Download Independent Origin of Sex Chromosomes in Two Species

Copyright Ó 2008 by the Genetics Society of America
DOI: 10.1534/genetics.107.085670
Note
Independent Origin of Sex Chromosomes in Two Species
of the Genus Silene
Martina Mrackova,* Michael Nicolas,† Roman Hobza,* Ioan Negrutiu,† Francxoise Monéger,†
Alexander Widmer,‡ Boris Vyskot* and Bohuslav Janousek*,1
*Laboratory of Plant Developmental Genetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, 612 65 Brno, Czech Republic,
†
CNRS-INRA-ENSL-UCBL Laboratoire Reproduction et Développement des Plantes, Ecole Normale Supérieure, 69364 Lyon,
France and ‡Plant Ecological Genetics, Institute of Integrative Biology, ETH Zurich, 8092 Zurich, Switzerland
Manuscript received December 11, 2007
Accepted for publication March 28, 2008
ABSTRACT
Here we introduce a new model species, Silene colpophylla, that could facilitate research of sex
chromosome evolution and sex-determining systems. This species is related to the well-established
dioecious plant model Silene latifolia. Our results show that S. colpophylla is, similarly to S. latifolia, a male
heterogametic species, but its sex chromosomes have evolved from a different pair of autosomes than in
S. latifolia. The results of our phylogenetic study and mapping of homologs of S. latifolia X-linked genes
indicate that the sex determination system in S. colpophylla evolved independently from that in S. latifolia.
We assert that this model species pair will make it possible to study two independent patterns of sex
chromosome evolution in related species.
M
EMBERS of the genus Silene compose an important model system for the study of the early phases
of sex chromosome evolution: several dioecious species
in this genus have evolutionarily recent sex chromosomes (10–20 MYA, according to Bergero et al. 2007).
Most Silene species are either gynodioecious or hermaphroditic and can therefore be used in comparative
studies that search for autosomal ancestors of the sex
chromosomes. To date, the attention of researchers has
been concentrated mainly on the study of dioecious
species possessing large heteromorphic sex chromosomes with an XX/XY sex-determining system as in Silene
latifolia, Silene dioica, and Silene diclinis (for a review see
Vyskot and Hobza 2004). Phylogenetic studies conducted on these species showed that they are closely
related and share a common origin of the sex chromosomes (Desfeux et al. 1996; Nicolas et al. 2005).
Studies focusing on S. latifolia have been particularly
successful in providing new insights into sex chromosome evolution. For example, sex chromosomes in
S. latifolia have evolved from a single pair of autosomes
(Filatov 2005) that have undergone a stepwise reduction of recombination, as shown by a stepwise-gradient of
1
Corresponding author: Laboratory of Plant Developmental Genetics,
Institute of Biophysics, Academy of Sciences of the Czech Republic,
Kralovopolska 135, CZ-612 65 Brno, Czech Republic.
E-mail: [email protected]
Genetics 179: 1129–1133 ( June 2008)
silent site divergence between X- and Y-linked copies of
genes from the pseudoautosomal region toward the
distal end of the X chromosome (Nicolas et al. 2005;
Bergero et al. 2007). In addition, the mechanism of
sex determination in S. latifolia has started to be elucidated (Westergaard 1958; Zluvova et al. 2006). Sex
determination in S. latifolia is based on the active role
of the Y chromosome that possesses genes for malepromoting functions necessary for anther development,
genes necessary for male fertility, and genes for the
gynoecium-suppressing function, which prevents the
development of female sex organs in males.
In addition to S. latifolia, S. diclinis, and S. dioica, there
is also another group of dioecious Silene species
consisting of Silene otites and its closely related species.
So far, the studied species of this group are characterized by the absence of heteromorphic sex chromosomes
(Sansome 1938; Westergaard 1958). This fact, together with the supposed nondioecious origin of the
genus Silene (Desfeux et al. 1996), suggests that the sex
chromosomes in S. otites and its closely related species
are evolutionarily younger than those in S. latifolia. It
was also suggested (on the basis of the sequencing of
rDNA internal transcribed spacer loci) that sex determination evolved independently in the group of
species around S. latifolia and in the group of species
around S. otites (Desfeux et al. 1996). Even though the
bootstraps supporting this hypothesis are rather low, the
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M. Mrackova et al.
hypothesis of the recent and independent origin of sex
determination in the species related to S. otites certainly
deserves great attention. If it is true, these two groups of
species could be suitable and complementary models
for the study of early sex chromosome evolution. This
type of system could also address important questions
relating to mechanisms of sex chromosome evolution
such as the suppression of recombination and evolutionary changes of sex-linked genes.
A problem complicating research in the group of
species closely related to S. otites is difficulties in
taxonomy. This group involves many species that have
been difficult to distinguish on the basis of morphology
alone, and a proper molecular characterization of these
species has not yet been done. We have decided to use
S. colpophylla (whose name is from the Greek kolpos,
meaning a fold, which refers to its typical leaf shape), a
species that is distinguishable from S. otites and other
closely related species by its typical leaf shape (see
supplemental Figure 1). The flowers of S. colpophylla are
relatively large compared to the small flowers of other
species related to S. otites (Wrigley 1986; see also
supplemental Figure 2 for comparison with S. otites).
The morphology of the rudiment of the gynoecium suggests that developmental arrest in S. colpophylla males
occurs later than in S. latifolia males. In S. colpophylla, the
pistil rudiment closely resembles the gynoecium (see
supplemental Figure 3a), while in S. latifolia it is a
nondifferentiated rod-like structure (Zluvova et al.
2007). In contrast to gynoecium development in males,
the anther development in S. colpophylla females is
reduced at an early stage of development similarly to
S. latifolia (see supplemental Figure 3b).
This article concentrates on three basic questions: (1)
Is the origin of sex determination in S. colpophylla
independent from that of S. latifolia?, (2) Have the sex
chromosomes in S. colpophylla evolved from the same
pair of autosomes as the sex chromosomes in S. latifolia?,
and (3) What is the type of sex determination in S.
colpophylla?
The phylogenetic analysis (Figure 1) using the
sequences of homologs of the gene SlX3 (for a characterization of this gene, see Nicolas et al. 2005) clearly
shows (the phylogenetic tree is characterized by much
higher bootstrap values than the tree published by
Desfeux et al. 1996) that the ancestral status in the
genus Silene was nondioecious (most probably gynodioecious) and that the sex determination evolved independently in the group including S. colpophylla and
S. otites and in the group including the other dioecious
species S. latifolia, S. diclinis, and S. dioica. In the hermaphroditic species, any recessive mutation in the nuclear
genome arresting the development of the sex organs
(male sex organs are supposed to be arrested first) can
promote the development of dioecy and sex chromosomes. In the next steps, nuclear gynodioecy is transformed to dioecy via evolution of the female-suppressing
Figure 1.—Tree for orthologs of the gene SlX3. The tree is
based on the sequence amplified using primers 11S18 and
11AS13 (Nicolas et al. 2005). This sequence includes both
the coding and noncoding part. The tree based on the neighbor-joining method (divergence observed, pairwise gap removal, 10,000 bootstrap replicates) was constructed using
Phylo_win software (Galtier et al. 1996). Branch lengths correspond to total sequence divergence under the model
assumed (see bar). Values indicated at the nodes are bootstrap values based on 10,000 replicates. Dioecious species
are in boldface underlined characters, the hermaphrodite
species are marked with an asterisk, and all the other included
species are gynodioecious. The tree shows that the distance of
S. colpophylla (and its close relative S. otites) from the dioecious
Silene species studied so far (S. diclinis, S. dioica, S. latifolia) is
relatively large and that there are nondioecious species that
are closely related to either S. colpophylla or S. latifolia.
function (Charlesworth and Charlesworth 1978).
Gynodioecy in the genus Silene is connected with nucleocytoplasmic male sterility. In the past, there was a widespread belief that dioecy cannot develop from the
nucleo-cytoplasmic sterility because linkage cannot develop between nuclear female sterility and cytoplasmic
male sterility (e.g., Ross 1978; Richards 1986). However, other authors (Charlesworth and Ganders 1979;
Maurice et al. 1994; Schultz et al. 1994) suggest that, in
the case of loss of the male fertile cytotype, male fertility
becomes completely controlled by a nuclear gene (fertility restorer), and so it is possible to expect that the
development of dioecy and sex chromosomes is feasible
in this case. The current mechanism of the suppression
of anther development in S. latifolia is not connected with
any kind of cytoplasmic sterility but originated via a lossof-function mutation (on the proto-X chromosome) in
the gene that was directly involved in the anther for-
Note
mation, as deduced from the data obtained from the
interspecific hybrid S. latifolia 3 S. viscosa (Zluvova et al.
2005). However, simultaneously some observations have
suggested that some nucleo-cytoplasmic system of male
sterility at the beginning could be involved (Zluvova
et al. 2005). Population studies indicate that nucleocytoplasmic male sterility systems are highly dynamic and
polymorphic even in the same species (Charlesworth
and Laporte 1998). The probability that two different
species of the same genus will have the same type of
cytotype and the same restorer therefore seems unlikely.
The hypothesis that related plant species of the plant
genus Silene possess completely different sex-determining pathways is therefore very likely.
In spite of the fact that sex determination evolved
independently, it is still relevant to ask whether there is
some region (or whole chromosome) in the Silene
genome with a higher tendency to be recruited for the
sex chromosome formation. To answer this question, we
have mapped the homologs of the genes that were
mapped in previously studied dioecious Silene species
and/or in the gynodioecious S. vulgaris (Filatov 2005;
Nicolas et al. 2005; Bergero et al. 2007). To continue
the nomenclature used in previous articles concerning
the genes of S. latifolia and their homologs that were
isolated from hermaphrodite species (Filatov 2005;
Nicolas et al. 2005; Bergero et al. 2007), we use the
terms ScolXY3, ScolXY4, ScolDD44XY, and ScolCypXY
for the homologs of the genes SlX3/SlY3, SlX4/SlY4,
SlDD44X/SlDD44Y, and SlCypX/SlCypY, respectively. The
sequences of primers used for the amplification of
homologs and the corresponding PCR profiles are summarized in the supplemental Table 1. The polymorphisms
used for the segregation analysis and recombination
mapping were based either on length polymorphisms
of the PCR products or on SNPs, which were identified
by sequencing PCR products. For the detection of SNPs,
we used the dCAPS method (Michaels and Amasino
1998; Neff et al. 1998).
Genetic analysis using the genes ScolXY3, ScolXY4,
ScolDD44, and ScolCyp showed that these genes are
located in one linkage group in S. colpophylla, similarly
to S. latifolia (the difference between the observed gene
orders in S. latifolia and S. colpophylla is represented only
by the inversion of the chromosomal part including
gene ScolXY3 and ScolXY4 (eventually SlX3 and SlX4;
Figure 2A). However, in contrast to S. latifolia for which
sex linkage of these genes has been shown (Moore et al.
2003; Nicolas et al. 2005; Bergero et al. 2007), none of
these genes show sex linkage in S. colpophylla. The results
obtained by Nicolas et al. (2005) and Bergero et al.
(2007) suggest that a number of markers (including
SlDD44X, SlX3, SlX4, and SlCypX whose homologs are
used in this study) are well spread over the X chromosome of S. latifolia. Therefore, we can conclude that the
pair of sex chromosomes in S. colpophylla probably
evolved from a different pair of autosomes than the
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Figure 2.—Results of the genetic mapping in S. colpophylla.
(A) Linkage group containing three AFLP loci and homologs
of the genes that are sex linked in S. latifolia compared with
the S. latifolia X chromosome map based on the data of
Bergero et al. (2007) (pseudoautosomal region is marked
as PAR; homologs are connected by dotted lines). (B) Linkage group containing the sex-determining region (sex) and
three AFLP loci.
sex chromosomes in S. latifolia. There is yet a rather
hypothetical possibility that sex-determining regions
evolved in S. colpophylla on a different pair of autosomes
but were subsequently translocated to the homolog of
the S. latifolia X chromosome. In this case, the sex
linkage of the studied genes could be undetectable in
spite of the fact that the sex-determining region would
be located on the same chromosome. However, even in
this case our data support the view that the evolution of
the sex determination in S. colpophylla proceeded independently on S. latifolia and started in a region that
was not genetically linked to the region homologous to
the current X linked region of S. latifolia.
A potential limitation of S. colpophylla as a future
model plant is a lack of knowledge concerning sex
determination in this species. The type of sex determination system (XX/XY or ZW/ZZ) has not yet been experimentally tested in this species and no exact data are
available for the related species. Studies on S. otites indicate even that both ZW/ZZ (Correns 1928; Sansome
1938) and XX/XY (Warmke 1942) sex-determining systems could be present in this species. We began research
in this area by studying mitotic chromosomes to test if
the sex chromosomes of S. colpophylla are homomorphic,
as they are in the related species S. otites. To obtain
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M. Mrackova et al.
Figure 3.—(A) Typical mitotic metaphase plate of S. colpophylla. The observed number of chromosomes (2n ¼ 24) suggests that S. colpophylla is not a polyploid species. No
heteromorphic sex chromosomes can be distinguished. (B)
Typical mitotic metaphase plate of the S. latifolia male (sex
chromosomes are marked by ‘‘X’’ and ‘‘Y’’). Bars, 5 mm.
karyotypes of high quality, we used a technique based
on the bulk preparation of mitotic protoplasts from root
tips of seedlings (for details see Lengerova et al. 2004).
Multiple metaphase plates (50) were evaluated to ensure
the presence of both sexes in the mixed sample. The results show either that there is a homomorphic sex chromosome pair in this species or that at least the difference
between heterochromosomes is much less prominent
than in S. latifolia. The total number of chromosomes is
24 as in most Silene species (for typical metaphase plate,
see Figure 3). Because of the supposed homomorphic
character of the sex chromosomes in S. colpophylla, we
decided to use molecular markers to further investigate
the sex-determining system. To obtain a sufficient number of markers, we used the AFLP technique as described
by Bratteler et al. (2006). In the initial experiment, we
tested 16 primer pair combinations (see supplemental
Table 2) on a representative group of males and females
and looked for markers that cosegregated with sex.
To determine the heterogametic sex, we used an approach that was based on the study of two subsequent
generations—the parental and F1 generation. This approach would enable us to identify the heterogametic sex
even if none of the markers were completely sex linked.
The results expected under two different models (XX/
XY and ZW/ZZ) are displayed in supplemental Figure 4.
We found 45 segregating AFLP markers, but only 3
showed sex linkage. Two markers (B4, C11) from the
pollen donor cosegregated predominantly with the
female sex in the progeny (supplemental Tables 3 and
4). This pattern of inheritance suggests that these
markers are linked to the X chromosome. One marker
(B5) showed cosegregation mostly with the male sex,
suggesting that the B5 marker is linked to the Y
chromosome (supplemental Table 5). In total, these
data reveal that males in S. colpophylla, rather than
females, are the heterogametic sex. Synthesis of the
data obtained by genetic mapping of the three chosen
genes and the AFLP markers (done using JoinMap 3.0
software; Van Ooijen and Voorrips 2001) enabled us
to define two separate linkage groups for S. colpophylla
(Figure 2). One of these linkage groups carries three
AFLP markers (A1, B13, and C7) and homologs of
genes that are sex linked in S. latifolia: genes ScolXY3,
ScolXY4, ScolDD44XY, and ScolCypXY (Figure 2A). The
second linkage group involves a sex-determining region
(marked ‘‘sex’’) and three AFLP markers: B4, B5, and
C11 (Figure 2B). None of these 3 sex-linked AFLP loci
shows linkage to the genes ScolXY3, ScolXY4, ScolDD44XY,
and ScolCypXY. These results further support the idea
that the sex chromosomes of S. colpophylla and S. latifolia
have evolved from different pairs of autosomes. In conclusion, the finding that sex chromosomes in S. colpophylla
have evolved from a different pair of autosomes further
supports the view that the genus Silene is an excellent
model for studying the evolution of sex chromosomes
and sex determination because of the diversity of evolutionary patterns represented within it.
This work was supported by the Grant Agency of the Academy of
Sciences of the Czech Republic (IAA600040801 to B.J.), the Grant
Agency of the Czech Republic (521/05/2076 to B.J. and 204/05/H505
to B.V.), the Ministry of Education of the Czech Republic (LC06004 to
B.V.), Institutional Research Plan (AVOZ50040507), and the Swiss
National Science Foundation grant 3100A0-116455 to A.W.
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Communicating editor: J. A. Birchler