Regular Article
Variable Chromosome Number and Ploidy Level of Five Phyllanthus Species in Bangladesh
2021 年 86 巻 2 号 p. 143-148
詳細
2021 年 86 巻 2 号 p. 143-148
Medicinally important five Phyllanthus species viz. P. acidus (L.) Skeels, P. emblica L. (small fruit form), P. emblica L. (large fruit form), P. niruri L., P. reticulatus Poir. and P. urinaria L. were cytogenetically studied through orcein-staining for authentic characterization. This genus showed variation in somatic chromosome numbers such as 2n=2x=26 (x=13) in P. acidus, P. niruri and P. reticulatus, 2n=6x=48 (x=8) in P. urinaria and 2n=10x=100 (x=10) in P. emblica (both small and large fruit forms), represented with multi-basic chromosome number. Formation of bivalents in addition to several multivalents at metaphase-I of P. emblica (both forms) indicated that this species has the possibility of being auto-allopolyploid or segmental-allopolyploid that are going through the process of diplodization.
The genus Phyllanthus belonging to the family Euphorbiaceae is distributed in almost all tropical and subtropical countries of Asia, Africa, and America (Bharatiya 1992, Burkill 1996, Abdulla et al. 2010). It has a remarkable diversity of growth forms including annual and perennial herbs, shrubs, trees, climbers, floating aquatics, and succulents (Abdulla et al. 2010). The genus comprises about 1,200 species of which 11 species reported from Bangladesh viz. P. acidus (L.) Skeels, P. boeobatryoides Wall., P. emblica L., P. maderaspatensis L., P. niruri L., P. pendulus Roxb., P. reticulatus Poir., P. roxburghii Muell. -Arg., P. sikkimensis Muell. -Arg., P. urinaria L. and P. virgatus Forst. f. (Ahmed et al. 2008). Among these, five species viz. P. acidus (L.) Skeels, P. emblica L., P. niruri L., P. reticulatus Poir. and P. urinaria L. are most commonly available in Bangladesh and are medicinally used (Abdulla et al. 2010).
However, Phyllanthus is recognized as one of the largest genera which represents such a vegetative and floral diversity that morphometric investigation of this group has always been controversial because the taxonomic data of these plants have not been made systematically. Most of the plants are classified based on external morphological structures such as flowers and fruits. These structures are not always observable on plants because of being seasonal. In contrast, such large genus Phyllanthus has been relatively little studied cytologically. The chromosomal studies of these species are difficult because of having small-sized and sticky chromosomes. Report on multiple basic chromosome numbers, different ploidy levels or cytotypes in the Phyllanthus also created a topic of interest to plant cytogeneticists.
Considering the importance of studying the genetic variability of this genus in the present study, cytogenetical methods will be used for an authentic characterization of Phyllanthus species for a comparative account among five species based on karyomorphological parameters.
Five Phyllanthus species viz. P. acidus (L.) Skeels, P. emblica L. (it included two distinct forms, one bearing small fruits and the other bearing large fruits), P. niruri L., P. reticulatus Poir. and P. urinaria L. of Bangladesh were used as plant materials (five individuals of each species). P. acidus and P. emblica (small fruit form) were collected from the National Botanical Garden of Bangladesh, BRAC (Bangladesh Rural Advancement Committee) Nursery of Bangladesh whereas P. emblica (large fruit form), P. niruri, P. reticulates, and P. urinaria were collected from Botanical Garden, Department of Botany, University of Dhaka. These plant materials were maintained in the Botanical Garden, Department of Botany, University of Dhaka, Bangladesh.
The young healthy roots were cut 0.5 cm away from the tip by a clean blade. The collected root tips (RTs) were pretreated with 2 mM 8-hydroxyquinoline for 20 min and were fixed in 45% acetic acid for 15 min at 4°C. The pretreated RTs were hydrolyzed for 2 min 30 s at 64°C in a mixture of 1 M HCl and 45% acetic acid (2 : 1). Then the hydrolyzed RTs were soaked on a filter paper and taken on a slide. The meristematic region was cut with a fine blade. A drop of 1% aceto-orcein was added to the material and the slide was kept in an acetic acid chamber for 2 h. A cover glass was placed on the material. At first, the materials were tapped gently by a toothpick and then squashed by placing thumbs. The slides were observed under a Micros microscope.
For studying meiosis, the young unopen buds of P. emblica (small fruit form), P. emblica (large fruit form) and P. urinaria were collected from the Botanic Garden, the University of Dhaka, Bangladesh at 9 a.m. and kept in a watch glass with distilled water. The bud was opened by a pair of forceps. Two-three anthers were taken on a slide. A drop of orcein was added to it and kept for 1 min. The anthers were gently tapped with a plastic taper. The debris was discarded. A cover glass was placed carefully on the materials. The slide was wrapped with filter paper and very gently pressed by thumbs. A drop of orcein was added to the corner of the coverslip and observed under a microscope. For studying pollen viability, mature flowers of the selected plants with yellow anthers were collected. One drop of orcein was placed on a glass slide. The mature anthers were touched to the orcein in such a way so that the pollen grains came in contact with orcein. A cover glass was placed on it and observed under a microscope.
The six specimens of Phyllanthus species were found to possess different somatic chromosome numbers and other karyomorphological features (Figs. 1–6, Table 1). Chromosomes (2n=26) were found in the somatic cells of P. acidus. The total length of the chromosome complement was 129.45±1.19 µm. The relative length of chromosome and individual chromosomal length were ranging from 0.84 to 2.10% and 3.86±0.11 to 6.33±0.19 µm, respectively (Fig. 1, Table 1). A pair of satellites (about 0.1 µm) was observed (arrows in Fig. 1). P. emblica (small fruit form) was found to possess 2n=100 chromosomes. The total length of diploid complements in this species was 140.03±2.20 µm. The relative length of chromosomes ranged from 0.71 to 1.64% whereas chromosomal length was ranging from 0.99±0.06 to 2.30±0.12 µm (Fig. 2, Table 1). 2n=100 chromosomes were also observed in P. emblica (large fruit form). The total length of chromosome complements of this species was 149.02±0.88 µm. This length was the largest among the five species. The relative length of the chromosomes was 0.64 to 1.47% and chromosome length from 0.95±0.09 to 2.19±0.16 µm (Fig. 3, Table 1). The somatic cells of P. niruri had 2n=26 chromosomes. The total length of the chromosome complements was 50.26±0.45 µm. This length was the shortest among the five species. The relative length of chromosome and chromosome length were ranging from 2.52 to 5.13% and 1.27±0.15 to 2.58±0.16 µm, respectively (Fig. 4, Table 1). P. reticulatus was found to possess 2n=26 chromosomes. The total length of diploid complements was 107.61±0.29 µm. The relative length of chromosomes ranged from 2.72 to 4.92% whereas chromosome length was ranging from 2.93±0.20 to 5.29±0.33 µm (Fig. 5, Table 1). 2n=48 chromosomes were observed in the somatic cells of P. urinaria. The total length of 2n chromosome complement of this species was 74.98±0.74 µm. The range of relative length of the chromosomes was 1.35 to 3.34% and chromosome length from 1.01±0.08 to 2.51±0.17 µm (Fig. 6, Table 1).
| Species | 2n | Range of chromosomal length (µm) (SD) | Total length of chromosome complements (µm) (SD) | Range of relative length (%) | Average chromosome length (µm) |
|---|---|---|---|---|---|
| P. acidus | 26 | 3.86 (±0.11)–6.33 (±0.19) | 129.45 (±1.19) | 0.84–2.10 | 4.98 |
| P. emblica (small fruit form) | 100 | 0.99 (±0.06)–2.30 (±0.12) | 140.03 (±2.20) | 0.71–1.64 | 1.40 |
| P. emblica (large fruit form) | 100 | 0.95 (±0.09)–2.19 (±0.16) | 149.02 (±0.88) | 0.64–1.47 | 1.49 |
| P. niruri | 26 | 1.27 (±0.15)–2.58 (±0.16) | 50.26 (±0.45) | 2.52–5.13 | 1.93 |
| P. reticulatus | 26 | 2.93 (±0.20)–5.29 (±0.33) | 107.61 (±0.29) | 2.72–4.92 | 4.14 |
| P. urinaria | 48 | 1.01 (±0.08)–2.51 (±0.17) | 74.98 (±0.74) | 1.35–3.34 | 1.56 |
It was observed that, aneuploidy and polyploidy played important role in the evolution of a series of new basic chromosome numbers (x=6, 7, 8, 10, 13, 14, 25) (Table 2), accompanied with the diversification of species within Phyllanthus. According to the previous chromosome number records, 2n=26 and 2n=28 chromosomes were reported for P. acidus (Table 2). The chromosome number of 2n=26 for P. acidus was determined in the present investigation which correlates with the previous report of Raghavan (1959) and indicate basic chromosome number x=13. However, 2n=28 (Thombre 1959) for this species might possess different basic chromosome number x=14.
On the basis of previous literatures (Table 2), P. emblica had different chromosome numbers such as 2n=28, 52, 98, 104, and 98–104. If the basic chromosome number was x=14, P. emblica with 2n=28 and 2n=98 could be regarded as diploid and heptaploid, respectively. In contrast, if the basic chromosome number was x=13, P. emblica with 2n=52 and 2n=104 might be considered as tetraploid and octaploid, respectively. On the other hand, 2n=99–103 (Janaki Ammal and Raghavan 1958) could be originated through different ways such as (i) Secondary modification (both hyper or hypo-aneuploidy) of polyploidy as proposed by Stebbins (1971), (ii) Presence of B-chromosome as found in other species belonging to the Phyllanthus such as 6–10Bs in P. pulcher (Soontornchainaksaeng and Chaiyasut 1999) and 1B in P. parvifolius (Sandhu and Mann 1989) or (iii) Numerical chromosomal aberration in the polyploidy generation because the effect of addition or deletion of few chromosomes could not be lethal in a polyploidy species due to possessing multiple copies of a basic set of chromosomes.
In P. niruri, 2n=26 chromosomes were observed in this research work (Fig. 4). Moreover, different chromosome numbers of this species were reported earlier such as 2n=14, 26, 28, and 36 (Table 2). Therefore, the diploid chromosome number of 2n=26 for P. niruri was determined in the present investigation supported the previous reports of Webster and Ellis (1962) and Chuang et al. (1963). If the basic chromosome number was x=7, species with 2n=14 and 2n=28 might be diploid and tetraploid, respectively. Species with 2n=2x=26 and 2n=6x=36 for this species might possess different basic chromosome number such as x=13 and x=6, respectively. However, according to previous literature and present investigation, x=7 and 13 were more frequent (Table 2).
In the case of the previous chromosome number records (Table 2), 2n=26 and 28 chromosomes were reported for P. reticulatus. The chromosome number of 2n=2x=26 with x=13 for P. reticulatus was determined in the present investigation which supports with the previous reports of (Krishnappa and Reshme 1980, Sarkar and Datta 1980).
In the present study, 2n=6x=48 (hexaploid) with x=8 was observed in P. urinaria. Different chromosome numbers of this species were reported earlier such as 2n=14, 24, 26, 28, 48, 50, and 52 (Table 2). Therefore, the chromosome number of 2n=48 for P. urinaria in the present investigation correlates with the previous report of Bancilhon (1971). However, P. urinaria have been found to be reported multiple basic chromosome number such as x=6, 7, 8, 10, 13, 14 and 25 (Table 2), among these, x=8 and 13 are more frequent than others, showing predominantly retain these two basic number linages. According to Haicour et al. (1994), the variable base number could be originated from the ancestral one i.e., x=13 where x=6, 7, 8, 10 formed by decreasing aneuploidy and x=14, 25 by upward disploidy. Polyploid complex with the presence of hexaploid (2n=6x=48) cytotypes of x=8 and diploid (2x=2x=26) and tetraploid (2n=4x=52) cytotypes of x=13 indicated the existence of two dominant polyploid complexes in nature.
Perry (1943) and Janaki Ammal and Raghavan (1958) hypothesized that the herbaceous species of Phyllanthus, with x=13, have a different basic number from the woody ones, with x=7. According to the present investigation, observations of Perry (1943) and Janaki Ammal and Raghavan (1958) substantiate to be incorrect and seem to have no correlation among basic chromosome number with the plant habit which supports the findings of Webster and Ellis (1962).
Inconstancy in the chromosome complement reported in Phyllanthus has revealed a number of interesting correlations regarding the basic numbers. Therefore, the diversity in basic chromosome number might be explained either to non-disjunction or possible hybridization within different cytotypes among these species or numerical chromosomal aberration in the polyploid generation.
Meiotic behavior and pollen fertilityMeiotic behavior of polyploid species of Phyllanthus varies sharply due to unstable somatic chromosome number such as 2n=100 (small and large fruit forms of P. emblica). During meiosis, some meiotic metaphase-I stage showed all bivalents (Fig. 8) while some metaphase-I stage had both bivalents and multivalent (Figs. 7, 9) in both small and large fruit forms of P. emblica. This feature indicated that chromosomes involved in the formation of multivalent might possess segmental homology and it can be pointed out the possibility of their being auto-allopolyploid or segmental allopolyploid. This cytogenetic observation such as the formation of bivalents and some irregular multivalent indicated the presently examined polyploid species as an interspecific hybrid which should be partly fertile. However, about 98% of pollens were viable in both forms of P. emblica since chromosomes were segregated regularly at anaphase-I during meiosis (Figs. 10, 11). Stebbins (1971) reported diplodization of polyploid in many ancient polyploid species. From the above features, it might be suggested that these species are going through the process of diplodization.
This research is dedicated by the authors to Late Professor Dr. Sheikh Shamimul Alam for his eminent contribution to Cytogenetics Laboratory, University of Dhaka, Bangladesh. The financial support from the University Grants Commission (UGC) of Bangladesh, for carrying out this study is gratefully acknowledged. Sincere thanks are due to Bangladesh Open University, Gazipur, Bangladesh for approving study leave for the first author and special thanks to the Cytogenetics Laboratory, Department of Botany, the University of Dhaka for providing research facilities.
