Cytologia 48: 543-550, 1983

Cytological Studies on Some Chlorococcoid Green Algae

U. N. Rai and Y. B.K. Chowdary Departmentof Botany,Banaras Hindu University, Varanasi-221005,India ReceivedSeptember 18, 1981

Cytological studies on chlorococcoid green algae are meagre. The reason for sucha limited work has been the relatively small size of chromosome which appears as small dots during nuclear division, making them inaccessible to light microscope (Rai 1980). Cytological studies made on this group of microorganism have shown only the basic chromosome number. Consequently no firm conclusions on the cytotaxonomy of this order have been achieved. We, therefore, investigated the morphology and cytology of nine chlorococcoid members available locally.

Materials and methods

Description of materials

Chlorococcum infusionum (Schrank) Meneghini

Cells spherical, 6-36ƒÊm in diameter, with a mucilaginous covering of 1.5-7.5ƒÊ

m thickness; chloroplast like a hallow sphere with a notch on one side and with a pyrenoid; reproduction by biciliate swarmers.

Trebouxia humicola (Treboux) West et Fritsch

Subareal, found as green patches in association with some blue-green algae on the bark of Schleichera triguga; cells spherical, 1.5-3.0ƒÊm in diameter, with a thin cell membrane; chloroplast central with lobed margin and a pyrenoid; repro

duction through biciliate zoospores.

Botryococcus braunii Kuetzing

Colonies 12-80ƒÊm in diameter, irregular in shape, free floating, with a muci

laginous envelope of irregular wrinkles or folds, often united into net-like aggregates

by means of long delicate mucilaginous projections from the colonial envelope;

cells 4-64 per colony, arranged in a zig-zag manner, with a solitary, parietal cup

shaped or laminate chloroplast with a pyrenoid, measuring 3.5-6.0ƒÊm broad and

5.5-11.0ƒÊm long; reproduction through mature cells producing 8 autospores per

cell.

Dactylococcus infusionum Naegeli

Cells solitary, attached end to end to form fragile colonies or chains, fusiform

or slightly curved with parietal chloroplast with a pyrenoid, 2-5ƒÊm broad, 6-11 um

long.

Ankistrodesmus spiralis (Turner) Lemmermann

Cells 1.5-4.5ƒÊm broad and 22-42ƒÊm long, acicular with pointed ends, occur ring in the form of bundle (32-74ƒÊm broad, 32-80ƒÊm long) consisting of 2-24

cells, usually twisted spirally around one another in the middle portion, while free 544 U. N. Rai and Y. B. K. Chowdary Cytologia 48

at the ends; chloroplast single, parietal in position, without pyrenoids.

Ankistrodesmus falcatus (Corda) Ralfs

Cells 1.5-3.5ƒÊm wide and 10-36ƒÊm long, mostly single, straight, acicular to

narrowly fusiform, with tapering to acute apices; chloroplast parietal, without any

pyrenoid. Nephrochlamys subsolitaria (G. S. West) Korshikov

Cells 3.5-6ƒÊm broad and 9-13.5ƒÊm long, solitary or 2-4 cells enclosed within

a persistant covering, crescent shaped having more or less rounded ends with one

end slightly broader than the other; chloroplast nearly filling the cell, without any

pyrenoid. Scenedesmus incrassatulus Bohlin

Cells 4.5-12ƒÊm broad, 12-32ƒÊm long, fusiform, with stumpy ends with

apical nodules; coenobia comprising 2-4 cells per coenobium solitary or in the form

of cells arranged in a linear or slightly subalternating series; peripheral cells of coenobium convex on their outer side and with more or less straight inner walls.

Chlorosarcina stigmatica Deason (Deason 1958)

Occurring as cubical packets containing 4-16 cells enclosed in a characteristic

pectic matrix; cells solitary, 4.5-12ƒÊm long and 3-7.5ƒÊm broad, nearly spherical but angular when under natural compression; chloroplast parietal, cup-shaped,

without any pyrenoid; reproduction by the production of 4 autospores.

All the above nine taxa were collected from nature, isolated and grown in modi fi ed Chu-10 medium (Safferman and Morris 1964) under controlled laboratory

conditions. All the algae were morphologically identified as per description given

in Brunthaler (1915), Prescott (1951), Korshikov (1953) and Philipose (1967).

Actively dividing materials of these taxa, from cultures,were fixed in 1: 3 acetic acid

alcohol mixture for about 25 hours. Cytological study was made on these materials

employing Godward's iron-alum acetocarmine squash technique (cf. Godward 1948).

Cytological observations All the nine taxa investigated in the present study are uninucleate to start with and become multinucleate at the time of reporduction. The number of nuclei in most of the taxa vary per cell depending upon the age of material under study (4-8 nuclei in Chlorococcum infusionum; 4 in Trebouxia humicola, 2-8 in Botryo coccus braunii, 2-4 in Dactylococcus infusionum; 4 in Nephrochalmys subsolitaria; 2-4 in Scenedesmus incrassatulus; 2-4 in Chlorosarcina stigmatica). The nuclear divisions are synchronous in colonial forms. Since the investigated taxa resembled in the details of the nuclear division,a general account is given here for all the isolates studied. The various karyological features for all the taxa have been summarized in Table 1.

Interphase nucleus Interphase nucleus is more or less spherical or ovoid in outline and the dia meter varies in different taxa (Table 1). Juvenile cells or colonies invariably ex hibited the interphase nuclei with smaller diameters than those in mature ones. 1983 Cytological Studies on Some Chlorococcoid Green Algae 545

Interphase nucleus takes intense stain and appears homogenous. As a result of this, chromocentres are not discernible in any case.

Nucleolus At interphase the nucleolar outline is not distinct as it did not take intense stain . Nucleolus readily takes intense stain in early prophase and becomes clearly visible. Its diameter was found to vary with the size of cells. Large nucleated forms do have bigger nucleoli. In all the taxa studied it disappeared earlier to late pro phase stage. In none of the materials nucleolar material perisisted beyond prophase.

Prophase At prophase, the nucleus increases about 2 times the size of its interphase one and the nucleoplasm becomes more granular. The granulated chromatin threads which appear in early prophase condense further to give rise to discrete late pro

Table 1. A comparative account of karyological features

phase chromosomes. By this time the nucleolus disintegrates and the nucleolar material and nuclear membrane are not visible.

Metaphsae

Late prophase chromosomes organize themselves into a clear metaphase plate whose diameter is more or less the same or little lesser than that of its prophase nucleus. Metaphase chromosomes are either dot-like or small dot-like. Centro meric positions are not clear in these chromosomes because of their minute sizes.

The chromosome size was found to vary from 0.4 to 1.0ƒÊm (Table 1).

Anaphase is normal; spindles are distinct in unsquashed preparations.

Cytokinesis takes place only when requisite number of nuclei are formed with in a cell. The chromosome numbers determined in the present study for all the taxa have been listed in Table 1 . (see Figs. 1, la, to 10, 10a). The lowest chromo some number reported is n=6 for Dactylococcus infusionum and Nephrochlamys 546 U. N. Rai and Y. B. K. Chowdary Cytologia 48

Figs. 1 and la to 10 and 10a. Cytology of chlorococcoid green algae. 1 and la, metaphase squash preparation and its drawing respectively of Chlorococcum infusionum showing 10 chromo somes. •~3500. 2 and 2a, metaphase squash preparation and its drawing respectively of Trebouxia humicola showing 10 chromosomes. •~3000. 3 and 3a, metaphase squash preparation and its drawing of Botryococcus braunii showing 8 chromosomes. •~4500. 4and 4a, metaphase squash preparation of Dactylococcus infusionum and its drawing showing 6 chromosomes. •~4500. 5 and 5a, metaphase squash preparation and its drawing respectively of Ankistrodesmus spiralis showing 10 chromosomes. •~4000. 6 and 6a, metaphase squash preparation of Ankistrodesmus falcatus 1983 Cytological Studies on Some Chlorococcoid Green Algae 547 subsolitaria, whereas the highest number recorded is n=10 for Chlorococcum in fusionum,Tribouxia humicola and Ankistrodesmus spiralis.

Discussion

The cytological work on chlorococcoid green algae is lagging far behind. The workers who contributed to this field are Timberlake (1902), Yamanouchi (1913),

Smith (1916, 1918), Bristol (1917), Mainix (1927), Reichardt (1927), Techermack

(1942), Proskauer (1952), Palik (1949), Sulek (1969), Sedova (1970) and Chan

(1974). The forms so far studied cytologically by Indian workers are Hydrodictyon

(Sarma 1962, Chowdary 1967, Sinha and Noor 1967, Sahay and Sinha 1971), Kirchneriella and Characium (Chowdary 1967), Characiosiphon (Petal and Francis

1967), Dimorphococcus (Rai and Chowdary 1980), Chlorocytrium (Rai and Chow

dary 1981). Mathew (1976) studied comparative nulcear cytology of nineteen

belonging to eleven genera of Chlorococcales.

All the taxa investigated in this study resemble in their cytological features.

In having a spherical, homogenously stained interphase nuclei without any con

spicuous chromocentres, the members investigated presently agree with the detailed

characters described by Mathew (1976). The heterochromatic bodies reported by

Sarma (1962) and Mathew (1976) in Hydrodictyon and in Gloeotaenium were not

observed in any taxa presently investigated. The diameter of the interphase nuclei

ranged between 1.5-3.5ƒÊm in most of the taxa. However, in Trebouxia humicola,

Dactylococcus infusionum and Chlorosarcina stigmatica, the size was a little more

than 4ƒÊm. All taxa are characterized by having single nucleolus per nucleus.

The size of nuclei ranges between 0.7-2.2ƒÊm at early prophase. The smallest

nucleolus is characteristic of Ankistrodesumus spiralis, while the largest of Chloro

coccum infusionum.

The prophase nuclei in different taxa studied here enlarge about 2 times the

size of their respective interphase nuclei. This is in conformity with the observa

tions made by Mathew (1976) in all the taxa studied by her.

The size of the metaphase plate was always the same or little lesser than the

size of prophase nucleus. Ring-like organization of metaphase chromosomes as

reported by Chan (1974) in a strain-280 of Coelastrum microsporum has not been

found in any member studied herein. Metaphase chromosomes are small dot

like or rod-like, measuring little less than 1.0ƒÊm in length in all the taxa observed.

Owing to small sizes, the chromosomes even at anaphase do not give any clue as

to the position of centromeres in them. They could be either telo or sub-telo

centric.

Among the members of Chlorococcaceae, only one species has been cytologi

and its drawing respectively showing 8 chromosomes. •~3000. 7 and 7a, metaphase squash

preparation and its drawing respectively of Nephrochlamys subsolitaria showing 6 chromoses. •~3500. 8 and 8a , metaphase squash preparation of Scenedesmus increassatuluts and its drawing showing 8 chromosomes . •~3500. 9 and 9a, metaphase squash preparation of Chlorosarcina stigmatica and drawing respectively showing 8 chromosomes. •~3000. 10 and 10a, metaphase

squash preparation of Chlorosarcina stigmatica and their drawing respectively showing 8 chromo

somes. •~ 4500 . 548 U. N. Rai and Y. B. K. Chowdary Cytologia 48 cally investigated so far. Sedova (1970) who studied cytology of Chlorococcum infusionum reported a chromosome number of 10 for this species. The present count of n=10 also confirms the earlier count for this species. Trebouxia humi cola, another investigated in the present study also exhibited a chromosome number of 10. No member of the family Botryococcaceae, other than Botryococcus braunii (present study) seems to have been worked out for its cytological details. This alga agreed with other Chlorococcalean members in its cytological features. The chromosome number determined for this alga is n=8. Among the members of Selenastraceae, Kirchneriella lunaris (Chowdary 1967, Mathew 1976), Actinastrum hantzschii (Sedova 1970), Ankistrodesmusfalcatus, A. convolutus and Selenastrum minutum (Mathew 1976) have been karyologically in vestigated. In the present study, four taxa of this family, Dactylococcus infusionum, Ankistrodesmus spiralis, A. falcatus and Nephrochlamys subsolitaria have been in vestigated and the chromosome numbers determined are 6, 10, 8 and 6, respectively. They agree with the other members of this family in their cytological characteristics. Mathew (1976) has reported 10 chromosomes for Ankistrodesmus falcatus. In the present study, a chromosome count as n=8 made for the same species clearly indicates the existence of cytological races in this species. So far, seven species of the genus Scenedesmus have been worked out cyto logically. Sulek (1969) studied the cytology of S. quadricauda and reported a chro mosome number of 12 for this species. Mathew (1976) in her investigation on six species of Scenedesmus reported chromosome nubmer 6 for S. obliquus, 8 for S. dimorphus and S. bijugatus var. graevenitzii, 4 for S. platydiscus, 10 for S. brasiliensis, 14 for S. quadricauda var. quadrispina. The morphologically distinct S. incrassa tulus agrees with S. dimorphus and S. bijugatus var. graevenitizii in having same chromosome number (n=8). A perusal of known chromosome numbers of the taxa investigated so far presents evidence for the role of aneuploidy to some extent in the speciation of the genus Scenedesmus. No members of the family Chlorosarcinaceae, other than the presently studied Chlorosarcina stigmatica have been karyologically investigated. In the cytological details this alga agrees with the other members of Chlorococcales. The chromo some number n=8 determined for this alga is a new record. However, at this stage, it is premature to draw any conclusion on the speciation and cytotaxonomy of the order Chlorococcales. It may be proposed that to some extent nutritional, physiological and biochemical characters of the organisms may be helpful as taxonomic tool in distinguishing various genera and species.

Summary In the present communication nine species belonging to eight Chlorococcalean genera and one belonging to Chlorosarcinaceae of Chlorosarcinales have been collected locally, isolated, and grown in cultures . All the algae have been sub jected to karyological investigation. Their studies have shown the existence of 1983 Cytological Studies on Some Chlorococcoid Green Algae 549 cytological races in the genus Ankistrodesmus. Based on the chromosome number and karyological features the cytotaxonomic aspects of the algae have been dis cussed. It was concluded that taxonomic treatment on the basis of cytological data is possible only when a large number of species of each genus were under taken and studied together.

Acknowledgements

We wish to acknowledge with gratitude the facilities provided for this work by Prof. E. R. S. Talpasayi, Head and Programme Coordinator, Centre of Advanced Study in Botany, Banaras Hindu University. We are also thankful to the Council of Scientific and Industrial Research, New Delhi for financial support.

References

Bristol, B. M. 1917. On the life history and cytology of Chlorochytrium grande sp. nov. Ann. Bot. 31: 107-126. Brunthaler, J. 1915. Protococcales. In A. Pascher: Die Susswasserflora Deutschlands, Oester reichs and der Schweiz. Hept 5. 2: 52-205. Chan, K. Y. 1974. Comparative nuclear cytology of Coelastrum. Canadian J. of Botany. 52: 2365-2367. Chowdary, Y. B. K. 1967. Cytological observations on some chlorococcoid green algae. Caryo logia 20: 233-238. Deason, T. R. 1958. Three chlorophyceas from Alabama soil. Amer. J. Bot. 46 (8): 572-578. Godward, M. B. E. 1948. The iron alum acetocarmine method for algae. Nature 161: 203. Korshikov, A. A. 1953. Viznacnik prisnovodnich Vodorostej USS-V. Protococcineae. 437 pp. 421 f. kiiv. Mainz, F. 1927. Untersuchungen uber Ernahrung and Zellteilung bei Eremosphaera viridis De Bary. Arch. Protistenk. 57: 1-13. Mathew, T. 1976. Cytological and physiological observations on some chlorococcoid green algae. Ph. D. Thesis (Banaras Hindu University). Palik, P. 1949. Kernteilung bei Hydrodictyon utriculatum Roth Index. Horti. Bot. Univ. Buda pestensis 7: 502-516. Patel, R. J. and Francis, M. A. 1967. Observation on morphology and cytology of Charciosiphon rivularis Iyengar. Phykos 6: 91-94. Philipose, M. T. 1967. Chlorococcales. Indian Council of Agricultural Research, New Delhi. Prescott, G. W. 1951. Algae of Western Great Lakes Area. Cranbook Inst. of Sciences, Bloom fi eld Hills, Mich., 946 p. 136 pl. (Gran. Book Inst. Sci. Bull. 31). Rai, U. N. 1980. Cytological and physiological studies on some common Indian Chlorococcus. Ph. D. Thesis, Banaras Hindu University. - and Chowdary. Y. B. K. 1980. Some observations on the cytology of Dimorphococcus lunatus A. Braun (Chlorococcales). Cell and Chromosome. News Letters 3 (1): 13-14. - and - 1981. Cytology of endophytic green alga Chlorochytrium limnathemum (Cunningham) G. S. West (Chlorococcales). Cell and chromosome News letters 4 (2): 35-37. Reichardt, A. 1927. Beitrage zur Cytologie der Protisten. Arch. Protestenk. 59: 301-338. Safferman, R. S, and Morris, M. E. 1964. Alga] virus isolation. Science 140: 679-680. Sahay, B. N. and Sinha, A. K. 1971. Cytological investigation in Hydrodictyon reticulatum Lagarh. Proc. 58th Ind. Sci. Congr. (Bot. Abst.). Sarma, Y S. R. K. 1962. Some observations on the karyology of Hydrodicyon reticulatum Lagh. and the effect of colchicine on the alga. Caryologia 15 (1): 131-138. Sedova,T. B. 1970. Certain results and prospects of comparative cytological investigation of uni 550 U. N. Rai and Y. B. K. Chowdary Cytologia 48

cellular green algae. Botanicheskii Zhurnal 35: 947-953. Sinha, J. P, and Noor, M. N. 1967. Chromosome numbers in some members of Chlorophyceae of Chota Nagpur (India). Phykos 6: 106-110. Smith, G. M. 1916. Cytological studies in the Protococcales, I. Zoospore formation in Chara cium sieboldii. A. Brown. Ann. Bot. 30: 459-466. - 1918. Cell structure and autospore formation. Tetraedron minimum (A. Br.) Hansj. Ann. Bot. 32: 459-465. Sulek, J. 1969. Nuclear division in Scenedesmus quadricauda. In Hecas. J. et Lhotsky, O. (ed.) Ann. Rep. Algolog. Lab. for 1968. Trebon. pp. 37-44. Timberlake, H. G. 1902. Development and structure of swarmers of Hydrodictyon. Trans. Wisconsin Acad. Sci. 13: 486-522. Yamanouchi, S. 1913. Hydrodictyon africanum. A new species. Bot. Gaz. 55: 74-79.