C 1999 The Japan Mendel Society Cytologia 64: 229-234, 1999

Cytogenetic Investigation of Some Euphorbiaceae in Thailand

P. Soontornchainaksaeng 1* and K. Chaiyasut 2

1 Department of Plant Science, Faculty of Science, Mahidol University, Rama VI Rd., Payathai, Bangkok, Thailand, 10400 2 Department of Botany, Faculty of Science, Chulalongkorn University, Payathai, Bangkok, Thailand

Accepted March 25, 1999

Summary Cytogenetic investigations of 36 species, 18 genera, Euphorbiaceae in Thailand were

discussed for the first time. The techniques of propionocarmine smear and Feulgen squash has been

developed for obtaining meiotic and mitotic chromosomes. The results revealed that there are the

great diversity of chromosome numbers between and within the genera from 2n=16 to 104. Most species have very small chromosomes ranging from 1.0 to 3.33 ƒÊm. Strophioblachia fimbricalyx

Boerl. is a distinct species which have the largest chromosomes with the length of bivalents about

5.33-8.67 ƒÊm. It was found polyploids in both natural groups and cultivated plants. Croton spp.

(2n=20, x=10) and Jatropha spp. (2n=22, x=11) showed the uniform chromosome number within the genus. The chromosomes of Euphorbia studied are very variable in sizes and numbers ranging

from 2n=16 to 60. New chromosome numbers for 16 species involving 12 genera are reported for

the first time. Phyllanthus emblica L. has 2n=104. It is varied from the previous recorded (2n=28,

98 and 104). B chromosomes were found in P pulcher Wall. ex Muell. Arg. uniquely. This prelimi-

nary study indicates that chromosome number, chromosome size and certain morphological differ-

ences may suggest that the Euphorbia could be broken up into several genera.

Key words Euphorbiaceae, Chromosome number, Meiotic configuration.

The Euphorbiaceae is recognized as one of a large family among the dicot plants. It is belong-

ing to 8,000 species of 300 genera (Perry 1978). Its members grow in varied habitats from tropical to temperate world and exhibit considerable diversity in growth types. The family is characterized

as follows: Trees or shrubs, more rarely herbs, a few twinning and frequently laticiferous; Leaves simple and usually entire margin and generally alternate; Flowers hypogynous, actinomorphic, mostly unisexual; androecium 1-•‡; ovary of 3 carpels, trilocular, with 1 or 2 suspended ovules in

each cell; micropyle directed upwards and outwards and covered with a fleshy outgrowth (carun-

cle); Fruit almost invariably a schizocarp-capsule, splitting into carpels, often elastically (Perry 1978, Ridley 1924). More than 400 species of 80 genera were found in Thailand. Many of them are important as medicinal plants and other economic uses. But the taxonomic data of these plants have

not been made systematically. The further interest of Euphorbiaceae is due to the great diversity of chromosome numbers

(2n=12 to 224) and chromosome sizes both between and within natural groups or cultivated plants. Very few chromosome numbers have been previously recorded. This is the first report of chromo-

some investigation of Euphorbiaceae in Thailand which will be continued by the project of the cy- togenetic study of Euphorbiaceae in Thailand. Results will provide in fundamental data for further researches especially it will be of value in future taxonomic study, plant conservation strategies and

improvement. This genetic diversity study will provide in database and a part of Chromosome Atlas of Plants in Thailand.

* Corresponding author. 230•@ P. Soontornchainaksaeng and K. Chaiyasut Cytologia 64

Materials and methods

Flower buds or immature inflorescences of Euphorbiaceae from natural populations in several sites of Thailand (Table 1) were harvested, fixed in Carnoy's solution and stored under refrigeration in 70% alcohol. Some species were grown from seed, from cutting or were obtained from various sources as small plants. Root tips were pretreated in saturated solution of alpha-bromonaphthalene, fixed in 90% acetic acid and stored under refrigeration in 70% alcohol. Slides were prepared with propionocarmine smear and Feulgen squash techniques (Darlington and La Cour 1966, Sharma and Sharma 1980). Cytogenetic study was made from well spread of chromosomes in various meiotic stages of pollen mother cells and mitotic metaphase of root meristems. Chromosome numbers were determined approximately from 25 cells under the light microscope of Olympus model BHA. The best cells were photographed at 100•~ oil immersion objective.

Voucher specimens were made for all samples and lodged in the department of plant science herbarium at Mahidol University and BKF herbarium in Bangkok Thailand.

Results

Cytogenetic investigations were made according to the collected samples and reported espe- cially identified plants of 36 species, 18 genera. The results are shown in Table 1 and Figs. 1-8.

Following the main taxonomic literatures (Shaw 1972, Smitinand 1980), chromosome num- bers of Jatropha and another genera consisting of one species such as Cladogynos, Hura, Ricinus,

Sampantaea, Strophioblachia and Thyrsanthera which were found in Thailand were determined. Acalypha, Bridelia, Croton, Euphorbia, Excoecaria, Hevea, Mallotus, Manihot, Pedilanthus, Phyl-

lanthus, Sauropus and the rest will be continued investigating. The results revealed that chromo- somes of Euphorbiaceae studied are relatively very small size ranging from 1.0 to 3.33 ƒÊm.

Cladogynos orientalis Zipp. ex Span. and Sauropus androgynos (L.) Merr. are unique species in which found 2n=32 and 2n=52, respectively. Strophioblachia fimbricalyx Boerl. and Euphorbia hirta L. have the same 2n=18 but the former distinctly consists of the largest bivalents about 5.33-

8.67 ƒÊm (Fig. 1). Crown was studied for 4 species showing the uniform of 2n=20. Their basic number was suggested x=10. It differs from that of the previous record x=8 (Darlington and Wylie

1955, Moore 1973). However, it is the same chromosome number as those of Acalypha indica L. and Ricinus communis L., but their chromosome sizes are different. The larger bivalents are found

in A. indica L. (about 4.0 ƒÊm), the smaller bivalents are those of R. communis L. (about 1.0 ƒÊm) and the length of bivalents. of Croton spp. are about 3.33 ,um respectively. Six cultivars of Jatropha are

quite uniform from the standpoint of chromosome number and chromosome morphology. They were found no deviation from 2n = 22 with x =11. Although, their chromosome numbers are similar

to those of Excoecaria cochinchinensis Lour., Mallotus barbatus Muell. Arg. and Sampantaea amentiflora Airy Shaw, but chromosome sizes seem to be smallest. Similar evidences occur in some

species of Euphorbia (E. cotinifolia L., E. heterophylla L., E. leucocephala Lotsy., E. pulcherrima Willd.) and Phyllanthus acidus (L.) Skeels which have 2n=28 and Hevea brasiliensis Muell. Arg.,

Manihot esculenta Crantz. and Pedilanthus tithymaloides Poit which have the same 2n=36 (Table 1). Most of them are different in chromosome size. The genus Euphorbia ,studied is extremely variable in chromosome number between species

ranging from 2n= 16 to 2n=60 with x=6, 7, 8, 9, 10 (Darlington and Wylie 1955). It was found that plants with morphological similarity have the same chromosome numbers and some are differ-

ent in sizes. The first group, are E. cotinifolia L., E. heterophylla L., E. leucocephala Lotsy. and E.

pulcherrima Willd. which have 2n=28 with x=7. Their chromosome sizes are different. The length of bivalents found in E. cotinifolia L. and E. pulcherrima Willd. are about 2.67 ,um but they are 2 ƒÊm and 3.33 ƒÊm for E. leucocephala Lotsy. and E. heterophylla L. respectively. More over, E. het- 1999 Cytogenetic Investigation of Euphorbiaceae in Thailand 231

Table 1. Summary of cytogenetic investigations in 36 species 18 genera, Euphorbiaceae in Thailand 232•@ P. Soontornchainaksaeng and K. Chaiyasut Cytologia 64

Figs. 1-8. 1) First meiotic metaphase chromosomes of Strophioblachia fiimbricaly Boerl. (n=9; 7ringII+2rodII indicated by arrows). They are the largest chromosomes in this study. 2) Diakinesis chro- mosomes of Euphorbia pukherrima Willd. (2n=28; 8II+3IV). Quadrivalents are indicated by arrows and n=nucleolus. 3) First meiotic metaphase chromosomes of Euphorbia cotiMfblia L. (n=14; 1VI+6II+ 10 divided chromosomes). Arrow and arrow heads indicate a chain of six and divided chro- mosomes. 4) First meiotic metaphase chromosomes of Excoecaria cochinchinensis Lour. (n=11; 1VI+IIV+6II). A ring of six chromosomes, a ring of four and a ring bivalent chromosomes are indicat- ed by arrow, long arrow head and short arrow head, respectively. 5) First meiotic metaphase chromo- somes of Euphorbia milii Desmoul. (n=20; 1XXIV+1IV+21I+8I). A chain of 24 chromosomes, a

quadrivalent, and a bivalent are indicated by arrow, long arrow head and short arrow heads, respectively. 6) First meiotic metaphase chromosomes of Hura crepitans L. (n=22; IVIII +4IV + 10II). A chain of 8 chromosomes and a quadrivalent are indicated by arrow and arrow heads. 7) First meiotic metaphase chromosomes of Sauropus androgynos (L.) Merr. (n=26; 1XII+2IV+16II). A chain of 12 chromo- somes and quadrivalents are indicated by arrow and arrow heads. 8) First meiotic metaphase chromo- somes of Phyllanthus emblica L. (n=25 + 7B; 25II+ 7B). B chromosomes are indicated by arrows. Bar represents 10 ƒÊm. 1999 Cytogenetic Investigation of Euphorbiaceae in Thailand 233 erophylla L., E. leucocephala Lotsy. and E. pulcherrima Willd. (Fig. 2) are found 1-3 quadrivalents at diakinesis and first meiotic metaphase. It can be pointed out the possibility of their being auto- or allotetraploid. On the other hand, it was found the chain of six chromosomes at first meiotic metaphase in E. cotinifolia L. (Fig. 3). These chromosomes are smaller than the rest which form normal bivalents. The second group are E. antiquorum L. and E. ligularia Roxb. with 2n= 60. Both of them have chromosomal similarity in number and size. Excoecaria cochinchinensis Lour. showed 5 rod II+ 1 ring II+ 1 ring IV+ 1 ring VI of 2n=22 (Fig. 4). Its chromosomes divide regu- lary in 11 : 11. Moreover, the irregular meiosis was found in E. milii Desmoul. and Hura crepitans L. which are both cultivated as ornamental plants. The earlier has 2n=40 and showed the highest multivalent chain of 24 chromosomes with 1 quadrivalents, 2 bivalents and 8 univalents at the first meiotic metaphase (Fig. 5). Its chromosomes divide in some of 20 : 20 and 19 : 21. On the other hand, Hura crepitans L. has 2n=44. Meiotic chromosomes form bivalents and some irregular mul- tivalents of 4 and 8 chromosomes (Fig. 6). This cytogenetic observation makes both cited species known as interspecific hybrid which are partly fertile. In addition, Sauropus androgynos (L.) Merr. has chromosome number 2n=52 which is different from another species studied. It showed some bivalents and multivalents of 4 and 12 chromosomes at the first meiotic metaphase (Fig. 7). Phyl- lanthus pulcher Wall. ex Muell. Arg. is a distinct species which found 6 to 10 B chromosomes (Fig. 8).

Discussion The diploid and polyploid plants were found in both natural groups and cultivated plants. However some of them are intraspecific or interspecific hybrids which are found as translocation heterozygotes, but sometimes their chromosomes divide regulary during meiosis. Thus the mecha- nism makes them fertile and stable. Some genera such as Jatropha, Croton, etc., have no any ob- servable cytological differences between species. This indicates that many of the taxonomic or mor- phological differences may be due to gene or molecular differences (addition, deletion, ...) rather than change in the whole chromosome complement. Perry (1943) suggested basic number of Eu- phorbia in two systems. The primary system is x=8 and the secondary system are x=6, 7, 9 and 10. This study agrees with Perry (1943) in a comparison of chromosome number in Euphorbia with life duration. A higher percentage of perennials were found among the species with secondary basic number of x=6, 7, 9 and 10 than for those belonging to the primary basic system of x=8. It is the first time to report that B chromosomes were found in an important medicinal plant (Phyllanthus pulcher Wall. ex Muell. Arg., 2n=50+6-10B).Their sizes are very small. B chromo- somes are generally found very small or about half as large as A chromosomes (Horiuchi et al. 1995, Moore 1976). Very few of them can synapse with A chromosomes. Thus it makes them move randomly during meiosis and sometime interferes of chiasmata frequency and plant fertility. It indi- cates that P pulcher Wall. ex Muell. Arg. may have a further change in cytological and morphologi- cal characters. Furthermore, the results point out a cytological analysis relating to plant improvement such as Croton which is very important in medicinal uses. Four species studied have a few chromosomes in a complement of 2n=20 with x=10. Most of them showed regulary meiosis. It has the possibility to double chromosome and/or to construct interspecific hybrid to improve medicinal yeilds. On the other hand, cytological comparison in each subfamily arranging by Shaw (1972), there is a correlation between some species studied. For example Sampantaea amentiflora Airy Shaw, Thyrsanthera suborbicularis Pierre ex Gagnep. and Mallotus barbatus Muell. Arg. are in subfamily Crotoneae. They contain the same 2n=22. Manihot esculenta Crantz. and Hevea brasiliensis Muell. Arg. are in Jatropheae. They have the same 2n= 36. In contrast, it was found highly variable in both Euphorbieae and Euphorbia. In this case, chromosome numbers, chromosome sizes and certain morphological differences, may suggest that the Euphorbia could be broken up into several genera. 234•@ P. Soontornchainaksaeng and K. Chaiyasut Cytologia 64

However this chromosomal analysis was still too little for the suggestion of neither basic num- ber for some genera nor certain phylogenetic relationships and evolution in the family.

Acknowledgements This work was supported by the TRF/BIOTEC Special Program for Biodiversity Research and Training grant BRT 140002. The authors are sincerely thankful to the BRT for financial assistance and also to the taxonomic researchers of the main project of Systematic Study of Family Euphor- biaceae in Thailand for plant taxonomic help.

References

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