Int. J. Plant Sci. 163(5):825–830. 2002.
䉷 2002 by The University of Chicago. All rights reserved.
1058-5893/2002/16305-0014$15.00
ENVIRONMENTAL SEX DETERMINATION IN THE GENUS EQUISETUM: SUGARS
INDUCE MALE SEX EXPRESSION IN CULTURED GAMETOPHYTES
Jean-Michel Guillon1,*,† and Christian Raquin*
*Laboratoire Ecologie, Systématique et Evolution, Unité Propre de Recherche de l’Enseignement Supérieur Associée au Centre National de la
Recherche Scientifique 8079, Université Paris-Sud, F-91405 Orsay Cedex, France; and †Conservatoire Botanique National du
Bassin Parisien, Muséum National d’Histoire Naturelle, 61 rue Buffon, F-75005 Paris, France
Horsetails (Equisetum, Sphenophyta) are homosporous, and sexual differentiation of Equisetum gametophytes is under the influence of environmental conditions. Still, the environmental cues responsible for sex
determination of Equisetum gametophytes in vitro and in the wild have remained elusive. Here, we show that
significantly different sex ratios are obtained when gametophytes are grown on media with or without sugar.
In our experimental conditions, male gametophytes outnumber females in the presence of 60–120 mM sucrose
and 120 mM glucose, whereas in the absence of sugar, most gametophytes differentiate as female. A similar
effect is also observed on already differentiated female gametophytes, which become hermaphroditic sooner
when cultured in the presence of sucrose in vitro. Interestingly, these results are reproducible within and across
species representative of the two subgenera Equisetum and Hippochaete, indicating that the entire genus may
share an identical pattern of environmental sex determination.
Keywords: sex determination, Equisetum, environment, gametophyte, in vitro culture, sugars.
Introduction
in each developing gametophyte, a series of threshold conditions appears to determine which sex is expressed (Duckett
1977).
To better understand sex determination in Equisetum gametophytes, several authors have studied their response to a
number of culture conditions in vitro (Mohan Ram and Chatterjee 1970; Hauke 1971, 1977). Studied environmental factors include light quality and level, crowding, pH, and sucrose
concentration in the culture media. Although each of these
factors seems able to affect the sex ratio, in many cases, the
effects are not consistent among different species. For example,
sucrose enrichment has been reported (i) to inhibit female sex
differentiation in Equisetum ramosissimum (Mohan Ram and
Chatterjee 1970), (ii) to decrease the proportion of males in
Equisetum arvense (Hauke 1971, 1977), (iii) to increase the
proportion of males in E. ramosissimum (Mohan Ram and
Chatterjee 1970) and Equisetum hyemale (Hauke 1971), or
(iv) to have no effect on sex expression in the same E. hyemale
(Hauke 1977). Hauke (1977) concluded: “A number of experiments utilizing sugar enrichment were attempted, but the
results were so inconsistent that no conclusions can be drawn
from them” (p. 29). Such discrepancies may be explained by
variations in culture conditions between experiments, leading
to the interaction of other important factors such as light quality and crowding. Alternatively, there may be variability among
spores collected from different individuals of the same species
and/or from individuals of different species.
In an attempt to characterize the pattern of sex expression
in Equisetum gametophytes from different species, we conducted repeated and controlled culture experiments in the presence of various sugar concentrations. Also, we examined the
stages during which sex differentiation can be modified by
sugar. Here, we present evidence that the male-promoting ef-
Seedless vascular plants are a paraphyletic group of plants
characterized by a haplo-diplophasic life cycle. Among these,
Isoetaceae, Selaginellaceae, and Marsileaceae are heterosporous, i.e., producing micro- and macrospores that differ in
morphology, whereas club mosses (Lycopodium), horsetails
(Equisetum), and most ferns (Filicophyta) are homosporous.
In Equisetum, perennial sporophytes (2n) produce sexually
undifferentiated spores (n) that are dispersed and germinate
to form photosynthetic male, female, or sometimes bisexual
gametophytes (n). Fertilizations between male and female gametes (n) result in the formation of new sporophytes.
The sexual determination of Equisetum gametophytes has
been problematic for many years, with various authors emphasizing genetic sex determination, environmental sex determination, or a mixture of both (Mohan Ram and Chatterjee
1970; Laroche et al. 1972, 1978; Hauke 1977). However, the
observations that (i) archegonial (female) gametophytes always
change to antheridial (male) lobe production when cultured
over a prolonged time, (ii) secondary archegonial branches can
be regenerated from otherwise purely male gametophytes, and
(iii) the proportions of male and female gametophytes developing from the same initial set of spores are highly sensitive
to growth conditions all indicate that the spores of Equisetum
are not individually predetermined for the expression of one
sex or the other (Duckett 1970, 1972, 1977, 1979). Instead,
1
Author for correspondence; address for correspondence: Laboratoire Ecologie, Systématique et Evolution, Université Paris-Sud, Bâtiment 362, F-91405 Orsay Cedex, France; telephone 33-1-69-15-6342; fax 33-1-69-15-73-53; e-mail [email protected].
Manuscript received October 2001; revised manuscript received March 2002.
825
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fect of sucrose and glucose in the culture medium is significant
and can be reproduced within and across species of Equisetum.
We also show that the addition of sucrose to the culture medium can induce mature female gametophytes to change sex.
The significance of this finding is discussed in relation to life
history in this ancestral lineage of plants and in the light of
evolutionary theories of sex determination.
Material and Methods
Spore Collection and Storage
Spores were collected from one cone per plant. For each
Equisetum species, plants from two distinct localities were used
as sources of spores. Equisetum palustre L.: P11, P12, Campus
University Paris XI, Orsay, France; P2, northern bank of the
Great Pond, Saint-Mars-La-Brière, France. Equisetum arvense
L.: A1, Campus University Paris XI, Orsay, France; A2, Campus University Paris XI, Bures-sur-Yvette, France. Equisetum
telmateia Ehrh.: T1, Campus University Paris XI, Orsay,
France; T2, Moulin de la Mare, Milon-La-Chapelle, France.
Equisetum variegatum Schleich.: plants cultivated in the Conservatoire National Botanique du Bassin Parisien and originating from (V1) Forest of Marly, Marly, France, and (V2)
Botanical garden, Samoens, France.
For E. variegatum and E. palustre, spores were collected
from mature cones and stored in hermetically closed tubes at
⫺70⬚C. For E. arvense and E. telmateia, mature but unopened
cones were harvested and sterilized for 5 min in 10 g L⫺1 of
calcium hypochlorite to which 50 mL of Teepol (Lever) detergent per 100 mL was added. Cones were rinsed in distilled
water, allowed to dry overnight, and then dissected to release
the spores, which were stored as above.
In Vitro Culture
G0 culture medium contained the macronutrients of Estelle
and Sommerville (1987): 2.5 mM KH2PO4 replaced by 2 mM
KH2PO4 and 0.5 mM K2HPO4 so that the medium was directly
buffered to pH 6.1, the mineral micronutrients of Murashige
and Skoog (1962) diluted by half, and 10 mM Fe EDTA. Phytagel (0.2% w/v; Sigma) and agar (0.4% w/v; Biofit, Fisher)
were used as gelling agents before autoclaving for 12 min at
120⬚C.
Whereas G0 contained no additional component, 120 mM
glucose was added to G120G, 30 mM sucrose was added to
G30S, 60 mM sucrose was added to G60S, 90 mM sucrose
was added to G90S, and 120 mM sucrose was added to G120S.
Sixty mM mannitol was added to G60ML for a control as an
unmetabolized osmotic agent. Sugars or mannitol were added
to the culture medium before sterilization: sucrose and glucose
(Prolabo, RP NORMAPUR) and mannitol (Merck).
Gametophyte cultures were placed in a growth chamber at
constant 24⬚ Ⳳ 1⬚C and 24 Ⳳ 3 mmol m⫺2 s⫺1 of photosynthetically active radiation for 16 h d⫺1 provided by fluorescent
lamps, a 1/1 mixture of Philips TLD36W82, 2700⬚K, and
TLD36W33, 4000⬚K.
For the primary sexual differentiation experiment, at t p
⫺10 d, spores were sown on G0 petri dishes and then placed
in the growth chamber. At t p 0, gametophytes were transferred to test-media dishes. Test media were G0, G30S, G60S,
G90S, G120S, G120G, and G60ML. Sixteen randomly chosen
gametophytes were transferred to each test-medium dish, with
each medium replicated in three dishes (for P11 and V1) or
four dishes (for P2, V2, A1, A2, T1, T2). The gametophytes
were observed under a binocular dissecting microscope every
week after transfer to the test media until the end of the experiment. For each gametophyte, viability and gender were
determined according to the presence or absence of antheridia
and archegonia, with each gametophyte recorded as female,
male, bisexual, undifferentiated, or dead. At the end of the
experiment, 5 wk for E. telmateia, 8 wk for E. arvense and
E. variegatum, and 9 wk for E. palustre, pairs of gametophytes
were transferred into a flooded G30S tube to assess the fertility
of some of the males and females grown on G0, G30S, G90S,
and G120S. In all cultures, a sporophyte developed after
3–4 wk, indicating that the sex organs and gametes were
functional.
For hermaphroditization experiments, spores were sown on
G0 dishes, and gametophytes were transferred to G0 for primary sex expression. When the gender of gametophytes could
be determined, female gametophytes were transferred to test
media and further grown as above. Nine randomly chosen
female gametophytes for P12 and 12 female gametophytes for
T2 and V1 were transferred to each test-medium dish, and
each medium was replicated over three dishes. The female gametophytes were observed every week after transfer to the testmedium plates, and the appearance of antheridia was recorded.
Statistical Analyses
The statistical analyses included only plates in which less
than three gametophytes failed to grow or to differentiate
(undifferentiated + dead ! 3). In the early sex-differentiation
experiment, the sex ratio was defined as (males + bisexuals)/
(males + bisexuals + females), accounting for the observation
of male sex expression both in initially male gametophytes
and in rare bisexuals deriving from initially female gametophytes. The sex ratio on each plate was square root– and
arcsine-transformed and then submitted to ANOVA, with
test medium and plant species as fixed effects and plant
origin (nested within species) as a random effect (general
linear models procedure; SAS Institute 1988). In the hermaphroditization experiment, where only one plant per species was used, the statistical treatment was the same as
above, except that the sex ratio on each plate was defined
as bisexuals/(bisexuals + females) and that the only effect included in the model was test medium. Tukey’s studentized
range test was performed for multiple comparisons. The
relation between sucrose concentration and the sex ratio
obtained on each dish was assessed by using the SAS regression procedure.
Results
To test the effect of sugar addition on sex expression in four
species of Equisetum, we performed two different experiments,
examining the influence of growth medium on sex expression,
starting with either 11-d-old undifferentiated Equisetum gametophytes (table 1; fig. 1) or older differentiated female Equisetum gametophytes (fig. 2).
GUILLON & RAQUIN—SEX DETERMINATION IN EQUISETUM
Table 1
Discussion
ANOVA of Test-Medium Effect, Plant Origin Effect (Nested within
Species), Plant Species Effect, and All Possible Two-Way
Interactions on the Sex Ratios Observed in the Early Sex
Differentiation Experiment (Fig. 1)
Sugar Addition Favors Maleness in Cultured
Equisetum Gametophytes
Effect
Test medium
Origin (species)
Species
Test medium # origin (species)
Test medium # species
Error
df
MS
F
P
6
4
3
24
18
133
1.476
0.303
8.270
0.069
0.114
21.88
4.43
27.50
2.81
1.67
!0.0001
0.0079
0.0039
!0.0001
0.12
In the first experiment, the addition of sugar to the culture
medium had a highly significant effect on the observed sex
ratio (P ! 0.0001; table 1). The species and the origin of plants
within each species were also found to be important (P p
0.0039 and P p 0.0079, respectively). Plants from a same species showed different responses to the test media (P ! 0.0001).
However, no significant interaction was found between testmedium effect and species effect (P p 0.12 ), indicating that
the general pattern of sex expression in response to sugar addition is conserved across the four studied Equisetum species.
For each plant, additional sucrose and glucose favored the
expression of male gender, whereas mannitol never had a significant effect, compared with the G0 mineral medium (fig. 1).
The sex ratio per plate increased with increasing sucrose concentration up to 120 mM, with all linear regressions significant
(P ! 0.05) with positive slopes. For T2 and V2 plants, 120 mM
glucose gave significantly higher sex ratios than both 60 mM
sucrose (same number of atom gram of carbon) and 120 mM
sucrose.
The addition of sugar, up to 120 mM sucrose and 120 mM
glucose, or of 60 mM mannitol in the test medium did not
affect the development of antheridia and archegonia as could
be seen under the microscope. At the end of the experiment,
5 wk for Equisetum telmateia, 8 wk for Equisetum arvense
and Equisetum variegatum, and 9 wk for Equisetum palustre,
pairs of gametophytes were transferred into a flooded G30S
tube to assess the fertility of some of the males and females
grown on G0, G30S, G90S, and G120S. In all cultures, a
sporophyte developed after 3–4 wk, indicating that the sex
organs and gametes were functional indeed.
In order to know whether the male-promoting effect of sugar
addition in the culture medium was restricted to an early stage
of gametophyte development, we transferred mature female
gametophytes to growth media with or without sucrose (60
mM and 90 mM; fig. 2). This experiment was carried out with
P12, T2, and V1 female gametophytes. The timing of hermaphrodite appearance was accelerated on sugar-containing
media (fig. 2). At the end of the experiment, the proportion
of hermaphrodites was significantly higher on G60S and G90S
(Tukey test; P ! 0.05) than on G0 (ANOVA for P12: R2 p
0.77, F2, 6 p 10.25, P p 0.0116; for T2: R2 p 0.78, F2, 6 p
10.73, P p 0.0104; for V1: R2 p 0.89, F2, 6 p 24.90, P p
0.0012). This result demonstrates that sugar addition not only
promotes male sex expression in young differentiating Equisetum gametophytes but also accelerates the onset of hermaphroditism in mature females.
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The results showed that sex ratios with an excess of males
are obtained when young gametophytes are grown on media
with added sugar. The pattern of sex expression described here
is in agreement with the now widely held opinion that Equisetum spores and gametophytes are potentially bisexual (Duckett 1977). Clearly, the sex ratios obtained on a given growth
medium differ when spores from different origins or species
are compared (fig. 1). This probably results from genetic differences between genotypes and species. The male-promoting
effect of sugar addition is significant and conserved across four
species representative of the two subgenera Equisetum (Equisetum arvense, Equisetum palustre, and Equisetum telmateia) and Hippochaete (Equisetum variegatum). It is not known
whether each of the two subgenera is monophyletic, but if this
were the case, it would indicate that the observed pattern of
environmental sex determination in response to sugar addition
would be general for most of the 16 Equisetum species. In
addition, the result that sucrose addition promotes the expression of male sex in both undifferentiated and mature female gametophytes indicates that a same mechanism of sex
determination could operate at these two developmental
stages.
To our knowledge, this is the first time that a same change
in culture conditions is reported to consistently modify the sex
ratio of Equisetum gametophytes from different species. In our
work, plants from two distinct localities were used as sources
of spores for each of four Equisetum species. Treatments were
replicated in order to allow statistical analysis, and we used a
rather stringent criterion (dead + undifferentiated ! 3) to minimize sex-biased mortality or inhibition as a possible cause of
variation in the analyzed sex ratios. The absence of such care,
as well as differences in culture conditions, such as light level,
light quality, and basic growth medium (Mohan Ram and
Chatterjee 1970; Hauke 1971, 1977), may account for the
discrepancies found across previous results. In addition, the
statistical analyses used in some of these investigations might
have confounded the growth-medium effect with the effect of
crowding, a factor that itself seems able to modify the sex ratio
of cultured Equisetum gametophytes (Hauke 1977).
We conclude that sugars act as nutrients in our experiments
because they enhance the growth rate of cultured gametophytes. However, the effect of sugar on Equisetum sex ratios
is contrary to the effect of light; in fact, the sex ratios are more
female biased with increasing light levels (J.-M. Guillon, unpublished data). Hauke (1971) already concluded that the
morphogenetic effect of sucrose on Equisetum gametophytes
was independent of any nutritional effect but, rather, superimposed on the morphogenetic effects of light. Two hypotheses
emerge from this picture: (i) the female-promoting effect of
light is not exclusively mediated by the photosynthesized carbohydrates, and/or (ii) the addition of exogenous sugars has
other physiological effects besides mimicking the photosynthesis of carbohydrates. Concerning the second proposition,
it is possible that the presence of exogenous sugars is a stress
to the young developing gametophyte. Specifically, osmotic
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Fig. 1 Sex ratios in cultures of Equisetum gametophytes. For each plant, bars represent the mean sex ratio ([male + bisexual]/
[male + bisexual + female]) and associated standard error obtained on the corresponding test medium. The number of repeats (n) and the result
of multiple comparisons (Tukey’s studentized range test) are also indicated. Treatments with the same letter do not significantly differ (P 1
0.05). Treatments that differ from G0 (P ! 0.05) are indicated with bars in black. The duration of growth on test media is specified with the
name of the plant. A1 and A2, Equisetum arvense; P11 and P2, Equisetum palustre; T1 and T2, Equisetum telmateia; V1 and V2, Equisetum
variegatum.
stress could account in part for the observed effect because the
addition of an osmotic agent, 60 mM mannitol, or a sugar,
60 mM sucrose, produced similar sex ratios (fig. 1). Higher
concentrations of mannitol should be used to test this
hypothesis.
When reviewing the variations in sex ratio of cultured Equisetum gametophytes, investigators have noticed that under
conditions favoring vigorous vegetative growth of young gametophytes, e.g., high light levels, most spores differentiate
into initially female individuals (Duckett 1970). However, less
GUILLON & RAQUIN—SEX DETERMINATION IN EQUISETUM
Fig. 2 Timing of hermaphrodite appearance in initially female
gametophyte cultures of P12 (Equisetum palustre), T2 (Equisetum telmateia), and V1 (Equisetum variegatum). For each plant and test medium, the mean sex ratio with its associated standard error is shown
as a function of time. Each treatment was replicated in three dishes.
Circles, G0 (no sucrose added); squares, G60S (60 mM sucrose); triangles, G90S (90 mM sucrose).
favorable regimes, e.g., crowding, drought, mineral deficiency,
or high temperatures, produce a majority of males (Duckett
1970; Mohan Ram and Chatterjee 1970; Hauke 1977). Observations of wild populations of Equisetum gametophytes
have shown that high frequencies of males correspond with
low growth rates, possibly resulting from environmental stress
(Duckett and Duckett 1980). Environmental factors are also
known to influence sex ratios in several seed plant species,
with males most typically tending to be overrepresented in
resource-limited or less favorable environments (Lloyd and
Bawa 1984; Korpelainen 1998). This general pattern of observations is consistent with the hypothesis that exogenous sugars could be a stress to cultured gametophytes of Equisetum.
Evolutionary Significance of Sex Determination
in Equisetum Gametophytes
Equisetum species have a haplo-diplophasic life cycle, characterized by the alternation of a perennial sporophytic gen-
829
eration and a short-lived gametophytic generation. Not all sporophyte populations produce spores, and they can propagate
efficiently through vegetative reproduction. New populations
may be founded either by broken-off or dispersed vegetative
parts of the sporophytes or by dispersed spores and sexual
reproduction between emerging gametophytes. Studies of genetic diversity in E. arvense and Equisetum hyemale populations, as well as high fertilization rates found in wild gametophyte populations, indicate that sexual reproduction occurs
and cannot be dismissed as marginal in Equisetum (Duckett
and Duckett 1980; Soltis et al. 1988; Korpelainen and Kolkkala 1996).
In the wild, observed differences in sex ratios between populations of the same species at the same time and between
populations at the same locality in different years all indicate
an important environmental component for sex determination
in Equisetum (Duckett and Duckett 1980). Evolutionary theories predict that environmental sex determination is favored
(i) when early in life, an individual enters a patchy environment
that has a large effect on its lifetime fitness (some patches being
relatively more beneficial to females than to males and some
being the opposite), (ii) when individuals have little control
over which patch they enter, and (iii) when individuals from
different patches mate randomly (Charnov and Bull 1977; Bull
1981). Equisetum gametophytes might fit this model if natural
populations inhabited patchy places made of microenvironments conferring more or less an advantage to one sex. It has
also been shown that short-lived species are less prone to
evolve environmental sex determination because environmental fluctuations may cause detrimental sex-ratio variations in
such a system (Bull and Bulmer 1989). For this reason, strong
sex-specific fitness effects of the environment would be expected in order to explain the occurrence of environmental sex
determination in the seasonal Equisetum gametophytes in the
wild. Since the literature does not provide such fitness-related
data, new work is clearly needed to examine whether the biology of Equisetum gametophytes conforms to the predictions
of the Charnov and Bull (1977) model.
As shown here, male sex expression can be induced in four
species of Equisetum by the addition of sugar to the growth
medium. However, the addition of sugar in our experimental
design does not mimic any specific environmental factor that
gametophytes would encounter in the wild. Hence, sex determination in response to sugar concentration is unlikely to have
evolved per se, but exogenous sugars must somehow interfere
with the physiology of the developing gametophyte in such a
way that sex determination is affected. Sex determination in
horsetails seems to integrate many physiological signals arising
from the interaction of the gametophyte with its environment.
Indeed, many environmental factors, such as light quality and
level, competition, and pH, are also able to modify sex expression in Equisetum gametophytes (Mohan Ram and Chatterjee 1970; Hauke 1971, 1977). In this context, it is quite
understandable that sugar concentration in the growth medium is one among many factors that influence sex determination of gametophytes cultured in vitro.
Lability of sex expression occurs in many plant taxa, but
only in homosporous pteridophytes is labile sex the rule (Korpelainen 1998). Indeed, sex determination in horsetails is reminiscent of that found in ferns, where undifferentiated game-
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INTERNATIONAL JOURNAL OF PLANT SCIENCES
tophytes respond to antheridogen hormones secreted by female
gametophytes. The evolutionary significance of this information system in fern gametophytes is still a matter of debate
(Willson 1981; Haig and Westoby 1988; Korpelainen 1998).
Because ferns and horsetails are sister clades (Kenrick and
Crane 1997; Pryer et al. 2001), sex determination in horsetails
could be evolutionarily related to that found in ferns, but antheridogen hormones have not been detected in horsetails
(Hauke 1971). Alternatively, environmental sex determination
might have evolved more than once in relation to the distinctive
life history of ferns and horsetails because they both are homosporous haplo-diplophasic land plants with perennial sporophytes and autonomous short-lived gametophytes. In particular, it has been proposed that the extreme lability in the
sex expression of homosporous pteridophytes may be primarily related to their peculiar mating system (Korpelainen
1998).
Acknowledgments
We would like to thank Jacques Moret and the people at
the Conservatoire Botanique National du Bassin Parisien for
their support of this work. We thank Claire Lavigne, Marc
Girondot, Bryan Epperson, and Jacqui Shykoff for discussions
and useful comments on the manuscript. Odylle Cudelou, Olivia Calvez, Alexandra Deville, and Amélie Pinot are acknowledged for their help in gametophyte culture.
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