6 CHLOROCOCCALES*a

. -~.0, 7~n

o

g

~j

fk 8m

p

SEXUAL REPRODUCTION (OOGAMY)IN THE MICRACTINIACEAE,OOCYSTACEAE, AND DICTYOSPHAERIACEAE

a-e, Golenkilliopsis minutissima (IYENG. ET BALAKR.) COMB. NOV.; a, MALECELL AFTER LIBERATION OF AN ANTHEROZOID; b, FUSION OF THE ANTHERCZCID WITH

THE EGG CELL; C, ZYGOTE ESCAPING OCT OF TilE FEMALE CELL; d-e, ZYGOTESATTACHED TO THE OOGONIAL WALL; d, WITH SMOOTH MEMBRANE; e, WITH A SPINY

WALL; f-m, Dictyosphcerium indicum IYENG. ET RAMANAT., f, PORTION OF A MALECOLONY WITH FULLY FORMED ANTHERIDIAL CELLS; g, DISCHARGE OF THE ANTHERC-ZOIDS FROM AN ANTHERIDIAL CELL; h, PORTION OF A FEMALE COLONY SHOWING PAIRS

OF DISCHARGED EGGS; i, TWO EGGS JUST DISCHARGED FROM THE MOTHER CELL (NOTE,

CONTRACTILE VACUOLES); j, ANTHEROZOIDS SWARMING ROUND AN EGG (PY, PYRE-NOID; n, NUCLEUS); k, ANTHEROZOID ABOUT TO FUSE WITH AN EGG; I, JUST AFTERFUSION OF ANTHEROZOID WITH EGG; m, a FULLY FORMED ZYGOTE; n, q; Oocystaeniumelegans GONZALVES ET MEHRA; n-o, FORMATION AND UBERATION OF ANTHERO-OIDS; p, OOSPHERE SURROUNDED BY ANTHEROZOIDS; q, MATURE ZYGOTE.

FIG. XIII,

(a-e, AFTER IYENGAR & BALAKRISHNAN,1956 (AS Go/enkinia minutusimaIYENG. ET BALAKR.); f-m, AFTER IYENGAR ~ RAM"'NATHAN, 1940; n-q, AFTERGON?;ALVES~ MEHRA, 1959).

REPRODUCTION 27

.t

Sexual Reproduction. (Figs. XII-XIII) In Chlorococcum,the swarmers some-times behave as gametes. The same phenomenon takes place also in Chlorochytriumlemnae, C. limnanthemum and Rhodochytrium. The functioning of swarmers either aszoospores or gametes is considered rather primitive (Fritsch, 1935).

The fusing gametes may be of equal size as in Rhodochytriumor they may be ofequal or unequal size as in Chlorococcumand ChlorlJchytrium. The zygote formed by thefusion of the gametes develops into a new individual. Fusion between similar ordissimilar gametes has also been knowl;l in Trebouxia when the gonidia taken from thelichen body of Parmelia are grown in pure. cultures. In Characiositphon,the biciliategametes are isogamous or anisogamous (Iyengar, 1936, 1954), theptant being diQe"cious. Isogamy may take place between larger gametes as well as between smal.lergametes. In Pediastrum and Hydrodictyon,gametes are produced in large numbers i~the parent cell and they are smaller than the zoospores and isogamous. Unlike thezoospoJes, they are liberated individually through a hole in the parent cell membrane.Hydrodictyonis monoecious and even gametes from the same coenocyte may copulate.The zygospores on germination give rise to four swarmers which develop intopolyhedral cells and these may be easily mistaken for Tetraedron cells (Fritsch,193?, Fig. 50, I, K). These polyhedral cells give rise to swarmers whith combine toform a new net.

Korshikoviellalimnetica (=Characium limneticum)'...d Rhopalosolensaccatus(=Characiumsaccatum)produc~ gametes which are distinctly anisogamous. In the latter, individualswhich are prpbably purely sexual, produce macro- and microgame_tes. In Phyllobium,the branched coenocytic threads swell up at places to form elongate or globoseresting cells or gametangia. These enlarge due to accumulation of the p.rotoplasmiccontents, and, on the leaves of the host th~y' appear as bright green nodal swellings(Fritsch, 1935). The gametangia produce a limited number of macrogametes ora large number of microgametes, both being biciliate. Sexual fusion takes place.between a micro- and macrogamete, the latter engulfing the former completely withthe result that the zygote:shmys only two cilia. .

Oogamous sexu~l reproduction of a fairly high order is known in some membersof-the Micractiniaceae and the Dictyosphaeriaceae._ Ii: was first reported inGolenkiniopsis longispina (=Golenkinia lqngisPina), G. solitaria (=Golenkinia solitaria)-and Micractinium pusillum oy Korsliikov in .1~37, and then in Dictyosphaeriumindicum(Iyengar and Ramanathan, 1940). Recently, it has also been reported in Golenkini...opsis minutissima (=Golenkinia minutissima Iyengar et Balakrishnan) and in OocystaeniumGonzalves et Mehra (Oocystaceae). In these genera, the gametangium is an unmodi-fied vegetative cell. In Golenkiniopsisand Micractiniumthe single egg formed from eachoogonium is retained within the oogonium at the time of fertilization. The anthero-zoid, which is probably formed singly from each male cell, as in G. minutissima or inhirger numbers as in the other species of Golenkiniopsisand Micractinium, is naked or.possesses a cell wall which is discarded just before fertilization. The oospores in.Golenkiniopsislongispina and G. minutissima are also provided with a firm spinous wall,as in Micractinium pusillum. In DictyosPhaeriumindicum, which is dioecious, each cellof a female colony produces two eggs which are discharged outside and the cells of malecolonies give rise to 16-32 naked antherozoids, which swarm round the egg. The

.

28 CHLOROCOCCALES

zygote is smooth-walled. In Oocystaenium,the male cells produce 16-32 antherozoidsand the female cells a single large egg which protrudes through an opening formed bythe rupture of the cell wall of the female cell. The zygote develops a fairly thickcell wall, the middle layer of which is verrucose. It is interesting that in the four generawith oogamous reproduction referred to above, asexual reproduction is azoosporic,DictyosPhaeriumterrestrebeing the only exception.

CYTOLOGY AND LIFE-HISTORY

The Chlorococcales are characterized by the lack of vegetative cell division, afeature shared by another order of the Chlorophyceae, the Siphonales. However,there is one family, the Chlorosphaeraceae (Fritsch; 1935), consisting of some imper-fectly known algae, which resembles the Chlorococcales in all respects except for thevegetative cell division. Fritsch (op. c.) appended it to the Chlorococcales, Lemmermann(1915) included the genera concerned under the Tetrasporales and Herndon (1958)created a new order, the Chlorosphaeraies, for-the same.

Formerly it was believed (Fritsch, 1935) that all the chromatin substance in thenucleus of the Chloropbyceae was restricted to the nucleolus (caryosome nucleus)with little or no chromatin substance-in the outer nucleus. Now, it has been establishedthat the chromatin present in the outer nucleus during the interphases occurs in amasked form which does not take up stains easily. In fact, a resting nucleus with adefinite nuclear reticulum is found_ only very rarely in green algae, including theChlorococcales. '

Even though there is no vegetative reproduction in the Chlorococcales, thedivision of the single nucleus takes place before reproduction. Cytokinesis of uni-nucleate cells is always preceded by a ~itotic division of the nucleus (G. M.' Smith,1950), Details of nuclear division are more or less as in other green algae. The

, chroIIlOsomes are usually short roas or merely small granules and not very large iIinumber, but in Eremosphaera (Fritsch, 1935), the chromosomes are long and looped.Cytokinesis in .the Chlorococcales, as in " other green algaetis usually 'not by the-formation of a cell plate, but by_the furrowing ofthe plasma..membrane mid-way betweenthe cell ends. This linear furrow deepens until it cuts through the cell and divides the .protoplast into two. However, in Eremosphaeracytokinesisprobably takes place by the"formation of a cell plate. " ~

, Two main types' of division 'of tlie~protopl~t to form neW individuals have beenrecognized in the Chlorococcales (Geitler, 1924; also Fritsch, 1935). In the first type(e.g., Tetraedron,Characium,Hydrodictyon,Pediastrum,Coelastrum,and probably Scmedesmus),the number of nuclei in each cell increases without accompanying division of the pro-toplast so that the mature cell is multinucleate. This is followed by ~'simultaneousdivision" of the protoplast by "progressive cleavage" so that a number of uninucleateprotoplasts _are formed (Fig. XIV). These protoplasts, which are angular at first,get rounded to' form reproductory units. In this type of division the pyrenoid doesnot divide, but persists in one of the daughter pro toplasts, only to disintegrate anddisappear sooner or later, new pyrenoids being formed by the daughter cells. In thesecond type of division (e.g., Chlorochytrium,Trebouxia, Chlorella,Dictyosphaerium, etc.)the nucleus divides "successively" and ~.ach division of the nucleus is accompanied.

..

t..

.-,

CYTOLOGY AND LIFE-HISTORY 29

,11'

by a division of the protoplast. The pyrenoid also divides during each division of theprotoplast so that each daughter cell receives a pyre.noid. This type of division takesplace in Characiosiphol1also, where the mature cell has a number of separate proto-

, plasmic units, each with a nucleus and a pyrenoid (Fig. IV, f).However, Fritsch (op.c.) stated that undue emphasis could not be laid on these two

-' types of d.ivision since in some genera both the types ?f division. migh t take \lace. Th~s,~ Characmm and Sorastrum, zoospores are formed eIther by sImultaneous pr successive

. division of the protoplast. In Sorastrum, division of the nucleus in uninucleate cells isfollowed by cytokinesis into uninucleate protoplasts. This is followed by a simultaneousdivision of the two 'protoplasts and these in their turn may divide two or three times insuccession(G. M. Smith, 1950). In Chlorococcum,where successive division had beenconsidered the general rule, some species (e.g., C. multinucleatumStarr) exhibit simul-taneous division of the protoplast by progressive cleavage of the adult multinucleatecell (Fig. XIV, a-f).. In the zoospore formation of Hydrodictyon, blepharoplast granules havo beenobserved (G. M. Smith, op.c.).~ . From studies on the pyren91d of Hydrodictyon, Timberlake. (1902) concluded that

the core of the pyrenoid becomes differentiated into two parts, the outer part becominga starch layer ultimately. According to this theory, cycle after cycle of starch grains~y be formed from the pyrenoid (Fig. XIV w). He disagreed with Klebs that starchcan be of two..kinds, viz., pyrenoid starch and stroma starch. According to B~ld(G. M. Smith, 1950), a somewhat similar process may take place in Chlorococcum.also,

..al!hough he ~ubted genetic continui'ty between-pyrenoid and starch. (see also Bold,1951). Though a number of workers have supported' TimbeTlake's theory, Fritsch(1935) held that it was not easy to conceive of the transformation of the protein matterof the pyrenoid into starch. Further, it would be difficult to explain the production ofstarch in some' green algae without pyrenoids. On the whole, he preferred to c::>IIsiderthe starch sheaths as being formed from outside.

Electron microscopidal studies (Desikachary, 1959) have revealed photosyntheticp!'od\!.cts between. the lamellae of the chloroplasts in non-pyrenoid' algae, therebyconfirming Klebs's (1891) findings which distinguish between pyrenoid starch andstroma starch~ Leyon (1954) also showed presence of chlorophyll in pyrenoids; and,that pyrenoids are permanent organelles which produce starch intensely but still retaintheir identity. ' ,

As in most Chlorophyceae, the majority of the Chloro<;occaies are haploid, thediploid condition being confined to the zygote only (Fig. XV). During germination,the zygote undergoes two successive divisions (Fig. XV d), the first being a reductiondivision, and the resulting fOUI cells develop into zoospores or aplanospores. Themeiosis is ~'zygotic" or "initial" (Bold, 1951). However, in a few Chlorococcales likeChlorochytriumlemnae(Fig. XVI), Apiococcusand probably Phyllobium(Fritsch, 1935)the first two divisions at the time of swarmer formation in the adult individual are.

meiotic so that the longest stage in their life-histories is a diploid one. In this respect,Chlorochytriumlemnaeand Apiococcusresemble the Siphonalesand the Bacillariophyceae(Fritsch, 1935). In these instances the zygote germinates directly and the meiosis is"gametic" (Bold, 1951).

30

a

~<~. g.;~~1!-~

. '~;:'r:~~

aI

CHLOROCOCCALES...:....

:.:~:::,\:;~":~}./}..::::~

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.:~.;,':.' ';':':::1\'".:". . ~&..;

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n

C I d I

~FIG. XIV. INCREASE IN NUMBER OF NUCLEI, CELL DIVISION, AND

STRUCTURE OF PYRENOID

b

n

v

bl

a-f, Chlorococcum multinucleatum STARR; a, YOUNG'. VEG. CELi. MEDIANOPTICAL SECTION; b-d, CELLS OF VARIOUS SIZES SHOWING INCREASE IN"NUMBER OFNUCLEI; e, CELL JUST BEFORE CLEAVAGE OF PRC.TOPLAST; f, q.EAVAGE;g, Spongiochloris excentrica STARR, COMPLETION CF CLEAVAGE BEFCRE ZOOSPOROGE-NESIS; h-I, Scenedesmus quadricauda (TURP.) BRE'B.; h-k, FOUR SUCCESSIVE S.TAGES INTHE FORMATION OF A DAUGHTER COENOBIUM; 1,MATURE COENOBIUld; p, PYRENOID;

n, NUCLEUS; m-o, Tetraedron minimum (~. BR.) HANsG.; m-n, CLEAVAGE OP THE

z

e I

~

~

4

.?~

8:+

ECOLOGY A:-iD PHYSIOLOGY 31

ECOLOGY A1~D PHYSIOLOGY

'.

The majority of the Chlorococcales are free living and planktonic, mostly in shal-low confined waters. Some are attached, endophytic, endozoic, parasitic or symbiotic,whereas a few are found in moist soil or on tree trunks. A few species are also found inbrackish water or the sea. Yet others form the constituents of snow floras. Some

species can also ~dapt themselves to the high temperatures obtaining in' hot springs.Quite a number of species are also easily grown in cultures.

Planktonic forms. A large number of the Chlorococcales, both unicellularand colonial, are planktonic, occurring mostly in ponds,. tanks, swamps, small lakes,reservoirs and in rivers with standing water; typical examples being the unicellulargenera, like Schroederia,Golenkinia, Tetraedron, Treubaria and Chodatella,and the colonialgenera, Micractin£um,Planktosphaeria,most species of Oocystis and Nephrocytium, most ofthe Selenastraceae, the Dictyosphaeriaceae, the Hydrodictyoideae; the Coelastraceae,the Scenedesmaceae, the Botryococcaceae and the Radiococcaceae. Most of these formsare adapted for a planktonic or pelagic habit (Fig. XVII, XVIII). Ih OocystisandNephrocytium the gelatinized parent memorane, and in - Radiococcus, -Kirchneriella,Gloeoactinium;DictyosPhaeriumand Tetrallal!tos the outer mucilaginous envelopes givethe necessary buoyancy to facilitate a pelagic habit. The flat disc-like or quadratecolonies of Pediastrum, Scenedesmusand Crucigenia, the irregular colonies of Dimorpho-coccus, Westella, Selenastrum and Ankistrodesmus, the star-shaped colonies of Adinastrumand the hollow net-like colonies of Coelastrum proboscideumall appear to serve thesame purpose. ~n Pediastrum-and Scenedesmusgelatinized bristles are occasion,!Jly found(Petersen, 1912, '1921) at the apices of the cells. These auginent the capacity forfloating. Some like Golenkinia, Micractinium, Polyedriopsis, Treubaria, Chodatella,Francezaand some species of Tetraedronand Scenedesmushave long spines, bristles or setae.Sometimes, as in some species of Treubaria, the setae are even gelatinous.

It is generally known that" the important genera of the Chlorococcales usuallyoccur during the Warm months of the year, though high summer temperatures are asunsuital.?le as l.ower ones" {Fritsch and Rich, 1913), and th~t they constitute one o~ theimportant components of the microflora of warm tropical waters. However, h~rdly anypublished data is available on the ecology of the Indian planktonic forms of Chlorococ-cales, 1)le only accounts available being in connection with taxonomical and morpho-

. logical.studies.. During 1937-40 the author (1940) studied. the ecology of the 'algae ofa pond at Madras, a number of which belonged to the Chlorococcales. Subsequently,a number of shallow waters in north-east and south India were studied for short or long

,..

~

-+ MULTINUCLEATE PROTOPLAST; 0, YOUNG AUTOSPORES WITHiN THE PARENT CELL;P-U, Pedias/rum boryanum(TURP.) MENE'GH.; p, YOUNG CELL \\lTH A SIKGLE Nt:CLEUS; q-s; COEKCCYTES \\lTH 2-16 NUCLEI;t, MULTINUCLEATE PROT<1I'LASTS FORMED BY FIRST CLEAVAGE FURRC.WS; U, COMPLE.TION OF CLEAVAGEINTO UNINUCLEATE PROTOPLASTS. NOTE THE SINGLE DISINTEGRATING PYRENOID; v, PROTOP4STSROUNDING UP. NOTE THE NUCLEUS AND NEW PYRENOID IN EACH PROTOPLASMIC BIT; y-d, Chlorococcumechinoz.ygo/um STARR, STAGES IN GERMINATION OF THE ZYGOTE FROM STAINED PREPARATIONS, SHOWIKGNUCLEAR DETAILS; W-X, OPTICAL SECTIONS OF TWO PYRENOIDS WITH ASSOCIATED STARCH GRAINS(s, STARCH; P, PYRENOID) IN Hydrodictyon re/icula/um (I..) LAGERH.

(a-g FROM STARR, 1955; h-1, m-o, AFTER G. M. SMITH; p-v, FROM G. M. SMITH,; y-ei,AFTER STARR, 1955; w-x, FROM TIMBERLAKE).

,"I

I

,

.-

FIG. XV. l.IFE-CYCLE OF Hydrodictyol/ reliClt/olulII (1..) LAGERHEIM

a-b, GAMETES;c-ci, ZYGOTE;d, FOUR SWARMERS FROM ZYGOTE;e, POLYHEDRON STAGE;f, NET FORMATION WITHIN POLYHEDRON;g, SWARMER FORMATION WITHIN SAME;.h, SMALL PORTION OF YOUNG COLONY (FROM TILDEN, 1935).

FIG. XVI. LIFE-CYCLE OF Chiorochylrillll/ [el1ll10e COliN

a, ZYGOTE: b, PENETRATION OF ZYGOTE (s) INTO TilE HOST;

C, RESTING CELLS (r) IN LEAF OF £ell/I/O; d, suOWING STRUCTURE OF RESTINGCELLS; e-g, DIV1SION STAGES OF SAME; h, LIBERATION OF SWARMERS (g') Ir-:TOVESICLE (b), m, OUTLINE OF MEMBRANE OF RESTING CELL; i, SWARMERS.

(d, AFTER BRISTOL; i, REDRAWN FROM KLEBS; THE REST AFTERKLEBS).

33£COLOGY AND PHYSIOLOGY32 CHLOItOCOCCAL'£S

M

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34 CHLOROCOCCALES

r ~I~~jI~~iI!B1~ J, '.

g

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~~ ~~~\q~J(1@Ji

. ..~!.~~..:<;S:'? 9.~'P.7,./.:->-.,." :~{".: \:...

~~l._-'~~:::::~6 A~ _ ).~.. '~9'-''''7i )1 <.it;'t'~( I~<J~.?

19:.~( \::.~:.j! >/Qf)",::~~"-~r11\'. . .,~.\::.~:-~~ i>£:::_"1:~," .~ ' - ..~ ~.,.. \'IJ ~.. '\\ ./.....~~C~'T \". M'\ifl'1:~ j~~~~) I.~~.. ~&~~/~::::J 'i!-....

FIG. XVII. ADAPTATIONS FOR PLANKTONIC HABIT

a, Tetraedronlimneticum BORGE.; b, ScenedesmUJ quadrieaudaVAR. longispina(CH~D.) G. M. SMITH; c, RadioeoeeUJ nimbatus (DE WILD.) SCHMIDLE;.d, Eehinosphaerella limnetiea G. M. SMITH;e, Seenedesmlls aeuminatUJI (LAGERH.)

CHODAT (SHOWING MUCILAGE BRISTLES); r, Franeeia ovalis (FRANCE) LEMM.;g, Crueigenia tetrapedia (KIRCHN.) WEST; h, Marthea tetras PASCHER;i, Treubariablanetoniea (G. M. SMITH) KORSH.; j, Pediastrum simplex VAR. duodenarium(BAILEY) RABENH. (SHOWING MUCILAGEBRISTLES); k, Micractinium pUJillum}IRES.; I, PeetodiclYon eubicum TAFT. . -+

ECOLOGY AND PHYSIOLOGY 35

<"1')..-

intervals with special reference to the Chlorococcales (Philipose, 1959, and unpub!.).Gonzalves and Joshi (1946) and V. P. Singh (1959) have also made some observationson the ecology of some planktonic forms.

(A) Types of waters predom.inated. Generally most of the planktonicChlorococcales are observed in shallow ponds and tanks, 30 em. to It m deep.Swamps with about 30 cm to a metre (or more) of water, paddy fields, and shallowfisherybunqhs used for the semi-natural spawning of carps also appear to be favouritehaunts .of the Chlorococcales, a large number of them being occasionally noted in asingle collection. Other bodies of waters in which they occasionally predominate areshallow rainwater ditches and pools, rock pools, small lakes, reservoirs and rivers inwhich the water remains almost static from December to May. Concrete cisterns usedfor experimental purposes also very often developed large number of Chlorococcales.Moats and tanks with decaying vegetation also sometimes provide a peculiar type ofChlorococcalean flora. W. & G. S. West (1902, 1907) have reported a number ofChlorococcales from paddy fields, swamps, tanks, streams and artificial ponds in Ceylonand Burma. Lemmermann (1907) and Holsinger (1955) recorded a few from theColombo Lakes, and Cro\v (1923) observed a number of.th~m in rock pools, tanks andlakes. Carter (1926) collected them in large numbers from ditches connected withpaddy fields and from streams in N.E. India. Handa (1927) and Skuja (1949) record-ed quite a nu.mber of Chlorococcales from ponds and lakes of Burma.

Planktonic Chlorococcales are us~ally rare in brackish and salt water. However,Moens (1958) has reported that a number of species like Ankistrodesmits Jalcatus,Coelastrum microporum, Cr..ucigenia emarg~nata, DictyosPhaerium ehrenbergianum; Kirchrzeriella

. lunaris, Oocystisgiga$, O. submarina;Pediastrumboryanum,P. duplex,P. integrum, Scenedem,usobliquus, S. quadrfcaudaand Tetrastrum multisetum could tolerate varying degrees of sali-nity obtaining in the North Sea Canal, which he investigated. Apart from the occa-sional presence of species of Ankistrodesmus, -Crucigenia, Dictyosphaerium, Oocystis,Pediastrum,Scenedesmus,Selenastrum, Tetrastrum and Telraedronin tidal rivers (Iyengar andVenkataraman, 1951; Dutta et al., 1954), no really.brackish water 'species have been re-ported from the Indian r_egio.n. Among the very few marine Chlorococcales kn(\wn,Chlorellaalone has been recorded from the Indian region (Svedelius, 1907).

(B) Species obse~ed in bloom.s. Though plankton collections from the--shallow waters in India referred to above invariably contained one or more of theChlorococcales, usually none of them occurred in very large numbers to constitute analmost exclusive bloom. However, some species like -Cloenkinia radiata, Micractinium

.pusillum, Tetraedron minimum, Selenastrum gracile, Pediastrum simplex val'. duodenarium,P. duPlex val's. clathratum, reticulatum and gracillimum, Botryococcus spp., Coelastrummicroporum,Scenedesmusbijugatus,S. quadricaudaval's. quadrispina,longispinaand westii wereoccasionally found in such large numbers that they imparted a distinct green colour tothe water. Others, though not so abundant, still occurred in fairly large numbers insome waters, often mixed with other algae. Instances of these were Lagerheimia

F.M

..

'.

-+ (a-b, ORIGINAL;c, AFTERDE WILDEMAN;d-k, FROMG. M, SMITH, 1950;e-j, AFTER PETERSEN (e, AS Pediastrum clathratllm (SCHROEDERLEMM.); r,AFTERFRANCE; g, AFTERSCHMIDLE;i, AFTERG. M. SMITH, 1923 (AS Borgeaplantoniea G. M. SMITH); I, FROMTAFT).

36 CHLOROCOCCALES

'/0

"

,

, FIG. XVIII. ADAPTATIONS FOR PLANKTONIC HABIT

a, Fraru:tia drotsehtri (LEMM.) G. M. SMITH SHOWING MUCILAGINOUS SHEATHS0' BRlSTUSj b, Tetrachlortlla ornata KORSH.; d, Micraetinium apptndiculatum ,KORSH.;C, DtSIII/llraclflm dtlicalissimum KORSH.; e-d, indutum (GEITLER) PASCHER; f, Botryo-sphDer/l'slIdlllea (LEMM.) CHODAT; g-h, Siderocystris fusea KORSH., SHOWING AUTO-SPORB 'ORMATION. NOTE THE MUCILAGINOUS ENVELOPES IN b,c, e, f.ALL AFTER KOR'lHIKOV, 1953).

...

ECOLOGY AND PHYSIOLOGY 37

:t

chodati, DictyosPhaeriumpulchellum, Pediastrum araneosum var. rugulosum, P. duPlex vars.coronatumand subgranulatum, P. muticum var. longicorne,Coelastrumreticulatum, C. scabrum,Crucigeniafinestrata, Hofmania lauterbornei, Scenedesmusopoliensis and S. perforatus.

(C) Seasons of abundance. The majority of the Chlorococcales observedby the author occurred during the sultry south-west monsoon period (June toSeptember), when the mean air temperatures usually ranged from 26'50 to 29'5°C(Table I), the monthly sunshine hours were lowest (120-180) and there was a rain-fall o~ ~bout 100-175 em. Some of these algae also showed a secondary maximumduring summer (March-May), when the mean air temperatures ranged from 26'5° to32' 5°C, the sunshine hours from 220-310, and the rain fall from 7' 5 to 30 em, rarelymore. There were, however, a few species which occurred in abundance during thecool months, particularly from November to February, when the mean air tempera-ture usually ranged from 20° to 25' 5°C., the sunshi~e from 120-230 and the rainfallfrom 12' 5 to 62' 5 em.

Sometimes; the altitude made a difference in the seasons of occurrence of some of

the species. Thus, in hill stations like Coonoor and Ootacamund (2,000-2,350 m),where the average temperatures obtaining in s,ummer were less than what they werein the plains during winter, a few species which occurred in abunaance in the plainsduring winter attained their maxima only il\ summer.

The Chlorococcales which were usually predominant during the south-west mon-soon months of Jl!ne to September were Tetraedron minimum, Lagerheimia. chodati,P~hycladon .umbrinus, Pediastum simPlex var. duodenarium, Codastrum microporum,DictyosPkaerium'pulckellum, Scenedesmusarmatus var. bjcaudatus, & quadricauda var.longisPinaand, rarely; S. opoliensis and S. tropicus. A number of others like .Vlicractiniumpusillum, Schroederia indica sp" nov., Actinastrum hantzschii, Pediastrum duPlex vars.reticulatumand gracillimum,Coelastrumscabrum,C. proboscideum,Crucigenia rectangularis,C. aPiculata, C" tetrapedia, Westella botryoides,Scenedesmusdimorphus and- S.' denticulatus,though not very abundant, 'very often became dominant during this period.

-A number of the algae mentioned above sometimes reappeared in fairly largenumbers 'in early or late' summer. Some like Pediastrum simplex var._ duodenarium,DictyosPhaeriumpulchellum and Micractiniumpusjilum be~ame even abundant occasionally.A few others like Sorastrum spinulosum,Pediastrum tetras; Coelastrum,cambricum'var. inter-

m!dium, Crucigenia fenestrata, Hofmania laute~borneiand Scenedesmus quadricauda var.,longisPina were 'also found dominant in sOIJ..lewater~ during summer. Of these,Sorastrumwas rarely observed. during other seasons.

The species which appeared to become dominant during the cool (and usuallydry) months of October to February were Kirchneriellalunaris, NePhrocytiumagardhianum,'N. lunatum, DimorPhococcuslunatus, Dictyosphaerium ehrenbergianum, Selenaslrum gracile,Pediastrumduplexvars. coronatum,reticulatum,clathratum, and gracillimum, P. araneosumvar.rugulosum, P. muticum var. longicorne,Coelastrumreticulatum and Botryococcusprotuberalls.Scen.edesmusaabundans. Some of these species like Kirchneriellalunaris and Dictyosphaeriumehrenbergianumwere observed in large numbers in waters at high' altitudes duringSummer instead of winter. .:;;'

(D) Physico-chemical features of the water and the Chlorococcales.

Apart from the differences in temperature, sunligl}t, rainfall and water level brou~nt

'"

oof

38 CHLOROCOCCALES

about by seasonal differences, the physico-chemical features of the water appeared tohave a great influence on the relative frequency and composition of the Chlorococca-lean flora. The physico-chemical features studied were, the water temperature,turbidity, pH value, total alkalinity and the NIP ratio, the nitrate nitrogen alone beingtaken into consideration.

The physico-chemical features of the waters in which the Chlorococcalespredominated were by no means uniform. Thus, 12 bodies of water from differentparts of north-east and south India (Table-II), in which the Chlorococcales were do-minant, showed great divergence in the ,relative frequencies and composition of theflora and in the physico-chemical features. The maximum number (53 species) ofChlorococcales in a single collection was obtained from a swamp in Puri district and thenext maximum (45 species) from nursery pond No. 38 at Cuttack. The shallow waterof the former was neutral and with very low total alkalinity (32), NIP ratio was lessthan unity (0' 52) and the period of collection was April, when the water was verywarm. In the latter, the water was more or' less alkaline (ph-8'4, T.A.-86), the NIPratio was less than unity (0' 06) and the period of collection was August, when the water

temperature was moderately high. 'The remaining waters (alkaline, neutral or slightlyacidic) supported ~arying numbers of Chlorococcales ranging from 13-21 species andvarieties, and the difference in the flora appearecl to be mainly due to the varyingphysico-.chemical features, Thus, in a series of nursery ponds situated side by side atCuttack' (only five of these are referred to in Table II) those with moderately low al-kalinity (36-78) showed a flora somewhat different from those of ponds with higheralkalinity (80-140). -No doubt there was a certain measure of'overl'!Pping in the t:aseof some' species, indicating a certain degree of toleranc~ to varying physico-chemicalfeatures by these species. Dyke's tank, Visakhapatnam, with a high concentrationof nitrates (10 ppm), high NIP ratio (77'9) and fairly high a~kalinity (135 ppm)showed mostly ScenedesmUsspp. On the other hand, the difference in flora betweenthe swamp in Puri and the bundh in Chandrakona Road; Midnapore, in which thephysico-chemical features were somewhat similar, was apparently due to the seasonal

_ factor alone, the collection from the swamp being taken in summer and the collection,I ' -. from the bundh in winter. "

By comparing the physico-chemical features--of'waters from different parts ofIndia in which one and the same species of Chlorococcales predominated (Table III),

it has been possible to arrive at certain tentative correlations between some speciesand certain physico-chemical features of the water.

WATER TEMPERATURE.A number of species and varieties like Micractinium

pusillum, Tetraedron minimum, Treubaria triappendiculata,Lagerheimiachodati, Ankistrodesmusfalcatus, Kirchneriellacontorta,Pediastrum simPlexval'. duodenarium, P.duPlexvar. subgranula-tum, P. biradiatum val'. longecomutum,Scenedesmusacuminatus,S. opoliensisval'. mononensis,S. carinatus, S. quadricaudaval's. longispina and westii have been observed in waters withthe temperature ranging from 30° to 37°C; Ankistrodesmusspiralis, Kirchneriella lunaris,Dimorphococcuslunatus, Pediastrum duplex vars. clathratum, reticulatum and gracillimum,Crucigenia tetrapedia, Scenedesmustropicus, S. acutiformis and Tetrallantos lagerheimii wereobserved-in fairly large numbers in waters with the temperature ranging from250 to 30°Cj and NePhroCJtiumagardhianum,N. lunatum, Pediastrum duPlex val'. coronatum,

ECOLOGY AND PHYSIOLOGY 39

P. muticum val'. longicorne,Dictyosphaeriumehrenbergianumand Scenedesmusarcuatus val'.capitatus under temperatures ranging from 19° to 25°C.

In this connection it may be mentioned that Petersen (1946) has reported speciesof Scenedesmusfrom hot springs of Kamtchatka, where S. dmticulatus occurred at 50°C,S. quadricaudaat 29°C, and S. rostrato-spinosus'at63°C. According to Elenkin .(1914,cr., Petersen, l.c.), S. quadricaudahas been found even in waters with a temperature ofup to 53°C.

TURBIDITY. High turbidity or even muddiness of the water appears to befavourable for the development in large numbers of many Chlorococcales, the effectof high turbidity b~ing that sunlight does not penetrate deep into the water therebysimulating conditions of low sunshine existing during the sultry monsoon months ofthe year. The turbidity in most cases depends on the rainfall and water level, shallo,~waters often becoming turbid even when there is no rain, but sometimes other algaealso contribute to the turbidity. .

Tetraedron caudatum, Dictyosphaeriump~chellum, Pediastrum tetras val'. tetraodon,Crucigeniafenestrata, Hofmania lauterbornei,Coelastrumreticulatum, and Scenedesmusopoliensis

_ were all collected in large numbers from waters which were very muddy (Iyengar andRamanathan (1940) also collected DiclJosphaeriumindicum from muddy rainwater poolsat Madras). A number of other founs like Schroederiaplanctonica, Tetraedron miniTr''''',T. limneticum, T. muticum,Lagerheimia chodati,Micractiniumpusillum, Coelastrummicroporum,Actinastrumhantz.schii,Ankistrodesmusspp., Westellabotryoides,-Pediastrumsimplex val'.duodenarium,Crucigenia apiculata, C. tetrapedia,'Scenedesmusabundans,S. quadricauda vars.longispina' and -westii .also appeared to be favoured by moderately high turbidity(50-250 ppm). However, a few Chlorococcales like Kirchneriellalunaris, Dimorphococcuslunatus, NePhrocytium lunatum, N. agardhianum, P.' duplex and its varieties, P. boryanum,

Coelastrumscabrum, and Scenedesmusartuatus val'. capitatus were usually observed in fairlyclear waters.

pH ANDTOTALALKA~INITY.Distinctly alkaline waters (total alkalinity well over100 and pH above 7' 7) were found suitable for the occurrence of certain Chlorococcalesin large nUJ!1bers. Schroederiaindi;a, NePhrocytiumagardhianum,Actinastrumhantz.s,hii,PediastrumsimPlex val'. duodenarium,fi: muticum val'. longicorne,Coelastrummicroporum,Soras-trum spinulosum,Scenedesmusdimorphus,S. acutiformis, S. abundans, and S. quadricau4aval'.longispina are some examples. Moderately alkaline waters (total alkalinity 60-100,pH 7' 7-8' 5) appeared suitable for the development of a number of other forIns likeMicractiniumpusillum, Tetraedron minimum, T. limneticum,Lagerheimia chodati, Botryococcusbraunii, Nephrochlamys subsolitaria, Pediastrum tetras val'. tetraodon, Coelastrum proboscideum,

Crucigeniaapiculata,Scenedesmusdenticulatus, S. opoliensis, and Tetrastrum punctatum. Onthe other hand, neutral or slightly acidic waters (total alkalinity 8-48, pH 6'2-7'1)appeared more favourable for the development of species like Treubaria triappendiculata,Nephrocytiumlunatum,Ankistrodesmusfalcatus, A. spiralis,Kirchneriellacontorta,DictyosPhaeriumehrenbergianum, Dimorphococcus lunatus, Pediastrum biradiatum.,' Scenedesmus acuminatu.r,Tetrastrum heteracanthum,Tetrallantos lagerheimii, and Botryococcusprotuberans.

Mention may be made here of a few species of the Chlorococcales from outsideIndia reported mostly from acidic waters or even acid bogs. The free-living Eremo-sphaera viridis and Desmatractum bipJramidatum (Lund, 1942), the epiphyte Octogoniella

40 CHLOROCOCCALES

and the endophyte Phyllobium sphagnicolaare typical examples. According to Lund(l.c.), the substances associated with low pH in nature may also be an important factordetermining the conditions of occurrence of Desmatractum. DimorPhococcuslunatus(Prescott, 1951) is also known to occcur in somewhat acidic waters.

NIP RATIO. A number of Chlorococcales (Table III) occurred abundantly inwaters with an NIP ratio less than unity whereas a number of others were observedwhen the ratio was higher than unity. Thus, Schroederiaindica, Microctiniumspp.,Tetraedronminimum, T. limneticum,Lagerheimiachodati,Actinastrumhantzschii, Pediastrumsim-

plex var. duodenarium,P. duplexvar. subgranulatum,P. araneosumvar. rugulosum,Nephrocytiumlunatum, Coelastrum microporum, ScenedesmusdimorPhus,S. denticulatus, S. opoliensis andTetrastrum punctatum occurred in waters with an NIP ratio usually less than 'unitywhereas, species like NePhrocytiumagardhianum, Kirchneriella lunaris, ScenedesmusasmatusDimorphococcus lunatus, Pediastrum duplex vars. coronatum, clathratum, reticulatum, andgracillimum, P. muticum var. longicorne,Botryococcusprotuberans,Coelastrum cambricum var.intermedium, Crucigeniatetrapedia, Scenedesmusabundans, S. perforatus, S. quadricaudavars.langispina and westii, and S. acutiformis occurred in waters with the NIP ratio usuallymore than unity. A number of others like Selenastrumgracile, Ankistrodesmus spiralisDictyosphaeriumpulchellum, Coelastrumscabrum, and Tetsallantos lagerheimii appearc:d to bemore or less indifferent whether the ratio was higher or less than unity.

Golenkinia radiata, Scenedesmusbijugatus yar. flexuosus, S. quadricaudavars. longispinaand quadrispi1}a,and Coelastrumscabrumalso came up in profusion in cement cisterns whichhad fairly high concentrations of ammoniacal nitrogen (and presumably with, NIPratio higher than unity) followingthe killing of submerged macroflora by the applica- .

tion of urea at 50-250 ppm. ~According to Ryther (1954), who studied the ecology of phytoplankton blooms

in a bay enriched by duck farm wastes, by simultaneous field and culture studies,green algae of fresh and brackish water grow much mor~ rapidly in water containingthree times as much phosphorus per atom of nitrogen as normal sea water, the normalratio of total' nitrogen to phosphorus in sea'water being 15: 1._ In the present observa-tions, in which the Chlorococcales alone' were considered, some algae aRpeared to befavoured by an NIP ralio (nitrate nitrogen alone taken into considerationf higher Jhanunity, wh~!eas others appeared to be. favoured by a ratio less than unity. Natarajan'(1959) reported an NIP value of 22Ll as necessary to obtain maximum grow~h ofSelenastrum westii in cultures, as against 15/1 reported for NePhrochlamys subsolitaria

(~Kirchneriella subsolitaria) by Potash, 1958 (Natarajan, l.c.).RESIDUAL FACTORS INFLUENCING THE PREDOMINANCE OF THE CHLOROCOCCALES.

A number of other factors not mentioned above are also probably responsible for theoccurrence and abundance of the Chlorococcales.

According to Kolkwitz and Marsson (cf. Brunnthaler, 1915), Chlorococcuminfusio-num is characteristic of highly organic waters; C. botryoides(now considered as a memberof the Xanthophyceae under t4e name Chlorobotrysregularis), C. hum~colo,Dictyosphaeriumehrenbergianum,D. pulchellum, Pediastrum ~oryanum,Ankistrodesmusfalcatus val'. acicularis,Scenedesmusacuminatus,S. obliquus, and S. quadricaudaare characteristic of waters withmedium organic content; and Dimorphococcuslunatus, Ankistrodesmusfalcatus, Westellabotryoides,Pediastrum duPlex, P. kawraiskyi, P. tetras, P. biradiatum, Actinastrum hant;:,schii,

""',f

ECOLOGY AND PHYSIOLOGY 41

CoelastrummicropiJrum,C. reticulatum, and Hydrodictyon reticulatum are characteristic ofwaters which are fairly pure, but with dissolved mineral substances and a little organicmatter.

Chick (1903) reported that Chlorellapyrenoidosaoccurred in sewage polluted water.Chlorellaand Scenedesmusare also commonly knO\yn to occur in sewage stabilization ponds(Raman, 1959).

Though no determinations of the organic matter of most waters studied by theauthor were ma<;le,it was observed that some of the Chlorococcales dominated in watersin which the abundant aquatic weeds present were killed by chemical treatment or inwhich the weeds were naturally decaying. Thus, forms like Golenkinia radiata,Micractinium pusillum, Ankistrodesmus sPiralis, Selenastrum gracile, Pediastrum tetras val'.tetraodon, Sorastrum spinulosum, Coelastrum cambricum val'. intermedium; C. proboscideum,C. scabrum,Crucigeniatetrapedia, Scenedesmusbijugatus var..flexuosus,and S. quadricaudaval's.quadrisPinaand longisPinawere frequently found occurring in fairly large ~umbers insuch waters.

It, thus,' appears that the_composition and, relative frequencies of the Chloro-coccales occurring in inland fresh-waters c!-ependon a number'of factors which ,operateseverally or collectively. Of these, the rainfall, temRerature, sunshine, water level, andturbidity; pH, total alkalinity, the nitrogen-phosphorus ratio and, probably, thedissolved organic matter appear to be important.' It is also fairly clear that a largenumber of Chlorococcales show their maxima during the sultry south-west monsoonE.eriod', l>articularly during the months of July and August, very often with secondarymaxima iri summer. A few forms. become preaominant during the winter monthsalso. ,The intense sunlight obtaining in shallow waters during bright periods is veryoften off-set by the turbidity of the water. 'Neutr~l or slightly acidic waters supporta parjicular Chlorococcalean flora which are usually differen~ from those occurringin alkaline waters. However, some species are tolerant of acidic as well as alkalineconditions. In waters richer in nitr!ltes than phosphates some species of Chlorococcalesappear to thrive better and vice versa.

Fritsch (1907), who studied the flora of the tanks, lakes and other inland watersof Ceylon" observed that the algal ,flora of tropical waters are greatly inft'fIenced bythe illumination, temperature, frequent changes in water level and to some ex:terit bythe chemical composition of the disso1ved matter, degree of movement of water, natureof the substratum' and the muddiness of the water. According to him, apart' fromthe frequent changes in water level brought about by rains and ,heat and which affectthe concentration of dissolved salts, the daily temperature range of 6°_10°C and theintense illumination taken along with turbidity (caused by suspended mud particlesor by algal blooms) are the most important factors which determine the flora of stag-nant waters of the tropics. Though his study was mostly with reference to the bluegreen algae and green algae other than the Chlorococcales, his observation.s appear tohold good in the case of the Chlorococcales as well, which form an important consti-tuent of tropical fresh-water algal flora. In addition, it appears that the seasonal aspectand some of the physico-chemical features of the water, which Fritsch could not studyowing to the short duration of his observations, are also equally impOl;tfmt in deter-mining the type of flora. .

42 CHLOROCOCCALES

~

t.-

g h k

c

FIG. XIX. LITHOPHVTIC, EPIPHVTIC, EPIZOIC, AND ENDOPHYTIC FORMS

a, CharaciosiPhon rivularis IVENG.,CLUSTER OF PLANTS ON A PEBBLE; b, CharaciumSP. ON Oedogonium; c, C. prinsheimii A. BRAUN ON Tabellaria flocculosaj d, C. anophelesi!VENG. ET !VENG. ON THE APPENDAGE OF A MOSQUITO LARVA; e, C. debaryanum (REINSCH)DE TONI ON THE ROTIFER Brachionus; f, Octogoniella sphagnicola PASCHER ON FRAGMENTOF A SPHAGNUM LEAF; g-k, Chlorochytrium dinobryonis LUND INSIDE Dinobryon divergens;g, CELL DEVELOPING INSIDE THE LORICA; h, FULL-GROWN CELL (st, STARCH MASSES);i.FORMATION OF ZOOSPORES: j, EXTRUSION OF PROTOPLAST FOR LmERATION OF ZOOSPORES:

k, WITH APLANOSPORES; I, Korshikoviella gracilipes (LAMBERT) SILVA ON THE CLADOCERANMoina; m, Chlorochytrium limnanthemum (CUNNINGH.) G. S. WEST INSIDE Limnanthemumindicum LEAF TISSUE AND SHOWING TWO ZOOSPORANOIA.

"

~

ECOLOGY ANI) PIIYSIOLOG'" 43

.'

Attached forms. There are three main categories of attached Chlorococcales:(a) attached to macroflora and filamentous algae, or epiphytes; (b) attached torotifers, copepods, cladocera, mosquito larvae or other animalcules, or epizoic (orepizootic) forms; and (c) attached to stones, pebbles and rocks, or lithophytes(Fig. XIX).

Epiphytes: Most. species of Characium and allied genera like Pseudocharacium,Pseudochlorothecium,Hydrianum and Hyalocharaciumocc~r epiphytically (attached by stalks)the substratum being usually filamentous algae,' rarely macroflora. Desmatractumbipyramidatumand Octogoniellasphagnicolaare two good instances of algae belonging tothe C,hlorococcales which occur attached to the leaves of aquatic macroflora, theformer by its outer mucilaginous envelope and the latter by its flat side. Ectogeronelodeaealso spreads itself flat on the leaves of submerged aquatic plants. Pulvinococcusand BicusPidella, on the other hand, are fixed with the aid of a gelatinous cushioq orshort thick stalk.

In the Indian region, Characium nasutum has been found on Oedogonium (R.N.Singh, 1939) and on Cladophora(Skuja, 1949); C. ambiguumon PithoPhora or Oedogonium(Dixit, 1937; G. S. Venkataraman, 1957); C. angustum on decaying algae (Turner,1892) and C. apiculatum on Chaei()tnorphaand Schizomens (Skuja, 1949). Trochisciagranulata val'. aeroPhila (Skuja, l.c.) has been reported as occurring on Euglena.

Sometimes certain .pedes of the Chlorococcales are found attached to floatingstems and twigs. Thus ChlorocoCGumvitiosum has been found (Skuja, 1949)~onfloating stems of bamboo in a pond and Characiosiphon'has been found (Agarkar; 1953)occasitmally on floating twigs, 'leaves or even pebbles. .

Ep{zoic forms (Fig. XIX d~, I): A number of Chlorococcalean genera likeKorshikoviellaSilva, RhopalosolenFott, GloxidiumKorshikov, HyaloraphidiumKorshikov, and

C~araciumA. Brau~ are ~nown to occur on various,animalcules like Cyclops, Branchypr:s'Dlaphanosoma,Moma, rotlfers and even on AnoPheles larvae. Amongst these, the specIesKorshikoviellagracilipes has been observed by the author on Moina in a nu~ber of pondsin Orissa. K, limnetica which ha.s been reported as occurring on DiaPhanosoma.01' !nthe' free floating condition (Skuja, 1948) has 'also been observed, but only in the

. detached condition. CharaciuTIJdebaryanumhas been observed by the author on rmifers,rarely on Cyclops. Another Indian species is C. anophelesiwhich occu'rs on sev(:ralspecies of' AnoPheles ~arvae. Apart from the fact that all these species are attached,there is probably some sort of symbiotic association between the animalcules and thealgal species concerned, as suggested by the usuJI occurrence of a' particular species' ofalga on a particular type of animalcule. It is also clear that one of the conditions. forthe predominance of these algae is the presence in large numbers of the animalculesconcerned in the plankton.

LithoPhytes: Characiosiphonand Dendrocystis are both examples of lithophytes..T~e former occurs in dusters on stones and pebbles (Fig. XIX a) in shallow streams(Iyengar, 1936), but; rarely, on other submerged strata as well. The latter remainsattached to stones of streams in the initial stages as well as in the dendroid condition.

s,r;jjIi '"

"

~ (a, AFTER IVENGAR, 1936; b, c, FROM G. S. WEST, 1916; f, AFTER PASCHER;g-k, FROM LUND, 1955; m, FROM CUNNINGHAM, 1888 (AS Stomatochytrium limnanthemumCUNNING.); e, FROMPHILlPOSE, 1940; 1,X 725). .

44 CHLOROCOCCALES

I

Rocky surfaces with dripping water often show a well defined association of algae,sometimes with Trochiscia and Oocystis (Tiffany, 1951). In India, Oocystis elliptica,

O. solitaria (Biswas, 1934) and O. kumaonensis(K. P. Singh, 1960) have been recorded

I from dripping rocks, often embedded in the mucilaginous masses of colonial bluegreen algae.

I Cryobionts. It has already been mentioned elsewhere that some of theChlorococcales form snow floras. Of these, Scotiella and some species of Trochiscia

are the most prominent. Chionaster,Mycacanthococcusand, rarely, Ankistrodesmus andTetraedronalso occur on snow and ice. Species of the foregoing genera are known fromthe Antarctic and Arctic regions or from the snow-clad mountains of Europe, Northand SQuth America. They are usually associated with unicellular Volvocales,Zygnemales and the Myxophyceae. Fritsch (1912) and Kol (1942) have givengood accounts of the snow and ice floras of the south Orkneys and Alaskarespectively.

According to Kol (l.c.), the cryobionts live very close to the surface and, so, thechanges taking place on the surface of the snow exert the greatest influence on theseorganisms. Since the source of mineral substances for these organisms is the surrQund-ing rocks, the nature of the rocks (whether composed of lime-stone or acidic) woulddecide the nature of the flora. Kol divided cryobionts into four classes, viz. 'glacialis- '

cryobionts ' or those which occur only on ice, , nivalis-cryobionts ' or those which occuronly on snoW, , mixo-cryobionts ' or those which are adapted to both snow and ice and, cryoxen ' or those which have their normallocabon on damp cliffs but get transferredto ice and snow. Trochiscia cryoPhilaf. longispina-Kol and brevispinaKol are examplesof -. glacialis-cryobionts '; Scotiella nivalis, S. antarctica,-Mycacanthococcus cellaris-f.antarctica, M. ovalis, and Tetraedron valdezii are examples of · nivalis-cryobionts-';Trochiscia antarctica and T. nivalis .are examples of · mixo-cryobionts' and Scotiella

po/ylJterais an example of a ' cryoxen '. Autospore formation appears to be the bestmode of reproduction in these cryobionts.

Species of Scotiella give an orange red to yellow colour to the snow.and Mycacanthococcuscellaris f. antarcticaWille ~ave also been observed in(Kol, l.c.). _ - . . -

No cryobionts have so far been recorded from the Indian region. However, thelong range of the Himalayas with its perpe~ual snow and .ice is bound to show anumber of these cryobiont~ including those pelonging to the Chlorococcales.. -

Terrestrial or aerophilous .forms. There are also members of the Chloro-coccales which occur in diverse terrestrial habitats. Thus, several members of theChlorococcaceae, including Chlorococcumhumicolo,and C. hypnosporum- and of theChlorochytriaceae like KentrosPhaerabristolae, are usually found in soils,' cultivated orotherwise, or, rarely, species of Chlorococcummay be found in stagnant waters also.These algae seem to be capable of tiding over unfavourable conditions of weatherlike drought in the soil, since they come up in cultures of dried soils. Tamiya (1959)reported that a Chlorellasurvived in a sample of desiccated soil for over ten years.Adaptation to desiccation is also seen in a number of Chlorococcales like Myrmeciaglobosa, Phaseolaria obliqua and .Chlorococcumvitiosum which are all aerophilous formsoccurring on the barks of trees.

S. polyptera

green snow

ECOLOGY AND PHYSIOLOGY .l5

Palmellococcusminiatus (=Chlorella miniata) and Chlorella conglomeratahave beenreported (G. S. West, 1916; Skuja, 1949) as' pale green patches on flower pots.Hansgirg's Mycacanthococcuscellaris, Mycotetraedroncellareand Myurococcusurococcusare allknown from damp walls. Palmellococcus protothecoides (=Chlorella protothecoides),Palmellococcussaccharophilus(=Chlorella saccharoPhila),Protothecamoriformis and P. zopfiiusually occur in the exudations of sap from trees (Brunnthaler,1915; Printz, 1927),though Palmellococcussaccharophilus is occasionally observed on flower pots (Sukja,1949). Petersen (1928) has reported several Chlorococcales like Myrmecia pyriformis,Trebouxia arboricolaand Chlorellarugosa from wooden beams and other wood works inIceland. Trochiscia granulata var. aeroPhila is another aerophilous alga known fromsub-aerial situations in Durban along with Phaseolaria, Chlorococcumvitiosum, etc.(Printz, 1921), but found by Skuja (1949) in Burma growing on Euglena. Rhodochy-trium occurs epiphytically (or endophytically) on the land plant Ambrosia.

Apart from Chlorella conglomerata, Palmellococcussaccharophilus and Trochisciagranulata var. aerophila.reported from terrestrial habitats in Burma by Skuja (l.c.), anumber of Chlorococcales have been reported from cultures of soils from the Indian

;.. region. These include Chlorococcumhumicolo(Biswas, 1934; Gonzalves and Gangla, 1949),C. infusionum (R. N. Singp, 1939), KentrosphfWa bristolae (as Chlorochytriumparadoxum,

,/tt >:-R. N. Singh, 1939), Characium acuminatum (Singh, 1939), C. nasutum (Khan, 1957),C. terrestris (Kanthamma, 1940) and Trochiscia aspera (Gonzalves and Gangla, 1949).

Algae like Chlorococcumhumicoloare also found in the rhizospheres of crop. plants(Gonzalves et Yalavigi, 1959).

An interesting instance of an alga', which, though norma11y found in the plankton,~ of ponds and rivers, can thrive healthily on moist sand near water, is Scenedesmus

arcuatusvar. capitatus. This alga was found by the author (Philipose, 1959) in the formof extensive patches on the sandy margins of River Daya in Orissa during November1952. The same alga was found in a sample (leg. Dr M. P. Motwani) collected duringthe same month under identical conditions from River Dehri-on-Sone, Bihar.Schroederiaplanctonica(=Characiumplanctonicum),which is a planktonic species, is alsofound sometimes on ~et ground (Skuja, 1949). .

., -,' - Several Chlorococcaleslike Trebouxiaand Chiorellahave also acquired a terrestrial.- habitat in association with fungi to form lichens. These will be dealt with under the

symbiotic forms. Tribouxia has also been reported (as Cystococcus-Skuja, 1949) fromthe barks of Anacardium occidentaleand Albizzia labbek in Burma.

Endophytic, endozoic and parasitic forlDs. (Fig. XIX g-m, XXI a-d).Endophytic forms usually occur within the tissues of higher aquatic plants, rarely within other algae. According to Fritsch (1935), it is only a short step from epiphytismto endophytism. Fritsch also stated that it is not clearly established whether generalike Ch~orochytriumare mere space parasites which damage the tissues of host plants intheir immediate neighbourhood only or whether they derive any nourishment fromthem. Since individual species are very often found confined to particular hosts, heconsiders some kind of interrelation between the two quite probable. In forms likeRhodochytriumthere is parasitism accompanied by loss of chlorophyll and, though the,damage to the host is not appreciable, the phloem is frequently locally damaged,

. Iyengar (1951) referred to Chlorochytriumas a space parasite and to Phyllobium.

,.

~.-

'..'

46 CHLOROCOCCALES

dimorphum, P. sphagnicola and Rhodochytrium spilanthidis as true parasites (also seeOltmanns, 1923).

The endophytic genera are all zoosporic and most of them belong to the familyChlorochytriaceae. Chlorochytriumlemnae is a common species in which the zoosporesor motile zygotes produced by sexual fusion settle down on the leaves of Lemna. Acell wall is developed and the cell produces a tubular .prolongation into the hostthrough a stomium or in between two epidermal cells. Once inside, the tube swellsup in the intercellular space, where it forms a large ellipsoidal to lobed cell whichreceives the protoplasmic contents from the cell settled outside. Then it forms a restingcell with thick and stratified wall having local excrescences. When the Lemna diesduring an unfavourable season and falls to the bottom mud, the resting cell of thealga is also carried with it. When the conditions are favourable again, the dormantresting cell gives rise to a large number of swarmers which escape by the rupture of thethick wall and the surrounding tissues of the host, the swarmers being enclosed withina wide mucilaginous .envelope. The swarmers very often fuse within the envelopeor outside. The swarmers or the quadriflagellate motile zygotes infest a fresh host.

Chlorochytriumiemnaeoccurs as an endophyte, also on other plants like CeratoPhyllumdemersum,Elodea canadensisand some mosses. C. limnanthemum(Cunning.) G. S. 'West(=Stomatochytrium iimnanthemumCunningham, 1887) occurs insid~ Limnanthemumindicum.C. cohnii and C. sarcophyci(Fritsch, 1935) occur endophyti::.lly in the marine algaeEnteromorpha and Polysiphonia and within the mucilaginous envelopes of the diatomSchiz;onema. Chlorochytriumdinobryonisis"another interesting species (Lund, 19.55) whichlives inside the empty lorica of Dinobryon divergens. KentrosphaeraJacciolae Borzi (incl.,K. gloeophiia (Bohlin) Brunnthaler) sometimes occurs within the -mucilaginous en-velopesof blue green algae. ApodochlorissimPlicissima(Korsh.) Komarek is found with-in the mucilaginous envelopes of Microcystisand Aphaniz;omenon.

Phyllobiumdimorphumforms its resting cells along the main veins of Ajuga andLysimachia and P. sPhagnicolaon Sphagnum. RhodochytriumsPilanthidisoccurs on the leavesof Ambrosia artimisiaefolia.

Some .species of CodWlum A. Braun, another endophytic genus, have beenconsidered as the sporophytes ~f Cladophorales while others are held under suspicion.

The thallus and reproductory structures of most of these algae are more or lessadapted for an endophytic or even parasitic habit. The Jhallus is'usually in the form ofa branching filamentous thread, which is coenocytic and it ramifies the intercellularspaces or sometimes breaks down a few cells of the tissues of the host plants. Hereand there, resting cells or gametangia, with thick stratified walls are formed andthese give rise to zoospores or gametes.

A few Chlorococcales are aiso known to occur within the tissues of various

animals, but these are all probably symbionts and will be dealt with under that head.Saprophytic fOnDS and forDls grown in cultures. A number of Chloro-

coccales, which are capable of a holophytic existence, exhibit a saprophytic tendencywhen grown in artificial cultures or in media rich in organic matter, and in thesemedia they might even show better development (Fritsch, 1935). Thus, Chiorellathrives in organic media like sewage, often accompanied by loss of chlorophyll.Chiorella, Ankistrodesmus,Scenedesmus,and several others also grow very well on agar

..

....

ECOLOGY .\;>;U PHYSIOLOGY ,p

~

supplied with mineral solution and the growth is luxuriant if glucose is also present.Fermenting cellulose is also useful in the place of glucose.

Some of the Chlorococcales can also be grown successfully in cultures in the dark.Usually the algal cells become colourless or yellow when grown in darkness, thoughthey are quite healthy in other respects, the decolorization being attributed to theinhibiting effect of glucose. When such algal cells are transferred to a medium havinga better balance of glucose and nitrogen, the cells might regain the green colour for ashort time. Some like ScClltdesmusobiiquushave also been grown in darkness for severalyears without loss of chlorophyll. These algae are probably capable of producingchlorophyll in darkness. Forms which live heterotrophically during darkness mayormay not change to autotrophic nutrition when exposed to sunlight.

Quite a number of members of the Chlorococcales easily lend themselves forculturing in the laboratory in mineral solutions containing the essential nutrient salts,including micro-nutrients under suitable pH, temperature, and light conditions.Chlorella,Selenastrum,and Scenedesmusare typical examples. Hopkins and Wann(1926, p. 353) demonstrated that in cultures the full effect of changes in pH onChiorellais seen only when iron, which gets.precipitated at.pH above 5'7, i.<tkept insolution by the omission of calcium and the addition of sodium citr~te. Under suchconditions the alga can grow at pH ranging fr!>m3'4to 7'~ with best growth at 'pH 7'5.Natarajan (1959) found both Fe-EDT A and Fe-tartrate proved to be satisfactory sourcesof iron for grow~ng Seunastrum westii in inorganic media. Hopkins (1930) 'has alsodemonstrated the. importance of manganese in getting better growth of Chiorella .incultures. I --.

According: to Witsch (1959), apart from the necessary -nutrition ana- properillumination, a regular supply of carbon dioxide and proper circulation of water aredesirable for mass cultures of algae. Ten times. the normal supply of nitrogen in anutrient solution is also stated to be favourable for maximum production of green algaelike Chlorellaand Scenedesmus.

Chodat (1999, 1913) carried out pure culture studies of many Chlorococcalesand concluded that many genera like Ankistrodesmus,Dactyiococcus,and Scenedesmusshowpolymorphism (Fig. XX). - Chlorella-likestages have also been shown to occur iQ.certaincultures of Scene.desmusand Goeiastrum._ According to Ohodat, !here are a number of

elementary species in a large number of genera of the Chlorococcales as shown bydifferences between individuals grown in cultures, and these differences are morpho-

. logical: New elementary species, according to him, are formed by small mutatiom.According to Fritsch (1935), it is very doubtful whether polymorphism exists in nature.Further, studies in pure cultures alone do not give an idea of form variations in algalspecies, since a given alga might very often assume a form in these cultures which

might be indistinguishable from another species. A distorted picture of the life-historymay also be obtained owing to abnormalities occurring in cultures. So, Fritsch-considers pure cultures reliable and useful only in supplementing field observations.

Pure culture work has also been useful in showing that the gonidia of Tdbouxiaextracted from different lichens belong to distinct races. These differences in race

might be due to the influence of the fungal partner, considerable physiological changesbeing brought about by the living together ,of an alga and a fungus. The alga ha~

48 CHLOROCOCCALES

i9jJ. .. :. .

::~ .~0~ ~~.::

".

FIG. XX. Ankislrodesmus falcalus (CORDA) RALFS GROWN ON AGAR-GLUCOSE, SHOWING POLYMORPHISM (FROM CHODAT, 1913).

been considered by some authors to lead a saprophytic existence, being supplied byorganic nutrient by the fungus, while the modern view is that it is an instance ofsymbiosis.

The saprophytic. tendency has 'probably ied to the. origin of permanentlycolourless forms like-- Hyaiocharacium, Prototheca, Hyaioraphidium, and MyciJtetraedron(Fritsch~ 1935). . .

The endophytism of space parasites like Chiorochytriumand the occurrence ofgreen cells within animals have also been sometimes considered as an expression of thesaprophytic .tendencies of the Chlorococcales. .

S}'IDbiODtS. There are also Chlorococales which occur in symbiotic rela-tionship (Fig. XXI) with other plants or animals (Oltmanns, 1923). TrJbouxia andChlorellaare examples of genera which live symbiotically with various fungi to form adefinite lichen thallus. Trebouxia occurring in the Parmeliaceae, Usneaceae and~oniaceae, and Chiarella (?) recorded in species of Ciadonia, all form lichen gonidia(Fntsch, 1935). There is also probably a symbiotic relationship between Chiorellaand the.ni~gen fixing:bacterium Azotobacter chroococcum(Lipman and Teakle, 1925).

According to Lazo (1961), Chiorellaenters into full association with Myxomyceteplasmodia of several species which have been freed from bacterial contamination andgrown on agar.

.-. ~

ECOLOGY AND PHYSIOLOGY 49

Giaucocystisis an interesting colourless member of the Oocystaceae (Fritsch, 1935;Tiffany, 1951) inhabited by a symbiotic rod-shaped member of the Chroococcaceae(Myxophyceae). Originally the curved radiating bodies inside the cells were takenfor the chloroplasts (Brunn thaler, 1915), but now they are definitely established asthe blue green component of an association between two algae (Fritsch, op.c.; G. M.Smith, 1950). Since the blue green component does not appear to lead independentexistence, Fritsch included Giaucocystisunder the Oocystaceae, whereas Smith, andPrescott (1951) restricted, the generic name to the blue green component and includedthe alga'tinder the Myxophyceae. Skuja (1949) placed it under a separate class, theGlaucophyta, between the Cyanophyta and the Chlorophyta. The formation ofautospores within the cells and the possible formation of swarmers in Giaucocystis(Fritsch, op.c.) have been advanced as reasons for not ignoring the importance of theChlorophycean component. .

,- a cb

a

..-...

..'8. "

b gFIG. XXI. ENDOZOIC AND SYMBIOTIC FORMS

i.a, LONGITUDINAL SECTION OF THE HYPOSTOME OF THE HYDROID Myrionema

amboinensis, WITH CELLS OF Chlorella IN THE ENDODERM CELLS (en); b, ChlorellaFROM THE SAME; c-d, Chlorella IN Hydra viridis; c, CELL OF ENDODERM WITHLIVING AND DISINTEGRATING GREEN CELLS; d, SECTION OF ENDODERM WITH PARTOF AN EGG BELOW INTO WHICH THE GREEN CELLS ARE SPREADING; e-f, GJauco-cysiis nostochinearum ITZlGs.; . e, SINGLE CELL; f, FOUR-CELLED COLONY;g, Cladoniafurcala BORNET, CROSS SECTION OF THALLUS.

a, e, GREEN ALGAL .CELLS; b, DISINTEGRATING ALGAL CELLS; e,

ECTODERM; en, ENDODERM; N, NUCLEUS); e-f, (b, BLUE-GREENSYMBIOl<TS); G (u,UPPER SURFACE 1, LOWER SURFACE).

(E-b, AFTER SVEDEUUS, 1907; c-d, AFTER BEIJERINCK (BOTH AS"ZOOCHLORELLA") ; e,f, AFMER GEITLER and HlERONYMOUS RESPECTIVELY;g, AFTER OLTMANNS, 1923).

;')0 CHLOROCOCC,\L£S

Several members of the Chlorococcales live in association with animals and they

appear as green cells within the tissues of these animals (Oltmanns, 1923; Fritsch, 1935).Freshwater animals like the Infusoria Stentor, Paramoecium, Ophyridium, some Foramini-

fera, the Coelenterate Hydra, the sponges Anodontaand Unio, mussels and snails, and someTurbellarians have all been shown to contain such green cells, and in most instances

the alga concerned appears to be species of Chlorella,though other green algae mightalso be found occasionally in some other animals. Scenedesmusquadricauda is a rareinstance of a colonial alga living symbiotically with the animal Carterius stepanowi.Chlorella occurring within animals has been termed by Brandt (1882) as :(,oochlorellawith two common species, viz. :(,. conductrix and :(,. parasitica. Beijerinck (1890)treated them as the respective species of Chlorella. Some authors consider these asidentical with C. vulgaris. However, Fritsch (op.c.) suspected different elementary speciesin different hosts. -

The relationship between the animals and the plants appears to be a symbioticone in most instances. The food manufactured by.the alga is utilized by the animaland the alga in turn gets a plentiful supply of carbon dioxide. Sometimes, a certainnum.ber of algaL cells are also digested by the animal. This symbiotic relationshipis not obligatory, since all the animals may not contain the algal cells and colourlessanimals can thrive independently.

HmNever, in the case of the green algae occurring in the mantle and gills offreshwater mussels and which are exposed to sunlight, the relationship between the

-alga and the mussel might very often 'range from true-symbiosis to parasitism (Fritsch,1935). Link (1911) has described a parasitic green alga occurri~g within the skin ofcarp. According to Fritsch (1935), it could probably be a species of Chlorochytrium.

How exactly the alga finds entry into the animal is not clearly established. Anumber of algae are taken as food by these animals, but the special symbiotic alga inall probability escapes digestion and establishes itself in _the tissues of the animal..Sometimes, the reproductory cells like the gemmulae of Euspongilla and the ova ofH;'dra are infected, and the former is usually green. Motile juvenile stages of manyanimals are also probably infected easily. Someti!iles, as in Stentor, the Chlorella cellsliberated from the dead anim_al-during' autumn remains in the plankton and reinfectnew animals during the following season..

Trochiscia zachariasii constantly occurs, in the mucus coveriI!-g the embryos andlarvae of the frog Rana, agilis. . OoPhila.amblystoml!tisoccurs witpin the:: envelopes sur-rounding the eggs of salamander (G. M. Smith; 1950). These, and the species ofCharacium, Korshikoviella, and Rhopalosolenoccurring on plankton animalcules and oninsect larvae may also be instances of symbiosis, but not of a marked nature" (Fritsch,1935). Protothecaportoricensishas been isolated from the stools of tropical spruepatients (Mariani, 1942).

Most of the instances of associations of algae with animals mentioned above are

reported froIll Europe, but there are also one or two instances known from the Indianregion. Thus, in Myrionema amboinensisfound by Svedelius (1907) near Galle, Ceylon,the entire body of the polyps were infested with" :(,oochlorella", the alga being foundin large numbers particularly in the hypostome. The algal cells found in theendoderm frequently divide into four or eight. Venkataraman (1957) refers to

~

..1.

ECONOMIC IMPORTANCE 51

Chlorellavulgaris as occurring within the freshwater sponge, but he does not refer to therelationship between the two. Strictly speaking, this species should be considered asChlorellaparasitica (Brandt) Beijerinck and that of Svedelius as C. conductrix (Brandt)Beijerinck.

ECONOMIC IMPORTANCE OF CHLOROCOCCALES

CHLOROCOCCALES AS FOOD OF FISH

It is generally known that a number of aquatic algae form the food offish eitherdirectly or indirectly. Diatoms, filamentous and some planktonic green algae and anumber of blue green algae, exclusive of species forming noxious blooms, are veryoften found in the guts of various species of fresh and brackish water fish and theyappear to be directly utilized as fish food. Apparently, there is also a certain degree of

.selective feeding by some fish' as indicated by the occasional presence of these algae ingood quantity in their guts even when the algae concerned are not dominant in thewater. The reserve food materials i!l these algae, viz. fats and volution in the diatoms,starch, often accompanied by oil, in ihe green algae; sugars and glycogen in the bluegreen algae, and polysaccharides in the Euglenineae, may have some bearing on thedigestibility of the cell contents of these algae since the diatoms (except their shells)'are the most easily digested and the Euglenineae the least.

Among the green algae other than th~ filamentous forms, t!Ie most important''" ~an.~ ubiquitous groups in the Indian region are the Volvocales, the Chlorococcales,

and tIu' Desmids. Of these, the Volvocales and some of the desmids found in themargins of shallow waters and occasionally in the plankton are sometimes encounteredin the guts of fish, but it is the Chlorococcales, which are more often found in fairlylarge numbers in ~the guts orfreshwater fish. In the case of the Siamese fish, Tilapiamossambica,which lias been experimentally introduced at Cutt~ck, and which isknown to be voracious feeder of filamentous algae and some tender macroflora, theChlorococcales have been occasionally found in their guts in such large riumbers, thatthe_gut ~ontents of this fish have been sometimes quite sufficient to obtain selectedgatherings of various species belonging to the order. According to Prowse (1957),though the gut contents of Tilapia mossambicastudied at' Malacca were very rich indiverse Chlorococcales, 'none of them were digested in contrast to the scattered diatomswhich were fully digested. *

However, the Chlorococcales appear to be more important as an indirect sourceof food to many fish than as a direct source.. Thus, the enteric cavities of rotifers, co-pepods, and cladocera are very often filled with various forms of algae, particularly theChlorococcales. There is also some experimental evidence to show th~i: some of theChlorococcales are utilized as food by these animalcules. In the LimnologicalLaboratory of Lund University, Sweden, a culture of Scenedesmuswas maintained over

\-a long period, the alga being supplied exclusively with a daily dose of fish meai.,Another culture exclusively of Daphnia was maintained in the same laboratory by

" feeding the animalcules daily on a certain quantity of Scenedesmustaken from the~.- .

*The author has also observed faecal pellets of the mullet Mugit corsuta,conSistingalmost exclusivelyof undigested Botryococcusbraunii floating in experimental filter beds at Barrackpore containing both theailla Anitth.. /;.h

52 CHLOROCOCCALES

cultures. Both Scenedesmusand Daphnia remained perfectly healthy as in nature andwent on multiplying rapidly. * It appears that many other plankton animalculescould be cultured in a similar manner. When it is remembered that planktonanimalcules are very useful items of food of fish, particularly in the early stages of thelife history of fish (Alikunhi et at., 1955), the potentialities of artificial feeding of fishand increasing the natural food supply in nursery and rearing ponds could be realized.Though the utility of phytoplankton as an indirect source of food is very often notappreciated by many fishery biologists, particularly}n India, on account of the fact that'the food cpa ins of many of our species of fish haye not been worked out, there is nodoubt that the phytoplankton, of which the Chlorococcales form a very prominent groupin the tropics, playa very important role. It is also known (Schwimmer and Schwim-mer, 1955) that several vitamins found in fish' can be ultimately traced t~.the phyto-plankton on which they feed.

Mention has already b.een made that many Chlorococcales have a cell wallwhich is orqamented with spines, bristles or setae. Apart from the fact that they helpthese algae to lead a pelagic life, it is also possible that'these armatures offer veryoften useful self-protection against the voraci?us plankton animalcules.. .

CHLOROCOCCALESAS A SOURCE OF FOOD AND FEED AND. IN INDUSTRY.\ Investigations on the use of algae as a source of food and feed and of industrial

raw materials have been receiving lot of attention all over the world since some tiIl}e.Many sea weeds, brown and red, and -a few green algae like Ulva- lactuca and

"u. fasciata, hav€,been used for a long time as a source'of food and feed -or as rawmate;ials for industry, particularly in Japan and China. It is only recently thatfresh-water members of the Chlorococcales, particularly Chlorella, and sometimesScenedumus and Ankistrodesmus, have been thought of for this purpose. Considerablework has been carried out at the Carnegie Institute of Washington (Burlew, 1953)and Germany (Witsch, 1959) on mass cultures ofChlorella.and its suitability ~ an alter-native source of anilI].al feed and of human vegetable food. (Quite recently Russianscientists are repor~ed to have made Use of Chlorella in space research, .the' effect ofradio-a.ctivity being studied on the alga). ,

Chlorelfapyrenoidosaand C. vulgaris are.~the two species' st~died in' detail. Thereasons why Chlorellawas selected for studies on its utility as food and feed were :(I) the alga is easily cultured on a mass scale under artificial conditions on account ofits rapid grow1h in mineral solutions; (2) ash analysis of Chlorellashows that its inorganiccontents are more or less on the same level as that of com; (3) the protein, carbotiydrate,and lipide contents and the R-value (which corresponds more or less to the energycontent) in Chlorella.show a great range of variation depending on the environment,and this range of variation is not exceeded by any other plant. matetial. Further,it is' possible to induce production of a fairly high content of these under controlledconditions of cultures; (4) all essential amino-acids are present in Chlorellato the extentof 42 per cent of its protein content. The amino-acid index for Chlorella is about62 which is more or less the same as in white flour, pea nut meal, etc.; (5) the pigments

Chlorophyll A, B, Carotene, and ~anthophyll are found in a higher proportion in

*Seen by the author by courtesy of Prof. Sven Thumnark.

ECONOMIC IMPORTANCE 53

Chlorellathan in many other plants; and (6) it is also considered a rich source of vitaminsA, C, and K. B, is also piesent in fairly large proportion, though it might vary with

, the age of cultures. Of these, only vitamin C is likely to be lost considerably in driedor frozen Chlorella. Witsch (1959) statea that vitamin B, values of young cultures ofalgae like Chlorella equals that of lemon juice.

Feeding experiments with dry Chlorella powder have shown that animals andhuman beings fed on it show a general improvement in health, though it has not beenpossible to determine whether it is due to its high protein content or high vitamincontent. However, though the powder has been considered as "food-like" and" food-satisfying ", its unpleasantly strong smell, general appearance, mildly unpleasantafter.;taste, and a noticeable tightening at 'the ~ck of the throat (" gag-factor ") havebeen considered disadvantages. This has been. generally overcome by mixing it with- - ~

other items of food like chicken soup so that any unpleasant taste, smell or other dis-advantages are maske<;:l. In Japan, powdered' Chlorella ellipsoidea has been usedsuccessfully after mixing it with green tea or in noodles made of wheat flour, even

tJ:1ough large qua~tities 'of th~ powder could not be' used o~""a5~~unt of theCiiso', -agreeable colour (also see Tamlya, 1959). i r~,

,... II" .',.. -. .. .1;

Thus, Chlorella has been used more as a food stabilizer (Randhawa;, 1956) than-. as a c~mplete item of food by itself. * It is possible to raise 20 to 30 time~iIiote dried

[~.o; .. . .. , ~ . _

. Chlorellafrom an, acre of ~ater one metre deep than maize from an acr.e~~ lanci... )

Because of these potentialities, in India the food, value of Chlorella i$<~entl}run~;c'.

. ~nvestig~tion at the Central.Food T?cQnological. Research I~stitq,te at ~so~e. 'T~~~' \ IS also a scheme at the Indian AgriculturaL Research InstItute, ~ew Delhi, to study ~,' .:

t" the genetic variability of useful algae, including Chlorella. . "":~. " ,<. Regarding the potentialities of the use of Chlorella as raw material'for various

. ..'.,~""~ indu;tries, *e only one which shows promise"is jts use as a potential source of chlorophyll ~

. -,' for deodOl:ant purposes. This pigment is present in the alga to the ix~nt of 4-6 per

..I. ~ent compared.to O'~ per c<,:ntin alfalfa, whi~~ is now commercially used. for its ~xtrac-tion (A. W. Fisher In Burlew, 1953). Residual by-products after the extractIOn of

- the pigment canaIso Qt used as)inimal feed: Though the vitamin contents ofChlorella.--are high (<;omp-arable to that of yeast, it is not, sufficient for extraction as concentrates.Similarly, ~xtraction of proteins, amino-acids an.d alginic acids also lI].ay not beeconomical. Chlorellais also ~nowii to 'have some antibiotic properties since it controlsbacteria to soine 'extent, but work along these lines has yet to be uidertaken in detail.According to Gupta and Srivastava (1963) Hydrodictyon reticulatum also produces an,'J' antibacterial substance. .

- Chlorella has also come in very handy for keeping the air in space vehiclespure on long interplanetary flights. Recent reports by the New York Times News~ervice indicate that three members of the life Sciences staff of Aerojet-General

"Corporation, Azusa, California, have developed an 'apparatus and pro<;ess for. thepurpose. The stale air in which the carbon dioxide has been concentfated i~ fed into

}a flood-lit container containihg a mixture of water and nutrient' chemicals andChlf!rella. The alga restores oxygen into the space vehicle by its photosynthesis.It is also expected that the additional alga grown in the process would serve as foodfor the astronaut.

~

, .

...

Ii!;

· Also, see j\ :ldendum.

CLASSIFICATION AND PHYLOGENY

HISTORICAL

(The classification of -the order Chlorococcales (= Protococcales) has been a verydifficult one.) A glance at the classifications suggested by various authors over the pastseven decades is sufficient to show the divergent views held from"time to time. This

has been partly due to the advancement in our knowledge of the order with the passageof time. In 1935, Fritsch pointed out that the classification could not remain natural -with the then state of our knowledge. Since that date the order has been in themelting pot and even today taxonomists are not agreed on ~fication which isacceptable to all. The classification givenpere is based on views expressed by some ofthe more important taxono~ists of the order.- '., .

Historical resume of theprogressivechangesin'.the classification: The order Pr~tococ- -cales, as conceived by Wille (1897), consisted of six families, viz. the Volvoca~eae,Tetrasporaceae, Chlorosphaerlfceae, Pleurococcaceae, Protococcaceae, and the Hydro-dictyaceae. The last family consisted of such genera .as Hydrodictyon, Pediastrum,Sorastrum, and Coelastrum. The evolu~ionary tendencies ~f these families in relation -to other orders of the Chlorophyceae were depicted by him as follows:

CONFERVOlDEAE

_ Hydrodictyaceae

~ ~~- _ ~~: Ch!orosphaeraceae

~ _ -/ ~ PleuroCoccaceae

P-Totoco~caceae _ _ ~-"" Tetrasporaceae' _

""~ >.

Conjugatae---V olvocaceaePROTOCOCCOlDEAE

By 1909, he recognized under the order ten families, viz. (1) 'Volvocaceae,(2) Tetrasporaceae (inc!. Dictydsphaerium, Dictyocystis, and Chlorosphaera),(3) Botryo-coccaceae, (4) Pleurococcaceae (inc!. Gloeotaenium,Acanthococcus,and the Myurococca-ceae), (5) Protococcaceae (Endosphaereae, Characieae, Halosphaereae, Botrydio-pseae, Chlorothc;cieae, and Rhodochytrieae),. (6) Ophiocytiaceae, (7) Hydrogastraceae

54

.CLASSIFICATION AND PHYLOGENY 55

(Protosiphon and Botrydium), (8) Oocystaceae [Eremosphaereae, Chlorelleae-incl.Tetracoccus (= Wesfella), Micractineae, Oocysteae, Nephrocytieae, Tetraedreae,Protothecaceae, and the unassigned gen~ra Desmatractum, Acanthosphaera,and Echino-sphaeridiumJ, (9) Hydrodictyaceae, and (10) Coelastraceae (Scenedesmeae, Sorastreae-incl. Coelastrum,SorastTum,Dimorphococcus,etc., Selenastreae, and the unassigned genusClosteriococcus). The phylogeny of the different families was represented by him asshown below.

SIPHONOCLADIALES' SIPHONALES CHAETOPHORALES

PROTOCOCCALES

....

Hyarogastraceae Coelastraceae

Ophiocytiaceae ~ HYdrO~ictYaceae .

- I"" '" Oocystaceae

"" protococcacea! (proto!hecaCeaC)I

\ / Pleurococcaceae

(RhodOChytria,ceae) -I (MYl!:~occace~e),

- - Botryococcaceae

Te'nupo,.ocae /C~NJUGATA~

Volvocaceae

','(H yalovolvocaceae)

._ Flagellata !'It was Brunnthaler (1915) who gi\ve the Protococcales the mo!e defined demarca-

tion which has come down to the pr:esent day. His classification of th; Protococcaleswas as follows :

Ser. I-Zoosporinae Brunnth., 1915,p 59Fam. Protococcaceae (Protococceae-incl., Ken/rosphaera;Endosphaereae),Fam. Characiaceae (incl. Ac/idesmium)Fam. ProtosiphonaceaeFam. Hydrodictyaceae

Ser. II~A~tosporinae Brunnth., 1915, p 107Fam. EremosphaeraceaeFam. Chlorellaceae (Chlorelleae-inc1. Te/racoccus;-Micractineae)Fam.Oocystaceae Oocysteaej --

Lagerheimieae;Nephrocytieae (incl. D'sma/rac/um);Tetraedreae;Scenedesmeae;Selenastreae (incl., Dictyosphaerium and Dimorphococcus)(incl., Soras/rum)

-

Faro. Scenedesmaceae

~.,.fl\m. Coe1astracel\e

'.

56 CHLOROCOCCALES

The main features of Brunnthaler's classification was the recognition of two broad

series, the Zoosporinae (reproducing by zoospores) and the Autosporinae (reproducingby autospores). Apart from excluding the Volvocaceae, Tetrasporaceae, Botryoco-ccaceae, Pleurococcaceae, and Ophiocytiaceae of Wille (1909) from the Protococcalesand reassigning some of the genera included in the Tetrasporaceae and Pleurococca-ceae to truly Protococcalean families, he also raised the status of the subfamiliesCharacieae, Eremosphaereae, Chlorelleae, and the Scenedesmeae to th~ of families.Botr)'dium was excluded from vVille's Hydrogastraceae and this family itself wassuppressed in favour of the monogeneric family' Protosiphonaceae.

The families Chlorangiaceae, Palmellaceae, Tetraspor~ceae, and Chlorosphae-raceae were separately dealt with by Lemmermann (1915) in th~ same volume undera separate order, the Tetrasporales.

Till 1915, the accepted name of the order' was Protococcales. Since Protococcus(=Pleurococcus) was no longer regarded as a member of the Protococcales (Pascher,1915) the term Chlorococcales suggested first by Marchand (1895) and later emendedby Pascher (1915) was considered the more appropriate name for the order. However,it was only from 1927 (West and "Fritsch, 1927) that the name was generally adopted.According to Papenfuss (1955), the name of the order should read as ChlorococcalesMarchand orth. mut. et emend. Pascher, 1915. .

G. S. '::est (1916) adopted the classifications of Wille (1909) and Brunnthaler(1915) with certain modifications. He recognized under his Protococcales three sub-orders, viz. the Volvocineae (with three families), the Tetrasporineae (with fivefamilies), and the - q,hlorococcineae (with two families). I( the Palmellaceae and .theChaetopeltidaceae are excluded from his Tetrasporineae, it corresponds: more or less toBrunnthaler's Autosporinae, and the Chlorococcineae to the Zoosporinae. Othersignificant changes were the removal of theProtosiphonaceae to the Siphonales and therecognition for the first time of a family for the genera Dictyosphaerium,Dictyocystis,- :$Dimorphococcus,and Westella. He als-o suggested the following evolutionary tendenciesin the order :

SIPHONALES .HIGHER ISOKONTAE

+

I-

rT

--------

~~ ~r~ ~:;;~~e~~ . ' . ~' ~

Planosporaceae . Protococcaceae «___r , II gHydrodictyaceae~.r.:: Palmellaceae 0

~ 0"'C"°c "';'- 0

~<% 0c .S <J (, Eo<C"? c:.. <J 5 ~...~ 0

'). ~C" g "0 ~o CI::

, - c ~., c.;\ ,:I.."" (,'<,; 0.(,~

(,~-,;

Chlamydomonas

Of these, West considered the Tetrasporine tendency the most important

.i

CLASSIFICATION AND PHYLOGENY 57

Oltmanns (1922) adopted a simple classification recognizing only four familiesin his Protococcales, viz. the Protococcaceae (Protococceae, Characieae, Endosphae-reae), the Protosiphonaceae, the Scenedesmaceae (Chlorelleae, Eremosphaereae,Scenedesmeae), and the Hydrodictyaceae. Sorastrumin which zoospores were recordedby Probst (1916) was again placed along with Pediastrum, Hydrodictyoll,and Euastropsisin its natural place in the Hydrodictyaceae. However, Coelastrum, DictyosPhaerium,Raphidium, and allied genera were all placed by him under the Scenedesmeae, andOocystisand allied genera under the Chlorelleae. According to him, the evolutionarytendencies of"the genera under the Protococcaceae are as'follows :

.. PhyllobiumI

Eremosphaera

IChlorocystis

I, Codiolum

~Chlorochyt rium D

. . hlcranoc aete

- 1,,/Sykidion

. I

Characium

.

Chlorococcum (incl. Cystococcum' USW)

Geidel' (1924) suggested the classification of the order into two major groupson the basis whether the contents of the cell divide gradually or simultaneously at thetime of reprodu~tion. - Howev,er, this has not been considered a satisfactory classifica-tion by most authors (G. M. Smith, 1933; Fritsch, 1935).

Korshikov (1926) classified the coccoid algae into two groups, the Vacuolatae(those with' contractile vacuoles) and the Protococ<,:ales (those without contractilevacuoles), and he depicted the phylogenetic connection among some of the unicellularforms as follows :

:t

..~ ~

?

Macrochloris

'p ROT 0 C 0 C C ALE S

VACUOLATAE \Hypnomonas

- - - - -- --- -

Apiococcus

IChlamy.domona~ "

58 CHLOROCOCCALES

Printz (1927) followed a classification which was essentially that of Wille (1897,1909) with some modifications. Apart from the reassignment of a few genera, impor-tant changes from Wille's classification were the exclusion of the Botryococcaceae(following in this respect Pascher, 1925, who tra~ferred it to the Heterokontae),Ophiocytiaceae, and the Hydrogastraceae, and the retention of the Protosiphonaceae.The Chlorosphaeraceae suppressed by Wille in 1909 was also revived. Printz's Tetra-poraceae included Myuroeoeeusetc., and the Pleurococcaceae, Desmatraetumand,Elakatothrix. The Eremosphaereae, Chlorelleae, Micractinieae, Oocysteae, Gloeo-taeniae (inc!. Tetraedron), and the Protothecaceae together formed the' Oocystaceae.The Dictyosphaerieae, Quatematae (= Westella), and Selenastreae were accommodatedalong with the Scenedesmeae, CIi1cigenieae, and Coelastreae under the CoelaStraceae.

The evolutionary tendencies and inter-relationships of his nine families weredepicted by him as follows :

~PHONOCLADIALES SIPHONALES CHAETOPHORALES l' .I ~

PROTOCOCCALES I ~~-:;:s.,I "Protosiphonaceae Pleurococcaceae ~/

\ Coelastraceae

I

/','"~ I' - ,/'.Oocystaceae Hydrodictyaceae Chlorosphaeraceae~ I . ,

- "~ (prth""' ) //Chlorococcaceae "I ",,"~

(Rhodochytriaceae)"~Tetrasporaceae "

I

HETEROCONTAE I

CONJUGATAE~II

~

Botryococcaceae

(Myurococcaceae),Volvocaceae

I(H yalovolvocaceae)

1>--'------------- :.; ~

FLAGELLATA

West and Fritsch (1921,) adopted, for the first time, the name Chlorococcales for'the order. Their classification was essentially that of Brunnthaler, the series Zoospor-inae and Autosporinae being retained. The former consisted of the Chlorococcaceae(Chlorococceae-inc!., Charaeium and ChlorosPhaeraand Chlorochytrieae), the Proto-siphonaceae, and the Hydrodictyaceae and the latter of the Eremosphaeraceae,

CLASSIFICATION AND PHYLOGENY 59

Chlorellaceae, Oocystaceae, Selenastraceae, Dictyosphaeriaceae, and the Coelastraceae.:1:1I The Selen.astreae was raised to the status of a family for the first time and the Coelas-

traceae was restricted to Coelastrum,Crueigenia,Scenedesmus,and allied genera.In 1935, Fritsch adopted a slightly modified classification in which the series

- Zoosporinaeand Autosporinae were not recognized. According to him, thoughBrunnthaler's two series were considered convenient at the time and continued to befollowed for about two decades, it was not a natural grouping showing the true affi-nities of the various families. Fritsch had also done away with all subfamilies (seealsoG. M. Smith, 1933). Like West (1916), the Protosiphonaceae was transferred tothe Siphonales. The Chlorosphaeraceae was appended to the order Chlorococcales

, as a special family. The Tetraed.reae included by West and Fritsch (1927) under'fig:' 'the Oocystaceae was also transferred to the Chlorellaceae.'J~r;:..f!~;: In his classification of the order into eight families Fritsch takes into consideration

" a number of characters, viz. the habitat (free-living, epiphytic, endophytic, etc.);',habit (solitary or colonial), when colonial, the presence or absence of gelatinized parent

i' '"cell.membranes, connecting threads or mucilaginous pads; the nature and number-of":!Chloroplasts and the chief mode of reproduction (by autospores, autocolonies or by"J 'zooSpores). Though Fritsch did not consider this classification as completely satisfac-" ,.tory, he was of the opinion that undue emphasis should not be laid on a single"\' character like autospore or zoospore formation. For, the' same reason he did not agree

"with Korshikov's (1926) classification into the Vacuolatae and the Protococcales, the:fo~e~ being placed between the Volvocales and the p!otococcales-to accommodate'~oid forms which have apparently given up reproduction by swarmei-s, but still.:etain contractile vacuoles and sometimes eye-spots.~; As regards phylogeny, Fritsch (op.e.)stated that, apart from the close affinity

,:to'the Volvocales, the diploid condition in some of the sexually reproducing Chloro-;f()Ccaleslike Chloroehytnumand probably Apioeoeeus,with tendency for direct germination

".of~ zygote, is significant in the hypothesis of a possible origin of the Siphonales

~fromthis order. Also, the coenocytic tendency <.>fsom~ of th~ zoosporic Chlorococcales~y have led to the evolution of forIns like ProJosiPhon,which serves as a proto~ypefor,the rest of the Siphonales.

i,:\-~ Tilden (i935) transferred not only the Protosiphonaceae but also the Hydro-}Iictyaceae and the entire subfamily Chlorochytrieae to the Siphonales, an order unde..'i~e subclass" Siphonae" (plant body a single coenocyte), the Chlorochytrieae being',accommodated in the family Phyllosiphonaceae. Her Chlorococcales, an order underthe subclass," Uninucleatae ", consisted of three sub-orders, the Volvocineae,~etrasporineae, and the Chlorococcineae. The Coelastraceae, Oocystaceae, and

§c~edesmaceae were placed in the Chlorococcirieae along with some non-Chloro-~calean families.

_ Smith (1933, 1950) followed a classification which Jas only a variation of thefystexiufollowed on the continent. In his earlier classification (1933) all subfamilies~ere dispensed with for the first time, raising the status of some to that of families and~ppressing others. He recognized eight families, viz. the Chlorococcaceae, Endos-

eraceae, Characiaceae, Protosiphonaceae, Hydrodictyaceae, Coelastraceae,

taceae, and the Scenedesmaceae. Of these, the prot,: "~ the

.

60 CHLOROCOqCALEs

Coelastraceae were mono generic. In 1950, he added two more families, the Micracti-niaceae and the Dictyosphaeriaceae (excl., Westella), the genera belonging to which

were previously accommodated in the Chlorococcaceae and Oocystaceae respectivelySmith (1950) admitted that the Hydrodictyaceae and Scenedesmaceae alone constitutedvery natural families, whereas families like the Chlorococcaceae a.nd Oocystaceae weremore or less artificial, the genera under them being grouped together on the basis ofthe method of reproduction only.

Prescott (1951) followed the same classification as that of Smith (1933) withthe difference that the Protosiphon'aceae was excluded from the order and the

Botryococcaceae was brought back from the Xanthophyceae.During the Second World \Var, Korshikov gave a completely new classification

in his Protococcineae, which was posthumously published in 1953. He divided

this large order into three series, viz. Vacuolales, Protococcales, and genera of doubt-ful systematic position. The series Vacuolales consisted of eight families, viz. theHypnomonadaceae, Actinochloridaceae, Palmellopsidaceae, Chlorangiopsidaceae,Chlorangiaceae, Gloeodendraceae,' Chaetochloridaceae, and the T etrasporaceae ; theseries Protococcales consisted of fourteen families, viz. the. Chlorococcaceae, Characia-ceae, Chlorosphaeraceae, Borodinellaceae, .Protosiphonaceae, Hormotilaceae, Pal-mellaceae, Hydrodictyaceae, Oocystaceae, Ankistrodesmaceae, Protococcaceae, Dic-tyosphaeriaceae, :6otryoc~ccaceae, and the Coelastraceae, and the third series comprisedthe genera Elakatothrix, Raphidonema,and Glaucosphaera. ,-

. .This classifiption is jn reality an extension and elabDration of the classificationgiven by the same author in 1926. In his Protococcineae of 1953",Korshikov not onlyincluded the Vacuolatae, but also a number of palmelloid (both zoosporic and non-

zoosporic) and dendroid genera traditionally included under the Volvocales or Tetras-porales. Other salient ,features of his classification were the inclusion .of theChlorosphaeraceae, Chaetochloridaceae, Botryococcaceae, and the Protococcaceaewithin the order; the Protococcaceae being revived to include genera like Protoc()CCUS,

Rl}diococcus,and Coenoc..occU4;the revision of the genus Characium and allied genera;the creation of a number of new families including the Hypnomonadaceae, Actinochlori-daceae Palmellopsidaceae; Borodinellaceae, and .the Ankistroaesmaceae; and theinclusion of the .subfamilies Golenkinioideae and Treubarioideae under the Chloro-cocc~ceae and the Micractinioideae' under the Coeh\straceae. Apart from reviving thesubfamilies themselves, he revived also their endings in "oideae" which is nowgenerally accepted as more correct than the endings " ieae " or " eae ".

While recording zoospores in Tet~aedronbitridens Beck-Mannegetta, Starr (1954)expressed the opinion that Tetraedron be transferred to the family Hydrodictyaceae, assuggested earlier by Probst (1926). He (1955) also revised the genus Chlorococcumtoinclude Hypnomonas Korshikov, accepting the views of Fritsch and John '(1942) thattheir separation into two genera based on the presence or absence of contractile vacuolesis questionable. 'His comparative study of nine allied genera in the same work hascontributed a great deal to a better understanding of the spherical Chlorococcaceae:

Herndon (1958), after making a detailed study of the Chlorosphaeracean algaeof soil, considered the vegetative division found in the Chlorosphaeraceae of funda-mental importance.to warrant the creation of a separate order, th~ Chlorosphaerales.

iiI

'.;

CLASSIFICATION ANI) PHYLOGENY 61

Under this order he recognized two families, viz. the Chlorosphaeraceae (zoosporic)and the Coccomyxaceae (non-zoosporic). He also suggested the abandoning of theorder Tetrasporales " inasmuch as, in the majority of its genera, cell multiplicationinvolves the formation of individual cells with completely new walls, the daughter cellsbeing quite independent of the parent cell membrane". Further, he suggested theinclusion of all unicellular and colonial Chlorophyceae which are non~motile duringthe vegetative phases of their life cycle and which have no capacity for vegetative celldivision ul}der the Chlorococcales and the delimi ting of the Volvocales to' those unicellulara.nd colonial green algae which are motile during the vegetative phases of theirlife history.

In his Algenkunde,Fott (1959) recognized 12 families under the order Chloroco-ccales, viz. the Chlorococcaceae, Chlorochytriaceae, Characiaceae, Hydrodictyaceae,Micractiniaceae, Gloeocystidaceae, Radiococcaceae, Botryococcaceae, Dictyosphaeria-ceae, Oocystaceae, Scenedesmaceae, and the Protosiphonaceae. Of these, theGloeocystidaceae consist of palmelloid zoosporic genera whereas the Radiococcaceaecomprise palmelloid non-zoosporic genera. 'He also recognized the order Tetrasporalesincluding in it the families Hypnomonadaceae, Chlorangiaceae, Chaetochloridaceae,Tetrasporaceae, and the Characiosiphonaceae. He did not refer to the Chlorosphaera-

-ceae at all. In 1960, he raised Korshikov's subfamily Treubarioideae to the statusof a family, the Treubariaceae, within the order Chlotococcales.

Bourrelly (1958 a, 1958 b, 1959) gave accounts of members of the order Telra-sporales under four families, viz. the Chlorangiaceae, Hypnomonadaceae, Palmellaceae,and Tetrasporaceae. His Chlorangiaceae included CharaciosiphonIyengar andCharaciochloris.Pascher. In 1961(a), he proposed a new classification qf the Chloro-coccales wherein he' recognized only seven families, viz. the Chlorococcaceae,Oocystaceae, Micractiniaceae, Dictyosphaeriaceae, Coelastraceae, Hydrodictyaceae,and the Coccomyxaceae. Of these, detailed account of only the Chlorococcaceae hasbeen published *. Besides Characium and allied genera and Chlorochytriumand alliedgenera, this large family of 45 genera include such genera as Tetraedron,Polyedriopsis,Desmatractum, and Disporopsis. . ,

Thus, the classifications suggested from 1915 onwards, when the order assumeda definite shape, fall under five broad categories': (I) that of Brunnt~aler (1915) inwhich the broad basis-of classification was the mode of repr:.oduc.tion,viz. by- zoosporesor by autospores, (2) that of Geitler (1924) in which the mode of division ofthe cell _

(gradual or simultaneous) at the time of reproduction was alone taken into account,(3) that of Fritsch (1935) in which a number of characters like habitat, habit, chloro-plast, mode of reproduction, etc. were all considered together, (4) that of Tilden (1935)in, which the uninucleate or coenocytic character of the cell was the main criterion,and (5) that of Korshikov (1926,1953) wherein the presence or absence of contractilevacuoles was used for separating.a large, order, the Protococcineae, into two majorseries, the Vacuolales and the Protococcales. Other authors, though mainly foJlowingmodifications of Wille (1897, 1909), Brunnthaler (1915), Fritsch (1935) or Korshikov

. *In the other families subsequently published (Bull. d. Microsc. Appl. (2), 13 (5): 113-43, 1963;.bid, 13 (6):155-86, 1963), interesting features are the inclusion of Botryococcus,Botryosphaera, Actinodesmium

.:.(? Actidesmium), Dictyochlorella, etc., under the Dictyosphaeriaceae, Paradoxia, Dicellula, Radiococcus,Actinastrum, Gloeoactinium, etc., under the Coelastraceae and Elakato/hrix under the Coccomyxaceae.

M CJtLOROCOCCALE~

(1953) have also contributed much, in their own way, in explaining the phylogeny ofthe different families and genera under the order.

Within the past two and a half decades some important additions to our knowledgeof the order have also been made. Iyengar (1936) discovered the unique genusCharaciosiphonwith a protocoenocytic thallus, for which he proposed a new family, theCharaciosiphonaceae. According to him, it has been probably derived from someunicellular ancestor resembling Characiochloris. Korshikov (1937) recorded oogamousreproduction in Golenkinia longispina, G. solitaria, and Micractinium pusillum, and on thisbasis he (1953) separated these species of Golenkinia into a new genus Golenkiniopsis.Oogamy has since been recorded in Dictyosphaeriumindicum Iyengar et Ramanathan(1940), in Golenkiniopsis minutissima (Iyengar et Balakr.) (=Golenkinia minutissimaIyeng. et Balakr.) and in a new genus OocystaeniumGonzalves et Mehra (Oocystaceae),having numerous chloroplasts. Iyengar (1962) has also recorded a new tlendroidattached genus, Dendrocystis, belonging to the Lagerheimioideae.

Zoospore formation has been recorded in a number of genera and species whichwere known to reproduce either by autospores or by autocolonies or in which re-production was not known at all. Thus, zoospores' were recorded iri AcanthosPhaera(G. M. Smith, 1950), DictyosPhaerium ierrestre (Fritsch and John, 1942), Tetraedronbitridens (Starr, 1954), PlanktosPhaetia spp. (Starr, 1954 a; Herndon, 1958 a) and Poly-edriopsis sPinulosa (Korshikov, 1953). It .is on the basis of zoospore formation inAeanthosPhqeraand possibly in allied genera that Smith (1950) has also raised the status ofthe Micractineae to that of a family_ KorshiKov (1953) has also recorded eye-spotsin the daughter cells, of Trochiscia reticltlaris (Fig. 22e), which genus he considers asallied to Golenkinia. According to Starr (1954 a), the discovery of zoospores in Plank-tosphaerlamakes it necessary to transfer this genus from the Oocystaceae, where Smithoriginally placed it, to the spherical Chlorococcaceae. The record of zoospOres inSchroederia(see Korshikov, 1953) also makes it possible t() keep this genus by the side ofCharacium. Schroederia-likealgae with a bifurcate stalk and reproducing by zoosporeshave been 'placed in a new genus Ankyra'by Fott (1957);

, K. P. Singh (1960) recorded macro- and miCro-autospores in a new spec!es of_..Oocystzs from Kumaop Hills, India. - -

01]._the _taxonomic side; though ,a number of new genera (particularly by Pascher,e- Korshikoy and others) have-been added to the Chlorococcales during the past 25,years,

life history studies have definitely proved that a' few genera which were originally -included under the Chlorococcales do not belong to this order at-all or sometimes evento the algae. Thus, Placosphaera Dangeard has been shown (Thompson, 1956) tobe a stage in the life history of Schizochlamys (Volvocales or Tetrasporales). Codiolumgregarium A. Braun and C. petrocelidis Kuckuck have been shown (Jorde, 1933; Fan,1959) to be the sporophytes of Urospora mirabilis Arechoug and SpongomorPhacoalita(Ruprecht) Collins respectively. Some other species are also under suspicion.Instances of wrong references {)f other plant material to the Chlorococcales are"Phytoniorula" Kofoid (19i4), "Thamniastrum" Reinsch (1867) and some speci~of" Cerasterias" (Copeland, 1937; Taft, 1945: G. M. Smith, 1950). These" genera"or "species" become naturally excluded from the Chlorococcales or suppressedaltogether as the case may be;.

CLASSIFICATION AND PHYLOGENY b3

As in the case of a number of species of Characium (Printz, 1927) which have beenfound to belong to Characiopsis (Xanthophyceae) a number of species of Tetraedronare under suspicion because of the existence of " Tetraedron-like" stages in the lifecycles of Pediastrum, Hydrodictyon, and Oocystis (West and Fritsch, 1927 and Fritsch,1935). Some of the described forms could also belong to the resting stages of otheralgae. Similarly," Trochiscia-,stages" are known to occur in the life history ofChlorococcum(seeFritsch and John, 1942). According to West and Fritsch (op. cit.)," the recording of such forms, even as new species. " . . . . . ,admits of their temporaryclassification until they are assigned elsewhere".

Since pyrenoids and starch have not been demonstrated in a number of speciesof Tetraedronand since the chromatophores are also several, Skuja (1948, 1949) referssuch species to new genera under the Xanthophyceae. Bourrelly (1951), Korshikov(1953), Fott (1959), and Fott and Komarek (1960) also do not recognize all the knownspecies of Tetraedron (also see under Tetraedron on plIO). More detailed observa-tions on these doubtful species appear to be warranted to make sure whether there areparallel forms of a}most identical structure belonging to the Chlorococcales as well~ the Xanthophyceae. - ',._

DichotomococcusKorshikov, formerly regarded -as one of the Dictyosphaeriaceae,is also now considered as a member of the Xanthophyceae (Fott and Komarek, 1960).Exactly the reverse has been the case with Botryococcus. Considered as a member of

the Chlorophyceae till 1925 and then transferred by Pascher to .the Heterokontae, thegenus is now 'more or less definitely broug~t back to the Chlorococcales on evidence

pUt forward' by Blackburn (1936), Belcher andFogg (1955) and several others (seeunder Botryococcus).

-' The advent of the electron microscopehas also contributed to a better knowledgeof the internal Structure of a number of algae including the Chloro.coccales (Desikachary,1957, [959). This has been particularly the case with regard to the cell wall, chloro-

. plast, and pyrenoid. ,

, Though these additions to our knowledge during the past quarter of-a century,.have made it necessary for a number of authors t!' revise the classification of theChlorococcales,- even today it cannot be said that a completely satisfactory classificationis available because the affinities of many genera are still imperfectly known. SeveralaUthors still find it convenient _ to separate predqminantly zoosporic families fromnon-zoosporic ones in spite of the fact that it is becoming more and more evident- that

'a number of genera which were formel-ly supposed to be autosporic do reproduceby zoospores under certain favourable conditions. It is, therefore, quite possiblethat the two types of reproduction known to occur in genera like Desmatractum, Octo-gonfella,PlanktosPhaeria,and Tetraedronmight exist in a number of other genera, but oneor other of the methods has apparently become more suitable'in the particular environ-.Dlentin which the alga lives. Sometimes, as in Actidesmiurnand Marthea,a zoospore-

;"'..like stage is passed through before the autospore or auto-colony is formed. The,: :Occasional formation of autospores in ,Characium terrestris (Kanthamma, 1940) and theif..formation of zoospores in DictyosPhaeriumterrestreare also revealing since the formation

,of ZOOsporesand auto-colonies are more common in the respective genera. Though~the aforesaid instances might help in tracing the evolutionary tendencies among the

6"~ CHLOROCOCCALES

showing

(see also,various families and genera, it does not necessarily follow that all gencra

reproduction by zoospores (or by autospores) should be grouped togetherFri tsch, 1935).

The very recent discovery of Trainor (1963) that Scelledesmusobliquusproducesbiflagellate swarmers with a parietal chloroplast, without any pyrenoid and apparentlywithout a cell wall, in basal media from which ammonium nitrate has been excludedand his subsequent finding (Trainor, ill press,from personal communication) that theseswarmers are re'ally gametes which are produced by individuals of different strains(Heterothallic) and which elump in pairs at low temperatures (15°C) in media low innitrogen to form quadriflagellatt:: zygotes, are extremely interesting since it changesour present concept of the genus and necessitates a reconsideration of its relationshipsand classification of this genus. Equally interesting is the record of motility in Coelastrum

(Trainor and Burg, 1964-not scenby the author). According to Trainor (l.c.), thefeasibility of maintaining two families of colonial or coenobial forms like the Hydro-dictyaceae and Coelastraceae (the latter including Scenedesmusand its allies) is q~eg...tionablc, as earlier hinted by Fritsch (1935). Since the above information was

recei,,~ed by the ~uthor after sending-the present work to the press, no changes in theclassification is attempted. It is sufficient to mention here that in three of the pre-dominantly autosporic families, viz. the Oocystaceae, Dictyosphaeriaceae, and theScenedesmaceae and possibly in the Coelastraceae, motile sexual cells have now beenestablished at least in some typical genera.

According to Bendix (1964), who studied the" phenotypic variability in certainChlOrellapyre/loidosQstrains", his observation that Chl;rellaproduces motile cells whichmight be gametes, as observed earlier by Pearsall and Loose (1937), and the record ofbiflagellate swarmers in Scenedesmusobliquusby Trainor (t.c.) support the possibility thatChlorella,an alga presently considered to be asexual, is in fact sexual~ Whether Chlorellareproduces sexually or not, he states that our present concept of its iife cycle must berevised after obtaining more information.

The classification adopted in this account is, one in which the vacu~late coccoidforms are ;etained within the order, whereas Chlorosphaeraand allied g:enera whichposSess definite vegetative cell division and which are placed by Herndon (1958)under the Chlorosphaerales are excluded from the order. - This- is due- to the factthat_ in genera like ChlorococcllTII,Actinochloris,Spongiococcus,and Bicuspidellathere arespe~es with or without contractile vacuoles. As regards Chlorosphaeraand its allies,not only they an: still imperfectly known but also no member has so far been reportedfrom the Indian region. Since the inclusion of truly palmelloid forms within theChlorococcales is a llluch larger question involving discussion of a part of the Volvo-

calc:s, they are also left out of the order as defined here. .The proposal by Starr (1954)to mclude Tetraedron under the Hydrodictyaceae (as originally suggested by 'Probst(1926) on the basis of his observation of zoospores in T. minimum)appears to be further

streng~en.ed by the record of zoospores by Korshikov (1953) in the allied genusPoge~su. Tetraedron and allied genera are, therefore, included here in theHydrodictyac~e as a separate subfamily. The families Radiococcaceae (Fott, 1959)and Tr~u~ceae (¥ott, .1960) are retained along with twelve other more wellknown families to make a ~tal of. 14 families within the order. Following Korshikov

CLASSIFICATION AND PHYLOGENY 65

1. Unicellular or in loose aggregates, rarely colonial; free-living, rarely in association with other plant.or animals; cells usually spherical and with or without contractile vacuoles; chloroplasts onc toseveral; reproduction by zoospores or gametes Fam. Chlorococwceae

2. Macroscopic clusters of club-shaped coenocytes without Cro.5 wal," between protoplasts; cellswith several contractile vacuoles; chloroplast stel1ate; reproduction by zoospores or gametes.. .. ..

Fam. Characiolipho1!aceae

3. Unicellular (rarely colonial); usually attached, rarely free-living; cells usually elongate andsometimes with contractile vacuoles; chloroplast parietal and laminate; reproduction by zoosporesor gametes.. . . . . Fam. Characiaceae

4-. Unicellular; usually endophytic, rarely epiphytic or free-living; cells large and irregularlythickened; chloro\-:...ls axial and massive; reproduction usually by numerous zoosporesorgametes.. . . . . . Fam. Chlorochylriaceae(Endosphaeraceae)

Unicellular or colonial; free-living; cells more or less spherical; ccll wall with ridges, warts,spines, bristles or setae; chloroplast single, parietal and cup-shaped or several and disc-shaped;reprod~ction usually oy autospores, rarely by zoospores or oogamous gametes.. . . . .. .

Fam. Micra£lilliaceae

6. Usually unicellular; free-living or rarely attached; cells spherical, ellipsoid, fusiform or tetrahedric'with projecting ridges at margins; often with setae from angles or ends; cell membrane differentiatedinto two layers, an inner thin firm one and an outer layer made up of two or more parts; reproduc-tion by zoospores (or aplanospores) or autospor~s.. Fam. Treubariaceae

7. Unicellular or in regular free-living colonies; cells tetrahedral or polygonal, sometimcs nearlyspnerical; chloroplast parietal-and laminate; reproduction by zoospores (which usually form auto-colonies) or by autos~ores or gametes Fam. Hydrodic£vaceae

(a) In regular free-living colonies (net-like, flat discs or spherical); celis-polygonal, sometimesnearly spherical; reproduction by zoospores which form auto-colonies or by gametes......

, Subfam. Hydrodictyoideae

Usually unicellular; free-living; cells usually tetrahedric, often with spines from angles,rarely semilunar; reproduction by autospores, rarely by zoospores.. -

Subfam. Telraedrolloideae

(b)

8. Unicellular or colonial; when colonial, usually within a gelatinous envelope; free-living, rarelysymbiotic; cells of different shapes; chloroplasts one to numerous; reproduction by autospores,rarely by oogamousgametes. . . . . . Fam:Oocyslaceae

(a) Usually solitary and planktonic, rarely in attached dendroid"colonies; cells spherical to elli-psoid; cell wall with bristles (rarely verrucose); chloroplasts one or more and parietal; repro-duction by autospores Subfam. Lagerheimioideae

Usually solitary; free-iiving or symbiotic; cells usually spherical or ellipsoid; cell wa1l5moothor sometimes ornamented; chloroplasts one or rarely more; reproduction by autospores......

Subfam. Chlorelloideae

(b)

(c) Unicellular; free-living; cells usually large, spherical, ellipsoia..'br naviculoid; with manydisc-shaped chloroplasts; reproduction by autospores or aplanoSpores, rarely by oogamousgametes Subfam. Eremosphaerdideae