed from the Herbarium of Botanical survey of India (BSI), Dehra Dun and were deposited in the Herbarium, Department of Botany, Punjabi University, Patiala

* Corresponding author e-mail: [email protected] ** PUN is the Herbarium Code of Department of Botany, Punjabi University, Patiala as per “Index Herbariorum” by Holmgren and Holmgren (1998). DOI: 10.1508/cytologia.77.187 188 S. Bala and R. C. Gupta Cytologia 77(2)

Table 1 Data on name of taxa, voucher data, accession number, occurrence with distribution, ﬂowering and fruiting period, meiotic chromosome number along with ploidy level in presently investigated species from the Kangra, Himachal Pradesh district in North India

Meiotic Occurrence Flowering Voucher data Accession chromosome Name of taxa with and distribu- and Ploidy (Locality with number Habitat number (2n) synonyms tion range in fruiting level altitude in meters) (PUN) (Figure meters period number)

Caryopteris odorata Andretta,1365 m 56040 Occasionally Hedges and March– 36 (Figs. 1–5) 4× (D. Don) Robinson Andretta,1365 m 56039 found, open slopes May 36 4× = C. bicolor (Hardw.) 1000–2000 m Mabberley =C. wallichiana Schau. = Clerodendron odoratum D. Don = Volkameria bicolor or Hardw.

Lantana camara Linn. Sakri-Bilaspur, 527 53181 Abundant, Forests and March– 44 (Figs. 6–9) 4× =L. aculeata Linn. Dharamshala, 1500 53215 500–1500 m shrubberies November 44 4× = L. camara Linn. var. aculeata (Linn.) Mold.

Verbena bonariensis Linn. Dharamshala, 1500 53177 Common, Along April– 42 6× Palampur, 1221 53178 1200–1500 m roadsides and June (Figs. 10–15) 8× ﬁelds 56

Verbena officinalis Linn. Dharamshala, 1500 53179 Common, Waste places March– 14 2× Jawali, 530 53180 500–1800 m and as an June (Figs. 16–18) 2× Boh, 1670 56036 undergrowth 14 2× in forests 12

Vitex negundo Linn. Masroor, 617 53183 Occasionally Forests and March– 32+0–2B 2× =V. paniculata Lam. found, shrubberies June (Figs. 19–20) 600–1500 m

(PUN**). Appropriate sized flower buds were fixed in Carnoy’s fixative (6 : 3 : 1=absolute alcohol : chloroform : glacial acetic acid, v/v) for 24 h and preserved in 70% alcohol at 4°C.

Cytomorphological analysis Morphological characters, including plant height, stem, leaf characters (texture, surface, shape, size and color), inflorescence type, flower and fruit characters, have been carefully studied in the presently investigated 10 accessions of 5 species. For meiotic studies, appropriate sized anthers were squashed in 1% acetocarmine. From meiotic preparations, chromosome numbers and detailed meiotic behavior in pollen mother cells (PMCs) were carefully examined. Pollen fertility was estimated by mounting mature pollen grains in glycero–acetocarmine (1 : 1). Normal well-filled and deeply stained pollens were taken as fertile when shriveled up and unstained pollens as sterile. Measurements of pollen size were taken using an ocular micrometer. Stomatal studies were made from epidermal peals obtained through 1% KOH treatment of mature leaves, stained with safranin and mounted in 50% glycerine. To determine the frequency and size of stomata, as well as trichomes from epidermal cells per unit area, an area of 70 mm2×15 was uniformally taken in all cases at 40× of objective. Stomatal index is calculated using the expression: Number of stomata Stomatal index = ×100 Number of epidermal cells+Number of stomata Photomicrographs of chromosome counts were made from freshly prepared slides using a 2012 Cytomorphological Studies̶North Indian̶Verbenaceae 189

Figs. 1–20. Meiosis in presently investigated taxa. Scale bar=10 μm. Caryopteris odorata (Figs. 1–5); 1. PMC (pollen mother cell) at diakinesis showing 18 bivalents; 2. PMC at anaphase I showing 18 : 18 chromosomes at each pole; 3. PMC at metaphase I with bivalents showing secondary associations; 4. Pollen mother cells at metaphase I with hyperploid and hypoploid chromosome number; 5. PMC at telophase I with chromatin bridges. Lantana camara (Figs. 6–9); 6. PMC at anaphase I with 2n=44; 7. PMC at telophase II with laggards; 8. Polyad with 8 nuclei; 9. Tetrad with 3 micronuclei. Verbena bonariensis (Figs. 10–15); 10. PMC at metaphase I with 21 bivalents; 11. PMC at metaphase I showing 28 bivalents; 12. PMC at metaphase I showing 2 unoriented bivalents; 13. Three PMCs at diakinesis involved in chromatin transfer; 14. Monad and a dyad; 15. Dyad with micronucleus. Verbena officinalis (Figs. 16–18); 16. PMC at metaphase I with 7 bivalents; 17. PMC at metaphase I with 6 bivalents; 18. PMC at metaphase I with n=4 II+1 IV. Vitex negundo (Figs. 19–20); 19. PMC at diakinesis with n=16+2B; 20. PMC at telophase I with laggards. 190 S. Bala and R. C. Gupta Cytologia 77(2)

Nikon Microscope Eclipse 80i system. The statistical tool involves the standard deviation, calculated for the number of observations for a particular character.

Results

Detailed observations were recorded for 10 accessions of 5 species under the Verbenaceae family, including taxa names along with synonyms, voucher data, accession number, taxa distribution range, habitat, ﬂowering and fruiting period in the study area, meiotic chromosome numbers and their ploidy levels (Table 1). The different morphotypes, cytotypes and morpho/cyto-types reported during the study are described.

Caryopteris odorata (D. Don) Robinson Commonly known as Bluebeard or Blue Mist Shrub, and of 2–3 m height. It is an Asian genus mainly distributed in the subtropical Himalayas up to 1700 m altitude. Presently, 2 accessions have been studied which are actually morphotypes differing on a ﬂower color basis: bluish-purple in Accession 1 and white in Accession 2. The accessions showed n=18 at diakinesis/metaphase I (M–I) (Fig. 1) with equal segregation of 18 : 18 chromosomes at each pole during anaphase I (A–I) in some pollen mother cells (PMCs) (Fig. 2). Two bivalents are bigger in size in comparison with the rest of the bivalents. Meiotic course is abnormal in both morphotypes. Secondary associations are quite common at diakinesis/metaphase I, which lead to the formation of variable number of groups with variable numbers of bivalents in each group; these have been noticed in 36.14% of cells (Fig. 3). Many cells are involved in the transfer of chromatin material through narrow 2–3 cy- tomictic channels at different stages of meiosis. As a result of cytomixis, some cells with polyploid and aneuploid chromosome numbers are also observed (Fig. 4). In addition to this, laggards and bridges are also reported in both accessions (Fig. 5). Pollen fertility is high with almost the same pollen size (Table 2).

Table 2 Data pertaining to meiotic abnormalities in different accessions of C. odorata

Cytomixis PMCs with PMCs with Pollen size laggards at bridges at Pollen Accessions (μm)±Standard Meiotic PMCs Anaphases/ Anaphases/ fertility Range deviation stage involved Telophases Telophases

Accession 1 Diakinesis 32/53 (60.37%) 2–6 4/54 (7.40%) 3/16 (18.75%) 494/504 (98.01%) 33.75–44.62±3.32 Telophase-I 18/49 (36.73%) 2–6

Accession 2 Diakinesis 43/89 (48.31%) 2–6 7/49 (14.28%)12/29 (41.37%) 422/426 (99.06%) 34.12–41.25±0.89

Table 3 Data pertaining to meiotic abnormalities in different accessions of Lantana camara

PMCs showing Total pollen PMCs with PMCs showing unequal PMCs with laggards at mother cells unoriented multipolar Accessions segregation of Pollen fertility (PMCs) bivalents at distribution at chromosomes observed Metaphase I T-II A-I/T-I A-II/T-II at A-I

Accession 1 Number 0 21/49 31/69 39/45 61/78 531/631 %age 0 42.85 44.92 86.66 78.20 84.15

Accession 2 Number 9/52 0 0 19/29 38/59 418/509 %age 17.30 0 0 65.51 64.40 82.12 2012 Cytomorphological Studies̶North Indian̶Verbenaceae 191

Lantana camara Linn. It is a signiﬁcant pantropical weed with 650 varieties (Howard 1969) due to its easy hybridiza- tion properties and is found in more than 60 counteries (Parsons and Cuthbertson 2001). It is native to tropical America and has a high invasion potential that makes it a terrible weed in most coun- tries. The 2 accessions presently investigated are morphotypes based on ﬂower color: orange in Accession 1 and white-pink in Accession 2. Both morphotypes depict n=22 and exist at the tetraploid level on x=11 (Fig. 6). Meiotic course is highly abnormal with the presence of various meiotic abnormalities (Table 3). A large frequency of cells shows irregular behavior in the form of unoriented bivalents at metaphase I, unequal/multipolar segregation and laggards during anaphase/telophase in both morphotypes (Fig. 7). Abnormal meiotic products lead to abnormal microsporogenesis with the formation of monads, dyads, triads, tetrads, polyads with or without micronuclei (Figs. 8, 9; Table 4). Pollen fertility is low with the production of heterogeneous sized pollens. There are 3 pollen size ranges: small (15.00–18.75±1.92 μm), medium (19.5–26.62±2.88 μm) and large (27.37–30.37±3.48 μm) have been noticed in Accession 2, while 20–30 μm size is found in Accession 1. As far as the frequency of pollen size ranges is concerned, 11.19% are small pollens, 13.94% are medium pollens and the remaining ones are large sized. Most of the small sized pollens are sterile.

Verbena bonariensis Linn. Present investigations on accessions from different geographical areas reveal the presence of n=21 in Accession 1 and n=28 in Accession 2, respectively (Figs. 10, 11). These 2 accessions are 2 different cytotypes: hexaploid (2n=6x=42) and octaploid (2n=8x=56) based on x=7. Further, meiotic course is normal in Accession 1, but microsporogenesis is abnormal. On the other hand, octa-

Table 4 Data pertaining to abnormal microsporogenesis in Lantana camara accessions

Total PMCs Dyad Triad Tetrad Polyad Accessions Monad observed M WM M WM M WM M WM

Accession 1 1 1 2 (1) 8 12 (1) 32 23 (1) 3 Pentad 1 Pentad (1) 11 (2) 14 (2) 2 Hexad 1 Hexad (1) 7 (3) 2 (3) 2 Octad

Number: 128 1 1 2 8 30 32 42 9 2

%age 0.78 0.78 1.56 6.25 23.43 25 32.81 7.03 1.56

Accession 2 0 0 0 3 3 (1) 94 22 (1) 4 Pentad 2 Pentad (3) 2 (2) 51 (2) 1 Hexad (2) 1 (3) 19 (3)

Number: 202 0 0 0 3 6 94 92 4 3

%age 0 0 0 1.48 2.97 46.53 45.54 1.98 1.48

* Abbreviations: M=without micronuclei; WM=with micronuclei; Value in parentheses represents the number of micronuclei.

Table 5. Data pertaining to abnormal microsporogenesis in Verbena bonariensis cytotypes

Total number of Dyad with Cytotypes Monad Dyad Triad Tetrad cells observed micronuclei

Hexaploid (2n=42) 30 1 (3.33%) 10 (33.33%) 3 (10%) 4 (13.33%) 12 (40%)

Octaploid (2n=56) 93 3 (3.22%) 76 (81.72%) 5 (5.37%) 0 9 (9.67%) 192 S. Bala and R. C. Gupta Cytologia 77(2)

Table 6 Comparison of some characters of hexaploid and octaploid cytotypes of Verbena bonariensis

Character Hexaploid (2n=42) Octaploid (2n=56)

Stomata size (μm): Upper epidermis 28.12–30.00×16.87–18.75 22.50–26.25×15.00–18.75 Lower epidermis 30.00–30.75×18.75–18.75 33.37–33.75×18.00–20.62 Stomata frequency/mm2: Upper epidermis 15.93 30.33 Lower epidermis 01.53 01.00 Stomatal index: Upper epidermis 32.89 52.22 Lower epidermis 03.59 03.33 Trichome size (μm): Upper epidermis 75.00–225.00×7.5–18.75 48.75–225×5.62–13.12 Lower epidermis 56.25–264.37×7.5–26.25 37.50–450×3.75–37.05 Trichome frequency/mm2: Upper epidermis 7.86 8.75 Lower epidermis 8.08 4.11

Microsporogenesis Abnormal Abnormal Pollen size (μm) 15.25–27.50 15.37–30.00 Pollen fertility 93 94 ploid cytotype show irregular meiotic course with the presence of unoriented bivalents as well as chromatin stickiness, resulting in the formation of groups (2–4) (Fig. 12). In addition to this, the transfer of chromatin material through narrow cytoplasmic channels is observed, which results in the complete transfer of chromatin mass in a few of and the partial transfer of chromatin mass in most of the cells, making a total contribution of 12.34% abnormality in the accession and ﬁnally leading to abnormal microsporogenesis (Figs. 13–15; Table 5). Pollen fertility is slightly low in both accessions (93–94%). Heterogeneous sized pollens are produced in both accessions with 15.00–27.50±5.18 μm size range in Accessions 1 and 2. The types of sizes are: small (15.37– 22.50±3.69 μm) in 22.50% frequency, and large (26.26–30.00±3.51 μm) in a 77.50% frequency in Accession 2. Morphologically, both cytotypes look alike. Observations on size and frequency of trichomes and stomatas are given in Table 6. The frequency of stomatas is almost double in the case of the octaploid population as compared to the hexaploid one. The rest of the observations are the same with little difference.

Verbena officinalis Linn. The common vervain is occasionally grown as an ornamental plant. Vervain is a perennial herb that grows throughout temperate North America and in tropical as well as subtropical regions in North India. Three accessions from different localities are 3 morphotypes that differ only on flower color; pink in Accession 1, light purple in Accession 2 and white in Accession 3. Meiotic studies have revealed that all the accessions exist at diploid level with the presence of n=7 in first 2 accessions and n=6 in Accession 3 (Figs. 16, 17). At metaphase I in Accession 3, quadrivalent (0–1) is noticed in 39.45% of cells (Fig. 18). Further course of meiosis is normal except for Accession 1, where 2–3 chromatin bridges and laggards have been observed during anaphase I in 6.66 and 9.33% of cells, respectively. Pollen fertility is high (97–99%) in Accessions 2 and 3, and relatively low (89%) in Accession 1. Heterogeneous sized pollens are observed in all morphotypes with constant size (31.87–33.75±0.66 μm) in Accessions 2 and 3, and 28.12–33.75±2.75 μm in Accession 1. 2012 Cytomorphological Studies̶North Indian̶Verbenaceae 193

Vitex negundo Linn. The 5-leaved chaste tree is a large aromatic shrub commonly distributed up to 2000 m in the Himalayas. Meiotic studies revealed the presence of n=16 in most of the cells. Some PMCs were observed with 1–2 B-chromosomes (Fig. 19). During anaphase I, these B-chromosomes lag behind and do not reach to the poles (Fig. 20). Microsporogenesis is normal, leading to 100% pollen fertility. Pollens are 15.00 μm in size.

Discussion

Morphological status Two types of variations, as described by Davis and Heywood (1963), are noticed in different populations; individual variation within the same population is seen in Caryopteris odorata (n=18) and collective variation between different populations is noticed in Lantana camara (n=22) and Verbena officinalis (n=6, 7). Morphological variation in Caryopteris odorata seems to be genic because these morphotypes are found within the same environmental conditions, and this variation conﬁrms previous observations made on populations from Garhwal Himalaya and Central India by Bedi et al. (1985). Both genic and environmental factors seem to be responsible for collective variation in both Lantana camara and Verbena officinalis.

Chromosomal status On the basis of cytological studies on 5 species of the family Verbenaceae, the chromosome number varies from 2n=12 in Verbena officinalis to 2n=56 in Verbena bonariensis. The report of n=18 in Caryopteris odorata, is a new chromosome count and does not agree with previous records from different parts of India (n=8, 11 by Bedi 1990), (n=20 by Bedi et al. 1985). Two bivalents are bigger in size which might have led to the report of n=20 by Bedi et al. (1985). The other species, C. foetida, is also known to have small, medium and large sized chromosomes. The occurrence of morphotypes in North Indian populations confirmed earlier observations. The presence of secondary associations among bivalents is reported for the first time in the species. The present chromosome count of n=22 in Lantana camara agrees with previous studies for Lantana camara populations from South Africa, the Caribbean and India, and shows that the species is tetraploid (x=11) and always has multivalents and univalents, (Sen and Sahni 1955, Raghavan and Arora 1960, Spies 1984, Sanders 1987, Ojha and Dayal 1992, Sinha et al. 1995). The present study does not report multivalents in the species but does report considerable amount of aberrant microsporads. Both chromosome counts of n=21 and 28 in Verbena bonariensis are new cytotypes added to the species, in addition to the diploid count (n=6, 7) reported by Kumar and Dutt (1989) from India and the tetraploid count (n=14) reported by Chaw et al. (1986) from Taiwan. The present chromosome count of n=7 in Verbena officinalis confirms the earlier report of Aryavand (1980) from Iran. However the chromosome number of n=6 and 7 both are new to Indian populations. The species is well known to have intraspecific cytotypes of x=7: 2n=14 and 28 from Indian populations and 2n=14, 28, 42 and 70 from outside India (Lewis and Oliver 1961). The report of n=16 in Vitex negundo confirms the previous chromosome count for the species (Sandhu and Mann 1988). Besides this, 2n=24, 26 and 34 are also known for the species. But no one has yet reported the presence of B-chromosomes in the species.

Meiotic abnormalities Multivalents in diploids: Presence of multivalents in diploids attributed to the either the hybrid nature of a taxon or the presence of heterozygosity for chromosomal interchanges. Presently, multivalents have been noticed in Verbena officinalis (n=6), where structural changes might be one of 194 S. Bala and R. C. Gupta Cytologia 77(2) the reasons for the morphological variation as reported by Gill (1979) in the case of Symplocos cra- taegoides. Secondary associations: Such types of associations seem to indicate a basic chromosome number for the taxon. In the present study, the phenomenon is observed in Caryopteris odorata. It is based on the idea that homologous bivalents have a tendency to attract each other (Heilborn 1936) due to more distantly related chromosomes, and this phenomenon is reported in a number of plants: Ocimum spp. (Mukherjee and Datta 2006), Physalis peruviana (Bala and Gupta 2011), Senecio and Gynura spp. (Gupta et al. 2010). In the 2010 Gupta et al. paper, the formation of 5 groups in Senecio nudicaulis due to secondary associations of bivalents clearly indicates x=5 as the original base number which supports the ideas of many researchers. Cytomixis: The phenomenon of the transfer of chromatin material from one PMC to another was named for the first time as ‘cytomixis’ by Gates in 1911. Different researchers gave different opinions as the possible cause of cytomixis, such as the effect of fixation (Haroun 1995); pathologi- cal factors (Brown and Bertke 1974, Omara 1976), physiological changes (Bahl and Tyagi 1988), temperature (Narain 1976) and genetic nifluence (De Mantu and Sharma 1983, Kaul and Nirmala 1991). This abnormality is noticed in the presently investigated accessions of Caryopteris odorata and Verbena bonariensis. The present findings are in accordance with previous observations made by Bedi et al. (1985) where 23.69% of PMCs of the C. odorata population were involved in cytomixis. However, a greater percentage of cells (48–60%) in both accessions is observed to be involved in chromatin transfer. It seems that cytomixis is under genetic control because populations from different localities showed similarly abnormal meiotic behavior. B-Chromosomes: These are genetically inert, extra or accessory chromosomes and are reported in a number of plant species (Jones and Rees 1982). During present investigations, B-chromosomes were observed in Vitex negundo. Previously there have been no such reports in the genus. The presence of high pollen fertility showed the chromosomes had no effect on pollen fertility.

Acknowledgements

The authors are thankful to DRS SAP-II, ASIST Programme and Rajiv Gandhi National Fellowship Scheme under UGC, New Delhi for ﬁnancial assistance.

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