358 Cytologia 19

Studies on the Morphology and Behaviour of the Reproductive Cells of Chaetomorpha Okamurai Ueda

Hiroyuki Hirose Department of Botany, Faculty of Science, Kobe University, Kobe Received April 5, 1954

Contents Page I. Introduction ...... 358 II. Materials and methods ...... 359 III. Observations and discussions ...... 359 1. Fertile filaments ...... 359 2. Gametangia and liberation pores ...... 359 3. Gametes ...... 361 4. Zoospores ...... 361 5. Copulations of gametes ...... 361 6. Types of copulations ...... 363 7. Planozygotes ...... 364 8. Morphological differences among gametes, planozygotes, and zoospores ...... 364 9. Remarks on the copulation phenomena ...... 36410 . Parthenoga metes ...... 365 11. Alternative appearance of reproductive cells ...... 365 12. Relation between sexuality of reproductive cells and phototaxis 366 13. Standstill and germination of planozygotes, zoospores and parthenogametes ...... 367 IV. Summary ...... 368 V. Literatures cited ...... 369 VI. Explanations of plates ...... 370

I. Introduction The algal genus Chaetofaorpha belongs to the family Cladoplioraceae in a class Chlorophyceae and the members of the present genus grow in both marine and fresh waters. Several contributions were accumulated as regards the morphology and behaviour of both sexual and asexual reproductive cells of marine members of this genus. The first report was made by L. K. Rosenvinge (1892a, b) in 1892 and next to this by V Jollos (1926) in 1926. Later in 1929 M. Hartmann (1929) reported on the reducing division of Cltaetoinorpha aerea (Dillw.) Kuetzing. Since 1949 the author made continual observations on the freshwater member of Chaetonzorpha, Ch. Okamurai Ueda (1932) with special reference to the process of the reproductive organ formations, the morphology and behaviour of reproductive cells, and the correlation between seasonal variation of their occurrences and the water temperature, the relation between the 1954 Studies on the Morphology and Behaviour of the Reproductive Cells 359 sexuality of reproductive cells and the phototaxis shown by them , the germi nation of zoospores and zygotes. The author wishes to report several note worthy results among them. Before going further, the author wishes to offer his hearty thanks to Dr . S. Kusunoki, Prof. of Kobe University for his kind suggestions to the present study. The author also must acknowledge that the pecunial support was given to the present study partially by the scientific research promotion fund of the Ministry of Education.

II. Materials and methods The materials used in the present study were those which grew in rivulets or ditches in Himeji and Kobe City in Hyogo Prefecture. The alga was cultured in a glass shell filled with ditch water which was boiled and filtered or Schreiber's solution. Slide glasses to which liberated zoospores or plano zygotes adhered were transferred into another glass shells and cultured . Noland's solution was used as for cilia-staining while Ziel's carbolfuchsin was used for making permanent preparates for the same purpose.

Ill. Observations and discussions 1. Fertile filaments Almost all cells which compose a filament only excepting one or several cells near base become fertile. The number of swarmers produced in a cell totals always more than hundred. Fertile cells are green colored being much deeper than that of vegetative cells. It can hardly be distinguishable whether the swarmers contained in a fertile cell are gametes or zoospores because of the morphological similarity of the shape of swariners unless the diameter of swarmers are measured and the number of cilia are counted.

2. Gametangia and liberation pores There appear mostly one sometimes two and rarely three liberation pores on a fertile cell (Pl. IX, 1). Shape of the pore is always circular, with a clearly defined outline (Pl. X, 2) and the opening of the pore always begins after being convexed outwardly at its position. More precisely speaking, as the swarmers become mature within a sporangium, outer membrane of two stratified cellwall begins to be elevated and finally mamillated (Pl. X, 8, 9). And then the inner membrane of cellwall begins to be opened through a narrow circular opening of small diameter at the place just below the mam illated portion of the outer membrane (Pl. X, 10). While the diameter of the pore which is already perforated through the inner membrane grows to become broader and broader, almost all area of the mamillated portion of the outer-membrane becomes gelatinous and finally melts away to accomplish a liberation pore through cellwall (Pl. X, 8-13). The swarmers just shed out Cytologia, 1954 Plate IX 1954 Studies on the Morphology and Behaviour of the Reproductive Cells 361

of the pore never aggregate densely and never be sorrounded with plasmic

matrix as often seen in Ulotricaceous or other algae , but mostly one-by-one (Pl. X, 1) swim away as soon as they pass through the pore or sometimes more or less forming a loose cluster for a while only when the swarmers

are shed very strongly and fast (Pl. IX , 2, 3). Whenever the diameter of swarmers may be broader than that of the pore, the diameter of the swarmers

easily become so narrower as they can pass through. When the diameter is

shorter than that of the pore, swarmers pass through the pore without any

deformation.

3. Gametes

Gamete shows mostly pear-, ovoid-, or other shapes and is furnished

with two equal cilia issued from anterior end of a cell and a single fusiform,

rod-shaped or narrow laminate stigma which can be easily observable and a

reticular chloroplast with many pyrenoids and moreover many starch grains are seen upon it (Pl. IX, 5-7). The body length is, on the average of 48

individuals, 9.3ƒÊ (minimum 7ƒÊ and maximum 13ƒÊ) and 8.6ƒÊ broad (almost

invariable). The present alga is isogamous just same as reported by L. K.

Rosenvinge (1892 a, b) and M. Hartmann (1929) upon marine Chaetomorpha.

4. Zoospores

Zoospores have just as same morphological characteristics (of same shape,

stigma and chloroplast) as those of gametes only excepting that their breadth

are broader, the length are longer and the number of cilia are always four

(Pl. X, 3-6, 33). The body length is, on the average of 21. individuals, 13.4ƒÊ

(minimum 7ƒÊ and maximum 17ƒÊ) and the breadth is about 12.3ƒÊ. Despite that the minimum body length 7ƒÊ of zoospores is far shorter than the

maximum length of gametes, the dirnentional difference can easily be felt

between the assemblage of zoospores and gametes' one. And the number of

cilia of zoospore is four.

5. Copulations of gametes

Zygote formations are always recognizable in a glass shell which contains a bundle of filaments. They can be also recognized in a glass shell which contains only two filaments and occurs even on a holed cover glass which contains but one filament. Whereas it can be concluded from the results of the above experiments that some portion of filament is occupied by those cells which produce female gametes and certain cells of another portion of the same filament produce male gametes. Therefore, it can be said that single filament becomes fertile both to be female and male.

Whether copulation will occur among those gametes which were produced in the same gametangium, it was examined too. For this purpose, presumably Cytologia, 1954 Plate X 1954 Studies on the Morphology and Behaviour of the Reproductive Cells 363

the most mature cell was cut off from a filament and put on a holed cover glass. Zygote formation was never observed until Sept. 29 in 1952 when the copulation among those gametes produced in the same gametangium was just observed. The author also could encounter, though it was but one occasion , such a gametangium whose liberation pore was bottled with mucous plasm matrix on the way of gamete liberation. Many gametes were moving actively in such a closed gametangium (Pl. IX, 29). With a precise observation on them, particularly large sized and common shaped or large sized and obtused triangular motile cells were seen within a gametangium. The existence of those large sized cells can not be explicable unless it is granted that they are such zygotes as have already copulated in a gametangium. Moreover, as the large sized cells are furnished with two clearly observable stigmas, it will be much affirmative that large sized cells may be zygotes. This phe nomenon must be rather abnormal, however it was verified that there exists sexual difference not only among respective cells on a same fertile filament, but also among those gametes which are produced in the same gametangium. This peculiar uncommon phenomenon was observed in two cases only. There fore further precise observations are necessary. As any morphological differ ences among gametes could not be perceivable whenever they were be libera ted, the present alga is clearly isogamous. M. Hartmann (1929) reported that the sex is defined clearly per each respective single filament of marine Chaetomor-pha, namely sex of all cells which compose a single filament is same. But the present study shows that there exists sexual difference not only among those gametangia which compose a single filament, but also sometimes among those produced in a single gametangium.

6. Types of copulations There are many types as regard wherefrom begins the copulation of two gametes. All types which have been observed are described as bellow. 1) Anterior with anterior: Two gametes adhere to each other at first with their respective anterior ends (Pl. IX, 8). And then fusion of bodies takes its place gradually towards posterior ends (Pl. IX, 9). This is the commonest type. Sometimes anterior portions of two gametes more or less cross each other at their touching place. 2) Anterior with ventral : Anterior end of one gamete adheres to a ventral side of another gamete as if the former was going to be inserted into the latter (Pl. IX, 10). This case is rather rare. 3) Posterior with posterior: Two gametes adhere to each other at first with their posterior ends (Pl. IX, 13-16). And then fusion of bodies takes its place gradually towards anterior ends. Sometimes two respective axes of fused gametes make an almost straight line, whereby fused cells become triangular or rhombic (Pl. IX, 11, 12). Though the process of the fusion of 364 H. Hirose Cytologia 19

this case could not be detected, these triangular or rhombic cells must become finally common pear shaped planozygotes. Next to the first type, this case is the commonest.

4) Posterior with anterior: Anterior end of one gamete adheres to the posterior end of another gamete. The moment of the fusion could not be caught, but the gourd shaped cells (Pl. IX, 17) just after fusion were encoun tered. This is rather rare type.

5) Random-touching: Two gametes gathers together. Keeping to contact with each other always at some portion of their bodies respectively without stopping to rotate on their respective axes, they fuse together gradually always rotating very fast. In this type, wherefrom they begin to fuse, from anterior, from posterior or from ventral sides, it is not perceivable (Pl. IX, 18-20). This type is not so uncommonly encountered. Be that as it may, in any type above-mentioned, two gametes fuse to become ultimately a com plete planozygote with two stigma and with four cilia which show as similar shape as those of zoospores (Pl. IX, 22-24).

7. Planozygotes

It was verified that the zygote of marine Chaetomorpha was planozygote by K. L. Rosenvinge in 1892. The zygote of Chaetonzorpha Okamurai is planozygote too (Pl. IX, 22-24). After the completion of gametic fusion, zygotes of the present alga become as same shapes as of gametes and swim about for a long period-several minutes to several hours. They also lack true cellwall, but are sorrounded with periplasts just like gametes so long as they are moving. Planozygotes are distinguishable from gametes because of their bodies being larger-sized and having four cilia. The average body-length of planozygote is 14.9ƒÊ and the breadth 16.4ƒÊ (the minimum breadth 11ƒÊ and the maximum one 27ƒÊ).

8. Morphological differences among gametes, planozygotes,

and zoospores

Comparative data concerned with the morphological differences among three kinds of swarmers are summarized as shown in the Table 1.

9. Remarks on the copulation phenomena

As soon as the gametes are liberated from gametangia, the almost all gametes begin to copulate among them before they reach not so far from mother filaments (Pl. IX, 4). Copulation was hardly observed among those gametes which gathered together already along the inner wall of a glass shell due to their positive phototaxis. This phenomenon is explicable if we presume that the unions of gametes have been completed before they swimmed up to the glass wall due to the fact that there exists sexual difference among respective cells of a single filament. In most cases, all motile cells which 1954 Studies on the Morphology and Behaviour of the R eproductive Cells 365

are swimming about along the inner wall of a glass shell j ust after liberation due to their positive phototaxis , are comprised of mostly supernumerary gametes of same sex and planozygotes which restored already their normal shape. Hence planozygotes here observed turn back to th e opposite side wall of glass shell soon, it is showed that the phototaxis of plano zygote is negative. Table 1. Comparative diagnosis of morphological characteristic s of swarmers

As both planozygotes and gametes are seen in the same field of vision under the microscope, planozygotes apt to be misunderstood as if they were female gametes due to their larger size than that of gametes . Contrally to this, gametes, due to their smaller size than that of planozygotes , apt to be misunderstood as if they were male gametes. Needless to say, planozygotes are equipped with four cilia and two stigma and gametes with two cilia and one stigma.

10. Parthenogametes For about two months from early September to late October, gametes become to show negative phototaxis more and more strongly and at the same time they copulate hardly or not and finally germinate parthenogenetically. The tendency to grow parthenogenetically become stronger clay by day from early September. It is very interesting that gametes are capable of copulating so far as they show positive phototaxis and if their copulation ability becomes weak or is lost, gametes begin to show negative phototaxis (Table 2).

11. Alternative appearance of reproductive cells The present alga grows all through a year and several generations alter nate during a year. Owing to the two years' observations since 1951, it 366 H. Hirose Cytologia 19 was disclosed that the kinds of reproductive cells (namely zoospores, gametes, and parthenogametes) alternate seasonally. The prosperity or dacay of them are shown in Table 2. As shown in Table 2, gametes are produced almost through a year, but zoospore formation is limited to very short period. Most of freshwater algae produce zoospores for a long period of their own life-cycle and the gamete formation occurs in a limited short period. For instance, according to Edna M. Lind (1932) Ulothrix rorda begins to appear in September and begins to disappear in April in the next year. During the proliferous period their reproduction is always by means of zoospore formation. But in late February, suddenly gamete formation occurs instead of zoospre

Table 2.

formation and this lasts until April. On the contrary, the period of gamete formation of the present alga is very much longer than that of zoospore. Zoospore formations were seen only in two separate months-May and Septem ber. From early September to late September the ability of gametic union is gradually faded and with the decrease of the cases of zygote formation, such increase as those which germinate parthenogenetically. The germination of parthenogametes are much slower than those of normal zygotes.

12. Relation between sexuality of reproductive cells and phototaxis With observations upon phototaxis shown by respective reproductive cells in each month, interesting results were obtained. The summation of these results are shown as below and also in Table 2. 1) Planozygotes show always negative phototaxis. 2) Gametes show positive phototaxis usually, however in September changes from positive to negative one. More precisely speaking, the negative character of phototaxis become stronger and stronger day by day and in late September finally it becomes completely negative. Those gametes whose phototaxis have already become negative never have the gametic union ability, but acquire a new character as parthenogametes. Henceforce the changes of sexual character of gametes to parthenogametic one must have an intimate 1954 Studies on the Morphology and Behaviour of the Reproductive Cells 367 correlation with the changes of physiological character of their own phototaxis . This fact was verified by observations in September of both 1951 and 1952 . This fact can not be explicable by any other than as follows . From April to September garnets never acquire the ability to germinate before they contact with their respective partners for copulation . Nevertheless in September gametes become to have ability to germinate without gametic union , namely partheno gametically. 3) Zoospores show negative phototaxis as a general rule , but this charac ter is not definite and sometimes changes into positive one . This reason is ambiguous. In most algae both gametes and zoospores show positive phototaxis and their zygotes negative. However it is not so in the present alga (Table 2). Until today those algae whose zoospores show negative phototaxis are fewly reported. The above two phenomena must be suggesting a certain principle behind. If above observations and suppositions are all correct , the character of phototaxis shown by every sort of reproductive cells of the present alga can be explicable according to the following hypotheses. Hypothesis I. The phototaxis shown by stigma-containing reproductive cells which have no ability to germinate by themselves is positive. In this case, the union of two cells are always necessary and indispensable as precursor of their germination. Gametes of the present alga belong to this category. Hypothesis II. The phototaxis shown by stigma-containing reproductive cells which have ability to germinate by themselves is negative. The zoospores, planozygotes and parthenogametes of the present alga belong to this category. Though phototaxis of zoospores shown by most other algae is generally positive, the examples of those algae whose zoospores show negative phototaxis will be increased. Hypothesis III. Stigmaless or almost stigmaless reproductive cells do not show definite phototaxis, but they keep random-movement untill they stand still. For example: zoospores of 11aucheria, Tetraspora gelationsa (manuscript) or others.

13. Standstill and germination of planozygotes, zoospores and parthenogametes Almost all planozygotes and zoospores lose their motility in several minutes or hours after being liberated. Those cells which have lost their motility are deprived of cilia and become spherical or subspherical, and cellwalls are newly formed sorrounding them (Pl. X, 29). It takes at least about six hours for zygote and about 3.5 hours for zoospores to complete their cellwall formation. As zygotes which come to a standstill are always furnished with two stigma and two chloroplasts (Pl. IX, 28) and zoospores which come to a

Cytologia 19, 1954 24 368 H. Hirose Cytologia 19

standstill contain single stigma and single chloroplast within (Pl. X, 7), these

two kinds of sporings are easily distinguishable. Both zygotes and zoospores

begin to germinate in four to six days after they stand still. The type of

germination of the present alga belongs to so-called "immediate Filamentous Type" named by S. Inch (1947). Germination type of sporings resulted from

both zygote and zoospore is quite similar merely except for the fact that

stigma disappears in zygote sporings after standstill in five days, but yet remains

in zoospore sporing seven after five days (Pl. X, 14-19, 23-28). For twenty days after the beginning of germination, cells grow to be elongated rhizoid

likely toward one side direction only and reach some 100ƒÊ in its average

length and up to 160ƒÊ long without any lamification, containing many

pyrenoids upon elongated laminate chloroplasts which expand along the inner wall of cell (Pl. X, 20, 21, 30-32). After 54 days sporings grow to be

elongated both towards rhizoidal portion and apical portion of filaments and are composed of a series of three to five elongated cylindrical cells which

are often singly ramified reaching to 30ƒÊ broad (15-20-25-30ƒÊ) and up to

200ƒÊ-300ƒÊ long (the longest one 665ƒÊ) (Pl. X, 22). The germination of

parthenogarnetes is much slower than that of zygotes or zoospores.

IV. Summary

Some new knowledges concerned with reproduction and reproductive cells of a freshwater alga, Chaetonwrpha Okamurai Ueda are summarized as follows:

1) The structure of fertile filaments of the present alga is described.

2) Developmental processes of liberation pore formation are observed.

Inner layer of cellwall firstly opens and later opens outer layer. Shedding out processes of swarmers are also described.

3) Shapes and structure of gametes and zoospores are described and their dimensions are measured too.

4) Sexual fusion takes its place not only among those gametes which are ejected from different filaments as commonly encountered as in most of algae, but also among those gametes ejected from a single filament. Moreoverr sometimes even among those gametes which are produced in a same game

tangium of a single filament sexual fusions are observed.

5) Several types concerned with gamete fusions are enumerated: i.

Anterior with anterior, ii. Anterior with ventral, iii. Posterior with pos

terior, iv. Posterior with anterior and v. Random-touching.

6) Zygotes always become planozygotes and their motility never be weakened for a comparatively long time.

7) In early autumn gametes often stand still and grow to become sporings parthenogenetically. 8) The present alga is perennial and her method of reproduction is

mainly by means of gamete formation through a year only excepting for by 1954 Studies on the Morphology and Behaviour of the Reproductive Cells 369

means of zoospores in early May and in early September.

9) Phototaxis of swarmers of the present alga is differently characterized

by each swarmer, namely positive by gametes and negative by zoospores,

planozygotes and parthenogametes.

10) Zygotes, zoospores and parthenogametes were cultured. The germi

nation type of these three sorts of reproductive cells all belong to Inoh's "Immediate Filamentous Type" After 54 days' culture of zygotes and

zoospores, their sporings grew to reach up to 665ƒÊ in length being composed

of two to five much elongated cells.

V. Literatures cited

Foyn, B. 1929. Untersuchungen uber die Sexualitat und Entwicklung der Algen IV. Vorlaufige Mitteilung uber dia Sexualitat und den Generationsweehsel von

Cladophora und Ulxa. Ber. Dtsch. Bot. Ges. 47: 495-506.

Fritsch, F. E. 1935. The structure and reproduction of the algae. Vol. I. Hartmann, M. 1929. Untersuchungen caber die Sexualitat und Entwicklung von Algen III. Uber die Sexualitat und den Generationswechsel von Chaetomorpha und

Enteroinorpha. Ber. Disch. Bot. Ges. 47: 485-494.

Hirose, H. 1938. On the Chaetomorpha. Okaoiurai found in Hyogo Prefecture (in Jap.).

H yogo Hakubutsu Gakkai Shi 15: 181-182. Inoh, S. 1947. Morphogenesis of marine algae (in Jap.). Jollos, V. 1926. Untersuchungen uber die Sexualitdtsverhaltnisse von Dasycladus

claxaeformis. Biol. Centralbl. 46: 279-295. Kusunoki, S. 1944. Zur Fortpflanzung der Konjugaten. Cytologia 13: 316-322.

Lind, E. M. 1932. A contribution to the life-history and cytology of the species of Ulothrix. Ann. of Bot. 45: 711-725

Okada, Y. 1939. Nippon Inkwasyokubutu Dukan. p. 111, fig. 4-8. (in Jap.).

Rosenvinge, L. K. 1892a. Om nogle vaextforhold hos slaegterne Cladophora og Chaeto morpha. Bot. Tidsskr. 18: 29-58.

- 1892b. Sur quelques phenomenes de croissance ches les Cladophora et (Jhactomorpha. Ibid. 18: 59-64.

Smith, G. M. 1951. Sexuality of algae in Manual of Phycology pp. 229-242.

Ueda, S. 1932. New freshwater species of Chaetornorpha. Journ. of Imp. Fisher. Inst.

27: 21-24 (in Jap. & Engl.).

24 Cytologia 19, 1954 370 11. Hirose Cytologia 19

Explanations of plates IX and X

Plate IX

1. Structure of gametangia; the lowest cell has a pore just before liberation and the upper two gametangia are already empty; the lower cell of empty gametangia has two pores and the upper one has a pore. •~ca. 100. 2. Gametangiurn just beginning of liberation of gametes. Neighbouring two cells are already empty. •~ca. 200. 3. Game tangium whose shedding of gametes just ended. •~130. 4. Copulation of gametes occurring at the place not so far from mother gametangial filament. •~ca. 80. 5-6. Gametes stained with Noland's solution. 7. Two cilia, one stigma and a chloroplast

filled with starch grains of gamete. 8. "Anterior with anterior" type of copulation of gametes. 9. More developed stage of the same. 10. "Anterior with ventral" type of copulation of gametes. 11, 12. "Posterior with posterior" type of copulation of gametes in the case of two respective axes of fused gametes making almost straight line. 13-16. "Posterior with posterior" type of copulation of gametes. 17. "Posterior with anterior" type of copulation of gametes. 18-20. "Random-touching" type of copu

lation of gametes. 18. Two gametes keeping to contact with each other. 19. They begin to fuse. 20. Much more fused stage. 21. Fused cell which is restoring normal shape. 22-24. Planozygotes with four cilia and two stigmas. 25-27. Three stages of gametic fusion showing two stigmas and many starch grains scattered within.

28. Standstill zygote, well defined cellwall already formed and two stigmas yet recog nizable within. 29. A portion of gametangium whose liberation pore is accidently bottled with plasmic substance. A rhombic planozygote is seen among subspherieal

gametes. 5-29, •~1050.

Plate X

1. Gametangia and liberation of gametes through a pore. •~264. 2. Liberation pore

of sporangium. •~200. 3. Schematic drawing of zoospore showing four cilia, one stigma and a reticular chloroplastt with many pyrenoids. •~ca. 2000. 4. Zoospore stained with Noland's solution. 5. Reticular chloroplast and pyrenoids of zoospore. 6. Four cilia and kinoplasm of zoospore. 7. Standstill zoospore with well defined cellwall already formed and a single stigma yet conspicuously recognizable within. 8-13. Developmental processes of liberation pore formation are schematically drawn. 14-19. Sporings re sulted from zygotes four days after liberation. Stigma already disappeared. 20-21.

Germlings resulted from zygotes 27 days after gametic fusion. Celldivision not yet occurred. 23. Germlings 54 days after gametic fusion, already ramified. 23-26.

Sporings resulted from zoospores, six days after they come to a standstill and a stigma

yet remained and clearly visible within. 27-28. Sporings resulted from zoospores whose cellwalls closely contacted with each other. 29. Standstill zygotes with their own cellwall just newly formed. 30-32. Sporings originated fromz ygote. 30. After twenty (lays since gametic fusion. 31 and 32. After 54 days since gametic fusion.

33. Zoospores stained with Noland's solution. •~530. 4-7, 23-26, •~1050. 14-22, 27-29,

•~ 600. 30, •~520. 31, •~66. 32, •~170.