DOI: 10.5061/dryad.6djh9w19n
title: Sex chromosome turnover in African annual killifishes of the genus Nothobranchius

description: Sex chromosomes of teleost fishes undergo frequent turnovers. Annual
 Nothobranchius killifishes provide a suitable system to study sex
 chromosome turnover as they comprise the XY sex chromosome system in the
 model turquoise killifish, N. furzeri, and X1X2Y multiple sex chromosomes
 in six other representatives scattered across the Nothobranchius
 phylogeny, nested within species without cytologically detectable sex
 chromosomes. We combined molecular cytogenetics and genomic analyses to
 examine the X1X2Y systems in four Nothobranchius spp. and their outgroup
 Fundulosoma thierryi. Fluorescence in situ hybridization with painting
 probes specific for three sex chromosome systems and N. furzeri bacterial
 artificial chromosomes (BAC) bearing orthologues of eight genes repeatedly
 co-opted as master sex determining (MSD) genes in fishes suggests at least
 four independent origins of sex chromosomes in the genus Nothobranchius.
 The synteny block carrying the amhr2 gene was shared by X1X2Y systems of
 N. brieni, N. guentheri, and N. lourensi, which, however, differ by their
 fused autosomes. The gdf6 gene is sex-linked in F. thierryi. None of the
 mapped MSD gene candidates was sex-linked in N. ditte. We further
 sequenced F. thierryi and N. guentheri genomes and
 performed analyses of male and female Pool-seq and coverage data to
 determine their non-recombining regions and their differentiation. Level
 of sex chromosome differentiation was low in F. thierryi, but we
 identified two distinct sex-linked evolutionary strata in N. guentheri.
 While the amhr2 gene represents a candidate for MSD in N. guentheri, its
 localization in the younger stratum and low allelic variation questions
 its role in sex determination in a common ancestor of N. brieni, N.
 guentheri, and N. lourensi. Recombination cold spots such as fusion
 breakpoints could have contributed to formation of sex chromosome
 evolutionary strata.
description: <strong>Genome de novo assembly and its quality
 evaluation</strong> The <em>F.
 thierryi</em> long reads were first corrected using the Illumina
 short reads. Then, several options were used for genome assembly using
 Flye v2.8 (Kolmogorov et al. 2019) with the "––nano-raw" and
 "<em>--min-overlap 5000</em>“ option giving the best
 assembly in terms of contiguity and BUSCO (Benchmarking Universal
 Single-Copy Orthologs) completeness. The assembly underwent one round of
 long read polishing via medaka (https://github.com/nanoporetech/medaka)
 followed by a round of short read polishing using NextPolish (Hu et al.
 2020) with Illumina reads. Haplotypic duplicates were then removed using
 the purge_dups v1.0.1 tool (Guan et al. 2020). Assembly quality was
 assessed using BUSCO v5 with the Actinopterygii database (odb_10) (Manni
 et al. 2021). Contamination checks were performed using BlobTools v1.0
 (Laetsch & Blaxter 2017), and any contigs associated with
 non-target organisms were eliminated using the
 "<em>seqfilter</em>" function. The best result for
 <em>N. guentheri</em> assembly was achieved using Flye v2.8
 (Kolmogorov et al. 2019) with the "––pacbio-raw" option, genome
 size not defined, and without further polishing or de-duplication.
 Assembly quality was verified with BUSCO v5 with the Actinopterygii
 database (odb_10). <strong>Genome
 annotation</strong> Functional and structural
 annotations were conducted using the GenSAS v6.0 pipeline (Humann et al.
 2019). Repetitive sequences were identified employing RepeatModeler2
 (Flynn et al. 2020) utilizing the RMBlast search engine and modules
 including TFR v4.09, RECON, and RepeatScout v1.0.5. Additionally, TAREAN
 (Novák et al. 2017) was utilized for satDNA annotation. All consensus
 sequences marked as satellites by TAREAN, regardless of confidence levels,
 were integrated into a custom database as dimers to enhance the satellite
 DNA annotation. RepeatMasker v4.1.1 (Smit et al. 2013–2015, available at
 http://www.repeatmasker.org) with the NCBI/RMBlast search engine was
 employed for repeat annotation. This involved a combination of the newly
 identified repeats from RepeatModeler2 and the custom database containing
 the satDNA sequences from TAREAN. RNA-seq data were
 used for <em>N. guentheri</em> genome annotation. To that end,
 total RNA was extracted from brain tissue dissected from <em>N.
 guentheri</em>. Subsequently, the 150 bp Illumina reads were aligned
 to the respective genomes using STAR v2.7.7 (Dobin & Gingeras
 2015). The genome index required for mapping was generated using the
 following command: <em>STAR --runThreadN 9 --runMode genomeGenerate
 --genomeDir ./genomedir --genomeFastaFiles &lt;genome&gt; 
 --genomeSAindexNbases 13.</em> Following index generation, mapping
 was executed using the command: <em>STAR --runThreadN 9 --genomeDir
 ./genomedir --readFilesIn ./Forward.fq</em>
 <em>./Reverse.fq</em>. The resulting SAM file was converted to
 BAM format using the SAMtools suite (v1.11) (Li et al. 2009). The
 generated BAM file was then utilized for gene prediction through BRAKER2
 with default settings, which incorporates Augustus and GeneMark-EP
 (Lomsadze et al. 2014; Brůna et al. 2021). For the annotation of
 <em>Fundulosoma thierryi </em>genome assembly, Augustus v3.3.1
 (Stanke & Morgenstern 2005) and GeneMark-ES were used directly,
 without RNA-Seq evidence to guide the process. Furthermore, tRNA and rRNA
 sequences were identified in all assemblies using tRNAscan-SE v2.0.7 (Chan
 & Lowe 2019) and RNAmmer v1.2 (Lagesen et al. 2007),
 respectively. <strong>Reference guided
 scaffolding</strong> To obtain pseudochromosome
 level assemblies of both <em>F. thierryi</em> and <em>N.
 guentheri</em>, we aligned genome assembly contigs to chromosomes of
 the <em>N. furzeri</em> reference (GenBank acc. no.
 GCA_014300015.1) using Minimap2 v2.17 (Li 2018) and sorted and oriented
 the contigs into pseudomolecules using RaGOO (Alonge et al. 2019) with
 “<em>-b -s</em>” options.
 <strong>Pool-seq analysis</strong>
 To identify male-specific (MSY) regions, we generated pooled
 samples of genomic DNA from <em>N. guentheri</em> (28 males,
 29 females) and <em>F. thierryi</em> (20 males, 11 females)
 and sequenced them in paired-end mode on the Illumina platform. The
 Illumina pools were quality checked with FastQC v0.11.5 (Andrews et al.
 2010) and filtered with the “<em>--nextseq-trim=20
 --minimum-length=100</em>” options using cutadapt v1.15 (Martin
 2011) and trimmed with Trimmomatic v0.36 (Bolger et al. 2014) with
 following parameters: “<em>SLIDINGWINDOW:4:25 MINLEN:100 HEADCROP:4
 CROP:140</em>”. The trimmed and filtered reads from female and male
 pools were mapped separately to the reference genomes using BWA-MEM
 v0.7.17 (Li & Durbin 2009) with default parameters. Using Picard
 Toolkit (https://broadinstitute.github.io/picard/), we sorted resulting
 bam files by coordinate (“<em>SortSam
 SORT_ORDER=coordinate</em>”) and removed PCR duplicates
 (“<em>MarkDuplicates REMOVE_DUPLICATES=true
 REMOVE_SEQUENCING_DUPLICATES=true</em>”). Subsequently, we generated
 a file with the nucleotide composition of all genomic positions using the
 <em>pileup</em> function of the software Pooled Sequencing
 Analysis for Sex Signal (PSASS; Feron & Jaron 2021). We used PSASS
 to identify non-overlapping 50 kb windows enriched in sex-specific SNPs,
 using the following parameters: “<em>--min-depth 10, --freq-het 0.5
 --range-het 0.15 --freq-hom 1 --range-hom 0.05 --window-size 50000
 --output-resolution 50000 --group-snps</em>”.
 <strong>Coverage analysis</strong>
 The short Illumina reads from three male and female samples of
 both <em>F. thierryi</em> and <em>N.
 guentheri</em> were quality checked with FastQC v0.11.5 (Andrews et
 al. 2010) and filtered with “<em>--nextseq-trim=20
 --minimum-length=100</em>” options using cutadapt v1.15 (Martin
 2011) and trimmed with Trimmomatic v0.36 (Bolger et al. 2014) with
 following parameters: “<em>SLIDINGWINDOW:4:25 MINLEN:100 HEADCROP:10
 CROP:130</em>”. Repetitive sequences pseudochromosome-level
 assemblies were identified by RepeatModeler v1.0.11 (Smit et al.
 2008–2015, available at http://www.repeatmasker.org) and annotated by
 RepeatMasker v4.0.7 (Smit et al. 2013–2015, available at
 http://www.repeatmasker.org) with the NCBI search. Filtered reads were
 mapped to the corresponding masked reference genome via Bowtie2 v2.2.9
 (Langmead & Salzberg 2012) with the
 “<em>--very-sensitive-local --no-discordant --no-mixed</em>”
 parameters and the outputs were compressed to BAM format using SAMtools
 <em>view</em> (v1.3.1; Li et al. 2009) and merged according to
 sex using SAMtools <em>merge</em>. The resulting BAM files
 were then parsed using utilities from the Bedtools suite v2.25.0 (Quinlan
 & Hall 2010). A genome file was parsed from the BAM files using
 SAMtools <em>view</em> and divided into 50 kbp sliding windows
 using Bedtools <em>makewindows</em> with “<em>-w 50000
 –s 50000</em>” parameters. The merged BAM files were sorted with
 SAMtools <em>sort</em> and converted to BED format using
 Bedtools <em>bamtobed</em> “<em>-split</em>”.
 Finally, the per-base coverage of aligned sequences within 50 kbp windows
 spanning the genome was computed using Bedtools
 <em>coverage</em>. In both sexes, coverage depths for each
 scaffold were normalized by mean coverage across scaffolds and compared
 between sexes, formulated as the Log2 of the male:female (M:F) coverage
 ratio. The resulting data, together with those from Pool-seq analysis,
 were visualised using “<em>SexGenomicsToolkit/sgt</em>r” R
 package (https://github.com/SexGenomicsToolkit/sgtr). <strong>Variant detection</strong> To analyse allelic variants in the <em>amhr2</em> gene, we mapped paired-end short read Illumina (Bentley et al., 2008) Pool-seq data to the reference <em>N. guentheri</em> genome and called the variants using Freebayes software (Garrison & Marth, 2012). Male and female Pool-seq data were trimmed and filtered using TrimGalore v0.6.2 (Krueger et al. 2023) using the following flags: “--fastqc --clip_R1 10 --clip_R2 10 --three_prime_clip_R1 10 --three_prime_clip_R2 10 --paired”. Effectivity of the trimming step was verified using FastQC v0.11.9 (2010). Trimmed reads were then mapped using Bowtie2 v2.3.5.1 (Langmead & Salzberg, 2012) with “--very-sensitive-local --no-mixed --no-discordant” flags. Resulting alignment files were used as an input for Freebayes v9.9.2 (Garrison & Marth, 2012) in default mode. By visualising the alignment files in Geneious Prime v2023.2.1, we obtained the compositions of the SNPs for males and females. <strong>Haplotype-specific assembly of chr 13</strong> To separately assemble male and female haplotypes of the putative sex determining region on the <em>N. guentheri</em> chromosome 13 (NguChr13), we have reassembled this chromosome using the trio binning mode. Original PacBio long reads used for the <em>N. guentheri</em> reference genome assembly were mapped back to it using Minimap2 v2.22 (Li, 2018) with the flags “-a -x map-pb”. From the resulting alignment file, only reads mapping in the chr13 were selected using Samtools v1.11 (Danecek et al. 2021). Subsequent conversion of alignment file into FASTQ format was done using Bedtools bamtofastq with default setting (Quinlan & Hall, 2010). Resulting reads were used as a sequence set for the new NguChr13 assembly performed using Canu v2.2 (Koren et al., 2017) with the following flags enabled “genomeSize=30m --pacbio [<em>filename</em>] -haplotypePAT [<em>patname</em>] -haplotypeMAT [<em>matname</em>]”. Parental Illumina short reads were used to enable the binning. Assembly process resulted in the creation of two chr13 haplotypes, one maternal and the other paternal. ############################################################################################################################################## Alonge M, Soyk S, Ramakrishnan S, Wang X, Goodwin S, Sedlazeck FJ, Lippman ZB, Schatz MC. (2019). RaGOO: fast and accurate reference-guided scaffolding of draft genomes. Genome Biology 20: 224. https://doi.org/10.1186/s13059-019-1829-6<br>Andrews S (2010). FastQC: a quality control tool for high throughput sequence data. http:// www.bioinformatics.babra ham.ac.uk/ proje cts/ fastqc/. 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description: # Description of the data and file structure **README for the Dryad
 repository**:
 [https://doi.org/10.1111/mec.70029](https://doi.org/10.1111/mec.70029)
 **Study:** Hospodářská M, Mora P, Chung Voleníková A, Al-Rikabi A,
 Altmanová M, Simanovsky SA, Tolar N, Pavlica T, Janečková K, Štundlová J,
 Bobryshava K, Jankásek M, Hiřman M, Liehr T, Reichard M, Krysanov EY, Ráb
 P, Englert C, Nguyen P, Sember A (2025) *Multiple Origins of Sex
 Chromosomes in *Nothobranchius* Killifishes*. **Molecular Ecology**,
 34:e70029.
 [https://doi.org/10.1111/mec.70029](https://doi.org/10.1111/mec.70029)
 Last updated: August 11, 2025 --- ## Data provided ### *Fundulosoma
 thierryi* #### Genome assembly *
 `Nthi_Corr-Uncor_Strict_Meta_Min5000_assembly_Medaka_Long_Next_Short_Purged.fasta`: FASTA format of the *Fundulosoma thierryi* female genome assembly. * `FTHI_NFU_ragoo.fasta`: FASTA of Fundulosoma thierryi pseudochromosomes scaffolded by RaGOO using *Nothobranchius furzeri* as reference. #### Gene annotation * `Fundulosoma-thierryi-v1.0.a1.65cb8ad34d6d8-publish.CDS.fna`: Nucleotide sequences of all CDS features annotated on the *Fundulosoma thierryi* assembly. * `Fundulosoma-thierryi-v1.0.a1.65cb8ad34d6d8-publish.genes.fna`: Gene models annotated on the *Fundulosoma thierryi* assembly. * `Fundulosoma-thierryi-v1.0.a1.65cb8ad34d6d8-publish.genes.gff3`: Annotation of genomic features (GFF3) in the *Fundulosoma thierryi* genome assembly (BRAKER v2.1.5 via GenSAS v6.0). * `Fundulosoma-thierryi-v1.0.a1.65cb8ad34d6d8-publish.protein.faa`: FASTA sequences of protein products annotated on the *Fundulosoma thierryi* assembly. #### Repeat annotation * `Fundulosoma-thierryi-v1.0.a1.65663ba64fbb4-repeatmodeler.repeats.gff3`: Generic Feature Format Version 3 (GFF3) of transposable elements annotated in the *F. thierryi* genome. * `Fundulosoma-thierryi-v1.0.a1.65663ba64fbb4-repeatmodeler.repeats.fna`: FASTA format of the annotated *F. thierryi* transposable elements. * `Fundulosoma-thierryi-v1.0.a1.654296049b4ef-repeatmasker.repeats.gff3`: Generic Feature Format Version 3 (GFF3) of tandem repeats annotated in the *F. thierryi* genome. * `Fundulosoma-thierryi-v1.0.a1.654296049b4ef-repeatmasker.repeats.fna`: FASTA format of the annotated *F. thierryi* tandem repeats. #### Gene-specific data * `Nthi_GDF6_XY_alleles.fas`: Manually curated sequences of X- and Y-linked alleles of the *Fundulosoma thierryi* gdf6 gene. * `gdf6_female_NTHI_poolseq_reads.bam`: gdf6 gene alignment file of female Pool-seq data (F. thierryi) mapped to pseudochromosomes (RaGOO, *N. furzeri* reference). * `gdf6_male_NTHI_poolseq_reads.bam`: gdf6 gene alignment file of male Pool-seq data (*F. thierryi*) mapped to pseudochromosomes (RaGOO, *N. furzeri* reference). * `hsd17b_female_NTHI_poolseq_reads.bam`: hsd17b gene alignment file of female Pool-seq data (*F. thierryi*) mapped similarly. * `hsd17b_male_NTHI_poolseq_reads.bam`: hsd17b gene alignment file of male Pool-seq data (*F. thierryi*) mapped similarly. * `GDF6_female_NTHI.vcf`: *Fundulosoma thierryi* gdf6 female variant calls (freebayes). * `GDF6_male_NTHI.vcf`: *Fundulosoma thierryi* gdf6 male variant calls (freebayes). * `HSD17b_female_NTHI.vcf`: *Fundulosoma thierryi* hsd17b female variant calls (freebayes). * `HSD17b_male_NTHI.vcf`: *Fundulosoma thierryi* hsd17b male variant calls (freebayes). --- ### *Nothobranchius guentheri* #### Genome assembly * `Ngue_Flye_run2-2.8_assembly.fasta`: FASTA of the *Nothobranchius guentheri* male genome assembly. * `NGU_NFU_ragoo.fasta`: Nothobranchius guentheri pseudochromosomes scaffolded by RaGOO using *Nothobranchius furzeri* as reference. #### Gene annotation * `Nothobranchius-guentheri-v1.0.a1.66266468babb3-publish.CDS.fna`: Nucleotide sequences of all CDS features annotated on the *Nothobranchius guentheri* assembly. * `Nothobranchius-guentheri-v1.0.a1.66266468babb3-publish.genes.fna`: Gene models annotated on the *Nothobranchius guentheri* assembly. * `Nothobranchius-guentheri-v1.0.a1.66266468babb3-publish.genes.gff3`: Annotation of genomic features (GFF3) in the *Nothobranchius guentheri* genome assembly (BRAKER v2.1.5 via GenSAS v6.0). * `Nothobranchius-guentheri-v1.0.a1.66266468babb3-publish.protein.faa`: FASTA sequences of protein products annotated on the *Nothobranchius guentheri* assembly. #### Repeat annotation * `Nothobranchius-guenterii-v1.0.a1.654b88373daeb-repeatmodeler.repeats.gff3`: Generic Feature Format Version 3 (GFF3) of transposable elements annotated in the *N. guentheri* genome. * `Nothobranchius-guenterii-v1.0.a1.654b88373daeb-repeatmodeler.repeats.fna`: FASTA format of the annotated *N. guentheri* transposable elements. * `Nothobranchius-guenterii-v1.0.a1.654b884662ddd-repeatmasker.repeats.fna`: FASTA format of the annotated *N. guentheri* tandem repeats. * `Nothobranchius-guenterii-v1.0.a1.654b884662ddd-repeatmasker.repeats.gff3`: Generic Feature Format Version 3 (GFF3) of tandem repeats annotated in the *N. guentheri* genome. #### Gene-specific data * `Ngu_AMHR2_XY_alleles.fas`: Manually curated sequences of X- and Y-linked alleles of the *Nothobranchius guentheri* amhr2 gene. * `NGU_female_poolseq_amhr2_extended.vcf`: *Nothobranchius guentheri* female variant calls (freebayes). * `NGU_male_poolseq_amhr2_extended.vcf`: *Nothobranchius guentheri* male variant calls (freebayes). * `NGU_male_poolseq_amhr2_extended_curated.vcf`: *Nothobranchius guentheri* male variant calls (freebayes), manually curated with unsupported variants assigned Qual 0. * `Ngu_male_poolseq_amhr2_reads_sorted.bam`: amhr2 gene alignment file of male Pool-seq data (*N. guentheri*) mapped to pseudochromosomes (RaGOO, *N. furzeri* reference). * `Ngu_female_poolseq_amhr2_reads_sorted.bam`: amhr2 gene alignment file of female Pool-seq data (*N. guentheri*) mapped to pseudochromosomes (RaGOO, *N. furzeri* reference).