じʻ尚ﾉ
ノ
謎
、
グ
鷺
〆
擬
；
&夢
I
窪
一
イ
ー
錨
、
〆
＝ 〆
?』
､
"
P H YC O L O G I C A L S T U D I E S
r謹
騒亀；ご-
IV. Some Soil Algae from Enchanted Rock and
Related AIgal Species
HARRY
W.
BISCHOFF
AND
HAROLD
C.
古
旬
BOLD
峨
守
THE
UNIVERSITY
OF
TEXAS
Publication N⑪・6318
P U B L I C AT I O N
September l5, 1963
熱
諺
心蝿
、
γ
躍
芽
、
渓
蕊鶏
薮
、
鰯
瀞
罫
奪
到
が
悪
態
込
可
蕊
蝿惣鐘郷； 蕊 蕊一
蓉
凸
…
．､些甚、
‫ب ـ ـ ـ ــاه‬،‫إةاخ ـ ـ ـ ــر‬
Ideological· Stadies‫؟‬
IV. Some Soil Algae from Enchanted Rock and
Related. Algal Species
Ijy
Harry Ỉ. BiscHOFF^ AND Harold C. Bold
1Department of Biology, Texas Lutheran College, Seguin, Texas.
2Department of Botany, The University of Texas, Austin, Texas.
Acknowledgments
The Investigations herein summarized were supported in part by grants from
the National Institutes of Health and National Science Foundation (NSF G-18746
and NIH GM-8211 to the junior author and an NSF Faculty Fellowship to the
senior author during 1959-60). The senior author also received astipend from the
NSF Research Participation Program Grant to the Department of Botany, University of Texas, during the summers of 1961-63.
We are grateful to Dr. Hannah Croasdale of Dartmouth College for translating
the speci c diagnoses into Latin.
The editorial assistance of Mrs. Frances Denny is acknowledged with gratitude.
Finally, we are grateful for support from the Publications Fund of the Graduate
fi
Sc.hool of The University of Texas.
Ta b l e o f C o n t e n t s
Page
Acknowledgment
3
Introductio
7
Materials and Method
9
Distidbutloii ol the soil algae on Enchanted Rock
14
Organisms new to science or O'therwise signi cant
19
Chlamydomonas megalis sp. no
19
ChloTococcunr ţ‫؛‬ol"١٠١moîi)h,uisp.Îvo١i
22
Chlorococcum minutum Star
24
Spongiochloris gigantea sp. no
25
Spongiochloris llanoensis sp. no
27
Summary ol speci c attributes i
.
Key to the currently known species o S t a r r
29
.
32
Neochloris pseudostigmatica sp. no
32
35
Key to the currently known species ol Aể0C٨ốrỉ'r Starr .
Planktosphaeria iexensis&v٠ nov. .
36
Planktosphaeria maxima sp. no
39
Ourococcus multisporus sp. no
42
The genus Bracteacoccus Tere
43
Bracteacoccus gigantens sp. no
4 4
Bracteacoccus medionucleaius sp. no
47
Bracteacoccus grandis sp. no
48
Bracteacoccus aerius sp. no
5 0
Bracteacoccus pseudominor sp. no
52
Bracteacoccus cohaerens sp. no
5 4
Summary ol speci c attributes in ßracieacoccMr
.
58
.
.
.
.
.
fi
.
l
v
v
v
v
v
v
v
n
g
v
v
v
v
66
v
Illustrations
r
60
fi
Literature Cited
fi
59
s
Summary
s
n
Key to the currently known species ol Bracteacoccus (Tereg) Starr
56
Introduction
Previous exploratory studies of the soil algal ora of the Carrizo Sands formation
of Texas by Deason and Bold (1960) suggested to the writers an investigation of
the soil algal ora from amore isolated and localized area. The station selected was
in Llano County, Texas, one-half mile north of the Llano-Gillespie County line,
on the summit of aporphyritic, leucogranitic, exposed batholith known as Enchanted Rock (Latitude 30. 30' 22" north and Longitude 98. 49' 06" west).
Enchanted Rock is adome-shaped, exposed area of granite worn smooth by weathering forces (Fig. 4). Sandy Creek separates this coarse granite area from the Packsaddle schist lying to the east. Enchanted Rock covers over asquare mile and rises
approximately 4-50 ft above Sandy Creek. The ecology of Enchanted Rock macrovegetation has been discussed by Whitehouse (1931).
The goals of this research were several. First, it was hoped that asystematic and
orderly surve)' of the algal ora of the several types of habitats on Enchanted Rock
might disclose different algal oras and reveal some signi cant pattern of distributlon of the soil organisms present. Second, in view of the extreme conditions with
respect to temperature, amount and duration of moisture, and the granitic substratum, it was anticipated that the soil algal ora might differ signi cantly from
that in the gypsum soils (Cain, 1963 ), Carrizo Sands formation (Deason and Bold,
1960), arid soils (Chantanachat and Bold, 1962), and prairie soils (Albert, un-
published) of the state of Texas. Finally, on the basis of past experience, it was
anticipated that new taxa would be encountered, and that isolation of these into
axenic culture and description and comparison of them with related species would
augment what is known of the soil algal ora in general.
Our knowledge has been increased during the last decade by the studies of Starr
(1953, 1955), Bold (1950, 1958), Trainor and Bold (1953), Herndon (I958a,
I958b), Arce and Bold (1958), Deason (1959), Deason and Bold (1960), Bold
and Parker (1962), Mattox and Bold (1962), Ghantanachat and Bold (1.962),
and Cain (1963). For future reference and comparison of the soil algal ora of
various parts of Texas and other areas, the algae isolated from the various sites at
Enchanted Rock have been tabulated (Table 1).
Ln addition to the general oristic studies, intensive, premonographic, comparative investigations were undertaken of the species of the genus Bracteacoccus Tereg.
The meagerness of present knowledge concerning the delimitation of species within
Bracteacoccus and the availability of living cultures suggested the present investiga-
fl
fi
fi
fl
fl
fl
fl
fl
fl
fl
tion of that genus.
Materials and Methods
Twenty-seven soil samples, from speci cally located stations on Enchanted Rock,
were collected asepticall)' into sterile paper bags. Five grams from each sample were
inoculated into a125-inl Erlenmeycr ask containing 50 ml of sterile modi ed
Bristol's solution (Bold, 1949, and further slightly modi ed). This medium was
used as the basic medium throughout this investigation, and is hereinafter known
as “Bold's Basal Medium” (BBM ). Inclusion in the investigation of some fastidious
organisms, with undetermined growth requirements, made it necessary to enrich
this basal medium with additional inorganic salts, goat manure and proteosepeptone.
“Bold’s Basal Medium” (BBM) was prepared as follows :
Six stock solutions 400 ml in volume were employed, each containing one of the
following salts in the concentration listed :
NaNOs
10.0 g
K H P O i
С а С ь г ш о
l.Og
Κ Η Ρ Ο *
MgSO.،٠7HO
3.0 g
NaCl ---
3.0 g
7.0g
l.Og
To 940 ml of distilled water were added 10 ml of each stock solution and 1.0 ml of
each of the 4stock, trace-element solutions prepared as follows :
(1) 50 gEDTA and 31 gKOH dissolved in 1liter Η2Ο.
(2) 4.98 gFeS0*-7H20 dissolved in 1liter acidi ed Hi0‫؛‬. (Acidi ed Η2Ο:
1.0ml H2SOİ added to 999 ml distilled water. )
(3) 11.42 gHBO^ dissolved ini liter Η2Ο.
(4) The following, in amounts indicated, all dissolved in 1liter Η2Ο :
ZnSO*-7H2Ơ, 8.82 g; MnCbTHO, I.44g; MoO.2, 0.71 g ,
1.57g;Co(NO.,)-6H20, 0.49 g.
Tire asks, after introduction of 5-g portions of each soil sample, were placed in
aculture room under "standard conditions”—Illumination 250 ft-c intensity for
12 hr followed by 12 hr of darkness, tempei'ature 22٥c. After 2weeks, growth was
visible in all the asks. Isolations of the organisms were made by plating-out and
streaking, and single-cell isolations were made by means of soft-glass micropipettes
(Bold, 1942; Pringsheim, 1946; Deason and Bold, 1960).
Other media used during this investigation for growing fastidious organisms were
prepared as follows:
luoĩganicalb enriclied Bold’s Basal Medium (IBBM)
To 910 ml of distilled Η2Ο were added 10 ml of each stock solution of BBM plus
10 ml of each of 3stock solutions prepared as follows :
10gCa(NO,٠١)2Ìn400 ml distilled Η2Ο
5gKNO.٩in 400 ml distilled HO
1gKCl in 400 ml distilled Η2Ο
fi
fi
fi
;
fi
fl
fi
fl
fl
]. .0 ml of each stock trace-element solution was also added.
Soll Algae ‫؛‬тот ETiclraTited Rock
i o
OTgaTTİcall‫ ؟‬eTiTİcked Bold’s Basal Medlem (OBBM)
Twenty- ve grams of dried goat manure were added to 1liter of distilled Η2Ο
and sterilized in the autoclave at 15 lb p.s.i. at I21٠c for 20 min. The extract was
cooled, ltered, and added (1 part) to (9 parts) of BBM. All organisms isolated
-from Enchanted Rock grew very well in this medium.
Pioteose Medlnm
This medium was prepared by adding l.Ogof proteose-peptone to each 1000 ml
of BBM. All of the above media were solidi ed by the addition of 1.5% agar or
1.0% “Ion agar”' before autoclaving.
The morphology of the various phases of the life cycles of the organisms isolated
into culture was studied by making fresh mounts and hanging-drop preparations
from cultures grown on agar or in liquid media under standard conditions. India
ink, aqueous Nigrosin, and aweak aqueous solution of Methylene blue were used
to determine the extent of the cell wall and presence of gelatinous matrices. Sudan
IV and Sudan Black-B were used to determine the presence of lipids. Various concentrations of aqueous Ιί-ΚΙ solution were used to determine the position of the
nucleus, number and length of the agella of the zoospores, and presence of starch
granules and pyrenoids. The acetocarmine stain technique was employed as an
additional cytological method when Ι2-ΚΙ solution did not satisfactorily reveal the
nuclei. The
xative for the acetocarmine stain was that of Johansen (194-0), as
modi ed by Cave and Pocock (1956), and further modi ed by Bischoff (1959).
The modi ed xative was prepared as follows :
Potassium iodide
0.25 g
1.00 g
Acetic acid. Glacial
4.0 ml
Iodine
Formalin
24.0 ml
Distilled water
400 ml
Slides were allowed to remain in the xative for approximately 3hr and then were
drained of excess uid. The preparations were then ooded with acetocarmine prepared according to the method of Cave and Pocock (1951), and placed upon a
hot-plate with the thermostat set at 300.1' or passed over alow ame of an alcohol
lamp until vapors arose from the stain. After 1¡/2-‫ ة‬min steaming, during' which the
stain turned adeep red, the slides were removed from the heat, drained and destained in 45% acetic acid for approximately 10 sec. Observations were made immediately and no permanent preparations were made.
In addition to the above-described materials and methods used to study the mor-
phological features of aparticular alga, other supplementary techniques proved
fruitful (Bold and Parker, 1962) and were used to help delimit species within a
genus. Before physiological attributes of an alga can be studied. It must be in the
axenic or pure-culture state. This was achieved by processing unialgal cultures
fl
fi
fl
fi
fi
fl
fl
fi
fi
fi
fi
fi
fi
'Oxoid Ion agar No. 2, Con.‫؟‬olidated Laboratories, Inc., Cliicago Heights, I‫¡اا‬noً ‫ا‬s.
Bìscko. aud Bold
11
through certain chemical and physical treatments (Brown and Bischoff, 1962).
Thick suspensions of algal cells were placed in heavy-walled, 12-ml, sterile centri-
fuge tubes containing a5% aqueous solution of “Tween-80” (a non-ionic surfaceactive agent). The tubes were immersed by hand into an ultrasonic water bath
(“Disontegrator, System 80” ;Model G-80C1) for treatment for from 1to 3min.
^٠١fter ultrasonic treatment, the algae were allowed to remain in the “Tween-80”
solution from 15 min to as long as 4hr. During this period, the algae were exposed
frequently to ultrasonic vibration, the duration of each perio'd not exceeding 10 sec.
Following the detergent and ultrasonic treatments, the cells were centrifuged only
to the degree that they became loosely packed in the bottom of the centrifuge tube,
.'kfter centrifugation, the supernatant was discarded. Sterile distilled water was then
added and the cells were re-suspended by short periods (10 sec each) of ultrasonic
vibration. It is important that the centrifuging and washing procedure be repeated
no fewer than 5times, since the removal of the physically separated bacteria seemingly occurs at this stage of treatment.
Basal agar medium was then cooled to 40.C and poured into sterile Petri dishes
(size 100 X15 inin) to which aquantity (0.5 ml or less) of thick algal suspension
from tlie washed ultrasonic treatment had been added previously. The plates were
swirled in both clockwise and counterclockwise directions in order to distribute the
algae thoroughly in the liquid agar phase, particularly in the surface layer, since
surface isolations are achieved with much greater ease than subsurface ones. Algae
isolated from the poured Petri dishes were transferred to dishes of proteose agar
which is favorable for the growth of algae as well as other microorganisms which
mav be present. This immediately revealed contamination of isolates previously
presumed to be bacteria-free.
One of the supplemental)' attributes studied was colony' characteristics, both
macroscopically and at amagni cation of 14- X, after 2-week and longer periods
on Basal agar medium, under standard conditions. It was necessary to adopt certain
descriptive terms to record attributes of the colonies on agar. Abrief glossary of
these terms follows:
I. Macroscopic appearance
A. Shiny
B. Dull-shiny
c. Dry (powdery)
If. Microscopic appearance (at 14Χ, stereoscopic binocular, re ected and transmitted light)
A. Smooth :homogeneous, individual cells not visible.
B. Gi'anular :homogeneous, but individual cells visible,
c. Rough :surface variously elevated and depressed, scabellate.
1. Glomerulate: surface composed of little mounds of cells, botryoid.
fl
fi
2. Reticulate: netted, veined.
12
Soil Algae ịĩom Euchauted Rock
3 . Ve r m i f o r m : w o r m - l i k e .
4. Radiately ridged :I'idges radiating from the center, like spokes of awheel.
III. Consistency (when transferred with aloop)
A, ButyiOus: growth of butterlike consistency, easily renroved from tlie surface
of the agar with an inoculating loop.
B. Viscid: growth adhei'ing to the inoculating loop when touched and withdi'awn.
c. Adherent: growth adliei'es to the surface of agar‫ ؛‬dif cult to remove witlr
the loop.
ช. Brittle: growth di'y, ci'umbles into small pieces when touched with the
inoculating loop.
Another useful series of attributes is the response of the algae in BBM enriched
with various organic compounds and grown under standard conditions as well as in
total darkness. Certain organic compounds inhibit, while others stimulate, growth.
Again, cultures used to determine these responses must be in axenic condition.
For these experiments, the culture tubes used were triple-washed and triple-rinsed
in distilled water, followed by triple-rinsing in de-ionized distilled water. The 01'ganic compounds were sterilized in an autoclave separatei)' in de-ionized distilled
water and then added aseptically to BBM. The tests were repeated and attention
was given to the time and temperature during the sterilization process, so that the
medium for repeated test runs would be as neaily as possible identical. Cool organic
solutions were added asepticall)' to cool sterile BBM to obtain a0.75% concentration. Sterile culture tubes plugged with nonabsorbent cotton were lled aseptically with aBrewer, Model 120-SS, automatic pipetting machine. The following
organic substances were added in concentration of 0.75% to BBM :arabinose, dextrose, fructose, sodium acetate, ribose and xylose.
Axenic cultures of the algae were inoculated into duplicate culture tubes containing 20 ml of medium. Five drops of inoculum were taken from athick sus-
pension of cells in BBM and inoculated by sterile disposable Pasteur capillary
pipettes. One set of duplicate tubes was placed under the standard conditions of
light and temperature, and the other duplicate set was stored in darkness at the
same temperature. After 14 days, both sets of cultures were observed for amount of
growth and recorded on basis of comparison with standards termed “none,”
“fair,” “good,” or “excellent” (Fig. 1).
Basal agar medium supplemented with starch was used to demonstrate comparatively the amylasic activity of the variou.s species investigated. This medium
was prepared by adding asmall amount (0.1 g) of soluble starch (Merck &Co.,
according to Linter) and lOgof Ion agar to 1000 ml of BBM. After autoclaving at
15 lb p.s.i. for 15 min, the medium was dispensed into disposable sterile (100 X15
mm) Petri dishes. Axenic cultures from agar slants were .streaked on the surface O'f
the solidi ed starch agar medium in duplicate. These plates were placed under
standard conditions of light and temperature. .After 14 days, the plates were ooded
svlth aweak solution of IrKI, which colors the unhydrolyzed starch agar blue.
fl
fi
fi
fi
trace.
BiscHo. aud Bold
13
Colorless zones surrounding the colonies indicate an area in which the starch has
been liydrolyzed. The clear zones were recorded on the basis of comparison with
standards as “’none,” “sliglit,” “good,” and “excellent” amylasic power (Fig. 2).
To deisionstrate the selective inhibitory action of certain antibiotics on the
growth of the algae. Multidisks (Consolidated Laboratories, Inc., Chicago Heights,
Illinois) were placed asepticall)-' on the surface of BBM Ion agar “pour” plates
which had been heavily inoculated with asuspension of the several algae from
axenic cultures. The plates were inverted and stored under standard conditions of
light and temperature. After 14 days, the plates were observed, and each antibiotic
was recorded as having negative or positive inhibitory action on the particular alga
tested (Fig. 3). Only clear zones of complete inhibition were recorded as positive
inhibition. The Multidisks had been impregnated with the following antibiotics in
the concentrations listed :
Chlortetracycline (‫؛‬A)
(AB() Amphotericin B----- ----- ..
(B.) Bacitracin ------------------ .---(Cl) Cliloramplienicol ----------- (СЛ+) Carbomycin ----------------------------------- (CS.) Colistin Sulfate
D+ )Denretliylclilortetracycline‫؛‬
(E.) Erythromycin -------------------(Κ+) Kanamycin ----- --------(Ν+) Neomycin
( N V. ) N o v o b i o c i n
(NS)) Nystatin
(OL+) Oleandomycin
Penicillin( )‫)دل‬
(РВ+)Ро1утух1п-В-------------------(ΡΜ+) Paromomycin.------- -- --(R() Ristocetin
(SY+) Syncillin---------(CY.) Cyclo.serinc
(S.) Dihydrostreptonrycln
Staphcillln
---(‫ (؛‬S C
--------------- (T.) Oxytetracycline
30 mc.g
10٥mcg
10 units
30 meg
15 meg
10 meg
30 meg
15 meg
30 meg
50 meg'
30 meg
100 units
15 meg
10 units
300 units
300 meg
30 meg
10 meg
30 meg
10 meg
30 meg
30 meg
(TAO.) Triacctyloleandomycin
(ΤΕ+) Tetracycline
(EL+) Sul somidine
(G+) Sul soxazole ---------------------(KY. )Sulfamethoxypyrldazine (TRI) Triclobisonium Cliloride
(V+)Viomycin---------------------------(VA+) Vancomycin -------------------(ΜΑ+ )Sulfadinietlioxine ----------(SD+) Sulfadiazine --------------------(SM+) Sulfanicrazine ----------------(SSS+) Triple Sulfa------(STI) Sulfatliiazole ----------- -------(TH+)Thiosulhl—
(ALI) Fur-iilttidone --------------- ----(Ε+ )Nitrofurantoin
{FCI)Nitrofurazone-------------
15 meg
30 meg
300 meg
300 meg
300 meg
1mg
10 meg
30 meg
300 meg
300 meg
300 nreg
300 meg
300 meg
300 meg
100 meg
100 meg
100 meg
100 meg
(FRI) Furazolidone
(ΙΗ+) Iso Nicotinic Acid Hydrazide 25 meg
(Μ+) Metlicnamine Mandelate
2.5 mg
(PS.) Para Amino Salicylic Acid -100 meg
All supplementary-attribute tests (colony characteristics, effects of organic compounds, amyla.sic activity, and inhibitor)' action of certain antibiotics) were performed in duplicate and repeated at least twice.
Observations of colon)' characteristics and isolation of single cells for axenic
cultures were made with aBausch and Lomb stereoscopic binocular microscope.
fi
fi
fi
fi
All drawings were prepared with the aid of aSpencer camera lucida and reduced
In reproduction. Photomicrographs were taken with a35-mm Zeiss-Winkel ‫ا‬2
camera attached to aBausch and Lomb microscope with apochromatic objectives.
The magni cations of all gures are given in the legends.
Soil Algae ‫'اأ‬0‫ ا‬Euchauted Rock
14
Distribution of the Soif Algae on Enchanted Rock
٥nly abrief account will be given of the distribution of the algae of Enchanted
Rock. The soil algal ora growing on this Pre-Cambrian rock was found to consist
of anumber of genera, and it seems worthwhile, for possible future reference, to describe brie )' the sampled sites and to list the genera isolated from cultured aliquots
of the samples.
٥n the summit and upper slopes of Enchanted Rock are approxinratcly 1‫هال‬
small pools, pot-holes and depressions containing enough soil to support the climax
genera, Andropogon, Yucca, opuntia, and Quercus.
These depressions vary in size from less than 3٥ cm in diameter to over 20 min
length (Fig. 5). The depth of soil is also variable. One depression supporting agood
stand of grasses has adepth of over 60 cm. Most of them are not this deep; many
are only 1-2 cm deep. There are anumber of ephemeral rain pools along lines of
drainage which are lacking in macroscopic vegetation. Most of these depre.ssions
contain water for several days after aheavy rain, but evaporation is rapid due to
exposure of the pools to the wind and heat. Several depressions or rain pools are
deep enough to contain water for longer periods. One such pool at the summit supports aluxuriant growth of Isoetes melanopoda (Fig. 6).
Crevices of various lengths and width occur throughout the summit and slopes of
Enchanted Rock (Fig. 7). These crevices, like the depressions, are lled with soil
which has accumulated by weathering of the granite and wind-blown soil and sue-
cessional changes of xerophytic vegetation, .supplemented by manure introduced by
goats which graze throughout this region.
The pH of the soil does not vary enough to be considered an important factor in
the distribution of the various soil algal genera. Arange of pH 6.3 to pH 6.9 was
r e c o r d e d f o r a l l s o i l s a m l e s taken from the summit or slopes of the rock proper.
However, the soil sample taken from the base of the rock had apH of 8.2.
Twenty-seven sites were selected, and soil samples were collected from each of
them. The sites were of 4types: (1)depressions containing soil mats supporting
growth of several genera of grasses—among them,Andropogon,Aristida and Panicum, and also opuntia, Yucca and Quercus (Fig. 5); (2) the dried soil from the
edge or bottom of ephemeral rain pools; (3) sandy soil on the west side of Sandy
Creek near the base of the rock near the “main trail"; (4) crevices containing soil
and supporting the growth of at least 2species of Selaginella, and several genera
of grasses (Fig. 7). The crevices selected were 5-15 cm ,in width. The depressions
containing soil and the ephemeral pools varied in diameter and soil depth. The
bearings and distances of the sites, where given (Table 1), were taken from the
Reference Mark 2, which is ush with the surface of bedrock at the summit of Enchanted Rock. The reference disk is stamped “ENCHANTED ROCK NO. 2
1956.”
fi
fl
fl
fl
Two-hundred and forty isolations were made from the 27 soil samples. Sixteen
Bischo. aud Bo^d
15
families and at least 30 genera were identi ed. It was noted that 3of the genera
were widely distributed, namely, Chlorococcum which occurred in 23 (it is possible
that some of these isolates could have been Neochloris, since the zoospores were not
ob::erved in all cultures) stations; Chlamydomonas from 21; and Hormidium
from 17 of the 27 sites. In contrast, anew species of Bracteacoccus and 2new
species of Planktosphaeria were found onl)' once each in 240 isolations. The
species of Bracteacoccus was isolated from acrevice on the northwest slope,
while both species of Planktosphaeria came from soil on the southeast slope.
Chlorococcum hypnosporum Starr was found in soil from 3of the 5crevices on
tlie northwest slope. Because of the location of these crevices, run-off ١yater is unquestionabl)' responsible for this distribution. Likewise, Hormidium was isolated
from soil taken from 6depressions that drain into each other sequentially. It was of
interest to observe that in the soil from which Stichococcus was isolated, Hormid-
ium was also present. Protosiphon was isolated 5times from soil having no drainage.
TMYj ١. Summary 0‫ ا‬Algal Geuera Preseut lu Ептгсkment Cultures 0‫ ا‬Errckauted
Rock Soll
Origin of soil sani)
ап(1 soil rode
Crevice on west slope
Abundance of algal
growth frotii so.‫؛‬
sample in (.BBM}
e x c e l l e n t‫'؛‬
300.38.7๓.
Oi.ganisin
Cklamydomoua.‫؟‬
cklorosai-cluoỊísls ٠>‫؟‬.
ckloTosarciua ‫؛‬p.
CkloTococcum kyinospoTum^tarr
Oscillatoria ‫؛‬p.
Anabaeixa‫؛‬p.
Hormldlumsp.
W C r - 1 .
Diatoms
Crevice on west slope
300.—32.0๓.
WGr-2.
sparse
Clrlorosarciuo5‫؛‬sls sp.
Spouglocklorls gigantea %p.nov.
Cklorococcum kypnospoTum
Lyngbya sp.
Diatoms
Crevice on west slope
300.—60.5
fair
๓.
W C r - 3 .
clilamytlomoiias uxegalls sp. ÏÏOV.
Chlamydomonas sp. (small)
CkloTosarclnopsis ‫؛‬p.
Cklamydomouas sp.
'"
Cklorococcumsp.
fi
'
fl
These descriptions of amount of algal growtk are qualitative estimates; “excellent” growth‫ن‬
designates asks with aheavy, green, algal mass; at tlie 0tiler extreme of growth, the presence
of algae could just be detected macroscopically (Fig. 1).
16
Soll Algae İTom Enchanted. Roch
TA B L E
{—Continued
0 o f H İ U Î Î İ ' ) 1٨‫ا‬, 1 1 1‫ا‬،‫ا;ا‬١
Origin of: soil sample
1soil٢‫ا‬٠01‫ ا‬growlll
anti soil eodc
(.SiiliJ)lß ill (Bß^I
Crevice on west slope
300.
66.6
fail-
m.
Oi'gimisiii
Chlamydomoiias sp. (gelatinous)
p.‫ ؛‬domonas'CKlam١
ρ . ΐ ν ο ν . ,‫ " " ؟‬g i g a n t e n s
W C r - 4 .
Scenedesmnssp.
Hormldinm ^accldnm
p.‫ ؛‬Stlchococcns
Anabaenasp.
Crevice on west slope
300.-67.8 m. pH 6.9
excellent
p.‫؛ ؟‬.Clilam^dotnona
Chlant^domonas ‫؛‬p. Vge\évou‫؟‬.١
p.‫؛‬hon(‫؛‬Protosl
p.‫ ؛‬Cklorosarciuopsis
Gleoc^stlssp.
Stlchococcussp.
W C r - 5 .
Depression lled witli
soil, grass and cactus
300
ChlorococctMTih^lmosliOTum
excellent
88 m.
W C - 1 .
Chlamydomonas sp. (gelatinous)
p.‫ ؛؟‬ChloTococcum
p.‫؛‬Scenesdesmns
HoTtirldlnm accldum
.Diatoms
Depression lled with
soil, grass and cactus
good
(north of WC-1) 86.4 m.
W C - 2 .
p.‫؛‬St)onglococcnm
p,.‫ ؟‬ChloTococcnnt
HoTTOİdlnnı^p,
Stlcliococcns^p.
Phormidium (delicate sp.)
Dominant species is small, sjrherical
chlorococcalean alga.
Depi'ession lled with
soil, gra.ss
spai'se
(north of WC-2) 91.2 m.
pH 6.9
w c - 3 .
p.,‫ ؟‬Chlorococcnm
sis sp.(‫؛‬chlor0.satclno
HoTmidinnvsp.
p .‫ ؛‬P k o u l d i i i
p■‫؛‬Anabaena
Diatoms
Dry soil from edge of
pool containing watei-
fair
hum sp. nov ‫ل‬50‫№الا‬0‫ ذا آ‬ChloTococcnm
Clilam^ .omonas sp.
p.‫؛‬Scene d.esmns
3 0 0٥_ 9 3 . 6 m .
P W - 1 .
Crevice neai' bronze plaque
on soutlieast slope, about
chaetoplioracean form
sparse
PhoTnridlunr sp.
.p‫ ؛‬ChloTOCoccurn
fair
Planktosjiliaeida maxima sp. ш\■.
ChtoTococcum ‫؛‬p.
Chlamydomonas sp.
ChloTosatcfna sp.
of the way up to the ‫ق‬/‫ل‬
summit of the rock.
Χ - 1 .
Crevice near base of rock
on southeast slope, left of
“main trail” neai' live-oak
fl
fi
fi
fi
fl
٠2.
17
Bische, aud Bold
ใแ
C o n t i n u e d
Aljundance of algal
Ii-‫؛‬g‫؛‬ii of soil saiii)
and soil rode
Deep depression on
growth froiii soli
«١n‫؛‬p l e i n ( B B M )
good
Organism
Chloiococcnm ‫؟‬.‫؟‬.
“ina,‫؛‬n trail” ]/2 of the
PleurastTumsp.
way np to the summit.
Protosiphon sp. (abundant)
Chlamydomonas ‫؛؟‬p.
HoTmidiumsp.
S-jtoııglochloTİs sp.
3
Sandy soil, on west
excellent
side of Sandy Creek
n e a r b o t t o m o f ‘‫؛‬m a i n
trail.” pH 8.2
Χ - 1 .
Chlanrydomoitas ^p. ‫\\ع ـ ـ ـ ـ ــا\ا‬١‫أ‬
Blear astrirmsp.
Stlchococcussp.
Hormidlam‫؛‬p.
Trlboiiema‫؛‬p.
CliloTOcoccum sp.
Chlorosarclnojrsis ‫؛‬p.
C h l o To c o c c a m
mlitum
‫؟‬i l a i T
Chlorogoniam ‫؛‬p.
OedogoalaTO‫؛‬p.
Chlamyclomonas sp. (gelatinous)
The
rst crevice on
excellent
southeast slope at light
side of “main ti'ail”
Almost unialgal culture of 2species:
Oscillatoria ‫؛‬p.
ChloTococcam sp.
way to summit. Β-1
Diled soil from edge
sparse
of 1'ain pool.
330.—8.1
m.
Small incipient pool, soil
has no visible vegetation.
25
sparse
1.2 m.
Ε-2.
Incipient pool.
fi
fi
fi
fi
180.—5.9
m.
spai'se
Sceriedesnias ‫؛‬p.
Oscillatoria sp.
Chlorella ‫؛‬p.
Chlamydomonas ‫؛‬p.
Airabaeaasp.
pseudo- lamentous green alga
branching lamentous green alga
Cli.lamydomonas ‫؛‬p. ١\‫؟‬,raa\\١j
Scenedesmus sp.
Chlamydomonas sp. (gelatinous)
Chlorococcwm ‫؛‬p.
ChloTosarclaoJi.sls ‫؟‬,p.
pseudo- lamentous green alga
Hormldlamsp.
Characlamsp.
Chlamydorrton.a.s ‫؟‬,p.
0.sclllatorla‫؛‬p.
Brotosi)ihon‫؛‬p.
Seleiiastramsp.
Scenedesmus٠.p,
Stlchococcws sp.
ChloTococcum sp.
Soil Algae ‫؛‬тот Enchanted Rock
]
TA B L E
[—Continued
Abiindamo (>{'alga!
Oi-‫؛‬g‫؛‬ii of soil saJiiple
g,-()wl،i from s،i‫!؛‬
and soil code
aample in (1‫ا‬1‫آلإ؟‬1)
Depression with soil,
excellent
Organism
Pto‫؛‬.osI-JíLow٩‫؟‬.
GliloTOCOCcam
cactus and grass.
2 1 0٥_ 1 1 . 6 m .
HoTmldlum siibtlllsslmam
Ε-4.
şp.‫؟‬,c٨ococcM¿،‫؟‬,
Chlamydomonas sp. (gelatinous)
GLlam^domoỉas ‫؟‬.‫؟‬.
goraema sp.¿،‫؟‬.
^Diatoms
Depression with soil,
good
cactus and grass.
Sticliococcussc).
HoTmldlwm subtilissimum
CbloTOcoccam
1 5 0٥— 9 . 2 m .
Oedogouium‫؟؛؟‬.
OedogoTclam‫؟؛‬.
Chlamydomonas sp. (gelatinous)
Scenedesmus 8‫دآ‬.
niatoms
Depression with soil
excellent
and grass.
1 5 0٠_ 1 6 . 2 m .
Neociiloris pseudostigmatica sp. nov.
sJoTTglococcTim
Clilamydomonas sp. (gelatinous)
C b l o To c o c c w m
p.<‫ ؟‬CbloTococcum
Planktos^baeTla texeiisli tvov.
sJoTtglocbloils llaTioeiTsl t v o v.
HoTmldmmsp,
CbloTosaTcliiopsls ‫؟‬.‫■؟‬
OedogoiTlam ‫؟>؛‬.
Depression witli soil
and grass.
sparse
150.—25.8
Ε-7.
Depression with soil,
grass and cactus.
good
1 5 0٠_ 4 2 . 0 m .
HoTmldlam^‫؟‬.
Lyngbya sp.
Cblam^domoiias
١t>.‫؟‬Z١gnema'
Chlamydomonas sp. (gelatinous)
S t l c l r o c o c c a s‫؟؛‬.
Chlam^domoaas ‫؟؛؟‬.
ClvloTococcum ‫؟>؟‬.
E S .
HoTmulium‫؟‬,‫؟‬.
Depression witli soil,
grass and cactus.
good
GhloTosaTcliro٠fsis ‫>؟؛‬.
Oedogomum‫؟؛؟‬.
150._53.8in.
O e d o g o u i u m‫؟؛‬.
Ε-9.
NeocbloTls‫؟؛‬.
Ghlam^domonas ‫؟>؟‬.
l l o i m l d l a m‫؟؛‬.
s
Large depression with
soil and grass.
fair
.٩١ GldoTococcam
.HoTmldlirm٩١
TA R L E ì — C o n t i n u e d
;ЧЬшкЛалсе of algal
gi4)١vlh from soil
sample in (RBM)
()ilgin of soil sample
aiitl soil totle
150.-59.0
Oj-ganlsm
Neochloiis‫؟‬,‫؟‬.
m.
CLlaTu١idomouas
E-l0pH6.3.
Scenedesmus sp.
PhoTiidium
ChloTosai ciuopsis
pseudo- lamentous green alga
Depression with soil
PiOtosipKonsp,
H o T m i d i u m‫؟؛‬,
ChloTococcum sp.
OscilltttoTİasp.
,fair
and grass.
1 5 0٥_ 6 2 . 7 m .
ρ,<١.1 ‫؟‬отоиа،١١.С1г1ат
Diatoms
Large depi'ession with
soil, grass and live-
spai'se
p.‫؛‬HoTmidium
p,‫ ؟‬Cklai'ydomonas
p.‫ ؛‬spouglococcum
p.‫؛‬CKloTococcum
Chlamydomonas sp. (gelatinous)
p.‫ ؛‬Chlorella
sparse
1 0 0 ٥ - -64.6 m.
p. UraaXV)‫ ؛‬Cklorococcuru
Horruldlauisp.
p.‫؛‬Neoclilorls
Oscillatoriaceae (tlominant)
Ε-13.
Diatoms
oak. 50
37.5 m.
E-12.pH6.6.
Large depi'ession with
soil, cactus, grass
and Тмссй.
Organisms New to Science or Otherwise Signi cant
Inasmuch as determinative taxonomic studies of soil algae have been few in the
past, an investigator in this eld expects to encounter previously undescribed taxa.
This has proven to be the case in the present study of the Enchanted Rock soil algal
ora. ٥f 240 isolates, 16 taxa new to science and otherwise signi cant are described
in the following .section.
Vo l v o c a l e . s
Chlainydomonadaceae
Chlamydomonas niegalis sp. nov. (Fig. 8-13 91-88 ‫؛‬, )
Cellular 2440 X18-27 μtruiicatae ovataeque. Papilla parva truncataque; cellula
hranam tenuem atquc 2 agella breviora quam corpus cellulae liabens. Chloroplastus solidus,
depi-essionlljus ssurlscjuc multis praedltus, multac vacuolae pulsantes Intel- super ciem plastidls
atque meuihranaiii plasmatlcain; 8-14 pyrenoldea lateralla 4ไ5 μdiam., per chloroplastům
fi
fi
fi
fi
fl
fi
m
fi
fl
19
Bìscho. aud Bold
e
m
-
Soil Algae ]тот Enchanted Roch
solidum fortuite distributa. Nucleus magnus, paululum anterioi- ad equatorialem; multa
corpora refractiva parva per cytoplasm di.stributa.
Reproductio asexualis pci' bipartitioirem endog'eiraiu plerumque iit 4cellulas- lias. Cellulae
in medio solido in matrice tenui aquosa gelatino.sa communal‫ ؛‬inclusae. Cellulae in culturis iit
incrementi periodo immobili semper virides.
Reproductio scxualis non observata
Origo: in solo ،' loco Enchanted Rock, !-,lauo Co., Texas dicto, m. .Jul. I960.
C e l l s 2 4 4 ٥ X 1 8 - 2 7 μ■■ ti'uircate and ovoidal. ])apilla sirtall and ti'uncate; celi wall tirili,
with 2 agella not as long as tire cell Irody. Chloropla.st massive, w-‫؛‬tli man)' depres.sions and
şsuresً ‫ ا‬many contractile vacuoles between tire plastid .surface iind plasma membrane14-8 ‫؛‬
latei'al pyrcnoids, each 4—5 μdiameter, distributed at random tlirougliout the mrissive chloroplast. Nucleus lai'gc, slightly anterloi- tt) equatorial; Iiraiiy small re 'active irodies distrilruted
tlirougliout the cytoplasm.
Asexual reproduction Iry endogenous bipartition, usually into 4daugliter cells. Cells on solid
media embedded in ath‫!؛‬i, watery, gelatinous ('omnion inati'ix. Cells remaining green in
cultures in stationary phase of growth.
Sexual reproduction not olrscrved.
Source: Soil from Enchanted Roc.k, Llano County, Texas: July, 19611.
The sample of soil from which this organism (WCr-3-7 )was isolated was collected from acrevice on the west exposure of Enchanted Rock. The alga has an
undetermined growth requirement so that it grows poorly in BBM. ft will grow
well, however, in OBBM and Proteose-peptone medium. Twenty clonal cultures
were started by introducing single cells into sterile ٥BBM. Good growth resulted .in
all 20 tubes. The absence of zygotes in the clonal cultures indicated the organism
was not homothallic. Pairs of clonal cultures were mixed in all possible combinations to determine the possibihty of heterothallism. No sexual reproduction has
been observed, and no zygotes were found in the mixed cultures after an appropriate interval.
The organism is truncate ovoidal (Eig. 8, 88, 89). The antei'ior pole is broadl)'
rounded and truncate, while the posterior is narrowed. The cell wall is thin and
closelv adpressed to the protoplast in motile cells. The very small papilla is elliptical
in outline with acurved surface (much like abread loaf) (Fig. 88, 89). .Actively
motile cells swim about slowl)' and in acumbersome manner. Vegetative cell size
ranges from 24- to 40 μin length and 18 to 27‫ مللم‬in width.
The chloroplast in median optical section and surface view is massive with many
depressions and ssures (Fig. 88, 89). The plastid is open at the anterior pole by
means of averv small canal extending from tlie region of the nucleus to the cell wall
fi
fi
fi
fi
adjacent to the papilla. The inner surface of the chloroplast encases the nucleus
and is more deepl).' ssured than the other (Fig. 84٦ .( 91-1‫؛‬he nucleus is relatively
large and slightlv' anterioi- to the equator (Fig. 9, 89). Many contractile ١'acuo!es,
approximately 2μ diameter, lie in small depressions of the outer- surface of the
chloroplast. As many as 15 have been olroserved pulsating simrtltaneously at one
focal level in surface view. Eight-14 or more spherical pyrenoid.s, approximately
fl
fi
20
Bischo. (md Bold
21
-diameter, lie in lateral positions, distributed at random throughout the mas ‫ل‬/4-5
sive chloroplast. The stigma is streaked-shaped and is embedded In the periphery of
the chloroplast near, or slightly anterior to, the equator of the cell. The size and
form of the stigma are constant in all cells observed. Many small refractive bodies
are distributed throughout the cytoplasm. The majority occur near the nucleus and
in the depressions containing the contractile vacuoles (Fig. 89-91 ).
The cells are constantly motile while in liquid medium and apellicle is not
formed at the surface of the culture vessel. The agella are not as long as the cell
and are not completel)' withdrawn or discarded when cells are grown on agar.
The only observed method of reproduction is by endogenous bipartition. The
protoplast rotates 90. within the cell wall in afashion similar to that observed in c.
microhalophila (Blschoff, 1959) and many other species of Chlamydomonas. Cytokinesis occurs during the nomotile phase and is Initiated by the appearance of a
furrow perpendicular to the longitudinal axis of the cell (Fig. 10, 11, 12). Cytokinesis is unilateral and often incomplete, for it is not uncommon to observe apartially divided cell with 2papillae and 4 agella. As cytokinesis progresses, the thin,
elastic mother cell wall begins to swell and increases to more than twice its original
size (Fig. 11). The 2daughter cells usuali)' undergo one more division which takes
place in the same manner (Fig. 12). No more than 4daughter cells have been observed in this species. The daughter cells, which are approximate!)' 20/İ long and
\ịμ wide, swim within the large, pliable mother cell wall until it ruptures and releases them.
Cells from 2-week-o!d cultures on Proteose-peptone agar are embedded in avery
thin, watery, gelatinous common matrix, demonstralile with India ink and Methylene blue. Colonies on this medium are smooth, viscid and shiny. The margin of the
colon)' is lobate, with the extreme outer edge consisting only of the watery matrix.
١Vith transmitted light at 14 Xmagni cation, the colonies are translucent and
motley, having dark- and light-green areas (Fig. 13). Three-month-old cultures
are still green, and still older cultures do not ever become orange-yellow. The only
noticeable difference between З-month-old cultures and 2-week-o!d cultures is an
fl
fi
fl
increase in the amount of the common matrix encasing the cells.
Chlamydomonas megalis is sensitive to all antibiotics tested (Table 2) except
bacitracin, chloramphenicol, erythromycin, novobiocin, oleandomycin, penicillin,
ristocetin, syncillin, cycloserine, staphcillin, Oxytetracycline, triacetyloleandomycin
and iso nicotinic acid hydrazide. Growth is onl)' in trace amounts in BBM 0.75%
glucose, fructose and xylose; no growth occurred in darkness in any of the carbon
sources used (Table 3). Growth is good on OBBM Ion agar containing 0.01%
soluble starclr, and the amylasic activity is excellent (Table 4).
Asearch of the literature (Pascher, 1927; Moewus, 1931; Gerloff, 1940; Etti,
I959a,b; I960a,'b; Etti and Etti, 1959a,b) failed to reveal an organism with a
combination of attributes like those of the organism here described. The alga is
somewhat suggestive of c. pseudogigantea Korschikoff, c. striata Korschikoff,
Soil Algae ịĩom Euchttuted Rock
22
and c. rubri liim Korschikoff but differs from them clearly in anumber of respects,
such as position and shape of the stigma, shape and structure of the massive chloroplast, shape of cell, thinness of cell wall, and position of the large nucleus, among
others. Therefore, this alga has been described as anew taxon, Chlamydomonas
megalis, amember of the Pleiochloris section of the genus.
Chlorococcales
Chlorococcaceae
Chlorococcum polymorphuni sp. nov. (Eig. 14-22; 92-1)8)
Cellulae vegctativac forma variantes: ovatae aut sarcatae aut ha.s!d¡o-forn١e.s ant p)i'‫؛‬forin،'s
ant ensiformes ant ellipsoideae aut tubulares, magnitudine usfjue ‫؛‬ld ‫ هـة‬μ. Membrana levis
tenuisque, aetate non spissescens. Cliloroplastus parietalis, super eie exteriőre parce dissect‫؛!؛‬
per des interioi' irregularis, formi vacnolaruin niagnarum cytoplasmatis incoloi'ati congruens;
[,yrenoideum slngulum, in parte cliloroplasti crassioi'e excentrice inclusuin. Ccllulae omnis
aetatis uninuclcatac. Vacuolac pulsantes in cellulis vegetativis non observatae.
Reproductlo asexualis per forniationem aplanosporariiin zoosporarumque. Zoosporac ovat‫!؛‬،',
8.0-8.5 X3.0-3.5 μ, 2vacuolas pulsantes anteriores, stig-ina anterius lineare ad disclforme,
nucleum postcìlorem, pyienoidcum singulum in chloroplasto parietali, et 2 agella longitudine
aequa, 12‫ ا؛‬pio longiora quani coi'pus cellulac liabentcs.
Reproductio sexualis non observata.
Coloniae actatc duaiiim Iicbdomadum in medio “agar” basali sin،‫ ؛‬matricibus gclatlnosis,
atrovirides, pei- 3menses saltem semper virides. Super cies culturíiruni actate 2liclidomaduin
granulosa (magni catione 14 pio); coloniile fusco-nitidae, macroscopicaliter visac butyraceac.
Origo: in solo eloco Enclianted Rock, Llano Co., Texas dicto. 111. Jul., li)60.
Vegetative cells of vai-ious forms: ovoidiil, saccate, liasldioforni, pyi-iforin, ciisiforin, ell ipsoldai or tuliular, reaching amaximum size of 50 μ. Cell wall smotitli and tilin, not thickeningwith age. Chloroplast parietal, its outer surface sparsely dissected‫ ؛‬iiiiiei- sui-fac،' irregular, con-
forming to shape of the large vacuoles of tlie coloiless cytoplasm, with asingle pyreiioid cmliedded in tliic.kcr part of cliloroplast in an excentric position. Cells uninucleate at all ‫؛‬Iges.
Contractile vacuoles not observed in vegetative cells.
Asexual repi'oduction liy aplanosporc and zoospore formation. Zoospores ovoidal, 8.8.5-‫ آل‬X
3.0-3.5 μ, witli 2anterior contractile vacuoles, aiiterioi- linear- to disc-sliapcd stigma, posterior
nucleus, single pyrenoid in aparietal c.Iiloroplast, and 2 agellii of equal length, 11/2 Xas long
as tine cell Inocly.
Sexual reproduction not observed.
Two-week-old colonies on Inasai agar medium exliilnitirig no gelatinous matrices‫ ؛‬darle green,
remaining green for at least 3niontlis. Surface of 2-week-o!d cultures granular (Ι4χ niagiii cation) colonies dull-shiny, and macroscopically Inutyroiis.
Source; Soil from Enchanted Rock, Llano County, Texas‫ ؛‬July, l6(‫(؛‬L
This organism (PW-1-5) was isolated from asoil sample collected from the
edge of alarge depression containing some water on the west slope of Enchanted
fi
fl
fi
fl
fi
fi
fi
fi
Rock. Cultures of this organism have been -maintained in abacteria-free state on
BBM agar and in BBM.
23
Biscko. and BoÎd
The vegetative cells of this species assume avariety of forms and shapes as they
mature. Most cells are ovoidal, saccate, basidioform, pyriform, ensiform, ellipsoidal
or tubular (Fig. 14). Asmall percentage of any population grown on agar medium
may be spherical to subspherical (Fig. 15). Cultures grown in BBM have ahigher
percentage of spherical forms. The maximum length of avegetative cell may be as
much as 50،u٠, with the population averaging 25-35 μin len^h and 8-15 μin width.
The cell wall is smooth and very thin and does not increase markedly in thickness as
the culture ages.
The protoplast contains aparietal chloroplast (Fig. 94, 95 ). The typical chloroplast of Chlorococcum, as described by Starr (1955), does not exist in every indi-
vidual of this species. Only' occasionally, in spherical to subspherical cells, may one
observe the typical "cup-shaped” chloroplast (Fig. 15). The outer surface of the
chloroplast is sparsel)' dissected. The inner surface is very irregular, and conforms
to the shape of the large vacuoles of the colorless cytoplasm (Fig. 16-18, 94, 95 ).
More than one pyrenoid never has been observed in any one cell, regardless of its
shape or size. The pyre'noid is embedded in the thicker part of the plastid in an excentric position (Fig. 92-94). Contractile vacuoles were not observed. The cells are
uninucleate at all ages. Preparations of cells that are in the process of aplanosporogenesis and zoosporogenesis stained with acetocarmine show that karyokinesis occurs between each successive cytokinesis (Fig. 19, 20, 96). Cells from 2-week-o!d
cultures on BBM agar exhibit no matrices with India ,ink or Methylene blue.
The only observed methods of reproduction are by infrequent aplanospore and
frequent zoospore formation. The zoospores are ovoidal and walled, failing to become immediately spherical at quiescence. They have 2small anterior contractile
vacuoles, an anterior linear to disc-shaped stigma, aposterior nucleus, asingle pyrenoid in aparietal chloroplast and measure 8.0-8.5 μin width. The agella are
equal in length, and are 1^ Xas long as the cell body (Fig. 98). The average
length of the zoosporangial cell is 17-22μ (Fig. 20, 21, 97). Usually 16 very
actively and rapidly swimming zoospores are released from each zoosporangium.
Each zoospore may remain active for an hour or longer before becoming quiescent.
All zoospores retain their ovoid shape upon quiescence (Fig. 17,20).
Sexual reproduction has not been observed.
It is very easy to evoke zoosporulation of this species by removing al-week-old
culture on agar medium from the dark after 12 hr, and exposing it to abright light
for at least 2hr before
ooding with distilled water.
Colonies on BBM agar are dark green after 2weeks and remain green through at
least 3months. The surface of a2-week-o!d colony, when observed at a14 Xmagni cation, is granular (Fig. 22), dull-shiny with re ected light, and the consistency
is butyrous.
fl
fi
fl
fl
fi
After comparison of this species with other known species of Chlorococcum
(Bold and Parker, 1962; Chantanachat and Bold, 1962), the organism described
above has been treated as anew taxon, c. polymorphum. The speci c name alludes
24
Soil Algae ^1'οτα EucKauted Rock
to the many forms of the vegetative cells which may enclose parietal, hollow spherical or parietal, irregularly shaped and dissected chloroplasts. .Although the shape
of the vegetative cells, the chloroplast and the large vacuoles present in the colorless
cytoplasm suggest attributes of Protosiphon, tire zoospores are the typical Chlamydomonas type (Starr, 1955) of Chlorococcum in that the)' remain ellipsoidal for
some time after quiescence. Chlorococcuni polymorphum is suggestive of Characium simplissimum Korschikoff (1953), from which it differs markedly in size.
Because Characium simplissimum lacks astalk and because its zoospores do not become spherical at quiescence, Korschikoff's alga, in the writers’ opinion, probably
should be transferred to Chlorococcum.
Other supplementary attributes of c. polymorphum are as follows: the organisnr
is inhibited by furaltadone, nitrofurazone, methenamine mandelate, nitrofurantoin, triclobisonium chloride and amphotericin B(Table 2). Growth is excellent
in light in BBM supplemented with 0.75% arabinose, dextrose, fructose, sodium
acetate, rlbo'c and xylose and occurs in trace amounts in arabinose, dextrose,
sodium acetate and xylose in darkness (Table 3). Only avery light trace of starch
hydroly.sis was observed using 0.01% soluble starch in BBM Ion agar (Tabled).
Chlorococcum hypnosporum Starr (1955) (Iffg. 24-31 )
To the best of the writers' knowledge, this organism has not been repol'ted since
its original description by Starr (1955). The organism Starr described as Chlorococcum hypnosporum was isolated from asoil sample fronr Bledsoe Count)', Tennessee, U.S.A.
An organism resembling c. hypnosporum was isolated 5times from soil which
was collected from 3different crevices on Enchanted Rock. ١٧ith the exception that
the zoospores are slightl)' larger on the average, the Enchanted Rocli isolate is very
similar to c. hypnosporum Starr, ft was observed that the spiny hypnospore of the
Texas isolates may give rise to 2or 4cells, instead of always only 2, as reported by
Starr (1955).
Supplementär)'- attributes such as colony characteristics, response in BBM enriched with various organic compounds, inhibitory action by certain antibiotics and
amylasic action on starch clearly established that the 5isolates from Enchanted
Rock and the Tennessee isolate are probably one and the same organism.
Chlorococcum minutum Starr (1955)
minutum was isolated from asoil sample from Bombay, India, by
the junior author and described as anew taxon by Starr (1955). This alga also
seems not to have been reported since its originili description.
An alga was isolated during the present investigation from asample of sandy soil
collected near the base of Enchanted Rock‫ ؛‬this organism closel)' re.sembles c.
25
Bischo. aud Bold
minutum. The morphology of the vegetative and reproductive cells and also the
method of asexual reproduction by zoospores and aplanospores and sexual reproduction by isogamy clearly substantiate their similarity.
Acomparison of supplementary attributes in which the Texas Isolate and atransfei- of c. minutum are similar also indicated that the 2isolates are one and the same
organism. Both are homothallic.
Spougiocliloi s
Two new species of Spongiochloris were isolated from Enchanted Rock soil and
compared with other known species. The generic attributes of Spongiochloris inelude: (1) vegetative cells with anetlike, reticulate chloroplast., both super cial
and internal; (2) with 1or more pyrenoids; and (3) with zoospores which become
spherical immediately at quiescence.
Spongiochloris gigantea sp. nov. (Eig. 23, 32,33; 99-1 ٥5 )
Cel!ulae vegetativae aetatis oitinis splicricae, dlarn. max. 1!0 μ. Membranae leves, aetate ad
7.5-15 μvel plus spissescentes. Chloroplastus cellularum maturarum reticulum typicum ligamentorum parletalium intcriorum tenuiin implexorum, in cellulis vetustloribus fere solidum
factum; pyrenoidca 1-5 excentrica, in ccllulis vetustioribus fracta. Guttuliic olci in cellulis in
incrementi periodo immobili saepe coacerva tae. Cellulae maturae multinucieatae; ccliulae
vegetativae sine vacuolis pulsantibus.
Reproductio asexualis per formationeni aplanosporarum zoosporarumque. Zoosporae fusi-
formes 8.٥-10.0 X2.5-3.о μ, 2 agella longitudine aequa, paillam parvam, stigma anterlus, 2
vacuolas pulsantes anteriores, nucleum medium, cliloroplastum parietalem dlssectum et pyrenoideum posterius, interdum non visible, liabentes.
Reproductio scxualis non observata.
Culturae duariim hebdomedum aetate in medio
'agar” basali atrovlrldes, post 3menses
lucide aurantiae. Super cies coloniac culturae duarum liebdomadum aetatc granulosa (magni catione 14 pio); coloniac nitidae macroscopicallter visae; butyraceae.
Origo: in solo eloco Enchanted Rock, Llano County, Texas diet¡; m. Jul. 1960.
Vegetative cells splierical at all ages, maximum diameter 110 μ. Gell walls smooth, tliickening with age to 7.5-15 μor more. Chloroplast of mature cells atypical network of ne. Interlacing parietal and internal strands, becoming almost solid in oldei- cells; with 1-5 cxccntrically located pyrenolds, fragmenting in older cells. Oil droplets often accumulating in cells
in stationary phase of growth. Cells multlnucleate when mature; contractile' vacuoles absent
in vegetative cells.
Asexual reproduction by aplanospore and zoospore formation. Zoospores fusiform, 8.010.0 X2.5-3.0 g, with 2 ag'ella of equal length, small papilla, anterior stigma, 2airterior
contractile vacuoles, median nucleus, parietal dissected chloroplast and aposterior pyrenoid,
the latter' sonretimes not visible.
Sexual reproduction not observed.
Two-week-old cultures on basal agar mediunt dark green, Irecoming bright oratrge ‫؛‬ifter 3
months. Colony surface of 2-weẹk-old culture granitlar (14-X irragni catlon); colonies slrltty
fi
fi
fi
fl
fl
fi
fi
fi
trtacroscopically; Irutyroirs.
.Source :Soil front Enchanted Rock, Llano County, Texas; July, 1960.
26
Soil Algae İTom E^c^a.ted Roc^
The alga (WCr-2-10) described here was isolated from soil taken from acrevice on the west exposure of Enchanted Rock, This isolate was dif cult to obtain in
axenic culture because of its thick cell wall which provided an excellent substrate
for bacteria and fuirgi. The organism grows best in BBM supplemented with organic nitrogen (OBBM).
The vegetative cells of this alga are spherical at all ages (Fig. 32, 33 ). Contractile
vacuoles were not observed in vegetative cells. The cell walls are smooth and thin
in young cells which sometimes exhibit atendency to cohere. However, no matrix
is present, as verihed by application of Methylene blue and India ink. Unlike s.
lamellata Deason and Bold (1960), the cells of this alga separate from each other
very easily. The cell wall thickens with age, attaining athickness of 7.5-1or more
in cultures in the stationary phase of growth (Fig. 105). In older cells, the wall
resembles that of s. lamellata Deason and Bold. Old cells transferred to fresh agar
medium resume growth and shed some of the outer layers of the cell wall. Cells may
attain amaximum diameter of 1ΙΟμ, but the average is 85-90‫ م‬in 4-week-o!d agar
cultures. Many of these larger cells found growing on agar medium are probably
cells which have failed to form aplanospores or zoospores through several transfers
and which have continued to enlarge.
The chloroplast of young cells is parietal and dissected (I٦ig. 33; 102-104). As
the cell increases in size, the chloroplast typically becomes anetwork of ne, inter-
lacing strands. The interstices of the network become smaller as the cell enlarges,
this causing the chloroplast to become almost solid, older cells are frequently lled
with an accumulation of oil droplets which mask the internal structure. Only very
young cells are uninucleate; aseries of successive mitotic divisions occurs as the cells
increase in size, this resulting in amultinucleate condition (Fig. 102-104). At
least 1pvrenoid is always embedded excentrically in the chloroplast. Occasionali)',
2-5 pyrenoids occur in young cells. Fragments, probabl)' of pyrenoid origin, are
frequently found near the pyrenoids of old cells.
Reproduction is accomplished by asexual aplanospores and zoospores; sexual
reproduction was not observed in this species. Young vegetative cells, 20-30μ in
diameter, undergo zoosporogenesis by aprogressive cleavage of the multinucleate
protoplast. It is characteristic of this alga to form 5٥ or more zoospores which are
liberated in avesicle through alai.ge pore in the parent wall. Large cells are also
capable of zoosporogenesis but do not form zoospores as frequently as the younger
cells.
The fusiform zoospores (Fig. 99) average 8-ΙΟμ in length and 2.5-3.0‫ دل ـ ـ ـ ـ ـ ــم‬in
width. Each zoospore bears 2 agella of equal length, asmall papilla, an anterior
stigma, 2anteilor contractile vacuoles, asingle, median nucleus, aparietal dissected chloroplast, and aposterior pyrenoid. Motility of the zoospores lasts for only
ashort time, rarely exceeding 15 min. As the quiescent phase approaches, the zoo-
spores withdraw their agella and the spherical form is rapidly achieved (Fig. 100-
fi
fi
fi
fl
fl
102).
The surface of 2-week-old cultures on BBM agar is shiny and when observed at
14 Xmagni cation is granular (Fig. 23 ). The colonies are of butyrous consistency.
Colonies on this medium are green at 2weeks, but become bright orange when 3
months old.
This alga is amember of the genus Spongiochloris since it clearly pO'Ssesses the
generic attributes described by Starr (1955). Comparison of the present organism
with other members of the genus, described by Starr (1955), Deason and Bold
(1960), and Chantanachat and Bold (1962), indicates that it is different in sev-
eral respects, ft is most similar to s. lamellata Deason and Bold, from which it differs in its larger size, thinner cell wall, isolated cells, and supplementary attributes
such as sensitivit)'- to certain antibiotics and growth in BBM supplemented with
certain carbon sources. The organism is inhibited by amphotericin B, nitrofurantoin, methenamine mandelate, furaltadone, nitrofurazone, paromomycin, and triclobisonium chloride (Table 2). Growth is fair in arabinose, dextrose, fructose and
xylose; trace in ribose; no growth in darkness (Table 3). The amylasic ability is
slight on BBM Ion agar containing 0.01% soluble starch (Table 4- ).
The organism has, therefore, been described as anew taxon, Spongiochloris gigautea.
Spongiochloris llanoensis sp. nov. (Fig. 34-38; 106—109)
Celliilae vegetativae aetatis omnls splieticae, diam. max. 35 μ. Membranae tenues levesque,
aetate non manifeste spissescentes. Cellulae vetustiores protrusionibus bulliformibus in membranis interdum praeditae. Cellulae invencs per membranas coJiaerentes. Chloroplastus eellularum maturarum ereticiilo tenui ligamentorum Implexorum ad super ciem constans, prope
centrum cfellulae grosslore facto; pyrenoideum singulum excentricum, in cellubs vetustioribus
saepe fractum. Cellulae vegetativae sine vacuolis pulsantibus. Cellulae matu rae multinucleatae.
Reproductio ascxualis per formationem zoosporarum aplanosporarumque. Singulum zoo-
sporangium usque ad 100 zoosopras formans. Zoosporae fusifoj-mes, 7.5 X3.0 μ, chloroplastům
parietalem intei'um dissectum, 2vacuolas pulsantes anteriores, nucleum anterius, Stigma
equatoriale ad posterius et 2f-lagella longitudine aequa habentes.
Reproductio sexualis non observata.
Culturae duarum hebdomadum aetatc in medio "iigar" basali atrovirides, post 3menses
avac ad aurantiacas factae. Super cies culturae duarum hebdomadum aetate granulosa vermi-
formlsquc (magni catione 14 pio); colonlae fusco-nitidae macroscopicaliter visae, butyraceae.
Origo: in solo cloco Enclianted Rock, Llano Co., Texas dicto, m. Jul. 1960.
Vegetative cells splicrical at 11‫ ا؛‬ages, maximum diameter 35 μ. Cell walls thin and smootli,
not tliickening mai'kedly with age. Older cells sometimes with bubble-like prolusions on tlieir
١١'alls. Young cells colierent by tlielr walls. Cliloroplast of mature cells consisting of a ne net-
fi
fi
fi
fi
١vork of interlacing strands at tlie surface, becoming coarser near the center of tlie cell, with a
single, cxccntrically located pyrenoid, the latter often fragmenting in older cells. Contractile
vacuoles absent in vegetative cells. Cells niultinucleate when mature.
Asexual reproduction by zoospore and aplanospore formation. Up to 100 zoospores formed
fi
fl
27
Bìscho. aud Bo‫؛‬d
28
Soil Algae ịĩom E^chalted Rock
by asingle zoosporangium. Zoospores fusiform, 7.5 X3.0 μ, their cliloroplast parietal, sometimes dissected; witli 2anterior contractile vacuoles, anterior nucleus, equatorial to posterior
stigma and 2 agella of equal length.
Sexual reproduction not observed.
Two-week-old cultures on basal agar medium dark green, becoming yellow to light (,range
after 3months. Surface of 2-week-old culture granular and vermiform (14 Xmagni cation),
macroscopically dull-shiny; butyrous.
Source: Soil from Enclianted Rock, Llano County, Texits; July, I O.
This alga (Ε-6-47) was isolated from the same soil sample as that from which
Neochloris pseudostigmatica was isolated and is maintained in bacteria-free condi-
tion on BBM, OBBM and Proteose-peptone agar media. The generic attributes of
this organism are clearly those of Spongiochloris (Starr, 1955).
The vegetative cells of actively growing cultures are spherical at all ages (Fig. 34,
35) and have an average diameter of 17-25‫ لءلم‬attaining amaximum size occasionally of 35۶. The cell wall is thin and smooth and does not increase in thickness
beyond 1.25-1.5μ. Bubble-like protrusions of the cell wall are frequently present in
older, inactive cultures in liquid medium (Idg. 36). Young cells will cohere by
their cell ١valls to form awater-repellent pellicle on the surface of liquid media (Fig.
34). No extensive individual matrix is indicated by India Ink or Methylene blue.
The chloroplast consists of a ne network of interlacing strands at the surface of
the protoplast and becomes coarse near t.he center of the cell. Asingle pyrenoid is
located excentrically in the spongy network (Fig. 106). In older cells, the pyrenoid
may disappear following fragmentation. No contractile vacuoles were observed in
the vegetative cells. Mature cells have many small nuclei which ultimately become
the centers about which aplanospores or zoospores are organized by progressive
cleavage of the protoplast (Fig. 107).
Asexual reproduction is accomplished by the formation of zoospores and aplano-
spores (Fig. 37). The zoospores (Fig. 108, 109) are fusiform when liberated from
the vesicle of the zoosporangium and become ovoid .shortly before they become
quiescent. The vesicle is twice the size of the zoosporangium before the former ruptures. The zoospores arC small, ranging up to 7.5/i in length and 3.0/Χ in width. Up
to approximately 100 zoospores may be formed in asingle zoosporangium. The
zoospore has aparietal, sometimes dissected, chloroplast, 2anterior contractile
vacuoles, an anterior nucleus, an equatorial-to-po.sterior stigma, and 2 agella of
equal length.
No sexual reproduction was observed.
The surface of 2-week-o!d cultures on BBM agar are dull-shiny and, when ob-
served at a14 Xmagni cation, are granular and vermiform (Fig. 38). They are
of butyrous consistency. Colonies on this medium are dark green after 2weeks, but
become yellow to light orang-e when 3months old.
fi
fl
fi
fl
fi
fi
fl
This alga differs from all the previously described species of Spongiochloris in
morphology and supplementary attributes. It is most similar to s. excentrica Starr
29
Bischo. and Bold
(1955), from which it differs in its smaller size and in the fusiform shape of zoospores, equatorial-to-posterior stigma, and supplementary attributes.
The organism is inhibited by amphotericin B, colistin sulfate, kanamycin, neomycin, polymyxin-B, paromomycin, dihydrostreptomycin, triclobisonium chloride,
nitrofurantoin, furazolidone and methenamine mandelate (Table 2). Growth is
excellent in BBM supplemented with 0.75% dextrose, fructose, sodium acetate;
fair in ribose and xylose; trace in arabinose. fn darkness, growth is good in BBM
with dextrose; trace in BBM with fructose and sodium acetate (Table 3). The
amylasic ability is good on BBM fon agar containing 0.01% soluble starch
( Ta b l e d ) .
To date, 6species of Spongiochloris have been described, namely s. Spongiosa
and >‫؟‬. excentrica Starr, s. lamellata Deason and Bold, s. minor and s. incrassata
Cliantanachat and Bold and s. typica Trainor and McLean. After close comparison of the speci c attributes of the present organism with those of the known species
of the genus, it was clear that these in totality differ considerably, and, accordingly',
the organism has been described as anew taxon, Spongiochloris llanoensis.
Summary 0١ speci c attributes iu Sfjongiocliloris
Although the 8species of Spongiochloris have been adequately characterized by
Starr (1955),Deason and Bold (1960), Chantanachat and Bold (1962),Trainor
and McLean (1964) and in the present study, for convenience of reference of
future investigators the morphological attributes of the 8species have been summarized in the following tabulation. Variations with respect to agiven attribute are
numbered in corresponding fashion.
Spongiocliloris Spongiosa (Vischer) Stai'r
(1)Vegetative cells spherical to subspherical, attain amaximum diametei' of
ΙΟΟμ.
(2)Cell wall smooth, not thickening with age.
(3) Chloroplast a ne network of interlacing strands; pyrenoid single in young
cells, fragmenting in old ones, occupying acentral to near-central position.
(4) Contractile vacuoles absent. Starch produced, with orange oil appearing in
old cells. Three-month-old cultures remaining green tinged with orange; 2wee'‫؛‬-old cultures homogeneous, but granular (at 14 Xmagni cation).
(5) Zoospores elongate to spindle-shaped; up to 15 X2/İ, usual size; stigma
anterior; 2anterior contractile vacuoles.
Source: Soil (from vicinity of
Fuorn, Switzerland)
Shongiochloris excentrica
fi
fl
fi
fi
fi
(1)Vegetative cells splierical, attaining amaximum diameter of 30،a.
Soil Algae ịĩom E^chai١,leá Rock
30
(2) Cell wall smooth, thickening slightly with age, forming abubble-like protrusion on one side. Cells sometimes secreting agelatinous substance demonstrable with dilute Methylene blue.
(3)Chloroplast acoarse network of interlacing strands, with asingle pyrenoid in
aparietal position.
(4) Contractile vacuoles absent. Cells remaining green to yellowish-green in
older cultures, not turning orange-red. Three-month-old cultures yellowishgreen. Two-week-old colonies very shiny, and smooth.
(5) Zoospores elongate to spindle-shaped; averaging 15 X2،แ; stigma anterior;
with 2anterior contractile vacuoles.
Source: Soil (from Roclc Island, Cane)' Fork River, Tennessee)
Spongiochloris lamellata Deason and Bold
(1)Vegetative cells spherical attaining arnaximunr of 80 .
(2) Cell wall composed of several layers, attaining athickness of 25 μ. Young
cells coherent by their cell walls, no extensive matrix present.
(3)Chloroplast more spongelike than netlike ;pyrenoids 1to several.
(4) Contractile vacuole absent. Cultures 3months old orange at edges of colon)'.
Two-week-old cultures dry and granular (at 14 Xmagni cation).
(5) Zoospores elongate to spindle-shaped; up to 13 X3.7 μ, average 9X3μ;
stigma anterior; with 2anterior contractile vacuoles.
Source: Soil (Caldwell County, Texas)
Spongiochloris minor Chantanachat and Bold
(1)Vegetative cells spherical at all ages, attaining amaximum size of 40 μ.
(2)Cell wall thin, thickening slightly in cultures 1month old, or older.
(3) Chloroplast of mature cells typically anetwork of coarse, interlacing
strands; single excentric pyrenoid, occasionally 2or more present.
(4) Contractile vacuoles absent, oil produced in large quantities in old cells.
Cultures 3months old entirel)' bright orange. Two-week-old colonies dr)',
minutely glomerulate.
(5) Zoospore ovoid; average length 8X3۶; nucleus anterior; chloroplast parietal with posterior pyrenoid; stigma anterior; 2anterior contractile vacuoles.
Source: Soil (6 miles from North St. John, Arizona)
Spongiochloris incrassata Chantanachat and Bold
fi
fi
fi
(1)Vegetative cells spherical, attaining amaximum diameter of 54 μ.
(2)Cell wall strati ed, thickened as much as 2-3 μin 2-week-o!d cultures.
Bischo. aud Bold
31
(3) Chloroplast a ne network of interlacing strands at the surface, coarse at
the center; pyrenoid single, 2or more, occasionally.
(4) Contractile vacuoles not observed. Oil in large quantities in older cells.
Three-month-old cultures having brick-red ecks on green. Two-week-old
colonies dry, scabellate, cracked into, irregular polygonal patches.
(5)Zoospores ovoid ;7-8 X2-4 μ;stigma anterior; 2anterior contractile vacuoles; nucleus anterior; chloroplast parietal with posterior pyrenoid.
Source :Soil (as-Sanam Lat. 21. 46' N. Long. 53° 1٥' E. Saudi Arabia )
Spoiigiochloris typica Trainor and McLean
(1)Vegetative cells spherical at all ages, attaining amaximum size of 80 μ.
(2)Cell wall smooth, not thickening with age.
(3) Chloroplast in mature cells netlike; while in older cells the pattern of the
chloroplast ma)' become less distinct resulting from the accumulation of
storage products within the cells.
(4) Many cells from 6-month-o!d cultures are vacuolate and yellow-orange.
(5) Zoospores variable in shape; up to 15 X4۶; stigma may be anterior or
posterior; 2contractile vacuoles; 2 agella of equal length which are withdrawn into the cell upon quiescence; single nucleus and aparietal chloroplast.
Source: Soil (corn eld in Storrs, Connecticut)
St)ongiodiloTÍs gigantea ‫آل؛؛‬.ً ‫اا‬0١‫ي‬.
(1)Vegetative cells spherical at all ages, attaining amaximum size of 110 ۶.
(2) Cell wall smooth and thin in young cells; cells sometimes coherent, but
readily separated. Cell wall thickening in cultures 3months old, up to 7.515 μ.
(3) Chloroplast of mature cells typically anetwork of interlacing strands; 1or
more excentric pyrenoids, often fragmenting'in older cells.
(4) Contractile vacuole not observed in vegetative cells; large quantities of oil
produced in older cells. Cultures 3months old are bright orange. Twoweek-old cultures are dull-shiny, granular, of butyrous consistency.
(5) Zoospores fusiform when liberated, becoming ovoid, with aminute papilla
between the 2equal agella, averaging 8-10 X2.5-3.0 A،; stigma anterior;
with 2contractile vacuoles; pyrenoid, when visible, posterior; chloroplast
parietal, dissected; nucleus median.
Source: Soil (west exposure of Enchanted Rock, Llano County, Texas)
Spongiochloris llanoensis sp. nov.
fl
fl
fl
fi
fi
(1)Vegetative cells spherical at all ages, attaining amaximum size of 35 μ.
Soil Algae ١ïom Enchanted Roch
32
(2) Cell wall thin and smooth, not thickening with age; may have bubhle-like
protrusions in older inactive cultures in liquid medium.
(3) Chloroplast a ne network of interlacing strands at the surface, becoming
coarse near the center; single excentrically located pyrenoid in young cells,
fragmenting in oldei- ones.
(4)Contractile vacuoles 'absent. Starch produced in large quantities in old cells.
Cultures 3months old yellow to light orange. Two-week-old cultures dark
green, dull-shiny, granular, vermiform and of butyrous consistency.
(5)Zoospores fusiform when liberated, becoming ovoid ;averaging 6.2 X 7 5 X
3μ; stigma equatorial or slightly posterior; 2contractile vacuoles; pyrenoid
not visible in zoospores; parietal chloroplast and an anterior nucleus present.
Source: Soil (southeast exposure of Enchanted Rock, llano County, Texas)
Ke"V to the cuïïentl١ кйоиеи species 0‫ أ‬Spoiiglochlorls StaiT
2
]. Cell walls thickening markedly with age -- .4,
1. Cell walls not thickening markedly with age' .
s
.
l
i
i
c
r
a
s
s
a
ta
2. Mature cells with 1pyrenoi
---------3
2. Mature cells with more than 1- pyrenoid --------------------------------------
3. Young cells coherent by the cell walls which persist throughout tire
lives of the cells; з-month-old cultures on BBM agar becoming
lamellata Deason and Bold (1960) .‫؟‬،
orange at the margin of the colony
3. Young cells isolated, separate ;з-month-old cultures on BBM agar
5
becoming bright orange all over ------------4. Pyrenoid central in mature cells ----- -4. Pyrenoid(s) excentric in mature cells
s. Spongiosa Stari' (1955)
6,
5. Vegetative cells attaining amaximum diameter of 40 μ; cell wall
thickening slightly in cultures 1month old, or older
S. minor Chantanacirat
and Bold (1962)
5. Vegetative cells attaining amaximum diameter of 11-0 μ; cell wall
s. gigantea sp. nov.
thickening up to 15 μin cultures 1month old, 01' older
s.typlca
6. Vegetative cells attaining amaximum diameter of 80 μ
Trainor and McLean (1964)
6. Vegetative cells attaining amaximum diameter of 35 ۶--------- 7. Zoospores elongate to spindle-slraped, avei.aging 15 μin length,
s. excentrica Starr (1955)
with anterior stigma
7. Zoospores fusiform to ovoid, averaging 6.2-7.5 μin length, with
s. llanoensis sp. nov.
an equatorial to posteriol' stigma
Neochloris pseudostigmatica sp. nov. (Fig. 39-49; 110-118)
fl
d
fi
fi
Cellulae omnis aetatis sphericae; 65 μdiam. max. Membrana cellulis in iuvenibus tenuis
levlsque, aetate spissescens. Protoplastus guttulas parvas avo-rubras ad locum eundem in
super cie exterioribus strati memlrranae successive secernens; cellulae vctustlores matricibus
BiscHo. a^d Bold
33
singulis communalibusque circumdatae. Chloroplastus parietalis cavus sphericus, ad unum
poluin aperturam forma irregulärem et 1-12 pyrenoidea excentrice inclusa praebens; vacuolae
pulsantes in cellulis vegetativis non observatae. Cellulae multinucleatae.
Reproductio asexualis per zoosporas aplanosporasque, per ssionein succcssivain protopiasti multinucleati formatas. Zoosporae tempore liebrationis ovatae, intra alic!uot minutas
sphericae quiescentesquc factae; zoosporae 6.8-8.0 X3.2-3.8 μ, nucleum medium parvuin, 2
vacuolas pulsantes anteriores, chloroplastům parietalem saepe dissectum habentes; Stigma
pyrenoideuinque non visa; agella dua longitudine aequa, aliquantuluin longiora quam corpus
cellulae.
Reproductio sexualis non observata.
Super cies cul turae 2hebdomaduin aetatc in medio “agar” basali nitida macroscopicallter
visa; glomerata (magnl catione 14 pio); viscida, culturls post 3menses nitido-aiirantiis factls.
٥rigo :in solo eloco Enclianted Rock, Llano Co., Texas dicto, m. Jul. ]960.
Cells spherical at all ages, maximum diameter 65 μ. Cell wall thin and smootli in young cells,
thickening with age. Protoplast secreting small orange-red droplets in succession at the same
point on the surface of the outci' wall layer; older cells surrounded by individual and common matrices. Chloroplast aparietal, hollow spliere, with an 1!'regularly shaped opening at one
pole with 1-12 pyrenoids embedded in excentric positions. Contractile vacuoles not observed
in vegetative cells. Cells multinucleate.
Λsexual reproduction by zoospores and aplanospores formed by progressive cleavage of the
multinucleate protoplast. Zoospores ovoidal wlien released, becoming spherical and quiescent
within several minutes; zoospores 6.8-8.0 X3.2-3.8 μ; with small median nucleus, 2antei'ior
conti'actile vacuoles, and aparietal, often dissected, cliloroplast; stigma and pyrenoid not
visible; with 2 agella of equal lengtli, slightly longer than tlie cell body.
Sexual reproduction not observed.
Surface of 2-week-o!d cultures on basal agar medium shiny inacroscopically; glomerate
(14 Xmagni cation) ‫؛‬viscid, cultures becoming bright 01'ange at 3months.
Soiii'ce: Soil from Eiiclianted Rock, Llano County, Texas; July, 1960.
This alga (Ε-6-41) was isolated from asoil sample collected from ashallow
depression on the southeast exposure of Enchanted Rock. Cultures of this organism
have been maintained in the bacteria-free condition in ٥BBM and IBBM and on
BBM agar.
The vegetative cells of this organism are spherical at all ages and may 1'each a
maximum diameter of 65 μ, with the cells of apopulation averaging between 20
and 28 μ. The cell wall is thin and smooth in )'Oung cells but thickens with age.
Mature vegetative cells have cell walls 3.5-7.7 μthick, surrounded by abroad,
lamellated individual matrix, which increases in thickness as the cells age (Eig.
110). Large numbers of cells adhere to one another by their sticky, individual
matrices (Eig. 39). One-12 or more excentrically placed pyrenoids are embedded
in the chloroplast which is aparietal, hollow sphere with an irregularly shaped
opening on one side (Fig. 40, 111).
The outstanding attribute of the present organism, which apparently is unique,
is the appearance of asmall, orange-red droplet on the outer surface of the cell wall.
fi
fl
fi
fl
fi
fi
This droplet, which is perhaps ametabolic waste, is secreted through the cell wall
and assumes various forms (Eig. 112). Large cells may have one of these droplets
3 4
Soil Algae İTom Eudianteđ Rock
on each concentric layer of their individual matrices (Fig. 113). The secretion of
this droplet is rst seen when aculture is 3-4 weeks old (Fig. 41 ). Some cells have
more than one pore through which the droplet is secreted. This pore also apparenti)weakens the wall in one area, thus allowing an older protoplast to slough off its
thickened wall and individual matrix (Fig. 42-44). older cultures in liquid BBM
may have abubble-like thickening at one pole of the cell similar to that of Neochloris terrestris Herndon (I958b) (Fig. 114). Contractile vacuoles were not
observed in the vegetative cells of this species.
The vegetative cells are already multinucleate at an early age and usually have
16 nuclei by the time progressive cleavage takes place (Fig. 45; 110, 115) in zoo٠
Commonly 16-32 zoospores are released in avesicle from the zoosporangium. Upon rupture of the vesicle, the active, ovoidal zoospores are released
into the surrounding medium and become quiescent and spherical within several
minutes. The zoospores range in size from 6.8 to 8.0 μin length and from 3.2 to
3.8 μin width. They are pointed at the anterior and rounded at the posterior pole
(k’ig. 116,117). Their very small nucleus is median, while the 2contractiles are
anterior. The chloroplast is parietal and may be dissected. No visible stigma or
pyrenoid was observed in the zoospores. Eacli of the latter has 2equal agella
slightl)' longer than the cell body.
Sexual reproduction was not observed.
The pyrenoid appears shortly after the motile phase (Fig. 118) ends. The vesicle
frequenti).- does not rupture, and the zoospores become quiescent, forming acluster
of aggregated cells (Fig. 46). This species, like N. gelatinosa Herndon (I958b),
grows rapidly on agar media by aplanospore formation. Mounds of vegetative cells
of various ages are enclosed in individual and common matrices (Fig. 47). Also
similar to TV. gelatinosa, zoospore production in liquid cultures was observed onl)-on rare occasions. However, asuccessful method for inducing zoosporulation was
found b).- transferring cells, heavily enveloped with gelatinous matrices, from an
agar medium into atube of distilled water. The latter was placed in an ultrasonic
water bath (“Disontegrator, System 80”; Model G-80C1) for 1min and then
stored in darkness for 12 hr followed b).' exposure to light for 2hr. Abundant zoo-
sporogenesis followed. The dif cult)' at rst experienced in evoldng zoosporogenesis
in the present alga suggests caution in using absence of zoospore production as a
taxonomic attribute without adequate prior experimentation.
This organism clearl)' possesses the 3distinguishing attributes of the genus Neochloris: (1) vegetative cells with ahollow, spherical chloroplast; (2) 1or more
pvrenoid.s .in the vegetative cells; and (3) production of zoospores with agella of
equal length which become immediately spherical upon quiescence.
The prominent individual and common gelatinous matrix, 1or more excentricall)' located pyrenoids, and multinucleate vegetative cells relate this species very
close').' to N. gelatinosa Herndon (I958b). However, convincing evidence that the
fl
fl
fi
fi
fi
present organism is adifferent taxon is suppoited by the fact that the outer wall
Bischo. aud Bo‫؛‬d
35
layer thickens markedly with age, extrudes red-orange droplets through the cell
wall, and lorms zoospores with pointed anterior poles and rounded posterior poles,
without visible pyrenoids and stigmata, tlpon this evidence, and, in addition, becau''C of the dissimilarities of supplementary attributes between the present organism and N. gelatinosa, the former is here described as anew taxon, Neochloris
pseudostigmatica sp. nov. The speci c name alludes to the colored ‫ص‬
body on the outer side of the cell wall.
Neochloris pseudostigmatica is inhibited by- amphotericin B, colistin sulfate,
kanamycin, neomycin, polymyxin-B, paromomycin, dihydrostreptomycin, sulfamethoxypyridazine, triclobisonium chloride, sul soxazole, sulfadimethoxine, sulfadiazine, triple sulfa, furaltadone, nitrofurantoin, furazolidone, methenamine mandelate, and para amino salicylic acid (Table 2). The surface of a2-week-o!d culture
on BBM agar, when observed at 14Χ magni cation, is gloinerulate (Fig. 48, 49).
Macro.scopically, it is shiny' and the consistency is viscid. Two-week-old colonies on
this medium are green but become bright orange when 3months old.
Growth is excellent in light in BBM supplemented with dextrose; good in BBM
with fructose; fair in BBM with ribose; occurs in trace amounts in BBM with xylose
(Table 3). In darkness, only dextrose supported atrace amount of growth. The
aiuylasic power of this organism is good on 0.01 %soluble starch in BBM Ion agar,
wide halos being present with the iodine test (Table 4-).
Key to the cunently kuoTjou species 0‫ ا‬NeochloTİs Starr
1. Cells, except those recently originating from zoospores, aggregated
in acommon matrix -------------
------- -- -
-
-
-
-
-
2,
3.
1. Mature cells lacking acommon matrix
2. Outei- wall layer (Deason and Bold, 1960 )thickening markedly
with age, exti'uding 1to several orange-red droplets
N. ()seudostlgmatica
s p . n o v.
2. Outer wall layer not thickening markedly with age, orange-red
droplets not extruded through the cell wall
- ΛΛ. gelatinosa (Hei'ndon,
19586).
3. Individual matrix of watery consistency, present in young cells;
cells attaini,ng 75 μ-------N. terrestris Hei'ndon (I958b)
3.
Individual
matrix
absent
4.
4. Outer wall layei- thickening markedly with age -----+. Outer wall layer not thickening markedly with age
5. Cells attaining amaximum size of 18 μ, zoospores almost spherical
N. minuta Arce &Bold
5. Cells exceeding 18 μ; zoospores elongate -- ----------- -----------------
5,
(1958)
6.
6. Cells up to 45 μin diameter; zoospores pointed anteriorly
fi
fi
fi
-
-
N. pyreiioidosa Arce &Bold (1958)
36
Soil Algae İTom Enchanted Rock
6. Cells up to 22 μin diameter; zoospores pointed at both ends
-- .и N .
jusispora
Arce
&Bold
(1958)
7. Mature cells miiltinucleate ‫ل‬٠‫ل‬٠‫للل‬
N. aquatica ‫اًل ـ ـ ـ ـ ـ ــةاة‬
7. Mature cells uninucleate ...... ........... ......
8.
8. Mature cells usually with 2pyrenoids
N. oleoabundans Chantanachat
&Bold (1962)
9.
8. Mature cells usually with 1pyrenoid
9. Colony dry, pyrenoid elongate-ellipsoidal
9. Colony dull-shiny, pyrenoicl isodiametric
.. N. alveolaris Bold (1958)
N. )}seudoalueolaTt.‫ ؟‬Iiea0<‫؟‬Ti
&Bold (1960)
Planktosţihaeîla
Smith (1918) reported anew planktonic genus and species, Planktosphaeria
gelatinosa, from alake in Wisconsin. The alga was described as reproducing only
by autospores. However, Smith (1933), suggested the possibility of reproduction
by zoospores as well, but later (1950) he reverted back to his original conception
of reproduction in the genus Planktosphaeria.
The rediscovery of the organism in soil at Woods Hole, Massachusetts, and the
observance of zoospore production by Starr (; 1954) impelled him to redescribe it
and transfer the genus from the ٥ocystaceae to the Chlorococcaceae. Skuja (1956 )
is of the opinion that it should be in the Tetrasporales near the genus Gloeocystis.
Up to date, 2species of Planktosphaeria have been described—T. gelatinosa Smith
(1918) and Starr (1954) and p. botryoides Herndon (I958b). The writers do not
recognize Planktosphaeria paradoxalis (Miller) comb. nov. (‫ ت‬Follicularia paradoxalis Miller, 1924-) as amember of the genus, as proposed by Starr (1954), for
the reasons given by Herndon (I958b).
Starr (1954, 1955 )distinguished the genus Planktosphaeria from other spherical
chlorococcaceous algae by the following attributes: the production of zoospores
without walls and with 2 agella of equal length; vegetative cells with many sepa1-ate, discoidal chloroplasts, eac.h containing ap)'reno!d.
Planktosphaeria texensis sp. nov. (Fig. 50-60; 119-121)
Cellulae vegetativae omnis aetatis .spherlcae, 50 μdiam. max. Cellulae vegetatlvae seiuiictae
aut in coloniis ealiquot ad 50 cellulas vel plurcs constantibus, in matricibus singulis communalibusque involutae. Membranae cellularum tenues levesque, aetate non manifeste spissescentes. Chloroplast! cellularum maturarum parietales prismatici, asupci' cle visi polygonales. Umnis chloroplastus unum pyrenoideum saltem habens; amyliim abundans, guttulis,
autem, ut videtur, omni aetate nullis, cytoplasma vacuolata; cellulae vegetativae sine vaeuolis
pulsantibus. Ccllulae maturae multlnucleatae.
Reproductio asexual is per formationen, zoosporarum aplanosporarumque. Sing'ulum zoosporangium usque ad 50 zoosporas formans. Zoosporae elongatae, ovatae factac, 8.3-11.6 X
fi
-
fl
.
2.5-3.5 μ, unum chloroplastům parietalem dissectum, 2vacuolas pulsantes anteriores, nucleum
37
Bischo. aud BoÎd
anteriorem magnum, stigma !‫؛‬neare, c٩uator‫؛‬ale ad posteriore, 2 agella longitudine aequa
halrentes. Pyrcnoidcum in zoospora non visibile.
Reproductio scxualis non observata.
Culturae duai'um liebdoniadum aetatc iir medio “agar"' basali atrovlrides, post 3menses
olivacco-virides ictae. Super cies culturae 2-hebdomadum aetate glomerulata (magni catione 14 pio) ;nitida macroscoplcaliter visa; viscida et substrato adhaerens.
Oi'igo :in solo eloco Enclianted Rock, Llano Co., Texas dicto, m. Jul. 19611.
Vegetative cells splierical at all ages, maximum diameter 50 /X. Vegetative cells isolated OI' in
colonies of several up to 50 or more, enveloped by individual and common matrices. Cell walls
thin and smootli, not tliickenlng markedly with age. Cliloroplasts of mature cells parietal, prismatic, appearing polygonal in surface view. Each cliloroplast witli at least 1pyrenoid; starch
abundant, but droplets apparently absent at all ages; cytoplasm vacuolated. Contractile vacuoles absent in vegetative cells. Cells multlnucleate wlien mature.
Asexual reproduction by zoospore and ‫؛‬iplanospore formation. Up to 50 zoospores formed
by asingle zoosporangium. Zoospores elongate, becoming ovoldal, 8.3-11.6 X2.5-3.5 μ; witli
1parietal, dissected chloroplast, 2anterior contractile vacuoles, nucleus anterior, large, linear
stigma, the latter equatorial to posterior; with 2 agella of equal length. Pyrenoid not visible
in zoospore.
Sexual reproduction not ob.served.
Two-week-old cultures on basal agar medium dark green, becoming olive-green after 3
months. Surface of a2-wcck-old culture glomerulatc (14 Xmagni cation); macroscopically
shiny; viscid and adlierent to .substratum.
Source: Soil from Enchanted Rock, Llano County, Texas; July, 1960.
An organism clearly belonging to the genus Planktosphaeria was isolated into
pure culture (Ε-6-25 )from asoil sample collected from adepression on the east
exposure of Enchanted Rock, Llano County, Texas. Axenic cultures have been
maintained on OBBM and Proteose agar. Young vegetative cells are spherical and
have 1or 2parietal chloroplasts, each with apyrenoid. In very young cells, 3.85.5 μ■ in diameter, which have just been formed from azoospore, the pyrenoid may
not be visible. However, staining with la-KI reveals large granules of starch present
in the chloroplast (Fig. 119). As the vegetative cell enlarges, the chloroplast divides
to form numerous parietal, platelike segments (Eig. 5٥). Each chloroplast contains
at least 1pyrenoid; starch is produced in abundance, but no oil droplets were seen
at any stage.
The colorless cytopIa.sm, which occupies the internal portion of the cell, may
contain several large volumes. Cells growing both on agar and in liquid media have
vacuolated cytoplasm. Like those of p. gelatinosa and p. botryoides, the chloroplaste
of mature cells are prismatic, appearing polygonal in surface view (Fig. 51 ).
The vegetative cells are spherical at all ages and occur singly or in colonies of
several up to 50 or more within agelatinous matrix (Fig. 52-54). The average
size of amature cell ranges between 20 and 25 ،a, amaximum diameter of 50 μ
being observed frequently. The cell wall is smooth and thin; however, it may increase to 2μin thickness in some large cells grown on agar media. Vegetative cells
are uninucleate only when young; they become multinucleate with increase in cell
fi
fl
fi
fl
fi
fi
size.
Soll Alette İTom Euchanted, Rock
s accomplished by the production of zoospores and‫ ؛‬ехиа1 reproduction‫^'\؟‬
aplanospores resulting from progressive cleavage of the multinucleate protoplast.
Zoosporogenesis was induced by submitting tubes containing thriving cultures in
liquid medium to ultrasonication followed by aperiod of darkness and then by
exposure to light. Release of zoospores from the zoosporangium is accomplished by
the swelling of the gelatinous vesicle within the zoosporangial wall until the latter
is broken into many pieces which lie attached to the periphery of this gelatinous
vesicle (Fig. 55, 120). Rupturing of the latter releases approximately 50 zoospores
into the surrounding medium. The majority of gelatinous vesicles do not rupture.
Instead, the zoospores are retained within them, and, after ashort period of limited
inotilitv within the vesicle (Fig. 56), they become quiescent, withdraw their
agella, and continue their existence in amanner similar to apianospores (Fig. 57 ).
The zoospores vary somewhat in size, being 8.3-11.6/xin length and 2.5-3.5 μ
in width. They are amorphous prior to escaping from the sporangial vesicle; after
being released, the)' are elongate at rst, later becoming ovoidal (Fig. 121), and
tlien spherical as the motile phase ceases (Fig. 58). Each zoospore possesses 1dissected parietal chloroplast, 2anterior contractile vacuoles, an anterior nucleus, a
large linear stigma embedded in the chloroplast near and slightly posterior to the
equator of the cell and 2 agella of equal length; apyrenoid usually is not visible.
The zoospores swim sluggishl)' for ashort period, after which they assume aspherical shape and withdraw their agella.
Aplanospores and zoospores not liberated from the gelatinous thick-walled ١'es‫؛‬cle
are indistinguishable In any given culture. Aplanospores are produced in groups of
Several to 50 or luore in asingle parent cell. Individual gelatinous matrices enclose
these young developing cells which are further enclosed by acommon matrix.
Τ١νο distinct gelatinous layers envelop any given group of aplanospores or zoospores. Ayoung vegetative cell developing from azoospore or aplanospore soon
begins secreting agelatinous la)'er. When this cell is mature the gelatinous layer is
not rm and appears to be of awatery consistency when stained with Methyleneblue solution. As aprotoplast undergoes progressive cleavage to produce zoospores
or aplanospores (Fig. 59), agelatinous layer develops within the cell wall, swells
and breaks the latter into many small pieces that lie on the peripher)' of this ex-
panding layer which appears like avesicle (Fig. 120). T'he zoospores or aplanospores are now enclosed by an outer, thin, delicate, gelatinous layer and an inner,
rm, gelatinous layer. The 2gelatinous layers are separated by fragments of the
original parent cell wall. This outer, gelatinous layer encroaches on and coheres to
other outer gelatinous layers of neighboring cells forming acommon matrix.
Sexual reproduction was not observed.
Two-week-old cultures (Irig. 60) on BBM agar are gloinerulate when observed
with 14Χ magni cation; macroscopically they are shiny, slightly viscid and adherent. Two-week-old cultures on this medium are dark green and remain so, even
fi
fl
fl
fi
when 3months old.
fi
fl
fi
38
39
Bischo. ttuđ Bold
Growth is excellent in glucose and occurs in trace amounts in dextrose in darkness (Table 3). Planktosphaeria texensis is inhibited by amphotericin B, colistin
sulfate, kanamycin, neomycin, polymyxin-B, paromomycin, triclobisonium chloride, furaltadone, nitrofurantoin, nitrofurazone, furazolidone, methenamine mandelate and para amino salicylic acid (Table 2). The amylasic power of this organism is good on 0.01 %soluble starch in BBM Ion agar (Tabled).
Planìítosphaeria texensis differs from p. botryoides Herndon (I958b) in various
supplementary' attributes, and in not producing droplets of oil. Its cytoplasm is
’which is not true of p. botryoides. The zoospores of p. texensis have a
linear stigma near the equator, while p. botryoides possesses asmall posterior
stigma. Furthermore, the zoospores of p. texensis have parietal, dissected chloroplasts instead of posterior chloroplasts. Planktosphaeria texensis differs from p.
gelatinosa, the species which it most closely' resembles morphologically, in that the
cy'toplasm is vacuolated. Vegetative cells reach amaximum of 50 /-،; also, vegetative
cells occur singly or in colonies of several up to 50 or more, while those of p. gelatinosa occur in colonies of d, 8or 16 cells.
Finally', comparison of supplementary attributes reveals that the alga isolated
from Enchanted Rock by the writers is not the same species as that described by'
Starr (I95d) from Woods Hole, Massachusetts.
In ١'iew of the distinctive dissimilarities among the morphological and .supplementary' attributes, the organism discussed above is here described as anew taxon,
Plauktoshhaeria texeusis.
Planklosphaeria maxima sp. nov. (Fig. 61-67; 122-126)
Collulac vegetativae oiiinis aetatis sphericae, saepe plus fjuam 200 ¡1 cliaii. Protopla.stus per
¡uvolucrum lamellatum usqu(' ad 30 μinterdiim incrassatuiii circiimdatus. Strata exteriora
involucil post rupturam ad polos oppositos abeuntia. Chloroplast! ecllularum maturaiiiui
parvi, laminiformes irregulares, et parietales et interiores. ٥innis cliloropla.stu.s cellularuiii iuvenuiii uniinr pyrenoideum saltem continens, pyi'enoideo cellulis in vetustioi'ilrus fracto. Ainylum omni in ecllula copio,sum. Cellulac vegetativac sine vaeriolis pulsaiitibu.s. Ccllulae !naturac multiniicleatac.
Reproductlo asexualis per zoosporas aplanospora.squc. Zoosporangia 35-1 do μdiam., maioilIjus plus quam 100 zoosporas ef cientibu.s. Zoospoi'ac fusiformes ad ovatiis, 9-11 X2.5-4.0 μ
nuclcunt anteriorem, Ivel 2eliloroplastos parietales stigma anterius et 2 agella longitudine
aequa habentes.
Reproductio .sexualis non obscivata.
Culturae duatum hebdomaduin aetatc in metilo "agar'۶ basiili atroviritles, post 3menses
aurantiae factac‫ ؛‬super cies cultui'ae duarum hebdornadum actate granulos‫ !؛‬et paululum reticulata (magni catione 14 pio); macroscopaliter visa fusca, substrato adhaerens.
Origo: In solo eloco Enclianted Rock, Llano Co., Texas dicto, ni. Jul. 1960.
Vegetative cells spherical at all ages, often reacliing diameters over 200 μ. Protopla.st surrounded by alamellated envelope which may tliickcn as mucli as to 30 μ. Outer layers of en-
fl
fi
fi
fi
velope slougliing off after I'upture at opposite poles. Cliloroplasts of mature cells small, plate-
Sot.1 Algae ţîom Ełicliaate Rock
sliapc, boti, parietal aud internili. Eacli chloroplast of young cells containing ً ‫اا‬١ ke, irregular ‫اًا‬
at least 1. pyrenoid, the latter fragmenting in older cells. Starcli abundant In all cells. Conti'actile Viicuolcs absent in vcgetiitive cells. Cells nniltinucleate when mature.
Asexual reproduction by zoospores and aplanospores. Zoosporangia 35-140 ‫ بم‬in diameter,
more than 100 zoospores produced by tlie larger ones. Zoospores fusiform to ovoidal, 9-11 X
interior stigma and 2‫ ؛‬,with anterior nucleus, 1or 2parietal cliloroplasts, anterior ,‫ ب ـ ـ ـ ـ ـ ــم‬2.5-4.0
agella of ecjual lengtli.
Sexual reproduction not observed.
Two-week-old cultures on basal ;igar medium dark green, liecoming orange at 3months;
surface of a2-week-o!d culture granular and slightly reticulate (14Χ magni cation); macroscopically dull; adherent to substratum.
Source: Soil from Encliantcd Rock, llano County, Texas; July, 1960.
Asecond species of Planktosphaeria was isolated into pure culture from asoil
sample collected from acrevice near the base of Enchanted Rock on the southeast
exposure, and it is unquestionably anew species. Planktosphaeria maxima has been
maintained in axenic culture on OBBM, and Proteose agar medium.
The vegetative cells of p. maxima are spherical at all ages and occur as solitary
or isolated individuals (Fig. 61) in comparison to the pseudocolonial habit of p.
gelatinosa, p. botryoides and p. texensis. However, the secretion of agelatinous
substance by the cell walls of thriving cultures causes groups of cells to cohere (Fig.
62). Methylene-blue solution indicates avery scant and delicate secretion of colloids on the exterior of the cell wall. Vegetative cells may reach adiameter up to
200۶(Fig. 63).
The chloroplasts of young cells are platelike and parietal; the internal portion
of the cell contains colorless cytoplasm. Evidence of ade nite pyrenoid is lacking
In some of the chloroplasts of very young vegetative cells; large starch granules are
present, however. As the young cells enlarge, the platelike chloroplast divides to
form many Slualler irregularly shaped plates. Cells 3days old have anumber of
small chloroplasts each containing apyrenoid. Mature cells also contain internal,
subsurface chloroplasts, so that the cell becomes almost solidly lled with chloroplasts, except for very small areas of colorless cytoplasm containing the nuclei (Fig.
122).
fi
fi
fi
The cells are uninucleate only when very young, becoming multinucleate with
increase In size and age. Starch is produced in abundance, but no oil accumulates
at any age in this species. The vegetative cells are surrounded by alamellated envelope which may thicken as much as to 30 μin older cells (Fig. 64). Five-day-old
cultures on agar show onl)' aslightly thickened envelope; 14-day-old cultures have
envelopes ranging between 20 and 30 μin thickness. The outer layers of the envelope often slough off after rupturing at opposite poles (Fig. 65, 66). The cells increase in size very rapidly in cultures between 7and 14 days old.
Planktosphaeria maxima reproduces asexuall)' by the formation of zoospores and
aplanospores, which result from progressive cleavage of the protoplast. Zoosporo-
fl
fl
4٧
41
Bischo. aud Bold
genesis is less dif cult to evoke in cultures of this species than in cultures of p.
texensis. Zoosporogenesis may be induced by transferring an old culture from agar
medium to atube of ٥BBM, which is then placed in darkness for 12 hr and subsequentl)' exposed to light for at least 2hr. Mature cells, which have recently sloughed
off their outer, thick lamellated wall, are the cells which have been observed "to
undergo zoosporogenesis. Zoosporangia range in size from 35 to 140 μin diameter.
The larger cells produce hundreds of zoospores, nlike other species of Planktosphaeria, the liberation of zoospores from the parent cell in p. maxima is accomplished by the formation of apore in the cell wall. Apparently because of the compactness of the chloroplasts in mature cells, it is not uncommon to observe abnormal
zoospores resulting from incomplete cleavage (Fig. 123-125).
Zoospores are fusiform when rst liberated (Fig. 126), becoming ovoidal with a
tapered posterior pole, and, nally near the end of the motile phase, they become
spherical. The)' range in size from 9to 11 ‫ ي ـ ـ ـ ـ ـ ــلم‬in length and from 2.5 to 4.0 ‫ أل ـ ـ ـ ـ ـ ــم‬in
width. Each zoospore has 2 agella which are equal in length, 1or 2parietal chloroplasts devoid of pyrenoids, an anterior nucleus and alarge, anterior stigma. Aplanospore formation is the usual method of reproduction on agar medium.
Sexual I’eproduction was not observed.
.'\mong the supplementary attributes recorded for this alga are the following:
2-week-o!d cultures on BBM agar are dark green, becoming orange after 3months.
The surface of a2-week-o!d culture, when observed under 14Χ magni cation, is
granular (Fig. 67) ;the colonies are dull macroscopically, and adherent to the substratum. Planktosphaeria maxima is inhibited by colistin sulfate, kanamycin, polymyxin-B, paromomycin, triclobisonium chloride, furaltadone, nitrofurantoin, fur-
azolidone, and methenamine mandelate (Table 2). ft grows excellently in BBM
supplemeirted with dextrose and fructose, and in trace amounts supplemented with
aralrinose, sodium acetate and ribose (Table 3). Growth is fair in dextrose and
fructose in darkness. Cultures in dextrose turn light orange at the surface of the
medium (Table 3). The amylasic power of this alga is excellent on 0.01 %soluble
starch in BBM Ion agar (Table4).
This organism differs from other recenti)' described species of this genus in the
following attributes: its vegetative cells OCCUJ- as separate or isolated individuals
and attain diameters up to 200 /x; mature cells become almost solidly lled with
small cliloroplasts and are surrounded b)' alanrellated envelope which may thicken
to as much as 30 ‫ ;يـ ـ ــلم‬and hundreds of zoospores are lilrerated from the zoosporangium through apore in the cell wall.
Upon this evidence, and, in addition, because of the dissimilarities in supplementar)'- attributes between the present organism and other species of Planktosphaeria, the alga discussed above is here described as anew taxon, Planktosphaeria
fi
fi
fl
fi
fi
fl
fi
-maxiiua.
Soil Algae ‫؛‬тот ETiehaated Rock
42
Tetrasporales
Goccomyxaceae
Ourococcus niultispoius sp. nov. (Eig. 68, 69; 127-130)
Gcllulac vegetativa, ovatae ad fusiformes 24 μlong, max.; umis polus ccllulae rotundatus
-cellulae praeditu.s. Chloro ‫؟‬.XlongİOTC quam corpu ‫_ل ـ ـ ـ ـ ـ ــةز‬liter acuminatus, spicula delicata 12‫؛‬
plastiis parietalis pyrenoideuni singulum liabens. Membrana levis tenuisque, aetate non spissescens. Cellulae nisl in medio liquido non coliaerentes, aggregatae per polos aennilnatos
eohaerentes.
Reproduetio ascxualis per forinationem 4-8 plernnique 16 autosporarum.
Ikeproductio scxualis atfjue formatlo zoosporarum non observata.
Coloniac duai'uni liebdoniadum aetate in medio '‫؛‬agar” ba.sall atroviride.s, nitidae macroscopicaliter visae, leves (magnilfcatione 14 pio); butyraeeae. Coloiiiae trium menslum aetate
nitido-aurantlae.
Origo: in .solo eloco Enchanted Rock, Elano Co., ICxas dlct(>, m.Jul. l6(‫(؛‬l.
Vegetative c.ells ovoidal lo fusiform, maximum lengtli 24 μ·, one cell pole rounded, tlie otlier
acuminate; witli adelicate .spicule 12-‫؛‬/‫ ل‬Xas long ‫؛‬.IS cell body attached to acuminate pole.
Cliloroplast parietal with asingle pyrenoid. Cells uninucleiite at all ages. Cell wall sinootli and
tliln, not thickening witli age. Cells not coherent, except In liquid medliint; gronp.s ol cells
(:(.')lierent by llieli' acuminate poles.
Asexual reproduction liy anto.spore formation, ‫^ا‬rodn('ing (u.snally) 4—8 ‫؛‬ind sont (.times 16
autospores.
Sexual reproduction and zoospot(' formation not oirserved.
l'١١O-week-old colonies on ba.sal agili medium dark green; mado.scoplcally .shiny; smootli
(14 Xniagnillcation); liutyrfjus. Three-montli-old (-olonles bright 01-ange.
Source: Soil from Enchanted Rock, !,lano County, Texas; July, 1660.
According to Chodat (1913), Mademoiselle -A. Grobèty studied an alga isolated
from watei' near Geneva, Switzeiland, and described it as anew gctius and specie.s,
O u To c o c c u s b i c a a d a t i A S .
Anothei. species of the genus, urococcus multisporus sp. nov., was isolated into
pure culture (Ε-9 )by the writers from an extremei)' dry soil sample collected from
adepression OIT the southeast exposure of Enchanted Rock, Llano County, L'exas.
Axenic cultures ha١'e been maintained on all types of media used during this invest‫؛‬gation, inasmuch as the alga seems to have no special growth requirements.
1'he cells vary in form from ovoidal to fusiform (l.’ig. 68). ٥ne pole is rounded
wliile the other is acuitiinate, the acumination terminating as asmall, ared apex.
Erom the latter extends asingle straight spicule which is delicatei)' attached; the
.spicule is 1i/r¡ Xas long as tlie cell bod)' (Idg. 127). Mature cells attain alength
of 24‫ ءلم‬and vary In width from ‫ة‬to 12μ. Cell size is varialrle in an)' given population
!')ccaiise of differences in the age of the individuals, the majority averaging between
12 and 1.5/.(.in length.
٦'he chloroplast is parietal and contains a'pyrenoid which is clearly visible wheiT
stained with f^-Kf solution. The clear cytoplasm contains asingle nucleus. The cell
fl
fi
walls are thin and smo tli. The cells are not coherent, an indication of the absence
43
Biseko٥ aud Βοίά
of an outer gelatinous layer. However,.in liquid medium, groups of cells are COherent by their acuminate poles. As mentioned above, the spicules are delicately
attached and are usuali)' disconnected from the cell in the proce.ss of maldng mounts
for stud).'(Fig. 68).
The onl)' method of reproduction observed is auto.spore formation (Fig. 128130). The chloroplast and pyrenoid divide preceding karyokinesi.s and cytokinesis.
The rst division of the protopla.st ma)' be transverse or oblique (I g. 128, 129).
The second, third and fourth division occur in an)' plane. An increase in growth of
the newl)' formed autospores causes the parent cell wall to rupture, this !.eleasing
them into the surrounding medium. The size of the autospores is inversely propoi'tional to the number produced by agiven parent cell (Fig. 130). Occasionally, a
bicaudate cell resemltling Ourococcus bicaudatus is seen, but in tills species this .is
an abnormalit)-- resulting fron! incomplete unilateral cytokinesis during autosporogenesis.
Among the supplementary attributes recorded for this alga are the following:
two-week-old cultui'es on BBM agar are dark green, shin).-, smooth and butyrous
(Fig. 69). Three-month-old cultures maintained on the same medium are liright
01-ange. Ourococcus multisporus is inhibited b)' neomycin, tricloliisonium chloride,
nitrofurantoin, fui'azolidone iind methenamine mandelate (Table 2). Growth is
good in dextrose, fructose; fair in arabinose and sodium acetate; occurs in tj'ace
amounts in ribose. Growth is fair In dextrose in darkness (Table 3). The alga, accordingl).', seems to be facultatively heterotrophic. No growth occurs in x)٠'lose in
light or darkness. The amylasic power of this alga is slight on 0.01 soluble starch
iiiBBM Ion agar (Tabled).
Ourococcus multisporus differs from o. bicaudciliis especiall)' in the following
attributes: the size of the vegetative cells is larger, up to 24/1 in length, and the
shape of the vegetative cells is relittivel)' uniform in any given culture (Fig. 68),
while polvmorphi.sm is said to be exces.sive in any given culture of o. bicaudaius.
Ourococcus midtisporus possesses along, delicate unipolar spicule, and produces
up to 16 autospores from aparent cell.
Ourococcus bicaudatus was not available for comparison of supplemental)' attributes.
The Genus Bracteacocciis Tereg
Bracteacoccus aggregatiis. (Chlorococcales, Clilorococcaceae )amonotypie new
genus and species, was described b)' Tereg (1923 )as follows :
fi
fi
Kol.: feucht glanzend griin, trocken stumpf gelbgrün, zart. Gt'. :4-14/1.
F.; kiigelig. I.: klares farbloses Plasma. K. :unsichtbar, p.: nicht vorhanden.
Ghr. :2bis ca. 10 oder mehr getrennte unregelmässig- begienzte Plättchen,
Soil· Algae İTom Enchanted Rock
44
zarte. M.: diinn, schwach verschleimend. V.: nicht vegetativ. Zoosporen
mit 2Zilien und 2Chromatophoren ;Akineten. Kügelig.
Protococcaceae, verwandt mit Dictyococcus (Gern.)
Starr (1955) added the following distinguishing characteristics to Tereg's de-
scription :"Zoospores, with two slightly unequal agella, without walls, rounding
up on becoming quiescent. Major reserve photosynthate is oil; starch Is present in
snrall amounts.”
Chlorococcacean algae belonging to the genus Bracteacoccus are characterized
bv possessing the following distinguishing attributes: (1) vegetative cells with
several to many separate polygonal, platelike chloroplasts, the thickness of which
may vary from one species to another, and also within the same species; (2) absence of pyrenoids in all phases of the life cycle (starch distributed throughout the
chloroplast is readily demonstrated by applying aweak solution of İ2-K1 to apreparation );(3) zoospores of various sizes, attened to various degrees dorsoventrally,
which become spherical at quiescence; each zoospore has 2 agella, slightly un-
equal in length, emerging from the anterior pole of the cell at alnrost the same location.
٥wing to the small size of the zoospore and the rapid withdrawal of the agella
at quiescence, the inequality of the agella is dif cult to perceive in certain species.
It is acommon experience for investigators of soil algae to isolate organisms
which are undoubtedly species of Bracteacoccus. Using the 5species available from
the Culture Collection of Algae at Indiana University (Starr, 1960) as the basis.,
and 21 isolates from various types of soils, plus 1isolate from the atmosphere‫ ؛‬for
comparative study, an investigation was made by the writer to determine how much
variation exists within the genus. The generic attributes, except perhaps for the in-
equalit)' of agellar leng'th, are unquestionably salient, while only minor differences
exist among the various species.
Six new species of Bracteacoccus are described below as aresult of these studies.
These 6organisms were selected from among the 21 isolates because they demonstrate morphological and physiological characters deemed to be important taxonomically at the species level.
Four of the 5species of Bracteacoccus from the Culture Collection of Algae, Indiana Universit)', namely, B. cinnabarinus, B. engadinensis, B. terrestris, and No.
345 B. sp. are not included in the following discussion. They apparenti)' do not pro-
duce zoospores, and afresh-mount preparation shows only vegetative cells of
similar sizes with autospores.
Bracteacoccus giganteus sp. nov. (Fig. 70-72; 131-136)
Csllulae vegetatlvae omiiis aetatls splieric.ae, 107 μmax. diam. Membrana levi,‫ ؟‬aetate spis-
fl
fl
fl
fi
fl
fl
fl
.are indelited to Mr. R. Maleolm Brown, Jr. for this isolate ‫؟‬.Tlie writer ‫ال‬
45
Bisekoฐ ciud Bo‫؛‬d
sescens. Chlorop!astl laminiformes, et parietales et interiores, cellulis in vetustioribus minores
farti. Vaeuolae pusantes in ccllulis vegetativis non observatae. Amyluin copiose factuni. Guttulac aurantiae In cellula senc.scente coacervatae. Cellulac maturae multinucleatac.
Reproductio asexualis per formationem zoosporarum aplanosporarumqtre, singulum zoosporangium usque ad 50 zoosporas ef ciens. Zoosporae fusiformes, 9.0-10.5 X3.54.2 ‫;ع ــلم‬
3-4 chloroplastos parietales, 2vacuolas pulsantes anteriores, nucleum anteriorem, Stigma
equatoriale et duo agella longitudine inaequalia habentcs.
Reproductio scxualis non observata.
Culturae duarum hebdomadum aetate in medio “agar” basali atiovirides, post 3menses
aurantiae factae. Super cies culttirarum 2hebdomadum aetate granulosa plicas minutas praebens (niagni catione 14-pio); fusco-nitida macroscopicallter visa; butyracea.
Origo: in solo eloco Enchanted Rock, Llano Co., Texas, m. July. 1960.
Vegtative cells splrerical at all ages, maximum diameter- 107 μ. Cell wall snrootlr, tlrickening
with age. Chloroplasts platelikc, both parietal and internal, becoitritrg strrallct- in older cells.
Contractile vacttoles irot observed in vegetative cells. Starclr produced in abundance; orange
droplets accumulating as cells age. Mature cells nrultirrucleate.
Asexital reproduction by zoospore and aplanospore fornration, up to 50 zoospores fornred by
asingle zoosporairgiitnr. Zoospore fusiform, 9.0-10.5 X3.5-4.2 ‫ ;ع ـ ـ ـ ـ ــلم‬with 34 parietal chloroplasts, 2anterior cotrtractile vacuoles, anterior nucleus, equatorial stigma, and 2 agella of
unequal length.
Sexual reproduction not observed.
Two-week-old cultures on basal medium agar dark green, becoming orange at 3months.
Surface of 2-week-old cultures granular with minute folds (14 Xmagni cation) :macroscopically dull-shiny; butyrous.
Sout-ce: Soil from Enchanted Rock, Llano County, Texas; July, 1960.
The sample of soil from which this organism (WCr-4-8) was isolated was collected from acrevice on the west exposure of Enchanted Rock. Clonal cultures have
been maintained in abacteria-free state on ٥BBM agar and Proteose agar. Transferring of an old culture from OBBM agar to Yeast Extract agar (prepared by
adding 1.0 gyeast extract to 1000 ml BBM agar) or Proteose agar had ade nite
stimulatory effect and resulted in the production of zoospores by many of the
mature cells.
Vegetative cells of all ages are spherical (Fig. 70). Mature vegetative cells of
this species are 2-5 times greater in diameter than any other species known at the
present time. The average size of the vegetative cells in agiven culture will range
between 50 and 75μ diameter. Older cells up to 107μ diameter were observed. The
cell wall is smooth and thin in young cells, but, as the cells age, the outer layers of
the multilayered wall slough off as inner layers are added. The cell wall varies in
thickness up to amaximum of 7.5/Ấ in stationary-phase cultures (Fig. 71). The cell
wall is characteristically smooth, but, occasionally, old cells may have bubble-like
thickenings at one or several poles of the cell.
fi
fl
fi
fi
fi
fi
fl
fi
Only ver)' young cells are uninucleate (Fig. 131). The rst nuclear division occurs in young cells as small as 7‫ ءل ـ ـ ـ ـ ـ ــم‬diameter (Fig. 132); mature cells are multinucleate. The protoplast contains several to many separate, platelike chloroplasts;
young cells have fewer, large chloroplasts, while older cells have many, small.
Soi‫'^ ؛‬VlRae ‫؛‬ΪΟΎΪΙ Enduiuled lock
4.()
íhicker ohlorop‫؛‬asts. No contractile vacuoles were observed. Orange droplets of
various sizes occur distributed throughout the colorless cytoplasm of maturing- vegetative cells. As the cells age, the droplets coalesce, forming alarge drop near the
center of the cell. This orange center is frequently’ obser١'ed in cells between 1and 2
months of age.
The zoospores are very pliable, as expected in the absence of acell wall (big.
133). Before the zoospores are liberated from azoosporangial ve.sicle they.’ are
spherical to o١٠'oidal in shape (Big. 134). Upon release from the vesicle they-' become
fusiform, with the antcriol' pole more blunt than the tapered posterior pole (Big.
135). .’-kftcr a١'ery’ short motile period, the zoospore becomes more blunt also pos-
tci'iorly’ (Idg. 136). The zoospores ultimately’ forn! spherical cells approximately 6/1
in diameter (Big. 131). Bifty or more zoospores are usually formed by asingle zoo-
sporangium. .·\ large motile zoospore may.' attain aleng'th of 10.5/i and awidth of
4.2μ. Thi-ee to 5parietal chloroplasts are present iir each cell; 2unequal agella,
a^iproximately 4‫ل‬/>-‫ ؤ‬as long as the cell body, an anterior nucleus, 2contractile
١'acuoles, and disc-shaped equatorial stigma are further attributes of the zoospores.
The more frequent method of asexual reproduction is by' apianospores. The entire mass of the latter cohei'es as it emerges from athick-walled mature cell
(Idg. 71).
.-\s the young cells increase in size, they’ separate, and continue their development
as isolated individuals.
Sexual reproducti n was not observed.
Two-weelc-old cultures on BBM agar are dark green, becoming orange at the
margins. The surface of a2-١٠veek-old culture on BBM agar is granular with few,
minute folds, ,just visible at 14 Xmagni cation (Big. 72). The colonies are dullshiny macroscopically and of buty.'rous consistency. Three-month-old cultures are
orange.
Additional supplementary.' attl'ibutes of this alga are: it is inhibited by' colistin
sulfate, kanamycin, neomycin, polymyxin-B, paromomycin, triclobisonium chloride, nitrofurantoin, and methenainine mandelate; slightly.’ inhibited by' amphotericin Band dihydrostreptomycin (Table 5). Growth is good in BBM with dextrose
and 'uctose; it is trace in amount in BBM with fructose in darkness (Table 6).
Amy'lasic power of B. giganteus on 0.01 soluble starch in BBM Ion agar is slight
(Table7).
This organism clearly possesses the 3distinguishing attributes of the genus Braeteacoccus: (1)vegetative cells with several to many separate polygonal, platelike
chloroplasts; (2) absence of pyrenoids in ‫؛‬ill phases of the life cycle; and (3) production of zoospores attened to various degrees dorsoventrally.’, with agella of
unequal length and without acell wall. It differs from previously described species
bv having mature veg'etative cells 2-5 times greater in diameter whose cell walls
thicken with age up to 7.5/1 in stationary-phase cultures. Upon this, and evidence
from otlier comparisons, such as supplementary attributes, WCr-4-8 is here de-
fl
fl
fi
fl
fi
fi
scribed as anew taxo٠n, B. gigantens.
4.7
BìscKoỊỊ (Ilid Bold
Bracleacoccus Iiiediomidealiissp. nov. 73-75; ]37, 138)
Celliilae vegetatlvac spherlcae ad subsphericas, secundum actatem; cellula(' inatuiae splieri-
cae usque ad 25 μdiam. Membiaiia levis tenuisqı‫ا‬e, aetate non miinifestc spissc.sccns. Clilorapiasti laminiformes, (:t parietales et iiiteriore.s. VacLiolae pul.sanle.s in cellulis vegetativis non
observatae. Cellulae maturac multinuclcatae, nucleis prope centru!n cellultie aggregatis..
Reproductio asexualls per formationem zoosporarum aplanospttrarumque. Zoosporae clon-
gatae, 5.5-5.(-١ ۶long., polis anterioriju.s acutl.s, po.sterioribu.s rotunclatis, 2cliloropla.stos parietales, tiucleum anteriorem, stigma parvum aequatorlale, 2 agella Iong٠ltudinc inacqitalia,
amboijus paululo longloi'ibus quani corpu.s cellulite, liabcnte.s.
Reproductio sexualis non observtita.
Culturae diiarum liebdoniadum actatc in medio "agar'٩ Ija.sali atrovirides, post 5nutnses
solum ad margines avae factae. Super cies cultura rum 2lieltdoniadum aetate, granulosa et
paululum reticulata (magni c‫!؛‬tl(,ne 14 pio); nitida ttiacrosco!,lcalitcr visa; butyracea et
paululum viscida.
Origo: esolo prati Inculti non corrupt! ptojre loculi Taylor, Wllliam.son Co., Texas dictum;
in. Jul. 1900.
Vegetative cells .spliCTİcal to sub.splierical, depending, on age; mature cell.s .splierical, attaining amaximum diameter of 25 μ. Cell wall siiiootli and tilin, not thickening, markedly with
age. Cliloroplasts platelikc, pai'ictal ‫؛‬and intcirial. No conti'actlle vacuoles olisei'ved In vegetative cells. Mature cells multlniicleiite, witli nuclei clustci'ed near tlic center of cell.
Asexiial reproduction by 'zoospore ‫؛‬and aplano.spore formation. Zoo.spores elong'ate, witli
acute ‫؛‬antei'lor, and rounded !losterior poles, 3.5-5.0 μlong, witli 2parietal cliloroplasts, 111‫؛‬terioi- nucleus, small equatorial stigma, and 2 itgella of uiiecjuitl Iciigtli liotli slightly longei'
tlian cell body.
Sexual rcproduc.tloii not observed.
Two-wcek-old cultures on basal ag٠ar medium dark g'reeii, lieconiing yellow only ‫؛‬It tile
niargin.s after 3Iiiontlis old. Surface of 2-week-o!d cultures .slightly granular and reticiiliate
(14- Xmagni cation); niacroscopically sliiiiy; liutyiOiis and sllglitl)' viscid.
Soui-ce: Soil f1-0111 undisturbed prairie neai- Taylor, IVIlliani.soii County, Texas; July, l‫( )؛‬١.
This organism (D48-7D) was isolated into axenic culture from asoil sample
collected by Miss LaVerne Albert^( from an Andropogon hay meadow near Taylor,
Texas. Axenic cultures have been maintained on BBM and Broteose agar.
Young cells are spherical at maturit)' (Idg. 73). The range in size of vegetative
cells of this organism is similar to that of B. grandis, the average diameter of matured cells being approximately 25 μ, attaining amaximum diameter of 36/1.. Two
additional attributes are also similar to B. grandis, namel).' :vegetative cells are separate, not coherent, and the cell wall is thin and smooth and 'does not increase
markedlv in thickness as the cells age. ]''reshly mounted preparations of these or-
ganisms are indistinguishable ;thus additional comparison is required to distinguish
them. Differences are stated under the description of B. grandis.
Yfaturing vegetative cells 12-14/Ζ diameter are nmitinucleate, with the nuclei
clustered near the center of the cell (Fig. 137). The protoplast of )'oting. cells contains several, large, distinct chloroplasts lled with visible starch granules. The
fi
fl
fi
fl
fi
fi
fl
fi
3Thu writers ‫؛‬arc indelrted to MİS.S Allrert foa- liais caaltaarc aaad ‫؛‬also for tlaat taf B. cohaerens.
48
Soil Algae |ϊθΐίΐ Εη,οΚαηΙβ(! Roek
platelike chloroplasts increase in number and decrease in size as the cell increases in
size. Vegetatile cells 16-18μ diameter contain internal chloroplasts arranged in a
netlike manner (tig. 74, 137). No contractile vacuoles were observed.
Progressive cleavage results In the formation of zoospores and apianospores. The
zoospores are typically elongate, the anterior pole acute and the posterior rounded;
the cell body exhibits de nite dorsoventralit)' (Fig. 138). Many zoospores are produced by asingle cell. The zoospores are small, averaging between 3.5 and 5.0μ in
length. Each zoospore contains 1or 2parietal chloroplasts, an anterior nucleus
and asmall equatorial stigma. Two unequal agella slightly longer tlian the cell
body length propel the cell as it swims. After ashort motile period, the active zoospores become quiescent and immediately assume aspherical shape; such cells are
but 3-4،a in diameter. Mature cells approximately 25 μdiameter produce 50 or
more aplanospores.
Sexual reproduction was not observed.
Two-week-old cultures on BBM agar are dark green. The surface of a2-weekold culture on BBM agar is slightly granular and reticulate when observed under
14- Xmagni cation (Fig. 75).The colonies are shiny macroscopicall)' and of butyrous, almost viscid consistency. Three-month-old cultures remain green with only a
sliglrt indication of yellow at the margins.
This alga is characterized by the following supplementar)' attributes: it is inhibited by amphotericin B, colistin sulfate, neomycin, polymyxin-B, paramomycln,
triclobisonium chloride, nitrofurantoin and methenamine mandelate (Table 5).
Growth is good in BBM tdextrose and fructose; fair in BBM tarabinose, sodium
acetate and ribose; occurs in trace amounts in BBM tdextrose and fructose in
darkness (Table 6). The ainylasic power of this organism is slight on ٥.01% soluble starch in BBM Ion agar (Table 7).
After close comparison of the speci c attributes of the present organism with
those of asimilar alga described in the following pages, the former is here described
.lEOYv, Bracteacoccus mediouiicleatus ‫؟\أةةة‬,١‫يب‬
Bracteacoccus graiidis sp. nov. (Fig. 76-79; 139)
Ccllulae vegetativae omnis actatis splierlcae, 35 μdiam. ìiax. Membrana Icvls ten‫اال‬scıı‫ا‬e‫ا‬
aetate non manifeste splssescens. Cliloroplastí par^'i laminiformes. Vacuolac pulsantes in
cellulis vcgetativls non observatae. Cellulae maturae miiltinucleatac.
Reproductio asexualis per formatioirem zoosporarum aplano.sporarumqiic. Zoosporac fusifoiincs, 8.5-10.0 X2-3.5 μ; 2-3 eliloroplastos parietales disscctos, 2vacuolas pulsantes antci'i-
fi
fl
fl
fi
fi
fi
fi
fi
ores, nucleum anteriorem, Stigma parvum aequatorlale et 2 agella longitudine inaequalia,
aeque longa ac corpus cellulae, habentes.
Reproductio sexualis non observata.
Cui turae duarum hebdomadum aetate in medio “agar)' basali atrovi ildes, post 3menses
solum ad margines aurantiae factae. Super cies culturac 2liebdomadum aetate granulosa
(magni catione 14- pio); nitida macroscopicaliter visa; butyracca.
Origo: esolo in latere orientali Viae 95 admodum septentrionalis nis mcridionalis loci
Williamson County, Texas dictl; m. Oct. 1957.
49
Vegetative ce‫اا‬s spherlca! at all ages, maximum diameter 35 μ. Cell wall smootli and thin,
not tltlckcning markedly with age. Cliloroplasts platellkc and snrall. No contractile vacuoles
observed in vegetative cells. Mature cells multinucleate.
Asexual reproduction by zoospore and aplanospore foirnation. Zoospore fusiform, 8.5-
10.0 X2-3.5 μ; with 2-3 parietal and dissected chloroplasts, 2anterior contractile vacuoles,
anterior, nucleus small equatorial, stigma and 2 agella of unequal length, the longer as long
as the cell body.
Sexual reproduction not observed.
Two-week-old cultures on basal agar medium dark green, becoming orange only at the
margins at 3months. Surface of 2-week-o!d culture sliglrtly granular (14 Xmagni cation);
macroscopicallj? shiny; butyrous.
Soui'ce: Soil, east side of Highway 95 just nortli of tire soutliern boundary of Williamson
Tounty, Texas; October, 1957.
Bracteacoccus grandis (Τ-1-2-Β) was isolated into pure culture front asample
of sandy soil in an open oak wood on the east side of Highway 95 just north of the
soutliern boundar)' of Williamson County, Texas. Axenic cultures have been maintained on BBM, Proteose and Soil-Extract agars.
Vegetative cells of all ages are spherical (Fig. 76), with an average size of 24but may become as large as 35 μdiameter on Proteose agar. Living cells freshly ،‫لم‬26
mounted from 2-week-o!d cultures on BBM agar are completely isolated from one
another. The cell wall is smooth and thin and does not increase markedl)' in thickness as the cells age. occasionally, mature and young cells may have abubble-like
thlc'ening at one pole of the cell (Fig. 77). It was observed tliat the bubble-like
thickening does not coincide with apoint of rupture when matured zoospores or
aplanospores are released from asporangial cell (Fig. 78).
Maturing vegetative cells are multinucleate. The protoplast contains 3to man)'
separate, distinct, platelike chloroplasts; the number in acell increases with the size
and age. These chloroplasts are arranged parietali)' and internally in both small
and large cells. The chloroplasts become less clearly de ned in large cells of old
cultures but give astrong positive starch reaction with I.‫ذ‬-KI. No contractile vac-
uoles were observed in the vegetative cells.
Reproduction is accomplished asexuall)'- b)' zoospore and aplanospore fornration.
Zoospores and aplanospores are formed by progressive cleavage of the protoplast.
Azoosporangium may contain 8-32 zoospores. The zoospores are fusiform, like
those of Bracteacoccus gigantens, with the anterior pole more blunt than the
tapered posterior pole (Fig. 139). Zoospores vary in size and may attain alength
of 8.5-10/Χ, and awidth of 2-3۶. Incomplete cleavage is frequently observed in
motile zoospores. Each zoospore contains 2or 3parietal chloroplasts, 2unequal
agella approximate!)' as long as the cell body, an anterior nucleus, and 2anterior
fi
fi
contractile vacuoles. Asmall stigma is located on the equator of the cell.
The prevalent method of reproduction is by aplanospores which average 5.5 μIn
diameter. Vegetative cells of any size are capable of becoming aplanosporangia.
Small cells produce as man)' as 25 aplanospores (Fig. 78).
fl
fl
Bischo.ttudBold
Soil Alitit ‫ا‬l'Olii Eiicliunled Rock
ScxLial reprocluctioii was not observed.
Two-week-old cultures on BBM agar ai'c dai'k green, becojning orange onl)' at
the margins when 3months old. The surface of a2-week-o!d culture on BBM agar
is .slightly granular when observed under 14 Xmagni cation (]' g. 79). Tlie
colonies are shiny macroscopically and of Ijutyrous consistency.
Bracteacoccus grandis possesses the following supplementary attributes: it is inhibited by colistin sulfate, polyniyxin-B, paramomycin, triclobisonium cliloride and
nitrofurantoin; slightly inhibited by kanatnycin and sulfadiazine (Table 5).
Growth is good in BBM supplemented with dextrose and fructose and trace in
BBM suppleniented with sodium acetate and ribose (Table 6). The anpla.sic
power of this species on 0.01 ً ‫ لجا‬soluble starch In BBM Ion agar is good (Table 7).
Bracteacoccus grandis differs from B. medionucleatus, the species which it most
closel)'- resembles, in that the nuclei are not clustered in the center of the cell as is
the case in B. medionucleatus; bubble-like protrusions may form on the cell wall of
B. grandis, while the cell wall of B. medionucleatus remains smooth; the zoo.spore.s
of B. grandis are large, up to If)μ in length, fusiform with the anterior pole more
blunt than the tapered posterior pole, while the zoospores of B. medionucleatus are
small, up to 5μin length, elongate, with the anterioi. pole acute and the posterie)!'
rounded.
Upon this evidence, and. In addition, bccau.se of the dissimilarities of .supplementary attributes between the present organism and B. medionucleatus, the
former is here described as anew taxon, Bracteacoccus grandis.
Bracteacoccus aerius sp. nov. (Iffg. 80, 81; 14-0-142)
Cellula(' vegetativae luvenes aut pyriformes aut ellipticac ‫؛‬lut sphcricac; eellulac niatuaie
sphericae, 28 μmax. diam. Membrana levis, aetate spissescens. Chloroplasti laminiformes
pai'ietalcs interioresque. Vacuolac pulsante.‫ ؟‬in ecllulis vegetativis non obsci'vatae. Giittuliic
auriantae cellula .senescente coacervatac. Cellulae maturae multinuc.lcatac.
"
asexuttlis per forni ‫؛‬Itionein zoospora rum aplanosporai'umque. Zoosporae ovatti('
ad ellipsoidcas, polo posteriore acutissimo 5.5-8.2 X3.5-4.2 μ, 1vel 2chloropltistos parietales,
2vacuolas pulsantes ‫؛‬interiores, Iiucleum anteriorem, Stigma po.sterius aut siili-po.stdius, et 2
agella longitudine inaequalia liabeiites.
"
scxiiiills non observata.
Ciillurae cluaruni lielrdomadiun aetate in 111 ('dio “agar” ba.sali llavo-viride.s, po.st 3meiises
aurantio-briiniieae factac. Super cies ciilturae 2-lieljdomaduni aetate IcvI.s (magiii catioiie 14
pio); fusco-nitida inacroscopicaliter visa; butyracea.
Origo :(Epulvere 111 turre Uiiivci'sltatis Texas), Ausi in, Texas. 111. .fun. I‫؛‬٠‫ا(ة‬.
Vegetative cells pyriform, elliptical 01' spliei'Ical
-lien young; mature c('lis .spherical, maxi-
Illuni diaiiietei' 28 μ. Cell w--all sniootli, thickening with age. Cliloroplasts platelike, parietal
-Iiid internal. No contractile vacuole.s obseivecl in vegetative cells. Orange dro!ilets accuiiiu‫؛‬
latiiig ‫؛‬IS cells age. Mature cells niultiniicleate.
Asexual reproduction liy zoospore ‫؛‬ind aplanospore formation. Zoospoi'cs ovoiclal and ellip-
fi
fi
fi
fi
.
soldai, with posterior pole pointed, 5.5-8.2 X3.5-4.2 μ; witli 101' 2parietal cliloroplasts, 2
.
fl
5(.)
51
ür stigma, aliti 2llagella‫ا‬oster‫ذاداا‬anlorior nudí'u.s, posterior or sı ,‫؛‬.‫ااااال‬-‫ '(ااًا'(ا؛‬١'‫'ا!الاأ'(أ؛‬,١ .i,)‫؛‬.,<)Ii١t‫؛‬
of unctjiial Iciigtli.
Scxiial reproduction not olrsereecl.
T١١'o-week-old cultures on Irasal agar media yellowish-green, heeoiiiiiig oraiige-hiOwni afteimontlis. Surface of 2-w'cek-old cultiii'e siiiootli (at 14Χ magni cation); iiiaciOscopically‫ة‬
dull-sliiny; butyrous.
Source: (Dust collected on University of lexas tower) ٨ustiii, Texas; June, 196(1.
This organism was not isolated from soil as were the othei" 5species descrilncd.
Bracteacoccus aerius was isolated from asample of air-borne diist collected by Mr.
R. Malcolm Brown, Jr. Prolonged comparative study of this organism has proven
it to be anew taxon. Axenic cultures are maintained on BBM and Proteose agar.
Cells from 2-week-o!d cultures on BBM agar, grown under standard conditions,
are mostly young, vegetative cells, pyriform, elliptical and spherical in shape. The
nonspherical shape of the growing vegetative cells is nnaintained while they are in-
creasing in size. Only fully mature cells, no longer Increasing in size, are spherical
(Fig. 80). Vegetative cells are isolated, not coherent like those of B. cohaerens, B.
minor, and B. pseudominor. The chloroplasts are large, parietal, platelike and few
in number. Cells 15/Χ in diameter, which is the size of the majority of vegetative
cells, contain 6-8 chloroplasts. Cells up to 22/Χ in diameter may occur in any given
culture. Youirg cells are uninucleate, becoming multinucleate as the cell ages and
increases in size. Contractile vacuoles were not observ'ed in the vegetative cells. The
cell wall is thin and smooth, but thickens slightly after 2weeks on BBM agar. At 6
weeks, the cell wall is up to 4.5 μin thickness, and in some cells the protoplast is
masked by large droplets of orange oil.
Asexual reproduction in this species is by zoospore and aplanospore formation
resulting from progressive cleavage of mature cells. Zoosporangia commonly are
in diameter; 16 zoospores are usually produced from each parent cell, but ‫مللم‬14-16
as in the formation of aplanospores, the number of cells produced varies with the
size of the parent cell. Upon rupture of the zoosporangial wall, the zoospores become motile and very active within the vesicle until released Into the surrounding
medium (Fig. 140), where they swim very rapidly with darting movements for a
short period. Tlie zoospores vary in size, being 5.5-8.2‫ مللم‬in width. The shape of the
zoospores during the motile phase is highly variable. Most are ovoidal or ellipsoidal
with the posterior pole somewhat pointed (Fig. 141-142). Each zoospore has 2
agella of unequal length, 2anterior contractile vacuoles, 1or 2parietal chloroplasts, an anterior nucleus and asmall posterior or subposterior stigma. The
zoospores are very pliable and probabl)' have no cell wall. On agar medih, man).'
odd-shaped cells occur, formed by young cells developing walls under crowded conditions. These cells maintain the small stigma for sev'eral days. Aplanospores pro-
duced by progressive cleavage average 5‫ مللم‬in diameter.
Sexual reproduction was not observed.
Two-week-old cultures on BBM agar are yellowish-green. Three-month-old cui-
fi
fl
and Hold 1‫؛؛‬.١(:‫الالأا‬
‫د' د‬
Soil Alette ١ïom Enchanted. Roch
tures maintained on the same medium are orange-brown. The surface of a2-week-
old culture on BBM agar is smooth when observed under Id Xmagni cation (Eig.
81). The colonies are dull-shin)' macroscojoically and of butyrous consistency.
Bracteacoccus aerius is inhibited by colistin sulfate, neomycin, polymyxin-B,
paromomycin, triclobisonium chloride, furaltadone, nitrofurantoin, furazolidone,
methenamine mandelate and para amino salicychc acid (Table 5). Unlike all other
species of Bracteacoccus investigated, B. aerius differs by making only fair growth
in BBM supplemented with dextrose and trace amounts in BBM with fructose.
Tubes with BBM and sodium acetate showed only atrace of growth. Trace
amounts of growth also were present in BBM with dextrose and sodium acetate in
darkness (Table 6). Bracteacoccus aerius is capable of good amylasic activity on
BBM Ion agar containing 0.01 c/c soluble starch (Table 7).
This organism differs from all other species of Bracteacoccus, except B. giganteus,
in having acell wall thickening markedly with age. The present organism differs
from B. gigantens by having vegetative cells never exceeding 22μ when grown on
BBM agar, under standard conditions. The vegetative cells of B. gigantens will
reach amaximum of 1Ομ. The zoospores of B. aerius are ovoidal or ellipsoidal, with
the posterior pole pointed, and stigma posterior, while those of B. gigantens are
fusiform with equatorial stigma.
With respect to supplementary attributes, this alga clearly differs in its combination of attributes from all other species in the genus, and is here described as a
Uxorv, Biacteacoccas aerius.
Bracteacoccus pseudominor sp. nov. (Fig. 82-84; 143)
Ccllulac vcgetativac sphericac ad subsphcidcas, secundum aetatem; ce^lulae maturai splieri-
cae, usque ad 25 μdiam., ce!!ul‫؛‬s vegetativis ad massas irregu!arcs formandas cohaercntibus.
Membrana !cvls tenulsque, aetate non manifeste spissescttns. Chloroplast‫ ؛‬laminiformes, magniparietales. Vacuolae pulsantes in cellulis vegetativis non observatac. Celltilae maturae multinucleatae.
Reproductio asexual¡s per formationem zoosporaruni aplanosporaruinquc. Zoosporae ovatae
6.5-7.5 X2.2-2.8 μ-, 2cliloroplastos parietales, 2vacuolas pulsantes anteriores, nucleuni
terioi'em, stigma posterius, et 2 agella longitudine inaequalia liabentes.
Reproductlo sexualis non observata.
Culturae duarum hcbdoniadum aetate in medio “agar" basali dilute virides, post 3menses
solum ad ntargines aurantiae factite. Supei' cies cuturae 2-hebdomadum aetate paululum
granulosa (magni cationc 14 pio); fusco-nitida macroscoplciiliter visa; butyracca.
Origo: esolo prope coniunctionem Ciaruin ISOetllllin loco Iluclspetli Co., Texas
dicto., m. Nov. 1961.
Vegetative cells spherical to sub.spherical, depending on age; mtiture cells spherical, attain-
ing amaximum diameter of 25 μ. Coherent vegetative cells forming irregular' masses. Cell
fi
fi
fl
fi
rvall srnootlr artd thin, not thicketring markedly witlr age. Chloroplasts platclike, large and
parietal. No cotrtractile vacuoles observed in vegetative cells. Matrrrc cells rrrutlnucleate.
Asexual rcproductiorr by zoospore and aplatrospore forrrration. Zoospores ovoidal, 6.5-7.5 X
Bischo. auđ Bold
53
-anterior contractile vacuoles, anterior nucleus, pos ‫؟‬, with 2parietaj chloroplast2 ,‫ علم‬2.2-2.8
terior stigma, and 2 agella of unequal length.
Sexual reproduction not observed.
Two-week-old cultures on basal agar medium liglit green, becoming orange only at tire
margins after 3months. Surface of a2-week-o!d ciiltui'C slightly granular (14 Xmagni cation); inacroscopically dull-shiny; butyrous.
Source: Soil from near junction of Highways 180 and 1111, Hudspetli County, Texas;
November 24, 1961.
This organism was isolated into axenic culture (W-25-B-1 )from asoil sample
collected in west Texas by Brother Joseph Cain, C.S.C., to whom the writers are
indebted for the culture for comparative study. The organism grows well on BBM
agar, and on all other media used.
Young vegetative cells of this alga are spherical to subspherical when young, and
contain large, distinct, platelike chloroplasts. Only 1nucleus is present in young
cells, but, as the cells ,increase in age and size, they become multinucleate. Mature
vegetative cells contain parietal, platelike chloroplasts (Fig. 82); anumber of
nuclei lie in the colorless cytoplasm. The majority of the mature cells range in size
from 12 to 18μ diameter; cells up to 25μ in diameter have Ireen observed. Contractile vacuoles are alxsent in vegetative cells. The cell wall is smooth and thin and
does not thicken markedly with age.
Ysexual reproduction is completed by the release of zoospores and aplanospores
formed by progressive cleavage. The aplanospores are produced in small numbers;
2-12 a
in diameter, commonly arise from asingle ‫مللم‬mospores, approximately 5‫؛‬
mother cell. Large irregular masses or clumps of cells result from the coherence of
aplanospores by theii- cells walls (Fig. 83). These clumps are unlike the spherical
clumps formed by B. cohaerens. No indication of an individual or common matrix
can be detected by fndia ink or Methylene blue.
The zoospores are ovoidal, slightl)' attened dorsoventrally and range in size be-
tween 6.5 and 7.5μ in length and 2.2 and 2.8‫ مللم‬in width. Thirty to 50 zoospores are
produced by asingle zoosporangium. The wall of the zoosporangium is very thin
and pliable. As the wall ruptures, the zoospores are released in adelicate gelatinous
vesicle which increases in size to twice its original diameter before releasing the
zoospores into the surrounding medium. Zoospores just emerging from the vesicle
are very active, but become spherical and quiescent after ashort motile phase.
Each zoospore (Fig. 143) has 2parietal chloroplasts, 2anterior contractile
v a c -
uoles, asingle nucleus slightly anterior to the equator of the cell, 2anterior agella
of slightly unequal length which are withdrawn immediately at quiescence, and
a
posterior, disc-shaped stigma.
Sexual reproduction was not observed.
Two-week-old cultures on BBM agar are light green, becoming dark green and
turning orange at the margins only when 3months old. The surface of a2-week-o!d
culture on BBM agar is slightly granular at 14 Xmagni cation (Fig. 84). The
fi
fl
fi
fl
fl
colonies are dull-shiny macroscopicall)' and have abutyrous consistenev.
Additional supplementary attributes of tilis alga are: it Is inhibited by triclO'blsonium chloride, furaltadone, nitrofurantoin, nitrofurazone, furazolidone, iso nico-
tinic acid hydrazide, methenamine mandelate, para amino salicylic acid, amphotericin B, colistin sulfate, neomycin, polymyxin-B, and paromomycin (Table 5).
Growth in BBM supplemented with dextrose and 'uctose is very good with the
culture turning abright orange and clinging to the sides of the tube. Growth is fair
in BBM with ribose, with only atrace of growth in BBM tarabin ose. In darkness,
dextrose and fructose supported fair growth, while only atrace amount occurred in
darkness in BBM with arabinose (Table 6). Th,e alga is capatile of good amylaslc
activity on, BBM Ion agar containing 0.01 %soluble starch (Table 7).
The present organism is similar to B. minor, described liy Starr (1955), in that
the vegetative cells are coherent, forming irregular masses, and in vegetative cell
size. However, because of dissimilarities of supplementary attributes and certain
morphological attl'ibutes, the present organism is here described as anew taxon,
Bracteacoccus ().‫؟‬eudoimoT. Bracteacoccmrs tmseudomiuoT ‫ >أاًعأ؛ه‬Нот в, тгиот тп
that the vegetative cells are spherical to subspherical, the chloroplasts large and distiimct, and the zoospores are ovoidal, with stigma clearl)' posterior in B. pseudominor. Brarteacoccus minor has vegetative cells that are spherical at all ages, the
clmloi'oplasts arc small and the zoospores ‫؛‬nre elongate with stlginna slightly posterior
to the equatoi" of the cell.
Bracteacoccus eohaerejis sp. nov. (Eig. 85-87; 144, 145)
Cellulac vegetativae oniiils aetatis spheilcae, 55 μdiam. max. Membrana levis tenuisque,
aetate non spisse.scens. Cellulae vegetatlvae cohacrentes ad ma.s.sa.s sphcricas formantes, alloro-
plast‫ ؛‬laminiloiìiies parietalesqiie, eellnlis in .seneseentibus minii.s distinct‫ ؛‬facti. Varuolae jniilsaintes in celltilis vegetativis non observatae. ^nmylnm copiose factiim. Celliilae matiirae
multiriucleatac.
Reproductio ascxualis per foinnatlonem zoo.sporariim aplanosporarumque. Ills auctoribii.s
solitis reprodiictionis. Zoosporae fusiformes 5.0-0.5 X3.0 μ-, ‫ا‬chloroplastům parietalem,
nucleunn paululo anteriorem ad equatorialem, stigma parvum .nosterius ad equatoriale, 2
agella longitudine inaequalia liabentes; vacuolae pulsantes non observatae.
Reproductio sexualis non 0Insellata.
Culturae duartim hebdomaclum aetate in medio “agar" basali dilutevirides, post 3Ilienses
aurantiae fattae. Super cies culturae 2-liebdomadum aetate paiiliilum granulosa (magni catlone 14 pio); fusco-nitida macroscoplcaliter visa; Iniityracca.
Origo: csolo loci Dripping Spi'ings, Hayes Co., Texas diet‫؛‬, m. Jiil. 1950.
Vegetative cells splierical at all ages, maximum diameter 55 μ. Cell evali snnootli and thin,
not thickening witli age. Colicrent vegetative cells forming splierlcal masses. CliloiOplasts
platclike and parietal, becoming- less distinct as cells age. No contractile vacuoles olnserved in
vegetative cells. Starch produced in alnundancc. Mature cells multiniicleate.
Asexual reproduction by zoospore and aplanospore formation, aplanospoi'es tine usual
agents of reproduction. Zoospores fusiform, 5.0-6.5 X3.0 ۶; with 1parietal chloroplast; contractile vac.uoles not observed; nucleus of zoosjnore sliglitly anteri01" to equatorial, stigma small,
fi
fi
fl
posterloi- to ecjiiiitorial; with 2 agella of unecjual length.
fi
fl
Soti Algae ịĩom Enchanted Roch
Sd
1Bold،‫آلأ‬، .Bischo
55
Sexua! reproduction not observed.
Two-week-old cultures on basal agar media arc liglit green, becoming orange after 3months.
Surface of 2-week-o!d culture sliglitly grtiniilar, (14 Xmagni cation); maeroseopleally dullshiny; butyrous.
Source: Soil Dripping Springs, Htiyes County, Texas, July, 1(‫؛ة)؛‬.
This alga(C-İ2-Ig) was isolated om asoil sample collected by Miss LaVerne
٤'\lbert from Dripping Springs, Texas. ٨xenic cultures of the ()rganism have been
maintained on BBM and Proteose agar.
The majority of young vegetative cells of B. cohaerens are spherical; it is uncontmon to nd )'oung cells frotti agiven culture to be sulispherical (Pig. 85).
Mature vegetative cells in actively growing cultures average approximate!)' 30μ in
diameter when grown on BBM agar under standard conditions. Cells up to 55 μ
diameter have been observed. The chloropla.sts in young cells from an acti١'e]y
growing culture are distinct, parietal, and platelike. Starch is produced in abundance (Fig. 14-4). Stained preparations of cells 12-15/Χ in diameter are inultinucleate. No contractile vacuole.s were observed. The mature protopla.sts are enclosed liy athin, smooth cell wall.
The most usual method of reproduction is by the formation of aplanospores. Cells
undergoing aplanosporogeiiesis range in size from 20 to 30/1· in diameter. ApproxiIiiately 50-75 aplaiiospores arc produced from each aplanosporangiulii wlien the
organism is growing in liquid BBM ;cells growing on BBM agar produce comparatively fewer aplanospores. The number- varie.s with the size of the mother cell, a
charactei'istic of most Chlorococcaceae. The aplanospore.s are small, 4.0-5.2/Ị in
diameter, and remain in clump.s after being released from the aplanosporaiigiiiiti.
This suggests in appearance the morula stage of animal development. Many cells
remain attached by their cell walls throughout theii- existence (I g. 86). No demonstrable gelatinous layer is present.
The zoospores are small, I'angiiig from 5.0 to 6.5/، in length and are 3.0,'J. in
width (Fig. 145). Many zoospores are produced from each zoosporangium, the
number again varying with the size of the mother cell, ft is not uncommon for 100
or more to be released by one zoosporangial cell, ไ-'he zoospores are fusiform, being
tapered at both poles, and do not show anoticeable drrrsoventrailty. They move
very rapid!)' during the short pei'iod of ,notility. When the mtitile period ceases, the
zoospores become spherical immediately and the ag'ella disappear. Each zoospore
has aparietal chloroplast, 2anterior contractile vacuoles, antej'ior nucleus, 2anterior agella of slightly unequal length, and avery small stigma embedded slightly
posterior to the equator of the cell. The stigma is retained for as long as 6days after
the cell has become quiescent (Fig. 144).
Sexual reproduction was not observed.
fi
fi
fi
fi
fi
fl
Two-week-old cultures on BBM agar are light green, becoming orange at the
margins onl)' after 3months. The .surface of a2-weelć-oId culture on BBM agar is
56
Soil Algae ịĩom Euchauteđ Rock
slightly granular when observed under 14 Xmagni cation (Fig. 87). The colonies
are dull-shiny macroscopicall)' and of butyrous consistency.
With respect to additional supplementary characteristics, B. cohaerens is inhibited by colistin sulfate, neomycin, polyinyxin-B, paromomycin, triclobisonium
chloride, nitrofurantoin, methenamine mandelate and para amino salicylic acid
(Table 5). Growth is good in BBM with dextrose and fructose, the mass turning
brown and clinging to the sides of the culture tube within 2weeks under standard
conditions. Only atrace of growth occurs in BBM with arablnose, sodium acetate,
ribose and xylose. Growth is fair in BBM with dextrose and fructose and trace In
amount in arabinose when cultures are placed in darkness (Table 6). The amylasic
power of this organism is good on 0.01 %soluble starch in BBM fon agar
( Ta b l e 7 ) .
This organism resembles B. minor and B. pseudominor more closely than any of
the other species, in that they all 3have vegetative cells coherent throughout their
existence, but it differs in that the coherent vegetative cells of B. cohaerens 'form
spherical masses, while the cells of B. minor and B. pseudominor form irregular
masses. Vegetative cells of B. cohaerens are twice as large (reaching amaximum
of 55μ) as those of B. minor and B. pseudominor.
The colony characteristics of B. cohaerens and B. pseudominor are almost identical, but growth in BBM, supplemented with organic compounds, and the inhibition
of growth by certain antibiotics clearly distinguish the 2species. The zoospores of
B. cohaerens are fusiform, with stigma slightl)' posterior to the equator, while the
zoospores of B. pseudominor are ovoidal with the stigma clearly posterior.
Upon this evidence of dissimilarities of morphological and supplementary attributes, the present organism is here described as anew taxon, Bracteacoccus COh a e t e u ,‫؟‬.
Summar'v 0‫ ا‬siieci c attributes mBracteacoccus
For the convenience of reference for future investigators, the morphological attributes of the 7known species of Bracteacoccus have been summarized in the follow-
ing tabulation. Variations with respect to agi١'en attribute are numbered in corresponding fashion.
Bracteacoccus minor (Chodat) Petrova (Starr [1955])
(1)Vegetative cells spherical at all ages, attaining amaximum diameter of 20μ.
(2)Cell wall smooth, not thickening with age.
(3) Zoospores spindle-shaped‫ ؛‬some have blunt posterior pole, 5.0-7.0‫ علم‬long;
1-3 parietal chloroplasts; stigma very small and slightly- posterior to equator
fi
fi
of c.ell.
BiscHoß auđ Bold
57
(4) Two-week-old cultures granular and slightly reticulate (14 X); dark green,
dull-shiny and butyrous. Threc-inonth-old cultures green.
Rracteacoccus gigauteus ‫آلة‬. IN.
(1) Vegetative cells spherical at all ages, attaining amaximum diameter of
107 μ.
(2) Cell wall smooth, with occasional bubble-like protrusions, thickening with
age up to 7.5 μin statlonary'-phase cultures.
(3) Zoospores fusiform, 9.0-10.5μ long, 3.5-4.2۶ wide; 3-5 parietal chloroplasts; 2anterior contractile vacuole.s; nucleus anterior; stigma equatorial; approximately 50 zoospores produced by' single zoosporangiunr.
(4) Two-week-old cultures granular with minute folds (14 X);dark green,
dull-shiny and butyrou.s. Three-month-old cultures orange.
Bracteacoccus grandis ‫؟>؟‬. τνοχ.
(1)Vegetative cells spherical at all ages, attaining amaximum diameter of 35 μ.
(2) Cell wall smooth and thin, not thickening markedly' with age; may have
bubble-hke protrusions at one pole of the cell.
(3) Zoospores fusiform, 8.5-10.0 ‫ ال ـ ـ ـ ـ ـ ــم‬long; 2.0-3.5 μwide; posterior pole more
tapered than anterior; 2-3 parietal and dissected chloroplasts; 2anterior
contractile vacuoles; nucleus anterior; small stigma equatorial.
(4) Two-week-old cultures are smooth (14 X);dark green, shiny and butyrous.
Three-month-old cultures orange only at the margins.
Bracteacoccus mediouucleatus ‫؛؟>؟‬. rvov.
(1)Vegetative cells .spherical to subspherical when young; mature cells spher-
leal, attaining amaximum diameter of 36 μ.
(2)Cell wall smooth and thin, not thickening markedly' with age.
(3) Zoospores elongate, with acute anterior and rounded posterior poles, 3.5-long; 2parietal chloroplasts; nucleus anterior; small stigma equa ‫ أل ـ ـ ـ ـ ـ ــم‬5.0
torial.
(4) Two-week-old cultures smooth (14 X); dark green, shiny, slightly viscid
and butyrous. Three-month-old cultures becoming yellow only' at the margins.
Bracteacoccus pseudominor sp. nov.
(1)Vegetative cells spherical to subspherical when young; mature cells spherleal, attaining adiameter of 25 μ. Vegetative cells coherent, forming irregular masses.
58
Soil Algae ịìom Ei(c^aaled Rock
(2)Cell wall thin and smooth, not thickening markedly with age.
(3) Zoospores ovoldal, 6.5-7.5 μlong, 2.3-2.8 μwide; with 2parietal chloroplasts; 2anterior contractile vacuoles; nucleus slightl)' anterior to equator
of cell; stigma disc-shaped and posteria]-.
(4) Two-week-old cultures slightly granuliir (14 X); light green, dull-shiny
and butyrous. Three-month-old cultures orange only at the margins.
Bracteacoccus aerius ‫!؟>؟‬. ً ‫\سا‬.
(1)Vegetative cells pyriform, elliptical or spherical when young; mature cells
spherical, attaining adiameter of 28 μ.
(2) Cell wall smooth, thickening with age up to 4.5 μ.in stationary-phase cuitures.
(3) Zoospores ovoidal 01' elliptical, with posterior pole pointed; shape variable,
5.5-8.2 μlong, 3.5-4.2 μwide; with 1or 2parietal chloroplasts; 2anterior
contractile vacuoles; anterior nucleus; small stigma at posterior pole of cell.
(4) Two-week-old cultures smooth (14 X); yellowish-green, dull-shiny and
butyrous. Three-month-old cultures orange-brown. Orange droplets accumulating as cells age.
Braclettcoccus cokaereus ‫؟ة‬. τ\ον.
(1)Vegetative cells spherical at all ages, attaining amaximum size of 55 μ,
cells coherent, forming spherical masses.
(2)Cell wall smooth and thin, not thickening markedl)'- with age.
(3)Zoospores fusiform, 5.0-6.5 μlong, 3.0 ‫ مل ـ ـ ـ ــلم‬wide ;with 1parietal chloroplast ;
contractile vacuole not observed; nucleus slightl)'- anterior to equator of cell;
small stigma posterior to equator. Usual method of reproduction by aplanospores.
(4) Two-week-old cultures slighti)' granular (14 X);light green, dull-shln)' and
butyrous. Three-month-old cultures orange only at the margins.
Key to the currentl).' known species of Bracteacoccus (Tei'cg) btarr
2
1. Cell wall thickening' markedly with age .. ........ .. ....
3
1. Cell wall not thickening markedly witli age. .. ...... .... ...
2. Vegetative cells large, up to 107 ،a diameter ... ....... B. giganteas ‫ألة\ا‬. ш\-.١
2. Vegetative cells never- exceeding 22 ‫ ملـ ـ ــلم‬in diameter .. ...
В. aerius {ĩ-ọ. nov.)
4.
3. Vegetative cells coherent throughout theii- existence .. .....
6.
3. Vegetative cells isolated, separate ........
.............. .. .
4. Coherent vegetative cells forming spherical masses, individual
.
cells up to 55 ‫ مللم‬diameter
В. cohaerens (sp. nov.)
4. Coherent veg-etative cells forming irregulai- masses, individual
5.
cells never exceeding 25 ‫ مل ـ ــلم‬diameter ..... ............ ....... ....... ..
5. Vegetative cells spherical at all ages, lip to 20 μin diameter‫؛‬
chloroplasts small ;2-week-o!d cultures on BBM agai' shiny and
slightly reticulate (Idx) ‫؛‬zoospores elongate with stigma sliglitly
B. minor (Chodat) Petrova (Starr [1955J)
posterior to equator of cell
5. Vegetative cells spherical to subspherical, up to 25 μin diameter;
chloroplasts larg'e and distinct; 2-week-o!d cultures on BBM agar
dull-shiny and slightly granular (14x); zoospores ovoidal, with
B. ‫)؛‬seudominoT ‫؟ة؛‬.‫آل‬0١'.١
stigma clearly posterior
6. Zoospores large, up to 10.0 μin length ;small number of
aplanospores produced by asingle aplanosporanglum; bubblelike protrusion may be present on cell wall of mature vegetative
cellsB. grandis {sp. nov.Ị
6. Zoospores small, up to 5.0 μin length; 50 or more aplanospores produced by asingle aplanosporangium; bubble-like
protrusion not pi'esent on cell ١vall of mature vegetative cells
---
B.
medioiiucleatu,s
w o v .٦
Summary
This paper reports on the soil algal ora of Enchanted Rock, Llano County,
Texas, as it is manifested in enrichment cultures of soil samples from that station.
The general aspects of the ora are discussed and summarized in tabular form
(Table 1).
Sixteen chlorophycean algae, new to science or otherwise signi cant, were isolated into axenic culture, studied morphologicall)' and physiologically, and com-
pared with related taxa available in culture. Such supplementary attributes as colony characteristics, color in the stationary phase of growth, differential inhibition by
certain organic compounds in light, and extracellular amylasic activity were studied
and recorded as taxonomic attributes.
The newly' described taxa are:
Vo l v o c a l e s
Chlamydomonadaceae
Chlamydomonas megalis sp. nov.
Chlorococcales
Chlorococcaceae
fi
fl
fl
Chlorococcum polymorphrim sp. nov.
Spongiochloris gigantea sp. nov.
Spongiochloris llanoensis sp. nov.
Neochloris pseufJostiginatica sp. nov.
Planktosphaeria texensis sp. nov.
Planktosphaeria nraxinia sp. nov.
Bracteacoccus gigantens sp. nov.
m e d i o n u c l e a t u s s p . n o v.
Bracteacoccus grandis sp. nov.
-
"
‫الة‬
BiscKo. ،I^d Bold
Soi‫ ؛‬Alatie ١Ϊ0ΊΎΙ EucHanled Rot^
‫الة‬
Bracteacoccus aerius sp. nov.
liracteacoccus pseutlominor sp, nov.
Bracteacoccus cohaerens sp. nov.
Tetrasporales
Coccomyxaceae
Ourococcus Íiultísporus sp. nov.
Keys to the species have been prepared for genera with several species, as have
correlative summaries of the speci c attributes.
Herbarium specimens of all newly described taxa have been deposited in the
Herbarium of the Chicago Natural History Museum, and axenic cultures of each
liave been deposited in the Culture Collection of Algae, Indiana University, Bloomington, Indiana.
Literature Cited
Arce, G., and H. c. BoLu. 1958. Some Clilorophyceae from Cuban soils. Amer. Joul'. Bot.
45: 492-503.
BiscHOFE, H. W. 1959. Some observations on Chlamydomonas microhalophila sp. nov.
B i o l . B u l l . 11 7 : 5 6 - 6 2 .
Bold, II. c. 1942. The cultivation of algae. Bot. Rev. 8: 70-138.
194-9. The morphology of Chlamydomonas chlamydogama sp. nov. Bull. Torrey
Bot. Club 76: 101-108.
1950. Some problems in tlic cultivation of algae. In The cultul'ing of algae, 114
pp. The Charles F. Kettering Foundation. Yellow Springs, Ohio.
1958. Tliree new chlorophycean algae. Anrer. Jour. Bot. 45: 737-743.
-, and B. c. Parke-r. 1962. Some supplementary attributes in tlic classi cation of
Chlorococcum species. Arcliiv für Mikrobiologie 4-2: 167-288.
Brown, r.'M., Jr., and II. w. BiscHOFF. 1962. Anew and useful inetliod for obtaining
axenic cultures of algae. Bliycol. News Bull. 15 (4) :43-44-.
Cain, Bro. Joseph. 1963. Tire morphology, taxonomy, ‫؛‬ind phy.siology of certain Chlaniydo„го„аг-11ке algae. Pli.D. Dissertation, Univ. of Texas.
Cave, M. S., and M. A. PococK. 1951. The acetocarmine teclinique iipplied to tlie colonial
V o l v o c a l e s . S t a i n Te c l i . 2 6 : 1 7 3 - 1 7 4 .
-,
and
1956. The variable cliroinosome number in Astrephomene
gubernaculifera. Amer. Jour. Bot. 43: 122-134.
Chantanachat, S.j and II. c. Bold. 1962. Phycological studies. II. Some algae from arid
s o i l s . U n i v. Te x a s P u b l i c a t i o n N o . 6 2 1 8 .
Chodat, R. 1913. Monographies d'algucs en culture pure. MatỂr. p. la Flore Crytog. Berne
(Suisse) 4(2): 1-266.
Deason, T. R. 1959. Tlij-ee Cliloropliyceac from Alabama soil. Amer. Joui'. Bot. 46: 572578.
-, and H. c. Bold. 1960. Exploratoi'y studies of Texas soil algae. Univ. of Texas
fi
fi
Pubi. No. 6022.
BiscHo. and Bold
61
Ettl, H. I959a. Zur Kenntnis der Klasse Volvophyceae. IV. Nova Hedwigia 1: 25-36.
-. I959b. Zur Kenntnis der Klasse Volvophyceae. V. Bemerkungen zur Artabgrenzting einiger Chlamydomonaden. Nova Hedwigia 1: 167-193.
I960a. Übet
ache Pytenoide bei Chlainydomonaden. Bot. Notiser 113: 38Ι-
384.
I960b. Zwei neue Chlamydomonaden. Arch Proti.stenk. 105: 273-284.
- , a n d ٥. E t t l . I 9 5 9 a . Z u r K e n n t n i s d e r K l a s s e Vo l v o p h y c c a e . I I . N e u e o d e r
wenig bekannte Clilamydomonaden. Arch. Protistenk. 104: 51-112.
-,
and
I959b. Zur Kenntnis der Klasse Volvophyceae. III. Einige Bemerkungen zur Gattung Gloeomonas Klebs. Arch. Protistenk. 104; 113-132.
Gerloff, j. 1940. Beiträge zur Kenntnis der Varlabilltlit und Systematik dci' Gattung Chlamyd o m o n a s . A r c l i . P r o t i s t e n k . 9 4 : 3 11 - 5 0 2 .
IIerndon, W. R. I958a. Studies on rhlorosphacracean algac Irom soll. Atner. Jour. Bot. 45:
298-308.
19581,.
Some new species ol chlorococcalean algae. Amer. Jour. Bot. 45: 308-
323.
Johansen, d. a. 1940. Plant microtechnique. McGraw-Hill Book Compan)', New York.
Korsciiikoff, a. a. 1953. Ere.shwater Indicator organisms from tlie Ukraine. V. Protocorcineae. (In Russian).
Mattox, K., and II. c. Boi.n. 1962. Phycological Studies. III. The taxonomy of certain ulotrichaceiin algae. Univ. of Texas Publication No. 6222.
Miller, W. 1924. Follicularia, eine neue Chlorophyceen-gattung. Arkli. Rus.sk. Protistol. 6:
131-146.
MOEWUS, F. 1931. Neue Clilainydomonaden. Arch. Protistenk. 75: 284-296.
Pascher, a. 1927. Die Siksswasser ora Deutschlands, Österreichs und dei- Scliweiz. Heft 4٠.
Gustav Fisc lier, Jena.
Pringshei^i, E. G. 1946. Pure ci.lltures of algae, tlieir preparation and maintenance. Univ.
Press, Cambridge.
Skuja, H. 1956. Taxonomische und biologische Studien liber das Phytoplankton .schwedisclier Binnengewässer. Nova Acta reg. Soc. sci. Upsal. 8.4,16(3): 1-104٠.
Smith, g. m. 1918. Asecond list of algae found in Wisconsin Lakes. Trans. Wisconsin Geol.
a n d N a t . H i s t . S u r v. 5 7 : 1 - 2 4 - 3 .
1933.
The fresh-water alg-ae of the United States. McGraw-Hill, New York.
1950. Tlie fresh-water algae of the United States. 2nd ed. McGraw-PIill, New
Yo r k .
Furtlier studies in the genus Chlorococcum Meneghini. Moydia 16: 142-
Starr, R. C. 1953.
148.
1954. Reproduc.tion l,y zoospores in Planktnsphaeria gelatinosa G. M. Smith.
Hydrobiologia 6: 292-397.
-1955.
Acomparative study of ChloTococcum Meneghini and otlier spherical,
zoospore-producing genera of the Chlorococcales. Indiana Univ. Puljl., Sci. Ser. No. 20.
1960.
Tlie culture collection of algae at Indiana University. Allier. Jour. Bot. 47:
fl
fl
67-86.
Soil Algae ١ïom EncKautecL Rock
62
Einige neue Grünalgen. Beih. z. Bot. Centralbi. 39: 179-195.
Te r e g , Ε . 1 9 2 3 .
Trainor, F. R., and H. c. Βοι.η. 1953. Three new unicellular Chlorophyceae from soli. Amer.
Jour. Bot. 40:758-767.
-, and R. .1. McLean. 1964. Astudy of anew species ol Spongiochloris introduced
into sterile soll. Amer. Joui'. Bot. 51(1). (In press).
WitiTEHOusE, Ειιι.α. 1931. The ecology of Enchanted Rock vegetation. Μ.Λ. Tliesis, Univ.
o f Te x a s .
"
0‫؟ ا‬،,ecles studied to certain antibiotic agents‫؛‬
Spongiodomonas Chiorococcum cHlorls
Chlamy٨‫؛ااإااا‬،١ 1 1 ‫ أ‬- .
A
glg(iT١lt’,a ‫ل‬,(١1)-‫اال‬،»٢‫ اااأاااا‬mcgalis
Spongio-
Npochioris
Plankto
ckloris
llanopusi.s'
p.s(4ulosligmnticn
sphaeri sphaeri O i i r o c o c c i i x
tP.xensis maxissima muitisporum
P i a n t o -
f
A R t
C A f
C S t
n
t
K
t
N
f
N S t
P B f
P M f
s
‫ا‬-
t
T E f
E L t
G
t
K Y t
T R I
-Ι-
V
Ι-
f
V A f
M A t
s n t
S M f
.SSSf
-(
S T t
Τ Η Ι A L I
)-
‫ا‬-
F t
-Ι-
-‫ا‬-
F R I
‫ا‬-
FC'
-Ι-
M
f
‫؛‬٠
Ι-Ι-
-Ι
t'
ً‫ ا‬-
P S I
tI'
.The antibiotics, tlieli' concentrations, and the abbreviations foi' them are listed on page 13 '‫؛‬
a
a
-
(t indicates inhibition and alilank no inhibition.)
BiscHo. aud Bold
63
โ№น1‫؟‬. Gïowth 0‫ ا‬s‫¡؛‬edes studied, a'‫؛‬teT Id da١١s,lu Bold’s Basal Medium IBBM )
euTlclied with oaĩlous 0‫أ‬gaule сатВои souTces^
‫إاا ' ؛‬،‫اااا‬١ á.íniona.s
C
lorococann
Spongiof
Snongio-
chlorìs
(:.iloris
llanocnsis
<;.\RRON SOURCE niegaHs polymorphum gigantra
Neockloris
DvSeudo-
Plauklo-
sphaBria
sphaerin
Ourococcus
stigmaiica
l<١;r.،١n.sis
m a x ì s s i ì ì i a
Plaiiklo-
Hiiillisponim
A R A B I N O S E
in light
N
E
F
T
N
N
T
in darkness
N
T
N
N
N
N
N
N
E
F
E
E
E
E
G
N
G
T
F
F
E
G
D E X T R O S E
in liglit
in darkness
N
F R U C T O S E
In light
T
E
F
E
in darkness
N
N
N
٦-
N
N
N
N
N
T
F
T
N
N
N
N
X
S O D I U M
A C E T A T E
in light
N
E
N
in darkness
N
T
N
R I B O S E
in lig'ht
N
E
T
F
F
N
T
in darkness
N
N
N
N
N
N
N
N
X Y L O S E
in light
T
E
F
F
T
N
N
N
in darkness
N
T
N
N
X
X
X
X
Grown under standard conditions as well as in total darkness; Eindicates excellent growtli;
Gindicates good growth; F‫؛‬ndicates fail- growth; Tindicates trace of grow'th; and Xindicates
no growth.
Τμέ, At. Am^laslc actlnlt^ 0‫ أ‬species, aiter 14 da^s, ou Bold’s Basal Medium (BBM)
lou. agar su|)|)lemeuted wltli 0.01 0‫ ؟‬soluble starch, aud growu
under standard conditions (See Fig. ‫)ة‬
Amylasic acU١٠it,v
Clilam^domonas me galls
CltloTococcum Jol١imorhlium
s‫؛‬,onglochloris glgtintea
excellent
s‫؛‬tonglochlorls llanocnsl.s
good
Xeochlorls ^seudostlgmatlca
good
good
Planktos haeTla texeirsls
fi
fi
Planktosphaerla maxls,slma
OuTococcus mulll.s()OTum
slight
slight
excellent
slight
Soil Algae Ιτοπι Enchanted Roch
Tmi ‫اة‬. SensltRlt^ 0‫ أ‬species 0‫ أ‬Bïacteacoccns to ceitaln antibiotic ‫آلن‬
. gigantcus
Antibiotic
. modiotxuclp-atus
. grov ls
, ٣ - ٠،١ t i ، s
. pseudomiaor
. coỉiacrcn.‫؟‬
Α Β +
C S I
κ
ι
Ν +
ΡΒ+
Ρ Μ +
s
.
T R I
S D P
A L I
F
l
F R I
I H I
M
l
P S I
.The antibiotics, tlieir concentrations and the abbreviations for them are listed on page 13‫؛؛‬
indicates slight inliibltion.)
(I indicates inhibition; ablank indicates no inllibition;
Тмя1‫>؟‬. Growth. 0‫ ؛‬species oị Bracteacoccns, aịter 14 da^s. In Bold’s Basal Medlnw
(BBM) enriched with oarlons organic carbon sources
(:ARBON
B. ịỊÌgari/eiỉs B. ine-dionuciealus
SOURCE
. gvatuỉis
a c r u،,‫؟‬
B. pseudominor B. cohaerens
A R A B I N O S E
in light
N
F
N
N
in darkness
N
N
N
N
in light
G
G
G
F
in darkness
N
T
R E X T R C S E
N
G
F
F
FRUCTCSE
G
in light
T
in darkness
G
T
G
G
N
N
F
F
T
N
N
N
N
S O D I U M
A C E T A T E
in light
N
F
T
in dai'kness
N
N
N
R I B O S E
in light
N
F
T
N
F
T
in darkness
N
N
N
N
N
N
in light
N
N
N
N
N
T
in darkness
N
N
N
N
N
N
X Y L O S E
aGrown under standard conditions as sveli as in total darkness; Gindicates good growth;
fi
fi
fi
Findicates fair growtli; Tindicates trace of growth; and Nindicates no growth.
fi
fi
fi
fi
fi
64
65
Bischo. ttud Bold
Τμ‫؛‬ιέ,٦. Amỉylasic actlolt‫ ؟‬oi Bracteacoccus, aỊteĩ 14 da١!‫؟‬,o^Bolďs Basal
Medlam (BBM) loa agai- supplemented lultli 0,01٠/ο soluble staicb,
aud giouiii uudei standard conditions (See Fig. 2)
Aiiiylaslc activily
B. grandis
slight
slight
good
B. aerlus
good
B. pseudomlnoT
good
good
B. gigantens
B. medlonucleatus
B. cohaerens
Soil Algae İTom E^clia^ted Rock
ÖÖ
Figs. 1-3
Fig. 1. Cu!‫؛‬٧res used as standards .f reference for estimating growth; left to right: “excellent,”
“ g o o d , " " f a i r, " “ t r a c e , " a n d “ n o n e . "
F i g . 2 . Va r i o u s o r g a n i s m s a f t e r 1 4 d a y s g r o w t h o n B B M I o n a g a r c o n t a i n i n g ٠/٠ 0 1 .‫ ه‬s o l u b l e
starch, and ooded with aweak solution o u p p e r - l e f t
culture
shows
no
amylasic
ability;
the
clear zone near the margin of the medium resulted from amold contaminant. Upper-right culture shows
sllgJit amyiasic activity. Lower-left culture shows good, while the lower-right culture illustrates excellent
a m y l a s l c a c t i v i t y.
F i g . 3 . E x a m p l e s o f I n h i b i t o r y a c t i o n o f c e r t a i n a n t i b i o t i c s o n t h e g r o w t h o f a l g a e ; n o t e t h a t ( K -‫؛‬- | ,
(Ν"Η), (PM"..} and (PBH )have Inhlbltoi^ ability on this particular organism, while the other δantibiotics
f
fl
have no effect.
67
Bischo. aud Bold
■
٠
ч
‫ط‬
ệ
*
I
'ậ٠
.
^ ‫خ‬٠‫ س‬- * *
®‫؛أديب‬١٣.
i
‫ر‬
68
Soil Algae ịĩom lii^e^a^teà Rock
Fig. 4. View of souiheasf s!٠pe of Enchonfed Rock, ‫؛‬aken from ‫؛‬he bank of Sandy Creek.
. : ‫ي‬
:
.‫ث ـ ــنج‬
. f f
- ٨'‫ي ـ ـ ـ ـ ـ ـه ـ ـ ـ ـ ـ ـص ـ ـ ـ ـ ـ ـغ ـ ـ ـ ـ ـ ــدي ـ ـ ـ ـ ـ ــي‬
-'/··.ч
---
Fig. 5. Adepression on .he west exposure of Enchanted Rock containing soil and vegetation.
‫‪60‬‬
‫‪BiscKo. and Bold‬‬
‫‪ ,‬س ـ ـ ـ ـ ـ ــد‪,‬‬
‫‪ů‬‬
‫"‬
‫‪ř‬‬
‫| ات‪٠‬غ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ــة ‪ ๅ :‬ب‪:‬تيه‪:‬ذة‬
‫ؤ‪:‬؛‪:‬غ‪;-‬ؤ§ة ‪١‬‬
‫‪I‬ت?‪4‬‬
‫'‪،‬صه‪:‬ل ‪-‬‬
‫‪-‬‬
‫‪ร‬‬
‫' أم‪٠‬ا‪٠٠‬س ‪ -‬ل ‪, „ .‬‬
‫ج ـ ـ ــمؤ‬
‫‪4‬‬
‫‪m:‬‬
‫‪Fig. 6. Rain‬‬
‫ا‪٠٠‬م ‪an summ‬؛‪c٠n.a .‬؛‪n‬؛‪ng adense gr.w.h .f Isoefes melanopoda; a‬؛‪m٠s. dry when piCure‬‬
‫‪.was .aken‬‬
‫؛ ‪، | ٠٠ ί ، Λ‬؛‪Ι ٠‬‬
‫;ض؛‪ ،.‬ف‬
‫‪■á - . - y u -‬‬
‫‪٦٠‬‬
‫‪.‬‬
‫‪-‬‬
‫‪.‬‬
‫‪ .‬ئ ـ ـ ـ ـ ـ ـ ـ ـ ــد‪I l ■٠‬‬
‫! ج ‘ ؟‬
‫ةي ــخ‪.‬؛‬
‫ن‬
‫‪-‬‬
‫؛‬
‫ب ـ ـ ـ ـ ـ ـ ــة؛اي‬
‫ث ـ ـ ــم‪-.-.-‬أم'‪-‬‬
‫ق ـ ـ ـ ـ ـ ــدة؟؛ ‪· :‬‬
‫‪-‬‬
‫‪۶ r‬‬
‫ـغ‬
‫ـ‬
‫ـ‬
‫ـ‬
‫ـ‬
‫ـ‬
‫ـ‬
‫ـ‬
‫ـ‬
‫ـ‬
‫ـ‬
‫ـ‬
‫ـ‬
‫ـ‬
‫ـ‬
‫ف‬
‫ا‬
‫‪Ếễ‬‬
‫‪1‬‬
‫‪Fig. 7. Crevices on west exposure; note goot droppings.‬‬
‫آ‬
‫‪® ,‬؛‬
70
Soil Algae !тот Eachaated Rock
Figs. 8-15
Figs. 8-13. Chlamydomonas msgalis.—Fig. 8. Vegetative cei.—Fig. 9. Acetocarmine stain prepara-
tion of avegetative tei.—Fig. 10. Cytoltinesis of vegetative cell during the nonmotile phase.Fig. 11. Two daughter cells inside distended mother cell wall.—Fig. 12. Four daughter cells and unilateral
cytolcinesis of vegetative cell.—Fig. 13. Portion of 2-weeh-old colony on Proteose-peptone agar.
Figs. 14-15. Chforococcum polymorphum. Vegetative cells from 2-week-old cultures growing on BBM
a g a r .
(Magni cations: X1000, except Fig. 8, which is Χ750‫ ؛‬Fig. 9, which is Χ500; and Fig. 13, which
fi
is Χ25.Ι
B/"伽ノα"d Bo"
側 , 蹄
蕊
灘
鱗
灘
蕊
1
／1
蕊
鶴
浄呂一由
弓
篝灘
寺
諾
澤
抄
可 , 部
内
,
①j
鍵蕊蕊溌蕊#議溌溌漣
:望ʻ､.．.::“．､3．
鍵
鉢
蕊
蕊
､禁．驚溌
蕊
⑥
鱗
…
̅唖聖宅垂騨…一轄酔grn,,者
二魂躍詞
…
2
審
So〃狐g" /ro"z E"c/m"ied Roch
72
Fig s、16­23
Figs、1 6-22. Chlorococcum polymorphum.­Figs.
1 6-1 8. Vegelq ve Eells; nofe ldrge vqcuoles.-
Fig.19．5u唾essive
. 1 9. 5uccessivecyfokinesis
cyfokinesis of
of vegefd
vege↑p ve
ve celIs.­Fig.
celIs.­rlg.zu･ApIqnospor回ngIum
20．Apldnospor@ngium wlm
with qpIqn
qplqnospores.­
Fig、21．Zoospordngium wifh zoosPores.­Fig. 22. Por on of 2-week-old colony on BBM qgqr
Fig. 23．Spongio[hioris gigqnfeQ. Porｻion of 2-week-old colony on BBM qgqr.
fi
fi
fl
fi
fi
fi
(Mqg ni cp Ons： 1 000, ex[ePf Figs. 22, 23 which口『e 25.）
岩
73
ノガ"伽ノα"〃；o〃
胡
P
蕊
鍵
驚蕊篭
識
獺
蛙
琴
寵
撹
翅
亙
呈
想
割
繊
霧
繍
鋳
難
蕊
蕊
I
藷
患
篭
…
弗
韓鮮
癖
蕊
騨,唱
織
̅
鐸
!
』
鍵
鐸
轄
I
蕊
無
挑
鍔
韓
与
綴
雛
●
蕊蕊
穂
騨
擬
寵 :灘蕊
唖
鐇
…
曾
識
潤
侭
潔
串
神
醜
一
蔦
』
鴬
識
＃
蕊
蕊蕊
蓬
-鍵
74
SoJ4/g('e /m'" E"Cﾉ'α"彫(J RoL･k
Figs. 24-31
Fi95．24-3 1 . Chloro[o[cum hypnosporum Sfqrr.­Fig. 24．Vegefq ve cells･一Fig．25. SIqgeS of suCCe5sI･ﾉe cy;o':ine3isサoform reProduC ve cellS･一Fig. 26. Eighl dplqnospores within cell wqll.­Fig．27. Eighf
hypnospores wifhin
cell wqII.-Fig.
28.
Echinqfe
hypnospores.­Fig.
29．Zoospordngium
liberqiing
zoosPores wi}hin q vesicle.­Fig. 30. Zoospores in vesicle5.-Fig. 31 . Two zoosPores immediqtely q er
mo)ile phq5e.
ff
fi
fi
fi
fi
(Mqgni cq ons : 1 000, excepf Fig. 31, which is 2000.）
75
ﾉ〃"加ノα"d Bo"
鍵
、
…
蕊
鶴
識:母
畢面
蕊
汽
毎
戯
ハ
謬
76
So" "9" /m'" E'"伽"/ed RocA
Figs、32­39
Figs. 32-33．Spongiochioris gigQnfed.­Fig. 32. Vegefq ve cell from 2-week-old cuifure.-Fig．33・
Young vegefq ve [ells; nofe Pdriefqlqnd disseC↑ed ChloropldS↑・
Figs. 34-38. Spongio[hloris〃口noensis.̲Fig．34. Young vegefo ve celIs・­Fig. 35．Mqfure vegefqtive
cell; nofe inferldEing strdnds of [hloroplqsｻ.­Fig. 36. Old vege↑｡↑ive ceIIs; nofe bubble-like profrusions
onEeIlwqll.­Fig. 37. Liberq on of qpldnospores.-Fig. 38．Por on of 2-week-old colony on BBM qgqr.
Fig．39. Neochioris PseudoS gmofに回. Vegefq ve cells embedded in fheir individuql mqfri[es; cells
Sfqined wifh Mefhylene blUe.
(Mqgni cq ons: Fig．32, 500; Figs. 33, 35 qnd 36, 1 000; Fig. 34, 100; Fig. 37, 750f
fi
fi
fi
fi
fi
fi
fi
fi
fi
Fig. 38, 25; Fig. 39, 350.）
句
戯“伽ノα7zd Bo"
匂
／ ／
,拶織J:嚥穀犀懸j,,,『:ʼ十?､、：
灘
I 掌 篭
鱗
驚
篭
蕊藷
鷺
鴬
錘
“
蕊鶏
鐸
篭 憩 辮 蕊 識
蕊
：,溌藍凝議蕊鱗
鱸
灘
關
里
蕊
弧
瀞
鶴
議蕊
-
、
'
'
灘
,
：
癖
芯
茸
- と
識
鵜
③
蕊
蕊謙鐇ʻ驚鐸錘
I
Figs. 40-47
Figs. 40-47. Neochloris pseusIos gmdjicq.-Fig．40. Vegefqlivece!Is sfCIined with l2-Kl.-Fig．41．
Three­week­old vegefq ve cells mounfed in lndiq ink; nole droplef on cell wqⅡOf lqrgesf cell.-Fi95.
42-44. 51qgeS of old tellS SIoughing o thickened Cell wqll.­Fig. 45．Progressive cleqvqge of vegefqve cell during process of zoosporogenesis.-Fig. 46. Liberq on of qpldnospores.-Fig. 47．VegeｻﾛｻIve
[ells enc!osed in individuql dnd common mqfrix; Mefhylene blue sfdin.
Mqgni CqlionS: Fig. 40, 1000; Fig. 41, 450; Figs. 42-44, 500; Fig. 45, 1250; Figs. 46, 47,
fi
ff
fi
fi
750，）
fi
fi
So" "9"' /'"o"z E"C/m""" Rock
78
Bischoft aud BoÎd
-
. i *
':แ
๏
,
‫ا‬
Soll· Algae พ01 Eachaated Rocii.
80
Figs. 48-55
Figs. 48—49. Neochloris pseudos gmah.co.—Fig. 48. Portion of 2-week-0!d co١ony growing on ΒΒΛΛ,
as seen with transmirted light.—Fig. 49. Same as Fig. 48, except photograph taken with re ected light.
Figs. 50—55. Planktosphoer،. texensis.—Fig. 50. Group of ٧egetatl٧e cells.—Fig. 51. Mature vegetative
cell; note prismatic chloroplasts.—Figs. 52, 53. Young vegetative cells showing arrangement of groups.—
Fig.
.Group of vegetative cells mounted In Methylene blue showing extent of gelatinous material.—Fig.
5 5 . Z o o s p o r e s s w i m m i n g w i th i n v e s i c l e .
(Magni cations: Figs. 48, 49, Χ25; Fig. 50, Χ500; Fig. 51, Χ1250; Fig. 52, χΐ25; Fig. 53,
fl
fi
fi
Χ500; Fig. 54, Χ2000; Fig. 55, Χ750.)
81
""加＃α"dBo〃
識
泡
⑨
聖
…
③
蕊
識 蝉 夢 搦 , 箇 蕊
:群鼠
灘
蕊＃
錘
患
蝿
鱒
僻
幹
趾
霊
篭
濯
③
蟻 篭鐙蕊
；
So" "g" /wonz E"c/Mi'jed Rock
Q < ）
U
皇
Figs. 56-63
Figs. 56-60. PIqnkfosphqerio fexensis.­Fig．56. Mo le zoospores wilhin vesi[le・­Fig. 57. This vesi[le
(Fig. 56) did noi ruplure, qnd q er q shorf period of mo lify fhe zoospores becdme quiesEenf, wifhdrew
their
qgelld｡nd [on nued fheir exisfen[e qs qpldnospores.-Fig. 58. Zoospores immediqiely qfter m。↑ile
phqsef nofe ihqf teIIs hQve become sPhericql buf fhqf fhey hqve no↑yet wifhdrqwn
qgellq回↑fhis stqge.­
Fig.59. Vegeiq ve [ell comPle ng progre35ive cleqvqge.­Fig. 60. Por on of 2-week-old [olony on BBM
qgo『.、
Figs. 61-63．PIqnkfosPhoeriq mQximo.-Fig. 61．Vegefq ve cells.-Fig. 62. Group of vege↑｡↑ive“lIs
Of vdriouS CIge5.-Fig. 63．Mqfure vegefq ve [ell wifhout thi[I< Iqmellqfed envelope.
(Mqgni cq ons; Figs. 56, 57 Qnd 59, 500; Fig．58, 1 000; Fig. 60, 25; Figs. 61 qnd 63, 275；
Fig．62, 45.）
fl
fi
fi
fi
fi
fi
ff
fi
fi
fi
fi
fi
fl
、
83
Bj妬加ノα7zdBo"
懲溌函
蕊
蕊
蕊
愚
霧 蕊
零
蕊
鼈
悪
灘鍵蕊
鐡蕊
爵
鰯
蹴
澤
蕊簿
蕊
謬管
̅
鍔
錘
睡
彦
鎌
参
唾一
㎡
曲
1
I
･ - - 』 , 、 や : 球 ， , 『
錘
鯵 ；蟻I織灘Iil: ③
・r"､、:#1
Mゞ灘ʻ＃
鑑
､ ザ
鰯
84
So" "9" /'･o"z E'"伽""d Rock
Figs. 64-7 1
Figs. 64-67. PIqnk#osphgerid mdximq.­Fig. 64. Vegefq ve cell surrounded by lqmellqfed envelope.
-Figs、65, 66. Vegelqlive celIs wifh lqmellqfed envelope rupfuring qf opposife poles prior1o sloughing
o .-Fig．67. Por on of 2-week-old colony on BBM qgqr.
FigS. 68-69. Ouro[◎に[us mul"sporus･-Fig. 68．Group of vegefq ve [ells; nofe defqched spicules.­
Fig．69．Por on of2­week-old colony on BBM qgqr,
I
Fig5、70­71．Br｡けed[occUs gigdnfeUs.­Fig．70. Vegefq ve cell.-Fig. 71. Liber回 on of Qpldnospores
f『om↑hi[k­wqIled mqfure celI．
IMqgni [qlions: Fig. 64, 200; Fig．65, 400; Figs. 66 qnd 68, 750ノFigs. 67 dnd 69， 25；
fi
fi
fi
fi
fi
fi
fi
ff
Figs，70 qnd 71, 500.）
ﾉﾀﾉjc伽ノα"。〃o〃
85
, 弓
̅
錨
4
…
溌瀬心吟
灘
蕊
鐵
中
ひ
霞
寺
⑤
…
■？■了J■■？＝母舞""･口百一了『̅­­
鯵
漉
塔
繩
堪
#
．､聡
#ʻ
！
爺
:
“
ʻ
役
奄
・
も
粒
Ⅸ
農蕊
鴬
鴬
Ａ
斗
考
灘型
軒
瀞
も
別
・
・
擬
頚
撫
陛
騨
聡
暇
馳
醗
蝿
Ⅱ
辮
鐸
轟
癖
鱗
Soル1Jg" /rom E"c/Z""jed Rock
86
Fig5、72­79
Fig．72. Brd[feqcoEcus gigqnfeus: por on of 2-weel(-old Colony on BBM qgq『・
Figs. 73-75．BrqcfeDcoEcus medionucleqfus.-Fig．73. Vege1｡↑ive tel1s; nofe young ceil whiEh is subsplfericcll.-Fig. 74. Vegefq ve cell; nofe subsurfqce chlorOPIqsf.-Fig．75．PC『↑ion of2­week­old
colony on BBM qgqr.
Figs. 76-79. Brq[fe口にo"Us grDndis.­Fig. 76．Mqfure vegetq ve tells・-Fig．77. Vegetq ve ceIIs qnd
qplqnosporqngium; nofe bubble-lil<e thickening qf one pole of lqrgesi ce11.一Fig. 78. Apldnospordngium
immedidiely口什er rupfure of wqll fo releqse dpldnospores.­Fig. 79．Por on of 2-weel<-old colony on
B B M q g q r.
fi
fi
fi
fi
fi
fi
fi
(Mqg ni [d ons: Figs. 72, 75 ond 79, 25; Figs. 73 qnd 74, 750; Figs. 76, 77 dnd 78, 1000.）
B/"加ノα"" Bo"
87
鑑..鐸.:]"．.'.､龍…曙.:.．．"....::．・
ヱ
ー
､
'
ぜ
,
麺
も
j
-
L
-
,
,
,
-
,
,
,
,
,
,
,
,
，
,
,
,
1
,
,
,
j 二 群 H 霊 , ̅ , ̅ - , , , , , - - , , , , % 『 ,
蕊“
：'ʼʼʼʼʼ唾ʼ錘ʼʼʼ心
､ ' ' 1 ' 唖 ､ ' ， ， ' ｼ ､ J ' ' ， ､
, , ' 1 , 1 1 , 諏 一 可 ,
, : ､ ' ' : ' 輿 ' 1 ' 造 習 , ず
Ⅱ
'
1
，
』
､
:
'
ʻ
,
1窯；
｣#
一
'
'
診
¥
』 ' ： , ,
鷲 議 I 鍵 認
諜
…
？
鯉
蕊
鱸
ぶ
鍵蕊
蕊
鋪？
灘
②
灘
〃
雛
ʻ
蕊
③
一'
《
So" "g" /ro'" E"cんα""d RocA
Figs. 80-87
Figs. 80 dnd 81．Brddeqto[rus qerius.-Fig．80. Mqfure vegefq ve teils.-Fig. 8 1．Por↑ionof2­week­
old colony on BBM qgdr.
Figs. 82-84．Brgcfeqcoccus Pseudominor.-Fig. 82. MQfure vegefq ve [ells.­Fig. 83. Group of vegeiq-
ve [ells showing fypicql qrrdngemenf of celIs forming irregulqr mdsses.-Fig．84．Por on of2­week­old
colony on BBM qgqr・
Figs、85­87．BrqEfeqEoc[ustoh⑥erenS.­Fig. 85．MQfure vegelq ve [e115.-Fig.
86. Aplqnospores
q q[hed by fheir cell wQIIs, forming spheri仁。l clumps of cells.­Fig. 87．Por on of 2-week-old [olony
on BBM qgq『.
(Mqgni [q ons: FigS. 80 dnd 86, 750; Fig5. 81, 84 dnd 87, 25; Fig. 82, 1000; Fig. 83, 100；
fi
fi
fi
fi
fi
fi
fi
Fig. 85, 300.）
ff
fi
88
Bisclioff aud Bold
๏
٠‫ال‬٠‫ل‬
0‫ت‬
■٥٥٠ i . . ;
٥
‫؟‬0
о
.
о «‫؛‬
■
พ
٥
‫ل‬٠
©;
Oo'lö
‫ه‬
о
٠
٠
во ... ١
»
· О
ч
ỈẬý '٠
@
/ ‫ا‬
®
‫ة‬
٥٠
‫اة‬
٠
١٥٥β.
.....‫"'ذا‬
‫اج ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ـ ــم‬
. ‫ا‬
‫اا‬
f j
· : | ฯ ' ) ๐
ì
๏
า
о
О
90
Soil Algae ịĩom Enchanled Rock
Figs. 88-105
Figs. 88—91. Chlamydomonas mega/i٠s.—Fig. 88. Vege‫؛‬٠٠i٧e ‫ !اجء‬In surface view,
٠F!g. 89. Ths
some in median .p cal section im.0.5).—Fig. 9٠. Transection of the cel! through level of nucleus.—
Fig. 91. Anterior pole view.
Ig. 94. Surface view‫؛‬
Fig. 92-98. Chforococcum po/ymorphum.—Figs. 92—93. Young vegetative cells.,
of mature vegetative cell showing dissected chloroplast, pyrenold and large vacuoles In colorless cyto-
plasm.—Fig. 95. Median optical section of mature vegetative cell showing the inner irregular surface of
the chloroplast.—Fig. 96. Mature vegetative cell during successive cytokineses (from acetocarmlne-
stained preparation).—Fig. 97. Formation of zoospores within parent cell.—Fig. 98. Liberated zoospore.
Figs. 99—105. Spongiochloris giganteo.-Fig. 99. Motile zoospores.—Figs. 100—102. Successive stages
In morphogenesis of zoospore to the spherical stage.—Figs. 103, 104. Young vegetative cells from
quiescent zoospores, showing Increase in number of nuclei and pyrenolds as cells increase in size.—
F i g . 1 0 5 . A c e t o c a r m l n e - s t a l n e d p r e p a r a t i o n ; n o t e‫ ؛‬i n c r e a s e I n n u c l e a r n u m b e r, o n s e t o f p r o g r e s s i v e
cleavage of the protoplast, and the thickened cell wall of avegetative cell In the stationary phase of
growth (m.o.s.).
(Magni cation: Figs. 88-91, Χ1500; Figs. 92-98, Χ2000; Figs. 99-104, Χ4000; Fig. 105,
fi
fi
Χ500.Ι
91
Bi"伽ノα"(I Bo"
一
I
I
ʼ
r》
' 0 J 品
⑬
So" "9" /"o'" E"c加"jed Rock
!)2
Fi95．106­121
FigS. 1 06-1 09. SpongioChIOrisⅡqnoensis.-Fig. 1 06. Medidn op [qI se[ on of vegelqlive celi showing
ne i!<e chloroplqsf, exfenfrit pyrenoid, qnd nuclei oc[upying colorless cyfopldsm in spongy dreds of
ChloroplqSf､-1 07. Acefocqrmine-sfqined prepdrq on of vegefqlive cell in process of progressive cleqvqge
（、.o､s.)-Fig．1 08. Liberqfed zoosPore.­Fig. 1 09. Zoospore shor y before quies[ence.
Figs、1 1 0-1 1 8. Neochloris pseudos gmq cq.­Fig. 110．Surfqce view of mqfure cell undergoing progrCSSive Cleqvqge in zoosporogenesi5, qnd surrounded by individuol mqfrix.-Fig. 1 1 1. Surfpにe view of
young vegefclive cell showing opening of Eup-shqped chloropldsf, nuclei in colorless [yloplqsm, qnd
periPherql pyrenoids.-Fig.
1 1 2. 0u ine drqwing lo illusfrqfe lhe vqrious forms of droplefs secreled
1hrough fhe cell woll.-Fig．1 1 3. Medidn opticQI sedior' of vegefq ve cell showing lqmellqfed individuql mdirix with droplef on eqch [onceniric ldyer.­Fig. 1 14. Mediqn op tqI sedion of vegefqtive
ceil from old cuIfure wifh bubble-li!<e wqll fhickening.--Fig. 1 1 5. Mul nutledfe vegefq ve cell iusl Prior
foprogressive [leqvoge
(qcefo(qrmine prepdrq on).­Fig. 1 1 6, 1 1 7. Liberq)ed zoospores.-Fig. 1 1 8.
Young veele!q ve tell from quiescenf zoosPore showing qppedrqn[e of pyrenoid qnd individuol mqfrix.
Figs. 1 1 9-1 21. PIqnl(fosPhqeriq fexensis.一Fig. 1 1 9. Young vegelqサive [ell from quiescenf zoospore
51q;ned wifh l.,-KI, showing lqrge grqnUles Of sfqrCh in fhe [hlorOplq5f.-Fig. 120. Mo le zoospores
wifhin vesiclei nole frqgmenfs of poreniql wqll on fhe periphery of expqnding vesicle, qnd qIsO↑hq he
zoospores qre now en[Iosed by qn ouler, fhin, delicQfe, gelq nous Iqyer qnd qn inner,
|qyer.­Fig. 121. LiberQfed zoospore.
rm, gelq nous
(Mqgn i Cq cns: Figs. 106, 107 qnd l l4, 1500; Fig5. 108, 109, 4000; Fig. 1 1 0, 1 800; Figs.
111, 113, 115 Qnd l20, 1000; Figs. 116-119 qnd l21, 2000.）
ff
fi
fi
fi
fi
fi
fi
fi
fi
fl
fi
fi
fi
fi
fi
fl
fi
fi
fi
fi
fl
1
93
Bi"加ガ""(J Bo"
ｒ
ｌ
ノ
⑩
、
１
１
●
I
⑩
一
I
淨口
一
⑫
⑪
一
o
9
C
9
●
⑭
口
？
⑬
⑰
⑬
鯵
③
So" ""e /ro"z E岬，加伽ted Roc&
94
Figs. 122-145
Figs、122-126.PIqnkfosphqeriqmoximd.-Fig．122．Mediqnop cqIsec onofyoungmqfurecell
showing internqI, subsurfq[e chloroplqs↑s wifh pyrenoids, qnd nuclei oEcupying colorless cyfoplqsm.­
Fig5．123­125．0u ine drqwings of qbnormdl mo le zoospores resul ng from incomplefe eleqvqge.Fi9. 126. Liberp↑ed zoospore.
Figs、127­130．Ouro[o[Eus mul sporus.­Fig．127．Vegefq ve cell wifh o qthed spicule.-Fig．128．
Au↑ospore formq on by frdnsverse ssion.-Fig．129．AuIospore formq on by oblique ssion.­Fig．130．
AufOspores wifhin pdrenfql wdⅡ、
Figs、1 31-1 36. BrqtfedcocEus gigqnieus.-Figs. 1 31, 1 32. Young vegeiq ve [ells showing inErecse in
nu[|edrnumber.­Fig. 133．PIiqble zoospore, exhibi ng dbsence of fell wqll.-Fig．1 34. Shqpe of zoospore
beforeliberp↑ion from zoosPordngiql veside.­Fig. 1 35. Liberqfed zoospore.-Fig. 136．Shqpe of zoospore,
iusf Prior fo quiescence.
Fi95. 1 37, 1 38. Brq[feqEoEEus medionucleofus.­Fig. 137．Mediqn oPticqI Section of muI nutleqle
vegefq ve cell; nole infernqi, subsurfq[e chloroplQsfs qnd fhe nuclei clustered nedr lhe cenfer of fhe cell.Fig．138．Liberqled zoospore.
Fig. 139. Br口鄙edcoccus grdndis: mo le zoospore.
Figs. 1 40­142．Brq[fed[ocEus qerius.-Fig．140．Mo le zoospores wifhin vesicle; nofe frdgmenfs of
pdrenfql wqll.-Figs. 141, 1 42. Motile zoospores.
Fig. 143．Brdcleq[oEcus pseudominor: mo le zoospore.
Figs、144­145．Brqcfed[oEcus EoI1derens.-Fig. 144. SuI･fqce view of mqfure vegefq ve [ell showing
Pldielike chloroplqsf; nofe stigmprefqined by chloroPldsf.-Fig. 145．Liberqfed zoospore
(Mqgni cd ons: Fig. 1 22, 1000; Fig. 138, 3000; Figs. 141-143 qnd l45, 4000; qll ofhers,
fi
fi
fi
fi
ff
fi
fi
fi
fi
fi
fi
fi
fi
fi
fi
fi
fi
fl
fi
fi
fi
fi
X 2000.）
腕施加'α"(I Bo"
〜
95
、
一
ﾉ
1
142）言聖延