Age group DNA methylation differences in lemon sharks (Negaprion brevirostris): Implications for future age estimation tools.

Age information is often non-existent for most shark populations due to a lack of measurable physiological and morphological traits that can be used to estimate age. Recently, epigenetic clocks have been found to accurately estimate age for mammals, birds, and fish. However, since these clocks rely, among other things, on the availability of reference genomes, their application is hampered in non-traditional model organisms lacking such molecular resources. The technique known as Methyl-Sensitive Amplified Polymorphism (MSAP) has emerged as a valid alternative for studying DNA methylation biomarkers when reference genome information is missing, and large numbers of samples need to be processed. Accordingly, the MSAP technique was used in the present study to characterize global DNA methylation patterns in lemon sharks from three different age groups (juveniles, subadults, and adults). The obtained results reveal that, while MSAP analyses lack enough resolution as a standalone approach to infer age in these organisms, the global DNA methylation patterns observed using this technique displayed significant differences between age groups. Overall, these results confer that DNA methylation does change with age in sharks like what has been seen for other vertebrates and that MSAP could be useful as part of an epigenetics pipeline to infer the broad range of ages found in large samples sizes.

Vertical space use and thermal range of the great hammerhead (Sphyrna mokarran), (Rüppell, 1837) in the western North Atlantic.

The great hammerhead (Sphyrna mokarran) is a highly mobile, large-bodied shark primarily found in coastal-pelagic and semi-oceanic waters across a circumtropical range. It is a target or by-catch species in multiple fisheries, and as a result, rapid population declines have occurred in many regions. These declines have contributed to the species being assessed as globally critically endangered on the IUCN Red List. Although conservation and management measures have yielded promising results in some regions, such as the United States, high levels of at-vessel and post-release mortality remain a major concern to the species population recovery. This examined the vertical space use and thermal range of pop-off archival satellite-tagged S. mokarran in the western North Atlantic Ocean, expanding the understanding of the ecological niche of this species and providing insight into by-catch mitigation strategies for fisheries managers. The results showed that S. mokarran predominantly used shallow depths (75% of records <30 m) and had a narrow temperature range (89% of records between 23 and 28°C). Individual differences in depth use were apparent, and a strong diel cycle was observed, with sharks occupying significantly deeper depths during the daytime. Furthermore, two individuals were confirmed pregnant with one migrating from the Bahamas to South Carolina, U.S.A., providing further evidence of regional connectivity and parturition off the U.S. East Coast. The findings suggest that S. mokarran may be vulnerable to incidental capture in the western North Atlantic commercial longline fisheries due to substantial vertical overlap between the species and the gear. The results can be incorporated into conservation and management efforts to develop and/or refine mitigation measures focused on reducing the by-catch and associated mortality of this species, which can ultimately aide S. mokarran population recovery in areas with poor conservation status.

Personality-driven life history trade-offs differ in two subpopulations of free-ranging predators.

Consistent individual differences in behaviour (i.e. personality) can be explained in an evolutionary context if they are favoured by life history trade-offs as conceptualized in the pace-of-life syndrome (POLS) hypothesis. Theory predicts that faster-growing individuals suffer higher mortality and that this trade-off is mediated through exploration/risk-taking personality, but empirical support for this remains limited and ambiguous. Equivocal support to the POLS hypothesis suggests that the link between life history and personality may only emerge under certain circumstances. Understanding personality-driven trade-offs would be facilitated by long-term studies in wild populations experiencing different ecological conditions. Here, we tested whether personality measured in semi-captivity was associated with a growth-mortality trade-off via risk-taking in the wild in two subpopulations of juvenile lemon sharks Negaprion brevirostris known to differ in their predator abundance. We expected stronger personality-driven trade-offs in the predator-rich environment as compared to the predator-poor environment. Sharks were captured yearly from 1995 onwards allowing us to obtain long-term data on growth and apparent survival in each subpopulation. We then used a novel open-field assay to test sharks for exploration personality yearly from 2012 to 2017. A subset of the tested sharks was monitored in the field using telemetry to document risk-taking behaviours. We tested (a) if fast explorers in captivity took more risks and grew faster in the wild and (b) if natural selection acted against more explorative, faster-growing sharks. In the subpopulation with fewer predators, more explorative sharks in captivity took more risks in the wild and grew faster. In turn, larger, fast-growing sharks had lower apparent survival. In the predator-rich subpopulation, despite finding selection on fast growth, we found no link between exploration personality and the growth-mortality trade-off. Our study demonstrates that the association between personality and life history is favoured in some ecological contexts but not in others. We identify predator and resource abundance as two main potential drivers of the personality-mediated trade-off and emphasize that future work on the POLS hypothesis would benefit from an approach integrating behaviour and life history across ecological conditions.

No apparent negative tagging effects after 13 years at liberty for lemon shark, Negaprion brevirostris implanted with acoustic transmitter.

An intact and uncompromised internal acoustic transmitter was non-lethally recovered from a lemon shark Negaprion brevirostris, after 13 years at liberty. The shark, first tagged at an estimated age of 2 years old near South Bimini, Bahamas in 2004, was recaptured in 2017 with a total length of 264 cm. The tagged shark displayed typical growth rate, pregnancy, natal homing and pupping behaviour of other individuals in this population. This observation provides important evidence regarding the effects from long-term retention of implanted acoustic transmitters in a carcharhinid shark.

Risky business for a juvenile marine predator? Testing the influence of foraging strategies on size and growth rate under natural conditions.

Mechanisms driving selection of body size and growth rate in wild marine vertebrates are poorly understood, thus limiting knowledge of their fitness costs at ecological, physiological and genetic scales. Here, we indirectly tested whether selection for size-related traits of juvenile sharks that inhabit a nursery hosting two dichotomous habitats, protected mangroves (low predation risk) and exposed seagrass beds (high predation risk), is influenced by their foraging behaviour. Juvenile sharks displayed a continuum of foraging strategies between mangrove and seagrass areas, with some individuals preferentially feeding in one habitat over another. Foraging habitat was correlated with growth rate, whereby slower growing, smaller individuals fed predominantly in sheltered mangroves, whereas larger, faster growing animals fed over exposed seagrass. Concomitantly, tracked juveniles undertook variable movement behaviours across both the low and high predation risk habitat. These data provide supporting evidence for the hypothesis that directional selection favouring smaller size and slower growth rate, both heritable traits in this shark population, may be driven by variability in foraging behaviour and predation risk. Such evolutionary pathways may be critical to adaptation within predator-driven marine ecosystems.

Modeling the population dynamics of lemon sharks.

BACKGROUND: Long-lived marine megavertebrates (e.g. sharks, turtles, mammals, and seabirds) are inherently vulnerable to anthropogenic mortality. Although some mathematical models have been applied successfully to manage these animals, more detailed treatments are often needed to assess potential drivers of population dynamics. In particular, factors such as age-structure, density-dependent feedbacks on reproduction, and demographic stochasticity are important for understanding population trends, but are often difficult to assess. Lemon sharks (Negaprion brevirostris) have a pelagic adult phase that makes them logistically difficult to study. However, juveniles use coastal nursery areas where their densities can be high. RESULTS: We use a stage-structured, Markov-chain stochastic model to describe lemon shark population dynamics from a 17-year longitudinal dataset at a coastal nursery area at Bimini, Bahamas. We found that the interaction between delayed breeding, density-dependence, and demographic stochasticity accounts for 33 to 49% of the variance in population size. CONCLUSIONS: Demographic stochasticity contributed all random effects in this model, suggesting that the existence of unmodeled environmental factors may be driving the majority of interannual population fluctuations. In addition, we are able to use our model to estimate the natural mortality rate of older age classes of lemon sharks that are difficult to study. Further, we use our model to examine what effect the length of a time series plays on deciphering ecological patterns. We find that-even with a relatively long time series-our sampling still misses important rare events. Our approach can be used more broadly to infer population dynamics of other large vertebrates in which age structure and demographic stochasticity are important. REVIEWERS: This article was reviewed by Yang Kuang, Christine Jacob, and Ollivier Hyrien.

Regional-scale migrations and habitat use of juvenile lemon sharks (Negaprion brevirostris) in the US South Atlantic.

Resolving the geographic extent and timing of coastal shark migrations, as well as their environmental cues, is essential for refining shark management strategies in anticipation of increasing anthropogenic stressors to coastal ecosystems. We employed a regional-scale passive acoustic telemetry array encompassing 300 km of the east Florida coast to assess what factors influence site fidelity of juvenile lemon sharks (Negaprion brevirostris) to an exposed coastal nursery at Cape Canaveral, and to document the timing and rate of their seasonal migrations. Movements of 54 juvenile lemon sharks were monitored for three years with individuals tracked for up to 751 days. While most sharks demonstrated site fidelity to the Cape Canaveral region December through February under typical winter water temperatures, historically extreme declines in ocean temperature were accompanied by rapid and often temporary, southward displacements of up to 190 km along the Florida east coast. From late February through April each year, most sharks initiated a northward migration at speeds of up to 64 km day(-1) with several individuals then detected in compatible estuarine telemetry arrays in Georgia and South Carolina up to 472 km from release locations. Nineteen sharks returned for a second or even third consecutive winter, thus demonstrating strong seasonal philopatry to the Cape Canaveral region. The long distance movements and habitat associations of immature lemon sharks along the US southeast coast contrast sharply with the natal site fidelity observed in this species at other sites in the western Atlantic Ocean. These findings validate the existing multi-state management strategies now in place. Results also affirm the value of collaborative passive arrays for resolving seasonal movements and habitat preferences of migratory coastal shark species not easily studied with other tagging techniques.

Two decades of genetic profiling yields first evidence of natal philopatry and long-term fidelity to parturition sites in sharks.

Sharks are a globally threatened group of marine fishes that often breed in their natal region of origin. There has even been speculation that female sharks return to their exact birthplace to breed ('natal philopatry'), which would have important conservation implications. Genetic profiling of lemon sharks (Negaprion brevirostris) from 20 consecutive cohorts (1993-2012) at Bimini, Bahamas, showed that certain females faithfully gave birth at this site for nearly two decades. At least six females born in the 1993-1997 cohorts returned to give birth 14-17 years later, providing the first direct evidence of natal philopatry in the chondrichthyans. Long-term fidelity to specific nursery sites coupled with natal philopatry highlights the merits of emerging spatial and local conservation efforts for these threatened predators.

Monogenoid infection of neonatal and older juvenile lemon sharks, Negaprion brevirostris (Carcharhinidae), in a shark nursery.

Fifty lemon sharks, Negaprion brevirostris , were captured in a shallow, mangrove-fringed shark nursery at Bimini, Bahamas and examined for the presence of skin-dwelling ectoparasitic monogenoids (Monogenoidea). Sixteen sharks were infected by Dermophthirius nigrellii (Microbothriidae); the youngest host was estimated to be 3- to 4-wk-old. Infection prevalence, mean intensity, and median intensity (0.32, 2.63, and 2.0, respectively, for all sharks) were not significantly different between neonates (estimated ages 3- to 10-wk-old) and non-neonatal juveniles (estimated ages 1- to 4-yr-old), suggesting that soon after parturition lemon sharks acquire infection levels of D. nigrellii matching those of juvenile conspecifics. Monogenoids were only found on the trailing portion of the first and second dorsal fins and upper lobe of the caudal fin. The prevalence of D. nigrellii was highest on the first dorsal fin; however, the mean and median intensities of D. nigrellii were similar between fins in all but 1 case. These results raise important husbandry implications regarding the practice of preferentially seeking neonatal and other small lemon sharks for captivity.

Social learning in juvenile lemon sharks, Negaprion brevirostris.

Social learning is taxonomically widespread and can provide distinct behavioural advantages, such as in finding food or avoiding predators more efficiently. Although extensively studied in bony fishes, no such empirical evidence exists for cartilaginous fishes. Our aim in this study was to experimentally investigate the social learning capabilities of juvenile lemon sharks, Negaprion brevirostris. We designed a novel food task, where sharks were required to enter a start zone and subsequently make physical contact with a target in order to receive a food reward. Naive sharks were then able to interact with and observe (a) pre-trained sharks, that is, 'demonstrators', or (b) sharks with no previous experience, that is, 'sham demonstrators'. On completion, observer sharks were then isolated and tested individually in a similar task. During the exposure phase observers paired with 'demonstrator' sharks performed a greater number of task-related behaviours and made significantly more transitions from the start zone to the target, than observers paired with 'sham demonstrators'. When tested in isolation, observers previously paired with 'demonstrator' sharks completed a greater number of trials and made contact with the target significantly more often than observers previously paired with 'sham demonstrators'. Such experience also tended to result in faster overall task performance. These results indicate that juvenile lemon sharks, like numerous other animals, are capable of using socially derived information to learn about novel features in their environment. The results likely have important implications for behavioural processes, ecotourism and fisheries.

Anthropogenic disturbance and evolutionary parameters: a lemon shark population experiencing habitat loss.

The level of genetic variation in natural populations influences evolutionary potential, and may therefore influence responses to selection in the face of future environmental changes. By combining long-term monitoring of marked individuals with genetic pedigree reconstruction, we assessed whether habitat loss influenced genetic variation in a lemon shark (Negaprion brevirostris) population at an isolated nursery lagoon (Bimini, Bahamas). We also tracked changes in the strength and direction of natural selection. Contrary to initial expectations, we found that after the habitat loss neutral genetic variation increased, as did additive genetic variance for juvenile morphological traits (body length and mass). We hypothesize that these effects might result from philopatric behavior in females coupled with a possible influx of male genotypes from other nursery sites. We also found changes in the strength of selection on morphological traits, which weakened considerably after the disturbance; habitat loss therefore changed the phenotypes favored by natural selection. Because such human-induced shifts in the adaptive landscape may be common, we suggest that conservation biologists should not simply focus on neutral genetic variation per se, but also on assessing and preserving evolutionary parameters, such as additive genetic variation and selection.

Novel acoustic technology for studying free-ranging shark social behaviour by recording individuals' interactions.

Group behaviours are widespread among fish but comparatively little is known about the interactions between free-ranging individuals and how these might change across different spatio-temporal scales. This is largely due to the difficulty of observing wild fish groups directly underwater over long enough time periods to quantify group structure and individual associations. Here we describe the use of a novel technology, an animal-borne acoustic proximity receiver that records close-spatial associations between free-ranging fish by detection of acoustic signals emitted from transmitters on other individuals. Validation trials, held within enclosures in the natural environment, on juvenile lemon sharks Negaprion brevirostris fitted with external receivers and transmitters, showed receivers logged interactions between individuals regularly when sharks were within 4 m ( approximately 4 body lengths) of each other, but rarely when at 10 m distance. A field trial lasting 17 days with 5 juvenile lemon sharks implanted with proximity receivers showed one receiver successfully recorded association data, demonstrating this shark associated with 9 other juvenile lemon sharks on 128 occasions. This study describes the use of acoustic underwater proximity receivers to quantify interactions among wild sharks, setting the scene for new advances in understanding the social behaviours of marine animals.

Long-term natal site-fidelity by immature lemon sharks (Negaprion brevirostris) at a subtropical island.

Although many sharks begin their life confined in nursery habitats, it is unknown how rapidly they disperse away from their natal area once they leave the nursery. We examine this issue in immature lemon sharks (Negaprion brevirostris) from the time they leave the nursery (approximately age 3) at a subtropical island (Bimini, Bahamas), through to the onset of sexual maturity (approximately age 12). From 1995 to 2007 we tagged and genotyped a large fraction of the nursery-bound sharks at this location (0-3 years of age, N = 1776 individuals). From 2003 to 2007 we sampled immature sharks aged from 3 to 11 years (N = 150) living around the island and used physical/genetic tag recaptures coupled with kinship analysis to determine whether or not each of these 'large immature sharks' was locally born. We show that many island-born lemon sharks remain close to their natal area for long periods (years) after leaving the nursery; more than half of the sampled sharks up to 135 cm total length ( approximately 6 years old) were locally born. The fraction of locally born sharks gradually declined with increasing shark size, indicating that dispersal is relatively slow and does not primarily occur after sharks reach a threshold size. Local conservation measures (e.g. localized fishery closures, marine protected areas) can therefore help protect island-born lemon sharks even after they leave the nursery habitat.

Population size and survivorship for juvenile lemon sharks (Negaprion brevirostris) on their nursery grounds at a marine protected area in Brazil

Sharks face a number of obstacles for surviving their first several years of life and many species occupy nursery areas. Although estimates of survival, particularly for young age classes, are essential for assessing, monitoring and effectively managing animal populations, there have been relatively few calculations of survival within shark populations and even fewer estimates based on direct methods for sharks on their nursery grounds. We used tag-recapture methods to estimate the population size and survival of juvenile lemon sharks (Negaprion brevirostris) on their nursery grounds at Atol das Rocas, a marine protected area in Brazil. Sharks were sampled from 1999 to 2003. Population size estimates ranged from 12 to 100 juvenile sharks and survival estimates ranged between 24-54% with a mean of 44.6% over the most robust sampling periods. The population of juvenile lemon sharks declined over the course of our study, whereas survival rates may have increased over the same time period. Even a modest level of fishing and removal of mature females in adjacent areas may dramatically affect small populations of sharks within a small and isolated nursery such as Atol das Rocas. The lower survival rates and population size at Atol das Rocas could be the result of differences in physical characteristics of this nursery in comparison to others used by lemon sharks in the northwestern Atlantic. Such comparatively lower populational parameters suggest that the population of young lemon sharks is fragile at the Atol das Rocas nursery.

Os tubarões enfrentam muitos obstáculos para sobreviver nos primeiros anos de vida e muitas espécies ocupam áreas de berçário. Embora estimativas de sobrevivência, particularmente para jovens, sejam essenciais para acessar, monitorar e manejar efetivamente populações animais, existem poucos cálculos destas estimativas para populações de tubarões e poucas estimativas baseadas em métodos diretos para estes animais em suas áreas de berçário. Métodos de marcação e recaptura foram utilizadosno presente estudo para estimar o tamanho populacional e a sobrevivência de jovens tubarões-limão (Negaprion brevirostris) em uma área de berçário na Reserva Biológica do Atol das Rocas, Brasil. Os indivíduos foram amostrados entre 1999 e 2003 e as estimativas de tamanho populacional variaram entre 12 a 100 indivíduos jovens e a taxa de sobrevivência entre 24 e 54%, com média de 44,6% durante o período de amostragem mais robusto. A população destes tubarões jovens diminuiu ao longo de nosso estudo, ainda que as taxas de sobrevivência tenham aumentado durante o mesmo período. Mesmo um nível moderado de pesca e a remoção de fêmeas maduras em áreas adjacentes podem afetar dramaticamente pequenas populações de tubarões num berçário pequeno e isolado como o Atol das Rocas. As taxas de sobrevivência e tamanho populacional relativamente mais baixos em Rocas podem ser resultado das diferenças nas características físicas deste berçário, comparadas a outros utilizados pela espécie no Atlântico norte-ocidental. Tais parâmetros comparativamente mais baixos no Atol das Rocas sugerem a fragilidade da população jovem de tubarões-limão neste berçário.

Evolutionary potential of a large marine vertebrate: quantitative genetic parameters in a wild population.

Estimating quantitative genetic parameters ideally takes place in natural populations, but relatively few studies have overcome the inherent logistical difficulties. For this reason, no estimates currently exist for the genetic basis of life-history traits in natural populations of large marine vertebrates. And yet such estimates are likely to be important given the exposure of this taxon to changing selection pressures, and the relevance of life-history traits to population productivity. We report such estimates from a long-term (1995-2007) study of lemon sharks (Negaprion brevirostris) conducted at Bimini, Bahamas. We obtained these estimates by genetically reconstructing a population pedigree (117 dams, 487 sires, and 1351 offspring) and then using an "animal model" approach to estimate quantitative genetic parameters. We find significant additive genetic (co)variance, and hence moderate heritability, for juvenile length and mass. We also find substantial maternal effects for these traits at age-0, but not age-1, confirming that genotype-phenotype interactions between mother and offspring are strongest at birth; although these effects could not be parsed into their genetic and nongenetic components. Our results suggest that human-imposed selection pressures (e.g., size-selective harvesting) might impose noteworthy evolutionary change even in large marine vertebrates. We therefore use our findings to explain how maternal effects may sometimes promote maladaptive juvenile traits, and how lemon sharks at different nursery sites may show "constrained local adaptation." We also show how single-generation pedigrees, and even simple marker-based regression methods, can provide accurate estimates of quantitative genetic parameters in at least some natural systems.

A genetic assessment of polyandry and breeding-site fidelity in lemon sharks.

We here employ 11 microsatellite markers and recently developed litter reconstruction methods to infer mating system parameters (i.e. polyandry and breeding-site fidelity) at a lemon shark nursery site in Marquesas Key, Florida. Four hundred and eight juvenile or subadult sharks were genotyped over eight complete breeding seasons. Using this information, we were able to infer family structure, as well as fully or partially reconstruct genotypes of 46 mothers and 163 fathers. Multiple litter reconstruction methods were used, and novel simulations helped define apparent bias and precision of at least some mating system parameters. For Marquesas Key, we find that adult female lemon sharks display high levels of polyandry (81% of all litters sampled) and stronger fidelity to the nursery site than do males. Indeed, few male sharks sired offspring from more than one litter during the course of the study. These findings were quite similar to previous results from another lemon shark nursery site (Bimini, Bahamas), suggesting conserved mating system parameters despite significant variation in early life-history traits (i.e. body size and growth) among sites. The finding of at least some site fidelity in females also supports the need for careful conservation of each nursery.

Are indirect genetic benefits associated with polyandry? Testing predictions in a natural population of lemon sharks.

Multiple mating has clear fitness benefits for males, but uncertain benefits and costs for females. We tested for indirect genetic benefits of polyandry in a natural population, by using data from a long-term genetic and demographic study of lemon sharks (Negaprion brevirostris) at Bimini, Bahamas. To do so, we followed the fates of individuals from six cohorts (450 age-0 and 254 age-1 fish) in relation to their individual level of genetic variation, and whether they were from polyandrous or monoandrous litters. We find that offspring from polyandrous litters did not have a greater genetic diversity or greater survival than did the offspring of monoandrous litters. We also find no evidence of positive associations between individual offspring genetic diversity metrics and our surrogate measure of fitness (i.e. survival). In fact, age-1 individuals with fewer heterozygous microsatellite loci and more genetically similar parents were more likely to survive to age-2. Thus, polyandry in female lemon sharks does not appear to be adaptive from the perspective of indirect genetic benefits to offspring. It may instead be the result of convenience polyandry, whereby females mate multiply to avoid harassment by males. Our inability to find indirect genetic benefits of polyandry despite detailed pedigree and survival information suggests the need for similar assessments in other natural populations.

Reconstruction of parental microsatellite genotypes reveals female polyandry and philopatry in the lemon shark, Negaprion brevirostris.

Because sharks possess an unusual suite of reproductive characteristics, including internal fertilization, sperm storage, relatively low fecundity, and reproductive modes that range from oviparity to viviparity, they can provide important insight into the evolution of mating systems and sexual selection. Yet, to date, few studies have characterized behavioral and genetic mating systems in natural populations of sharks or other elasmobranchs. In this study, highly polymorphic microsatellite loci were used to examine breeding biology of a large coastal shark, the lemon shark, Negaprion brevirostris, at a tropical lagoon nursery. Over six years, 910 lemon sharks were sampled and genotyped. Young were assigned into sibling groups that were then used to reconstruct genotypes of unsampled adults. We assigned 707 of 735 young sharks to one of 45 female genotypes (96.2%), and 485 (66.0%) were assigned to a male genotype. Adult female sharks consistently returned to Bimini on a biennial cycle to give birth. Over 86% of litters had multiple sires. Such high levels of polyandry raise the possibility that polyandry evolved in viviparous sharks to reduce genetic incompatibilities between mother and embryos. We did not find a relationship between relatedness of mates and the number of offspring produced, indicating that inbreeding avoidance was probably not driving pre- or postcopulatory mate choice. Adult male sharks rarely sired more than one litter at Bimini and may mate over a broader geographic area.

The breeding biology of lemon sharks at a tropical nursery lagoon.

Surprisingly little is known about the reproductive behaviour and breeding biology of most shark species, especially in natural populations. Here, we characterize reproductive patterns and use of a natal nursery at Bimini, Bahamas by lemon sharks, Negaprion brevirostris. We systematically and exhaustively sampled young lemon sharks at Bimini annually from 1995 to 2000 and opportunistically sampled adults over the same period. Out of the 897 young sharks sampled, 119 could be assigned to five sampled mothers using microsatellite genotyping. Reproductive females showed strong philopatry to the nursery, returning to Bimini every two years to give birth. Each of these females may rely entirely on the Bimini nursery for recruitment. The protection of known nursery grounds should therefore figure prominently in conservation efforts for large coastal shark species. The reconstruction of paternal genotypes indicates that litters are sired by multiple males, and females mate with different males nearly every breeding cycle. The ubiquitous polyandry reported here raises the possibility that genetic incompatibility and post-copulatory paternity-biasing mechanisms may operate in viviparous sharks.