_??_1987 by Cytologia, Tokyo Cytologia 52: 343-356, 1987

Karyomorphological Studies in South Indian

R. Selvaraj Department of Botany, Annamalai University, Annamalainagar -608 002, Tamil Nadu, India

Accepted March 5, 1986

The Rubiaceae is popularly known as Madder family. Rubiaceae consist of 450 genera including 5,500 and is one of the largest families of Dicotyledons. Majority of the taxa of Rubiaceae distributed both in tropical and temperate regions. There are 216 species coming in 45 genera in South India (Gamble 1957). Many workers have studied this family for cytological and cytogenetical investigations as well, but there has been no proper cyto genetical investigation as far as the South Indian taxa concerned. An attempt is made in the present work to understand the cytotaxonomical relationship in the light of our findings. Besides, in some of the taxa under investigation no record of cytological work is available and in others, the present study is for the sake of revision and or confirmation.

Materials and methods

The selected healthy fresh excised root tips were pretreated in 0.002M aqueous solution

of 8-hydroxyquinoline kept at 4•Ž for 3 hours. After thorough washing, the root tips were fi xed in 1:3 acetic alcohol mixture (1:3) for at least three hours or overnight. Then they were squashed following Marimuthu and Subramaniam's (1960) iron alum haematoxylin squash schedule. Important plates were drawn with Abbe's camera lucida and some of them photo

graphed. The chromosome numbers of taxa determined in the present study are listed in Table 1.

Observations

The following categorization of chromosomes has been made, with a view to describe the karyotype and to represent the same by karyotype formulae. This facilitates the understading of interspecific and intergeneric relationships of the taxa analysed. Type: A-Long chromosome (more than 5ƒÊ) with median or nearly median centromere. B-Long chromosome (more than 5ƒÊ) with sub-median centromere.

C-Long chromosome (more than 5ƒÊ) with sub-terminal centromere. D-Medium chromosome (3ƒÊ to 5ƒÊ) with median or nearly median centromere. E-Medium chromosome (3ƒÊ to 5ƒÊ) with sub-median centromere. F-Medium chromosome (3ƒÊ to 5ƒÊ) with sub-terminal centromere.

G-Short chromosome (1ƒÊ to 3ƒÊ) with median or nearly median centromere. H-Short chromosome (1ƒÊ to 3ƒÊ) with sub-median centromere. I-Short chromosome (1ƒÊ to 3ƒÊ) with sub-terminal centromere. J-Very short chromosome (less than 1ƒÊ) with median or nearly median centromere. K-Very short chromosome (less than 1ƒÊ) with sub-median centromere.

L-Very short chromosome (less than 1ƒÊ) with sub-terminal centromere. B•L-B-chromosomes 344 R. Selvaraj Cytologia 52

Table 1. Chromosome numbers of the species investigated (Vide Fedorov 1974) 1987 Karyomorphological Studies in South Indian Rubiaceae 345

Table 1. continued 346 R. Selvaraj Cytologia 52

Table 1. continued

Superscript S-Denotes the presence of satellite The diploid chromosome number of taxa; absolute chromosome length; range; karyotype and special remarks are the main features of observations in the present study listed in Table 2.

Discussion

The chromosome counts of 41 species belonging to 24 genera resolved in the present study are listed in Table 1. They range from 2n=18 to 2n=72. Of the 41 taxa studied, first record of chromosome counts have been made in Oldenlandia wightii, Neanotis indica, Ophiorrhiza pectinata, Mussaenda tomentosa, Knoxia mollis, K. wigh tiana, Pavetta tomentosa, P. indica var. montana, arabica var. San Ramon, Psychotria nudiflora and Luculia gratissima. Deviant chromosome counts have been observed as against the previous reports in Oldenlandia biflora and Serissa foetida. In the rest of the species studied, the present report of chromosome numbers confirms the earlier records of chromosome num bers. The present report of diploid number in Cinchona calisaya var. ledgeriana confirms that of Mendes (1939), in Dentella repens that of Raghavan and Rangaswamy (1941), in Oldenlandia corymbosa that of Lewis (1965), in O. herbacea that of Lewis (1965), in O. umbellata that of Raghavan and Rangaswamy (1941),in O. biflora that of Raghavan and Rangaswamy (1941), in Mussaenda hirsutissima that of Khoshoo and Bhatia (1963), in M. erythrophylla that of Fagerlind (1937), in that of Lewis (1962), in Tarenna asiatica that of Raghavan and Rangaswamy (1941), in Canthium parvii forum that of Raghavan and Rangaswamy (1941), in Ixora coccinea that of Sharma and Chatterji (1960), in I. singaporensis that of Sharma and Chatterji (1960), in I. chinensis that of Sharma and Chatterji (1960), in Pavetta laeta that of Homeyer (1932), in Coffea arabica that of Mendes (1938b) and Krug (1936, 1937), in C. robusta that of Heyn (1936), Orlido and Capinpin (1957), in C. excelsa that of Fagerlind (1937), in Morinda coreia that of Raghavan and Rangaswamy (1941), in Borreria articularis that of Raghavan and Rangaswamy (1941), in B. pusilla that of Raghavan and Rangaswamy (1941), Ruaia cordifolia that of Khoshoo and Bhatia (1963), in Galium rotundifolium that of Khoshoo and Bhatia (1963), in G. asperifolium that of Fagerlind (1937), in Pentas lanceolata that of Lewis (1962), in Guettarda speciosa that of Raghavan and Srinivasan (1941b), in Coprosma lucida that of Homeyer (1935), in C. baueri that of Poucques (1949a), in Rondeletia amoena that of Fagerlind (1937), and in Serissa foetida that of Fagerlind (1937). 1987 Karyomorphological Studies in South Indian Rubiaceae 347

Out of the 41 species studied here, 2n= 18 chromosomes have been observed in Oldenlandia herbacea, 2n=20 chromosomes have been observed in Pentas lanceolata, 2n=22 chromosomes have been observed in the following species like Mussaenda tomentosa, M. hirsutissima, M. erythrophylla, Tarenna asiatica, Ixora coccinea, I. singaporensis, I. chinensis, Pavetta laeta, P. tomentosa, P. indica var. montana, Coffea robusta, Psychotria nudiflora, Morinda coreia, Rubia

Figs. 1-41a. Drawings of mitotic metaphase plates (except. Figs. 32, 35).•~1250. 1, Cinchona calisaya var. ledgeriana (2n=34). 2, Dentella repens (2n=36). 3, Oldenlandia corymbosa (2n= 36). 4, O. herbacea (2n=18). 5, O. umbellata (2n=36). 6, O. wightii (2n=36). 7, O. biflora

(2n=54). 7a, O. biflora (2n=72). 8, Neanotis indica (2n=72). 9, Ophiorrhiza pectinata (2n=44). 10, Mussaenda tomentosa (2n=22). 11, M. hirsutissima (2n=22). 1la, M. hirsutissima (2n=

22). 12, M. erythrophylla (2n=22). 13, Hamelia patens (2n=24). 14, Tarenna asiatica (2n=22). Table 2. Table pertaining the details of the name of the taxa, its absolute chromosome lengths, range, karyotype and remarks of the various species of Rubiaceae studied here 1987 Karyomorphological Studies in South Indian Rubiaceae 349 350 R. Selvaraj Cytologia 52 cordifolia, Galium rotundifolium, Coprosma baueri and Serissa foetida, 2n=24 chromosomes have been observed in Hamelia patens, 2n=34 chromosomes have been observed in Cinchona calisaya var. ledgeriana, 2n=36 chromosomes have been observed in the following species of

Figs. 14a-30. 14a, Tarenna asiatica (2n=22). 15, Knoxia mollis (2n=44). 16, K. wightiana (2n=44). 17, Canthium parviflorum (2n=44). 18, Ixora coccinea (2n=22). 19, IL singaporensis (2n=22). 20, IL chinensis (2n=22). 21, Pavetta laeta (2n=22). 22, P. tomentosa (2n=22). 23, P. indica var. montana (2n=22). 24, Coffea arabica (2n=44). 25, C. arabica var. San Ramon (2n=44). 26, C. robusta (2n=22). 26a, C. robusta (2n=44). 26b, C. robusta (2n=40). 27, C. excelsa (2n=44). 28, Psychotria nudiflora (2n=22). 28a, P. nudiflora (2n=44). 29, Morinda coreia (2n=22). 30. Borreria articularis (2n=56). 1987 Karyomorphological Studies in South Indian Rubiaceae 351

Dentella repens, Oldenlandia corymbosa, O. umbellata and O. wightii, 2n=40 chromosomes have been observed in Rondeletia amoena, 2n=44 chromosomes have been observed in the species of Ophiorrhiza pectinata, Knoxia mollis, K. wightiana, Canthium parviflorum, Coffea arabica,

Figs. 31-41a. 31, Borreria pusilla (2n=56). 32, Rubia cordifolia (2n=22)-prometaphase. 32a, R. cordifolia (2n=22). 33, Galium rotundifolium (2n=22). 34, G. asperifolium (2n=44). 34a, G. asperifolium (2n=66). 35, Pentas lanceolata (2n=20)-prometaphase. 35a, P. lanceolata (2n=20). 36, Guettarda speciosa (2n=44). 37, Coprosma lucida (2n=44). 38, C. baueri (2n=22). 39, Rondeletia amoena (2n=40). 40, Luculia gratissima (2n=44). 41, Serissa foetida (2n=22). 41 a, S. foetida (2n=44). 352 R. Selvaraj Cytologia 52

C. arabica var. San Ramon, C. excelsa, Galium asperifolium, Guettarda speciosa, Coprosma lucida and Luculia gratissima, 2n=54 chromosomes have been observed in Oldenlandia biftora, 2n= 56 chromosomes have been observed in Borreria articularis and in B. pusilla, and 2n=72 chro

Figs. 42-55. Photomicrographs of mitotic metaphase plates. 42, Cinchona calisava var . ledgeriana (2n=34). 43, Ophiorrhiza pectinata (2n=44). 44, Hamelia patens (2n=24) . 45, Ixora chinensis (2n=22). 46, Pavetta laeta (2n=22). 47, Ixora coccinea (2n=22). 48, Mus

saenda erythrophylla (2n=22). 49, Tarenna asiatica (2n=22). 50, Mussaenda tomentosa (2n=22) . 51, Pavetta indica var. montana (2n=22). 52, Neanotis indica (2n=72). 53, Ixora singaporensis

(2n=22). 54, Coffea robusta (2n=22). 55, Borreria pusilla (2n=56). •~1250. 1987 Karyomorphological Studies in South Indian Rubiaceae 353 mosomes have been observed in Neanotis indica. A common survey of the chromosome numbers in Rubiaceae reveals the existence of graded series of haploid numbers, namely, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 30, 32, 33, 34, 35, 36, 41, 42, 43, 44, 48, 50, 51, 52, 54, 66, 77, 91, 94 and 110 (the so-called Carex type and Antirrhinum type of Tischler (1937), suggesting an increase by a few chromosome or by one chromosome). Of these haploid numbers, n=11 and n=22 represented the highest frequency among the taxa so far investigated in the family and in this present investigation the taxa showed the same haploid numbers, n=11 and n=22, representing

Figs. 56-61. Photomicrographs of mitotic prometaphase plates. 56, Cinchona calisaya var.

ledgeriana (2n=34). 57, Ixora coccinea (2n=22). 58, Coffea arabica var. San Ramon (2n=44). 59, Rubia cordifolia (2n=22). 60, Serissa foetida - tetraploid cell (2n=44). 61, Serissa foetida diploid cell (2n=22). •~1250.

the highest frequency (Fig. 62). Thus, of the 41 species including a few varieties studied here, a maximum of 17 species possesses 11 as the haploid number and a maximum of 11 species possesses 22 as the haploid num ber (Fig. 62). It is logical to assume the original primary basic number to be 11 which should have given rise to the derived primary basic number, that is 22. In Rubiaceae, the higheset frequency of haploid chromosome numbers, that is 11 and 22, are nearly equal (vide Fedorov 1974). So it may be inferred that 11 may be the original primary basic number and 22 the derived primary basic number. This fact would perhaps appear Fig. 62. Graph showing frequency distribution of to suggest that the haploid number 11 may haploid chromosome numbers. 354 R. Selvaraj Cytologia 52

be considered as the original primary basic number of this family, from which the other hap loid number, high and low, might have been derived. Increase or decrease in chromosome numbers may be brought about by various karyological mechanisms.

Babcock (1947) and Togby (1943) have reported in Crepis the progressive decrease in chromosome basic number from 6 to 3. In the present study, in the family Rubiaceae, a similar process of chromosomal reduction might have been in operation so that a basic number of n=11 might have got reduced to n=10, n=9 and finally to n=6 by a series of unequal transiocations involving concurrent loss of inert heterochromatin parts of the chromosomes.

The haploid numbers, 6, 9 and 10 represent secondary basic numbers from which primary haploid numbers might have derived through aneuploidy or euploidy. This view is evidenced by statement of Heywood (1967) that in the Kingdom even 14 may be considered as a

polyploid number in as much as that the herbaceous dicotyledons have been reported to show frequency curves of n=7, 8 and 9. As revealed by karyotype analysis of 41 species of Rubiaceae, in the present investigation,

there exists a close correlation between the size and number of somatic chromosomes. For instance, the diploid species of Coffea namely C. robusta has fewer number of somatic chromo

somes (2n=22). But at the same time, the chromosomes are comparatively larger in size (size range 2.8ƒÊm to 5.5ƒÊm). The two taxa of Coffea namely C. arabica and C. arabica var. San Ramon possess more number of somatic chromosome (2n=44). But at the same time the chromosomes are comparatively smaller in size (size ranges from 1.0ƒÊm to 1.8ƒÊm and 0.8ƒÊm to 2.6ƒÊm respectively). Still further, Coffea excelsa possesses combination of 66 and 88 somatic chromosomes as rare occurrences along with usual occurrence of 44 somatic chromosomes. When compared to the cells having 44 chromosomes, the cells whith 66 and 88 chromosomes show comparatively smaller chromosomes. In other words, the more the number of chromo somes the smaller is the size of the chromosomes in Coffea excelsa. So also the size range of the somatic chromosomes of the diploid species of Rubia cordifolia (ranging from 4.0ƒÊm to 8.0ƒÊm) is the maximum size range observed in this study. The same phenomenon may be visualized in other species of Rubiaceae studied. Therefore, there is a colse correlation be tween the size and number of somatic chromosomes among the species of Rubiaceae studied. Different theories have been proposed to account for this decrease in chromosome size

(Bennett and Rees 1967). The concept of the karyotype analysis has been reviewed by many authors in the recent

past (Stebbins 1950, Swanson 1957, Sharma and Sharma 1959). Babcock, Stebbins and Jenkins (1937) found that primitive species of Crepidinae have many chromosomes nearly equal in size with median centromeres and that with advancing evolution, the chromosomes become

unequal in size with subterminal centromeres. In the present study, critical karyotype analyses of as many as 41 species of Rubiaceae have been made. Of these, almost all the taxa studied showed asymmetrical karyotype. It is one of the advanced characters. Excepting Pavetta (P. indica var. montana) all other genera studied here showed typical asymmetry in karyotypes. It is a fact that the family belongs to the advanced category and is further evidenced by their shortest chromosome/longest chromo some ratios, short arm/long arm ratios, their relative lengths, and also by the common occur rence of subterminal kinetochores. This family, therefore, may be considered as one of the most evolved families of angiosperms.

Summary

The chromosome number of 41 species belonging to 24 genera of Rubiaceae from South India has been studied. The chromosome numbers range from 2n=18 to 2n=72 . First record 1987 Karyomorphological Studies in South Indian Rubiaceae 355 of chromosome numbers have been made as many as 11 species and deviant records of chromo some number as against the previous reports have been worked out in 2 species. In the rest of the species studied, the present report of chromosome numbers confirms the previous records. A common survey of the chromosome numbers in Rubiaceae reveals the existence of graded series of haploid numbers from 6 to 110. Of those, haploid numbers n=11 and n=22 repre sented the highest frequency among the taxa studied. Therefore, it may be assumed that the original primary basic number may be 11 and it should have given rise to derived primary basic number 22. A process of chromosomal reduction as observed among the species of Crepis might have been in operation so that the basic number I 1 might have got reduced to n=10, n=9 and finally to n=6 by a series of unequal translocations involving concurrent loss of inert heterochromatin parts of the chromosomes. The other higher haploid numbers above the level of n=11 should have been arisen by means of aneuploidy and euploidy. As revealed by karyotype analyses of 41 taxa of Rubiaceae studied it is clear that, there is a close correlation between the size and the number of somatic chromosomes. Karyotypes in Rubiaceae also show differences in absolute chromosome size indicating changes in nuclear DNA in evolution. No definite trend of either phylogenetic increase or decrease of chromo some size is indicated by the available limited data. In the present study, critical karyotype analyses of as many as 41 species of Rubiaceae showed asymmetrical karyotype. Along side this advanced character, and by the common occurrence of subterminal kinetochores showed that this family may be considered as one of the most highly evolved families of Angiosperms.

Acknowledgements Indeed, it gives me immense pleasure to place on record my deep sense of gratitude to Dr. D. Subramanian, M. Sc., Ph. D., F. B.S, in Botany, Annamalai University, under whose valuable guidance, this investigation was carried out. I extend my sincere thanks to Dr. R. Genesan, B. Sc. (Hons.), M. Sc., Ph. D., Professor and Head of the Department of Botany, for the facil ities which were so kindly offered to me. My thanks are also due to authorities of Annamalai University for their kind help in offering me all facilities and to the UGC, for the award of Financial Assistance to Teachers.

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