Evolution of sexual systems, sex chromosomes and sex-linked gene transcription in flatworms and roundworms – Nature.com

Identifying sex-linked regions in schistosomes and nematodes

To systematically identify the sex-linked regions in each ofthese two deeply diverged phyla, we compiled sexual system information as well as publishedgenomic (references shown inSupplementary Data1) and transcriptomic (Supplementary Data2) data from sexed individuals (when available) for 13 platyhelminths and 41 nematodesspecies (Fig.1a). Genome sequencing quality varied markedly among these published datasets (Supplementary Data1, with references of all used data included). For 11 nematodes and 3 platyhelminth species, chromosome-level genome assemblies are available, and 4 nematode chromosomal genome assemblies (from Trichuris muris in clade I, Brugia malayi in clade III, Strongyloides ratti in clade IV and C. elegans in clade V) were used as references to create chromosome assemblies from scaffold-level draft genomes of 19 other related species (Supplementary Data3; and between 60% and 99% of the sequences can be incorporated into the assemblies). Previous cytogenetic studies suggested a ZZ/ZW sex system in the ancestor of schistosomes41, and an XX/XO system in the ancestor of nematodes42,43. If extensive Y or W-linked regions of species in either phylum have become highly degenerated due to lack of recombination27,28, the X or Z-linked regions can be identified from low genomic read coverage in the heterogametic sex (females in schistosomes, and males in nematodes). Such low coverage results from substitutions accumulated in Y- or W-linked sequences, together with Y- or W-specific transposable element insertions, both of which hinder mapping of sequencing reads to their counterpart X- or Z-linked regions, plus the direct effect on coverage of large deletions28. Coverage analysis, without linkage map data for the sex chromosomes, can thus detect highly divergent sex-linked regions44, and is widely used, including in birds23, teleosts22, reptiles and theLepidoptera45. It has also previously been used to detect sex-linked regions in schistosomes20, and to identify the X-linked regions in several nematode species34,46. Our analysis looked for bimodal distributions of male or female read coverage, with separate peaks corresponding to autosomal and sex-linked regions. For all 8 species whose sex chromosomes have previously been identified, our coverage pipeline yielded results in agreement with the published ones33,34,46,47,48,49,50,51, confirming that this approach is reliable (Supplementary Data1). Physically small or non-differentiated sex-linked regions, however, may not be detectable by our approach.

Among all the platyhelminthspecies studied, only the schistosome genome sequences exhibited the signature indicating presence of sex chromosomes, and (as expected since males are ZZ) it was not seen when only male reads were available (Fig.1b; Supplementary Data4). Hermaphroditic trematodes and cestodes also exhibited similar coverage for all chromosomes or a unimodal coverage distribution, again as expected. By comparing the previously identified oldest evolutionary stratum (stratum 0 or S0) of Schistosoma japonicum52 to the sex-linked region of the distantly related S. haematobium (Supplementary Fig.1), we confirmed the previous conclusion that they share the same S0 region. Therefore, recombination was first suppressed between the Z and W chromosomes in the ancestor of all schistosomes20, estimated to be about 21 MYA53, and the younger strata evolved subsequently. Our results also support previous data5,20 suggesting that gonochorism and the presence of sex-linked degenerated regions have originated only once in flatworms, with only a single ancestral linkage group having evolved to become a sex chromosome pair.

In the nematodes, different chromosomes became sex-linked in different clades. Our analyses newly identified the sex-linked regions and/or allowed the annotation of their corresponding Nigon elements in 17 species (indicated by red arrowheads in Fig.1c). This was possible for almost all of the species with the expected bimodal read coverage distributions in males (Fig.1b, Supplementary Figs.24), whereas most parthenogenetic species did not show bimodal read coverage(see below), or species with reads available only in females, or with both sexes pooled (Fig.1b, c). In addition to identifying sex-linked sequences in the homogametic sex of the species studied, we also sought to identify Y- or W-linked genes. As such genes may not be well assembled, due to the presence of repetitive sequences or very long introns, we further identified genes whose de novo assembled transcriptome sequences showed sex differences in genomic read coverage values from sexed animals (Supplementary Fig.5). Combined with genes directly assembled from genomic reads, we annotated between 6 and 219 Y-linked genes in 17 individual nematode species studied, and 48 W-linked genes for S. mansoni (Fig.1c, Supplementary Data4).

Previous studies reported translocations between sex chromosomes and autosomes in some nematode species34,36,38. Our findings confirm these results, but also identify new translocations, and we now characterise the order and timing of these rearrangements in relation to the phylogenetic relationships, demonstrating independent events over a broad evolutionary time scale (Fig.2).

a Whole-genome alignments of sex chromosome sequences between six nematode species with chromosome-level genome sequence assemblies. The X chromosome of each species is indicated by a red bar, and blue bars represent the autosome numbers shown after the species names. The abbreviations are as follows: Srat: S. ratti, Tmur: T. muris, Bmal: B. malayi, Ovol: O. volvulus, Hcon: H. contortus. Each line represents an orthologous gene pair, with red lines indicating NX element genes and blue lines NN ones. b Venn diagrams118 showing the numbers of shared sex-linked genes belonging to the same Nigon element in different species, with the same abbreviations as in (a). c Summary of the sex chromosome constitutions and turnovers among six nematode species with chromosome-level genome assemblies, other than C. elegans, organised according to the phylogeny. Nodes inferred to have had XY systems (which we infer to be the ancestral state, see the section of Evolution of sex-linked gene transcription in the main text) are indicated with dark blue dots, and nodes with XO systems with light brown dots. The estimated divergence times54 are also shown at the nodes. The X chromosomes of different species are indicated in red at the edge of each circos plot119, and different colours indicate each Nigon element that contributes part of each species X (these are also indicated by the distinct colours of the branches of the phylogeny). d Homologous Nigon elements detected in the sex chromosomes of all studied nematode species (except those shown in gray, where the contributions could not be determined); contributions are indicated by squares with the same colours as in part c. These results allowed us to infer the nodes at which different Nigon elements became part of the sex chromosome. The parentheses under the element names at the top indicate the number of times each Nigon element became part of a sex chromosome. Rhabditophanes sp.KR3021 (yellow squares) has no sex chromosomes. The dotted blocks represent reversion of sex chromosomes back to autosomes in Dracunculus medinensis.

We first focused on seven representative species with chromosome-level genome assemblies available (six from the orderRhabditida, i.e., clade III, cladeIVand clade V species, one from clade I species) (Supplementary Data1), and compared their X-linked gene contents and genome sequences. The autosomes of C. elegans (clade V species) correspond to Nigon elements NA to NE, and its X chromosome corresponds to two elements, NX and NN (X+N)36. The X+N composition is based on alignments of C. elegans X-linked genes to those in the genomes of five other Rhabditida nematodes: 103 C. elegans genes orthologs are also located on the X chromosomes of all five species, and are classified as NX-derived genes (Fig.2a, b; Supplementary Fig.6). The orthologs of the other 417 genes are classified as the NN-derived genes. These orthologs are either autosomal (in another clade V species- Pristionchus pacificus, in clade IV species- Strongyloides ratti, and clade III species- O. volvulus; Fig.2a; Supplementary Fig.7), or are found on the X chromosome, but in a distinct region from that occupied by the NX-derived ones (in the clade V species- Haemonchus contortus, and the clade III species- B. malayi). These findings suggest that the NX element was already a sex chromosome in the ancestor of rhabditid species 241 MYA (http://www.timetree.org/) or even earlier (see Fig.2c below), and that sex chromosome-autosome fusions or translocations added the NN element independently in some clade III and clade IV species (Fig.2a). The sex chromosomesof the Rhabditida thus originated much earlier than the origin of the oldest sex-linked region in extant schistosomes.

The only non-rhabditid species in Fig.2, Trichuris muris from clade I, has a sex chromosome composition of NA and NB elements (A+B), and lacks genes derived from the NX element. The divergence between clade I and the Rhabditida is dated at 400 million years ago54. During this time, either separate sex chromosomes originated independently in clade I and the ancestorof theRhabditida, or an ancestral NX element was replaced in a turnover event in clade I, with either the NA or NB element subsequently becoming the sex chromosome, followed by a fusion with the other element, creating a neo-sex chromosome (see below).

Based on seven species with chromosomal genome assemblies shown in Fig.2, we infer that, in the non-clade I or rhabditid species, the NX element has undergone independent translocations involving other elements, repeatedly forming neo-sex chromosomes. In clade V species, the NN element has translocated to the NX element, forming an X+N sex chromosome in C. elegans and H. contortus, but not in the P. pacificus lineage. In clade III, the NE and ND elements have translocated to the NX in O. volvulus (X+E+D)33, and the NN and ND elements have translocated to the NX in B. malayi (X+N+D)34 (Fig.2c). As the X+D translocation was found in species within clade III and clade IV, but not in the clade V species studied, it could have occurred either in the common ancestor of the Rhabditida, or independently in the ancestors of the respective clades. The former scenario seems unlikely, as it requires an X+D translocation followed by a complete fission and reversion of the ND element to a separate autosome in the ancestor of clade V. By further examining the orthologous genes shared by the autosomal ND element of C. elegans (clade V species) and clade III and cladeIV species, all with the X+D translocation, we estimated that only ~60% of the S. ratti (clade IV) ND-derived genes orthologs are also X-linked in O. volvulus or B. malayi (clade III), much less than the proportion of at least 85% (and up to 98%) of the NX-derived genes found among X-linked genes in pairwise comparisons among all ofthe Rhabditida species studied (Fig.2b). This supports independent X+D translocations in the ancestors of the clade III or cladeIV species. Similarly, the NB element corresponds to part of the X chromosomes of both T. muris (clade I) and S. ratti (clade IV), but fewer than half of the NB-derived orthologs are shared between these two species (Fig.2b), also suggesting its independent translocation.

The Nigon element compositions of the X chromosomes of nematode species, in addition to the seven species studied above, suggest even more translocations (Fig.2d, Supplementary Figs.810, Supplementary Data5). As only scaffold-level draft genome assemblies are available for these species, we estimated the coverage of their orthologous genes on different Nigon elements in male or pooled-sex genomic sequences, to identify patterns that indicate sex-linked regions (see above).

Among clade I species, in addition to T. muris, the inferred X-linked genes of two other Trichuris species (T. trichiura and T. suis) also include orthologs in C. elegans on both the NA and NB elements. X-linked genes of Romanomermis culicivorax, an outgroup to the Trichuris species, had orthologs only on the NA element (Fig.2d, Supplementary Figs.8b, 10). Most likely, therefore, the NA element was a sex chromosome pair in the ancestor of clade I nematodes and a translocation created the A+B state in the ancestor of Trichuris. If the NX element was the sex chromosome in the ancestor of all nematodes, a turnover event must have occurred, and resulted in the NA element becoming a sex chromosome in clade I.

Among the clade III species, we inferred that, after the ancestral X+D translocation in a clade III ancestor, the NA and NB elements were translocated to the X chromosome in the ancestor of the infraorder Ascaridomorpha (creating an X+D+A+B chromosome that is found in species including Anisakis simplex and Ascaris suum). An independent translocation of the NE element (X+D+E) also occurred in the ancestor of Onchocerca species including O. volvulus, and a translocation of the NN element (X+D+N) in the ancestor of both B. malayi and Wuchereria bancrofti. Other lineage-specific changes include translocations in Thelazia callipaeda (X+D+B) and Dirofilaria immitis (X+D+N). Interestingly, the X+D ancestral sex chromosome configuration has been replaced by the NE element in Dracunculus medinensis in a turnover event which occurred long enough ago to be detected by our coverage analysis (Fig.2d, Supplementary Figs.8a10).

Among the clade IV species, we note that Strongyloides species are parasites, and have environmental sex determination in which XX females reproduce parthenogenetically in the parasitic stage, but can produce XO males in response to the host immune response. The mechanism involves loss of one copy of the X-linked sequences during oocyte mitosis46,55, suggesting that this is a derived state in these species, and that the X is ancestral. Our inference of sex chromosomes in clade IV (Fig.2d), including these species, suggested that, following the X+D translocation in the ancestor of clade IV species (Fig.2c), a second translocation involving the NB element created the (X+B+D) sex chromosomes in both the free-living Parastrongyloides trichosuri and all Strongyloides species studied here, as the sex chromosome of their outgroup species P. redivivus does not include NB element genes. It follows that these translocations occurred in a common ancestor of P. trichosuri and Strongyloides, consistent with the transition to parasitism, and environmental sex determination, in the latter being more recent.

We identified translocations only when the Nigon elements involved exhibited reduced male read coverage. Our analyses therefore inferred only regions that have been non-recombining for such a long evolutionary time that either Y-linked genes have been lost or have undergone so much sequence evolution that they do not map to their X-linked alleles. Importantly, however, the results suggest that (barring the unlikely possibility of insertion of large autosomal regions into a non-recombining part of the X) each of the translocation events led to a formerly autosomal region becoming completely sex-linked.

Despite the large number of rearrangements involving sex chromosomes that we identified, it is therefore currently not possible to ask whether sex chromosomes are more often involved in translocations than autosomes, because coverage analysis does not detect events involving only autosomes.

The time since a translocated Nigon element became non-recombining can be inferred if it still carries enough Y-linked sequences to estimate the divergence level from their X-linked counterparts. If recombination becomes completely suppressed in a formerly recombining region, the region will start to diverge in sequence (as explained above)56. Events causing loss of recombination at different times are detectable from different levels of divergence (termed evolutionary strata) between sequences on the two sex chromosomes. Strata may either occur through successive chromosome inversions (e.g., some strata in mammals24, birds23 and sticklebacks21), or by other mechanisms affecting local crossover rates56. Strata could also arise on neo-sex chromosomes either (i) over time after their translocation (as in mammals), or (ii) immediately, as a direct consequence of the fusion in a species without crossing over in the heterogametic sex (as in Drosophila, see above). Unlike Drosophila, nematode males undergo crossovers. In both sexes, C. elegans chromosomes exhibit distinct recombination domains: a low recombination central region, flanked by high recombination arms, and very low recombination at the tips and other nematodes show similar patterns57,58 (though exceptions are reported, including in H. contortus59). In P. pacificus, strong localisation of crossovers to one chromosomeend specifically in males was observed for elements NC and ND, though not for the three other autosomes60. If such a sex difference exists, it could prevent recombination across most of the genome in males, similar to the proposed situation in the guppy61. In species with a recombination landscape similar to that of C. elegans, an X-autosome fusion creates males heterozygous for the fused chromosome and the free autosome, and the protein machinery that ensures crossover localisation may re-position crossovers away from the fused chromosome end of the former autosome. Such a change was reported for an X-IV fusion in C. elegans62, which has a decreased recombination rate in the fusion-proximal former chromosome arm region, but an increased rate in the distal region. To discover whether, and how, the fusions affected recombination on the neo-X chromosomes, and whether the cessation of recombination occurred in a single or multiple steps, we examined the translocated Nigon elements in different nematode lineages.

In extant clade IV and cladeV species with XX/XO sex systems (Fig.2c), the homologous chromosome (the neo-Y chromosome) is no longer present, and only the translocated X-linked Nigon element (the neo-X) remains. In such systems, the only approach available to detect multiple recombination suppression events is to test for accumulation of repetitive sequences in the neo-X. Regions in which recombination was suppressed longest ago are likely to have higher accumulated repetitive content. However, deletions can occur in degenerated regions, and accumulation differences may be obscured by intrachromosomal rearrangements, so repeat distribution is unlikely to reliably reflect the pattern of distinct evolutionary strata. Not surprisingly, no clear evidence of evolutionary strata was found (e.g., in H. contortus, Fig.3, Supplementary Fig.4). The absence of neo-Y-linked genes shows only that these elements have completely stopped recombining, followed by genetic degeneration involving complete loss of genes, consistent with these neo-sex chromosomes having evolved long ago (Fig.2d).

For the X of each species, the component Nigon elements correspond to strata inferred based on the presence of mappable Y-linked sequences, male vs. female ratios of mapped read coverage and SNP densities, and repeat densities. All metrics were estimated for 50-kb long, 10-kb overlapping windows. For Brugia malayi and Onchocerca volvulus, the orthologous genes (vertical lines) are colour-coded according to their different Nigon elements. For Trichuris muris, B. malayi and O. volvulus, the Y-linked sequences that could be assembled along the X are colour-coded according to their pairwise sequence divergence between the X/Y, and the log2 male vs. female SNP density ratios and the repeat density on the X are indicated by colour-codes. Strata are labeled S0 for the oldest stratum, followed by S1, S2, etc.). PAR stands for the pseudoautosomal region.

Three other clade I andclade III species have XX/XY sex systems with retained neo-Y chromosome sequences, and we expect to find neighboring regions belonging to different strata, reflecting their different Nigon elements, and different times during which these have been sex-linked. First, recently translocated Nigon element(s) are expected to be either pseudoautosomal regions (PARs), where recombination still occurs, or young strata. PAR boundaries with the adjacent fully sex-linked regions should be revealed by sharply increased male read coverage to levels similar to that of autosomal sequences. Second, strata that evolved at different time points, via translocations or inversions, will have different Y-X sequence divergence levels (or, with less clarity, sex differences in SNP density), or, even less clearly, in the lengths of contiguously assembled Y-linked fragments. Repeat densities may also show increases at strata boundaries within fully sex-linked regions, particularly in the Y-linked haplotype (but also potentially in the X-linked one), as repeat elements are expected to accumulate rapidly after recombination stops63. We defined statistically significant transitions, and inferred strata boundaries, using change-point analyses (see Methods, Supplementary Fig.11).

Across almost all X chromosomes of the two clade III species, B. malayi and O. volvulus, read coverage in females is nearly uniformly twice that in males, except at the end of the neo-X chromosome, where the lack of any coverage difference identifies a single probable PAR (or a very young stratum) in both species (6.3% of the total assembly length in B. malayi, and 12.9% in O. volvulus). Nearly half of the X chromosome length consists of intermingled ND- and NX-element derived genes (Fig.3), indicating massive intrachromosomal rearrangements after the translocation of ND element genes to the NX element in the clade III ancestor (Fig.2d). Similarly, the X chromosomes of C. elegans and S. ratti are also derived from two Nigon elements, and genes from the two elements are also intermingled (Fig.2). The translocation boundary between the ND and NX elements in B. malayi or O. volvulus cannot be determined precisely due to the rearrangements, but we inferred in the previous section that the NX element must have been a sex chromosome for longer than the ND element. Therefore, each element should represent at least one stratum (labeled together as S0+S1 in Fig.3). S0 and S1 are shared by all clade III species, and S1 probably stopped recombining in males before the species divergence (estimated to be less than 50 MYA64; Fig.2d).

The other X-linked genes are almost entirely derived from the NN element in B. malayi, and from the NE element in O. volvulus, neither of which is intermingled with ND or NX element sequences, consistent with more recent translocations compared with the ND element that formed the S1 stratum (Fig.3, Supplementary Fig.12). Parts of the ND, NE or NN elements may have continued recombining with their autosomal homologs after the translocation, as discussed above. However, their lower coverage in males than autosomes indicates that the translocated regions have now completely lost recombination and become degenerated Y-linked regions.

Nematode chromosome end regions often have high repeat content, probably indicating that the terminal regions recombine very rarely57,58,60. Interestingly, in both B. malayi and O. volvulus, the translocation breakpoint between the S0+S1 region (ND+NX element) and the NE or NN element has a higher repeat content than other X-linked regions (P<2.21016, Chi-squared test), as do both ends of the fused chromosome (Fig.3). It is not clear whether this reflects accumulation of repeats after recombination was reduced by a translocation in a formerly recombining chromosome end region, similar to the case of X+IV fusion in C. elegans mentioned above62, or is simply a feature retained from the pre-translocation ancestral chromosome. If less distantly related species pairs differing by fusions can be found, it might be possible to distinguish between these possibilities.

Although few X-Y gametolog pairs were retained in either of B. malayi or O. volvulus (Supplementary Data6), and Y-X divergence cannot be reliably estimated, there are some signs that recombination might not have been suppressed directly by the fusions (as explained above, recombination suppression is not expected, in contrast to the effect of X-A fusions in Drosophila). Evolutionary strata may therefore have evolved since the fusions occurred. As expected if strata younger than S0+S1 subsequently evolved, the NE or NN elements have more assembled Y-linked sequences (indicating less degeneration) than the ND+NX element region (P<2.21016, Chi-squared test). Moreover, the NN element in B. malayi can be divided into a stratum S2 region next to S0+S1, based on genomic coverage indicating male hemizygosity (and pronounced gene loss from the Y), and a region with similar coverage in both sexes at the end of the chromosome (Fig.3). The latter can be further divided into an S3 region, with a significantly higher SNP density in males than females (P<0.001, Wilcoxon test, Supplementary Fig.11), suggesting some divergence between the X- and Y-linked sequences without pronounced Y gene loss (Fig.3), and a PAR with no SNP density difference between the sexes. Overall, we conclude that the B. malayi sex chromosomes could have at least four evolutionary strata and a PAR. A similar approach suggests five evolutionary strata and a PAR in O. volvulus (Fig.3, Supplementary Fig.11). Only 1% of O. volvulus X-linked S2 (element NE) sequences have corresponding Y-linked sequences, whereas the S3 carries 12% of the genes found in the counterpart X-linked region, suggesting that it formed more recently.

Analysis of the lengths of contiguously assembled Y-linked fragments failed to identify either PARs or evolutionary strata in the XY sex systems of the clade Ispecies T. muris and T. suis. Only 0.06% of the T. muris X-linked sequences have Y-linked counterparts. Their Y chromosomes have probably become completely degenerated, as suggested by a previous cytogenetic study of T. muris65, so that few fragments can be assembled.

Given that non-recombining regions have recently formed between sex chromosomes of both nematodes and schistosomes, it is interesting to ask whether genes in these regions followed the evolutionary trajectory of canonical ancestral sex chromosomes, including evolution of dosage compensation (DC), and a different distribution of sex-biased genes relative to autosomes66,67.

Genetic degeneration of Y-linked genes is expected to reduce transcript abundances of hemizygous X-linked genes below the ancestral level, favouring the evolution of dosage compensation (DC), either in individual genes (incomplete DC, iDC) or of the entire X chromosome (chDC)68. Dosage compensation likely initially involves an upregulation of X-linked genes in males (as in Drosophila69), but this may be followed by downregulation in females (as in mammals70 and C. elegans71). The opposite changes are expected in species with ZW systems68. S. mansoni was reported to have chDC in the cercarial stage, but iDC after sexual differentiation20,72. Meiotic sex chromosome inactivation (MSCI) in male gonads can also lead to evolution of an underrepresentation of male-biased genes on X chromosomes, as reported in C. elegans73 and Drosophila74. Because of its inheritance pattern and selection differences between the sexes66,75, the X chromosome is also expected to accumulate genes with female-biased expression (feminisation) and become depleted for genes with male-biased expression (demasculinisation) if the mutations are expressed in heterozygotes (wholly or partially dominant), while the Y chromosome should become masculinised. These changes occur either through expression changes, or relocation of individual genes between chromosomes. Both feminisation and demasculinisation have been reported for numerous insects29,74 and some nematode species76, and affect relative expression levels of sex-linked and autosomal genes, in addition to the effects of MSCI and dosage compensation.

Comparisons of the transcriptomes from whole adults between fully X-linked versus autosomal genes in males (X/A ratios), and between the sexes (M/F ratios for X-linked genes), suggest very diverse forms and extents of DC in our seven representative nematode species (Fig.4a, b). All the nematode species studied showed X/A expression ratios below 1 in males (based on between 782 to 2742 X-linked genes and 5398 to 10,490 autosomal genes, depending on the species, and Wilcoxon tests yielding P<0.05 for all species, see Supplementary Data7). These patterns were the same, regardless of whether we included strongly sex-biased genes (2-fold transcriptional difference between sexes) or not (Supplementary Fig.13). This supports the genomic coverage data showing degeneration of the Y chromosome, though, because the tissues analysed included gonads, MSCI of the male X chromosome may also contribute (Fig.4a).

a Transcription levels of autosomal (blue) and X-linked genes (red) of different nematode species in males and females. The dotted line shows the median log2 normalised transcription levels of autosomal genes. b Male/female transcription ratios of autosomal (blue) and X-linked genes (red) in nematodes. c Transcription levels of B. malayi and O. volvulus genes divided into different chromosome regions, with autosomes (A) in blue, pseudoautosomal regions (PAR) in green, and different evolutionary strata (S0-S4) in different colours. The dotted line is the median log2 normalised transcription level of autosome genes. Overall the whole-genome transcription level is male-biased. The two-sided Wilcoxon rank-sum tests yielding P<0.05 in all species (based on between 782 to 2742 X-linked genes and 5398 to 10,490 autosomal genes, see Supplementary Data7). The boxplots show the 25th percentile, median, and 75th percentile, and whiskers are set within 1.5 times the interquartile range. d Percentages of male- (blue) and female- (red) biased genes, showing, respectively, deficiency and enrichment on the X relative to the autosomes across the studied nematode species (based on between 7568 to 13,129 genes). Significant deficiencies or enrichments by two-sided Chi-square tests are indicated with blue asterisks (male-biased gene), and with red asterisks (female-biased gene): *P<0.05; **P<0.01; ***P<0.001. e Transcription patterns for autosomal O. volvulus genes (Ovol_A) that are homologous to the Y-linked genes of B. malayi (Bmal_Y) in males (M) and females (F), or vice versa. Species abbreviation: Tmur: T. muris, Tsui: T. suis, Spap: S. papillosus, Bmal: B. malayi, Ovol: O. volvulus, Hcon: H. contortus, Ppac: P. pacificus.

However, in clade I and cladeIII species, most X-linked genes in females, despite having twice the genomic copy number compared with males, exhibited whole-body transcription levels similar to, or even lower than, in males. This suggested either upregulation of X-linked gene expression in males, or adownregulation in females for nematodesin these clades (Fig.4a). While autosomal genes have the same genomic copy numbers between sexes, they are expected to exhibit an equal transcription level between sexes. However, autosomal genes of all studied species of these two clades exhibited a significantly (P<0.05, Wilcoxon test) lower transcription level in females than in males. Particularly in O. volvulus, the median transcription level of autosomal genes was four times higher in males than in females. In addition, the transcription levels of autosomal genes in male O. volvulus were similar to those of their orthologs in B. malayi or other species in clade I groups (see Methods, Fig.4c, Supplementary Fig.14). These findingssuggested that genome-wide downregulation of transcription levels in females not restricted to the X chromosome, probably accounted for a similar or even male-biased transcription pattern on the X chromosome (Fig.4a, Supplementary Fig.14). The female-biased sex ratio of O. volvulus77 may reflect selection for lower female transcription to resolve sexual conflict, as suggested in ref. 75. Intriguingly, in both B. malayi and O. volvulus females, estimated M/F transcription ratios of X-linked S0 region genes were closer to 1 than for autosomal genes (Fig.4c) and appeared to show iDC.

In the clade IV and clade V species studied, however, transcription levels of X-linked genes in females were lower than those of autosomal ones (Fig.4b), except for H. contortus, suggesting a chDC mechanism similar to that inC. elegans. All clade IV and cladeV species M/F transcription ratios were, nevertheless, lower for X-linked genes than autosome ones probably due to MSCI in males66,75.

The X chromosomes of most nematode species studied consistently showed adepletion of male-biased genes (using the criterion of 2-fold higher transcriptional levels in males than in females), and anenrichment in female-biased genes, compared to autosomes (all species P<0.05 by Chi-square tests; Fig.4d). Feminisation of the O. volvulus X chromosome seems to have evolved more slowly than demasculinisation, as all evolutionary strata have become demasculinised, but only S0 is enriched in female-biased genes. The Y chromosomes of both O. volvulus and B. malayi show signatures of masculinisation, involving recruitment of gene duplications from autosomes. Among the total 91 O. volvulus and 219 B. malayi Y-linked genes (Supplementary Data4), we identified 10 and 13 Y-linked genes, respectively, with no homologs elsewhere in the genome, but with an autosomal homolog in the other species as their potential progenitors, and these mostly have male-biased transcript levels (Fig.4e). This suggests that the Y chromosomes of both species have preferentially fixed relocated autosomal genes with pre-existing male-related functions (Supplementary Fig.15). Among the other Y-linked genes, we found two genes (Bm2848: WBGene00223109 and Ovo-sma-5: WBGene00242293) with X-linked homologs in the S0 region of the NX element. This suggests that the ancestor of clade III species had a Y chromosome homologous to the NX element (Supplementary Data8), which has now become degenerated. The sex chromosome system of the nematode orancestor of the Rhabditida ancestor might therefore have been XX/XY rather than XX/XO (Fig.2c), contradicting the currently accepted view42,43.

Finally, we also found evidence of masculinisation of the S. mansoni Z chromosome. The overall median expression level of S. mansoni Z-linked genes in testes was significantly higher (P<0.05, Wilcoxon test) than that of autosomal genes (Supplementary Fig.16).

Having defined the ancestral sex-determining regions in which suppressed recombination first arose in schistosomes (Supplementary Fig.1), we used the available somatic and gonad tissue transcriptome data of the (hermaphroditic) trematode Clonorchis sinensis (liver fluke)78, a relatively close outgroup to schistosomes, and the (hermaphroditic) taeniid cestode Taenia multiceps79, to suggest transcription patterns in the hermaphroditic ancestor of flatworms before gonochorism evolved. C. sinensis and T. multiceps were estimated to have diverged from the gonochoristic schistosomes about 70 and 100 MYA, respectively53,79. We first examined the chromosomal rearrangements that produced the extant schistosome sex chromosomes, then looked for candidate sex-determining genes that might be responsible for the transition to gonochorism, and finally studied genome-wide transcriptional changes after the transition. Our inferences of genes potentially participating in the sex-determination pathways of flatworms and nematodes other than C. elegans used the well-characterised C. elegans genes as a reference. However, it is known that sex-determining pathway genes can undergo rapid turnovers between even related species80,81; therefore, orthologs of C. elegans genes in other nematode species, and particularly the deeply diverged flatworms do not necessarily have a sex-determining function, and need to be functionally validated in the focal species in the future. In the following, we mainly focus our description on certain genes that have functional evidence in both schistosomes and C. elegans.

Alignments of sequences of the chromosomal assemblies of S. mansoni with C. sinensis (Fig.5a, Supplementary Fig.17a) and T. multiceps (Supplementary Fig.17b) indicate that the S. mansoni Z chromosome evolved through a translocation, as first proposed in the 1980s41. Our analyses clarified that this involved two ancestral chromosomes homologous to two (CM030370 and CM030373) of the seven C. sinensis linkage groups, and to LG3 and one part of LG1 of T. multiceps, followed by intrachromosomal rearrangements (Supplementary Fig.17b). These rearrangements might have led to the suppressed recombination of the oldest schistosome stratum, S0, which is expected to carry the candidate sex-determining genes of all Schistosoma species, since the translocation boundary overlaps the S. mansoni S0/PAR boundary. Within the Z-linked S0 region of S. mansoni, we found the ortholog of the C. elegans gene mag-1, whose knockdown in C. elegans causes the hermaphrodites to produce sperm instead of oocytes82; in S. japonicum, its knockdown causes apparent cell proliferation in testicular lobes83. Functional experiments in Drosophila84 have indicated an important and evolutionarily conserved role of mag-1 during oogenesis. If gonochorism originated in a non-gonochoristic schistosome ancestor via two mutations, a possible scenario might be that recessive female-suppressing mutations have first affected mag-1s likely feminising function in the germline, leading to a transition from hermaphroditism to androdioecy as the first step in the evolution of gonochorism2. The second mutation might have given rise to the dominant demasculinising/feminising gene U2AF2, which was recently identified on the S0 region of W chromosome of S. mansoni85,86.

a Gene synteny relationships between Schistosoma mansoni chrZ (Sman-chrZ) and Clonorchis sinensis linkage group CM030370 (Csin_CM030373, red) and CM030373 (Csin_CM030373, blue). The evolutionary strata of S. mansoni are shown in different colours, and the positions of the candidate sex-determining genes U2AF2 and mag-1 on the chrZ are indicated by arrows. b The distribution of the reported genes in the C. elegans sex determination pathway on different Nigon elements. c Lineage-specific duplication of C. elegans sex-determining genes across the phylogeny of nematodes. d Density plots comparing the testis or ovary (e) transcription levels, or the testis (f) or ovary (g) transcription specificity (measured by the transcript level ratios of gonad vs. soma) of gonad-biased genes of C. sinensis (light blue or red) and their S. mansoni orthologs (dark blue or red). Masculinisation, i.e., increase of testis transcription levels, is indicated with a larger male sign, and vice versa for feminisation and demasculinsation etc. Comparison of whole-body male (h) and female (or hermaphrodite, i) transcriptomes between C. elegans (dotted line) vs. H. contortus (solid line). j Masculinised S. mansoni ortholog of C. sinensis gonad-enriched genes (defined as the former having at least 2-fold higher transcription level in testis than the latter), and demasculinised, feminised, or defeminised genes. Their testis or ovary transcription levels of each gene are shown in the heatmap, as well as its functional category, chromosome location in S. mansoni, and information about the knockdown phenotype of its C. elegans ortholog (Supplementary Data10). k A proposed model for the transition of sexual systems in platyhelminths and nematodes. We hypothesised a 2-step model for the transition from a hermaphroditic ancestor to the gonochoristic schistosomes (see text). The recent independent transitions from gonochorism to androdioecy in different Caenorhabditis species have been studied previously88,120, as have the genes reported here. Changes in gonad transcription levels in relation to the sexual systems are indicated using larger or smaller male or female/hermaphrodite symbols.

The schistosome Z-linked S0 region also had a duplication of fox-1. In C. elegans, fox-1 communicates the different ratios of X vs. autosome copy numbers between sexes to the downstream switch gene xol-1, and directs male or hermaphrodite development87. However, the duplication of fox-1 in the S0 region of the Z chromosome is specific to S. mansoni and has not been detected in S. japonicum or S. haematobium. Therefore, the fox-1 paralog of S. mansoni may not have a sex-determining function.

Duplication of orthologs of genes involved in the sex-determination pathway of C. elegans88 also seems to have occurred in other nematodes, as we found lineage-specific duplications of 12 sex-determination pathway genes on different Nigon elements (Fig.5c). For instance, the ortholog of xol-1, the direct target gene of fox-1, is present only in Caenorhabditis species89,90 but not in the other nematode species (or in the schistosome species studied here, Supplementary Data9). The fog-3 gene is critical for spermatogenesis in C. elegans91, and is duplicated in both B. malayi and O. volvulus (Supplementary Fig.18), probably in the ancestor of clade III nematode species. Interestingly, O. volvulus also has a duplication of fox-1, similar to S. mansoni.

We found no clear relationship between the numbers of sex-determining gene orthologs and the probability of particular Nigon elements being translocated to an ancestral sex chromosome pair. The NB element carries the largest number of orthologs of C. elegans sex-determining genes and has been more frequently translocated onto the ancestral sex chromosome than other Nigon elements (Fig.2d). However, the NC element, with orthologs of seven C. elegans sex-determining genes, was not involved in such a translocation in any nematode species so far studied.

Next, we compared the transcriptomes among the gonochoristic S. mansoni vs. the hermaphrodites C. sinensis and T. multiceps, and between the gonochoristic H. contortus vs. androdioecious C. elegans (Fig.5d-i, Supplementary Fig.19), in order to reveal genome-wide changes of transcription following transitions in sex systems. We focused our comparison on gonad tissues whose transcriptomes were available for the three flatwormspecies and are probably more likely to change than somatic tissues in response to the transition. We defined gonad genes as those with at least 2-fold higher transcription levels in gonads than the soma. For the two nematode species, we analysed sexed whole-body transcriptome data, as tissue-specific transcriptomes were not available.

C. sinensis and T. multiceps gonad genes had unimodal distributions of transcription levels and specificities (measured by the gonad/soma transcription ratios); in contrast, their S. mansoni orthologs exhibited bimodal distributions of both values in testes, predominantly with lower values of both measures in ovaries (Fig.5dg, j, Supplementary Figs.19, 20). In particular, there were 202 S. mansoni genes that became masculinised (i.e., had increased testis transcription levels) relative to C. sinensis, compared with 231 genes that became demasculinised (Fig.5j). In contrast, only 114 genes became feminised (i.e., had increased ovary transcription levels) compared with 389 defeminised genes (Fig.5j). A similar pattern is observed in comparisons to the more distantly related T. multiceps vs. S. mansoni (Supplementary Figs.19, 20). These transcriptome patterns suggest that the transition from a hermaphroditic ancestor (represented by C. sinensis or T. multiceps) to the ZW system in the gonochoristic S. mansoni involved mostly defeminisation, a lesser or similar extent of masculinisation and demasculinisation, and an even lower extent of feminisation of gonad gene expression. C. elegans orthologs of feminised genes in S. mansoni are enriched for mutant phenotypes related to female reproduction (e.g., egg laying, vulva development) and embryonic or germ cell development (e.g., embryonic lethal and germ cell development variant) (P<0.05, Chi-square test, Supplementary Data11). While male-related mutant phenotypes (e.g., male behaviour variant and male response to hermaphrodite variant) show enrichment only in masculinised, but not in feminised, genes (Supplementary Data12). Interestingly, we identified 65 S. mansoni genes that were both masculinised and defeminised, and 3 genes that were feminised and demasculinised relative to C. sinensis in the gonads, suggesting possible resolution of sexual conflict after the evolution of gonochorism (Fig.5j).

Similarly, the reverse transition from a gonochoristic ancestor represented by H. contortus in relation to the androdioecious C. elegans seems mainly to have involved defeminisation and masculinisation of the gene transcription (Fig.5h, i, Supplementary Figs.21, 22).

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