# Plant sex-determining genes and the genetics of the evolution towards dioecy

Gabriel a B Marais, Catarina Branco, Margarida Rocheta, Mathilde Dufaÿ,

Jeanne Tonnabel

► To cite this version:

Gabriel a B Marais, Catarina Branco, Margarida Rocheta, Mathilde Dufaÿ, Jeanne Tonnabel. Plant sex-determining genes and the genetics of the evolution towards dioecy. Journal of Experimental Botany, 2025, 76 (14), pp.3896-3911. 10.1093/jxb/eraf224. hal-05294133

HAL Id: hal-05294133

https://hal.science/hal-05294133v1

Submitted on 2 Oct 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
1 Plant sex-determining genes

2 and the genetics of the

3 evolution towards dioecy

4 Gabriel AB Marais¹,²,³*, Catarina Branco¹,³,⁴, Margarida Rocheta⁵,⁶, Mathilde Dufay⁷,†, Jeanne

5 Tonnabel⁸,†

6
7 1. LBBE - Laboratoire Biométrie et Biologie Evolutive, CNRS / Univ. Lyon 1, Villeurbanne, F-69622

8 France.

9 2. GreenUPorto - Sustainable Agrifood Production Research Centre, Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, 4485-661 Vairão, Portugal

10

11 3. Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, 4099-002, Porto, Portugal.

12

13 4. CIBIO - Centro de Investigação em Biodiversidade e Recursos Genéticos, InBIO, Laboratório Associado, Campus de Vairão, Universidade do Porto, 4485-661 Vairão, Portugal.

14

15 5. LEAF - Linking Landscape, Environment, Agriculture and Food Research Center, Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, 1349-017, Lisbon, Portugal.

16

17 6. Forest Research Centre, TERRA Associate Laboratory, School of Agriculture, University of Lisbon, Tapada da Ajuda, 1349-017 Lisboa, Portugal.

18

19 7. CEFE - Centre d'écologie fonctionnelle et évolutive, CNRS / Univ. Montpellier, Montpellier, F-34293 France.

20
1

8. ISEM - Institut des Sciences de l'Évolution de Montpellier, CNRS / Univ. Montpellier,

2

Montpellier, F-34095 France.

3

4

*Corresponding author. Email gabriel.marais@cnrs.fr

5

†equal contribution

6

7
1 **Highlight (stating the primary objective of the review)**

2 Since 2014 and the discovery of the first plant sex-determining gene, more than 20 other genes

3 have been identified thanks to breakthroughs in plant genomics and bioinformatics. Here we

4 review what this tells us about the evolution of separate sexes (dioecy) in angiosperms.

5

6 **Abstract (stating the primary objective of the review and the reason for focusing on this objective, the principal findings and conclusions)**

8 Thanks to significant advances in genomics and bioinformatics, research on plant sex-

9 determining genes has made remarkable progress over the past decade. Since the discovery of
10 the OGI-MeGI sex-determination system in persimmons in 2014, candidate sex-determining
genes have been identified in a dozen flowering plant species. In this review, we examine these
11 newly discovered genes and explore what they reveal about the genetic basis of dioecy
evolution. While these genes are diverse, many belong to key developmental pathways
13 previously described in other species. Two primary evolutionary routes from hermaphroditism
14 to dioecy have been proposed: gynodioecy and monoecy. The gynodioecy pathway has been
modeled with two genes and two sterility mutations, whereas recent theoretical work on the
16 monoecy route suggests a gradual differentiation of sexual morphs involving multiple genes.
17 Current data align with these models, particularly in the expected number of sex-determining
genes. The gynodioecy pathway is typically associated with two or more genes, while the
19 monoecy route varies, involving either one or two genes. From a molecular evolution
perspective, loss-of-function mutations are frequent, but gene duplication appears to be the
21 most common mechanism driving the emergence of new sex-determining genes. Future
23 research should aim to characterize additional systems to gain a comprehensive view of plant
sex determination and employ functional approaches to validate proposed candidate genes.
24

25

26 **Keywords (6–10, alphabetically)**

27 Dioecy, Gynodioecy route, Monoecy route, Recombination suppression, Sex determination, Sex
28 chromosomes, Two genes model
1 Introduction

2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Sex determination and sex-determining genes are the genetic mechanisms that establish and regulate the development of separate sexes. More than 95% of animals have distinct male and female individuals within the same species (Fruchard and Marais, 2021 and references therein).

In contrast, approximately 85% of angiosperm species are hermaphroditic (with bisexual flowers), while only 6% are dioecious—meaning that they have separate male and female individuals (Renner, 2014; Fruchard and Marais, 2021). Hermaphroditism is thought to be the ancestral sexual system in angiosperms (Sauquet et al., 2017), with most transitions to dioecy being relatively recent (Renner, 2014). As many as 871 to 5000 independent transitions to dioecy may have taken place during the evolution of angiosperms (Renner, 2014). For decades, research on plant sex determination lagged behind that of animals. However, despite dioecy being less common in angiosperms, dioecious species are numerous—more so than the total number of bird and mammal species combined (Renner, 2014; Fruchard and Marais, 2021).
Furthermore, 13% of crops are dioecious or derived from dioecious progenitors, including economically important species such as grapevine, strawberries, kiwifruit, papaya, hemp, and hop (Ming et al., 2011; Fruchard and Marais, 2021). Understanding plant sex determination is essential not only for developing a broader perspective on sex determination across multicellular organisms—moving beyond an animal-centered view—but also for unraveling the genetic basis of transitions between sexual systems in flowering plants, and for advancing tools for managing and improving crop reproduction (Ming et al., 2011; Charlesworth, 2015; Käfer et al., 2017; Fruchard and Marais, 2021; Masuda and Akagi, 2023; Charlesworth and Harkess, 2024).

Current cytogenetic data from approximately 150 angiosperm species suggest that sex
chromosomes may represent the predominant mechanism of sex determination in flowering
plants (Ming *et al.*, 2011; Garcia *et al.*, 2023). To understand the genetic networks involved in
plant sex determination, it is essential to identify the sex-determining genes located on sex
chromosomes. However, the genomic characterisation of Y and W chromosomes present
1 significant challenges for assembly using short-read sequencing technologies because of their
2 non-recombining nature, particularly when the non-recombining regions are large and
3 repetitive (Muyle et al., 2017). Moreover, homomorphic sex chromosomes have been observed
4 in most dioecious species studied, suggesting a widespread low differentiation between sex
5 chromosomes in plants (Ming et al., 2011; Garcia et al., 2023). Even with high-quality genome
6 assemblies, identifying the sex chromosome pair in homomorphic systems can be difficult
7 without additional data such as those discussed right below (Muyle et al., 2017).
8

9 Over the past decade, there has been a surge in innovative approaches to studying sex
10 chromosomes, many of which were developed by the plant research community (Muyle et al.,
11 2017). Methods such as analyzing SNPs (Single Nucleotide Polymorphisms) or k-mers (substring
12 of length k within a DNA sequence) for sex linkage, comparing male-to-female read depth
13 ratios, and other techniques have provided critical insights into the presence of sex
14 chromosomes, the boundaries of non-recombining regions, and the genes they contain (Muyle
15 et al., 2017). These approaches have revolutionized the study of plant sex chromosomes,
16 leading to major breakthroughs (Renner and Muller, 2021). This progress began with studies on
17 *Silene latifolia*, a model species in plant sex determination research (Chibalina and Filatov,
18 2011; Charlesworth and Bergero, 2011; Muyle et al., 2012), and has since expanded to dozens
19 of dioecious plants (Figure 1). The revolution continues with the advent of long-read
20 sequencing technologies, which allow for the assembly of large and highly-repetitive Y or W
21 chromosomes, as recently demonstrated in *Rumex hastatulus* (Sacchi et al., 2024) and S.
22 *latifolia* (Akagi et al., 2025; Moraga et al., 2025).
23

24 These novel methods have led to the discovery of the first plant sex-determining gene in 2014
25 (Akagi et al., 2014). This gene, OGI, is a Y-linked non-coding RNA that represses the autosomal
26 gene MeGI, which regulates stamen and carpel development in persimmons (Akagi et al., 2014,
27 2016; Yang et al., 2019). To date, potential sex-determining genes have been proposed in over
28 10 plant systems (Table 1). However, in many cases, these remain candidate genes, as their
29 functional roles have not yet been confirmed through technologies like CRISPR-Cas9 eliciting
1 knockout or via overexpression experiments (but there are notable exceptions, such as OGI,
2 ARR17, and a few other genes, which have been experimentally confirmed as sex-determining
genes). In this review, we discuss how sex-determining genes might have evolved and their
3 consequences on plant phenotypes, focusing on three key aspects: (i) the developmental
4 pathways modified in dioecious plants, (ii) the genetic pathways involved in the evolution of
5 dioecy, and (iii) the molecular evolutionary mechanisms that gave rise to these genes.
6

8 What are the plant sex-determining genes and
their functions?
9

10 The process of sex determination in plants regulates the development of male or female
11 flowers. The mechanisms underlying this process are expected to vary across plant species, as
12 dioecy has evolved independently in most of them, and the opportunities to affect the sex
13 phenotype, a highly polygenic trait, are many. Table 1 provides a comprehensive list of
14 currently known sex-determining genes in angiosperms; some of them are candidates, others
15 confirmed by experiments. These genes have been identified in various species, including
16 *Amborella trichopoda*, *Asparagus officinalis*, *Actinidia chinensis* (kiwi), several species of
17 *Diospyros* (persimmons), multiple *Fragaria* species (strawberries), *Humulus lupulus* (hop),
18 *Cannabis sativa* (hemp), *Carica papaya* (papaya), *Phoenix dactylifera* (date palm), several
19 *Populus* and *Salix* species (poplars and willows), *S. latifolia* (white campion) and *Vitis* (wild and
20 domesticated grapevines) (see Table 1 for references). Many of these sex-determining genes
21 and their functions have already been reviewed elsewhere (Masuda and Akagi 2023;
22 Charlesworth and Harkess, 2024). The current pace of discovery in this field is rapid, with new
23 sex-determining genes identified annually in approximately two–three angiosperm species.
24 Below, we provide an in-depth presentation of the sex-determining genes discovered most
25 recently.
26
1 The long-read sequencing of the *S. latifolia* male genome using both ONT and PacBio HiFi
technologies has confirmed Clavata3 (CLV3) as a potential sex-determining gene in this species
2 (Akagi *et al.*, 2025; Moraga *et al.*, 2025). CLV3 was initially identified as a candidate sex-
determining gene through partial genome sequencing and functional analysis (Kazama *et al.*,
3 4 2022). Moraga *et al.* (2025) further supported this hypothesis by demonstrating that all
5 hermaphrodite mutants, which have Y deletions, lack the CLV3 gene. Kazama *et al.* (2023)
6 refined these findings by revealing a Clavata3-Wuschel1 (WUS1) balance as the mechanism
7 underlying sex determination in *S. latifolia*. Both CLV3 and WUS1 are well-established
8 developmental genes involved in meristem maintenance (Kazama *et al.*, 2023, and references
9 therein). In *S. latifolia*, CLV3 has a functional copy on the Y chromosome and a pseudogene on
10 the X chromosome, while WUS1 shows the reverse pattern, with a functional copy on the X and
11 a complete deletion from the Y. CLV3 contributes to the masculinization of flowers during
12 development, whereas WUS1 promotes feminization. The XX and XY genotypes exhibit
13 different CLV3/WUS1 gene dosages, which may trigger sex determination. The sequences of the
14 sex chromosomes have placed CLV3 and WUS1 on stratum 1, which is the most diverged region
15 of the non-recombining portion of the sex chromosomes (Yue *et al.*, 2023; Akagi *et al.*, 2025;
16 Moraga *et al.*, 2025). Knockout experiments that would definitively confirm CLV3 and WUS1 as
17 sex-determining genes are still needed; the ability to transform this plant suggests that such
18 experiments are feasible in the future (Fujita *et al.*, 2019; Hudzieczek *et al.*, 2019). Classical
19 genetics had previously identified three sex-determining regions on the *S. latifolia* Y
chromosome (Westergaard, 1958; Zluvova *et al.*, 2007; Bergero *et al.*, 2008). Using a mutant
21 22 23 24 25 26 27
20 collection, Moraga et al. (2025) found sex-determining genes in all three regions, indicating that
multiple genes may ultimately contribute to the determination of sex during the transition
between sexual systems. While CLV3 is presumably the oldest sex-determining gene in *S. latifolia*, studying the other sex-determining genes is crucial for understanding both function
and evolutionary history of the Y chromosome, especially the sequential steps leading to the
evolution of dioecy (see Box 1).
28
ZW chromosomes have been identified in *A. trichopoda* using one of the bioinformatic approaches mentioned earlier (Käfer *et al.*, 2021). While the *A. trichopoda* genome has been available for some time, the PacBio HiFi sequencing successfully produced a diploid genome with independently assembled Z and W chromosomes (Carrey *et al.*, 2024). The sex-determining region in *A. trichopoda* is significantly smaller than that of *S. latifolia* (~3 Mb compared to ~480 Mb), and, unlike *S. latifolia*, no collection of mutants was used to identify potential sex-determining genes. Instead, sex-biased gene expression combined with functional annotation provided sufficient evidence to pinpoint several candidate genes. Among the candidates, the WUS1 gene reappears, but in contrast to its role in *S. latifolia*, where it promotes carpel development, in *A. trichopoda* it might suppress carpel formation as it is a male-biased gene. Another gene, MNT1-2, exhibiting female-biased expression, is considered a potential candidate due to its involvement in the development of non-dehiscent anthers and non-viable pollen in *Arabidopsis thaliana*, which resemble the staminodes in female flowers of *A. trichopoda*. Both MNT1-2 and WUS1 are located in the region of highest Z-W divergence, suggesting that they may be among the oldest sex-determining genes. Although knockout experiments were not conducted, they would be challenging to perform in this shrub species. Nonetheless, the identification of these candidate genes, along with their location in the most divergent regions of the Z and W chromosomes, provides important insight into the evolution of sex determination in *A. trichopoda*.

In *Humulus* and *Cannabis*, sex determination does not rely on an “active” Y (or W) but on the X-to-autosome balance as in *Drosophila melanogaster* (Ming *et al.*, 2011). Theory suggests that this type of sex determination should evolve secondarily in a well-differentiated system in which the Y is very degenerated (Charlesworth *et al.*, 2005; Ming *et al.*, 2011). Using methods mentioned earlier, studies in *C. sativa* and *H. lupulus* found that they share the same XY system that is therefore effectively ~30 My old, the oldest documented so far in plants (Prentout *et al.*, 2020, 2021). Most of the Cannabaceae sex chromosome system is non-recombining and contains several evolutionary strata. The long-read sequencing of the *H. lupulus* (hop) genome confirmed these observations and focused on the oldest stratum to locate the sex-determining
1 candidates. Based on gene expression and functional annotation, they identified ETR1-like X-

2 encoded Ethylene Receptor (EXER) as an interesting candidate, with ethylene being a

3 phytohormone known to be key in flower development in many plants (Akagi et al., 2024).

4 Transformation of tobacco with EXER led to flower feminization, while Virus-Induced Gene

5 Silencing (VIGS) of EXER in female hop resulted in flower masculinization, demonstrating a clear

6 dosage-dependent effect of EXER (Akagi et al., 2024). A transcriptomic-based systems biology

7 analysis of male and female flower development, using a female *C. sativa* (hemp) reference

8 genome, identified 18 and 111 candidate sex-determining genes on the X and Y chromosomes,

9 respectively (Shi et al., 2025). Among these, several stand out due to their roles as expression

10 hubs, strong sex-biased expression, and functional annotations suggesting involvement in

11 flower development. Notably, homologs of the transcription factors FLOWERING LOCUS D (FD)

12 on the Y chromosome and REPRODUCTIVE MERISTEM 16 (REM16) on the X chromosome are of

13 particular interest (Shi et al., 2025). However, the relationship between these candidate genes

14 and the expected X-to-autosome balance in *C. sativa* remains to be investigated. Future studies

15 should also aim to produce a complete Y chromosome assembly and perform functional

16 validation through experimental analysis.

17 The sex-determining region of octoploid strawberries was one of the first to be deciphered, just

18 four years after the OGI-MeGI system (Tennessen et al., 2018). Two key genes were identified:

19 GMEW, a GDP-mannose 3,5-epimerase that converts GDP-mannose to GDP-L-galactose, playing

20 a role in vitamin C and cell wall biosynthesis, which influences both fruit development and

21 pollen production; and RPP0W, a ribosomal protein-coding gene, which may function as a long

22 non-coding RNA (lncRNA) whose function has not yet been determined (Tennessen et al., 2018;

23 Cauret et al., 2022). These genes are part of a genetic cassette that has been repeatedly

24 translocated between chromosomes during the evolutionary history of octoploid strawberries,

25 each time incorporating bits of flanking regions (Tennessen et al., 2018). A more recent study

26 on tetraploid strawberries, specifically *Fragaria moupinensis*, also identified a genetic cassette,

27 but it contained a different gene, AFT (Qiao et al., 2024). This gene appears to promote carpel

28 development via the auxin pathway while potentially inhibiting anther formation (Qiao et al.,
1 2024). The role of AFT was further validated using VIGS in domesticated strawberries, which
2 showed aborted carpels but normal anthers, reinforcing its involvement in sex determination
3 and floral development.

4
5 In poplars and willows, a single gene—duplicate of ARABIDOPSIS RESPONSE REGULATOR 17
6 (ARR17)—has been identified as the primary sex-determining factor (Muller *et al.*, 2020; Xue *et
7 al.*, 2020; Leite-Montalvão *et al.*, 2022; He *et al.*, 2021; He *et al.*, 2024). ARR17 produces small
8 RNAs (sRNAs) that silence the intact ARR17-like gene, thereby indirectly activating PISTILLATA
9 (PI), which is required for stamen development, while suppressing female development.

Despite extensive sex chromosome turnover in these species, ARR17 remains a conserved sex-
determining gene, except in *Populus deltoides*, where another gene, FERR-R, has been
identified as the sex-determining factor (Xue *et al.*, 2020). A recent study explored the sex
chromosomes of two early-diverged species from the Salix and Vetrix clades, *Salix triandra* and
*Salix mesnyi* (Wang *et al.*, 2024). The study located ARR17 in the sex-determining region,
alongside another gene, MSF, which corresponds to a duplicate of an autosomal gene.
However, no functional analysis was conducted on the MSF duplicate, and its role in sex
determination remains unclear, making it a candidate for further investigation.

18
19 To date, the determination of sex in plants reveals an enormous level of complexity, involving
20 multiple factors that act through diverse mechanisms and pathways, shaped by the respective
21 evolutionary histories of different species. Many of the key genes identified in this process are
22 developmental genes, often transcription factors, which are also described in other plant
23 species for their roles in growth and development. Moreover, a significant number of these sex-
24 determining genes are involved in phytohormone pathways, highlighting the intricate
25 connection between hormonal regulation and sex determination in plants.
26

26
1 What genetic changes underlie the transition
to dioecy? Theoretical models versus empirical
data

Several modelling studies have explored the conditions for the evolution of dioecy and its
genetics basis (Figure 2). Historically, the first modeled pathway was the gynodioecy pathway,
which begins with the emergence of female (male-sterile) individuals co-occurring with
hermaphrodites. Over time, hermaphrodites are replaced by pure males. Charlesworth and
Charlesworth (1978) proposed a seminal model investigating this scenario and showed that a
nuclear male-sterility mutation could spread if male-sterile plants compensate for the loss of
male function by increasing seed production. Once females have emerged, selection favors
hermaphrodites that invest more in male reproduction, effectively facilitating the spread of a
female-sterility allele at a second locus. A dominant female-sterility and recessive male-sterility
mutation will produce a XY system, while a dominant male-sterility and a recessive female-
sterility mutation will produce a ZW one. Selection favours recombination suppression between
both genes, leading to the production of sex-specific haplotypes through genetic linkage
(Charlesworth and Charlesworth, 1978). In line with this, selection should favour Y chromosomes
with male-fertile and a female-sterile haplotype, and X chromosomes with male-sterile and
female-fertile haplotype. In many systems, gynodioecy appears to be associated with a
cytoplasmic male sterility (CMS) mutation, which can be counteracted by a nuclear allele
restoring male function (i.e. restorer allele) (Saumitou-Laprade et al., 1994; Chase, 2007). This
CMS-based mechanism does not necessarily contradict a scenario involving two nuclear genes.
Under certain conditions, CMS can become fixed in a population, effectively leading to a nuclear
genetic basis of gynodioecy (Dufay et al., 2004; Dufay and Pannell, 2010). In such cases, the
maintainer allele at the nuclear restorer locus (i.e. the one not restoring the male function)
becomes functionally equivalent to the nuclear male sterility mutation from the Charlesworth
and Charlesworth (1978) model.
Besides the gynodioecy pathway, another scenario proposes that dioecy would evolve from
monoecy (i.e. a case of hermaphroditism where individuals exhibit separated male and female
flowers) through disruptive selection (Barrett, 2002). The underlying idea of the monoecy
pathway is that evolution of dioecy is promoted by the selection of individuals that allocate
resources more efficiently to either the male or female function instead of both (Pannell, 2017).

Accordingly, any selective force, including both natural and sexual selection, rendering sexual
specialization beneficial should in principle promote the evolution of separate sexes from
monoecy. In contrast to the gynodioecy pathway, the monoecy pathway is supposed to involve
a gradual separation of male and female phenotypes with a large number of associated
mutations. These mutations could potentially impact not only the multiple genes involved in
male vs. female flower production but also any gene influencing sex-specific performance. This
monoecy pathway has received less attention from modelling studies, or at least with less
explicit hypotheses about its genetics basis. The situation has recently changed with Lesaffre et
al.'s models (2024a,b) demonstrating that two groups of genotypes with unbalanced resource
allocation to female (alternatively male) reproduction could gradually emerge in a large set of
conditions. When the allocation to female vs. male function was modelled as a polygenic trait,
as generally expected when the evolving trait is quantitative, such a scenario of disruptive
selection also evolved. In addition, when a modifier locus determining the contribution of each
locus to sex allocation was allowed to evolve, the model showed that the dioecious system
ended up with a simpler genetic basis consisting of one or two major loci (Lesaffre et al.,
2024a). Although such a gene controlling the relative contribution of several loci to sex
allocation has not been documented by empirical studies to our knowledge, this demonstrates
that selection could favor transition from polygenic to simpler sex determination of sex along
the monoecy-dioecy pathway. Taken together, theory on sexual system transitions predicts that
dioecy evolving via the gynodioecy pathway is typically associated with a two-gene sex-
determination while the monoecy-dioecy pathway can lead to more diverse genetic
architectures, ranging from single-locus to multi-locus systems. The specific genetic basis may
thus depend on the evolutionary stage along the monoecy-pathway.
1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

How does genomic knowledge of the genetic basis of dioecy advance the testing of predictions from evolutionary models of sexual systems transitions? The advent of genomic studies on sex determination in plants has revealed that many identified sex-determination systems do not strictly rely on the two sex-determining genes that remained the dominant model for explaining sexual system transitions for decades (i.e. persimmons, poplars and willows, see Table 1). This has triggered some criticism regarding the validity and generality of the two genes model (e.g., Renner, 2016; Renner and Müller, 2021). Lesaffre et al.'s model presents a compelling alternative for explaining the evolution of dioecy through the monoecy pathway (Lesaffre et al., 2024a,b). One of its most relevant aspects is that the genes modeled are not directly involved in fertility but rather influence the allocation of resources to male and female functions, thereby modifying the expression of monoecy. This aligns closely with findings about the genetics of monoecy in cucurbits (Boualem et al., 2008; Martin et al., 2009; Boualem et al., 2015; Zhang et al., 2022). In their 2015 study, Bendahmane's team successfully generated an artificial dioecious line by sequentially mutating ACS-11, an ethylene pathway gene that regulates the coexistence of male and female flowers, and its target gene WIP1, a male-promoting transcription factor that functions as a switch to determine floral sex (Boualem et al., 2015).

Remarkably, in this artificially derived dioecious population, the ACS-11 mutation became fixed, while the WIP1 mutation remained polymorphic, creating the illusion that the system is controlled by a single genetic factor. In one of their models, Lesaffre et al. (2024a) proposed a system in which sex phenotype is controlled by a single locus, while a second locus functions as a cis or trans factor, regulating the expression of the first gene and determining its dominance.

This model bears a striking resemblance to the OGI-MeGI system, where MeGI, an autosomal gene that governs sex phenotype, is regulated by OGI, a Y-linked locus that controls its expression (Akagi et al., 2014). The striking similarities between this newly modeled pathway and recent genomic discoveries highlight the need for further modelling efforts that explicitly incorporate genetic mechanisms. Expanding theoretical frameworks to better reflect empirical findings will be crucial for refining our understanding of the evolution of dioecy.
1 Models of the evolution of dioecy predict that the gynodioecy pathway should involve a two-
2 gene system, whereas the monoecy pathway could be governed by more diverse genetic
3 systems. However, these predictions apply specifically to the de novo evolution of sex
4 chromosomes. In cases where pre-existing sex chromosomes are replaced by a new pair—a
5 process known as a turnover event—this transition can occur through the evolution of a single
6 sex-determining gene (Van Doorn and Kirkpatrick, 2007). In order to provide a first test of these
7 ideas, we combined available data on the number of sex-determining genes identified with the
8 route followed by a given lineage to evolve towards dioecy i.e. the gynodioecy and monoecy
9 pathways, as reported in Table 1. To infer the route to dioecy, we used previously published
datasets informing on the presence of monoecious and gynodioecious species in dioecious
10 genera, families and orders (Renner, 2014; Dufay et al., 2014; Figure 3). Although these
11 inferences have limitations—particularly at the family and order levels, and a formal test of
12 their association is not possible due to the limited characterization of genetic systems to date,
13 we observe that the gynodioecy pathway tends to be associated with two-gene systems, while
14 the monoecy pathway is associated with both one and two-gene systems. In *Humulus*, where
15 the turnover is documented (a secondary X-to-autosome balance sex-determination system has
16 evolved, see Akagi et al. 2024), a single master gene has been identified. The empirical data
17 thus seems to support so far the available theoretical models.
18

19
20
21
22
23
24
25
26
27
28
29
Beside the sex-determining genes, sex chromosomes are expected to accumulate sexually-
antagonistic genes, i.e. with alleles beneficial in one sex but detrimental to the other
(Charlesworth et al., 2005). Unlike sex-determining genes, sexually-antagonistic genes do not
define sexual phenotypes but instead enhance sex-specific performance. Sexually-antagonistic
genes can emerge through fecundity selection, favoring differential male and female gamete
production, or through sexual selection, which is defined as selection arising from 'fitness
differences associated with non-random success in competition for access to reproductive
resources provided by potential mating partners' (Janicke, 2024). In animals, numerous studies
have shown that sexually-selected traits are disproportionately located on sex chromosomes,
contributing to iconic phenotypes such as fish colour patterns (Lindholm and Breden, 2002;
Reinhold and Engqvist, 2013; Layh et al., 2023). Additionally, the strength of sexual selection

has been shown to affect the extent of the recombination arrest between sex chromosomes

(Wright et al., 2017) and to increase the signature of selection on the X chromosome (Wiberg et

al., 2021). Theoretical predictions further predict that genes underlying female choice

mechanisms may be located on sex chromosomes (Rice, 1984), a hypothesis supported by

limited empirical evidence (Lindholm and Breden, 2002). In flowering plants, sexual selection

has traditionally been studied through the impact of pollen traits on ovule fertilization.

However, recent studies have identified mate acquisition and female choice mechanisms as

potential mechanisms applying to plants (Tonnabel et al., 2019, 2021, 2022; Barbot et al.,

2023), though their prevalence in plants remains poorly understood. The study of sex

chromosome evolution and sexual selection are two expanding fields of research in plants

which may gain from cross-talk exchanges. Beyond sex chromosomes, sexual selection is

expected to shape genome architecture more broadly. Because sexual selection is expected to

act in a condition-dependent manner i.e. selecting individuals in overall greater condition with

ability to develop sexually-selected traits (Rowe and Houle, 1996), it may lead to genetic

signatures beyond sex-linked regions. This effect has been documented in two animal studies,

where sexual selection significantly affected signatures of selection at the whole genomic scale

(Dugand et al., 2019; Parrett et al., 2022). Distinguishing between sex-determining genes and

genes on sex chromosomes subject to sexual selection —such as those involved in pollen

function—can be challenging (see Box 1).

A particularly important phenomenon to study is the reversion to hermaphroditism that has
occurred during domestication in several crops, including grapevine, papaya, and strawberries.

A common assumption is that this transition is driven by modifications in sex-determining

genes. However, a detailed comparison of wild and cultivated persimmons revealed that

hermaphroditism was reinvented through the recruitment of a small-Myb RADIALIS-like gene

specifically in cultivated hexaploid persimmons (Masuda et al., 2020, Masuda et al. 2022;

Masuda and Akagi, 2023). In grapevine, the identity of the sex-determining genes in wild

populations remains controversial. However, sequencing of the sex-determining region in both
1 wild and cultivated grapevines has highlighted the role of recombination between female and

2 male haplotypes in producing hermaphrodite ones (see Box 2). Identifying the genes

3 responsible for these reversions has significant economic implications, as it could provide

4 valuable tools for manipulating sexual systems in crops. Depending on the context, dioecy could

5 be favored for controlled crosses and the development of new varieties, while

6 hermaphroditism may be preferable for maximizing fruit production.

## What kind of mutations have occurred in the sex-determining genes?

9 The original gynodioecy model proposed that sex chromosome evolution is driven by reciprocal
10 loss-of-function mutations in two fertility genes—one causing male sterility and the other
11 female sterility, while the monoecy one is somewhat blind to the type of mutations occurring at
12 the sex locus (see above). As shown in Table 1, some species, such as *A. trichopoda*, *S. latifolia*,
13 and *V. vinifera*, align with this scenario. In certain cases, genes have been entirely lost from one
14 haplotype (e.g., CLV3 and WUS1 in *S. latifolia*, INP1 in *V. vinifera*). In others, both alleles have
15 remained functional, but differences in gene expression have evolved between haplotypes
16 (e.g., MTN1-2 and WUS1 in *A. trichopoda*, YABBY or APT3 in *V. vinifera*).

17 In theory, sex-determining mutations could also arise through gain-of-function changes, not
18 just loss-of-function. Indeed, Table 1 highlights that in most species, sex-determining genes
19 have originated via gene duplication, supporting the idea that gain-of-function mutations play a
20 critical role. In some cases, these duplicated genes function by silencing their parental gene, as
21 observed in OGI (*Diospyros*) and ARR17 (*Populus* and *Salix*). These non-protein-coding gene
22 duplicates exert epigenetic control over their parental genes (e.g., MeGI silencing by OGI,
23 ARR17-like silencing by ARR17 duplicates). The prevalence of duplication in the evolution of
24 sex-determining genes remains an open question, but it may be linked to the general
25 propensity for gene duplications in plant genomes (Soltis *et al.*, 2015).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16

Sex-determining gene turnover represents a unique case, where gain-of-function mutations are expected to dominate, as new genes must arise to replace old ones as modelled by Van Doorn and Kirkpatrick (2007). Turnover events have been documented in a number of plant genera (e.g. Balounova et al., 2019; Müller et al., 2020; Yang et al., 2021; Akagi et al., 2023; Wang et al., 2022, 2024). In that context, the genetic cassette system found in Fragaria (strawberries) is very intriguing. In both tetraploid and octoploid species, the sex-determining region is flanked by repetitive DNA, and remarkably, the same genetic region, containing the same genes, has been found on different chromosomes, suggesting repeated translocations during evolution (Tennessen et al., 2018). This mobile sex-determining system is so far unique to strawberries, and its evolutionary drivers remain unclear. Theory links sex chromosome turnover events to sexually antagonistic selection (Van Doorn and Kirkpatrick, 2007). However, in plants, sexually antagonistic selection is supposedly weaker than in animals (Barrett and Hough, 2013), where similar mobile cassettes have not been reported on sex chromosomes. The Fragaria system remains an evolutionary mystery, raising fundamental questions about the forces shaping sex determination in plants.

17

Conclusion

18
19
20
21
22
23
24
25

Significant advances in plant sex determination research have been made over the past decade, both at the mechanistic and evolutionary levels. While important insights have been gained, current conclusions remain tentative, as only a limited number of systems have been characterized. Moreover, most sex-determining genes identified so far are still candidates that require validation through functional analyses, particularly knockout (KO) or overexpression experiments. Future research should aim at characterising more systems and validating candidate genes. A good idea could be to focus on the lineages not currently covered (see Figure 1).
1 Acknowledgements

2 We thank Editor Rainer Melzer for his kind invitation to contribute to the FNL special issue

3 Conflict of interest

4 We declare no conflict of interest

5 Funding

6 GABM and CB acknowledge the support of Agence Nationale de la Recherche (ANR grants ANR-20-CE20-0015-01 and ANR-20-CE02-0015). MR acknowledges the support of FCT – Fundação para a Ciência e a Tecnologia (UID/04129 LEAF-Linking Landscape, Environment, Agriculture and Food Research Center and UID/00239 CEF-Forest Research Centre).

10

11 References

12 Akagi T, Fujita N, Shirasawa K, et al. 2025. Rapid and dynamic evolution of a giant Y chromosome in Silene latifolia. Science 387, 637–643.

14 Akagi T, Henry IM, Ohtani H, Morimoto T, Beppu K, Kataoka I, Tao R. 2018. A Y-Encoded Suppressor of Feminization Arose via Lineage-Specific Duplication of a Cytokinin Response Regulator in Kiwifruit. The Plant Cell 30, 780–795.

17 Akagi T, Henry IM, Tao R, Comai L. 2014. A Y-chromosome-encoded small RNA acts as a sex determinant in persimmons. Science 346, 646–650.

19 Akagi T, Kawai T, Tao R. 2016. A male determinant gene in diploid dioecious Diospyros, OGI, is required for male flower production in monoecious individuals of Oriental persimmon (D. kaki). Scientia Horticulturae 213, 243–251.

22 Akagi T, Pilkington SM, Varkonyi-Gasic E, et al. 2019. Two Y-chromosome-encoded genes determine sex in kiwifruit. Nature Plants 5, 801–809.

23
1

Akagi T, Segawa T, Uchida R, et al. 2024. Evolution and functioning of an X-A balance sex determination system in hops. bioRxiv, 2024.11.04.621975.

2

3

Akagi T, Shirasawa K, Nagasaki H, Hirakawa H, Tao R, Comai L, Henry IM. 2020. The persimmon genome reveals clues to the evolution of a lineage-specific sex determination system in plants. PLOS Genetics 16, e1008566.

4

5

6

Akagi T, Varkonyi-Gasic E, Shirasawa K, et al. 2023. Recurrent neo-sex chromosome evolution in kiwifruit. Nature Plants 9, 393-402.

7

8

Badouin H, Velt A, Gindraud F, et al. 2020. The wild grape genome sequence provides insights into the transition from dioecy to hermaphroditism during grape domestication. Genome Biology 21, 223.

9

10

Balounova V, Gogela R, Cegan R, et al. 2019. Evolution of sex determination and heterogamety changes in section Otites of the genus Silene. Scientific Reports 9, 1045.

12

13

Barbot E, Dufaü M, De Cauwer I. 2023. Sex-specific selection patterns in a dioecious insect-pollinated plant. Evolution 77, 1578-1590.

14

Barrett SCH. 2002. The evolution of plant sexual diversity. Nature Reviews Genetics 3, 274-284.

16

Barrett SCH, Hough J. 2013. Sexual dimorphism in flowering plants. Journal of Experimental Botany 64, 67-82.

17

18

Bergero R, Charlesworth D. 2011. Preservation of the Y Transcriptome in a 10-Million-Year-Old Plant Sex Chromosome System. Current Biology 21, 1470-1474.

19

20

Bergero R, Charlesworth D, Filatov DA, Moore RC. 2008. Defining Regions and Rearrangements of the Silene latifolia Y Chromosome. Genetics 178, 2045-2053.

21

22

Boualem A, Fergany M, Fernandez R, et al. 2008. A Conserved Mutation in an Ethylene Biosynthesis Enzyme Leads to Andromonoecy in Melons. Science 321, 836-838.

23

24

Boualem A, Troadec C, Camps C, et al. 2015. A cucurbit androecy gene reveals how unisexual flowers develop and dioecy emerges. Science 350, 688-691.

25

26

Carey SB, Aközbek L, Lovell JT, et al. 2024. ZW sex chromosome structure in Amborella trichopoda. Nature Plants doi: 10.1038/s41477-024-01858-x.

27
1 Carrasco D, Bellido A, Vargas AM, Picq S, Bacilieri R, This P, Arroyo-Garcia R. 2020.

2 Comparative analysis of the expression of sex candidate genes in flower of dioecious and

3 hermaphrodite grapevine (Vitis vinifera L. ssp.). Scientia Horticulturae **274**, 109639.

4 Cauret CMS, Mortimer SME, Roberti MC, Ashman T-L, Liston A. 2022. Chromosome-scale

5 assembly with a phased sex-determining region resolves features of early Z and W chromosome

6 differentiation in a wild octoploid strawberry. G3 Genes | Genomes | Genetics **12**, jkac139.

7 Chae T, Harkess A, Moore RC. 2021. Sex-linked gene expression and the emergence of

8 hermaphrodites in Carica papaya. American Journal of Botany **108**, 1029–1041.

9 Chaïb J, Torregrosa L, Mackenzie D, Corena P, Bouquet A, Thomas MR. 2010. The grape

10 microvine – a model system for rapid forward and reverse genetics of grapevines. The Plant

11 Journal **62**, 1083–1092.

12 Charlesworth D. 2015. Plant sex chromosomes. Annual review of plant biology **67**, 397–420.

13 Charlesworth D. 2021. When and how do sex-linked regions become sex chromosomes?

14 Evolution **75**, 569–581.

15 Charlesworth B, Charlesworth D. 1978. A Model for the Evolution of Dioecy and Gynodioecy.

16 The American Naturalist **112**, 975–997.

17 Charlesworth D, Charlesworth B, Marais G. 2005. Steps in the evolution of heteromorphic sex

18 chromosomes. Heredity **95**, 118–128.

19 Charlesworth D, Harkess A. 2024. Why should we study plant sex chromosomes? The Plant Cell

20 **36**, 1242–1256.

21 Chase CD. 2007. Cytoplasmic male sterility: a window to the world of plant mitochondrial–

22 nuclear interactions. Trends in Genetics **23**, 81–90.

23 Chibalina MV, Filatov DA. 2011. Plant Y Chromosome Degeneration Is Retarded by Haploid

24 Purifying Selection. Current Biology **21**, 1475–1479.

25 Coito JL, Ramos MJN, Cunha J, Silva HG, Amâncio S, Costa MMR, Rocheta M. 2017. VviAPRT3

26 and VviFSEX: Two Genes Involved in Sex Specification Able to Distinguish Different Flower Types

27 in Vitis. Frontiers in Plant Science **8**.

28 Coito JL, Silva HG, Ramos MJ, Cunha J, Eiras-Dias J, Amâncio S, Costa MM, Rocheta M. 2019.

29 Vitis flower types: From the wild to crop plants. PeerJ **7**, e7879.
1 Dalla Costa L, Malnoy M, Lecourieux D, Deluc L, Ouaked-Lecourieux F, Thomas M, Torregrosa LJ-M. 2019. The state-of-the-art of grapevine biotechnology and new breeding technologies (NBTS). Oeno One 53, 189-212.

2
3

4 Dong Y, Duan S, Xia Q, et al. 2023. Dual domestications and origin of traits in grapevine evolution. Science 379, 892-901.

6
7
Dufaÿ M, Hossaert-McKey M, Anstett M-C. 2004. Temporal and sexual variation of leaf-produced pollinator-attracting odours in the dwarf palm. Oecologia 139, 392-398.

8
9
Dufay M, Pannell JR. 2010. The effect of pollen versus seed flow on the maintenance of nuclear-cytoplasmic gynodioecy. Evolution 64, 772-784.

10
11
Dugand RJ, Tomkins JL, Kennington WJ. 2019. Molecular evidence supports a genic capture resolution of the lek paradox. Nature Communications 10, 1359.

12 Fruchard C, Marais GAB. 2021. The Evolution of Sex Determination in Plants. In: Nuño de la Rosa L, Müller GB, eds. Evolutionary Developmental Biology: A Reference Guide. Cham: Springer International Publishing, 683-696.

15 Fujita N, Kazama Y, Yamagishi N, Watanabe K, Ando S, Tsuji H, Kawano S, Yoshikawa N, Komatsu K. 2019. International Journal of Molecular Sciences 20, 1031.

17 García A, Aguado E, Garrido D, Martínez C, Jamilena M. 2020. Two androecious mutations reveal the crucial role of ethylene receptors in the initiation of female flower development in Cucurbita pepo. The Plant Journal 103, 1548-1560.

20 Garcia S, Janousek B, Pascual-Díaz JP, Renner SS. 2023. Sex-chrom v. 2.0: a database of green plant species with sex chromosomes. Chromosoma 132, 55-58.

22 Grassi F, De Lorenzis G. 2021. Back to the Origins: Background and Perspectives of Grapevine Domestication. International Journal of Molecular Sciences 22.

24 Harkess A, Huang K, van der Hulst R, Tissen B, Caplan JL, Koppula A, Batish M, Meyers BC, Leebens-Mack J. 2020. Sex Determination by Two Y-Linked Genes in Garden Asparagus[OPEN]. The Plant Cell 32, 1790-1796.

27 Harkess A, Zhou J, Xu C, et al. 2017. The asparagus genome sheds light on the origin and evolution of a young Y chromosome. Nature Communications 8, 1279.
1 **He L, Jia K-H, Zhang R-G, et al.** 2021. Chromosome-scale assembly of the genome of Salix dunnii
2 reveals a male-heterogametic sex determination system on chromosome 7. Molecular Ecology
3 Resources **21**, 1966–1982.

4 **He L, Wang Y, Wang Y, Zhang R-G, Wang Y, Hörandl E, Ma T, Mao Y-F, Mank JE, Ming R.** 2024.
5 Allopolyploidization from two dioecious ancestors leads to recurrent evolution of sex
6 chromosomes. Nature Communications **15**, 6893.

7 **Iocco-Corena P, Chaïb J, Torregrosa L, Mackenzie D, Thomas MR, Smith HM.** 2021. VviPLATZ1
8 is a major factor that controls female flower morphology determination in grapevine. Nature
9 Communications **12**, 6995.

10 **Janick J.** 2007. Genetic Alteration Associated with Fruit Domestication. Acta Horticulturae.
11 International Society for Horticultural Science (ISHS), Leuven, Belgium, 27–36.

12 **Janicke T.** 2024. Sexual selection: competition for resources provided by mating partners.
13 Behavioral Ecology **35**, arae016.

14 **Käfer J, Lartillot N, Marais GAB, Picard F.** 2021. Detecting sex-linked genes using genotyped
15 individuals sampled in natural populations. Genetics **218**, iyab053.

16 **Käfer J, Marais GAB, Pannell JR.** 2017. On the rarity of dioecy in flowering plants. Molecular
17 Ecology **26**, 1225–1241.

18 **Kazama Y, Kitoh M, Kobayashi T, Ishii K, Krasovec M, Yasui Y, Abe T, Kawano S, Filatov DA.**
19 2022. A CLAVATA3-like Gene Acts as a Gynoecium Suppression Function in White Campion.
20 Molecular Biology and Evolution **39**, msac195.

21 **Kui L, Tang M, Duan S, Wang S, Dong X.** 2020. Identification of Selective Sweeps in the
22 Domesticated Table and Wine Grape (Vitis vinifera L.). Frontiers in Plant Science **11**.

23 **Layh S, Nagarajan-Radha V, Lemos B, Dowling DK.** 2023. Y chromosome-linked variation affects
24 locomotor activity in male Drosophila melanogaster and is robust to differences in thermal
25 environment. Heredity **130**, 312–319.

26 **Leite Montalvão AP, Kersten B, Kim G, Fladung M, Müller NA.** 2022. ARR17 controls dioecy in
27 Populus by repressing B-class MADS-box gene expression. Philosophical Transactions of the
28 Royal Society B **377**, 20210217.
1 Lesaffre T, Pannell JR, Mullon C. 2024a. An explanation for the prevalence of XY over ZW sex determination in species derived from hermaphroditism. Proceedings of the National Academy of Sciences **121**, e2406305121.

4 Lesaffre T, Pannell JR, Mullon C. 2024b. The joint evolution of separate sexes and sexual dimorphism. Journal of Evolutionary Biology, voae136.

6 Liao Z, Zhang T, Lei W, et al. 2023. A telomere-to-telomere reference genome of ficus (Ficus hispida) provides new insights into sex determination. Horticulture research **11**, uhad257.

8 Lindholm A, Breden F. 2002. Sex Chromosomes and Sexual Selection in Poeciliid Fishes. The American Naturalist **160**, S214–S224.

10 Little HA, Papadopoulou E, Hammar SA, Grumet R. 2007. The influence of ethylene perception on sex expression in melon (Cucumis melo L.) as assessed by expression of the mutant ethylene receptor, At-etr1-1, under the control of constitutive and floral targeted promoters. Sexual Plant Reproduction **20**, 123–136.

14 Ma X, Yu L, Fatima M, et al. 2022. The spinach YY genome reveals sex chromosome evolution, domestication, and introgression history of the species. Genome Biology **23**, 75.

16 Manzano S, Martínez C, Domínguez V, Avalos E, Garrido D, Gómez P, Jamilena M. 2010. A Major Gene Conferring Reduced Ethylene Sensitivity and Maleness in Cucurbita pepo. Journal of Plant Growth Regulation **29**, 73–80.

19 Martin A, Troadec C, Boualem A, Rajab M, Fernandez R, Morin H, Pitrat M, Dogimont C, Bendahmane A. 2009. A transposon-induced epigenetic change leads to sex determination in melon. Nature **461**, 1135–1138.

22 Massonnet M, Cochetel N, Minio A, et al. 2020. The genetic basis of sex determination in grapes. Nature Communications **11**, 2902.

24 Masuda K, Akagi T. 2023. Evolution of sex in crops: recurrent scrap and rebuild. Breeding Science **73**, 95–107.

26 Masuda K, Fujita N, Yang H-W, Ushijima K, Kubo Y, Tao R, Akagi T. 2020. Molecular Mechanism Underlying Derepressed Male Production in Hexaploid Persimmon. Frontiers in Plant Science **11**.
1 Masuda K, Ikeda Y, Matsuura T, Kawakatsu T, Tao R, Kubo Y, Ushijima K, Henry IM, Akagi T.

2 2022. Reinvention of hermaphroditism via activation of a RADIALIS-like gene in hexaploid

3 persimmon. *Nature Plants* **8**, 217–224.

4 Maurice S, Belhassen E, Couvet D, Gouyon P-H. 1994. Evolution of dioecy: can nuclear–

5 cytoplasmic interactions select for maleness? *Heredity* **73**, 346–354.

6 Mibus H, Tatlioglu T. 2004. Molecular characterization and isolation of the F/f gene for

7 femaleness in cucumber (Cucumis sativus L.). *Theoretical and Applied Genetics* **109**, 1669–

8 1676.

9 Ming R, Bendahmane A, Renner SS. 2011. Sex Chromosomes in Land Plants. *Annual Review of*

10 Plant Biology **62**, 485–514.

11 Moraga C, Branco C, Rougemont Q, et al. 2025. The Silene latifolia genome and its giant Y

12 chromosome. *Science* **387**, 630–636.

13 Müller NA, Kersten B, Leite Montalvão AP, et al. 2020. A single gene underlies the dynamic

14 evolution of poplar sex determination. *Nature Plants* **6**, 630–637.

15 Muyle A, Shearn R, Marais GA. 2017. The Evolution of Sex Chromosomes and Dosage

16 Compensation in Plants. *Genome Biology and Evolution* **9**, 627–645.

17 Muyle A, Zemp N, Deschamps C, Mousset S, Widmer A, Marais GAB. 2012. Rapid De Novo

18 Evolution of X Chromosome Dosage Compensation in Silene latifolia, a Plant with Young Sex

19 Chromosomes. *PLOS Biology* **10**, e1001308.

20 Pannell JR. 2017. Plant Sex Determination. *Current Biology* **27**, R191–R197.

21 Parrett JM, Chmielewski S, Aydogdu E, Łukasiewicz A, Rombauts S, Szubert-Kruszyńska A,

22 Babik W, Konczal M, Radwan J. 2022. Genomic evidence that a sexually selected trait captures

23 genome-wide variation and facilitates the purging of genetic load. *Nature Ecology & Evolution*

24 **6**, 1330–1342.

25 Prentout D, Razumova O, Rhoné B, Badouin H, Henri H, Feng C, Käfer J, Karlov G, Marais GA.

26 2020. An efficient RNA-seq-based segregation analysis identifies the sex chromosomes of

27 Cannabis sativa. *Genome research* **30**, 164–172.

28 Prentout D, Stajner N, Cerenak A, Tricou T, Brochier-Armanet C, Jakse J, Käfer J, Marais GA.

29 2021. Plant genera Cannabis and Humulus share the same pair of well-differentiated sex

30 chromosomes. *New Phytologist* **231**, 1599–1611.
1 Qiao Q, Cao Q, Zhang R, Wu M, Zheng Y, Xue L, Lei J, Sun H, Liston A, Zhang T. 2024. Genomic

2 analyses provide insights into sex differentiation of tetraploid strawberry (Fragaria

3 moupinensis). Plant Biotechnology Journal **22**, 1552–1565.

4 Reinhold K, Engqvist L. 2013. The variability is in the sex chromosomes. Evolution **67**, 3662–

5 3668.

6 Renner SS. 2014. The relative and absolute frequencies of angiosperm sexual systems: Dioecy,

7 monoecy, gynodioecy, and an updated online database. American Journal of Botany **101**, 1588–

8 1596.

9 Renner SS. 2016. Pathways for making unisexual flowers and unisexual plants: Moving beyond

10 the “two mutations linked on one chromosome” model. American Journal of Botany **103**, 587–

11 589.

12 Renner SS, Müller NA. 2021. Plant sex chromosomes defy evolutionary models of expanding

13 recombination suppression and genetic degeneration. Nature Plants **7**, 392–402.

14 Rice WR. 1984. Sex Chromosomes and the Evolution of Sexual Dimorphism. Evolution **38**, 735–

15 742.

16 Rowe L, Houle D. 1996. The lek paradox and the capture of genetic variance by condition

17 dependent traits. Proceedings of the Royal Society of London. Series B: Biological Sciences **263**,

18 1415–1421.

19 Sacchi B, Humphries Z, Kružlicová J, et al. 2024. Phased Assembly of Neo-Sex Chromosomes

20 Reveals Extensive Y Degeneration and Rapid Genome Evolution in Rumex hastatulus. Molecular

21 Biology and Evolution **41**, msae074.

22 Saumitou-Laprade P, Cuguen J, Vernet P. 1994. Cytoplasmic male sterility in plants: molecular

23 evidence and the nucleocytoplasmic conflict. Trends in Ecology & Evolution **9**, 431–435.

24 Sauquet H, von Balthazar M, Magallón S, et al. 2017. The ancestral flower of angiosperms and

25 its early diversification. Nature Communications **8**, 16047.

26 Schultz ST. 1994. Nucleo-cytoplasmic male sterility and alternative routes to dioecy. Evolution

27 **48**, 1933–1945.

28 She H, Liu Z, Li S, et al. 2023. Evolution of the spinach sex-linked region within a rarely

29 recombining pericentromeric region. Plant Physiology **193**, 1263–1280.
1 Shi J, Toscani M, Dowling CA, Schilling S, Melzer R. 2024. Identification of genes associated with sex expression and sex determination in hemp (Cannabis sativa L.). Journal of Experimental Botany, erae429.

4 Soltis PS, Marchant DB, Van de Peer Y, Soltis DE. 2015. Polyploidy and genome evolution in plants. Genomes and evolution **35**, 119–125.

6 Spigler RB, Lewers KS, Johnson AL, Ashman T-L. 2010. Comparative Mapping Reveals Autosomal Origin of Sex Chromosome in Octoploid Fragaria virginiana. Journal of Heredity **101**, S107–S117.

9 Tao Q, Niu H, Wang Z, Zhang W, Wang H, Wang S, Zhang X, Li Z. 2018. Ethylene responsive factor ERF110 mediates ethylene-regulated transcription of a sex determination-related orthologous gene in two Cucumis species. Journal of Experimental Botany **69**, 2953–2965.

12 Tennessen JA, Wei N, Straub SCK, Govindarajulu R, Liston A, Ashman T-L. 2018. Repeated translocation of a gene cassette drives sex-chromosome turnover in strawberries. PLOS Biology **16**, e2006062.

15 Tonnabel J, Cosette P, Lehner A, Mollet J-C, Amine Ben Mlouka M, Grladinovic L, David P, Pannell JR. 2022. Rapid evolution of pollen and pistil traits as a response to sexual selection in the post-pollination phase of mating. Current Biology **32**, 4465-4472.e6.

18 Tonnabel J, David P, Janicke T, Lehner A, Mollet J-C, Pannell JR, Dufay M. 2021. The Scope for Postmating Sexual Selection in Plants. Trends in Ecology & Evolution **36**, 556–567.

20 Tonnabel J, David P, Pannell JR. 2019. Do metrics of sexual selection conform to Bateman's principles in a wind-pollinated plant? Proceedings of the Royal Society B **286**, 20190532.

22 Torregrosa LJ-M, Rienth M, Romieu C, Pellegrino A. 2019. The microvine, a model for studies in grapevine physiology and genetics. OENO One **53**.

24 Torres MF, Mathew LS, Ahmed I, Al-Azwani IK, Krueger R, Rivera-Nuñez D, Mohamoud YA, Clark AG, Suhre K, Malek JA. 2018. Genus-wide sequencing supports a two-locus model for sex-determination in Phoenix. Nature Communications **9**, 3969.

27 Van Doorn GS, Kirkpatrick M. 2007. Turnover of sex chromosomes induced by sexual conflict. Nature **449**, 909–912.

29 Wang D, Li Y, Li M, et al. 2022. Repeated turnovers keep sex chromosomes young in willows. Genome Biology **23**, 200.
1 Wang Y, Zhang R-G, Hörandl E, Zhang Z-X, Charlesworth D, He L. 2024. Evolution of Sex-linked

2 Genes and the Role of Pericentromeric Regions in Sex Chromosomes: Insights from Diploid

3 Willows. Molecular Biology and Evolution **41**, msae235.

4 Westergaard M. 1958. The Mechanism of Sex Determination in Dioecious Flowering Plants. In:

5 Demerec M, ed. Advances in Genetics. Academic Press, 217–281.

6 Wiberg RAW, Veltsos P, Snook RR, Ritchie MG. 2021. Experimental evolution supports

7 signatures of sexual selection in genomic divergence. Evolution Letters **5**, 214–229.

8 Wright AE, Darolti I, Bloch NI, Oostra V, Sandkam B, Buechel SD, Kolm N, Breden F, Vicoso B,

9 Mank JE. 2017. Convergent recombination suppression suggests role of sexual selection in

10 guppy sex chromosome formation. Nature Communications **8**, 14251.

11 Xue L, Wu H, Chen Y, et al. 2020. Evidences for a role of two Y-specific genes in sex

12 determination in Populus deltoides. Nature Communications **11**, 5893.

13 Yang H-W, Akagi T, Kawakatsu T, Tao R. 2019. Gene networks orchestrated by Me: a single-

14 factor mechanism underlying sex determination in persimmon. The Plant Journal **98**, 97–111.

15 Yang W, Wang D, Li Y, et al. 2021. A General Model to Explain Repeated Turnovers of Sex

16 Determination in the Salicaceae. Molecular Biology and Evolution **38**, 968–980.

17 Yue J, Krasovec M, Kazama Y, Zhang X, Xie W, Zhang S, Xu X, Kan B, Ming R, Filatov DA. 2023.

18 The origin and evolution of sex chromosomes, revealed by sequencing of the Silene latifolia

19 female genome. Current Biology **33**, 2504-2514.e3.

20 Zhang S, Tan F-Q, Chung C-H, et al. 2022. The control of carpel determinacy pathway leads to

21 sex determination in cucurbits. Science **378**, 543–549.

22 Zhang X, Wang G, Zhang S, et al. 2020. Genomes of the Banyan Tree and Pollinator Wasp

23 Provide Insights into Fig-Wasp Coevolution. Cell **183**, 875–889.e17.

24 Zhou R, Macaya-Sanz D, Schmutz J, Jenkins JW, Tuskan GA, DiFazio SP. 2020. Sequencing and

25 Analysis of the Sex Determination Region of Populus trichocarpa. Genes **11**.

26 Zluvova J, Georgiev S, Janousek B, Charlesworth D, Vyskot B, Negrutiu I. 2007. Early Events in

27 the Evolution of the Silene latifolia Y Chromosome: Male Specialization and Recombination

28 Arrest. Genetics **177**, 375–386.
1 Zluvova J, Nicolas M, Berger A, Negrutiu I, Monéger F. 2006. Premature arrest of the male flower meristem precedes sexual dimorphism in the dioecious plant Silene latifolia. Proceedings of the National Academy of Sciences 103, 18854-18859.

4 Zou C, Massonnet M, Minio A, et al. 2021. Multiple independent recombinations led to hermaphroditism in grapevine. Proceedings of the National Academy of Sciences 118, e2023548118.
# 1 Tables

2

3 Table 1. Summary of characterized sex determination systems, including proposed sex-determining genes and their underlying molecular mechanisms. Each entry specifies the species used for characterization, noting that multiple systems may exist within the same genus. The function of each sex-determining gene is indicated as a male (+)/female (+) promoter or male (-)/female (-) suppressor.

<table><thead><tr><td>Genus (Family)</td><td>Evolutionary routes†</td><td>Sex determination system (species)</td><td>Sex-determining genes</td><td>Molecular evolution</td><td>Known molecular mechanism</td><td>Refs</td></tr></thead><tbody><tr><td>Actinidia (Actinidiaceae)</td><td>Gynodioecy?</td><td>XY (A. rufa x A. chinensis)</td><td>Y-linked SyGI (female -) and FrBy (male +)</td><td>SyGI results from a duplication event</td><td>SyGI regulates the Cytokinin signaling pathway</td><td>Akagi et al., 2018; Akagi et al., 2019</td></tr><tr><td>Amborella</td><td>?</td><td>ZW (A. trichopoda)</td><td>W-linked MTN1-2 (male -) and WUS1 (female +)</td><td>Sex-biased expression of the W-linked and Z-linked alleles</td><td>MTN1-2 regulates anther development; WUS1 regulates flower development and carpel formation</td><td>Carey et al., 2024</td></tr><tr><td>Asparagus (Asparagaceae)</td><td>Gynodioecy</td><td>XY (A. officinalis)</td><td>Y-specific SOFF (female -) and TDF1 (male +)</td><td>SOFF results from a duplication and posterior neofunctionalization</td><td>SOFF suppresses pistil development; TDF1 promotes antherdevelopment</td><td>Harkess et al., 2017; Harkess et al., 2020</td></tr><tr><td>Cannabis (Cannabaceae)</td><td>Gynodioecy?</td><td>XY (C. sativa)</td><td>Y-linked FD and X-linked REM16</td><td>Unknown</td><td>FD and REM16 are transcript factors implicated in the flower development</td><td>Shi et al., 2025</td></tr></tbody></table>
<table><tr><td>Carica (Caricaceae)</td><td>Gynodioecy</td><td>XY (C. papaya)</td><td>Y-specific SVP-like (female -)</td><td>Unknown</td><td>SVP-like gene is a MADS-box transcription factor that regulates gynoecium development</td><td>Chae et al., 2021</td></tr><tr><td>Diospyros (Ebenaceae)</td><td>Monoecy</td><td>XY (D. kaki, D. lotus)</td><td>Y-linked OGI (male +) MeGI (female +)</td><td>OGI results from a duplication of the autosomal gene MeGI</td><td>OGI encodes a sRNA that suppresses MeGI</td><td>Akagi et al., 2014; Akagi et al., 2016; Yang et al., 2019; Akagi et al., 2020</td></tr><tr><td>Ficus (Moraceae)</td><td>Monoecy</td><td>XY (F. hispida)</td><td>Y-linked AG2, AG3 (male +) and FT1, FT2 (female -)</td><td>Duplication of the AGAMOUS (AG) gene</td><td>Activation of MYB, NAC, and WRKY in female, and CRC, MYB33, and YABBY1 among others in male</td><td>Zhang et al., 2020; Liao et al., 2023</td></tr><tr><td>Fragaria (Rosaceae)</td><td>Gynodioecy</td><td>ZW (tetraploid species)</td><td>W-specific AFT (female + and male -)</td><td>Only gene located in the SDR that duplicated from an autosome and transposed to a proto-W</td><td>Carpel promoting effect (Auxin pathway) and potentially a male-sterility effect</td><td>Qiao et al., 2024</td></tr><tr><td></td><td></td><td>ZW (octoploid species)</td><td>GMEW and RPPOW genes located within a short SDR W-specific transposable cassette (unknown sex function)</td><td>RPPOW and GMEW moved from an autosomal by retrotransposition and transposition, respectively</td><td>Unknown, but RPPOW could be a lncRNA</td><td>Spigler et al., 2010; Tennessen et al., 2018; Cauret et al., 2022</td></tr><tr><td>Humulus (Cannabaceae)</td><td>Gynodioecy?</td><td>XY (H. lupulus)</td><td>X-linked EXER</td><td>Unknown</td><td>Ethylene receptor 1-like gene</td><td>Akagi et al., 2024</td></tr></table>
<table><tr><td>Phoenix (Arecaceae)</td><td>Monoecy</td><td>XY (all 14 species)</td><td>Y-linked CYP703 (male +), GPAT3 (male +) and LOG (female -)</td><td>LOG Y-gene arose after duplication of an autosomal gene</td><td>Unknown</td><td>Torres et al., 2018</td></tr><tr><td rowspan="3">Populus (Salicaceae)</td><td rowspan="3">Monoecy</td><td>XY (P. deltoides)</td><td>FERR-R (female -) and MSL (male +)</td><td>Duplication of autosomal copy (FERR)</td><td>Y-specific FERR-R generates siRNAs that suppress the autosomal gene FERR. MSL is a TE that generates lncRNAs</td><td>Xue et al., 2020</td></tr><tr><td>XY (P. tremula and P. trichocarpa)</td><td>ARR17 (female +)</td><td>Duplication of autosomal copy</td><td>Y-specific duplicates of ARR17 result in sRNA that silences the autosomal ARR17 copy</td><td>Müller et al., 2020; Zhou et al., 2020</td></tr><tr><td>ZW (P. alba)</td><td>W-specific ARR17 (female +)</td><td>Loss of ARR17 gene from Z chromosome</td><td>ARR17 is involved in a network that regulates floral organ identity</td><td>Müller et al., 2020; Leite Montalvão et al., 2022</td></tr><tr><td rowspan="2">Salix (Salicaceae)</td><td rowspan="2">Monoecy</td><td>XY (S. chaenomeloides and S. arbutifolia)</td><td>ARR17 (female +)</td><td>Y-specific duplicates of ARR17 duplicated and translocated from an autosomal copy</td><td>Y-specific duplicates of ARR17 result in sRNA that silences the autosomal ARR17</td><td>Wang et al., 2022</td></tr><tr><td>XY (S. tiandra and S. mesnyi)</td><td>ARR17 (female +) and MSF (male +)</td><td>Y-specific duplicates of ARR17 and MSF originate from the duplication and translocation of</td><td>Y-specific duplicates of ARR17 result in sRNA that silences the autosomal ARR17. Role of MSF is unknown</td><td>Wang et al., 2024</td></tr></table>
<table><tr><td>Silene (Caryophyllaceae)</td><td>Gynodioecy</td><td>XY (S. latifolia)</td><td>CLV3-like (female -) and WUS1 on the X (male -)</td><td>X copy of the CLV3-like gene has a loss-of-function mutation</td><td>CLV3-like gene is part of the WUS-CLV pathway. WUS1 (X-specific, no Y copy) activates transcription of CLV3 (a carpel inhibitor) that then represses WUS1 (a carpel promoter)</td><td>Kazama et al., 2022; Akagi et al., 2025; Moraga et al., 2025</td></tr><tr><td>Spinacia (Amaranthaceae)</td><td>Monoecy</td><td>XY (S. oleracea and S. turkestanica)</td><td>Y-specific NRT1, bZIP transcription factor, EIF3A, and FCF1 (male +)</td><td>Duplication of an autosomal gene into the Y</td><td>NRT1 (Y-specific) may transport gibberellins and jasmonates that regulate B-class genes AP3 and PI to suppress carpel development and promote stamen initiation</td><td>Ma et al., 2022; She et al., 2023</td></tr><tr><td>Vitis (Vitaceae)</td><td>Monoecy</td><td>XY (V. sylvestris)</td><td>INP1 and PLATZ (male +), YABBY and APRT3 (female -)</td><td>INDEL in INP1 gene specific to females</td><td>INP1 evolved through deletion. YABBY and APRT3 evolved though neofunctionalization</td><td>Badouin et al., 2020; Massonnet et al., 2020; Zou et al., 2021</td></tr></table>

1. The evolutionary route was inferred from the presence of monoecious or gynodioecious species in the same genus, family or order (appears with a ?) from data provided in Renner (2014) and Dufay et al., (2014)
1 **Boxes**

2

3 **Box 1: *Silene latifolia* and its multiple sex-determining genes**

4 Classical genetics has identified three sex-determining regions on the *S. latifolia* Y chromosome:

5 the gynoecium-suppression factor (GSF), the stamen-promoting factor (SPF) and the male-
6 fertility factor (MFF). It was known that GSF and SPF included sex-determining genes acting
7 early in the development because deletions in these regions resulted in early developmental
defects, while the genes in the MFF acted much later and were involved in pollen production.
8

9 Genomics has identified Clavata3 in the GSF region (Kazama et al., 2022, Akagi et al., 2025, Moraga et al., 2025), the immunophilin-like FKBP42/TWISTED DWARF1 (TWD1) gene (Akagi et al., 2025) or a SCARECROW-LIKE 4/7 (SCL4-7) pseudogene (Moraga et al., 2025) in the SPF region - all developmental genes, and genes including CYP704B1, a gene involved in the sporopollenin synthesis, a key component of the pollen wall, and THI-1, another tapetum-expressed gene critical for pollen maturation in the MFF region (Moraga et al., 2025).

10

11

12

13

14

15 As explained in the main text, Clavata3 may be involved in a balance with Wuschel1 controlling
16 sex determination, as suggested by gene copy number, expression and functional analysis of
17 those genes.

16

17

18 The location of those sex-determining genes on the sex chromosomes showed that Clavata3
19 and Wuschel1 are stratum 1 gene (diverged first), while the other genes are from the other
20 strata (diverged later on in the evolution of the sex chromosomes).

20

21

21 Recombination suppression between X and Y thus initiated around Clavata3 and Wuschel1, a
22 relatively small region in *S. latifolia* (while stratum 2 is much larger). Synteny analysis of X, Y and
23 outgroup genomes identified an inversion on the X as potential cause for recombination
24 suppression event forming stratum 1. This can be interpreted in the light of the Charlesworth
25 and Charlesworth (1978)'s model (also called the two-gene model, see Figure 2A for details),
26 with Clavata3 and Wuschel1 being the two fertility genes assumed by the model. The X
27 chromosome carries the Clavata3 male-sterility mutation (WUS-biased expression), while the Y
28 chromosome carries the female-sterility Wuschel1 one (CLV3-biased expression), which sets up
29 an male-determining Y chromosome.

22

23

24

25

26

27

28

29
1. How to interpret the other sex-determining genes? Given the size of the Y chromosome, which includes hundreds of genes, there had to be genes essential for male development on that chromosome. The genes without any pleiotropic effects in females could have been lost neutrally on the X, passively enriching the Y for that kind of genes. Another possibility is sexually-antagonistic selection that favored the loss of male genes from the X chromosome in an "active" manner (through selection). Pollen genes for example could fall in this category as they are often the target of sexual selection in plants (Tonnabel et al., 2021).

9. *Figure box 1 here*

10. Genetic model for sex determination in *Silene latifolia*. Three sex-determining regions on the Y are indicated (see text for more details). Candidate genes found in those regions are indicated (see Text for more details). Effects on the male and female flower development are shown. (Picture from Zluvova et al., 2006. Copyright (2006) National Academy of Sciences.)
1 **Box 2: Sex determination in grapevine, and how domestication can break dioecy**

2 *Vitis sylvestris*—the wild grapevine—is a dioecious species with a homomorphic XY system,
3 likely involving two sex-determining genes: a male-determining gene and a female-suppressing
4 gene (Coito et al., 2019). Most *Vitis vinifera* (domesticated grapevine) varieties are
5 hermaphroditic, possessing a modified Yh haplotype (reviewed in Coito et al., 2019). In the
6 grapevine literature, X, Y and Yh haplotypes are called F, M and H. Several studies have
7 explored sex determination in both wild and cultivated grapevines using comparative genomics,
8 transcriptomics, and bioinformatics/GWAS approaches and have identified a small sex-
9 determining region of about 100 Kb with several candidate genes (Coito et al., 2017; Massonnet
10 et al., 2020; Badouin et al., 2020; Zou et al., 2021; Dong et al., 2023).

11 INP1, a pollen-specific gene with a partially deleted and inactive allele on the F haplotype, has
12 been proposed as a male-determining candidate gene. There is however an ongoing debate
13 regarding the identity of the female-suppressing gene. Some studies have suggested YABBY3,
14 one of the Yabby genes—an important developmental gene family—as the key candidate
15 (Massonnet et al., 2020; Zou et al., 2021; Dong et al., 2023). YABBY3 is clearly associated with
16 female-fertility in GWAS including *V. vinifera* cultivars (Zou et al., 2021). However, it is not in
17 the non-recombining region defining the sex-determining region in *V. sylvestris* (Massonnet et
18 al., 2020; Badouin et al., 2020 but see Zou et al. 2021). Others have pointed to APRT3—a gene
19 involved in the cytokinin pathway (Coito et al., 2017; Badouin et al., 2020). Cytokinin treatment
20 is known to stimulate carpel formation in grapevines (discussed in Coito et al., 2019). In *Vitis
21 amurensis*, APRT3 seems to be involved in sex-determination, although its expression patterns
22 seem to differ from that of *V. sylvestris* (Men et al., 2021).

23 ETO1, also known as Ethylene Overproducer-1, is another candidate gene. It plays a role in
24 inhibiting the enzyme activity of ACS (1-aminocyclopropane-1-carboxylic acid synthase), which
25 is crucial for ethylene biosynthesis (Carrasco et al., 2020; Badouin et al., 2020). In other plant
26 species like Cucurbitaceae, ethylene is a well-known regulator of sex determination, influencing
27 the development of male and female flowers through the regulation of ethylene biosynthesis
28 genes such as ACS (Garcia et al., 2020; Little et al., 2007; Mibus et al., 2004; Boualem et al.,
29 2015; Tao et al., 2018; Manzano et al., 2018). Mutations affecting ethylene sensitivity can lead
1 to changes in flower sex expression, as seen in *Cucurbita pepo*, where ethylene-insensitive
2 mutations result in androecy (male-only flowers) (Garcia *et al.*, 2020; Manzano *et al.*, 2018).

3 Further work is necessary to disentangle the role of these different genes. Research on Platz, on
4 the other hand, has demonstrated its role in reflexed stamens—a key phenotype of female
5 grapevine flowers, using CRISPR-Cas9 and microvines - dwarf grapevines that flower multiple
6 times a year and serve as a model for grapevine functional genomics (Chaib *et al.*, 2010;
7 Torregrosa *et al.*, 2019; Dalla-Costa *et al.*, 2019; Iocco-Corena *et al.*, 2021).

8 This demonstrates that in dioecious species, the sexual morph phenotypes can be influenced by
9 more than two genes, even when the sex-determining region is small and includes a limited
10 number of genes, as is the case in grapevines. The domestication process of this remarkable
11 crop had a substantial impact on grapevine dioecy, resulting in the fixation of favourable traits
12 and the selection of plants with hermaphrodite flowers, thereby enhancing yields and
13 facilitating cultivation (Zou *et al.*, 2021; Kui *et al.*, 2020; Grassi *et al.*, 2021). This selection
14 process led to the breakdown of dioecy, allowing for self-pollination and higher fruit production
15 (Zou *et al.*, 2021; Janik *et al.*, 2007). Sequencing of the M, F and H haplotypes in many
16 accessions has provided very useful information on the evolution of the H haplotypes, many of
17 which derive from recombination events between M and F haplotypes (Badouin *et al.*, 2020;
18 Zou *et al.* 2021; Dong *et al.*, 2023). These changes have direct implications for grapevine
19 production and quality traits, highlighting the ecological and evolutionary consequences of
20 grapevine sex determination and domestication.

21

22 *Figure box 2 here*

23

24 Gene content and annotation of the wild grapevine sex-determining region. Both M (= Y) and F (= X)
25 haplotypes are shown. The "≠" symbol denotes differences in expression levels (RNA-seq) among sexes
26 (male, female or hermaphrodite). The "aa" symbol denotes amino acid replacements between M and F
27 coding sequences. The "+++" and "++" denote "very strong" and "strong" association to sex phenotype
28 in GWAS analysis, respectively (Zou *et al.*, 2021). Red asterisks point to the best candidates highlighted
29 in the literature so far and discussed in the text. Adapted from Massonnet et al. (2020) and Badouin et
30 al., (2020).
1

A list of figure legends

Figure 1: Advances in characterizing plant sex chromosomes using genomics in the last 15 years.

Most of the systems have been characterised using non-standard methods (see text). Data on
*Laurus azorica* are unpublished. The phylogenetic tree was adapted from Ming et al. (2011).

Taxa in bold include dioecious species with documented sex chromosomes by cytogenetics
listed in Ming et al. (2011).

7

Figure 2: Models for the evolution of dioecy and sex chromosomes (A) The classical gynodioecy model (Charlesworth and Charlesworth, 1978). This model describes the evolution of dioecy via the gynodioecy pathway, involving two fertility genes: M (male function) and F (female function), with fertility alleles (Mf and Ff). Dioecy evolves in two steps: (i) Female emergence: A male-sterility mutation (Msm) arises, which is recessive, leading to the formation of females, (ii) Male emergence: A dominant female-sterility mutation (Fsm) appears, producing males.
Selection favors recombination suppression, ultimately leading to the formation of X and Y haplotypes. (B) The Cytoplasmic Male Sterility (CMS) Model. This model, based on Schultz (1994) and Maurice et al. (1994) (see also Fruchard and Marais, 2021), starts with a CMS factor in the mitochondrial genome, which suppresses male function, creating females. A nuclear restorer-of-fertility gene (Rf) counteracts CMS, restoring hermaphroditism. A female-sterility mutation (Fsm) later arises on the same chromosome as Rf, leading to the evolution of males.

As in the classical model, selection favors recombination suppression, eventually forming X and Y haplotypes. (C) The monoecy pathway model (Lesaffre *et al.*, 2024a,b). This model builds on the idea that sex differentiation evolves gradually in monoecious species (see Pannell, 2017).

The initial step involves two plant types: Male-biased monoecious plants (producing more male than female flowers), Female-biased monoecious plants (producing more female than male flowers). Through multiple steps, involving multiple genes (annotated Q1, Q2, etc... because of their quantitative effect on sex phenotype) possibly on different chromosomes, sex determination becomes more canalized (Lesaffre *et al.*, 2024a). In the final stage, a single major-effect locus can emerge, replacing the original polygenic sex determination system.

However, if multiple sex-determining genes are already linked on the same chromosome, the
system may evolve into a two-gene (or multi-gene) system rather than a purely single-locus model.

Figure 3: Evolutionary routes to dioecy and single- vs. two-gene systems. Data summarized in Table 1 highlights different evolutionary trajectories leading to dioecy, particularly in relation to whether sex determination is governed by a single-gene or two-gene system. 2+ genes: two genes or more, 1-2 genes: one or two genes (depending on the species), 1 gene: single-gene system. *Populus* and *Salix* are categorized as single-gene systems, as nearly all studied species rely on a single dominant sex-determining gene. However, in two species, an additional candidate gene has been proposed, though its role remains unconfirmed. *Humulus* represents a clear case of sex chromosome turnover, as it exhibits an X-to-autosome balance system, which is considered a later stage of sex chromosome evolution. Consistent with theoretical predictions (Van Doorn and Kirkpatrick 2007; Charlesworth and Harkess, 2024), turnover events are expected to result in single-gene sex determination systems. *Cannabis* was not included in the analysis, as it is currently difficult to conclude about the number of genes involved.

Gynodioecy-derived dioecy tends to be associated with two-gene systems, aligning with models that propose a stepwise evolution involving male-sterility and female-sterility mutations.

Monoecy-derived dioecy, on the other hand, can lead to either one-gene or two-gene systems, reflecting the more gradual and flexible nature of sex determination in monoecious lineages.

20
21
<figure><img src="image_2.png" /><figcaption>Figure 1
159x105 mm (x DPI)</figcaption></figure>

1

2

3

4
A

<figure><img src="image_3.png" /></figure>

B

<figure><img src="image_4.png" /></figure>

C

<figure><img src="image_5.png" /><figcaption>Figure 2
159x65 mm (x DPI)</figcaption></figure>

1
2
3
4
<figure><img src="image_6.png" /><figcaption>Figure 3
159x227 mm (x DPI)</figcaption></figure>

1

2

3

4
<figure><img src="image_7.png" /><figcaption>Figure box 1
159x134 mm (x DPI)</figcaption></figure>

1
2
3
4
<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>Figure box 2
159x114 mm (x DPI)</figcaption></figure>

1
2
3