Epigenomic changes are a hallmark of aging, and DNA methylation (DNAm) has emerged as the most reliable molecular marker of an individual's age. Genome-wide patterns of age-associated hypo- and hypermethylation have been applied to generate predictive models (i.e., "epigenetic clocks") capable of estimating chronological age in an increasingly diverse set of species including many mammals, a few birds, a reptile, and several bony fishes. Elasmobranchs (sharks, skates, and rays) are underrepresented in comparative investigations of epigenetic aging despite exhibiting exceptional life history variation, occupying a key basal position in the vertebrate phylogeny, and encompassing a large proportion of threatened species lacking accurate, non-lethal age determination methods. Here, we characterize epigenome-wide aging signals in the zebra shark (Stegostoma tigrinum), a long-lived elasmobranch of conservation concern, from whole-genome enzymatic methyl-sequencing of whole blood. Using a cohort of 51 known-age aquarium-bred individuals, we develop several epigenetic clock models capable of predicting chronological age with a median absolute error of 1.03-1.99 years (3.32%-6.42% of lifespan) based on the methylation status of as few as ten cytosines. We further apply our models to 19 individuals of unknown age originating from the wild. By profiling the broader age-associated methylome we demonstrate that these patterns not only predict age with high accuracy but also exhibit striking similarities in their genomic distributions to those observed in mammals pointing to conservation of the processes underlying epigenetic aging across vertebrates.

The Vertebrate Genomes Project (VGP) aims to produce complete and near-error-free reference genomes for all ~70,000 extant vertebrate species1. Organized in four phases, it progressively targets all vertebrate orders, families, genera, and eventually all species. Here we present the completion of VGP Phase I, delivering reference genomes for ~95% of vertebrate orders, along with additional lineages within those orders, totaling 816 species and 1.6 trillion base pairs of main haplotype sequence. These genomes were assembled and annotated over an 8-year period (2018-2026) of rapid advances in genome sequencing, assembly, and annotation methods2-4, alongside the growth of associated consortium initiatives and international collaborations5-9. They represent some of the highest-quality vertebrate genomes currently available, and most have become the primary reference for their respective species in public databases. Comparative analyses across a subset of 579 species when we reached a threshold of 85% of orders allowed us to reconstruct the genome of the last common ancestor of all vertebrates 500 million years ago, identify diverse modes of sex chromosome evolution, reveal clade-specific three-dimensional genome architecture, discover methylated epigenetic landscapes across vertebrates, and provide a framework for studying gene and pseudogene evolution, immune loci, cancer-associated genes, and other trait-associated loci. Approximately a quarter of this subset are listed as Vulnerable to Critically Endangered by the IUCN Red List of Threatened Species, and have enabled more advanced genomic investigations of extinction risk. VGP Phase I delivers a reference backbone for vertebrate genomics, enabling discoveries that would otherwise remain out of reach across evolution, conservation, and medicine.

The tetraploid smiliogastrines (Pseudobarbus & allies) constitute a small group of cyprinids that are endemic to freshwaters of southern Africa. How and when these polyploids originated has remained a mystery to researchers for years. Their phylogenetic relationships and biogeographical history also need further investigation. In this study, we reconstructed the phylogenetic relationships of this group using DNA sequences from both mitochondrial and nuclear genes. The initial split occurred between Namaquacypris hospes and all other tetraploid smiliogastrines. Amatolacypris is sister to the clade formed by Sedercypris and Cheilobarbus, which are reciprocally monophyletic. The clade formed by these three genera is sister to the monophyletic Pseudobarbus. For the three nuclear genes (i.e., RAG1, IRBP2, and EGR2B) used, we attempted to separate the two gene copies for each gene and each polyploid species. On the phylogenetic trees inferred from individual nuclear genes, one gene copy of Pseudobarbus & allies was clustered with African Enteromius & allies, whereas the other gene copy seen to be more closely related to Asian Puntius & allies. These results suggest that this tetraploid group likely originated through hybridization, although additional nuclear genomic data will be needed to rigorously test this hypothesis. The maternal progenitor of this group is likely a lineage from African Enteromius & allies, while the paternal progenitor likely came from an extinct Asian lineage ("Lineage P") closely related to Puntius & allies. Our results suggest that the Lineage P might have originated earlier than the common ancestor of all African smiliogastrines. The tetraploid smiliogastrines may have originated at late Paleogene to Eocene (58.9-36.5 Ma). They established themselves in the Orange River and later colonized all major rivers and lakes to the south. During this process, the tetraploid ancestor diverged into the current generic and specific diversity.

The Thorny Skate (Amblyraja radiata) is a vulnerable species displaying a discrete size-polymorphism in the northwest Atlantic Ocean (NWA). We conducted whole genome sequencing of samples collected across its range. Genetic diversity was similar at all sampled sites, but we discovered a ~ 31 megabase bi-allelic supergene associated with the size polymorphism, with the larger size allele having introgressed in the last ~160,000 years B.P. While both Gulf of Maine (GoM) and Canadian (CAN) populations exhibit the size polymorphism, we detected a significant deficit of heterozygotes at the supergene and longer stretches of homozygosity in GoM population. This suggests inbreeding driven by assortative mating for size in GoM but not in CAN. Coalescent-based demographic modelling reveals strong migration between regions maintaining genetic variability in the recombining genome, preventing speciation between morphs. This study highlights short-term context-dependent evolutionary consequences of a size-determining supergene providing new insights for the management of vulnerable species. The thorny skate is a vulnerable species in the northwest Atlantic ocean with a discreet size polymorphism. Here, the authors have sequenced 49 thorny skate individuals, finding a supergene locus that is associated with skate size.

Botiidae is a small family of freshwater fishes distributed across Southeast Asia, South Asia, and East Asia. It comprises two subfamilies: the diploid Leptobotiinae and the tetraploid Botiinae. Whether species in the Botiinae are autotetraploids or allotetraploids and how many polyploidization events occurred during the evolution of this subfamily remain open questions. The phylogenetic relationships and biogeography of the Botiidae also require further investigation. In the current study, we compared phylogenetic trees constructed using DNA sequences from the mitochondrial genome and five phased nuclear genes. We also performed whole genome sequencing for two tetraploid species: Chromobotia macracanthus and Yasuhikotakia modesta. Genome profiling of five botiine species suggests that they are likely of allotetraploid origin. Nuclear gene tree topologies indicate that the tetraploidization of the Botiinae occurred only once in the common ancestor of this subfamily. Although the possible maternal progenitor and paternal progenitor of the Botiinae cannot be determined, the subfamily Leptobotiinae can be excluded as a progenitor. The gene trees built in this study generally agree on the following sister group relationships: Leptobotiinae/Botiinae, Leptobotia/Parabotia, Chromobotia/Botia, Yasuhikotakia/Syncrossus, and Sinibotia/Ambastaia. Clades formed by the last two generic pairs are also sisters to each other. Timetree analyses and ancestral range reconstruction suggest that the family Botiidae might have originated in East Asia and Mainland Southeast Asia approximately 51 million years ago and later dispersed to South Asia and the islands of Southeast Asia.

The first molecular-assisted assessment of shark and batoid taxonomy in the waters of south-eastern Arabia was undertaken almost a decade ago, at a time when many species lineages lacked clear resolution. Consequently, only tentative identifications could be offered in some cases. There has been a considerable amount of focus on elasmobranch taxonomy since then, and this has resulted in numerous revisions to species identities and distributions. Therefore, in light of these more recent findings, mitochondrial NADH2 sequences from the original south-eastern Arabia study were re-assessed and taxonomically updated. This resulted in the recovery of 28 distinct shark lineages, of which all but one could be attributed to currently-recognised species. The remaining lineage most likely represents an undescribed Echinorhinus sp., which has been flagged in previous studies. Twenty-eight batoid lineages were also recovered, 13 of which have undergone taxonomic revision since the original study. Although most of these could be attributed to currently-recognised species, certain lineages remain problematic. Consequently, further work is required to provide a comprehensive assessment of elasmobranch taxonomy in the region.

While sex-determining mechanisms have been extensively characterized in many vertebrates, they have not been explored in chondrichthyan fishes until relatively recently. In the present study, we used high-quality whole genome reference assemblies to examine the putative sex chromosomes of 14 elasmobranch species spanning nine orders. We describe four newly assembled reference genomes belonging to the white shark Carcharodon carcharias , the Atlantic stingray Hypanus sabinus , the smalltooth sawfish Pristis pectinata , and the zebra shark Stegostoma tigrinum . We conducted sex chromosome identification and verification using short-read sequence data collected for multiple individuals for three of the species. This revealed putative pseudoautosomal regions (PARs) and, in one instance, a candidate sex chromosome reassignment. A synteny analysis revealed an ancient and shared origin of the chromosomes within elasmobranchs considerably older than any previously proposed scenario, and a potential candidate gene involved in sex determination shared across all examined species. The synteny analysis also revealed a historical fusion and the formation of neo-Y chromosomes between two myliobatiform species. Our results show that there has been strong conservation and homology of the X chromosomes among elasmobranchs in spite of their varied features and different evolutionary histories. ### Competing Interest Statement The authors have declared no competing interest.

Phylogenomics, which uses genome-scale data for phylogenetic inference, has clarified many controversial nodes in the tree of life. Such extensive data improve tree resolution and better reflects organismal history compared to analyses based on single or a few genetic loci. However, some relationships within the tree of life remain unresolved, as increased data can yield high node support without ensuring accuracy due to systematic errors. For example, the order-level relationships among chondrichthyans are still contentious despite the use of phylogenomic data. To address systematic errors, complex models have been developed, and filtering for less erroneous data shows great promise. Current metric-based filtering methods rank loci based on overall tree statistics, but problematic signals are often local; and topology-based data filtering approaches struggle with circular assumptions. In this study, we introduced two novel metric-based data filtering methods based on the ratio of local branch length or GC content between problematic clades. We applied these methods to a dataset of 4,452 single-copy exons extracted from 98 chondrichthyan species. The results using all loci showed that the Hexanchiformes was positioned at the root of Elasmobranchii, pulling other squalomorphs to the basal position and rendering Squalomorphii paraphyletic. Contrastingly, filtering for loci with more even branch length, the branch ratio method (absRatioLen) strongly supported the monophyly of all superorders of the chondrichthyans as well as their higher classification grouping, such as Selachii and Batoidea. By concentrating on problematic nodes, our assumption-free filtering methods demonstrate significant potential in resolving contentious relationships in the tree of life. ### Competing Interest Statement The authors have declared no competing interest.

Otodus megalodon (Lamniformes: Otodontidae) is an iconic Neogene shark, but the lack of well-preserved skeletons has hampered our understanding of various aspects of its biology. Here, we reassess some of its biological properties using a new approach, based on known vertebral specimens of O. megalodon and 165 species of extinct and extant neoselachian sharks across ten orders. Using the median neurocranial and caudal fin proportions relative to the trunk proportion among non-mitsukurinid/non-alopiid lamniforms, we show that O. megalodon could have had a slender body and possibly reached about 24.3 m in length. Allometric considerations indicate that a stout body plan like the extant white shark (Carcharodon carcharias) for O. megalodon could have incurred excessive hydrodynamic costs, further supporting the interpretation that O. megalodon likely had a slenderer body than C. carcharias. A 24.3-m-long O. megalodon may have weighed around 94 t, with an estimated cruising speed of 2.1-3.5 km h-1. A reanalysis of vertebral growth bands suggests a size at birth of 3.6-3.9 m for O. megalodon, supporting the previous interpretations of its ovoviviparity and embryos' intrauterine oophagous behavior, but less likely the need for nursery areas. Additional inferred growth patterns corroborated by the known fossil record support the hypothesis that the emergence of C. carcharias during the Early Pliocene is at least partly responsible for the demise of O. megalodon due to competition for resources. These interpretations are working hypotheses expected to serve as reasonable reference points for future studies on the biology of O. megalodon.

Mitonuclear discordance has been observed in several shark species. Female philopatry has often been invoked to explain such discordance but has never been explicitly tested. Here, we focus on the white shark, for which female philopatry has been previously proposed, and produced a chromosome-level genome, high-coverage whole-genome autosomal, and uniparental datasets to investigate mitonuclear discordance. We first reconstructed the historical population demography of the species based on autosomal data. We show that this species once comprised a single panmictic population, which experienced a steady decline until recent times when it fragmented into at least three main autosomal genetic groups. Mitochondrial data depict a strikingly different picture, inconsistent with the spatial distribution of autosomal diversity. Using the demographic scenario established from autosomal data, we performed coalescent and forward simulations to test for the occurrence of female philopatry. Coalescent simulations showed that the model can reproduce the autosomal variability, confirming its robustness. A forward simulation framework was further built to explicitly account for a sex-biased reproduction model and track both autosomal and uniparental markers (Y chromosome and mitochondrial DNA). While our model generates data that are consistent with the observed Y chromosome variation, the mitochondrial pattern is never reproduced even under extreme female philopatry (no female migration), strongly suggesting that demography alone cannot explain the mitonuclear discordance. Our framework could, and perhaps should, be extended to other shark species where philopatry has been suggested. It is possible that the proposed widespread occurrence of female philopatry in sharks should be revisited.

Fishes in the cypriniform family Catostomidae (suckers) are evolutionary tetraploids. The use of nuclear markers in the phylogenetic study of this important group has been greatly hindered by the challenge of identifying paralogous copies of genes. In the present study, we used two different methods to separate the gene copies of five single-copy nuclear genes (i.e., RAG1, EGR2B, EGR3, IRBP2, and RAG2). For each gene, all sequences of Copy I formed a clade that was sister to the clade formed by all sequences of Copy II in the phylogenetic trees. The maternal and paternal progenitor of the tetraploid ancestor of the Catostomidae could not be determined. We also constructed a mitochondrial tree to reflect the maternal relationships among major catostomid lineages. Our data appear to support a sister relationship between Catostominae and a monophyletic group composed of Myxocyprininae, Cycleptinae, and Ictiobinae. However, within Catostominae, there is significant conflict between mitochondrial and nuclear data regarding the relationships among Erimyzonini, Catostomini, and Moxostomatini/Thoburnini. Many indels, unexpected stop codons, and possible gene loss were identified in one gene copy of RAG1, RAG2, and IRBP2. We believe that additional nuclear genome data are needed to better resolve the phylogenetic relationships within the family Catostomidae.

Leptocharias smithii has been poorly explored in anatomical terms. This species bears a mosaic of morphological characters and is considered to represent an intermediate condition between other carcharhiniform clades. In the present paper, the anatomy of the appendicular skeleton of the species is thoroughly investigated and compared with other representatives of the order Carcharhiniformes. Leptocharias bears exclusive characteristics, such as the visible separation of the pro- and mesopterygia but it also has an aplesodic pectoral fin, a condition shared with carcharhiniforms placed at the base of the phylogenetic tree and at the same time a chevron-shaped coracoid bar, a condition characteristic of charcharhiniforms placed at the apex of the phylogenetic tree. Additionally, in an attempt to understand the evolution of its appendicular skeleton and of other carcharhiniforms, 20 characters of the paired fins and girdles are explored and discussed in light of two recent phylogenetic hypotheses. Most of these characters were not previously explored and support not only the monophyly of Carcharhiniformes, such as the mesopterygium overlapping the metapterygium in ventral view, but also the monophyly of the less inclusive clade Hemigaleidae + (Galeocerdonidae + (Carcharhinidae+Sphyrnidae)), such as the morphology and arrangement of the distal radials, which are pointed and spaced.

Aim: Biogeographic boundaries and genetic structuring have important effects on the inferences and interpretation of effective population size (N-e) temporal variations, a key genetics parameter. We reconstructed the historical demography and divergence history of a vulnerable coastal high-trophic shark using population genomics and assessed our ability to detect recent bottleneck events.Location: Western and Central Indo-Pacific (IPA), Western Tropical Atlantic (WTA) and Eastern Tropical Pacific (EPA).Taxon: Carcharhinus leucas (Muller & Henle, 1839).Methods: A DArTcap (TM) approach was used to sequence 475 samples and assess global genetic structuring. Three demographic models were tested on each population, using an ABC-RF framework coupled with coalescent simulations, to investigate within-cluster structure. Divergence times between clusters were computed, testing multiple scenarios, with fastsimcoal. N-e temporal variations were reconstructed with STAIRWAYPLOT. Coalescent simulations were performed to determine the detectability of recent bottleneck under the estimated historical trend for datasets of this size.Results: Three genetic clusters corresponding to the IPA, WTA and EPA regions were identified, agreeing with previous studies. The IPA presented the highest genetic diversity and was consistently identified as the oldest. No significant within-cluster structuring was detected. N-e increased globally, with an earlier onset in the IPA, during the last glacial period. Coalescent simulations showed that weak and recent bottlenecks could not be detected with our dataset, while old and/or strong bottlenecks would erase the observed ancestral expansion.Main Conclusions: This study further confirms the role of marine biogeographic breaks in shaping the genetic history of large mobile marine predators. N-e historical increases in N-e are potentially linked to extended coastal habitat availability. The limited within-cluster population structuring suggests that N-e can be monitored over ocean basins. Due to insufficient amount of available genetic data, it cannot be concluded whether overfishing is impacting Bull Shark genetic diversity, calling for whole-genome sequencing.

The megatooth shark, dagger Otodus megalodon, which likely reached at least 15 m in total length, is an iconic extinct shark represented primarily by its gigantic teeth in the Neogene fossil record. As one of the largest marine carnivores to ever exist, understanding the biology, evolution, and extinction of dagger O. megalodon is important because it had a significant impact on the ecology and evolution of marine ecosystems that shaped the present-day oceans. Some attempts inferring the body form of dagger O. megalodon have been carried out, but they are all speculative due to the lack of any complete skeleton. Here we highlight the fact that the previous total body length estimated from vertebral diameters of the extant white shark (Carcharodon carcharias) for an dagger O. megalodon individual represented by an incomplete vertebral column is much shorter than the sum of anteroposterior lengths of those fossil vertebrae. This factual evidence indicates that dagger O. megalodon had an elongated body relative to the body of the modern white shark. Although its exact body form remains unknown, this proposition represents the most parsimonious empirical evidence, which is a significant step towards deciphering the body form of dagger O. megalodon.

The increased availability of reference genome assemblies of sharks and rays has contributed greatly to our understanding of their biology, including their sex-determination mechanisms. However, several publicly available genome assemblies of sharks and rays appear to be missing information about the sex of the source individuals. This can confound the investigation into genetic sex-determining elements and hinder the discovery of sex-specific patterns. Herein, we highlight the importance of clear and accurate sex identification in sharks and rays for future genome assemblies, using an example of a white shark (Carcharodon carcharias) genome, in which the phenotypically assigned sex conflicts with the genetic information. This genome assembly was reported to be sourced from a juvenile female (BioSample: SAMN01915239). We analyzed the assembly by mapping its available genome sequences to the current white shark reference genome assembly and compared the read coverage to sequences collected from other samples. Evidence suggests that this specimen is genetically male, which contradicts its assignment based on phenotype. Therefore, we urge researchers to provide as much accurate information (e.g., sex, sampling localities, and life history) as possible when publishing genome assemblies for sharks and rays (or for any other organism).

Stingrays genus Hypanus currently encompasses nine valid species from the Atlantic and Pacific oceans, though the phylogenetic relationships amongst some of them were based on a single mitochondrial gene and did not involve all putative Hypanus species. To address the monophyly of the genus and its relationship to other Dasyatinae genera, we sequenced the whole mitochondrial genomes of all species that supposedly belong to this genus and representatives of Dasyatinae, Neotrygoninae, and, as an outgroup, Fontitrygon (Urogymninae). Based on phylogenetic analyses, Hypanus is the sister-genus to all other Dasyatinae, and this subfamily is closely-related to Neotrygoninae within the family Dasyatidae. The species F. geijskesi is closely related to H. guttatus rather than to its congeners and should be allocated to Hypanus as H. geijskesi for the genus monophyly. After lineage delimitation analyses, we identified three species complexes composed of H. americanus, H. guttatus, and H. say, with two distinct evolutionary lineages within each, leaving the genus with 13 evolutionary units, of which six are currently under threat and only H. sabinus is of least concern. The urgency in identifying these new lineages lies in the fact they might already be under threat before being formally described.

The genus Dichichthys was resurrected for five species previously allocated to the genus Parmaturus in the family Pentanchidae. Supraorbital crests on the chondrocranium distinguish Dichichthys from Parmaturus and other members of the family Pentanchidae. A new family, Dichichthyidae, has been proposed to contain Dichichthys. The sequence of the NADH2 mitochondrial gene confirms the placement of Dichichthys outside of the Pentanchidae family, as well as separate from the Atelomycteridae and Scyliorhinidae families. Dichichthys albimarginatus was described using a holotype collected off the coast of New Caledonia. A second juvenile specimen collected off the coast of Papua New Guinea was tentatively assigned as D. cf. albimarginatus. Dichichthys bigus is known from the holotype collected in the Coral Sea off the coast of Queensland, Australia. A new, parasite-afflicted underwater observation was reported further north of Queensland. The type species Dichichthys melanobranchus, previously only known from juvenile specimens, was redescribed based on adult specimens. Dichichthys nigripalatum is known from the holotype collected off Sumbawa, Indonesia, and a tentatively identified photo record from West Java. Dichichthys satoi n. sp. is described from the West Norfolk Ridge and off the North Island of New Zealand. Members of the genus Dichichthys have unique curved egg cases which have pliable ridges made up of numerous fibres and long coiled tendrils on the posterior end.

Abstract Understanding the relationship between census size, recent demography and genetic diversity is central for effective conservation and management of threatened species. Thorny skate (Amblyraja radiata) biomass in the northwest Atlantic has declined significantly in the past 50 years, prompting strict conservation measures . Curiously, population recovery has not occurred in the Gulf of Maine. We conducted whole genome sequencing of thorny skate samples collected from across its range. Genetic diversity was similar in all sampled locations, but we discovered a ~31 megabase supergene bi-allelic locus associated with a discrete size polymorphism occurring in the northwest Atlantic. Historical demographic modelling reveals that the allele associated with larger size originally introgressed into the ancestral thorny skate population ~160,000 years ago. Off Newfoundland (Canada), where population recovered, supergene genotypes are in Hardy-Weinberg equilibrium. In contrast, the Gulf of Maine population, exhibiting the most acute non-recovery, displays a significant deficit of heterozygotes. This strongly implies sub-regional fitness effects associated with the supergene, hindering recovery efforts in the Gulf of Maine. At the same time, regional migration sustains genetic variability in the recombining genome component, preventing speciation between morphs. This study highlights a rarely considered significance of context-dependent genetic compatibilities in the conservation of threatened populations and reconcile census size trajectory with genetic diversity estimates through accurate evolutionary modelling.

Management of thorny skate (Amblyraja radiata) in the Northwest Atlantic has posed a conservation dilemma for several decades due to the species' lack of response to strong conservation efforts in the US Gulf of Maine and the Canadian Scotian Shelf, confusion over the relationship between two reproductive size morphs of differing life histories that are sympatric in the Northwest Atlantic, and conflicting data on regional population connectivity throughout the species' broader range. To better assess potential A. radiata regional population differentiation and genetic links to life-history variation, we analysed complete mitochondrial genome sequences from 527 specimens collected across the species' North Atlantic geographic range, with particular emphasis on the Northwest Atlantic region. A high level of genetic diversity was evident across the North Atlantic, but significant genetic differentiation was identified between specimens inhabiting the Northwest (Gulf of Maine and Newfoundland) and Northeast (Greenland, Iceland, North Sea, and Arctic Circle) Atlantic. In the Northwest Atlantic, significant differentiation between the Gulf of Maine and Newfoundland regions was revealed; however, the overall level of differentiation was very low. No genetic difference was identified between the large and small reproductive morphs. The results of this study advance our understanding of A. radiata population structure in the North Atlantic but do not resolve all the questions confounding our understanding of the species' biology and evolutionary history.

Abstract Sawfishes (Pristidae) are large, highly threatened rays named for their tooth‐studded rostrum, which is used for prey sensing and capture. Of all five species, the smalltooth sawfish, Pristis pectinata, has experienced the greatest decline in range, currently found in only ~20% of its historic range. To better understand the genetic underpinnings of these taxonomically and morphologically unique animals, we collected transcriptomic data from several tissue types, mapped them to the recently completed reference genome, and contrasted the patterns observed with comparable data from other elasmobranchs. Evidence of positive selection was detected in 79 genes in P. pectinata, several of which are involved in growth factor/receptor tyrosine kinase signaling and body symmetry and may be related to the unique morphology of sawfishes. Changes in these genes may impact cellular responses to environmental conditions such as temperature, dissolved oxygen, and salinity. Data acquired also allow for examination of the molecular components of P. pectinata electrosensory systems, which are highly developed in sawfishes and have likely been influential in their evolutionary success.