BULLETIN OF MARINE SCIENCE, 34(3): 330-334, 1984
BATOPHORA LARGOENSIS NEW SPECIES
(CHLOROPHYTA, DASYCLADACEAE) FROM SOUTH
FLORIDA: GENETIC CONFIRMATION
J, S Prince and S Baker
ABSTRACT
Crosses conducted between Batophora oerstedi J, Agardh and B, largoensis Prince and
Baker, found that they were reproductively isolated, The crosses also showed that individual
plants and gametangia of both B, oerstedi and B, largoensis produced cysts of two mating
types. Individual cysts of B, oerstedi and B. largoensis, however, contained gametes of only
a single mating type. Pretreated cysts of B. oerstedi showed a considerable delay in releasing
gametes while this delay was markedly shortened for cysts of B. largoensis.
Morphological traits have traditionally been used to separate algal organisms
at the species level. As a result of the accumulation of evidence of the phenotypic
plasticity of many algae, particularly marine macroalgae, taxonomists have become increasingly aware of the danger of relying too heavily on morphological
criteria to characterize species (Mathieson et al., 1981). Fjeld and Lovlie (1976)
suggest that reproductive isolation is the only really valid criterion for the species
taxon in algae.
Prince and Baker (1984) compared the morphological and ultrastructural characteristics of a new species of Batophora, B. largoensis Prince and Baker, with B.
oerstedi, the only other species of this green, siphonaceous genus recorded for the
waters off South Florida. This paper reports the results of crossing experiments
which attempted to determine whether or not B. largoensis is reproductively
isolated from B. oerstedi.
MATERIALS
AND METHODS
The collecting sites for Batophora oerstedi and B. largoensis, characterization of mature gametangia,
the isolation of cysts, and the culture medium employed, have been described elsewhere (Prince and
Baker, 1984).
Different combinations of temperature and light were tested for their influence on the release of
gametes by cysts. Cysts from mature gametangia on 10 reproductive individuals of either Batophora
oerstedi or B. largoensis were dispensed into culture dishes (5 x I cm). Four replicate dishes for both
taxa each containing 30-40 cysts were exposed for 48 h to either: (1) darkness at 15° ± 1°C, (2)
continuous light (950 lux; Westinghouse 20 W, fluorescent lights, Weston Illumination Meter) at 15°C
or (3) darkness at 21°C ± IOC.The culture dishes were then transferred to 21°C, continuous light (950
lux). The number of empty cysts with an open operculum was recorded after 8 days with at least 20
cysts being examined in each dish. Statistical parameters were calculated after the percent release was
transformed to normally distributed data by means of the arcsine transform (Zar, 1974).
Multiple culture dishes each containing at least 30 to 40 cysts from mature gametangia on 10
reproductive individuals of Batophora oerstedi or B. largoensis were exposed to continuous darkness
for 48 h at 15°C. Four replicate dishes for both taxa were then transferred to each of four temperatures
(20,25, 30, or 35°C) under continuous fluorescent light of 2750 ± 250 lux (40 W, Sylvania Life Line).
The number of cysts that released gametes was recorded every 3 days with at least 20 cysts being
examined in each dish.
The crossing experiments involved the isolation of individual, mature gametangia or cysts using
drawn pasteur pipettes. The culture dishes were checked for cyst or gametangial presence after isolation,
The dishes were sealed with parafilm and placed at 30°C under continuous light of 2750 ± 250 lux.
The presence of germlings was recorded after 2 to 3 weeks. The release of gametes by cysts in four
replicate cultures of both Batophora oerstedi and B. largoensis was recorded concurrent with the
crossing experiments involving one or two cysts. Crosses were conducted between mid fall, 1980 to
330
PRINCE AND BAKER: BATOPHORA LARGOENS/S
Table 1. Number ofreproductive
SP. NOV. (CHLOROPHYTA):
GENETIC CONFIRMATION
331
plants and trials per cross
No. Reproductive Plants with
Mature Gametangia Used
per Trial
Type of Cross
Cyst from the same plant
Cyst from the same gametangium
Single cyst
Two cysts from the same species
Two cysts, one from Batophora oerstedi.
one from Batophora largoensis
No. Trials
Balophora
Balophora
oersted;
largoensls
10-20
5
4
10
I
I
10
10
3
4
2
2
4
10
No. Crosses
per Trial
10-20
20
40
40-60
40-60
late spring, 1981. Table I lists the number of reproductive individuals of B. oerstedi or B. /argoensis
with mature gametangia which served as sources of cysts or gametangia for each trial, the number of
crosses conducted on each trial, and the total number of trials conducted for each type of cross.
RESULTS
AND DISCUSSION
Maximum release of gametes by cysts of both Batophora oerstedi and B. largoensis occurred after 48 h in the dark at 15°C (Table 2). Gamete release decreased,
however, by approximately 30% when cysts were exposed for 48 h to either 15°C
continuous light or 21°C continuous dark.
In the experiments described below cyst release was maximized by first pretreating them for 48 h under continuous darkness at 15°C.
The effect of temperature on gamete release by pretreated cysts is depicted in
Figures 1 and 2. Cysts of Batophora oerstedi (Fig. 1) showed a 6 day lag in the
release of gametes at all temperatures. By the 12th day, however, 83% of the cysts
at 30°C had released gametes while cyst release at the other temperatures was
markedly reduced. A three day lag in gamete release occurred for cysts of B.
largoensis under 20 and 35°C, but no lag period was evident for cysts at 25 or
30°C (Fig. 2). Nine days after pretreatment, cyst release was apparently completed
at all temperatures, with 75% and 90% of the cysts releasing gametes by this time
at 30 and 25°C, respectively.
The crossing experiments described below were conducted at 30°C to maximize
the release of gametes by cysts of both Batophora oerstedi and B. largoensis. In
addition, gamete release by B. oerstedi and B. largoensis was more closely synchronized by pretreating the cysts of B. largoensis three days after those of B.
oerstedi and then combining the two for the crossing experiments.
Similar studies of gamete release by cysts have been conducted using Acetabularia (Koop, 1975). This genus is closely related to Batophora and representatives
of the two genera are frequently found growing together. These studies of AceTable 2. Conditions and percentages of cysts released after 8 days
Releases
Temperature
Cyst'
Batophora
Batophora
Batophora
Batophora
Batophora
Batophora
oerstedi
oerstedi
oerstedi
largoensis
/argoensis
/argoensis
• 4 replicates (30 cysts in each) per treatment.
(CO)
Light
15
15
21
15
15
21
Continuous
Dark
Dark
Continuous
Dark
Dark
so
12 (11-14)
49 (43-55)
10 (7-14)
13(11-16)
51 (45-57)
12 (9-15)
332
BULLETIN OF MARINE SCIENCE, VOL. 34, NO.3,
1984
70
30
"
OArs
OArs
Figure I. (Left) The percentage of pretreated cysts of Balophora oersledi that released gametes at
several temperatures. Pretreatment described in text. Mean ± SE.
Figure 2. (Right) The percentage of pretreated cysts of Batophora largoensis that released gametes
at several temperatures. Mean ± SE.
tabularia found that the percentage of cysts that released gametes, as well as the
time it took 50% of the cysts to release gametes were both optimal at 21°C, and
generally decreased at lower and higher temperatures. In the current study the
temperature that resulted in the optimal rate of release, as well as percentage of
release was elevated by approximately 9°C above that of Acetabularia. The current
study employed mature reproductive individuals collected from in situ populations while cultured material was used for studies on Acetabularia.
Crosses between cysts isolated from the same reproductive plant or gametangium (Table 3) indicated that both plants and gametangia of Batophora oerstedi
and B. largoensis produced cysts of two compatible mating types. The fertility
(percentage of reproductive plants with mature gametangia which produce progeny
in vitro) of B. oerstedi was lower than that of B. largoensis which is consistent
with previous observations (Prince and Baker, 1984).
Cultures of single isolated cysts of either Batophora oerstedi or B. largoensis
failed to produce offspring (Table 3). The mean percentage of cysts that released
gametes was 79% and 85% for B. oerstedi and B. largoensis, respectively (based
upon Figs. 1 and 2 and also including the information on cyst release obtained
during the crossing experiments). Prince and Baker (1984) report that the fertility
of B. oerstedi and B. largoensis averaged 40% and 95%, respectively. Based upon
fertility and cyst release the frequency of offspring production by single cysts of
either B. oerstedi or B. largoensis should have been 32% and 81%, respectively;
but offspring were not produced by either. These crosses, therefore, suggest that
cysts of B. oerstedi and B. largoensis produce gametes of only one mating type.
Table 3. Compatibility of plants, gametangia and single cysts
Batophora
Type of Cross
Cyst from the same plant*
Cysts from the same gametangium*
Single cyst
oersted;
Balophora
With
Germlings
Total
Crosses
With
Germlings
(No.)
(%)
(No.)
(%)
70 (5)t
20 (I)t
120 (3)t
40
60
0
60 (4)t
20 (1)t
80 (2)t
95
85
0
0.40 x 0.79 = 32%*
• Each cross (culture dish) contained more than 20 cysts.
t No. of trials shown within parentheses.
Expected frequency of germlings based upon percent fertility x percent of cysts releasing gametes.
*
largoensis
Total
Crosses
0.95 x 0.85 = 81%*
PRINCE AND BAKER: BATOPHORA
Table 4. Compatibility
Cross
LARGOENSIS
SP. NOV. (CHLOROPHYTA):
With Germlings (%)
180 (4)*
Batophora largoensis
333
of two cysts from the same species
Total Crosses (no.)
Batophora oerstedi
GENETIC CONFIRMATION
2
X
0.5
X
0.5
X
0.5
X
0.4
80 (2)*
2
X
0.5
X
0.95
4 (2.2%)t
X 0.4 X 0.79
12 (15%)t§
X 0.95 X 0.85
X
X
0.79 = 5%t
0.85
=
32%t
• No. of trinls in parentheses.
t Observed frequency.
:t: Expected frequency of germlings based upon cyst compatibility (=2 x 0.5 x 0.5) x percent fertility x percent release of each cyst.
§ Observed frequencies are significantly different (X'. P < 0.01) from expected frequencies.
The frequency of offspring production by crosses between two cysts of Batophora
largoensis or B. oerstedi was 15 and 2.2% respectively (Table 4). The frequency
of offspring production based upon cyst compatibility, fertility and release was
calculated to be 5% for B. oerstedi and 32% for B. largoensis. Statistical analysis
indicated that the observed production of offspring by two cysts of B. oerstedi
was consistent with the calculated value. Production of progeny by B. largoensis,
however, was statistically different from the calculated value (Table 4). This suggests that some condition other than those mentioned above must also affect the
production of progeny or the fusion of gametes of B. largoensis.
No progeny were produced in 180 crosses containing one cyst of Batophora
oerstedi and B. largoensis while approximately 13% ofthese crosses were expected
to produce offspring based upon cyst compatibility, release and fertility (Table
5). The results of crosses between two cysts of B. oerstedi or B. largoensis (Table
4) were used, in addition, to calculate the percentage of crosses between single
cysts of B. oerstedi and B. largoensis that should have produced progeny (Table
5). This second calculation suggested that 5.7% of these crosses should have
produced offspring. The production of progeny by crosses between B. oerstedi and
B. largoensis was, therefore, significantly lower than the expected frequency based
upon two separate methods of calculation. The time course of gamete release and
the percentage of cysts that released gametes during these crosses approximated
that depicted by Figures 1 and 2 for both B. oerstedi and B. largoensis, respectively.
A reproductive barrier must therefore prevent fusion of gametes of B. largoensis
with those from B. oerstedi.
Prince and Baker (1984) describe several morphological and ultrastructural
Table 5. Compatibility
Cross
of two cysts, one from Batophora oerstedi. one from B. largoensis
Total Crosses (No.)
With Germlings (No.)
180 (4)*
o
Batophora
oerstedi
X
Batophora
/argoensis
2 X 0.5 X 0.5 X 0.95 X 0.4 X 0.85 X 0.79 = 12.8%tt
2(G2)§ = 0.022, G = 0.105; 2(R 2)§ = 0.15,
R = 0.274; 2(R X G) = 5.7%tll
• No. of trials.
t Expected frequency of germ lings based upon cyst compatibility (=2 x 0.05 x 0.05) x percent fertility of each cyst x percent release
for each cyst.
:j: Observed frequencies are significantly (x', P < 0.01) different from expected frequencies.
§ G B. oersledi; R = B. largoelJsis.
II Expected frequency of gcrmlings based upon the observed crosses between two cysts of B. aemed; or B. largaensis (Table 4).
r-...:l
334
BULLETIN
OFMARINESCIENCE,
VOL.34,NO.3. 1984
traits which readily distinguish Batophora oerstedi from B. largoensis and which
allow B. largoensis to be considered a new species of the genus. The results of the
crossing experiments between B. oerstedi and B. largoensis that are reported in
this paper indicate that B. largoensis is reproductively isolated from B. oerstedi.
The results of these experiments are, therefore, consonant with the morphological
distinctions recorded for B. oerstedi and B. /argoensis and verify that the two are
distinct species.
The crossing experiments suggest that the cytological life cycle of both Batophora oerstedi and B. largoensis is similar to that of Acetabularia Lamouroux
(Green, 1976). Several nuclei that originated from the splitting of the primary
nucleus in the rhizoidal base of B. oerstedi (Liddle et al., 1976) must migrate into
each gametangium otherwise a gametangium would produce cysts of only one
mating type. This complements Bonotto's (1979) observation that each developing
gametangium of B. oerstedi had several secondary nuclei. Gametangia of another
related genus, Cympo!ia barbata (Linnaeus) Lamouroux, produce gametes of only
one mating type (Liddle, 1979), but unlike Batophora or Acetabularia gametes
are not apparently enclosed in cysts. Cysts of Batophora like those of Acetabu/aria
(Green, 1976) release gametes of only one mating type which suggests that meiosis
not only occurs before the enclosure of the nucleus by the cyst wall but that only
one, secondary nucleus is enclosed by each cyst. Mitosis of the secondary nucleus
during maturation of the cyst results in the production oflarge numbers of gametes
by each cyst. In the case of Acetabularia, Schulze (1939) found over 1,000 gametes
in each of two cysts. The crossing experiments do not localize the site of meiosis
in Batophora, but it is probable that like Acetabu/aria (Koop, 1975) meiosis occurs
during the fragmentation of the primary nucleus.
ACKNOWLEDGMENT
Dr. J. Heywood kindly reviewed and criticized the manuscript.
LITERATURE CITED
Bonotto, S. 1979. Observations on the morphogenesis of Batophora oerstedi. Pages 27-34 in S.
Bonotto, V. KefeH and S. Puiseux-Dao, eds. Developmental biology of Acetabu/aria. Elsevier/
North Holland Biomedical Press. Amsterdam, New York, Oxford.
Fjeld, A. and A. Lovlie. 1976. Genetics of multicellular marine algae. Pages 219-235 in R. A. Lewin,
ed. The genetics of the algae. Univ. of Calif. Press, Berkeley and Los Angeles.
Green, B. R. 1976. Approaches to the genetics of Acetabu/aria. Pages 236-256 in R. A. Lewin, ed.
The genetics of the algae. Univ. of Calif. Press, Berkeley and Los Angeles.
Koop, H. U. 1975. Germination of cysts in Acetabu/aria mediterranea. Protoplasma 84: 137-146.
Liddle, L. 1979. Modes of reproduction in Cympo/ia barbata (Chlorophyta, Dasycladaceae). Pages
19-26 in S. Bonotto, V. Kefeli and S. Puiseux-Dao, eds. Developmental biology of Acetabu/aria.
Elsevier/North Holland Biomedical Press. Amsterdam, New York, Oxford.
--,
S. Berger and H-G. Schweiger. 1976. Ultrastructure during development of the nucleus of
Batophora oerstedi (Chlorophyta; Dasycladaceae). 1. Phycol. 12: 261-272.
Mathieson, A. c., T. A. Norton and M. Neushul. 1981. The taxonomic implications of genetic and
environmentally induced variations in seaweed morphology. Bot. Review 47: 313-347.
Prince, J. S. and S. Baker. 1984. Batophora /argoensis new species (Chlorophyta, Dasycladaceae)
from South Florida: morphological and ultrastructural evidence. Bull. Mar. Sci. 34: 321-329.
Schulze, K. L. 1939. Cytologische Untersuchunger an Acetabu/aria mediterranea und Acetabu/aria
wettsteinii. Arch. Protistenk. 92: 179-225.
Zar,1. H. 1974. Biostatistical analysis. Prentice Hall, Inc., New Jersey.
DATEACCEPTED: April 19, 1983.
ADDRESS: Bi%gy
Department, University of Miami, Coral Gab/es, F/orida 33124.