Sponge genomes reveal a pre-metazoan origin of the sex determination toolkit and sex chromosomes

Sponge genomes reveal a pre-metazoan origin of the sex determination toolkit and sex chromosomes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Sponge genomes reveal a pre-metazoan origin of the sex determination toolkit and sex chromosomes Ana Riesgo, Jose Lorente-Sorolla, Aida Verdes, Irene De Sosa, and 17 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8554461/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Sex has profoundly shaped animal biology and evolution, yet sex determination is strikingly diverse and its evolutionary origins remain poorly understood outside bilaterians. We investigate the molecular basis of sex determination in eight gonochoristic sponge species using integrated genomic and transcriptomic analyses. We identify sex chromosomes in two species, likely arising through chromosomal rearrangements, and find widespread polygenic sex determination across all taxa. Sponge sex determination relies on a deeply conserved genetic toolkit with sex-specific loci in DNA repair and recombination genes, syntenic across species and traceable to unicellular ancestors, indicating that core components of sexual reproduction predate animals. We also uncover a younger metazoan-specific complement and show independent evolution of XY and ZW systems, revealing recurrent shifts in heterogamety. Biological sciences/Evolution Biological sciences/Genetics/Evolutionary biology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Main text Sex occupies a central role in the evolution of eukaryotes. It may have emerged as a repair mechanism to prevent damage to single DNA strands 3 , possibly triggered by rising oxygen levels in early environments 4 . Since its emergence, sex has profoundly shaped animal biology and ecology. The evolution of separate sexes has occurred multiple times independently both in plants and animals 1 , and there is a bewildering number of molecular mechanisms underpinning sex determination 1 . These range from entirely genetic sex determination (GSD) to environmentally-induced sex determination (ESD) and a combination of both. Within GSD, there is also a panoply of genetic mechanisms to specify females and males, from sex chromosomes to polygenic systems 5 . Revealing the molecular basis of sex in animals is crucial for explaining their evolution and diversification. Yet, most research has concentrated on a limited number of arthropods and vertebrates 1,2 , leaving early-diverging animal lineages largely unexplored 6 . Among them, sponges (phylum Porifera) hold a pivotal position as the most likely sister-group to the rest of animals 7 for investigating the origins of sex, but current knowledge on this phylum is fragmentary. The last common ancestor of sponges was hermaphroditic, but multiple independent transitions to gonochorism have occurred 8 , suggesting repeated evolution of GSD and/or ESD systems. However, evidence for GSD in sponges is restricted to the identification of a few candidate genes in a handful of species (e.g., DMRT1 and FEM1 ) 9 . To address this gap, we conducted a phylogeny-wide genome sampling of eight gonochoristic species from two classes and seven different orders of sponges (Fig. S1). We used genome resequencing of males and females to identify sex-linked regions and chromosomes (Table S1), six of which had chromosome-level reference genomes (Table S2; Supplementary text Sections 1, 2). Sex was identified through cytological surveys of male and female gametes (Figs S2–S3) and patterns of sex-biased gene expression were quantified through RNA-seq (Table S2). Finally, we traced the evolutionary origins of genes harboring sex-specific loci and analyzed their synteny across sponge genomes to uncover macroevolutionary patterns underlying sex determination. Sponges have independently evolved male and female heterogametic sex chromosomes. Using a combination of genomic approaches, we detected clear sex-linked chromosomes/regions in only two of the eight sponge species analysed: Chondrosia reniformis and Oscarella lobularis (Fig. 1, Fig. S4). In C. reniformis , a region spanning 2.6–3.8 Mb on chromosome 11 showed significantly higher heterozygosity in females but higher coverage in males (Fig. 1A–B; Fig. S5A-B; Table S3–S6). This pattern is consistent with a female heterogametic (ZW) sex-determination system, in which reduced mapping of W reads to the Z chromosome results in increased male coverage, while W-specific SNPs increase female heterozygosity. It also indicates intermediate differentiation between Z and W chromosomes. This region, hereafter called sex determining region (SDR), coincides with an area with significant sequence divergence between males and females, where 12 out of the 25 loci with highest (over 0.20) sex-specific F ST values were located (Table S3). The SDR exhibits significantly higher SNP density in females (i.e., number of SNPs per unit of genome length; Table S7) and a significantly higher accumulation of sex-specific loci (i.e., short DNA fragments generated by RADseq that are present/absent in one sex or the other) than that in other chromosomes (X-squared = 24.339, df = 13, p -value = 0.02815) (Fig. 1C, Fig. S6A; Table S8B-C). These sex-specific loci in the SDR were predominantly female loci. Finally, k-mer analysis identified female-specific k-mers assembled into 2178 contigs within the SDR (Fig. 1D), while only 38 were significantly associated to males (Fig. S7A). We also found pronounced sex differences in genome coverage (strongly male-biased), but not in heterozygosity, across a 3.6-4.7 Mb region of chromosome 2 in C. reniformis (Fig. 1A–C; Fig. S5; Table S3–S6). This region contained only a few sex-specific loci (Fig. 1C; Fig. S6) and kmers (343 contigs assembled from female-specific k-mers and only 20 from male-specific kmers; Fig. S7A–B). The genomic signature of chromosome 2 is consistent with an older Z chromosome 10 . To test this, we blasted the main haplotype to the alternate and found a scaffold in the alternate haplotype (CAMBO01000013.1) that blasts against chromosome 2 but displays the opposite pattern. It has coverage bias towards females (Fig. S5C–D) and contains 2437 contigs assembled with female-specific k-mers (Fig. S7C-D), pointing to the complementary W. In this scenario, we suggest that C. reniformis might possess two pairs of sex chromosomes (ZW and zw), with chromosome 2 being an older Z and 11 a younger z (Supplementary Text Section 1), similar to that observed in several animals 11–14 . Because chromosomal inversions suppress recombination in many organisms 15,16 , we next explored whether an inversion in these regions of chromosomes 2 and 11 could be driving divergence between the Z and W chromosomes. Since the reference genome derived from a female individual (heterogametic ZW), we investigated structural variation between sex chromosomes by mapping the alternate haplotype against the reference assembly. This revealed a ~0.7 Mb chromosomal inversion on chromosome 2 (3.7 to 4.4 Mb; Fig. 1E), that co-localizes with the SDR and confirms the reference specimen female sex. We propose that this inversion in chromosome 2 was a primary step in establishing recombination suppression and initiating the evolution of this sex chromosome in C. reniformis , as in other organisms 17 . Then we examined read-pair mapping orientation in chromosome 11 across all samples, and we recovered a cluster of paired reads mapping distantly and collinearly in positions 1.9 to 3.1 Mb (Fig. S8A–B), but without any vicariant signal between males and females (Fig. S8A). While a potential very recent inversion in the 1.9–3.1 Mb region of chromosome 11 cannot be ruled out, the signature observed can be due to accumulation of transposable elements (TEs) (see below), highlighting the diverse mechanistic origins of sex chromosomes in sponges. We then investigated whether the in SDRs of chromosomes 2 and 11 contained sex-related genes by doing GO enrichments, and found that in the category Biological Process functions related to regulation of the mitotic cell cycle, DNA recombination, reproductive process, and genitalia morphogenesis were among the most enriched (Fig. 1F). Finally, given that accumulation of TEs and specifically LINEs (Long Interspersed Nuclear Elements) is often associated with sex chromosome evolution in many species (e.g., in cephalopods) 18 , we analysed TE and LINE density across C. reniformis chromosomes. We found accumulations of TEs in the central region of all chromosomes, including sex chromosomes and their SDRs, but no particular pattern of LINE accumulation in any of the two putative sex chromosomes (Fig. S9, Fig. S10A). Such accumulations frequently follow inversion events, as suppressed recombination in heterokaryotypes (eg: ZW or XY) prevents TE purging, consistent with the sex-driving inversion hypothesis. Nonetheless, TE accumulation alone may also drive SDR origination or promote inversion events, scenarios that cannot be ruled out here. In Oscarella lobularis , we found sex-specific loci, using RADSex, exclusively in males (Table S8B, D; Fig. 2A; Fig. S6B), indicating the presence of an XY-like SD system. Consistent with this, males showed higher heterozygosity and higher coverage within the 2–3 Mb region of chromosome 5 (Fig. 1G–I; Fig. S11A–B), with a significant accumulation of male-specific loci (Fig. S6B; Table S8) compared to other chromosomes (X² = 101.69, df = 19, p -value = 2.641e-13) and 1213 contigs assembled from male-specific k-mers covering the same region (Fig. 1J; Fig. S11C). These findings are consistent with chromosome 5 being the sex chromosome. This would mean that the SDR has failed to assemble separately in the reference genome given its relatively low divergence, resulting in a co-assembly of the X and Y (Supplementary Text Section 2; Figs. S11, S12). We then analysed haplotype divergence between sexes in the entire genome with iDlG, and found a clear pattern only in this SDR of chromosome 5 (Fig. 1K, Fig. S8C–D), consistent with an inversion promoted by physical hindrances during recombination 19 . To further validate the presence of an inversion in chromosome 5, which potentially led to sex determination in O. lobularis , we evaluated the karyotype of the reference specimen by mapping the alternate haplotype against the reference assembly, since the reference genome derived from a male individual (heterogametic XY). This analysis revealed a ~1.3 Mb region flanked by two chromosomal inversions on chromosome 5, from 1.8 to 3.1 Mb, that co-localized with the limits of the SDR (Fig. 1L). We propose this pair of inversions flanking the SDR as the primary mechanism producing the recombination suppression that propelled the evolution of sex chromosomes in O. lobularis . Sequence pairs mapping distantly in the same direction on the same strand across all specimens showed that the chromosome 5 SDR had a higher density of reads mapping collinearly at long distances in the same strand than the rest of the chromosome (Fig. S8D), further supporting the presence of an inversion. As for C. reniformis , we evaluated the TE and LINE density across all chromosomes, and found that one of the greatest accumulations of TEs occurred in the SDR region of chromosome 5 (Fig. S13), similar to the sex chromosomes of other organisms 18 . However, chromosome 5 showed the fewest number of LINEs per Mb (Fig. S10B). Both collinear reads and the high abundance of TEs within the SDR further provides evidence on a potential sex-driving inversion. In this case, the segregation of alleles as found with iDlG (Fig. 1J) indicates that this inversion is older than the one identified in C. reniformis , and could be a driver for the emergence of a sex chromosome given that it contains several sex-specific loci. To understand the functions of the genes present in the SDR of chromosome 5, we looked into the enriched GO terms for Biological Processes, finding regulation of the mitotic cell cycle, DNA recombination and reproductive process among the most enriched (Fig. 1L). None of the other four species with reference genomes showed significant biases in their heterozygosity, coverage or SNP density between females and males (Figs. S4, S6; Table S4–S7) that could be indicative of a large sex-linked region. In addition, no accumulation of LINEs was detected for any of the genomes of these four species (Fig. S10). Evidence for a convergent polygenic sex determination toolkit across sponges. With RADSex, we identified 7,657 sex-specific loci across all eight surveyed species (Table S8). In every case, both the total number of sex-specific loci and their distribution between sexes were significantly different from random expectations, as shown by 1,000 permutations in which individual sexes were randomly reassigned (Fig. S14). Except for C. reniformis and O. lobularis , where sex-specific loci clustered in SDRs, in the other species they were widely distributed across their genomes (Fig. S6; Table S8). This is consistent with polygenic sex determination (PSD), in which multiple loci contribute additively or epistatically to sexual fate, as reported in cichlids, zebrafish, and houseflies 5,20 . Although sex-specific loci of both XY- and ZW-like type were retrieved in all sponge species, one system appears to predominate in each of them: in P. ficiformis , and H. panicea , the excess of female-specific loci suggests dominance of ZW-like systems, whereas in the remaining species the prevalence of male-specific loci points to dominance of XY-like systems. This interpretation is further supported by sex ratio biases observed in these sponge species (Table S1), which, together with variance in sex ratios among families and the genetic mapping of multiple sex-specific markers, are considered indicators of PSD 20 , with dominance of one mode of inheritance over the other 5 . The sponge sex-specific loci identified by RADSex in the eight species mapped on to 1,437 genes, with 15–25% of them known to be involved in animal sex determination (Fig. 2A) and a few further annotated within the GO term category sex determination (GO:0007530) (Fig. 2C; Table S9C–D; Supplementary Text Section 3.1). Around 22% of the sex-specific loci were shared by two or more species (Fig. 2D, F; Fig. S15A) (Table S9A–B). This is similar to what is found in sex determination of insects and vertebrates, where a fraction of the sex determination gene complement is shared, like the DMRT1 or FEM1 genes, but there is usually a larger fraction of sex determination genes specific to each lineage 21,22 . In any case, genes regulating sex determination in sponges seem to converge on the same cohort of genetic pathways that maintain and repair DNA structure and determine gonad fate, as well as regulate the meiotic cell cycle checkpoint signaling and the MAPK cascade, with minor contributions of other genes involved in metabolic and/or structural processes (Figs. 2B–C, F–G, Fig. S15). Similar observations have been found in mammalian Y chromosome genes 23 and those of other organisms 2 . Among the shared genes with sex-specific loci, we found several transposable elements or transposon-related genes, such as YRD6, RTBS and POL2–5 (Tables S9D, S10). These transposons harbored male-specific SNPs and are known for their established involvement in spermatogenesis 24,25 . Also, two helicases were largely shared by most sponges (Tables S9D, S10), the ATP-dependent helicase PIF1 and the RNA binding protein ZNFX1 , which are known to be associated to males and differentially expressed in testicles 24,26,27 . Besides these, some of the shared sponge genes containing sex-specific loci were involved in conserved sexual reproductive machineries and regulation pathways of animals (Fig. 2A–C). For example, transcription factors (TFs) are key players in vertebrate sex determination, where SOX9 , SRY , NR5A1 and GATA TFs are crucial to initiate the development of testes 28,29 . Here, we found that GATA4 contains female-specific SNPs in both Geodia spp. and H. panicea (Tables S9D, S10). This is important because GATA4 is an upstream effector of SRY and plays a predominant role in both primary sex determination and sex differentiation via steroidogenic function activation of SF-1 and Star in mammals 30 , and thus indicates an important, evolutionary conserved role for this transcription factor in animal gonadal development and function 30 . In addition, a male-specific SNP was identified in Star (Steroidogenic acute regulatory protein) genes in P. ficiformis (Tables S9D, S10). Also, Fem genes, here with loci specific to males in both C. reniformis and Axinella damicornis (Tables S9D, S10), have been identified as sex-determining genes in nematodes and insects 31,32 . Sex determination genes in sponges are largely lineage-specific. Almost 70% of all the genes with sex-specific loci (either female or male) were restricted to a single sponge species (Fig. 2D, F; Table S9A–D), even though orthologous sequences of these genes were present in all species (see examples in Figs. S16–S17). Genes with species-specific sex-loci that were unique to each lineage, were mostly involved in DNA repair, chromosome organization, and cell cycle checkpoint signaling (Fig. 2E, G). In this category of lineage-specific genes with sex-specific loci fell those identified in the SDRs of C. reniformis and O. lobularis (Table S11A-B). In C. reniformis , the SDR of chromosome 2 mostly contained transposable elements with low levels of expression (Table S11A–S12A), while that of chromosome 11 contained both transposable elements (with low expression), and genes with sex-specific loci involved in cell regulation and homeostasis ( PAR14, LORF2, GARS, PTPRD ), cyclins ( CDK13 ), mitotic checkpoint ( CENP-E ) and cell cycle regulators ( ADAS ) that were highly expressed (Table S11A–S12B). Several of these genes have known functions in sexual reproductive processes (See Supplementary Text Section 3.2), such as PTPRD (with male-specific loci in our dataset, Table S8), CENP–E and CDK13 (with female-specific loci in chromosome 11, Table S8, See Supplementary Text Section 3.2). Interestingly, sponge CENP-E sequences appear in different clades of the phylogenetic tree of the eukaryotic protein family, suggesting divergent protein conformations that might confer different functions (Fig. S16). In O. lobularis , six genes with male-specific loci (Table S11B) were located in the candidate SDR of chromosome 5 ( G2E3, PIF1, HMCN1, MRC1, DAPK1, RECQ ), and showed higher expression in females than in males (Fig.1O–P; Table S12C), although not significantly. Notably, all of these genes have known functions related to sex determination in other organisms (Tables S11B, S10), such as fruit flies and sea cucumbers 26,33 . Among the genes with sex-specific loci unique to each species, we also identified several associated with DNA damage repair and chromatin or structural maintenance, along with a diverse array of signaling pathways (Fig. 2E, G; Tables S9–S10). A fundamental aspect of sexual reproduction is the capacity of recombination to exchange genetic material during meiosis. While homologous autosomes pair and recombine completely during meiosis, sex chromosomes have evolved to restrict this process 34 . For instance, the DNA repair genes RAD51 and RAD52 , whose protein products recognize meiotic double-strand breaks (DSBs) and repair them, are described to be fundamental for chromosome pairing 35 , and are thought to be the primary sexual genetic machinery that arose to facilitate sexual cycles in eukaryotes 3 . Here, RAD52 in P. ventilabrum and RAD51 in H. panicea contained male and female-specific loci respectively (Tables S8, S9A–D, S10). Other DNA repair and meiotic genes also contained sex-specific loci in different species, including BRCA2/FANCD1, BRWD1, AGO2, PKRDC, HAP2 and MEIG1 (Fig. 2A, Fig. S18; Tables S9, S10; Supplementary Text Section 3.3). Although meiotic genes are critical for making functional gametes 36 , they usually do not determine whether the gamete should be female or male. However, they are essential for gametogenesis and mediate membrane fusion between gametes in a broad range of eukaryotes, ranging from algae and higher plants to protozoans and cnidaria 37,38 . Several signaling pathways, including SRY , WNT, JNK and MAPK have critical roles in sex determination 39 , and these and embryonic morphogenetic pathways were enriched among the sex determination complements of several sponges (Fig. 2E; Table S10; Fig. S18; Supplementary Text Section 3.3). In O. lobularis we identified male-specific loci in genes that are part of the SRY cascade in mammals and marsupials 40–42 , including ATRX , INSRR , and UBE3A (Table S10, Supplementary Text Section 3.3). Although ATRX was present in all species except P. ficiformis , a sex-specific locus was only found on this gene in O. lobularis (Fig. S17). The WNT pathway, fundamental in mammalian female development through the activation of WNT4 41,43 , was also identified as crucial in P. ficiformis, O. lobularis, and H. panicea , with female-specific loci in WNT4 , APC and NRARP respectively (Fig. S18; Tables S9D–S10). Finally, the MAPK cascades, that are important for growth, proliferation, differentiation, motility, stress response, survival and apoptosis 44 , were also found enriched among the genes with male-specific loci in C. reniformis , P. ficiformis , P. ventilabrum , and H. panicea, with genes directly activating the MAPK cascade that were shared such as RYK , and some that were unique, such as GSK or ROR1 (Fig. 2E; Fig. S18; Tables S9D–S10). Alternative splicing underlies sex-specific gene expression in polygenic sponge sex determination. Differentiated (heteromorphic) sex chromosomes often exhibit a non-random accumulation of sex-biased genes 45 . By comparison, in homomorphic systems, sex-linked genes are sometimes expressed at similar levels in males and females 46 , and sex-specific regulation may instead arise through alternative splicing (AS). AS provides a plastic mechanism for generating isoform diversity from a shared genetic background, enabling the fine-tuning of expression to meet male and female developmental needs without requiring gene duplication or large-scale divergence. In this way, AS plays a central role in regulating development and is essential for sex determination in both vertebrates 47 and invertebrates 48 , where it underlies processes such as sexually dimorphic cell differentiation and dosage compensation 49 . Given that sponges do not show sexual dimorphism, the sexes likely share very similar trait optima, and thus, following the predictions of Flintham (2025) 50 , we did not expect to observe strong sex-biased expression even at genes with sex-specific loci within their polygenic sex determination system. Here, we tested whether genes with sex-specific loci were more likely to show differential expression or alternative splicing, as such regulatory differences can help restrict sex-specific fitness effects to the appropriate sex. We first examined chromosome-wide expression patterns and found similar F:M expression ratios for autosomes and sex chromosomes in both C. reniformis and O. lobularis (Fig. S19A, D), even in the SDR (Fig. S19B–C, E; Tables S9-S10). Then, we looked at genes with sex-specific loci in particular, and found that only 0.3 to 11.5% of these genes were differentially expressed between sexes (Figs. 3A; Table S10, S14; Supplementary Text Section 3). Among those, we found that the PTPRD gene, which contains a male-specific loci and is located in the SDR region of chromosome 11 in C. reniformis , was significantly upregulated in males (logFC=-5.72 logCPM=5.33, p -value=4,78E-07 FDR=0,00013) (Table S14B). This gene is a well-known candidate for temperature dependent sex determination in reptiles, and its upregulation can help stabilize male fate commitment during ESD in turtles 51 . Then, we observed that most genes with sex-specific loci produce several transcripts with few nucleotide differences (detected with StringTie), and the different transcripts show higher expression in one sex than the other (Fig. S18, Table S15). To test whether those differences were statistically significant, we performed a Welch Two Sample t-test comparing genes with sex-specific loci that produce a single transcript with those that produce several. We found significant differences in gene expression between sexes ( p -value=0.0045) only in genes with sex-specific loci that produce several transcripts in C. reniformis (Fig. 3C–J; Fig. S18; Table S13-S15). In this case, the expression was consistently biased towards females (Fig. 3D, Table S15). We then looked at AS in the six sponges with reference genomes, and found that all showed AS events (ASE), ranging from 3.5% of genes in P. ficiformis to 60.3% in C. reniformis. These ASE were identified in both autosomal genes and those with sex-specific loci, with exon skipping (SE) being the most common type, followed by alternative 3’ splice sites (A3SS) (Table S16, Supplementary Text Section 4), unlike what has been previously found in other sponges 52 . Interestingly, significantly sex-biased ASE in genes with sex-specific loci were found across all species except O. lobularis , again with SE being the predominant event (Fig. 3B, Table S16). Again, it was in C. reniformis where we found the greatest proportion of genes with sex-specific loci and ASE (3.1%, Table S16A). In many cases, the isoforms produced by exon skipping in genes with sex-loci resulted in different proteins, like the case of CENP-E in C. reniformis (Fig. S16B–C). Overall, these findings indicate that in sponges, where sexual dimorphism is absent and trait optima are likely shared between sexes, strong sex-biased levels of gene expression are rare even at genes with sex-specific loci. Instead, sex-specific regulation appears to arise primarily through alternative splicing, providing a flexible mechanism for achieving sex-specific regulation that has been described in polygenic sex determination systems without requiring large-scale shifts in gene expression 53 . Sponge sex chromosomes show synteny and chromosomal fusions. To quantify shared genomic complements across sponges, we first compared the genomes of our six sponges and found they share around 75% of protein-coding genes, with 25% conserved across species and 50% shared by at least two species, and with the remaining 25% of each genome being species-specific (Fig. S15B–C; Supplementary Text Section 4). Then, to investigate how macrosyntenic regions have been rearranged during sponge evolution, we analyzed 941 single-copy orthologs shared among the six sponge species (Fig. 4A). This analysis showed well-conserved synteny and colinearity, with 18 ancient linkage groups or ALGs shared between O. lobularis and the other five sponges (Fig. 4A). This indicates a remarkable conservation of chromosome architecture across sponge classes that diverged more than 600 million years ago 54 , consistent with previous reports of macrosynteny conservation in sponges 55 . Despite strong syntenic and collinear patterns across sponges, only 65 out of the 828 syntenic genes had sex-specific loci in at least one species (Fig. 4B; Table S17; Supplementary Text Section 5), which is partly due to the fact that many genes with sex-specific loci had several paralogs. We then performed the analysis only with the two species with sex chromosomes, and found 115 syntenic genes with sex-specific loci among the total 4,582 (Fig. S20; Table S17). Although most of them were distributed across many chromosomes (Fig. 4B), 60% of them were concentrated on chromosome 11 in C. reniformis , which appears to result from a fusion between two ancestral chromosomes (Fig. 4A–B). The genomic instability of this fusion could have prompted an inversion (Fig. S8B), altering recombination between this pair of chromosomes and leading to the emergence of a sex chromosome in C. reniformis . Our results support the idea that sex chromosomes evolve independently from autosomes 15 , with frequent turnovers due to the moderate divergence of sponge sex chromosomes, which could make them more prone to revert back to autosomes. Deep evolutionary origin of genes with sex-specific loci. We investigated the evolutionary age of sponge genes across the six species with reference genomes by assigning each gene to its most probable taxonomic origin based on their homology (Table S18A–B). Around 60% of the genes traced back to ancestral nodes (Fig. 4C; Table S18A), highlighting their conservation across animals and deeper eukaryotic lineages 54,56 , while only 11.1% were classified as species-specific. We then examined specifically the evolutionary history of sponge genes with sex-specific loci (Table S18C), and found that the majority were assigned to deep evolutionary nodes (Fig. 4C; Table S18A). Genes assigned to unicellular organisms and Eukaryota were predominantly enriched in functions related to DNA repair and recombination, cell-cycle regulation, cytoskeleton organization, membrane fusion and adhesion, stress response, and apoptosis (Fig. 4D, Table S18E). Genes with sex-specific loci assigned to Metazoa were enriched in terms related to transcriptional regulation and reproductive processes, consistent with the emergence of specialized, multicellular reproductive systems 3 . Previous studies suggest that sex determination mechanisms have been conserved for more than 300 million years 57 , but our results suggest an even earlier origin, given the conserved set of genes across metazoans that underlie sex determination in sponges. Our results suggest that the same genetic toolkit that facilitated sex determination in sponges was already present in ancestral eukaryotes and early metazoans 58 , with conserved functions in sexual development 38 . Finally, the modest contribution of more recent, lineage-specific genes (Fig. 4D, Table S18E) likely reflects functional fine-tuning or clade-specific adaptations, rather than de novo evolution of entirely new sexual pathways. Patterns of evolution of sex determination systems in animals. A major challenge in understanding the evolution of sexual systems across Metazoa is resolving their distribution and diversity among extant lineages. Here we show that the transitions from hermaphroditism to gonochorism that occurred repeatedly during sponge evolution 8 were accompanied by the independent evolution of a striking variety of sexual systems (Fig. 5A). Such diversity mirrors the situation across the Tree of Life (Fig. 5B; Supplementary Text Section 7), where gonochorism can be achieved through a wide array of molecular mechanisms and sex chromosome systems, with shifts documented at the phylum, class, order, and even family level 2,26,59 (Fig. 5B). Most information about sex chromosomes is known for well-established bilaterian model species 2 , with non-model organisms remaining comparatively understudied. Recent advances in sequencing techniques, computational tools, and algorithms are now enabling rapid progress in uncovering sex determination in other invertebrate groups 6,18 . Given that sex determination systems vary widely across animals, expanding research into diverse and early-diverging lineages is vital to understand its origins and evolution. Our study broadens knowledge in early-splitting animals by identifying sex determination systems in seven orders of sponges— one XY, another ZW, and five polygenic (both XY-like and ZW-like). We reveal both the persistence of a conserved set of genes involved in sex determination inherited from ancestral eukaryotes and early metazoans and the independent origin of sponge sex chromosomes, likely shaped by unstable chromosomal rearrangements. Together, these findings underscore sponges as key to understanding the evolutionary forces generating the extraordinary diversity of sexual systems across animals. Materials and Methods 1. Sampling, assessment of reproductive activity and sex assignment: Samples of eight gonochoristic species were collected from the North Atlantic Ocean between 2017-2019, the Mediterranean Sea in the summer of 2021 and 2022, and the Kiel Bay in summer of 2021 (Table S1, Supplementary Fig.1). We collected approximately 3-5 cm 3 of sponge tissue per specimen and divided the sample in 4 pieces that were later preserved in three different preservatives: 1. For traditional histology, in 4% formaldehyde in seawater, 2. For further construction of DNA libraries (for both RADseq and WGS), in absolute ethanol and stored at -20 °C, and 3. For transcriptomic analyses, a tissue piece of about 2 cm 3 was preserved in RNAlater at 4ºC for 24 h and then frozen at -20 °C until further processing. Since sponge sex is only possible to determine through presence of gametes, we processed the samples for histology. Samples of sponges with siliceous spicule content ( Petrosia ficiformis , Geodia hentscheli, Geodia barretti , Axinella damicornis , Halichondria panicea , and Phakellia ventilabrum ) went through a step of desilicification in 5% hydrofluoric acid overnight and then rinsed with distilled water at least twice. Then, their tissues and those of the sponges without spicules ( Chondrosia reniformis and Oscarella lobularis ) were processed for light microscopy Tissues were dehydrated through an increasing ethanol series and later embedded in paraffin after a brief rinse in xylene. Then, paraffin blocks were sectioned at 5 μm with an HM 325 rotary microtome (ThermoFisher-Scientific) and sections stained with hematoxylin and eosin, using standard protocols, and mounted in slides with DPX. Slides were observed with an Olympus microscope (BX43) with a UC50 camera at the Museo Nacional de Ciencias Naturales de Madrid (MNCN-CSIC). Samples with oocytes were coded as females and samples with any spermatogenic stage as males. We did not find any case of hermaphroditism among our samples. 2. DNA and RNA extraction, RADseq and RNAseq library preparation: DNA was extracted from all samples using the DNeasy Blood & Tissue kit (Qiagen) following the manufacturer’s protocol, except for the cell lysis time which was conducted overnight. Double-stranded DNA was quantified with Qubit dsDNA HS assay (Life Technologies). For RADseq library preparation we followed a protocol from Peterson et al. (2012) 60 with modifications described in Taboada et al . (2022) 61 . RNA was extracted separately from all samples using the Invitrogen RNA mini kit (Thermofisher) following the manufacturer’s protocol using TRIzol for cell lysis and quantified with NanoDrop. Further, mRNA libraries were constructed using the Illumina Stranded mRNA Prep kit, quantified using Qubit dsDNA HS assay (Life Technologies) and checked for size and quality using a TapeStation 2200 (Agilent Technologies, USA). They were then pooled and sequenced using paired-end 150-bp reads on an Illumina NovaSeq6000 at Novogene Europe (Cambridge, UK). Our final dataset of genomic and transcriptomic resources for assessing sex determination in sponges can be found in Table S2. 3. WGS library preparation: DNA extracted as above from tissues of the sponges (see Supplementary Table 2) and prepared to obtain WGS libraries. The genomic DNA was randomly sheared into short fragments using enzymes, and the obtained fragments were end-repaired, A-tailed, and further ligated with an Illumina adapter. The fragments with adapters were PCR amplified, size selected, and purified. The library was checked with Qubit 3.0 and real-time PCR for quantification, and Bioanalyzer for size distribution detection. Quantified libraries were pooled and sequenced on the Illumina platform NovaseqX plus, according to effective library concentration and data amount required (6 Gb/sample), done at Novogene. 4 Genome annotation : The chromosome-level genomes of Oscarella lobularis , Chondrosia reniformis , Petrosia ficiformis, Axinella damicornis, and Phakellia ventilabrum, Halichondria panicea were sequenced by the Aquatic Symbiosis Genome Consortium (Table S2). We annotated the genes in the assemblies of O. lobularis , C. reniformis and P. ficiformis using a combination of tools for de novo and evidence-based gene prediction (BRAKER2 2.1.6 62 , Augustus 3.5.0 63,64 , StringTie 2.2.1 65 , and GenomeThreader 1.7.1 66 ) and optimal gene model selection (Mikado 2.3.4 67 ). This procedure is described below. First, we mapped bulk paired RNA-seq libraries to the reference genome using the read aligner STAR 2.7.10b 68 without multi-mapping reads (flag: --outFilterMultimapNmax 1), only considering uniquely mapping reads for splice junctions (--outSJfilterReads Unique), reporting splice junction-supporting reads and keeping only the reads with junctions that passed filtering (--outFilterType BySJout, --alignSJDBoverhangMin 1, --alignSJoverhangMin 8), and reporting the alignment strand based on intron motifs (--outSAMstrandField intronMotif). The resulting coordinate-sorted BAM file (--outSAMtype BAM SortedByCoordinate) was used to produce an initial set of transcript predictions using StringTie in conservative mode (-t -c 1.5 -f 0.0 flags), from which open reading frames (ORFs) were predicted using TransDecoder 5.7.1 69 . Predicted peptides were collapsed by sequence similarity using CD- HIT 4.8.1 (-c 0.95) and complete genes (i.e. with start and end codons) of non-extreme lengths (>600 and <10,000 amino-acids) were retrieved for later use in the de novo gene prediction step. Specifically, these were used to train Augustus iteratively within BRAKER2, by aligning them to the reference genome using GenomeThreader (BRAKER2 flag: --prg=gth –trainFromGth), and using the original STAR-produced alignments as further evidence (--bam=). Second, we used Mikado to select the best gene predictions from each locus, selecting from the output of BRAKER2/Augustus (all exons and coding exons [CDS] were used separately), an unguided Stringtie assembly, and the filtered set of GenomeThreader training peptide alignments to the reference genome. To build the Mikado hints file, (i) all sources of evidence were considered as strand-specific; (ii) a score of 1 was associated with the BRAKER2/Augustus predictions and 0 for the others; (iii) the CDS from BRAKER2/Augustus were considered as a reference annotation; (iv) redundant models were excluded from all samples; and (v) CDS sequences with errors were removed from the model set. To build the Mikado configuration file (mikado conFig), we clustered transcripts with a minimum cDNA overlap of 20% (--min-clustering-cdna-overlap 0.2) and any for CDSs (--min-clustering-cds-overlap 0.0), set the programme to permissive mode with regards to ORF splitting policy (--mode permissive). After preparing the transcripts sets with the mikado prepare submodule, we prepared further evidence sources to be considered by Mikado: (i) predicted ORFs for all Mikado models, using TransDecoder; (ii) evidence-based splice junction coordinates from STAR (see above), obtained using the junctools convert 1.2.4 module of Portcullis 1.1.2.; and (iii) homology models obtained with diamond blastx 70 against the 2022-05 release of UniRef50 71 , adding the percentage of positive-scoring alignments and the traceback operations fields to the reported output (flag: -f 6 qseqid sseqid pident length mismatch gapopen qstart qend sstart send evalue bitscore ppos btop). These additional sources of evidence were considered by Mikado (mikado serialise submodule, disabling start codon adjustment with –no-start-adjustment). The best gene model for each locus was selected using the mikado pick module, prioritising reference models (--reference-update). Finally, transcript and peptide sequences for each gene model were retrieved using gffread 0.11.7 72 . The completeness of the resulting gene set was evaluated using BUSCO 5.5.0 73 in protein mode (-m proteins) against the Metazoa database of universal orthologs (-l metazoa_odb10). Annotation of P. ventilabrum, Axinella damicornis, and Halichondria panicea was performed elsewhere, and the details can be found in the references in Table S2. 5. Repeatome analysis: The density of transposable elements (TEs) per chromosome was calculated in 100 Kbp windows for each species with chromosome-level reference genome ( C. reniformis and O. lobularis ). First, the Repeatmodeller 74 output was filtered to only include TEs, removing simple repeats, low-complexity regions, rRNAs, and unknown repeats. Subsequently, bedtools v2.30.0 75 was used to create a bed file with 100 Kbp windows for each reference genome using the options “makewindows -w 100000”, and to calculate the density per window using the options “intersect -a genome_windows.bed -b ALL_TEs.bed -c”. Finally, TE distribution plots per chromosome were created in R using the ggplot2 package v3.5.1 76 . Additionally, we repeated these analyses exclusively including long interspersed nuclear elements (LINEs), which were extracted from the Repeatmodeller output and plotted as LINE density per Mbp. 6. Sex chromosome identification: To identify putative sex chromosomes and sex determining regions (SDR) we employed several complementary approaches. First, we ran the FindZX 10 pipeline with default parameters for detecting and visualising sex chromosomes based on genome coverage and heterozygosity differences. The strength and nature of sex-specific genomic signals depend on the degree of sex chromosome differentiation and Y/W degeneration. In homomorphic systems, heterozygosity provides the clearest signature, whereas in heteromorphic systems, coverage differences are most informative, expected to be approximately the double on the X/Z chromosome in the homogametic sex compared to the heterogametic sex. Restricting the number of allowed mismatches enhances detection by preventing reads from the sex-limited chromosome from aligning to its gametolog, and heterozygosity can appear higher in either the heterogametic or homogametic sex depending on whether reads from the sex-limited chromosome successfully align or not. The analyses were conducted using WGS reads in 50kb, 100kb and 500kb windows, unfiltered, 0.2 and 0.0 mismatches allowed and their species reference genome in the no-synteny mode. We also calculated differences in coverage with mosdepth [v0.3.2] 77 , SNP density relative to the reference and F ST values between sexes.SNP density and F ST values are expected to be higher in the heterogametic sex chromosomes due to the differentiation and reduced recombination. k-mer–based analyses provide a complementary and highly sensitive approach, as they are independent of a reference genome and can detect sex-linked differences even when high sequence homology allows equal read mapping across sexes and at the same time can recover signals that may be overlooked by coverage- or SNP-based approaches when read mapping differs between sexes. WGS reads of each individual and also in male and female datasets were aligned to the reference chromosome-level genome with bowtie2 78 and indexed with samtools [v1.14] 79 . SNP calling was done with bcftools [v1.12] 79 and calls filtered with vcftools [v0.1.16] 80 to only keep biallelic sites that pass the bcf_filter. Male/Female mapped read files were used to perform a coverage analysis after normalization. The analysis of the difference in coverage was performed with mosdepth [v0.3.2] in 1 Mb windows. SNP density relative to the reference genome in 100kb windows was calculated with vcftools and differences between male and female with the R script SNPdensity_permutations.R from the SexFindR pipeline 81 that calculates true means and difference and carries out a permutation test that determines p-values for SNP density differences windows. Kernel-smoothed F ST was calculated with the populations program of Stacks from the RADseq reads. K-mer analyses were performed following the SexFindR pipeline. kmersGWAS 82 was used for k-mer counting, and PLINK 83 for sex-association testing of k-mers with p-value < 0.00001. Significant k-mers were subsequently assembled into short contigs using ABySS 84 , which were then aligned to the reference genome with BLAST to determine their genomic positions 7. Intraspecific inversion detection in sex-chromosomes: To evaluate the presence of putative inversions in the sex-determining gene regions, we conducted a multi-evidence approach based on (i) mapping alternative haplotypes to the reference assemblies, (ii) genotypes and (iii) mapping quality for those species with male and female specimens. Since the reference genome for both C. reniformis and O. lobularis derived from the heterogametic sex, we mapped the alternative haplotypes to the reference assemblies using minimap2 85 and visualized the dotplots in D-GENIES 86 . For the genotype approach, we used the R script “individual Detection of linkage by Genotyping (iDlG)” 87 , which identifies linked regions across chromosomes at the individual level based on mean genotype calculated in sliding windows. This makes it possible to not only identify inversions but other regions with high linkage such as sex chromosomes. We established windows of 5,000 SNPs with sliding steps of 1,000 SNPs, and graphically visualized the iDlG results with the R package “ggplot2”. To further support the presence of inversions in those regions, we kept from each specimen “sam” file those paired-reads mapping colinearly 88 (forward–forward; reverse–reverse) in the same chromosome. From those, we retained only those pairs mapping at a distance above the expected from library preparation (400 bp), and subsequently visualized the relative position of both collinear reads with “ggplot2”. 8. Detection of sex-specific loci using RADseq data: RADseq is a crucial tool to study the genetic basis of sex determination in species without heteromorphic sex chromosomes 89 . Although RADseq alone can identify sex chromosome systems, it cannot determine sex chromosome linkage or the size of the non-recombining region. However, when used with a reference genome, sex-specific loci can be mapped to identify linkage groups and sex determining regions. RADseq data also allows us to calculate SNP density, and F ST between males and females across the genome, both predicted to be higher in the sex-determining, non-recombining regions of sex chromosomes. These indicators are expected to converge in the sex determining regions of the sex chromosomes. To this end, we used the RADsex pipeline [v1.2] 90 . The quality of raw reads was assessed with FastQC before and after demultiplexing with the process_radtags module of Stacks [v2.61] 91 . In addition, the detection and mapping of sex-specific loci was conducted with RADsex, which compares presence/absence of non-polymorphic markers between individuals in two groups, with a minimum marker depth of 1 for the process step, and 5 for the distribution, significance and mapping steps, keeping only the significative loci present in just one sex (i-e-, sex-specific loci). To assess the significance of the male/female distribution of markers we conducted a 1,000 permutations test followed by a chi-square test comparing the observed results against the permutations-based expected frequencies. The sex-specific loci were blasted against a reference transcriptome with local blast 92 and then aligned to their genome with bwa 93 to map them to reference genes. 9. Gene expression patterns: We aligned the RNAseq data from each male and female of each species of those with chromosome-level genomes with HISAT2 94 and the alignments were passed to StringTie for transcript assembly 65 . We filtered transcripts with expression lower than 2 TPM and those expressed in less than 2 samples per condition when possible, and we then tested for differential gene expression (DE) between males and females with edgeR after normalisation 95 . In both Geodia spp., given there is no chromosome-level genome, we used de novo transcriptomes built previously 96,97 , and also we built de novo transcriptomes for the rest of the species for further annotation. Then reads that were quality filtered with Prinseq lite v 0.20.4 98 , and decontaminated from rRNA with SortMeRNA v 4.3.6 99 with the provided SILVA 119 Ref NR 99 database 100 were mapped with Bowtie2 aligner 78 and expression levels estimated with RSEM v 1.2.21 101 . Expected counts (number of reads mapped) and the normalised trimmed mean of M values (TMM) were used for testing for differential gene expression (DE) between males and females with edgeR after normalisation and using the glmQLFit function. Expression levels of genes with sex-specific loci were retrieved from the count table for plotting. Then, genes with sex-specific loci identified were locally blasted against the genomes and de novo transcriptomes (retrieving all isoforms with a hit) of their corresponding species using BLAST and then annotated using Blast2GO PRO 102 . The gene annotations from the sex-loci were retrieved and implemented in ShinyGO 103 against the human database to perform Gene Ontology (GO) enrichments (with Benjamini-Hochberg FDR corrections of 0.05) and then plotted using ggplot in R. 10. Identification of sex-biased Alternative Splicing: We used rMATs 4.3.0 104 to identify Alternative Splicing (AS) events in our transcriptomes. We used the trimmed reads from our RNA-seq data for each species (Table S2). Besides assessing annotated splice junctions in the reference genome, rMATs can also evaluate differential splicing between two groups of samples, which in this case were females and males. rMATs measures splicing at each splice site with the percentage spliced in or PSI, which indicates the proportion of two alternative isoforms at each splice site, being 1 and 0 the extremes at which only one of the two alternatives is expressed, and 0.5 indicating equal expression. To compare splicing between sample groups, rMATS calculates the inclusion level difference (ΔPSI), defined as the average PSI in males minus the average PSI in females. ΔPSI ranges from +1 (the isoform is expressed exclusively in males) to −1 (the alternative isoform is expressed exclusively in females). rMATS uses a likelihood-ratio test to identify significant differences in ΔPSI between males and females, and we used the criterion of an FDR P value <0.05 to call differential splicing events. This was calculated both for all genes present in the genome and then for genes with sex-specific loci. 11. Macrosynteny analyses: We used Orthofinder v.2.5.4 105 to identify and compare orthologous groups shared between the six sponge species with chromosome-level genomes: Oscarella lobularis , Chondrosia reniformis , Petrosia ficiformis , Axinella damicornis , Halicondria panicea , and Phakelia ventilabrum . With the set of orthologous groups we then looked at conservation of syntenic regions across sponge genomes with the R package macrosyntR 106 . Then, genome location information for each ortholog was extracted from genome annotation files and used to identify regions of synteny by inferring significantly ancient linkage groups (ALG) which were ordered and displayed on Oxford grids for pairwise species comparisons. To evaluate inter-chromosomal rearrangements and conservation of sex-related genetic regions across sponges, we mapped the genomic location of CLGs across sponge chromosomes and generated ribbon diagrams with macrosyntR. 12. Evolutionary trajectories of genes with sex-specific loci: To analyse the presence and evolution of genes associated with sex determination in sponges, we conducted a comprehensive search of an extensive metazoan taxonomic database, with a special emphasis on early-branching animal lineages such as Ctenophora, Cnidaria, Placozoa, and Porifera. To avoid redundancies arising from the use of transcriptomes and ensure better quality and completeness, we included only complete sponge genomes (Porifera). The analysis was performed using a pipeline developed specifically for this study (EvoDomainSearch). This tool allows a systematic and controlled search for genes based on the presence of conserved domains. First, searches were performed with HMMER (v3.4), using HMM models generated from representative sequences of target genes related to sex determination in metazoans. The recovered sequences were subsequently filtered to retain only those containing the target domains of each gene. The final sequences were aligned using MAFFT (v7.525) 107 , employing the L-INS-i strategy for high-quality alignment. Subsequently, ambiguous alignment regions were removed with BMGE (v1.12) 108 using the parameters -m BLOSUM30 -b 3 -h 0.8 -g 0.8, with the aim of retaining only informative and phylogenetically reliable positions. Phylogenetic inferences were then performed using two maximum likelihood methods: FastTree (v2.1.11) 109 for rapid family tree exploration, and IQ-TREE (v2.3.6) 110 for more accurate analyses, employing automatically selected substitution models and statistical assessment using ultrafast bootstrap (UFBoot). This approach allowed us to identify candidate genes in sponges with high confidence and assess their phylogenetic relationship with homologs from other metazoan groups and, when present, their unicellular relatives. 13. Evolutionary age and diversification of sex-specific loci: To investigate the evolutionary origins and diversification of sex-specific genes in sponges, we implemented a multi-step analytical approach that combined ortholog inference with phylostratigraphic reconstruction. We first looked into the shared genome complements of the six species with chromosome-level genomes with OrthoVenn 3 111 . Then, orthologous genes were collected with OrthoFinder 3 as before to infer orthologous groups (OGs) and to establish homologous relationships across species. To assign evolutionary ages to genes, we applied phylostratigraphic analysis using GenEra v1.4.2 112 (Barrera-Redondo et al. 2023). Protein sequences predicted from each sponge species were queried against the NCBI non-redundant (NR) protein database using DIAMOND v2.1.9 70 in sensitive mode. The search employed an e-value threshold of 1e-5 and used the -a flag to input both the species-specific fasta files and corresponding NCBI Taxonomy IDs. GenEra automatically traced phylogenetic relationships using NCBI Taxonomy to determine the most basal taxonomic node associated with each gene hit. Genes were then assigned to discrete evolutionary strata based on their inferred taxonomic age. Among the annotated gene sets, we extracted the genes with sex-specific loci previously identified with RADSex in each species. These genes were mapped to their respective phylostrata to assess the relative timing of their evolutionary origin. We further analysed whether sex-related genes within the same stratum were associated with shared or distinct OGs, enabling insights into patterns of gene duplication and lineage-specific diversification. To evaluate the functional characteristics of genes with sex-specific loci across evolutionary strata, we performed Gene Ontology (GO) enrichment analysis using ShinyGO as before. Enriched GO terms were then categorized into broader semantic groups reflecting known ancestral functions (e.g., DNA repair, cell cycle regulation, cytoskeleton organization, apoptosis). This allowed us to assess the extent to which genes originally involved in ancient cellular processes were subsequently co-opted into core sexual reproduction functions such as meiosis, gametogenesis, and fertilization. A full list of enriched GO terms and their classifications by stratum is provided in Table S18F. Lastly, we characterized the evolutionary origin of syntenic genes with sex-specific loci identified following the methodology outlined in the Methods Section 10. These analyses facilitated the exploration of conserved genomic architecture and potential linkage patterns associated with sex-related gene clusters. Declarations Acknowledgments: We are indebted to Manuel Maldonado and Konstantina Mitsi for help in sampling, and Astrid Böhne, Arnau Sebé-Pedrós, and Manuel Irimia for fruitful discussions. We are also thankful to Gonzalo Giribet, Sally Leys, and April Horton for critical reading of the manuscript. This research could be done thanks to the funding obtained by several people: A.R. obtained funding from the grants RYC2018-024247-I and PID2019-105769GB-I00, both funded by the Spanish agencies MCIN/AEI/10.13039/50110001103 and EI “FSE invierte en tu futuro.” Also, A.R. acknowledges funding from the European grant “Biodiversity Genomics Europe” (Grant agreement ID: 101059492). C.D.-V. received the support of a fellowship from “la Caixa” Foundation (ID 100010434), with the fellowship code LCF/BQ/PI22/11910040. I.S. was supported by a JdC (Juan de la Cierva 2024) personal grant (JDC2024-054954-I). M.T. was supported by a JdC (Juan de la Cierva Formación, 2020) personal grant (FJC2020-043677-I) and MSCA Postdoctoral Fellowship (HORIZON-MSCA.2022-PF-01, Project 101105716). The work by J.A.H was supported by grants PID2023-153118OB-I00 funded by MCIN/AEI/10.13039/501100011033 and CRSII5_198737/1 by the Swiss National Science Foundation. S.T. received funding from the grants PID2020-117115GA-100 of the Spanish Ministry of Science and Innovation and CNS2023-144572 and by the Ramón y Cajal grant RYC2021-03152-I, funded by the MCIN/AEI/10.13039/501100011033 and the European Union NextGenerationEU/PRTR. A.V. was supported by a European Union NextGenerationEU/PRTR grant (IJC2020-045256), a fellowship from “la Caixa” Foundation (ID 100010434), with fellowship code LCF/BQ/PR24/12050011 and a Ramón y Cajal grant RYC2023-044466-I. PÁ-C was supported by MCIN/AEI/10.13039/501100011033 and by the European Union “Next Generation EU”/PRTR (CNS2023-145193). M.Á-P. received funding from a fellowship from the Fundación General CSIC’s ConFuturo under the Marie Skłodowska-Curie grant agreement 101034263 and a Ramón y Cajal grant RYC2023-043807-I. A.E.W is funded by a UKRI grant EP/X041921/1 and Philip Leverhulme Prize grant. Finally, part of this research was supported through the SponBIODIV project (A.R. and S.T.), a 2021-2022 BiodivProtect joint call for research proposals, under the Biodiversa+ Partnership co-funded by the European Commission, and with the funding organization “Fundación Biodiversidad”. Support for sponge genome sequencing from the Aquatic Symbiosis Genomics (ASG) project was provided by the Gordon and Betty Moore Foundation through a grant (GBMF8897) to the Wellcome Sanger Institute and U.H. Author contributions: Conceptualization: AR, JLS, PAC, AV, ST, AEW Formal analysis: JLS, CDV, CGC, AV, CL, VT, MAP, JAH, AR, MT, XGB Investigation: JLS, AR, AV, VT, MC, CGC Visualization: JLS, CDV, CGC, AV, CL, VT, MAP, JAH, AR, MT, PAC, MC Funding acquisition: AR, ST, CDV, AV, PAC, UH, JAH, MAP, MT, AEW Supervision: AR, ST, PAC Writing – original draft: JLS, AR, AEW, PAC, VT, CGC, CL Writing – review & editing: all authors Competing interests: Authors declare that they have no competing interests. Data and materials availability: Raw genomic and transcriptomic sequences are deposited in NCBI Bioprojects PRJNA1336057 (RADseq, WGS and RNAseq of Chondrosia reniformis, Oscarella lobularis, Petrosia ficiformis, Halichondria panicea, Axinella damicornis, Phakellia ventilabrum, Geodia hentschelli and Geodia barreti Raw sequence reads) and PRJNA1274490 (RNAseq of Halichondria panicea ). No original code was developed for this work. Code used is available at https://github.com/Jose-LSP/Sponges-sex-scripts and https://github.com/CGaliaCamps/iDlG.git References Bachtrog, D. et al. Sex determination: why so many ways of doing it? PLoS Biol. 12 , e1001899 (2014). https://doi.org/10.1371/journal.pbio.1001899 Zhu, Z., Younas, L. & Zhou, Q. Evolution and regulation of animal sex chromosomes. Nat. Rev. Genet. (2025). https://doi.org/10.1038/s41576-024-00757-3 (in the press). Bernstein, H., Bernstein, C. & Michod, R. E. 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Supplementary Files TableS2genomicresources.xlsx Table S2 TableS4Coveragebed.xlsx Table S4 TableS3fststacks.xlsx Table S3 TableS17Syntenysexloci.xlsx Table S17 TableS13sexlocienrichments.xlsx Table S13 TableS1sampling.xlsx Table S1 TableS6FindZXheterozigosity.xlsx Table S6 TableS10sexlociannotations.xlsx Table S10 TableS9Sexlocigenes.xlsx Table S9 TableS16AS.xlsx Table S16 TableS14DEGstringtie.xlsx Table S14 TableS18phylostratanalysesvt.xlsx Table S18 TableS11sexlociSDR.xlsx Table S11 TableS15transcriptexpressionsexlocistringtie.xlsx Table S15 LorenteSorollaSexchromosomesspongesNatcommsupplementarymaterials.pdf Supplementary Materials TableS7SNPdensityallsp.xlsx Table S7 TableS5FindZXcoverage.xlsx Table S5 TableS8SummaryRADSEXFINALJL.xlsx Table S8 TableS12expressionSDR.xlsx Table S12 r1.pdf Reporting Summary SupplementaryMaterials.docx Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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1","display":"","copyAsset":false,"role":"figure","size":844199,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenome-wide patterns of sex-linked chromosomes in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eChondrosia reniformis \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(A–F) and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eOscarella lobularis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(G–M). A \u0026amp; G.\u003c/strong\u003e Sex differences (female–male) in heterozygosity and genome coverage (50,000 bp windows). The grey background marks the 95% confidence interval (CI), calculated with bootstrapping from the genome-wide mean value for each metric expected under no sex differences, and the colour scale indicates significant bias to females (red) and to males (blue). Genome coverage was calculated with 0 mismatches when mapping. \u003cstrong\u003eB \u0026amp; H.\u003c/strong\u003e Genome coverage in sex-linked chromosomes (50,000 \u0026amp; 25,000 bp windows, respectively). \u003cstrong\u003eC \u0026amp; I.\u003c/strong\u003e Number of significant sex-specific loci identified using RADsex. \u003cstrong\u003eD \u0026amp; J.\u003c/strong\u003ePosition of contigs assembled from female- (D) and male-specific (J) k-mers in sex-linked chromosomes. \u003cstrong\u003eK. \u003c/strong\u003eSegregation of alleles as found with iDlG. Average genotype for each specimen (5,000 SNPs windows). 0 = fixation of reference SNPs, 1 = non-fixation, 2 = fixation of alternative SNPs. Red squares of figures D and J, and vertical grey shaded areas in all other panels identify SDRs.\u003cstrong\u003e \u003c/strong\u003e\u0026nbsp;\u003cstrong\u003eE \u0026amp; L.\u003c/strong\u003e Syntenic dot plots of the alignments between alternate haplotypes and reference genomes for the sex-linked chromosomes, illustrating overall collinearity and chromosomal inversions. Colors represent the percentage of identity between the two haplotypes and vertical grey shaded areas identify the SDRs. \u003cstrong\u003eF \u0026amp; M.\u003c/strong\u003eGO enrichments for genes in the SDRs\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8554461/v1/3cf04b294516f131caaa6b90.png"},{"id":102494104,"identity":"5f859136-b537-4ba2-a4ac-1f013e0c2583","added_by":"auto","created_at":"2026-02-12 09:15:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":956654,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenes with sex-specific loci. A. \u003c/strong\u003eNumber of sex-specific loci identified by RADSex in each species. Among those, genes with a known sexual function are shown in circles coloured in blue with size proportional to number of genes.\u003cstrong\u003e B. \u003c/strong\u003eMajor Gene Ontology categories enriched within the catalog of genes with sex-specific loci in each species. \u003cstrong\u003eC.\u003c/strong\u003e Number of genes that are annotated under the GO category “sex differentiation” among the genes with sex-specific loci in each species. \u003cstrong\u003eD.\u003c/strong\u003e Number of shared and unique genes with male-specific loci by the species studied.\u003cstrong\u003e E. \u003c/strong\u003eGO enrichments of genes with male-specific loci shared by at least 2 species or unique for each of the species.\u003cstrong\u003e F.\u003c/strong\u003e Number of shared and unique genes with female-specific loci by the species studied. \u003cstrong\u003eG.\u003c/strong\u003e GO enrichments of genes with female-specific loci shared by at least 2 species or unique for each of the species. Olob=\u003cem\u003eOscarella lobularis\u003c/em\u003e, Cren=\u003cem\u003eChondrosia reniformis\u003c/em\u003e, Pfic=\u003cem\u003ePetrosia ficiformis\u003c/em\u003e, Gbar=\u003cem\u003eGeodia barretti\u003c/em\u003e, Ghen=\u003cem\u003eGeodia hentscheli\u003c/em\u003e, Pven=\u003cem\u003ePhakellia ventilabrum\u003c/em\u003e, Adam=\u003cem\u003eAxinella damicornis\u003c/em\u003e, Hpan=\u003cem\u003eHalichondria panicea\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8554461/v1/4128555f2b44a88c2be2d6f1.png"},{"id":102494105,"identity":"67b00770-9efa-4d9d-b647-4a01f511a2d2","added_by":"auto","created_at":"2026-02-12 09:15:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":568266,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression patterns of genes with male– and female–specific loci.\u003c/strong\u003e \u003cstrong\u003eA. \u003c/strong\u003eProportion of genes with sex-specific (S) and autosomal (A) loci showing expression of different isoforms. Darker shades indicate loci with isoforms and lighter indicate loci without. Pie charts show the proportion of differentially expressed genes that contain sex-specific loci (lighter shade). \u003cstrong\u003eB.\u003c/strong\u003eProportion of genes with alternative splicing (AS) events among the autosomal genes (A) and those with sex-specific loci (S). The colour gradient represents different AS event types (SE, A5SS, A3SS, MXE, and RI) in decreasing intensity respectively. Pie charts show the number of AS events in lighter shades in both autosomal genes (A) and those with sex-specific loci (S). \u003cstrong\u003eC–J. \u003c/strong\u003eAverage (log) expression in TPMs for genes with male and female-specific loci with and without expression of different isoforms for the different species. Olob=\u003cem\u003eOscarella lobularis\u003c/em\u003e, Cren=\u003cem\u003eChondrosia reniformis\u003c/em\u003e, Pfic=\u003cem\u003ePetrosia ficiformis\u003c/em\u003e, Gbar=\u003cem\u003eGeodia barretti\u003c/em\u003e, Ghen=\u003cem\u003eGeodia hentscheli\u003c/em\u003e, Pven=\u003cem\u003ePhakellia ventilabrum\u003c/em\u003e, Adam=\u003cem\u003eAxinella damicornis\u003c/em\u003e, Hpan=\u003cem\u003eHalichondria panicea\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8554461/v1/a90e63d7c6ebf3f99f3c261e.png"},{"id":104397161,"identity":"d7f76c27-8348-43d6-b3e5-d475a9b109a5","added_by":"auto","created_at":"2026-03-11 11:37:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1888572,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eConserved synteny across sponges. Colored vertical lines connect orthologous genes across the six species with significantly enriched conservation of synteny. Each color represents a distinct ALG. Two or more colors converging on a chromosome indicate fusion events. \u003cstrong\u003eB. \u003c/strong\u003eConserved synteny of genes with sex-specific loci across sponges. Colored vertical lines indicate genes with sex-specific loci and grey vertical lines are all ALGs shown in Fig. 4A. \u003cstrong\u003eC.\u003c/strong\u003e Phylostratigraphic patterns in sponges. Loci were mapped to phylostrata from cellular organisms to sponges; circle size indicates loci number, and pie charts show species contributions and origins of sex-linked genes. \u003cstrong\u003eD.\u003c/strong\u003e Functional categories of genes with sex-specific loci assigned to different evolutionary strata. Abbreviations: B+A+S=Bubarida, Agelasida and Suberitida, A+S=Agelasida and Suberitida.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8554461/v1/a1d00e78cad6e5fb014f5804.png"},{"id":102494108,"identity":"901aff04-3d9d-4ca9-b5ba-411ba9ccb86d","added_by":"auto","created_at":"2026-02-12 09:15:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":303190,"visible":true,"origin":"","legend":"\u003cp\u003eEvolution of sex determination systems, sex chromosomes and sexual modes in A. Porifera and B. Metazoa, using\u003cstrong\u003e \u003c/strong\u003ecomposite trees for Porifera and Metazoa from previously published studies (modified from Cárdenas \u0026amp; Morrow, 2012\u003csup\u003e60\u003c/sup\u003e and from Giribet \u0026amp; Edgecombe, 2020\u003csup\u003e61\u003c/sup\u003e).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8554461/v1/7eb3452837ff14d61b328e88.png"},{"id":106401423,"identity":"d5b88867-acf4-4283-b747-8ae8bb667e21","added_by":"auto","created_at":"2026-04-08 08:49:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6925245,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8554461/v1/391780b3-0e2b-46f9-b30c-e037888254fd.pdf"},{"id":102494113,"identity":"3e0729af-cbab-4d04-b5a6-a01252caa9d8","added_by":"auto","created_at":"2026-02-12 09:15:53","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13129,"visible":true,"origin":"","legend":"Table S2","description":"","filename":"TableS2genomicresources.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8554461/v1/4ca2a13e04e1a9feaa0fecc0.xlsx"},{"id":102494102,"identity":"c5f0bf5b-ade0-4ddf-9fb0-7a0b1ea78f58","added_by":"auto","created_at":"2026-02-12 09:15:53","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":65017,"visible":true,"origin":"","legend":"Table S4","description":"","filename":"TableS4Coveragebed.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8554461/v1/1471997ea4f766122fd0be70.xlsx"},{"id":102494109,"identity":"6d4a6326-713a-4af9-bfc4-75cd29142635","added_by":"auto","created_at":"2026-02-12 09:15:53","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":347233,"visible":true,"origin":"","legend":"Table S3","description":"","filename":"TableS3fststacks.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8554461/v1/1b7a2eb4d4fea89a0224ea6d.xlsx"},{"id":102746869,"identity":"449ce5d7-62c1-4883-9378-ccaa931672f6","added_by":"auto","created_at":"2026-02-16 09:02:18","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":234779,"visible":true,"origin":"","legend":"Table S17","description":"","filename":"TableS17Syntenysexloci.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8554461/v1/867662aa275da0aba612d8d1.xlsx"},{"id":102746221,"identity":"c5d93ce3-fe89-446b-a7bf-b474eef5e91b","added_by":"auto","created_at":"2026-02-16 08:56:09","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":413149,"visible":true,"origin":"","legend":"Table S13","description":"","filename":"TableS13sexlocienrichments.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8554461/v1/8b4ae556cc5e00ae0ee35787.xlsx"},{"id":102494106,"identity":"cb52bce3-b5c7-4796-8e6f-f33b1c6858c1","added_by":"auto","created_at":"2026-02-12 09:15:53","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":10591,"visible":true,"origin":"","legend":"Table S1","description":"","filename":"TableS1sampling.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8554461/v1/329e0ceac6b224f6fa77c1dd.xlsx"},{"id":102746291,"identity":"06a8c964-f5b2-4015-9919-1e992edd61ff","added_by":"auto","created_at":"2026-02-16 08:56:28","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":1497223,"visible":true,"origin":"","legend":"Table S6","description":"","filename":"TableS6FindZXheterozigosity.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8554461/v1/0c30f2029d82f3e62246e81a.xlsx"},{"id":102962353,"identity":"419f0115-bab6-4138-bbf3-e2c544cbb697","added_by":"auto","created_at":"2026-02-19 04:07:24","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":104663,"visible":true,"origin":"","legend":"Table S10","description":"","filename":"TableS10sexlociannotations.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8554461/v1/824f1e78e283973d8ebec180.xlsx"},{"id":102494114,"identity":"579bdcfc-d895-45a0-88b4-583cbf42551f","added_by":"auto","created_at":"2026-02-12 09:15:53","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":110129,"visible":true,"origin":"","legend":"Table S9","description":"","filename":"TableS9Sexlocigenes.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8554461/v1/4aca2b8619357c95b58c6a29.xlsx"},{"id":102494115,"identity":"39e6c29f-4447-4642-94c5-adda8704fe11","added_by":"auto","created_at":"2026-02-12 09:15:53","extension":"xlsx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":3398118,"visible":true,"origin":"","legend":"Table S16","description":"","filename":"TableS16AS.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8554461/v1/7781bce442bde5bdb51eaf41.xlsx"},{"id":102494118,"identity":"31997983-19fa-4e1a-8de5-333ea6619d15","added_by":"auto","created_at":"2026-02-12 09:15:53","extension":"xlsx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":1155503,"visible":true,"origin":"","legend":"Table S14","description":"","filename":"TableS14DEGstringtie.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8554461/v1/7cfc26f89f0109a72c118a6a.xlsx"},{"id":102746098,"identity":"88b0da84-ac13-48ed-9565-7666d2c7e021","added_by":"auto","created_at":"2026-02-16 08:55:41","extension":"xlsx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":3205964,"visible":true,"origin":"","legend":"Table S18","description":"","filename":"TableS18phylostratanalysesvt.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8554461/v1/809fead73461d16728d80d50.xlsx"},{"id":102746693,"identity":"be3c720c-b099-4c42-8bad-6ab1f3a15c03","added_by":"auto","created_at":"2026-02-16 08:59:15","extension":"xlsx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":22234,"visible":true,"origin":"","legend":"Table S11","description":"","filename":"TableS11sexlociSDR.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8554461/v1/2c68ef936f1600eaa0efd676.xlsx"},{"id":102494116,"identity":"0c8a1930-24fb-4875-a394-35be8524668c","added_by":"auto","created_at":"2026-02-12 09:15:53","extension":"xlsx","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":1268781,"visible":true,"origin":"","legend":"Table S15","description":"","filename":"TableS15transcriptexpressionsexlocistringtie.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8554461/v1/de6b84e7aeec98adc9a9dd02.xlsx"},{"id":102494121,"identity":"17fe5e5d-4bed-4c1a-8f4a-b5755141509b","added_by":"auto","created_at":"2026-02-12 09:15:54","extension":"pdf","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":5136641,"visible":true,"origin":"","legend":"Supplementary Materials","description":"","filename":"LorenteSorollaSexchromosomesspongesNatcommsupplementarymaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8554461/v1/f5cd86daec3561e168b52606.pdf"},{"id":102494124,"identity":"dfd26615-2741-478a-b8f1-4cf546886dca","added_by":"auto","created_at":"2026-02-12 09:15:54","extension":"xlsx","order_by":16,"title":"","display":"","copyAsset":false,"role":"supplement","size":207829,"visible":true,"origin":"","legend":"Table S7","description":"","filename":"TableS7SNPdensityallsp.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8554461/v1/4eedcdf7035b43634ffffac7.xlsx"},{"id":102494119,"identity":"bb757d9e-102f-47f4-b8ac-728c6c2c569a","added_by":"auto","created_at":"2026-02-12 09:15:53","extension":"xlsx","order_by":17,"title":"","display":"","copyAsset":false,"role":"supplement","size":1873949,"visible":true,"origin":"","legend":"Table S5","description":"","filename":"TableS5FindZXcoverage.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8554461/v1/7f86d649219daaca0bff0708.xlsx"},{"id":102494122,"identity":"c0c99ae6-23b9-4f3f-808f-3e26cfa5cb45","added_by":"auto","created_at":"2026-02-12 09:15:54","extension":"xlsx","order_by":18,"title":"","display":"","copyAsset":false,"role":"supplement","size":1274154,"visible":true,"origin":"","legend":"Table S8","description":"","filename":"TableS8SummaryRADSEXFINALJL.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8554461/v1/c559267c127be3526c11433a.xlsx"},{"id":102494123,"identity":"84ce5243-0540-4058-8cc7-34fadb5894aa","added_by":"auto","created_at":"2026-02-12 09:15:54","extension":"xlsx","order_by":19,"title":"","display":"","copyAsset":false,"role":"supplement","size":4188408,"visible":true,"origin":"","legend":"Table S12","description":"","filename":"TableS12expressionSDR.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8554461/v1/3c21abda7727fcb4cf33b9fb.xlsx"},{"id":102494125,"identity":"076cc1e8-2172-487c-891d-6151b56566c9","added_by":"auto","created_at":"2026-02-12 09:15:54","extension":"pdf","order_by":20,"title":"","display":"","copyAsset":false,"role":"supplement","size":103695,"visible":true,"origin":"","legend":"Reporting Summary","description":"","filename":"r1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8554461/v1/6d78589f29eeb4458ddbd44f.pdf"},{"id":102494127,"identity":"8e5430f5-51a4-42e0-a0df-f526883a528e","added_by":"auto","created_at":"2026-02-12 09:15:55","extension":"docx","order_by":21,"title":"","display":"","copyAsset":false,"role":"supplement","size":26883939,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-8554461/v1/2cc5e346c9adee2ca38888f2.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Sponge genomes reveal a pre-metazoan origin of the sex determination toolkit and sex chromosomes","fulltext":[{"header":"Main text","content":"\u003cp\u003eSex occupies a central role in the evolution of eukaryotes. It may have emerged as a repair mechanism to prevent damage to single DNA strands\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e3\u003c/span\u003e\u003c/sup\u003e, possibly triggered by rising oxygen levels in early environments\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Since its emergence, sex has profoundly shaped animal biology and ecology. The evolution of separate sexes has occurred multiple times independently both in plants and animals\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e1\u003c/span\u003e\u003c/sup\u003e, and there is a bewildering number of molecular mechanisms underpinning sex determination\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e1\u003c/span\u003e\u003c/sup\u003e. These range from entirely genetic sex determination (GSD) to environmentally-induced sex determination (ESD) and a combination of both. Within GSD, there is also a panoply of genetic mechanisms to specify females and males, from sex chromosomes to polygenic systems\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Revealing the molecular basis of sex in animals is crucial for explaining their evolution and diversification. Yet, most research has concentrated on a limited number of arthropods and vertebrates\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e1,2\u003c/span\u003e\u003c/sup\u003e, leaving early-diverging animal lineages largely unexplored\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Among them, sponges (phylum Porifera) hold a pivotal position as the most likely sister-group to the rest of animals\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e7\u003c/span\u003e\u003c/sup\u003e for investigating the origins of sex, but current knowledge on this phylum is fragmentary. The last common ancestor of sponges was hermaphroditic, but multiple independent transitions to gonochorism have occurred\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e8\u003c/span\u003e\u003c/sup\u003e, suggesting repeated evolution of GSD and/or ESD systems. However, evidence for GSD in sponges is restricted to the identification of a few candidate genes in a handful of species (e.g., \u003cem\u003eDMRT1\u003c/em\u003e and \u003cem\u003eFEM1\u003c/em\u003e)\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e9\u003c/span\u003e\u003c/sup\u003e. To address this gap, we conducted a phylogeny-wide genome sampling of eight gonochoristic species from two classes and seven different orders of sponges (Fig. S1). We used genome resequencing of males and females to identify sex-linked regions and chromosomes (Table S1), six of which had chromosome-level reference genomes (Table S2; Supplementary text Sections 1, 2). Sex was identified through cytological surveys of male and female gametes (Figs S2\u0026ndash;S3) and patterns of sex-biased gene expression were quantified through RNA-seq (Table S2). Finally, we traced the evolutionary origins of genes harboring sex-specific loci and analyzed their synteny across sponge genomes to uncover macroevolutionary patterns underlying sex determination.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSponges have independently evolved male and female heterogametic sex chromosomes.\u003c/strong\u003e Using a combination of genomic approaches, we detected clear sex-linked chromosomes/regions in only two of the eight sponge species analysed: \u003cem\u003eChondrosia reniformis\u003c/em\u003e and \u003cem\u003eOscarella lobularis\u003c/em\u003e (Fig. 1, Fig. S4). In \u003cem\u003eC. reniformis\u003c/em\u003e, a region spanning 2.6\u0026ndash;3.8 Mb on chromosome 11 showed significantly higher heterozygosity in females but higher coverage in males (Fig. 1A\u0026ndash;B; Fig. S5A-B; Table S3\u0026ndash;S6). This pattern is consistent with a female heterogametic (ZW) sex-determination system, in which reduced mapping of W reads to the Z chromosome results in increased male coverage, while W-specific SNPs increase female heterozygosity. It also indicates intermediate differentiation between Z and W chromosomes. This region, hereafter called sex determining region (SDR), coincides with an area with significant sequence divergence between males and females, where 12 out of the 25 loci with highest (over 0.20) sex-specific \u003cem\u003eF\u003c/em\u003e\u003csub\u003eST\u003c/sub\u003e values were located (Table S3). The SDR exhibits significantly higher SNP density in females (i.e., number of SNPs per unit of genome length; Table S7) and a significantly higher accumulation of sex-specific loci (i.e., short DNA fragments generated by RADseq that are present/absent in one sex or the other) than that in other chromosomes (X-squared = 24.339, df = 13, \u003cem\u003ep\u003c/em\u003e-value = 0.02815) (Fig. 1C, Fig. S6A; Table S8B-C). These sex-specific loci in the SDR were predominantly female loci. Finally, k-mer analysis identified female-specific k-mers assembled into 2178 contigs within the SDR (Fig. 1D), while only 38 were significantly associated to males (Fig. S7A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe also found pronounced sex differences in genome coverage (strongly male-biased), but not in heterozygosity, across a 3.6-4.7 Mb region of chromosome 2 in \u003cem\u003eC. reniformis\u0026nbsp;\u003c/em\u003e(Fig. 1A\u0026ndash;C; Fig. S5; Table S3\u0026ndash;S6). This region contained only a few sex-specific loci (Fig. 1C; Fig. S6) and kmers (343 contigs assembled from female-specific k-mers and only 20 from male-specific kmers; Fig. S7A\u0026ndash;B). The genomic signature of chromosome 2 is consistent with an older Z chromosome\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e10\u003c/span\u003e\u003c/sup\u003e. To test this, we blasted the main haplotype to the alternate and found a scaffold in the alternate haplotype (CAMBO01000013.1) that blasts against chromosome 2 but displays the opposite pattern. It has coverage bias towards females (Fig. S5C\u0026ndash;D) and contains 2437 contigs assembled with female-specific k-mers (Fig. S7C-D), pointing to the complementary W. In this scenario, we suggest that \u003cem\u003eC. reniformis\u003c/em\u003e might possess two pairs of sex chromosomes (ZW and zw), with chromosome 2 being an older Z and 11 a younger z (Supplementary Text Section 1), similar to that observed in several animals\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e11\u0026ndash;14\u003c/span\u003e\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBecause chromosomal inversions suppress recombination in many organisms\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e15,16\u003c/span\u003e\u003c/sup\u003e, we next explored whether an inversion in these regions of chromosomes 2 and 11 could be driving divergence between the Z and W chromosomes. Since the reference genome derived from a female individual (heterogametic ZW), we investigated structural variation between sex chromosomes by mapping the alternate haplotype against the reference assembly. This revealed a ~0.7 Mb chromosomal inversion on chromosome 2 (3.7 to 4.4 Mb; Fig. 1E), that co-localizes with the SDR and confirms the reference specimen female sex. We propose that this inversion in chromosome 2 was a primary step in establishing recombination suppression and initiating the evolution of this sex chromosome in \u003cem\u003eC. reniformis\u003c/em\u003e, as in other organisms\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Then we examined read-pair mapping orientation in chromosome 11 across all samples, and we recovered a cluster of paired reads mapping distantly and collinearly in positions 1.9 to 3.1 Mb (Fig. S8A\u0026ndash;B), but without any vicariant signal between males and females (Fig. S8A). While a potential very recent inversion in the 1.9\u0026ndash;3.1 Mb region of chromosome 11 cannot be ruled out, the signature observed can be due to accumulation of transposable elements (TEs) (see below), highlighting the diverse mechanistic origins of sex chromosomes in sponges.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe then investigated whether the in SDRs of chromosomes 2 and 11 contained sex-related genes by doing GO enrichments, and found that in the category Biological Process functions related to regulation of the mitotic cell cycle, DNA recombination, reproductive process, and genitalia morphogenesis were among the most enriched (Fig. 1F). Finally, given that accumulation of TEs and specifically LINEs (Long Interspersed Nuclear Elements) is often associated with sex chromosome evolution in many species (e.g., in cephalopods)\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e18\u003c/span\u003e\u003c/sup\u003e, we analysed TE and LINE density across \u003cem\u003eC. reniformis\u003c/em\u003e chromosomes. We found accumulations of TEs in the central region of all chromosomes, including sex chromosomes and their SDRs, but no particular pattern of LINE accumulation in any of the two putative sex chromosomes (Fig. S9, Fig. S10A). Such accumulations frequently follow inversion events, as suppressed recombination in heterokaryotypes (eg: ZW or XY) prevents TE purging, consistent with the sex-driving inversion hypothesis. Nonetheless, TE accumulation alone may also drive SDR origination or promote inversion events, scenarios that cannot be ruled out here.\u003c/p\u003e\n\u003cp\u003eIn \u003cem\u003eOscarella lobularis\u003c/em\u003e, we found sex-specific loci, using RADSex, exclusively in males (Table S8B, D; Fig. 2A; Fig. S6B), indicating the presence of an XY-like SD system. Consistent with this, males showed higher heterozygosity and higher coverage within the 2\u0026ndash;3 Mb region of chromosome 5 (Fig. 1G\u0026ndash;I; Fig. S11A\u0026ndash;B), with a significant accumulation of male-specific loci (Fig. S6B; Table S8) compared to other chromosomes (X\u0026sup2; = 101.69, df = 19, \u003cem\u003ep\u003c/em\u003e-value = 2.641e-13) and 1213 contigs assembled from male-specific k-mers covering the same region (Fig. 1J; Fig. S11C). These findings are consistent with chromosome 5 being the sex chromosome. This would mean that the SDR has failed to assemble separately in the reference genome given its relatively low divergence, resulting in a co-assembly of the X and Y (Supplementary Text Section 2; Figs. S11, S12). We then analysed haplotype divergence between sexes in the entire genome with iDlG, and found a clear pattern only in this SDR of chromosome 5 (Fig. 1K, Fig. S8C\u0026ndash;D), consistent with an inversion promoted by physical hindrances during recombination\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e19\u003c/span\u003e\u003c/sup\u003e. To further validate the presence of an inversion in chromosome 5, which potentially led to sex determination in \u003cem\u003eO. lobularis\u003c/em\u003e, we evaluated the karyotype of the reference specimen by mapping the alternate haplotype against the reference assembly, since the reference genome derived from a male individual (heterogametic XY). This analysis revealed a ~1.3 Mb region flanked by two chromosomal inversions on chromosome 5, from 1.8 to 3.1 Mb, that co-localized with the limits of the SDR (Fig. 1L). We propose this pair of inversions flanking the SDR as the primary mechanism producing the recombination suppression that propelled the evolution of sex chromosomes in \u003cem\u003eO. lobularis\u003c/em\u003e. Sequence pairs mapping distantly in the same direction on the same strand across all specimens showed that the chromosome 5 SDR had a higher density of reads mapping collinearly at long distances in the same strand than the rest of the chromosome (Fig. S8D), further supporting the presence of an inversion. As for \u003cem\u003eC. reniformis\u003c/em\u003e, we evaluated the TE and LINE density across all chromosomes, and found that one of the greatest accumulations of TEs occurred in the SDR region of chromosome 5 (Fig. S13), similar to the sex chromosomes of other organisms\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e18\u003c/span\u003e\u003c/sup\u003e. However, chromosome 5 showed the fewest number of LINEs per Mb (Fig. S10B). Both collinear reads and the high abundance of TEs within the SDR further provides evidence on a potential sex-driving inversion. In this case, the segregation of alleles as found with iDlG (Fig. 1J) indicates that this inversion is older than the one identified in \u003cem\u003eC. reniformis\u003c/em\u003e, and could be a driver for the emergence of a sex chromosome given that it contains several sex-specific loci. To understand the functions of the genes present in the SDR of chromosome 5, we looked into the enriched GO terms for Biological Processes, finding regulation of the mitotic cell cycle, DNA recombination and reproductive process among the most enriched (Fig. 1L).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNone of the other four species with reference genomes showed significant biases in their heterozygosity, coverage or SNP density between females and males (Figs. S4, S6; Table S4\u0026ndash;S7) that could be indicative of a large sex-linked region. In addition, no accumulation of LINEs was detected for any of the genomes of these four species (Fig. S10).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvidence for a convergent polygenic sex determination toolkit across sponges.\u0026nbsp;\u003c/strong\u003eWith RADSex, we identified 7,657 sex-specific loci across all eight surveyed species (Table S8). In every case, both the total number of sex-specific loci and their distribution between sexes were significantly different from random expectations, as shown by 1,000 permutations in which individual sexes were randomly reassigned (Fig. S14). Except for \u003cem\u003eC. reniformis\u003c/em\u003e and \u003cem\u003eO. lobularis\u003c/em\u003e, where sex-specific loci clustered in SDRs, in the other species they were widely distributed across their genomes (Fig. S6; Table S8). This is consistent with polygenic sex determination (PSD), in which multiple loci contribute additively or epistatically to sexual fate, as reported in cichlids, zebrafish, and houseflies\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e5,20\u003c/span\u003e\u003c/sup\u003e. Although sex-specific loci of both XY- and ZW-like type were retrieved in all sponge species, one system appears to predominate in each of them: in \u003cem\u003eP. ficiformis\u003c/em\u003e, and \u003cem\u003eH. panicea\u003c/em\u003e, the excess of female-specific loci suggests dominance of ZW-like systems, whereas in the remaining species the prevalence of male-specific loci points to dominance of XY-like systems. This interpretation is further supported by sex ratio biases observed in these sponge species (Table S1), which, together with variance in sex ratios among families and the genetic mapping of multiple sex-specific markers, are considered indicators of PSD\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e20\u003c/span\u003e\u003c/sup\u003e, with dominance of one mode of inheritance over the other\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe sponge sex-specific loci identified by RADSex in the eight species mapped on to 1,437 genes, with 15\u0026ndash;25% of them known to be involved in animal sex determination (Fig. 2A) and a few further annotated within the GO term category sex determination (GO:0007530) (Fig. 2C; Table S9C\u0026ndash;D; Supplementary Text Section 3.1). Around 22% of the sex-specific loci were shared by two or more species (Fig. 2D, F; Fig. S15A) (Table S9A\u0026ndash;B). This is similar to what is found in sex determination of insects and vertebrates, where a fraction of the sex determination gene complement is shared, like the \u003cem\u003eDMRT1\u003c/em\u003e or \u003cem\u003eFEM1\u003c/em\u003e genes, but there is usually a larger fraction of sex determination genes specific to each lineage\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e21,22\u003c/span\u003e\u003c/sup\u003e. In any case, genes regulating sex determination in sponges seem to converge on the same cohort of genetic pathways that maintain and repair DNA structure and determine gonad fate, as well as regulate the meiotic cell cycle checkpoint signaling and the MAPK cascade, with minor contributions of other genes involved in metabolic and/or structural processes (Figs. 2B\u0026ndash;C, F\u0026ndash;G, Fig. S15). Similar observations have been found in mammalian Y chromosome genes\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e23\u003c/span\u003e\u003c/sup\u003e and those of other organisms\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Among the shared genes with sex-specific loci, we found several transposable elements or transposon-related genes, such as \u003cem\u003eYRD6, RTBS\u003c/em\u003e and \u003cem\u003ePOL2\u0026ndash;5\u003c/em\u003e (Tables S9D, S10). These transposons harbored male-specific SNPs and are known for their established involvement in spermatogenesis\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e24,25\u003c/span\u003e\u003c/sup\u003e. Also, two helicases were largely shared by most sponges (Tables S9D, S10), the ATP-dependent helicase \u003cem\u003ePIF1\u003c/em\u003e and the RNA binding protein \u003cem\u003eZNFX1\u003c/em\u003e, which are known to be associated to males and differentially expressed in testicles\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e24,26,27\u003c/span\u003e\u003c/sup\u003e. Besides these, some of the shared sponge genes containing sex-specific loci were involved in conserved sexual reproductive machineries and regulation pathways of animals (Fig. 2A\u0026ndash;C). For example, transcription factors (TFs) are key players in vertebrate sex determination, where \u003cem\u003eSOX9\u003c/em\u003e, \u003cem\u003eSRY\u003c/em\u003e, \u003cem\u003eNR5A1\u003c/em\u003e and GATA TFs are crucial to initiate the development of testes\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e28,29\u003c/span\u003e\u003c/sup\u003e. Here, we found that \u003cem\u003eGATA4\u003c/em\u003e contains female-specific SNPs in both \u003cem\u003eGeodia\u003c/em\u003e spp. and \u003cem\u003eH. panicea\u003c/em\u003e (Tables S9D, S10). This is important because \u003cem\u003eGATA4\u003c/em\u003e is an upstream effector of \u003cem\u003eSRY\u003c/em\u003e and plays a predominant role in both primary sex determination and sex differentiation via steroidogenic function activation of \u003cem\u003eSF-1\u003c/em\u003e and \u003cem\u003eStar\u003c/em\u003e in mammals\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e30\u003c/span\u003e\u003c/sup\u003e, and thus indicates an important, evolutionary conserved role for this transcription factor in animal gonadal development and function\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In addition, a male-specific SNP was identified in \u003cem\u003eStar\u003c/em\u003e (Steroidogenic acute regulatory protein) genes in \u003cem\u003eP. ficiformis\u003c/em\u003e (Tables S9D, S10). Also, \u003cem\u003eFem\u003c/em\u003e genes, here with loci specific to males in both \u003cem\u003eC. reniformis\u003c/em\u003e and \u003cem\u003eAxinella damicornis\u003c/em\u003e (Tables S9D, S10), have been identified as sex-determining genes in nematodes and insects\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e31,32\u003c/span\u003e\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSex determination genes in sponges are largely lineage-specific.\u0026nbsp;\u003c/strong\u003eAlmost 70% of all the genes with sex-specific loci (either female or male) were restricted to a single sponge species (Fig. 2D, F; Table S9A\u0026ndash;D), even though orthologous sequences of these genes were present in all species (see examples in Figs. S16\u0026ndash;S17). Genes with species-specific sex-loci that were unique to each lineage, were mostly involved in DNA repair, chromosome organization, and cell cycle checkpoint signaling (Fig. 2E, G). In this category of lineage-specific genes with sex-specific loci fell those identified in the SDRs of \u003cem\u003eC. reniformis\u0026nbsp;\u003c/em\u003eand \u003cem\u003eO. lobularis\u0026nbsp;\u003c/em\u003e(Table S11A-B). In \u003cem\u003eC. reniformis\u003c/em\u003e, the SDR of chromosome 2 mostly contained transposable elements with low levels of expression (Table S11A\u0026ndash;S12A), while that of chromosome 11 contained both transposable elements (with low expression), and genes with sex-specific loci involved in cell regulation and homeostasis (\u003cem\u003ePAR14, LORF2, GARS, PTPRD\u003c/em\u003e), cyclins (\u003cem\u003eCDK13\u003c/em\u003e), mitotic checkpoint (\u003cem\u003eCENP-E\u003c/em\u003e) and cell cycle regulators (\u003cem\u003eADAS\u003c/em\u003e) that were highly expressed (Table S11A\u0026ndash;S12B). Several of these genes have known functions in sexual reproductive processes (See Supplementary Text Section 3.2), such as \u003cem\u003ePTPRD\u003c/em\u003e (with male-specific loci in our dataset, Table S8), \u003cem\u003eCENP\u0026ndash;E\u003c/em\u003e and \u003cem\u003eCDK13\u003c/em\u003e (with female-specific loci in chromosome 11, Table S8, See Supplementary Text Section 3.2). Interestingly, sponge \u003cem\u003eCENP-E\u003c/em\u003e sequences appear in different clades of the phylogenetic tree of the eukaryotic protein family, suggesting divergent protein conformations that might confer different functions (Fig. S16). In \u003cem\u003eO. lobularis\u003c/em\u003e, six genes with male-specific loci (Table S11B) were located in the candidate SDR of chromosome 5 (\u003cem\u003eG2E3, PIF1, HMCN1, MRC1, DAPK1, RECQ\u003c/em\u003e), and showed higher expression in females than in males (Fig.1O\u0026ndash;P; Table S12C), although not significantly. Notably, all of these genes have known functions related to sex determination in other organisms (Tables S11B, S10), such as fruit flies and sea cucumbers\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e26,33\u003c/span\u003e\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong the genes with sex-specific loci unique to each species, we also identified several associated with DNA damage repair and chromatin or structural maintenance, along with a diverse array of signaling pathways (Fig. 2E, G; Tables S9\u0026ndash;S10). A fundamental aspect of sexual reproduction is the capacity of recombination to exchange genetic material during meiosis. While homologous autosomes pair and recombine completely during meiosis, sex chromosomes have evolved to restrict this process\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e34\u003c/span\u003e\u003c/sup\u003e. For instance, the DNA repair genes\u003cem\u003e\u0026nbsp;RAD51\u003c/em\u003e and \u003cem\u003eRAD52\u003c/em\u003e, whose protein products recognize meiotic double-strand breaks (DSBs) and repair them, are described to be fundamental for chromosome pairing\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e35\u003c/span\u003e\u003c/sup\u003e, and are thought to be the primary sexual genetic machinery that arose to facilitate sexual cycles in eukaryotes\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Here, \u003cem\u003eRAD52\u003c/em\u003e in \u003cem\u003eP. ventilabrum\u0026nbsp;\u003c/em\u003eand \u003cem\u003eRAD51\u003c/em\u003e in \u003cem\u003eH. panicea\u0026nbsp;\u003c/em\u003econtained male and female-specific loci respectively (Tables S8, S9A\u0026ndash;D, S10). Other DNA repair and meiotic genes also contained sex-specific loci in different species, including \u003cem\u003eBRCA2/FANCD1, BRWD1, AGO2, PKRDC, HAP2\u0026nbsp;\u003c/em\u003eand \u003cem\u003eMEIG1\u003c/em\u003e (Fig. 2A, Fig. S18; Tables S9, S10; Supplementary Text Section 3.3). Although meiotic genes are critical for making functional gametes\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e36\u003c/span\u003e\u003c/sup\u003e, they usually do not determine whether the gamete should be female or male. However, they are essential for gametogenesis and mediate membrane fusion between gametes in a broad range of eukaryotes, ranging from algae and higher plants to protozoans and cnidaria\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e37,38\u003c/span\u003e\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSeveral\u003cem\u003e\u0026nbsp;\u003c/em\u003esignaling pathways, including \u003cem\u003eSRY\u003c/em\u003e, \u003cem\u003eWNT, JNK\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;MAPK\u0026nbsp;\u003c/em\u003ehave critical roles in sex determination\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e39\u003c/span\u003e\u003c/sup\u003e, and these and embryonic morphogenetic pathways were enriched among the sex determination complements of several sponges (Fig. 2E; Table S10; Fig. S18; Supplementary Text Section 3.3). In \u003cem\u003eO. lobularis\u003c/em\u003e we identified male-specific loci in genes that are part of the SRY cascade in mammals and marsupials\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e40\u0026ndash;42\u003c/span\u003e\u003c/sup\u003e, including \u003cem\u003eATRX\u003c/em\u003e, \u003cem\u003eINSRR\u003c/em\u003e, and \u003cem\u003eUBE3A\u003c/em\u003e (Table S10, Supplementary Text Section 3.3). Although \u003cem\u003eATRX\u003c/em\u003e was present in all species except \u003cem\u003eP. ficiformis\u003c/em\u003e, a sex-specific locus was only found on this gene in \u003cem\u003eO. lobularis\u003c/em\u003e (Fig. S17). The WNT pathway, fundamental in mammalian female development through the activation of \u003cem\u003eWNT4\u003c/em\u003e\u003cem\u003e\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e41,43\u003c/span\u003e\u003c/sup\u003e\u003c/em\u003e, was also identified as crucial in \u003cem\u003eP. ficiformis, O. lobularis,\u003c/em\u003e and \u003cem\u003eH. panicea\u003c/em\u003e, with female-specific loci in \u003cem\u003eWNT4\u003c/em\u003e, \u003cem\u003eAPC\u003c/em\u003e and \u003cem\u003eNRARP\u003c/em\u003e respectively (Fig. S18; Tables S9D\u0026ndash;S10). Finally, the MAPK cascades, that are important for growth, proliferation, differentiation, motility, stress response, survival and apoptosis\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e44\u003c/span\u003e\u003c/sup\u003e, were also found enriched among the genes with male-specific loci in \u003cem\u003eC. reniformis\u003c/em\u003e, \u003cem\u003eP. ficiformis\u003c/em\u003e, \u003cem\u003eP. ventilabrum\u003c/em\u003e, and \u003cem\u003eH. panicea,\u0026nbsp;\u003c/em\u003ewith genes directly activating the MAPK cascade that were shared such as \u003cem\u003eRYK\u003c/em\u003e, and some that were unique, such as \u003cem\u003eGSK\u003c/em\u003e or \u003cem\u003eROR1\u003c/em\u003e (Fig. 2E; Fig. S18; Tables S9D\u0026ndash;S10).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAlternative splicing underlies sex-specific gene expression in polygenic sponge sex determination.\u003c/strong\u003e Differentiated (heteromorphic) sex chromosomes often exhibit a non-random accumulation of sex-biased genes\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e45\u003c/span\u003e\u003c/sup\u003e. By comparison, in homomorphic systems, sex-linked genes are sometimes expressed at similar levels in males and females\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e46\u003c/span\u003e\u003c/sup\u003e, and sex-specific regulation may instead arise through alternative splicing (AS). AS provides a plastic mechanism for generating isoform diversity from a shared genetic background, enabling the fine-tuning of expression to meet male and female developmental needs without requiring gene duplication or large-scale divergence. In this way, AS plays a central role in regulating development and is essential for sex determination in both vertebrates\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e47\u003c/span\u003e\u003c/sup\u003e and invertebrates\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e48\u003c/span\u003e\u003c/sup\u003e, where it underlies processes such as sexually dimorphic cell differentiation and dosage compensation\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Given that sponges do not show sexual dimorphism, the sexes likely share very similar trait optima, and thus, following the predictions of Flintham (2025)\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e50\u003c/span\u003e\u003c/sup\u003e, we did not expect to observe strong sex-biased expression even at genes with sex-specific loci within their polygenic sex determination system. Here, we tested whether genes with sex-specific loci were more likely to show differential expression or alternative splicing, as such regulatory differences can help restrict sex-specific fitness effects to the appropriate sex. We first examined chromosome-wide expression patterns and found similar F:M expression ratios for autosomes and sex chromosomes in both \u003cem\u003eC. reniformis\u003c/em\u003e and \u003cem\u003eO. lobularis\u0026nbsp;\u003c/em\u003e(Fig. S19A, D), even in the SDR (Fig. S19B\u0026ndash;C, E; Tables S9-S10). Then, we looked at genes with sex-specific loci in particular, and found that only 0.3 to 11.5% of these genes were differentially expressed between sexes (Figs. 3A; Table S10, S14; Supplementary Text Section 3). Among those, we found that the \u003cem\u003ePTPRD\u0026nbsp;\u003c/em\u003egene, which contains a male-specific loci and is located in the SDR region of chromosome 11 in \u003cem\u003eC. reniformis\u003c/em\u003e, was significantly upregulated in males (logFC=-5.72 logCPM=5.33, \u003cem\u003ep\u003c/em\u003e-value=4,78E-07 FDR=0,00013) (Table S14B). This gene is a well-known candidate for temperature dependent sex determination in reptiles, and its upregulation can help stabilize male fate commitment during ESD in turtles\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Then, we observed that most genes with sex-specific loci produce several transcripts with few nucleotide differences (detected with StringTie), and the different transcripts show higher expression in one sex than the other (Fig. S18, Table S15). To test whether those differences were statistically significant, we performed a Welch Two Sample t-test comparing genes with sex-specific loci that produce a single transcript with those that produce several. We found significant differences in gene expression between sexes (\u003cem\u003ep\u003c/em\u003e-value=0.0045) only in genes with sex-specific loci that produce several transcripts in \u003cem\u003eC. reniformis\u003c/em\u003e (Fig. 3C\u0026ndash;J; Fig. S18; Table S13-S15). In this case, the expression was consistently biased towards females (Fig. 3D, Table S15).\u003c/p\u003e\n\u003cp\u003eWe then looked at AS in the six sponges with reference genomes, and found that all showed AS events (ASE), ranging from 3.5% of genes in \u003cem\u003eP. ficiformis\u003c/em\u003e to 60.3% in \u003cem\u003eC. reniformis.\u003c/em\u003e These ASE were identified in both autosomal genes and those with sex-specific loci, with exon skipping (SE) being the most common type, followed by alternative 3\u0026rsquo; splice sites (A3SS) (Table S16, Supplementary Text Section 4), unlike what has been previously found in other sponges\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Interestingly, significantly sex-biased ASE in genes with sex-specific loci were found across all species except \u003cem\u003eO. lobularis\u003c/em\u003e, again with SE being the predominant event (Fig. 3B, Table S16). Again, it was in \u003cem\u003eC. reniformis\u003c/em\u003e where we found the greatest proportion of genes with sex-specific loci and ASE (3.1%, Table S16A). In many cases, the isoforms produced by exon skipping in genes with sex-loci resulted in different proteins, like the case of \u003cem\u003eCENP-E\u003c/em\u003e in \u003cem\u003eC. reniformis\u003c/em\u003e (Fig. S16B\u0026ndash;C). Overall, these findings indicate that in sponges, where sexual dimorphism is absent and trait optima are likely shared between sexes, strong sex-biased levels of gene expression are rare even at genes with sex-specific loci. Instead, sex-specific regulation appears to arise primarily through alternative splicing, providing a flexible mechanism for achieving sex-specific regulation that has been described in polygenic sex determination systems without requiring large-scale shifts in gene expression\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e53\u003c/span\u003e\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSponge sex chromosomes show synteny and chromosomal fusions.\u0026nbsp;\u003c/strong\u003eTo quantify shared genomic complements across sponges, we first compared the genomes of our six sponges and found they share around 75% of protein-coding genes, with 25% conserved across species and 50% shared by at least two species, and with the remaining 25% of each genome being species-specific (Fig. S15B\u0026ndash;C; Supplementary Text Section 4). Then, to investigate how macrosyntenic regions have been rearranged during sponge evolution, we analyzed 941 single-copy orthologs shared among the six sponge species (Fig. 4A). This analysis showed well-conserved synteny and colinearity, with 18 ancient linkage groups or ALGs shared between \u003cem\u003eO. lobularis\u003c/em\u003e and the other five sponges (Fig. 4A). This indicates a remarkable conservation of chromosome architecture across sponge classes that diverged more than 600 million years ago\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e54\u003c/span\u003e\u003c/sup\u003e, consistent with previous reports of macrosynteny conservation in sponges\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Despite strong syntenic and collinear patterns across sponges, only 65 out of the 828 syntenic genes had sex-specific loci in at least one species (Fig. 4B; Table S17; Supplementary Text Section 5), which is partly due to the fact that many genes with sex-specific loci had several paralogs. We then performed the analysis only with the two species with sex chromosomes, and found 115 syntenic genes with sex-specific loci among the total 4,582 (Fig. S20; Table S17). Although most of them were distributed across many chromosomes (Fig. 4B), 60% of them were concentrated on chromosome 11 in \u003cem\u003eC. reniformis\u003c/em\u003e, which appears to result from a fusion between two ancestral chromosomes (Fig. 4A\u0026ndash;B). The genomic instability of this fusion could have prompted an inversion (Fig. S8B), altering recombination between this pair of chromosomes and leading to the emergence of a sex chromosome in \u003cem\u003eC. reniformis\u003c/em\u003e. Our results support the idea that sex chromosomes evolve independently from autosomes\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e15\u003c/span\u003e\u003c/sup\u003e, with frequent turnovers due to the moderate divergence of sponge sex chromosomes, which could make them more prone to revert back to autosomes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeep evolutionary origin of genes with sex-specific loci.\u0026nbsp;\u003c/strong\u003eWe investigated the evolutionary age of sponge genes across the six species with reference genomes by assigning each gene to its most probable taxonomic origin based on their homology (Table S18A\u0026ndash;B). Around 60% of the genes traced back to ancestral nodes (Fig. 4C; Table S18A), highlighting their conservation across animals and deeper eukaryotic lineages\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e54,56\u003c/span\u003e\u003c/sup\u003e, while only 11.1% were classified as species-specific. We then examined specifically the evolutionary history of sponge genes with sex-specific loci (Table S18C), and found that the majority were assigned to deep evolutionary nodes (Fig. 4C; Table S18A). Genes assigned to unicellular organisms and Eukaryota were predominantly enriched in functions related to DNA repair and recombination, cell-cycle regulation, cytoskeleton organization, membrane fusion and adhesion, stress response, and apoptosis (Fig. 4D, Table S18E). Genes with sex-specific loci assigned to Metazoa were enriched in terms related to transcriptional regulation and reproductive processes, consistent with the emergence of specialized, multicellular reproductive systems\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Previous studies suggest that sex determination mechanisms have been conserved for more than 300 million years\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e57\u003c/span\u003e\u003c/sup\u003e, but our results suggest an even earlier origin, given the conserved set of genes across metazoans that underlie sex determination in sponges. Our results suggest that the same genetic toolkit that facilitated sex determination in sponges was already present in ancestral eukaryotes and early metazoans\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e58\u003c/span\u003e\u003c/sup\u003e, with conserved functions in sexual development\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Finally, the modest contribution of more recent, lineage-specific genes (Fig. 4D, Table S18E) likely reflects functional fine-tuning or clade-specific adaptations, rather than \u003cem\u003ede novo\u003c/em\u003e evolution of entirely new sexual pathways.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatterns of evolution of sex determination systems in animals.\u003c/strong\u003e A major challenge in understanding the evolution of sexual systems across Metazoa is resolving their distribution and diversity among extant lineages. Here we show that the transitions from hermaphroditism to gonochorism that occurred repeatedly during sponge evolution\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e8\u003c/span\u003e\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003ewere accompanied by the independent evolution of a striking variety of sexual systems (Fig. 5A). Such diversity mirrors the situation across the Tree of Life (Fig. 5B; Supplementary Text Section 7), where gonochorism can be achieved through a wide array of molecular mechanisms and sex chromosome systems, with shifts documented at the phylum, class, order, and even family level\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e2,26,59\u003c/span\u003e\u003c/sup\u003e (Fig. 5B).\u003c/p\u003e\n\u003cp\u003eMost information about sex chromosomes is known for well-established bilaterian model species\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e2\u003c/span\u003e\u003c/sup\u003e, with non-model organisms remaining comparatively understudied. Recent advances in sequencing techniques, computational tools, and algorithms are now enabling rapid progress in uncovering sex determination in other invertebrate groups\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e6,18\u003c/span\u003e\u003c/sup\u003e. Given that sex determination systems vary widely across animals, expanding research into diverse and early-diverging lineages is vital to understand its origins and evolution. Our study broadens knowledge in early-splitting animals by identifying sex determination systems in seven orders of sponges\u0026mdash; one XY, another ZW, and five polygenic (both XY-like and ZW-like). We reveal both the persistence of a conserved set of genes involved in sex determination inherited from ancestral eukaryotes and early metazoans and the independent origin of sponge sex chromosomes, likely shaped by unstable chromosomal rearrangements. Together, these findings underscore sponges as key to understanding the evolutionary forces generating the extraordinary diversity of sexual systems across animals.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e1. Sampling, assessment of reproductive activity and sex assignment:\u0026nbsp;\u003c/strong\u003eSamples of eight gonochoristic species were collected from the North Atlantic Ocean between 2017-2019, the Mediterranean Sea in the summer of 2021 and 2022, and the Kiel Bay in summer of 2021 (Table S1, Supplementary Fig.1). We collected approximately 3-5 cm\u003csup\u003e3\u003c/sup\u003e of sponge tissue per specimen and divided the sample in 4 pieces that were later preserved in three different preservatives: 1. For traditional histology, in 4% formaldehyde in seawater, 2. For further construction of DNA libraries (for both RADseq and WGS), in absolute ethanol and stored at -20 \u0026deg;C, and 3. For transcriptomic analyses, a tissue piece of about 2 cm\u003csup\u003e3\u003c/sup\u003e was preserved in RNAlater at 4\u0026ordm;C for 24 h and then frozen at -20 \u0026deg;C until further processing.\u003c/p\u003e\n\u003cp\u003eSince sponge sex is only possible to determine through presence of gametes, we processed the samples for histology. Samples of sponges with siliceous spicule content (\u003cem\u003ePetrosia ficiformis\u003c/em\u003e, \u003cem\u003eGeodia hentscheli,\u003c/em\u003e \u003cem\u003eGeodia barretti\u003c/em\u003e, \u003cem\u003eAxinella damicornis\u003c/em\u003e, \u003cem\u003eHalichondria panicea\u003c/em\u003e, and \u003cem\u003ePhakellia ventilabrum\u003c/em\u003e) went through a step of desilicification in 5% hydrofluoric acid overnight and then rinsed with distilled water at least twice. Then, their tissues and those of the sponges without spicules (\u003cem\u003eChondrosia reniformis\u003c/em\u003e and \u003cem\u003eOscarella lobularis\u003c/em\u003e) were processed for light microscopy Tissues were dehydrated through an increasing ethanol series and later embedded in paraffin after a brief rinse in xylene. \u0026nbsp;Then, paraffin blocks were sectioned at 5 \u0026mu;m with an HM 325 rotary microtome (ThermoFisher-Scientific) and sections stained with hematoxylin and eosin, using standard protocols, and mounted in slides with DPX. Slides were observed with an Olympus microscope (BX43) with a UC50 camera at the Museo Nacional de Ciencias Naturales de Madrid (MNCN-CSIC). Samples with oocytes were coded as females and samples with any spermatogenic stage as males. We did not find any case of hermaphroditism among our samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. DNA and RNA extraction, RADseq and RNAseq library preparation:\u0026nbsp;\u003c/strong\u003eDNA was extracted from all samples using the DNeasy Blood \u0026amp; Tissue kit (Qiagen) following the manufacturer\u0026rsquo;s protocol, except for the cell lysis time which was conducted overnight. Double-stranded DNA was quantified with Qubit dsDNA HS assay (Life Technologies). For RADseq library preparation we followed a protocol from Peterson \u003cem\u003eet al.\u003c/em\u003e (2012)\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e60\u003c/span\u003e\u003c/sup\u003e with modifications described in Taboada\u003cem\u003e\u0026nbsp;et al\u003c/em\u003e. (2022)\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e61\u003c/span\u003e\u003c/sup\u003e. RNA was extracted separately from all samples using the Invitrogen RNA mini kit (Thermofisher) following the manufacturer\u0026rsquo;s protocol using TRIzol for cell lysis and quantified with NanoDrop. Further, mRNA libraries were constructed using the Illumina Stranded mRNA Prep kit, quantified using Qubit dsDNA HS assay (Life Technologies) and checked for size and quality using a TapeStation 2200 (Agilent Technologies, USA). They were then pooled and sequenced using paired-end 150-bp reads on an Illumina NovaSeq6000 at Novogene Europe (Cambridge, UK). Our final dataset of genomic and transcriptomic resources for assessing sex determination in sponges can be found in Table S2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. WGS library preparation:\u0026nbsp;\u003c/strong\u003eDNA extracted as above from tissues of the sponges (see Supplementary Table 2) and prepared to obtain WGS libraries. The genomic DNA was randomly sheared into short fragments using enzymes, and the obtained fragments were end-repaired, A-tailed, and further ligated with an Illumina adapter. The fragments with adapters were PCR amplified, size selected, and purified. The library was checked with Qubit 3.0 and real-time PCR for quantification, and Bioanalyzer for size distribution detection. Quantified libraries were pooled and sequenced on the Illumina platform NovaseqX plus, according to effective library concentration and data amount required (6 Gb/sample), done at Novogene.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4 Genome annotation\u003c/strong\u003e: The chromosome-level genomes of \u003cem\u003eOscarella lobularis\u003c/em\u003e, \u003cem\u003eChondrosia reniformis\u003c/em\u003e, \u003cem\u003ePetrosia ficiformis, Axinella damicornis,\u0026nbsp;\u003c/em\u003eand \u003cem\u003ePhakellia ventilabrum,\u003c/em\u003e \u003cem\u003eHalichondria panicea\u003c/em\u003e were sequenced by the Aquatic Symbiosis Genome Consortium (Table S2). We annotated the genes in the assemblies of \u003cem\u003eO. lobularis\u003c/em\u003e, \u003cem\u003eC. reniformis\u0026nbsp;\u003c/em\u003eand \u003cem\u003eP. ficiformis\u003c/em\u003e using a combination of tools for \u003cem\u003ede novo\u003c/em\u003e and evidence-based gene prediction (BRAKER2 2.1.6\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e62\u003c/span\u003e\u003c/sup\u003e, Augustus 3.5.0\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e63,64\u003c/span\u003e\u003c/sup\u003e, StringTie 2.2.1\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e65\u003c/span\u003e\u003c/sup\u003e, and GenomeThreader 1.7.1\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e66\u003c/span\u003e\u003c/sup\u003e) and optimal gene model selection (Mikado 2.3.4\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e67\u003c/span\u003e\u003c/sup\u003e). This procedure is described below. First, we mapped bulk paired RNA-seq libraries to the reference genome using the read aligner STAR 2.7.10b\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e68\u003c/span\u003e\u003c/sup\u003e without multi-mapping reads (flag: --outFilterMultimapNmax 1), only considering uniquely mapping reads for splice junctions (--outSJfilterReads Unique), reporting splice junction-supporting reads and keeping only the reads with junctions that passed filtering (--outFilterType BySJout, --alignSJDBoverhangMin 1, --alignSJoverhangMin 8), and reporting the alignment strand based on intron motifs (--outSAMstrandField intronMotif). The resulting coordinate-sorted BAM file (--outSAMtype BAM SortedByCoordinate) was used to produce an initial set of transcript predictions using StringTie in conservative mode (-t -c 1.5 -f 0.0 flags), from which open reading frames (ORFs) were predicted using TransDecoder 5.7.1\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Predicted peptides were collapsed by sequence similarity using CD- HIT 4.8.1 (-c 0.95) and complete genes (i.e. with start and end codons) of non-extreme lengths (\u0026amp;gt;600 and \u0026amp;lt;10,000 amino-acids) were retrieved for later use in the \u003cem\u003ede novo\u003c/em\u003e gene prediction step. Specifically, these were used to train Augustus iteratively within BRAKER2, by aligning them to the reference genome using GenomeThreader (BRAKER2 flag: --prg=gth \u0026ndash;trainFromGth), and using the original STAR-produced alignments as further evidence (--bam=\u0026amp;lt;file\u0026amp;gt;). Second, we used Mikado to select the best gene predictions from each locus, selecting from the output of BRAKER2/Augustus (all exons and coding exons [CDS] were used separately), an unguided Stringtie assembly, and the filtered set of GenomeThreader training peptide alignments to the reference genome. To build the Mikado hints file, (i) all sources of evidence were considered as strand-specific; (ii) a score of 1 was associated with the BRAKER2/Augustus predictions and 0 for the others; (iii) the CDS from BRAKER2/Augustus were considered as a reference annotation; (iv) redundant models were excluded from all samples; and (v) CDS sequences with errors were removed from the model set. To build the Mikado configuration file (mikado conFig), we clustered transcripts with a minimum cDNA overlap of 20% (--min-clustering-cdna-overlap 0.2) and any for CDSs (--min-clustering-cds-overlap 0.0), set the programme to permissive mode with regards to ORF splitting policy (--mode permissive). After preparing the transcripts sets with the mikado prepare submodule, we prepared further evidence sources to be considered by Mikado: (i) predicted ORFs for all Mikado models, using TransDecoder; (ii) evidence-based splice junction coordinates from STAR (see above), obtained using the junctools convert 1.2.4 module of Portcullis 1.1.2.; and (iii) homology models obtained with diamond blastx\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e70\u003c/span\u003e\u003c/sup\u003e against the 2022-05 release of UniRef50\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e71\u003c/span\u003e\u003c/sup\u003e, adding the percentage of positive-scoring alignments and the traceback operations fields to the reported output (flag: -f 6 qseqid sseqid pident length mismatch gapopen qstart qend sstart send evalue bitscore ppos btop). These additional sources of evidence were considered by Mikado (mikado serialise submodule, disabling start codon adjustment with \u0026ndash;no-start-adjustment). The best gene model for each locus was selected using the mikado pick module, prioritising reference models (--reference-update). Finally, transcript and peptide sequences for each gene model were retrieved using gffread 0.11.7\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e72\u003c/span\u003e\u003c/sup\u003e. The completeness of the resulting gene set was evaluated using BUSCO 5.5.0\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e73\u003c/span\u003e\u003c/sup\u003e in protein mode (-m proteins) against the Metazoa database of universal orthologs (-l metazoa_odb10). Annotation of \u003cem\u003eP. ventilabrum, Axinella damicornis,\u0026nbsp;\u003c/em\u003eand \u003cem\u003eHalichondria panicea\u0026nbsp;\u003c/em\u003ewas performed elsewhere, and the details can be found in the references in Table S2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. Repeatome analysis:\u003c/strong\u003e The density of transposable elements (TEs) per chromosome was calculated in 100 Kbp windows for each species with chromosome-level reference genome (\u003cem\u003eC. reniformis\u003c/em\u003e and \u003cem\u003eO. lobularis\u003c/em\u003e). First, the Repeatmodeller\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e74\u003c/span\u003e\u003c/sup\u003e output was filtered to only include TEs, removing simple repeats, low-complexity regions, rRNAs, and unknown repeats. Subsequently, bedtools v2.30.0\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e75\u003c/span\u003e\u003c/sup\u003e was used to create a bed file with 100 Kbp windows for each reference genome using the options \u0026ldquo;makewindows -w 100000\u0026rdquo;, and to calculate the density per window using the options \u0026ldquo;intersect -a genome_windows.bed -b ALL_TEs.bed -c\u0026rdquo;. Finally, TE distribution plots per chromosome were created in R using the ggplot2 package v3.5.1\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e76\u003c/span\u003e\u003c/sup\u003e. Additionally, we repeated these analyses exclusively including long interspersed nuclear elements (LINEs), which were extracted from the Repeatmodeller output and plotted as LINE density per Mbp.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. Sex chromosome identification:\u0026nbsp;\u003c/strong\u003eTo identify putative sex chromosomes and sex determining regions (SDR) we employed several complementary approaches. First, we ran the FindZX\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e10\u003c/span\u003e\u003c/sup\u003e\u0026nbsp; pipeline with default parameters for detecting and visualising sex chromosomes based on genome coverage and heterozygosity differences. The strength and nature of sex-specific genomic signals depend on the degree of sex chromosome differentiation and Y/W degeneration. In homomorphic systems, heterozygosity provides the clearest signature, whereas in heteromorphic systems, coverage differences are most informative, expected to be approximately the double on the X/Z chromosome in the homogametic sex compared to the heterogametic sex. Restricting the number of allowed mismatches enhances detection by preventing reads from the sex-limited chromosome from aligning to its gametolog, and heterozygosity can appear higher in either the heterogametic or homogametic sex depending on whether reads from the sex-limited chromosome successfully align or not. The analyses were conducted using WGS reads in 50kb, 100kb and 500kb windows, unfiltered, 0.2 and 0.0 mismatches allowed and their species reference genome in the no-synteny mode. We also calculated differences in coverage with mosdepth [v0.3.2]\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e77\u003c/span\u003e\u003c/sup\u003e, SNP density relative to the reference and \u003cem\u003eF\u003c/em\u003e\u003csub\u003eST\u003c/sub\u003e values between sexes.SNP density and \u003cem\u003eF\u003c/em\u003e\u003csub\u003eST\u003c/sub\u003e values are expected to be higher in the heterogametic sex chromosomes due to the differentiation and reduced recombination. k-mer\u0026ndash;based analyses provide a complementary and highly sensitive approach, as they are independent of a reference genome and can detect sex-linked differences even when high sequence homology allows equal read mapping across sexes and at the same time can recover signals that may be overlooked by coverage- or SNP-based approaches when read mapping differs between sexes. WGS reads of each individual and also in male and female datasets were aligned to the reference chromosome-level genome with bowtie2\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e78\u003c/span\u003e\u003c/sup\u003e and indexed with samtools [v1.14]\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e79\u003c/span\u003e\u003c/sup\u003e . SNP calling was done with bcftools [v1.12]\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e79\u003c/span\u003e\u003c/sup\u003e and calls filtered with vcftools [v0.1.16]\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e80\u003c/span\u003e\u003c/sup\u003e to only keep biallelic sites that pass the bcf_filter. Male/Female mapped read files were used to perform a coverage analysis after normalization. The analysis of the difference in coverage was performed with mosdepth [v0.3.2] in 1 Mb windows. SNP density relative to the reference genome in 100kb windows was calculated with vcftools and differences between male and female with the R script SNPdensity_permutations.R from the SexFindR pipeline\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e81\u003c/span\u003e\u003c/sup\u003e that calculates true means and difference and carries out a permutation test that determines p-values for SNP density differences windows. Kernel-smoothed \u003cem\u003eF\u003c/em\u003e\u003csub\u003eST\u003c/sub\u003e was calculated with the \u003cem\u003epopulations\u003c/em\u003e program of Stacks from the RADseq reads. K-mer analyses were performed following the SexFindR pipeline. kmersGWAS\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e82\u003c/span\u003e\u003c/sup\u003e was used for k-mer counting, and PLINK\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e83\u003c/span\u003e\u003c/sup\u003e for sex-association testing of k-mers with p-value \u0026lt; 0.00001. Significant k-mers were subsequently assembled into short contigs using ABySS\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e84\u003c/span\u003e\u003c/sup\u003e, which were then aligned to the reference genome with BLAST to determine their genomic positions\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7. Intraspecific inversion detection in sex-chromosomes:\u0026nbsp;\u003c/strong\u003eTo evaluate the presence of putative inversions in the sex-determining gene regions, we conducted a multi-evidence approach based on (i) mapping alternative haplotypes to the reference assemblies, (ii) genotypes and (iii) mapping quality for those species with male and female specimens. Since the reference genome for both \u003cem\u003eC. reniformis\u003c/em\u003e and \u003cem\u003eO. lobularis\u003c/em\u003e derived from the heterogametic sex, we mapped the alternative haplotypes to the reference assemblies using minimap2\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e85\u003c/span\u003e\u003c/sup\u003e and visualized the dotplots in D-GENIES\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e86\u003c/span\u003e\u003c/sup\u003e. \u0026nbsp;For the genotype approach, we used the R script \u0026ldquo;individual Detection of linkage by Genotyping (iDlG)\u0026rdquo;\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e87\u003c/span\u003e\u003c/sup\u003e, which identifies linked regions across chromosomes at the individual level based on mean genotype calculated in sliding windows. This makes it possible to not only identify inversions but other regions with high linkage such as sex chromosomes. We established windows of 5,000 SNPs with sliding steps of 1,000 SNPs, and graphically visualized the iDlG results with the R package \u0026ldquo;ggplot2\u0026rdquo;. To further support the presence of inversions in those regions, we kept from each specimen \u0026ldquo;sam\u0026rdquo; file those paired-reads mapping colinearly\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e88\u003c/span\u003e\u003c/sup\u003e (forward\u0026ndash;forward; reverse\u0026ndash;reverse) in the same chromosome. From those, we retained only those pairs mapping at a distance above the expected from library preparation (400 bp), and subsequently visualized the relative position of both collinear reads with \u0026ldquo;ggplot2\u0026rdquo;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8. Detection of sex-specific loci using RADseq data:\u0026nbsp;\u003c/strong\u003eRADseq is a crucial tool to study the genetic basis of sex determination in species without heteromorphic sex chromosomes\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e89\u003c/span\u003e\u003c/sup\u003e. Although RADseq alone can identify sex chromosome systems, it cannot determine sex chromosome linkage or the size of the non-recombining region. However, when used with a reference genome, sex-specific loci can be mapped to identify linkage groups and sex determining regions. RADseq data also allows us to calculate SNP density, and \u003cem\u003eF\u003c/em\u003e\u003csub\u003eST\u003c/sub\u003e between males and females across the genome, both predicted to be higher in the sex-determining, non-recombining regions of sex chromosomes. These indicators are expected to converge in the sex determining regions of the sex chromosomes. To this end, we used the RADsex pipeline [v1.2]\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e90\u003c/span\u003e\u003c/sup\u003e. The quality of raw reads was assessed with FastQC before and after demultiplexing with the process_radtags module of Stacks [v2.61]\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e91\u003c/span\u003e\u003c/sup\u003e. In addition, the detection and mapping of sex-specific loci was conducted with RADsex, which compares presence/absence of non-polymorphic markers between individuals in two groups, with a minimum marker depth of 1 for the process step, and 5 for the distribution, significance and mapping steps, keeping only the significative loci present in just one sex (i-e-, sex-specific loci). To assess the significance of the male/female distribution of markers we conducted a 1,000 permutations test followed by a chi-square test comparing the observed results against the permutations-based expected frequencies. The sex-specific loci were blasted against a reference transcriptome with local blast\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e92\u003c/span\u003e\u003c/sup\u003e and then aligned to their genome with bwa\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e93\u003c/span\u003e\u003c/sup\u003e to map them to reference genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e9. Gene expression patterns:\u0026nbsp;\u003c/strong\u003eWe aligned the RNAseq data from each male and female of each species of those with chromosome-level genomes with HISAT2\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e94\u003c/span\u003e\u003c/sup\u003e\u0026nbsp; and the alignments were passed to StringTie for transcript assembly\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e65\u003c/span\u003e\u003c/sup\u003e. We filtered transcripts with expression lower than 2 TPM and those expressed in less than 2 samples per condition when possible, and we then tested for differential gene expression (DE) between males and females with edgeR after normalisation\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e95\u003c/span\u003e\u003c/sup\u003e. In both \u003cem\u003eGeodia\u003c/em\u003e spp., given there is no chromosome-level genome, we used \u003cem\u003ede novo\u003c/em\u003e transcriptomes built previously\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e96,97\u003c/span\u003e\u003c/sup\u003e, and also we built \u003cem\u003ede novo\u003c/em\u003e transcriptomes for the rest of the species for further annotation. Then reads that were quality filtered with Prinseq lite v 0.20.4 \u003csup\u003e\u003cspan lang=\"EN-US\"\u003e98\u003c/span\u003e\u003c/sup\u003e, and decontaminated from rRNA with SortMeRNA v 4.3.6\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e99\u003c/span\u003e\u003c/sup\u003e with the provided SILVA 119 Ref NR 99 database\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e100\u003c/span\u003e\u003c/sup\u003e were mapped with Bowtie2 aligner\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e78\u003c/span\u003e\u003c/sup\u003e and expression levels estimated with RSEM v 1.2.21\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e101\u003c/span\u003e\u003c/sup\u003e. Expected counts (number of reads mapped) and the normalised trimmed mean of M values (TMM) were used for testing for differential gene expression (DE) between males and females with edgeR after normalisation and using the glmQLFit function. Expression levels of genes with sex-specific loci were retrieved from the count table for plotting. Then, genes with sex-specific loci identified were locally blasted against the genomes and \u003cem\u003ede novo\u003c/em\u003e transcriptomes (retrieving all isoforms with a hit) of their corresponding species using BLAST and then annotated using Blast2GO PRO\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e102\u003c/span\u003e\u003c/sup\u003e. The gene annotations from the sex-loci were retrieved and implemented in ShinyGO\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e103\u003c/span\u003e\u003c/sup\u003e against the human database to perform Gene Ontology (GO) enrichments (with Benjamini-Hochberg FDR corrections of 0.05) and then plotted using ggplot in R.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e10. Identification of sex-biased Alternative Splicing:\u0026nbsp;\u003c/strong\u003eWe used rMATs 4.3.0\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e104\u003c/span\u003e\u003c/sup\u003e to identify Alternative Splicing (AS) events in our transcriptomes. We used the trimmed reads from our RNA-seq data for each species (Table S2). Besides assessing annotated splice junctions in the reference genome, rMATs can also evaluate differential splicing between two groups of samples, which in this case were females and males. rMATs measures splicing at each splice site with the percentage spliced in or PSI, which indicates the proportion of two alternative isoforms at each splice site, being 1 and 0 the extremes at which only one of the two alternatives is expressed, and 0.5 indicating equal expression. To compare splicing between sample groups, rMATS calculates the inclusion level difference (\u0026Delta;PSI), defined as the average PSI in males minus the average PSI in females. \u0026Delta;PSI ranges from +1 (the isoform is expressed exclusively in males) to \u0026minus;1 (the alternative isoform is expressed exclusively in females). rMATS uses a likelihood-ratio test to identify significant differences in \u0026Delta;PSI between males and females, and we used the criterion of an FDR P value \u0026lt;0.05 to call differential splicing events. This was calculated both for all genes present in the genome and then for genes with sex-specific loci.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e11. Macrosynteny analyses:\u0026nbsp;\u003c/strong\u003eWe used\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eOrthofinder v.2.5.4\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e105\u003c/span\u003e\u003c/sup\u003e to identify and compare orthologous groups shared between the six sponge species with chromosome-level genomes: \u003cem\u003eOscarella lobularis\u003c/em\u003e, \u003cem\u003eChondrosia reniformis\u003c/em\u003e, \u003cem\u003ePetrosia ficiformis\u003c/em\u003e, \u003cem\u003eAxinella damicornis\u003c/em\u003e, \u003cem\u003eHalicondria panicea\u003c/em\u003e, and \u003cem\u003ePhakelia ventilabrum\u003c/em\u003e. With the set of orthologous groups we then looked at conservation of syntenic regions across sponge genomes with the R package macrosyntR\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e106\u003c/span\u003e\u003c/sup\u003e. Then, genome location information for each ortholog was extracted from genome annotation files and used to identify regions of synteny by inferring significantly ancient linkage groups (ALG) which were ordered and displayed on Oxford grids for pairwise species comparisons. To evaluate inter-chromosomal rearrangements and conservation of sex-related genetic regions across sponges, we mapped the genomic location of CLGs across sponge chromosomes and generated ribbon diagrams with macrosyntR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e12. Evolutionary trajectories of genes with sex-specific loci:\u0026nbsp;\u003c/strong\u003eTo analyse the presence and evolution of genes associated with sex determination in sponges, we conducted a comprehensive search of an extensive metazoan taxonomic database, with a special emphasis on early-branching animal lineages such as Ctenophora, Cnidaria, Placozoa, and Porifera. To avoid redundancies arising from the use of transcriptomes and ensure better quality and completeness, we included only complete sponge genomes (Porifera). The analysis was performed using a pipeline developed specifically for this study (EvoDomainSearch). This tool allows a systematic and controlled search for genes based on the presence of conserved domains. First, searches were performed with HMMER (v3.4), using HMM models generated from representative sequences of target genes related to sex determination in metazoans. The recovered sequences were subsequently filtered to retain only those containing the target domains of each gene. The final sequences were aligned using MAFFT (v7.525)\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e107\u003c/span\u003e\u003c/sup\u003e, employing the L-INS-i strategy for high-quality alignment. Subsequently, ambiguous alignment regions were removed with BMGE (v1.12)\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e108\u003c/span\u003e\u003c/sup\u003e using the parameters -m BLOSUM30 -b 3 -h 0.8 -g 0.8, with the aim of retaining only informative and phylogenetically reliable positions. Phylogenetic inferences were then performed using two maximum likelihood methods: FastTree (v2.1.11)\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e109\u003c/span\u003e\u003c/sup\u003e for rapid family tree exploration, and IQ-TREE (v2.3.6)\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e110\u003c/span\u003e\u003c/sup\u003e for more accurate analyses, employing automatically selected substitution models and statistical assessment using ultrafast bootstrap (UFBoot). This approach allowed us to identify candidate genes in sponges with high confidence and assess their phylogenetic relationship with homologs from other metazoan groups and, when present, their unicellular relatives.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e13. Evolutionary age and diversification of sex-specific loci:\u0026nbsp;\u003c/strong\u003eTo investigate the evolutionary origins and diversification of sex-specific genes in sponges, we implemented a multi-step analytical approach that combined ortholog inference with phylostratigraphic reconstruction. We first looked into the shared genome complements of the six species with chromosome-level genomes with OrthoVenn 3\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e111\u003c/span\u003e\u003c/sup\u003e. Then, orthologous genes were collected with OrthoFinder 3 as before to infer orthologous groups (OGs) and to establish homologous relationships across species. To assign evolutionary ages to genes, we applied phylostratigraphic analysis using GenEra v1.4.2\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e112\u003c/span\u003e\u003c/sup\u003e (Barrera-Redondo et al. 2023). Protein sequences predicted from each sponge species were queried against the NCBI non-redundant (NR) protein database using DIAMOND v2.1.9 \u003csup\u003e\u003cspan lang=\"EN-US\"\u003e70\u003c/span\u003e\u003c/sup\u003e in sensitive mode. The search employed an e-value threshold of 1e-5 and used the -a flag to input both the species-specific \u003cem\u003efasta\u003c/em\u003e files and corresponding NCBI Taxonomy IDs. GenEra automatically traced phylogenetic relationships using NCBI Taxonomy to determine the most basal taxonomic node associated with each gene hit. Genes were then assigned to discrete evolutionary strata based on their inferred taxonomic age.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong the annotated gene sets, we extracted the genes with sex-specific loci previously identified with RADSex in each species. These genes were mapped to their respective phylostrata to assess the relative timing of their evolutionary origin. We further analysed whether sex-related genes within the same stratum were associated with shared or distinct OGs, enabling insights into patterns of gene duplication and lineage-specific diversification.\u003c/p\u003e\n\u003cp\u003eTo evaluate the functional characteristics of genes with sex-specific loci across evolutionary strata, we performed Gene Ontology (GO) enrichment analysis using ShinyGO as before. Enriched GO terms were then categorized into broader semantic groups reflecting known ancestral functions (e.g., DNA repair, cell cycle regulation, cytoskeleton organization, apoptosis). This allowed us to assess the extent to which genes originally involved in ancient cellular processes were subsequently co-opted into core sexual reproduction functions such as meiosis, gametogenesis, and fertilization. A full list of enriched GO terms and their classifications by stratum is provided in Table S18F. Lastly, we characterized the evolutionary origin of syntenic genes with sex-specific loci identified following the methodology outlined in the Methods Section 10. These analyses facilitated the exploration of conserved genomic architecture and potential linkage patterns associated with sex-related gene clusters.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We are indebted to Manuel Maldonado and Konstantina Mitsi for help in sampling, and Astrid B\u0026ouml;hne, Arnau Seb\u0026eacute;-Pedr\u0026oacute;s, and Manuel Irimia for fruitful discussions. We are also thankful to Gonzalo Giribet, Sally Leys, and April Horton for critical reading of the manuscript.\u003c/p\u003e\n\u003cp\u003eThis research could be done thanks to the funding obtained by several people: A.R. obtained funding from the grants RYC2018-024247-I and PID2019-105769GB-I00, both funded by the Spanish agencies MCIN/AEI/10.13039/50110001103 and EI \u0026ldquo;FSE invierte en tu futuro.\u0026rdquo; Also, A.R. acknowledges funding from the European grant \u0026ldquo;Biodiversity Genomics Europe\u0026rdquo; (Grant agreement ID: 101059492). C.D.-V. received the support of a fellowship from \u0026ldquo;la Caixa\u0026rdquo; Foundation (ID 100010434), with the fellowship code LCF/BQ/PI22/11910040. I.S. was supported by a JdC (Juan de la Cierva 2024) personal grant (JDC2024-054954-I). M.T. was supported by a JdC (Juan de la Cierva Formaci\u0026oacute;n, 2020) personal grant (FJC2020-043677-I) and MSCA Postdoctoral Fellowship (HORIZON-MSCA.2022-PF-01, Project 101105716). The work by J.A.H was supported by grants PID2023-153118OB-I00 funded by MCIN/AEI/10.13039/501100011033 and CRSII5_198737/1 by the Swiss National Science Foundation. S.T. received funding from the grants PID2020-117115GA-100 of the Spanish Ministry of Science and Innovation and CNS2023-144572 and by the Ram\u0026oacute;n y Cajal grant RYC2021-03152-I, funded by the MCIN/AEI/10.13039/501100011033 and the European Union NextGenerationEU/PRTR. A.V. was supported by a European Union NextGenerationEU/PRTR grant (IJC2020-045256), a fellowship from \u0026ldquo;la Caixa\u0026rdquo; Foundation (ID 100010434), with fellowship code LCF/BQ/PR24/12050011 and a Ram\u0026oacute;n y Cajal grant RYC2023-044466-I. P\u0026Aacute;-C was supported by MCIN/AEI/10.13039/501100011033 and by the European Union \u0026ldquo;Next Generation EU\u0026rdquo;/PRTR (CNS2023-145193). M.\u0026Aacute;-P. received funding from a fellowship from the Fundaci\u0026oacute;n General CSIC\u0026rsquo;s ConFuturo under the Marie Skłodowska-Curie grant agreement 101034263 and a Ram\u0026oacute;n y Cajal grant RYC2023-043807-I. A.E.W is funded by a UKRI grant EP/X041921/1 and Philip Leverhulme Prize grant. Finally, part of this research was supported through the SponBIODIV project (A.R. and S.T.), a 2021-2022 BiodivProtect joint call for research proposals, under the Biodiversa+ Partnership co-funded by the European Commission, and with the funding organization \u0026ldquo;Fundaci\u0026oacute;n Biodiversidad\u0026rdquo;. Support for sponge genome sequencing from the Aquatic Symbiosis Genomics (ASG) project was provided by the Gordon and Betty Moore Foundation through a grant (GBMF8897) to the Wellcome Sanger Institute and U.H.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: AR, JLS, PAC, AV, ST, AEW\u003c/p\u003e\n\u003cp\u003eFormal analysis: JLS, CDV, CGC, AV, CL, VT, MAP, JAH, AR, MT, XGB\u003c/p\u003e\n\u003cp\u003eInvestigation: JLS, AR, AV, VT, MC, CGC\u003c/p\u003e\n\u003cp\u003eVisualization: JLS, CDV, CGC, AV, CL, VT, MAP, JAH, AR, MT, PAC, MC\u003c/p\u003e\n\u003cp\u003eFunding acquisition: AR, ST, CDV, AV, PAC, UH, JAH, MAP, MT, AEW\u003c/p\u003e\n\u003cp\u003eSupervision: AR, ST, PAC\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; original draft: JLS, AR, AEW, PAC, VT, CGC, CL\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; review \u0026amp; editing: all authors\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e Authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability:\u003c/strong\u003e Raw genomic and transcriptomic sequences are deposited in NCBI Bioprojects PRJNA1336057 (RADseq, WGS and RNAseq \u003cem\u003eof Chondrosia reniformis, Oscarella lobularis, Petrosia ficiformis, Halichondria panicea, Axinella damicornis, Phakellia ventilabrum, Geodia hentschelli\u003c/em\u003e and \u003cem\u003eGeodia barreti\u003c/em\u003e Raw sequence reads) and PRJNA1274490 (RNAseq of \u003cem\u003eHalichondria panicea\u003c/em\u003e). No original code was developed for this work. Code used is available at https://github.com/Jose-LSP/Sponges-sex-scripts and \u0026nbsp;https://github.com/CGaliaCamps/iDlG.git\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBachtrog, D. et al. Sex determination: why so many ways of doing it? \u003cstrong\u003ePLoS Biol.\u003c/strong\u003e \u003cstrong\u003e12\u003c/strong\u003e, e1001899 (2014). https://doi.org/10.1371/journal.pbio.1001899\u003c/li\u003e\n\u003cli\u003eZhu, Z., Younas, L. \u0026amp; Zhou, Q. Evolution and regulation of animal sex chromosomes. \u003cstrong\u003eNat. Rev. Genet.\u003c/strong\u003e (2025). https://doi.org/10.1038/s41576-024-00757-3 (in the press).\u003c/li\u003e\n\u003cli\u003eBernstein, H., Bernstein, C. \u0026amp; Michod, R. E. 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The chromosomal genome sequence of the stone sponge Petrosia ficiformis (Poiret, 1789) and its associated microbial metagenome sequences. \u003cstrong\u003eWellcome Open Res.\u003c/strong\u003e \u003cstrong\u003e10\u003c/strong\u003e, 70 (\u003cstrong\u003e2025\u003c/strong\u003e). https://doi.org/10.12688/wellcomeopenres.24743.1\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8554461/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8554461/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Sex has profoundly shaped animal biology and evolution, yet sex determination is strikingly diverse and its evolutionary origins remain poorly understood outside bilaterians. We investigate the molecular basis of sex determination in eight gonochoristic sponge species using integrated genomic and transcriptomic analyses. We identify sex chromosomes in two species, likely arising through chromosomal rearrangements, and find widespread polygenic sex determination across all taxa. Sponge sex determination relies on a deeply conserved genetic toolkit with sex-specific loci in DNA repair and recombination genes, syntenic across species and traceable to unicellular ancestors, indicating that core components of sexual reproduction predate animals. We also uncover a younger metazoan-specific complement and show independent evolution of XY and ZW systems, revealing recurrent shifts in heterogamety.","manuscriptTitle":"Sponge genomes reveal a pre-metazoan origin of the sex determination toolkit and sex chromosomes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-12 09:15:48","doi":"10.21203/rs.3.rs-8554461/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"747f862d-8ff6-444a-9c0a-d3794dcf58ad","owner":[],"postedDate":"February 12th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"revise","date":"2026-05-06T08:54:06+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-04-29T10:13:19+00:00","index":2,"fulltext":"This content is not available."}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":60892239,"name":"Biological sciences/Evolution"},{"id":60892240,"name":"Biological sciences/Genetics/Evolutionary biology"}],"tags":[],"updatedAt":"2026-05-06T09:06:27+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-12 09:15:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8554461","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8554461","identity":"rs-8554461","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}