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.

Abstract Although cartilage in tetrapod skeletons is typically said to lack blood vessels, this is only true for adult cartilage. In young bird and mammal cartilage, a dense network of vasculature-containing tunnels —cartilage canals— perforate the growing skeleton, helping nourish the cartilage and develop the ossification centers that will later form the skeleton’s epiphyseal bone. As the canals and their rich vascular network typically recede as animals age, the healthy cartilage of adult animals is typically known to be avascular. Here, however, we use a range of tissue characterization and visualization techniques —including light/electron microscopy and microCT— to show that the skeletons of rays and sharks (elasmobranch fishes) not only possess cartilage canals, but that these structures persist in the adult skeleton. The morphology and tissue composition of elasmobranch cartilage canals argues homology with mammalian cartilage canals and an ancient invasion of the vascular system into cartilage. However, the anatomical location of canals —extending away from mineralized tissue not toward it— and the lack of endochondral ossification in ray and shark cartilage suggest that cartilage canals developed early in vertebrates as a transport system for nutrients and mesenchymal cells into the growing skeleton. We describe distinctive features and variation in elasmobranch cartilage canals, discuss their possible roles and their potential for tissue mineralization, and the biomedical implications for their presence in a clade of animals with continuously growing cartilaginous skeletons.

Protein electrophoresis is a tool used in the health assessments of non-mammalian vertebrates. In elasmobranchs, agarose gel electrophoresis (AGE) has been described in various species and a newer method called capillary zone electrophoresis (CZE) has been developed and implemented in the undulate skate (Raja undulata) and nursehound shark (Scyliorhinus stellaris). The study goals were to implement AGE and CZE methods on plasma samples from the sand tiger shark (Carcharias taurus) and examine differences in resolution as well as to calculate reference intervals (RI). Plasma was obtained from aquarium sharks (n = 23) and free-ranging sharks (n = 62) sampled during field research conducted from 2017 to 2023. As with previous reports, CZE was found to provide superior resolution with definition of two major globulin migrating fractions compared to AGE. Overall, the alpha and beta migrating fractions were well correlated between the methods (r = 0.92, 0.89, respectively, p < 0.0001). The correlation for the gamma fraction was weaker (r = 0.42, p = 0.002) as the CZE fraction was lower in concentration versus AGE. There were minor, but significant, differences between the concentration of some of the fractions in samples from sharks under managed care versus free-ranging animals which necessitated the production of two sets of RI. In total, this information may help in further studies to address the applicability of these tools in the management of this species under human care as well as in health assessments of free-ranging sharks.

As mid to upper trophic level predators, elasmobranchs (sharks, skates, and rays) are known to have elevated levels of a potent neurotoxicant, methylmercury (MeHg). The lethality of MeHg across different vertebrate taxa is well established, but its sublethal effects have only been investigated in depth at the scope of human health. The sublethal effects of exposure to MeHg, which are known to impact animal physiology and behavior, remain largely undescribed in elasmobranchs. In this study, a new approach was tested for assessing bioaccumulation and the sublethal effects of MeHg in a coastal shark species over extended time periods (five months) in a spatially constrained research setting. By the end of the experiment, shark muscle in the high-dose treatment had 24.03 μg/g MeHg, while low-dose shark muscle had 10.11 μg/g MeHg, compared to the 1.18 μg/g MeHg in the muscle of control sharks. Regardless of treatment, the highest concentrations of MeHg were found within the muscle, brain, heart, and kidneys while the lowest concentrations were found in the liver, intestine, and stomach. This work provides insight into how biological processes may respond to MeHg exposure, a widespread contaminant in aquatic environments.

Predator tissues can accumulate specific signatures of persistent organic pollutants (POPs) based on their feeding ecology. Likewise, stable isotopes, which are also incorporated into tissues via diet, are complementary to POPs as they provide additional geographic and trophic information into the ecology of consumers. The Common Thresher Shark (Alopias vulpinus) and the Bigeye Thresher (Alopias superciliosus) are closely related but have distinct spatial habitat use, with Common Threshers having shallower and more coastal distributions compared to Bigeye Threshers, which occur in deeper offshore waters. This study used two types of chemical markers (stable isotopes and POPs) to understand how ecological differences in nearshore use may influence their propensity to accumulate PCBs and DDXs. Liver samples were obtained from thresher sharks incidentally captured in southern California (a known hotspot for DDX and its metabolites) and analyzed for a suite of organic contaminants and two stable isotopes (δ13C and δ15N). Common Thresher Sharks had significantly higher concentrations for every contaminant group examined compared to Bigeye Threshers, and species were distinguishable based on their contaminant signatures. Contaminant levels significantly declined with length in Common Thresher Sharks, although the dataset was biased towards mature females, but levels significantly increased with length in Bigeye Threshers, which were skewed towards more mature males. Isotopic niche space had little overlap between species, and DDX concentrations positively correlated with δ13C (a proxy of nearshore use). The results suggest that proximity to shore may play a key role in hepatic POP accumulation, but intrinsic factors, such as sex, may also be influential.

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.

Ultrasonography is widely used to monitor pregnancy in viviparous species, but it is underutilized as a tool to characterize embryonic development in oviparous species. Currently, a multi-institutional effort is underway to re-wild the endangered zebra shark (Stegostoma tigrinum) to locations where this species was previously extirpated by leveraging the reproductive efforts of aquarium sharks as a source of brood stock. Zebra sharks are oviparous and fecund, but a large percentage of their yolked eggs do not result in hatchlings. Therefore, ultrasonography represents a potential tool for distinguishing fertile eggs with developing embryos from degrading eggs, and to diagnose changes in early embryonic development predictive of poor outcomes. The objectives of the current study were to use ultrasonography to assess egg fertility, monitor early embryonic development, and identify morphological indicators that may be predictive of early embryonic mortality. Freshly laid eggs from four female zebra sharks were collected and inventoried daily at Aquarium of the Pacific. Eggs were incubated undisturbed for 2 to 4 weeks and subsequently examined weekly via ultrasound to assess fertility and monitor embryo development. Among 120 fertile eggs, embryos were identified as early as 8 days post-oviposition, with average (±SD) time to first observation at 30 ± 7 days. Morphological and behavioral abnormalities were observed for most embryos (n = 84, 70%) as early as 16 days and up to 95 days post-oviposition. Common abnormalities included: bent or curled tails, vesicle(s) at the base of the yolk stalk, and slow or weak movement. Only one embryo survived to hatch during the study and was genetically-confirmed parthenogenetic, suggesting hatching success for parthenotes is low (<1%). Ultrasonography was demonstrated to be an effective and non-invasive method to determine egg fertility, identify embryos with developmental abnormalities, and monitor embryo growth.

Comparing co-occurring species may provide insights into how aspects of ecology may play a role in influencing their microbial communities. During the 2019 commercial shrimp trawl season off coastal Georgia, swabs of skin, gills, cloaca, and gut were taken for three species of batoids (Butterfly Ray, Bluntnose Stingray, and Atlantic Stingray) and one shark species (Atlantic Sharpnose) for high-throughput sequencing of the V4 region of the bacterial 16S rRNA gene. White muscle was analyzed for stable isotopes (δ13C and δ15N) to evaluate potential niche overlap in these four sympatric mesopredators. Significant differences were found in both δ13C and δ15N signatures across species, suggesting a degree of resource partitioning. When examined within tissue type, the host species had a weak effect on β-diversity for cloaca and skin, with no differences found for gill and gut samples. However, network analysis metrics demonstrated a stronger species-specific effect and distinct microbial community relationships were apparent between the shark and batoids, with the former having tighter networks for both internally- and externally-influenced tissues (gut/cloaca and skin/gills, respectively). Despite overlapping habitat use, species’ microbiomes differed in their organizational structuring that paralleled differences in stable isotope results, suggesting a mediating role of species-specific ecology on bacterial microbiomes.

Ontogenetic changes in area use, habitat use, and trophic interactions play an important role in the ecology, demography, and ultimately population dynamics of many species. Assumed to be driven by shifting life-history requirements, trophic niche shifts in white sharks (Carcharodon carcharias) are well documented, but the timing of the spatial niche shift that is hypothesized to occur with the trophic niche shift remains poorly understood. To document how fine-scale area use varies as sharks age and the timing of the ontogenetic spatial shift of this top predator, we tracked individual white sharks tagged as young-of-the-year or young juveniles over multiple years. Using data from juvenile white sharks detected over multiple years in a nursery habitat with a high-density receiver array, we found no difference in area use with age. However, using a coast-wide receiver array including nursery and adult habitat, we found the probability of detecting a juvenile white shark in nursery habitat decreased with age, with a concurrent increasing probability of detection in adult habitat. As the conservation and management of this species relies on understanding nursery habitat use and age-related movements, data presented here address an important knowledge gap for the understudied juvenile to subadult life stages and the ontogenetic habitat shift of this species.

Age data are essential for estimating life history parameters and are thus critical for population assessment, management, and conservation. Traditional vertebrae-based age estimation in elasmobranchs can be costly, time intensive, of low accuracy, and is by necessity lethal. Herein, epigenetic clocks were developed for an elasmobranch, the cownose ray (Rhinoptera bonasus), using aquarium-born individuals (n = 42) with known dates of birth (age range: 7-7,878 days or 0-21 years) and two tissue types (fin clips and whole blood) that can be sampled in a relatively non-invasive manner. Enzymatically-converted restriction site-associated DNA sequencing (ECrad-seq) was used to identify CpG sites that exhibited age-correlated DNA methylation. The epigenetic clocks developed were highly accurate (mean absolute error, MAE, < 0.75 years) and precise (R2 > 0.98). Age-associated CpG sites were identified across tissues, and a multi-tissue clock was also highly accurate (MAE < 1 year) and precise (R2 = 0.97). Using the developed fin clip clock, three wild-caught individuals of unknown age but managed in aquariums for > 22 years were predicted to be 22.10-23.49 years old. Overall, the results have important implications for future epigenetic clock development and noninvasive age estimation in elasmobranchs.

Few studies have documented microplastics (<5 mm) in shark gastrointestinal (GI) tracts. Here, we report microplastic contamination in the tiger shark (Galeocerdo cuvier), an apex predator and generalist feeder, at several different life stages. We examined seven stomachs and one spiral valve from eight individuals captured off the United States Atlantic and Gulf of Mexico coasts (eastern US) and conducted a literature review of publications reporting anthropogenic debris ingestion in elasmobranchs. Specimens were chemically digested in potassium hydroxide (KOH) and density separated using calcium chloride (CaCl2) before quantifying and categorizing suspected anthropogenic particles (>45 μm) by size, morphology, and colour. Anthropogenic particles were found in the stomachs and spiral valve of all sharks. A total of 3151 anthropogenic particles were observed across all stomachs with 1603 anthropogenic particles observed in a single specimen. A subset of suspected anthropogenic particles (14%) were chemically identified using Raman spectroscopy and μ-Fourier Transform Infrared spectroscopy to confirm anthropogenic origin. Overall, ≥95% of particles analyzed via spectroscopy were confirmed anthropogenic, with 45% confirmed as microplastics. Of the microplastics, polypropylene (32%) was the most common polymer. Diverse microparticle morphologies were found, with fragments (57%) and fibers (41%) most frequently observed. The high occurrence and abundance of anthropogenic particle contamination in tiger sharks is likely due to their generalist feeding strategy and high trophic position compared to other marine species. The literature review resulted in 32 studies published through 2022. Several methodologies were employed, and varying amounts of contamination were reported, but none reported contamination as high as detected in our study. Anthropogenic particle ingestion studies should continue in the tiger shark, in addition to other elasmobranch species, to further understand the effects of anthropogenic activities and associated pollution on these predators.

Carolina hammerheads Sphyrna gilberti and scalloped hammerheads S. lewini are cryptic species with an overlapping distribution, and young-of-the-year (YOY) use similar coastal and estuarine nursery areas along the US Southeast coast. The diet of scalloped hammerheads has been widely studied throughout their global distribution; however, little is known about their diet in the western North Atlantic Ocean. Carolina hammerheads have only been recently described, and as such, their trophic ecology is largely unstudied. Stomach content analysis of genetically identified YOY Carolina and scalloped hammerheads revealed both species to be generalist feeders with diverse diets and no apparent resource partitioning between them. By contrast, multi-tissue (muscle and plasma) stable isotope analyses showed significant interspecific differences in signatures, particularly in muscle delta 13C values for the youngest hammerheads. Due to slow tissue turnover rates, YOY muscle signatures were assumed to reflect maternal isotopic signatures, suggesting habitat or resource partitioning between mature female Carolina and scalloped hammerheads. In particular, the data are consistent with mature Carolina hammerheads inhabiting more offshore waters or consuming a higher proportion of pelagic prey relative to scalloped hammerheads. YOY muscle and plasma delta 15N values decreased drastically across the sampling season, reflecting a loss of the maternal signal as the YOY hammerheads fed and grew rapidly in these productive nursery habitats.

The elemental composition of mineralized vertebral cartilage has proven useful for reconstructing the movement and environmental history of elasmobranchs. However, the effects of intrinsic and extrinsic factors on vertebral chemistry have not been well explored, making meaningful interpretations of results difficult. This study quantified trace element incorporation along the corpus calcareum of 116 Round Stingray ( Urobatis halleri ) vertebrae and explored the influence of intrinsic (i.e., sex) and extrinsic (i.e., location and year) factors on vertebral edge chemical variations. We quantified eight element/Ca ratios (Li/Ca, Mg/Ca, Mn/Ca, Co/Ca, Cu/Ca, Zn/Ca, Sr/Ca, Ba/Ca) by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Behavioral sexual segregation by Round Stingrays explained differences found in Mn/Ca, Zn/Ca, Sr/Ca, and Ba/Ca ratios. Higher vertebral Mn/Ca and Zn/Ca in females likely originate from the use of salt marsh habitats as a refuge as well as higher reproductive hormone concentrations during gestation, respectively. Female and male Round Stingrays were also found to have an inverse relationship between Sr/Ca and Ba/Ca, likely due to environmental differences (e.g., temperature) between their sexually segregated habitats. This study demonstrates how multielement signatures in vertebral cartilage may improve understanding of habitat use and migration throughout life and advances the development of biomineral usage as an environmental proxy in elasmobranchs.

The zebra shark Stegostoma tigrinum , a popular aquarium fish, is an endangered species that is known to readily reproduce both sexually and through facultative parthenogenesis while in human care. Artificial insemination trials that took place between 2011 and 2013 resulted in the hatching of 2 sexually produced (herein heterozygotes) and 10 parthenogenetic sharks that allowed for a retrospective comparison of growth, feeding and longevity between offspring produced from 2 distinct reproductive modes. Parthenogenetic offspring were generally smaller at hatch than their heterozygous counterparts and, after the first several months post-hatch, failed to increase in mass and length at the same rate as heterozygotes. Parthenogenetic offspring exhibited non-normal swimming behaviors such as spiraling, spy hopping and head standing, which may have been correlated with a gradual decline in the ability of some sharks to properly suction feed. Median lifespan for the parthenotes was 1.05 yr (range: 0.27-6.64 yr); one of the heterozygotes lived to 2.37 yr of age, and the other was alive at the time of this writing in August 2022 and had reached reproductive maturity. By contrast, the 2 longest surviving parthenotes perished just prior to reaching sexual maturity (~5.5 and ~6.5 yr). Parthenogenesis has been documented among ex situ S. tigrinum maintained in aquariums across the globe, and this study demonstrates substantial negative costs to fitness in parthenogenetic offspring compared with their heterozygous siblings. The reduced fitness of parthenotes has implications for managing populations in human care as well as for in situ conservation efforts.