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Learn more: PMC Disclaimer | PMC Copyright Notice Genome Biol Evol . 2025 Oct 9;18(4):evaf191. doi: 10.1093/gbe/evaf191 Search in PMC Search in PubMed View in NLM Catalog Add to search Before the East African Radiation: Sex Chromosome Systems in Basal Haplotilapiine Cichlids Kristen A Behrens Kristen A Behrens 1 Department of Biology, University of Maryland, College Park, MD 20742, USA Find articles by Kristen A Behrens 1 , Zexuan Zhao Zexuan Zhao 2 Department of Biology, University of Maryland, College Park, MD 20742, USA Find articles by Zexuan Zhao 2 , Michael R Kidd Michael R Kidd 3 Department of Biology and Chemistry, Texas A&M International University, Laredo, TX 78041, USA Find articles by Michael R Kidd 3 , Alfred Maluwa Alfred Maluwa 4 Malawi University of Science and Technology, Limbe, Malawi Find articles by Alfred Maluwa 4 , Avner Cnaani Avner Cnaani 5 Department of Poultry and Aquaculture, Institute of Animal Sciences, Agricultural Research Organization, Volcani Institute, Rishon LeZion, Israel Find articles by Avner Cnaani 5 , Stephan Koblmüller Stephan Koblmüller 6 Institute of Biology, University of Graz, Graz 8010, Austria Find articles by Stephan Koblmüller 6 , Thomas D Kocher Thomas D Kocher 7 Department of Biology, University of Maryland, College Park, MD 20742, USA Find articles by Thomas D Kocher 7, ✉ Editor: Esther Betran Author information Article notes Copyright and License information 1 Department of Biology, University of Maryland, College Park, MD 20742, USA 2 Department of Biology, University of Maryland, College Park, MD 20742, USA 3 Department of Biology and Chemistry, Texas A&M International University, Laredo, TX 78041, USA 4 Malawi University of Science and Technology, Limbe, Malawi 5 Department of Poultry and Aquaculture, Institute of Animal Sciences, Agricultural Research Organization, Volcani Institute, Rishon LeZion, Israel 6 Institute of Biology, University of Graz, Graz 8010, Austria 7 Department of Biology, University of Maryland, College Park, MD 20742, USA ✉ Corresponding author: E-mail: [email protected] . Roles Esther Betran : Associate Editor Accepted 2025 Oct 1; Collection date 2026 Apr. © The Author(s) 2025. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License ( https://creativecommons.org/licenses/by-nc/4.0/ ), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact [email protected]. PMC Copyright notice PMCID: PMC13076031 PMID: 41063647 This article has been corrected. See Genome Biol Evol. 2026 Apr 14;18(4):evag076 . Abstract Cichlid fishes have undergone an extraordinary diversification in East Africa. They also have a high rate of sex chromosome turnover. This clade provides an opportunity to study the rates and patterns of sex chromosome turnover, and the interactions of sex chromosome turnover with adaptation and speciation. Here we investigate the evolution sex chromosomes in the tribes Tilapiini, Coptodonini, Heterotilapiini, Gobiocichlini, Pelmatolapiini, and Oreochromini. We assembled chromosome-scale genomes of male and female Pelmatotilapia mariae . We then mapped pooled sequencing reads for males and females of P. mariae and 12 additional species on several genome assemblies to identify sex chromosomes. Tilapia sparrmanii and Oreochromis aureus share a ZW system on LG3 that overlaps the ZW system identified in P. mariae . Heterotilapia buettikoferi , Tilapia brevimanus , and Coptodon bakossiorum share an XY system mapping to another region of LG3. Coptodon zilli , Sarotherodon galilaeus , Sarotherodon melanotheron , and Oreochromis niloticus share an XY system on LG1. Finally, Oreochromis mossambicus and O. shiranus share an XY system on LG14 and we find evidence of an XY system on LG20 in Danakilia sp. “shukoray”. The phylogenetic distribution of these sex determination systems suggests a long period of polymorphism for the systems on LG1 and LG3 and a generally lower rate of sex chromosome turnover in these lineages compared to the lacustrine lineages of the East African radiation. Our data is not consistent with the recent suggestion of figla and banf2 as candidate genes for the LG1XY and LG3ZW systems. We suggest a possible role for ubiquitination in the XY systems on LG3. Keywords: cichlid fish, sex chromosome turnover, genome assembly Significance. Sex chromosome turnovers are frequent in the radiation of cichlid fishes in the lakes of East Africa. To better understand the patterns of sex chromosome turnover in the ancestors of the lacustrine radiations we assembled genomes for male and female Pelmatolapia mariae and used pooled sequencing to characterize the sex chromosome systems of 13 species representing six lineages that are outgroups to the lacustrine radiations. Oreochromis aureus and Tilapia sparrmanii share a ZW system on LG3 that overlaps the system in Pelmatolapia mariae . Three species from the tribes Heterotilapiini, Gobiocichlini, and Coptodonini shared an XY system that maps to a different location on LG3. We also narrowed the locations of XY systems on LG1 and LG14 in the Oreochromini. The rate of sex chromosome turnover in these lineages is considerably slower than in the rapidly diversifying haplochromine cichlids of the East African radiation. Introduction Sex chromosomes have long been recognized as playing an important role in adaptation and speciation. Haldane (1922) was the first to notice that sterility and inviability in hybrid crosses most often affects the heterozygous sex. The X chromosome carries a disproportionate number of genes affecting hybrid fitness ( Coyne 1992 ). A variety of explanations for these observations have been proposed, including the dominance, “faster-X” and “faster male” theories (reviewed in Cutter 2024 ). A recent hypothesis suggests that divergent evolution of cis- and trans-regulators of gene expression on the sex chromosomes may provide a single explanation for these several observations ( Lenormand and Roze 2025 ). Sex chromosomes often include large regions experiencing little or no recombination. These large haplotype blocks may carry variants affecting multiple phenotypic traits and thus act as “supergenes” ( Schwander et al. 2014 ). Evidence suggests a more rapid accumulation of adaptive substitutions on the sex chromosomes ( Meisel and Connallon 2013 ; Irwin 2018 ). Thus, sex chromosomes may carry a disproportionate number of genes involved in the adaptive differences among species. Finally, ecological factors may play an important role in the origin, evolution, and turnover of sex chromosome systems ( Meisel 2022 ). Sexually antagonistic selection and sexual selection play an important role in shaping the evolution and turnover of sex chromosomes ( van Doorn and Kirkpatrick 2010 ). The intersection of sex chromosomes, adaptation, and speciation is therefore an area of active research ( Payseur et al. 2018 ). The radiation of cichlids in the lakes and rivers of East Africa now includes more than 1,000 species, representing the highest rate of speciation known in extant vertebrates ( Wagner et al. 2014 ). Studies have revealed an enormous diversity of sex chromosomes in this group ( El Taher et al. 2021 ). Sex loci have been identified on at least 18 of 23 chromosomes in East African ( Behrens et al. 2024a ) but the sex chromosome state of the ancestor of the East African radiation has yet to be identified. Considerable effort has been made to map sex determiners in an outgroup to the East African radiation—the Oreochromine tilapias, which are important in aquaculture. To date, major genetic loci affecting sex determination in tilapia have been identified on at least four chromosomes. The first was the mapping of an XY system on linkage group (LG) 1 in a strain of Oreochromis niloticus ( Lee et al. 2003 ). Other strains of O. niloticus were found to be segregating an XY system on LG23 ( Eshel et al. 2012 ), which was eventually determined to involve a duplication of amh ( Li et al. 2015 ). A ZW system on LG3 was found to be epistatically dominant to the LG1 system in a strain of Oreochromis aureus ( Lee et al. 2004 ). Oreochromis mossambicus may segregate a ZW system on LG3 and/or an XY system on LG14 ( Gammerdinger et al. 2019 ; Tao et al. 2021a ). Unravelling the origins and distribution of these several systems is complicated by the frequent hybridization of different strains for aquaculture, and a complex history of introgression after aquaculture strains have been transported and released worldwide ( Ciezarek et al. 2024 ). Sex determination is also influenced by environmental temperature during the critical stages of gonad development ( Baroiller et al. 1995 ) and this temperature sensitivity can be selected as a quantitative trait ( Baroiller et al. 2009 ). The Importance of High-quality Genome assemblies Alongside the efforts to genetically map these sex determiners have been efforts to assemble high-quality genome sequences to support research on a variety of traits. Previously, assembly of the heterogametic sex has been avoided due to the high concentration of repeats and low gene content on the Y or W chromosomes ( Carey et al. 2022 ). The homogametic sex was preferred for genome assembly, which meant that valuable information about sex determination was not captured. A complete assembly of the human Y chromosome was only recently achieved ( Rhie et al. 2023 ). Long-read sequencing platforms, such as PacBio HiFi and Oxford Nanopore, allow the generation of high-quality assemblies that can span the highly repetitive regions found on the Y and W sex chromosomes. These resources give us unprecedented opportunities to study the biology of sex chromosomes ( Rhie et al. 2021 ; Carey et al. 2022 ). The first high quality genome assemblies for African cichlid fishes were developed for O. niloticus ( Conte et al. 2017 ; Tao et al. 2021b ) and Metriaclima zebra ( Conte et al. 2019 ). High quality genome assemblies have also been published for O. aureus ( Tao et al. 2021b ), O. mossambicus ( Tao, Cao et al. 2021a ), and Chromidotilapia guntheri ( Behrens et al. 2024b ). Goals of This Study A series of cichlid clades diverged between the emergence of the Oreochromini and the East African radiation ( Astudillo-Clavijo et al. 2023 ). These clades are sometimes referred to as “former tilapiines”, as many were originally assigned to the genus Tilapia ( Dunz and Schliewen 2013 ). They include the tribes Tilapiini, Pelmatolapiini, Heterotilapiini, Gobiocichlini, and Coptodonini. These clades are important for understanding the origin of the hyperdiverse radiation of cichlids in East Africa. The first study to examine sex chromosomes in these clades identified a ZW system on LG3 in Pelmatolapia mariae , and an XY system on LG1 in Coptodon zilli ( Gammerdinger et al. 2019 ). The goals of the present study were first, to assemble chromosome-scale genomes of a male and female P. mariae to improve the analysis of the sex chromosomes in this species and second, to identify the sex chromosomes in species from each of several tribes of the “former tilapiines” by mapping pooled whole genome sequence reads of males and females on the phylogenetically close assemblies of P. mariae . Finally, we take the opportunity to reexamine sex chromosomes in the Oreochromini to evaluate their homology to sex systems in the “former tilapiines”. Results and Discussion Genome Assemblies We obtained 96.2 Gb of sequence reads for the male, and 73.1 Gb of reads for the female P. mariae . The circular consensus reads had an N50 of 15.71 kb for the male and 12.66 kb for the female. The base accuracy (medianQ) of the reads was 33 for the male and 35 for the female. The long and highly accurate reads facilitated a highly contiguous assembly. Compared to many previous assemblies, the P. mariae assemblies have a significantly larger contig N50 (>30 Mb) ( Table 1 ). This is on par with the average size of cichlid chromosomes (∼45 Mb) and largely obviates the need for additional datatypes to scaffold the assembly. Table 1. Genome assembly statistics. BUSCO scores calculated with compleasm is in parentheses O. aureus (ZZ male) ( Tao et al. 2021b ) P. mariae (ZZ male) Pm_UMD_M (this study) P. mariae (ZW female) Pm_UMD_F_2 (this study) Genome ID GCF_013358895.1 GCA_036321365.1 GCF_036321145.2 Sequencing type Nanopore with Hi-C PacBio HiFi PacBio HiFi Genome size (bp) 1,005,590,959 977,654,343 972,201,687 Genome coverage 85× 93× 75× Number of chromosomes 22 20 20 GC % 40.5 40.82 40.76 Scaffold N50 (bp) 40,723,988 43,036,702 42,111,825 Scaffold L50 10 6 8 Contig N50 (bp) 4,404,323 34,428,017 31,656,669 Contig L50 65 11 13 Genes and pseudogenes 36,085 … 32,558 Protein-coding genes 27,686 … 25,233 Noncoding genes 7,104 … 6,153 Complete BUSCOs 99.5% (99.8%) 96.2% (97.6%) 97.8% (98.9%) Complete and single-copy BUSCOs 97.5% (99.2%) 95.1% (97.3%) 96.8% (97.79%) Complete and duplicated BUSCOs 2.0% (0.6%) 1.1% (0.4%) 1.0% (1.1%) Fragmented BUSCOs 0.2% 0.4% 0.5% Missing BUSCOs 0.2% (0.2%) 3.4% (2.3%) 1.7% (1%) Consensus quality (QV) … 66.18 66.47 K-mer completeness … 92.58% 94.93% Open in a new tab A previous karyotype study found that P. mariae has 2 n = 40 chromosomes, indicating that it has two fusions relative to the O. niloticus genome ( Poletto et al. 2010 ). However, the identity of the chromosomes involved in the fusions were not known. Here, we suggest that fusions occurred between LGs 10 and 11, and between LGs 16 and 19 as we assembled contigs spanning each of these proposed fusions in both sexes ( Fig. S1 ). These proposed fusions will need to be further validated with additional data, but we found no evidence for the involvement of other chromosomes. Pelmatolapia mariae has a ZW system on LG3 ( Gammerdinger et al. 2019 ). The Z scaffold was assembled from five contigs and has a total length of 142,753,887 bp. The W scaffold is shorter than the Z, likely because of unplaced shorter contigs, and includes seven contigs totaling 94,765,109 bp. We compared the Z and W haplotypes to characterize the sex chromosomes. A dotplot comparison of the W and Z scaffolds shows large regions of repetitive sequence and extensive rearrangement ( Fig. S2 ). Assembly of LG3 has proved difficult in every African cichlid sequenced to date. Three major regions of the chromosome were identified in O. aureus ( Conte et al. 2020 ). Region 3a (0 to 40 Mb) is gene rich, with extensive homology to medaka chr18. Region 3a′ (40 to 90 Mb) is gene poor but also show homology to medaka chr18. Region 3b (90 to 135 Mb) is gene-poor and highly repetitive, with no significant homologies to medaka chr18. Region 3b may represent an ancestral fusion between LG3 and a highly repetitive B chromosome ( Conte et al. 2020 ). The assembly of LG3 in O. niloticus is less complete but follows the same ground plan, albeit with numerous inversions and translocations ( Fig. 1 ). Fig. 1. Open in a new tab Synteny plot highlighting the similarities in structure of P. mariae LG3 to O. aureus and O. niloticus . The contig N50 for our new assemblies of P. mariae are about 8× larger than the O. aureus assembly (∼30 vs. ∼4 Mb), but LG3 is still comprised of multiple contigs (seven contigs for the W and five contigs for the Z), and we still found it useful to scaffold the P. mariae assemblies on the O. aureus reference. The overall structure of these chromosomes in P. mariae shows some of the features of the O. aureus assembly ( Fig. 1 ). The gene dense region (LG3a) is still apparent, though about half of this region appears to be involved in an inversion relative to O. aureus . But the differences between the assemblies in regions LG3a′ and LG3b are difficult to unravel. Some of these differences are real, but others may reflect errors in the assembly of these repetitive regions across all of the species. Unravelling the complex evolutionary history of this large chromosome across the tilapias will require additional high-quality assemblies, as well as other datatypes (e.g. Hi-C) that can confirm long-range structure. NCBI annotated the female genome using the Eukaryotic Genome Annotation Pipeline ( https://www.ncbi.nlm.nih.gov/books/NBK169439/ ). A total of 32,558 genes and pseudogenes were identified, including 25,233 protein-coding and 6,153 noncoding genes. The BUSCO scores ( Manni et al. 2021 ) are presented in Table 1 . This annotation was lifted over to the male genome, to allow comparisons of the male LG3 (Z) to the female LG3 (W). The liftover successfully transferred 92% of the female annotations to the male genome ( Table S1 ). Using the liftover annotation, we compared the synonymous divergence (dS) and the nonsynonymous divergence (dN) of the 619 orthologous genes that could be detected between the Z and W ( Table S2 , Fig. S3 ). Comparison of the Z and W sequences revealed median values of dN = 0.0009286 and dS = 0.002926. We found no evidence for evolutionary strata, as there were no peaks in dN or dS that corresponded with the peak of sex-specific SNPs around 58 Mb. Pooled Sequencing To identify sex-linkage in each species we constructed separate pools of DNA from males and females and obtained Illumina sequence from each pool ( Table S3 ). We discuss the pool-seq results for each population below. Note: Smith and Kukowka (2025) analyzed our unpublished data for Coptodon bakossiorum , Steatocranus casuarius , Tilapia sparrmanii , and Sarotherodon galilaeus . Their analysis pipeline appears to have swapped the identity of the male and female sequence files, leading to reversed inference of the heterogametic sex in C. bakossiorum , T. sparrmanii , and S. galilaeus . LG3 ZW Systems Three species, P. mariae , T. sparrmanii , and O. aureus , appear to share a ZW system on LG3. We initially mapped the pooled sequencing reads on the P. mariae female (ZW) genome assembly. Both families of P. mariae showed strong differentiation between the sexes on LG3 ( Figs. S4 and S5 ). The top 1% 100 kb windows of sex-patterned SNPs for family 1 were 41 ZW and 37 XY windows, all on LG3. For family 2 the numbers were 50 ZW and 28 XY windows on LG3 ( Table S4 ). The presence of both XY and ZW patterned windows on the same chromosome is typical of older sex chromosomes and likely arises when W reads do not map to the reference, causing heterozygous sites in the ZZ males to appear as an XY system. Because of our concerns that the assembly of the LG3 W haplotype is incomplete, we also mapped the pooled reads to the Z haplotype from the male assembly ( Fig. 2 ). There is a large region of differentiation extending from ∼40 to 80 Mb. The highest sex-patterned SNP density is in a 100 kb block from 57.9 to 58.0 Mb ( Table S5 ). Fig. 2. Open in a new tab Sex patterned SNP density for the ZW systems on LG3 mapped on the P. mariae male and O. aureus male genome assemblies. The GWAS plots for T. sparrmanii on the P. mariae reference also suggest a ZW system on LG3 ( Fig. S6 ). The top 1% windows are 67 ZW and 10 XY windows on LG3 and one ZW window on LG13 ( Table S4 ). Mapped on the Z haplotype, the highest SNP density is a 100 kb window from 52.9 to 53.0 Mb ( Table S5 ) which is part of a broader region of differentiation from 51 to 54 Mb ( Fig. 2 ). The F ST plot for O. aureus also shows strong signal on LG3 ( Fig. S7 ). The top 1% windows are all on LG3 and include 24 XY-patterned and 55 ZW-patterned windows indicating a ZW system ( Table S4 ). Mapped on the P. mariae Z haplotype, the highest ZW SNP density is 1,515 SNPs in 100 kb block from 54.0 to 54.1 Mb ( Table S5 ). Most of the differentiation occurs from 47 to 78 Mb ( Fig. 2 ). A previous study proposed a duplication of the barrier-to-autointegration factor 2 ( banf2_w ) as the causative locus for sex determination in O. aureus ( Curzon et al. 2021 ). To evaluate this claim we remapped the poolseq data for all three LG3 ZW species on the O. aureus ZZ genome assembly ( Tao et al. 2021b ) ( Fig. 2 ). The peaks of sex differentiation in T. sparrmanii (51.4 to 55.0 Mb) and O. aureus (54.2 to 55.3 Mb) map to the same narrow region of LG3. The differentiation in P. mariae extends over a much broader region, but also peaks ∼53.3 to 54.1 Mb. The region we have identified is approximately 30 Mb distant from the region identified by Tao et al. ( 2021a , 2021b ), who suggested the sex locus was located in a 600 kb region from 82.8 to 83.4 Mb in the O. aureus genome. This conclusion was based on the identification of a region with a high-density of ZW-patterned SNPs universally shared by several different O. aureus populations. Curzon et al. (2021) relied on the localization by Tao et al. (2021b) . They focused on duplications of the banf2 gene, and deconvoluted Sanger sequencing chromatograms to postulate a variety of multicopy haplotypes in different strains of tilapia. Finally, they developed a series of genetic markers in the region and genotyped them in a hybrid O. aureus stock. Markers in banf2_w were not perfectly associated with sex and surprisingly, the sex association was significantly reduced for markers just 19 and 216 kb away from the banf2_w marker. Despite this, they claim to have reduced the critical interval for sex determination to a region of 235 kb. We find it difficult to reconcile our results with these previous studies. While we cannot be sure all three species are segregating the same sex locus, the plots of sex-patterned variation on both the T. sparrmanii and O. aureus genomes are quite consistent in showing the highest ZW SNP density around 54 Mb ( Fig. 2 ). The spatial heterogeneity in SNP density suggests there is still much to learn about the structural differences between the Z and W chromosomes, but it nevertheless seems worthwhile to explore candidate genes in the region of highest ZW SNP density ( Table S6 ). LG3 XY Systems Three species, Heterotilapia buettikofferi , Tilapia brevimanus , and C. bakossiorum , appear to share an XY system around 76.5 Mb on LG3. The GWAS plots for H. buettikoferi on the P. mariae female genome assembly show strong evidence for an XY system on LG3 ( Fig. S8 ). All 78 of the top 1% blocks are XY-patterned on LG3 ( Table S4 ). The plot of sex-SNP density shows two regions of differentiation ( Fig. 3 ). The first is a broad region of differentiation from 45 to 58 Mb on LG3 that roughly corresponds to the region of differentiation in the LG3 ZW systems. The second region, from 76 to 78 Mb, includes a 100 kb block at 76.6 Mb that contains 1,096 sex-patterned SNPs. The highest density of XY-patterned SNPs is located within a 10 kb window at 76.63 Mb that contains 277 XY SNPs ( Table S5 ). Fig. 3. Open in a new tab Sex-patterned SNP density on LG3 in H. buettikoferi , T. brevimanus , and C. bakossiorum mapped on the P. mariae female genome. The GWAS plots for T. brevimanus also show an XY signal on LG3 ( Fig. S9 ). The analysis of the top 1% windows identifies 72 XY and 6 ZW windows on LG3 ( Table S4 ). The highest SNP density is a 10 kb window with 132 XY SNPs at 76.63 Mb on LG3 ( Table S5 ), the same location as the peak density of ZW-patterned SNPs in H. buettikoferi ( Fig. 3 ). The GWAS plots for C. bakossiorum also suggest an XY system on LG3 ( Fig. S10 ). This is supported by the top 1% windows analysis in which 70 of the top 78 windows are XY windows on LG3 ( Table S4 ). The remaining 8 windows are scattered on seven different chromosomes. The top 100 kb block contains 1,053 XY-patterned SNPs at 57.0 Mb on LG3, very near the ZW peak in T. sparrmanii and an XY peak in H. buettikoferi . However, the highest SNP density is a 10 kb block with 262 XY SNPs at 76.36 Mb on LG3, near the peak in XY peaks in H. buettikoferi and T. brevimanus ( Fig. 3 ). The gene annotations at 76.36 Mb include ubiquitin carboxyl-terminal hydrolase 17-like protein B (USP17L2) and E3 ubiquitin-protein ligase ARI5. Immediately upstream is a sequence homologous to Nedd8 , which is annotated in the O. aureus genome, but not in P. mariae ( Table S7 ). The annotations at 76.63 Mb include E3 ubiquitin-protein ligase RNF144A-A and a lncRNA. The presence of several genes in the ubiquitination and neddylation pathways is interesting, given the known roles of these processes in gonad development ( Racca et al. 2016 ; Kikuchi et al. 2020 ; Yu et al. 2020 ; Windley et al. 2022 ; Chahdi et al. 2025 ). LG1 XY Systems Four species, C. zilli , S. galilaeus , S. melanotheron , and O. niloticus , showed evidence of an XY system on LG1. The GWAS analysis for C. zilli on the P. mariae female assembly suggests an XY system on LG1 ( Fig. S11 ). All 78 of the top 1% windows are XY on LG1 ( Table S4 ). The sharp peak in SNP density falls in two adjacent 100 kb windows centered at 26.1 Mb ( Table S5 ). In order to determine the homology of the C. zilli peak with S. galilaeus and O. niloticus , we repeated the GWAS analysis on the O. niloticus UMDNMBU reference and found the peak at 25.256 Mb ( Fig. 4 ). Fig. 4. Open in a new tab XY-patterned SNP density on LG1 in C. zilli, S. galilaeus, S. melanotheron , and O. niloticus on the O. aureus ZZ male genome assembly. The GWAS analysis of S. galilaeus on the O. niloticus genome identified differentiation on both LG3 and LG1 ( Fig. S12 ). The peak of differentiation on LG1 is located in a 200 kb interval (25.2 to 25.4 Mb) centered on tspan15 ( Fig. 4 ). This indicates homology of the LG1 XY systems in C. zilli and S. galilaeus . There is some additional differentiation located nearby (25.7 to 26.0 Mb) which includes intersectin2b and cathepsin B, both potentially involved in the function of granulosa cells. The F ST plot for S. melanotheron also shows a strong signal on LG1 ( Fig. S13 ). Among the top 1% windows there are 43 XY and 15 ZW windows on LG1. There are also 7 XY windows on LG22 ( Table S4 ). The number of XY-patterned SNPs per window is lower than either C. zilli or S. galilaeus . The signal on LG1 is centered at 25 Mb but spread over a much broader region ( Fig. 4 ). We note that Curzon et al. (2022) suggest our S. melanotheron, collected from Lake Guiers, Senegal ( Gammerdinger et al. 2016 ), is actually S. galilaeus . Although these fish had S. galilaeus mtDNA, they had pigmentation and salt tolerance typical of S. melanotheron (H. D'Cotta, pers. comm.). Regardless of the specific identity, these fish represent an independent sample of Sarotherodon segregating a LG1 XY system. The O. niloticus data also show clear evidence of an XY system on LG1 ( Fig. S14 ) and all 78 of the top 1% windows of sex-patterned SNPs are on XY on LG1 ( Table S4 ). The region of differentiation encompasses the region 19.5 to 28.4 Mb and has sharp boundaries suggestive of an inversion ( Fig. 4 ). The differentiation is relatively even across this region, but encompasses the same region identified in C. zilli and S. galilaeus . There is no sex signal on LG23, which has been identified as an XY system in another strain of O. niloticus ( Li et al. 2015 ). A previous study suggested that variation in figla-like is responsible for sex determination in O. niloticus ( Curzon et al. 2022 ). Instead of using the O. niloticus reference assembly, this study relied on an assembly of the O. aureus genome ( Tao et al. 2021b ). They identified 66,372 variants in a 3.3 Mb region between the flanking microsatellite markers BYL012 and BYL018 (25.4 to 28.7Mb on the O. aureus assembly) and identified six consistent with an XY sex system in O. niloticus (Amherst), S. melanotheron and C. zilli . With our new data for the LG1XY system in S. galilaeus , and the new assembly of P. mariae , we can reevaluate these claims. Gene content and order are generally consistent across the three genome assemblies ( O. niloticus UMDNMBU, O. aureus and P. mariae ) with the exception of figla-like , which is found only in the O. aureus assembly ( Table S8 ). The sex linkage demonstrated by Curzon et al. (2022) can be taken as evidence that insertion of the figla-like gene between csmd1 and chs1 is not an assembly artifact. However, they provided scant evidence to exclude other candidate genes in the 1 Mb interval identified as sex-linked in previous studies. Their argument relies primarily on the identification of six sites concordant with an XY model that are shared among three samples. Of these, two are presence/absence markers of the figla-like insertion, three are intergenic sites, and the last is a synonymous site in depdc7a . Thus, the insertion of figla-like is the most significant variant shared by the three samples. The sharp peaks we identified in C. zilli and S. galilaeus are roughly 300 kb from the location of figla-like in the O. aureus assembly. Twelve genes are annotated within this 200 kb region ( Table S8 ). Two genes, tacr2 and tspan15 , stand out as potential candidates for sex determination. The tachykinin receptor 2 ( tacr2 ; aka nk2r ) is a seven transmembrane domain G-protein coupled receptor that preferentially binds neurokinin A (Substance K) ( Campo et al. 2022 ). Knockout of tacr2 in mice reduces LH expression ( Torres et al. 2021 ). Knockout of LH in zebrafish affects female reproductive function ( Chu et al. 2014 ) and steroidogenesis ( Xie et al. 2017 ). Tacr2 also modulates Wnt/β-catenin signaling in prostate cancer ( Jianfeng et al. 2021 ). If it has similar activity in fish, the changes in Wnt signaling could affect sex determination ( Zhou et al. 2016 , Harris et al. 2018 ). Tetraspanins are membrane proteins that organize microdomains on the cell surface ( Broadbent et al. 2024 ). Tetraspanin 15 ( tspan15 ) is a component of the ADAM10 scissor complex ( Koo et al. 2020 , Lipper et al. 2022 ) that regulates both Notch signaling and cadherins ( Tang et al. 2008 ). Cadherins are particularly important during gonad development ( Piprek et al. 2020 ). LG14 XY Systems We previously suggested an XY system on LG14 in O. mossambicus ( Gammerdinger et al. 2019 ) and this result was confirmed in samples of O. mossambicus from China ( Tao et al. 2021a ). In the current study, we found an apparently homologous LG14 XY system in O. shiranus from Malawi. We have three datasets for O. mossambicus . Two are full-sib families first reported by Gammerdinger et al. (2019) . The third dataset consists of 5 males and 5 females sampled from the Pearl River Fishery Research Institute ( Tao et al. 2021a ) which we pooled for our analyses. Each of these datasets shows large blocks of linkage disequilibrium due to inbreeding of the parental stocks ( Figs. S15 to S17 ). Family 1 has 40 XY windows on LG14. Family 2 has 22 XY and 19 ZW windows on LG3, and only 1 XY and 1 ZW on LG14. The individuals from the Pearl River Institute have 33 XY windows on LG12 and 17 ZW windows on LG3 ( Table S4 ). However, all three datasets show significant differentiation around 35 Mb on LG14 ( Fig. 5 ). Fig. 5. Open in a new tab Sex-patterned SNP density on LG14 in O. mossambicus and O. shiranus mapped on the O. niloticus UMDNMBU reference assembly. Oreochromis shiranus is closely related to O. mossambicus and the F ST plot shows a narrow peak of XY differentiation on LG14 ( Fig. S18 ). The peak on LG14 contains only one XY-patterned window but corresponds to the sex-linked region identified in O. mossambicus ( Fig. 5 ). Genes in the 200 kb region from 35.5 to 35.7 Mb in the O. niloticus UMDNMBU assembly include pak1 , aqp11 , and clns1a ( Table S9 ). The top 1% windows also include 27 ZW windows on LG18 as well as 10 XY and 18 ZW windows on LG3 ( Table S4 ). The signal on LG3 is typical of artifacts arising from the highly repetitive sequence on this chromosome. The signal on LG18 might be indicative of a polygenic system, but more likely represents an artifact of inbreeding in our sample. LG20 XY Systems The F ST plot for Danakilia sp. “shukoray” shows differentiation on LG20 ( Fig. S18 ). Among the top 1% windows there are 18 XY and 15 ZW windows on LG20 ( Table S4 ). The XY differentiation is strongest in the last 10 Mb of the chromosome ( Fig. 6 ; Table S5 ). Fig. 6. Open in a new tab Sex-patterned SNP density on LG20 in Danakilia sp. “shukoray” mapped on the O. niloticus UMDNMBU reference assembly. A QTL for an XY system on LG20 was previously reported in O. niloticus across the same interval using RAD markers mapped to the Oni1.1 genome assembly ( Palaiokostas et al. 2015 ). SNP marker Oni3161 showed the highest association in this interval and appears to be located in the emilin3a gene at 16.1 Mb in the O. niloticus UMDNMBU assembly, not the emilin3-like gene at 8.89 Mb as reported previously. The Orenil1.1 assembly is scrambled through this region relative to the more recent UMDNMBU assembly used for our analysis. The QTL region identified by Palaiokostas et al. (2015) corresponds to the last 20 Mb of LG20 in the UMDNMBU assembly ( Table S10 ). Conclusion Our results are placed in phylogenetic context in Fig. 7 . The most significant observation is the broad distribution of the LG1 and LG3 systems across the tree. Such results have often been ascribed to misidentification of samples or hybridization among the Oreochromini, either in aquaculture, or due to introductions into wild populations ( Curzon et al. 2021 , 2022 ). We do not doubt that such introgression has occurred, and it may be difficult to reconstruct its history. Hybridization and introgression have likely been occurring since ancient times, as these fishes have been cultivated for millennia ( Ciezarek et al. 2024 ). From a practical standpoint, what is most important is to identify the genes contributing to sexual development, so that breeding of optimal genotypes can be performed. Fig. 7. Open in a new tab Phylogenetic relationships of the species studied. Topology follows Astudillo-Clavijo et al. (2023) . Nevertheless, it is apparent that ZW systems on LG3 are found in clades that diverged at least 25 MY ago and it is unlikely this distribution is the result of introgression among such divergent lineages. If the sex determination systems in these clades are indeed homologous, it implies a long period in which these two systems were polymorphic in the ancestors of the modern species. Alternatively, the same loci may have been repeatedly recruited to act as Mendelian factors in sexual development. What might explain our failure to detect evolutionary strata in P. mariae ? First, the LG3 ZW system might not be 25MY old, but instead a gene or genes on LG3 might have independently taken up a ZW role in each lineage. Second, evolutionary strata may not have evolved even on old and/or highly diverged sex chromosomes. The peaks of ZW SNPs are relatively narrow, suggesting that recombination has not been significantly reduced around the sex locus in any of the three species. Detecting the signature of narrow evolutionary strata might not be possible when only ∼600 shared annotated genes are available. Finally, our ability to detect strata may have been compromised by any remaining errors in the genome assemblies. The LG3 XY system is also found in divergent lineages, including the Heterotilapiini, Gobiocichlini, and Coptodonini. The common ancestor of these clades diverged about 20MY ago ( Irisarri et al. 2018 ; Ronco et al. 2021 ), raising the same questions about ancestral polymorphisms versus convergent recruitment. Only a detailed investigation of the causative polymorphisms in each lineage will address these issues. The remaining systems are restricted to relatively recent lineages. So far, the LG23 XY system has only been found in some strains of O. niloticus , and the LG14 XY system is limited to Oreochromis lineages in southeastern Africa. Uncertainty in the age of these lineages makes it difficult to estimate the rate of sex chromosome turnover, but it is clearly lower than that estimated for the East African cichlid radiation ( El Taher et al. 2021 ; Behrens et al. 2024a ; Behrens et al. 2024c ). Whether this is related to the substrate spawning habits of many of these species, or differences in the level of sexually antagonistic selection, will require characterization of sex chromosomes in many more independent lineages. With the exception of the LG23 XY system that involves a duplication of the amh gene ( Li et al. 2015 ), none of the sex-linked chromosome segments we have characterized contain candidate genes typically associated with teleost sex determination ( Curzon et al. 2023 ). The absence of these “usual suspects” suggests there is a much larger set of genes can become Mendelian factors affecting sexual development. We note in particular the possible role of ubiquitination pathways in the LG3 XY system. We hope these examples will stimulate new models of regulatory interactions in the development of cichlid gonads. Materials and Methods Whole Genome Sequencing of P. mariae One male and one female P. mariae from a lab-maintained population were used for genome sequencing. Animal experiments were conducted in accordance with the Guide for Care and Use of Laboratory Animals. All animal use was approved under IACUC protocol R-OCT-19-48 (U. Maryland). DNA was extracted from heart tissue using the Nanobind Tissue and Short Read Elimination kits (Pacific Biosciences, Menlo Park, CA, USA). DNA concentrations were quantified by fluorescence spectroscopy using a Quant-iT PicoGreen assay (ThermoFisher, Waltham, MA, USA). Sequencing libraries were constructed and extra-long HiFi PacBio sequencing was conducted on the PacBio Revio machine by Maryland Genomics (University of Maryland School of Medicine, Baltimore, MD, USA). Genome Assembly Prior to assembly, HiFiAdapterFilt ( Sim et al. 2022 ) was used to inspect sequencing reads for internal PacBio HiFi adapters. The assemblies were conducted using hifiasm ( Cheng et al. 2021 ; Wang 2022 ) with the default purge_dups setting on. Bandage ( Wick et al. 2015 ) was then used to visualize connections among primary contigs and make supported merges. Contigs for each sex were then aligned against the O. aureus (ZZ) assembly (GCF_013358895.1) ( Tao et al. 2021b ) using D-Genies ( Cabanettes and Klopp 2018 ) to determine placement of contigs. This initial alignment was used to inform preliminary manual merges between contigs, where 50 “N” were used between two contigs to indicate a gap. When necessary, contigs were reverse-complemented with either seqtk ( https://github.com/lh3/seqtk ) or AliView ( Larsson 2014 ), depending on contig size. After this initial assignment of contigs, the male and female assembly were aligned against each other and visualized in D-Genies. Telomere Identification Toolkit (Tidk, v0.2.31) ( https://github.com/tolkit/telomeric-identifier ) was used to detect repeats with a pattern consistent with a telomeric identity. Once the repeat was detected (AACCCT and its reverse complement), this was used to guide correct orientation of contigs when scaffolding. To assemble the W in the female, all female haplotigs generated by hifiasm were aligned against the preliminary assemblies for each sex. Haplotigs belonging to LG3 that aligned well against the female LG3 and poorly against the male LG3 were selected, as these presumably represent the W chromosome. These haplotigs were then merged to form the W. Based on karyotypic data ( Poletto et al. 2010 ), P. mariae should have two fewer chromosomes relative to the reference (2 n = 40), O. aureus (2 n = 44). Both the male and female assemblies featured contigs that spanned two chromosomes, indicating a fusion, and the validity of these fusions produced by hifiasm were validated by aligning reads and contigs of both sexes against each of the assemblies using minimap2 with the flags -x asm20 -a ( Li 2018 ). Alignments were visually inspected in IGV ( Thorvaldsdóttir et al. 2013 ) to determine if the contig produced by hifiasm was supported by the reads. For a fusion of LG10 to LG11, and of LG16 to LG19, the fusion was supported in both sexes. In the male, the contig belonging to LG12 was incorrectly fused to the LG11 contig and had to be manually broken. Contamination of the sequence data by Lunularia cruciata , a species of liverwort, was detected in the male unplaced contigs. To remove this contamination, the unplaced contigs were aligned against the L. cruciata genome using minimap2 and -x asm20 setting ( Li 2018 ), and then reads that were unmapped against the genome were selected using samtools view -f 4 ( Danecek et al. 2021 ). These unplaced contigs were then considered to be contamination free. Assembly Quality Assessment Genome assembly quality was assessed using the Genome Evaluation Pipeline (GEP) ( https://git.imp.fu-berlin.de/cmazzoni/GEP ), which contains the following programs: GenomeScope2 ( https://github.com/tbenavi1/genomescope2.0 ) ( Vurture 2017 ), meryl and merqury ( Rhie et al. 2020 ), BUSCOv5 ( Manni et al. 2021 ), and a modified version of assembly_stats (v0.1.4) ( http://doi.org/10.5281/zenodo.3968775 .). To investigate why BUSCO scores were lower in the P. mariae male genome, the scores were re-calculated with the more sensitive program compleasm (v.0.2.7) ( Huang and Li 2023 ). The O. aureus ( Tao et al. 2021b ) assembly was used as a guide for expected chromosome size. Coverage was calculated using samtools depth ( Danecek et al. 2021 ). Synteny plots were generated by aligning sequences with minimap2 and visualized with the R package SVbyEye ( Porubsky et al. 2024 ). Annotation Annotation of the female P. mariae genome using their Eukaryotic Genome Annotation Pipeline ( https://www.ncbi.nlm.nih.gov/books/NBK169439/ ). The annotations are available under accession number GCF_036321145.2-RS_2024_05. This annotation was lifted over to the male P. mariae genome using liftoff ( Shumate and Salzberg 2021 ). Finally, dS and dN divergence between the P. mariae male Z and the P. mariae female W was calculated using the R package orthologr ( https://github.com/drostlab/orthologr ) dN_dS command with the settings delete_corrupt_cds = TRUE, ortho_detection = “RBH”, aa_aln_type = “pairwise”, aa_aln_tool = “NW”, codon_aln_tool = “pal2nal”, dnds_est.method = “Comeron”. Pooled sequencing Several sampling designs have been used to construct pooled sequence comparisons of males and females. The collection of males and females from the same population directly from the wild provides the best signal to noise ratio, as the genetic background is randomized by multiple generations of recombination. Collection of males and females from a single lab-reared family can also randomize the background noise, but spatial resolution of the sex-linked region will be reduced. However, lab-reared families from inbred lines or commercial stocks may produce artifacts from large blocks of linkage disequilibrium. In any case, samples of at least 20 individuals of each sex are usually required to maximize the signal to noise ratio. We sequenced seven new species for this study: T. sparrmanii (20 males and 26 females, full-sibs reared at the University of Graz); H. buettikoferi (23M, 29F, full-sibs, U. Graz); T. brevimanus (22M, 24F, full-sibs—U. Graz); C. bakossiorum (20M, 22F, full sibs—Texas A&M International University); S. galilaeus (40M, 41F, wild-caught from the Sea of Galilee); O. shiranus (30M, 30F, sampled from the National Aquaculture Centre, Domasi, Malawi); D. sp. “shukoray” (19M, 31F, full-sibs—U. Graz). The parents of the fish reared at U. Graz were obtained from the aquarium trade (parents likely F2 or more). Animal use at U. Graz was approved under animal care protocol BMWFW-66.007/004-WF/V/3b/2016 and carried out with the approval of the U. Graz ethics committee (permit number GZ: 39/11563 ex 2022/23). The accession numbers for these samples, and the sequences reanalyzed from previous publications, are listed in Table S3 . Read mapping The pool-seq reads from each species were aligned against one of several reference genome assemblies. The Heterochromini, Gobiocichlini, and Coptonini were first aligned against the female assembly of P. mariae (GCF_036321145.2), while the Oreochromini were first aligned against the O. niloticus UMDNMBU assembly (GCF_001858045.2). Additional alignments, including to the O. aureus ZZ male assembly (GCF_013358895.1), were made to compare particular sex systems in a common genome coordinate space. The sequence reads were aligned with BWA version 0.7.12 using the default parameters along with read group labels ( Li and Durbin 2009 ). The alignments were sorted, marked for duplicates, and indexed using Picard version 1.119 ( http://broadinstitute.github.io/picard/ ). Alignments were then converted into an mpileup file using Samtools version 0.1.18 ( Li et al. 2009 ) and subsequently into a sync file using Popoolation2 ( Kofler et al. 2011 ). Base calls with a PHRED score less than 20 were filtered out of the data set. We then used Sex_SNP_finder_GA.pl ( https://github.com/Gammerdinger/sex-SNP-finder ) to calculate FST between the male and female pools to identify both XY and ZW patterned SNPs ( Gammerdinger et al. 2016 ). The results were plotted using R ( R Core Team 2019 ). The Bedtools ( Quinlan and Hall 2010 ) make windows and coverage commands were used to calculate the density of sex-patterned SNPs per 100 kb window. The top 1% windows with the highest sex-patterned SNP density were examined as previously described ( Kocher et al. 2022 ). The log2(XY:ZW) ratio of SNP density was then calculated for each window ( Darolti et al. 2022 ). The Kruskal–Wallis test on the ranked data was conducted in R to determine if the log ratio differed among chromosomes with Benjamini–Hochberg correction for multiple tests. If the differences were statistically significant, the Dunn's test was conducted post-hoc to determine which chromosomes significantly differed from each other using the rstatix R package ( Kassambara 2023 ). Analysis of Candidate Genes The previously proposed candidate sex-determining genes, banf2 and paics-1 from the O. niloticus annotation (Release 104) ( Conte et al. 2017 ) were aligned against the P. mariae assemblies first using blastn ( Camacho et al. 2009 ) and then using minimap2 with the flags -x asm20 -a ( Li, 2018 ). These were then visualized in IGV ( Thorvaldsdóttir et al. 2013 ). Supplementary Material evaf191_Supplementary_Data evaf191_supplementary_data.zip (111.8MB, zip) Contributor Information Kristen A Behrens, Department of Biology, University of Maryland, College Park, MD 20742, USA. Zexuan Zhao, Department of Biology, University of Maryland, College Park, MD 20742, USA. Michael R Kidd, Department of Biology and Chemistry, Texas A&M International University, Laredo, TX 78041, USA. Alfred Maluwa, Malawi University of Science and Technology, Limbe, Malawi. Avner Cnaani, Department of Poultry and Aquaculture, Institute of Animal Sciences, Agricultural Research Organization, Volcani Institute, Rishon LeZion, Israel. Stephan Koblmüller, Institute of Biology, University of Graz, Graz 8010, Austria. Thomas D Kocher, Department of Biology, University of Maryland, College Park, MD 20742, USA. Supplementary Material Supplementary material is available at Genome Biology and Evolution online. Funding This work was supported by the U.S. National Science Foundation [grant number DEB-1830753 to T.D.K.]. Data Availability The data underlying this article are available in the GenBank Nucleotide Database at https://www.ncbi.nlm.nih.gov . 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[ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Associated Data This section collects any data citations, data availability statements, or supplementary materials included in this article. Supplementary Materials evaf191_Supplementary_Data evaf191_supplementary_data.zip (111.8MB, zip) Data Availability Statement The data underlying this article are available in the GenBank Nucleotide Database at https://www.ncbi.nlm.nih.gov . The Pelmatolapia genome assemblies can be accessed under BioProject PRJNA1031194 and the new pooled sequencing reads can be accessed under BioProject PRJNA802233. 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