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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
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.

SUMMARY

T r i c h o s a r c i n a polymorpha gen. e t sp. nov. i s newl y described as a m e m b e r of the Ulvales, Schizomeridaceae. The life cycle includes a uniseriate,
Horrnidium-like phase; a p l u r i s e r i a t e stage, and,
finally, a chain o f sarcinoidpackets which m a y d i s sociate. The c e l l s a r e uninucleate, with a parietal
chloroplast and single pyrenoid. Quadriflagellate
z o o s p o r e s a r e produced singly by c e l l s of the pluris e r i a t e and sarcinoid stages. Sexual reproduction
was not observed.

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

The alga herein described a s Trichosarcina polymorpha occurs i n shallow, temporary pools in granitic
rocks in Llano County, Texas. It was first encountered
a t 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 r e intermittently filled with rain water and harbor
such other aquatics a s 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

Figs. 1-1 7. T r i c h o s a r c i n a 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 1 7 1 5. Figs. 13-1 5. Variation i n pluriseriate holdfasts, X 857. Fig. 16. R u r i s e r i a t e filament; note early sarcinoid appearance, X 857. Fig. 17. Plurisenate filament with loosely associated packets of cells, X 857.

To 940 ml of distilled water were added 1 0 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 H 2 0 .
(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; M o G ,
0.71 g; CuS04.5Ha0, 1.57 g; C o ( N 4 ) . 6Ha0, 0.49 g.

'For a general account of the physiography of the Enchanted


Rock region and i t s 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 containing one of the following salts in the concentration listed:

. .lO.Og
. . . 1.Og
. . 3.0g

NaN4
... ..
CaC1,.ZH20. . .
MgS0+.7H20.. . .

. . . , - . . . .3.Og
. . . . . . . . .7.Og
. . . . . . . . . .l.Og

&HPO,
KH2PO+
NaCl .

34

0
3

10

35

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


longitudinal and transverse divisions of original cell
of the primary filament and i t s 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 completion of dissociation, all evidence of filamentous
organization may be lost (Fig. 28).
The uniseriate filaments a r e 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
T r i c h o s a r c i n a polymorpha a r e by zoospores and by
fragmentation and dissociation of the pluriseriate filaments, especially in the sarcinoid stage. Only the pluriseriate filaments and sarcinoid packets have been observed 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 forming 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, a s expected, (Fig. 18) a massive, lamellate
chloroplast, (ch) with a single pyrenoid, mitochondria, (m)
Golgi bodies, endoplasmic reticulum, vacuoles and additional 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.

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 a s 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, a s revealed
by the India ink test, while the erect system is devoid
of such a matrix. The filaments a r e unbranched throughout development.
Cellular organization of T r i c h o s a r c i n a polymorppha
is Hormidium-like inthe young filaments (Fig. 8,10, 21 23). Each cell contains a prominent, incomplete, bandlike 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 longitudinal 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 r e separated by intercalary, 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 c h o s a r c i n a 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 1666. Figs. 20,Z 1. Zoospore germination, 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 Bolds 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.

37

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


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
e t 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 chloroplast 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.

DISCUSSION
After search of the literature, discussion with several
phycologists, and appraisal of the attributes ofthe organism herein described. the authors were impelled to
assign it to a new taxon, Trichosarcina. Pluriseriate,
zoosporiferous Chlorophyceae a r e 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, S c h i z o m e r i s
and F r i t s c h i e l l a , a s examples of the pluriseriate
condition.
T r i c h o s a r c i n a is, perhaps, most like S c h i z o m e r i s
and the terrestrial F r i t s c h i e l l a but differs from the
former in lacking ring-like wall thickenings, in having
cells with a single pyrenoid, and in i t s characteristic
Sarcina-like configuration and dissociation of the latter
into packets, which suggest strikinglythe chlorosphaeracean genus, C h l o r o s a r c i n o p s i s (6, 1). It is unlike
F r i t s c h i e l l a in habitat and in lack of branching and
differentiation into prostrate and aerial branches. Furthermore, unlike other algae in Trichosarcina, termination 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 T r i c h o s a r c i n a r (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 its own.
The writers, at present, prefer the first alternative.
The affinities of T r i c h o s a r c i n a a r e probably with the
filamentous Ulotrichales and the Chlorosphaerales both
of which undergo true vegetative (5, 6, 4) or parechymatous cell division which is lacking in the Volvacales,
Tetrasporales and Chlorococcales.
The new organism is described as follows:
T r i c h o s a r c i n a polymorpha gen. et sp. nov.
Plantae in tribus formis sequentialiter affinibus repertae; primum filamento uniseriato Hormidio simili;
deinde filamento pluriseriato; demum catena fasciculorum. Plantae conditionibus novioribus affixae, postea per
fragmentationem libere fluitantes. Cellulae uninucleatae,
chloroplasto parietali unicum pyrenoideum continente
praedito.
Reprcductio per fragmentationem fasciculorum s a r cinoideorum atque per zoosporas in conditionibus pluriseriatis sarcinoideisque effectas; zoosporae una unaquaque in cellula, sphericales ad ovatas (6 x 4 p),

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.
3. 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. P r e s s , Cambridge.
6. Herndon, W. R. 1958. Studies on Chlorosphaeracean algae
from soil. Amer. Jour. Bot. 45,298-308.

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