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New Ideas and Directions in Botany
Pathways for making unisexual flowers and unisexual plants:
Moving beyond the “two mutations linked on one
chromosome” model1
Susanne S. Renner2,3
KEY WORDS developmental genetics; dioecy; monoecy; retrotransposon; sex determination genes; unisexual
Sex determination in higher plants is of fundamental biological
interest and has great practical significance for fruit yield and
high-quality seed production. The last decades have witnessed an
explosion in the genetic-developmental understanding of a typical
hermaphroditic flower; however, the genes controlling dicliny
(unisexual flowers) and dioecy (unisexual individuals) are only now
being revealed (Akagi et al., 2014; Boualem et al., 2015). These
genetic-developmental insights, from distantly related groups, now
need to be brought together with the large body of empirical observations showing that dicliny and dioecy each evolved independently many thousands of times, but often in the same genera and
families suggesting related evolutionary pathways (Darwin, 1877;
Lloyd, 1972, 1975a, b, 1980; Renner and Ricklefs, 1995; Renner,
2014). Here I highlight how the recent insights may redirect our
understanding of pathways to dioecy in flowering plants.
The fundamental model for the evolution of dioecy in angiosperms has long been that “at least two gene mutations are necessary to transform an hermaphroditic or monoecious species into
one with separate sexes; one mutation must affect ovule production, and the other the production of pollen” (Charlesworth and
Charlesworth, 1978: p. 975), with the corollary that “different genes
determining sex [are] grouped together in one region of one chromosome in most species” (p. 976). This model disregards that (1) in
monoecious species, mutations suppressing male and female function do not need to arise since they already exist and that (2) there
is little evidence that sex-determining genes in dioecious plants are
grouped together on one chromosome. The developmental-genetic
1
Manuscript received 16 January 2016; revision accepted 11 February 2016.
Systematic Botany and Mycology, University of Munich (LMU), Menzinger-Str. 67 80638
Munich, Germany
3
E-mail: [email protected]
doi:10.3732/ajb.1600029
2
insights summarized in the next two sections now suggest it is time
to abandon strict adherence to the “two major mutations linked on
one chromosome” model of angiosperm dioecy.
PATHWAYS TO UNISEXUAL FLOWERS AND INDIVIDUALS IN
DIOSPYROS AND CUCUMIS
The Ebenaceae genus Diospyros includes about 475 species of tropical or subtropical trees that as far as is known are dioecious, with
the flowers having either fertile stamens and arrested carpels, or
reduced stamens and normal carpels. The family has a crown age
of ca. 54 Myr and four genera, with Diospyros diverging from
Actinidia about 52 Ma (Akagi et al., 2014). Akagi et al. (2014)
searched the genome of Diospyros lotus, a species with XY males
and homomorphic sex chromosomes, for short completely sexlinked sequences and discovered a sex-determining gene that they
named OGI for Oppressor of MeGI and Japanese for “male tree”.
OGI is expressed only in male flower buds and controls an autosomal gene named MeGI (=Male Growth Inhibitor gene and Japanese
for “female tree”). MeGI encodes male-suppressing regulatory
RNA and has female-biased bud and flower-specific expression.
The two genes result from gene duplication, with one MeGI copy
having moved to the Y chromosome. Y-linked OGI sequences were
also detected in other species of Ebenaceae, suggesting that the duplication happened a long time ago. Since the MeGI male-suppressing
gene is autosomal it does not match the model of “two mutations
becoming linked on the proto-sex-chromosome” (Charlesworth
and Charlesworth, 1978). OGI could instead be an example of a
single-locus sex-determining gene (Charlesworth, 2015), and
Akagi et al. (2014: p. 649) suggested that perhaps it is, “a single master regulator of sex determination, such as SRY in humans.” SRY is
the male-determining gene on the Y-chromosomes of mammals
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(Foster and Graves, 1994). Put simply, autosomal MeGI makes
RNA that suppresses anther function in female flowers and plants;
its duplicate copy OGI, on the Y chromosome, makes RNA that
suppresses MeGI in male flowers and plants.
Cucurbitaceae may be a bit older than the Ebenaceae (Schaefer
et al., 2009: ca. 63 Myr), and the suppression of either stamens or
carpel in their flowers happens earlier during floral development,
so that mature flowers are readily recognized as staminate or pistillate (Fig. 1). About 50% of the 950–980 species of Cucurbitaceae are
monoecious and 50% dioecious; no wild species have only bisexual
flowers, although a few have individuals with bisexual flowers and
others with staminate flowers (Schaefer and Renner, 2011). Dioecy
appears to be ancestral, with much back and forth between dioecy
and monoecy (Zhang et al., 2006; Schaefer and Renner, 2010;
Volz and Renner, 2008), and dioecy may even go back to the common ancestor of Begoniaceae, Cucurbitaceae, Datiscaceae, and
Tetramelaceae, all of which have normally unisexual flowers
(Zhang et al., 2006). Spontaneous mutations that modify flower sex
phenotype are common and were favored by breeding targeted
toward femaleness. In Cucumis, a genus of 66 species, most of them
monoecious but a few dioecious (Sebastian et al., 2010), breeders
have selected gynoecious or “all-female” cultivars of C. sativus
because of their high fruit yield, with the plants in greenhouses
then either parthenocarpic or else requiring a few androecious
plants (with only male flowers) or monoecious plants with both
types of flowers to ensure fertilization. Their sexual lability and economic importance have made cucurbits, which also include melon
(Cucumis melo) and watermelon (Citrullus lanatus), an important
system for the developmental genetics of sex determination.
The laboratory group of Abdel Bendahmane, building on work
by Tova Trebitsh and colleagues, has isolated the sex determining
genes of C. melo and C. sativus (CmWIP1, CmACS11, CmACS7;
CsWIP1, CsACS11, CsACS2) and has published a model that explains both the development of unisexual flowers and the transition
from monoecy to gynoecy (the aforementioned all-female plants),
androecy (the aforementioned all-male plants), and dioecy (two
sexes in equal ratios). To show that a combination of alleles of these
genes can lead to artificial dioecy, Boualem et al. (2015), in two rounds
of crossing, mated female plants homozygous for the recessive
alleles Cmwip1 and Cmacs11 and male plants of Cmwip1/CmWIP1
A M E R I C A N J O U R N A L O F B OTA N Y
or Cmacs11/Cmacs11 genotypes. They found no significant deviation from a 1:1 sex ratio, demonstrating that the combination of
alleles of genes controlling monoecy (repression of bisexual flowers)
can lead to dioecy (repression of bisexual individuals). The sexdetermining genes G (WIP), A (ACS11), and M (ACS7) sit on three
different chromosomes (A. Bendahame, Institute of Plant Sciences
Paris-Saclary, Université Paris-Sud, personal communication, 6
February 2016), and the androecy gene, ACS11, controls the development of pistillate flowers by producing an enzyme that is rate‐
limiting for the biosynthesis of ethylene (Trebitsh et al., 1997;
Boualem et al., 2008). When ACS11 is active and ethylene is produced at the correct location, pistillate flowers develop. When a
mutation causes ACS11 to be inhibited, the result is a lower level of
ethylene in the developing flower bud, resulting in staminate flowers. Gynoecy, by contrast, is due to suppression of a C2H2 zincfinger transcription factor of the WIP protein subfamily due to
retrotransposon-mediated DNA methylation (Martin et al., 2009).
This natural and heritable epigenetic change, caused by the insertion
of a transposon, leads to the spread of DNA methylation to the WIP1
promoter. If WIP1 is not methylated, but instead expressed, carpels
are aborted, and flowers become staminate (Martin et al., 2009).
COMPARISON OF SEX DETERMINATION IN DIOECIOUS
DIOSPYROS AND MONOECIOUS/DIOECIOUS CUCUMIS WITH THE
TWO TRADITIONAL MODELS OF DIOECY EVOLUTION
In Diospyros, the key steps to dioecy were the duplication of an autosomal gene encoding a regulatory small-RNA, with one copy becoming Y-linked and repressing function of the autosomal copy. The system
apparently goes back to the ancestor of modern Diospyros, a genus
with homomorphic sex chromosomes (Akagi et al., 2014), yet the
OGI gene alone determines sex in persimmons. In Cucumis, monoecy and dioecy (the latter manmade in C. sativus but occurring
naturally in other Cucumis species) are caused by differential ethylene sensitivity of male and female meristems and the insertion of
a retrotransposon affecting a carpel promoter. The sex-determining
genes appear conserved across the genus (Boualem et al., 2015), and
there is no evidence of sex chromosomes. Dioecious cucurbits in
related genera, such as Coccinia grandis, however have hugely
FIGURE 1 Male (left) and female (right) flowers of Sicyos angulatus, the star-cucumber, an annual vine in the Cucurbitaceae native to eastern North
America. Cucurbitaceae, with 950–980 species, have ancestrally unisexual flowers and roughly equal proportions of monoecious and dioecious species. Dioecy appears to be ancestral. Photographs by Anil Kumar Thakur.
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heteromorphic sex chromosomes (Sousa et al., 2013). Neither
the Diospyros nor the Cucumis system conforms to the “two mutations linked on one chromosome” model of the evolution of dioecy.
The search for a dominant female-suppressing mutation, which
in the standard model becomes tightly linked to recessive malesterility mutations on the proto-sex chromosome (Charlesworth and
Charlesworth, 1978), of course continues in other systems. So does
the search for support for the gynodioecy pathway (Charlesworth
and Charlesworth, 1978), which involves the transition to nuclearcontrolled dioecy from normally cytoplasmically controlled gynodioecy, a sexual system named by Darwin (1877: p. 298) in which
populations include individuals with only female flowers and others with bisexual flowers (e.g., Delph, 1990: Veronica section Hebe;
McCauley and Bailey, 2009; Miller and Bruns, 2016). As considered by Darwin (1877: p. 298), species “which I have called gynodioecious are found in various widely distinct families, but are
much more common in the Labiatæ. [These species] rarely show
any tendency to be dioecious, as far as can be judged from their
present condition and from the absence of species having separated
sexes within the same groups.” In spite of the sparse genetic and
phylogenetic evidence for transitions from gynodioecy to dioecy,
this pathway has received vast attention.
Determining whether some dioecious angiosperms perhaps
do have male and female sex-determining genes linked in a
recombination-suppressed region (i.e., follow the Charlesworth and
Charlesworth (1978) model of plant sex chromosome evolution) is
technically very difficult (Charlesworth, 2015). Even in dioecious
Silene latifolia, with a distinct Y chromosome but no fully sequenced genome, current evidence suggests that the “Y is not heavily weighted with genes involved in male development and does not
fit the criteria for functional coherency” (Moore et al., 2003: p. 332;
Kazama et al., 2016: new map of the Silene Y). Many old and speciesrich dioecious clade, such as Baccharis (300 species, Asteraceae),
Ilex (400–600 species, Aquifoliaceae), Litsea (400 species of trees,
Lauraceae), Pandanus (600 species, Pandanaceae), or Smilax (210
species; Smilacaceae), however, have not yet been investigated in
terms of the developmental genetics of their unisexual flowers and
individuals, and given the thousands of times that dioecy has
evolved and been lost in flowering plants, it is perhaps to be expected that the genetics of sex determination takes paths other than
just the “two sequential mutations linked on a chromosome” model.
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ACKNOWLEDGEMENTS
I thank Lynda Delph and two anonymous reviewers for critical
comments. This work is supported by DFG grant RE 603/19-1.
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