Turnovers of sex-determining systems represent important diversifying forces across eukaryotes. Shifts in sex chromosomes—but conservation of the master sex-determining genes—characterize distantly related animal lineages. Yet in plants, in which separate sexes have evolved repeatedly and sex chromosomes are typically homomorphic, we do not know whether such translocations drive sex-chromosome turnovers within closely related taxonomic groups. This phenomenon can only be demonstrated by identifying sex-associated nucleotide sequences, still largely unknown in plants. The wild North American octoploid strawberries (Fragaria) exhibit separate sexes (dioecy) with homomorphic, female heterogametic (ZW) inheritance, yet sex maps to three different chromosomes in different taxa. To characterize these turnovers, we identified sequences unique to females and assembled their reads into contigs. For most octoploid Fragaria taxa, a short (13 kb) sequence was observed in all females and never in males, implicating it as the sex-determining region (SDR). This female-specific “SDR cassette” contains both a gene with a known role in fruit and pollen production and a novel retrogene absent on Z and autosomal chromosomes. Phylogenetic comparison of SDR cassettes revealed three clades and a history of repeated translocation. Remarkably, the translocations can be ordered temporally due to the capture of adjacent sequence with each successive move. The accumulation of the “souvenir” sequence—and the resultant expansion of the hemizygous SDR over time—could have been adaptive by locking genes into linkage with sex. Terminal inverted repeats at the insertion borders suggest a means of movement. To our knowledge, this is the first plant SDR shown to be translocated, and it suggests a new mechanism (“move-lock-grow”) for expansion and diversification of incipient sex chromosomes.

Sex chromosomes frequently restructure themselves during organismal evolution, often becoming highly differentiated. This dynamic process is poorly understood for most taxa, especially during the early stages typical of many dioecious flowering plants. We show that in wild strawberries, a female-specific region of DNA is associated with sex and has repeatedly changed its genomic location, each time increasing the size of the hemizygous female-specific sequence on the W sex chromosome. This observation shows, for the first time to our knowledge, that plant sex regions can “jump” and suggests that this phenomenon may be adaptive by gathering and locking new genes into linkage with sex. This conserved and presumed causal sex-determining sequence, which varies in both genomic location and degree of differentiation, will facilitate future studies to understand how sex chromosomes first begin to differentiate.

Here, we use whole-genome sequencing and molecular evolutionary analysis of multiple octoploid Fragaria taxa to characterize and compare SDRs that are found on different chromosomes ( Fig 1B and Table 2B ). Our goal is to determine whether a single W-specific sequence has translocated among genomic locations. We find an “SDR cassette” shared by females across taxa and never detected in male plants. The SDR cassette contains two putatively functional sex-determining genes and has moved at least twice, together with flanking sequences that reveal the order of the translocation events. Because the moved regions are hemizygous, each translocation has created a wider hemizygous region than formerly existed. Therefore, we report the first case, to our knowledge, of a repeatedly translocating SDR in plants and propose a new hypothesis for sex-chromosome differentiation.

(A) There are seven haploid Fragaria chromosomes. Diploids (e.g., F. vesca ssp. bracteata, used to generate the reference genome Fvb) have two copies of each. Octoploids have eight copies of each, within four homoeologous subgenomes (Bi, B2, B1, Av) showing high synteny with each other and with Fvb. (B) In multiple independent linkage crosses across octoploid taxa, the SDR (colored circles) had been previously mapped to three locations on different chromosomes of homoeologous group 6, corresponding to three positions (1 Mb, 13 Mb, or 37 Mb) on Fvb6. The “Linkage Cross” column indicates the taxon in which each SDR has been fine-mapped. Sex always showed ZW inheritance, but no sex-specific sequence had been previously identified. Fc, F. chiloensis; Fvb, diploid reference genome assembly informed by F. vesca ssp. bracteata; Fvp, F. virginiana ssp. platypetala; Fvv, F. virginiana ssp. virginiana; Mb, megabase; SDR, sex-determining region.

The SDR of Fragaria octoploids has been mapped in three geographically distinct octoploid taxa (here in order from eastern to western North America): F. virginiana ssp. virginiana [ 42 ], F. virginiana ssp. platypetala [ 40 ], and F. chiloensis [ 39 , 43 ] (Tables 1 and 2A ). Each SDR occurs at a unique section of a chromosome from the same homoeologous group, i.e., the group that corresponds to Fvb6 in the diploid reference, but each from a different subgenome ( Fig 1B ) [ 40 ]. Specifically, the mapped SDR locations match Fvb6 position 1 Mb on subgenome B2 in a cross of two F. virginiana ssp. virginiana parents ([ 42 ], results herein), 13 Mb on subgenome B1 in a cross of two F. virginiana ssp. platypetala parents [ 40 ], and 37 Mb on subgenome Av in three crosses involving pairs of F. chiloensis parents [ 39 , 43 ] ( Table 2A ). Moreover, genetic maps in the natural hybrid (F. × ananassa ssp. cuneifolia) of two of these taxa corroborate these map locations [ 48 ] ( Table 2A ). Though the chromosomes harboring the various SDRs are all homoeologous, they are distinct: Fragaria subgenomes show little evidence of recombination with each other [ 41 ], and the positions of the various SDR locations are too far apart (several Mb) for normal recombination ( Fig 1B ). All SDRs occur far from centromeres in gene-dense regions, and although early stages of recombination suppression may be evolving, pseudoautosomal recombination still occurs between the Z and W along most of their lengths, allowing for fine-scale mapping [ 39 ]. The recent evolutionary origin of dioecy and the extensive recombination still occurring on the sex chromosomes suggest that there is very little sex-specific sequence other than the causal gene(s). However, despite extensive previous work mapping the chromosomal locations of Fragaria SDRs [ 39 , 40 , 42 , 43 , 48 – 50 ] as well as conjecture that autosome Fvb6 may possess sexually antagonistic genes that predispose it to become a sex chromosome [ 51 ]—as seen in other systems [ 52 – 54 ]—no candidate causal genes have been identified, and nothing is known of the molecular mechanism beyond very broad inferences (e.g., that control is nuclear rather than cytoplasmic). Therefore, identifying sex-determining gene(s) and inferring whether they are shared across the octoploid Fragaria will provide a unique opportunity for testing whether sex chromosome turnovers represent translocations of the same SDR.

The octoploid (8x) strawberries (Fragaria) stand out as model system for studying plant sex chromosomes [ 36 – 40 ] and polyploidy [ 36 , 41 ] in an evolutionary context because they show recently evolved dioecy from within a group of closely related, predominantly hermaphroditic diploid (2x) taxa. The octoploid taxa all possess homomorphic, female heterogametic (ZW) sex chromosomes with a single SDR explaining the majority of variation in male and female function, though the degree of sexual dimorphism varies across taxa [ 36 , 39 , 42 – 45 ]. Male function (sterile versus fertile), in particular, is a binary trait showing simple Mendelian inheritance (1:1). Male sterility (“female”) is dominant to male fertility (“male”), determined entirely by the SDR, and here we use it to define sex phenotype. All octoploid species share a recent polyploid origin involving four diploid ancestors (now coexisting as “subgenomes” [Av, Bi, B1, and B2] within the octoploid genome, Fig 1A ) [ 41 , 46 ]. The homologous chromosomes from each subgenome (homoeologs) are genetically distinct and are inherited disomically. Nevertheless, homoeologs show high synteny with each other and with the Fragaria reference genome (“Fvb”) derived from the hermaphroditic diploid F. vesca with seven haploid chromosomes (named Fvb1 through Fvb7, Fig 1A ) [ 41 ]. Therefore, the octoploids have seven homoeologous groups, each with eight chromosomes (2N = 8x = 56). The approximately 700 megabase (Mb) octoploid genome is slightly smaller, however, than four times the approximately 200 Mb diploid Fvb reference genome, likely due to numerous small deletions [ 41 , 47 ]. The diploid and octoploid genomes are largely collinear [ 41 ], and we refer to all genome positions by their location along Fvb chromosomes in Mb.

Turnovers of SDRs are likely to be quite common in plants, in which genetic control of sex appears to be poorly conserved [ 28 , 29 ]. Flowering plant SDRs may be diverse because dioecy (separate males and females) has evolved repeatedly from hermaphroditism (combined male and female function) and many sex chromosomes are relatively young and homomorphic [ 28 , 29 , 30 ]. Additionally, approximately one-third of flowering plant species are estimated to have a recent polyploid ancestry [ 31 ]. These whole-genome duplications provide a larger substrate for potential sex-determining genes or rearrangements [ 32 ]. Yet despite the potential of dioecious plants for yielding evolutionary insights, there are few systems with mapped SDRs [ 28 , 29 ] or known causal genes [ 33 , 34 ], although long-standing theory predicts that two linked genes, one controlling male function and one controlling female function, are involved [ 1 , 35 ]. Moreover, even when observed, the pattern and mechanism of turnovers remain entirely unexplored.

Fundamental questions about SDR turnovers therefore remain unanswered. Do turnovers typically involve mutations in new loci that take control of an existing sex-determining mechanism [ 18 , 19 ], functionally independent mutations [ 20 ], or translocations of the existing sex-determining gene(s) to new chromosomes [ 21 – 24 ]? Similarly, do turnovers typically restart the process of SDR divergence, maintaining “ever-young” sex chromosomes [ 25 ], or do they contribute to increasing chromosome heteromorphy via loss or gain of sequence [ 11 , 14 , 26 ]? And ultimately, is there an adaptive basis for these turnovers? Although master sex-determining genes like SRY and DMRT1 are highly conserved in some animal systems, the causal SDR loci or gene cassettes remain unknown for most dioecious eukaryotes [ 27 ]. Even less is known about the temporal order of turnovers in any taxon and thus directional trends in sex-chromosomal rearrangement [ 2 ].

Sex chromosomes can be a strikingly diverse and evolutionarily labile component of eukaryotic genomes [ 1 ]. The defining feature of a sex chromosome, the sex-determining region (SDR), has experienced similar restructuring in multiple independent instances of autosomes evolving into heteromorphic sex chromosomes [ 2 ]. Specifically, recombination is suppressed, and an increasingly greater proportion of the chromosome becomes hemizygous, which is thought to involve existing and/or newly acquired linkage to loci under sexually antagonistic selection [ 3 ]. The mechanisms of this chromosome restructuring may involve modifying crossover sites and/or successive inversions of the SDR or translocations of large or small sequences on and off the sex chromosome [ 3 , 4 ]. Turnovers that change the genomic location of the SDR have been revealed in the evolution of animal sex-determining systems [ 1 , 5 – 8 ], where they may be important drivers of sexual dimorphism and speciation [ 9 , 10 ]. While theory on the processes driving these transitions is growing [ 11 – 14 ], few systems exist in which the mechanisms of turnovers can be empirically inferred [ 15 – 17 ].

Results and discussion

Possible mechanisms of DNA movement Although the mechanism of translocation of sex-determining sequence remains unknown, a striking sequence pattern suggests transposon-mediated movement. Specifically, we observe a 25 bp sequence that is inverted and repeated at the very distal ends of the flanking sections, where sequence homologous to Fvb6 13 Mb meets sequence homologous to Fvb6 37 Mb (Fig 4). On the distal end of each segment, we observe the dinucleotide motif TA. Pairs of terminal inverted repeats of 10 bp or more in length, adjacent to short duplications, are hallmarks of Class 2 transposable elements [68,69]. Therefore, this sequence signature is consistent with the hypothesis that a mobile element transported the 23 kb of SDR cassette and flanking sequence from the β clade location at Fvb6 13 Mb to the γ clade location at Fvb6 37 Mb. Terminal inverted repeats also occur in foldback elements, which can cause chromosomal rearrangement via ectopic recombination [70], and this mechanism could also facilitate movement of the SDR among homoeologs of Fvb6. We do not see terminal inverted repeats at the border between the SDR cassette and the flanking sequence, but this may have been lost, perhaps explaining why adjacent sequence was then also moved during the second translocation. Most transposable elements are under 23 kb in size, and we see no evidence of either an intact transposase, a Helitron transposon, or any known plant repetitive sequences other than stretches of dinucleotide repeats under 50 bp. Therefore, although the full W-specific haplotype remains incompletely assembled and could harbor a transposase (Fig 4), we hypothesize that the SDR movements do not involve a classic, active transposon but rather are relatively rare events that leverage active transposases that may be encoded elsewhere, as with miniature inverted-repeat transposable elements [68,69]. Consistent with the scenario of relatively few SDR movements, no female appears to have more than a single SDR cassette. Although we cannot assemble paralogous autosomal sequence due to high similarity among subgenomes, we can identify autosomal read pairs that align to the W haplotype but are spaced too far apart (>1 kb) to have originated in the SDR. The nonadjacent sections of the W haplotype where these paired reads align must therefore be contiguous in autosomes as they are in Fvb, though not in the SDR (S7 Fig and S4 Table). Coverage depth for these reads does not differ between males and females (Student t test, p > 0.1), and in females, coverage is 8-fold higher than for W-specific read pairs, suggesting that these reads originate from autosomal or pseudoautosomal regions on all four subgenomes. Therefore, there is no evidence that any autosomal homoeolog possesses an insertion representing a degraded or partial SDR. After an SDR translocation event, there would have been little or no co-occurrence of two SDR cassettes in the same female because the SDRs would occur on distinct subgenomes that segregate separately. Once separated, two SDR cassettes can never rejoin the same genome because two female plants cannot mate. Therefore, it appears that the former sex chromosomes, which have reverted to autosomes due to SDR turnover events, are descended from Z chromosomes and not W chromosomes (Fig 5). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 5. Model of sex-chromosome evolution in Fragaria. The eight homoeologs of Fvb6 on four subgenomes (Av, B1, B2, and Bi) are shown in a temporal sequence, starting with a presumed hermaphrodite octoploid ancestor (left). Dotted arrows indicate evolutionary descent of chromosomes. Solid arrows indicate inferred translocation or retrotransposition events. Following the move-lock-grow model, hemizygosity increases with each jump, from the retrotransposed RPP0W (red), to the SDR cassette including sequence homologous to the first SDR location (orange), to the SDR cassette plus flanking sequence (purple) representing the largest hemizygous region that is observed in the final SDR location. SDR, sex-determining region. https://doi.org/10.1371/journal.pbio.2006062.g005 Repeated translocation of the SDR is the only explanation consistent with all observations. Shared sequence across disparate SDRs could be explained if the shared sequences were repetitive motifs common throughout the genome, but this is not the case. Indeed, such motifs would be present in multiple copies in all individuals and thus would not be female specific. If female-specific 31-mers were false positives due to chance co-occurrence of some sequences in our female samples, we would expect to see a similar quantity of male-specific false positives, which we do not (S2 Table). Similarly, if control of sex were polygenic, then several distinct sequences could all show a correlation with sex without being physically adjacent, but this explanation can also be ruled out. Not only does sex map to a single genomic location in each of several linkage crosses [39,40,42,43,48], but under polygenic architecture, no one sequence would show a perfect correlation with sex. Furthermore, we observe sequencing reads spanning the junctions between distinct sections of the female-specific haplotype (S7 Fig), confirming that these sequences occur side by side. Therefore, the distinct sections of the SDR are adjacent only in females and must have been brought together by translocation.