Genomic basis of Y‐linked dwarfism in cichlids pursuing alternative reproductive tactics

Sexually antagonistic selection, which favours different optima in males and females, is predicted to play an important role in the evolution of sex chromosomes. Body size is a sexually antagonistic trait in the shell‐brooding cichlid fish Lamprologous callipterus, as “bourgeois” males must be large enough to carry empty snail shells to build nests whereas females must be small enough to fit into shells for breeding. In this species, there is also a second male morph: smaller “dwarf” males employ an alternative reproductive strategy by wriggling past spawning females into shells to fertilize eggs. L. callipterus male morphology is passed strictly from father to son, suggesting Y‐linkage. However, sex chromosomes had not been previously identified in this species, and the genomic basis of size dimorphism was unknown. Here we used whole‐genome sequencing to identify a 2.4‐Mb sex‐linked region on scaffold_23 with reduced coverage and single nucleotide polymorphism density in both male morphs compared to females. Within this sex region, distinct Y‐haplotypes delineate the two male morphs, and candidate genes for body size (GHRHR, a known dwarfism gene) and sex determination (ADCYAP1R1) are in high linkage disequilibrium. Because differences in body size between females and males are under strong selection in L. callipterus, we hypothesize that sexual antagonism over body size initiated early events in sex chromosome evolution, followed by Y divergence to give rise to bourgeois and dwarf male reproductive strategies. Our results are consistent with the hypothesis that sexually antagonistic traits should be linked to young sex chromosomes.

sexes is believed to be the acquisition of a sex-determining gene on the Y or W chromosome (Charlesworth & Charlesworth, 2000;Brian Charlesworth, 1996). When this is followed by recombination suppression between the new sex chromosome pair, possibly via an inversion, degeneration of the Y or W chromosome occurs, leading to heteromorphic sex chromosome pairs (Bachtrog, 2013;Charlesworth et al., 2005).
Several models to explain the repeated loss of recombination on sex chromosomes have been proposed (reviewed by Ponnikas et al., 2018). The prevailing model stipulates that sexually antagonistic selection can drive recombination suppression because it favours linkage between the sex-determining gene and a locus with sexually antagonistic effects (i.e., beneficial in one sex and detrimental in the other; Charlesworth & Charlesworth, 1980;Rice, 1987). However, the broad applicability of the sexual antagonism hypothesis of sex chromosome evolution is still debated as it has been challenging to demonstrate empirically (Ironside, 2010;Ponnikas et al., 2018). One challenge is that in old and/or heteromorphic sex chromosome systems, it is difficult to disentangle whether recombination suppression was initiated due to the presence of sexually antagonistic loci or whether sexually antagonistic loci accumulated after recombination was suppressed. Younger and/or less differentiated (i.e., homomorphic) sex chromosomes are more promising systems to test whether sexually antagonistic selection has played a role in the evolution of sex chromosomes (Bachtrog, 2013;Ponnikas et al., 2018). Indeed, sexually antagonistic traits are linked to young sex chromosomes in guppies (Almeida et al., 2020;Sandkam et al., 2021;Wright et al., 2017), stickleback (Kitano et al., 2009) and cichlids (Roberts et al., 2009). These studies highlight the value of studying traits that are known to be sexually antagonistic and then determining whether they are associated with young sex chromosome systems.
Adaptive radiations of cichlid fish harbour hundreds of recently diverged species that have young and homomorphic sex chromosomes (Ozouf-Costaz et al., 2017;Poletto et al., 2010), with a high turnover of sex determination mechanisms even among closely related species (El Taher et al., 2021;Gammerdinger & Kocher, 2018).
We hypothesize that sexually antagonistic selection may play an important role in sex chromosome evolution in cichlid fishes, as many sexually dimorphic traits related to body colour, body size and behaviour are found in this lineage (Lande et al., 2001;Taborsky, 2001).
Indeed, in several sexually dimorphic cichlid species from Lake Malawi, the sexually antagonistic "orange blotch" body colour patterning is associated with the evolution of a novel sex determination locus (Roberts et al., 2009). Furthermore, as cichlid sex chromosomes are young and homomorphic, studies will not be confounded by the effects of recombination suppression that happened a long time in the past. Thus, cichlids are an excellent system to investigate the sexual antagonism theory of sex chromosome evolution.
Here, we focused on the cichlid Lamprologous callipterus from Lake Tanganyika in East Africa (Figure 1a) that exhibits the most extreme sexual size dimorphism so far known among animals in which males exceed females in size. Nest-building "bourgeois" males have a 12-fold difference in body mass compared to females (Schütz & Taborsky, 2000;see Taborsky, 1998 for terminology). These males must exceed a minimum size threshold in order to carry empty gastropod shells to build a nest, and females must be small enough to fit into these shells for spawning and brood care (Schütz et al., 2006;Schütz & Taborsky, 2005). Consequently, body size is a sexually antagonistic trait in this species due to differential fitness effects of body size for shell-brooders. In addition to the bourgeois males, socalled "dwarf males" that average only 2.4% of the mass of bourgeois males also exist in this species (Figure 1a). These males compete with bourgeois nest owners for egg fertilization by applying an alternative reproductive strategy that involves wriggling past spawning females into the shell to fertilize eggs from this advantageous position ( Figure 1a; Wirtz-Ocana et al., 2014). Thus, dwarf males need to be even smaller than females (Sato et al., 2004). Bourgeois males acquire more mates than dwarf males, but dwarf males are more successful during direct sperm competition in laboratory trials and in the field (Schütz et al., 2010;Taborsky et al., 2018;Wirtz-Ocana et al., 2014). Dwarf males are much rarer than bourgeois males in natural populations Wirtz-Ocana et al., 2014). Negative frequency-dependent selection is thought to be responsible for the relative numbers of dwarf and bourgeois males in nature, as these tactics are fixed for life and hence should yield similar fitness returns (Brockmann & Taborsky, 2008;Parker, 1984;Taborsky & Brockmann, 2010).
Experimental pedigrees have shown that reproductive tactic and body size in L. callipterus males adhered to the expectation of a Y-linked Mendelian trait controlled by a single locus (Wirtz-Ocana et al., 2014). Bourgeois males only sire bourgeois sons and dwarf males only sire dwarf sons, while daughters do not differ in any respect between these two types of males (Wirtz-Ocana et al., 2014).
These results suggest that Y-linkage has resolved sexual antagonism over body size between males and females. However, neither the sex chromosomes nor the genes controlling male body size have yet been identified in this species. It is also unclear which of the two male types is the ancestral state.
In this study, we investigate the genomic basis of male body size in L. callipterus using whole-genome resequencing data from dwarf and bourgeois males, and their daughters (Table 1). Dwarf males initially grow at a faster rate than bourgeois males, but growth halts early in ontogeny (Wirtz-Ocana et al., 2013), and bourgeois males continue to grow indefinitely (Taborsky, 2001;Figure 1b). Thus, we hypothesized that the dwarf-determining gene may encode a growth factor.
We tested whether such a factor was linked to the sex-determining region, as the sexual antagonism hypothesis outlined above would predict. To investigate this hypothesis, we first needed to characterize the sex-determining region. The karyotype of L. callipterus has not been studied, but karyotypes of other Lamprologini and related cichlids show no sex chromosome differentiation (Ozouf-Costaz et al., 2017). However, genomic data provide higher resolution into the subtle molecular differences between sexes in species with homomorphic sex chromosomes (Fontaine et al., 2017;Gammerdinger & Kocher, 2018). We compared whole genomes of males and females to identify regions of differentiation between the sexes and the morphs. In addition to L. callipterus samples, we included males and females of six related species from the Lamprologini tribe (see Table 1) for a comparative phylogenomic perspective on sex determination and body size evolution.

| Sampling and sequencing
Sampling was conducted at the breeding facility at the Ethologische Station Hasli, University of Bern, Switzerland. Genomic DNA was extracted from fin clips of males and females of five species and males only of two species for which females were not available (Table 1). The stock population of the target species Lamprologous callipterus originated from the southern end of Lake Tanganyika near Mpulungu. Two L. callipterus dwarf males and two bourgeois males, as well as one daughter per male were sampled (see Data S1 for genetic and biological relatedness) because it is easier to identify sex chromosomes in related individuals (Palmer et al., 2019).
The TrueSeq DNA Library Preparation Kit was used for library preparation, and samples were sequenced at the Vienna BioCentre Sequencing Facility in Austria. The libraries were sequenced on eight lanes of an Illumina HiSeq 2500 machine generating ~125 million paired-end reads (120 bp in length) per sample.

| Reference-based assembly and characterization of sex-determining region coverage
Raw Illumina reads were trimmed using fastx-trimmer (http://hanno nlab.cshl.edu/fastx_toolkit v0.13). Reads were mapped to the F I G U R E 1 The Lamprologous callipterus cichlid system from Lake Tanganyika and genomic patterns across male and female individuals of this species. (a) The shell-brooding cichlid species L. callipterus from Lake Tanganyika in East Africa has large "bourgeois" males that collect empty snail shells and build nests, small females that enter the snail shells to lay eggs, and even smaller dwarf males that sneak past brooding females to fertilize laid eggs in close contact. Normalized genome-wide coverage differentiation between (c) bourgeois males (n = 2) vs. females (n = 4) and (e) dwarf males (n = 2) vs. females (n = 4). Genomic coverage is calculated in 1.0 Mb windows with a step-size of 0.5 Mb. Dots represent normalized log 2 (average male coverage/average female coverage). Solid red/blue lines represent the moving average. Grey dashed horizontal lines represent the 1st and 99th genome-wide percentiles. Black dashed horizontal lines represent the genome-wide mean. Coloured vertical shades demarcate the proposed sex-linked region of L. callipterus on scaffold_23. Data from (c) and (e) are presented as density plots in (d) and (f) Neolamprologous brichardi genome (Brawand et al., 2014), which was chosen because it is from the most closely related cichlid species to our focal species (Darolti & Mank, 2022). However, to verify our results, we also mapped our data to the high-quality genome of Oreochromis niloticus (Conte et al., 2017). See Section 3.1 for details.
While N. brichardi is the most closely related reference genome, it is not the best assembled reference cichlid genome and there are trade-offs that must be considered when picking the best reference genome for sex chromosome mapping. As we are attempting to identify a young and homomorphic sex chromosome, we chose to use a more closely related reference. Read mapping was conducted using bowtie2 version 2.2.7 (Langmead et al., 2009) in the "very-sensitive" mode with no mixed alignments and retaining only concordantly mapped reads. We obtained an average coverage of ~25× across the genome (Appendix S1). Coverage of windows across the genome was calculated using bedtools version 2.17 (Quinlan & Hall, 2010). The genome-wide coverage analysis was performed using 1-Mb moving windows with a 0.5-Mb step-size for all scaffolds with more than three windows. represented the two peaks identified in the coverage density plots shown in Figure 1(d,f): (i) the region from 1 bp to 2.4 Mb and (ii) the region from 2.4 to 6.8 Mb. The permutation test was also repeated after dividing scaffold_36 into two regions that represented the two peaks identified in the coverage density plots shown in Figure 1d: (1) the region from 2.0 to 3.5 Mb and (ii) the region from 1.0 to 2.0 Mb and 3.5 to 4.9 Mb. Altolamprologus sp. "Sumbu Shell" (obligate shell-brooder) 1 male 5.5

| Phylogenomics
We applied a gene tree vs. species tree approach to investigate the evolution of sex chromosomes in the lamprologini clade. A tailored FASTA genome file was built for each individual using angsd version 0.930 -dofasta function (Korneliussen et al., 2014), which assembles FASTA-format genome sequences by picking the most common base per position in the mapped reads for each individual. Gene sequences for every second gene in the reference genome were extracted using bedtools version 2.17 (Quinlan & Hall, 2010) and aligned using mafft version 7.273 (Yamada et al., 2016). Using every second gene provided a genome-wide overview with reduced computation time.
A genome phylogeny was estimated using a coalescent approach using astral-ii (Mirarab & Warnow, 2015) with the 11,855 gene trees and N. brichardi as the outgroup. Alignments of genes in the most diverged sex region of scaffold_23 from 1.18 to 1.28 Mb were built as outlined above but with 1000 bootstrap replicates.

| Signatures of selection
As body size is an important trait across the whole Lamprologini clade, we tested for signatures of selection on the candidate dwarfism gene GHRHR. Exon sequences of the gene were extracted from the reconstructed fasta genomes generated for the phylogenies using bedtools version 2.17 (Quinlan & Hall, 2010). The exons were concatenated, and aligned using mafft version 7.273 (Yamada et al., 2016). End stop codons were removed, and pairwise dN/dS was calculated using random models in paml (Yang, 2007).

| Identification of sex-linked scaffolds in Lamprologous callipterus
To identify the sex-determining region in Lamprologous callipterus, we analysed the genomic coverage of mapped reads from eight individuals (two bourgeois males, two dwarf males and four females; genome (Brawand et al., 2014). Approximately 80% of the ~120 million paired-end reads per sample mapped to the reference genome with average genome-wide coverage of 20-30× across samples (Table S1).
We compared normalized genomic coverage between males and females to identify regions of reduced coverage in males, as diverged Y reads will no longer map to the X chromosome. Coverage analysis using a 1.0-Mb moving window and 0.5-Mb step-size revealed a small region on scaffold_23 with significantly reduced coverage outlier windows (below the bottom 1% of the genome-wide average), compared to the rest of the genome, in the two bourgeois males vs.
the four females (Figure 1c), as well as in the two dwarf males vs. the four females (Figure 1e) Combining genomic coverage and SNP density can be useful for characterizing sex-linked regions (Palmer et al., 2019;Wright et al., 2017). In regions where there is some differentiation (i.e., sequence divergence) between the X and the Y but not yet degeneration to the point where sequence reads from the Y chromosome no longer map to the reference genome, there will be more SNPs in males than in females. However, if the Y has degenerated, SNP density will be lower in males. We  Mbp, see Figure S2) we discovered that the N. brichardi sex region on scaffold_23 may represent a subset of a putatively larger sex region on LG18 (Figures S3 and S4). However, the genome-wide coverage results were noisier with many coverage outliers ( Figure S5). This is not surprising given the large divergence times (~24 million years ago, Irisarri et al., 2018) between O. niloticus and L. callipterus, but also because the sex region in L. callipterus is young and not strongly differentiated. O. niloticus LG18:19.3-26.1 Mb was a significant outlier in the dwarf males:females comparison ( Figure S5).

| Refining patterns of sex linkage and candidate genes on scaffold_36
To further examine the pattern of divergence across scaffold_36 in L. callipterus bourgeois males vs. females, we calculated SNP density and F ST using 30-kb windows ( Figure S6). There was a decrease in M:F SNP density beyond the 5th percentile of genome-wide SNP density at 2.0-2.4 Mb and 3.6-3.9 Mb ( Figure S6A). There was, however, no clear increase in F ST beyond the genome-wide 95th percentile across scaffold_36 ( Figure S6B). Of the 28 genes annotated in the region of reduced coverage on scaffold_36, none had a function that could putatively be associated with a phenotype that delineates the bourgeois male reproductive tactic (Data S2). However, one gene in this region, endothelin-2 (EDN2) is associated with ovary F I G U R E 2 Distribution of M:F coverage and SNP density for all scaffolds in Lamprologous callipterus. Comparisons between (a) bourgeois males and females and (b) dwarf males and females. Scaffold_23 and scaffold_36 are divided based on the bimodal peaks in Figure 1(d,f): windows between scaffold_23: 1 bp to 2.4 Mb are pink, windows between scaffold_23: 2.4-6.8 Mb are black, windows between scaffold_36: 2-3.5 Mb are green and windows between scaffold_36: 1 bp to 2 Mb and 3.5-4.9 Mb are blue. Windows from all other scaffolds are grey. Circles denote the mean and vertical and horizontal lines depict the standard deviation. Dotted lines denote coverage and SNP density means across all scaffolds development in mice (Ko et al., 2006). Because this region only had reduced coverage in bourgeois males vs. females and did not have strong evidence of genes with sex-associated phenotypes, the region of reduced coverage on scaffold_23 was the best candidate for the L. callipterus sex region, and we therefore concentrated our further analyses on this scaffold.

| Refining patterns of sex linkage on scaffold_23
To further examine the spatial pattern of divergence across scaf-  Figure S7B). This suggests that the sex region on scaf-fold_23 is distinct between the dwarf and bourgeois males.
When SNP density was analysed together for all windows within 2.3 Mb that was higher between bourgeois males and females and exceeded the genome-wide 95th percentile. We also compared F ST between bourgeois males and dwarf males on scaffold_23 to identify intrasexual differentiation in the sex-determining region ( Figure S8). The most significant F ST between the two male-types was in windows spanning 1.08-1.11 Mb with F ST = 0.49 (Grubbs's outlier test p < 6.3 e −11 ), which also exceeded the 95th percentile of genome-wide F ST . There were also 20 other F ST outliers (>genomewide 95th percentile) spread across scaffold_23.

| Recombination suppression on scaffold_23
The sexual antagonism model of sex chromosome evolution posits that recombination suppression between the sex-determining loci and sexually antagonistic loci ensures that they cosegregate (Charlesworth & Charlesworth, 1980;Rice, 1987). To determine whether there is recombination suppression on scaffold_23, we did not find evidence of inversions on scaffold_23 using two different bioinformatics approaches (see Section 2: Methods). Our negative results could just reflect the limitation of the short-read data for identifying inversions. However, the LD pattern of inversions is expected to be more block-like and not "V" shaped as we observe in our data.

| Identification of candidate sex determination and body size genes on scaffold_23
By investigating the function of genes in the sex-linked regions of scaffold_23 using www.genec ards.org and a thorough literature review, we identified several genes that have known functions associated with sexual development, growth and alternative reproductive behaviours (  (Vaudry et al., 2009) and are involved in oogenesis (Apa et al., 1997) and spermatogenesis in rats (Romanelli et al., 1997). little phenotype in laboratory mice (Baumann & Maheshwari, 1997;Godfrey et al., 1993). Thus, GHRHR is a strong candidate for body size determination in L. callipterus. Both ADCYAP1R1 and GHRHR are in high LD with the genes at the centre of LD-Block 5 (Figure 4), and they also known to interact directly in protein-protein interaction networks in humans ( Figure S10).
There were 15 genes in LD-Block 5, with two genes (THEM6 precursor and THEM6 [2 of 2]) at the centre of perfect LD at ~1.1 Mb F I G U R E 4 Linkage disequilibrium (LD) measured using r 2 on scaffold_23. Heatmap of LD on part of scaffold_23 (the entire scaffold is shown in Figure S9). Identified LD blocks B1-B5 are indicated; B5 is the largest LD block, spanning 1-1.4 Mb. (b) Genes from 1.05 to 1.26 Mb in LD block B5 are shown. The location of SNPs used to calculate r 2 is denoted by grey lines; location of SNPs in candidate genes for sex determination and body size is denoted by lines coloured by the gene that they are found in (Table 2; Data S2). Just upstream of THEM6 (2 of 2) lies THEM6 (1 of 2). All three of these genes are members of the thioesterase superfamily, which has been associated with regulating energy expenditure in animals (Zhang et al., 2012). A detailed discussion of other interesting genes in the LD-block can be found in the Supporting Results and Discussion (Appendix S2).

| Turnover of sex chromosomes in the Lamprologini
Lamprologous callipterus belongs to a large, substrate-breeding clade of cichlids in Lake Tanganyika constituting the tribe Lamprologini, where shell-brooding and small body size, particularly in females, have co-evolved multiple, independent times (Koblmüller et al., 2007;Sato & Gashagaza, 1997;Schütz et al., 2006). To resolve the evolutionary history of the sex-linked region, we compared the phylogeny of the scaffold_23 sex-linked region to the whole-genome phylogeny for L. callipterus and six other Lamprologini species sequenced in this study (Table 1). The whole-genome phylogeny was constructed using 11,855 genes across the genome, and it confirmed the strongly supported (bootstrap values = 100) monophyly of all included species ( Figure 5). The tree suggests that obligate shellbrooding evolved three times in these species, or that it evolved in the common ancestor of these species after it split from N. brichardi and was subsequently lost three times.
In the phylogeny of the candidate body size gene, GHRHR ( Figure 5, inset), the species monophyly is maintained but the position of L. lemairii (facultative shell-brooder) and L. callipterus is switched compared to the whole-genome phylogeny. This results in a cluster of all obligate-shell brooders, except for N. brevis. The Altolamprologus calvus "Congo," which is not a shell-brooder, is also in this cluster. The switch in topology between the whole-genome phylogeny and the sex-region phylogeny could reflect either the coalescent history of this region or introgression. In fact, F 1 hybrids between L. callipterus and other snail shell dwellers have been observed in nature (Koblmüller et al., 2007).
To determine whether the sex determination system in L. callipterus is shared with other Lamprologini, we placed the L. callipterus sex-linked region in a phylogenomic context by analysing the coverage of scaffold_23 in males and females of four related species ( Figure S11). The facultative shell-brooders N. multifasciatus and L. lemarii did not have outlier coverage windows (below the genomewide 5th percentile) on scaffold_23. The obligate shell-brooders L. ocellatus had reduced coverage (below the genome-wide 5th percentile) on scaffold_23 from 1.29 to 1.34 Mb ( Figure S11) (2012) Note: For a full list of genes in the sex-linked region see Data S2.
scaffold_23 in the shell-brooding Lamprologini tribe, which needs to be further investigated in a follow-up study.

| No evidence for rapid evolution of the GHRHR gene across the Lamprologini
We screened the GHRHR gene for signatures of selection and found evidence for purifying selection (dN/dS <1) in all the ingroup species (L. callipterus, A. calvus "Congo," A. sp. "Sumbu shell," L. ocellatus) compared to the L. lemairii outgroup (Table S2). All ingroup species were obligate shell-brooders, with the exception of A. sp. "Calvus Congo," a sister species of A. sp. "Sumbu shell." The strongest purifying selection was in the L. callipterus lineage (dN/dS = 0.069). This was significantly lower than the other species (p = 4.0 e −8 , 1.19 e −10 and 7.0 e −6 Welch's t tests; see Table S2), which had dN/dS values >0.12. We found no malespecific nonsynonymous mutations in the GHRHR gene, but we did identify two synonymous mutations ( Figure S12). It may be that gene regulation and not sequence variation in GHRHR is controlling body size determination in L. callipterus. In the future it would be valuable to study the gene expression networks and alternative splicing patterns of candidate sex and body size genes of L. callipterus (Singh & Ahi, 2022;Tian et al., 2019).

| DISCUSS ION
The shell-brooding cichlid species Lamprologous callipterus is a unique system to test the sexual antagonism hypothesis of sex chromosome evolution. The presence of bourgeois and dwarf male morphs that are strictly paternally inherited also offers the opportunity to study origins of Y haplotype diversity. Here we present first insights into the genomic basis of sex-linked body size determination in L. callipterus, which is under sexually antagonistic selection and the foundation of two very different alternative male reproductive tactics. We identify a small sex region in L. callipterus, with candidate genes for sex determination and sexual antagonism ( Figure 6) and report the presence of two distinct Y haplotypes. We hypothesize that the L. callipterus sex chromosome may have evolved to resolve sexual antagonism over body size in males and females, followed by the evolution of Y-haplotype diversity that gave rise to two male reproductive tactics.

| Lamprologous callipterus has a young sex chromosome
Using multiple lines of evidence (coverage, SNP density, LD, F ST ) we found scaffold_23 to be the most promising sex-linked region of L.  (Darolti et al., 2019). In addition, we found elevated F ST between dwarf males and females from 1 bp to 2.4 Mb. Higher genetic differentiation is predicted around sexually antagonistic loci that are linked to sex-determining loci (Kasimatis et al., 2017;Kirkpatrick & Guerrero, 2014), but high F ST can arise as a result of several other factors (Bissegger et al., 2020;Rowe et al., 2018). Overall, the differentiation in the sex-linked region of L. callipterus was not as extreme as that found in older therian sex chromosomes (Vicoso, 2019), probably because it is a comparatively younger sex chromosome that evolved <3 million years ago (Irisarri et al., 2018). In the context of East African cichlid radiation, however, the L. callipterus sex region would be considered old and thus shows more differentiation than that observed in other studied cichlid sex chromosomes (Gammerdinger & Kocher, 2018). Interestingly, scaf-fold_23 of L. callipterus maps to linkage group 18, which is one of the candidate sex chromosomes of the riverine cichlid Astatotilapia burtoni (Böhne et al., 2016;Roberts et al., 2016). Since scaffold_23 is a smaller scaffold that has been anchored to a larger linkage group in the high-quality cichlid reference genome of Oreochromis niloticus, by remapping our data to O. niloticus we found that scaffold_23 may be a subset of a putatively larger sex region. In future studies it would be important to generate long-read data for L. callipterus to enhance the resolution of its sex region.
As no orthologues of known sex-determining genes such as SRY or Dmrt1 (Doris Bachtrog, 2013) were found in the L. callipterus sexlinked region, we determined ADCYAP1R1 as the best candidate for the master sex-determining gene due to its role in gametogenesis (Apa et al., 1997;Romanelli et al., 1997;Vaudry et al., 2009). The location of ADCYAP1R1 in the most degenerated region of scaf-fold_23 (1.18-1.28 Mb) is consistent with predictions that a novel sex-determining gene can initiate sex-chromosome evolution and differentiation. This would point to a dose-dependent mechanism, whereby fish with two copies become female and fish with one copy become male (unless there are actually more copies of this gene).
This is very different from most sex-determination loci identified so far in fish, which are either genes that have divergent alleles on the X and Y chromosomes or Y-specific gene duplications (Pan et al., 2021).
However, Y-specific gene duplications cannot be identified by our analytical approach, which mapped reads to the reference genome.
The ADCYAP1R1 gene is a receptor of the ADCYAP gene, which has sex-specific expression associated with sexual behaviour in nestbuilding vs. non-nest-building males and female oocyte maturation in gourami fish (Levy & Degani, 2012). ADCYAP1 was located just downstream at 1.8 Mb on scaffold_23 (Table 2; Data S2). Given how remarkably conserved this gene family is across vertebrates (Vaudry et al., 2009), it is likely that ADCYAP1R1 and ADCYAP1 play a functionally conserved role in L. callipterus sexual development.
However, more detailed work is needed to determine whether and how these genes contribute to sex determination in L. callipterus.

| Linkage between the sex-determining locus and a candidate sexually antagonistic locus
The L. callipterus candidate sex-determining gene, ADCYAP1R1, was in linkage with the candidate gene for body size determination, GHRHR (Figures 4 and 6). The sexual antagonism model of sex chromosome evolution postulates that novel sex-determination genes may arise in response to conflict between sexes over traits such as body size (van Doorn & Kirkpatrick, 2007). Since differences in body size between males and females affect reproductive fitness in the shell-brooding L. callipterus (Schütz et al., 2006;Schütz & Taborsky, 2000 and higher dwarf male-female F ST (a possible indicator of antagonism) was found in the sex-determining region with the candidate body size gene (Figure 6), we propose that sexual antagonism over body size may have driven the evolution of a novel sex chromosome in L. callipterus. It is possible that this sex chromosome on scaffold_23 is ancestral to the Lamprologini cichlid tribe, but our results were unclear ( Figure S11). We also cannot rule out that it may have arisen in the lamprologini ancestor and was subsequently lost in some species.
Although inversions are often found on sex chromosomes and are proposed to mediate recombination suppression (Lahn & Page, 1999;Peichel et al., 2020;Ponnikas et al., 2018), we did not find evidence of an inversion. It is possible that strong selection for low recombination facilitated the establishment of sex-specific and morph-specific polymorphisms as observed with guppy alter- Thus, the sex-linked region of L. callipterus may govern multiple sex and male morph-related phenotypes, consistent with "supergene" architectures found in other species with alternative male reproductive tactics (Sandkam et al., 2021;Schwander et al., 2014;Thompson & Jiggins, 2014).

| Male morphs are associated with two Yhaplotypes
While the sex region on scaffold_23 was shared by both L. callipterus male morphs, there were clear differences in this region between the two males, suggesting that there are two distinct Y haplotypes in L. callipterus (Figure 3). The bourgeois males have an additional region of low coverage on scaffold_36 that is not found in dwarf males, which further supports that there are two distinct Y haplotypes. Within the shared sex region on scaffold_23, the dwarf male Y haplotype has lower coverage and may therefore be more degenerated than the bourgeois male Y haplotype, which can have important gene dosage effects (Raznahan et al., 2018).
F ST patterns across the sex region also suggested that the region of recombination suppression is larger in the dwarf male Y haplotype compared to bourgeois males. This would be the case if selection for linkage was stronger between the sex-determining locus and a sexually antagonistic locus in dwarf males, perhaps because the dwarf male body size was under stronger selection pressure than the bourgeois male body size. This is conceivable as the dwarf male mating tactic is strictly applicable for males small enough to enter the shell and wriggle past the female (Sato et al., 2004), whereas the bourgeois male mating tactic may be applied by males of a range of body sizes, even if passing a size threshold to carry shells greatly improves the efficiency of this mating tactic (Schütz & Taborsky, 2005). It is also possible that the much smaller population size of dwarf males compared to bourgeois males (Wirtz-Ocana et al., 2014) resulted in greater Y degeneration. Indeed, differences in the rate and extent of Y degeneration have been observed in two closely related stickleback species that share the same ancestral Y chromosome, with more rapid and extensive degeneration in the species with the smaller effective population size (Sardell et al., 2021).
But how did the second Y haplotype evolve and how is it maintained in the population? Given that the reproductive potential and evolutionary fitness of the dwarf males is critically dependent on the fitness of the bourgeois males, we hypothesize that the bourgeois male type is ancestral. It is possible that a mutation in the GHRHR candidate body size gene gave rise to dwarf males that had higher fitness due to their efficient parasitic reproductive tactic (Taborsky et al., 2018;Wirtz-Ocana et al., 2014). This high fitness allowed the new dwarfism mutation to establish and be maintained by negative frequency-dependent selection in the population (Clark, 1987;Gadgil, 1972), as the fitness of each type of males is dependent on their relative frequency in the population (Taborsky & Brockmann, 2010;Taborsky, 2008;. Such Y haplotype diversity has also been observed in guppies (Poecilia reticulata) that experience negative frequencydependent selection on Y-linked colour patterns, as well as in the closely related species Poecilia parae in which there are five behaviourally and morphologically distinct reproductive male morphs that each have a distinct Y-haplotype (Almeida et al., 2020;Sandkam et al., 2021).

| CON CLUS ION
Our study reveals a young sex chromosome in the Lake Tanganyika cichlid adaptive radiation that supports the sexual antagonism model of sex chromosome evolution. Our results suggest that the suite of complex traits that differ between the bourgeois and dwarf male morphs in body size, growth rate, testis/sperm size, behaviour and reproductive strategy are delineated by two distinct Y haplotypes. Future functional studies of candidate genes and long-read exploration of the L. callipterus sex-linked region provide exciting avenues to further investigate the sexual antagonism hypothesis and the evolution of male alternative reproductive tactics.

AUTH O R CO NTR I B UTI O N S
MT and CS conceived the study. CLP contributed to interpretation of the results. PS designed and conducted all analyses, made the figures, and drafted the manuscript, which was edited by MT, CS and CLP. All authors have read and approved the final manuscript.

ACK N O WLE D G E M ENTS
We thank the ZMF facility at Med-Uni Graz for library preparation, W. Gessl for fish photographs and M. Koller for figure editing.

FU N D I N G I N FO R M ATI O N
This work was supported by the Swiss National Foundation grant (SNF Project 310030B-138660) to MT and the Austrian Science Fund grant (FWF P29838) to CS. PS was funded by a PhD scholarship from the Austrian Centre of Limnology, University of Graz.

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.