
==== Front
Cell Genom
Cell Genom
Cell Genomics
2666-979X
Elsevier

S2666-979X(24)00201-5
10.1016/j.xgen.2024.100607
100607
Short Article
Comparative genomics illuminates karyotype and sex chromosome evolution of sharks
Wu Jiahong 18
Liu Fujiang 28
Jiao Jie 1
Luo Haoran 13
Fan Shiyu 1
Liu Jiao 1
Wang Hongxiang 1
Cui Ning 1
Zhao Ning 14
Qu Qingming 2
Kuraku Shigehiro 56
Huang Zhen 7
Xu Luohao luohaox@gmail.com
19∗
1 Integrative Science Center of Germplasm Creation in Western China (Chongqing) Science City, MOE Key Laboratory of Freshwater Fish Reproduction and Development, School of Life Sciences, Southwest University, Chongqing 400715, China
2 State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, China
3 Key Laboratory of Ministry of Education for the Coastal and Wetland Ecosystems, College of the Environment and Ecology, Xiamen University, Xiamen 361102, China
4 Key Laboratory of Animal Genetics and Breeding and Molecular Design of Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China
5 Molecular Life History Laboratory, Department of Genomics and Evolutionary Biology, National Institute of Genetics, Shizuoka, Japan
6 Department of Genetics, Sokendai (Graduate University for Advanced Studies), Mishima, Japan
7 Fujian-Macao Science and Technology Cooperation Base of Traditional Chinese Medicine-Oriented Chronic Disease Prevention and Treatment, Innovation and Transformation Center, Fujian University of Traditional Chinese Medicine, Fuzhou 350108, China
∗ Corresponding author luohaox@gmail.com
8 These authors contributed equally

9 Lead contact

11 7 2024
14 8 2024
11 7 2024
4 8 10060715 1 2024
1 5 2024
19 6 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Summary

Chondrichthyes is an important lineage to reconstruct the evolutionary history of vertebrates. Here, we analyzed genome synteny for six chondrichthyan chromosome-level genomes. Our comparative analysis reveals a slow evolutionary rate of chromosomal changes, with infrequent but independent fusions observed in sharks, skates, and chimaeras. The chondrichthyan common ancestor had a proto-vertebrate-like karyotype, including the presence of 18 microchromosome pairs. The X chromosome is a conversed microchromosome shared by all sharks, suggesting a likely common origin of the sex chromosome at least 181 million years ago. We characterized the Y chromosomes of two sharks that are highly differentiated from the X except for a small young evolutionary stratum and a small pseudoautosomal region. We found that shark sex chromosomes lack global dosage compensation but that dosage-sensitive genes are locally compensated. Our study on shark chromosome evolution enhances our understanding of shark sex chromosomes and vertebrate chromosome evolution.

Graphical abstract

Highlights

• The assembled white-spotted bamboo shark genome is the largest in Chondrichthyes

• Chondrichthyes chromosomes, including microchromosomes, are evolutionarily stable

• Sharks have conserved XY sex chromosomes that are 181 million years old

• The sex chromosome dosage compensation is incomplete in sharks

Wu et al. compared the genomes of six cartilaginous fishes and found that the chromosomes evolve at a very slow rate. The authors identified and characterized the sequences of the highly decayed Y chromosomes of sharks that originated more than 180 million years ago.

Keywords

cartilaginous fish
microchromosome
sex chromosome
vertebrate karyotype
shark
dosage compensation
Published: July 11, 2024
==== Body
pmcIntroduction

Chondrichthyes (cartilaginous fishes) is an early branching lineage of vertebrates and often used for reconstructing the evolutionary history of early jawed vertebrates.1,2,3 Despite being an ecologically and evolutionarily important lineage, very few high-quality genome assemblies are available for Chondrichthyes. Because of their large sizes and repetitive nature,4 chondrichthyan genome assemblies are often highly fragmented and incomplete. The rapid development of long-read sequencing in recent years has allowed for substantial improvement in genome assemblies, as seen in the recent efforts for several Chondrichthyes genomes.5,6,7,8,9,10

The diploid number of chromosomes varies among chondrichthyan lineages (ranging from 28 to 106), but the within-lineage variation is relatively small.11 Some lineages constantly have large diploid chromosome numbers, including Orectolobiformes (carpet sharks) and Rajiformes (rays and skates). The white-spotted bamboo (WSB) shark (Chiloscyllium plagiosum) and brownbanded bamboo shark (Chiloscyllium punctatum) are known to have a diploid number of 106,11 the largest among chondrichthyans (Figures 1A and 1B). Various studies suggest that the gnathostome ancestor likely had 90–108 chromosomes.2,12,13 Whether the large chromosome number of bamboo sharks reflects the maintenance of the ancestral gnathostome karyotype or is a derived feature remains unclear. Intriguingly, recent cytogenetic6,11 and genomic7 studies on chondrichthyans reveal the presence of microchromosomes, which have been inferred to originate in the gnathostome ancestor.14,15 This provides a clue that chondrichthyans may maintain some ancestral gnathostome karyotype features (such as microchromosomes), as bird genomes do.13Figure 1 Phylogenetic and comparative analyses of 11 gnathostome genomes

(A) The divergence time is labeled at each node with a 95% confidence interval. Black asterisks denote the fossil calibration nodes.

(B) The haploid number is shown for each species. Asterisks denote estimated haploid numbers according to Hi-C scaffolding without cytogenetic evidence.

(C and D) The WSB shark has the largest assembled genome sizes (C) and repeat content (D).

(E) Synteny between chicken and WSB shark is conserved. Chromosome IDs marked in red represent microchromosomes shared by the two species, while blue and violet mark chicken- and WSB-shark-specific microchromosomes, respectively.

(F) Gene-order synteny between chicken (chr10 and chr6), WSB shark, and amphioxus homologous chromosomes. WSB shark chr42 has mixing orthologs of two different amphioxus chromosomes, while chr43 is homologous with a single amphioxus chromosome. Blue, red, lilac, and green colors represent genes on chromosomes 1, 20, 2, and 16, respectively.

(G) The proposed model of microchromosome changes across vertebrate phylogeny.

Recent cytogenetic and genomic investigations also revealed the presence of XY sex chromosomes in sharks.7,11 According to the cytogenetic observations, the X-Y chromosome pairs are highly differentiated, suggesting a possible ancient origin of XY sex chromosomes; alternatively, but less likely, the Y chromosome may have rapidly decayed once it independently originated in separate lineages. A recent genomic study on zebra shark (Stegostoma tigrinum) and whale shark (Rhincodon typus) X chromosomes suggests that at least Orectolobiformes share the common origin of sex chromosomes.7 Whether the sex chromosomes are shared by a wider range of shark species is unclear, and it requires the inclusion of additional shark sex chromosomes for analyses. Moreover, to date, sequence and structural characterization of the shark Y chromosomes, which is crucial for understanding the evolutionary trajectories of sex chromosome differentiation, is still lacking.

In this study, we acquire the haplotype-resolved high-quality genome sequences of a male WSB shark at the chromosomal level, including an X and a Y chromosome. We performed comprehensive chromosome syntenic analyses by including six chromosome-level chondrichthyans genomes, covering all three major lineages: Selachimorpha (sharks), Batomorpha (rays and skates), and Holocephali (chimaeras) (Figure 1A).

Results

Haplotype-resolved genome assembly for WSB shark

We generated 211.3 Gb PacBio HiFi sequencing data for a male WSB shark (Table S1). Combining Hi-C data produced from the same individual, we assembled two phased haploid genomes: hap1 and hap2, with the sizes of 4.74 and 5.04 Gb, respectively (Table S2). The haploid assemblies are at least 33.2% larger than a previous draft genome (3.56 Gb) of the WSB shark,16 with over 118-fold improvement in contig N50 lengths (4.93 and 4.78 Mb vs. 39.9 kb). Our assembled genome size is closer to the estimated size based on k-mer (4.99 Gb)17 and flow cytometry (4.85 Gb)18 analyses. The assembled genome of the WSB shark becomes the largest among the sequenced chondrichthyans (Figure 1C) and is one of the largest haplotype-resolved vertebrate genome assemblies. Chondrichthyan genomes usually have a large proportion of repetitive sequences,16,19,20 which reaches 68.6% in the WSB shark (Table S3), the largest among all available chromosome-level chondrichthyan genomes we sampled in this study (Figure 1D). The LINE is the major component of repetitive repeats in chondrichthyans (Figure 1D), and its recent expansion likely contributed to the enlargement of the WGS shark genome (Figure S1).

We used Hi-C data to anchor contigs of both hap1 and hap2 contigs into chromosomes. As a result, we identified 52 chromosomes in each haploid genome, including an X chromosome in the hap1 assembly and a Y chromosome in the hap2 assembly (Figure S2). Over 98.4% of the sequences were included in chromosomes, but according to the known karyotype of the WSB shark (2n = 106),11 one chromosome is missing in each haploid genome. In the following analyses, hap1 was used as the reference genome.

In the WSB shark genome, we annotated 26,372 protein-coding genes, and the assembled genome comprises 92.5% of vertebrate BUSCO genes (Table S4). We further searched for orthologous genes among six chondrichthyans and five representative bony vertebrates (Euteleostomi; see STAR Methods), utilizing 3,222 single-copy orthologs to construct the phylogenetic tree of those 11 gnathostome species (Figure 1A). Our divergence time analysis showed that the common ancestor of sharks diverged from skates about 237 million years ago and the common ancestor of Orectolobiformes diverged from other sharks about 181 million years ago (Figure 1A).

Origin and evolution of chondrichthyan microchromosomes

Vertebrate microchromosomes commonly found in birds or sometimes in other non-mammalian lineages share some common features.13,14 We found that WSB shark chromosomes can be grouped into two, more effectively by GC content and repeat content than by chromosome length or gene density (Figure S3A). The group with smaller chromosomes was considered to encompass putative microchromosomes. Those putative microchromosomes show significantly higher GC content, repeat content (Figure S4), and more intense interchromosomal interactions (Figure S5), properties observed in microchromosomes of other vertebrates.14,21,22 They also exhibit different landscapes of repeat and GC content along the chromosomes compared with the putative macrochromosomes (Figures S3B–S3E). While the expansions of repeats at the chromosomal ends increase the total sizes of microchromosomes (even larger than some small macrochromosomes), they retain the typical features of microchromosomes mentioned above, though gene density becomes lower (Figure S4).

Out of the 21 WSB shark microchromosomes, we found that 18 have orthologous relationships with chicken microchromosomes (Figure 1E). This suggests that most chondrichthyans likely share a common origin of microchromosomes with Euteleostomi. We found evidence that a few WSB-shark-specific microchromosomes were formed due to fissions, including chromosomes 39, 40, 42, and 50 (Figures 1E and 1F). Taking chr39 and chr42 for example, while they are homologous with a single chicken chromosome (chr10), they have mixing gene content of homologous amphioxus chr1 and chr20 (Figure 1F). This is likely because chr39 and chr42 were formed due to a fission event of an ancestral chromosome that had sufficient time to allow intrachromosomal gene-order reshuffling before the fission. On the contrary, chr43 is primarily homologous with a single amphioxus chromosome (chr16) (Figure 1F), thus likely descending from an ancestral microchromosome.

Our evolutionary comparative analysis of chromosome synteny between chicken, WSB shark, and amphioxus indicates that the vertebrate common ancestor likely had 18 pairs of microchromosomes, corroborating previous understandings of the origin of vertebrate microchromosomes.14,15 We propose a model in which the microchromosome members continued to expand by 11 in Euteleostomi due to large-scale sequence losses following the divergence of Chondrichthyes and Euteleostomi (Figure 1G). In Chondrichthyes, only a limited number of chromosomes further developed into microchromosomes, consistent with a larger degree of ohnolog retention in Chondrichthyes.10

Chondrichthyan chromosomes are evolutionarily stable

To investigate the karyotype variation within Chondrichthyes, we included six chromosome-level Chondrichthyes genomes for synteny analyses (Figure 2). Among them, three species do not have known karyotypes (Figure 1B), so we selected the largest chromosome-scale scaffolds for analyses. Overall, chondrichthyan chromosomes are stable but with varying degrees of conservation across lineages (Figures 2, S6, and S7). Elasmobranchii species (e.g., sharks and skates) have similar karyotypes, while Holocephali (e.g., elephant fish, also known as elephant shark) has several unique chromosomal fusions (e.g., chr8) (Figure 2). We also note that the elephant fish retains a few proto-vertebrate chromosomes (e.g., chr3) that are homologous with chicken counterparts but were split into smaller chromosomes in elasmobranch (Figure S8). Our evolutionary comparative approach allowed us to infer 46 chromosomes at the chondrichthyan common ancestor. Following the divergence of Elasmobranchii and Holocephali, different lineages accumulated fusions that were not shared by others, with the most frequent one found in the great hammerhead shark with seven fusions (Figure 2).Figure 2 Chromosomal synteny across cartilaginous fishes

Any homologous chromosome involving chromosomal changes in elephant fish is highlighted in colors. The homologs of shark X chromosomes are highlighted in red. The chromosome IDs of the elephant fish are renamed according to the size rank, with their original IDs shown in Table S5. The inferred ancestral chromosomal number is labeled at each node.

Conserved sex chromosomes with a highly decayed Y chromosome

The WSB shark, among other sharks, is known to have a cytogenetically differentiated pair of XY sex chromosomes where the Y is about one-third the size of the X.11 It is thus expected that the X chromosome should show reduced male (XY) sequencing depth, while the Y chromosome should have sparse coverage by female (XX) sequencing reads.23 We identified a 28.4 Mb X chromosome showing a female-to-male depth ratio of nearly two (Figure S9) and a 12.5 Mb Y chromosome showing a female-to-male coverage ratio of almost zero (Figures 3A and 3B). We detected very few SNPs on the X chromosome for male but not female sequence data (Figure 3C), consistent with the hemizygous status of the X in males. Similar analyses on a female genome assembly did not detect any female-specific contig (Figure S10). At the termini of both X and Y chromosomes, we identified ∼1.5 Mb PARs (pseudoautosomal regions) that show autosome-like sequencing coverage (Figures 3B and S9), similar to the cases in other sharks.7 Our approach also led to the identification of 4.8 Mb Y-linked sequences of the epaulette shark from its recently published genome6 (Figure S11).Figure 3 Complex evolutionary history of shark sex chromosomes

(A) The Y chromosome has a female-to-male (f/m) coverage ratio close to zero, but this ratio is close to one for the X. The X also shows an almost 2-fold f/m depth ratio, consistent with the hemizygous status of the X.

(B) The top shows the Hi-C interaction map for the Y chromosome. The bar charts on the bottom show male and female HiFi sequencing coverage and satellite DNA percentage. The gene ZBTB39 is located in a young evolutionary stratum but very close to the PAR. In the first identity image, colored vertical bars represent the sequence identity between the X and Y chromosomes. The second identity image shows the alignment of the Y chromosome segments with 23 autosomes, with blue, orange, and red lines representing sequencing identities of 85%–90%, 90%–95%, and 95%–100%, respectively. The light blue and light red backgrounds indicate the young evolutionary stratum and the PAR, respectively.

(C) Almost absent of SNPs on the X and Y chromosomes in males while the X chromosome shows a comparable SNP density relative to autosomes (chr12 is shown as one example) in females.

(D) Fluorescence in situ hybridization (FISH) verification for Y chromosome. Blue fluorescence represents DAPI, and red fluorescence represents the signal of a Y-linked satellite DNA.

(E) The gene SHARKY1 shows zero coverage by female HiFi or RNA sequencing (RNA-seq) coverage but is covered by male HiFi (hemizygous) and RNA-seq reads. m, male; f, female.

(F) The gametologous tree for ZBTB39 shows that the gametologs are clustered by species rather than by chromosomes, suggesting an independent origin of the non-recombining region in the WSB shark, epaulette shark, and great white shark.

About 51.2% of the WSB shark Y chromosome is covered by satellite DNA (Figures 3B and S12; Table S6). We used one of the most abundant satellite DNA for FISH experiments and detected a strong hybridization signal on a single chromosome in a male chromosome spread (Figures 3D and S13).

The X chromosome of the WSB shark is the third smallest chromosome in terms of the assembled size, next to chr49 and the Y. Its orthologous chromosome in chicken (chr33) is also one of the smallest.13 Intriguingly, the X chromosome is homologous with other known shark X chromosomes, including those of zebra shark, whale shark,7 epaulette shark,6 small-spotted catshark (Scyliorhinus canicula), and great white shark (Figure S14). The X chromosome of the thorny skate8 is, however, not homologous with the shark X chromosomes (Figure S14), suggesting an independent origin of the skate XY sex chromosome.

Our in silico prediction detected 13 putative protein-coding genes on the Y, of which only two are not in the PAR (Figure 3B). These two genes, ZBTB39 and SHARKY1 (named in this study), do not seem to be the usual suspects of sex-determining genes documented in other vertebrate species and are widely expressed across male tissues (Figures 3B–3E and S15). The gene SHARKY1 does not have a gametologous copy on the X, while ZBTB39 has an X-linked gametolog and is very close to the PAR boundary (Figure 3B). We failed to detect amplicons that are often found in Y chromosomes of other species.24

Highly differentiated sex chromosomes usually have experienced multiple recombination suppression events, leading to the formation of so-called “evolutionary strata.”25 We analyzed the presence and number of evolutionary strata of the sex chromosomes by comparing the divergence between the X and Y. We identified a ∼1 Mb region adjacent to the PAR exhibiting X-Y similarity of 89.5% on the nucleotide sequence level (Figures 3B and S16), indicating a very young evolutionary stratum. This region contains only one gene, the abovementioned ZBTB39. Our phylogenetic analysis for ZBTB39 gametologs shows the clusterings by species rather than by chromosome (Figure 3F), supporting independent origins of this young evolutionary stratum among shark lineages. Apart from this stratum, the rest of the non-recombining region of the Y contains very few homologous segments with the X, and the X-Y homologs have large sequence divergence, suggesting very old evolutionary strata (Figures 3B and S16). It is unclear how many strata have evolved due to sparse X-Y divergence information in the highly differentiated region. Intriguingly, this region carries many segments with autosomal origins (Figure 3B). Unlike the scenario in Drosophila,26 none of these autosomal transpositions added protein-coding genes to the Y chromosome.

Incomplete sex chromosome DC in sharks

In vertebrates, the vast majority of sex chromosome systems are incompletely compensated, including birds,27 snakes,28 and stickleback fishes,29 among many others. Despite sex chromosomes having evolved independently hundreds of times within vertebrates, complete mechanisms of dosage compensation (DC) are thus far known only in mammals, Poecilia fish,30 and Anolis lizards.31 In WSB sharks, males have an imbalanced dosage between the X chromosome and autosomes due to massive gene loss on the Y chromosome. To assess whether the expression of the X-linked genes is compensated in males, we calculated the male-to-female (m/f) expression ratios for 12 different tissues. In all these tissues, the X-linked genes have lower expression in males than in females, with m/f expression ratios ranging from 0.46 to 0.68; in contrast, the m/f expression ratios for autosomal genes were close to 1 (ranging from 0.88 to 0.98) (Figure 4A; Table S7). This suggests that global DC has not evolved in sharks.Figure 4 Shark sex chromosomes evolved gene-by-gene dosage compensation

(A) The X-linked genes have a lower m/f expression ratio relative to autosomal genes.

(B) In male tissues, the X-linked genes have lower expression levels relative to autosome genes.

(C) 26 genes (denoted by orange bars) exhibit balanced gene expression between males and females, termed DC genes.

(D) DC genes are more dosage sensitive than non-DC genes.

(E) The expression levels of DC genes are significantly higher than non-DC genes in both females and males.

(F) DC genes tend to be more broadly expressed but without a significantly lower tau value.

(G) Comparison between mammal, avian, sturgeon, and shark sex chromosome features. The bottom and top horizontal lines in the boxplot represent the quartiles, while the middle horizontal lines represent the median. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 (Wilcoxon rank-sum test).

We further compared the expression levels between autosomal genes and X-linked genes in both male and female tissues. While the expression levels are comparable between autosomal and X-linked genes in females, the X-linked genes show lower expression levels in male tissues relative to autosomal genes by a scale of 0.38–0.73 (Figures 4B and S17; Table S8). The approximately halved expression of male X-linked genes is consistent with their reduced dosage. We also investigated whether one of the female X chromosomes was inactivated by calculating the allele frequency of SNPs of the X-linked transcript. Our analysis suggests that the parental alleles are expressed at a similar level (Figure S18), excluding the X-inactivation scenario.

We then calculated the m/f expression ratio for each X-linked gene to detect individually compensated genes. Among the 109 X-linked genes, we identified 26 genes showing balanced gene expression between males and females (Figure 4C), with the m/f expression ratios larger than 0.8. Those genes with local DC (DC genes) have significantly higher (p < 0.05, Wilcoxon rank-sum test) haploinsufficiency scores than those without DC (non-DC genes) (Figure 4D). Moreover, the DC genes have much higher (p < 0.01, Wilcoxon rank-sum test) expression levels than the non-DC genes (Figure 4E) and tend to be more broadly expressed (Figure 4F).

Discussion

Though recent cytogenetic studies have revealed many features for chondrichthyan karyotypes, including large chromosome numbers and the presence of microchromosomes and XY sex chromosomes,11 it has been unclear whether those properties are conserved among chondrichthyans. Our comparative genomic study shows that all chondrichthyan lineages have a slow rate of chromosomal changes. This is consistent with the recent estimate of low mutation rates in sharks.6 We inferred that the vertebrate ancestor likely had around 18 microchromosomes, similar to most chondrichthyans, while birds evolved ∼10 more microchromosomes, likely through additional sequence deletions following the divergence from other lineages.

Despite the overall high degree of chromosome conservation, certain Elasmobranchii lineages experienced more interchromosomal rearrangements, e.g., great hammerhead shark. All interchromosomal changes are fusions without a single fission event, similar to what has been reported in other vertebrate lineages with extensive interchromosomal changes.21,32

Though the X chromosome sequences of the zebra shark and whale shark have been characterized in a recent genomic study,7 it has been unclear when and at what rate the XY sex chromosome evolved in sharks. We characterized the first Chondrichthyes Y chromosomes and revealed a common origin of the XY sex chromosome system in sharks. Previous cytogenetic study suggests that many skates also have XY sex chromosomes,33 but more genomic studies of skate sex chromosomes are needed to confirm whether their XY chromosomes are homologous with the sharks’ ones. If sharks and skates share the XY chromosome system, then it would be older than most other vertebrate sex chromosome systems, including those of therian XY (∼180 Ma)34 and sturgeon ZW (∼180 Ma).35

The shark sex chromosomes, on the other hand, exhibit distinct evolutionary patterns compared with other highly differentiated XY chromosomes. First, while the Y chromosome encompasses abundant satellite DNA, we failed to identify amplicons that are frequently found in mammalian36 and Drosophila24 Y chromosomes. Second, the Y-linked genes do not seem to have a testis-biased expression pattern but are widely expressed across male tissues, in contrast to the Y chromosomes of other lineages.37,38 Third, we found that the shark sex chromosome lacks a mechanism of global DC, which has independently evolved in many sex chromosome systems.39 Finally, we failed to detect a male-determining factor on the Y chromosome, indicating a possible role of an X-linked gene in sex determination given its dosage difference between male and female. All these properties suggest that the shark XY chromosomes, to some degree, remarkably resemble the avian ZW chromosome (Figure 4G), including an absence or sparsity of amplicons25 and non-gonadal biased expression40 of the sex-limited chromosomes, an absence of complete DC,41 and possibly dosage-based mechanism of sex determination.42

Limitations of the study

While we included six chromosome-level chondrichthyan genomes, this does not fully represent the biodiversity of Chondrichthyes. Many other lineages likely have independently experienced chromosomal fusions leading to fewer diploid numbers. Large-scale, high-quality shark genome assembly, as proposed by the Squalomix project,43 will promote our understanding of shark karyotype evolution.

Our Y chromosome assembly may be incomplete, so it is possible that a male-determining gene exists on the Y. Further efforts in improving the Y chromosome assembly and investigations for the role of the X-linked genes in female determination are needed to elucidate the sex-determining mechanism in sharks.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER	
Chemicals, peptides, and recombinant proteins	
	
Phytohemagglutinin (PHA)	SIGMA	Cat#L8902	
Colchicine	MCE	Cat#HY-16569	
Gel Extraction Kit	Omega	Cat#D2500-02	
Nick Translation Kit	Roche	Cat#10976776001	
4′,6-diamidino-2-phenylindole (DAPI)	Invitrogen	Cat#2680173	
QIAGEN® Genomic Kit	QIAGEN	Cat#13343	
	
Deposited data	
	
Assembly results (male)	This study	NCBI: JAWILY000000000, JAWILZ000000000	
Assembly results (female)	This study	NCBI: JBDMBR000000000 and JBDMBS000000000	
Sequencing data (male and female)	This study	NCBI SRA: PRJNA1023715, Table S1	
Raw data of FISH experiment results	This study	Mendeley Data: https://dx.doi.org/10.17632/nt6kx9pkv3.2	
	
Software and algorithms	
	
Hifiasm (v0.16.1)	Cheng et al.42	https://github.com/chhylp123/hifiasm	
Purge_haplotigs (v1.1.2)	Roach et al.43	https://bitbucket.org/mroachawri/purge_haplotigs/src/master/	
Juicer (v1.6)	Durand et al.44	https://github.com/aidenlab/juicer	
3D-DNA (v201008)	Dudchenko et al.45	https://github.com/aidenlab/3d-dna	
RepeatModeler (v2.0.3)	Hubley and Smit46	https://www.repeatmasker.org/RepeatModeler/	
SRF	Zhang et al.47	https://github.com/lh3/srf	
RepeatMasker (v4.1.2-p1)	Smit and Hubley48	http://www.repeatmasker.org/	
Liftoff (v1.6.3)	Shumate and Salzberg49	https://github.com/agshumate/Liftoff	
PASA (v2.5.2)	Haas et al.50	https://github.com/PASApipeline/PASApipeline	
Trinity (v2.15.0)	Grabherr et al.51	https://github.com/trinityrnaseq/trinityrnaseq	
OrthoFinder (v2.5.4)	Emms and Kelly52,53	https://github.com/davidemms/OrthoFinder	
MUSCLE (v3.8.1551)	Edgar54	https://www.drive5.com/muscle/	
Trimal (v1.4.rev22)	Capella-Gutiérrez et al.55	https://github.com/inab/trimal	
IQ-TREE 2 (v2.2.0.3)	Minh et al.56	https://github.com/iqtree/iqtree2	
PAML (v4.9j)	Yang57	https://github.com/abacus-gene/paml	
BWA (v0.7.17-r1188)	Li58	https://github.com/lh3/bwa	
HiCExplorer (v3.6)	Wolff et al.59	https://github.com/deeptools/HiCExplorer	
ggplot2 (v3.4.4)	Wickham60	https://ggplot2.tidyverse.org/	
R (v4.3.2)	R Core Team	https://www.r-project.org/	
MUMmer (v4.0.0rc1)	Marçais et al.61	https://github.com/mummer4/mummer	
odp (v.0.3.0)	Simakov et al.15	https://github.com/conchoecia/odp	
JCVI	Tang et al.62	https://github.com/tanghaibao/jcvi	
BEDtools (v2.30.0)	Quinlan and Hall63	https://bedtools.readthedocs.io/en/latest/	
Minimap2 (v2.26-r1175)	Li64,65	https://github.com/lh3/minimap2	
SAMtools (v1.18)	Li et al.66	http://samtools.sourceforge.net/	
Picard (v.2.27.4)	Broad Institute67	https://broadinstitute.github.io/picard/	
GATK4 (v.4.2.6.1)	Broad institute68	https://gatk.broadinstitute.org/hc/en-us	
BCFtools (v1.16)	Narasimhan et al.69	http://samtools.github.io/bcftools/bcftools.html	
Meryl (v1.4)	Rhie et al.70	https://github.com/marbl/meryl	
StainedGlass (v0.5)	Vollger et al.71	https://github.com/mrvollger/StainedGlass	
Tidehunter (1.5.4)	Gao et al.72	https://github.com/Xinglab/TideHunter	
LASTZ (v1.04.03)	Harris73	https://github.com/lastz/lastz	
Hisat2 (v2.2.1)	Kim et al.74	https://github.com/DaehwanKimLab/hisat2	
edgeR (v3.38.4)	Robinson et al.75	https://bioconductor.org/packages/release/bioc/html/edgeR.html	
ggfortify (v 0.4.16)	Yuan Tang76	https://github.com/sinhrks/ggfortify	

Resource availability

Lead contact

Further information and requests for resources should be directed to and will be fulfilled by the lead contact, Luohao Xu (luohaox@gmail.com).

Materials availability

This study did not generate new unique reagents.

Data and code availability

The HiFi, Hi-C, and RNA-seq reads are deposited under NCBI BioProject PRJNA1023715 (Table S1). The male diploid genomes are deposited under NCBI JAWILY000000000 and JAWILZ000000000, while the female diploid genomes are deposited under NCBI JBDMBR000000000 and JBDMBS000000000. These accession numbers are also listed in the key resources table. All software utilized in this study is listed in the key resources table. The raw images of the FISH experiments presented in Figures 3D and S12 are deposited on Mendeley Data: https://dx.doi.org/10.17632/nt6kx9pkv3.2. This paper does not report original codes.

Experimental model and study participant details

Animals

WSB sharks used in this study were purchased from a fish market in Xiang’an, Xiamen, Fujian province. One male and one female WSB shark were sacrificed for genome sequencing. Two additional male WSB sharks and three female WSB sharks were dissected for gonadal RNA-seq. One additional male WSB shark was sacrificed for the FISH experiment. The Institutional Animal Care and Use Committee (IACUC) of Southwest University has approved the animal ethics.

Method details

Long-read sequencing

High molecular weight genomic DNA was extracted from the muscles of a male and a female WSB shark and was prepared using the CTAB method, followed by purification with QIAGEN Genomic Kit (QIAGEN, Cat#13343) for regular sequencing, according to the standard operating procedure provided by the manufacturer. For HiFi (CCS) sequencing, SMRTbell target size libraries were constructed according to PacBio’s standard protocol (Pacific Biosciences) using the 15 kb preparation solutions. A total of 15 μg DNA per muscle sample was used for the DNA library preparations. The library was treated by nuclease with SMRTbell Enzyme Cleanup Kit and purified by AMPure PB Beads. Sequencing was performed on a PacBio Sequel II instrument with Sequencing Primer V2 and Sequel II Binding Kit 2.0 at Grandomics.

Hi-C sequencing

The muscle tissue from the male individual was chopped and fixed with formaldehyde,44 and was ground into powder. Subsequently, it was re-suspended in nuclei isolation buffers to obtain a nuclear suspension for further processing. The purified nuclei were digested with 100 units of MboI and marked by incubating with biotin-14-dCTP. The ligated DNA was sheared into 300 to 600 bp fragments and then was blunt-end repaired and A-tailed, followed by purification through biotin–streptavidin-mediated pull-down. The Hi-C libraries were quantified and sequenced using the Illumina Hiseq platform (Illumina, San Diego).

RNA-seq

Total RNA from the testis and ovary tissues was extracted by grinding tissue in TRIzol reagent (TIANGEN) on dry ice and processed following the protocol provided by the manufacturer. mRNA molecules were purified from total RNA using oligo(dT)-attached magnetic beads. First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a certain period at the proper temperature. The synthesized cDNA was subjected to end-repair and then was 3′adenylated. Adapters were ligated to the ends of these 3′adenylated cDNA fragments. After quality-checking the library, DNA circularization was performed to generate the final library. The library was amplified with phi29 to make DNA nanoball (DNB) which had more than 300 copies of one molecular. The DNBs were loaded into the patterned nanoarray and paired-end 150 bp reads were generated in the way of combinatorial Probe-Anchor Synthesis (cPAS).

Chromosome-level assembly with Hi-C data

Hifiasm45 (v0.16.1) with Hi-C integration mode was used for genome assembly. This mode produced two sets of haplotype-resolved contigs, named hap1 and hap2. Purge_haplotigs46 (v1.1.2) was used to remove haplotigs with default parameters. The Hi-C sequencing reads were then mapped to the contigs using the Juicer47 (v1.6) pipeline. Following this, the 3D-DNA48 (v201008) pipeline was used to create the ".hic" file. Subsequently, we visualized the Hi-C heatmap using Juicebox Assembly Tools for manual curations. We demarcated chromosome boundaries and adjusted the contig order based on the Hi-C interaction heatmap in Juicebox. We did not perform scaffolding analysis for the female contigs.

Repeat and gene annotation

We used RepeatModeler49 (v2.0.3) to predict repeat consensus sequences and SRF50 (Satellite Repeat Finder) to predict the units of tandem repeats. RepeatMasker51 (v4.1.2-p1) was utilized to annotate the repeat elements using the combined repeat library produced by RepeatModeler and SRF. Gene models were obtained by lifting existing annotations of a published WSB genome (GCA_004010195.1). To do so, we used Liftoff52 (v1.6.3) to generate homology-based gene models. The PASA53 (v2.5.2) pipeline was used to polish the gene models using genome-guided transcripts assembly by Trinity54 (v2.15.0). The RNA-seq data used for this purpose is listed in the Table S9.

Phylogenetics and divergence time estimation

The genomic and coding sequences (CDS) of whale shark (Rhincodon typus, GCA_021869965.1), zebra shark (Stegostoma tigrinum, GCA_022316705.1), WSB shark, great hammerhead (Sphyrna mokarran, GCA_024679065.1), thorny skate (Amblyraja radiata, GCA_010909765.2), elephant fish (Callorhinchus milii, GCA_018977255.1), human (Homo sapiens, GCA_020881995.2), mouse (Mus musculus, GCA_000001635.9), chicken (Gallus gallus, GCA_024206055.2), spotted gar (Lepisosteus oculatus, GCA_000242695.1), and Nile tilapia (Oreochromis niloticus, GCA_001858045.3) were used to construct a phylogenetic tree and perform comparative genomics analysis. The phylogenetic tree was constructed using 3222 single-copy orthologous genes identified by OrthoFinder55,56 (v2.5.4). The CDS sequences were aligned using MUSCLE57 (v3.8.1551), and the alignments were further filtered by the Trimal58 (v1.4.rev22) with the parameter set to “-gt 0.1”. IQ-TREE 259 (v2.2.0.3) was used to construct the maximum likelihood tree based on concatenated single-copy orthologous genes, and the best substitution model GTR+F+R5 automatically selected by ModelFinder for CDS alignments.

The divergence time of the selected representative species was calculated using the third position base of codons from the single-copy orthologous genes. The MCMCTree software in the PAML60 (v4.9j) package was used to estimate the divergence time among different species. The following parameter settings of MCMCTree were used: “clock = 3, RootAge = <5.0, model = 4, BDparas = 1 1 0, kappa_gamma = 6 2, alpha_gamma = 1 1, rgene_gamma = 2 2, sigma2_gamma = 1 10”. The fossil calibration nodes used are: 65.6–164.6 million years for the human and mouse split, 318–332.9 million years for the chicken and mammal split, and 378.2–422.4 million years for the zebrafish to spotted gar split.61 The root calibration node used is 420.7–460.0 million years.62

Hi-C chromatin interaction

We used BWA-MEM63 (v0.7.17-r1188) algorithm to map Hi-C reads with the parameters “-A 1 -B 4 -E 50 -L 0 -t 16”. Subsequently, the hicBuildMatrix function from the HiCExplorer64 (v3.6) was used with the parameters “--binSize 100000, --inputBufferSize 400000, --restrictionSequence GATC”, resulting in an output matrix file. Then, the Hi-C matrix was corrected using hicCorrectMatrix with default parameters to normalize the number of restriction sites per bin and remove open chromatin biases. The resulting h5 file was converted to ginteractions data using hicConvertFormat, which was used as input to calculate the frequency of chromosomal interactions with custom Perl scripts (https://github.com/lurebgi/chicken-T2T/blob/main/Hi-C_analysese).

Principal component analysis (PCA)

We calculated the GC content, log10-transformed chromosome sizes, CDS density and the percentage of repeat content for each chromosome for PCA analysis. The PCA analysis was performed using the ggfortify65 (v 0.4.16) package in R (v4.3.2). The source data is available in Table S10.

Chromosomal synteny

Six chromosome-scale chondrichthyan genomes (Table S11) were used for analyses. We used the MCscan pipeline (implemented in JCVI66) to identify and visualize the chromosome synteny in a pairwise manner. Homologous chromosomes were identified through pairwise comparison. We discarded the unanchored scaffolds, and then connected the five pairwise synteny into a multiway synteny. MCscan synteny inference relies on orthologous gene blocks. To further validate the homologous chromosomal pairs identified by MCscan, we used MUMmer67 (v4.0.0rc1) tool nucmer to perform the pairwise whole-genome alignment with the parameter “-b 500”. The alignments were filtered to retain the one-to-one best hits using delta-filt from the MUMmer package. To infer the chondrichthyan ancestral chromosomes, we added the chicken genome as the outgroup. When a chromosome was shared by all chondrichthyan members without interchromosomal changes, it was directly inferred as an ancestral chromosome; when there was disparity between the two main lineages of chondrichthyes (Elasmobranchii and Holocephali), we relied on chicken to determine the ancestral status. Similarly, we inferred the ancestral chromosomes for all internal nodes of Elasmobranchii. To investigate the evolutionary history of chromosomal changes in chondrichthyes, we collected the previously identified proto-chordate68 and proto-vertebrate13 linkage groups. We mapped the 23 proto-chordate and 45 proto-vertebrate chromosomes to the genomes of WSB shark, elephant fish and chicken. We performed synteny analysis using odp15 (v.0.3.0) to generate Oxford dot-plot using an amphioxus genome (GCA_019207075.1) as the outgroup.

Identification of sex chromosomes

We used short-reads generated from a female and a male (Table S1) to calculate the ratio of female-to-male (f/m) sequencing depth to identify the sex chromosomes. The reads were mapped to the assembled genome using BWA-MEM63 (v0.7.17-r1188), and then BEDtools69 (v2.30.0) was used to calculate the counts of covered sites and sequencing depth in 50 kb windows. Subsequently, the f/m ratios of covered sites (coverage ratio) and sequencing depth were calculated. Given that the Y chromosome is highly differentiated from the X, it was expected to have f/m ratios for covered sites and depth close to zero, while the X chromosome was expected to have an autosome-like f/m mapped site ratio but a 1/2 f/m depth ratio. To visualize the coverage pattern on the Y, we mapped the male and female HiFi reads using Minimap270,71 (-x map-hifi, v2.26-r1175), and calculated the depths over 50 kb windows. We also visualized the intra-chromosomal chromatin interaction pattern of the Y using the heatmap produced by the juicebox program.

We further called SNP for validating the X and Y chromosomes using short-reads, expecting that the Y chromosome was effectively hemizygous in males. To increase the specificity of read mapping, we used BWA (v0.7.17-r1188) aln, instead of the mem algorism. Subsequently, the aligned bam files were sorted using SAMtools72 (v1.18), and duplicates generated during library preparation PCR were marked using the MarkDuplicates module of Picard73 (v.2.27.4). SNP calling was performed using the HaplotypeCaller module of GATK474 (v.4.2.6.1) with the filtering criteria "QD < 2.0, MQ < 40.0, FS > 60.0, SOR >3.0, MQRankSum < −12.5, and ReadPosRankSum < −8.0". SNPs with an allelic fraction (AF) equal to 0.5 were retained. The filtered variants were then further processed using BCFtools75 (v1.16) to remove sites with read depth (DP) less than one-third of the average depth or greater than three times the average depth. Subsequently, SNP counts were calculated for each chromosome using a 50 kb window size. We randomly selected an autosome (chr12) as a control.

K-mer analyses

Meryl76 (v1.4) was utilized to extract k-mers from male and female HiFi reads. The data is initially filtered using the "meryl greater-than 3″ parameter to select k-mers present in more than three instances. Subsequently, the "meryl difference" parameter was employed to determine the sex-specific k-mers. Contigs smaller than 0.5 Mb were discarded. For each contig, intersections with male-specific k-mers are performed using the "meryl intersect" parameter. This process identifies the male-specific k-mers present on each contig. The number of male-specific k-mers of each contig was further scaled by the contig length. A similar methodology is applied to analyze k-mers for female contigs.

Chromosome preparation

We obtained a male WSB shark and prepared chromosomes using the phytohemagglutinin (PHA)-colchicine intraperitoneal injection method. Initially, we injected PHA (SIGMA, Cat#L8902) at a dose of 10 μg/g, followed by an injection of colchicine (MCE, Cat#HY-16569) at a dose of 4 μg/g after 24 h. After 4 h, we anesthetized and dissected the shark, extracted the kidney, and washed it with physiological saline. The kidney was then placed in a beaker with an appropriate amount of physiological saline, chopped finely, and allowed to stand for 3-5 min to collect the cell suspension. The cell suspension was then centrifuged at 1000r/min for 10 min, the supernatant was discarded, and the cells were collected. Subsequently, the cells were subjected to a 40-min treatment with 0.075 mol/L KCl at room temperature, followed by centrifugation at 1000r/min for 10 min, and removal of the hypotonic solution. Next, the cells were fixed twice with freshly prepared Carnoy’s fixative (methanol to glacial acetic acid ratio of 3:1), each time for 40 min. After fixation, the cells were thoroughly mixed with an appropriate amount of fixative. Finally, the prepared cell suspension was dropped onto glass slides and air-dried.

FISH experiment

StainedGlass77 (v0.5) was used to visualize the distribution of satellite DNA, and Tidehunter78 (1.5.4) was used to identify the units of satellite DNA. We selected one of the satellite DNA units 46_rep254 (Figure S11) specific to the Y chromosome to prepare the FISH experiment probes: GATAGGGCGGCGCTCCTAGGTCTCGATGTATTGAGTCCTCCTGGCC. Primers used are shown in the Table S12. Initially, the target segment was amplified via PCR. followed by agarose gel electrophoresis to confirm the target bands. Subsequently, the target DNA fragments were recovered and purified using the Gel Extraction Kit (Omega, Cat#D2500-02). Probe labeling was conducted using the Nick Translation Kit (Roche, Cat#10976776001). Chromosomal slides were first subjected to enzymatic digestion, following by washing, fixation, dehydration, chromosome denaturation, probe denaturation, and hybridization steps, with specific procedures described in Xue et al.79 Then, the slides were stained with DAPI (Invitrogen, Cat#2680173) for 10 min at room temperature, followed by washing three times with 1×PBS. Finally, images were captured using a laser confocal microscope (Olympus FV3000, Japan).

Evolutionary strata

The X and Y chromosome sequences were masked for repeats before performing the LASTZ80 (v1.04.03) alignment. A relaxed set of parameters (--hspthresh = 2200 --inner = 2000 --ydrop = 3400 --gappedthresh = 10000) was used to align the Y chromosome to the X chromosome. Alignment chains and nets were further produced to join the syntenic fragmented alignments into longer blocks. Subsequently, alignments with a sequence similarity lower than 60% were filtered out to reduce the false positives. Next, we calculated the sequence similarity between the X and Y chromosomes using 50-kb sliding windows along the Y chromosome. Finally, the ggplot2 (v3.4.4) package was used for visualizing the X-Y sequence similarity.

Dosage compensation

RNA-seq data from 12 tissues of the male and female WSB sharks (Table S13) were used to assess the presence and extent of sex chromosome dosage compensation. The RNA-seq data all came from Zhang et al. (2020).16 We used Hisat281 (v2.2.1) to map the RNA-seq reads to our assembled genome. The male-to-female (m/f) gene expression ratio was analyzed in R (v4.1) using edgeR82 (v3.38.4). Genes with less than 10 counts in all samples were removed. This filtered gene expression matrix was then normalized by the GETMM83 method. Normalization factors were then calculated by the calcNormFactors function. The estimateCommonDisp followed by the estimateTagwiseDisp function was used to estimate negative binomial dispersions. Normalized counts per million (CPM) values were then calculated by the cpm function. The fold change ratios (logFC) of male over female gene expression levels were extracted from the exactTest function results. Genes with an average expression level greater than 0.8 across m/f in each tissue were considered to have local dosage compensation. Human haploinsufficiency scores84 were extracted and projected to the WSB shark according to the 1:1 orthologous relationship between WSB shark and human genes. We only retained the genes that have reported human haploinsufficiency scores.

To determine whether both X chromosomes of the female bamboo sharks are expressed, we calculated the allele frequency of SNPs located on the transcripts of X-linked genes. We mapped RNA-seq data from female tissues (Table S13) to the hap1 genome using Hisat2.81 Then, we performed SNP calling with GATK,74 filtering out SNP sites with depths less than 20. We extracted the allelic depth (AD) values of SNP sites located on X chromosome exons to calculate allele frequency. The chromosome 32 was used as a control.

Supplemental information

Document S1. Figures S1–S18 and Tables S1–S5 and S7–S13

Table S6. Excel file containing additional data too large to fit in a PDF, related to Figure S12

Document S2. Transparent peer review records for Jiahong Wu et al

Document S3. Article plus supplemental information

Acknowledgments

We thank Kun Wang (Northwestern Polytechnical University) for his helpful comments. This work is supported by the 10.13039/501100001809 National Natural Science Foundation of China (32370445 ), the Chongqing Science Fund for Distinguished Young Scholars, the special fund for youth teams of the 10.13039/501100006250 Southwest University (SWU-XJPY202302 ), and Science Foundation of School of Life Sciences SWU (20232022020201 ) to L.X. and the Chongqing Innovation Program for Graduate Research (CYB23114 ) to J.W.

Author contributions

L.X. conceived and designed the project. F.L. collected the experimental samples. J.W., J.J., H.L., S.F., H.W., and N.C. performed data analysis. J.W. and J.L. conducted the fluorescence in situ hybridization experimental analysis. L.X. and J.W. wrote the manuscript. S.K., Q.Q., and Z.H. participated in revising the manuscript.

Declaration of interests

The authors declare no competing interests.

Supplemental information can be found online at https://doi.org/10.1016/j.xgen.2024.100607.
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