Evolution of Neo-Sex Chromosomes in Silene diclinis

A small cluster of dioecious species in the plant genus Silene has evolved chromosomal sex determination and sex chromosomes relatively recently, within the last 10 million years (MY). Five dioecious Silene species (section Elisanthe) are very closely related (1–2 MY of divergence) and it was previously thought that all five have similar sex chromosomes. Here we demonstrate that in one of these species, Silene diclinis, the sex chromosomes have been significantly rearranged, resulting in the formation of neo-sex chromosomes. Fluorescence in situ hybridization with genic and repetitive probes revealed that in S. diclinis a reciprocal translocation has occurred between the ancestral Y chromosome and an autosome, resulting in chromosomes designated Y1 and Y2. Both Y1 and Y2 chromosomes are male specific. Y1 pairs with the X chromosome and with the autosome (the neo-X), which cosegregates with X. Y2 pairs only with the neo-X, forming a chain X-Y1-neo-X-Y2 in male meiosis. Despite very recent formation of the neo-sex chromosomes in S. diclinis, they are present in all surveyed individuals throughout the species range. Evolution of neo-sex chromosomes may be the cause of partial reproductive isolation of this species and could have been the isolating mechanism that drove speciation of S. diclinis.PAIRING of homologous chromosomes during meiosis, in the majority of diploid plants and animals, leads to the formation of bivalents at first metaphase and subsequently the correct segregation of the chromosomes. Chromosomal translocations that produce multivalents usually result in unbalanced segregation, which consequently affects fertility. However, chain or ring configurations appear to be stably inherited in some species. An extreme example is found in the plant genus Oenothera, where many species display a ring involving all 14 chromosomes (Cleland 1972). In animals these configurations may include sex chromosomes, resulting in the formation of multiple X and Y chromosomes. For example, the monotreme platypus possesses five X and five Y chromosomes that form a chain of alternating X and Y chromosomes in male meiosis (Bick and Sharman 1975; Gruetzner et al. 2006). Such chains are formed due to several interchromosomal translocation events, including sex chromosome–autosome translocations (Gruetzner et al. 2006). Since sex chromosomes are rare in plants, examples of plant sex-linked chromosome multiples have been reported on only a few occasions. A chain of four X and five Y has been identified in an East African mistletoe Viscum fischeri (Wiens and Barlow 1975) and a chain of two X and two Y has been found in Humulus lupulus ssp. cordifolius (Shephard et al. 2000). Trivalent formation comprising Y1 X Y2 has been observed both in H. japonicus (Shephard et al. 2000) and in a number of dioecious species in the genus Rumex (Cunado et al. 2007; Navajas-Perez et al. 2009). Here we report that the plant species Silene diclinis has multiple sex chromosomes that form a chain of four during meiosis metaphase I.S. diclinis is a member of a small group of dioecious species (having separate male and female plants) in section Elisanthe in the plant genus Silene (Caryophyllaceae). The other members of this group are S. latifolia, S. dioica, S. heuffelii, and S. marizii (Prentice 1978). The presence of large heteromorphic sex chromosomes in S. latifolia and S. dioica has been known for many years (Westergaard 1958). Due to the ease of cytogenetic identification of the sex chromosomes, the clear morphological difference between the sexes and the short generation time, S. latifolia was used in early genetic research concerning sex determination in plants. The male was shown to be the heterogametic sex (XY) with the larger Y chromosome having a decisive role in sex determination (Westergaard 1958). Since then, S. latifolia has become a species of choice for studies in plant genetics, ecology, and evolution (Bernasconi et al. 2009). It is particularly useful for studies of sex chromosome evolution because the sex chromosomes in Silene are of relatively recent origin compared to those of mammals (Charlesworth 2002; Ming and Moore 2007; Marais et al. 2008).Experimental crosses involving all five dioecious species in Silene section Elisanthe in various pairwise combinations have produced viable hybrids and, although some combinations were less successful than others, the formation of these hybrids suggests a close relationship within this group (Prentice 1978). This close relationship is also illustrated by DNA sequence comparisons that show that interspecific silent divergence between these species does not exceed 2%, which is comparable to intraspecific polymorphism in S. latifolia (Ironside and Filatov 2005). S. diclinis is a rare and restricted endemic, found only in Southern Valencia, Spain in an area smaller than 18 × 9 km (Prentice 1976; Montesinos et al. 2006). Of the other four Elisanthe species, only S. latifolia occurs in this region, and experimental crosses between these two species are the least successful (Prentice 1978). Hybrids between S. latifolia and S. dioica occur naturally in regions where their populations coincide (Baker 1948) but no natural hybrids of S. diclinis and S. latifolia have been reported.Cytogenetic analysis of S. diclinis has been limited. Examination of mitotic metaphase spreads in root tip squash preparations from adult male and female plants indicated that the male had one X and one Y chromosome. Both chromosomes were large but the difference between them was slight (van Nigtevecht and Prentice 1985). Regular pairing of chromosomes with 12 bivalents at metaphase I in pollen mother cells has been reported (Morisset and Bozman 1969). However, these observations were made without the benefit of a marker for the Y chromosome. Recently, sequences with homology to an Ogre retrotransposon have been isolated from S. latifolia and used as probes in fluorescence in situ hybridization (FISH) experiments on mitotic (Cermak et al. 2008) and both mitotic and meiotic (Filatov et al. 2009) chromosome spreads. The pattern of hybridization showed that these sequences are widespread over the X chromosome and all of the autosomes but are mainly confined to a small section at the pairing region of the Y chromosome in S. latifolia. Therefore, these probes “paint” all the chromosomes apart from the Y, providing a “negative paint” for the Y chromosome. By using one of these probes (clone 4.2) on meiotic spreads of S. dioica and S. marizii, we confirmed that these species have sex chromosomes similar to those of S. latifolia (Filatov et al. 2009). The X and Y formed a rod bivalent and the Y chromosome was larger than both the X and autosomes.In this article we report our FISH experiments with S. diclinis using the negative paint probe together with probes containing S. latifolia sex-linked gene sequences. We demonstrate that S. diclinis males have two Y chromosomes that differ in the distribution of the paint signal and these gene sequences. In meiotic metaphase I, one Y pairs with the X and an autosome while the second Y pairs with the other arm of this autosome, forming a chain of four chromosomes. We suggest that an autosome–Y reciprocal translocation was involved in the evolution of neo-sex chromosomes in this species.