New insights into population structure, demographic history, and effective population size of the critically endangered blue shark Prionace glauca in the Mediterranean Sea.

The blue shark, Prionace glauca, is the most abundant pelagic shark in the open ocean but its vulnerability remains poorly understood while being one of the most fecund sharks. In the Mediterranean Sea, the blue shark is listed as Critically Endangered (CR) by the International Union for Conservation of Nature. The species is facing a strong decline due to fishing, and scientific data regarding its genetic structure and vulnerability are still lacking. Here, we investigated the genetic diversity, demographic history, and population structure of the blue shark within the Mediterranean Sea, from samples of the Gulf of Lion and Malta, using sequences of the mtDNA control region and 22 microsatellite markers. We also compared our mitochondrial data to previous studies to examine the Atlantic-Mediterranean population structure. We assessed the blue shark's genetic vulnerability in the Mediterranean basin by modelling its effective population size. Our results showed a genetic differentiation between the Atlantic and the Mediterranean basins, with limited gene flow between the two areas, and distinct demographic histories making the Mediterranean population an independent management unit. Within the Mediterranean Sea, no sign of population structure was detected, suggesting a single population across the Western and Central parts of the sea. The estimated effective population size was low and highlighted the high vulnerability of the Mediterranean blue shark population, as the estimated size we calculated might not be sufficient to ensure the long-term persistence of the population. Our data also provide additional evidence that the Gulf of Lion area acts as a nursery for P. glauca, where protection is essential for the conservation strategy of the species in the Mediterranean.

Maternal investment evolves with larger body size and higher diversification rate in sharks and rays.

Across vertebrates, live bearing evolved at least 150 times from ancestral egg laying into diverse forms and degrees of prepartum maternal investment.1,2 A key question is how reproductive diversity arose and whether reproductive diversification underlies species diversification.3,4,5,6,7,8,9,10,11 To test this, we evaluate the most basal jawed vertebrates: the sharks, rays, and chimaeras, which have one of the greatest ranges of reproductive and ecological diversity among vertebrates.2,12 We reconstruct the sequence of reproductive mode evolution across a phylogeny of 610 chondrichthyans.13 We reveal egg laying as ancestral, with live bearing evolving at least seven times. Matrotrophy evolved at least 15 times, with evidence of one reversal. In sharks, transitions to live bearing and matrotrophy are more prevalent in larger-bodied tropical species. Further, the evolution of live bearing is associated with a near doubling of the diversification rate, but there is only a small increase associated with the appearance of matrotrophy. Although pre-copulatory sexual selection is associated with increased rates of speciation in teleosts,3 sexual size dimorphism in chondrichthyans does not appear to be related to sexual selection,14,15 and instead we find increased rates of speciation associated with the colonization of novel habitats. This highlights a potential key difference between chondrichthyans and other fishes, specifically a slower rate of evolution of reproductive isolation following speciation, suggesting different rate-limiting mechanisms for diversification between these clades.16 The chondrichthyan diversification and radiation, particularly throughout shallow tropical shelf seas and oceanic pelagic habitats, appear to be associated with the evolution of live bearing and proliferation of a wide range of maternal investment in developing offspring.

Antigen specific VNAR screening in whitespotted bamboo shark (Chiloscyllium plagiosum) with next generation sequencing.

IgNAR exhibits significant promise in the fields of cancer and anti-virus biotherapies. Notably, the variable regions of IgNAR (VNAR) possess comparable antigen binding affinity with much smaller molecular weight (∼12 kDa) compared to IgNAR. Antigen specific VNAR screening is a changeling work, which limits its application in medicine and therapy fields. Though phage display is a powerful tool for VNAR screening, it has a lot of drawbacks, such as small library coverage, low expression levels, unstable target protein, complicating and time-consuming procedures. Here we report VANR screening with next generation sequencing (NGS) could effectively overcome the limitations of phage display, and we successfully identified approximately 3000 BAFF-specific VNARs in Chiloscyllium plagiosum vaccinated with the BAFF antigen. The results of modelling and molecular dynamics simulation and ELISA assay demonstrated that one out of the top five abundant specific VNARs exhibited higher binding affinity to the BAFF antigen than those obtained through phage display screening. Our data indicates NGS would be an alternative way for VNAR screening with plenty of advantages.

Shark genome size evolution and its relationship with cellular, life-history, ecological, and diversity traits.

Among vertebrates, sharks exhibit both large and heterogeneous genome sizes ranging from 2.86 to 17.05 pg. Aiming for a better understanding of the patterns and causalities of shark genome size evolution, we applied phylogenetic comparative methods to published genome-size estimates for 71 species representing the main phylogenetic lineages, life-histories and ecological traits. The sixfold range of genome size variation was strongly traceable throughout the phylogeny, with a major expansion preceding shark diversification during the late Paleozoic and an ancestral state (6.33 pg) close to the present-day average (6.72 pg). Subsequent deviations from this average occurred at higher rates in squalomorph than in galeomorph sharks and were unconnected to evolutionary changes in the karyotype architecture, which were dominated by descending disploidy events. Genome size was positively correlated with cell and nucleus sizes and negatively with metabolic rate. The metabolic constraints on increasing genome size also manifested at higher phenotypic scales, with large genomes associated with slow lifestyles and purely marine waters. Moreover, large genome sizes were also linked to non-placental reproductive modes, which may entail metabolically less demanding embryological developments. Contrary to ray-finned fishes, large genome size was associated neither with the taxonomic diversity of affected clades nor with low genetic diversity.

Insights into the nuclear and mitochondrial genome of the Lemon shark Negaprion brevirostris using low-coverage sequencing: Genome size, repetitive elements, mitochondrial genome, and phylogenetic placement.

The Lemon shark Negaprion brevirostris is an important species experiencing conservation issues that is in need of genomic resources. Herein, we conducted a genome survey sequencing in N. brevirostris and determined genome size, explored repetitive elements, assembled and annotated the 45S rRNA DNA operon, and assembled and described in detail the mitochondrial genome. Lastly, the phylogenetic position of N. brevirostris in the family Carcharhinidae was examined using translated protein coding genes. The estimated haploid genome size ranged between 2.29 and 2.58 Gbp using a k-mer analysis, which is slightly below the genome size estimated for other sharks belonging to the family Carcharhinidae. Using a k-mer analysis, approx. 64-71 % of the genome of N. brevirostris was composed of repetitive elements. A relatively large proportion of the 'repeatome' could not be annotated. Taking into account only annotated repetitive elements, Class I - Long Interspersed Nuclear Element (LINE) were the most abundant repetitive elements followed by Class I - Penelope and Satellite DNA. The nuclear ribosomal operon was fully assembled. The AT-rich complete mitochondrial genome was 16,703 bp long and encoded 13 protein coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. Negaprion brevirostris is closely related to the genera Carcharhinus, Glyphis and Lamiopsis in the family Carcharinidae. This new genomic resources will aid with the development of conservation plans for this large coastal shark.

Genetic identification of three CITES-listed sharks using a paper-based Lab-on-a-Chip (LOC).

Threatened shark species are caught in large numbers by artisanal and commercial fisheries and traded globally. Monitoring both which shark species are caught and sold in fisheries, and the export of CITES-restricted products, are essential in reducing illegal fishing. Current methods for species identification rely on visual examination by experts or DNA barcoding techniques requiring specialist laboratory facilities and trained personnel. The need for specialist equipment and/or input from experts means many markets are currently not monitored. We have developed a paper-based Lab-on-a-Chip (LOC) to facilitate identification of three threatened and CITES-listed sharks, bigeye thresher (Alopias superciliosus), pelagic thresher (A. pelagicus) and shortfin mako shark (Isurus oxyrinchus) at market source. DNA was successfully extracted from shark meat and fin samples and combined with DNA amplification and visualisation using Loop Mediated Isothermal Amplification (LAMP) on the LOC. This resulted in the successful identification of the target species of sharks in under an hour, with a working positive and negative control. The LOC provided a simple "yes" or "no" result via a colour change from pink to yellow when one of the target species was present. The LOC serves as proof-of-concept (PoC) for field-based species identification as it does not require specialist facilities. It can be used by non-scientifically trained personnel, especially in areas where there are suspected high frequencies of mislabelling or for the identification of dried shark fins in seizures.

Molecular identification of sharks from the genus Sphyrna (Elasmobranchii: Chondrichthyes) in Maranhão Coast (Brazil).

Sharks of the genus Sphyrna are under intense exploitation globally. In Brazil's northern coast, this genus represents a high proportion of fisheries landings and comprises four species. However, due to difficulty of specific identification when specimens are landed, most of the records are limited to the genus level. Here we analyzed the effectiveness of ITS2 (Internal Transcribed Spacer 2 of rDNA) fragment length protocol (Abercrombie et al., 2005) for identifying hammerhead shark species, comparing with the analysis of COI (Cytochrome oxidase subunit I) and ITS2 sequences. We evaluated samples of muscle tissue acquired in the main fishing ports of Maranhão: Carutapera, Raposa e Tutóia. Sampling was conducted between March 2017 to March 2018 and complemented with material deposited in collection (2015). COI results indicated the occurrence of endangered species which are prohibited to be landed. These include Sphyrna mokarran (67%), S. lewini (15%), S. tudes (3%), and S. tiburo (15%). For the ITS2 marker, we investigated the optimization of the protocol developed by Abercrombie (2005) for to improve the use in this geographical area througout design of a new primers.

First molecular identification and phylogenetic illustration of Sarcocystis species infection in Red Sea shortfin mako shark (Isurus oxyrinchus Rafinesque, 1810).

BACKGROUND: members of the genus Sarcocystis are intracellular obligate protozoan parasites classified within the phylum Apicomplexa and have an obligate heteroxenous life cycle involving two hosts. A more comprehensive understanding of the prevalence and geographic range of different Sarcocystis species in marine ecosystems is needed globally and nationally. Hence, the objective of this study was to document the incidence of Sarcocystis infection in sharks within the aquarium ecosystem of Egypt and to identify the species through the characterization of the SSU rDNA gene. METHODS: All organs of the mako shark specimen underwent macroscopic screening to detect the existence of a Sarcocystis cyst. Ten cysts were collected from the intestine and processed separately to extract the genomic DNA. The polymerase chain reaction (PCR) was accomplished by amplifying a specific 18S ribosomal RNA (rRNA) gene fragment. Subsequently, the resulting amplicons were subjected to purification and sequencing processes. RESULTS: Macroscopic examination of the mako shark intestinal wall sample revealed the presence of Sarcocystis cysts of various sizes and shapes, and sequencing of the amplicons from Sarcocystis DNA revealed a 100% nucleotide identity with the sequence of Sarcocystis tenella recorded from sheep in Iran; The mako shark sequence has been deposited in the GeneBank with the accession number OQ721979. This study presents the first scientific evidence demonstrating the presence of the Sarcocystis parasite in sharks, thereby documenting this specific marine species as a novel intermediate host in the Sarcocystis life cycle. CONCLUSIONS: This is the first identification of Sarcocystis infection in sharks, and we anticipate it will be an essential study for future screenings and establishing effective management measures for this disease in aquatic ecosystems.

De novo transcriptome for Chiloscyllium griseum, a long-tail carpet shark of the Indian waters.

Sharks have thrived in the oceans for 400 million years, experienced five extinctions and evolved into today's apex predators. However, enormous genome size, poor karyotyping and limited tissue sampling options are the bottlenecks in shark research. Sharks of the family Orectolobiformes act as model species in transcriptome research with exceptionally high reproductive fecundity, catch prominence and oviparity. The present study illustrates a de novo transcriptome for an adult grey bamboo shark, Chiloscyllium griseum (Chondrichthyes; Hemiscyllidae) using paired-end RNA sequencing. Around 150 million short Illumina reads were obtained from five different tissues and assembled using the Trinity assembler. 70,647 hits on Uniprot by BLASTX was obtained after the transcriptome annotation. The data generated serve as a basis for transcriptome-based population genetic studies and open up new avenues in the field of comparative transcriptomics and conservation biology.

Diversity and genetic structure of freshwater shark Wallago attu: an emerging species of commercial interest.

Pakistan has natural freshwater resources acting as a hotspot for diverse fish fauna. However, this aquatic fauna is declining at an alarming rate due to over-exploitation, habitat degradation, water pollution, climate change, and certain anthropogenic activities. The freshwater shark, Wallago attu, is a popular edible catfish inhabiting these freshwater ecosystems. Habitat degradation, overfishing, and human activities are heavily impacting the natural population of this species. So, sound knowledge about its population structure is necessary for its proper management in natural waters. The current study involves utilizing two mtDNA markers (COI, Cytb) to assess the genetic structure and differentiation among W. attu populations of Pakistani Rivers. Genetic variability analysis indicated a high haplotype (0.343 ± 0.046-0.870 ± 0.023) and low nucleotide diversity (0.0024 ± 0.012-0.0038 ± 0.018) among single and combined gene sequences, respectively. Overall, River Indus was populated with more diverse fauna of Wallago attu as compared to River Chenab and River Ravi. Population pairwise, Fst values (0.40-0.61) were found to be significantly different (p < 0.01) among three Riverine populations based upon combined gene sequences. The gene flow for the combined gene (COI + Cytb) dataset among three populations was less than 1.0. The transition/transversion bias value R (0.58) was calculated for testing of neutral evolution, and it declared low genetic polymorphism among natural riverine populations of Wallago attu. The current study's findings would be meaningful in planning the management and conservation of this economically important catfish in future.

DNA barcoding continues to identify endangered species of shark sold as food in a globally significant shark fin trade hub.

Shark fins are a delicacy consumed throughout Southeast Asia. The life history characteristics of sharks and the challenges associated with regulating fisheries and the fin trade make sharks particularly susceptible to overfishing. Here, we used DNA barcoding techniques to investigate the composition of the shark fin trade in Singapore, a globally significant trade hub. We collected 505 shark fin samples from 25 different local seafood and Traditional Chinese Medicine shops. From this, we identified 27 species of shark, three species are listed as Critically Endangered, four as Endangered and ten as Vulnerable by the International Union for Conservation of Nature (IUCN). Six species are listed on CITES Appendix II, meaning that trade must be controlled in order to avoid utilization incompatible with their survival. All dried fins collected in this study were sold under the generic term "shark fin"; this vague labelling prevents accurate monitoring of the species involved in the trade, the effective implementation of policy and conservation strategy, and could unwittingly expose consumers to unsafe concentrations of toxic metals. The top five most frequently encountered species in this study are Rhizoprionodon acutus, Carcharhinus falciformis, Galeorhinus galeus, Sphyrna lewini and Sphyrna zygaena. Accurate labelling that indicates the species of shark that a fin came from, along with details of where it was caught, allows consumers to make an informed choice on the products they are consuming. Doing this could facilitate the avoidance of species that are endangered, and similarly the consumer can choose not to purchase species that are documented to contain elevated concentrations of toxic metals.

A comprehensive phylogenomic study unveils evolutionary patterns and challenges in the mitochondrial genomes of Carcharhiniformes: A focus on Triakidae.

The complex evolutionary patterns in the mitochondrial genome (mitogenome) of the most species-rich shark order, the Carcharhiniformes (ground sharks) has led to challenges in the phylogenomic reconstruction of the families and genera belonging to the order, particularly the family Triakidae (houndsharks). The current state of Triakidae phylogeny remains controversial, with arguments for both monophyly and paraphyly within the family. We hypothesize that this variability is triggered by the selection of different a priori partitioning schemes to account for site and gene heterogeneity within the mitogenome. Here we used an extensive statistical framework to select the a priori partitioning scheme for inference of the mitochondrial phylogenomic relationships within Carcharhiniformes, tested site heterogeneous CAT + GTR + G4 models and incorporated the multi-species coalescent model (MSCM) into our analyses to account for the influence of gene tree discordance on species tree inference. We included five newly assembled houndshark mitogenomes to increase resolution of Triakidae. During the assembly procedure, we uncovered a 714 bp-duplication in the mitogenome of Galeorhinus galeus. Phylogenetic reconstruction confirmed monophyly within Triakidae and the existence of two distinct clades of the expanded Mustelus genus. The latter alludes to potential evolutionary reversal of reproductive mode from placental to aplacental, suggesting that reproductive mode has played a role in the trajectory of adaptive divergence. These new sequences have the potential to contribute to population genomic investigations, species phylogeography delineation, environmental DNA metabarcoding databases and, ultimately, improved conservation strategies for these ecologically and economically important species.

A singular shark bitter taste receptor provides insights into the evolution of bitter taste perception.

Many animal and plant species synthesize toxic compounds as deterrent; thus, detection of these compounds is of vital importance to avoid their ingestion. Often, such compounds are recognized by taste 2 receptors that mediate bitter taste in humans. Until now, bitter taste receptors have only been found in bony vertebrates, where they occur as a large family already in coelacanth, a "living fossil" and the earliest-diverging extant lobe-finned fish. Here, we have revisited the evolutionary origin of taste 2 receptors (T2Rs) making use of a multitude of recently available cartilaginous fish genomes. We have identified a singular T2R in 12 cartilaginous fish species (9 sharks, 1 sawfish, and 2 skates), which represents a sister clade to all bony fish T2Rs. We have examined its ligands for two shark species, a catshark and a bamboo shark. The ligand repertoire of bamboo shark represents a subset of that of the catshark, with roughly similar thresholds. Amarogentin, one of the most bitter natural substances for humans, also elicited the highest signal amplitudes with both shark receptors. Other subsets of ligands are shared with basal bony fish T2Rs indicating an astonishing degree of functional conservation over nearly 500 mya of separate evolution. Both shark receptors respond to endogenous steroids as well as xenobiotic compounds, whereas separate receptors exist for xenobiotics both in early- and late-derived bony vertebrates (coelacanth, zebrafish, and human), consistent with the shark T2R reflecting the original ligand repertoire of the ancestral bitter taste receptor at the evolutionary origin of this family.

Evolution of reproductive modes in sharks and rays.

The ecological and life history drivers of the diversification of reproductive modes in early vertebrates are not fully understood. Sharks, rays and chimaeras (group Chondrichthyes) have an unusually diverse variety of reproductive modes and are thus an ideal group to test the factors driving the evolution of reproductive complexity. Here, using 960 species representing all major Chondrichthyes taxa, we reconstruct the evolution of their reproduction modes and investigate the ecological and life history predictors of reproduction. We show that the ancestral Chondrichthyes state was egg-laying and find multiple independent transitions between egg-laying and live-bearing via an intermediate state of yolk-only live-bearing. Using phylogenetically informed analysis, we also show that live-bearing species have larger body size and larger offspring than egg-laying species. In addition, live-bearing species are distributed over shallow to intermediate depths, while egg-layers are typically found in deeper waters. This suggests that live-bearing is more closely associated with pelagic, rather than demersal habitats. Taken together, using a basal vertebrate group as a model, we demonstrat how reproductive mode co-evolves with environmental conditions and life-history traits.

Genetic stock structure of the silky shark Carcharhinus falciformis in the Indo-Pacific Ocean.

The silky shark, Carcharhinus falciformis, is a cosmopolitan species commonly caught as a bycatch for longline fisheries. However, the genetic stock structure for the Indo-Pacific Ocean is not well-defined yet. Here, we used eight microsatellite loci to examine the genetic stock structure and effective population size of 307 silky sharks across 5 Indo-Pacific sampling locations. A major genetic break was found between Aceh and the remaining locations (FST = 0.0505-0.0828, p = 0.001). The Indian Ocean displayed a slightly lower effective population estimate (Ne) compared to the Pacific Ocean, potentially due to the higher fishing pressure in the Indian Ocean region. The lowest Ne was found in the Aceh population (Ne = 2.3), suggesting it might be a small and endemic population. These findings offer valuable information for the conservation and management of the silky shark. We suggest that the population around Aceh waters constitutes a distinct stock and should be managed independently. Further investigations into migratory and movement patterns are needed to define the boundaries of different stocks, ensuring effective management the silky shark across the Indo-Pacific region.

Low mutation rate in epaulette sharks is consistent with a slow rate of evolution in sharks.

Sharks occupy diverse ecological niches and play critical roles in marine ecosystems, often acting as apex predators. They are considered a slow-evolving lineage and have been suggested to exhibit exceptionally low cancer rates. These two features could be explained by a low nuclear mutation rate. Here, we provide a direct estimate of the nuclear mutation rate in the epaulette shark (Hemiscyllium ocellatum). We generate a high-quality reference genome, and resequence the whole genomes of parents and nine offspring to detect de novo mutations. Using stringent criteria, we estimate a mutation rate of 7×10-10 per base pair, per generation. This represents one of the lowest directly estimated mutation rates for any vertebrate clade, indicating that this basal vertebrate group is indeed a slowly evolving lineage whose ability to restore genetic diversity following a sustained population bottleneck may be hampered by a low mutation rate.

Endangered shark species traded as "cação" in São Paulo during the COVID-19 lockdown: DNA-barcoding a snapshot of products.

BACKGROUND: Elasmobranch populations are declining, predominantly driven by overfishing, and over a third of global sharks, rays, and chimeras are estimated to be threatened with extinction. In terms of trade, Brazil is ranked the eleventh-largest shark producer and the top importer of shark meat in the world. Research has shown that elasmobranchs are sold in Brazil under the name "cação" (a generic designation for cartilaginous fish) to overcome consumer resistance. METHODOLOGY AND RESULTS: This study used DNA barcoding to investigate the sale of sharks in the State of São Paulo during the COVID-19 lockdown. A total of 35 samples of "cação" were analysed, revealing six different shark species on sale, including Carcharhinus falciformis, Carcharhinus signatus, Carcharias taurus, Isurus oxyrinchus, and Isurus paucus, that are threatened with extinction according to the IUCN red list. This study demonstrates that vulnerable elasmobranchs are being commercialised under the label "cação" in the São Paulo State and Brazil. CONCLUSIONS: Comparison of shark products traded before and during the COVID-19 pandemic showed no significant difference, suggesting lockdown did not affect patterns of species commercialisation. Effective fisheries and sale monitoring, correct product labelling legislation and increased consumer awareness that "cação" is shark are needed for appropriate conservation and management of shark populations in Brazil.

Genetic evidence for plastic reproductive philopatry and matrotrophy in blacktip reef sharks (Carcharhinus melanopterus) of the Moorea Island (French Polynesia).

The exploitation of sharks and the degradation of their habitats elevate the urgency to understand the factors that influence offspring survival and ultimately shark reproductive success. We monitored and sampled blacktip reef sharks (Carcharhinus melanopterus) in nursery habitats of Moorea Island (French Polynesia), to improve knowledge on shark reproductive behavior and biology. We sampled fin clips and morphometrics from 230 young-of-the-year sharks and used microsatellite DNA markers to process parentage analysis to study the reproductive philopatric behavior in female sharks and the matrotrophy within litters. These traits are driving the success of the local replenishment influencing selection through birth site and maternal reserves transmitted to pups. Parentage analysis revealed that some female sharks changed their parturition areas (inter-seasonally) while other female sharks came back to the same site for parturition, providing evidence for a plastic philopatric behavior. Morphometrics showed that there was no significant relationship between body condition indices and nursery locations. However, similarities and differences in body condition were observed between individuals sharing the same mother, indicating that resource allocation within some shark litters might be unbalanced. Our findings further our understanding of the reproductive biology and behavior that shape shark populations with the aim to introduce these parameters into future conservation strategies.

DNA metabarcoding of cloacal swabs provides insight into diets of highly migratory sharks in the Mid-Atlantic Bight.

The abundances of migratory shark species observed throughout the Mid-Atlantic Bight (MAB) during productive summer months suggest that this region provides critical habitat and prey resources to these taxa. However, the principal prey assemblages sustaining migratory shark biomass in this region are poorly defined. We applied high-throughput DNA metabarcoding to shark feces derived from cloacal swabs across nine species of Carcharhinid and Lamnid sharks to (1) quantify the contribution of broad taxa (e.g., invertebrates, fishes) supporting shark biomass during seasonal residency in the MAB and (2) determine whether the species displayed distinct dietary preference indicative of resource partitioning. DNA metabarcoding resulted in high taxonomic (species-level) resolution of shark diets with actinopterygian and elasmobranch fishes as the dominant prey categories across the species. DNA metabarcoding identified several key prey groups consistent across shark taxa that are likely integral for sustaining their biomass in this region, including Atlantic menhaden (Brevoortia tyrannus), Atlantic mackerel (Scomber scombrus), and benthic elasmobranchs, including skates. Our results are consistent with previously published stomach content data for the shark species of similar size range in the Northwest Atlantic Ocean, supporting the efficacy of cloacal swab DNA metabarcoding as a minimally invasive diet reconstruction technique. The high reliance of several shark species on Atlantic menhaden could imply wasp-waist food-web conditions during the summer months, whereby high abundances of forage fishes sustain a diverse suite of migratory sharks within a complex, seasonal food web.

Elasmobranch genome sequencing reveals evolutionary trends of vertebrate karyotype organization.

Genomic studies of vertebrate chromosome evolution have long been hindered by the scarcity of chromosome-scale DNA sequences of some key taxa. One of those limiting taxa has been the elasmobranchs (sharks and rays), which harbor species often with numerous chromosomes and enlarged genomes. Here, we report the chromosome-scale genome assembly for the zebra shark Stegostoma tigrinum, an endangered species that has a relatively small genome among sharks (3.71 Gb), as well as for the whale shark Rhincodon typus Our analysis using a male-female comparison identified an X Chromosome, the first genomically characterized shark sex chromosome. The X Chromosome harbors the Hox C cluster whose intact linkage has not been shown for an elasmobranch fish. The sequenced shark genomes show a gradualism of chromosome length with remarkable length-dependent characteristics-shorter chromosomes tend to have higher GC content, gene density, synonymous substitution rate, and simple tandem repeat content as well as smaller gene length and lower interspersed repeat content. We challenge the traditional binary classification of karyotypes as with and without so-called microchromosomes. Even without microchromosomes, the length-dependent characteristics persist widely in nonmammalian vertebrates. Our investigation of elasmobranch karyotypes underpins their unique characteristics and provides clues for understanding how vertebrate karyotypes accommodate intragenomic heterogeneity to realize a complex readout. It also paves the way to dissecting more genomes with variable sizes to be sequenced at high quality.