Evolution of vertebrate gill covers via shifts in an ancient Pou3f3 enhancer.

Whereas the gill chambers of jawless vertebrates open directly into the environment, jawed vertebrates evolved skeletal appendages that drive oxygenated water unidirectionally over the gills. A major anatomical difference between the two jawed vertebrate lineages is the presence of a single large gill cover in bony fishes versus separate covers for each gill chamber in cartilaginous fishes. Here, we find that these divergent patterns correlate with the pharyngeal arch expression of Pou3f3 orthologs. We identify a deeply conserved Pou3f3 arch enhancer present in humans through sharks but undetectable in jawless fish. Minor differences between the bony and cartilaginous fish enhancers account for their restricted versus pan-arch expression patterns. In zebrafish, mutation of Pou3f3 or the conserved enhancer disrupts gill cover formation, whereas ectopic pan-arch Pou3f3b expression generates ectopic skeletal elements resembling the multimeric covers of cartilaginous fishes. Emergence of this Pou3f3 arch enhancer >430 Mya and subsequent modifications may thus have contributed to the acquisition and diversification of gill covers and respiratory strategies during gnathostome evolution.

Novel developmental bases for the evolution of hypobranchial muscles in vertebrates.

BACKGROUND: Vertebrates are characterized by possession of hypobranchial muscles (HBMs). Cyclostomes, or modern jawless vertebrates, possess a rudimentary and superficial HBM lateral to the pharynx, whereas the HBM in jawed vertebrates is internalized and anteroposteriorly specified. Precursor cells of the HBM, marked by expression of Lbx1, originate from somites and undergo extensive migration before becoming innervated by the hypoglossal nerve. How the complex form of HBM arose in evolution is relevant to the establishment of the vertebrate body plan, but despite having long been assumed to be similar to that of limb muscles, modification of developmental mechanisms of HBM remains enigmatic. RESULTS: Here we characterize the expression of Lbx genes in lamprey and hagfish (cyclostomes) and catshark (gnathostome; jawed vertebrates). We show that the expression patterns of the single cyclostome Lbx homologue, Lbx-A, do not resemble the somitic expression of mammalian Lbx1. Disruption of Lbx-A revealed that LjLbx-A is required for the formation of both HBM and body wall muscles, likely due to the insufficient extension of precursor cells rather than to hindered muscle differentiation. Both homologues of Lbx in the catshark were expressed in the somitic muscle primordia, unlike in amniotes. During catshark embryogenesis, Lbx2 is expressed in the caudal HBM as well as in the abdominal rectus muscle, similar to lamprey Lbx-A, whereas Lbx1 marks the rostral HBM and pectoral fin muscle. CONCLUSIONS: We conclude that the vertebrate HBM primarily emerged as a specialized somatic muscle to cover the pharynx, and the anterior internalized HBM of the gnathostomes is likely a novelty added rostral to the cyclostome-like HBM, for which duplication and functionalization of Lbx genes would have been a prerequisite.

The intraspecific diversity of tooth morphology in the large-spotted catshark Scyliorhinus stellaris: insights into the ontogenetic cues driving sexual dimorphism.

Teeth in sharks are shed and replaced throughout their lifetime. Morphological dental changes through ontogeny have been identified in several species and have been correlated with shifts in diet and the acquisition of sexual maturity. However, these changes were rarely quantified in detail along multiple ontogenetic stages, which makes it difficult to infer the developmental processes responsible for the observed plasticity. In this work, we use micro-computed tomography and 3D geometric morphometrics to describe and analyze the tooth size and shape diversity across three ontogenetic stages (hatchling, juvenile, and sexually mature) in the large-spotted catshark Scyliorhinus stellaris (Linnaeus, 1758). We first describe the intra-individual variation of tooth form for each sex at each ontogenetic stage. We provide a tooth morphospace for palatoquadrate and Meckelian teeth and identify dental features, such as relative size and number of cusps, involved in the range of variation of the observed morphologies. We then use these shape data to draw developmental trajectories between ontogenetic stages and for each tooth position within the jaw to characterize ontogenetic patterns of sexual dimorphism. We highlight the emergence of gynandric heterodonty between the juvenile and mature ontogenetic stages, with mature females having tooth morphologies more similar to juveniles' than mature males that display regression in the number of accessory cusps. From these data, we speculate on the developmental processes that could account for such developmental plasticity in S. stellaris.

Presence of the protein indoleamine 2,3-dioxygenase (IDO) in the maternal-fetal interface of the yolk sac placenta of blue shark, Prionace glauca.

Indoleamine 2 3-dioxygenase (IDO) is a protein usually described in mammals, which, among other functions, participates in the maternal-fetal tolerance process. The blue-shark, Prionace glauca (Linnaeus, 1758) is a viviparous placentary species in which the yolk sac develops during the pregnancy, turning into a placenta for matrotrophic nutrition of the embryo. The purpose of this study was to investigate the expression of IDO in the P. glauca maternal-fetal interface along three gestation phases and describe its distribution and the meaning of its presence. The results showed IDO labelling during the yolk sac/placenta development in the ectoderm on the three development phases and in the endoderm at the two first phases. In the uterine epithelium, IDO was observed in the last two phases. These interface tissues are major contact areas between the mother and the semiallogeneic conceptus and this relation could induce an immunological response against the fetus. Therefore, the presence of IDO may indicate that it could have a similar role in the mechanism of maternal-fetal tolerance in the P. glauca placental interface, as described in eutherian mammals.

Ontogenetic Shifts in Brain Size and Brain Organization of the Atlantic Sharpnose Shark, Rhizoprionodon terraenovae.

Throughout an animal's life, species may occupy different environments and exhibit distinct life stages, known as ontogenetic shifts. The life histories of most sharks (class: Chondrichthyes) are characterized by these ontogenetic shifts, which can be defined by changes in habitat and diet as well as behavioral changes at the onset of sexual maturity. In addition, fishes experience indeterminate growth, whereby the brain and body grow throughout the organism's life. Despite a presupposed lifelong neurogenesis in sharks, very little work has been done on ontogenetic changes in the brain, which may be informative about functional shifts in sensory and behavioral specializations. This study quantified changes in brain-body scaling and the scaling of six major brain regions (olfactory bulbs, telencephalon, diencephalon, optic tectum, cerebellum, and medulla oblongata) throughout ontogeny in the Atlantic sharpnose shark, Rhizoprio-nodon terraenovae. As documented in other fishes, brain size increased significantly with body mass throughout ontogeny in this species, with the steepest period of growth in early life. The telencephalon, diencephalon, optic tectum, and medulla oblongata scaled with negative allometry against the rest of the brain throughout ontogeny. However, notably, the olfactory bulbs and cerebellum scaled hyperallometrically to the rest of the brain, whereby these structures enlarged disproportionately as this species matured. Changes in the relative size of the olfactory bulbs throughout ontogeny may reflect an increased reliance on olfaction at later life history stages in R. terraenovae, while changes in the relative size of the cerebellum throughout ontogeny may be indicative of the ability to capture faster prey or an increase in migratory nature as this species moves to offshore habitats, associated with the onset of sexual maturity.

Identification of a nursery area for the critically endangered hammerhead shark (Sphyrna lewini) amid intense fisheries in the southern Gulf of Mexico.

Since the 1980s, there has been growing concern in the Mexican Atlantic regarding high catches of neonate and juvenile sharks in small-scale fisheries. Fishery-dependent data from 1993 to 1994 and 2007 to 2017 and fishers' ecological knowledge from 2017 were used to identify nursery areas for scalloped hammerhead, Sphyrna lewini, in the southern Gulf of Mexico. Catch records and fishing areas of neonates, YOYs, juveniles and adults of S. lewini (N = 1885) were obtained from calcareous and terrigenous regions in the western Yucatan Peninsula. The results suggest that a nursery for scalloped hammerhead is found in the terrigenous region, characterized by relatively shallow and turbid waters due to rivers' discharges. Neonates and YOYs (96% and 86% of their total records, respectively) were commonly found there over the years in May-August in multiple fishing areas identified by fishers, although mainly between isobaths 10-30 m. The enforcement of management measures is necessary because the nursery is located in a region with intense fishing effort.

Holocephalan (Chondrichthyes) dental plates with hypermineralized dentine as a substitute for missing teeth through developmental plasticity.

All extant holocephalans (Chimaeroidei) have lost the ability to make individual teeth, as tooth germs are not part of the embryonic development of the dental plates or of their continuous growth. Instead, a hypermineralized dentine with a unique mineral, whitlockin, is specifically distributed within a dentine framework into structures that give the dental plates their distinctive, species-specific morphology. Control of the regulation of this distribution must be cellular, with a dental epithelium initiating the first outer dentine, and via contact with ectomesenchymal tissue as the only embryonic cell type that can make dentine. Chimaeroids have three pairs of dental plates within their mouth, two in the upper jaw and one in the lower. In the genera Chimaera, Hydrolagus and Harriotta, the morphology and distribution of this whitlockin within each dental plate differs both between different plates in the same species and between species. Whitlockin structures include ovoids, rods and tritoral pads, with substantial developmental changes between these. For example, rods appear before the ovoids and result from a change in the surrounding trabecular dentine. In Harriotta, ovoids form separately from the tritoral pads, but also contribute to tritor development, while in Chimaera and Hydrolagus, tritoral pads develop from rods that later are perforated to accommodate the vasculature. Nevertheless, the position of these structures, secreted by the specialized odontoblasts (whitloblasts), appears highly regulated in all three species. These distinct morphologies are established at the aboral margin of the dental plate, with proposed involvement of the outer dentine. We observe that this outer layer forms into serially added lingual ridges, occurring on the anterior plate only. We propose that positional, structural specificity must be contained within the ectomesenchymal populations, as stem cells below the dental epithelium, and a coincidental occurrence of each lingual, serial ridge with the whitlockin structures that contribute to the wear-resistant oral surface.

Non-lethal assessment of reproductive stage for female blue sharks Prionace glauca using sex steroid hormones.

Plasma concentrations of progesterone (P4 ) and 17ß-oestradiol (E2 ) in juvenile, pre-ovulatory, early, mid- or late pregnancy stages of female blue sharks Prionace glauca were analysed. Concentrations of P4 were significantly higher in pregnant than in non-pregnant individuals, whereas E2 concentrations increased with embryonic and follicular development. A highly accurate (86.1%) random forest classification model was developed to predict shark pregnancy. It is proposed that hormone concentrations could be used for the subsequent non-lethal determination of female P. glauca reproductive state.

Periodicity of the growth-band formation in vertebrae of juvenile scalloped hammerhead shark Sphyrna lewini from the Mexican Pacific Ocean.

The age of 296 juvenile scalloped hammerhead sharks Sphyrna lewini caught by several fisheries in the Mexican Pacific Ocean from March 2007 to September 2017 were estimated from growth band counts in thin-sectioned vertebrae. Marginal-increment analysis (MIA) and centrum-edge analysis (CEA) were used to verify the periodicity of formation of the growth bands, whereas elemental profiles obtained from LA-ICP-MS transect scans in vertebrae of 15 juveniles were used as an alternative approach to verify the age of the species for the first time. Age estimates ranged from 0 to 10+ years (42-158.7 cm total length; LT ). The index of average percentage error (IAPE 3.6%), CV (5.2%), bias plots and Bowker's tests of symmetry showed precise and low-biased age estimation. Both MIA and CEA indicated that in the vertebrae of juveniles of S. lewini a single translucent growth band was formed during winter (November-March) and an opaque band during summer (July-September), a period of faster growth, apparently correlated with a higher sea surface temperature. Peaks in vertebral P and Mn content spatially corresponded with the annual banding pattern in most of the samples, displaying 1.19 and 0.88 peaks per opaque band, respectively, which closely matched the annual deposition rate observed in this study. Although the periodicity of growth band formation needs to be verified for all sizes and ages representing the population of the species in the region, this demonstration of the annual formation of the growth bands in the vertebrae of juveniles should lead to a re-estimation of the growth parameters and productivity of the population to ensure that it is harvested at sustainable levels.

Size frequency, dispersal distances and variable growth rates of young sharks in a multi-species aggregation.

During a mark-recapture survey from November 2014 until April 2017, 333 neonatal and juvenile blacktip reef sharks Carcharhinus melanopterus and 302 neonatal and juvenile sicklefin lemon sharks Negaprion acutidens were tagged and measured at the uninhabited and isolated St. Joseph Atoll (Republic of Seychelles). Both species demonstrated seasonal reproductive synchronicity and relatively large sizes at birth. Despite the extended times at liberty > 2.5 years, the majority of recaptures were found in close proximity to the initial tagging location (< 500 m). Annual growth rates of C. melanopterus (n = 24) and N. acutidens (n = 62) ranged from 6.6 to 31.7 cm year-1 (mean ± SE; 16.2 ± 1.2 cm year-1 ) and 0.2 to 32.2 cm year-1 (11.8 ± 1 cm year-1 ), respectively and are to date the most variable ever recorded in wild juvenile sharks. High abundances of both species coupled with long-term and repeated recaptures are indicative of a habitat where juveniles can reside for their first years of life. However, large variability in annual growth rates in both species may suggest high intra and interspecific competition induced by a possibly resource limited, isolated habitat.

Captive parturition and neonatal growth of the dwarf ornate wobbegong (Orectolobus ornatus de Vis, 1883).

Neonatal predation in multispecies aquarium exhibits can prevent detection of captive breeding by wobbegong sharks. We used ultrasonography and isolation strategies to prevent neonatal predation and maximize survival/growth of the dwarf ornate wobbegong (Orectolobus ornatus de Vis, 1883). We captured seven free-living wobbegongs (two males and five females) and subjected each animal to a health assessment which led to the euthanasia of one female with a retained hook. Ultrasonography showed that females were pregnant, one was preovulatory, and one was in a resting phase. Two females (one pregnant) and one male were placed in isolation in each of two tanks. In October 2006, 25 neonates were born overnight with the two litters placed into separate neonate tanks. Over the ∼6.5-month monitoring period, four neonates with reduced body condition died without premonitory signs resulting in a 63.0% annual survival rate. Finite growth rates did not differ between sexes or litters and averaged (±SE) 12.2 (1.5) cm/year and 156.4 (26.4) g/year. At the cessation of monitoring, total length had increased by ∼30%, whereas total weight had almost doubled with neonatal body condition in line with free-living wobbegongs. Our efficacious, six-step manipulative, the approach should be applicable with all wobbegongs given their reproductive similarities, but we recommend that efforts focus on the dwarf ornate, tasselled and Japanese wobbegongs because all are small in size and have bred in aquaria. Ultimately, this approach should produce self-sustaining aquarium populations, place less reliance on the wild acquisition and provide animals for other aquaria, population restocking, or scientific research.

Elements of time and place: manganese and barium in shark vertebrae reflect age and upwelling histories.

As upper-level predators, sharks are important for maintaining marine food web structure, but populations are threatened by fishery exploitation. Sustainable management of shark populations requires improved understanding of migration patterns and population demographics, which has traditionally been sought through physical and/or electronic tagging studies. The application of natural tags such as elemental variations in mineralized band pairs of elasmobranch vertebrae cartilage could also reveal endogenous and exogenous processes experienced by sharks throughout their life histories. Here, elemental profiles were characterized in vertebrae encompassing complete life histories (birth-to-death) of shortfin mako (Isurus oxyrinchus), common thresher (Alopias vulpinus) and blue shark (Prionace glauca) of known tag and recapture locations in the eastern North Pacific Ocean. All sharks were injected with oxytetracycline at initial capture, released and subsequently recaptured, with individual liberty times ranging from 215 days to 6 years. Vertebral band pairs forming over the liberty intervals were verified by counting the number of band pairs deposited since the oxytetracycline band. Regular oscillations in vertebrae manganese (Mn) content corresponded well with the number of validated band pairs, suggesting that Mn variation could be used to age sharks. Increases in vertebrae barium concentration were correlated with times when individuals occupied areas with high coastal upwelling indices, the timing and spatial intensity of which varied from year to year. Interspecific relationships were probably influenced by behavioural differences in horizontal and vertical habitat use, feeding habits and thermoregulatory physiology. These results indicate that vertebral sclerochronology has the potential to advance our knowledge of elasmobranch life history including age and growth estimation and environmental reconstruction.

Development and evolution of tooth renewal in neoselachian sharks as a model for transformation in chondrichthyan dentitions.

A defining feature of dentitions in modern sharks and rays is the regulated pattern order that generates multiple replacement teeth. These are arranged in labio-lingual files of replacement teeth that form in sequential time order both along the jaw and within successively initiated teeth in a deep dental lamina. Two distinct adult dentitions have been described: alternate, in which timing of new teeth alternates between two adjacent files, each erupting separately, and the other arranged as single files, where teeth of each file are timed to erupt together, in some taxa facilitating similarly timed teeth to join to form a cutting blade. Both are dependent on spatiotemporally regulated formation of new teeth. The adult Angel shark Squatina (Squalomorphii) exemplifies a single file dentition, but we obtained new data on the developmental order of teeth in the files of Squatina embryos, showing alternate timing of tooth initiation. This was based on micro-CT scans revealing that the earliest mineralised teeth at the jaw margin and their replacements in file pairs (odd and even jaw positions) alternate in their initiation timing. Along with Squatina, new observations from other squalomorphs such as Hexanchus and Chlamydoselachus, together with representatives of the sister group Galeomorphii, have established that the alternate tooth pattern (initiation time and replacement order) characterises the embryonic dentition of extant sharks; however, this can change in adults. These character states were plotted onto a recent phylogeny, demonstrating that the Squalomorphii show considerable plasticity of dental development. We propose a developmental-evolutionary model to allow change from the alternate to a single file alignment of replacement teeth. This establishes new dental morphologies in adult sharks from inherited alternate order.

Condition analysis of the Brazilian sharpnose shark Rhizoprionodon lalandii: evidence of maternal investment for initial post-natal life.

The present study analysed the condition factor (K) and the hepato-somatic index (I H ) of the Brazilian sharpnose shark Rhizoprionodon lalandii during its entire life cycle. Discontinuity of liver growth was observed after sexual maturity. High condition values were recorded in the length classes of 300-350 mm (neonates), when the livers represented about 8% of total mass, decreasing until the length classes of 400-450 and 450-500 mm in females and males, respectively. Seasonal analysis exhibited low I H values in the summer, while for K, low values were in spring, indicating greater investment for body growth during spring and for gonad maturation in summer. Such findings indicate that this small coastal shark species accumulates reserve substances in the liver that will be used during reproduction. The decline in I H values in juveniles suggests that reserves provisioned by the mother to the neonate liver can be used in the first months of life.

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.

The Greenland shark: A new challenge for the oxidative stress theory of ageing?

The free radical theory of ageing predicts that long-lived species should be more resistant to oxidative damage than short-lived species. Although many studies support this theory, recent studies found notable exceptions that challenge the generality of this theory. In this study, we have analysed the oxidative status of the Greenland shark (Somniosus microcephalus), which has recently been found as the longest living vertebrate animal known to science with a lifespan of at least 272years. As compared to other species, the Greenland shark had body mass-corrected values of muscle glutathione peroxidase and red blood cells protein carbonyls (metric of protein oxidative damage) above 75 percentile and below 25 percentile, respectively. None of the biochemical metrics of oxidative status measured in either skeletal muscle or red blood cells were correlated with maximum lifespan of species. We propose that the values of metrics of oxidative status we measured might be linked to ecological features (e.g., adaptation to cold waters and deep dives) of this shark species rather to its lifespan.

The tooth, the whole tooth and nothing but the tooth: tooth shape and ontogenetic shift dynamics in the white shark Carcharodon carcharias.

Results from this study of the white shark Carcharodon carcharias include measurements obtained using a novel photographic method that reveal significant differences between the sexes in the relationship between tooth cuspidity and shark total length, and a novel ontogenetic change in male tooth shape. Males exhibit broader upper first teeth and increased distal inclination of upper third teeth with increasing length, while females do not present a consistent morphological change. Substantial individual variation, with implications for pace of life syndrome, was present in males and tooth polymorphism was suggested in females. Sexual differences and individual variation may play major roles in ontogenetic changes in tooth morphology in C. carcharias, with potential implications for their foraging biology. Such individual and sexual differences should be included in studies of ontogenetic shift dynamics in other species and systems.

Reproductive aspects of the Atlantic angel shark Squatina dumeril in the southern Caribbean Sea.

The maturity and reproduction of the Atlantic angel shark Squatina dumeril were assessed using 77 females (29·2-110·4 cm total length; LT ) and 269 males (58·7-108·2 cm LT ) harvested by artisanal gillnetters off Venezuela. The biased sex ratio implied segregation or sex-specific gear selectivity. Based on the development of the reproductive tract, 50% LT at sexual maturity (LT50 , mean ± s.e.) for females and males were estimated at 86·14 ± 0·64 and 81·55 ± 0·12 cm, respectively. Uterine fecundity ranged between one and six and with a maximum embryo size of 25·7 cm LT . Gravid females were observed from August to December, including those close to parturition and while the gestation period was not confirmed, the size of ovarian follicles among some specimens implied protraction. The low fecundity of the species supports close monitoring of catches.

Population biology of the little gulper shark Centrophorus uyato in Lebanese waters.

A total of 38 individuals of the heavily exploited little gulper shark Centrophorus uyato were collected from Lebanese coastal waters using bottom longlines and trammel nets of different meshes at depths ranging from 115 to 600 m between May 2013 and February 2014. Their total lengths were between 45 and 94 cm and their total mass was from 870 to 6700 g. The sex ratio was not significantly different from 1:1, with 20 males and 18 females, but bathymetric sexual segregation occurred. Catch per net setting (CNS) was used as a relative abundance index to detect spatial distribution; trammel nets showed largest CNS ranging from 4·9 to 5·45 kg per unit effort in the north and south, at depths from 120 to 200 m, during spring and summer. The mass-length relationships demonstrated negative allometric growth (b < 3) (males: MT = 0.3585LT2·071 , r2 = 0·94; females: MT = 0.0239LT2·735 , r2 = 0·64). The condition factor as well as the gonado-somatic and hepato-somatic indices of C. uyato in the study area were also calculated. The results are discussed in relation to the distribution, growth and reproduction as well as the management of C. uyato.

Biological observations on the broadfin shark Lamiopsis temminckii (Carcharhiniformes: Carcharhinidae).

Biological information was collected from 214 individuals of the broadfin shark Lamiopsis temminckii measuring 418 to 1782 mm total length, LT . Size at maturity (L50 ) for females and males was estimated at 1430 and 1368 mm LT , respectively, while mature and gravid females were observed from 1350 mm LT with litter sizes 2-8 and size at birth 418-650 mm LT . Analysis of stomach contents revealed a variety of prey, primarily crustaceans (54·0%), teleosts (42·7%) and cephalopods.