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Background The evolutionary processes that shape patterns of species richness in marine ecosystems are complex and may differ between organismal groups. There has been considerable interest in understanding the evolutionary processes that led to marine species richness being concentrated in specific geographical locations. In this study we focus on the evolutionary history of a group of small-to-medium sized sharks known as carpet sharks. While a few carpet shark species are widespread, the majority of carpet shark species richness is contained within a biodiversity hotspot at the boundary of the Indian and Pacific oceans. We address the significance of this biodiversity hotspot in carpet shark evolution and speciation by leveraging a rich fossil record and molecular phylogenetics to examine the prehistoric distribution of carpet sharks. Results We find that carpet sharks species richness was greatest in shallow seas connected to the Atlantic Ocean during the Late Cretaceous, but that there was a subsequent loss of biodiversity in Atlantic waters. Fossil evidence from sites in close geographic proximity to the current center of carpet shark diversity are generally restricted to younger geologic strata. Conclusions From this data we conclude that (1) center of carpet shark biodiversity has shifted during the last 100 million years, (2) carpet sharks have repeatedly dispersed to nascent habitat (including to their current center of diversity), and (3) the current center of carpet shark biodiversity conserves lineages that have been extirpated from this prehistoric range and is a source of new carpet shark species. Our findings provide insights into the roles of marine biodiversity hotspots for higher-tropic level predators and the methods applied here can be used for additional studies of shark evolution.

Karyotyping, traditionally performed using cytogenetic techniques, is indispensable for validating genome assemblies whose sequence lengths can be scaled up to chromosome sizes using modern methods. Karyotype reports of chondrichthyans are scarce because of the difficulty in cell culture. Here, we focused on carpet shark species and the culture conditions for fibroblasts and lymphocytes. The utility of the cultured cells enabled the high-fidelity characterization of their karyotypes, namely 2n = 102 for the whale shark (Rhincodon typus) and zebra shark (Stegostoma fasciatum), and 2n = 106 for the brownbanded bamboo shark (Chiloscyllium punctatum) and whitespotted bamboo shark (C. plagiosum). We identified heteromorphic XX/XY sex chromosomes for the two latter species and demonstrated the first-ever fluorescence in situ hybridization of shark chromosomes prepared from cultured cells. Our protocols are applicable to diverse chondrichthyan species and will deepen the understanding of early vertebrate evolution at the molecular level., Shark cells are difficult to culture in the laboratory as the high osmolality physiological conditions do not reflect those of commonly cultured mammalian or teleost fish cell lines. Uno et al. present a new method of culturing shark fibroblasts and lymphocytes, and characterized the karyotypes of four carpet shark species, enabling whole genome analysis of these species.

Examination of seven specimens of an undescribed species of bamboo shark (Orectolobiformes: Hemiscylliidae), currently referred to as Chiloscyllium sp. 1, from Sri Lanka yielded three new species of 'tetraphyllidean' cestodes, one each in the genera Carpobothrium Shipley et Hornell, 1906, Spiniloculus Southwell, 1925, and Yorkeria Southwell, 1927. Carpobothrium marjorieae Caira, Otto et Jensen sp. n. differs from its three valid congeners in total length, number of testes, and size of the apical sucker of its anterior bothridial flap. Like Carpobothrium eleanorae Koontz et Caira, 2016 it possesses spherical eggs with highly elongate bipolar filaments, but its eggs are conspicuously larger than those of the latter species. Spiniloculus akshayi Caira, Otto et Jensen sp. n. can be distinguished from its five congeners in total length and number of proglottids. Yorkeria sachiniae Caira, Otto et Jensen sp. n. differs from its 16 congeners in number of proglottids and testes, total length, terminal proglottid, cirrus sac, and cephalic peduncle length, hook size, genital pore position, and bothridial shape. The eggs of Y. sachiniae Caira, Otto et Jensen sp. n. are spindle-shaped and bear a single long, polar filament. All three species were found to exhibit microthrix patterns on their scolices like those of the majority of their congeners that have been examined to date with scanning electron microscopy. Microtriches on the distal surfaces of the bothridial pouches of a species of Carpobothrium were characterised for the first time; C. marjorieae Caira, Otto et Jensen sp. n. was found to possess a modified form of gladiate spinithrix on this surface. Among the seven members of the genus, this is the first species of Chiloscyllium Muller et Henle that has been found to host representatives of all three of these 'tetraphyllidean' genera. However, unlike most of its congeners, a representative of the non-hooked genus Scyphophyllidium Woodland, 1927 was not found infecting Chiloscyllium sp. 1.

Exposure to anoxia leads to rapid ATP depletion, alters metabolic pathways and exacerbates succinate accumulation. Upon re-oxygenation, the preferential oxidation of accumulated succinate most often impairs mitochondrial function. Few species can survive prolonged periods of hypoxia and anoxia at tropical temperatures and those that do may rely on mitochondria plasticity in response to disruptions to oxygen availability. Two carpet sharks, the epaulette shark (Hemiscyllium ocellatum; ES) and the grey carpet shark (Chiloscyllium punctatum; GCS) display different adaptive responses to prolonged anoxia: while the ES enters energy conserving metabolic depression, the GCS temporarily elevates its haematocrit prolonging oxygen delivery. High-resolution respirometry was used to investigate mitochondrial function in the cerebellum, a highly metabolically active organ that is oxygen sensitive and vulnerable to injury after anoxia/re-oxygenation (AR). Succinate was titrated into cerebellar preparations in vitro, with or without pre-exposure to AR, then the activity of mitochondrial complexes was examined. Like most vertebrates, GCS mitochondria significantly increased succinate oxidation rates, with impaired complex I function post-AR. In contrast, ES mitochondria inhibited succinate oxidation rates and both complex I and II capacities were conserved, resulting in preservation of oxidative phosphorylation capacity post-AR. Divergent mitochondrial plasticity elicited by elevated succinate post A/R parallels the inherently divergent physiological adaptations of these animals to prolonged anoxia, namely the absence (GCS) and presence of metabolic depression (ES). Since anoxia tolerance in these species also occurs at temperatures close to that of humans, examining their mitochondrial responses to AR could provide insights for novel interventions in clinical settings.

We compared the hematological responses of wild and captive populations of two closely related sharks to a standardized anoxic challenge and during a 12 hr recovery period in normoxia: the epaulette shark (Hemiscyllium ocellatum, Bonnaterre, 1788) and the grey carpet shark (Chiloscyllium punctatum, Muller and Henle, 1838). Compared to normoxic controls, a significant increase in hematocrit (captive 22.3%; wild 35.9%) coupled with a decline in mean corpuscular hemoglobin concentration occurred in epaulette sharks indicating erythrocyte swelling in response to anoxia. However, the grey carpet shark had a significantly increased hematocrit (captive 27.2%; wild 29.3%), erythrocyte count (captive 37.6%; wild 46.3%) and hemoglobin concentration (captive 31.9%; wild 31.5%), suggesting a release of erythrocytes into the circulation and/or hemoconcentration in response to anoxia. Plasma glucose concentrations were maintained in both wild and captive epaulette sharks and in wild grey carpet sharks during anoxia but increased significantly after 2 hr of re-oxygenation (epaulette: captive 55.8%; wild 50.1%; grey carpet shark: wild 70.3%) and remained elevated for 12 hr. Captive grey carpet sharks had an immediate increase in plasma glucose concentrations after anoxia (96.4%), which was sustained for 12 hr of re-oxygenation. Lactate concentrations significantly increased in captive and wild animals of both species after anoxia, reaching a peak at 2 hr of re-oxygenation. Both species showed significant, yet divergent, hematological changes in response to anoxia and re-oxygenation, which may not only prolong their survival and assist in recovery but also reflect their respective ecophysiological adaptations to the extreme environments that they inhabit. J. Exp. Zool. 311A:422–438, 2009. © 2009 Wiley-Liss, Inc.

We examined gill morphology and ventilatory function in the carpet shark family Parascylliidae using 14 preserved specimens of Parascyllium ferrugineum, P. variolatum, P. collare and Cirrhoscyllium japonicum, and two live specimens of P. ferrugineum and P. variolatum. Morphological examinations revealed eight morphological characteristics related to the fifth gill, based on comparisons with other elasmobranchs, viz. large fifth gill slit without gill filaments, anatomical modifications in the fourth ceratobranchial cartilage and coraco-branchialis muscle, and the hypaxialis muscle associated with the fifth gill arch. Ventilation examinations using dyed seawater and prey items showed different water flows through the gill slits for respiration and prey-capture actions. For respiration, water sucked into the mouth was expelled equally through the first to fourth gill slits via a "double-pump" action, there being no involvement of the fifth gill slit. In prey-capture, however, water sucked into the mouth was discharged only via the widely opened fifth gill slit. This form of water flow is similar to that in other benthic suction-feeding sharks (e.g., Chiloscyllium plagiosum), except for the active water discharge by wide expansion and contraction of the fifth parabranchial cavity. The latter is dependent upon the morphological modifications of the fourth and fifth gill arches, derived phylogenetically as a mechanistic suction specialization in Parascylliidae.

The epaulette shark (Hemiscyllium ocellatum) and the grey carpet shark (Chiloscyllium punctatum) are commonly found in periodically hypoxic environments. The ecophysiological time available for these animals to safely exploit these niches during different seasonal temperatures was examined. The time to loss of righting reflex (T LRR) was examined in response to an open ended anoxic challenge at three seasonal temperatures (23, 25 and 27°C). Ventilation rates were measured in an open ended anoxic challenge at 23°C and during 1.5 h of anoxia followed by 2 h of re-oxygenation at 23 and 25°C. The mean T LRR of epaulette and grey carpet sharks was inversely proportional to temperature. The T LRR was similar between species at 23°C; however, grey carpet sharks had significantly reduced T LRR at higher temperatures. During the standardised anoxic challenge, epaulette sharks entered into ventilatory depression significantly earlier at 25°C. During re-oxygenation, epaulette sharks exposed to anoxia at 23°C had no significant increase in ventilation rates. However, after anoxic challenge and re-oxygenation at 25°C, epaulette sharks showed a significant increase in ventilation rates during re-oxygenation. Grey carpet sharks displayed no evidence of ventilatory depression during anoxia. However, during re-oxygenation, grey carpet sharks had significantly elevated ventilation rates above pre-experimental levels and control animals. These data demonstrate that the anoxia tolerance times of both species were temperature dependent, with a significant reduction in the T LRR occurring at higher temperatures. Epaulette sharks had a significantly greater T LRR at higher temperatures than grey carpet sharks, which did not enter into a ventilatory depression.

This study utilizes retrograde axonal transport of cobaltous-lysine, and conventional silver and Golgi staining techniques to study the abducens motor nucleus innervating the external rectus muscle of the carpet shark. The nucleus consists of 300-400 motoneurons located immediately ventrolateral to the medial longitudinal fasciculus (MLF), distributed over about 1.25 mm in a rostrocaudal direction at the level of exit of the VI nerve. The axons of the motoneurons form seven or eight discrete ventrally directed fascicles which, having exited from the brainstem, group together to form the abducens (VI) nerve. The motoneurons are on average about 16 micron in diameter, are bipolar, and their dendrites have a transverse orientation. Typically one set of dendrites penetrates the MLF and the other set extends ventrally into the reticular formation.

1. Carpet Shark haemoglobin shows co-operative equilibrium CO binding at pH 7.0 (n = 1.8). 2. Flash photolysis shows that the T form of the protein is stabilised at low pH and in the presence of Inositol Hexaphosphate (I.H.P.). 3. At high pH the R form of the protein is stabilised and exhibits kinetic heterogeneity, assigned to different chain reactivities. 4. Stopped flow and flash photolysis under various conditions allows estimates to be made of the association and dissociation rates of CO binding for the two forms of the protein. 5. The functionality of the protein, with regard to CO binding, can be described by a modified two state model.