Karyotype Analysis in Three Morphological Forms of Lasia Spinosa (L.) Thwaites (Araceae)

Karyotype Analysis in Three Morphological Forms of Lasia spinosa (L.) Thwaites (Araceae)

Sharmin Sultana, Rubaiyath Bin Rahman and Sheikh Shamimul Alam*

Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

Received July 24, 2006; accepted September 12, 2006

Summary Three morphological forms based on the leaf character of Lasia spinosa (L.) Thwaites viz. i) sagittate, ii) lamina dissected and iii) a mixed of sagittate and lamina dissected were cytogenet- ically investigated. The sagittate form had 2n27. A minute subtelocentric chromosome was found in the sagittate form. Both lamina dissected and mixed form possessed 2n26 chromosomes. The centromeric formula of sagittate form was 19m7sm1st. It was 9m15sm2st in the lamina dissected form and 14m11sm1t in the mixed form. The total length of 2n chromosome complement was 102.23 mm in the sagittate form, 57.78 mm in the lamina dissected form and 74.46 mm in the mixed form. In orcein-staining, one satellite was found in sagittate form, 2 in the lamina dissected form and 5 in the mixed form. In CMA-staining, 2 big CMA-positive satellites were found in the sagittate form. Two small CMA-positive satellites were found in the lamina dissected form. The mixed form had 1 big and 1 small satellite. Two CMA-positive bands were found in sagittate, 5 in lamina dissected and 3 in mixed form. The percentage of GC-rich region was 2.88 in the sagittate, 11.23 in the lamina dissected and 6.79 in the mixed form. The above features indicated that the mixed form might be a natural hybrid between the sagittate and the lamina dissected form. Each form possessed speciﬁc karyotype which could be applied to elucidate the taxonomic rank of these 3 forms in Lasia spinosa.

Key words CMA banding, Karyotype, Lasia, Araceae.

The genus Lasia Lour belongs to the family Araceae. Unlike the other genus of Araceae, Lasia has only 2 species so far been reported (Hosne Ara 2001). These are i) Lasia spinosa (L.) Thwaites and ii) L. concinna Alderw. Lasia spinosa has wide distribution from India (Sikkim, The Himalayas and Assam), Myanmar, Sri Lanka, Singapore, Malaysia and China where as Lasia concinna is con- fined in Indonesia (Hosne Ara 2001, Hay 1988). Although Lasia has only 2 species, Lasia spinosa was found to possess 4 different forms in Bangladesh which mainly based on the morphology of leaves (Hosne Ara 2001). These are i) sagittate form, the leaves are sagittate i.e. arrow shape, ii) lamina dissected form, the leaves have several dissection on margin, iii) entire lamina margin form, the leaves have entire margin and iv) a mixed form of sagittate and lamina dissected, this form possesses both sagittate and lamina dissected leaves. Hossain and Sharif (1984) were tried to distinguish taxonomically the different forms of Lasia spinosa. However, they could not find any valid reason of these variations and recognized these as “ecophenic variations”. Identification problems were also found in other members of Araceae. For examples, Coloca- sia esculenta has 3 and Xanthosoma violaceum has 2 forms. Taxonomists faced problems to identi- fy these taxa authentically (Hosne Ara 2000). Latter extensive cytological investigation had been carried out in these taxa. The cytological data indicated sharp differences among the different forms of Colocasia esculenta and Xanthosoma violaceum (Deen and Alam 2002, Alam and Deen 2002).

* Corresponding author, e-mail: [email protected] 360 S. Sultana, R. B. Rahman and S. S. Alam Cytologia 71(4)

It is well-known that where the taxonomic parameters are insufficient, karyotype analysis often plays an important role in determining the taxonomic status of a taxon. However, when the different taxa possess same chromosome number and almost similar karyotypic features, it is hard to distinguish between such taxa by conventional karyotype analysis. In such a situation, some modern methods may be used. Staining with fluorochromes (CMA and DAPI) is one of such methods. With the help of fluorescent staining it was possible to characterize different karyotypes. (Schweizer 1976, Alam and Kondo 1995, Alam et al. 1995, Kondo and Hizume 1982, Jessy et al. 2005, Akter and Alam 2005). Therefore the karyotype analysis with both conventional and fluorescent staining might be helpful to distinguish the different forms of Lasia spinosa. In this study, 3 forms of Lasia spinosa viz. i) sagittate form, ii) lamina dissected form and iii) mixed form were investigated cytologically. The aim was to i) compare the karyotypes of 3 forms after staining with orcein and CMA, ii) ascer- tain the status of the mixed form and iii) elucidate whether the different forms of Lasia spinosa could be splited on the basis of differentially stained karyotypes.

Materials and methods The 3 forms of Lasia spinosa viz. sagittate, lamina dissected, and mixed were studied in this investigation. These forms were collected from the greater Sylhet and Mymensingh districts of Bangladesh. The plants were grown and maintained in the Botanic garden, Department of Botany, University of Dhaka, Bangladesh. Healthy roots were collected and pretreated with 2 mM 8-hydroxyquinoline for 2.5 h at room temperature followed by 15 min fixation in 45% acetic acid at 4°C. These were then hydrolyzed in a mixture of 1 N HCl and 45% acetic acid (2 : 1) at 60°C for 7 s. The root tips were stained and squashed in 1% aceto orcein. For fluorescent banding, Alam and Kondo’s (1995) method was used with slight modification. After hydrolyzing and dissecting, the materials were squashed with 45% acetic acid. The cover glasses were removed quickly on dry ice and allowed to air dry for at least 48 h before study. The air-dried slides were first pre-incubated in McIlvaine’s buffer (pH 7.0) for 30 min followed by Distamycin A (0.1 mg/ml) treatment for 10 min. The slides were rinsed mildly in McIlvaine’s buffer supplemented with MgSO4 (5 mM) for 15 min. One drop of CMA (0.1 mg/ml) was added to the materials for 30 min and rinsed with McIlvaine’s buffer with Mg2 for 10 min. Slides were mounted in 50% glycerol and kept at 4°C for overnight before observation. These were observed under Nikon (UFX-IIA) fluorescent microscope with Blue Violet (BV) filter cassette.

Results and discussion Orcein stained karyotype Although the lamina dissected form and the mixed form were found to possess 2n26 chromosomes (Figs. 2, 3), and 2n27 chromosomes were observed in the sagittate form. The extra chromosome in this form was subtelocentric and much smaller than the smallest chromosome (Fig. 1, arrowhead). This chromosome was stable and found in every cell, therefore could not be considered as B-chromosome. Srebniak et al. (2002) found a small chromosome in potato. They have considered this kind of chromosome as mini chromosome. Mini chromosome might be eu- or hetero- chromatic in nature. Sometimes they played vital role in expressing phenotypic characters. Here the function of this mini chromosome was unknown. However, it was a characteristic feature of the karyotype in the sagittate form. The centromeric formula of 19m7sm1st was determined in the sagittate form, 9m15sm2st in the lamina dissected form and 14m11sm1t in the mixed form (Table 1). The 2006 Karyotype Analysis in Lasia 361

Figs. 1–9. Differential staining in three forms of Lasia spinosa. 1) Orcein stained metaphase chromosomes of the sagittate form, 2) Orcein stained metaphase chromosomes of the lamina dissected form, 3) Orcein stained metaphase chromosomes of the mixed form, 4) CMA-stained metaphase chromosomes of the sagittate form, 5) CMA-stained metaphase chromosomes of the lamina dissected form, 6) CMA-stained metaphase chromosomes of the mixed form, 7) CMA-stained karyotype of the sagittate form, 8) CMA-stained karyotype of the lamina dissected form, 9) CMA- stained karyotype of the mixed form. Bar10 mm. mixed form showed an intermediate feature in respect of the number of metacentric and sub-metacentric chromosomes. The 3 forms had metacentric, sub-metacentric, sub-telocentric and telocentric chromosomes in their karyotypes. This data revealed that these forms had asymmetric karyotype. Stebbins (1971) stated that the asymmetric karyotype was advanced character. Thus these 3 forms might be considered as advanced in this regards. 362 S. Sultana, R. B. Rahman and S. S. Alam Cytologia 71(4)

Table 1. Comparative orcein and CMA-karyotype analysis in 3 forms of Lasia spinosa (L.) Thwaites

Total No. of CMA- GC-rich No. of satellites chromatin Orcein positive bands portion (%) Specimens length karyotypes (including (including Orcein CMA (mm) satellites) satellites)

Sagittate form 1 2 102.23 19m7sm1st 4 2.88 Lamina dissected form 2 2 57.78 9m 15sm2st 7 11.23 Mixed form 5 2 74.46 14m11sm1t 5 6.79

mmetacentric chromosome, smsubmetacentric chromosome, stsubtelocentric chromosome, ttelocentric chromosome.

One satellite was found in the sagittate form, 2 in the lamina dissected form and 5 in the mixed form (Figs. 1–3, arrow). The satellite-bearing chromosomes were different in different forms. Therefore, the number and location of satellites were another characteristic feature of the karyotypes in 3 forms. Heteromorphicity in respect of centromeric type and chromosomal length was recorded in pair XI in the sagittate form, pair I, IV, XIII in the lamina dissected form and pair XIII in the mixed form (Figs. 7–9). The probable reason for this heteromorphicity might be due to deletion from the respective chromosomes.

CMA staining Satellites Two big CMA-positive satellites were observed in the sagittate form. These 2 satellite-bearing chromosomes were the members of pair V (Figs. 4, 7, arrow). In lamina dissected form a pair of small satellites was observed in the short arm of pair XIII. One of them was brighter than the other (Figs. 5, 8, arrow). One big CMA-positive satellite was observed in the mixed form. It was a member of pair V (Fig. 9, arrow). The satellite portion and the respective chromosome were ex- actly same to that of the sagittate form (Fig. 7, arrow). A member of pair XIII in the mixed form possessed a terminal satellite (Fig. 9, arrow). This chromosome looked similar to that of pair XIII in the lamina dissected form. Therefore, the mixed form might have a CMA-positive sat-chromosome like the sagittate form and the lamina dissected form. One thing may be noted here that the number of satellite varied in different staining (Figs. 7, 8, 9). The reason was unknown however, the satellite portions might have stain-speciﬁcity. CMA-banding Excluding the CMA-positive satellites, the 3 forms showed some other CMA- positive bands. The sagittate form had 1 CMA-positive band on the long arm of a member in pair XI (Fig. 7, thick arrow). The extra chromosome of this form was ﬂuoresced well (Figs. 4, 7, thin arrow). The lamina dissected form had 5 CMA-positive bands in different chromosomes (Fig. 8, thick arrow). Three CMA-positive bands were found in the mixed form (Figs. 6, 9, arrow). CMA banded regions indicated the positions of GC-rich repeats in the genome. Each form thus possessed a distinct karyotype in respect of CMA-positive band. The percentage of total CMA banded region (including satellite) was 2.88 in the sagittate form, 11.23 in the lamina dissected form and 6.79 in the mixed form (Table 1). It showed that the mixed form possessed an intermediate percentage of CMA- banded regions.

The mixed form, a natural hybrid? A morphological form of Lasia spinosa possessed both sagittate and lamina dissected leaves. On this basis it was called the ‘mixed form’. The so called mixed form showed certain intermediate cytogenetical features of the sagittate and the lamina dissected form. These were: 2006 Karyotype Analysis in Lasia 363

i) The centromeric formulae of the sagittate and lamina dissected form were 19m7sm1st and 9m15sm2st, respectively. It was 14m11sm1t in the mixed form (Table 1). The mixed form possessed almost intermediate feature in respect of centromeric formula. ii) The total length of 2n chromosome complement was 102.23 mm and 57.78 mm in the sagittate and lamina dissected form, respectively. It was 74.46 mm in the mixed form (Table 1). The length of 2n complements in the mixed form was in between the 2 extremes. iii) Two big CMA-positive satellites were found one in each member of pair V in the sagittate form (Fig. 7, arrow). Two small CMA-positive satellites were present in the members of pair XIII in the lamina dissected form. One of them was brighter than the other (Fig. 8, arrow). In the mixed form, 1 big CMA-positive satellite and a small satellite were found (Fig. 9, arrow). The size of satellites and satellite bearing chromosomes were same as those of sagittate and lamina dissected form. iv) The percentage of CMA-banded region was 2.88 in the sagittate form, 11.23 in the lamina dissected form and 6.79 in the mixed form (Table 1). The percentage of CMA-banded region of the mixed form was in between the 2 forms. The above results indicated that the so called ‘mixed form’ might be a natural hybrid between the sagittate and lamina dissected forms, which underwent some minute structural changes in its karyotype.

Taxonomic status Hossain and Sharif (1984) did not ﬁnd any stable taxonomic reason to categorize the morphological variation in Lasia spinosa. They recognized those as ‘ecophenic variations’. However in the present study, marked differences were found in the karyotypes of 3 forms. These were: i) the 2n chromosome number of sagittate form was different from the rest 2, ii) presence of a minichromo- some in only the sagittate form, iii) presence of distinct centromeric formulae, iv) different length in 2n chromosome complement, v) different number of satellites ( both in orcein and CMA), vi) different number of CMA-positive bands and different percentage of CMA-banded region. The above discussion showed that each form had speciﬁc karyotype which could be character- ized by differential staining with orcein and CMA. Therefore, the cytological data may be applied to elucidate the taxonomic status of these 3 forms in Lasia spinosa.

Acknowledgement We are grateful to Dr. M. Akhtaruzzaman, Professor of Botany, University of Dhaka, Bangladesh for constructive criticism and going through the manuscript. Special thanks to Hosne Ara, Senior Scientiﬁc Ofﬁcer, Bangladesh National Herbarium, Chiriakhana Road, Mirpur-1, Dhaka-1216, for providing the research materials and necessary informations about Lasia spinosa.

References

Akter, S. and Alam, Sk. S. 2005. Differential ﬂuorescent baning pattern in three varieties of Cicer arietinum L. (Fabaceae). Cytologia 70: 441–445. Alam, Sk. S. and Kondo, K. 1995. Differential staining with Orcein, Giemsa, CMA and DAPI for comparative chromosome study of 12 species of Australian Drosera (Droseraceae). American J. Bot. 82: 1278–1286. —, Kondo, K. and Hoshi, Y. 1995. Study on diffused centromeric nature of Drosera chromosomes. Cytologia 60: 43–47. — and Deen, S. S. 2002. Karyotype and isozyme analysis in three forms of Colocasia esculenta (Araceae). Bangladesh J. Bot. 31: 95–98. Deen, S. S. and Alam, Sk. S. 2002. Comparative study in two forms of Xanthosoma violaceum (Araceae) through karyotype and isozyme analysis. Bangladesh J. Bot. 31: 45–47. Hay, A. 1988. Lasia concinna Alderw: An Engima in the Kebun Raya at Bogor, Java. Aroideana 11: 4–6. 364 S. Sultana, R. B. Rahman and S. S. Alam Cytologia 71(4)

Hossain, E. and Sharif, M. 1984. A preliminary study on the ecophenic variation in Lasia heterophylla Schott. (Araceae). Bangladesh J. Bot. 13: 167–178. Hosne Ara 2000. Colocasia falax Schott. (Araceae)-A new angiospermic record for Bangladesh. Bangladesh J. Plant Tax- onomy 7: 85–87. — 2001. An annotated checklist of Aroids in Bangladesh. Bangladesh J. Plant Taxonomy 8: 19–34. Jessy, N. S., Begum, R., Khatun, M. and Alam, Sk. S. 2005. Differential ﬂuoresent chromosome banding of four species in Haworthia Duval (Aloaceae). Cytologia 70: 435–440. Kondo, T. and Hizume, M. 1982. Banding for the chromosomes of Cryptomeria japonica D. Don. J. Jpn Forestry Soc. 64: 356–358. Schweizer, D. 1976. Reverse ﬂuorescent chromosome banding with Chromomycin and DAPI. Chromosoma 58: 307–324. Srebniak, M., Rasmussem, O. and Maluszynska, J. 2002. Cytogenetic analysis of an asymmetric potato hybrid. J. Appl. Genet. 43: 19–31. Stebbins, G. L. 1971. Chromosomal Evolution in Higher Plants. Addison-Wesley Pub. Co, USA.