_??_1989 by Cytologia, Tokyo Cytologia 54: 255 -262 , 1989

Nuclear Cytology of Phacus Dujardin

R. N. Prasad1 and B. R. Chaudhary

Centre of Advance Study in Botany, Banaras Hindu University, Varanasi-221005, India

Accepted March 2, 1988

Phacus, a eugleniod flagellate is well-distributed throughout the world , but our contribu tions to the karyology of the are neglegible. Dangeard (1902) was the first to investigate the cytology of Phacus pyrum (Ehrb.) Stein, who recorded n=30-40 chromosomes for the . Later, Leedale (1958) reported n=42 chromosomes for Phacus pusillus Lemmerman. Recent ly, Chaudhary and Prasad (1983) followed the course of nuclear events in four Phacus taxa from

India and ascertained their chromosome numbers during mitosis . As far as the authors are aware there seems to be no work on the genus thereafter . in this communication the present authors, in continuation to their earlier report, present the karyology of 16 local isolates of

Phacus, intending better understanding of cytology and cytotaxonomy of the genus .

Materials and methods

The organisms used in the present study were collected from Varanasi and its environs. The morphological characteristics agreed with those described for respective by Forest

(1954) and Huber-Pestalozzi (1955). Modified Carnoy's fluid i. e. mixture of 1 part of glacial acetic acid saturated with ferric acetate and 3 parts of absolute ethyl alcohol (cf. Cave and Pocock 1951) gave better results. Concentrated algal suspensions achieved through centrifugation were added with few drops of 1% iodine solution to achieve instantaneous killing of organisms. The supernatent was de

canted and the algal material was fixed in the above fixative following recentrifugation. For cytological preparation, a drop of fixed material was taken on a slide, the fixative was allowed to evaporate and then one to two drops of acetocarmine solution (2%) were added to it. Staining and squashing were done as per methods devised by Godward (1948) for algae.

Observations

Karyological features as revealed by light microscopy are as under:

1. Phacus cylindrus Pochmann (Figs. 10A, B) Nucleus spherical, situated in the anterior region of the , 2.5 ƒÊm in diameter; endosome one, spherical, 1.2ƒÊm across; chromosomes small dots or short rods, 0.4 to 0.7ƒÊm long; chromosome number: n=10. 2. Phacus pusillus Lemmermann (Figs. 12A, B) Nucleus spherical in shape, situated slightly in the anterior region of the cell, measuring 2

to 3ƒÊm in diameter; endosome one, spherical, 0.5 to 0.7ƒÊm across; chromosomes small dots measuring 0.5 to 0.83ƒÊm in diameter; chromosome number: n=12. 3. Phacus inflexus (Kisselew) Pochmann (Figs. 13A, B) Nucleus spherical in shape, situated slightly in the posterior region, 3.2 to 3.6ƒÊm in di ameter; endosome one, spherical, measuring 1.3ƒÊm across; chromosomes small, dot-like,

1 Senior Research Assistant, Central Sericultural Research and Training Institute, Research Extension Centre, Sirsi-581 401, North Kanara, Karnataka, India. 256 R. N. Prasad and B. R. Chaudhary Cytologia 54 ranging from 0.4 to 0.8ƒÊm across; chromosome number: n=12.

4. Phacus agilis Skuja (Figs. 8A, B) Nucleus spherical to ovoid, slightly posterior in position, measuring 5ƒÊm in length and 3ƒÊm in diameter; endosome one, subspherical, 0.8ƒÊm across; chromosomes small, dot or

rod-shaped, 0.4 to 1.47ƒÊm long; chromosome number: n=13.

Figs. 1-13. Microphotographs of squashed interphase nuclei and respective outline drawings

of chromosomes. IA, B, Phacus pseudonordstedii n=40+3. 2A, B, P. curvicauda n=35•}2 . 3A, B, P. triqueter n=108•}2. 4A, B, P. acuminatus Strain 'B' n=38. 5A , B, P. acuminatus Strain 'A' n 19. 6A, B, P. indicus n=135•}6. 7A, B, P. pyrum n=50•}3. 8A, B, P. agilis

n=13. 9A, B, P. segretii n=22. 10A, B, P. cylindrus n=10. 11 A, B, P. angulatus n=43•}3 . 12A, B, P. pusillus n=12. 13A. B, P. infexus n=12. 1989 Nuclear Cytology of Phacus Dujardin 257

5a. Phacus acuminatus Stokes Strain 'A' (Figs. 5A, B) Nucleus spherical, situated slightly in the posterior region of the cell, measuring 3.5 to 4.5ƒÊm in diameter; endosome one, spherical, 1.2ƒÊm across; chromosomes small dots or thread-like, 0.6 to 1.9ƒÊm in length; chromosome number: n=19.

5b. Phacus acuminatus Stokes Strain 'B' (Figs. 4A, B) Nucleus spherical, slightly posterior in position and 4.8 to 6.4ƒÊm in diameter; endosome one, spherical and 1.9ƒÊm across; chromosomes small, dot to rod-shaped and 0.57 to 1.7ƒÊm long; chromosome number: n=38. 258 R. N. Prasad and B. R. Chaudhary Cytologia 54

Figs. 14-17. Microphotographs of squashed interphase nuclei and/or respective outline drawings of chromosomes. 14A, B, Phacus pleuronectes n=114•}5. 15A, B, P. brevicaudatus n=40. 16, P. circumftexus n=152±7. 17, P. longicauda n=226•}8. 1989 Nuclear Cytology of Ph acus Dujardin 259

6. Phacus segretii Allorge et Lefevre (Figs . 9A, B) Nucleus spherical to oblong , slightly posterior in position, 6.5 to 7.8ƒÊm in diameter; en dosome one, spherical, measuring 2 .1ƒÊm across; chromosomes small dot to rod-like, 0.37 to 1.67ƒÊm long; chromosome number: n=22 . 7. Phacus curvicauda Swirenko (Figs . 2A, B) Nucleus spherical to oblong, situated slightly in the posterior side of the cell , 4.8 to 6.0ƒÊm i n diameter; endosome one, spherical , 1.5ƒÊm across; chromosomes small dots or short rods, 0.5 to 1.25 ƒÊm long; chromosome number: n=35•}2 . 8. Phacus brevicaudatus (Klebs) Lemmermann (Figs . 15A, B) Nucleus spherical in shape, slightly posterior in position , 6.0 to 8.0ƒÊm in diameter; end osome one, spherical, 2.5ƒÊm across; chromosomes small dots or short rods , 0.4 to 1.34•}m l ong; chromosome number: n=40 . 9. Phacus pseudonordstedii Pochmann (Figs . I A, B)

Nucleus spherical, situated centrally in the cell , 10.4ƒÊm in length and 8.4ƒÊm in diameter; endosome one, spherical, 2.9 to 4.0ƒÊm across; chromosomes small dot to rod-like , 0.35 to 1.34 ƒÊm long; chromosome number: n=40•}3 . 10. Phacus angulatus Pochmann (Figs . I IA, B) Nucleus spherical to oblong, situated in the middle region of the cell , 6 to 8ƒÊm in diameter; endosome one, spherical, 2.9 to 3.1ƒÊm across; chromosomes small , dot to rod-shaped, ranging from 0.46 to 1.67ƒÊm in length; chromosome number: n=43•}3 . 11. Phacus pyrum (Ehrb.) Stein (Figs. 7A, B) Nucleus spherical to oblong, situated in between two paramylon granules in the centre, sometimes slightly posterior in position, measuring 10.0ƒÊm in length and 5.0ƒÊm in diameter; endosome one, spherical to oboid, measuring 1.5 to 1.9ƒÊm across; chromosomes small dot to rod-shaped, 0.4 to 2.0ƒÊm long; chromosome number: n=50•}8. 12. Phacus triqueter (Ehrb.) Dujardin (Figs. 3A, B) Nucleus spherical to oblong, situated centrally in the cell, 10.4ƒÊm in length and 8.4ƒÊm in diameter; endosome one, spherical, 2.9 to 4.0ƒÊm across; chromosomes small, dot to rod like, 0.35 to 1.34ƒÊm long; chromosome number: n=108•}2. 13. Phacus pleuronectes (0. F. M) Dujardin (Figs. 14A, B) Nucleus spherical to oblong, slightly posterior in position, 12ƒÊm in diameter; endosome one, spherical, 4.5ƒÊm across; chromosomes small dots or thread-like, 0.4 to 1.5ƒÊm in length; chromosome number: n=135•}6. 14. Phacus indicus Skvortzov (Figs. 6A, B)

Nucleus spherical to oblong, located centrally in the cell, 8 to 11.5ƒÊm in diameter; en dosome one, spherical, 3.5ƒÊm across; chromosomes small dots or thread-like, ranging from 0.6 to 2.5ƒÊm in length; chromsome number: n=135•}6. 15. Phacus circumflexus Pochmann (Fig. 16) Nucleus spherical, centrally located, 11 to 14ƒÊm in diameter; endosome one, spherical, measuring 4.5ƒÊm across; chromosomes small dot to rod-like, 0.5 to 1.34ƒÊm long; chromo some number: n=152•}7.

16. Phacus longicauda (Ehrb.) Dujardin (Fig. 17)

Nucleus spherical, located centrally just behind the central paramylon granule, 17 to 22.5ƒÊm in diameter; endosome one, spherical, 5 to 6ƒÊm across; chromosomes small dots or thread-like, measuring 0.4 to 2.75ƒÊm in length; chromosome number: n=226•}8.

Discussion

In the genus Phacus, so far, only two species have been worked out cytologically by Dang- Table 1. Karvological features of the taxa of Phacus studied 1989 Nuclear Cytology of Phacus Dujardin 261

eard (1902) and Leedale (1958) , who reported chromosome numbers of n=30-40 for P. pyrum and n=42 for P. pusillus, respectively . The karyological features of the presently investigated taxa of Phacus are summarised in Table 1. The nuclei were spherical, oblong or ovoid and possesssed dot-to rod-or thread-like chromosomes, which varied from 0.35 to 2.75ƒÊm in length. The structure and behaviour of nucleus did not conform to the standard pattern of mitosis met with in typical (cf. Leedale 1966, Chaudhary and Prasad 1983, 1985, 1986, Prasad and Chaudhary 1987).

The chromosome numbers recorded for all the 16 taxa of Phacus are new to science . While

chromosome numbers ascertained for 14 species are first reports , those for the remainder two referred to as above (n=12 for P. pusillus and n=50•}3 for P . pyrum) are new additions to the existing ones (Table 2). The number of chromosomes ranged from n=10 in P. cylindrus to n=226•}8 in P. longicauda, the latter taxon exhibiting the highest chromosome number so far reported for the genus Phacus and also for the whole group of euglenoid flagellates.

Table 2. Taxa of Phacus so far studied karyologically with their chromosome counts

Looking at chromosome numbers it can tentatively be stated that the high chromosome forms are eupoloids of Phacus cylindrus (n=10) wherein aneuploidy occurred at different ploidy levels in due course of time. The chromosome number n=10 determined for Phacus cylindrus

might be tentatively considered as the base number for the genus Phacus. The chromosome numbers reported for Phacus pusillus (n=12) and Phacus pyrum (n=

50•}3), in the present study, differ from earlier counts of Leedale (1958) for Phacus pusillus (n= 42) and of Dangeard (1902) for Phacus pyrum (n=30-40), suggesting thereby the presence of cytological races in these taxa. The present chromosome counts of n=19 and n=38 for

Phacus acuminatus further indicate the existence of cytotypes in one and the same species through polyploidy. Besides, the chromosome count of n=12 both for Phacus pusillus and Phacus inflexus is indicative of the role of chromosomal rearrangements in speciation. 262 R. N. Prasad and B. R. Chaudhary Cytologia 54

Summary

Karyological investigations accomplished on sixteen local isolates of Phacus revealed the structure and pattern of mitotic division of nuclei to be atypical. The chromosome numbers ranged from n=10 in P. cylindrus to n=226•}8 in P. longicauda, representing the widest range of chromosome numbers reported so far for euglenoid flagellates. All the chromosome counts made in this study proved to be new to science. Cases of the existence of cytological races are well-documented. The possible role of chromosomal rearrangements, euploidy and more specifically aneuploidy in speciation of the genus is suggested.

Acknowledgements

The authors are grateful to the Head of the Department of Botany for laboratory facilities and to the authorities of C. S. I. R., New Delhi for financial help.

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