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J. Phycol. 1,34-38 (1965). Trichosarcina polymorpha Gen. et Sp. Nov. H. WAYNE NICHOLS AND HAROLD C. BOLD Departments of Botany, Washington University, St. Louis, Missouri, and The University of Texas, Austin, Texas SUMMARY Trichosarcina polymorpha gen. et sp. nov. is new- ly described as a member of the Ulvales, Schizo- meridaceae. The life cycle includes a uniseriate, Horrnidium-like phase; a pluriseriate stage, and, finally, a chain of sarcinoidpackets which may dis- sociate. The cells are uninucleate, with a parietal chloroplast and single pyrenoid. Quadriflagellate zoospores are produced singly by cells of the pluri- seriate and sarcinoid stages. Sexual reproduction was not observed. The alga herein described as Trichosarcina poly- morpha occurs in shallow, temporary pools in granitic rocks in Llano County, Texas. It was first encountered at Enchanted Rock' in November, 1960, was collected again in October, 1961 , from the same general area, near Balanced Ro&, and has been collected several times since especially during the winter months. These pools are intermittently filled with rain water and harbor such other aquatics as Zsoetes melampoda and several sedges. The pH of the water usually ranges between 6.3 and 6.9. Unialgal cultures, in which the plants corresponded in all respects to those studied from natural collections, were isolated by removing single, pluriseriate branches from the natural collections, rinsing them in distilled water and inoculating them into tubes of soil-water medium and Bold's Basalmedium2. The alga grows luxuriantly in the inorganic medium and less so in the same medium with agar and in media with soil-extracts. Unialgal and axenic cultures were 'For a general account of the physiography of the Enchanted Rock region and its soil algal flora see (2.3) and Bold (1 963). a "Bold's Basal Medium" (BBM) was prepared as follows (3): Six stock solutions 400 ml involume wereemployed, each contain- ing one of the following salts in the concentration listed: NaN4 . . . . . . . . .lO.Og &HPO, . . . , - . . . .3.Og CaC1,.ZH20. . . . . . 1.Og KH2PO+ . . . . . . . . .7.Og MgS0+.7H20.. . . . . 3.0g NaCl . . . . . . . . . . .l.Og achieved by the familiar techniques of streaking and/or plating, followed by selection of bacteria-free colonies. Repeated transfer of axenic culture in the basal medium (without agar) during several years has demonstrated the absence of special growth-factor requirements. OBSERVATIONS Inasmuch as Trichosarcina produces zoospores in natural habitats and in culture, it is convenient to des- cribe ontogeny beginning with these agents of asexual reproduction The individual quadriflagellate zoospores range from spherical to ovoid (Fig. 1, 19) and are approximately 6 x 4 p. Each has 4 anterior contractile Figs. 1-1 7. Trichosarcina polymorpha gen et sp. nov. Fig. 1. Motile zoospore, X 2666. Fig. 2. Quiescent zoospores. X 2166. Figs. 3-7. Successively older germlings from zoospores, X 1200. Fig. 8. Cellular organization in uniseriate filament, X 1715. Fig. 9. Apex of uniseriate filament, X 857. Fig. 10. Cellular detail in apical region of uniseriate filament, X 857. Fig. 11. Early sporogenesis inyoungpluriseriate filament; note appearance of stigma before zoospore release, X 1715. Fig. 12. Young pluriseriate filament, diagrammatic, X 171 5. Figs. 13-1 5. Varia- tion in pluriseriate holdfasts, X 857. Fig. 16. Ruriseriate fila- ment; note early sarcinoid appearance, X 857. Fig. 17. Pluri- senate filament with loosely associated packets of cells, X 857. To 940 ml of distilled water were added 10 ml of each stock solution and 1.0 ml of each ofthe4 stock. trace-element solutions prepared as follows: (1 ) 50 g EDTA and 31 g KOH dissolved in 1 liter Ha 0. (2) 4.98 g FeS04.7Ha0 dissolved in 1 liter acidified H20. (Acidified HaO: 1.0 ml &SO4 added to 999 ml distilled water.) (3) 11.42 g H 3 B 4 dissolved in 1 liter H20. (4) The following, in amounts indicated, all dissolvedinl liter HaO: ZnSO4.7HaO. 8.82 g; MnCla.4Ha0, 1.44 g; MoG, 0.71 g; CuS04.5Ha0, 1.57 g; Co(N4). 6Ha0, 0.49 g. 34

Trichosarcina polymorpha Gen. et Sp. Nov

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Page 1: Trichosarcina polymorpha Gen. et Sp. Nov

J . Phycol. 1,34-38 (1965).

Trichosarcina polymorpha Gen. e t Sp. Nov.

H. WAYNE NICHOLS AND HAROLD C. BOLD

Departments o f Botany, Washington University, St. Louis, Missouri, and The University of Texas,

Austin, Texas

S U M M A R Y

Tr ichosa rc ina polymorpha gen. e t sp. nov. is new- l y described as a member of the Ulvales, Schizo- meridaceae. The life cycle includes a uniseriate, Horrnidium-like phase; a pluriseriate stage, and, finally, a chain o f sarcinoidpackets which m a y dis- sociate. The cel ls are uninucleate, with a parietal chloroplast and single pyrenoid. Quadriflagellate zoospores are produced singly by cel ls of the pluri- seriate and sarcinoid stages. Sexual reproduction was not observed.

The alga herein described as Trichosarcina poly- morpha occurs in shallow, temporary pools in granitic rocks in Llano County, Texas. It was first encountered at Enchanted Rock' in November, 1960, was collected again in October, 1961 , from the same general area, near Balanced Ro&, and has been collected several times since especially during the winter months. These pools a re intermittently filled with rain water and harbor such other aquatics as Zsoetes melampoda and several sedges. The pH of the water usually ranges between 6.3 and 6.9. Unialgal cultures, in which the plants corresponded in all respects to those studied from natural collections, were isolated by removing single, pluriseriate branches from the natural collections, rinsing them in distilled water and inoculating them into tubes of soil-water medium and Bold's Basalmedium2. The alga grows luxuriantly in the inorganic medium and l e s s so in the same medium with agar and in media with soil-extracts. Unialgal and axenic cultures were

'For a general account of the physiography of the Enchanted Rock region and i ts soil algal flora see (2.3) and Bold (1 963).

a "Bold's Basal Medium" (BBM) was prepared as follows (3): Six stock solutions 400 ml involume wereemployed, each contain- ing one of the following salts in the concentration listed:

N a N 4 . . . . . . . . .lO.Og &HPO, . . . , - . . . .3.Og CaC1,.ZH20. . . . . . 1.Og KH2PO+ . . . . . . . . .7.Og M g S 0 + . 7 H 2 0 . . . . . . 3.0g NaCl . . . . . . . . . . .l.Og

achieved by the familiar techniques of streaking and/or plating, followed by selection of bacteria-free colonies. Repeated transfer of axenic culture in the basal medium (without agar) during several years has demonstrated the absence of special growth-factor requirements.

OBSERVATIONS

Inasmuch a s Trichosarcina produces zoospores in natural habitats and in culture, i t is convenient to des- cribe ontogeny beginning with these agents of asexual reproduction The individual quadriflagellate zoospores range from spherical to ovoid (Fig. 1, 19) and a re approximately 6 x 4 p. Each has 4 anterior contractile

Figs. 1-1 7. T r i chosa rc ina polymorpha g e n et sp. nov. Fig. 1. Motile zoospore, X 2666. Fig. 2. Quiescent zoospores. X 2166. Figs. 3-7. Successively older germlings from zoospores, X 1200. Fig. 8. Cellular organization in uniseriate filament, X 1715. Fig. 9. Apex of uniseriate filament, X 857. Fig. 10. Cellular detail i n apical region of uniseriate filament, X 857. Fig. 11. Early sporogenesis inyoungpluriseriate filament; note appearance of stigma before zoospore release, X 1715. Fig. 12. Young pluriseriate filament, diagrammatic, X 171 5. Figs. 13-1 5. Varia- tion in pluriseriate holdfasts, X 857. Fig. 16. Ruriser ia te fila- ment; note early sarcinoid appearance, X 857. Fig. 17. Pluri- senate filament with loosely associated packets of cells, X 857.

To 940 ml of distilled water were added 10 ml of each stock solution and 1.0 ml of each ofthe4 stock. trace-element solutions prepared as follows:

(1 ) 50 g EDTA and 31 g KOH dissolved in 1 liter Ha 0. (2) 4.98 g FeS04.7Ha0 dissolved in 1 l i ter acidified H20 .

(Acidified HaO: 1.0 ml &SO4 added to 999 ml distilled water.)

(3) 11.42 g H 3 B 4 dissolved in 1 l i ter H20. (4) The following, in amounts indicated, all dissolvedinl l i ter

HaO: ZnSO4.7HaO. 8.82 g; MnCla.4Ha0, 1.44 g; M o G , 0.71 g; CuS04.5Ha0, 1.57 g; Co(N4) . 6Ha0, 0.49 g.

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8 4

6

10

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NICHOLS & BOLD: Trichosarcina polymorpha Gen. et Sp. Nov.

vacuoles and a parietal chloroplast with single pyrenoid and prominent equatorial stigma. A single equatorial to slightly anterior, nucleus is embedded in the colorless cytoplasm. Motility of zoospores is brief and rarely exceeds 3 min from the time of emergence.

Upon quiescence, the zoospores withdrawtheir flagella, elongate slightly (Fig. 2, 20), and within an hour undergo a transverse division (Fig. 3, 4). The 2-celled germling usually exhibits polarity in that the proximal cell elongates as a primary holdfast (Fig. 4-6, 21 ), while the other continues to divide transversely to form a distal uniseriate filament (Fig. 8, 22, 23).in which growth is generalized. The apical cell of each filament is bluntly rounded, never acuminate (Fig. 9 , 22). The component cells a r e cylindrical with straight sides or markedly tumid (Fig. In older filaments the primary holdfast grows into a simple or forked. pluriseriate structure (Fig. 13-15, 25). The prosuate holdfast secretes copious colloidal material, as revealed by the India ink test, while the erect system is devoid of such a matrix. The filaments are unbranched through- out development.

Cellular organization of Trichosarcina polymorppha is Hormidium-like inthe young filaments (Fig. 8,10, 21 - 23). Each cell contains a prominent, incomplete, band- like chloroplast which, in turn, contains a single pyrenoid. The latter typically i s surrounded by two starch segments (Fig. 8, 11 ). The plastid usually is less than cell length A single nucleus lies in the colorless cytoplasm.

Although the uniseriate condition may continue for long periods in actively growing cultures in liquid media, the pluriseriate condition arises ultimately by longitudin- al divisions in some or all of the cells of each filament (Fig. 11, 12, 26). Individuals a r e occasionally observed in which pluriseriate regions a re separated by inter- calary, uniseriate segments. Two longitudinal divisions in perpendicular planes result in delicate cylinders composed of 4 rows of cells (Fig. 11, 12). Continuing

Figs. 18-33. T r i chosa rc ina polymorpha. Fig. 18. Electron micrograph of segment of pluriseriate filament. Note organization of cell walls; ch, chloroplast; 1. w., outer wall layer along lateral surface; m, mitochondrion; n. nucleus; s , starch grain; t. w., outer wall layer of transverse wall, X 8400. Fig. 19. Living zoospores at quiescence X 1 666. Figs. 20,Z 1. Zoospore germina- tion, X 1666. Fig. 22. Apical region of uniseriate filament, x 857. Fig. 23. Segment of uniseriate stage; note band-shaped chloroplasts, each with a pyrenoid. X 857. Fig. 24. Low-power view of a fresh mouth from a month-old culture in Bold’s Basal medium; note uniseriate and variously pluriseriate stages, X 142. Fig. 25. Holdfast supporting divergent uniseriate filaments, 1000. Fig. 26. Stages in attainment of pluriseriate condition, 428. Fig. 27. Late sarcinoid stage, dissociation of packets in

Progress, X 142. Fig. 28. Enlarged view of sarcinoid packets, their dissociation more advanced than in Fig. 27, X 570.

longitudinal and transverse divisions of original cell of the primary filament and i ts derivatives transform the filament into a series of sarcinoid packets (Fig. 16-1 7, 24, 26) which ultimately may dissociate (Fig. 17,27, 28). Dissociation occurs also in agar cultures. Upon com- pletion of dissociation, all evidence of filamentous organization may be lost (Fig. 28).

The uniseriate filaments a re about 7 p, while the pluriseriate stages may exceed 200 p in diameter. The filaments may become several hundred cells long. Filamentous plants may become detached from their holdfasts, apparently by dissolution of their colloidal matrices, or by fragmentation and become free-floating in the culture vessel and in the granitic pools where the plant was collected.

The sole methods of reproduction so far observed i n Trichosarcina polymorpha are by zoospores and by fragmentation and dissociation of the pluriseriate fila- ments, especially in the sarcinoid stage. Only the pluri- seriate filaments and sarcinoid packets have been ob- served to undergo zoosporogenesis. Cells of intercalary, uniseriate regions of mature plants disintegrate when the pluriseriate regions form zoospores.

Zoosporogenesis can be evoked readily by transfer of pluriseriate filaments o r their sarcinoid derivatives to fresh culture media. They respond within 24 hr by form- ing zoospores (Fig. 11 ). Impending zoosporogenesis i s manifested by the appearance of stigmata and contractile vacuoles in the vegetative cells. Each of the latter usually liberates a single zoospore, and the empty parenchymatous mother cell walls persist for some time in axenic cultures. Zoospore release may be almost simultaneous i n some instances.

Electron micrographs of certain stages in the life cycle have provided information regarding cellular organization, especially with respect to the cell walls (Fig. 18). From such micrographs, it i s clear that in the uniseriate filament, each protoplast is surrounded completely by an inner wall layer. This, in turn, is covered by an outer layer continuous over the lateral cell surfaces but less-well developed between contiguous cells which have recently arisen by division. In the older, multiseriate stages (Fig. 1 a), the ‘‘outer” wall layer (1.w.) also is deposited on the transverse (t.w.) and, subsequently on the internal longitudinal, cell faces. In older packets of the sarcinoid stages, limits between outer and inner wall layers become obliterated.

Special study was not made of the cellular organelles, but it may be mentioned that the uninucleate cells contain, as expected, (Fig. 18) a massive, lamellate chloroplast, (ch) with a single pyrenoid, mitochondria, (m) Golgi bodies, endoplasmic reticulum, vacuoles and ad- ditional unidentified particles. Both autochthonous (Fig. 18, 5) and pyrenoid starch occur. The pyrenoid is characteristically bisected by 2 double lamellae with intrude from the chloroplast.

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NICHOLS 81 BOLD: Trichosarcina polymorpha Gen. e t Sp. Nov.

DISCUSSION

After search of the literature, discussion with several phycologists, and appraisal of the attributes ofthe organ- ism herein described. the authors were impelled to assign i t to a new taxon, Trichosar’cina. Pluriseriate, zoosporiferous Chlorophyceae a re unknown, except i n the Ulvales and Chaetophorales. One immediately recalls such genera a s P e r c u r s a r i a , the early stages of Ulva and Enteromorpha, and especially, Schizomeris and Fri tschiel la , a s examples of the pluriseriate condition.

Trichosarcina is, perhaps, most like Schizomeris

and the terrestrial F r i t s ch ie l l a but differs from the former in lacking ring-like wall thickenings, in having cells with a single pyrenoid, and in i ts characteristic Sarcina-like configuration and dissociation of the latter into packets, which suggest strikinglythe chlorosphaera- cean genus, Chlorosarcinopsis (6, 1). It is unlike F r i t s ch ie l l a in habitat and in lack of branching and differentiation into prostrate and aerial branches. Fur- thermore, unlike other algae in Trichosarcina, termin- ation of increase in plant length at the onset of the pluriseriate condition and restriction of zoosporogenesis to the sarcinoid phase emphasize that the latter i s a stage in maturation and completion of ontogeny.

Three possibilities occurred to the writers regarding the affinities of the genus Trichosarcinar (1 ) It might be considered a s a member of the Schizomeridaceae; (2) It might be assigned to the Ulvaceae; or, (3) It might be classified in a new family of i ts own.

The writers, at present, prefer the first alternative. The affinities of Trichosarcina a re probably with the filamentous Ulotrichales and the Chlorosphaerales both of which undergo “true vegetative” (5, 6, 4) or parechy- matous cell division which is lacking in the Volvacales, Tetrasporales and Chlorococcales.

The new organism is described as follows: Trichosarcina polymorpha gen. et sp. nov.

Plantae in tribus formis sequentialiter affinibus re- pertae; primum filamento uniseriato Hormidio simili; deinde filamento pluriseriato; demum catena fasciculor- um. Plantae conditionibus novioribus affixae, postea per fragmentationem libere fluitantes. Cellulae uninucleatae, chloroplasto parietali unicum pyrenoideum continente praedito.

Reprcductio per fragmentationem fasciculorum sar- cinoideorum atque per zoosporas in conditionibus pluri- seriatis sarcinoideisque effectas; zoosporae una una- quaque in cellula, sphericales ad ovatas (6 x 4 p),

quadriflagellatae, quattuor vacuolas pulsantes anteriores, unicum pyrenoideum, unicum nucleum necnon stigma equatoriale habentes.

Reproductio sexualis non observata. Plantae in stagnis graniticis in loco Enchanted Rock

et Balanced Rock, Llano County, Texas dicto, an. 1960, 1961 repertae.

plants occurring in three sequentially related forms: first a uniseriate Hormidium-like filament; second, a pluriseriate filament; and third, a chain of sarcinoid packets. Earlier stages attached, later free-floating by .fragmentation. Cells uninucleate, with a parietal chloro- plast containing a single pyrenoid.

Reproduction by fragmentation of sarcinoid packets and by zoospores produced in pluriseriate and sarcinoid stages; zoospores one per cell, spherical to ovoid (6 X 4 p), quadriflagellate with 4 anterior contractile vacuoles, single pyrenoid and nucleus, and equatorial stigma.

Sexual reproduction not observed. Enchanted Rock and Balanced Rock, granitic pools;

Llano County, Texas - 1960. 1961. Axenic cultures of the organism ave been deposited

in the Culture Collection of Algae, Indiana University. Herbarium specimens have been deposited in the Chicago Natural History Museum.

REFERENCES 1. Arce, G., & Bold, H. C. 1958. Some Chlorophyceae from

Cuban Soils. Amer. Jour. Bot. 45,492-503. 2. Bischoff. H. W. 1963. Phycological Studies. I . The Soil

flora of Enchanted Rock. PhD. Cissertation. The University of Texas.

Bischoff, H. W.. & Bold, H. C. 1963. Phycological Studies. I V. Some soil algae from Enchanted Rock and related algal species. Univ. Texas Publ. No. 6318.

4. Deason, T. R., & Bold, H. C. 1960. Phycological Studies. I . Exploratory studies of Texas soil algae. Univ. Texas Publ. No. 6022.

5. Fritsch. F. E. 1935. The structure and reproductionof the algae. I . Univ. Press , Cambridge.

6. Herndon, W. R. 1958. Studies on Chlorosphaeracean algae from soil. Amer. Jour. Bot. 45,298-308.

3.

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