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Biological Journal of the Linnean Society, 2010, 100, 737–752. With 3 figures REVIEW ARTICLE Sex chromosomes and sex determination pathway dynamics in plant and animal models BOHUSLAV JANOUSEK* and MARTINA MRACKOVA Institute of Biophysics, Kralovopolska 135, Brno, CZ 612 65, Czech Republic Received 23 November 2009; revised 17 March 2010; accepted for publication 17 March 2010 bij_1470 737..752 In this review, we discuss and compare data obtained from animal and plant models, focusing our attention on the mechanisms that affect sex linkage and changes in sex-determining pathways. Patterns in data across taxa suggest that sex bias and the dynamics that occurs within hybrid zones can play an important role in these processes that enable the spread of some otherwise handicapped genotypes. We discuss the data obtained from several main plant model species in the light of the patterns demonstrated in animal models. In several plant models, we discuss possible differences in the age of their sex-determining pathways and the age of their current sex chromosomes. We also address an open question: how can an X/A ratio based sex-determining system evolve from a sexdetermining system based on two genes on the Y chromosome that control two separate sex-determining pathways (for the control of gynoecium suppression and anther promotion)? Taking inspiration from the well described mechanisms involved in sex determination dynamics in animals, we suggest a hypothetical stepwise scenario of change of the plant sex-determining system based on two separate sex-determining pathways (for the control of gynoecium suppression and anther promotion) into the other sex-determining systems. We suppose that an intermediate step occurs before shift to X/A based sex determination. At that phase, sex determination in plants is still based on an active Y chromosome, although there exists already a connected control of both sex-determining pathways. We suggest that this connection is enabled by the existence of the genes that control sexual dimorphism in the vegetative state of plant development, and that, in some circumstances, these genes can become sexdetermining genes. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100, 737–752. ADDITIONAL KEYWORDS: evolution – hybrid zones – sexual dimorphism. INTRODUCTION The mechanisms involved in sex determination are some of the most dynamic from an evolutionary point of view. Gonochorism prevails in current animal model species, although hermaphroditism is prevalent in angiosperm plants (approximately 90%). However, many plant families also include dioecious species, and dioecy is present in several crop species (Grant et al., 1994). The sex chromosomes in plants and animals have evolved independently, although the mechanisms of *Corresponding author. E-mail: [email protected] their evolution are probably very similar. The necessity of a synthesis of data obtained from various model species can be demonstrated by considering the research of the stepwise arrest of sex chromosome recombination. In general, it is hypothesized that sex chromosomes evolved from a specific pair of autosomes carrying some sex-determining gene(s). Subsequently, the newly-formed sex chromosomes stopped recombination in a small region around the sex-determining locus. Sex chromosomes in this early stage of evolution are not cytologically distinguishable (homomorphic). The process of recombination suppression then progresses through almost the entire sex chromosome. The results of the human genome project have © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100, 737–752 737 738 B. JANOUSEK and M. MRACKOVA revealed a correlation between the localization of genes along the X chromosome and silent site sequence divergence from their Y homologues. This silent site sequence divergence serves as a measure of the time since the X and Y copy stopped recombining. The regions containing genes with similar levels of divergence were named ‘evolutionary strata’ based on a metaphorical similarity to geological strata (Lahn & Page, 1999). Originally, the differences in silent site sequence divergence were explained by several large chromosome rearrangements (Lahn & Page, 1999), although later research showed that the boundaries between some strata are blurred in mammals, suggesting that epigenetic mechanisms or small inversions should also be taken into account (Skaletsky et al., 2003). However, discrete strata, suggesting the role of chromosomal rearrangements in the arrest of recombination in birds, were identified in chickens (Nam & Ellegren, 2008). In humans, computer simulations of inversions followed by the analysis of putative breakpoints conducted by Lemaitre et al. (2009) indicate that inversions played a crucial role in the origin of strata 4 and 5. Unfortunately, it was not possible to obtain clear results for stratum 3 because its age precludes successful use of the method. Results from nonvertebrate models suggest that the stepwise spread of a nonrecombining region is generally widespread process, probably connected with the existence of any nonrecombining region. Gradients in the silent site divergence have been found in plants (Silene latifolia; Nicolas et al., 2005) and fungi (Microbotryum violaceum; Votintseva & Filatov, 2009). The dioecious plant S. latifolia has much younger sex chromosomes compared to humans but the strata are already present in these chromosomes (Nicolas et al., 2005; Bergero et al., 2007), which suggests that S. latifolia is a promising model for the study of the initial mechanisms of recombination arrest. Despite substantial progress in the knowledge of sex chromosome evolution, there are still topics that are not well understood. One of them is why sex determination (including sex determination systems on a chromosomal level) is conserved in some taxonomic groups, whereas, in others, it is highly dynamic. In this review, we present the current status of knowledge of sex determination and sex determination plasticity in animal and plant models. We also discuss possible causes of differences and similarities between animal and angiosperm models. We then suggest a possible model of shifts in the sex-determining systems in plants from a typical plant sex determination system evolved from gynodioecy (Charlesworth & Charlesworth, 1978) into sexdetermining systems more similar to the systems found in animal models. SEX DETERMINATION PATHWAYS AND SEX CHROMOSOME DYNAMICS IN ANIMALS THE VARIABILITY OF SEX CHROMOSOMES AND SEX-DETERMINING PATHWAYS IN INSECTS Sex-determining systems in insects exhibit a wide range of diversity. In addition to XX/XY and ZW/ZZ sex-determining systems, some taxa have evolved systems that have not been found in any other classes: for example, the haploid/diploid sexdetermining system or, more exactly, the complementary sex-determining system, present in social insects such as Hymenoptera and Isoptera (Sánchez, 2008). Surprising findings from recent years point to a common origin of these extremely diverse sexdetermining systems. The role of the doublesex gene is evolutionary well conserved even in species with complementary sex determination (Sánchez, 2008). The doublesex (dsx) gene was originally described in Drosophila melanogaster. It is involved in the control of somatic sexual differentiation in both sexes. Two functional products are encoded by dsx: one product is expressed in females and represses male differentiation, and the other is expressed in males and represses female differentiation (Burtis & Baker, 1989). In the order Diptera (most often studied), the prevailing sex-determining systems are of the XX/XY type (either with the active role of the Y-chromosome or X/A ratio based). These systems diversified into different types of sex determination based on the selective elimination of paternal chromosomes in some fraction of offspring. In at least one species, Musca domestica, extreme variability and plasticity of the sex determination is present. Sex linkage change is controlled by the presence of the gene modifier M, which transforms individuals without the Y chromosome into males. In some populations, this modifier is fixed and sex determination is under the control of another unlinked gene, FD, which causes females to arise even in the presence of the male factor. This sex linkage change is therefore connected with the switch to female heterogamety (Dübendorfer et al., 2002). In the main Dipteran model species, D. melanogaster, this extent of sex determination plasticity is not present. In this species, the probable role of dosage compensation and Sex-lethalbased sex determination was suggested by Schütt & Nöthiger (2000) as an explanation of the stability of sex determination. Sex-lethal (Sxl) is a master gene that regulates the splicing of doublesex, so that its early expression in females (based on the X/A ratio) leads to a female type of doublesex splicing (Bell et al., 1988). Simultaneously, this early expression of Sex-lethal in females prevents the expression of male-specific lethal 2 (msl-2) that controls dosage compensation by doubling the expression levels of the X-linked genes in D. melanogaster males (McDowell, Hilfiker & Lucchesi, © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100, 737–752 SEX CHROMOSOMES AND SEX DETERMINATION PATHWAY DYNAMICS 1996). A great variability of the sex chromosomes is, however, observed in the various species of the genus Drosophila (Charlesworth & Charlesworth, 2005). In Drosophila affinis, fertile male individuals without a Y chromosome can be found (Voelker & Kojima, 1971). The sex determination of another often studied order, Lepidoptera, was ancestrally based on the ZO/ZZ system. This system, however, evolved in many Lepidopteran species, including the main Lepidopteran model Bombyx mori to the ZW/ZZ sex determination system (Traut, Sahara & Marec, 2007). The important mechanism for the spatial spread of the novel sex chromosomes was revealed based on the data obtained in grasshopper Podisma pedestris (Veltsos, Keller & Nichols, 2008). In the hybrid zone, the neo Y-chromosome can spread despite the harm that it causes its carriers because sexually antagonistic effects of the neo-Y chromosome induce selection in favour of the neo X-chromosomes causing their spread, such that they simultaneously remove the original X chromosome from the population. This selection then helps to the neo Y-chromosome to spread in the population. Paradoxically, processes of Y-chromosome degeneration can theoretically support its expansion in metapopulations (Veltsos et al., 2008). This finding suggests that hybrid zones can enable evolutionary processes that would not be possible in a single unstructured population. It is also possible to speculate that similar or related processes, with a different focus of antagonistic selection, may explain the spread of other types of chromosomal rearrangements considered to be subject to positive frequency-dependent selection (Pannell & Pujol, 2009) SEX-DETERMINING SYSTEMS IN FISH AND THE HYPOTHESES EXPLAINING THE DYNAMICS OF SEX-DETERMINING PATHWAYS Fish are an important group of models that have undergone dynamic evolution in sex determination pathways and sex determination. Sex determination in fish is predominantly established by genotype but it can be often influenced by environmental factors (Baroiller, D’Cotta & Saillant, 2009). Although gonochorism is common, there are also several hundreds of hermaphroditic species (Devlin & Nagahama, 2002). Moreover, in some species, individuals undergo a sex change (males to females or vice versa) during their life in response to age, social factors or temperature (Schartl, 2004). The variability of the sexdetermining systems in fish is representented not only by the presence of both of the two common heterogametic systems (XY male heterogamety and ZW female heterogamety), but also by occurrence of several different modifications of these primary systems (e.g. in genus Xiphophorus; Kallman, 1984). Recently, two 739 main model genera have been used in studies of sex determination: Oryzias and Xiphophorus (especially the species Xiphophorus maculatus). All known species from the genus Oryzias belong to one of three phylogenetically distinct groups: latipes, javanicus, and celebensis (Takehana, Naruse & Sakaizumi, 2005). The species Oryzias latipes and Oryzias curvinotus from the group latipes have an XY sex-determining system. In these species, the sex-determining gene is DMY (also called dmrt1bY, according to its origin from the duplicated autosomal gene dmrt1; Matsuda et al., 2002; Nanda et al., 2002; Matsuda et al., 2003). However, this sex-determining gene is not present in all Oryzias species. Genetic mapping has revealed that different genes and different linkage groups are involved in sex determination in the other two groups (Kondo et al., 2003; Takehana et al., 2007a). Moreover, the sex-determining system in Oryzias hubbsi, a species from the javanicus group, is not XX/XY as in closely-related Oryzias dancena, but ZZ/ZW (Takehana et al., 2007b). Genetic mapping also showed that sex chromosomes of O. hubbsi and O. dancena evolved from different autosomal ancestors (Takehana et al., 2007b). Another remarkable model is the genus Xiphophorus (Schartl, 1995). Studies in this genus (Morizot et al., 1991) revealed sex determination systems that vary from simple XX/XY or ZZ/ZW systems to multifactorial sex determination (Volff & Schartl, 2001). The species X. maculatus has an unusual sex-determining system with three different homomorphic sex chromosomes: X, Y, and W (Kallman, 1984). Different combinations of sex chromosomes determine whether fish become males or females: males have XY or YY, and females have XX, WX or WY gonosomal pairs (Volff & Schartl, 2001). Several models have attempted to explain this three-chromosomes system. According to Volff & Schartl (2001), each sex chromosome could have a different copy number of genes ‘pushing’ the development in male direction (male determining gene): Y, two copies; X, one copy, and W, no copies. According to the number of sex-genes an individual becomes male or female. The evolutionary events leading to this type of sex determination system are still unclear but, according to experimental data, it is possible to surmise that the mechanisms of sexdetermining pathway evolution in fish are similar to the mechanisms taking place in insects. According to hypothesis of Ezaz et al. (2006), X. maculatus is caught in a transition between the XY and ZW systems. Quantitative basis of sex determination was recently described also in related species Poecilia reticulata, which becomes a widely studied model. Cross of the individuals from different populations revealed the X chromosomes differring in the strenght of their male (respectively female) tendency. Male organ develop- © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100, 737–752 740 B. JANOUSEK and M. MRACKOVA ment is therefore possible even in the absence of the Y chromosome (Tripathi et al., 2009). Schartl (2004) presented a theory explaining the mechanisms the sex linkage change in fish that can also be applied to other taxonomic groups. This theory is derived from the theory of Wilkins (1995) concerning the evolution of sex-determining cascades. Wilkins’ theory was deduced from the data obtained in Caenorhabditis elegans. Wilkins (1995) hypothesized that the evolution of sex-determining cascades involves the sequential acquisition of new gene functions in reverse order (the last acquired gene function performs the first step in the cascade). Schartl (2004) adopted the theory of Wilkins (1995), but also stressed the role of gene duplications in the evolution of sex-determining pathways. According to Schartl’s theory, some of the genes in the sex-determining pathway can be changed into the master sex-determining gene by duplication. Important progress in the theory of the turnover of sex chromosomes was achieved by van Doorn & Kirkpatrick (2007), who showed that sexually antagonistic autosomal gene can under some conditions stimulate ‘hijacking’ of sex determination by a new sex expression-influencing mutation that occurs in its proximity. This process leads to the formation of the neo-X and neo-Y from the autosome carrying sexually antagonistic locus, whereas the original Y-chromosome is lost from the population and the original X becomes an autosome. The great evolutionary stability in some taxonomic groups can be therefore explained by presence of the genes important for male fertility on the original Y that are absent on the X, by the presence of dosage compensation, and/or by the presence of the sexualy antagonistic genes on the original sex chromosomes. The conditions for the hijacking of sex determination are then more restrictive. Sex-limited expression can, however, lead to the loss of polymorphism at the ancestral sex chromosomes and make the turnover of sex chromosomes easier. THE ROLE OF HYBRIDS ZONES IN THE PLASTICITY OF SEX DETERMINATION AND DYNAMICS OF SEX CHROMOSOMES: LESSONS FROM AMPHIBIANS The role of sex bias in the evolution of sex-determining pathways and sex chromosomes is exhibited in the Japanese frog, Rana rugosa. This species forms population groups varying in type of heterogamety and morphology of sex chromosomes. The main four groups are named according to geographical locations (Fig. 1), and a recently discovered group is called ‘Neo-ZW’ according to phylogeny (Ogata et al., 2008). Three of them have the XX/XY sex determination system, whereas the north-west Japan and the Neo-ZW show female heterogamety (ZZ/ZW) (Miura et al., 1998; Ogata et al., 2008). West Japan and Kanto groups have Figure 1. Scheme explaining the role of sex bias in sex linkage change and heteromorphic chromosome formation in Rana rugosa. Big arrows show the sex and direction of movement of individuals founding the derived populations. The male bias in the north-west population is suppressed by the newly arisen locus responsible for sex change (x to W transformation). In the central population, the original female bias is compensated for by the accumulation of male advantageous genes on the original y chromosome (y to Y transformation) (sensu Ogata et al., 2003). cytologically indistinguishable sex chromosomes, whereas central and north-west Japan groups possess heteromorphic sex chromosomes (Miura et al., 1998). According to sequence similarities, it can be deduced that the chromosomes Y and Z originated from the west Japan type of sex chromosomes, whereas X and W originated from the Kanto type of sex chromosomes. These results suggest that small group(s) of individuals were isolated geographically from the original population and their sex chromosome evolution pathways diverged. Afterwards, these two groups (west Japan and Kanto) could have met each other again and reciprocally hybridized (Fig. 1). Subsequently, subpopulations of progeny could have been separated from their parental populations and evolved independently. This theory was experimentally tested by artificial crossings between these ‘parental’ groups with homomorphic sex chromosomes (Ogata et al., 2003). The crossings between west Japan females and Kanto males produced progeny with a male-biased sex ratio, whereas the reciprocal crossings produced progeny with a female-biased sex ratio. These results suggest that the sex bias could cause a strong positive selective pressure for the minor sex-favouring gene to reestablish the 1 : 1 sex ratio. This hypothesis is also supported by the results of phylogenetic analysis based on mitochondrial sequences. This example illustrates that intense sex bias can promote accumulation of © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100, 737–752 SEX CHROMOSOMES AND SEX DETERMINATION PATHWAY DYNAMICS sexually antagonistic genes, leading to formation of heteromorphic sex chromosomes or even the acquisition of a new dominant sex determiner that caused the evolution of a novel sex-determining system (ZW/ZZ). These data well support the theoretical studies of van Doorn & Kirkpatrick (2007). SEX CHROMOSOMES AND PLASTICITY OF SEX DETERMINATION IN MAIN PLANT MODEL SPECIES Although most angiosperm plant models are reported as having stable genetically based sex-determining systems, even in angiosperm plants, some cases of nongenetic sex determination and fluctuating sex occur (e.g. the genus Arisaema: Policansky, 1981; Renner, Zhang & Murata, 2004). Moreover, even in species with well established genetic sex determination, a certain level of instability of sexual phenotype can be found (Shull, 1911; Winge, 1931; Ainsworth et al., 2005). The research of this problem in plants is rare in comparison with animal models. So far, no cases of change of XX/XY to ZW/ZZ system (or other sexdetermining system) have been described in plants. Here, we present an overview of the data concerning the plasticity of the sex chromosomes and sexdetermining pathways that were obtained in a main angiosperm model species. As described above, the sex-determining pathways in animals (or at least in their large taxonomic units) are highly conserved. In all chordates and lancelets, sex organ differentiation is likely based on the action of the sex steroid hormones (Mizuta & Kubokawa, 2007) and their receptors (Baker & Chang, 2009) and, as decribed above, a large proportion of invertebrates have sex-determining pathways based on the gene doublesex (Sánchez, 2008). On the other hand, dioecy evolved independently in plants more than 100 times to account for 160 plant families that include dioecious species (Charlesworth and Guttman, 1999) and, thus, it is very unlikely that the sex-determining pathways in all dioecious plants are of the same origin. Moreover, several different routes leading to dioecy have been described: from hermaphroditism through gynodioecy (Charlesworth & Charlesworth, 1978), from selfincompatibility through polyploidization (e.g. species of the genus Lycium; Miller & Venable, 2000), and directly from monoecy (e.g. species of the genus Siparuna, Renner & Won, 2001). The evolutionary routes to androdioecy are probably very rarely seen in the nature. The route from hermaphroditism through androdioecy is unlikely to evolve because the conditions for the invasion and spread of males among hermaphrodites are severe (Charlesworth & Charles- 741 worth, 1978; Pannell, 2002). The most famous proposed example of this route, the genus Datisca, has not been confirmed by more extensive phylogenetic studies (Zhang et al., 2006). Similarly, the genus Acer as an example of the route from heterodichogamy through androdioecy (Pannell & Verdú, 2006) was not further confirmed (Renner et al., 2007). A putative candidate for the origin of the dioecy through androdioecy could be subdioecious Fragaria virginiana (incipient ZW/ZZ system), as indicated by the presence of the dominant suppressor of male fertility on the proto-W chromosome and by the presence of the recessive allele causing female sterility on the proto-Z chromosome (Spigler et al., 2008; Moore, 2009). However, it is not possible to reject the hypothesis that this status is secondary unless extensive phylogenetic studies are carried out. Because of the frequently independent origin of sex determination in individual plant families or even genera, we discuss the topics of sex determination plasticity and sex chromosome changes separately in the chosen model genera and species. SILENE LATIFOLIA AND SILENE DIOICA: SPECIES CONSIDERED TO HAVE STABLE SEX DETERMINATION SHOW SOME PLASTICITY OF SEX EXPRESSION A small cluster of dioecious species in the genus Silene have evolved chromosomal sex determination and sex chromosomes relatively recently, within the last 10 Myr. Five dioecious Silene species (Silene heuffelii, S. dioica, S. latifolia, Silene diclinis, and Silene marizii; the former section Elisanthe) are very closely related (1–2 Myr of divergence), and it was previously considered that all five species had morphologically similar sex chromosomes (Nicolas et al., 2005). Recently, neo-sex chromosomes originated by reciprocal translocation between the original Y chromosome and an autosome were reported in S. diclinis (Howell, Armstrong & Filatov, 2009). Silene latifolia (Fig. 2A, B) is a widely studiedj dioecious model. The mechanism of sex determination in this species is based on the presence of three kinds of genes that are located in the nonrecombining region of the Y-chromosome: stamen promoting gene(s) [often called stamen-promoting function to emphasize that the exact molecular principle of its (or their) action is not yet known] promote the development of anthers and filaments (Donnison et al., 1996; Farbos et al., 1999), male fertility gene(s) (called male fertility function) enable the production of the fertile pollen (Donnison et al., 1996; Farbos et al., 1999), and gynoecium suppressing gene(s) (often called gynoeciumsuppressing function) control the arrest of the gynoecium development in males (Donnison et al., 1996; Lardon et al., 1999). © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100, 737–752 742 B. JANOUSEK and M. MRACKOVA A B C D Figure 2. Examples of two dioecious models from the genus Silene. A, Silene latifolia male. B, Silene latifolia female. C, Silene colpophylla male. D, Silene colpophylla female. A good illustration of the fact that even plant species with a well established sex determination system based on the active Y chromosome and with a good phenotypic stability of the sexual phenotype (van Nigtevecht, 1966) are prone to changes in sex determination is the finding of a dominant autosomal mutation in S. latifolia that causes transformation of males to androhermaphrodites (Lardon et al., 1999). The androhermaphroditism in S. latifolia can be also induced by the action of the DNA hypomethylating (Janousek, Siroky & Vyskot, 1996; Janoušek, Grant & Vyskot, 1998), or histone H4 hyperacetylating drugs (Zluvova et al., 2008a). The assumed mechanism of these agents is DNA hypermethylation of the Y-linked gynoecium suppressor that occurs as a consequence of a genome hypomethylation (Janousek et al., 1996; Janoušek et al., 1998). This hypermethylation is probably the result of defence mechanisms that serve to inactivate transposable elements activated by overall DNA hypomethylation (or histone hyperacetylation) (Zluvova et al., 2008a). There are already some experimental data concerning the mechanism of gynoecium suppression in males. Histological studies have revealed a reduction of cell division in the central part of the male flower meristem (Matsunaga, Uchida & Kawano, 2004). Molecular studies have revealed the role of homologues of Arabi- dopsis thaliana SHOOTMERISTEMLESS (STM) and CUP SHAPED COTYLEDON (CUC) 1 and CUC2 genes in the arrest of the gynoecium development in S. latifolia males (Zluvova et al., 2006). The data (Matsunaga et al., 2004; Zluvova et al., 2006) suggest that an absence of SHOOTMERISTEMLESS (STM) and the presence of CUP SHAPED COTYLEDON (CUC) 1 and CUC2 transcripts in the central part of the male flower meristem are the cause of reduced meristematic activity in this region. The results obtained by Kazama et al. (2009) indicate a possible role of SUPERMANlike gene in the suppression of the anther development in S. latifolia females. Interestingly, some sex-specific differences in gene expression patterns were detected in plants before flowering, suggesting a hidden sexual dimorphism in S. latifolia (Zluvova et al., 2008b; J. Zluvova, unpubl. data). The existence of such a dimorphism is also supported by the data showing quantitative differences between males and females in photosynthesis (Gehring & Monson, 1994) and in other quantitative traits (Laporte & Delph, 1995; Delph et al., 2005). Because of the large number of the sexually dimorphic traits, it is reasonable to suppose that a master gene(s) for their control is located on the Y chromosome and that autosomal genes with sex-limited expression are involved in sexual dimorphism in S. latifolia. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100, 737–752 SEX CHROMOSOMES AND SEX DETERMINATION PATHWAY DYNAMICS SILENE, SECTION OTITES: SEX LINKAGE CHANGE OR INDEPENDENT EVOLUTION OF DIOECY? Section Otites is a group of morphologically similar dioecious species (Wrigley, 1986) that are evolutionary distant from S. latifolia (Desfeux et al., 1996; Mrackova et al., 2008). Some evidence suggests that the sex-determining system of S. otites, the most studied species of this group, could be ZW/ZZ (Correns, 1928; Sansome, 1938). However, work by Warmke (1942) suggested an XX/XY sex determination system. Genetic mapping performed in a closelyrelated species S. colpophylla (Fig. 2C, D) has shown that the sex chromosomes of this species evolved from different pair of autosomes than dioecious species from the section Elisanthe, and that sex determination in this species is based on male heterogamety (XX/XY type) (Mrackova et al., 2008). Phylogenetic analysis proved that the sex chromosomes developed independently in S. colpophylla and in the species from the section Elisanthe that are closely related to S. latifolia (Mrackova et al., 2008). Apart from the genus Silene, only a few cases of independent origins of sex chromosomes within one genus have been reported in plants (Renner & Won, 2001; Renner et al., 2007). However, extensive phylogenetic studies analyzing the evolution of dioecy within one genus have only been performed a few cases (Navajas-Pérez et al., 2005; Volz & Renner 2008), so it is possible to expect that more cases of independent origins of sex determination in related plant species will be found in the near future. GENUS RUMEX The reproductive systems in Rumex are very diverse and include hermaphroditism, polygamy, gynodioecy, monoecy, and dioecy (Navajas-Pérez et al., 2005). In dioecious Rumex species, two different sexchromosomal systems and sex-determining mechanisms have been described: (1) XX/XY with an active Y chromosome (e.g. Rumex acetosella) and (2) XX/XY1Y2 with sex determination based on the X/A ratio (e.g. Rumex acetosa) (Navajas-Pérez et al. (2005). There is one exceptional species Rumex hastatulus, which has two chromosomal ‘races:’ the Texas race with XX/XY and the North Carolina race with XX/X Y1Y2. In this species, the X/A ratio controls sex determination, although the presence of the Y chromosome is necessary for male fertility (Smith, 1963). In R. acetosa, repetitive sequence similarity between both Y chromosomes suggests that they probably originated from one Y chromosome that underwent centromere fission and gave rise to a pair of metacentric chromosomes possessing identical chromosomal arms (isochromosomes). These isochromosomes were subsequently modified by deletions (Rejón et al., 1994). A recent 743 phylogenetic study (Navajas-Pérez et al., 2005) indicates that all dioecious Rumex species evolved from a common hermaphroditic ancestor. This conclusion suggests that a switch from a sex-determining mechanism based on the active role of the Y chromosome to a mechanism based on the X/A ratio occurred during the evolution of this genus. Such a switch occurred at least twice independently according to Navajas-Pérez et al. (2005). The role of the X/A ratio in the sex determination of R. acetosa resembles the sex-determining system of Drosophila, where the primary genetic sexdetermining signal is provided by the ratio of X-linked genes to autosomal genes (Pomiankowski et al., 2004). DIOECIOUS MERCURIALIS ANNUA: TO M. DOMESTICA? SIMILARITY A relatively complex and variable system of sex determination is found in M. annua and its closely related species. Mercurialis annua is a ruderal species of pan-European distribution. Three sex-controlling loci have been described in the dioecious race of M. annua. The dominant allele at the sex-determining locus A, together with the dominant allele(s) in at least one of the other two loci (B1 and/or B2), causes a male phenotype in the plant. If the recessive allele is present at the A locus, the plant becomes female independently of the genotypes at the B1 and B2 loci (Louis, 1989). It is therefore possible to speculate that the A locus is the original sex-determining locus and the B1 and B2 loci are modifiers that developed later. It is known that plant hormones play an important role in sex determination in M. annua. Auxins produce a masculinizing effect on females and cytokinins produce a feminizing effect on males (independent of the genotype at B loci; Hamdi, Teller & Louis, 1987). The plasticity of the sexual phenotype is in apparent contradiction to the pronounced sexual dimorphism that is reflected in sex specific gene expression (Khadka et al., 2005). Sexual dimorphism evolves step by step as a result of sexually antagonistic selection (Rice, 1984); therefore, in species where the sex determination was established recently, the level of sexual dimorphism should be minimal. Studies of M. annua could therefore contribute to an understanding of the mechanisms controlling sexual dimorphism and its evolution. Genes controlling sexual dimorphism should be linked to the A/a locus. The first steps towards the isolation of this sex-determining region have been initiated by Khadka et al. (2005), who isolated one completely male linked marker. This system could therefore be interpreted as an XX/XY sex-determining system with autosomal modifiers (similar to M. domestica; Dübendorfer et al., 2002). The importance of M. annua for the evolutionary studies of sex determination can be illustrated by the © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100, 737–752 744 B. JANOUSEK and M. MRACKOVA results obtained by Dorken & Pannell (2009). Their data show that the absence of males in experimental populations provoked greater male allocation (measured as male reproductive effort; a proportion of the total above ground biomass allocated to the staminate flowers). The results obtained in this experiment support the model for the origin of gynoecium suppressing mechanisms that was proposed by Charlesworth & Charlesworth (1978). CHANGE IN THE SEX-DETERMINING MECHANISM OF MERCURIALIS: ANDRODIOECIOUS RACE OF M. ANNUA Differences in the body plans and life-history strategies can favour higher plasticity of both the genotype and phenotype in angiosperms compared to animal models (Kejnovsky, Leitch & Leitch, 2009). Because the plant body is usually formed by several relatively independent meristems, the coexistence of branches with different sexual phenotypes should not lead to the detrimental effects seen in gonochoristic animal species (e.g. mammals), where the simultaneous presence of both sexual organs can cause damage in both sex functions because of hormonal imbalances (Krob, Braun & Kuhnle, 1994). Despite this prerequisite, there are just a few examples of changes in the sex-determining mechanisms in plants. According to Obbard et al. (2006), androdioecy in hexaploid populations of M. annua may have evolved as a result of hybridization between dioecious Mercurialis huetii and monoecious tetraploid M. annua, an event that brought together the genes for specialist males with those for hermaphrodites. Interestingly, the results of a study by Pannell (1997) indicate that male sex in the androdioecious populations is determined by the presence of the dominant allele of a single sex-determining locus (that suppresses development of female organs) but that males are prone to sex change in response to environmental influences (low plant density). It is argued that these findings help to explain the maintenance of androdioecy in M. annua. BRYONIA DIOICA AND DIOECY IN CUCURBITACEAE Species of the Cucurbitaceae family are, in contrast to most angiosperms, characterized by the presence of unisexual flowers. Of the approximately 800 species in this family, 460 are monoecious and 340 are dioecious. Some species produce a mixture of bisexual, female, and male flowers in various intra- and inter-individual patterns, and populations can be andromonoecious, androdioecious, gynomonoecious or gynodioecious (Kocyan et al., 2007). Phylogenetic studies suggest that the Cucurbitaceae family has a dioecious origin (Zhang et al., 2006). Various switches to monoecy or other types reproductive systems such as androdioecy occurred frequently during the evolution of Cucurbitaceae, and thus it is difficult to precisely ascertain how old the sex chromosomes in a given species are (Renner et al., 2007). The best studied Cucurbitacean genus containing dioecious species is Bryonia. Genetic crosses between the dioecious B. dioica and the monoecious Bryonia alba in 1903 provided the first clear evidence for Mendelian inheritance of sexual phenotypes (dioecy) and made B. dioica the first organism for which the XY sex-determination was experimentally proven (Correns, 1907). Applying molecular tools to this system, Oyama, Volz & Renner (2009) showed that size of the nonrecombining region differs between the north-European southern,European populations. Because of availability of whole genome sequencing data from the family (see below), the genus Bryonia could become a good model for the study of the evolution of sex-determining pathways and sex chromosomes. Important data concerning the possible mechanisms of sex determination in Cucurbitaceae were recently obtained in melons (Cucumis melo). In this mostly monoecious species, sex determination is governed by the genes andromonoecious (a) and gynoecious (g). Dominant allele of the a locus (CmACS-7 gene; 1-aminocyclopropane-1-carboxylic acid synthase) causes arrest of the stamen development (Boualem et al., 2008). The dominant allele of the g locus causes arrest of the gynoecium development. Monoecious (A-G-) and andromonoecious (aaG-) plants bear male flowers on the main stem and, respectively, female or hermaphrodite flowers on the axillary branches, whereas gynoecious (AAgg) and hermaphrodite individuals (aagg) only bear female or hermaphrodite flowers, respectively. The insertion of the Gyno-hAT transposon in proximity of the g gene (CmWIP1) was shown to be a cause of the gynoecious phenotype of several lines (G to g change by hypermethylation of promoter of the g gene, i.e. CmWIP1). The occasional presence of the flowers with stamen and reduced ovaries suggests that the DNA hypermethylations of the CmWIP1 can be reduced during somatic development of gynoecious plants (Martin et al., 2009). Surprisingly, both CmACS-7 and its homologue from Cucumis sativa are specifically expressed in female buds. Role of 1-aminocyclopropane-1-carboxylic acid synthase in anther arrest appears to be indirect and inter-organ communication is probably responsible for the anther arrest (Boualem et al., 2009). The analysis based on the knowledge of the whole genome sequence of C. sativa revealed that the evolution of unisexual flowers in cucurbits may have involved the acquisition of two ethylene-responsive elements (AWTTCAAA) and one flower meristem identity gene LEAFYresponsive element (CCAATGT) of the ACS genes. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100, 737–752 SEX CHROMOSOMES AND SEX DETERMINATION PATHWAY DYNAMICS Extensive expressed sequence tag analysis in unisexual and bisexual flower buds (using 454 sequencing) revealed that six auxin-related genes (auxin can regulate sex expression by stimulating ethylene production) and three short-chain dehydrogenase or reductase genes (homologues to the sex determination gene ts2 in maize41) are more highly expressed in unisexual flowers (Huang et al., 2009). CARICA PAPAYA Carica papaya has become an important model for the studies of sex determination and sex chromosome evolution because of its relatively small genome and extremely young sex chromosomes (Liu et al., 2004; Yu et al., 2008). There are some signs indicating ongoing degeneration of the areas surrounding the sex-determining locus in papaya. This pattern, together with phylogenic data, which indicate a possible dioecious origin of the Caricaceae family, has lead some researchers to doubt the proposed age of papaya sex chromosomes (Charlesworth, Charlesworth & Marais, 2005). If the hypothesis of the young age of papaya chromosomes is accepted, it means that processes of degeneration in the nonrecombining region must have worked extremely quickly in this species. However, recent estimates of the divergence between the X- and Y-linked genes further support the young age of the sex chromosomes in C. papaya (Yu et al., 2008). In addition, specific heterochromatinized DNA-hypermethylated knobs are present in the nonrecombining region, suggesting a possible role of epigenetic mechanisms in the sex chromosome evolution (Zhang et al., 2008). The apparent contradiction of the young age of sex chromosomes and prevailing dioecy in Caricaceae is proposed by Yu et al. (2008) to be the product of multiple independent origins of sex chromosomes in this family. It should be stressed that an old sex determination system need not be in a contradiction with the young sex chromosomes. The possibility that a new sex-determining locus was recruited, as in the known cases in Oryzias species (Nanda et al., 2002), cannot be so far excluded. A big advantage of C. papaya as a model for the future research is the availability of the draft sequence of its genome (Ming et al., 2008). GENUS POPULUS Genus Populus became an important model object in the studies of sex determination in the last years. Primarily, the economic importance of several of its species promoted genetic mapping studies. The interest of reserchers is mainly focused on the species Populus trichocarpa as a model tree for whole genome sequencing studies. A main goal has been to study the 745 genes involved in the synthesis of wood but studies of the sex determination can profit from the knowledge of the genomic sequence (Tuskan et al., 2006). Multiple lines of evidence point to a ZW sex determination system in Populus, with the female being the heterogametic sex. First, the sequenced tree, Nisqually-1, is a female, and it showed highly divergent haplotypes in the sex determination region. Second, suppressed recombination in this region was only observed in the female parent of the cross studied by Yin et al. (2008). Finally, the female heterogamety is also according to Haldane’s rule (‘When in the F1 offspring of two different animal races one sex is absent, rare, or sterile, that sex is the heterozygous [currently = heterogametic] sex’; Haldane, 1922) in accordance with the overall male bias in populations of various species of the genus Populus (Grant & Mitton, 1979; Rottenberg, Nevo & Zohary, 2000). CONCLUSIONS AND PERSPECTIVES A comparison of the data for animal models with the data for plant models that were collected in this review suggests that the sex-determining pathways in these two kingdoms evolved by a similarly diverse set of mechanisms. An overview is provided in the Supporting information (Tables S1, S2). All variants of the basic sex determination systems found in animal models were reported also in plants with exception of the complementary sex determination (haplodiploidy) that is specific for social insects and aneuploidy-based sex determination with female heterogamety (ZO/ZZ), which is found in butterflies. The XX/XO type (also found in insects) was reported in plants but it has not yet been sufficiently experimentally supported (in Dioscorea sinuata; Smith, 1937). The absence of the total loss of W or Y chromosomes, respectively, can be explained by an overall younger age of sex chromosomes in plants. Differences between animals and plants can be found in the form in which plasticity of sex-determining pathways is manifested. In many animal species, it is often possible to observe female to male changes or male to female changes as a result of environments effects or the manifestation of new sexdetermining genes. In the plant species studied so far, the cases of sex change to the opposite sex are rare (Policansky, 1981). The widespread manifestation of plasticity of sex expression in plants takes the form of instability of the gynoecium suppression in male plants (subdioecy). This phenomenon is probably selectively advantageous because it enables species that have no possibility of active movement to overcome lack of pollinators and other problems that could otherwise lead to extinction of strictly dioecious species (Delph & Wolf, 2005). © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100, 737–752 746 B. JANOUSEK and M. MRACKOVA The question of somatic sex determination and germline sex determination (often discussed in animal models) has not yet been studied in plants. Plants do not set aside a germline in strictu senso. There is, however, a process enabling plants to limit the number of necessary cell divisions preceding meiosis. The cells in the center of the vegetative meristem possess hypermethylated DNA, and they divide sparsely. This mechanism lowers the mutation rate and also reduces the epigenetic influence of environmental factors (Zluvova, Janousek & Vyskot, 2001). There is therefore at least some similarity to the germline found in animals. Another so far neglected question concerns how a system based on two separate sex controlling pathways controlled by genes present on the Y chromosome (Charlesworth & Charlesworth, 1978) is transformed into a system controlled by the pathway based on the X/A ratio in some species. The following hypothetical scenario of sex-determining pathway evolution, based on the observation of the early sex dimorphism in species with an active Y chromosome, as well as on the knowledge of the mechanisms involved in the change of sex linkage in animal models, can be suggested. In the first step, dioecy is established in plants from gynodioecy by the evolution of a gynoecium suppressing gene in the proximity of the male fertility controlling gene (Fig. 3A). This process is promoted by sexually antagonistic selection (Charlesworth & Charlesworth, 1978). The original theory (Charlesworth & Charlesworth, 1978) assumes just one male sterility mutation (i.e. that the male fertility locus of gynodioecious ancestor is identical with the anther promoting gene of the resulting dioecious species). The possibility of the stepwise shift in the stage of male organ arrest toward earlier stages was discussed by Zluvova et al. (2005) but, even if present, this does not influence our next considerations. The scheme of sex determination at this stage is outlined in the Supporting information (Figure S1a). This figure stresses the independence of the male-promoting and female-suppressing pathways. There are a lack of data concerning the origin of sex determination in animals. Although there are indications that sex determination systems of both insects and vertebrates could have a common origin (Koopman, 2009), no hypotheses concerning the sex determination mechanism in their common ancestor were suggested. A scenario based on two mutations is also possible in animal models (Charlesworth & Charlesworth (2005). In this case, the difference between plants and animals is only in the nomenclature (dioecy versus gonochorism, etc.). In the scheme, we use universal terms (e.g. male organ promoter instead of anther promoter) to stress that this processes can occur not only in plants. In the second step, sexually antagonistic selection continues and improves the linkage of the sexdetermining loci. Sex chromosomes that are created by this process can continue to accumulate sexually antagonistic alleles (Rice, 1984) (Fig. 3B). Even in species that are at this stage of sex determination, it is possible to find early expressed sexual dimorphism (e.g. S. latifolia; Zluvova et al., 2008b; J. Zluvova unpublished data). In the third step, the sex-determining genes start to accommodate to the sex specific gene expression patterns controlled by the sexually antagonistic gene(s) and their expression starts to be controlled by these genes. Eventually, the gene(s) that were previously controlling only sexual dimorphism become(s) a sex-determining gene(s). For reasons of space, Figure 3C shows the situation in males only. The situation in females is shown in the Supporting information (Fig. S1). It is known that sexually antagonistic genes are evolving fast (Qvarnström & Bailey, 2009). A good example of the fast evolution of Y-linked genes in plants was revealed in S. latifolia, where a lack of the Y chromosome can not be completely compensated for by the presence of the genome of the related species Siline viscosa, and anther defects in the hybrid between S. latifolia and S. viscosa resemble two different mutants lacking part of Y chromosome (Zluvova et al., 2005). The active role of the Y chromosome in sex determination is still preserved because the new sex-determining locus is still located on the Y chromosome. The important difference from the previous stages is that connection between the control of the stamen promotion and anther suppression is established. This opens new possibilities for the evolution of the sexdetermining system. In the fourth step, two alternative scenarios are possible. The original sex-determining loci can be lost from the Y chromosome just by chance or, more parsimoniously, the translocation of the original sexdetermining region to the autosomes could be supported by Y chromosome degeneration as a result of the absence of recombination (Fig. 3D). Because male organ promotion and female organ suppression are already controlled by the single controlling pathway, the genotypes possessing translocation of these genes to autosomes can be selected for because they can escape from the process of degeneration. The change of the position of these genes does not influence the sexual phenotype because the genes show sexlimited expression, and they are controlled by the gene derived from the gene originally controlling only sexual dimorphism). In the fifth step, the secondary sex-determining locus loses its controlling role as well, and control of sex determination is coopted by alternative © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100, 737–752 SEX CHROMOSOMES AND SEX DETERMINATION PATHWAY DYNAMICS 747 A female organ suppressor male organ promoter male organ promoter female hermaphrodite gynodioecy female male dioecy B SAG-F SAG-M female organ suppressor female organ suppressor male organ promoter proto-X proto-Y C male organ promoter X female organ suppressor: present Y Early expressed SAG-M present male organ promoter: present Male specific expression profiles female organ absent male organ present Male specific expression profiles female organ suppressor: on female organ absent D SAG-F SAG-M female organ suppressor male organ promoter autosomes X Y E SAG-F male organ present SAG-F female organ suppressor. male organ promoter autosomes SAG-M (sex determining) X Y SAG-F SAG-M (sex determining) SAG-M autosomes X Y male organ promoter: on SAG-M autosomes X Y Figure 3. The evolution of the sex-determining pathways in plants. A, theory of origin of dioecy via male sterility suggested by Charlesworth & Charlesworth (1978). Both the female organ (gynoecium) suppressor and the male organ (anther) promoter promoter act independently but their coordination is achieved by their close location on the Y-chromosome or by their location in the nonrecombining region of the Y-chromosome. B, formation of sex chromosomes. Accumulation of sexually antagonistic genes and reduction of recombination frequency between female organ (gynoecium) suppressor and male fertility controlling genes creates sex chromosomes. For simplification, only one sexually antagonistic gene (SAG) is presented. SAG-F means sexually antagonistic alelle advantageous for females and SAG-M means male advantageous sexually antagonictic allele of the same gene. C, sexually antagonistic gene(s) based switch in the sex-determining pathway. Sexual dimorphism (i.e. controlled by SAG-M) is improved step by step and starts to act before the Y-linked genes involved in female and male organ development control. At certain stage, the expression of both the female organ suppressor and the male organ promoter becomes sex-limited as a consequence of their adaptation to sex specific expression profiles of other genes. D, restructuration of sex chromosomes. Female organ suppressor and male organ promoting gene(s) are lost from the Y chromosome and transferred to autosome(s). E, origin of the X/A based sex-determining system. SAG-M is lost from Y-chromosome and transferred to an autosome. The X/A ratio becomes crutial for sex determination because SAG-M pushes development toward the male direction in contrast to SAG-F that pushes development toward female direction. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100, 737–752 748 B. JANOUSEK and M. MRACKOVA mechanisms such as an X/A ratio-based mechanism (Fig. 3E). The switch could be based on the mechanisms described in fishes and insects, in which one of the genes, ‘a slave’ in the sex-determining pathway located on the X chromosome, can become ‘a master’. Simultaneously, the function of this new master gene is influenced by the genes located on autosomes. An important prerequisite of this kind of transformation of sex-determining system is that male organ promotion and female organ suppression are controlled by the same pathway. Veltsos et al. (2008) described processes that may influence the spread of new sex-determining mechanisms. In this case, the invasion of the degenerated Y chromosome (in the hybrid zone) can cause selection in favour of the new type of X chromosome that is able, in combination with autosomal loci, to determine sex in a given species. It is apparent that, in most plant species and in many animal species, this evolution is not complete because many species still rely on an active role of the Y chromosome in sex determination. Instability of sex expression and/or cytologically homomorphic sex chromosomes are sometimes taken as a sign of the primitive status of the evolution of sex-determining systems in some plant species (Vyskot & Hobza, 2004). Data obtained in the animal models suggest that even very advanced sexdetermining systems (as in mammals) can show a considerable plasticity of sex expression (Bianchi, 2002); thus, the sequencing of the sex-determining regions of the studied species should be the method of choice used to ascertain the age of sex chromosomal systems. Additionally, a more detailed phylogenetic approach should answer the question of the age of sex-determining pathways in model dioecious species. 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Premature arrest of the male flower meristem precedes sexual dimorphism in the dioecious plant Silene latifolia. Proceedings of the National Academy of Sciences of the United States of America 103: 18854– 18859. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Figure S1. Sexually antagonistic gene(s) based switch in the sex-determining pathway changes in females: changes in females. Table S1. Overview of mentioned fungal and animal species. Table S2. Overview of the mentioned plant species. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100, 737–752